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J_Autism_Dev_Disord-4-1-2175022

Brief Report: Adults with Mild Autism Spectrum Disorders (ASD): Scores on the Autism Spectrum Quotient (AQ) and Comorbid Psychopathology

While knowledge about symptom presentation of adults with mild ASD, including comorbid psychopathology, is limited, referral of adults with suspected mild PDD is increasing. We report on pilot research investigating whether patients diagnosed with mild ASD (n = 15) and patients who were not diagnosed with ASD (n = 21) differed in terms of (a) AQ scores and (b) Axis I and II disorders, assessed by the SCAN and the IPDE. Additionally, AQ scores were compared with those from non-ASD patients referred to a general outpatient clinic (n = 369). The results showed very few differences between ASD patients and non-ASD patients. Self-report may not differentiate mild ASD patients from non-ASD patients and Axis I and II disorders seem equally prevalent among these two groups. Introduction Although there is consensus (APA, 1994) about DSM IV criteria for children and adolescents with Autistic Disorder and Asperger’s disorder, the largest category, PDDNOS, remains difficult to classify. The latter is even more true for adult patients since the DSM-IV criteria are not formulated with adult age in mind (Gillberg, 1998; Tamtam, 1991; Vermeulen, 2002). The Autism Diagnostic Interview – Revised (ADI-R, Lord, Rutter, & Le Couteur, 1994) and the Autism Diagnostic Observation Schedule-Generic (ADOS-G; Lord et al., 2000) are validated only for children and adolescents. Follow-up studies of adolescents and adults with autism indicate that a slow decrease in symptoms across time occurs in a small group, particularly the less severely affected patients (Seltzer, Shattuck, Abedutto, & Greenberg, 2004). However, there is a lack of knowledge about adults with mild ASD, i.e., Asperger’s Disorder and PDDNOS. In light of this limited knowledge on symptom presentation in adults, the present paper reports on a pilot research-project that was done with two aims: (a) to test the Autism Spectrum Quotient (AQ; Baron-Cohen, Wheelwright, Skinner, Martin, & Clubley, 2001) as an instrument for screening adult non-mentally retarded patients for ASD. We hypothesised that scores would be higher for an ASD group than for two non-ASD comparison groups; (b) there is “a clinical feeling” that mild ASD symptoms often mix with other disorders (e.g., Obsessive Compulsive Disorder or Schizoid Personality Disorder). Therefore, in patients referred to an ASD expertise-centre, prevalences of Axis I and II comorbid disorders were estimated to test the hypothesis that certain diagnoses are more prevalent among ASD patients than non-ASD patients. Method Participants Two groups of patients were investigated; the first group (n = 36) consisted of patients referred to the “Autism Team North Netherlands” (ATN). The ATN is the ASD outpatient center of expertise for the three Northern provinces of the Netherlands (1.5 million inhabitants). On a yearly basis, about 80 new adult patients are referred for diagnostic evaluation and (advice for) treatment. Since the ATN is a “third line” center of expertise, all patients were referred by psychiatrists and psychologists, working in the field of adult (but not forensic) psychiatry. Only patients with parents who were able to give the required information were included in the present study. They were 18 years or older, with an estimated IQ higher than 80. Over a span of 10 months from February 2003 through October 2003, 60 eligible patients were asked to participate, of which 35 patients and their parents consented. The patients were randomly assigned to two experienced (child) psychiatrists (CK or EH) for diagnostic assessment. The second group (n = 369) consisted of patients from the same region, who were referred in this same period to the general outpatient psychiatric clinic of the University Medical Centre Groningen. This group from the general outpatient clinic (GOC) enabled us to compare the scores on the AQ screening instrument. Instruments The clinical standardized diagnostic protocol that was used in the intake of ATN-referred patients consisted of a semi-structured interview (taken from at least one of the parents), and observations from the ADI-R, ADOS-G, as well as clinical experience. Actual ASD symptoms were translated in the interview as much as possible to the adult-world situation. Questions about the past were used to reconstruct the developmental history of the patient as adequately as possible. The information derived from the diagnostic protocol was subsequently used to score the DSM-IV criteria on ASD. Intelligence was assessed by using a short version of the “Groninger Intelligentie Test” (GIT, Luteijn, 1966). For classification of PDDNOS, the minimal amount of positive DSM-IV criteria was two, of which at least one derived from the social interaction domain. For Asperger’s Disorder at least two items on the social domain and at least one item on the stereotypic domain needed to be present with no early delay of language. For High Functioning Autism at least six items were needed to be present, with at least two items on the social domain and at least one positive item on each of the other two domains, with onset of abnormal functioning before the age of three years. The reliability of the diagnostic protocol was tested by assessing a series of seven patients simultaneously by two experienced (child) psychiatrists (CK and EH). These independent classifications differed only on one patient (PDDNOS versus non-ASD). The seven patients were not included in the present study, which started after having determined diagnostic reliability. The AQ was translated in Dutch and was named AQ-D (Dutch). This self-report screening questionnaire has 50 items, which score on 5 domains of behavior: social skill, attention switching, attention to detail, communication, and imagination. Each domain has a maximum score of 10 (for details on this instrument, see Baron-Cohen et al., 2001) The AQ-D was completed by the patient before the clinical procedure started. The investigators were blind to the outcome of AQ-D scores. Internal consistency reliability estimates (Cronbach’s α) for AQ subscales were acceptable for the ASD-group but somewhat on the lower side for the non-ASD groups. Averaged across the five subscales Cronbach’s α was .67 for the ASD group, .62 for the non-ASD group referred to the ATN, and .59 for the GOC group. For the total scale internal consistency reliability was acceptable in all three groups: i.e., .85, .82, and .79, respectively. Present and life-time comorbidity were assessed by using the Schedules for Clinical Assessment in Neuropsychiatry (SCAN-2.1, Giel & Nienhuis, 1996; World Health Organisation, 1992) and the International Personality Disorder Examination (IPDE, Loranger et al., 1994). IQ, SCAN and IPDE assessments were done by a psychologist (JB) who was blind to the outcome of the ASD psychiatric diagnosis. JB was formally trained by the Groningen WHO Training Center for administering the SCAN. Statistical Analysis Chi-square tests and analyses of variance were conducted to analyze possible differences between the groups. Results Fifteen of the 36 patients were diagnosed with ASD. In this ASD-group, 10 patients classified as PDDNOS, 4 patients as Asperger’s Disorder and 1 patient as High Functioning Autism. The number of positive DSM-IV items ranged from 2 to 8, with an average score of 3.8. Table 1 shows gender, age, and IQ of ASD and non-ASD patients. Also included in this table are gender and age of the GOC patients. The significant differences between the groups were (a) the younger age of the ASD patients relative to the other patients groups; (b) the younger age of the non-ASD group referred to the ATN relative to the GOC patients; and (c) the predominance of males in the ATN-referred patients compared with the GOC patients. Table 1Characteristics of participantsDiagnosisASD (n = 15)No ASD (n = 21)General outpatient clinic (n = 369)Male/femaleam = 12, f = 3m = 18, f = 3m = 180, f = 189 Average ageb (range, sd)22 years (18–24, 5)27 years (18–55, 9)35 years (14–73, 11)Mean IQ (sd)104 (10)105 (12)a Difference between GOC group and non-ASD group (chi-square = 14.2, df = 1, p < 0.0001); difference between GOC group and ASD group (chi-square = 5.8, df = 1, p < 0.05); no difference between ASD group and non-ASD groupb GOC group differs from non-ASD group (p < 0.01) and from ASD group (p < 0.0001); no difference between ASD group and non-ASD group Table 2 shows AQ-D sub-domain and total scores for the three groups. Generally, there were no significant differences between the three groups. The one significant difference was on the sub-domain Communication between the ASD group and the general outpatient group. Further, within the ASD group, average AQ-D scores varied with the severity of ASD: High Functioning Autism: 31 (n = 1); Asperger’s Disorder: 24 (n = 4), and PDDNOS: 21 (n = 10). In light of the small sample sizes the latter differences were not tested for statistical significance. Table 2Mean scores AQ-D (sd) and ANOVA tests for differences in mean scoresASD (n = 15)No ASD (n = 21)General outpatient clinic (n = 369)Social skill5.0 (2.8)4.2 (2.6)4.1 (2.6)Attention switching5.1 (2.6)5.3 (2.4) 5.3 (2.3) Attention to detail3.5 (2.3)4.4 (2.2)4.2 (2.3)Communication*4.5 (2.1)3.5 (2.2)2.8 (1.8)Imagination4.4 (2.4)4.3 (2.0)3.5 (1.9)Total22.5 (8.4)21.8 (7.6)19.9 (7.0)* Significant difference between ASD group and GOC group at p < 0.01 Table 3 reports Axis I diagnoses in ASD and non-ASD patients as assessed by the SCAN. With one exception, there were no differences in either Axis I or Axis II diagnoses between the two groups. This held for both actual and lifetime (not reported) Axis I diagnoses. The one exception was that more patients were diagnosed with actual psychotic disorder NOS in the non-ASD group than in the ASD group. When we aggregated separate categories into more broad diagnostic categories, i.e., anxiety disorders, psychotic disorders, and mood disorders, there were no differences between the two groups in past-month or lifetime diagnoses either, except for actual psychotic disorders (results not shown). Table 3SCAN-derived actual (past month) DSM IV Axis I diagnosesASD (N = 15)n (%)No ASD (N = 21)n (%)No disorder7 (47)7 (33)Mood disorder with psychotic symptoms2 (13)–Mood disorder without psychotic symptoms2 (13)3 (14)Substance abuse 3 (20)2 (10)Sleep disorders4 (27)6 (29)Psychotic disorder NOS–4 (19)*Schizophrenia––Social Phobia3 (20)4 (19)Panic attacks/Agoraphobia2 (13)1 (5)Other anxiety disorders1 (7)–Obsessive compulsive disorders1 (7)1 (5)Other disorders1 (7)2 (10)* Significant difference between ASD group and no ASD group at p < 0.05 Table 4 reports Axis II diagnoses as assessed by the IPDE. ASD and non-ASD patients did not differ on individual Axis 2 diagnoses. Also, when we tested whether ASD and non-ASD groups differed with regard to the total number of patients with any complete, partial, or either of these two, Axis 2 disorders (bottom two rows, Table 4), we found no difference between the two groups. Table 4IPDE derived Axis II diagnoses by ASD statusASD (N = 15)No ASD (N = 21)Completen (%)Partialn (%)Completen (%)Partialn (%)Paranoïd–––1 (5)Schizoïd 1 (7)2 (13)1 (5)1 (5)Schizotypical–1 (7)––Antisocial–1 (7)––Borderline 1 (7)––1 (5)Avoidant 1 (7)1 (7)2 (10)–Obsessive compulsive––3 (14)1 (5)Personality disorder NOS1 (7)2 (13)3 (14)1 (5)Any personality disorder3 (20)6 (40)8 (38)3 (14)Any personality disorder partial or complete7 (47)10 (48) Discussion Thus far, publications about screening instruments for adult ASD use the Autism-Spectrum Quotient (AQ; Baron-Cohen et al., 2001) or the Autism Spectrum Disorder in Adults Screening Questionnaire (ASDASQ; Nylander & Gillberg, 2001). With the help of the latter instrument, Nylander & Gillberg (2001) and Chang et al. (2003) estimated a 1.4 and 0.6 % ASD prevalence in a general psychiatry outpatient clinic for adults. The AQ was investigated by Baron-Cohen in 2001 in Asperger’s Disorder or High Functioning Autism patients and compared with several subgroups in the general population, and was tested again in 2005 at the national diagnostic clinic for Asperger’s Disorder (Woodbury-Smith, Robinson, Wheelwright, & Baron-Cohen, 2005). The AQ differentiated the ASD patients adequately from healthy controls, and ASD patients from non-ASD patients. The present study tried to replicate the findings of Baron-Cohen. However, our results showed that the AQ-D did not differentiate between an ASD group (predominantly PDDNOS) and two non-ASD patient groups. Only for the communication domain, the ASD patients had higher scores relative to the general outpatient group. It is of interest to mention here that this domain was also one of the two domains that differentiated parents of autistic patients from healthy controls (Bishop et al., 2004). The most probable explanation of this discrepant outcome is the fact that the Dutch patients were less severely affected than the British patients. The ASD patients but also the non-ASD patients in the Baron-Cohen papers had higher total mean scores on the AQ (for ASD: 35.8 and 35.6 and for non-ASD: 26.2) than our patients (ASD: 22.5, non-ASD and general outpatients: 21.8 and 19.9). By comparison, British, non-psychiatric, control groups typically have total scores of around 16–17 (Baron-Cohen et al., 2001). Interestingly, the mean score on the recent Japanese version of the AQ was 29.4 in ASD patients and 22.2 in non-psychiatric controls (Kurita, Koyoma, & Osada, 2005). The Japanese authors, like we, reasoned that part of the Japanese patients may have been less severely affected relative to the British patients. They also argued that it is possible that autistic related behavior as assessed by the AQ is more prevalent in the Japanese population than in the British. In the absence of the actual diagnoses of the non-ASD patients in both the British and one of the Dutch samples, it remains difficult to pinpoint why scores between the British and Dutch non-ASD patients (26.2 and 21.8/19.9, respectively) differ. The absence of differences between scores of the AQ-D in the present study suggests that there might be “ASD-like” symptoms, as felt by the patients themselves, present in the non-ASD patients in the ATN-referred group and in the patients of the general outpatient clinic, and revealed by self-report. Indeed, the individuals in the non-ASD group were referred to the ATN for the possible presence of an ASD diagnosis, so ASD-like symptoms must have been voiced by these patients even if these could not be diagnosed as such on the basis of the standardized diagnostic protocol. In sum, although there are still many open questions, for example about differences in scores cross-culturally, the present data suggest that self-report questionnaires are not adequate for differentiating less severe ASD patients from other patient groups. Another study is needed to investigate the merits of an alternative approach to this problem; i.e., a parents/caregivers questionnaire as a screening tool for adult ASD. Our second aim was to look into possible differences in comorbidity of Axis I and Axis II DSM IV classifications in ASD and non-ASD groups. This because of the notion that mild ASD symptoms often mix with symptoms of other disorders such as Obsessive Compulsive Disorder or Schizoid Personality Disorder. There is a scarcity of research on this topic. For example, (comorbid) diagnoses of the British patients were not reported in the paper of Woodbury-Smith et al. (2005). For the Dutch, ATN-referred group, however, we took a systematic approach to diagnosing DSM IV Axis I and II disorders. The results indicated that, except for Psychotic Disorder NOS which was diagnosed in roughly 20% of the non-ASD group and not in the ASD group, there were no significant differences in the pattern of diagnoses between the ASD and non-ASD patients. Possibly, the difference in Psychotic Disorder partly accounts for the referral to the ATN and for the “ASD-like” symptoms in these patients as discussed in the above paragraph. However, the general picture indicated by our data is that clinical accounts of a relatively high prevalence and/or a specific profile of comorbid disorders in the ASD group relative to the non-ASD group could not be confirmed. There are important limitations to this study, among which is the small number of patients in the ATN-referred group. This major drawback precludes any definite conclusions. Of note is further that the ASD patients in this investigation belonged to the less-severe side of the spectrum. This makes it difficult to compare results with the investigations by Baron-Cohen and collegues. Clinical experience indicates that ASD patients, compared to non-ASD patients, benefit more from treatment by structuring, long-term repetitive treatment, and adaptations of the environment. This may hold even more when comorbid conditions are present. Since the prevalence of patients with less severe ASD is relatively high compared to the more severe ASD categories, efficient and valid screening of this group and charting possible comorbidity is consequently an even more important issue. The present study provided a modest contribution in improving our knowledge in this direction; clearly, much more research is needed.

Int_J_Biochem_Cell_Biol-1-5-1906734

Cytosolic and ER J-domains of mammalian and parasitic origin can functionally interact with DnaK

Both prokaryotic and eukaryotic cells contain multiple heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) proteins, which cooperate as molecular chaperones to ensure fidelity at all stages of protein biogenesis. The Hsp40 signature domain, the J-domain, is required for binding of an Hsp40 to a partner Hsp70, and may also play a role in the specificity of the association. Through the creation of chimeric Hsp40 proteins by the replacement of the J-domain of a prokaryotic Hsp40 (DnaJ), we have tested the functional equivalence of J-domains from a number of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1). An in vivo functional assay was used to test the functionality of the chimeric proteins on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant Escherichia coli strain (OD259). The Hsp40 chimeras containing J-domains originating from soluble (cytosolic or endoplasmic reticulum (ER)-lumenal) Hsp40s were able to reverse the thermosensitivity of E. coli OD259. In all cases, modified derivatives of these chimeric proteins containing an His to Gln substitution in the HPD motif of the J-domain were unable to reverse the thermosensitivity of E. coli OD259. This suggested that these J-domains exerted their in vivo functionality through a specific interaction with E. coli Hsp70, DnaK. Interestingly, a Hsp40 chimera containing the J-domain of ERj1, an integral membrane-bound ER Hsp40, was unable to reverse the thermosensitivity of E. coli OD259, suggesting that this J-domain was unable to functionally interact with DnaK. Substitutions of conserved amino acid residues and motifs were made in all four helices (I–IV) and the loop regions of the J-domains, and the modified chimeric Hsp40s were tested for functionality using the in vivo assay. Substitution of a highly conserved basic residue in helix II of the J-domain was found to disrupt in vivo functionality for all the J-domains tested. We propose that helix II and the HPD motif of the J-domain represent the fundamental elements of a binding surface required for the interaction of Hsp40s with Hsp70s, and that this surface has been conserved in mammalian, parasitic and bacterial systems. 1 Introduction While many classes of molecular chaperones exist, members of the heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) families form chaperone pairs that are amongst the most ubiquitous (Fink, 1999). The diverse cellular processes involving these chaperones include the correct folding of nascent polypeptide chains, prevention of protein denaturation and misfolding during cellular stress, degradation of proteins, protein translocation, and quaternary assembly/disassembly (Hennessy, Nicoll, Zimmermann, Cheetham, & Blatch, 2005b). The major molecular chaperone, Hsp70, consists of an N-terminal ATPase domain and a C-terminal substrate-binding domain. The affinity of Hsp70 for protein client is modulated by ATP binding and hydrolysis. In the ATP bound state, affinity of the substrate-binding domain for the client protein is low and exchange rates are high. Hydrolysis of ATP to ADP results in high affinity for the substrate and low exchange rates, effectively locking the substrate into the binding pocket (Schmid, Baici, Gehring, & Christen, 1994). This integral step in assisted protein folding is directly modulated by the binding of Hsp40 proteins (Cheetham, Jackson, & Anderton, 1994; Liberek, Marszalek, Ang, Georgopoulos, & Zylicz, 1991). Furthermore, there is evidence that some Hsp40 proteins bind client protein first and then target client to Hsp70 (Han & Christen, 2003; Suh, Lu, & Gross, 1999). Hsp40s are defined by the presence of an approximately 70 amino acid region known as the J-domain, which is essential for interaction with Hsp70. The J-domain is a highly conserved α-helical structure that interacts with the Hsp70 ATPase domain and possibly also with the Hsp70 substrate-binding domain (Auger & Roudier, 1997; Hennessy et al., 2005b; Suh et al., 1999). Hsp40s are divided into three groups based on their possession of domains in addition to the J-domain (Cheetham & Caplan, 1998). Type I Hsp40s contain four primary domains: an N-terminal J-domain, a glycine/phenylalanine (GF)-rich region, a zinc finger domain and a C-terminal domain. Type I Hsp40s have been shown to bind protein substrates at their C-terminal domain and to have independent chaperone activity by inhibiting denaturation and aggregation (Langer et al., 1992; Rüdiger, Schneider-Mergener, & Bukau, 2001). Type II Hsp40s contain an N-terminal J-domain, a GF-rich region and a C-terminal domain. Type III Hsp40s contain the J-domain, and this may occur at any position within the protein. Other than the J-domain, the type III Hsp40s are highly divergent in size, sequence and structure and tend to serve highly specialized functions. Specific Hsp40-Hsp70 partnerships have been identified that are dedicated to the correct folding of distinct subsets of client proteins. We and others have proposed that the J-domain makes important contributions to the affinity and specificity of binding of a specific Hsp40 protein to its partner Hsp70 (Garimella et al., 2006; Hennessy et al., 2005b). The structures of the J-domain from six Hsp40 and Hsp40-like proteins have been published: E. coli DnaJ (Huang, Flanagan, & Prestegard, 1998; Pellecchia, Szyperski, Wall, Georgopoulos, & Wüthrich, 1996); human HDJ1 (Qian, Patel, Hartl, & McColl, 1996); E. coli Hsc20 (Cupp-Vickery & Vickery, 2000); the large T antigen from murine polyomavirus (Berjanskii et al., 2000); the large T antigen from SV40 in conjunction with the retinoblastoma tumour suppressor (Kim, Ahn, & Cho, 2001); and bovine auxilin (Gruschus, Greene, Eisenberg, & Ferretti, 2004; Jiang et al., 2003). The J-domain structures reveal the presence of four α-helices (helices I–IV) and a loop region between helices II and III. Helix I is usually seen as a short helix in type I Hsp40s. However, although there are a number of highly conserved hydrophobic residues in helix I, the tertiary structure of helix I, as seen from X-ray and NMR studies, becomes divergent in types II and III Hsp40s. The helices II and III are structurally conserved in all known J-domains, in particular helix II which bears an overall positive charge and is thought to interact with the negatively charged underside of the ATPase domain of Hsp70. Of particular importance is the HPD tripeptide that resides in the transhelix loop between helices II and III. Alteration of these residues always results in loss of functional interaction between Hsp40 and Hsp70 (Genevaux, Wawrzynow, Zylicz, Georgopoulos, & Kelley, 2001; Laufen et al., 1999; Mayer, Laufen, Paal, McCarty, & Bukau, 1999; Tsai & Douglas, 1996; Wittung-Stafshede, Guidry, Horne, & Landry, 2003). Apart from the HPD motif, the other amino acids on the J-domain of Hsp40 proteins that are involved in the binding to a partner Hsp70 are less precisely defined. However, as a result of our work (Hennessy, Cheetham, Dirr, & Blatch, 2000; Hennessy, Boshoff, & Blatch, 2005a) and that of other researchers (Garimella et al., 2006; Genevaux, Schwager, Georgopoulos, & Kelley, 2002; Genevaux et al., 2003; Lu & Cyr, 1998; Suh et al., 1999), other residues and regions outside the HPD motif, especially helices II, III and IV, are gradually being implicated in the general binding and specificity of interaction of Hsp40 proteins with Hsp70 proteins. The high resolution structure of the E. coli DnaJ J-domain suggested that J-domain stabilization occurred through a buried core of hydrophobic residues, primarily Ile9, Leu10, Val12, Ile21, Ala53 and Leu57 (Hill, Flanagan, & Prestegard, 1995; Pellecchia et al., 1996; Szyperski, Pellecchia, Wall, Georgopoulos, & Wuthrich, 1994). Amino acid sequence alignment analysis of over 200 type I Hsp40s showed that Tyr7, Ala53 and Leu57 were conserved in over 98% of all sequences, and that Leu10 was absolutely conserved in all sequences (Hennessy et al., 2000). Tyr7 projects upward in the E. coli DnaJ J-domain tertiary structure and potentially makes contacts with residues of helices II, III and IV. Leu10 projects toward the helix II–helix III inter-helical space, potentially interacting with residues of helices II and III. Therefore, Tyr7 and Leu10 may be critical in ensuring the stability of the helix-loop structure of helix II–helix III for presentation to Hsp70 (Hennessy et al., 2005a). Arg26 has been shown to be critical for J-domain function in E. coli DnaJ and Agrobacterium tumefaciens DnaJ (Agt DnaJ), and has been proposed to be part of a network of residues on helix II (and possibly helix III) that form an Hsp70-binding site on the J-domain (Genevaux et al., 2002; Hennessy et al., 2005a,b). In addition, Genevaux et al. (2002) described Tyr25 of E. coli DnaJ as a candidate catalytic residue that potentially comprises part of this binding site. His33 of the HPD motif in the loop region has been extensively documented as being a critical residue in regulation of Hsp70 ATPase stimulation, and substitution of this residue results in loss of functional interaction between Hsp40 and Hsp70 (Genevaux et al., 2002; Kelley & Georgopoulos, 1997; Laufen et al., 1999; Mayer et al., 1999; Tsai and Douglas, 1996). Genevaux et al. (2002) revealed that a glycine substitution at Arg36 (Lys36 in Agt DnaJ) resulted in loss of J-domain function and suggested that the critical cluster for J-domain function is not only the HPD tripeptide but could also include Arg36 and Asn37 to form the critical HPD-R/K-N pentapeptide. Helix III appears to contain a so-called KFK motif (Hennessy et al., 2000), and a number of amino acid substitutions have been conducted on this motif. In particular, the F47A substitution in the J-domain of E. coli DnaJ resulted in loss of in vivo function (Genevaux et al., 2002), suggesting this was an essential amino acid for J-domain structure and function, potentially by forming interactions with His33 of the HPD motif. Interestingly, an F47L substitution in Agt DnaJ had no detectable effect on its in vivo function, suggesting that the Leu residue could sufficiently contribute to the contacts originally made by the Phe residue (Hennessy et al., 2005a). The highly conserved Leu57 of helix III projects into the J-domain interior and is likely to be a key residue in holding helices II and III together. Furthermore, experimental evidence has suggested that Leu57 was essential for J-domain function (Hennessy et al., 2005a). While residues of helices II and III and the loop region linking the two helices are crucial to J-domain function (Pellecchia et al., 1996), it has been proposed that the residues of helix IV are not essential to the co-chaperone function of DnaJ (Genevaux et al., 2002). However, recent studies involving the J-domain of Agt DnaJ identified residues on helix IV that were important for its in vivo function, suggesting a structural or functional role of this helix in other DnaJ homologues, potentially in the enhancement of the affinity or specificity of interactions with Hsp70 (Hennessy et al., 2005a,b). Asp59 and Arg63 are part of a conserved charged cluster of residues in helix IV, with Arg63 being part of the conserved QKRAA motif on helix IV of the J-domain of DnaJ. Data from studies on E. coli DnaJ (Suh et al., 1999) and Agt DnaJ (Hennessy et al., 2005a) suggested that substitutions of Asp59 and Arg63 partially disrupted the structure and function of these proteins, and recently it has been suggested that helix IV may contribute to the specificity of J-domains for their Hsp70 partners (Garimella et al., 2006; Hennessy et al., 2005b). Hsp40 proteins are not completely interchangeable with respect to their interaction with distinct Hsp70s. The cytosolic Hsp70s, Hsc70, yeast Ssa1 and E. coli DnaK, are not interchangeable with BiP with respect to protein translocation into the endoplasmic reticulum (Brodsky, Hamamoto, Feldheim, & Schekman, 1993; Wiech, Buchner, Zimmermann, Zimmermann, & Jakob, 1993). Furthermore, E. coli DnaJ is capable of stimulating the ATPase activity of mammalian Hsc70, whereas mammalian Hdj1 is incapable of stimulating the ATPase activity of DnaK (Minami, Höhfeld, Ohtsuka, & Hartl, 1996). Hence, the J-domain may contain sequence and structural features that mediate the specificity of binding between Hsp40s and partner Hsp70s, and a number of J-domain swapping experiments have been conducted to establish the elements of specificity (reviewed in Hennessy et al., 2005b). In general, J-domains appear to be interchangeable when they are derived from Hsp40 proteins that interact with functionally equivalent or homologous Hsp70 proteins, or are involved in similar cellular processes (Deloche, Kelley, & Georgopoulos, 1997; Genevaux et al., 2001). However, J-domains appear to be less interchangeable when derived from Hsp40 proteins that are involved in very different cellular processes; for example, interchanging J-domains of type I Hsp40s with those of membrane-bound Hsp40s (Schlenstedt, Harris, Risse, Lill, & Silver, 1995) or viral Hsp40-like proteins such as the T antigen (Kelley and Georgopoulos, 1997; Sullivan et al., 2000). This suggests that two broad classes of J-domains may have evolved; those J-domains that have evolved to specifically interact with Hsp70s involved in assisted protein folding, and those J-domains that have evolved to specifically interact with Hsp70s involved in more specialized cellular processes. To date no systematic analysis has been conducted on the interchangeability of J-domains between all the types I, II and III Hsp40-like proteins from any one cell type, compartment or system. In this study we have conducted domain swapping of the J-domains from a selection of divergent Hsp40s of mammalian and parasitic origin (malarial Pfj1 and Pfj4, trypanosomal Tcj3, human ERj3, ERj5, and Hsj1, and murine ERj1) in an attempt to identify system specific and common factors in Hsp40–Hsp70 interactions. The similarities and differences in the structure and function of Hsp40s of parasites and their hosts have yet to be determined, and therefore from this broader perspective we were interested in a comparative analysis of the J-domains of parasitic and human origin. An in vivo functional assay was used to assess the ability of the J-domains to substitute for the J-domain of a prokaryotic type I Hsp40. Furthermore, the functional importance of specific residues was addressed through single amino acid substitution analysis. The data suggested that cytosolic and ER J-domains of mammalian and parasitic origin can interact with DnaK using a common mechanism, and that a fundamental binding surface appears to be conserved in J-domains of Hsp40s of mammalian, parasitic and bacterial origin. 2 Materials and methods 2.1 Materials E. coli OD259 (MC4100 araD139 Δara714 ΔcbpA::kan dnaJ::Tn10-42) was kindly provided by Dr. Olivier Deloche (University of Geneva, Switzerland). The pGEX-4T-ERj1, pGEX-4T-ERj3 and pGEX-4T-ERj5 plasmids encode mouse ERj1, human ERj3 and human ERj5. The pET23b-Tcj3 construct encoding Tcj3 has been described (Edkins, Ludewig, & Blatch, 2004), while the pCMV-Tag3a(Hsj1a) construct encodes Hsj1a. Mutagenesis was performed using the QuikChange site directed mutagenesis kit (Stratagene, USA) as per the manufacturer's instructions. Mutagenesis and PCR primers were synthesised by IDT (USA) and Inqaba Biotec (SA). 2.2 Creation of the Agt DnaJ chimera proteins The pQE30-derived pRJ30 vector containing the Agt DnaJ coding sequence (Hennessy et al., 2005a) served as a base vector for all domain swapping and subsequent mutagenesis. A silent mutation encoding a BstBI restriction site was introduced directly downstream of the J-domain at residue Phe74 of Agt DnaJ to produce vector pRJ-B (Fig. 1A). Removal of the coding region of the Agt DnaJ J-domain and insertion of the respective coding regions of the J-domains under investigation into the Agt DnaJ coding region backbone was achieved through use of the BstBI restriction site and a BamHI restriction site immediately upstream of the start of Agt DnaJ coding region (Fig. 1A). E. coli codon optimized versions of the Plasmodium falciparum Pfj1 J-domain (Fig. 1B) and full length Pfj4 (Nicoll et al., 2006) were synthesized using Polymerase Chain Reaction (PCR) assembly (Stemmer, Crameri, Ha, Brennan, & Heyneker, 1995). The gene design process was also utilized to introduce the BamHI and BstBI sites to allow subsequent domain swapping. Subcloning of the coding regions for the Pfj1 (residues 60–128) and Pfj4 (residues 1–79) J-domains into the pRJ-B vector resulted in the creation of the expression constructs encoding the chimera Pfj1-J-Agt-DnaJ and Pfj4-J-Agt-DnaJ, respectively. The coding region for the Tcj3 (residues 1–74) J-domain was amplified by PCR from pET23b-Tcj3 with BamHI and BstBI restriction sites to allow subcloning into pRJ-B. Insertion of the coding region for the Tcj3 J-domain into the pRJ-B vector resulted in the creation of the expression construct encoding the Tcj3-J-Agt-DnaJ chimera. Similarly, the coding regions for the J-domains of Hsj1a (residues 1–72), ERj1 (residues 56–128), ERj3 (residues 23–97) and ERj5 (residues 35–100) were PCR amplified from pCMV-Tag3a (Hsj1a), pGEX-4T-ERj1, pGEX-4T-ERj3 and pGEX-4T-ERj5, respectively, and subcloned into pRJ-B to give expression constructs encoding Hsj1-J-Agt-DnaJ, ERj1-J-Agt-DnaJ, ERj3-J-Agt-DnaJ and ERj5-J-Agt-DnaJ. All mutations were produced by the whole-plasmid linear amplification approach using complementary oligonucleotides (QuikChange site directed mutagenesis kit, Stratagene, USA). Primers were designed so as to introduce or eliminate a restriction endonuclease site to facilitate the identification of successful mutants by restriction analysis. Mutations were confirmed by subsequent DNA sequencing. 2.3 In vivo complementation assays Complementation assays were performed in the thermosensitive E. coli dnaJ cbpA strain, OD259 (Deloche et al., 1997; Kelley and Georgopoulos, 1997). Agt DnaJ has been shown to functionally replace CbpA and DnaJ in E. coli OD259 at 40 °C similarly to E. coli DnaJ (Hennessy et al., 2005a). Thus E. coli OD259 cells exogenously producing Agt DnaJ from a pQE30-based plasmid served as the positive control for the functional in vivo assays. Substitution of His33 of the HPD motif of Agt DnaJ is known to abolish interactions of the protein with DnaK (Hennessy et al., 2005a), therefore E. coli OD259 cells exogenously producing Agt DnaJ-H33Q from a pQE30-based plasmid served as the negative control. Plasmids were transformed into competent E. coli OD259, and single colonies were used to inoculate 5 ml yeast-tryptone (YT) broth containing 100 μg/ml ampicillin for plasmid selection and 50 μg/ml kanamycin for strain selection. The cultures were grown overnight at 30 °C, before being diluted 1:100 with YT broth containing 100 μg/ml ampicillin and 50 μg/ml kanamycin, and grown further at 30 °C until an A600 of approximately 2.0 was reached. Cultures were diluted to an A600 of 0.3 and serial dilutions were performed to a final dilution of 1 × 10−8. Aliquots (3 μl) of each of these dilutions were spotted onto agar plates containing 50 μM IPTG. Plates were grown at 30 °C, 40 °C and 42 °C, respectively, to determine the ability of Agt DnaJ, Agt DnaJ-H33Q and the chimera proteins to reverse the thermosensitivity of E. coli OD259. 2.4 Western analysis for the detection of chimeras Western analysis was performed on whole cell lysates of E. coli OD259 and its transformants producing His6-tagged Agt DnaJ and chimera proteins. Proteins were resolved on a 12% (acrylamide, w/v) sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to nitrocellulose membrane. Proteins of interest were detected using a mouse anti-His antibody (Amersham, UK) and horseradish-peroxidase-conjugated anti-mouse secondary antibody (Amersham, UK) using chemiluminescence-based detection (ECL Western blotting kit, Amersham, UK). Images were captured using a Chemidoc chemiluminescence imaging system (Biorad, USA). 2.5 Binding studies with ERj1-J and BiP The pGEX-4T-1-based construct containing the coding region for the J-domain of murine ERj1 (originally called Mtj1; Brightman, Blatch, & Zetter, 1995) fused downstream of the coding region for glutathione S-transferase (GST) has been described previously (Dudek et al., 2002). The heterologous overproduction and purification of the GST-ERj1-J fusion protein was carried out as described previously for other GST-J-domain fusion proteins (Tyedmers et al., 2000). Hamster BiP was generously provided by Dr. Martin Jung (Universität des Saarlandes, Germany). For the ERj1-BiP pull-down binding assays, purified GST-ERj1-J and its mutant derivatives were buffer exchanged into phosphate buffered saline (PBS) at 4 °C so as to remove GSH from the purified protein. A sample (80 μl) of a 50% slurry of GSH-Sepharose beads was equilibrated in PBS (200 μl). Sufficient GST-ERj1-J was added to the GSH-Sepharose suspension to give 0.5 μM final concentration. The suspension was incubated for 1 h at 4 °C to allow binding of GST-ERj1-J to the GSH-beads, before washing twice with PBS (300 μl). For the binding reaction, the bead-bound GST-ERj1-J was reconstituted in 200 μl of PBS, with and without ATP (2 mM), and with BiP (0.5 μM), and binding allowed to occur for 1 h at 4 °C. The beads were washed twice with PBS (200 μl), and the proteins eluted from the beads by treatment with SDS-PAGE sample treatment buffer (40 μl), and analysed by SDS-PAGE. Surface plasmon resonance spectroscopy was carried out in a BIAlite upgrade system. Monoclonal goat anti-GST-antibodies (BIACORE, Uppsala, Sweden) were immobilized on a sensor chip CM5 (BIACORE, Uppsala, Sweden) by amine coupling according to the manufacturer's protocol. The sensor chip was equilibrated with running buffer (phosphate buffered saline containing 3 mM KCl, 1 mM MgCl2, 0.1% Tween 20 and 2 mM ATP). GST was bound to the immobilized antibodies in the reference cell, while the GST-ERj1-J fusion proteins were immobilized separately in the measuring cell (400 response units; flow rate 5 μl/min). Subsequently, solutions containing increasing concentrations of purified BiP (0.25–2 μM) were passed over the chip in the presence of ATP (flow rate 20 μl/min). Each BiP application was followed by the application of running buffer until baseline was reached. The analysis of the data were carried out using the BIAevaluation software version 2.2.4. 3 Results 3.1 Bioinformatic analysis of the J-domain and the identification of structurally and functionally important residues The J-domain sequences analyzed in this work covered a wide range of diverse Hsp40 types (Fig. 2). Agt DnaJ, a prokaryotic type I Hsp40 that has 57% identity to the E. coli DnaJ, was previously shown to be able to reverse the thermosensitivity of a dnaJ cbpA mutant E. coli strain (OD259), suggesting that it was capable of functionally replacing DnaJ and CbpA (Hennessy et al., 2005a). Furthermore, Agt DnaJ-H33Q was unable to reverse the thermosensitivity of E. coli OD259, suggesting that Agt DnaJ reversed the thermosensitivity of E. coli OD259 through J-domain-based regulation of the chaperone activity of E. coli DnaK (Hennessy et al., 2005a). Due to the consistency and reproducibility of the results produced in functional in vivo complementation assays of E. coli OD259 producing Agt DnaJ, this protein was chosen as the type I Hsp40 backbone molecule for the creation of chimeras. It should be noted that J-domains chimeras were created using the E. coli DnaJ backbone, and the same complementation trends were observed as for the Agt DnaJ chimeras; however, the results were not as reproducible (data not shown). The lack of reproducibility of complementation assays using the E. coli DnaJ chimeras could possibly be related to the toxic effects that have been observed for the overproduction of E. coli DnaJ (Pröls et al., 2001). Tcj3 is a type I Hsp40 from Trypanosoma cruzi, the protozoan causative agent of Chagas’ disease (Edkins et al., 2004), while Pfj1 is a type I Hsp40 and Pfj4 is a type II Hsp40 identified from the protozoan malarial parasite P. falciparum (Watanabe, 1997). While no experimental evidence exists for the subcellular localization of Tcj3, the knowledge-based subcellular localization program PSORTII (Nakai & Kanehisa, 1992) predicted a cytosolic localization. Pfj1 has a potential mitochondrial import signal (RRKVCS; Watanabe, 1997), while Pfj4 was predicted to have a nuclear localization signal by PSORTII. HSJ1 is a human Hsp40 that is preferentially expressed in neuronal cells (Cheetham, Brion, & Anderton, 1992). Two forms of HSJ1 are found in vivo, both of which contain an identical J-domain. One form is localized to the cytosolic face of the ER (HSJ1b) while the other is cytosolic and nuclear (HSJ1a) (Chapple & Cheetham, 2003). ERj1 (also called Mtj1) is a mouse type III Hsp40 that has been shown to be enriched in microsomal and nuclear fractions of murine cells (Brightman et al., 1995). In particular it has been shown to be present in the endoplasmic reticulum (ER) in close association with active ribosomes (Dudek et al., 2002, 2005), and to interact with the ER Hsp70, BiP (Chevalier, Rhee, Elguindi, & Blond, 2000; Dudek et al., 2002). ERj3 (also called HEDJ) is a soluble Type I/II Hsp40 that has a Cys-rich region instead of a typical Cys-repeat region, and been shown to be present in the ER lumen and to functionally interact with BiP (Bies et al., 1999, 2004; Yu & Haslam, 2005). ERj5 (also called JPDI) is a type III Hsp40 that contains thioredoxin motifs characteristic of a protein disulfide isomerase (Cunnea et al., 2003; Hosoda, Kimata, Tsuru, & Konho, 2003). ERj5 is located in the ER lumen and has been shown to interact with BiP in an ATP-dependent manner (Cunnea et al., 2003). The sequences of the J-domains used in this study were aligned, phylogenetically analysed, and key conserved residues identified and highlighted on the three-dimensional structure of the E. coli DnaJ J-domain (Fig. 2). We derived a J-domain consensus sequence for the highly conserved sequences, and also identified the positions of highly conserved charged residues (Fig. 2A). The numbering used for residues in the ensuing text will be the J-domain consensus numbering which is equivalent to the E. coli DnaJ J-domain numbering (Fig. 2A). All of the J-domain sequences exhibited conservation of most of the residues previously identified to be conserved from a study of over 200 J-domains (Hennessy et al., 2000). Phylogenetic tree analysis (Fig. 2B) revealed the relative relatedness of the J-domains of the prokaryotic Hsp40s (Agt DnaJ and E. coli DnaJ), certain parasitic Hsp40s (Pfj1 and Tcj3) and certain mammalian Hsp40s (HSJ1 and ERj3; ERj1 and ERj5). The J-domain of Pfj4 appeared to be relatively divergent from all the other J-domains. In this study, the amino acid residues targeted for substitution were chosen based on their conservation (Fig. 2A), predicted orientation in three dimensions (Fig. 2C), and predicted structural and functional roles as determined from previous studies on DnaJ from our group and others (Genevaux et al., 2002; Hennessy et al., 2005a). 3.2 Characterization of the chimeras Each of the Hsp40 J-domain chimera-encoding plasmid constructs were transformed into E. coli OD259, and the ability of the chimeras to reverse the thermosensitivity of this strain was assessed by comparing growth at 30 °C and 40 °C, respectively. For the E. coli OD259 cells producing Agt DnaJ (positive control), growth at 30 °C occurred up to the highest dilution (10−8), indicating that Agt DnaJ was not toxic to the cells. Furthermore, the growth profile observed at 40 °C was similar to that observed at 30 °C, suggesting that Agt DnaJ was able to reverse the thermosensitivity of E. coli OD259 (Fig. 3). This result was consistent with our previously published data that demonstrated Agt DnaJ was able to replace DnaJ and CbpA in E. coli OD259 and reverse the thermosensitivity of this strain (Hennessy et al., 2005a). As expected, E. coli OD259 producing Agt DnaJ-H33Q (negative control), was able to grow at 30 °C, but was unable to grow at 40 °C. E. coli OD259 transformants producing the chimeras HSJ1-J-Agt-DnaJ, Pfj1-J-Agt-DnaJ, Pfj4-J-Agt-DnaJ, Tcj3-J-Agt-DnaJ, ERj3-J-Agt-DnaJ and ERj5-J-Agt-DnaJ were all able to grow at 30 °C, and reversed the thermosensitivity of this strain at 40 °C similar to the control (Fig. 3). Furthermore, the H33Q mutant version of all these chimeras disrupted their in vivo functionality (Table 1 and data not shown). These data indicated that each of the J-domains were able to functionally replace the J-domain of Agt DnaJ in this prokaryotic system, and most likely exerted their in vivo function through a functional interaction with E. coli DnaK. The only chimera that was unable to reverse the thermosensitivity of E. coli OD259 was the ERj1-J-Agt-DnaJ. Western analysis to determine the protein production levels of the various chimeras in E. coli OD259, demonstrated that the chimeric proteins were produced and detectable in all the transformed strains (Fig. 3). This indicated that the inability of the ERj1-J-Agt-DnaJ chimera to reverse the thermosensitivity of E. coli OD259 was due to a lack of functionality rather than an absence of protein. 3.3 Pfj1, Pfj4 and Hsj1 J-domain mutants The ability of this wide range of J-domains to functionally substitute for the Agt DnaJ J-domain in the E. coli system suggested that an underlying commonality existed in their mode of interaction with the E. coli DnaK. Therefore, a comparative analysis was conducted of the functional effects of the substitution of certain conserved amino acids of the J-domains of these proteins (Table 1). Production of the helix I mutant protein Pfj4-J-Agt-DnaJ-Y7A in E. coli OD259 partially reversed its thermosensitivity indicating this protein was partially functional in this assay, while the Pfj4-J-Agt-DnaJ-L10A was unable to reverse the thermosensitivity of this strain indicating it was non-functional in this assay (Table 1). Similar results were observed for the equivalent helix I mutant proteins of HSJ1-J-Agt-DnaJ. The helix II mutant protein Pfj4-J-Agt-DnaJ-R26A was partially functional (Table 1), while the HSJ1-J-Agt-DnaJ-R26A, Pfj1-J-Agt-DnaJ-R26A, and Pfj1-J-Agt-DnaJ-F25A mutant proteins were all non-functional (Table 1). As discussed in the previous section, the H33Q substitution in the loop HPD motif was found to disrupt the functionality of all the chimeras. By contrast, substitution of the basic residue of the so-called HPD-R/K-N pentapeptide in Pfj4-J-Agt-DnaJ-K36A, did not affect its functionality in the in vivo assay (Table 1). The so called KFK motif of helix III is replaced by a KMA motif in the J-domain of Pfj1. Substitution of Met47 with Phe47 (the KMA to KFA mutation) resulted in a mutant Pfj1-J-Agt-DnaJ protein that was only partially functional in the in vivo assay (Table 1). Further substitution of the KMA motif of the Pfj1 J-domain to produce KMK and KFK, resulted in mutant Pfj1-J-Agt-DnaJ proteins that were fully functional. The RFK motif on helix III of the J-domain of Pfj4 was modified to RAK, and the resultant mutant protein Pfj4-J-Agt-DnaJ-F47A was found to be non-functional. Furthermore, substitution of the conserved helix III L57 residue in Pfj4-J-Agt-DnaJ-L57A resulted in a non-functional mutant protein (Table 1). The roles of key conserved residues (e.g. D59) and motifs (e.g. the “QKRAA” motif) in helix IV were also investigated in this study (Table 1). The equivalent helix IV mutant proteins Pfj4-J-Agt-DnaJ-D59A and Hsj1-J-Agt-DnaJ-D59A were both found to be functional. The Pfj4-J-Agt-DnaJ-R63A mutant protein (Arg63 of the KRRRK motif of Helix IV in Pfj4, corresponding to the QKRAA motif of Agt DnaJ) was found to be functional, while in contrast the Pfj1-J-Agt-DnaJ-K63A mutant protein (substitution of Lys63 of the KKKEF motif of Helix IV in Pfj1) was only partially functional. Mutation of the Pfj1 KKKEF motif to the more conserved QKRAA motif produced a functional Pfj1-J-Agt-DnaJ mutant protein. Production of all the mutant chimeric proteins was detected by Western analysis, suggesting that the inability of certain mutant proteins to reverse thermosensitivity was the result of a lack of protein functionality rather than a lack of protein production (Table 1). As has been previously observed (Hennessy et al., 2005a), levels of protein production in E. coli OD259 often vary between mutants and do not necessarily correlate with levels of functional recovery. 3.4 ERj1 J-domain Since it was not possible to characterize the ERj1 J-domain any further through an in vivo assay, an in vitro analysis was conducted. Using a GST-ERj1-J fusion protein and mutant derivatives, GST-ERj1-J-R26A and GST-ERj1-JH33Q, affinity pull-down assays were conducted to evaluate the interaction of the J-domains with BiP in the presence and absence of ATP (Fig. 4A). While GST-ERj1-J functionally interacted with BiP in an ATP-dependent manner, GST-ERj1-J-H33Q had no significant interaction with BiP since the levels of BiP detected in the pull-down assay were equivalent in the presence and absence of ATP. The GST-ERj1-J-R26A protein exhibited an ATP-dependent interaction with BiP, but at reduced levels compared to GST-ERj1-J. The interaction of GST-ERj1-J-R26A was further assessed using surface plasmon resonance spectroscopy (SPR; Fig. 4B). Using a range of BiP concentrations (0.25–2.0 μM), the apparent affinity of GST-ERj1-J-R26A for BiP was found to be reduced by 50% compared to the affinity of GST-ERj1-J for BiP (Fig. 4B). The results of the in vitro study on GST-ERj1-J-R26A were consistent with the results of the in vivo assays on mutant J-domain chimeras, where we have found that substitution of the equivalent residue at position 26 on helix II of other J-domains disrupted their functionality in the in vivo assay (J-domains of Pfj1, Pfj4 and HSJ1). 4 Discussion Apart from the J-domain of the integral-membrane-bound ER Hsp40, ERj1, our findings suggested that all the J-domains tested exerted their functionality in the in vivo assay through a specific interaction with E. coli Hsp70, DnaK. Therefore, we propose that cytosolic and ER J-domains of mammalian and parasitic origin can interact with DnaK using a common mechanism. Furthermore, we found that substitution of a basic residue at position 26 of the helix II of the J-domain compromised functionality in all the J-domains in which this mutation was investigated (J-domains of Pfj1, Pfj4, HSJ1 and ERj1). Similar results have been found for E. coli DnaJ (Genevaux et al., 2002) and Agt DnaJ (Hennessy et al., 2005a,b). Therefore, we propose that this basic residue of helix II together with the HPD motif of the loop region are important elements of a fundamental binding surface required for J-domain-based Hsp40-Hsp70 interaction. This fundamental binding surface appears to be conserved in J-domains of Hsp40s of mammalian, parasitic and bacterial origin. 4.1 J-domain interchangeability The number of different Hsp40 proteins in any organism generally outweighs the number of different Hsp70 proteins identified; for example, there are 6 Hsp40s and 3 Hsp70s in E. coli (Hennessy et al., 2005a), while there are 43 Hsp40s and 6 Hsp70s in P. falciparum (Matambo, Odunuga, Boshoff, & Blatch, 2004; Sargeant et al., 2006). This suggests the Hsp70 protein is the more promiscuous member of the Hsp40/Hsp70 pair and that the Hsp40 protein provides the specificity to this chaperone partnership. The J-domain may provide some of the molecular determinants of this specificity (Garimella et al., 2006; Hennessy et al., 2005a,b). In this study we have analyzed the J-domains from a number of diverse Hsp40s to assess their ability to functionally replace the J-domain of an exogenously produced Agt DnaJ in the prokaryotic E. coli OD259 in vivo complementation system. Interestingly, while Pfj1, Pfj4 and Tcj3 were all of parasitic origin and HSJ1, ERj3, and ERj5 were of mammalian origin, the J-domains of each protein were able to functionally replace the J-domain of the prokaryotic Agt DnaJ J-domain. In contrast, the J-domain of ERj1, the only integral membrane-bound Hsp40 tested, was unable to substitute for the J-domain of Agt DnaJ in the in vivo complementation assay. Two other membrane-bound ER Hsp40s, ERj2 and ERj4, have been investigated previously by others. Using a yeast complementation assay, Schlenstedt et al. (1995) found that the J-domains from the yeast Hsp40s Sis1p (cytosolic) and Mdj1p (mitochondrial lumen) were unable to functionally replace the J-domain of the integral membrane-bound Hsp40, Sec63p (ERj2 homologue). By contrast, the J-domain of the membrane-associated ERj4 (also called Mdg1) was shown to functionally replace the J-domain of E. coli DnaJ in an E. coli complementation assay (Pröls et al., 2001). Membrane-bound Hsp40s have also been investigated in prokaryotic systems. Kluck et al. (2002) showed that the J-domain of a membrane-bound E. coli Hsp40, DjLC, could not replace that of cytosolic E. coli DnaJ. This result may reflect that fact that DjLC was shown to interact with a specialized E. coli Hsp70 called HscC, which did not interact with E. coli DnaJ (Kluck et al., 2002). The lack of interaction of the J-domain of ERj1 with DnaK in the in vivo complementation assay, suggested that it may have reduced affinity for Hsp70s other than its partner BiP. It is well established that ERj1 can functionally interact with BiP, and that this association is important for the role of ERj1 in protein translocation into the ER (Chevalier et al., 2000; Dudek et al., 2002). It has also been shown that the ERj1 J-domain could stimulate E. coli DnaK ATPase activity in vitro, however, only at levels greater than that required for similar stimulation by E. coli DnaJ (Chevalier et al., 2000). This finding suggests that the ERj1 J-domain has low affinity and specificity for DnaK, and is consistent with our findings. It would be worthwhile conducting a gain-of-function analysis on the ERj1 J-domain by changing certain divergent residues of the ERj1 J-domain to those found in other J-domains (e.g. Phe9 to Ile or Leu), and evaluating which residues promote a functional interaction with DnaK using both in vitro and in vivo assays. 4.2 Targeted mutagenesis 4.2.1 Helix I Published J-domain structures have revealed that while helix IV is relatively mobile, helix I is relatively fixed in position (Berjanskii et al., 2000; Cupp-Vickery & Vickery, 1997, 2000; Huang et al., 1998; Pellecchia et al., 1996; Qian et al., 1996). The side-chains of the helix I residues Tyr7 and Leu10 may be involved in core contacts with helices I, II and III thereby stabilizing the J-domain scaffold for interactions with Hsp70 (Berjanskii et al., 2000). Substitution of these residues to Ala resulted in partial and complete reduction of J-domain functionality, respectively, in both the Pfj4-J-Agt-DnaJ and HSJ1-J-Agt-DnaJ chimeras. This was consistent with the findings of Hennessy et al. (2005a,b) where a lack of functionality was shown for Y7A and L10A mutant Agt DnaJ proteins in the same in vivo functional assay. These substitutions most likely destabilized the J-domain, resulting in a destabilized mutant protein with compromised functionality. Therefore, while these data suggest that Y7 and L10 of helix I have primarily a structural role, they do not exclude the possibility that these residues have an indirect functional role ensuring that the J-domain is correctly orientated for interactions with Hsp70 (Hennessy et al., 2005a). 4.2.2 Helix II and the loop Based on structural data from the J-domain of polyomavirus T antigen (Berjanskii et al., 2000), residues Tyr25 and Lys26 of the positively charged helix II of the J-domain of DnaJ were predicted to form part of the interaction surface that contacts the ATPase domain of DnaK. When substituted, these residues were found to disrupt in vivo functionality of E. coli DnaJ and Agt DnaJ (Genevaux et al., 2002; Hennessy et al., 2005a,b). Our findings were consistent with these data, in that the R26A mutations in the J-domains of Pfj1-J-Agt-DnaJ, Pfj4-J-Agt-DnaJ, HSJ1-Agt-DnaJ and GST-ERj1-J, and the Y25A mutation of the J-domain of Pfj1-J-Agt-DnaJ J-domain disrupted functionality. Lys36 (equivalent to Arg36 in E .coli DnaJ) is part of the functionally critical pentapeptide (HPD-R/KN) predicted by Genevaux et al. (2002). While it has been shown that a R36G mutation in E. coli DnaJ resulted in loss of function (Genevaux et al., 2002), we have demonstrated that a K36A mutation in Pfj4-J-Agt-DnaJ had no effect on its in vivo functionality. This suggested that while Arg36 appeared to be a structurally and/or functionally critical residue in E. coli DnaJ, it did not appear to be critical in all Hsp40s. 4.2.3 Helix III Phe47 of the KFK motif has been predicted to be important in J-domain function and was previously the only residue in close proximity to His33 that abolished Hsp40 function when mutated (Genevaux et al., 2002). Since Phe47 protrudes into the inter-helical space of helices II and III, it is tempting to propose that it has a largely structural role in maintaining the orientation of the J-domain and particularly the loop region. Indeed, it has been suggested that Phe47 may sterically constrain the movement of the helix II–helix III inter-helical loop (Genevaux et al., 2002); however, its conserved nature and proximity to His33 also suggest a potential mechanistic role in Hsp40–Hsp70 interactions (Hennessy et al., 2000; Landry, 2003). Here we have demonstrated that the mutation F47A in the Pfj4-J-Agt-DnaJ chimera completely abolished functionality of the chimera, as is seen for the equivalent mutation in E. coli DnaJ, yeast Ydj1 and an Hdj1 E. coli DnaJ chimera (Genevaux et al., 2002; Hennessy et al., 2005a,b; Johnson & Craig, 2000). While the function of Phe47 is unclear, these findings demonstrate that this residue is necessary for the functioning of certain diverse Hsp40s. Interestingly, the F47L substitution in Agt DnaJ had no adverse effect on its in vivo functionality (Hennessy et al., 2005a,b), suggesting that the minimum requirement for functionality at this position was the presence of a large hydrophobic residue. This was further supported by our data in which the M47F substitution of the KMA motif of the Pfj1-J-Agt-DnaJ chimera preserved functionality of the mutant protein, albeit at reduced levels. This was similarly the case when the neighbouring hydrophobic residue Ala48 was mutated to the more frequently encountered conserved residue Lys48, and when the double mutation M47F/A48K (producing a KFK motif) was performed on the Pfj1-J-Agt-DnaJ chimera. Leu57 is predicted to be a key residue involved in maintaining the structural integrity of helices II and III, since its side chain projects directly into the helix II–helix III cleft (Hennessy et al., 2005a,b). The L57A mutation of the Pfj4-J-Agt-DnaJ chimera, and the L57S mutation of Agt DnaJ (Hennessy et al., 2005a,b) abolished in vivo functionality of these Hsp40 proteins, suggesting that Leu57 was indeed required for the structure and function of the J-domain. 4.2.4 Helix IV Residue Asp59 is predicted to be of structural importance in the J-domain due to its location on the border of helices III and IV. Structural data suggests that helix IV is mobile, and its exact functional location is unclear. Hennessy et al. (2005a,b) showed that a D59A mutation of Agt DnaJ abolished its in vivo functionality; however since the mutant protein was undetectable, the inability of the protein to function in vivo may be attributed to a lack of protein production. Conversely, Genevaux et al. (2002) demonstrated that a T58A/D59A double mutation in E. coli DnaJ had no effect on its in vivo functionality. Our data showing that the Pfj4-J-Agt-DnaJ-D59A and Hsj1-J-Agt-DnaJ-D59A chimeras were functional in the in vivo assay, were consistent with the E. coli DnaJ data (Genevaux et al., 2002). Arg63 is part of the conserved QKRAA motif of helix IV that has been proposed to play a role in enhancing the affinity or specificity of interactions with Hsp70 (Garimella et al., 2006; Hennessy et al., 2005a,b). In this study, Pfj1-J-Agt-DnaJ-K63A was fully functional in the in vivo assay, and Pfj4-J-Agt-DnaJ-R63A was partially functional. While this result suggested that a basic residue at position 63 was not absolutely critical for J-domain-based interactions with Hsp70, it was consistent with its proposed role in the affinity or specificity of interaction. 4.3 Conclusion and future perspectives This study has analysed cytosolic and ER Hsp40s of mammalian and parasitic origin, and found that certain key features of the J-domain appear to be fundamental to the function of all the J-domains studied, and perhaps to the function of J-domains in general. Interestingly, the differences appear to be subtle (e.g. the effects of the Y7A and R26A substitutions on the function of the J-domains of Hsj1 versus Pfj4), and may reflect slight differences in affinity or specificity of these J-domains for DnaK. These differences need to be probed further using quantitative in vitro assays, and incorporating an analysis of the less-conserved J-domain residues shown by NMR analysis to occur at J-domain-DnaK/Hsp70 binding interfaces. Furthermore, the possibility that there are specialized features unique to the J-domains of integral-membrane-bound Hsp40s needs to be investigated more extensively.

Arch_Sex_Behav-3-1-1914258

Sexual Decision-Making in HIV-Positive Men Who Have Sex with Men: How Moral Concerns and Sexual Motives Guide Intended Condom Use with Steady and Casual Sex Partners

Determinants of intended condom use with steady and casual sex partners were examined among Dutch HIV-positive men who have sex with men (MSM) (N = 296). Given the proposition that safer sex behavior among HIV-positive people is a form of prosocial behavior, the present study extended the general framework of the Theory of Planned Behavior with Schwartz’s norm-activation theory and tested the assumption that personal norms would mediate the effects of other psychosocial factors on intended condom use for anal sex. In addition, it was hypothesized that, depending on the context in which sex occurs, specific motives for unprotected anal sex may have a negative influence on intended condom use and, as such, undermine a prosocial tendency to practice safer sex. Therefore, we also investigated the influence of sexual motives for unprotected anal sex on intended condom use with steady and casual sex partners. Results indicated that the Theory of Planned Behavior adequately predicted condom use intentions (for casual sex partners and steady sex partners, the explained variance was 52% and 53%, respectively). However, our proposed model of sexual decision-making significantly improved the prediction of behavioral intentions. For steady and casual sex partners, the assumption of the mediating role of personal norms on condom use intention was confirmed empirically. Additionally, sexual motives for unprotected anal sex exerted, as expected, a direct, negative effect on condom use intention with casual sex partners. The implications of the findings for future research and the development of HIV-prevention programs for HIV-positive MSM are discussed. Introduction Studies on the effects of HIV counseling and testing show that most individuals who are tested HIV-positive respond by reducing their sexual risk behavior (Weinhardt, Carey, Johnson, & Bickman, 1999). Nevertheless, it has been found that approximately one third of HIV-positive men who have sex with men (MSM) engaged in unprotected anal sex in the last two to three months (for a review, see Kalichman, 2000). Moreover, increased prevalence rates of gonorrhea and syphilis have been reported among HIV-positive and HIV-negative MSM in several western regions (e.g., Anon, 2002; Macdonald et al., 2004; Van de Laar & Op de Coul, 2003). These findings suggest a rise in unprotected anal sex among HIV-negative and HIV-positive MSM. Given that at least a subgroup of HIV-positive MSM, for various reasons, engage in risky sexual behavior, an understanding of why this occurs remains a matter of significant public healthy concern. Studies have been conducted that examined possible antecedents of (un)safe sexual behavior in HIV-positive MSM (Crepaz & Marks, 2002). One of the factors that has been identified as promoting sexual behavior change is perception of risk, i.e., awareness that unprotected sex increases the risk for HIV infection and STDs. Accordingly, studies have indicated that HIV-positive MSM who believe that HIV-superinfection and other STDs may have negative consequences for their own health are more likely to use condoms for anal sex (Colfax et al., 2004; McConnell, Grant, & Greenwood, 2002). However, other studies suggest that a person’s own risk of HIV-superinfection and STDs is not a key concern. Of great importance is the finding that safer sex in HIV-positive MSM is primarily motivated by concerns about the risks for the other and that concerns about one’s own risks are subordinate (Keogh, Weatherburn, & Stephens, 1999; Van Kesteren, Hospers, Kok, & Van Empelen, 2005). The notion that HIV-positive MSM engage in safer sex because of concerns about the welfare of sex partners can be characterized as a form of prosocial behavior. Prosocial behavior “represents a broad category of acts…that are defined as generally beneficial to other people” (Penner, Dovidio, Piliavin, & Schoeder, 2005, p. 366). If HIV-positive MSM indeed engage in safer sex to benefit others, or more specifically, are motivated to prevent harm to others, it is of particular importance to understand why this is the case and to determine which factors promote such behavior. The literature offers several explanations to account for prosocial behavior. One explanation for a wide range of moral and prosocial behaviors, which has received considerable attention, is the so-called personal standards approach (e.g., Manstead, 2000). This approach emphasizes how internalized, self-reinforced standards, such as altruism, can promote prosocial behavior as people strive to maintain a positive self-image or achieve their ideals (Penner et al., 2005). Indeed, some qualitative studies suggest that internalized values or personal standards play an important role in the practice of safer sexual behavior. For example, Nimmons and Folkman (1999) showed that HIV-positive MSM described moral and altruistic values in regard to the need to practice safer sex. A study by Wolitski, Bailey, O’Leary, Gómez, and Parsons (2003) found that many HIV-positive MSM perceived that they had a particular responsibility for protecting their partners and that this perception influenced safer sexual decision-making. Participants cited altruism and self-imposed standards as the motivations underlying their beliefs about personal responsibility. Similarly, Van Kesteren et al. (2005) found that a greater concern about potential HIV transmission to sex partners was related to enhanced feelings of personal responsibility for safer sex. While these studies have contributed greatly to a preliminary understanding of the role of intrinsic motivation in safer sexual behavior, the factors that may motivate HIV-positive MSM to adopt safer sex practices for the protection of others warrants further examination. This study represents such an attempt, examining social-psychological factors of condom use in the context of steady and casual sex. For this reason, we combined the Theory of Planned Behavior (Ajzen, 1991), a well-established framework used to understand behavior in a broad range of contexts, with the norm-activation theory (Schwartz, 1977; Schwartz & Howard, 1981), a framework specifically developed to understand prosocial behavior. In spite of the supposed importance of prosocial motivation in safer sexual behavior, it is possible that other motivations (e.g., sexual motives) for unprotected anal sex may have a negative influence on intended condom use in certain circumstances (Cooper, Shapiro, & Powers, 1998). Therefore, for both steady and casual sex partners, we examined the extent to which sexual motives for unprotected anal sex negatively affect intended condom use. Theory of Planned Behavior The Theory of Planned Behavior (TPB) (Ajzen, 1991) is one of the most important social psychological theories for predicting and understanding behavior. The TPB posits that the most proximal determinant of whether or not a person performs a behavior is his intention to do so. According to the theory, behavioral intention is determined by attitude, subjective norm, and perceived behavioral control. Attitude is the person’s overall evaluation of the advantages and disadvantages of a particular behavior whereas subjective norms are determined by the perceived social pressure to perform the behavior. Perceived behavioral control is the person’s conviction about whether or not the required skills and resources to perform the behavior are at his disposal and is closely related to Bandura’s (1986) concept of self-efficacy. The TPB has received significant support from research on the adoption of many health-related behaviors (Godin & Kok, 1996), including safer sex behavior (Albarracín, Fishbein, Johnson, & Muellerleile, 2001; Rye, Fisher, & Fisher, 2001; Sheeran, Abraham, & Orbell, 1999). Essentially, the TPB stresses the importance of cognitive, information processing mechanisms in explaining behavior. However, not every act of safer sex is necessarily the result of deliberate thought processes. Of particular interest are the growing number of studies that suggest that HIV-positive MSM are motivated by concerns about the effect that their sexual behavior may have on others; that is, that they feel personally responsible for protecting their partners from HIV. Although we agree that the TPB provides a valuable framework for predicting safer sex practices, we argue that this perspective is too narrow and may not fully account for feelings of personal responsibility that may promote safer sexual behavior. More specifically, we believe that safer sexual behavior in HIV-positive MSM is based on concerns that go beyond a purely rational weighing of personal advantages and disadvantages, i.e., that safer sex is simply the right thing to do. One theory that may help explain why HIV-positive MSM are motivated to adopt safer sexual practices for the protection of others is Schwartz’ (1977) norm-activation theory. Norm-Activation Theory The norm-activation theory (Schwartz, 1977; Schwartz & Howard, 1981) seeks to predict and understand prosocial or altruistic behavior. According to norm-activation theory, personal norms are the immediate determinant of behavior. Personal norms are considered as strong intrinsic motivators, as they trigger an individual’s internal value system and are tied to one’s self-concept. In the case of sexual behavior, these aspects reflect the feeling of moral obligation to practice safer sex because of concern about the welfare of sex partners. That personal norms may play an important role in safer sexual behavior in HIV-positive MSM was shown in a study by Godin, Savard, Kok, Fortin, and Boyer (1996). Their study showed that personal norms, in addition to perceived behavioral control, are important predictors of intended condom use for anal sex. The important relationship between personal norms and condom use intention underscores the assumption that safer sexual behavior is influenced by feelings of moral obligation. However, Schwartz argued that there will be no moral motivation unless: (1) an individual is aware of the specific action that is needed and the consequences of action or inaction to oneself and others (awareness of need); (2) an individual ascribes responsibility to himself for the consequences of the action or inaction (ascription of responsibility); (3) a person identifies actions that might be effective in handling the specific situation (efficacy); and (4) a person believes that he is capable of performing the required actions or behavior (ability). As such, we expect that, in predicting safer sex, awareness of need and ascription of responsibility may be important determinants of behavioral intention to use condoms for anal sex with steady and casual sex partners. Note that in this context, efficacy and ability are not differentiated because both concepts seem to be covered by the theoretical paradigm of the TPB (i.e., attitude and self-efficacy, respectively). Sexual motives for unprotected anal sex Notwithstanding our argument that personal norms play a central role in sexual decision-making, we expect that, depending on the social context in which sex occurs, strong motives to engage in unprotected sex may conflict with one’s personal norm for safer sex. For instance, Cooper et al. (1998) have demonstrated that sexual behaviors, whether risky or safe, may serve a range of psychological functions that have little to do with health protection and disease avoidance. Indeed, the study by Van Kesteren et al. (2005) indicated that the extent to which HIV-positive MSM acted in accordance with their feelings of personal responsibility to practice safer sex depended, in part, on their sexual motives for engaging in unprotected anal sex. Typically, HIV-positive MSM reported having engaged in unprotected anal sex to express emotions related to love or because they were “sexually turned-on” by their sex partners. Thus, sexual motives appear to have a negative influence on intended condom use, which may undermine a prosocial motivation to practice safer sex. However, it can be argued that a conflict such as this may play a role more in casual sex encounters than in steady sexual relationships, as it is likely that the benefits of unprotected sex are more salient in the context of casual sex (i.e., when one does not necessarily know one’s sex partner and is less likely to be confronted with the potential adverse longer term social consequences of having engaged in unprotected sex) than with steady partners (i.e., when one is emotionally involved with a partner and committed to an ongoing relationship) (Flowers, Marriott, & Hart, 2000; Van Kesteren et al., 2005). It has been shown that when people perceive increased benefits of a specific behavior (unsafe sex), it leads to a more favorable affective impression, resulting in lower judgments of risk (so-called affect-heuristic; Finucane, Alhakami, Slovic, & Johnson, 2000). Therefore, we expect that sexual motives may have a negative impact on intended condom use, particularly in casual sex encounters. Proposed model of sexual decision-making Safer sex in HIV-positive MSM can be seen as a prosocial behavior, making it vital to integrate variables specific to the TPB with variables specific to the norm-activation theory. In accordance with the TPB, we propose a model (see Fig. 1) in which intention is the most proximal determinant of behavior. However, in line with the norm-activation theory, we assume that the decision to use condoms for anal sex largely depends on one’s intrinsic motivation to engage in safer sex. Accordingly, we expect personal norms to be the most direct determinant of intention, thereby mediating the effects of the other determinants specific to the TPB and the norm-activation theory. Following Cooper et al. (1998), we further assume that sexual motives for unprotected anal sex may have a direct and negative impact on intended condom use and, as such, may compete with a prosocial tendency to engage in safer sex. However, due to the social context in which sex occurs, we hypothesize that sexual motives will contribute to the explanation of intended condom use with casual sex partners, whereas this is unlikely to be the case in the context of steady sex partners.Fig. 1Proposed model of sexual decision-making Method Participants Data were obtained by means of a self-administered questionnaire between July 2002 and April 2003. Participants were recruited through AIDS consultants working in 15 Dutch hospitals. Participants were asked to fill out the questionnaire at home and return the questionnaires directly to the research institution by means of a stamped, pre-addressed envelope. Consequently, no information was available about the response rate per hospital. Participation was on a voluntary and anonymous basis. A pilot study indicated that completion of the questionnaire took about 30 to 45 min. Participants received no compensation for filling out the questionnaires. Approval for the study was obtained from the Ethics Committee of Maastricht University Hospital. Participants who met the following criteria were included in the study: (1) tested positive for HIV-antibodies; (2) identified themselves as homosexual or bisexual; (3) aged between 20 and 65 years; and (4) were sufficiently fluent in Dutch to complete the questionnaire. A total of 1,050 questionnaires were distributed, of which 296 were completed and returned (response rate = 28.2%). Table 1 summarizes the demographic characteristics of the participants. Participants were predominantly Dutch nationals, were mainly from the Western part of the Netherlands, including Amsterdam, and ranged in age from 25 to 59 years, with a mean age of 42.1 years. Most participants had a medium or high level of education and were employed full-time or part-time. Most participants identified themselves as exclusively homosexual. More than half of the men had known themselves to be HIV-positive for 6 years or less. The majority of men was aware of their CD4 counts and viral load and indicated that they were currently on some form of HIV antiretroviral therapy.Table 1Sociodemographic characteristics of the sample (maximum N = 296)CharacteristicsN%Nationality Dutch25185.7 Surinam/Antillean 51.7 Other3712.6Geographic area of the Netherlands Amsterdam7325.3 West (excluding Amsterdam)11038.0 South6723.2 Northeast3913.5Age 25–355518.9 36–4513646.7 46–558228.2 >56186.2Education Higher vocational education or university12443.1 Secondary vocational training or high school12041.7 Primary school or basic vocational training3411.8 Other103.5Employment status Full-time11743.5 Part-time8029.7 Unemployed7226.8Self-identification Homosexual24482.4 More homosexual than heterosexual3812.8 Bisexual 82.7 More heterosexual than bisexual 41.4 Other 20.7Years knowing HIV-positive status <2 yrs5920.1 2–6 yrs11639.6 7–11 yrs7124.2 12–16 yrs3712.6 >16 yrs103.4Self-reported CD4+ <2003311.1 200–50012040.5 >5009231.1 Don’t know5117.2Self-reported viral load Detectable [median=10.000, range 50–2.080.0007225.9 Undetectable17663.3 Don’t know3010.8Antiviral treatment No5920.1 Yes23479.9 Measures The questionnaire was based on earlier studies (Hogeweg & Hospers, 2000; van Kesteren et al., 2005) and consisted of several questions on determinants for explaining condom use behavior: attitudinal beliefs, subjective norms, self-efficacy, personal norms, awareness of consequences, ascription of responsibility, sexual motives, intention and demographic variables. Separate scales were used for condom use with steady and casual sex partners for all determinants. The questionnaire was pre-tested among a group of 12 HIV-positive MSM for comprehension and completeness. Attitudinal beliefs were measured through 16 items drawn from interviews with HIV-positive MSM (van Kesteren et al., 2005) and from an earlier quantitative study among MSM (Hogeweg & Hospers, 2000). Four items were used to assess response efficacy (e.g., “By using condoms when having anal intercourse, I protect my steady partner/casual sex partners against the AIDS virus/HIV-superinfection”). In addition, seven possible pros (e.g., “By using condoms during anal sex with my steady partner/casual sex partners, I worry less about his/their well-being”), and five possible cons of condom use behavior (e.g., “Using condoms for anal sex with my steady partner/casual sex partners reminds me of HIV/AIDS”) were included in the questionnaire. Items were indexed on 5-point scales (1 = totally disagree, 5 = totally agree), and had an alpha of .81 for steady and .76 for casual sex partners. Subjective norms were measured through the use of three items on 5-point scales. Two items assessed the perception of participants of the opinions of other important people on whether or not they should use condoms when having anal intercourse (1 = certainly not, 5 = certainly). Following Schwartz (1973), one item assessed perceived social sanctions regarding not using condoms during anal intercourse and was expressed as follows: “How do you think important others would react if you told them that you don’t use condoms when having anal intercourse with your steady partner/casual sex partners?” (1 = very approving, 5 = very disapproving). Alpha for steady and casual sex partners was. 76 and .63, respectively. Self-efficacy was measured with seven items based on van Kesteren et al. (2005) and Hogeweg and Hospers (2000) (e.g., “Suppose you want to use condoms when having anal intercourse, will you be able to discuss condom use with your steady partner/casual sex partners?”). Items were measured on 5-point scales (1 = certainly not, 5 = certainly), and had an alpha of .89 for steady and .87 for casual sex partners. Personal norms were measured by means of three items on 5-point scales (Godin et al., 1996; Parker, West, Stradling, & Manstead, 1995), such as: “As a matter of principle, I use condoms every time I have anal intercourse with my steady partner/casual partners.” Items were rated on 5-point scales (1 = totally disagree, 5 = totally agree) and had an alpha of .96 and .90 for steady and casual sex partners, respectively. Both awareness of consequences for self and awareness of consequences for others were assessed separately for condom use with steady and casual sex partners by a single item on a 5-point (1 = totally agree, 5 = totally disagree) scale. Awareness of consequences for self measured the participant’s view about whether the consequences for his health were negligible when not using condoms during anal sex. Awareness of consequences for others measured the participant’s views about whether the consequences for the health of his partner were negligible when not using condoms during anal sex. Ascription of responsibility to self was measured by means of four items on 5-point scales concerning responsibility for condom use behavior and one item concerning transmission of HIV. For example: “How responsible are you yourself for negotiating condom use?” (1 = not at all, 5 = completely). Alpha for steady and casual sex partners was .91 and .89, respectively. Sexual motives for unprotected anal sex were measured using seven affective states on 7-point scales (1 = considerably less, 7 = considerably more), such as lust, love, excitement, and feeling good. Following Nelissen, Dijker, and De Vries (in press), participants were asked to indicate the extent to which they would feel a particular emotion when engaging in unprotected anal sex with steady (alpha = .89) and casual sex partners (alpha = .85). Intention to use condoms when having anal intercourse was measure by the following three items on 5-point scales: “I intend to use a condom when having anal intercourse with my steady partner/casual partners”; “I will try to use a condom when …”; and “I plan to use a condom when …” (1 = certainly not, 5 = certainly). Alpha for both steady and casual sex partners was .93. With respect to sexual risk behavior, participants were asked whether or not they had engaged in receptive and insertive oral sex with ejaculation and in receptive and insertive anal sex in the past six months (separate for steady and casual sex partners). For each sexual behavior they had engaged in, the frequency of condom use (1 = never, 5 = always) was assessed. Results Sexual behavior Table 2 shows that about 60% of the participants reported a male steady partner in the preceding six months. Among these, about one third (30.3%) reported that they had not had oral or anal sex with their steady partner. Among those who had had oral sex with ejaculation with their steady partners (23.6%), only one person reported that he consistently had used a condom. Among those who had had anal sex with their steady sex partners (51.1%), about half (48.4%) reported that they had not used condoms consistently. Approximately 72% of the participants reported male casual sex partners in the preceding six months. When oral intercourse was considered, none of the men who had engaged in oral sex with ejaculation (31.8%) reported consistent condom use. When anal sex was considered, about half (45.6%) of the participants who had engaged in anal intercourse with casual sex partners (74.8%) had not consistently used condoms.Table 2Descriptives of sexual behavior and disclosure in casual sex encounters of the sample HIV-positive MSM (N = 296)N%Steady partner(s) in the preceding  6 months17860.1 Anal or oral sex with steady sex  partner124 (178)69.7 Oral intercourse with ejaculation  with steady sex partner42 (178)23.6 Unprotected oral intercourse  with steady sex partner41 (42)97.6 Anal intercourse with steady sex  partner91 (178)51.1 Unprotected anal intercourse with  steady sex partner44 (91)48.4Casual partner(s) in the preceding  6 months21472.3 Anal or oral sex with casual sex  partners167 (214)78.0 Oral intercourse with ejaculation  with casual sex partners68 (214)31.8 Unprotected oral intercourse  with casual sex partners68 (68)100.0 Anal intercourse with casual sex  partners160 (214)74.8 Unprotected anal intercourse with  casual sex partner73 (160)45.6Disclosure of HIV status in casual  sex encounters Has never/rarely informed  casual sex partners about  HIV-positive status177 (214)82.8 Was never/rarely informed about  HIV-status of casual sex partners189 (214)88.3 Among men who had anal sex with their steady sex partner, unprotected sex occurred more often within the context of HIV-seroconcordant relationships than within HIV-serodiscordant relationships: 23.3% (7 out of 30) of the men with an HIV-positive partner reported consistent condom use in the preceding six months compared to 61.0% (25 out of 41) of the men with an HIV-negative partner and 73.7% (14 out of 19) of the men with a partner whose HIV status was unknown, χ2(2) = 14.7, p < .01. Although no data were available on the HIV-status of casual sex partners, participants were asked two questions regarding disclosure of HIV-status. The majority of the participants (82.8%) reported that they never or rarely had informed casual sex partners about their HIV-status in the preceding six months. Similarly, most participants (88.3%) reported that casual sex partners had never or rarely informed them of their HIV-status (see Table 2). Thus, accurate knowledge of each others HIV-status appears to be minimal, which makes the use of effective partner selection strategies (i.e., “serosorting”) as an explanation for the occurrence of unprotected sex within casual sex encounters less likely. Descriptive statistics and correlations Means, SDs, and correlations for the determinants of condom use for anal sex are provided in Table 3. A missing value analysis was conducted for independent variables relating to condom use with steady and casual sex partners. Participants with missing values exceeding 10% of all items were excluded from further data analyses. For participants with less than 10% missing values, the item score was replaced with the mean of the remaining participants for the respective item. As a result, analyses of intention to use condoms were conducted among 149 out of 178 participants who reported a steady sex partner and 188 out of 214 participants who reported casual sex partners in the preceding six months. Of the participants who were included in the analyses with steady and casual sex partners, there were 22 and 24, respectively, who had one or more missing items (range 1–4). In both samples, however, the majority was missing only one of the questionnaire items.Table 3Means and correlations for determinants of condom use with steady (N = 149; below diagonal) and casual sex partners (N = 188; above diagonal)ScaleRange(1)(2)(3)(4)(5)(6)(7)(8)(9)Intention (1)1–5–.56**.57**.67**.83**.36**.25**.64**−.56**Attitudinal beliefs (2)1–5.60**–.49**.48**.57**.28**.18*.53**−.55**Subjective norms (3)1–5.77**.57**–.45**.62**.17*.07.45**−.43**Self-efficacy (4)1–5.74**.62**.69**–.62**.36**.24**.56**−.49**Personal norms (5)1–5.88**.64**.80**.74**–.32**.22**.64**−.53**Awareness consequences self (6)1–5.23*.30**.21*.26**.22**–.72**.26**−.29**Awareness consequences others (7)1–5.39**.35**.38**.37**.41**.53**–.23**−.23**Ascription of responsibility (8)1–5.60**.60**.51**.71**.60**.18*.35**–−.49**Sexual motives (9)1–7−.57**−.62**−.57**−.59**−.59**−.16*−.31**−.47**–Steady  M4.13.54.34.24.13.34.04.23.5  SD1.30.71.00.91.41.61.40.71.6Casual  M4.33.84.34.24.33.84.24.33.4  SD1.10.60.60.81.11.21.20.81.4*p < .05.**p < .01. The descriptive statistics indicates that, in general, participants’ intentions to use condoms during anal sex with steady and casual sex partners were high. In addition, the means of most of the other determinants were on the positive side of the scale. The exception was for sexual motives for unprotected anal sex, where participants scored negative with regard to both steady and casual sex partners. Consistent with our proposed model (see Fig. 1), attitudinal beliefs, subjective norms, self-efficacy, personal norms awareness of consequences for self and others, and ascription of responsibility to self were all positively correlated with behavioral intention, with the exception of sexual motives for unprotected anal sex. As expected, the correlation between sexual motives for unprotected anal sex and intended condom use was negative for both steady and casual sex partners. Predictors of intended condom use Because intention for both steady and casual sex partners was positively skewed (more than 50% of the sample scored 5 on a 5-point scale), intended condom use with steady and casual sex partners was dichotomized on its median into high versus low intention. To test our proposed model (see Fig. 1), two hierarchical logistic regression analyses were conducted with intended condom use with steady and casual sex partners as the outcome variables. Personal characteristics and partner variables were controlled in both analyses and were entered first. In order to examine the relative contribution of the TPB variables (i.e., attitudinal beliefs, subjective norms and self-efficacy), these variables were entered in the second step. Awareness of consequences for self and others and ascription of responsibility were entered in the third step, followed by sexual motives for unprotected anal sex in the fourth step. Personal norms were entered in the final step of the regression analyses. In addition, we examined the mediating role of personal norms following the standard procedure specified by Baron and Kenny (1986). Prior to analyses, collinearity between all variables in the regression analyses was checked by computing the Variance Inflation Factor (VIF) of each variable. Values above 10 were regarded as an indication of near-collinearity (Kleinbaum, Kupper, Muller, & Nizam, 1998). With respect to steady and casual sex partner variables, all VIFs were <5 and <3, respectively. Additionally, initial regression analyses were conducted to minimize the number of potential personal characteristics and partner variables used in the logistic regression. Only those personal characteristics and partner variables making significant contributions to prediction of intended condom use with steady or casual sex partners were included in subsequent analyses. Steady sex partners First, a regression of intended condom use with steady sex partners on personal characteristics (age, education, antiviral treatment, and years knowing HIV-positive status) and partner variables (steady partner only versus casual partners as well and HIV-negative or unknown status partner versus HIV-positive status) showed education level and partner HIV status to be the only significant predictors. Therefore, these variables were entered in the first step of the subsequent regression analysis, yielding an explained variance of 13% (see Table 4). When the TPB variables were entered in the second step, a further 40% (p <.001) of variance was explained, with self-efficacy as a significant predictor. Subjective norms were borderline significant (p = .05). As shown in Table 4, there was no significant increase in variance explained with the addition of awareness of consequences for self and others and ascription of responsibility in Step 3, or with sexual motives for unprotected anal sex in Step 4. At this stage of the analysis, self-efficacy significantly contributed to the prediction of intention, whereas a marginally significant effect was found for subjective norms (p <.10). When personal norms were entered in the final step of the analysis, a further 9% (p <.001) of variance was explained. In this model, the effect of personal norms was significant; however, the effects of subjective norms and self-efficacy dropped below significance, which suggested that the effects of subjective norms and self-efficacy were mediated through personal norms. Testing this suggestion required that three conditions be met (see Baron & Kenny, 1986). The first condition-that subjective norms and self-efficacy be related to intended condom use-was assessed in the above analysis (see Step 4, Table 4). The second condition requires that the mediator (i.e., personal norms) predict intended condom use and that subjective norms and self-efficacy have weaker effects when controlling for personal norms. This was also confirmed in the above analysis (see Step 5, Table 4). The third condition requires that subjective norms and self-efficacy significantly predict the mediator. To test this condition, personal norms were regressed on subjective norms and self-efficacy, while controlling for educational level and partner HIV status. Results revealed that subjective norms (OR = 4.6, p <.001) and self-efficacy (OR = 16.3, p <.001) were significantly related to personal norms. Thus, there was evidence that personal norms mediated the effects of self-efficacy on intended condom use. In addition, there was evidence that personal norms mediated the tentative influence of subjective norms. Casual sex partners Initially, a regression of condom use intention with casual sex partners on personal characteristics (age, education, antiviral treatment, and years knowing HIV-positive status) and partner variables (casual partners only versus steady partners as well) showed antiviral therapy to be the only statistically significant predictor. Therefore, only this variable was subsequently entered in Step 1 of the logistic regression analysis, yielding an explained variance of 6% (see Table 5). When the TPB variables were entered in the second step, a further 46% (p <.001) of the variance was explained. Attitudinal beliefs, subjective norms and self-efficacy significantly predicted intention, whereas a marginally significant effect was found for antiviral therapy. The entry of awareness of consequences for self and others and ascription of responsibility in Step 3 led to a further 8% (p <.001) of explained variance. Step 4 also accounted for a significant increase of variance explained with 3% (p <.01). In this step of the analysis, antiviral therapy, self-efficacy, ascription of responsibility and sexual motives significantly contributed to the prediction of intention, whereas a marginally significant effect was found for subjective norms (p <.10). When personal norms were entered in the final step, a further 5% (p <.001) of variance was explained. As shown in Table 5, antiviral therapy and personal norms positively and sexual motives negatively predicted behavioral intention. Furthermore, the effects of subjective norms, self-efficacy and ascription of responsibility became non-significant, which suggested that these variables were mediated through personal norms. To test this notion, we again assessed the three conditions specified by Baron and Kenny (1986). First, the analysis above revealed that self-efficacy and ascription of responsibility significantly predicted intended condom use, whereas a marginally significant effect was found for subjective norms (see Step 4, Table 5). Second, the above analysis showed that adding personal norms gave a significant regression coefficient for personal norms, and non-significant regression coefficients for subjective norms, self-efficacy, and ascription of responsibility (see Step 5, Table 5). Finally, in an analysis predicting personal norms while controlling for antiviral therapy, subjective norms (OR = 2.7, p <.05), self-efficacy (OR = 3.0, p <.01), and ascription of responsibility (OR = 10.5, p <.001) emerged as significant predictors. Taken together, these results provided evidence that personal norms were a mediator of the self-efficacy-intention and ascription of responsibility-intention relationships. In addition, evidence was found that personal norms mediated the tentative influence of subjective norms.Table 4Logistic regression analyses predicting high intention (0 = no, 1 = yes) to use condoms with steady sex partners (N = 149)Model 1Model 2Model 3Model 4Model 5BOR95% CIBOR95% CIBOR95% CIBOR95% CIBOR95% CIStep 1 Education level.251.3*1.1–1.6.111.1.86–1.5.071.1.81–1.4.071.1.81–1.4.01 1.0.74–1.4 Partner HIV statusa−1.2.30**.14–.64−.43−.65.24–1.8−.40.67.24–1.9−.40.67.23–1.9−.35.70.22–2.3Step 2 Attitudinal beliefs.491.6.67–4.0.351.4.55–3.7.371.5.53–4.01.1.37–3.1 Subjective norms.672.01.0–3.8.611.8.92–3.7.621.9.92–3.7.07.64.20–2.1 Self-efficacy1.75.4***2.1–13.71.64.9**1.7–13.61.64.9**1.7–14.3−.442.7.82–8.9Step 3 ACSb−.02.98.71–1.4−.02.98.70–1.4−.01.99.70–1.4 ACOc.241.3.88–1.8.241.3.88–1.8.221.3.85–1.8 ARd.291.3.50–3.5.281.3.50–3.5.211.2.43–3.5Step 4 Sexual motives.021.0.70–1.5−.041.0.65–1.4Step 5 Personal norms2.410.8**2.0–57.5 Nagelkerke R2.13.53.54.54.62 Model χ215.874.577.177.192.1Note. At each step, variables were included in the equation simultaneously.aVariable coding: 0 = HIV-negative or unknown, 1 = HIV-positive.bAwareness consequences self.cAwareness consequences others.dAscription of responsibility.*p <.05.**p <.01.***p <.001 (two-tailed).Table 5Logistic regression analyses predicting high intention (0 = no, 1 = yes) to use condoms with casual sex partners (N = 188)Model 1Model 2Model 3Model 4Model 5BOR95% CIBOR95% CIBOR95% CIBOR95% CIBOR95% CIStep 1 Antiviral treatmenta.992.7**1.4–5.2.742.1.88–5.01.12.9*1.1–7.31.13.1*1.2–8.41.23.3*1.2–9.2Step 2 Attitudinal beliefs1.23.2**1.4–7.5.842.3.92–5.8.321.4.50–3.8.251.3.45–3.6 Subjective norms1.12.9**1.3–6.5.902.51.0–6.1.862.4.91–6.1.391.5.50–4.4 Self-efficacy1.54.2***2.3–7.91.12.9**1.5–5.9.862.4*1.1–5.0.531.7.76–3.8Step 3 ACSb.241.3.85–1.9.251.3.85–2.0.131.1.72–1.8 ACOc.341.4.89–2.2.341.4.88–2.2.341.4.86–2.3 ARd1.13.0*1.2–7.51.13.0*1.1–7.9.641.9.65–5.5Step 4 Sexual motives−.61.54**.35–8.3−.57.57*.36–.89Step 5 Personal norms1.33.8**1.6–8.9 Nagelkerke R2.06.52.60.63.68 Model χ28.991.4111.8120.5132.3Note. At each step, variables were included in the equation simultaneously.aVariable coding: 0 = no, 1 = yes.bAwareness consequences self.cAwareness consequences others.dAscription of responsibility.*p <.05.**p <.01.***p <.001 (two-tailed). Discussion The present study showed that the majority of HIV-positive MSM reported engaging in safer sexual behavior either though abstinence or consistent condom use for anal sex. Nonetheless, a high number of men who had had anal sex reported that they had not used condoms consistently with steady and casual sex partners. Results further indicated that HIV-positive MSM were more likely to engage in unprotected anal intercourse in the context of casual sex encounters than in steady sexual relationships (overall 34.1% and 24.7%, respectively). This finding was consistent with patterns of sexual risk behavior observed in other populations of HIV-positive MSM (cf. Crepaz & Marks, 2002). In this study, we tested a model of factors that influence intended condom use for anal sex with steady and casual sex partners. The results showed that, compared with the TPB (Ajzen, 1991), our proposed model of sexual decision-making led to a significant increase of explained variance from 53% to 62% for steady sex partners and from 52% to 68% for casual sex partners. Furthermore, evidence was obtained for the mediating role of personal norms on intended condom use with both steady and casual sex partners, suggesting that moral issues play an important role. However, the study also showed that sexual motives may undermine the influence of personal norms on intended condom use in the context of casual sex, with the likelihood of unprotected sexual intercourse. Taken together, the results revealed strong support for our model of sexual decision-making. In this study, the finding that personal norms appeared to exert a strong direct effect on intention to use condoms with both steady and casual partners was consistent with results of earlier studies that suggest that safer sex in HIV-positive MSM is largely determined by moral concerns or feelings of personal responsibility (e.g., Godin et al., 1996). Moreover, this finding lends further indirect support to the proposition that condom use behavior among HIV-positive MSM is a form of prosocial behavior (e.g., Kok, 1999; Nimmons, 1998). For steady partners, personal norms were identified as the most proximal determinant of intended condom use, whereas an indirect effect was found for self-efficacy on intended condom use through personal norms. Furthermore, some evidence that personal norms mediate the tentative effect of subjective norms on intended condom use with steady sex partners was found. Similar results were found for intended condom use with casual sex partners. However, we also found an additional indirect effect of ascription of responsibility on intended condom use through personal norms. These findings are important because they underscore the need to take into account the role of social expectations, self-efficacy expectations, and attributions about responsibility for condom use in promoting feelings of moral obligation-a process that has not been identified in previous research on sexual risk behavior in HIV-positive MSM. Furthermore, in the case of casual sex partners, condom use intention was not only directly related to personal norms, but was also negatively influenced by sexual motives for unprotected anal sex. Thus, the findings from this study suggest that the need to engage in unprotected anal sex for physical and emotional satisfaction may supercede the prosocial motivation for safer sex. The fact that sexual motives contributed toward explaining intended condom use in casual sex encounters but not in steady relationships does not imply that sexual motives are not important in the context of steady sex. More likely, there are some differences between steady and casual sex that encourage HIV-positive MSM to suppress sexual motives within the context of steady relationships but not within casual sex encounters. As discussed previously, a possible explanation may lie in the so-called affect-heuristic. In the context of casual sex, the benefits of unprotected sex may be more salient, thus resulting in a decreased assessment of risk. It is worth noting that the affect-heuristic is especially likely to color judgments under time pressure, which may play a more important role in the context of casual sex (Finucane et al., 2000). The “Coolidge effect” may also explain why sexual motives play a role in the casual sexual context, but not in steady sexual relationships. The Coolidge effect refers to enhanced sexual arousal that is felt when sexual stimuli and partners are novel (Gregoire, 1999).1 A final explanation may be the greater ambivalence that HIV-positive MSM experience in casual sex encounters as opposed to steady relationships. Greater ambivalence is likely to result in a reduction in both behavioral intention and actual behavior. In addition, people who experience ambivalence are more easily persuaded (Conner & Armitage, 2000). It may be that pointing out the potential for casual sex partners to become steady partners in the future could increase the perceived benefits of safer sex, thus facilitating prosocial motivation to engage in protected sex. Indeed, the study by van Kesteren et al. (2005) suggests that such a mechanism exists. Further, the results of this study demonstrated that the use of antiviral therapy was associated with a greater intention to use condoms with casual sex partners. To date, inconclusive evidence exists regarding the association between the use of antiviral therapy and unprotected sex (for a meta-analysis, see Crepaz, Hart, & Marks, 2004). Some study considerations and limitations and implications for practice should be mentioned. First, as Ajzen and Fishbein (1970) held, it may be argued that the important role of personal norms as a predictor of intention can be attributed to a certain amount of overlap between the measures of personal norms and intention. In this study, high correlations were found between personal norms and intention. On theoretical grounds, however, it seems imperative to distinguish personal norms from intentions. As Manstead (2000) argued, holding the belief that something is morally “right” or “wrong” is not the same as the perceived likelihood of performing certain behaviors. Consequently, an individual may feel a personal obligation to act in a certain way, but intend to behave in a way that is contradictory to his personal norm because the personal or social advantages of acting inconsistently with his personal norm outweigh the personal or social advantages of acting consistently with the norm. Second, it should be noted that the data were cross-sectional, which hampers conclusions about causality. Due to the cross-sectional design, we did not specifically address the relationship between the psychosocial factors and behavior. It should be noted, however, that tentative support for our model was found in an additional analysis when intention was examined as a mediator of the personal norms-behavior relationship, both for steady and casual sex partners. Yet, longitudinal studies are needed to determine causative relationships between the measured constructs. A third limitation of the present study concerns the use of a single-item approach to measure awareness of consequences, as single-item measures are known to be less reliable. This issue merits attention in future research. Finally, the sample used in this study warrants some concern. More than 70% of those who received questionnaires did not respond. This raises the question as to whether these participants were predominantly more aware of the need to practice safer sex and, as such, are not a representative sample of Dutch HIV-positive MSM. On the other hand, Swanborn (2002) found that 30% is a common response rate for this type of research in the Netherlands. It is possible that the sensitivity of the subject and the fact that the participants suffer from a chronic disease may create barriers to participation. Moreover, substantial efforts were made to recruit HIV-positive MSM from throughout the Netherlands. Although this approach did not ensure that the sample was representative, it did enhance its diversity and therefore strengthens the external validity of the study results. The findings reported in this study carry several implications for HIV-prevention efforts to promote safer sexual behavior among HIV-positive MSM. When addressing condom use behavior with steady and casual sex partners, it seems particularly crucial to enhance feelings of moral obligation to use condoms for anal sex. Feelings of moral obligation could be addressed by inducing HIV-positive MSM to reflect on their own personal risk and personal standards for safer sex (Kalichman, 1998). The findings of this study further suggest that desirable changes in subjective norms and self-efficacy expectations might lead to corresponding changes in personal norms and intentions (e.g., Deci & Ryan, 2000; van Empelen, Kok, Jansen, & Hoebe, 2001; van Empelen, Schaalma, Kok, & Jansen, 2001). Therefore, preventive interventions should focus on helping HIV-positive MSM to cope with an unsupportive social environment and to encourage them to build the skills and confidence required for communicating and negotiating condom use. For the promotion of condom use with casual sex partners, personal norms may be further increased by urging HIV-positive MSM to accept responsibility for safer sexual behavior. Moreover, especially within casual sex encounters, it is imperative to help HIV-positive MSM to deal with feelings that may conflict with the goal of safer sex. Possible methods that can help HIV-positive MSM to identify and control high risk situations are, for example, action planning and coping planning (Sniehotta, Schwarzer, Scholz, & Schüz, 2005). Action plans and coping plans are detailed plans of what a person needs to do when a specific situation occurs. When such situation arise, it is likely to function as a cue for the execution of those precise plans of implementation. Additionally, stimulating HIV-positive MSM to imagine how they would feel if they were in their sex partners shoes (i.e., perspective taking) may evoke a mixture of egoistic and altruistic motivation and, as such, lead to more protected sex (Batson, Early, & Salvarani, 1997). That such an approach is feasible has been shown in a study by van Kesteren, Kok, Hospers, Schippers, and De Wildt (2006), in which the above-mentioned methods have been integrated in an intervention to promote sexual health in HIV-positive MSM.

Osteoporos_Int-3-1-1766477

The prevalence of vertebral fracture amongst patients presenting with non-vertebral fractures

Introduction Despite vertebral fracture being a significant risk factor for further fracture, vertebral fractures are often unrecognised. A study was therefore conducted to determine the proportion of patients presenting with a non-vertebral fracture who also have an unrecognised vertebral fracture. Introduction Osteoporosis may be defined as a ‘skeletal disorder characterized by compromised bone strength predisposing a person to an increased risk of fracture’ [1]. Overall, osteoporotic or low trauma fractures are common - it has been estimated that one in two women and one in five men aged over 50 years will sustain at least one low trauma fracture in their lifetime [2]. The fracture sites which most commonly come to clinical attention include the distal radius (Colles’), proximal femur (hip), humerus and ankle. In the Fracture Liaison Service covering Glasgow, 82% of presenting fractures were at these sites [3]. The spine is another key fracture site; however, it has been estimated that only 30% of vertebral fractures receive clinical attention - which means that the majority of patients with vertebral fractures remain undetected [4]. It appears that only those patients with the most severe vertebral fractures come to clinical attention—it is likely that this is due to higher levels of back pain and disability [4]. The European Prospective Osteoporosis Study (EPOS) assessed vertebral fracture incidence in 3,174 men (mean age 63.1 years) and 3,614 women (mean age 62.2 years) over a mean of 3.8 years. The age-standardised incidence of morphometric fracture was 10.7/1,000 person years in women and 5.7/1,000 person years in men [6]. Vertebral fracture incidence increased markedly with age in both men and women. Indeed, the incidence in women aged 50–54 was 3.6/1,000 person years, rising to 29.3/1,000 person years in women aged 75–79 [5]. Lateral dorsal and lumbar X-rays were used in the European Vertebral Osteoporosis Study (EVOS) population to identify vertebral fracture over an 8-year period. The investigators found an incidence rate of 9.85/1,000 person years compared with 3.06/1,000 person years for hip fracture, 4.28/1,000 person years for Colles’ fracture and 4.76/1,000 person years for other fractures [6]. Low trauma fractures have substantial consequences. Although hip fracture has the greatest mortality and morbidity, vertebral fracture is also associated with significant mortality and morbidity. In one study, patients with vertebral deformity had a substantially higher risk of death compared to patients without vertebral deformity, regardless of age, sex and BMD (mortality rate 30.3% versus 10.7% over a 14-year follow-up). The risk of death was highest in those patients with a vertebral deformity who had a subsequent symptomatic fracture [HR 9.0 (3.1–26.0)] [7]. In terms of morbidity, around 70% of patients with symptomatic vertebral fracture complain of difficulty in standing and 65% of difficulty in bending, and 41% complain of constant pain [8]. It is well-established that the presence of one low trauma fracture significantly increases the risk of a further fracture [9–11]. In a systematic review carried out by Klotzbuecher et al. [9], women with a single pre-existing vertebral fracture, identified in eight studies at baseline by vertebral morphometry and in another seven as clinical (symptomatic) fractures, were 4.4-times more likely to have another vertebral fracture than women without vertebral fracture. Prior vertebral fracture also predicted non-vertebral fracture: in women with vertebral fracture, the risk of hip fracture was increased by 2.3-times, of Colles’ by 1.4-times and of any non-vertebral fracture by 1.8-times. Fracture risk increased even further with each additional vertebral fracture; for example, the odds of a new vertebral fracture in women with five or more prior vertebral fractures were 35-times greater than for women without prior vertebral fracture. Given that vertebral fracture is a significant risk factor for further fracture, and that vertebral fractures often go undiagnosed, this study set out to determine the proportion of patients attending a Fracture Liaison Service following a non-vertebral fracture who also have an unrecognised vertebral fracture. The standard method to assess vertebral fracture is radiography of the thoraco-lumbar spine [12]. However, there is no gold standard for the definition of osteoporotic vertebral fracture. A number of methods have been developed for interpretation of spinal X-rays, including the Genant semi-quantitative method [13], which has been used as a surrogate ‘gold standard’ in a number of key osteoporosis studies. This approach is more objective and reproducible than other qualitative methods [12]. Vertebral morphometry using dual-energy X-ray absorptiometry (DXA) also known as morphometric X-ray absorptiometry (MXA) is a fast, low-radiation technique which produces images that are of sufficient quality to be used to diagnose the presence of vertebral deformity consistent with fracture [14]. Methods The Fracture Liaison Service in South Glasgow has been previously described [3]. This service assumes responsibility for all patients who present with a clinical low trauma fracture and ensures that appropriate assessment, diagnosis and treatment recommendations are made to prevent further fracture. A low trauma fracture is defined as one sustained from a standing height or less, and not occurring as a result of a road traffic accident. Patients with skull or facial fractures are not routinely offered assessment for osteoporosis [3]. All patients aged 50 years or over identified by an Osteoporosis Specialist Nurse have a DXA scan carried out if they are likely to be candidates for bisphosphonate therapy. It should be noted that patients aged over 70 years presenting to this service after hip fracture do not have a DXA scan carried out, since most of these patients have osteoporosis and require therapy. All patients who presented with a low trauma fracture and who underwent DXA evaluation with vertebral morphometry from DV5/6 to LV4/5 were included in this prospective study. All scans were carried out on a Lunar Prodigy densitometer using the Dual Energy Vertebral Assessment (DVA) morphometry imaging software (version 6.3). Vertebral fractures were identified by direct visualisation using the Genant semi-quantitative grading scale [17]. All scans were reviewed by and vertebral deformities reported by the same physician (SJG). Although Genant’s semi-quantitative grading is based on analysis of X-rays and is not validated for MXA, it was used in this study to assess vertebral status on MXA since it provides a practical and pragmatic option. Statistical analysis Baseline differences that potentially explained observed differences in morphometric vertebral fracture rates (sex, age, body mass index, T-score and prior fracture history) were clustered into pre-defined categories: Sex: male/femaleAge: 50–64, 65–74, 75+Body mass index:  ≤18 kg/m2, 19–24, 25–29, ≥30 kg/m2; also compared by quartilesT-score: ≥−1, −2.4 to −1.1, ≤−2.5Prior fracture history: yes/no Contingency tables for each set of categories were prepared and chi-squared values calculated to test for overall interaction. Yates continuity correction was used where 2×2 tables resulted. Where the overall result was significant, individual pairwise comparisons and t-testing were then carried out. Results Data was collected during the routine work of the FLS between September 2004 and March 2005. The data was analysed between July 2005 and July 2006. Demographics Data were available for 337 patients presenting with low trauma fracture; 77.4% (n=261) were female. Of all patients, 10.4% were aged 50–64 years, 53.2% were aged 65–74 years and 36.2% were aged 75 years or over. Mean body mass index (BMI) was 24 kg/m2 (SD 32); 6% (n=20) had a BMI ≤18 kg/m2, 33% (n=112) between 19–24 kg/m2, 35% (n=118) between 25–29 kg/m2 and 26% (n=87) ≥30 kg/m2 According to WHO definitions, 35.0% of patients had normal lumbar spine BMD (T-score −1 or above), 37.4% were osteopenic (T-score −1.1 to −2.4) and 27.6% were osteoporotic (T-score −2.5 or lower). Humerus (n=103, 31%), radius–ulna (n=90, 27%) and hand/foot (n=53,16%) were the most common presenting fractures (see Fig. 1). Other fracture sites included hip (n=16, 5%), clavicle (n=11, 3%), rib (n=7, 2%), tibia-fibula (n=8, 2%), pelvis–pubic rami (n=7, 2%) and olecranon, patella, scapula, sternum and shaft of femur (n=7, 2%). Fig. 1Presenting fracture and prevalence of vertebral deformity for each type of fracture. *Other includes olecranon, patella, scapula, sternum and shaft of femur The presenting fracture was the first non-vertebral, low trauma fracture to come to clinical attention in 72% of patients (n=241), the second in 25% (n=85), the third in 1% (n=5) and the fourth in 2% (n=6). Information regarding risk factors for fracture—including prior history of fracture, early menopause, BMI, history of smoking or excess alcohol, thyrotoxicosis, history of maternal hip fracture, family history of osteoporosis, steroid use, rheumatoid disease, history of recurrent falls and partial gastrectomy—was also collected. Of all patients, 63% had one fracture risk factor, 23% had two risk factors, 11% had three risk factors and 3% had more than three risk factors. Risk fractures for fracture were assessed after patients had presented to the FLS with a non-vertebral fracture; therefore, every patient had a prior history of fracture. Vertebral deformity consistent with fracture The overall prevalence of vertebral deformity established by MXA was 25%. The prevalence of vertebral deformity by type of presenting fracture is shown in Fig. 1. Patients with a hip fracture were significantly more likely to have a vertebral deformity identified on MXA than were patients who presented with a non-hip fracture (p=0.009). Overall, 55.4% of patients with vertebral deformities had deformities in the thoracic spine and 26.4% had lumbar spine deformities, whilst 18.2% had deformities in both regions. Of the patients with vertebral deformities identified by MXA, 45% had deformities of more than one vertebra. Multiple vertebral deformity prevalence varied considerably according to the site of the presenting fracture (see Fig. 2). Fig. 2Presenting fracture and prevalence of multiple (two or more) vertebral deformities Of the patients with vertebral deformities, 3 (3.6%) had unreadable scans for grading. Of the 80 remaining patients, 58 (72.5%) had Grade 2 or Grade 3 deformities. Thirty-two (55.2%) of the patients with vertebral deformity of Grade 2 and Grade 3 had multiple vertebral fractures. Of the sub-group of patients with vertebral deformity of Grade 2 and 3, 56.9% had deformities in the thoracic spine, 22.4% in the lumbar spine and 20.7% in both regions. There was no significant difference between the vertebral deformity population and the sub-population of patients with Grade 2 or 3 deformities, in terms of the proportion of patients with multiple vertebral deformities or in the site of deformity. Table 1 shows the prevalence of vertebral deformity and multiple vertebral deformities by lumbar spine T-scores. Patients whose lumbar spine T-scores were consistent within the osteoporosis range (T-score ≤2.5) were significantly (p<0.05) more likely to have vertebral deformities (42%) than patients whose T-scores were in the osteopenic or normal ranges (20% and 16% respectively). They were also significantly more likely to have multiple vertebral deformities than those with a normal T-score (54% versus 14%, p<0.001). Table 1Impact of lumbar spine T-score on prevalence of vertebral deformityLumbar spine T-scoreTotalVertebral deformityMultiple vertebral deformitiesFracture Grade 2 or 3n%n%%an%%bn%%aOsteoporotic (T-score ≤−2.5).93283947.041.9205451.33255.234.4Osteopenic (T-score −2.4 − −1.1).126372530.119.8123248.01627.612.7Normal118351922.916.151426.31017.28.5Totals337100831003710058100Vertebral deformity: Chi-squared=13.471, p=0.0012Multiple vertebral deformities: Chi-squared=16.304, p=0.0003a% within each T-score group with vertebral deformityb% of vertebral deformity group with multiple deformities within each T-score group Patients with lower BMI (≤18 kg/m2) were significantly more likely to have vertebral deformity than patients with the highest BMI (≥30 kg/m2), (40% versus 17%, p<0.0001) (Table 2). A very similar picture was seen when BMI was divided into quartiles: 45% of patients in the lowest quartile had a vertebral deformity, compared with 18% of those in the highest quartile (p<0.0001). The mean BMI for patients with and without vertebral deformity did not differ significantly (24.35±4.78 kg/m2 vs 25.22±3.90 kg/m2), nor did the mean BMI of patients with multiple vertebral deformities (23.70±4.78 kg/m2). Table 2Impact of BMI on prevalence of vertebral deformity, n=336BMITotalVertebral deformityMultiple vertebral deformitiesFracture Grade 2 or 3n%n%%n%%n%%Underweight <18206.089.640.0513.562.5813.840.0Normal 19–2411133.03947.035.12054.151.32441.421.625–2911835.12226.518.6616.227.31729.314.430+8725.91416.916.1616.242.9915.510.3Totals336100831003710058100Vertebral deformity: Chi-squared=16.572, p=0.0009Multiple vertebral deformities: Chi-squared=15.288, p=0.0016 Patients with a history of one or more non-vertebral fractures prior to the fracture they presented with were significantly more likely to also have vertebral deformity(ies) (Table 3), (43% versus 20%, p=0.008). Table 3Impact of prior fracture history on prevalence of vertebral deformity TotalVertebral deformityMultiple vertebral deformitiesFracture Grade 2 or 3n%n%%n%%n%%024171.54959.020.32464.949.03560.314.51 or more9628.53441.042.71335.138.22339.724.0Totals337100831003710058100Vertebral deformity: Chi-squared=7.131, p=0.0078Multiple vertebral deformities: Chi-squared=0.237, p=0.63 (ns) Table 1 also includes analysis of the sub-group of patients with vertebral deformities of Grade 2 and Grade 3 Patients with an osteoporotic T-score were significantly more likely to have vertebral deformities than those with a normal or osteopenic T-score (34% vs 12.7% [osteopenic] and 8.5% [normal] p<0.0001)Patients with BMI<18 kg/m2 were significantly more likely to have vertebral deformities than those with BMI of over ≥30 kg/m2 (40% vs 10.3%, p<0.0001)Patients with a history of one or more prior non-vertebral fractures were more likely to have vertebral deformity although this did not reach significance (24% vs 15%, p=0.055) Older patients were slightly (although not statistically significantly) more likely to have a vertebral deformity: 30% of patients aged over 75 had a vertebral deformity, compared with 23% and 22% of patients aged 50–64 years and 65–74 years respectively. Gender did not appear to affect the likelihood of identifying a vertebral deformity (25% of women and 24% of men), although women were somewhat more likely to have multiple vertebral deformities (45% vs 40%, p=ns). Discussion One-quarter of patients presenting to our Fracture Liaison Service with a non-vertebral fracture had a previously undiagnosed vertebral deformity, of which almost one-half (45%) were multiple vertebral deformities. This is similar to the prevalence seen in previous studies using radiographic or MXA detection in women with and without non-vertebral fracture. Sub-analysis of patients by grade of deformity revealed that almost three-quarters of the patients had vertebral deformities of Grade 2 and Grade 3. These are more likely to be vertebral fractures, rather than vertebral deformities of non-osteoporosis aetiology, which might be more likely where Grade 1 deformities are present. In the Study of Osteoporotic Fractures (SOF), a prospective study of almost 10,000 women, the prevalence of vertebral fracture was 20% (42% of which were multiple) in women aged over 65 years who did not have a non-vertebral fracture [11]. In another study of 482 women aged over 65 years without a non-vertebral fracture, the prevalence of vertebral fracture was 18.3% [15]. In a smaller study of 58 women with Colles’ fracture, vertebral fractures were identified by MXA in 19% of patients [16]. In this present study, patients presenting with hip fracture, spine T-score ≤−2.5, low BMI (either in the lowest quartile or ≤18 kg/m2) or with more than one prior non-vertebral fracture were all significantly more likely to have evidence of a prevalent vertebral deformity. However, we found that 16% of patients with normal BMD at the lumbar spine had a prevalent vertebral deformity (26% of which were multiple). This is comparable to that seen in an earlier study conducted in women aged over 65 years without a non-vertebral fracture and with normal BMD at the spine, which revealed a prevalence of vertebral fracture of 18.7% [15]. In the UK, current treatment decisions for patients with low trauma fracture are based on fracture status and BMD as determined by DXA [17, 18]. In England, patients aged under 75 receive treatment if they have a fracture and osteoporosis is confirmed by DXA scanning [17]. In Scotland, patients with vertebral fracture and BMD outside the osteoporotic range, as defined by the WHO classification, do not routinely receive treatment unless they have two or more vertebral fractures, irrespective of BMD, or one vertebral fracture and an osteopenic BMD score [18]. Identification of those patients who already have an (often unrecognised) prior vertebral fracture is particularly important, as they are at high risk of further fracture by virtue of their prior vertebral fracture. Although these patients might not fulfil the criteria for therapy on the basis of their (non-vertebral) fracture history and BMD, they might be candidates for therapy if their underlying positive vertebral fracture status was known. In this study, five of the 118 patients with normal BMD (4.2%) had two or more previously undiagnosed vertebral deformities plus a new non-vertebral fracture, and 25 of the 126 patients with osteopenic BMD (19.8%) had one or more previously undiagnosed vertebral deformities plus a non-vertebral fracture. These patients would not have been candidates for anti-resorptive treatment if MXA had not been performed and their vertebral deformities identified. Therefore, one in 20 patients with a normal T-score and almost one in five patients with an osteopenic T-score would experience a change in management by virtue of their underlying vertebral deformity or deformities. Overall, 30 (8.9%) of the patients in this study would be eligible for treatment by virtue of their newly identified vertebral deformity., which means that 11 patients who present with a non-vertebral fracture would need to undergo vertebral morphometry in order to identify one patient who ought to be managed differently. This ‘Number Needed to Screen’ gives an indication of the number of patients for whom treatment recommendations would change after the identification of vertebral fracture. These data also provide the first attempt to quantify the overlap between patient populations with non-vertebral and vertebral fracture. Estimates suggest that 1.28 million post-menopausal women in the UK have experienced a prior non-vertebral fracture [19] and 1.3 million have at least one prior vertebral fracture [20]. In this study, 72% of patients presented with their first non-vertebral fracture, and vertebral morphometry subsequently identified vertebral deformities in one in five (21%) of these patients. Nearly one-half of vertebral deformities were multiple (48%). It is highly likely that these vertebral fractures had occurred prior to the first non-vertebral fracture, suggesting that a substantial number of patients will have experienced a vertebral fracture prior to any non-vertebral fracture event. Our results support the recommendation to perform vertebral morphometry in any patient who is referred for DXA testing after experiencing a non-vertebral fracture. Treatment decisions will then better reflect any given patient’s future absolute fracture risk. The 'Number Needed to Screen' if MXA is used in this way would be seven to identify one patient with vertebral deformity, and 14 to identify one patient with two or more vertebral deformities. Routinely performing lateral vertebral morphometry does raise a number of issues. Firstly, there is an increase in radiation exposure for the patient. A standard AP spine and hip DXA scan is associated with a radiation exposure [entrance surface dose (ESD)] of around 0.037 mGy, and results in an effective dose of less than 2 μSv (microsieverts). The ESD associated with lateral vertebral morphometry is 0.083 mGy [21],corresponding to an estimated effective dose of about 2.5 μSv. These are relatively small doses compared to the UK natural background radiation level of approximately 2.5 mSv. The small carcinogenic potential associated with these scans has to be balanced against both the prognostic importance (in terms of future fracture risk) that is associated with the identification of unrecognised vertebral deformities and also the much higher radiation exposure risk associated with plain spine radiology (effective dose ∼0.6 mSv). Lateral vertebral morphometry does have limitations; for example, it is less reliable than conventional lateral spine X-rays at the upper thoracic spine and in Grade 1 deformities. A recent study [22] comparing MXA with lateral spine X-rays found that vertebral morphometry using MXA allowed diagnosis of vertebral fracture in the lumbar and mid thoracic spine, where vertebral fractures are most common. MXA was less reliable at the upper thoracic spine, which is also an established limitation of X-rays. An earlier study [23] compared identification of vertebral fracture on MXA using the Genant semi-quantitative method using MXA with conventional X-rays in 80 post-menopausal women. Of the 1,027 potentially evaluable vertebrae, 81% were adequately visualised on MXA to allow grading. Prevalent vertebral fracture was identified in 40 vertebral bodies using X-ray; of these, 28 (70%) were correctly identified on MXA, 17/18 (95%) Grade 2 or 3 and 11/22 (50%) of Grade 1 fractures. Of the non-fractured vertebrae, 96.2% were correctly classified as normal by MXA. Although the Genant semi-quantative method is not validated for MXA, it has been used in other studies [22, 23], and was used in this study as a pragmatic option. To carry out quantitative analysis on each scan would have been time-consuming and impractical. Given that the sample population had already had at least one non-vertebral fracture (28% had more than one prior fracture), were drawn from the FLS population, and that almost three-quarters had Grade 2 or 3 vertebral deformity, it seems reasonable to expect the vertebral deformity to be due to osteoporosis, rather than other diagnoses such as degenerative change or Scheurmann’s disease. The sensitivity of detection of a vertebral fracture is lower both where there is significant spine scoliosis and also where there is marked disc-space osteoarthritic change. In these settings, proceeding to plain spine radiology would be recommended [24]. Plain spine X-rays were not carried out in this study. Furthermore, in keeping with our local protocol, bone densitometry was not carried out in older patients (over the age of 70) after hip fracture. Both of these features may mean that the overall prevalence of vertebral deformity in this population has been underestimated. In conclusion, undiagnosed vertebral deformity is highly prevalent in patients aged over 50 years who present with a new non-vertebral fracture. Given that prior vertebral fracture significantly increases the risk of further fracture and because newer diagnostic techniques make identification of vertebral deformity consistent with fracture relatively straightforward with minimal radiation exposure, we recommend that all patients undergoing DXA should also undergo vertebral morphometry to identify prior vertebral deformities consistent with fractures.

Int_J_Colorectal_Dis-4-1-2386753

Fibrin glue and transanal rectal advancement flap for high transsphincteric perianal fistulas; is there any advantage?

Backgrounds and aim In recent decades, fibrin glue has appeared as an alternative treatment for high perianal fistulas. Early results seemed promising, with high success rates being reported. However, with increasing follow-up, the enthusiasm was tempered because of disappointing results. The aim of this retrospective study was to assess the additional value of fibrin glue in combination with transanal advancement flap, compared to advancement flap alone, for the treatment of high transsphincteric fistulas of cryptoglandular origin. Introduction Perianal fistulas of cryptoglandular origin cause considerable discomfort and arise from infections in anal glands lying in the intersphincteric space [1]. To delineate the fistula tract, magnetic resonance imaging (MRI) and anal endosonography are nowadays readily available and are increasingly used for fistula imaging [2]. The essence of surgical treatment of perianal fistulas is to eradicate the fistula tract and at the same time preserve continence. Low fistulas, where the fistula tract is submucosal, intersphincteric or located in the lower third of the external anal sphincter can be treated by fistulotomy with low recurrence rates and relatively little impact on continence [3]. In patients with high perianal fistulas, the fistula tract is located in the upper two-thirds of the external sphincter. Fistulotomy performed on high fistulas results in loss of sphincter function in a considerable number of patients due to the interference of the external sphincter complex [4, 5]. There are various alternative surgical options for high fistulas, namely, rectal advancement, fibrin glue, and seton drainage. In 2006, Johnson et al. [6] reported a new biologic anal fistula plug to treat high transsphincteric perianal fistulas. The anal fistula plug is biologic absorbable and consists of lyophilized porcine intestinal submucosa. In their series of 46 patients treated with the anal fistula plug, a success rate of 83% was achieved at a median follow-up of 12 months [7]. After this publication, several authors have reported their experience with the anal fistula plug, resulting in success rates ranging from 41–88% [8–10]. Currently, the transanal rectal advancement flap (AF) remains the “gold standard” in the treatment of high transsphincteric perianal fistulas of cryptoglandular origin. The rationale behind the AF is that the open internal opening is the cause of the persisting fistula tract. By advancing the tissue over the internal opening, it would be impossible for fecal material to be forced into the fistula tract during defecation. However, recurrence rates of the advancement flap found in literature vary considerably and extend up to 63% [11–15]. Roughly, one out of every four patients requires multiple surgical interventions to close the fistula tract successfully. In recent decades, fibrin glue has appeared as an alternative treatment for high perianal fistulas. As a result of the obliteration of the fistula tract and the closure of the internal opening, the fistula might heal. Early results seemed promising, with high success rates being reported. However, with increasing follow-up, the enthusiasm was tempered because of disappointing results [16–20]. Recently, Zmora et al. [21] conducted a retrospective study including 37 patients with high perianal fistulas. In a subset of 13 patients with fistulas of various etiologies, the advancement flap was used in addition to the fibrin glue installation. The results showed a recurrence rate of 46%. The aim of this study was to assess the additional value of fibrin glue to the transanal rectal advancement flap in a well-defined group of patients with high transsphincteric fistulas of cryptoglandular origin. Patients with previous fistula surgery are a surgically more challenging group as the result of scar tissue and sometimes anal stenosis. Therefore, patients were matched for the presence of a history of fistula surgery. Materials and methods Patient characteristics Between January 1995 and January 2006, a consecutive series of patients were treated by AF. Only patients with high transsphincteric perianal fistulas of cryptoglandular origin were analyzed. High perianal fistulas were defined as patients with fistulas running through the upper two thirds of the external sphincter complex, which is confined by the puborectal sling and the end of the anal canal. Patients in whom the internal fistula opening was not detectable and patients with perianal fistulas as a result of Crohn’s disease, HIV, and other causes were excluded. Patients over 18years of age were included. A consecutive series of patients were operated on with fibrin glue (Tissucol Duo®, Baxter International) in addition to the AF in an attempt to decrease the recurrence rate of the AF. This series of patients were treated on between February 2003 and January 2006. Patients were matched for previous surgery and divided into two groups; one group with previous fistula surgery and the other without. Preoperatively, no routine imaging was performed. Only in selected case when the fistula was complex and/or recurrent, MRI or anal endosonography was used to outline the fistula tract. In The Netherlands and Belgium, non-experimental clinical case series of patients treated with a CE-approved device do not require approval of the local Medical Ethics Commission. A subset of patients from this series were also included in a study where the aim was to assess the long-term functional outcome and identify risk factors for the development of recurrence in patients surgically treated for cryptoglandular fistulas [22]. Surgical technique On the day of surgery, an enema was administered to the patient to clean the proctum. All procedures were performed under general or locoregional anesthesia in the lithotomy position, and broad spectrum antibiotics were administered perioperatively. Subsequently, the internal opening was located by probing the external opening. During surgery, the amount of sphincter involved was judged by palpation of the puborectal sling and the inferior edge of the external sphincter complex. In cases where the internal opening was not found by probing, hydrogen peroxide was injected to locate the internal opening. In case of active sepsis, a seton was placed for a period of at least 3 months insuring adequate drainage. In the group that was operated with the AF, the internal opening was excised followed by mobilization of the mucosa, submucosa, and a small amount of muscular fibers from the internal sphincter complex. The rectal flap was mobilized to sufficiently cover the internal opening with overlap. Hemostasis was performed to prevent a hematoma under the flap. The base of the advancement flap was kept wide enough to ensure adequate circulation in the flap. The internal opening was not closed before advancing the flap over the internal opening. This was followed by suturing the flap in the distal anal canal with Vicryl 2/0 after the fistula tract had been curetted. In the AF + G group, the identical procedure was carried out. In addition to the advancement procedure, fibrin glue was installed retrogradely in the fistula tract after the AF was completed and the fistula tract had been curetted. Installation of the fibrin glue was performed via the external opening, under direct vision of the advancement flap to prevent flap dislocation and subsequent failure. No specific postoperative instructions were given to the patients. Data collection Chart review was performed on age, gender, tertiary referral, previous fistula surgery, smoking habits, complications, and fistula recurrence. All patients visited the outpatient’s clinics on a regular basis (every 2–4 weeks) until full closure of the fistula tract was achieved. The fistula was considered closed if the external opening was closed and no discharge or pain was experienced; otherwise, it was considered as a persistent or recurrent fistula. No routine postoperative imaging or proctoscopy was performed to confirm the closure of the fistula tract. The data was collected retrospectively and the outcome was compared between groups. Statistical analysis Data are presented as median values with ranges, unless otherwise specified. Categorical data are presented as frequencies or percentages. Differences between groups were tested using Mann–Whitney U test for continuous data. Chi-square test or Fisher’s exact test were used when appropriate to compare groups in case of categorical or dichotomous variables. All reported p values are two-sided. A p value of 5% or less was considered as statistical significant. Statistical analysis was done using the SPSS v.12.0 package (SPSS, Chicago, IL, USA). Results In the study period, a total of 127 patients were operated for high perianal fistulas. Inflammatory bowel disease (n = 30), HIV (n = 12), or no internal opening found during surgery (n = 5) were the reason of exclusion in 47 patients. In total, 80 patients were analyzed in this comparative study. Of these, 54 patients were treated with the AF and 26 patients underwent AF combined with the installation of fibrin glue. Furthermore, patients were matched for a history of fistula surgery. Patient characteristics for both groups are shown in Tables 1 and 2. The groups were comparable for patients’ characteristics as sex, age, smoking, seton drainage, and number of tertiary referrals. Table 1Characteristics of patients with high anorectal fistula without previous fistula surgeryVariableAF (n = 32)aAF + G (n = 9)bp valueM/F (n)18:146:30.711Age (median, in years)42 (21–67)41 (29–55)0.653Tertiary referral26 (81%)7 (78%)1.000Smoking43%71%0.232Seton drainage18 (56%)6 (67%)0.711aRectal advancement groupbRectal advancement group with fibrin glueTable 2Characteristics of patients with high anorectal fistulas with previous fistula surgeryVariableAF (n = 22)aAF + G (n = 17)bp valueM/F (n)18:411:60.282Age (median, in years)43 (22–62)47 (35–72)0.136Tertiary referral15 (68%)8 (47%)0.209Smoking53%50%1.000Seton drainage10 (46%)12 (71%)0.193aRectal advancement groupbRectal advancement group with fibrin glue Clinical outcome All patients were operated in day case setting. There were no intraoperative complications. In two patients out of the AF group, a postoperative complication was encountered, consisting of a minor bleeding (n = 1) and a bradycardia for which the patient was observed overnight (n = 1, patient with cardial history). In the AF + G group, there were no postoperative complications recorded. The minimal follow-up after surgery was 13 months with a median of 67 months (range, 13–127). There were no patients lost to follow-up. The overall recurrence rate was 26% (n = 21; Table 3.). In 17% of the patients, the fistula persisted in the AF group compared to 46% in the AF + G group (p = 0.05). In the matched group without previous fistula surgery, the result was significantly worse for the AF + G group compare to the AF group (p = 0.014). The recurrence rates were 56% (n = 5) and 13% (n = 4), respectively. In the group with a history of fistula surgery, the recurrence rate was 23% (n = 5) compared to 41% (n = 7) in the AF and the AF + G group, respectively (p = 0.216). Table 3Recurrence rates for the matched group analysisGroupAFaAF + Gbp valueOverall (n = 80)9/54 (17%)12/26 (46%)0.050No previous fistula surgery (n = 41)4/32 (13%)5/9 (56%)0.014Previous fistula surgery (n = 39)5/22 (23%)7/17 (41%)0.216aRectal advancement groupbRectal advancement group with fibrin glue Discussion High perianal fistulas remain a surgical challenge. There are various treatment options for treating high transsphincteric fistulas, e.g., the rectal and anodermal advancement flap, loose and cutting seton, fibrin glue, and potentially the newly developed anal fistula plug [3]. However, the results from these therapies vary. Transanal rectal advancement flap is nowadays the treatment of choice because of its sphincter-saving approach. Due to the low recurrence rate of only 30%, which leaves a lot of room for improvement [11, 14], fibrin glue has been widely studied. Fibrin glue was developed to obliterate the fistula tract by stimulating fibroblasts, which leads to permanent closure of the fistula tract. Unfortunately, the long-term results were not as good as expected [19, 20]. In the present series, an attempt was made to decrease the recurrence rate of the surgical treatment of high transsphincteric perianal fistulas of cryptoglandular origin by combining the two methods, i.e., fibrin glue and the rectal advancement flap in a consecutive series of patients. Overall, although not significant, a clear trend was found consisting of a worse outcome for patients from the AF + G group. The recurrence rate was 46% compared to 17% in the AF + G and AF group, respectively. In the group without a history of fistula surgery, patients in the AF + G group did significantly worse than the AF group. In the group with a history of fistula surgery, no significantly different recurrence rates were found. In 2003, Zmora et al. [21] described a small retrospective series of 13 patients with perianal fistulas of different origins treated with fibrin glue in combination with the AF [14]. In their series, a recurrence rate of 46% was found after a mean follow-up of 12.1 months. The group contained patients with fistulas of cryptoglandular origin and fistulas associated with Crohn’s disease or surgical trauma. In addition, two patients with rectovaginal fistulas were included. More recently, Ellis and Clark [23] reported on a series of 58 patients randomized into advancement flap repair alone or advancement flap repair combined with fibrin glue. Selected were patients with perianal fistulas where the fistula tract comprised more than 30 to 50% of the sphincter complex. Furthermore, patients were included when the fistula was located anteriorly in women or when the patient had a history of incontinence. In two thirds of the patients, the mucosal advancement flap was used and the remaining patients were treated by anodermal advancement flap. The recurrence rate was significantly higher in the group where fibrin glue was combined with the advancement flap compared to the group treated only by advancement (46% vs 20%). Two techniques, i.e., mucosal advancement flap and anodermal advancement flap, were used. Furthermore, no information was provided on the distribution of causes of the fistulas in both groups making the results difficult to interpret. The effectiveness of the AF is the result of the closure of the internal opening [24]. The reason why addition of fibrin glue fails to decrease the recurrence rate and even seems to worsen the result is still unclear. After AF, the fistula tract acts as a drainage canal for any remaining sepsis, with the external opening left open. With the installation of the fibrin glue, a temporary closure of the fistula tract is theoretically achieved. After a few weeks, when the clot resolves, the fibroblasts activated by the matrix should provide collagen syntheses for a definitive closure of the tract [25]. A possible explanation to why the AF + G group does worse is that the closure of the fistula tract with the fibrin glue leads to a situation where insufficient drainage from the primary and eventual secondary fistula tracts occurs. Sentovich reported on a prospective series of 48 patients (75% of cryptoglandular origin) treated with fibrin glue [20]. In their technique of using the fibrin glue, the procedure was combined with closure of the internal opening with only a figure eight suture without an advancement flap. After a median follow-up of 22 months, a recurrence rate of 31% was found. Surprisingly, the patients with longer fistula tracts did significantly worse than those with short fistula tracts. Loungnarath et al. [26] reported on 39 patients with perianal fistulas treated with fibrin glue. The overall recurrence rate was 69%. In 6 of the 39 patients, the internal opening was closed using a figure eight suture to avoid clot extrusion because of high pressure from the anal canal during defecation. Four of these patients had a recurrence (33%). This retrospective study, in contrast to earlier studies [21, 23], assessed the additional value of fibrin glue to the transanal rectal advancement flap of only patients with high transsphincteric fistulas of cryptoglandular origin with a long follow-up. The rectal advancement flap combined with fibrin glue installation was associated with a significantly higher recurrence rate, compared to the advancement flap treatment alone, in patients without previous fistula surgery. This observation must be interpreted carefully because of the small sample size of the AF + G group. As the costs of the fibrin glue are considerable and the therapeutic effect very doubtful, it cannot be recommended routinely in the adjunct of transanal rectal advancement flap treating high perianal fistulas. The rectal advancement flap remains the treatment of choice for high transsphincteric perianal fistulas of cryptoglandular origin until novel methods like the anal fistula plug are studied sufficiently in randomized trials.

J_Mol_Med-4-1-2374880

Prorenin anno 2008

For many years, prorenin has been considered to be nothing more than the inactive precursor of renin. Yet, its elevated levels in diabetic subjects with microvascular complications and its extrarenal production at various sites in the body suggest otherwise. This review discusses the origin, regulation, and enzymatic activity of prorenin, its role during renin inhibition, and the angiotensin-dependent and angiotensin-independent consequences of its binding to the recently discovered (pro)renin receptor. The review ends with the concept that prorenin rather than renin determines tissue angiotensin generation. Introduction Despite nearly 40 years of research on prorenin, the renin precursor is still the least well-understood component of the renin–angiotensin system (RAS). Initially, it was thought to have no function at all. Yet, it circulates in human plasma in excess to renin, sometimes at concentrations that are 100 times higher, and in plasma of anephric subjects, prorenin, but not renin, is still present [1, 2]. This suggests that prorenin, in contrast with renin, is also of extrarenal origin. Prorenin is particularly elevated in diabetic subjects with microvascular complications [3]. Moreover, the renal vasodilator response to captopril in diabetic subjects correlated better with plasma prorenin rather than with plasma renin [4]. Thus, (circulating) prorenin may have a function after all. Possibly, it is prorenin (and not renin) which is responsible for tissue angiotensin generation. Obviously, this would require local prorenin–renin conversion, for which no evidence exists [5]. In support of this concept, however, transgenic rodents with (inducible) prorenin expression in the liver display increased cardiac angiotensin (Ang) I levels, cardiac hypertrophy, and/or vascular damage [6, 7]. Origin and regulation of prorenin The juxtaglomerular epithelioid cells, located in the walls of renal afferent arterioles, are the main source of renin in the body. Renin is synthesized as preprorenin. Preprorenin is converted to prorenin upon insertion into the endoplasmatic reticulum. The majority (75%) of prorenin is secreted constitutively, while the remainder is targeted to dense core secretory granules. In these granules, an acidic pH is created to optimize the activity of the proteases (cathepsin B, prohormone convertases) that cleave off the prosegment to yield renin. Prorenin and renin levels are highly correlated but do not alter in parallel under all circumstances [1]. Acute stimuli of renin will not affect prorenin levels, whereas chronic stimuli (like a decrease in Ang II) increase both renin and prorenin. This suggests that renin is stored as active enzyme and is released immediately upon stimulation of the juxtaglomerular apparatus. Prorenin is released constitutively, and no acute responses occur. Chronic stimulation causes more prorenin to be converted to renin, leading to an increased renin/prorenin ratio in plasma. However, some exceptions to this rule exist. A well-known example is, as mentioned above, diabetes mellitus complicated by retinopathy and nephropathy [3]. Pregnant women also have high plasma prorenin levels, derived from the ovaries [8]. The function of this prorenin is unknown, as is the function of prorenin in amniotic fluid, in which prorenin was discovered. The reproductive organs, together with the adrenal, eye, and submandibular gland, are sites of extrarenal renin gene expression [9]. For reasons that are not understood, these tissues predominantly, if not exclusively, synthesize and release prorenin. Enzymatic activity of prorenin? A 43-amino-acid N-terminal propeptide explains the absence of enzymatic activity of prorenin. This propeptide covers the enzymatic cleft and obstructs access of angiotensinogen to the active site of renin. Prorenin can be activated in two ways: proteolytic or nonproteolytic [10]. Proteolytic activation is irreversible: it involves actual removal of the propeptide. Nonproteolytic activation of prorenin is reversible. It can best be imagined as an unfolding of the propeptide from the enzymatic cleft. Nonproteolytic activation can be induced by exposure to low pH (pH = 3.3) or cold (4°C) [10]. Nonproteolytically activated prorenin is enzymatically active and can be recognized by monoclonal antibodies that are specific for the active site. Kinetic studies of the nonproteolytic activation process have indicated that an equilibrium exists between the closed (inactive) and open (active) forms of prorenin. The inactivation step is highly temperature dependent and occurs very rapidly at neutral pH and 37°C. Consequently, under physiological conditions, <2% of prorenin is in the open and active form, i.e., displays enzymatic activity, and >98% is closed and inactive. Prorenin receptor(s)? The beneficial effects of RAS blockers are due, at least in part, to blockade of the generation or action of Ang II at tissue sites [11]. In tissues that are believed not to express the renin gene, like the heart and vascular wall, such angiotensin generation depends on renin/prorenin taken up from the circulation. Simple diffusion cannot explain the relatively high renin levels in these organs, and thus a receptor-mediated mechanism may exist. Two candidates currently have been proposed: the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) [12–14] and the (pro)renin receptor [15]. The M6P/IGF2R nonselectively binds M6P-containing proteins like renin and prorenin. However, such binding did not result in angiotensin generation, and it is now believed that the M6P/IGF2R is a clearance receptor for renin/prorenin [16]. This leaves the (pro)renin receptor as the most promising candidate for tissue uptake of circulating renin/prorenin. This receptor, a 350-amino-acid protein with a single transmembrane domain, binds prorenin with higher affinity than renin [17] and, unlike the M6P/IGF2R, does not internalize these proteins. Interestingly, binding to the receptor allowed prorenin to become catalytically active without proteolytic cleavage of the prosegment [15, 17]. Apparently, therefore, binding induces a conformational change in the prorenin molecule, similar to the change occurring after exposure to cold or low pH. Angiotensin-independent effects of prorenin? After the discovery of the receptor, (pro)renin receptor antagonists were designed based on the idea that the prosegment contains a ‘handle region’ which binds to the receptor [18]. These (peptidic) antagonists (also known as ‘handle region peptides’, HRP) mimic the handle region and thus may bind to the receptor instead of prorenin. In support of this concept, HRP infusion normalized the elevated renal angiotensin content in diabetic rats [18] and simultaneously prevented the development of diabetic nephropathy. Surprisingly, identical effects occurred in diabetic angiotensin II type 1A receptor-deficient mice [19]. Since such mice no longer display the normal (constrictor) response to Ang II [20], the effect of the (pro)renin receptor antagonist in these mice cannot be due to suppression of local angiotensin generation. Thus, prorenin may also exert direct angiotensin-independent effects, possibly via the above described (pro)renin receptor (Fig. 1). Indeed, prorenin (and renin) induced p42/p44 mitogen-activated protein kinase (MAPK) activation and transforming growth factor-β1 release in mesangial cells [15, 21], and these effects did not occur following deletion of the receptor with siRNA [21]. Moreover, in cardiomyocytes, prorenin concentration dependently activated p38 MAPK and phosphorylated heat shock protein 27 [22]. Fig. 1Model depicting prorenin activation by the (pro)renin receptor (P)RR, allowing prorenin to generate angiotensin I from angiotensinogen. In addition, prorenin binding to the receptor results in effects (intracellular signaling) that are independent of angiotensin generation. HRP is assumed to block both prorenin activation and the direct prorenin-induced effects (see text for further explanation) Overexpression of the human (pro)renin receptor in rats resulted in elevated blood pressure, increased plasma aldosterone, and/or glomerulosclerosis [23, 24]. Since such overexpression was not accompanied by changes in renin or Ang II, angiotensin-independent effects of the receptor may underlie this phenotype. HRP prevented the development of glomerulosclerosis in (pro)renin receptor transgenic rats [25]. Yet, transgenic rats with inducible hepatic prorenin expression (resulting in a >200-fold rise in plasma prorenin) did not develop glomerulosclerosis [26], although such animals did develop hypertension. Moreover, HRP blocked neither prorenin binding to cells overexpressing the human (pro)renin receptor [17] nor prorenin-induced signaling in U937 monocytes [27]. Thus, it is uncertain to what degree the beneficial in vivo effects of HRP are solely due to prorenin blockade. Such effects are unlikely to involve renin, since HRP will not block renin–(pro)renin receptor interaction. Prorenin and renin inhibition Prorenin levels will rise during renin inhibition, as they do during any type of RAS blockade. Renin inhibitors affect the equilibrium between the open and closed forms of prorenin, because such drugs (due to their high affinity for the active site) prevent inactivation [10]. Therefore, renin inhibitors, like low pH, cold, and the (pro)renin receptor, are capable of nonproteolytically ‘activating’ prorenin although, of course, due to the presence of the renin inhibitor, this open prorenin cannot display enzymatic activity. Open prorenin will however be recognized by the active site-directed antibodies applied in renin immunoradiometric assays, thus leading to an overestimation of the renin rise during renin inhibition [28]. Theoretically, the elevated (pro)renin levels during renin inhibition might result in (pro)renin receptor activation. In vitro studies suggest that renin inhibitors do not interfere with this process. The consequence of such overstimulation is unknown. However, since such a rise also occurs during other types of RAS blockade, its detrimental effects, if present, should have been known by now. A possible explanation comes from the work of Schefe et al. [29], who showed that, on activation of the receptor, the transcription factor promyelocytic zinc finger is translocated to the nucleus and represses transcription of the (pro)renin receptor itself, thus creating a short negative feedback loop. In other words, high (pro)renin levels, as occurring during RAS blockade, will suppress (pro)renin receptor expression, thereby preventing excessive receptor activation. Furthermore, the members of a family with a mutated renin allele, resulting in high plasma prorenin levels, were phenotypically normal [30]. Conclusion After many years, it now seems that a function for prorenin has been found. The ‘inactive’ renin precursor gains Ang I-generating activity by binding to a receptor, without undergoing proteolytic cleavage. This mechanism explains how prorenin might contribute to tissue angiotensin generation, even when no prorenin–renin conversion occurs outside the kidney. Renin inhibitors will bind to such open activated prorenin, to the same degree as they bind to renin, and may thus be the ideal tools to block tissue angiotensin generation. In vitro studies suggest that prorenin also acts as an agonist of the (pro)renin receptor, inducing intracellular signaling pathways in an angiotensin-independent manner. However, given the contradictory data obtained with the (pro)renin receptor blocker HRP, more work is needed to verify the in vivo importance of such prorenin-induced (pro)renin receptor activation.

Dysphagia-3-1-1914227

Viscosity Is Not a Parameter of Postdeglutitive Pharyngeal Residue: Quantification and Analysis with Scintigraphy

The aim of this study was to explore the influence of viscosity on pharyngeal residue in normal healthy volunteers. Scintigraphy was used to measure pharyngeal residue in 11 healthy volunteers after swallowing three different substances (age = 20.2–48.3 years). The first substance was a 10-ml solution of tap water with 0.5% xanthan with a viscosity of 4500 mPa s, comparable to a yogurt drink. The second and third substances were a 0.75% xanthan and a 1.00% xanthan solution, with viscosities of 10,500 and 21,000 mPa s, comparable to low-fat yogurt and 3% fat yogurt, respectively. Tap water was used as the control substance. Mean pharyngeal residue after swallowing tap water was 2.3% (SD = 1.2) of the initial volume in the oral cavity. Pharyngeal residue after swallowing 0.5% xanthan solution was 1.8% (SD = 0.8), after swallowing 0.75% xanthan solution 2.6% (SD = 2.2), and after swallowing 1.00% xanthan solution 2.8% (SD = 1.7). No significant correlation between increase of viscosity and pharyngeal residue was found. In healthy persons viscosity does not seem to be a significant parameter for pharyngeal residue for boluses with viscosities ranging from tap water to solutions having a viscosity comparable to 3% fat yogurt. The residual amount of food or beverage in the pharynx after swallowing (pharyngeal residue) is an important parameter in diagnostic procedures in the treatment of swallowing disorders. In common practice of management of swallowing disorders, the amount of pharyngeal residue is determined either by videofluoroscopy or by transnasal flexible endoscopy (FEES). With the help of logistic regression, Perlman et al. [1] predicted the chance of aspiration (food or liquid that gets into the lungs) based on the amount of pharyngeal residue. An odds ratio of 1.4 for aspiration was found for patients with pharyngeal residue. In this study 330 videofluoroscopies of dysphagic patients were analyzed and the amount of pharyngeal residue was rated on a four-point scale (absent, mild, moderate, severe). The study also showed that a patient with severe pharyngeal residue has an increased risk of aspiration (odds ratio of 4.0) and underlined the importance of quantifying pharyngeal residues. Dejaeger et al. [2] analyzed videofluoroscopies of 25 healthy elderly and clearly described localization of pharyngeal residue; unfortunately, they did not attempt to quantify the amount of residue. Few attempts have been made to quantify pharyngeal residue in normal and dysphagic populations, mainly because of the limited availability of appropriate diagnostic tools. Videofluoroscopy or FEES has been used to explore physiologic parameters of swallowing [3,4]. One of the limitations of the use of these techniques is the lack of reliable interpretation of physiologic parameters. Stoeckli et al. [5] analyzed the inter- and intrajudge reproducibility of the interpretation of videofluoroscopy and suggested that the agreement for assessing pharyngeal parameters is minimal to moderate (κ = 0.01–0.56). Scintigraphy allows quantitative measurement by using a technetium-99m (99mTc)-labeled colloid. 99mTc colloid is used in routine clinical practice for investigating gastrointestinal motility disorders of the esophagus and stomach [6,7]. The scintigraphic technique allows quantitative assessment because the amount of radioactivity (radioactive counts) can be measured with a gamma camera. Scintigraphy in dysphagia mainly is used for detection of aspiration in young children because the radiation doses are less than those of videofluoroscopy [8]. It is used less frequently in adults, although recently it has been described as a useful and reliable diagnostic tool in adult patients with oropharyngeal dysphagia [9]. Only three studies have been performed with scintigraphy to examine pharyngeal parameters of normal swallowing physiology: Cook et al. [10] used scintigraphy to study the influence of age on swallowing efficiency. Hamlet et al. [11] reported on the differences in bolus transit times and oropharyngeal residues in healthy volunteers, while Shaw et al. [12] studied the influence of bolus volume on pharyngeal residue. All these studies examined different parameters of swallowing and found different outcomes. The aim of this study was to explore the influence of viscosity on pharyngeal residue in normal healthy volunteers, where our hypothesis was that swallowing a more viscous product would result in more pharyngeal residue in healthy volunteers. Methods Volunteers Eleven healthy volunteers (9 female, 2 male, mean age = 29.1 years, standard deviation [SD] = 8.9), recruited by public advertisement, were invited to participate. All volunteers reported no history of swallowing problems. All volunteers gave written informed consent to participate in the study, which was approved by the Medical Ethics Committee of the Academic Medical Center of the University of Amsterdam. Bolus Characteristics Before the examination, three different substances with three different viscosities were prepared in a research laboratory. All viscosities were measured with a viscosity meter (Brookfield LVT Viscometer, model DV-II+) after 24 h of storage in a refrigerator at a constant temperature of 4°C. Measurements were performed with spindle No. 63 at a velocity of 3 rpm and viscosities were recorded after 30 s. The substances consisted of tap water thickened with different percentages of xanthan, a polysaccharide commonly used in the food industry to thicken fluids. The first substance was a solution of tap water with 0.5% xanthan resulting in a substance with a viscosity of 4500 mPa s, comparable to a yogurt drink. The second and third substances were tap water with 0.75% and 1.00% xanthan, resulting in viscosities of 10,500 and 21,000 mPa s, comparable to low-fat yogurt and 3% fat yogurt, respectively. As reference, tap water with a viscosity of 1 mPa s (per definition) was used. For all solutions 200 ml was labeled with 260 MBq of 99mTc colloid (Hepatate, GE Health, Eindhoven, The Netherlands) dissolved in 1 ml 0.9% saline solution and prepared in such a way that for 10 ml of each solution the radiation dose was 13 MBq. After labeling, 44 syringes (4 per volunteer) were used to collect 10 ml of each of the four solutions (tap water, 0.5%, 0.75%, and 1.00% xanthan). The three xanthan solutions, stored for more than 24 h in a refrigerator at a stable temperature of 4°C, were mixed with the 99mTc colloid 1 h before the study. Data Collection For this study a gamma camera (Orbiter, Siemens Medical Systems, Germany) was fitted with a low-energy all-purpose collimator. A dynamic acquisition using 0.25-s frames was performed for 30 s. Data were acquired and processed on a Hermes workstation (Nuclear Diagnostics, Sweden). Volunteers were asked to sit in front of the gamma camera and hold their right cheek against the collimator. The oral cavity was placed in the middle of the field-of-view. When the volunteer was placed in the right position, 10 ml of one of the solutions was emptied in the mouth of the volunteer with a syringe. Volunteers were instructed to hold the bolus in their mouth and swallow on command. They were specifically instructed to swallow the whole bolus in one swallow and to breath through the mouth to inhibit a second swallow. Three cobalt markers were placed on the skin of the volunteers using a pen pointer: one on the mastoid bone, one on the hyoid bone, and one underneath the thyroid cartilage. The exact placement for the marker was found by palpation of the head and neck. First, the volunteers were asked to swallow the water with 99mTc colloid. After recording the swallow for 30 s, the volunteers were asked to rinse their mouth with unlabeled tap water and to swallow 200 ml unlabeled tap water to wash away any residue from the previous recording. The second recording was done with 10 ml of the 0.5% xanthan solution, the third with the 0.75% xanthan, and the last with the 1.00% xanthan. Between recordings the volunteers were instructed to rinse their mouth and swallow 200 ml unlabeled tap water to clear any possible residue. Each volunteer swallowed a consistency labeled with a dose of 13 MBq four times, for a total of 52 MBq, which is equal to a radiation dose of less than 1 milliSievert (mSv). According to the ICRP62 guidelines, this is a minor radiation dose (category IIa). After data collection the results were stored and processed on a Hermes workstation. Based on the markers, two regions of interest (ROI) were defined: the oral cavity (ROI-1), bordered by a straight line between the hyoid marker and the mastoid marker, and the pharynx (ROI-2), bordered by a straight line between the hyoid marker and the mastoid marker and a horizontal line from the thyroid marker, as described by previous studies [10,11]. To estimate background activity during the examinations, a third ROI with a comparable surface area to ROI-2 was defined to the right of the field of view. Figure 1 shows the three ROIs. Collected data were transferred to a spreadsheet file (Excel, Microsoft Corp., Redmond, WA), which showed the number of counts within the specific ROI at each frame (0.25 s) of the total acquisition. For our analysis the average count of 8 frames (2 s) before swallowing was used to determine the average number of counts in the oral cavity. After swallowing the average count of 4 s (16 frames) was used to determine the average amount of pharyngeal residue, expressed in counts. Background activity was estimated by a measurement of 4 s; this was subtracted from the average count in the oral cavity and the pharyngeal residue. Hereafter, the number of pharyngeal counts was divided by the number of counts in the oral cavity, thus providing a percentage of pharyngeal residue. Fig. 1.Scintigraphic evaluation with three regions of interest (ROI). 1= oral cavity, 2 = pharynx, 3 = background radiation, a = mastoid marker, b = hyoid marker, 3 = thyroid marker. Statistical Analysis The calculated percentages of pharyngeal residue were transferred to SPSS v12.0 (SPSS Inc., Chicago, IL) for further analysis. Distribution of the percentages was formally tested with the Kolmogorov–Smirnoff test for normality and was not found to be normally distributed. To explore differences in median percentages of pharyngeal residues of the three xanthan solutions to the reference (tap water) on a group level, a nonparametric (Friedman) test was used. All statistical tests were two-tailed and differences were evaluated at the 5% level of significance. Results The mean pharyngeal residue after swallowing tap water was 2.3% (SD = 1.2; range = 1.1%–4.5%) of the initial volume in the oral cavity. The percentage of pharyngeal residue after swallowing the 0.5% xanthan solution was 1.8% (SD = 0.8; range = 0.9%–2.8%), after swallowing the 0.75% xanthan solution the residue was 2.6% (SD = 2.2; range = 0.7%–8.1%), and after swallowing the 1.00% xanthan solution the pharyngeal residue was 2.8% (SD = 1.7; range = 1.2%–6.4%). Median percentages of pharyngeal residue were analyzed. Figure 2 shows by boxplot the distribution of residue for the four different viscosities in the 11 nondysphagic volunteers. No significant difference in the amount of residue between the four groups was found (χ2 = 0.927, df = 3, p = 0.819).Fig. 2.Distribution of percentages residue on group level. Although the sample size was limited, post hoc analysis showed that this study had a power of 0.96 (n = 11 with an effect size of 0.47 based on a SD and correlation between measurements occasions of 1.1 and 0.7, respectively). Based on these results, a power calculation showed that in future research (with a power of 0.80 and alpha of 0.05) at least 137 healthy volunteers would be needed to detect any possible difference in the amount of pharyngeal residue between the four groups (tap water, 0.5%, 0.75%, and 1.00% xanthan) (Table 1). Table 1.Pharyngeal residue in percentages with 10ml bolusesNo.Water0.5% Xanthan0.75% Xanthan1.00% Xanthan11.11.20.71.221.81.21.01.731.30.78.13.441.41.51.71.354.12.32.66.462.91.51.51.271.20.91.22.482.42.55.35.493.12.52.42.4102.02.52.12.7114.52.82.12.4Mean2.3 (SD ± 1.2)a1.8 (SD ± 0.8)a2.6 (SD ± 2.2)a2.8 (SD ± 1.7)aaNo significant difference (p > 0.05). Discussion Our study attempted to investigate the correlations between viscosity and pharyngeal residue in healthy volunteers; we did not find evidence for a positive correlation between an increase of viscosity and an increase in pharyngeal residue. Because Shaw et al. [12] established that bolus volume does not influence pharyngeal residue, allowing extrapolation of our findings to other volumes, only one bolus volume was used in this study. It might be suggested that our volunteers drank 200 ml tap water after the first labeled bolus and thus the mucosa of the pharynx was relatively dry before the first estimation of pharyngeal residue. Because the larger proportion of our volunteers had drunk coffee, tea, or water in the last hour before the study, differences in dryness of the pharyngeal mucosa is not considered a confounder. Only one bolus per viscosity was used, therefore no data on intrasubject differences could be provided. If multiple boluses per viscosity had been used in this study, the radiation exposure for our volunteers would have been much higher. Because none of the volunteers had swallowing disorders and all were used to swallowing tap water and yogurt, any adaptation in swallowing during the study to the circumstances during data collection or intrasubject differences can be considered negligible. Raut et al. [13] examined with manometry the influence of increased viscosity on pharyngeal pressures. It was concluded that an increased bolus viscosity led to increased amplitude of the bolus wave and clearing contractions within the pharynx. The increase in contraction of the pharyngeal muscles supports our findings of equivalent percentages of residue for the different viscosities. Hamlet et al. [11] reported on the differences in bolus transit times and oropharyngeal residues in healthy volunteers. In that study 20 healthy volunteers (age = 39–65 years) swallowed tap water and a thickened substance labeled with 99mTc. The mean pharyngeal residue was found to be 4.0% (standard error [SE] = 0.5) for tap water and 10.5% (SE = 0.1) for a substance with a viscosity of 1070 mPa s. Differences in the Hamlet et al. study and our study might be explained by temperature influences and different approaches to measure viscosity. In the Hamlet study [11] the bolus temperature was set at 25°C, whereas in our study syringes were stored in a chilled room. Therefore, temperature is not considered a confounding factor in our study. Also, in the Hamlet study [11] a Brookfield Viscometer type RVT was used, which is more suitable for measuring the viscosity of low-viscosity products like milk. In our study we used a Brookfield Viscometer type LVT, which is more suitable for high-viscosity substances, making an adequate comparison between measured viscosities of the two studies not possible. Literature suggests that body positioning, bolus temperature, and taste affect swallowing [3,4,14]. In our study the volunteers had to rotate their heads to the right side to obtain adequate measurements, following the protocol used in the Hamlet study [11]. This might have reduced the amount of pharyngeal residue because the bolus passes the pharynx mainly on one side and the contact area between bolus and pharyngeal wall is decreased. It is known that in some dysphagic patients (i.e., patients with unilateral pharyngeal weaknesses) rotating the head decreases the amount of residue, but the effects of head rotation on healthy individuals has not been investigated. Looking at the wide distribution of percentages of residues in volunteers (Table 1) and the slightly lower percentage (2.3%) found in our study compared with that of Shaw [12] (2.5%), where measurements were made in a right lateral position, one might conclude that the influence of head rotation in our study cannot be seen as a confounding factor on the outcome. Conclusion In 11 healthy volunteers, mean pharyngeal residue after swallowing tap water was 2.3% (SD = 1.2) of the initial volume in the oral cavity. The percentage of pharyngeal residue after swallowing 0.5% xanthan solution was 1.8% (SD = 0.8), after swallowing 0.75% xanthan solution the residue was 2.6% (SD = 2.2), and after swallowing 1.00% xanthan solution the residue was 2.8% (SD = 1.7). Our data do not show a significant increase in pharyngeal residue in healthy volunteers as a result of an increasing viscosity of viscous solutions, ranging from tap water to full-fat yogurt. Although some trends may be seen, (confounding) factors other than viscosity may play a role in the build up of pharyngeal residue.

Planta-4-1-2270920

Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in Plantago major, Plantago maritima, Prunus persica, and Apium graveolens

Sugar and sugar alcohol concentrations were analyzed in subcellular compartments of mesophyll cells, in the apoplast, and in the phloem sap of leaves of Plantago major (common plantain), Plantago maritima (sea plantain), Prunus persica (peach) and Apium graveolens (celery). In addition to sucrose, common plantain, sea plantain, and peach also translocated substantial amounts of sorbitol, whereas celery translocated mannitol as well. Sucrose was always present in vacuole and cytosol of mesophyll cells, whereas sorbitol and mannitol were found in vacuole, stroma, and cytosol in all cases except for sea plantain. The concentration of sorbitol, mannitol and sucrose in phloem sap was 2- to 40-fold higher than that in the cytosol of mesophyll cells. Apoplastic carbohydrate concentrations in all species tested were in the low millimolar range versus high millimolar concentrations in symplastic compartments. Therefore, the concentration ratios between the apoplast and the phloem were very strong, ranging between 20- to 100-fold for sorbitol and mannitol, and between 200- and 2000-fold for sucrose. The woody species, peach, showed the smallest concentration ratios between the cytosol of mesophyll cells and the phloem as well as between the apoplast and the phloem, suggesting a mixture of apoplastic and symplastic phloem loading, in contrast to the herbal plant species (common plantain, sea plantain, celery) which likely exhibit an active loading mode for sorbitol and mannitol as well as sucrose from the apoplast into the phloem. Introduction In order to understand carbon partitioning, the regulation of photosynthesis, phloem loading, or sink–source interactions, it is important to know how photoassimilates are distributed between the subcellular compartments of mesophyll cells of source leaves. Much of the information concerning subcellular metabolite concentrations is derived from studies of species such as spinach, barley, tobacco, and potato. In such plant species carbohydrates assimilated from CO2 are exported into the phloem exclusively in the form of sucrose (Riens et al. 1991; Winter et al. 1992; Lohaus et al. 1998). However, it has been shown that in several plant species, sugar alcohols like mannitol, sorbitol or dulcitol are also translocated in addition to sucrose (Zimmermann and Ziegler 1975). The advantage of transporting sucrose and sugar alcohols is that these compounds are highly soluble and chemically inert. In addition to functioning as a transport form for reduced carbon, sugar alcohols have been also shown to serve as storage for reduced carbon, as compatible solute synthesized in response to abiotic or biotic stress, or as osmoprotectants. It has been estimated that up to 30% of the carbon fixed by plants on earth may be present in the form of polyols (Bieleski 1982). Mannitol is the most-widely distributed sugar alcohol and has been reported in more than 100 species of vascular plants, including most species of the Apiaceae (celery, parsley, and carrot), Oleaceae (olive, privet), Rubiaceae (coffee), and Scrophulariaceae (snapdragon) (Zimmermann and Ziegler 1975; Bieleski 1982). In higher plants, mannitol is synthesized by the action of an NADPH-dependent mannose-6-phosphate reductase that catalyzes the conversion of mannose-6-phosphate to mannitol-1-phosphate, followed by dephosphorylation by a phosphatase (Rumpho et al. 1983). The activity of mannose-6-phosphate reductase was only observed in mature leaves. In different plant species, mannitol synthesis occurs simultaneously with either sucrose synthesis, as in celery (Rumpho et al. 1983), or with raffinose oligosaccharide synthesis, as in Oleaceae (Zimmermann and Ziegler 1975). To date, there are only few reports on the intracellular distribution of mannitol (Keller and Matile 1989; Moore et al. 1997) and these studies do not include measurements of phloem concentrations of mannitol. Another sugar alcohol present in higher plants is sorbitol. Sorbitol is a major photoassimilate translocated in addition to sucrose in the phloem of woody Rosaceae, including all members of the economically important genera Malus (apple), Pyrus (pear) and Prunus (stone fruits such as peach, cherry, plum and apricot; Zimmermann and Ziegler 1975; Bieleski 1982; Moing et al. 1997). Sorbitol is synthesized in source organs from glucose-6-phosphate by the activity of aldose-6-P-reductase (Negm and Loescher 1981) converting glucose-6-phosphate to sorbitol-6-phosphate. Sorbitol-6-phosphate is further converted to sorbitol by a specific phosphatase. Sinks have little or no capacity to synthesize sugar alcohols like mannitol or sorbitol (Loescher and Everard 1996). To our knowledge, there is only one report on the intracellular distribution of sorbitol in leaf cells (Moore et al. 1997) and with the exception of peach (Moing et al. 1997) there are no data on polyol concentrations in the phloem sap of sugar–alcohol-translocating species. In addition to mannitol and sorbitol, most plant species also contain the cyclitol myo-inositol (Zimmermann and Ziegler 1975) which is also produced from glucose-6-phosphate (Loewus and Loewus 1983). Myo-inositol and its derivatives are involved in membrane formation, cell wall synthesis, seed germination, and hormone responses as well as stress responses (Loewus and Loewus 1983). As an integral part of galactinol, myo-inositol also acts as a carrier of activated galactose that is transferred to sucrose, yielding raffinose and its polymeric derivates of higher order. Knowledge of concentrations of sugar alcohols and sugars in subcellular compartments of leaf cells and in the phloem is important to understand possible modes of phloem loading. Therefore, we examined the subcellular concentrations of sorbitol, mannitol, myo-inositol, hexoses and sucrose in peach (P. persica, Rosaceae), celery (A. graveolens, Apiaceae), common plantain (P. major, Plantaginaceae), and sea plantain (P. maritima, Plantaginaceae) as well as the corresponding concentrations in the apoplast and in the phloem sap. Celery and common plantain were selected because much of what is known about the transport of mannitol and sorbitol has come from studies of these plant species, and more recently, the relevant transporters for sucrose as well as for sugar alcohols were cloned from these species. In addition, sea plantain and peach were selected because both species also translocate sorbitol but the former belongs to the group of halophytes and the latter to the group of woody plants. In this manner, different groups of sorbitol-translocating plants are included in the comparison. The sugar alcohols and sugars listed above are the main soluble carbohydrates in these plants. Based on the subcellular distribution, we calculated the concentration ratios of soluble carbohydrates between the different subcellular compartments as well as in compartments which are involved in phloem loading. In combination with results from morphological studies and data about sucrose and polyol transporters available in the literature the results are compared to predictions based on models of symplastic and apoplastic phloem loading that are currently discussed in the field. Material and methods Materials Plants of A. graveolens var dulce cv Vert d´Elne (celery; supplied by R. Lemoine, University of Poitiers cedex, France), P. major (common plantain; supplied by N. Sauer, University of Erlangen, Germany) and P. maritima (sea plantain; supplied by N. Sauer, University of Erlangen, Germany) were grown in 2-l pots in compost soil in a green-house with supplemental illumination at a 15 h photoperiod and a 23/18°C day–night thermoperiod. Two-month-old plants were taken for the experiments. P. persica (peach), cv. Red Haven (Jenssen, Göttingen, Germany) was grown in 5-l pots in compost soil in a green house. Three-year-old plants were used for the experiments. Leaf samples were harvested in May and June. Non-aqueous fractionation of leaf tissue Leaves were cut from the plants after 9 h of the light period. The middle rib and larger veins were removed, and the samples were ground to a fine powder in liquid nitrogen in a precooled mortar. The leaf tissue was lyophilized at −25°C. The dry leaf powder was suspended in 20 ml of heptane:tetrachloroethylene mixture (density 1.27 g ml−1, the solvents were dried and stored over molecule sieve beads from Merck, Darmstadt, Germany) and ultrasonicated with 5-s pulses and 5-s breaks for a total sonication period of 95 s (Cell Disrupter B15, Bronson Sonifer). The suspension was then poured through a polyester monolen sieve with a pore size <40 μm, diluted 3-fold with heptane, and centrifuged for 10 min at 2,200g (Centrifuge 5810R, Eppendorf). The clear supernatant was discarded and the sediment resuspended in 3 ml of a heptane:tetrachloroethylene mixture (density 1.27 g ml−1). Two 200 μl aliquots were withdrawn for the determination of enzyme activity and carbohydrates in the unfractionated material. Two milliliter of the remaining material were added to an exponential heptane-tetrachlorethylene gradient with a density difference between 1.27 and 1.48 g ml−1 for common plantain and sea plantain, and between 1.27 and 1.45 g ml−1 for peach and celery. The bottom of the centrifugation tube contained a 2 ml cushion of a heptane:tetrachlrethylene mixture with the highest density (1.58 g ml−1). After centrifugation (25,000g, 2.5 h) six or seven fractions were collected from the centrifuge tube content. Each of these fractions was divided into two portions, one for assay of marker enzymes and the other for assay of carbohydrates. NADP-glycerine aldehyde phosphatase, PEP-carboxylase and α-mannosidase were taken as marker enzymes for stroma, cytosol and vacuole. Chloroplastic material (including stroma) appeared in the lower region of the gradient, the cytosolic compartment was found to be enriched in the middle region of the gradient, whereas vacuolar material was mainly found in the fraction of highest density. Chloroform methanol extracts were prepared from the aliquots for the determination of the carbohydrate concentrations (see next section: “Extraction of sugars and sugar alcohols from non-aqueous fractions”). For the evaluation of the subcellular distribution of sugars and sugar alcohols between the stromal, cytosolic, and vacuolar compartment, a calculation procedure according to Riens et al. (1991) was used. This calculation method is based on the assumption that the metabolites are confined to the three compartments as indicated by the corresponding marker enzymes. The evaluation is done by a computer program testing all possible cases for the distribution of a certain metabolite between the three compartments, using increments of 1%; for example: (1) vacuole 100%, stroma 0%, and cytosol 0%; (2) vacuole 99%, stroma 1%, and cytosol 0%; (3) vacuole 99%, stroma 0%, and cytosol 1%. There are 5,151 possibilities for the distribution of a metabolite between the three compartments and the program uses possibility yields to calculate the best fit (agreement) with the experimental results. To avoid the results being falsified by analytical errors, the calculations are based on mean values obtained from measurements of at least five independent density gradient fractionations. Extraction of sugars and sugar alcohols from non-aqueous fractions Chloroform:methanol extracts were prepared to extract sugars and sugar alcohols from samples after non-aqueous fraction. The aliquots (see above) were dried and 5 ml chloroform:methanol (3:7, v/v) was added to the powder. The samples were homogenized until completely thawed and kept on ice for 30 min. The homogenate was then extracted twice with 3 ml water. The aqueous phases were combined and evaporated in a rotary evaporator. The dried residue was dissolved in 0.7 ml ultrapure water (Millipore), syringe-filtrated (0.45 μm cellulose-acetate; Schleicher and Schuell, Dassel, Germany) and stored at −80°C. Collection of sieve tube sap Sieve tube sap was obtained from severed stylets of the green-peach aphid, Myzus persicae (Sulz.). About 10 aphids were caged for about 5 h on the leaf. Their stylets were cut by a laser beam (Lohaus et al. 1995). The exuding phloem sap (10–500 nl) was collected in micro-capillaries (total volume 0.5 μl) and the volume of the exudate was determined by measuring the length occupied by the solution. Evaporation of the phloem sap was prevented by bringing the front edge of the capillary in close contact with the leaf surface and surrounding the end with a plastic cap. The humidity around the capillary was about 80%. Under these conditions no evaporation from reference capillaries was detectable. The samples were ejected into 50 μl of distilled sterile water and stored at −80°C. Extraction of apoplastic washing fluids from leaves Leaves were cut 7–8 h after the onset of illumination and carefully washed with deionized water. Leaves were placed into a syringe filled with 40 ml deionized water and infiltrated by pulling the plunger, producing a reduced pressure of approximately 20 kPa. Thereafter, intact leaves were blotted dry, positioned with the xylem wound up in a 10 ml vessel and centrifuged immediately at 200g, for 4 min at 4°C. Due to the fact that infiltration of the apoplastic air space leads to a dilution of the apoplastic fluid, the solute concentrations in the apoplastic washing fluid were corrected by the ratio volume of the infiltration solution (which corresponds to the volume of the apoplastic gas space Vgas) to the volume of the apoplastic water space (Vwater). The ion concentration in the apoplast was calculated by multiplying the ion concentration in the apoplastic washing fluid by the dilution factor (=(Vwater + Vgas )/Vwater). The mean dilution factor of several C3 herbal dicot plant species is about six (Lohaus et al. 2001). Therefore, the concentrations of the apoplastic fluid were diluted about 6-fold by the infiltration procedure in common plantain, sea plantain and celery. The dilution factor in peach was only 2.7 because the apoplastic water space was 17%, expressed as percentage of total leaf water content (Moing et al. 1997) which corresponds to about 113 μl g−1 leaf water space (Table 3), and the apoplastic gas space was 190 μl (g FW)−1 (Table 3). Cellular contamination of apoplastic washing fluids was quantified by comparing the activity of malate dehydrogenase in the apoplastic wash fluid with that in leaf extracts (Lohaus et al. 2001). According to this test, cellular cross-contamination was always below 0.1%. Metabolite analysis Sugars and sugar alcohols in the samples after non-aqueous fractionation, apoplastic washing fluids, and phloem sap were analyzed by HPLC. An ion exchange column (CarboMA10; Dionex Corp, Sunnyvale, CA, USA) was eluted isocratically with 600 mM NaOH (0.4 ml min−1) buffer for 60 min. Sugars were detected by a pulse amperometric detector with a gold electrode (ESA, Model 5200, Coulochem II, Bedford, MA, USA). Pulse setting was at 50, 700 and −800 mV for 500, 540 and 540 ms, respectively. Sugar standards were measured daily, and plant samples were diluted sufficiently to provide signals within the linear range of the detector response (50–500 μM). The metabolite amount was calculated from the peak area using Peaknet 5.1 software (Dionex, Idstein, Germany). Electron microscopy and determination of subcellular volumes Source leaves were cut into pieces of approximately 2 mm2 and prefixed in 5% glutaraldehyde (v/v), 5 CaCl2, and 50 mM sodium-cacodylate buffer, pH 7.0. After 3 h at 4°C, the fixed slices were washed four times for 15 min each in cold 50 mM sodium-cacodylate buffer, pH 7.0, fixed in 2% (w/v) osmium tetraoxide in cacodylate buffer and 0.8% (w/v) K4Fe(CN)6 for 3 h at 4°C. The samples were washed five times in distilled water and stained with 5% uranyl acetate. Samples were dehydrated by subsequent incubation in higher concentrations of acetone [30, 50, 70, 90 and 100% (w/v)]. The samples were then incubated in an acetone/epoxyresin mix (Spurr 1969) with increasing concentrations of epoxyresin [33, 66 and 100% (w/v)] for 1 h per concentration, and overnight and 4 h at 100% (w/v). Sample blocks were than trimmed with a razor and sectioned in an ultra-microtome (Leica Microsystems, Wetzlar, Germany) with a diamond knife. Slices of approximately 60 nm were placed onto copper grids (Plano, Marburg, Germany), stained with uranylacetate and 0.3% lead citrate in NaOH and observed in a transmission electron microscope (EM 400, Philips, Eindhoven, NL). Electron micrographs of these sections were used for the evaluation of subcellular volumes according the principle of Delesse (1847): “the areal density of profiles on sections is an unbiased estimate of the volume density of structures” (Weibel and Bolender 1973; Winter et al. 1993; Farré et al. 2001). About 50 electron micrographs were taken from 2 independent fixation procedures and from each 6 different leaves. Low magnification and high magnification (>x1,000) were used to determine the areas of most compartments. The cross-sectional areas of subcellular compartments were quantified using analysis software (NIH image, public domain software, developed at US National Institutes of Health, available at http://rsb.info.nih.gov/nih-image/). The relative volumes (percent of total) were converted to absolute volumes per unit mass by taking into account the water content per mass fresh weight. Results Subcellular distributions of sugars and sugar alcohols in leaf cells Subcellular distributions of sugar alcohols, mono- and di-saccharides were measured by non-aqueous fractionation and HPLC in common plantain, sea plantain, peach and celery. The chosen method is based on the comparison of metabolite and marker enzyme distributions using a three-compartment calculation program (Riens et al. 1991). The method gives highly reproducible results for metabolites which are exclusively located (e.g., hexoses that are predominantly located in the vacuole). A higher variation is found for metabolites located in more than one compartment. The variability is greatest when the proportion found in a particular compartment is low. Farré et al. (2001) estimated that the limit of detection of a compound in a particular compartment is around 5% of the total amount in the tissue. HPLC analysis of gradient fractions of common plantain, sea plantain, and peach showed that sucrose, hexoses (glucose and fructose), myo-inositol, and sorbitol were present in leaf cells in these three plants (Fig. 1a–c). Celery contains mannitol instead of sorbitol (Fig. 1d). In all four species the subcellular distribution of sorbitol and mannitol, respectively, was very different from those of hexoses and sucrose (Fig. 1a–d). Sorbitol (in common plantain and peach) and mannitol (in celery) were located in all three compartments, but predominantly in the vacuole (48–73%), followed by the stroma (19–27%) and the cytosol (8–25%). The distribution in sea plantain was different, and sorbitol was found mainly in the vacuole (88%) with only small portions in the stroma and in the cytosol. In contrast, in all four plants glucose and fructose were confined entirely to vacuoles (96–98%), and in common plantain and sea plantain sucrose was mainly distributed among the vacuolar and the cytosolic compartments (Fig. 1a, b). In peach and celery, a greater proportion of sucrose was found in the vacuolar compartment and a smaller part in the cytosolic compartment (Fig. 1c, d). The cyclitol, myo-inositol, was mostly present in the stroma of common plantain and sea plantain. In peach and celery the proportion was high in the stroma as well as in the vacuole, while the proportion found in the cytosol was always the lowest (Fig. 1c, d). Fig. 1Percentage distribution of sugars and sugar alcohols among the vacuolar, stromal and cytosolic compartments of leaf cells from a common plantain, b sea plantain, c peach, and d celery. Data represent mean values ± SD from five to six independent fractionations Subcellular volumes The further conversion of the percentage distribution of sugars and sugar alcohols into concentrations requires an estimation of the volumes of the subcellular compartments of mesophyll cells. Leaves contain different tissues but in dicots about 75% of the aqueous volume of the leaf is occupied by mesophyll cells (Winter et al. 1993, 1994; Leidreiter et al. 1995). Table 1 shows the relative proportions of the vacuolar, chloroplastic and cytoplastic (sum of cytosol, peroxisomes, mitochondria, and nucleus) compartments of TEM micrographs of mesophyll cells. In all plant species analyzed the most voluminous compartment was the vacuole, occupying 68–85% of the total cell, followed by the chloroplasts (10–21%) and the cytoplasm (5–12%). The proportion of the vacuole was lowest in the woody plant peach whereas in common plantain and sea plantain mesophyll cells were dominated by the vacuole (82–85%). Accordingly, the relative proportions of chloroplasts and cytoplasm were smallest in these plants. The relative proportion of the combined volumes of mitochondria, peroxisomes, and nuclei of the total cytoplasmic volume has been estimated to be about 20% (Winter et al. 1993, 1994) and the proportion of the stroma volume of the total chloroplast volume about 50% (Winter et al. 1993, 1994). From the relative proportions mentioned above, the data from Winter et al. (1993, 1994) and the average water contents of leaf cells (Table 2) the volumes of the vacuolar, stroma, and cytosolic compartments can be estimated (Table 2). Table 1Relative volumes (%) of the subcellular compartments within the total volume of mesophyll cells from common plantain, sea plantain, peach and celeryVacuole %Chloroplast %Cytoplasm %Common plantain82 ± 512 ± 46 ± 1Sea plantain85 ± 510 ± 45 ± 2Peach68 ± 721 ± 611 ± 4Celery72 ± 516 ± 412 ± 3Data were obtained from TEM micrographes (n = 37–42). Cytoplasm is defined as cytosol, peroxisomes, nucleus and mitochondria. Mean values ± SDTable 2Dry weight (n = 8), water and gas space (n = 12) of leaves and the volumes of subcellulare compartments of mesophyll cells from common plantain, sea plantain, peach and celeryCommon plantainSea plantainPeachCeleryDry weight (mg g−1 FW)147 ± 9124 ± 15336 ± 30140 ± 8Water space (μl g−1 FW)853 ± 9876 ± 15664 ± 30860 ± 8Gas space (μl g−1 FW)226 ± 48222 ± 21190 ± 44213 ± 33Volume vacuole (μl g−1 FW)701743452860Volume stroma (μl g−1 FW)50437068Volume cytosol (μl g−1 FW)42385882Mean values ± SD Subcellular sugar and sugar alcohol concentrations From the data on leaf carbohydrate contents, the subcelluar volumes, and the distribution of metabolites between the subcellular compartments the corresponding metabolite concentrations in the subcellular compartments were calculated. Sorbitol or mannitol, respectively, made up more than twothird of the total leaf content of soluble carbohydrates in all the plants (Table 3). The concentrations of sorbitol and mannitol, respectively, were high in all compartments of peach and celery (Table 3). Most of the sorbitol and mannitol were located in the vacuole (Fig. 1a–d) but due to the smaller volume cytosolic concentrations were calculated to be similar to those in the vacuole, and the highest level was found in the stroma (Table 3). The sorbitol concentration in the cytosol of sea plantain was lower (13 mM) than in the vacuole (87 mM) whereas in common plantain the vacuole showed the lowest sorbitol concentration (15 mM). Table 3Whole leaf contents as well as sugar and sugar alcohol concentrations in the vacuolar, stromal and cytosolic compartments of leaf cells from common plantain, sea plantain, peach, and celeryWhole leaf content μmol (g FW)−1Concentration (mM)VacuoleStroma CytosolCommon plantain Sorbitol22 ± 3.115 ± 2.3118 ± 34133 ± 48 myo-Inositol0.7 ± 0.20.1 ± 0.18.8 ± 1.36.1 ± 0.7 Glucose and fructose2.4 ± 1.13.2 ± 0.21.4 ± 2.00.6 ± 0.7 Sucrose1.2 ± 0.10.9 ± 0.20.1 ± 0.212 ± 2.5Sea plantain Sorbitol73 ± 9.187 ± 8.0201 ± 14013 ± 8.4 myo-Inositol0.8 ± 0.20.2 ± 0.115 ± 0.40 ± 0 Glucose and fructose11 ± 2.515 ± 0.50.2 ± 0.50 ± 0 Sucrose1.4 ± 0.20.8 ± 0.30.1 ± 0.120 ± 4.9Peach Sorbitol148 ± 0.1220 ± 15461 ± 165290 ± 62 myo-Inositol1.5 ± 0.11.5 ± 0.48.5 ± 3.64.1 ± 3.3 Glucose and fructose30 ± 6.165 ± 15.0 ± 5.05.0 ± 5.0 Sucrose38 ± 2.164 ± 4.539 ± 26106 ± 15Celery Mannitol108 ± 12127 ± 7.0294 ± 30100 ± 39 myo-Inositol1.4 ± 0.11.1 ± 0.39.0 ± 1.91.7 ± 0.9 Glucose and fructose17 ± 2.028 ± 0.32.5 ± 1.72.9 ± 1.2 Sucrose35 ± 4.345 ± 3.35.2 ± 3.286 ± 28Mean values ± SD In each plant species sucrose was mainly concentrated in the cytosol, followed by the vacuole and was lowest in the stroma, however, concentrations varied widely between the species (cytosol 12–106 mM, vacuole 0.8–64 mM, and stroma 0.1–39 mM; Table 4). These differences between the plant species reflect the differences in the total leaf contents (Table 3). The sucrose content in both plantain species was much lower than in peach and celery. Table 4Carbohydrate concentrations in the cytosol of mesophyll cells of leaves as well as in the apoplast and in the phloem sap from common plantain, sea plantain, peach and celeryConcentration (mM) RatioRatioCytosolApoplast Phloem sapPhl/apoPhl/cytCommon plantain Sorbitol133 ± 485.5 ± 1.2422 ± 129773.2 Sucrose12 ± 2.50.3 ± 0.1645 ± 2252,15040 Sorbitol/sucrose11180.7Sea plantain Sorbitol13 ± 8.47.9 ± 2.5315 ± 1054024 Sucrose20 ± 4.90.3 ± 0.1355 ± 1121,18318 Sorbitol/sucrose0.7260.9Peach Sorbitol290 ± 6224.4 ± 9.5582 ± 90242.0 Sucrose106 ± 161.2 ± 0.6207 ± 511732.0 Sorbitol/sucrose2.7202.8Celery Mannitol100 ± 396.7 ± 3.3732 ± 1311097.3 Sucrose86 ± 281.2 ± 0.9389 ± 843244.5 Mannitol/sucrose1.25.61.9Mean values of n = 3–9 independent measurements are shown. Mean values ± SD Of the compartments analyzed the highest concentration of glucose and fructose was consistently found in the vacuole. As the evaluation of the subcellular fraction was performed in increments of 1%, glucose and fructose concentrations found in the cytosol or stroma represent an upper limit of 1% for these compounds. The concentration of myo-inositol was highest in the stroma, with intermediate values in the cytosol and lowest in the vacuole (Table 3). Myo-inositol, sorbitol and mannitol, respectively, made up between 90 and 99% of the total soluble carbohydrates in the stroma of the plant species. Apoplastic and phloem sap concentrations Carbohydrates found in the phloem sap of the four plant species investigated were sucrose and mannitol or sorbitol (Table 4). Concentrations were high in all cases, ranging between 200 and 700 mM. In the two Plantaginaceae sucrose concentrations, measured directly, were slightly higher than sorbitol concentrations, whereas in peach and celery the sorbitol and mannitol concentrations, respectively, were 3- and 2-fold higher than the sucrose concentration. Therefore, the ratios of sugar alcohol to sucrose concentrations for the four plants differ between 0.7 in common plantain and 2.8 in peach. No or only traces of glucose and fructose were found in the phloem, and the concentration of myo-inositol was in the low millimolar range (data not shown). Sorbitol and mannitol, respectively, dominated the apoplast of all four plants, while sucrose was present at concentrations at or below 1 mM (Table 4). Nevertheless, the polyol as well as the sucrose concentrations in the apoplast were much lower than in phloem sap (Table 4). The concentration differences between the apoplast and the phloem were about 100-fold for mannitol, between 20- and 80-fold for sorbitol and between 170- and 2,200-fold for sucrose. Discussion Despite the central importance of the compartmentation of sugar alcohols in plant cells in relation to phloem transport, up to now the sugar alcohol distribution in subcellular compartments has been the subject of very few studies. We have used non-aqueous fractionation in addition to HPLC to study subcellular sugar alcohol distributions in common plantain, sea plantain, peach, and celery which translocate large amounts of sorbitol or mannitol within the phloem. It should be pointed out that the non-aqueous fractionation technique was developed for determination of subcellular concentrations of metabolites that are exclusively located in the mesophyll (Gerhardt and Heldt 1984). For other metabolites such as sugars, which are also present in tissues other than the mesophyll, the method tends to somewhat overestimate concentrations in the mesophyll cell compartments, because the amounts of metabolites in other tissues are assumed to be negligible. However, Voitsekhovskaja et al. (2006) have shown that concentrations of hexoses and sucrose in mesophyll cells measured either by single-cell technique or by non-aqueous fractionation were similar. A possible explanation is that e.g., the dense phloem tissue remains intact and will be removed from the other tissue during fractionation process (either in the sieve or in the bottom of the gradient; see chapter “Non-aqueous fractionation of leaf tissue”). Sorbitol and mannitol concentrations are high in stroma and cytosol despite of a large vacuolar polyol depot The main proportion of sorbitol and mannitol was found in the vacuolar compartment (48–87%; Fig. 1a–d) whereas in most species analyzed the highest concentrations were found in the stroma (Table 3). High concentrations of polyols in the stroma require either polyol-uptake or polyol-synthesis inside this compartment. Unfortunately, little is known about the intracellular distribution of the enzymes involved in sorbitol synthesis. Experiments with apple (Yamaki 1981) support a predominantly chloroplastic location for sorbitol-6-phosphate dehydrogenase. On the other hand, the intracellular immunocytochemical localization data for mannose-6-phosphate reductase, the key enzyme for mannitol synthesis, show that this enzyme is primarily cytosolic (Loescher and Everard 1996). So far no polyol transporter has been demonstrated to be localized in the envelopes of chloroplasts. The function of high concentrations of polyols in the stroma could be e.g., for osmoregulation because the concentration of several other metabolites or ions in this compartment is much lower than in the cytosol and in the vacuole (Table 3; Winter et al. 1993; Lohaus et al. 1998; Voitsekhovskaja et al. 2006). Of the plants analyzed which produce sorbitol, only in sea plantain was the sorbitol concentration in the vacuole of mesophyll cells higher by about 7-fold than that in the cytosol (Table 3). The only previous report about subcellular distribution of sorbitol was of snapdragon (Moore et al. 1997) where up to 100% of sorbitol was found in the vacuole. However, the total leaf content of sorbitol was much lower in snapdragon than in plants in our study. A higher sorbitol concentration in the vacuole than in the cytosol, as in sea plantain, demonstrates that there is an active transport of sorbitol into the vacuole, because most likely sorbitol is either produced in the cytosol or in other cell compartments. So far no sorbitol transporter has been demonstrated to be localized in the tonoplast of mesophyll cells. The high sorbitol concentration in the vacuoles of sea plantain indicates that sorbitol may have more functions in that species than just serving as a transport-form for carbon. Sea plantain is a more salt tolerant plant species than common plantain (Ahmad et al. 1979). In the study presented here no salt stress was given but the plants have taken up sodium from the soil used for plant culture. As a result the sodium content in common plantain leaves was 4.3 ± 1.1 μmol (g FW)−1, whereas sea plantain contained 36 ± 6.5 μmol (g FW)−1 which corresponds to the higher sorbitol content in the leaves in sea plantain (Table 3). Stoop et al. (1996) have discussed that mannitol may accumulate in the vacuole of salt-tolerant plant species for redistribution to the cytosol in response to stress, and recently Pommerrenig et al. (2007) demonstrated increased sorbitol contents in leaf cells of common plantain in response to salt treatment. In leaves of celery three quarters of mannitol was located in the vacuole (Fig. 1d). In A. barclaiana (Voitsekhovskaja et al. 2006), parsley and snapdragon (Moore et al. 1997) the vacuole, stroma and the cytosol contained each onethird of leaf mannitol, but the total leaf content of mannitol was much lower in these three plants than in celery. Probably the higher leaf content of mannitol requires the deposition of a greater portion of mannitol in the vacuole. Despite the high proportion of mannitol in the vacuole, cytosolic and stromal mannitol concentrations were calculated to be similar to be higher than those in the vacuole (Fig. 1d and Table 3). Mannitol is synthesized in the cytosol of leaf cells (Rumpho et al. 1983) and similar concentrations of mannitol in the vacuole and the cytosol indicate that diffusion of mannitol between both compartments could be responsible for the pattern observed. On the other hand, mannitol is transiently stored in the vacuoles of parenchyma cells of fleshy petioles (Greutert et al. 1998). Similarly, Keller and Matile (1989) estimated a 3-fold higher mannitol concentration in the vacuole than in the cytosol of petiole cells, suggesting that an active uptake system of mannitol is present in the tonoplast. The vacuolar mannitol concentration higher in storage petioles than in source leaves may be a reflection of the different functions of these two organs in celery. A large pool of myo-inositol is located in the stroma A great portion of myo-inositol is localized in the stroma (Fig. 1a–d) which corresponds to earlier findings (Voitsekhovskaja et al. 2006; Moore et al. 1997). The high proportion of myo-inositol in the stroma also resulted in the highest myo-inositol concentration in this compartment (8–15 mM, Table 3), compared to other compartments. This stromal pool probably originates from its synthesis by the stromal isoform of the myo-inositol synthesizing enzyme, myo-inositolphosphate synthase (Adhikari et al. 1987). In the halophyte, Mesembryanthemum crystallinum, myo-inositol synthesis is increased in response to salt stress (Ishitani et al. 1996). Leaves of sea plantain contained a 8-fold higher salt content than those of common plantain (see above). Despite this difference, the content of myo-inositol was very similar in both plants in contrast to the sorbitol content (Table 3). Therefore, it seems for plantain that the sorbitol concentration is correlated with the sodium concentration rather than the myo-inositol concentration, even under low sodium conditions. The subcellular distribution of hexoses and sucrose is similar in plant leaves with and without sugar alcohols Hexoses are often confined to the vacuole (Riens et al. 1991; Winter et al. 1992). Figure 1a–d shows that these findings are also true for the four species analyzed here: concentrations of glucose and fructose were always higher in the vacuole than in the cytosol or in the stroma (Table 3). Both facilitated diffusion and active transport have been described for vacuolar uptake of glucose. Facilitated diffusion has been discussed for the transport of glucose across the tonoplast in celery (Daie 1987). Heineke et al. (1994) suggested that leaf vacuoles may contain transporters for the active uptake of hexoses. However, Martinoia et al. (2000) noted that accumulation of hexoses within the vacuole is not a proof for energized hexose uptake, because the vacuole contains acid invertases, allowing for enzymatic production of hexoses on the vacuoles. Between 50 and 80% of the sucrose detected in the four species analyzed were located in the vacuole (Fig. 1a–d). However, due to the smaller volume of the cytosol relative to that of the vacuolar compartment, cytosolic sucrose concentrations were calculated to be similar to or even higher than those in the vacuole (Table 3). A similar distribution has also been reported for leaves of other species (Riens et al. 1991; Winter et al. 1992). The lowest vacuolar sucrose concentration was found in the two Plantaginaceae and may be related to the overall low sucrose content in the leaves of these two species (Table 3). Recently Endler et al. (2006) have localized sucrose transporters in the tonoplast of barley (HvSUT2) and Arabidopsis (AtSUT4) and proposed that sucrose transporters are involved in vacuolar transport. Concentration ratios between the cytosol of mesophyll cells and the phloem were similar for sucrose and sugar alcohols Sugar alcohols and sucrose are the major compounds translocated in the four species analyzed. The phloem concentrations of sorbitol and mannitol (Table 4) were in the same range as that of sucrose (300–700 versus 200–700 mM) although the ratio of sorbitol, or mannitol, to sucrose varied between 0.7 and 2.8. High concentrations of sugar alcohols in the phloem indicate that a high proportion of assimilated carbon is exported in this way. Moing et al. (1997) estimated that sorbitol accounts for 60–90% of the carbon exported from the leaf in peach and in celery, while mannitol was shown to account for 10–60% (Daie 1986). In the four plants the sucrose concentration was always higher in the phloem sap than in the cytosol of mesophyll cells although the concentration ratio of sucrose between the cytosol of mesophyll cells and the phloem varied between 2- and 40-fold (Table 4). The different ratios arose from variable cytosolic sucrose concentrations rather than by different phloem concentrations in the four plants (Table 4). With the exception of common plantain the ratios of both, sorbitol and mannitol, between the phloem and the cytosol of mesophyll cells were similar to the corresponding ratios of sucrose. In common plantain the overall sucrose concentration was relatively low which resulted in a higher ratio for sucrose than for sorbitol. Concentration ratios between the apoplast and the phloem for sucrose and sugar alcohols with relation to the phloem loading mode Gamalei (1989) has shown that the members of Plantaginaceae display a virtually complete symplastic isolation of the sieve element-companion cell complex, as indicated by a very low plasmodesmal abundance between phloem companion cells and bundle-sheath cells. Sucrose and sorbitol were present in the apoplast of both Plantaginaceae, however, the concentrations were much lower than in phloem sap (Table 4). Therefore, the loading of both sucrose and sorbitol into the phloem must be energized. This is consistent with the observation that sucrose transporters (PmSUC1, PmSUC2, and PmSUC3; Gahrtz et al. 1994; Barth et al. 2003) as well as polyol transporters (PmPLT1 and PmPLT2; Ramsperger-Gleixner et al. 2004) were cloned from P. major. The function of the putative transporter proteins in phloem loading was corroborated by their specific localization in companion cells of source leaf phloem (PmSUC2, Stadler et al. 1995; PmPLT1 and PmPLT2 Ramsperger-Gleixner et al. 2004). The Km value of PmSUC2 for sucrose was about 1 mM (Gahrtz et al. 1994) and the Km value of PmPLT1 for sorbitol was about 12 mM (Ramsperger-Gleixner et al. 2004), which are in the same range as the apoplastic sucrose and sorbitol concentrations (Table 4). No data of transporters exists for sea plantain, but the concentration gradients for sucrose as well as those for sorbitol strongly suggest active transport systems for both carbohydrates. Together, the data support apoplastic transfer of sucrose and sorbitol in the members of Plantaginaceae. Pathway and mechanism of phloem loading in peach are still a matter of debate (Moing et al. 1997). The phloem structures of Rosaceae trees were shown to be of the intermediate-type (moderate numbers of plasmodesmal connections between mesophyll and companion cells; Gamalei 1989). Therefore, the mechanism of phloem loading in Prunus species could be symplastic, apoplastic or mixed. In apoplastic loaders, sucrose or sorbitol destined for phloem transport has to be released from the mesophyll cells into the apoplast, whereas in symplastic loaders, sugars and sugar alcohols synthesized in the mesophyll and destined for the phloem transport, are expected to stay in the symplast. Thus, it could be expected that the percentage of these carbohydrates in the cytosol of mesophyll cells in putative symplastic loaders would be much higher than in apoplastic loaders. Sorbitol concentrations were high in the phloem sap of peach (Table 4, Moing et al. 1997) and in addition to sucrose sorbitol is in general a major photoassimilate translocated in the phloem of woody Rosaceae (Bieleski 1982). The concentration ratios for sucrose and sorbitol between the cytosol of mesophyll cells and the phloem were about 2-fold for each carbohydrate (Table 4). These are the lowest ratios among the species analyzed and much lower than those determined for apoplastic phloem loaders (ratio of sucrose 5–30; Lohaus et al. 1995; Lohaus and Möllers 2000). A 2-fold concentration ratio for sucrose corresponds to the gradient found in Alonsoa meridionalis, a Scrophulariaceae with a symplastic phloem loading mode (Knop et al. 2001). A lower concentration ratio is obviously easier to balance or even reverse, depending on the metabolic situation, than a steeper ratio. Therefore, the possibility of phloem loading of sucrose and sorbitol by simple diffusion cannot be ruled out. Turgeon and Medville (1998) were unable to detect accumulation of sucrose against the concentration gradient in minor veins of willow leaves, representing another woody species. On the other hand, leaf infiltration with p-chloromercuribenzenesulfonic acid (PCMBS) was found to inhibit sugar phloem transport in peach and Moing et al. (1992) further concluded from this result that apoplastic phloem loading predominates in peach. Unfortunately up to now no sucrose or sorbitol transporters were cloned from peach as they had from other Rosaceae (Prunus cerasus, Gao et al. 2003; Malus domestica, Watari et al. 2004). Watari et al. (2004) reported the existence of three sorbitol transporters in apple source leaves with apparent Km values for sorbitol between 0.7 and 3.2 mM. The apoplastic sorbitol concentration in apple leaves was not detected but the corresponding concentration in peach was about 25-fold higher (Table 4). Sucrose and sorbitol uptake in plasma membrane vesicles from peach leaves exhibited saturated kinetics (Marquat et al. 1997). These results suggest that uptake of sorbitol and sucrose is carrier mediated. Active absorption of sucrose was completely inhibited by PCMBS, contrary to the absorption of sorbitol (Marquat et al. 1997). The activities of sorbitol transporters from Rosaceae heterologously expressed in yeast were also not or only slightly inhibited by PCMBS (Gao et al. 2003; Watari et al. 2004). Ramsperger-Gleixner et al. (2004) have shown that sorbitol transport by PmPLT1, but not that driven by PmPLT2, is inhibited by PCMBS. Therefore, PCMBS sensitivity of sorbitol transport can vary and the insensitivity of transport processes against PCMBS conclusively does not rule out that carriers are involved. Unfortunately, the type of phloem loading in peach is still an open question; the available data are consistent with either an apoplastic or a symplastic mode of loading or, more likely, a combination of both. Celery is the only plant species analyzed here which translocates mannitol in addition to sucrose. The concentration ratio of sucrose between the apoplast and the phloem sap was about 300-fold and the ratio for mannitol was about 100-fold (Table 4). Studies of metabolite uptake into plasma membrane vesicles from phloem tissues of celery petioles by Salmon et al. (1995) showed mannitol- as well as sucrose-proton-co-transport, indicating that in this species mannitol and sucrose transporters may be involved in phloem loading. Noiraud et al. (2000) have cloned two sucrose transporters from celery (AgSUT1, AgSUT2). AgSUT1 is expressed in mature leaves and phloem of petioles. Noiraud et al. (2001) have also cloned a cDNA of a mannitol transporter (AgMaT1) of celery which is not sensitive to PCMBS. The expression profile for AgMaT1 in source leaves and phloem was in agreement with a role in phloem loading of mannitol in celery. The AgMaT1 protein expressed in yeast cells exhibited a Km value for mannitol uptake of about 0.3 mM (Noiraud et al. 2001). The apoplastic mannitol concentration was 25-fold higher (Table 4). In summary, celery shows all characteristics of a typical apoplastic phloem loader. The comparison of common plantain, sea plantain, peach, and celery indicates that different modes of phloem loading are employed by different plant families, which may in part be related to the respective ecophysiological requirements.

Evid_Based_Complement_Alternat_Med-2-4-1297501

Complementary and Alternative Medicine for Chronic Prostatitis/Chronic Pelvic Pain Syndrome

To discuss challenges concerning treatment for chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) and review complementary and alternative medical (CAM) therapies being evaluated for this condition, we performed a comprehensive search of articles published from 1990–2005 using the PubMed, Medline databases. Data from the articles were abstracted and pooled by subject. Keywords cross-searched with CP/CPPS included: complementary, alternative, integrative, therapies, interventions, nutrition, antioxidants, herbs, supplements, biofeedback and acupuncture. Listed articles with no abstracts were not included. Various CAM therapies for CP/CPPS exist including biofeedback, acupuncture, hyperthermia and electrostimulation. Additionally, a variety of in vitro and in vivo studies testing herbal and nutritional supplements were found. Saw palmetto, cernilton and quercetin were the most frequently tested supplements for CP/CPPS. Although many CAM therapies demonstrate positive preliminary observations as prospective treatments for CP/CPPS, further exploratory studies including more randomized, controlled trials are necessary for significant validation as treatment options for this complex disorder. Introduction and Characterization of Prostatitis Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is of significant interest in urology and accounts for up to 2 million office visits per year (1). Currently, there are multiple approaches to the management of CP/CPPS depending on the classification of the related symptoms. However, there are no absolute findings or laboratory tests employed and diagnosis is often one of exclusion. In 1995, the National Institutes of Health established an International Prostatitis Collaborative Network in order to construct a new classification of prostatitis syndromes and better define chronic prostatitis (2,3). The categories are now documented as follows: Category 1. Acute bacterial prostatitisCategory 2. Chronic bacterial prostatitisCategory 3. Chronic prostatitis/chronic pelvic pain syndrome A. InflammatoryB. Non-inflammatoryCategory 4. Asymptomatic inflammatory prostatitis. While most patients report a primary subjective symptom of local pain and/or dysuria, the clinical presentation of acute versus chronic prostatitis varies. Laboratory cultures are standardly employed to detect bacterial involvement and this testing along with other diagnostic criteria determines each classification. Men with Category 1, acute prostatitis, frequently present with dysuria, fever, malaise, myalgia (non-specific) and positive culture analysis that often reveals coliform bacteria. This imparts to standard antibiotic treatment with good prognosis for recovery and minimal recurrence. Patients with Category 2, chronic bacterial prostatitis, present with similar symptoms as those with acute prostatitis. However, the frequency of symptoms (duration >3 months), recurrent urinary tract infections and additional diagnostic tests including analysis of lower urinary tract cultures contribute to its diagnosis as Category 2 prostatitis (2). Men with Category 4, asymptomatic inflammatory prostatitis, do not present with subjective symptoms. This diagnosis is often discovered via laboratory findings such as the positive presence of white blood cells in prostatic secretions or in prostate tissue during routine evaluation for other disorders (1). Comparative to the total number of prostatitis cases reported, the majority of representative cases are Category 3, CP/CPPS (1,4). This diagnosis is usually one of exclusion, as bacterial etiology acute or chronic is ruled out. Other exclusion criteria include urogenital cancer, urethral stricture and neurologic disease affecting the bladder. However, the patient may still present with polyuria, dysuria, generalized myalgia or specific pelvic pain, urethral discharge, voiding dysfunction, sexual dysfunction and negative impact on quality-of-life (QOL). The presentation of this symptom set is now termed Category 3, CP/CPPS. Categories 3A and 3B are further differentiated by the presence or absence of inflammatory blood cells in prostatic secretions and seminal fluid, respectively (4,5). Table 1 further outlines the characteristics and treatment options of Category 3 prostatitis. Due to the complexity in diagnosing CP/CPPS, the National Institutes of Diabetes and Digestive and Kidney Diseases funded the Chronic Prostatitis Collaborative Research Network (CPCRN) in 1995 (2,3). This network was fundamental in the construction and validation of the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI), which was implemented in 1999 (3). The index has become a valid measure that quantifies the qualitative experience of men with CP/CPPS and addresses three different aspects of CP/CPPS: pain, function and QOL (3,6). The formation of the CPCRN and the advent of the NIH-CPSI have better characterized diagnosis and treatment for CP/CPPS, but challenges still exist. While standard treatment options including anti-inflammatory agents, analgesics and alpha-blockers are often prescribed, impact on QOL is another factor often overlooked in the treatment and management of CP/CPPS (1,4,5,7). The focus on QOL, anecdotal data, epidemiological studies and the increased popularity and validation of herbal, complementary and alternative medicine (CAM) has led to exploration of the utility of CAM therapies as treatments for CP/CPPS (8). CAM therapies including biofeedback, acupuncture, heat therapy, electrostimulation, herbal and nutritional supplements will be discussed below. CAM Background CAM, as defined by the National Center for Complementary and Alternative Medicine (NCCAM), is a group of diverse medical and health care systems, practices and products that are not presently considered part of conventional medicine. Conventional medicine is further defined as medicine as practiced by holders of MD (medical doctor) or DO (doctor of osteopathy) degrees (9). Though the list of what therapies or practices considered to be CAM changes continually, the pool of both practitioners of CAM modalities and patients utilizing CAM services continues to grow within the United States and globally (9). The inclusion of CAM practices in urology is also being implemented in the clinic. Many groups such as the Committee of Complementary and Alternative Medicine within the American Urological Association (AUA) recognize the integration of non-conventional therapies into urological clinical practice. Additionally, both public demand for CAM therapies and their testing and validation within health science research centers has increased (8,10,11). It has been suggested that many urological conditions possessing subjective and QOL components such as in benign prostatic hyperplasia (BPH), chronic prostatitis, voiding, erectile dysfunction and cancer prevention and survivorship might be particularly amenable to CAM treatment strategies (10). This review will focus on current CAM therapies found in the literature for CP/CPPS. CAM Therapies for CP/CPPS Biofeedback Biofeedback therapy is considered a mind–body technique that utilizes a monitoring machine to assist people in controlling bodily functions such as heart rate, blood pressure and muscle tension. This therapy has been studied for its efficacy in urological conditions such as incontinence, prolapse, pediatric voiding dysfunction and CP/CPPS (12–17). The hypothesis of biofeedback's mechanism of action in treating CP/CPPS is based on the principle that maximum muscle contraction prompts maximum muscle relaxation. This mechanism addresses the chronic pain aspect of CP/CPPS and focuses on muscular reeducation, which may ultimately provide symptom relief (13–15). Two studies testing the value of biofeedback therapy for CP/CPPS yielded positive results. The first study assessed 62 patients who were refractory to conventional therapy (such as antibiotics and/or alpha-blockers) for greater than half a year. These patients were treated utilizing the Urostym Biofeedback equipment five times a week for 2 weeks with a stimulus intensity of 15–23 mA and duration of 20 min. The NIH-CPSI index noted a significant overall reduction in score (P < 0.01) and no side effects were reported during the trial (13). A second pilot study evaluated biofeedback therapy in 19 men with pelvic floor tension and CP/CPPS. These results demonstrated significant improvement in pain scores as measured by the AUA symptom index (P = 0.001). While this study focused on testing the effect of biofeedback therapy in treating the symptoms associated with CP/CPPS, it also implicated the presence of pelvic floor tension contributing to pain and the paramount importance of muscular reeducation for its treatment (15). These initial, positive biofeedback studies may warrant larger randomized clinical trials to confirm safety and efficacy as well as explore the mechanism of action of biofeedback therapy. Acupuncture Acupuncture is a traditional Chinese method of medical treatment involving the insertion of fine, single-use, sterile needles in acupoints according to a system of channels and meridians that was developed by early practitioners of Traditional Chinese Medicine (TCM) over 2000 years ago. The needles are stimulated by manual manipulation, electrical stimulation or heat (18). Currently, acupuncture is often used with TCM and it is a recognized health profession with strict licensure and regulatory status in 40 states (19). Common applications include acupuncture as a complementary therapy for cancer patients undergoing chemotherapy or radiotherapy, for conditions involving pain such as migraines and back pain, and for relieving the impact of stress among patients with chronic conditions. The precise physiological mechanism of action of acupuncture is unknown but a variety of hypotheses exist. For example, acupuncture analgesia is thought to be mediated by central nervous system (CNS) mechanisms of pain control via the release of specific neurotransmitters, such as endorphins (20–23). Additionally, there are significant data which suggest that acupuncture treatment can decrease inflammation and relieve pain (24,25). Data suggesting the ability of acupuncture treatment to decrease pain, positively impact QOL and potentially modulate inflammation and/or affect the CNS has suggested it as potential therapeutic option for men with CP/CPPS. While a number of studies listed in other journals test the utility of acupuncture treatment for CP/CPPS (26) only two medline listed pilot studies are shown testing acupuncture treatment in patients with CP/CPPS. The first study examined whether acupuncture improved pain, voiding symptoms and QOL in 12 men with CP/CPPS. This study reported a significant decrease in total NIH-CPSI pain, urinary and QOL scores (P < 0.05) over 6 weeks of treatment and an average 33 weeks of follow-up (27). The mechanism of action addressed in this paper suggests a neuropathic model of CP/CPPS and the hypothesis that acupuncture, if considered a neuromodulatory therapy, may provide a therapeutic option for men with CP/CPPS (27). A second study tested acupuncture treatment for CP/CPPS patients with intrapelvic venous congestion. This study of 10 patients receiving 5 weeks of acupuncture treatment also reported a significant decease in NIH-CPSI pain and QOL scores (P < 0.05, P < 0.01). While the study reported no serious adverse events, the mechanism of action was not addressed (28). The promising clinical outcome of both studies testing acupuncture for CP/CPPS implies that larger studies are required to confirm the utility of acupuncture in this patient population. High Frequency Electrostimulation Only one study in English was found utilizing electrostimulation for chronic prostatitis. This study tested a new, high frequency, urethral–anal prototype stimulation device in men with CP/CPPS twice weekly for 5 weeks. The results demonstrated a significant decrease in the NIH-CPSI (P = 0.0002) with no urethral, anal complaints or other side effects (29). The authors suggest that due to the positive results, simple technology and ability to be self-administered, this new device may have utility in the treatment of CP/CPPS. However, further studies and standardization of the electrostimulation device are essential. Hyperthermia Anecdotal evidence and a few clinical trials have suggested heat therapy or hyperthermia as a treatment option for men with CP/CPPS. Its mechanism of action is based on the application of heat to the prostate to relieve pain. An excellent review by Zeitlin (30) discusses the lack of literature concerning hyperthermia and CP/CPPS. The review notes a variety of pitfalls in hyperthermia research including variation and lack of standardization of treatment. These concerns are applicable to both the type of heat utilized such as interstitial heat or microwaves and variation in its application, either transrectally or transurethrally. The review also suggests that the hyperthermia instruments used were not validated and outcome measures were subjective. However, the review implies that utilization of a quantitative assessment tool, applied statistics and greater documentation of therapy type may offer hyperthermia a better opportunity to be evaluated as a potential therapy for CP/CPPS (30). We also discovered the paucity of literature described by Zeitlin and only three listed clinical trials utilizing hyperthermia. The first study analyzed a group of 45 men with chronic abacterial prostatitis or prostadynia who underwent 6 weekly, 1 h sessions of local deep microwave hyperthermia (42.5 +/−0.5°C) to the prostate. Although the authors report encouraging results in the decrease of pain, these subjective patient assessments were not quantified by the NIH-CPSI or other index for CP/CPPS (31). A second abstract discussed a randomized, sham-controlled comparative study utilizing transrectal microwave hyperthermia in 80 men with CP/CPPS. While this abstract noted a 75% symptomatic improvement in the treatment group, the study was available as an abstract only with no statistical significance or descriptive methodology reported (32). A third study also tested transrectal microwave hyperthermia for both chronic non-infectious and infectious prostatitis. While the study design incorporated obtaining measurement of prostatic secretions, uroflowmetry and transrectal color Dopplerographic mapping, the results from the study were not abstracted as the article was in Russian (33). Herbal and Nutritional Supplements Herbal and nutritional supplement therapies have been most widely investigated for their utility in CP/CPPS and other prostate conditions such as BPH (34–40). While many formulations have been cited for their use in a wide variety of urological conditions, saw palmetto, pollen extract and quercetin were the supplements found with specific application to CP/CPPS. While most herbal and nutritional supplements contain a wide variety of synergistic ingredients upon compositional analysis, some of the active components such as phytosterols or antioxidants are listed in Fig. 1. Saw palmetto Saw palmetto (Serenoa repens) garnered much attention in urology based on a great deal of anecdotal evidence regarding its prostate specific properties. It is widely used in many Asian, African and European countries and compositional analysis of the berry of S. repens exhibits sterols and free fatty acids as its major constituents (41). Initial studies suggested that the efficacy of saw palmetto may be similar to that of the pharmaceutical enzyme inhibitors such as finasteride. This preliminary data prompted the exploration of mechanism, utility and efficacy of saw palmetto in in vitro analysis and in clinical trial settings. While a number of trials have examined saw palmetto use for symptoms related to BPH (34–39), only a few have focused on it specifically for CP/CPPS (42–44). The first study compared the safety and efficacy of saw palmetto berry supplement versus finasteride in men with Category 3, CP/CPPS. This prospective, open label 1 year study randomized 64 men to the saw palmetto or finasteride group, respectively. After 1 year of treatment, the NIH-CPSI score decreased from 23.9 to 18.1 in the finasteride group (P < 0.003) and from 24.7 to 24.6 in the saw palmetto group (P = 0.41). While significance was only achieved in the finasteride treatment arm, it was notable that at the end of the trial 41 and 66% of participants opted to continue the therapies of saw palmetto and finasteride, respectively, regardless of achieved statistical significance (42). The second clinical trial from China examined the effects of prostadyn sabale capsules containing saw palmetto berry in patients with CP/CPPS. While 125 men reported positive outcome and the NIH-CPSI was used as a primary end point, the article is only available in Chinese and the manufacturer, active constituents of the capsules and statistical significance was not reported (43). A third multicenter study testing a saw palmetto abstract called Permixon analyzed the response of Permixon therapy in 61 patients with Category 3B prostatitis. While 65% of the Permixon group reported improvement based on the Patients Subjective Global Assessment (SGA), the total NIH-CPSI and the pain, voiding and QOL/impact domains of the NIH-CPSI, statistical significance was not reported (44). Additionally, prostate volume was unchanged in both the treatment and control groups. While this multicenter study suggests that Permixon may provide clinical benefit for CP/CPPS 3B, the dosages and components of the Permixon product were not listed in the abstract. The assessments of saw palmetto studies for CP/CPPS are far fewer than those for BPH. However, marked and continued progress in molecular studies, increased mechanistic data and more clinical trials in CP/CPPS are warranted to ascertain the utility and reproducibility of saw palmetto use in men with chronic prostatitis. Pollen Extract: Cernilton Pollen extract is traditionally collected from the flowers of various plant types and it contains carbohydrates, fat, protein, vitamins and minerals (41). The particular pollen extract named cernilton has been suggested to benefit a variety of urological conditions. Anecdotal evidence and references from traditional herbal texts have implicated cernilton's potent anti-inflammatory properties and potential in treating symptomatic relief of urinary pain and dysfunction often present in both CP/CPPS (41) and BPH (45,46). In vitro studies demonstrate a variety of experimentation on this particular extract including histopathological analysis of its effect on cell proliferation, apoptosis, serum cytokines and testosterone (47,48). The literature also lists quite a few clinical trials on pollen extract; however, five are in the Japanese language and one in the German language (45,48–52). While many of these studies report the positive activity of pollen extract and suggest its usefulness for CP/CPPS, data from these studies were not abstracted due to unavailability and translation of the articles. One available study testing pollen extract reported a 78% favorable response of men with chronic prostatitis taking Cernilton® pollen extract at a dosage of 1 tablet TID for 6 months (53). While this study reported favorable results, the study was published in 1993 and similar subsequent larger phase clinical trials are not evident to further elucidate the possible utility of pollen extract in men with CP/CPPS. Quercetin Quercetin is known chemically as a mixture of 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one and 3,3′,4′,5,7-pentahydroxy flavone. It belongs to a group of polyphenolic substances known as flavonoids and is a member of the class of flavonoids called flavonols. It is commonly found in the plant kingdom in the rinds and barks of certain foods such as onions, grapes and green tea (41). Since quercetin is thought to have antioxidant, anti-inflammatory, antiviral, immunomodulatory, anticancer, gastroprotective and antiallergy activities it has been studied for a variety of conditions (54–56). One prospective, randomized, double-blind, placebo-controlled trial was performed to test the action of this bioflavonoid in men with CP/CPPS. This placebo-based study assessed 30 men with CP/CPPS to receive the bioflavonoid quercetin, 500 mg twice daily or the placebo pill for 1 month. Significant change in the NIH-CPSI score was observed in the quercetin (P = 0.003) versus the placebo group, who had an insignificant mean improvement in the NIH-CPSI score (57). While this was the only clinical trial found testing quercetin for CP/CPPS, the positive outcome supports the need for further study including cost analysis of quercitin therapy in this patient population. Other Herbal Agents for Prostatitis Finally, a wide variety of herbal compounds not previously discussed were found during this review, many of which are commonly used in the TCM herbal material medica. Some of the compounds are Nan mi qing capsules containing Rheum palmatum and Rx. astragalus memberanaceus, Ye Ju Hua Shuan, an herbal suppository of Flos Chrysanthemi Indici and a variety of other formulae or capsules of which the ingredients were not listed (58–62). Initial in vitro studies examining some of the herbal compounds effects on markers of inflammation such as thromboxanes (TBX2) and 6-keto-PGF1-α are promising as the link between chronic inflammation in CP/CPPS is still under investigation (58). While the preliminary reports of additional herbal compounds are encouraging, a number of difficulties exist in the extraction of this data. Namely, the articles are in the Chinese or other language and data including the ingredients, constituents and other practices including good manufacturing are unable to be assessed. While this conundrum exists for many herbal agents despite their historical use in a variety of traditional herbal medicine systems, testing of safety and utility are still necessary. However, the variety of herbal agents available offers a large source to draw from and the possibility that other herbal products might be beneficial in the treatment of CP/CPPS. Conclusions A summary of the reviewed literature is outlined in Table 2 and a variety of CAM modalities tested in patients with CP/CPPS include practitioner-based therapies such as biofeedback, hyperthermia, acupuncture and electrostimulation, and biological-based therapies including herbal and nutritional supplements. As the challenges in treating this complex and chronic disorder remain, further evidence of efficacious CAM treatment options for men with CP/CPPS is needed. Thus far, promising data on the function and efficacy of certain CAM therapies suggest their potential as treatment options for this patient population. Further exploratory studies including more in vitro studies of herbal products, mechanistic data, cost analysis and randomized, controlled trials will assist in validation of certain CAM therapies as permanent treatment options for men with CP/CPPS (63,64).

Dig_Dis_Sci-4-1-2413116

Abuse in Women and Men with and without Functional Gastrointestinal Disorders

We aimed to investigate the history of abuse in childhood and adulthood and health-related quality of life (HRQL) in women and men with FGID in the general adult population. A cross-sectional study in a random population sample (n = 1,537, 20–87 years) living in Östhammar municipality, Sweden, in 1995 was performed. Persons with FGID (n = 141) and a group of abdominal symptom-free controls (SSF, n = 97) were selected by means of a validated questionnaire assessing gastrointestinal symptoms (the ASQ). Abuse, anxiety and depression (the HADS) and HRQL (the PGWB) were measured. Women with FGID had a higher risk of having a history of some kind of abuse, as compared with the SSF controls (45% vs.16%, OR = 2.0, 95% CI: 1.01–3.9; SSF = 1), in contrast to men (29% vs. 24% n.s.). Women with a history of abuse and FGID had reduced HRQL 91 (95% CI 85–97) as compared with women without abuse history 100 (95% CI 96–104, P = 0.01, “healthy” = 102–105 on PGWB). Childhood emotional abuse was a predictor for consulting with OR = 4.20 (95% CI: 1.12–15.7.7). Thus, previous abuse is common in women with FGID and must be considered by the physician for diagnosis and treatment of the disorder. Introduction Maltreatment, threats or violence from another person, often an intimate partner, is psychosocially traumatic and a growing problem in society. During the last decade, 1997–2006, the number of assaults reported to the police in Sweden increased by 40%, to 1,079/100,000 inhabitants, and the number of sexual offences increased by 57% to 137/100,000, of which rapes increased by 142% to 46/100,000. The latter high percentage is partly attributable to a change in the Swedish legislation that lowered the threshold at which sexual offence is classified as rape. For children (0–15 years) the number of assaults reported to the police has increased by 64% to 97/100,000 and the number of rapes has increased during the last decade by 300% to 12/100,000 [1]. About half of the women in Canada and Sweden have been described as having experienced at least one incident of violence by a man since the age of 16 [2, 3], and in a Japanese study three quarters of the women said they had experienced sexual, physical or emotional intimate partner violence [4]. A population survey in Los Angeles reported a childhood (lifetime for age <16 years) sexual assault prevalence of 6.8% in women and 3.8% in men [5], and 10.5% in adults [6]. Functional gastrointestinal disorders (FGIDs) are a group of digestive diseases with a chronic or recurrent course in the absence of organic illness likely to explain the symptoms [7]. The most frequent FGIDs are irritable bowel syndrome (IBS), with an average estimated prevalence of 12%, and functional dyspepsia with a prevalence of 25% in the general adult population, but with a considerable proportion of persons with overlapping symptoms [8]. Most studies on IBS report more sufferers among women than men [9]. The difference in prevalence of dyspepsia between women and men is inconsistent; some studies report a higher prevalence of symptoms of dyspepsia in women than in men [10, 11], whereas others do not find any sex difference [12, 13]. Although FGID is common, only about 50% of people who have symptoms of dyspepsia ever seek medical attention for it [14, 15], while the consulting figures for IBS are somewhat higher [15]. FGIDs constitute about 5% of all consultations in primary care [16], and only a minority is referred to secondary care [17, 18]. Nevertheless, FGID accounts for up to 50% of all gastroenterology consultations [19]. The pathogenesis of FGID is at present often explained using the biopsychosocial model in which biologic and psychosocial factors participate in the origin of symptoms [20]. An overrepresentation of a history of past abuse has been reported in both patients and non-patients with IBS and among non-patients with functional dyspepsia [21, 22]. However, investigating abuse is difficult among patients with abdominal distress, since sexual and physical abuses are seldom reported to doctors by their patients [23]. Our aims were to investigate the occurrence of a history of sexual, physical or emotional abuse experienced in childhood or later in life among women and men with FGID in the general population and the possible association with consultation rate, as compared with subjects free from FGID, controlling for age, sex, education and psychological distress. Methods Setting and sampling A population sample (n = 1,537) drawn from the National Swedish Population Registry in 1995 involved men and women 20–87 years of age, born on day 3, 12 or 24 of each month living in Östhammar municipality. The sampling method is equivalent to random sampling since there is no reason to believe that date of birth is associated with the variables measured. The subjects were sent a validated postal questionnaire, called “The Abdominal Symptom Questionnaire” (ASQ) [24]. The responders (n = 911) in the population sample were classified according to their responses in the ASQ as having either functional gastrointestinal disorder (FGID n = 244), i.e., functional dyspepsia or irritable bowel syndrome, but not predominant symptoms on gastroesophageal reflux, or being strictly symptom free (SSF n = 219), i.e., having reported no gastrointestinal (GI) symptoms and also no GI symptoms reported by subjects who also participated in two previous population studies in 1988–1989 [25]. In 1996, the FGID (n = 244) and SSF (n = 219) sample groups were invited by mail to an appointment at one of their six local health centres. About 187 (77%) with FGID and 156 (71%) SSF accepted the invitation. At the health centre they filled in the ASQ once again together with other questionnaires about health-related quality of life (HRQL) and their abuse history. A nurse assisted only when needed. With the same criteria for FGID applied to this ASQ response, 141 with FGID (IBS and dyspepsia: 99; only dyspepsia: 40; only IBS: 2) and 97 SSF remained, and thus constituted the study groups. Thus the FGID symptomatics were classified as FGID twice, in 1996 and 1995, and the symptom-free SSF group up to four times in 1996, 1995, 1989 and 1988. This sampling procedure assured persistent symptom status in the two study groups, as has been presented earlier [26] and in Fig. 1. Fig. 1Formation and sampling procedure for the study groups from the population of Östhammar Questionnaires The Abdominal Symptom Questionnaire (ASQ) has previously been validated and found reliable and reproducible [27–29]. The definition of FGID used in this study is dyspepsia, IBS or both, as taken from the ASQ. Solitary heartburn and/or regurgitations were symptoms not included in our definition of dyspepsia, which is in agreement with the current ROME III definition [30]. In the Abuse Questionnaire, the questions about sexual abuse were those initially developed for the National Population Survey of Canada [2] from survey questionnaires that fulfill reliability criteria [31]. The questions were translated into Swedish and back-translated into English for validation. Questions were written separately for childhood ≤13 years and adulthood (>13), including six categories of abuse: sexual, physical and emotional, each as a child or as an adult [23]. The Psychological General Well-Being (PGWB) index is a health-related quality of life instrument including 22 items divided into six domains: anxiety, depressed mood, positive well-being, self-control, general health and vitality. Items are scored on a six-grade Likert scale, with higher scores indicating better HRQL. The total score with a high responsiveness and validity for dyspepsia [32] was used in this study. HRQL values varying between 102 and 105 have been observed in a normal healthy population [33]. The total score was dichotomized at the median with a cut-off point 107/108 at the upper 95% CI for functional dyspepsia. Consequently, a good HRQL was considered to be a total score of 108 or more, which was reached by 117 subjects (score 108–132; mean 117.5, SD = 6.3), while a poor HRQL was reached by 120 subjects (score 49–107; mean 90.8, SD = 12.6). The Hospital Anxiety and Depression Scale (HADS) is a validated, reliable instrument with subscales for measuring anxiety and depression [34–36]. The questionnaire has seven items, graded 0–3, with possible ranges of 0–21 for each subscale (total minimum score of 0, total maximum score of 42). A score of 7 or less on each subscale (out of a maximum of 21) denotes a non-case, 8–10 a doubtful case, and 11 or more a definite case of anxiety or depression. The cut-off point 10/11 was set for identifying sufferers in this study. All participants per definition completed the ASQ, 231 (97.1%) the Abuse Questionnaire, 237 (99.6%) the PGWB questionnaire and 217 (91.1%) the Hospital Anxiety and Depression Questionnaire. Educational background was registered at five levels and dichotomized, with low including elementary, comprehensive, secondary level and high upper secondary, university level. Data of previous consultations for GI symptoms and educational background were taken from questions added to the 1995 ASQ. This study was approved by the Ethics Committee of the Medical Faculty, Uppsala University, on 5 June 1996. Statistical analyses A Spearman rank correlation test was performed for the variables anxiety, depression and quality of life. A multiple logistic regression analysis was performed with FGID/SSF and consulters/non-consulters as outcome variables, each of the abuse variables as exposure variables and the possible confounding factors: age (−40/40- years), sex (female/male), education level, anxiety (1-HADS subscale >10, 0-HADS subscale <11), depression (1-HADS subscale >10, 0-HADS subscale <11) and HRQL score (1-PGWB >108, 0-PGWB ≤108) as explanatory variables. In order to adjust for the influence of explanatory variables the variables were added one by one with a forward stepwise multiple logistic regression technique [37], and if the variable affected the odds ratios of the outcome variables less than ±10% and with P > 0.10, the variable was eliminated from the model. The Hosmer–Lemeshow goodness-of-fit test was performed for each model, and model improvements were tested with the likelihood-ratio test. Tests for interactions were completed between the abuse variables and age, sex and HRQL. No significant interactions were found. STATA version 9.2 statistical package [38] was used for the analyses. Results Demography and sexual, physical and emotional abuse The distribution of age, sex, education, GI consultation, anxiety, depression, HRQL and sexual, physical and emotional abuse for persons with FGID and SSF is presented in Table 1. Forty-one percent of those with FGID had experienced some kind of abuse, as compared with 20% (P < 0.01) among SSF controls. Impaired anxiety and HRQL were significantly more common among subjects exposed to prior abuse, while depression was not. Table 1Distribution of age, sex, education, previous GI consultation and abuse for women and men with functional gastrointestinal disorder (FGID) and strictly GI symptom free (SSF) and previous consulters/non-consulters for FGIDFGID (n = 141) n (%) SSF (n = 97) n (%)Statistic PConsulters GI (n = 99) n (%)Non-consulters GI (n = 39) n (%)Statistic PMean age years (SD)b45.7 (14.3)52.4 (15.4)0.000746.7 (13.4)41.4 (14.2)0.041Femalea93 (66.0)51 (52.6)0.038 66 (66.7)26 (66.7)01Completed high schoola66 (47.1)28 (29.5)0.00743 (43.4)23 (60.0)0.100Consulted for GI evera99 (71.7)8 (8.3)<0.001Not relevantNot relevantPsychological general well-beingb96.8114.6<0.000196.896.60.963Anxietya13 (10.0)0 (0.0)0.0025 (7.5)8 (13.1)0.507Depressiona5 (4.8)1 (1.7)0.3193 (5.4)2 (4.3)0.942Childhood abuseChildhood sexual abusea19 (13.9)3 (3.1)0.00616 (16.5)3 (7.9)0.196Childhood physical abusea15 (11.0)3 (3.2)0.02914 (14.4)1 (2.6)0.050Childhood emotional abusea37 (27.4)15 (16.1)0.04632 (33.7)5 (13.2)0.017Any childhood abusea43 (31.4)18 (18.8)0.03137 (38.1)6 (15.8)0.012Adulthood abuseAdult sexual abusea17 (12.4)2 (2.1)0.00514 (14.4)3 (7.9)0.303Adult physical abusea3 (2.2)1 (1.1)0.5133 (3.1)0 (0.0)0.273Adult emotional abusea32 (23.7)5 (5.4)<0.00124 (25.3)8 (21.1)0.608Any adulthood abusea40 (29.2)8 (8.3)<0.00130 (30.9)10 (26.3)0.598Any childhood, not adulthood, abusea16 (14.6)11 (12.4)0.65415 (20.0)1 (3.0)0.022Any adulthood, not childhood, abusea13 (11.8)1 (1.1)0.0038 (10.7)5 (15.2)0.509Any abuse (child or adult)Sexual abusea30 (21.9)4 (4.2)<0.00125 (25.8)5 (13.2)0.113Physical abusea17 (12.4)3 (3.2)0.01416 (16.5)1 (2.6)0.029Emotional abusea48 (35.6)15 (16.1)0.00139 (41.1)9 (23.7)0.060Any abusea56 (40.9)19 (19.8)0.00145 (46.4)11 (29.0)0.064aPearson chi2-test, b Student’s t-test Women There were 144 women, 93 with FGID and 51 SSF in the study. Almost half of the women, 42 out of 93 (45%) with FGID, had a history of abuse, in contrast to the 8 out of 51 (16%, P < 0.01) SSF controls. Women with FGID had a higher prevalence than SSF women for sexual, physical and emotional abuse in childhood and for sexual and emotional abuse in adulthood, as presented in Table 2. Table 2The number of people and the proportion of individuals (%) with a history of sexual, physical and emotional abuse in women and men with a functional gastrointestinal disorder (FGID) and strictly GI symptom free (SSF). The total number of FGID/SSF for both childhood and adulthood abuse in women was 93/51 and 48/46 in menChildhood abuse number FGID/SSF n(%) PAdulthood abuse number FGID/SSF n(%) PSexualPhysicalEmotionalAnySexualPhysicalEmotionalAnyWomen18(19)/3(6) 0.02711(12)/1(2) 0.04826(28)/5(10) 0.02132(34)/7(14) 0.00614(15)/2(4) 0.0442(2)/0(0) 0.40324(26)/2(4) 0.00131(33)/4(8) <0.001Men1(1)/0(0) 1.004(8)/2(4) 0.87611(23)/10(22) 0.93711(23)/11(24) 1.003(6)/0(0) 0.3311(2)/1(2) 1.008(17)/3(7) 0.3439(19)/4(9) 0.382P value computed with Fisher’s exact two-sided test Out of the 50 women with a history of abuse, 24 (48%, FGID n = 21, SSF n = 3, P < 0.01) had experienced sexual, physical or emotional abuse both in childhood and as adults. In the FGID group, 42 (46%) women reported abuse in childhood and/or adulthood, as compared with 8 (16%, P < 0.01) in the SSF group. Moreover, in the FGID group, 32 (35%) women had a history of childhood abuse as compared with 7 (14%, P < 0.01) in the SSF group. Men In contrast to the women, the men had no significant difference for a history of abuse: 14 out of 48 (29%) men with FGID versus 11 out of 46 (24%, P = 0.69) SSF men. Similarly, no statistically significant difference was found for a history of sexual, physical or emotional abuse in the subgroups childhood and adulthood for men; see Table 2. Anxiety and depression Women and men with FGID significantly more often reported anxiety (P < 0.01), consultations for GI problems (P = 0.02), childhood abuse (P < 0.01) and adulthood abuse (P < 0.01) than SSF women and men. In contrast, depression had no significant univariate association with FGID, GI consulting, childhood abuse or adulthood abuse. FGID (OR = 2.1, 95% CI: 1.1–4.0) and anxiety (OR = 10.7, 95% CI: 2.3–51) were the only remaining independent predictors for abuse when adjusted for age, sex, education and depression when tested with the multiple logistic regression technique. Quality of life Subjects with FGID had a significant reduction in HRQL, as measured by the PGWB, with a mean value of 97 (95% CI: 94–99), as compared with SSF controls, who scored 115 (95% CI: 112–116). There was no significant difference in HRQL between women and men either for the FGID subjects (women 96; 95% CI: 92–99; men 99; 95% CI: 95–102) or for the SSF group (women 114; 95% CI: 110–117; men 115; 95% CI: 112–119). Women with a history of some kind of abuse and FGID had significantly reduced HRQL, with a mean value of 91 (95% CI: 85–97) as compared with a mean value of 100 (95% CI: 96–104) for women without abuse history. Similarly, men with a history of some kind of abuse and FGID had significantly reduced quality of life, with a mean value of 90 (95% CI: 82–99) as compared with a mean value of 102 (95% CI: 98–105) for men without abuse history. The HRQL for FGID consulters and non-consulters was the same; both groups had a mean value of 97 with 95% CI 93–100 and 92–102, respectively. However, FGID consulters with a history of abuse had significantly lower HRQL, with a mean value of 92 (95% CI: 86–97), than FGID consulters without a history of abuse with a mean value of 100 (95% CI: 97–104, P < 0.05). There was a significant negative correlation between HRQL and anxiety [r = −0.61 (P < 0.01)] and between HRQL and depression [r = −0.54 (P < 0.01)]. Multivariate risk modeling Persons with FGID had higher odds ratio 2.2 (95% CI: 1.1–4.4) of a history of some kind of abuse (in childhood or adulthood, sexual, physical or emotional) as compared with the SSF controls adjusted for age, sex and HRQL (main model, adding the variable anxiety or education did not improve the model). Some kind of abuse in adulthood had odds ratio 2.8 (95% CI: 1.1–7.1) with the same strategy. Emotional abuse in adulthood had the highest (and only significant) odds ratio 3.1 (95% CI: 1.0–9.4), while sexual abuse did not reach significance. In childhood, physical abuse seems to have the highest odds ratio 2.9 (95% CI: 0.7–12) for future FGID, although significance was not reached in this study (data not shown). With the main model, women had significantly high odds ratios for FGID: from some kind of abuse 3.13 (95% CI: 1.21–8.10), emotional abuse 3.66 (95% CI: 1.22–11.0), physical abuse 5.07 (95% CI: 0.55–47.1) and sexual abuse 3.03 (95% CI: 0.89–10.3); the latter two types of abuse did not reach significance, as presented in Table 3. There were significant high odds ratios in women with the crude model for FGID from all types of abuse in childhood and adulthood, except adult physical abuse (probably owing to the small number of observations). Similarly, with the main model, there were high odds ratios in women, but they did not reach significance, see Table 3. Table 3Odds ratio and 95% confidence interval (CI) for having a functional gastrointestinal disorder (FGID) (logistic regression) in different groups of abuse, women and men, in crude model and main model adjusted for age and HRQLVariableCrude modelMain model (adjusted for age and HRQL)WomenMenWomenMenChildhood abuseNo childhood sexual abuse11Childhood sexual abuse3.95 (1.10–14.1)*2.44 (0.60–10.0)*No childhood physical abuse1111Childhood physical abuse 6.88 (0.86–54.9)1.95 (0.34–11.2)4.93 (0.53–46.3)1.61 (0.21–12.3)No childhood emotional abuse1111Childhood emotional abuse 3.71 (1.32–10.4)1.00 (0.38–2.68)2.71 (0.87–8.4)1.14 (0.37–3.55)No childhood abuse1111Any childhood abuse3.41 (1.38–8.43)0.94 (0.36–2.47)2.50 (0.91–6.85)1.10 (0.36–3.38)Adulthood abuseNo adult sexual abuse111Adult sexual abuse4.45 (0.97–20.5)*3.20 (0.60–16.9)1.51 (0.53–4.29)No adult physical abuse11Adult physical abuse *0.93 (0.06–15.4)*0.84 (0.03–25.3)No adult emotional abuse1111Adult emotional abuse 8.86 (2.00–39.3)2.74 (0.67–11.1)3.42 (0.70–16.9)2.78 (0.55–14.0)No adulthood abuse1111Any adulthood abuse6.07 (2.00–18.4)2.43 (0.69–8.57)3.10 (0.92–10.3)2.41 (0.55–10.5)Any abuse (child or adult)No sexual abuse111Sexual abuse3.57 (1.34–9.5)*3.03 (0.89–10.3)1.49 (0.52–4.24)No physical abuse1111Physical abuse7.59 (0.96–60.2)2.5 (0.46–13.6)5.07 (0.55–47.1)1.84 (0.26–13.0)No emotional abuse1111Emotional abuse5.83 (2.11–16.1)1.26 (0.48–3.28)3.66 (1.22–11.0)1.31 (0.43–3.95)No abuse1111Any abuse4.61 (1.95–10.9)1.31 (0.52–3.32)3.13 (1.21–8.10)1.32 (0.45–3.90)1 = Reference, * too few observations In contrast, men did not have the same elevation of odds ratio for FGID from any type of abuse as compared to women. The highest odds ratio in men for FGID was for some kind of adulthood abuse and was 2.41 (95% CI: 0.55–10.5), while adult emotional abuse was 2.78 (95% CI: 0.55–14.0), but neither were significant; see Table 3. Consulters and non-consulters with FGID Consulters with FGID more often (46% of them) had a history of abuse as compared with non-consulters (29%, P = 0.06), although it did not reach significance as shown in Table 1. Some kind of abuse in childhood was more prevalent: 38% in consulters versus 16% in non-consulters (P = 0.01) and correspondingly for childhood emotional abuse 34% and 13% (P = 0.02) and for childhood physical abuse 14% and 3% (P = 0.05). Moreover, there was a significantly higher prevalence of physical abuse in childhood or adulthood in consulters, 17%, versus non-consulters, 3%, P = 0.03. Female consulters with a history of some kind of abuse had an odds ratio of 2.47 (95% CI: 0.92–6.7), and with a history of childhood emotional abuse this was significant, 4.20 (95% CI: 1.12–15.7.7), in contrast to male consulters, with an odds ratio of 1.66 (95% CI: 0.37–7.50) for some kind of abuse and 2.10 (95% CI: 0.37–12.0) for childhood emotional abuse. Discussion Women, but not men, with longstanding FGID often have a history of abuse. Women with FGID reported past sexual, physical or emotional abuse in 45% of the cases, as compared with 16% for women without FGID, 29% in men with FGID and 25% in men without FGID. In this study emotional and sexual abuse are the most common type of threats to women’s health related to FGID. This study also shows that childhood emotional abuse is a predictor for consulting for GI problems. Moreover, longstanding FGID is associated with a significantly reduced HRQL, and a history of abuse further reduces the HRQL in women and men. The findings reveal that a history of abuse is an important psycho-social factor linked to FGID in women. In contrast, the association in men is less clear, although men with a history of some kind of abuse and FGID had significantly reduced HRQL as compared with men with FGID without any history of abuse. Our study does not support the idea that consulters with FGID generally have a poorer HRQL than non-consulters, but a history of abuse had a negative effect on HRQL for consulters with FGID. Most studies that investigate FGID use a technique that implies questioning the subjects of the experiment at a single point in time. One of the strengths of this study is the repetitive sampling procedure, which makes it possible to compare individuals with longstanding FGID with persistently symptom-free individuals in the same population. We consider that our findings can be generalized to the general population, as the study groups were sampled from a well-defined and thoroughly investigated population [24, 26]. The individuals answered the abuse questionnaire in a quiet, safe environment, in reality anonymously, with the support of a nurse only when needed, which was an advantage since patients tend to underreport abuse when asked face to face by a doctor [23]. The abuse questionnaire has not been thoroughly validated in a Swedish population, but the questions are straightforward and the questionnaire was translated into Swedish and back-translated into English. The definition of functional dyspepsia and IBS used is not exactly the same as the now recommended Rome III criteria [39]. The definitions of dyspepsia and IBS used in this study were those used in the original study from 1988 [25], before current recommendations were available. We opted to retain our original study definition since our IBS definition was in accordance with the Rome III criteria and our definition of dyspepsia was more restricted in terms of symptoms, but wider in terms of abdominal location. We consider the overall prevalence of FGID in this study and the concordance on an individual level to be applicable within the current definition [39], and thus we consider our results conclusive. Previous studies in the field have reported that about half the patients referred to a gastroenterology clinic had a history of sexual or physical abuse [23, 40]. Moreover, 71% of women who reported domestic violence to the police had FGID according to Perona et al. [41]. In the literature, a history of childhood or adult abuse characterizes patients who present with a variety of functional symptoms apart from FGID [42]: headaches [43], pelvic pain [44], panic disorders [45], non-epileptic attack disorders [46] and back pain [47]. Psychological factors such as anxiety [48], as well as physiological factors such as enhanced visceral sensitivity, all contributed to a poor health outcome [42]. Only a few studies have focused on abuse and FGID in the general population, where Koloski et al. [22] found abuse to be significantly associated with IBS and/or functional dyspepsia, but less important when psychosocial factors were controlled for, a finding confirmed by others [49, 50]. The same research team [51] found that past sexual, physical and emotional abuse was not a significant predictor for seeking health care, in contrast to our finding that a history of childhood emotional abuse was associated with consulting, and anxiety and FGID were independent predictors for previous abuse. It is clear from our study that many subjects with traumatic memories and uncomfortable symptoms never seek health care and that when they do it is important to take a thorough medical history. There is a bi-directional communication between the gut and the brain through the autonomic nervous system and the hypothalamic–pituitary–adrenal axis [51] that can mediate an emotional trauma such as previous abuse. Common links suggested are neuroticism [49], generally diminished pain threshold [52] and current depression [53]. There is a higher incidence of childhood abuse reported for women than for men in this study as in others [5]. This could explain the significant relation of consulting and childhood emotional abuse for women, but not for men found in this study, suggesting that FGID in some patients might be a sequel of previous abuse. The treatments available to date for FGIDs are of limited value. Creed et al. [54] have reported that psychological treatment and antidepressants are effective in patients with IBS and a history of sexual abuse, which stresses the importance of discovering past abuse in patients with FGID. Future studies must focus on how the knowledge of a patient’s previous exposure to different kinds of abuse and the effects of different kind of psychotherapy can improve the treatment for at least a subset of FGID sufferers. Conclusions We conclude that women with longstanding FGID in many cases have a history of physical, emotional or sexual abuse in childhood or adulthood, which is associated with a poor HRQL and increased health care seeking. This is important for physicians to consider when diagnosing and treating FGID in women.

Eur_Spine_J-2-2-1602182

A rare case of complete C2–C3 dislocation with mild neurological symptoms

The authors report a rare case of complete C2–C3 dislocation with unexpectedly mild neurological symptoms in a 57 year old man involved in a motor vehicle accident, who had previously undergone posterior laminectomy from C3 through C7. A retrospective chart analysis and a thorough radiographic review were performed. X-rays and CT of the cervical spine demonstrated a complete dislocation at the C2–C3 level. Computed tomographic angiography revealed disruption of both vertebral arteries; however, blood flow was evident in the basilar artery. After radiologically guided placement in cervical traction with tongs that reduced the subluxation by approximately 50% the patient had spontaneous eye opening and was able to follow commands. A two-stage 360o stabilization and fusion was performed and the patient was finally discharged 24 days after admission with his neurological status essentially unchanged. In conclusion, our patient presented with surprisingly mild neurological symptoms. The previously performed laminectomy could have both predisposed to injury as well as protected his spinal cord from potentially fatal trauma. Introduction Subluxation of the cervical spine in adults usually occurs at its lower segments (C4–C7) [12, 14]; 50% of anterior subluxation of the neck has been reported to involve the highly mobile C5–C6 junction [11, 14]. In the pediatric population (up to 9 years old), the upper cervical spine is more susceptible to injury due to the anatomical differences in the developing spine [3]. Subluxation at the C2–C3 level is a particularly uncommon injury. We report a case of complete dislocation at the C2–C3 level in a patient involved in a motor vehicle accident who presented with surprisingly mild neurological symptoms. Case report A 57 year old African American man under the influence of alcohol and benzodiazepines was involved in a severe motor vehicle accident including one fatality. The patient was wearing a seat belt. He was transferred to our clinic via ambulance from a community hospital, fully immobilized with a hard cervical collar on, while the spinal cord injury solumedrol protocol had been initiated. On arrival to our Emergency Department, the patient had a Glascow Coma Scale (GCS) score of 11 and was thrashing all extremities. He responded to pain. He was intubated and thorough radiologic evaluation was undertaken. The CT of the brain revealed a slender tentorial subdural hematoma not requiring surgical evacuation. X-rays and CT of the cervical spine revealed a complete dislocation at the C2–C3 level with acute angulation between the two cervical vertebral segments (Fig. 1). Additionally, the imaging studies demonstrated multi-level degenerative spondylosis, anterior bridging osteophytes from C3 through C7 consistent with Forestier’s disease and extensive previously performed posterior laminectomy from C3 to C7. Laminectomy was performed, according to the patient, because of underlying degenerative disease. Computed tomographic angiography (CTA) revealed disruption of the vertebral arteries bilaterally at the level of C2–C3 (Fig. 2). However, flow was identified in the basilar artery indicating a patent posterior circle of Willis providing back flow to the basilar artery and the posterior circulation. No MRI could be obtained due to the presence of a previously implanted cardiac pacemaker. Fig. 1Plain lateral X-rays of the cervical spine demonstrating a complete C2–C3 dislocation upon the patient’s admissionFig. 2Computed tomographic angiography obtained at patient’s admission demonstrating bilateral complete disruption of vertebral arteries The patient was placed in cervical traction with tongs under radiologic guidance for verifying alignment. The weight applied was gradually increased up to twelve pounds; at that point, fluoroscopic imaging demonstrated a significant improvement in the angulation of the two separate segments and a reduction of the degree of subluxation. However, a subluxation of approximately 50% of the cervical spine vertebral body length was still present (Fig. 3). Upon reversal of sedation, the patient had spontaneous eye opening and was able to follow commands. His upper and lower extremities’ muscle strength was 3/5. The treatment plan for this patient consisted of a two stage 360o stabilization and fusion. Fig. 3Plain lateral X-rays of the cervical spine on the same patient obtained after applying external traction via tongs, demonstrating sub-optimal realignment of his cervical spine Shortly after his arrival to our hospital, the patient started to experience respiratory difficulties, leading the authors to the decision to delay surgery until the respiratory condition of the patient was stabilized. Two days after his admission, anterior cervical discectomy and fusion (ACDF) with the standard Smith-Robinson technique and bone allograft was performed at the C2–C3 level. A 24 mm Synthes (Paoli, PA, USA) statue plate was utilized after appropriate bending in order to contour the curvature of the cervical spine. Intraoperatively, disruption of the anterior longitudinal ligament at the C2–C3 level as well as fracture of the anterior osteophytic bony bridges, were evident. The vertebral endplates of the vertebral bodies at that level were severely degenerated. At the conclusion of the case, adequate reduction, satisfactory alignment and stabilization of the anterior cervical spine were accomplished. A halo vest was applied post-operatively for additional stabilization, necessary for the aggressive physical therapy that the patient’s respiratory condition demanded. Post-operative X-rays demonstrated reduction of the dislocation of C2 with respect to C3 and anatomic alignment of the structures of the cervical spine. The patient’s neurological condition remained unchanged during the following days. Respiratory distress was however noted, and a few days later, the patient was diagnosed with pneumonia. Appropriate treatment with antibiotics was initiated and it was decided to delay the second, posterior surgical approach until the patient’s respiratory condition was stabilized and his pneumonia resolved. Eighteen days after the initial operation, a second, posterior approach was employed. Under general endotracheal anesthesia, through a midline skin incision, a posterior cervical fusion and instrumentation (Synthes, Paoli, PA, USA) with pedicle screws at C2 level (20 mm long and 4 mm in diameter) and lateral mass screws (12 mm in length and 3.5 mm in diameter) at C3 level was undertaken. After optimal position of the screws was fluoroscopically verified, a 40 mm rod was used, which was attached and secured on a C2 and a C3 screw. Allograft bone chips were then appropriately placed (Fig. 4). The patient was placed back in the halo post-operatively due to his age and some concerns regarding osteoporosis. The halo vest was kept for four weeks after the second operation. He was finally discharged from the hospital to a rehabilitation clinic 24 days after admission. His neurologic status was essentially unchanged, with a muscle strength 3/5 in the upper and lower extremities. Fig. 4Post-operative lateral X-rays on the same patient after completion of anterior and posterior fusion The patient was followed at a different institution. At the last follow-up visit, 12 months postoperatively, his neurological status showed improvement with muscle strength of 4/5 in the upper and lower extremities.(Fig. 5a, b) Fig. 5a Post-operative CT (3D reformatted midsagittal image) of the cervical spine demonstrating adequate anatomical alignment at the C2–C3 level. b Post-operative CT (3D reformatted midsagittal image) of the cervical spine demonstrating adequate anatomical alignment at the C2–C3 level Discussion Injury of the cervical spine is a potentially fatal and debilitating incident due to the risk of damage of the cervical spinal cord. Although the degree of subluxation does not necessarily correlate with clinical symptoms and neurological signs, one would agree that the patient described above presented with unusually mild symptomatology in regards to the complete dislocation of the cervical vertebrae of the C2–C3 level, evident in his radiological examination. In reviewing the possible parameters related to this patient’s favorable outcome, attention should be drawn to the posterior cervical laminectomy previously performed on our patient due to underlying degenerative disease. It is quite likely that the lack of dorsal spinal elements of the C3 level, provided for adequate space for the posterior dislocation of the spinal cord to occur without sustaining the anticipated injury due to compression. This must have been of great importance both during the injury itself, as well as, after the dislocation had been established. Interestingly, Fountas et al. [5] noted in their study using postmyelogram computed tomographic measurements, that the cervical spinal cord might be smaller than considered. Specifically, they found that the diameter of the cervical spinal cord was 15–20% smaller than has been reported by autopsy studies. A smaller cervical spinal cord would also be less susceptible to injury due to compression. Although this is an interesting point that should perhaps be taken into consideration when referring to compression of the cervical spinal cord, its role in our case of a complete dislocation of the cervical spine could not have been, by itself, determinant without the history of previous laminectomy. On the other hand, the removal of the posterior arc of the vertebral bone may have predisposed for the development of such a complete dislocation. Hansen-Schwartz et al. [7] observed that 26% of patients developed static subluxation at the cervical spine 7.7±0.6 years after cervical laminectomy. Cusick et al [2], in a cadaveric study examining the biomechanical responses of the cervical spine to laminectomy, concluded that multilevel cervical laminectomy induces increases in total column flexibility, allowing additional motion in the flexion-extension plane. This observation may well describe the mechanism of injury in our case in which extension of C2–C3 induced a posterior dislocation and was not limited to a severe sprain. Likewise, Hong-Wan et al. [8] noted in their biomechanical study a significant increase in intersegmental motions following cervical laminectomy. Furthermore, Fields et al. [4] observed in their randomized study comparing the effects of laminectomy and laminoplasty in the rabbit, that laminectomized animals had poorer clinical outcome at 3 months post-operatively, associated with statistically significant angular deformity. Similarly, Baisden et al. [1], using a goat model, concluded that laminoplasty is superior to laminectomy in maintaining sagittal cervical alignment and preventing spinal deformities. The removal of bony and ligamentous structures of the posterior cervical spine might alter the biomechanics of the vertebral column and predispose to instability. The involvement of the C2–C3 level in our patient which coincides with the transition from non-laminectomized to laminectomized segments raises further suspicion regarding the putative role of laminectomy in the mechanism of injury in our case. Finally, the presence of osteophytes in the anterior cervical column in our patient (Forestier’s disease) could predispose to injury; more severe neurologic deficits have been described in patients with diffuse idiopathic skeletal hyperostosis even following minor trauma [15]. It is interesting to note that our patient had no signs of posterior fossa infarction despite the fact that both of his vertebral arteries were disrupted at the C2–C3 level. We assume that this is because of the presence of fetal circulation in this patient providing back flow to the basilar artery as this was demonstrated in his CTA, thus maintaining adequate perfusion of the posterior fossa [9]. Treatment of subaxial cervical spinal injury remains controversial. Both the anterior and posterior procedures have serious advantages and disadvantages [6, 10, 13]. In our case, we decided to perform a combined anterior and posterior surgical procedure, since both the anterior and posterior longitudinal ligaments were disrupted and we felt that a circumferential approach would sufficiently restore the lost stability. Furthermore, the presence of disc fragments compromising the spinal canal could not be ruled out, due to the inability to obtain an MRI in our patient. A posterior approach, thus, without anterior stabilization was considered to harbor an increased risk of spinal cord compromise. It needs to be emphasized that further multiinstitutional prospective clinical studies are required in order to establish guidelines for the management of subaxial cervical spine injury. Conclusion In conclusion, we have reported a rare case of complete C2–C3 dislocation with unexpectedly mild neurological symptoms in a patient who had previously undergone a C3–C7 laminectomy. The previously performed laminectomy might have both predisposed and protected the patient’s spinal cord from severe injury.

Eur_J_Health_Econ-_-_-1388084

The “Essential Levels of Care” in Italy: when being explicit serves the devolution of powers

The definition of an explicit health benefit package in Italy has gained importance because of devolution of powers from the national level to the regions. The set of services to be guaranteed by the public sector are defined at national level, while regions are accountable for their provision. This contribution discusses the entitlements and the decision criteria adopted by Italian policy-making bodies. Entitlements to services are clearly defined for few sectors (mainly outpatient specialist care); for hospital care the benefit catalogue is vague. The definition of the health benefit package in Italy is an essential element of the relationship between the central government and the regions. It is argued that adequate monitoring systems and accountability procedures are still needed to make the essential levels of care an effective pivotal element of the Italian National Health Service. Italy has a National Health Service based on principles of universalism and comprehensiveness [1, 2]. Established in 1978, the Italian National Health Service (INHS) absorbs 76.4% of total health care expenditure and it is financed by general taxation (OECD health data, 2005). As a consequence most care is provided free of charge at point of delivery, although user charges apply for outpatient services and procedures and, to a lesser extent, on pharmaceuticals [3]. A major critical feature of the INHS rests on the distribution of powers between the central government and the regions. Mainly created to reverse the concentration of state power occurring during the Fascist regime, the 20 regions are the emerging institutional tier of the country as they benefit from a substantial, although controversial, devolution of powers from the State. They have almost full control over approx. 200 Local Health Units and 100 Independent INHS Hospitals and they are expected to cover their deficit [4]. The Italian Constitution, revised in 2001, reserves to the central government the exclusive power to set the so-called “essential levels of care” (Livelli Essenziali di Assistenza, LEAs), which must be guaranteed to all residents. Regions have virtually exclusive powers over regulation, organization, administration, and funding of publicly financed healthcare. The Italian Constitution of 1948 specifies the citizen’s right to health. This constitutional guarantee is expressed in very general terms. Article 32 of the Constitution says that “The Republic protects health as a fundamental right of the individual and as a concern of collectivity and guarantees free care to the indigent.” The principle of a package of benefits available to all citizens irrespective of age, social condition, or income was stated later, in the law introducing the INHS in 1978. The expression “levels of care” was mentioned for the first time with the objective to guarantee equal health care coverage throughout the country: “the State is to set objectives for eliminating geographical differences in social and health care conditions” (Art. 2) and “and to determine levels of care to be guaranteed to all citizens” (Art. 3). The same legislation also introduced another major feature of the INHS: the patient’s right to choose “provider and place of treatment.” Although the 1978 reform listed the areas in which treatments are to be delivered under the INHS, it did not define the benefits to be included and excluded in detail. The concept of levels of care gained prominence in the legislation reforming the INHS in 1992–1993. The delegation of new powers to regions was coupled with tighter accountability systems. On the provision side the regions must deliver uniform levels of care, while on the funding side regions are mandated to cover any deficit required to provide the LEAs and to use their own resources to provide services above those guaranteed by national laws. The reform laid out a new logical framework, but it took time to establish the LEAs. Significant progress was made in the late 1990s with the approval of the National Health Plan 1998–2000 and a new reform approved in 1999. These emphasized the importance of the principle of equality in the access of care and introduced LEA system; they also clearly stated the criteria that should inform their definition: human dignity, effectiveness, appropriateness, and efficiency. The LEAs notion was at risk of remaining an abstract concept as political costs and implementation difficulties of clarifying and limiting INHS coverage were paramount. However, substantial progress in the definitions of LEAs was necessary to make sense of the overall strategy of redistribution of powers between the central government and the regions. This progress was made with the agreement between the regions and the central government on 8 August 2001 which was followed by a governmental decree (the LEA decree). At present this decree is the pivotal element of the Italian health benefit catalogue (Fig. 1). It defines the main areas of healthcare services to be guaranteed by the INHS (positive list), those completely excluded by public coverage (negative list), and those partially covered (only available for specific clinical conditions).Fig. 1 Decision making on benefits in Italy The positive list is based on the recognition and systematization of current legislation (other decrees, laws, guidelines, etc.), i.e., it includes all the services that the INHS is actually providing categorized in three macrolevels of care: (a) public health services, (b) community care, and (c) hospital care (Fig. 2). Table 1 displays the main elements of LEA decree and of the other benefit-defining laws and decrees currently in force in Italy. The present contribution focuses on curative services.Fig. 2 DPCM, 29 November 2001. Definition of national standards of care (LEAs). MoH Ministry of HealthTable 1 Benefit-defining laws/decrees and catalogues for curative care services in Italy (LEAs DPCM, 29 November 2001, “Definition of Essential Levels of Care”; Specialist DM, 22 July 1996, “Specialist outpatient services”; NPF National Pharmaceutical Formulary; Prostheses DM 332/1999: “Prosthetic devices, tariffs and provision modalities”; Rehab Guidelines on Rehabilitative Care adopted on May 7th 1998; Primary National Contract for Primary Care)LEAsSpecialistNPFaProsthesesaRehabaPrimaryInpatient curative care+–––––Day cases curative care+–––––Primary care+––––+Outpatient dental care+–––––Specialist outpatient care++––––Alternative medicine+–––––Rehabilitative care+–––+–Long-term nursing care+–––––Clinical laboratory–+–––Emergency rescue+–––––Pharmaceuticals+–+–––Therapeutic devices+––+––Prevention and public health services+–––––Legal statusLegislative decreeMinistry DecreeAgency administrative actLegislative decreeLawPresidential decreeDecision makerPermanent State-Regions Conference; MoHMinistry of HealthNational Drug AgencyMinistry of HealthMinistry of healthMinistry of health; Trade-Unions of GPsOriginal purposeEntitlementsFee-schedulePositive listFee-scheduleGuidelinesEntitlementsPositive/negative definition of benefitsP and NPPPPPDegree of explicitnessb233311/2If itemized: goods, procedures only; linked to indicationsVaries by area of careProcedures, sometimes linked to indicationsGoods, for certain categories specific indications (prescription notes)GoodsNoPartiallyUpdatingForeseenNo at national level; regular at regional levelRegularly (annually)No (foreseen)NoEvery 2–3 yearsCriteria––––––  Need+––+–+  Costs++++––  Effectiveness–++––+  Cost-effectiveness––––––  Budget+––––+  Appropriateness+–––++aThese sections (noncurative care services) are not discussed in detail in the present contributionb1, “all necessary”; 2, areas of care; 3, items The decree also defined a system for monitoring LEA implementation across the country. Responsibility for this was assigned to a special technical body established in April 2002 and composed of representatives of the Ministry of Health, the Treasury, and the regional governments. The main objective of the commission is to “monitor and evaluate the actual provision of services included in the LEAs and their costs.” In 2004 a new technical body (the National LEA Commission) was established to update LEAs on the basis of scientific, technological, and economic evidence (Ministry decree of 25 February 2005). The Commission is set up of 14 members: 6 experts of healthcare management, planning, and organizational sciences are nominated by the Ministry of Health, 7 are regional representatives, and one is appointed by the Treasury. Services of curative care in the health benefit basket Inpatient curative care Traditionally the services to be provided in hospital settings were never explicitly defined by the INHS. It has been rather implicitly recognized that all types of services deemed to be appropriately delivered at hospital level must be available to citizens. The LEA decree defines seven chapters as broad categories of services to be delivered in the hospital: (a) emergency services, (b) ordinary admissions (including rehabilitative and long-term inpatient care), (c) day hospital, (d) day surgery, (e) curative home-care, (f) collection, diffusion, control of blood-components and transfusion services, and (g) organ and tissue transplantation services. In addition, it is explicitly recognized that some benefits are available in hospitals although not included in the positive list of outpatient services provided under public coverage (e.g., certain pharmaceuticals or diagnostic tests). While the detailed spectrum of services to be provided by the hospitals is not explicitly defined, national and regional fee-schedules regulate their funding. Since January 1995 Italian hospitals have been financed mainly according to nationally predetermined rates based on classification into diagnosis-related groups (DRGs) [4, 5, 6]. Regions are free to modify the rates according to their own standards but must take the national rate as the maximum level [1]. To a certain extent DRG lists may be interpreted as the catalogue of hospital services and benefits covered by the INHS. This interpretation, however, should be done with some caution. DRGs are divided in two major groups: surgical and medical. The interventions to which surgical DRGs refer are expected to be offered and funded. Therefore surgical DRGs define a sort of list of services available to patients. For medical DRGs the situation is different as the classification includes all possible diagnoses, including those for which hospital admissions may not been appropriate. Therefore medical DRGs do not define a list of services to be guaranteed but rather economic constraints according to which providers act. Thus, although implicitly, the tariff values assigned to DRGs influence the specific content of the services provided in each diagnosis category. In Italy as well as in other countries, determining tariffs for specific diagnosis and treatments is seen as detrimental to the adoption of innovative technologies. Fixed and outdated tariffs may discourage the adoption of new expensive technologies and may force hospitals to look for alternative source of funding, often resulting in wide disparities of their availability to citizen. Regional authorities may react to the deficiencies in national policy with a variety of measures as it is illustrated in the case of Drug Eluting Stents (DES). Since their introduction into the European market in 2002, DES have been gradually implemented in clinical practice in Italy, mainly by hospitals in the north of the country. Several position papers and recommendations have been provided by scientific societies (e.g., Italian Group for Heamodynamics Studies, GISE), study groups, local committees (e.g., Emilia-Romagna Cardiology-Cardiosurgery Committee) in order to guide the adoption of DES. Early updating of reimbursement policies has been advocated by Italian cardiologists, hospital administrators, and patients to allow this technology to be economically sustainable by the hospitals [7]. Faced with continuously growing clinical and economic evidence, some regional authorities started to adopt different policy measures [8]. In 2002 the Lombardy Region revolutionized its DRG classification by creating three new DRGs to cover stent reimbursement and to encourage utilization. Other regions (Emilia Romagna, Lazio, Marche, Puglia) allowed for particular DRG tariff increase while the Campania Region established a special regional fund for DES reimbursement. In addition, Emilia-Romagna and recently Sicily established a regional registry to monitor the rate of adoption of the new technology and its effectiveness in real-life conditions. The final aim is to identify the target populations for which the new technology would be most beneficial. Most of the regions still have not updated in any way their reimbursement policies, thus limiting to certain extent the diffusion of the device on their territory. The example on DES illustrates the existence of wide variations across the regions concerning how to fund innovative technologies. Similar situations may be found in other sectors of health care. However, the issue is not that simple when we consider that one of the fundamental rights of Italian citizens is the “freedom of choice of provider and place of care” [9], making regions responsible for the cost of treatment provided to their residents in other regions. Day cases of curative care Services available in day hospital regime are defined as “diagnostic, rehabilitation, and curative care, delivered as alternative to ordinary hospitalization, when the services to be provided require, due to their nature or complexity of provision, medical or/and nurse continuous assistance, not available in the outpatient (ambulatory) setting” (Presidential decree, 20 October 1992). Regarding inpatient care services available in day hospital are not explicitly defined, but rather there are specific criteria for the service to be considered appropriately delivered in this regime. Guidelines are defined at national level and they refer mostly to organizational aspects of services’ provision rather than their specific types (i.e., number of beds assigned for day hospital services must be at least 10% of the total). The situation is very similar as regards day surgery care. National guidelines provide a list of possible interventions that may be performed in day care setting, as an alternative to normal hospital care. The list includes about 780 interventions, itemized by service delivered and grouped according to the organ or system of organs they refer to (e.g., respiratory system interventions, cardiovascular system interventions) Outpatient curative care: primary care All patients in Italy are registered with a general practitioner (GP) or a pediatrician who is in charge for providing most primary care, referring to specialists, and prescribing diagnostic interventions and drugs. The citizen’s can freely choose his or her own GP, given the limit of maximum number of enrolled patients. Primary care services provided by GPs are outlined broadly in the National Contract for General Practitioners, which is the most important document regulating various aspect of primary care. The National Contract is a result of negotiations between the government and representatives of general practitioners organized in various trade unions. Once reached, the content of the National Contract is legislated through a Decree approved by the Ministry of Health (i.e., the agreement is a binding by law). Regions are autonomous in establishing further agreements (Accordi Integrativi Regionali) aimed mainly at identifying the most appropriate organizational arrangements for the provision of services set at national level. The regional agreements may define additional services to be provided in primary care. The categories of services that primary care physicians are obliged to provide under the National Contract are defined broadly as: (a) essential services: acute and chronic disease management, in line with best practice indications and in agreement with the patient; (b) health promotion activities; (c) patient management within programmed and integrative domiciliary care coordinated with providers of specialist and rehabilitative care services; and (d) community services defined on the basis of regional agreements. The National Contract also encourages various forms of integration between primary care physicians and district services such as social and home care. Additionally, the National Contract obliges Local Health Units to guarantee continuity of care, i.e., primary care services 24 h a day, 7 days a week. Organizational arrangements are decided at regional level. Outpatient dental care Public coverage of dental care services has always been a debated issue in the Italian NHS. Current legislation explicitly excludes almost all types of dental services from the nationally defined benefit package. Some limited care is available to special groups of patients defined according to age and to specific clinical conditions. This explicit and rather broad exclusion of dental care services at national level had an impact on regional health policies. Numerous regions have adopted measures to guarantee some types of dental care services to citizens. An example of regionally defined benefit catalogue for dental care services is that in Veneto region, defined by Regional decree 2227/02. The regional decree defines the list of services available free of charge or subject to copayment for special patient categories. The list of services is available under set conditions only for the residents of Veneto Region, while nonresidents are fully charged. Beneficiaries are identified according to age, income, employment status, and presence of specific disease criteria: (a) dental health care in developmental age (0–16 years): preventive, diagnostic, and curative services for patients under the age of 16 years, orthodontic care for patients under the age of 12, nonsurgical treatment of paradental pathology for patients under age 16; (b) dental and prosthetic care to very low-income residents (<€ 8,500 per year) affected by chronic (e.g., cardiac insufficiency, psychosis) and rare (e.g., metabolic diseases, immunodeficiency) conditions; and (c) specialist curative care (excluding prosthetics) for anthalgic emergency cases caused by infections of caries and of paradental pathologies of traumatic events. Specialized outpatient care Specialized ambulatory services, including specialist visits, diagnostic, and curative interventions, are provided either by LHUs or by accredited public and private facilities. Patients are allowed to access specialist care only after approval by their general practitioner, who is responsible for the referral. Once the general practitioner has authorized the visit or the procedure, the patient is free to choose any provider among those accredited by the NHS anywhere in Italy. A list of outpatient services, including diagnostic procedures, specialist visits and laboratory tests was drawn up in 1996, and its original purpose was to define reimbursement fees of providers. Since then the national list of services has not been updated. However, regional authorities have often revised their fee schedules. The main criteria including services are effectiveness (based on solid scientific evidence) and costs. The benefits are classified in three different sections: (a) specialist outpatient care (including clinical laboratory and diagnostic imaging) provided under INHS coverage (a positive list of services, explicitly defined and enumerated, mainly without specific link to clinical conditions); (b) specialist services available only for specific indications (positive list of services limited to special patient categories); and (c) specialist outpatient care not covered by the INHS (negative list). Regions are free to deliver additional services for which they are financially responsible. These services should be marked separately in the fee schedule and added to the list in accordance with the coding system in place. The positive list of specialist outpatient services is itemized by service delivered. The items (approx. 2,000) are grouped into 16 categories on the basis of system of organs the intervention refers to (e.g., respiratory system interventions). Each category is further divided according to the specific organ (e.g., trachea and larynges interventions). Finally, each subcategory contains a list of specific services (e.g., laryngoscope). Some services in the positive list are limited to special settings (e.g., where special equipments are available). Services that are available only to special patients categories (i.e., limited for specific clinical conditions) include about 20 items, mainly laboratory and diagnostic examinations that are very costly (e.g., positron emission tomography) or in some way controversial (palliative pain treatments). All other outpatient curative care Numerous services of physiotherapy are excluded from the national benefit package. Many regions, however, have approved their inclusion in the regional benefits so to generate substantial variability across the country. Lombardy, for example, includes all services listed on the national negative list while Veneto and Friuli Venezia Giulia authorize “water rehabilitation.” Almost all regions provide anthalgic electrotherapy, ultrasound therapy, mesotherapy, and laser therapy. The national benefit package explicitly excludes all types of alternative and complementary medicine, leaving it to the regions to decide whether to provide some of these services to their citizens. Only 4 of 21 the regions have invested in this category of services: (a) acupuncture is available in Piedmont, Valle d’Aosta, Umbria, and Tuscany; (b) homeopathy is available in Valle d’Aosta (limited to specific clinical conditions); and (c) chiropractic services are available in Valle d’Aosta only for spinal cord pathologies. Spa treatment is available for a limited number of pathologies, identified as those for which thermal treatment may provide actual benefits (based on scientific evidence). The list of pathologies is explicitly defined in a Ministry decree (1994) and includes: rheumatic diseases (e.g., osteoarthrosis and other degenerative forms, extrajoint rheumatisms), respiratory diseases (e.g., chronic pulmonary diseases), and dermatological diseases (e.g., psoriasis). Discussion Almost 40 year since its inception, the Italian NHS has an explicit system of national services guaranteed to all its citizens. The concept of providing a limited set of services under the INHS is now well established. Entitlements to services in particular areas (mainly outpatient care) are now clearly defined, and some services (e.g., dental and thermal care) are explicitly excluded. In the area of hospital care entitlements remain broad and general, although a strong reference to appropriateness criteria and the use of DRGs contribute to make benefits more explicit in this setting as well. As in other countries, in Italy a clear definition of the benefits provided by the statutory system is thought to be beneficial for several reasons: it can contribute to a better allocation of resources, help to reassure patients about their rights and responsibilities, and facilitate the development of supplementary insurance [9]. In addition, the definition of the health benefit package in Italy is also an essential element in the relationship between the central government and the regions. The system of LEAs is the means to keep management and policy powers at regional level while assuring national guarantees. In this sense the basic package is primarily a policy devise to keep regions accountable to national standards. A constitutional reform aimed at a new redistribution of powers between the central and regional authorities is presently under parliamentary discussion. This specifies that powers on health matters are exclusively in the hands of regions, provided that national principles are respected. If this reform is approved, the basic package would gain even more importance. Therefore we foresee the need for substantial investments to further specify the content of the package and, more importantly, to develop adequate monitoring systems and accountability procedures. In our opinion, two issues are particularly critical in this respect. First, the coherence between benefits and resources made available requires an adequate governance system. At present Italy does not have a higher Chamber (it may be introduced in the Constitution reform under discussion) in which regions are represented, and where negotiations between them and the central government can take place within an appropriate institutional framework. At present, the devolution process lacks adequate rules to govern negotiations and conflicts. The risk of institutional conflicts, endless negotiations, legal disputes, and lack of coordination is very real and hitherto affects mainly the health care sector. It should be clear that without adequate governance mechanisms conflicts between the two central and regional authorities and between the regions themselves may result in further acceleration of the fragmentation of the INHS. The second issue concerns an adequate infrastructure to sustain the national government as guarantor of health care rights. Without an appropriate information system and new jurisdictional powers the national tier cannot ensure its guarantor role. To implement effective national guarantees the central government needs to develop the benefit package, to implement an effective monitoring system, and to design appropriate rules to force regions to act adequately. On the other hand, these conditions are also needed to ensure that resources available to the regions are compatible with the cost of the provision of services included in the benefit package [10]. It has been suggested that LEAs serve two main policy goals: equality and cost-containment [11]. An explicit definition of the guarantees provided Italians is a major mechanism to promote equality in the access of care, while limiting coverage may be a strong tool to contain costs. In theory the two objectives are compatible as explicitness of coverage can coexist with a different extent of the benefit package. In practice, however, LEAs may results in an overall system to defend the basic principle of the INHS from the risk of poor funding. Should radical policies aimed at reducing the ambitious goals of the INHS prevail, LEAs may become the main pillar of the system. A few reflections can be made about the criteria adopted in Italy to build the benefit package. Effectiveness (in most cases efficacy), as confirmed by scientific evidence, is the dominant criterion in defining the package. Need criteria have been also used; ailments for minor conditions (cough, sore throat, minor headache), cosmetic surgery, and ritual circumcisions) are excluded on the basis of various interpretations of the need criteria. In general, clinical and organizational appropriateness is promoted as well. As the former requires that treatments and procedures are applied only to patients with particular clinical conditions, the latter tries to assure that patients are treated in the most adequate (and often cheapest) setting. Overall the use of appropriateness criteria suggests that benefit catalogues should be made of detailed lists of services for particular clinical conditions rather than simple lists of services.

Clin_Rheumatol-3-1-1847465

Measuring quality of life in rheumatic conditions

Musculoskeletal disorders often have associated pain, functional impairment and work disability, and, not surprisingly, are the most common reasons for utilizing healthcare resources. Rheumatoid arthritis (RA) and fibromyalgia (FM) are causes of musculoskeletal pain and disability. Research indicates that there is a widespread impact of RA and FM on physical, psychological and social factors in affected individuals, and thus, outcome measures that encompass multiple aspects of quality of life are needed. Generic measures of quality of life identify associations between physical conditions and mental health and highlight the need to address psychological functioning to ultimately improve the individuals’ quality of life. Musculoskeletal disorders often have associated pain, functional impairment and work disability, and, not surprisingly, are the most common reasons for utilizing healthcare resources [1, 2]. Rheumatoid arthritis (RA) and fibromyalgia (FM) are among the most common causes of musculoskeletal pain and disability. They are estimated in community studies to affect 0.5–1 and 2–6% of the population, respectively [3–5]. These two conditions differ in a number of ways: In particular, RA has a clear pathophysiology with a range of clinical measures being able to indicate its presence, whereas the biological basis for FM is less defined with no “gold standard” test available. Despite this difference, these two rheumatic conditions share a range of associated outcomes. These include not only pain and fatigue but also difficulties with activities of daily living, ranging from basic and intermediate functions through to more complex tasks such as paid employment and social roles, e.g. child rearing [2, 6]. Further, RA and FM are associated with poor psychological functioning with 20 to 60% reporting depressive symptoms [7–9]. It is clear that there is a widespread impact of RA and FM on physical, psychological and social factors in affected individuals, and thus, outcome measures that encompass multiple aspects of quality of life are needed. In this volume, Birtane et al. [10] study the use of a generic measure of physical and psychological functioning in patients with RA and FM and thus provide insights on the quality of life outcomes in these disorders. Generic and self-report measures of quality of life provide a wealth of information regarding the experience of individuals with rheumatic conditions that medical and observational measures cannot capture [11]. One such measure is the Medical Outcomes Survey 36 Items (SF-36) [12], which is a self-report multidimensional measure of quality of life that has been used extensively in RA and FM research [13, 14]. The SF-36 includes subscales across eight dimensions of health and well-being, including physical functioning, role physical, role emotional, bodily pain, general health, vitality, social functioning and mental health. Scores for all subscales are expressed on a scale of 0 to 100, with a higher score indicating a better state of health. It has good reliability and validity for both clinical and healthy community-based samples [15]. A generic measure of quality of life can offer a number of insights into the impact of rheumatic conditions on everyday functioning. The SF-36 provides the opportunity for interesting comparisons of the quality of life between healthy individuals and those with rheumatic conditions and other chronic physical conditions. Studies indicate that those with RA and FM, almost invariably, have reduced or compromised quality of life compared to healthy age-matched controls. Both RA and FM have a similar impact on psychological and physical functioning and social roles as other chronic conditions that contribute to the global burden of diseases, such as heart disease, chronic obstructive pulmonary disease and diabetes [10, 16, 17]. These findings have implications on health policy and allocation of funding for both healthcare and research. For instance, musculoskeletal conditions affect more than 17% of the community and 60% of working-aged adults, with projections indicating that arthritis will affect up to 20% of people by 2020 [18]. The prevalence, cost and burden of musculoskeletal disorders indicate that policymakers need to address the impact on economies, healthcare systems and society at large. For instance, if a public health intervention in 2005 enabled the onset of arthritis to be delayed by 10 years, the age-related incidence rates would be reduced by 11.1% before 2020 [18]. The burden of musculoskeletal disorders and issues of impaired quality of life have become an international health priority with initiatives such as the Bone and Joint Decade launched by the World Health Organization (WHO). Birtane’s use of a generic measure of quality of life highlights the fact that FM, a condition with unclear pathophysiology, is associated with impaired quality of life to a similar extent as RA. This is in keeping with the recent models of quality of life or disability that place less emphasis on disease processes, pathophysiology and/or structural damage. Previous models of outcomes for rheumatic conditions have a strong biomedical focus with many models of disability or quality of life having an implicit assumption that there is a linear relationship between disease processes and disability. Attempting to use such models of disability by reading from left to right has obvious shortcomings because there are many instances such as FM that do not have a fully identifiable disease process [19]. Indeed, focusing on pathophysiology and functional ability in a limited context without consideration of the individual’s everyday tasks and roles sheds little light on the quality of life [20]. Newer approaches to examining disability acknowledge the roles of demographic, physiological, psychological, social and environmental factors acting as buffers and exacerbators of poor outcomes for chronic physical conditions [2]. The International Classification of Functioning (ICF) promoted by the WHO incorporates a broader biopsychosocial framework of disability, health and health-related states [21]. Indeed, the ICF takes a neutral stand with regard to aetiology and allows researchers to arrive at causal inferences using appropriate scientific methods. The ICF attempts to dislodge assumptions that the body level dysfunctioning, classified as impairments, is the fundamental dimension of disability and quality of life [21–23]. In other words, the quality of life in both RA and FM is not contingent on aetiology. The presence of a disability implies a cause, but the cause may not be sufficient to explain the resulting quality of life [21, 24, 25]. Measures such as the SF-36 provide the building blocks to create models of quality of life and study the different perspectives of this process across a range of chronic physical conditions as well as healthy individuals. Within this framework, using generic measures of quality of life can offer opportunities to frame research and interventions that appropriately target the quality of life of individuals with musculoskeletal disorders. Using this approach, Birtane et al. [10] identified in patients with RA and FM that the incidence of poor physical and psychological functioning is almost invariably greater than for healthy controls. Additionally, higher depression levels are evident in patients with FM compared to those with RA. It is not surprising that FM is associated with the worst psychological functioning, given that depression is often evident in individuals with FM [26]. These observations are consistent with previous findings, which indicate that significant depressive symptoms are reported by up to 60% of individuals with chronic physical conditions. Addressing poor psychological functioning, especially depression, in musculoskeletal conditions is critical due to its impact on the condition itself. Reciprocal relationships can occur between depression and pain and disability leading to a cycle of poor mental and physical health. Depression is related to poor treatment compliance and increased morbidity and mortality in rheumatic conditions [27]. Measures such as the SF-36 clearly identify the associations between chronic physical conditions and mental health and further highlight the importance of addressing psychological functioning in both research and interventions to ultimately improve the individuals’ quality of life. RA and FM are of course not mutually exclusive. The pathophysiology of each disorder is different, and many patients with RA also have FM, as do many other patients with a variety of chronic rheumatic diseases. The substrate of FM lies in the pain system, which can be functionally activated by distress, a common feature of chronic painful and disabling disorders. How much of the FM mechanism contributes to the low quality of life of RA? Further exploration of the mechanisms causing FM, with its resultant highly significant effect on the quality of life, will have a great impact on the management of not only FM itself but also RA and related musculoskeletal conditions.

Plant_Cell_Rep-4-1-2413084

Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce (Lactuca sativa)

The MYB transcription factors play important roles in the regulation of many secondary metabolites at the transcriptional level. We evaluated the possible roles of the Arabidopsis R2R3-MYB transcription factors in flavonoid biosynthesis because they are induced by UV-B irradiation but their associated phenotypes are largely unexplored. We isolated their genes by RACE-PCR, and performed transgenic approach and metabolite analyses in lettuce (Lactuca sativa). We found that one member of this protein family, AtMYB60, inhibits anthocyanin biosynthesis in the lettuce plant. Wild-type lettuce normally accumulates anthocyanin, predominantly cyanidin and traces of delphinidin, and develops a red pigmentation. However, the production and accumulation of anthocyanin pigments in AtMYB60-overexpressing lettuce was inhibited. Using RT-PCR analysis, we also identified the complete absence or reduction of dihydroflavonol 4-reductase (DFR) transcripts in AtMYB60- overexpressing lettuce (AtMYB60-117 and AtMYB60-112 lines). The correlation between the overexpression of AtMYB60 and the inhibition of anthocyanin accumulation suggests that the transcription factorAtMYB60 controls anthocyanin biosynthesis in the lettuce leaf. Clarification of the roles of the AtMYB60 transcription factor will facilitate further studies and provide genetic tools to better understand the regulation in plants of the genes controlled by the MYB-type transcription factors. Furthermore, the characterization of AtMYB60 has implications for the development of new varieties of lettuce and other commercially important plants with metabolic engineering approaches. Introduction Flavonoid metabolism is unique to plants and produces a great number and variety of compounds including flavones, flavonols, anthocyanins, and proanthocyanins (Winkel-Shirley 2001; Park et al. 2004). These molecules play important roles in many fundamental processes in plants, such as the pigmentation of flowers and fruits, UV resistance, pollen fertility, responses to wounding and fungal attacks, and environmental stress responses (Shirley et al. 1995; Weisshaar and Jenkins 1998; Harborne and Williams 2000; Dixon and Piava 1995; Moyano et al. 1996; Pietta 2000; Cominelli et al. 2005). The regulation of the anthocyanin pigments in plants is particularly important in metabolic engineering applications because these compounds act as visual signals that attract the insects and animals required for pollination and seed dispersal (Holton and Cornish 1995). The metabolic pathways involved in anthocyanin biosynthesis are well established, and the central pathways in this process are highly conserved in plants (Shih et al. 2006). Moreover, studies in model plants, such as Arabidopsis and maize, have facilitated a sound understanding of the genes involved and the assembly of the relevant enzyme complexes (Winkel-Shirley 2001). Two groups of genes are required for anthocyanin biosynthesis: structural genes that encode the enzymes that directly participate in the biosynthetic reactions, and genes for transcription factors that regulate the expression of these structural genes and the accumulation of flavonoid metabolites. Transcription factors can act as activators or repressors of gene expression, and mediate either increases or decreases in transcription through sequence-specific DNA binding and protein-protein interactions (Broun 2004). MYB proteins have been identified in a large number of eukaryotes, including fungi, plants and vertebrates (Ohi et al. 1994; Martin and Paz-Ares 1997; Slamon et al. 1986), and have roles in a wide array of cellular processes. These include the regulation of secondary metabolism, signal transduction, cell division, and responses to plant diseases and various forms of stress (UV-B light, cold and drought) (Larkin et al. 1994; Borevitz et al. 2000; Stracke et al. 2001; Vailleau et al. 2002; Cominelli et al. 2005). The R2R3-MYB-related proteins activate the transcription of structural genes that function in different branches of phenylpropanoid metabolism (Martin and Paz-Ares 1997). The promoters of these structural genes each contain potential MYB protein recognition motifs and also bind with the basic helix-loop-helix (bHLH) domain and conserved WD40 repeat proteins (Park et al. 2007a, b; Martin and Paz-Ares 1997; Winkel-Shirley 2001). The classification of MYB transcription factors is based on the strong conservation of imperfect repeats in the MYB DNA-binding domain of the member proteins (Martin and Paz-Ares 1997). In contrast to animals (R1R2R3), the MYB domain transcription factors found in plants are characterized by the R2R3-type MYB domain, comprising a DNA-binding domain and an activation/repression domain. Moreover, the balance between activators and repressors in this transcription factor family in plants may provide extra flexibility in terms of transcriptional control (Jin et al. 2000). There have been 126 R2R3 MYB genes identified in Arabidopsis thaliana, all of which exhibit strong homology within their N-terminal MYB DNA-binding domains and can be divided into 24 subgroups on the basis of their sequences (Stracke et al. 2001; Riechmann and Ratcliffe 2000; Kranz et al. 1998). To determine the biological roles of the MYB-related transcription factors in the production of phenylpropanoids, we isolated six R2R3-MYB proteins from Arabidopsis based on available information indicating that these genes are induced by environmental stress (UV-B irradiation) (Kranz et al. 1998). However, they do not have an informative phenotype, because of the structural and functional redundancy among these factors (Bouche and Bouchez 2006). Furthermore, although there have been other studies of anthocyanin accumulation in lettuce, one of the most popular and commercially important vegetables in the world, this process has not been characterized before at the level of transcriptional regulation. The regulation of anthocyanin accumulation in plants is also a key issue in our understanding of the regulation of leaf color. The identification of the factors that exert this control will provide valuable tools to moderate the extent and distribution of anthocyanin-derived pigmentation in plant tissues. In this study, we overexpressed the AtMYB60 gene in lettuce plants (“Jinjachuckmyun” cultivar) that are highly pigmented with a red color throughout their leaf tissues. Because the loss of these pigments is easily detected, the activity and repression of the MYB-type transcription factors that affect these pathways can be investigated. In the leaves of lettuce, coordinated transcriptional regulation controls virtually each step of the anthocyanin biosynthetic pathway (Park et al. 2007a). We compared the expression of the major anthocyanin biosynthetic genes and the AtMYB60 gene by RT-PCR. We show that this transcription factor is functionally active in repressing anthocyanin accumulation and can thus generate green lettuce leaves. We also show, by functionally repressing the accumulation of this compound, that AtMYB60 plays a significant role in controlling anthocyanin biosynthesis via the inhibition of a key target gene encoding dihydroflavonol reductase (DFR). Thus, we report here for the first time that anthocyanin biosynthesis and the transcription of the DFR gene are repressed through the overexpression of Arabidopsis R2R3-MYB transcription factors in transgenic lettuce plants. We also discuss the function of transcriptional regulators in the control of the expression of the structural protein that are required for anthocyanin biosynthesis in lettuce. Materials and methods Plant materials and growth condition To investigate the effects of exogenous AtMYB proteins on anthocyanin biosynthesis in lettuce leaves at the molecular level, Jinjachuckmyun plants, supplied by Syngenta (Seoul, Korea), were used. The plants were grown at the National Institute of Agricultural Biotechnology in Suwon, South Korea. Fresh lettuce leaves were plucked from the plants, frozen immediately in liquid nitrogen and stored at −80°C until required. Isolation of AtMYB cDNAs To isolate the AtMYB genes, cDNAs were isolated from UV-B-irradiated Arabidopsis plants using the BD SMART RACE cDNA Amplification Kit (Clontech, USA) and then used as templates for PCR cloning. The following gene-specific primers were used for RACE-PCR: AtMYB4 (forward: 5′-GTATGGGAAGGTCACCGTGCTGTGAGAAA-3′; reverse: 5′-TATTATTTCATCTCCAAGCTTCGAAAGCC-3′), AtMYB29 (forward: 5′-AAATGTCAAGAAAGCCATGTTGTGTGGGA-3′; reverse: 5′-GATCATATGAAGTTCTTGTCGTCATAATC-3′), AtMYB30 (forward: 5′-TAATGGTGAGGCCTCCTTGTTGTGACAAA-3′; reverse: 5′-CTTCAGAAGAAATTAGTGTTTTCATCCAA-3′), AtMYB34 (forward: 5′-AGATGGTGAGGACACCATGTTGCAAAGAA-3′; reverse: 5′-CGTCAGACAAAGACTCCAACCATATTGTC-3′), AtMYB51 (forward: 5′-GAATGGTGCGGACACCGTGTTGCAAAGCC-3′; reverse: 5′-ACTCATCCAAAATAGTTATCAATTTCGTC-3′) and AtMYB60 (forward: 5′-AGATGGGTAGGCCTCCATGCTGTGACAAG-3′; reverse: 5′-AATTAAAGCATATTAGAGAGCTCCATCAA-3′). The resulting PCR products were then cloned into the pGEM-T Easy vector (Promega, USA) and sequenced on both strands. Vector construction AtMYB coding regions were cloned into an expression vector containing the CaMV 35S promoter and the nopaline synthase 3′ terminator. The resulting constructs were then introduced into the binary vector 22103 (a derivative of pCAMBIA3301; Cambia, Australia). The complete coding sequence of each AtMYB cDNA was amplified with a specific forward primer designed to introduce an MluI restriction site and a reverse primer designed to introduce an XbaI restriction site to allow subcloning, as follows: AtMYB4 (forward: 5′-ATACGCGTATGGGAAGGTCACCGTGCTGTG-3′; reverse: 5′-TATCT AGATTATTTCATCTCCAAGCTTCGA-3′), AtMYB29 (forward: 5′-ATACGCGTATGTCAAGAAAGCCATGTTGTG-3′; reverse: 5′-TATCTAGATCATATGAAGTTCTTGTCGTCA-3′), AtMYB30 (forward: 5′-ATACGCGTATGGTGAGGCCTCCTTGTTGTG-3′; reverse: 5′-TATCTAGATCAGAAGAAATTAGTGTTTTCA-3′), AtMYB34 (forward: 5′- ATACGCGTATGGTGAGGACACCATGTTGCA-3′; reverse: 5′-TATCTAGATCAGACAAAGACTCCAACCATA-3′), AtMYB51 (forward: 5′-ATACGCGTATGGTGCGGACACCGTGTTGCA-3′; reverse:5′-TATCTAGATCATCCAAAATAGTTATCAATT-3′), AtMYB60 (forward: 5′-ATACGCGTATGGGTAGGCCTCCATGCTGTG-3′; reverse: 5′-TATCTAGATTAAAGCATATTAGAGAGCTCC-3′). After PCR and digestion, the MluI/XbaI fragments were cloned into the binary vector 22103 between CaMV 35S promoter and the nopaline synthase poly(A) addition site, creating the binary vector 22103-AtMYB plasmids (Fig. 2). These constructs were then introduced into the Agrobacteriumtumefaciens EHA105 strain using the freeze–thaw method. Generation of AtMYB transgenic lettuce Transgenic lettuce plants were generated using the Agrobacterium-mediated transformation of cotyledons. Lettuce seeds of the Jinjachuckmyun cultivar were supplied by Syngenta (Korea) and were surface sterilized in 25% (v/v) bleach for 7 min, washed (three changes) in sterile water, and placed on phytogel-solidified (0.2%, w/v) MS medium at pH 5.8. The seeds were germinated and maintained at 25°C (16 h photoperiod, 200 μmol m−2 s−1, daylight fluorescent tubes). The cotyledons were excised from the 4-day-old lettuce seedling and inoculated with freshly grown A. tumefaciens. The inoculated cotyledons were then co-cultivated on MS salt medium solidified with 0.2% purified phytogel (Sigma, USA), for 2 days at 24°C in the dark. The cotyledon explants were then transferred to shoot initiation medium supplemented with 400 mg l−1 carbenicillin (Duchefa, the Netherlands). After a washing step, these explants were cultured (ten per plate) on selection medium (MS salt 4.43 g l−1, sucrose 30 g l−1, phytogel 2 g l−1, kinetin 0.5 mg l−1, carbenicillin 400 mg l−1, phosphinothricin 1.0 mg l−1 and cysteine 10 mg l−1) and subcultured every 2 weeks on the same medium. Shoots that regenerated from explants on medium containing phosphinothricin were rooted in rooting medium (MS salt 4.43 g l−1, sucrose 30 g l−1, phytogel 2 g l−1 and phosphinothricin 1.0 mg l−1), before transfer to the greenhouse, where they were allowed to self-pollinate and to set seed. The seeds were harvested and stored at 4°C. Anthocyanin extraction and HPLC analysis Anthocyanin in the lettuce plants was quantified by HPLC at a detection wavelength of 510 nm, comparing the sample retention times and peaks with those of known standards. The relative quantification of each phenolic compound samples was expressed as the equivalent quantity of purified standards for each treatment. About 0.2 g of ground and freeze-dried leaves was accurately weighed and extracted with 1 ml of a solution of methanol:water (80:20, v/v) at 80°C for 1 h, then sonicated for 20 min. The extracted solution was filtered into a 50-ml volumetric flask, and the flask and filter were rinsed with a solution of methanol:water (80:20, v/v). The filtrate was then made up to the required volume with the same solvent. Approximately 1 mL of the sample solution was passed through a 0.4 μm filter before analysis by HPLC. A Shimadzu HPLC system with 10AD dual pumps was used, with an RP18 (LiChrospher, 250 mm × 4 mm × 5 μm) column. The HPLC parameters were as follows: column temperature 30°C, solvent A = 0.1% trifluoroacetic acid in water, solvent B = 98% acetonitrile with 0.1% trifluoroacetic acid, solvent gradient, 0 min = 0% B, 3 min = 6% B 12 min = 18% B, 25 min = 25% B, 35 min = 100% B, 40 min = 100% B. The flow rate was 0.35 mL min−1. The peaks were classified as either cyanidin or delphinidin derivatives by UV spectral analysis. RT-PCR analysis Total RNA from the transgenic and wild-type lettuce leaves was extracted using RNeasy Plant Mini Kit (Qiagen, USA), according to the manufacturer’s instructions. To estimate the transcript levels of the exogenous AtMYB4 and AtMYB60 genes in lettuce, we used the primers 5′-ATGGGAAGATCGCCTTGTTGTGAA-3′ (F) and 5′-TCATTTCATCTCTAAGCTTCTGTAGTCCAAAA-3′ (R) for AtMYB4 and 5′-ATGGGGAGGCCTCCTTGTTGTG-3′ (F) and 5′-TCAATTATCGAAAAAATTAGGGTTTTCATCA-3′ (R) for AtMYB60. The mRNA expression patterns of the CHS, F3H, DFR and UFGT genes were analyzed by RT-PCR with the All-in-one RT/PCR Premix (SuperBio P7003, Suwon, Korea). The amplification of rRNA was used as the internal control. Total RNA (100 ng) in a volume of 20 μL containing 20 mM Tris–HCl (pH 7.9), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, M-MLV reverse transcriptase (RNaseH Minus), Super Taq Plus DNA polymerase and 10 pmol of each gene-specific amplification primer was used for RT-PCR, according to the manufacturer’s instructions. Gene-specific primers for CHS (forward: 5′-GGTTTGCTCTGAGATTACAGCGGTTACC-3′, reverse: 5′-TCCTTGAGACCAAGCTTGAGCTCCACCT-3′), F3H (forward: 5′-GAGATCTTATCAGAGGCAATGGGCCTTG-3′, reverse: 5′-ACAACGGCCCGATGGTCTGCGTTCTTAA-3′), DFR (forward: 5′-GGTCTATGACGAGTCTCATTGGAGCGAT-3′, reverse: 5′-CTATCAATTGCTCCTTTGAACATCTCCT-3′), UFGT (forward: 5′-TGGAGAAGCGGGTTAGACAGTTGATGGA-3′, reverse: 5′-TATAGCTACCATGATTCAACCAACTTCG-3′), rRNA (forward: 5′-TACGGCACTGAAGGTGCCAAGCTCGTG-3′, reverse: 5′-CATCCTCTTGGCAGTCTTGGCGTAGGG-3′) and bar (forward 5′-GCCGCAGGAACCGCAGGAGT-3′, reverse: 5′-AGCCCGATGACAGCGACCAC-3′) were used to confirm the expression levels of these genes in the lettuce plants. Reverse transcription of the total RNA was carried out at 50°C for 30 min, and the reaction was inactivated at 96°C for 3 min. The amplification conditions comprised 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 2 min, and a final extension at 72°C for 10 min. PCR was performed using a GeneAmp PCR system 9700 Cycler (Perkin-Elmer, MA, USA). The resultant RT-PCR products were resolved on a 1% agarose gel, stained with ethidium bromide, and photographed. The amplified products of the CHS, F3H, DFR, UFGT, rRNA and bar transcripts were 398, 319, 534, 153, 386 and 267 bp, respectively. Results Cloning of MYB-type transcription factor genes from Arabidopsis To isolate and characterize the MYB-type transcription factors in Arabidopsis (AtMYB) that play a role in phenylpropanoid metabolism, primers were designed to amplify the entire coding regions of the AtMYB genes that are induced by environmental stress (UV-B irradiation) (Meissner et al. 1999). Previous studies of Arabidopsis have suggested that the R2R3-MYB genes of this plant have important functions in the regulation of secondary metabolism, disease resistance, and hormonal responses (Kranz et al. 1998). We isolated six Arabidopsis R2R3-MYB transcription factor genes based on available information concerning the AtMYB genes induced by UV-B irradiation (Kranz et al. 1998). These were AtMYB4 (AF062860, AT4G38620), AtMYB29 (AF062872, At5G07690), AtMYB30 (AF062873, AT3G28910), AtMYB34 (U66462, AT5G60890), AtMYB51 (AF062887, AT1G18570) and AtMYB60 (AF062895, AT1G08810), with apparent full-length cDNAs of 849, 1011, 972, 885, 1059 and 843 bp, respectively, encoding proteins of 283, 337, 324, 295, 353 and 281 amino acids, respectively (Fig. 1). The amino termini of these proteins contain the R2R3 repeats responsible for their binding to target DNA sequences and which are highly conserved among the R2R3-MYB proteins. Fig. 1Multiple alignment of AtMYB proteins. Amino acids identical in all six proteins are marked in black, amino acids found in four proteins are marked in gray. The line above indicates the R2 and R3 MYB repeats. GenBank accession numbers for the sequences: AtMYB4 (AF062860), AtMYB29 (AF062872), AtMYB30 (AF062873), AtMYB34 (U66462), AtMYB51 (AF062887) and AtMYB60 (AF062895) Expression of AtMYB genes in lettuce plants To examine the effects of the six isolated AtMYB genes on leaf color changes and on the anthocyanin composition of lettuce, cotyledons of the cultivar “Jinjachuckmyun” were transformed using the Agrobacterium tumefaciens strain EHA105 with the binary vector 22103-AtMYB (a derivative of pCAMBIA3301; Fig. 2), carrying these genes and a barstar selection marker. Shoots were regenerated from the callus of phosphinothricin-resistant transformatants. To confirm the introduction and expression of the exogenous AtMYB genes in the Jinjachuckmyun cultivars, total RNA was extracted from the leaves of six-week-old wild-type and transformed lettuce plants (T1 generation) and analyzed by RT-PCR. Fig. 2Schematic diagram of the T-DNA region of the 22103-AtMYB binary vector used in the transformation of lettuce cultivars. LB left border, RB right border, P35S Cauliflower mosaic virus 35S RNA promoter, PolyA Cauliflower mosaic virus 3′UTR terminator, Tnos nopaline synthase terminator Transgenic plants were confirmed by their resistance to a barstar spray and by subsequent RT-PCR with specific primers for six AtMYB genes. Because the T1 plants of the AtMYB4, AtMYB29, AtMYB30, AtMYB34 and AtMYB51 transformants were similar in leaf color and in the anthocyanin accumulation in their leaves, the AtMYB4 transformant (AtMYB4-101) and the AtMYB60 transformant (AtMYB60-117 and AtMYB60-112) were selected for further RT-PCR analysis to detect the expression of the structural genes that are involved in anthocyanin biosynthesis (Fig. 3). RT-PCR analysis was performed using six-week-old lettuce leaves of wild-type and transgenic lettuce plants. The wild-type lettuce plants had no integrated bar gene whereas each of the transgenic plants expressed bar mRNA. Analysis of the exogenously expressed AtMYB4 and AtMYB60 genes showed that AtMYB4 was specifically expressed in AtMYB4-101 and AtMYB60 in AtMYB60-117 and AtMYB60-112. Ribosomal RNA levels were used as a normalization control. Fig. 3RT-PCR analysis confirming the transcription of the AtMYB4, AtMYB60 and Bar genes in the indicated transgenic lettuce lines and not in the wild-type (WT) plants. rRNA was used as the control Plant morphology in AtMYB-transformed lettuce In our previous study, we demonstrated that the wild-type lettuce leaf accumulates anthocyanin, with cyanidin and delphinidin representing the major and minor components, respectively (Park et al. 2007a). To further characterize the functions of the AtMYB genes in anthocyanin biosynthesis, we compared the morphologies of transgenic lettuce plants overexpressing these genes with that of wild-type lettuce. More than 20 independent transgenic plants, selected with barstar spray and subsequently confirmed by PCR analysis (data not shown), were obtained with each A. tumefaciens strain. There was a large variation in the leaf color phenotypes of these transgenic lines. The AtMYB4-101 line showed normal anthocyanin accumulation and a red coloration phenotype, similar to that of the wild-type, but the AtMYB60-117 line showed different levels and patterns of anthocyanin accumulation. Under standard growth conditions, transgenic lettuce plants of the T1 generation that overexpressed AtMYB60 under the control of the cauliflower mosaic virus (CaMV) 35S promoter showed an inhibition (AtMYB60-117) of anthocyanin accumulation (green leaves), compared with that of wild-type plants (red leaves) (Fig. 4a, b). Apart from this inhibition of anthocyanin biosynthesis, no other morphological or developmental abnormalities were detected in this line under standard growth conditions. These results confirm that the exogenous AtMYB60 protein was responsible for the repressed anthocyanin phenotype. Fig. 4Morphological analysis showing the inhibition of anthocyanin biosynthesis in lettuce leaves, indicated by the lack of red pigmentation, following the introduction of the Arabidopsis MYB gene, AtMYB60: a young (20 days after sowing) wild-type (WT) lettuce and the AtMYB60 transgenic lettuce line, AtMYB60-117; b morphologies of both the WT and AtMYB-overexpressing lettuce plants at a later stage of growth. Both the AtMYB4-101 and AtMYB60-117 transgenic plants are shown and reveal that AtMYB4 does not inhibit anthocyanin production High-performance liquid chromatography (HPLC) analysis of AtMYB60 transgenic lettuce shows that accumulation of anthocyanin compounds is inhibited To confirm the identity of the anthocyanins that are synthesized in lettuce after transformation with AtMYB genes, lettuce leaf tissues were harvested and analyzed by HPLC for the presence of soluble anthocyanins. The results confirmed the predominance of cyanidin in the wild-type lettuce leaves, with traces of delphinidin also evident, as we previously described (Park et al. 2007a). The cyanidin and delphinidin peaks were detected in the AtMYB4-overexpressing lettuce (AtMYB4-101) and wild-type lettuce. The AtMYB29-, AtMYB30-, AtMYB34- and AtMYB51-overexpressing lettuce plants that showed a similar red-colored phenotype also produced corresponding cyanidin and delphinidin peaks on HPLC analysis (data not shown). However, clear differences were evident in the patterns of anthocyanin accumulation in the AtMYB60-overexpressing lettuce plants (Fig. 5). With HPLC, we found that the cyanidin (red pigment) peak was absent or sharply reduced in AtMYB60-117 and AtMYB60-112. The delphinidin (blue pigment) peak was absent in every AtMYB60-overpxpressing lines. Hence, the anthocyanin derivatives detected in lettuce leaves by HPLC can be correlated with the morphologies of the plants and with the inhibition of anthocyanin biosynthesis by the AtMYB60 protein. Fig. 5HPLC analysis of methanolic extracts from both wild-type control (WT) and AtMYB transgenic lettuce leaves (AtMYB4-101, AtMYB60-117 and AtMYB60-112). HPLC chromatograms were recorded at 510 nm. Peak C corresponds to cyanidin and D represents delphinidin. Each chromatogram was generated using the same quantity of injected sample RNA expression profiles of anthocyanin biosynthetic enzymes suggest their coordinated regulation in AtMYB60 transgenic lettuce plants Based on the correlation between the plant color morphology and our HPLC analysis of the anthocyanins, we undertook to verify the effects of the AtMYB proteins on the expression of the structural genes encoding the enzymes responsible for anthocyanin biosynthesis. As shown in Figs. 4 and 6, a dramatic leaf color change was evident in the AtMYB60-117 (green leaf) and -112 (mosaic phenotype) lines, in which anthocyanin accumulation is inhibited, and these plants showed a strong green leaf color phenotype. The enzymes that act in the anthocyanin biosynthetic pathway have been well characterized in lettuce in our previous study (Park et al. 2007a), in which we also analyzed the expression of the structural genes encoding the relevant enzymes. Following environmental stimuli such as UV-B irradiation, the expression of chalcone synthase (CHS), flavanone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR) is positively induced, and the accumulation of anthocyanin is increased in lettuce. We also performed RT-PCR analysis using gene-specific primers in that study to analyze the expression of these structural genes. They were identified from young UV-B-irradiated lettuce leaves and designated as CHS (CV700441), F3H (CV700152), DFR (CV700105) and UFGT (CV700246) (Park et al. 2007a). The transcript levels of these four anthocyanin biosynthetic genes were compared between the wild-type, AtMYB4- and AtMYB60-overexpressing lettuce plants in the present study. Fig. 6RT-PCR analysis of the genes involved in anthocyanin biosynthesis in the leaves of wild-type and AtMYB4- and AtMYB60-overexpressing lettuce plants. The transcripts of four biosynthetic genes involved in general anthocyanin metabolism (CHS, F3H, DFR and UFGT) were analyzed by RT-PCR in the AtMYB4-101 and AtMYB60-117 transgenic lettuce lines and were compared with the wild-type expression patterns. rRNA was used as the quantitative control Total RNA extracts were prepared from the leaves of six-week-old lettuce plants of wild-type, AtMYB4-101 and AtMYB60-117 lines and were analyzed by RT-PCR for the expression of the previously determined structural genes that are involved in the major biochemical pathways of anthocyanin biosynthesis (Fig. 6b). No reduction in the expression of any of these genes was observed in wild-type or AtMYB4-101 lines, both of which exhibited red coloration in their leaves. Moreover, the mRNA levels of the CHS, F3H and UFGT genes, which catalyze the synthesis of chalcones, flavanones and anthocyanin, respectively, were unchanged in any of the plants. In contrast, the mRNA levels of DFR, the production of which catalyzes the conversion of dihydroquercetin and dihydrokaempferol to leucocyanidin and leucopelargonidin (of the leucoanthocyanidins), respectively, was inhibited in the AtMYB60-117 line, which exhibits green-colored leaves with no red pigmentation. Discussion To isolate the MYB-type transcription factor genes involved in phenylpropanoid metabolism, the transgenic expression of six AtMYB genes was analyzed in lettuce. To date, a large number of transcription factors containing DNA-binding domains that are similar to those of the MYB proteins have been identified across the eukaryotic kingdom (Kranz et al. 1998). Several MYB transcription factors are known to regulate phenylpropanoid metabolism, and the quantities of the various end-products of the associated pathways that accumulate in specific cells, tissues and organs are thought to respresent the balanced expression of several interacting MYB proteins (Tamagnone et al. 1998; Espley et al. 2007). Recently, biochemical studies have also suggested that the MYB-type transcription factors are involved in regulating the branches of the phenylpropanoid metabolic networks in higher plants. Multiple MYBs are also thought to regulate the expression of the structural proteins that drive several downstream pathways of flavonoid metabolism (Moyano et al. 1996). A previous study has also shown that MYB proteins can regulate flavonoid biosynthesis via the transcriptional regulation of the gene encoding CHS, an enzyme that catalyzes the first committed step in flavonoid biosynthesis (Feldbrugge et al. 1997). The lettuce cultivar “Jinjachuckmyun”, which we used in our current transformation experiments, shows anthocyanin pigment accumulation in its leaves during development. We have shown in a recent study that environmental stresses upregulate the expression of the genes that encode anthocyanin biosynthetic enzymes in this same lettuce variety, as demonstrated by their responsiveness to UV-B irradiation (Park et al. 2007a). The lettuce has merit as a model plant system to further characterize the functions of those gene products in anthocyanin metabolism, because of its leaf color is red under field conditions and green under chamber condition when exposed to UV-B irradiation. The findings described in this study further validate the use of lettuce as a model plant because it offers experimental versatility in terms of its genetic regulation of the inhibition of leaf pigmentation. The function of the MYB proteins as activators (green to red in a growth chamber) or repressors (red to green in the field) of anthocyanin biosynthesis can be characterized directly from a phenotypic analysis of the lettuce leaf color. This system has potential applications as a research tool in a number of areas of plant molecular biology. We show in our present experiments that the anthocyanin accumulation in leaves is strongly inhibited in AtMYB60 transgenic plants compared with that in wild-type plants (Fig. 4a, b). These results suggest that AtMYB60 functions as a repressor of anthocyanin biosynthesis. Based upon our current findings of the inhibition of anthocyanin biosynthesis and our metabolite analysis of AtMYB60-overexpressing lettuce plants compared with the wild-type, we speculate that the synthesis of anthocyanin is principally controlled by the AtMYB60 transcription factor. The production of anthocyanin is inhibited in the AtMYB60-117 and AtMYB60-112 lines, and this is likely to underlie the green leaf phenotype of these plants. Hence, this characterization of the function of AtMYB60 represents a crucial step in furthering our understanding of the molecular regulation of anthocyanin biosynthesis and the transcription of related genes in lettuce and other plants. It is noteworthy in this context that MYB-related proteins generally act as transcriptional activators (Foos et al. 1994). The R2R3-MYB-related proteins have so far been shown to activate the transcription of structural proteins that act in different branches of the phenylpropanoid metabolism machinery (Martin and Paz-Ares 1997) or that play roles in the plant response to stress signals (Cominelli et al. 2005). Anthocyanin expression in maize is dependent on the presence of both the MYB and MYC types of regulatory factors. It is of some interest, therefore, that in this study, the expression of AtMYB60 alone, under the control of the CaMV 35S promoter, was sufficient to repress anthocyanin accumulation in a red lettuce variety. We also investigated the effects of AtMYB60 on the expression of the different structural proteins involved in anthocyanin production, which we have identified previously in lettuce. Our results reveal that this MYB protein represses the expression of the DFR gene in the AtMYB-117 and -112 lines. Significantly, DFR is a crucial structural protein that facilitates anthocyanin pigmentation in lettuce and that no such reduction in the expression of this gene was found in the wild-type or AtMYB4-101 lines that also develop red-colored leaves. The DFR protein represents an important branching point within the anthocyanin biosynthetic pathway and catalyzes the commitment step in this process, which is the formation of leucoanthocyanidins from the substrate dihydroflavonol. This indicates that AtMYB60 specifically inhibits the flux of flavonoid intermediates toward the production of leucoanthocyanins by repressing the transcription of DFR, with a resulting green leaf phenotype (Fig. 7). Fig. 7Schematic representation of the relationship between AtMYB60 expression and the structural proteins that play roles in anthocyanin biosynthesis It is interesting to note that the transcriptional response to UV-B irradiation that operates through AtMYB60 focuses on the regulation of DFR expression and anthocyanin accumulation. Recently there has been some debate about the identity of the phenylpropanoids involved in the response to UV-B, which may provide the most effective components of sunscreen formulations. MYB transcription factors that are known to regulate the transcription of genes in the phenylpropanoid biosynthetic pathway have been studied previously (Jin and Martin 1999). The overexpression of PAP1 in Arabidopsis results in the up-regulation of the genes encoding phenylalanine ammonia-lyase (PAL), CHS and DFR (Borevitz et al. 2000; Tohge et al. 2005). Moreover, mutations in the CHS, DFR and LDOX genes reduce the pigmentation of plants (Shirley et al. 1995; Abrahams et al. 2003), suggesting that these proteins are required for the synthesis of both anthocyanidin and proanthocyanidin. Because MYB-binding sites are present in the promoter regions of these flavonoid biosynthetic genes (Nesi et al. 2001; Debeaujon et al. 2003), it is possible that the AtMYB60 transcription factor might also specifically and directly target them. It is also possible that additional target genes that are related to phenylpropanoid metabolism can be negatively regulated by AtMYB60 when it is overexpressed in lettuce. The effects of AtMYB60 suggest that this MYB protein acts as a direct transcriptional regulator by binding to the MYB motifs common to the promoters of many structural genes in the same metabolic pathway, thus resulting in the downregulation of this pathway. The precise determination of this regulatory system at the molecular level remains to be achieved. Recently, Cominelli et al. (2005) reported that AtMYB60 is involved in the plant response to stress and its expression is negatively modulated during conditions of drought (Cominelli et al. 2005). A null mutation in AtMYB60 results in a constitutive reduction in stomatal openings and in decreased wilting under water-stress conditions. Therefore, we hypothesize that AtMYB60 has multiple functions in the regulation of anthocyanin biosynthesis and in the plant responses to different environmental conditions, including drought and exposure to UV-B light. AtMYB60 is the first MYB protein identified that functions as a transcriptional repressor of the lettuce DFR gene in anthocyanin biosynthesis, and thus extends the known roles of the R2R3-MYB proteins in secondary metabolism. Evidence from our transgenic lettuce experiments suggests that the direct repression exerted by AtMYB60 protein produces phenotypic effects in transgenic plants. The engineering of the anthocyanin biosynthetic pathway is potentially of great commercial significance. Because the AtMYB60 transcription factor should act effectively in most plant species, it can potentially be used to control anthocyanin biosynthesis in agriculturally and industrially important plants. This method of modifying the anthocyanin content in plant tissues also opens up new avenues to engineering improvements in commercial crops by altering metabolic pathways via the regulation of different structural genes in these pathways.

Cancer_Immunol_Immunother-3-1-2150634

Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI)

The Cancer Vaccine Consortium of the Sabin Vaccine Institute (CVC/SVI) is conducting an ongoing large-scale immune monitoring harmonization program through its members and affiliated associations. This effort was brought to life as an external validation program by conducting an international Elispot proficiency panel with 36 laboratories in 2005, and was followed by a second panel with 29 participating laboratories in 2006 allowing for application of learnings from the first panel. Critical protocol choices, as well as standardization and validation practices among laboratories were assessed through detailed surveys. Although panel participants had to follow general guidelines in order to allow comparison of results, each laboratory was able to use its own protocols, materials and reagents. The second panel recorded an overall significantly improved performance, as measured by the ability to detect all predefined responses correctly. Protocol choices and laboratory practices, which can have a dramatic effect on the overall assay outcome, were identified and lead to the following recommendations: (A) Establish a laboratory SOP for Elispot testing procedures including (A1) a counting method for apoptotic cells for determining adequate cell dilution for plating, and (A2) overnight rest of cells prior to plating and incubation, (B) Use only pre-tested serum optimized for low background: high signal ratio, (C) Establish a laboratory SOP for plate reading including (C1) human auditing during the reading process and (C2) adequate adjustments for technical artifacts, and (D) Only allow trained personnel, which is certified per laboratory SOPs to conduct assays. Recommendations described under (A) were found to make a statistically significant difference in assay performance, while the remaining recommendations are based on practical experiences confirmed by the panel results, which could not be statistically tested. These results provide initial harmonization guidelines to optimize Elispot assay performance to the immunotherapy community. Further optimization is in process with ongoing panels. Introduction Elispot is a widely used assay for immune monitoring purposes [6, 17, 25, 39]. Measuring immune responses has been accepted as an important endpoint in early clinical trial settings in order to prioritize further vaccine or other immunotherapy development [11, 22, 24, 26, 38]. Despite the overwhelming use of various immune assays for exactly that purpose, reported results are often met with skepticism, caused mainly by two reasons: (1) high variability among results from the same laboratories and/or among different laboratories, and (2) the lack of demand to report standardization, validation and training strategies as well as assay acceptance criteria by the laboratories conducting immune testing. This is surprising since the reporting of results for other clinical endpoints, e.g., side effects, has to follow strict guidelines and definitions. The Clinical Laboratory Standard Institute (CLSI) clearly phrased the demand for internal validation of immune assays as well as external validation in proficiency panels [28]. Proficiency panels are a common strategy for clinical laboratories to prove their ability to perform clinical tests at a level that permits patient testing. Defined as a program in which multiple specimens are periodically sent to laboratories for analysis, and in which each laboratory’s results are compared with those of the other laboratories and/or with an assigned value [27], proficiency panels serve various purposes:To provide regulatory and sponsoring agencies with confidence that reported data are generated following necessary standards and rigor that supports product licensure.To provide an external validation tool for individual labs.To provide proof to patients and volunteers that necessary measures have been taken to allow successful study participation [3].Some reports about comparison of Elispot performances among laboratories have been published [5, 32, 33]. A two-step approach to assay harmonization is being described by the C-IMT monitoring panel in this issue [2]. The earliest report, a four-center comparative trial in 2000, showed that most participants were able to correctly detect low frequency responses or absence of response against specific peptides in PBMCs from six donors, following their own protocol [33]. Another four-center comparison was conducted among members of the Elispot standardization group of the ANRS in France [32]. This study demonstrated overall good qualitative and quantitative agreement in Elispot results in the participating labs when testing HIV negative and positive donors for reactivity against a variety of peptides. All labs used their own protocol, but shared a high level of Elispot experience. An important program was launched by the NIAID for 11 international laboratories participating in HIV-1 clinical trials [5]. The panel demonstrated good concordance in qualitative detection of specific immune responses in previously defined and tested donors, but also notable inter-laboratory and intra-sample variability in spot counts, cell recovery and viability. This observation was met with strict standardization strategies across all panel members, and the panel was repeated twice with all laboratories following a standardized protocol. Variability was decreased under these conditions, but not abrogated (Cox, personal communication). Justification for the strict standardization approach among participating HIV vaccine laboratories can be found in the nature of HIV vaccine testing programs. Immune monitoring for these vaccine trials is performed at many sites simultaneously. Further, results from different trials need to be comparable in order to identify most suitable vaccine candidates. Importantly, the experimental Elispot setup is similar or identical in most laboratories, where PBMCs are tested against a variety of peptides [25, 31]. In contrast, immune monitoring approaches in the cancer vaccine field are more heterogeneous, based on the vast variety of vaccine design, type of cancer, and availability of antigen presenting cells. Standardization of the entire Elispot protocol across laboratories is therefore not feasible. We set out to devise a strategy to identify issues and deficiencies in current Elispot practices, and to identify common sources of assay variability within and between laboratories, with the extended goal of standardizing the identified factors in an assay harmonization effort across laboratories. In 2005, the Cancer CVC/SVI initiated an Elispot proficiency panel program to achieve this goal. In addition to offering an external validation program, the CVC addressed the need for such strategy by comparing assay performance across the field, identifying critical protocol choices and gaining an overview of training and validation practices among participating laboratories. For this program, predefined PBMCs from four donors with different ranges of reactivity against two peptide pools were sent to participants for Elispot testing. Laboratories had to further provide cell recovery and viability data, as well as respond to surveys describing their protocol choices and training and validation status. In response to the survey results, the CVC/SVI established requirements for laboratories to participate in future proficiency panels, which included the existence of a Standard Operating Procedure (SOP) prior to joining the program. Further, individualized assay performance assessment was offered to all laboratories, together with suggestions for implementation of protocol optimization steps. The results of the second Elispot proficiency panel conducted a year later demonstrated a significant improvement. The results and survey data allowed the identification of critical protocol choices for a successful assay performance (Fig. 1). Fig. 1Initial guidelines for harmonization of the Elispot assay to optimize assay performance and reproducibility derived from two international proficiency panels, based on their findings and trends observed Materials and methods Participants and organizational setup All participants were members of the Cancer Vaccine Consortium or its affiliated institutions, the Ludwig Institute for Cancer Research (LICR) and the Association for Immunotherapy of Cancer (C-IMT). Laboratories were located in ten countries (Australia, Belgium, Canada, Germany, Italy, Japan, France, Switzerland, UK, and USA). Each laboratory received an individual lab ID number. Panel leadership was provided by a scientific leader experienced in Elispot, in collaboration with the CVC Executive office. SeraCare BioServices, Gaithersburg, MD, served as central laboratory, providing cells, pretesting and shipping services, as well as logistical services like blinding of panelists. IDs were not revealed to panel leader, CVC or statistician during the panel. Thirty-six laboratories including the central lab participated in panel 1, 29 including the central lab in panel 2. Twenty-three laboratories participated in both panels. Six new panelists were added to the second testing round. Thirteen dropped out after the first panel. Main reasons for drop out were switch of assay priorities and not meeting criteria for panel participation. Various groups stated that one time participation fulfilled their need for external validation. PBMCs and peptides PBMCs from healthy donors were obtained from a commercial donor bank, manufactured and processed under GMP conditions using established Standard Operating Procedures at SeraCare. PBMCs were frozen using a rate controlled freezer and transferred to the vapor phase of liquid nitrogen. A three lot validation study was performed [20] in which the following was validated: cell separation procedure, freezing media, dispensing effect on function, freezing procedure, and shipping on dry ice. It was demonstrated that functionality and viability were maintained throughout the procedure. In addition, Elispot values from fresh and frozen PBMCs, shipped on dry ice, were nearly equivalent. Each vial of PBMCs contained enough cells to ensure a recovery of 10 million cells or more under Seracare’s SOP. PBMCs were pretested at the central laboratory for reactivity against the CEF [7] and CMV pp65 peptide pools [23]. The CEF peptide pool was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. It contains 32 8-11mers known to elicit CD8-restricted responses, and serves as a widely used control in IFNγ Elispot assays [7]. The CMV pp65 peptide pool was a generous gift of the NIAID and Becton Dickenson. This pool consists of 135 15mers overlapping by 11 amino acids, and has been shown to elicit CD8- and CD4-restricted responses [23]. PBMCs were selected for no, low, medium and strong responses against both peptide pools, and repeatedly Elispot-tested at Seracare in order to confirm responder status. Response definition was set arbitrarily for the spot number/well (200,000 PBMC): no responder: average median/panel 1 + 2 = 1 (CMV and CEF); low responder: average median/panel 1 + 2 = 18 (CMV) and 40 (CEF); medium responder: average median/panel 1 + 2 = 98 (CMV) and 127 (CEF); and high responder: average median/panel 1 + 2 = 396 (CMV) and 398 (CEF). Peptide pools were resuspended in DMSO and further diluted with PBS to a final concentration of 20 μg/ml. Aliquots of 150 μl of peptide pool were prepared for final shipment to participants. Corresponding PBS/DMSO aliquots for medium controls were also prepared. Participants were blinded to the content of these vials, which were labeled as “Reagent 1, 2 or 3”. All cells and reagents sent to participants in both panels were obtained from the same batches. Cells and reagent vials were shipped to all participants for overnight delivery on sufficient dry ice for 48 h. Shipping was performed by Seracare under their existing SOPs. Elispot Participants received a plate layout template and instructions for a direct IFNγ Elispot assay which had to be performed in one Elispot plate. Each donor was tested in six replicates against three reagents (medium, CEF and CMV peptide pool). Further, 24 wells were tested for the occurrence of false positive spots by the addition of T cell medium only. About 200,000 PBMC/well were tested against 1 μg/ml peptide pool or the equivalent amount of PBS/DMSO. All other protocol choices were left to the participants, including choices about: Elispot plate, antibodies, spot development, use of DNAse, resting of cells, T cell serum, cell counting and spot counting method. All plates were reevaluated at ZellNet Consulting (Fort Lee, NJ) with a KS Elispot system (Carl Zeiss, Thornwood, NY), software KS Elispot 4.7 (panel 1) and KS Elispot 4.9 (panel 2) in blinded fashion. Each well in each plate was audited. Since the focus of this study was on assay performance and protocol evaluation and not on the definition of a positive response, we prospectively defined the ability to detect a response (independent of magnitude) by using the following “empirical” method: the antigen-specific spot counts per 2 × 105 PBMCs had to be >10, and at least 3× as high as the background reactivity. Similar approaches have been described elsewhere [5, 8]. Statistical analysis The following parameters were calculated for the overall panel and the individual participant’s performance, using either lab-specific counts or central recounts: the mean, standard deviation, and coefficient of variation (CV), the median, minimum, and maximum spot counts for each donor and reagent and the media only wells. Box plots were used to illustrate the distribution of spot counts across the panel per given test condition. Further, individual results were represented as box plots comparing lab counts with recounts, central lab results and overall panel results. For panel 2, results from repeating laboratories were also compared in box plot format for each donor and condition. The Fisher’s Exact test was used to compare the proportions of laboratories which missed to detect responses in each panel. For the comparison of recovery and viability, the Student’s t test was applied. Results Feasibility In the first proficiency panel, shipping and Elispot testing among 36 laboratories from 9 countries were conducted without delays. The success of this panel demonstrates the feasibility of such large international studies, the biggest of such format as of today, under the described organizational setup. The second panel with 29 participating laboratories from 6 countries followed the approach of panel 1. However, customs delays of dry ice shipments to some international sites required repeated shipments of cells and antigens to these destinations. Based on this experience, the use of dryshippers with liquid nitrogen is being implemented for international destinations in the third CVC panel round in 2007. Recovery and viability of PBMCs in panel 1 and 2 Participants were asked to record recovery and viability of cells immediately after thawing (Table 1). The mean and median for both parameters were almost identical in both panels, and for all four donors (mean cell recovery between 11.5 and 13.3 million cells and mean viability between 85 and 91%). Only a small percentage of laboratories recovered less than 8 million cells (13% in panel 1 and 14% in panel 2). The percentage of groups reporting less than 70% viability was 7% for both panels. Table 1Cell recovery and viability in both proficiency panelsDonoraMean P1/P2bMedian P1/P2Minimum P1/P2Maximum P1/P2Cell recovery in 106 cells112.5/11.512.0/12.06.8/1.026.4/18.8213.3/12.313.2/12.65.5/0.328.4/22.4313.1/12.813.6/12.86.7/1.725.1/24.3412.8/12.411.9/12.95.6/6.234.4/22.7Viability (%)188/8589/8948/58100/98287/8889/9057/67100/98391/8793/9054/69100/100486/8990/9243/75100/98aPBMCs from same donors and batches were used in both panelsbP1/P2 refer to data from panel 1 (P1) and panel 2 (P2) Interestingly, only 4/10 laboratories in panel 1, and 4/7 laboratories in panel 2 with recoveries below 8 million cells were from international locations. Similar, only 1/5 laboratories in panel 1 and 2/5 in panel 2 reporting viabilities less than 70% belonged to international sites. This clearly demonstrates that location for dry ice shipment had no effect on overall cell recovery and viability. We also investigated whether low (<8 million) or very high (>20 million) cell recovery had an influence on spot counts, assuming that these were potential erroneous cell counts, leading to too low or too high cell dilutions, respectively, what in turn would lead to too high (in case of underestimating cell number) or too low (in case of overestimating cell number) spots counts. However, except for a few sporadic incidents, there was no correlation between cell counts and spot counts (data not shown) in either direction. Only one laboratory with low recovery and low viability was found to have peptide pool-specific spot counts for all donors much below the panel median. The use of an automated cell counter (Guava) did not reveal any trend in recovery for this group (recovery ranged from 6.7 to 25.1 million cells), nor a difference compared to the overall panel recovery data. In contrast, the mean cell viability per donor measured by Guava counter users was significantly lower than the overall cell viability in users of a hemocytometer (P < 0.01, see table in Fig. 5). Elispot results in panel 1 All 36 laboratories completed testing of all 4 donors against medium, CEF and CMV peptide pool. Spot appearance and size as well as occurrence of artifacts differed dramatically among laboratories (not shown). Four outlier laboratories were identified, which detected less than half of the responses correctly. In all four cases, detected responses were well below the panel median, and often, there was high background reactivity (up to 270 spots/well) in medium controls. No obvious protocol choices could be identified which could have been responsible for the suboptimal performance. One out of the four laboratories had little experience at the date of the panel. Another group reported a less experienced scientist performing the assay. A third outlier repeated the assay, and was able to perform adequately. No feed back was available from the fourth group. Thirty-two out of 36 labs were able to detect medium and strong responders as well as the non-responder. However, almost 50% of participants were challenged with the detection of the low responder. The responses measured for that particular donor (mean spot counts/well for CEF = 42 and for CMV = 22) has been chosen for illustration purposes. Figure 2 demonstrates the distribution of responses measured including intra-laboratory variability for this donor against all three reagents and across both panels. Fig. 2Laboratory spot counts and variability for low responder across panel participants. Panel 1 left column, panel 2 right column. The reagent tested is indicated. Lab-specific spot counts per 200,000 cells are depicted as box plots with the box presenting the interquartile range, the triangle the mean and the horizontal line the median. Maximum and minimum spot counts are illustrated through the upper and lower mark. The horizontal line across a graph demonstrates the overall panel median. The central laboratory performed the assay under two different conditions in panel 1. Results from both experiments are presented; therefore 37 laboratories for panel 1. Laboratory ID numbers do not correlate in both panels. In panel 1, laboratory #18 reported spot counts for the medium control as high as 270 per well (mean 81, median 34.5). For proper illustration of all other panel data, these data were omitted from the graph. Intra-laboratory variability and variability among participants as well as reactivity against medium are representative for all responder PBMCs tested In panel 1, 17 labs did not detect the response against the CMV peptide pool. In one case, the response was missed due to high reactivity against medium. Three other labs missed to detect the response due to inaccurate spot counting, specifically in the medium control wells. Artifacts were erroneously included in counts (Fig. 3). Reevaluation of those plates revealed that those labs had spot counts indicating a response against CMV. Fig. 3Elispot assay results can be confounded by plate evaluation accuracy. The table demonstrates spot counts for PBMC/medium control wells with many artifacts from three different laboratories (Lab X, Y, Z). Respective well images are shown below each column for that specific group. Differences in lab-specific spot counts (“own”) and counts from reevaluation in an independent laboratory (“central”) including resulting variability measures are presented in the table A similar scenario was found for response detection against the CEF peptide pool. Thirteen laboratories missed to detect the response, one of which due to high reactivity against medium, and one due to inaccurate spot counting. An interesting observation was that 23 groups reported false positive spots in a range of 1–26 spots/well. Reevaluation revealed that the actual number of groups with false positive spots was lower (12), and the false positive spot number range per well fell between 0 and 8. Survey results about protocol During the first panel, participants had to provide information about their protocol choices: plates, potential prewetting of PVDF, antibodies, enzyme, substrate, use of DNAse during PBMC thawing, resting of cells, serum used, cell counting, and plate reader. There was a wide range of protocol choices across the panel participants. The most common choices for the parameters listed above were as follows: use of PVDF plates (64%) prewetted with Ethanol (52%), coated with Mabtech antibodies (67%) at 0.5–0.75 μg/well (33%); spot development with HRP (53%) from Vector Laboratories (33%) using AEC (44%) from Sigma (42%); no use of DNAse when thawing cells (83%), and no resting period for cells prior to the assay (53%); use of human serum (64%); cell counting with trypan blue exclusion using a hemocytometer (78%); and plate evaluation with a Zeiss reader (36%). There were some clear trends for international sites with preferred use of the AP/BCIP/NBT development system (83 via 29% in the US), the use of nitrocellulose plates (75 via 21% in the US), and the use of Mabtech antibodies (83 via 58% in the US). On the other hand, 7/8 laboratories using an automated cell counter were located in the US. We stratified our panel participants into groups consisting of: (1) outliers (failed to detect more than 50% of responses correctly), (2) labs which missed weak response detection only, and (3) labs with correct qualitative response detection in all four donors. We then checked for specific parameters that could be potentially responsible for the groups’ panel performance. We were not able to detect any parameter that seemed to be responsible for missed response detection. In contrast, laboratories with the same overall protocol choices (except serum) could have very different assay performance, whereas laboratories with different protocol choices could have almost identical spot counts (Fig. 4). Reasons for the failure to detect protocol choices which significantly proved the advantage or disadvantage of their use can be found in (1) the study design with open protocol choices allowing participants to follow their own SOP, (2) the overall wide range in choices of reagents and materials (e.g., 7 different sources for the coating antibody with 10 different total antibody amounts reportedly used, 10 different enzyme sources, 7 different Elispot reader systems, cell resting times between 0.5 and 20 h, serum choices unique for each laboratory etc.), and (3) the small sample sizes resulting from this dispersion of protocol choices. These widely spread individual protocol choices among participants did not allow the aggregation of data for an adequately powered statistical analysis. Fig. 4Demonstration of examples of concordance of spot counts among three laboratories (Lab I–III) using different Elispot protocols (a, high responder), or of disagreement of spot counts for two laboratories (Lab IV–V) using almost identical protocol choices (b, medium responder). Lab-specific spot counts per 200,000 cells are depicted as box plots with the box presenting the interquartile range, the triangle the mean and the horizontal line the median. Maximum and minimum spot counts are illustrated through the upper and lower mark. The triangle refers to the overall panel mean for that specific condition. Results of Lab I–III are shown in a, and results of IV–V in b. The table contains reference to the figure above, and information about specific protocol choices One trend observed was that the users of an automated cell counter (7 labs used a Guava cell counter) had an overall better assay performance (Fig. 5), with no outliers in this group and only two laboratories who missed to detect the weak responders (29 vs. 52% in non-Guava users). Fig. 5Distribution of mean spot counts (upper two graphs) and cell viability (table) among the users of a Guava automated cell counter (a) and users of a hemocytometer (b). Spot counts per well (200,000 cells) are represented on a logarithmic scale. D1–4 refer to the donor tested with D1 being the strong, D2 the low, D3 the medium and D4 the non-responder. The tested reagent is indicated (med medium). The mean viability per donor reported by Guava users and users of other cell counting methods (one lab used an automated cell counter from Beckman Coulter, all others used a hemocytometer with trypan blue exclusion) is presented in the table below the figure Almost half of the participants (47%) introduced a resting period for cells before adding them to the Elispot plate. The time frame varied from 0.5 to 20 h. A valuable side-by-side comparison was performed by the central laboratory, in which two tests were run in parallel using the lab’s SOP with the exception that their standard resting period of 20 h was replaced by a shorter resting period of 2 h in the second test. The results are summarized in Fig. 6, demonstrating a significant increase in spot numbers for all peptide pool-specific responders when cells were rested for 20 h, without an increase in background reactivity (P < 0.05 for the weak responder against CMV; for all other antigen-specific responses in all donors P < 0.01). Fig. 6Comparison of the effect of 2 h (checkered bars) and 20 h (solid bars) resting periods for cells after thawing, before adding to the assay. D1–4 CEF/CMV refer to the specific donor and peptide pool tested. Background reactivity for all donors and testing conditions was between 0 and 5 spots (not shown). The standard error is shown. * Indicates a statistical significant difference of P < 0.01 in spot counts between 2 and 20 h resting periods for a given donor and reagent; # indicates in statistical difference with P < 0.05 (Student’s t test) Survey results about validation and training practices During the lively discussion of the results of panel 1 and its protocol survey at the Annual CVC meeting in Alexandria in November 2005, it was suggested that the level of experience, standardization and validation of participating laboratories might have been the cause for the variability and performances observed. In response, we conducted a survey among panelists, in which 30 laboratories participated. As expected, the experience and Elispot usage varied significantly. Some laboratories had the Elispot assay established less than one year before panel testing, whereas others used the assay for more than 10 years. The experience of the actual performer of the panel assay also varied widely. Interestingly, even though 2/3 of participants reported to have specific training guidelines for new Elispot performers in place, more than 50% never or rarely checked on the scientist’s performance after the initial training. Almost all laboratories indicated that they use an SOP that had been at least partially qualified and/or validated. Validation tools and strategies varied widely. Only 12 groups monitored variability, whereas 23 reported the use of external controls of some kind (e.g., T cell lines, predefined PBMC, parallel tetramer testing). Thirteen groups were found to have some kind of criteria implemented for assay acceptance. Among the 20 different criteria reported, not one was described by more than one lab. Mirroring these survey results, 20 laboratories believed that they need to implement more validation steps. All except one group expressed their strong interest in published guidelines for validation and training strategies for Elispot. Elispot results in panel 2 Based on the experience from the first panel, acceptance criteria for participation in the program were redefined. Only laboratories with established SOPs were accepted. The second panel was repeated with identical experimental setup as the first panel, and with the same batches of PBMCs, peptides and control reagents. Twenty-nine laboratories participated including the central lab. This time, there were no outlier performers identified. The number of groups that did not detect the weak responder dropped dramatically (P < 0.01) from 47% (17/36 labs) in panel 1 to 14% (4/29) in panel 2 (Fig. 2). The overall panel median for the CMV-response in the weak responder increased from 14 spots in panel 1 to 21 spots in panel 2, and for the CEF response from 30 to 51, respectively. Of the 23 labs repeating the panel, 8 had changed their protocol before panel 2, 3 of which as an immediate response to results from the first panel. One outlier lab from panel 1 participated in panel 2, and improved its performance by detecting all responses correctly as per reevaluation counts. Only their lab-specific evaluation did not detect the low CMV responder. Overall, 4/23 panel-repeating labs (17%) did not detect the low CMV-responder, 3 of which also did not detect this response in panel 1. About 10/23 groups missed the low CMV responder in panel 1, but 7 of these laboratories were able to detect it in panel 2. Only one repeater detected the low CMV response in panel 1, but not panel 2. This is a clear performance improvement for that group (47% missed this response in panel 1), and highlights the usefulness of multiple participation in panel testing as an external training program. We ran an in-depth analysis of the results and previous survey responses, where available, from participants who missed the weak responder, as well as from laboratories with marginal detection of response, including personal communication. We were able to narrow down the possible sources for these performances. Two laboratories missed responses due to inaccurate evaluation, during which they either included artifacts into spot counts or simply did not count the majority of true spots, as central reevaluation revealed. One laboratory did not follow the assay guidelines. The majority of laboratories, however, followed common protocol choices, but had either very low response detection across all donors and antigens, or detected very high background reactivity in some or all donors. This pattern pointed to serum as the possible cause for suppressed reactivity or non-specific stimulation. Three of these laboratories shared with the CVC that retesting their serum choice indicated that they had worked with a suboptimal serum during the panel; and that they now successfully introduced a different serum/medium to their protocol with improved spot counts. Serum choices included human AB serum, FCS, FBS, and various serum-free media. There was no difference in assay performance detectable between these groups. Discussion The CVC conducted two large international Elispot proficiency panels in which participants tested four batches of predefined donor PBMC for CEF and CMV peptide pool reactivity. Common guidelines like number of cells per well and amount of antigen had to be followed, in order to allow result comparison and reduce variability due to the known influence of cell numbers plated [2, 14]. A surprising finding from the first panel was that almost half of the participants were not able to detect the weak responder. The initial protocol survey did not allow the detection of common sources for this sub-optimal performance. In some cases, laboratories with identical protocol choices performed very differently, whereas others with distinct protocols had almost identical spot counts (Fig. 4). This observation supports the premise that many common Elispot materials and reagents (e.g., plates, antibodies, spot development reagents) perform equally or similarly well; and that there are other factors which influence the outcome of the Elispot assay. Various laboratories missed the response detection by inaccurately evaluating their Elispot plate, independent of the reader used, as reevaluation revealed (Fig. 3). This observation is in contrast to results from the first NIAID proficiency panel, which describes good agreement in spot counts from participants and experienced, independent centers [5]. Operator-dependent variability in Elispot evaluation results is a known phenomenon [15]. Despite the availability of high resolution readers and software features for automated spot gating and other potentially helpful options, it is essential to employ well trained operators for spot counting, to audit all plates, and to implement changes of reading parameter in cases when well and spot appearances differ from the overall assay, typically for technical reasons. For that, SOPs used for plate evaluation might require revision. The use of available certification and training services can be helpful. Cell counting is a protocol step known for introducing variability. Because of the wide range in PBMC recovery, we investigated whether cell counts could have been potentially erroneous, which would lead to wrong final cell dilutions, and can therefore lead to too high or low spot counts. Despite large differences in cell recovery (from 5.5 to 34.4 million cells per vial in panel 1), we did not find a correlation between cell recovery and spot counts. Very low (<8 million) and very high (>20 million) cell counts were consistently found for all donors among the same few laboratories. Both, hemocytometers and automated counters were used in those laboratories. Various automated cell counters have been introduced to the market, which do not only offer automated spot counting features, but also the discrimination of apoptotic, viable and dead cells. The use of such systems can potentially decrease variability in cell counts, and most importantly increase the accuracy of viable cell counts [4, 21]. Seven laboratories in panel 1 used the Guava counter, which allows the discrimination of apoptotic cells. Even though our panel did not reveal a difference in cell counts between Guava and hemocytometer users, it could be demonstrated that the overall cell viability reported by Guava users was significantly lower. This could likely be attributed to the ability to discriminate apoptotic cells with this method. Smith et al. [35] recently reported the usefulness of apoptosis acceptance criteria that allowed the separation of PBMC samples by their ability to respond to an antigenic stimulus or not. The introduction of a resting period for thawed cells is known to be advantageous since apoptotic cells die during the resting period, and final dilutions for the assay are based on a more homogenous population of viable cells [16, 18]. In contrast, the addition of a mixture of viable and apoptotic cells, which are prone to die during assay incubation time, directly after thawing leads to lower spot counts. During panel 1, the central laboratory performed a side-by-side comparison of the influence of a 2 and 20 h resting period on final spot counts, and demonstrated that a 20 h resting period yielded significantly higher counts (Fig. 6). Proficiency testing results from the C-IMT also support the introduction of a cell resting period for Elispot assays [2]. Even though almost half of participants in panel 1 let cells rest before addition to the plate, there was no clear correlation to the magnitude of peptide-specific spot counts. This might have been due to the variation of resting times between 0.5 and 20 h, and other protocol variables, which included the actual resting protocol. Factors like serum and serum concentration, cell concentration, and actual storage condition (e.g., tissue culture flasks or plates can lead to cell adherence and therefore loss of professional antigen-presenting cells) are known to influence the success of cell resting. There are no scientific studies published about the effect of serum choices on immune assay results, one of the best known “secrets” in immunology [16]. It is critical to choose serum that supports low background reactivity, but strong signals. The leading choice in this panel to use human AB serum reflects the historic preference for human immune assays. Each serum batch, however, is unique in its ability to support optimal assay resolution, and may potentially contain mitogenic or immune suppressive factors. There was some anecdotal evidence that the serum choice among our panelists was responsible for suboptimal performance. Interestingly, six laboratories preferred to work with serum-free medium. None of these groups observed high background reactivity, but two failed to detect the weak responder. A survey conducted among participating laboratories shed light on validation and training practices. The wide experience range of participating laboratories, in combination with various levels and approaches to validation and training, correlated with the overall high variability in panel results. In response, new criteria for panel participation were introduced for panel 2. The outlier lab number decreased from 4 to 0 in the new panel, however 3/4 outliers from panel 1 did not participate in panel 2. The percentage of laboratories that did not detect the weak responder decreased significantly from 47 to 14% (P < 0.01). This improvement might have been partially due to the stricter participant selection in panel 2. However, 7/10 labs repeating the panel improved their performance by correctly detecting the low CMV responder in panel 2, while missed in panel 1. A striking finding was that 2/3 of all laboratories stated that they believed they needed to implement more validation. And all but one group expressed their wish for published guidelines for Elispot assay validation and training. The outcomes from these two proficiency panels first provide Initial Elispot Harmonization Guidelines for Optimizing Assay Performance (Fig. 1) that can fulfill this need and may provide—if implemented widely—the grounds for substantial improvement of assay utility for research applications and development of immune therapies. This can be implemented in accordance with assay recommendations made for cancer immunotherapy clinical trials by the Cancer Vaccine Clinical Trial Working Group [12]. Further optimization is aimed for through ongoing proficiency panel work conducted by the CVC. Validation of assays is now a requirement for all endpoint parameters in clinical trials [30]. There are an increasing number of publications available describing validated Elispot assays [1, 19, 25, 34]. These papers contain valuable scientific information, but only limited referral to FDA regulations. The FDA guidelines for validation of analytical procedures [9] describe validation as the process of determining the suitability of a given methodology for providing useful analytical data, which consists of analyzing or verifying the eight or nine assay parameters as described in the US pharmacopeia or the ICH guidelines [13, 37]. Only few publications address validation of bioassays and Elispot in FDA terms [10, 16, 29, 36]. And even these few publications give only limited advice on how to validate the Elispot assay in a given laboratory setting, not to mention specific training guidelines. Furthermore, acceptance criteria for assay performance were only used by a limited number of laboratories, and each criterion was unique for the laboratory that used it. These observations should be a wake-up call for the immune monitoring community, which does not only include the cancer vaccine field, but also the infectious disease and autoimmunity field and others. General assay practices for the detection of antigen-specific T cells are comparable across all fields. The CVC as part of the Sabin Vaccine Institute is intending to develop and tighten collaborations with groups from other research and vaccine development areas. Published documents with specific criteria for Elispot assay validation, assay acceptance criteria and training guidelines will be most valuable for the immune monitoring field, and are now being established as CVC guidelines as a result of the described studies. Continuous external validation programs need to be a part of these efforts in order to check upon the success of inter-laboratory harmonization including assay optimization, standardization and validation as well as of laboratory-specific implementation of guidelines and protocol recommendations. These efforts are essential to establish the Elispot assay and other immune assays as standard monitoring tools for clinical trials.

Histochem_Cell_Biol-4-1-2228376

Topology of molecular machines of the endoplasmic reticulum: a compilation of proteomics and cytological data

The endoplasmic reticulum (ER) is a key organelle of the secretion pathway involved in the synthesis of both proteins and lipids destined for multiple sites within and without the cell. The ER functions to both co- and post-translationally modify newly synthesized proteins and lipids and sort them for housekeeping within the ER and for transport to their sites of function away from the ER. In addition, the ER is involved in the metabolism and degradation of specific xenobiotics and endogenous biosynthetic products. A variety of proteomics studies have been reported on different subcompartments of the ER providing an ER protein dictionary with new data being made available on many protein complexes of relevance to the biology of the ER including the ribosome, the translocon, coatomer proteins, cytoskeletal proteins, folding proteins, the antigen-processing machinery, signaling proteins and proteins involved in membrane traffic. This review examines proteomics and cytological data in support of the presence of specific molecular machines at specific sites or subcompartments of the ER. Introduction The ER is a dynamic organelle essential for cell life. The main subdomains of the ER include the nuclear envelope (NE), rough ER (rER), and transitional ER (tER), each with its own characteristic structure and function. The NE forms a shell around the nucleus. The shell typically consists of a flattened saccule closely applied to nuclear chromatin and showing multiple discontinuities or nuclear pores (Fig. 1a). The rER is continuous with the NE and consists mostly of stacked, flattened saccules. Each saccule is limited by membranes with attached ribosomal particles (Fig. 1b). The tER is composed of two distinct but continuous membrane domains: a rough domain characterized by the presence of attached ribosomes and a smooth domain (sER), giving rise to membrane buds and tubules (Fig. 1c). Clusters of vesicles, and tubules are often observed closely associated with the tER and represent the intermediate compartment, also called ER-Golgi Intermediate Compartment (ERGIC), vesiculo-tubular clusters (VTCs) or pre-Golgi intermediates (Bannykh et al. 1996; Hauri et al. 2000; Saraste and Kuismanen 1992; Fan et al. 2003). In certain cell types (such as steroid-synthesizing cells or hepatocytes) the smooth ER is extensive and consists of a network of interconnecting tubules limited by smooth membranes devoid of attached ribosomes. The tubules are linked by tripartite junctions and limit cytoplasmic regions often devoid of large organelles (Fig. 1d). Fig. 1Different subcompartments of the endoplamic reticulum. The ER is composed of continuous but distinct subdomains. a The nuclear envelope (NE) is shown with nuclear pores and ribosomal particles attached to the outer membrane. b The rough ER (rER) is continuous with the NE and consists of stacked flattened saccules, whose limiting membranes have numerous attached ribosomal particles. c Transitional ER (tER) is composed of a rER subdomain continuous with the rER and a smooth ER (sER) subdomain consisting of buds and tubules devoid of associated ribosomes (arrowhead points to a coated bud). d In some cells (e.g., steroid secreting cells and hepatocytes) the sER is composed of a large network of interconnecting tubules showing tripartite junctions (arrows) and fenestrations. Micrograph in C is courtesy of Christian Zuber and Jurgen Roth The diversity of ER structures parallels its many functions. The numerous ribosomes associated with the flattened saccules of the rER indicate its high capacity for protein synthesis, translocation, and folding. The two domains of the tER indicate an additional function within this compartment: that of the formation of tubules and vesicles allowing cargo exit and transport to the Golgi Apparatus, and the tubules of the sER, which are continuous with the rER indicate an increased ER volume thus providing enhanced capacity for calcium storage, drug handling, detoxification, and lipid and steroid synthesis (reviewed in Baumann and Walz 2001; Shibata et al. 2006). Based on these considerations, the subcompartments of the ER are predicted to have a presence and enrichment of very specific proteins. Such predictions can be analyzed by proteomics analysis of each ER subcompartment. A number of proteomics studies were done on the subcompartments of the ER providing an ER protein dictionary with new data being made available on many molecular protein complexes of relevance to the biology of the ER. As summarized in Table 1, proteomics studies were done on the NE, total ER microsomes (mixture of rough and smooth ER membranes), ribosomes, purified rough microsomes, purified smooth microsomes, and the ERGIC compartment. Because of the paucity of proteomics data on pure outer nuclear envelope membrane, we have restricted comment on studies of this ER compartment to that within Table 1. Subcompartments of the ER were isolated by subcellular fractionation using centrifugation and various sucrose gradients and some were isolated by immuno-isolation. The degree of purity of these fractions was analyzed by morphology and/or morphometry and in many cases biochemically by measuring the level of enrichment of expected proteins (markers) compared to the starting material (Table 1). Identifying the proteins present in these ER subfractions is an important step towards better understanding the specialized functions of the ER subcompartments and molecular mechanisms governing these functions. Table 1Assessment of published endoplasmic reticulum proteomics datasetsOrganelleTissue/cellsSpeciesER purificationMass spectrometryProteins detectedReferenceNENeuroblastoma N2a cellsMouseNE preparation (Triton-X-100 treatment)2D-BAC gels, MALDI MS148Dreger et al. (2001)NELiverMouseSubstractive proteomics (NE fraction-MM fraction)MudPIT, LCQ-Deca ion-trap MS, Tandem MS566Schirmer et al. (2003)NELiverRatNuclear pore complex fraction (enriched in nucleoporins) 1D gels, MALDI-QqTOF MS, Tandem MS 94Cronshaw et al. (2002)NESaccharomyces cerevisiaeYeastPurified nuclear pore complexHPLC, 1D gels, MALDI-TOF MS, Tandem MS174Rout et al. (2000)ERaLiverRatbMembrane proteins1D and 2D gels, MALDI-TOF MS68 (1D)39 (2D)Galeva and Altermann (2002)ERaLiverHamstercTenfold enrichmentj (calnexin marker, IB)2D gels, MALDI-Q-TOF MS39Morand et al. (2005)ERdLiverMousePCP-fraction co-sedimenting with calnexinLC, linear ion-trap Fourier transform MS, Tandem MS 229Foster et al. (2006)RERSaccharomyces cerevisiaeYeastPurified ribosomesMultidimensional LC, LCQ ion-trap MS, Tandem MS95Link et al. (1999)RERLiverMouse75% RMe, luminal proteins 2D gels, MALDI-TOF MS, Tandem MS141Knoblach et al. (2003)RERPancreasDogRibosome-associated membrane proteinBlue Native gels, LCQ ion- trap MS, Tandem MS30Shibatani et al. (2005)RER LiverRat4.0-fold enrichment j (G6Pase enzyme assay) 77% RMe, f1D gels, LC, QTOF-2 MS, Tandem MS787Gilchrist et al. (2006)SER LiverRat4.5-fold enrichment j (G6Pase enzyme assay) 58% SMe, 39% RMe, g1D gels, LC, QTOF-2 MS, Tandem MS998Gilchrist et al. (2006)ERGIChHepG2 cellsiHuman110-fold enrichmentj (ERGIC-53 marker, IB)1D gels, LC, Tandem MS24Breuza et al. (2004)ER-Golgi derived vesiclesdLiverMousePCP-fraction co-sedimenting with p115LC, linear ion-trap Fourier transform MS, Tandem MS220Foster et al. (2006)BAC 16-benzyldimethyl-n-hexadecyl ammonium chloride, ERGIC ER-golgi intermediate compartment, G6Pase glucose-6-phosphatase, HPLC high pressure liquid chromatography, IB immuno-blot, LC liquid chromatography, MALDI matrix-assisted laser desorbtion ionization, MM microsomal membrane, MS mass spectrometry, MudPIT Multidimensional protein identification technology, PCP protein correlation profiling, RM rough microsomes, SM smooth microsomes, TOF time of flightaTotal microsomes (containing both rough and smooth ER membrane derivatives)bUntreated and phenobarbital treated ratscModel of insulin resistance and metabolic dislipidemia fructose-fed animaldFractions obtained by rate-zonal centrifugation of the postnuclear supernatanteMorphometric characterization by electron microscopyfPresence of ≥11 ribosomal particles/vesiclegPresence of 1–4 ribosomal particles/vesiclehImmuno-affinity purified ERGIC membranesiCells were treated with brefeldinA to accumulate cycling proteins in the ERGICjEnrichment over homogenate Protein synthesis and secretion The ER is a key organelle of the secretion pathway involved in the synthesis of both proteins and lipids destined for multiple sites within and without the cell. Ribosomal proteins Because ribosomes define the rough domain of the ER, studies of the proteins of purified ribosomes are relevant to the proteome of the rough ER. Link et al. (1999) reported on the proteome of ribosomes purified from Saccharomyces cerevisiae. Multidimensional chromatography and tandem MS were used to identify 70 of the 78 predicted ribosomal proteins in yeast. The YMR116p protein (homologue of human RACK1 an intracellular receptor for activated protein kinase C) was found to be associated with the 40S ribosomal subunit implicating this protein in translation in PKC-mediated signal transduction. RACK1 is now known to be a bona fide ribosomal protein and to play an important role in regulating eukaryotic translation (Nilsson et al. 2004). Of the proteomics studies carried out so far on mammalian liver ER subcompartments, Gilchrist et al. (2006) have identified most of the ribosomal proteins in purified rough microsomes from rat liver including 33 proteins of the 40S ribosomal subunit and 45 proteins of the 60S ribosomal subunit. This number is close to the 84 proteins isolated from rat ribosomal particles (Wool et al. 1995). The study by Gilchrist et al. (2006) revealed that the concentration of ribosomal proteins was greatest in high-density rough microsomes (HDM) when compared with the ribosomal protein concentration in a smooth microsomal fraction containing low-density rough microsomes (LDM). This is consistent with previous morphometric studies showing more ribosomal particles associated with more vesicles in the HDM fraction compared to that in the LDM fraction from normal rat liver (Gilchrist et al. 2006; Lavoie et al. 1996). Foster et al. (2006) identified a comparable number of ribosomal proteins in ER fractions enriched with the marker proteins calnexin and p115. Proteins involved in RNA metabolism Rough microsomal derivatives of the ER are expected to contain a variety of proteins involved in RNA metabolism because they contain and translate associated messenger RNA (Lerner and Nicchitta 2006). A variety of proteins known to be involved in the metabolism of mRNA and in translation of mRNA have been detected in ER membrane derivatives by mass spectrometry (Foster et al. 2006; Gilchrist et al. 2006). These include heterogeneous nuclear ribonucleoproteins (hnRNPs, hnRNP A1, hnRNP A2/B1, hnRNP D, and hnRNP K), Poly(A)-binding protein 1 and many proteins that are involved in translation (eEF-1A, eEF-2, several subunits of eIF-3, and eIF-5A). Heterogeneous nuclear ribonucleoproteins are involved in mRNA metabolism both inside the nucleus and in the cytoplasm. In the cytoplasm hnRNPs are known to regulate mRNA localization, mRNA translation, and mRNA turnover (Dreyfuss et al. 2002; Shyu and Wilkinson 2000). Since hnRNP family proteins are known to accompany RNA from the gene through nuclear pores and into polysomes (Visa et al. 1996) the hnRNP family proteins detected in ER maybe associated with membrane-bound polysomes and modulate translation of specific proteins relevant to protein synthesis. Proteins mediating targeting, co-translational translocation, and processing of nascent polypeptide chains Secretory and transmembrane proteins are synthesized on polysomes that are attached to the rough domain of the ER (Palade 1975). Ribosomes are bound to the ER membrane and are associated with a molecular complex involved in protein translocation (Gilmore 1993; Rapoport et al. 1996). Sec61p made up of the three subunits alpha, beta, and gamma comprises the main protein-conducting channel, whereas signal-recognition particle receptor, signal peptidase, oligosaccharyltransferase, TRAM, TRAP, p180, p34, Sec63, and BIP comprise associated protein complexes that assist in the signal sequence-mediated targeting, co-translational translocation, and processing of nascent polypeptide chains (Johnson and van Waes 1999; Nilsson et al. 2003; Osborne et al. 2005). As would be expected because of the continuity between rough and smooth ER membrane compartments and because of the capacity of translocon components to diffuse laterally along the ER membrane (Nikonov et al. 2002) translocon components were detected in microsomal-derivatives of both rough and smooth ER (Gilchrist et al. 2006). However, despite such membrane continuity polysomes are retained concentrated in the rough domain of the ER. Cytoskeletal elements (i.e., microtubules) known to interact with specific receptors of the ER (i.e., CLIMP-63) might play a role in mechanisms that restrict the lateral mobility of membrane-bound polysomes (Farah et al. 2005; Vedrenne et al. 2005; Nikonov et al. 2007). Proteins involved in glycosylation and the calnexin cycle During the process of protein translocation, the asparagine residues in Asn-x-Ser/Thr motifs on the nascent polypeptide chain are glycosylated in the lumen of the ER by a multisubunit membrane protein complex called oligosaccharyltransferase (OST). Although several subunits (mainly the ribophorins I and II) of OST have previously been identified in proteomics studies of ER subfractions the most complete analysis of these was reported by Shibatani et al. (2005) who carried out proteomics analysis of isolated ribosome-associated membrane protein from canine rough microsomes. These investigators identified five known subunits of OST (STT3-A, ribophorin I, ribophorin II, OST48, and DAD1) and two previously uncharacterized proteins that co-purified with these subunits DC2 and KCP2. Oligosaccharyltransferase transfers preassembled glucose3-mannose9-N-acetylglucosamine2 core oligosaccharides from the ER membrane lipid donor dolichol pyrophosphate to nascent polypeptide chains. Glycosylation facilitates protein folding by increasing the solubility of yet-unstructured nascent chains and allows nascent chains to enter the folding sensor system by recruiting the lectin chaperones calnexin and calreticulin (Ellgaard et al. 1999). Access to the calnexin/calreticulin system requires modification of the N-glycans by the enzymes glucosidase I (GI), glucosidase II (GII), UDPglucose: glycoprotein glucosyltransferase (UGT1), ER-mannosidase 1 and EDEMs (ER degradation enhancing-mannosidase-like protein) (reviewed by Ruddock and Molinari 2006). Polypeptide released from this sensor system fulfils quality control requirements and can exit ER and transport to their final destination. However, misfolded polypeptides are retrotranslocated into the cytosol and degraded (described in “Ubiquitin metabolizing enzymes”). A number of enzymes involved in N-glycan modification have been reported in association with ER subfractions including GI (Gilchrist et al. 2006), GII (Knoblach et al. 2003; Foster et al. 2006; Gilchrist et al. 2006) and UGT1 (Gilchrist et al. 2006) following analysis by mass spectrometry. In addition to the expected detection in the rough microsomes (Knoblach et al. 2003; Gilchrist et al. 2006), these enzymes have also been detected in smooth microsomes (Gilchrist et al. 2006). This is consistent with the localization of GII and UGT in smooth ER and also ERGIC by immuno-electron microscopy (reviewed by Roth et al. 2002). These data are also consistent with the detection of chaperones calnexin and calreticulin, in rough and smooth microsomes from rat liver (Gilchrist et al. 2006) as well as the presence the ERAD machinery in smooth microsomes (Gilchrist et al. 2006). Thus, glycoprotein quality control not only takes place in the rER but also may occur to a significant extent in the sER. Biosynthetic cargo Many hepatic secretory proteins were detected by proteomics analyses within ER fractions from normal liver. These include albumin, serotransferrin, apolipoproteins A, B, and E, Complement C3, alpha-2u-globulin, and transthyretin (Foster et al. 2006; Galeva and Altermann 2002; Gilchrist et al. 2006; Knoblach et al. 2003). The association of these proteins with the endoplasmic reticulum is assumed to be biogenetic. The proteins are thought to be in the lumen of the ER en route for secretion via the early secretion apparatus. Consistent with this suggestion quantitative proteomics revealed two to fivefold higher concentrations of these proteins in a Golgi fraction when compared with that in rough and smooth ER fractions from the same tissue (Gilchrist et al. 2006). Although transmembrane proteins destined for transport out of the ER to the Golgi Apparatus and to the plasma membrane are expected to be part of the biosynthetic cargo of the ER such proteins maybe either too low in abundance for detection by mass spectrometry or they could be considered as membrane contaminants of the ER preparations. Confirmation of the latter suggestion would require double labeling for specific membrane markers using imunocytochemistry of the subcellular fractions used for proteomics analysis. Proteins involved in cargo exit and membrane traffic Interactions between membrane derivatives of the ER and the Golgi Apparatus are well known and this includes formation of ER exit sites to permit transport of newly synthesized protein out of the ER towards the Golgi (Palade 1975) and the formation of the ER-Golgi intermediate compartment to permit membrane recycling between the ER and the Golgi (Lee et al. 2004). Proteomics studies of different fractions of the ER have detected a number of proteins involved in membrane traffic. Coat proteins of the COPI and COPII protein complexes were detected in smooth microsomes (Gilchrist et al. 2006) and in ER/Golgi/vesicles (Foster et al. 2006). The smooth microsomes used in the studies of Gilchrist et al. (2006) are considered derivatives of the tER based on previous molecular, histochemical and morphological characterizations (Lavoie et al. 1996, 1999; Roy et al. 2000). As would be expected for proteins that recycle between the ER and the cis Golgi P24 family members (p24A, gp25L2, and TMP21) ERGIC-53, Rab1a, Sec22b, and the KDEL receptor were detected in smooth microsomes (Gilchrist et al. 2006) in ERGIC (Breuza et al. 2004) and in ER/Golgi-derived vesicles marked by the cis Golgi marker p115 (Foster et al. 2006). The tER plays a key role in the formation of cargo exit sites and since the extent and amount of cargo exit sites may vary between different cell types (Fan et al. 2003; Bannykh et al. 1996) the relative content of cargo and membrane traffic proteins is expected to vary in proteomics studies using membrane-derivatives of the tER from different cell types. ER chaperones The ER functions to both co- and post-translationally modify newly synthesized proteins and lipids and sort them for housekeeping within the ER and for transport to their sites of function away from the ER (Palade 1975). A variety of proteins both membrane-bound and luminal are involved in the control of maturation of nascent proteins and lipids and include proteins involved in folding, in the regulation of calcium homeostasis, in oligomerization, and glycosylation (Hebert and Molinari 2007). Many such proteins have been detected by proteomics analysis of fractions representing different domains of the ER. Knoblach et al. (2003) have concentrated their efforts on the analysis of luminal proteins in purified rough microsomes from mouse liver and have detected BiP, GRP94, lectin-like chaperones (calnexin, calreticulin), peptidylprolyl isomerases, thiol disulphide oxidoreductases (PDI, P5 (CaBP1), ERp72, ERp57, ERp44, ERp29, and ERp46). Quantitative proteomics revealed these same chaperones to be in similar concentrations in rough and smooth microsomes from rat liver (Gilchrist et al. 2006). Detergent extraction of the rough and smooth microsomes after salt wash revealed the presence of the ER chaperones in the aqueous phase of the detergent extracts, thus confirming the luminal localization for all these proteins (Gilchrist et al. 2006). The subunits of the chaperonin containing TCP-1 were detected associated with ER subcellular fractions (Foster et al. 2006; Gilchrist et al. 2006). Although tubulin and actin are well-known substrates for chaperonin t-complex proteins, new substrates are being defined and include proteins involved in cell cycle events (Liu et al. 2005). A role for chaperonin containing TCP-1 in protecting proteolytic intermediates in the MHC class I antigen-processing pathway has been described (Kunisawa and Shastri 2003). Whether this chaperone complex participates in MHC class I antigen processing at the cytosolic surface of a specific ER subcompartment remains to be confirmed. Calcium-handling proteins The ER plays an important role in Ca2+-homeostasis. This organelle has been described as a heterogeneous compartment with respect to the distribution of its Ca2+-handling proteins including the Ca2+-binding proteins, the Ca2+-pumps, and the Ca2+-release channels (Papp et al. 2003). Whereas the quantitative proteomics analysis of Gilchrist et al. (2006) revealed the Ca2+-pump SERCA2 and the Ca2+-release channel inositol 1,4,5-trisphosphate (InsP3) receptor to be enriched in smooth microsomes most Ca2+-binding proteins including calreticulin, glucose-regulated protein 78 and 94 (Grp78/BiP and Grp94), and protein disulfide isomerase, were similarly distributed between rough and smooth microsomes. Electron energy loss imaging analysis confirmed the heterogeneity of calcium distribution in ER of PC12 cells but was unable to distinguish distributions with respect to rough and smooth ER domains (Pezzati et al. 1997). The relative capacity for calcium storage and release of the rough and the smooth domains of the ER may vary between different cell types and the volume of these two subcompartments may influence this capacity. Enzymes of lipid and glucose metabolism The endoplasmic reticulum plays an important role in lipid biosynthesis and in glucose metabolism. Because of the large amount of sER in steroid-producing cells, lipid biosynthesis has often been attributed to the smooth domain of the ER. Indeed enzymes involved in lipid biosynthesis have been localized to the sER in such cells (Frederiks et al. 2007). However, smooth microsomes purified from the steroid-producing adrenocortical cells revealed not only enzymes involved in lipid synthesis but surprising high levels of translocation apparatus, and oligosaccharyltransferase complex proteins (Black et al. 2005). The implication of this finding is that lipogenic enzymes and other proteins involved in lipid metabolism should be easily recognized in both sER and rER domains. Lipogenic enzymes (ex. fatty acid synthase, acetyl-CoA carboxylase 1, and ATP citrate lyase) and lipid transfer proteins (ex. microsomal triacylglycerol transfer protein) indeed have been detected by proteomics analysis in both rough microsomal (Foster et al. 2006; Gilchrist et al. 2006; Knoblach et al. 2003) and smooth microsomal (Gilchrist et al. 2006) fractions. In terms of the distribution of proteins involved in lipid metabolism the data suggests a lack of distinction between sER and rER membrane domains. This suggestion is consistent with the previous morphological demonstration of lipid droplet formation in association with rER domains in situ (Robenek et al. 2006) and the demonstration of the formation of lipid droplets using ribosome-depleted rough microsomes in a cell-free reconstitution system (Paiement et al. 1994). A number of proteins involved in the pentose-phosphate pathway (PPP) have been reported in association with ER subfractions including transketolase (Foster et al. 2006; Gilchrist et al. 2006), glucose-6-phosphate dehydrogenase (G6PD) (Gilchrist et al. 2006) and transaldolase (Gilchrist et al. 2006) following analysis by mass spectrometry. The detection of the transketolase and G6PD in the ER by mass spectrometry is consistent with previous electron microscope cytochemical studies showing these proteins in association with the ER in situ (Boren et al. 2006; Frederiks and Vreeling-Sindelarova 2001). Thus PPP enzymes may play a role in glucose metabolism at the level of the ER in specific cell types. Proteins of detoxification and drug protein targets of the ER The xenobiotic metabolism pathway of the ER includes functioning cytochrome p450 family (CYPs) proteins, UDP- glucuronosyltransferases, and carboxylesterases. Extensive biochemical and cytochemical data exist showing enrichment of such proteins in ER (reviewed in Seliskar and Rozman 2007) but little data differentiates between the rER and sER subcompartments. In the quantitative proteomics study of Gilchrist et al. (2006) over 30 different cytochrome P450s were detected as well as nine carboxylesterases and over 10 different glucuronosyltransferases and most of these proteins were in similar concentrations in rough and smooth microsomes. Continuity between the rough and smooth ER domains may endow specific ER proteins with the capacity to diffuse freely along the membranes (Nikonov et al. 2002) and within the lumen (Snapp et al. 2006), thus explaining equal distribution of enzymes involved in xenobiotic metabolism in rER and sER domains. Endoplasmic reticulum proteins of detoxification and drug protein targets are now being examined quantitatively by proteomics in diseased states and under controlled drug treatments. Proteomics studies of liver microsomes under controlled drug treatments have revealed differential expression of specific ER proteins. Cytochromes P450 2B1, and 2B2, protein disulfide isomerase A3 and A6, and 78-kDa glucose-regulated protein were differentially expressed following phenobarbitol treatment (Galeva and Altermann 2002). p450 family proteins CYP1A2, −2A4/5, −2B10, −2B20, −2C29, −2C37, −2C38, −3A11, and −39A1 were up-regulated, and CYP2C40, −2E1, −3A41, and −27A1 were down-regulated by treatment with 1,4-bis-2-(3,5-dichloropyridyloxybenzene) (Lane et al. 2007). Ubiquitin metabolizing enzymes Endoplasmic reticulum-associated degradation (ERAD) is a regulated process whereby misfolded and unassembled proteins are recognized and retained in the ER by the quality-control apparatus and subsequently extracted, polyubiquitinated, and finally degraded in the cytoplasm by the multi-subunit 26S proteasome complex (reviewed by Meusser et al. 2005 and Romisch 2005). Ubiquitin and ubiquitin-activating enzymes were shown by electron microscope immuno-gold labeling to be associated with the post-ER/pre-Golgi network consisting of anastomosing tubulated and fenestrated membranes (Raposo et al. 1995). More recently misfolded protein was detected in an ER subdomain and in enlarged pre-Golgi intermediates by electron microscope immuno-gold labeling (Zuber et al. 2004). The quantitative proteomics analysis of Gilchrist et al. (2006) revealed proteasome subunits as well as ubiquitin metabolizing enzymes only in smooth microsomes. This data is consistent with a previous report showing that proteasomes were associated with the smooth endoplasmic reticulum and that they were practically absent from the rough endoplasmic reticulum (Palmer et al. 1996). Smooth microsomes used in the study of Gilchrist et al. (2006) may correspond to the compartment identified and described by Raposo et al. (1995) and Zuber et al. (2004). The smooth ER could be an important subcellular site for proteasome-dependent degradation of misfolded protein? Proteins involved in antigen processing Major histocompatibility complex (MHC) class I molecules present short, perfectly cleaved peptides on the cell surface for immune surveillance by T lymphocytes (Hammer et al. 2007). The intracellular processes that take part in creating the antigen-presenting machinery of the plasma membrane occur at the level of the ER and involve protein degradation, membrane translocation, and protein folding (reviewed by Elliott 2006 and Hammer et al. 2007). Protein degradation is carried out on the cytosolic side of the ER by the proteasome and the enzyme tripeptidyl peptidase II. The group II chaperonin TRiC (TCP-1 ring complex) is thought to chaperone the peptide between proteases and TAP the peptide transporter of the ER. TAP is composed of two transmembrane proteins, TAP1 and TAP2, and participates in the translocation of peptide to the lumen of the ER in an ATP-dependent step. On the luminal side of the ER the peptide is further processed by ERAAP, the ER aminopeptidase associated with antigen processing. The multicomponent MHC class I peptide-loading complex (PLC) includes MHC class I molecules, beta 2 microglobulin, the chaperone calreticulin, the oxidoreductases ERp57, and protein disulfide isomerase, the class I-specific accessory molecule tapasin and the peptide transporter TAP. This complex ensures the establishment of proper conformation of MHC class I molecules for peptide loading in the ER. A number of the components of the PLC have been reported in proteomics studies of ER fractions (Foster et al. 2006; Gilchrist et al. 2006). The study of Gilchrist et al. (2006) revealed higher amounts of beta 2 microglobulin, tapasin, and ERAAP in smooth microsomes compared to rough microsomes. Electron microscope immuno-gold labeling has previously revealed Class I molecules and beta 2 microglobulin in association with the post-ER/pre-Golgi network consisting of anastomosing tubulated and fenestrated membranes (Raposo et al. 1995). Whether the transitional zone of the ER, which contains the smooth ER is a major site for antigen processing and peptide loading onto the PLC remains to be confirmed. Endoplasmic reticulum proteins were previously detected in purified phagosomes using proteomics analysis (Garin et al. 2001). This observation prompted consideration that the ER maybe involved in phagocytosis and indeed electron microscope cytochemical, electron microscope immuno-gold labeling, and biochemical data were obtained to testify to this possibility (Gagnon et al. 2002). Endoplasmic reticulum involvement in phagocytosis has been confirmed independently by other laboratories (Ackerman et al. 2006; Becker et al. 2005) and the findings have led to additional proteomics studies and the important conclusions explaining antigen cross-presentation (Guermonprez et al. 2003; Houde et al. 2003). Therefore even though organelle proteomics studies may often suggest the presence of organelle contaminants using intuitive reasoning and corollary experiments as was done by Michel Desjardins and colleagues, in the case of ER proteins associated with phagosomes such studies may also provide information leading to new paradigms. Cytoskeletal proteins The ER interacts with the cytoskeleton to maintain its position within sedentary cells and to establish new intracellular positions in motile cells. The ER contains a receptor for specific interaction with microtubules (Farah et al. 2005; Klopfenstein et al. 1998) and can move along both microtubules and actin cables (Allan and Vale 1994; Kachar and Reese 1988; Terasaki and Reese 1994). Based on these reports one might expect proteomics studies of the ER to reveal the presence of associated cytoskeletal proteins. Indeed, protein components of microtubules but not of microfilaments have previously been detected in association with ER subfractions from mammalian liver (Foster et al. 2006; Gilchrist et al. 2006). In addition a number of molecular motors including protein members of the myosin, kinesin, and dynein protein families have been detected in association with these same membrane fractions. Subcellular membrane movements are directed by molecular motors including myosin, dynein, and kinesin family molecular motors (Mallik and Gross 2004). Other cytoskeletal proteins, which have previously been detected in ER fractions include filamin A and B, the actin-binding proteins profilin 1, and transgelin 2. Filamin A and B were detected in smooth ER fractions (Gilchrist et al. 2006). Filamin A has been implicated in intracellular traffic of several cell-surface receptors and was observed to bind one of these at the level of the endoplasmic reticulum (Feng et al. 2005; Liu et al. 1997). Therefore filamin A may bind yet unidentified nascent receptors in the ER in hepatocytes and promote traffic of these molecules to the cell surface. The actin-binding proteins profilin 1 and transgelin 2 were detected in the ER fractions. Higher amounts of these proteins were found in association with smooth microsomes compared with that in rough microsomes (Gilchrist et al. 2006). Profilin 1 is able to bind membrane phosphoinositides and thus affect cell signaling and has been implicated in membrane traffic (Witke 2004), thus it maybe involved in ER membrane traffic in hepatocytes. Transgelin 2 mRNA was reported overexpressed in hepatocellular carcinoma and this protein has been proposed as a potential diagnostic marker for this disease (Shi et al. 2005). The role of transgelin 2 at the level of the ER remains to be determined. Cytoskeleton-associated protein 4/CLIMP-63 was reported predominantly in rough microsomes of rat liver by the studies of Gilchrist et al. 2006. CLIMP-63 serves as a specific ER- microtubule receptor (Klopfenstein et al. 1998) and was shown to link the ER to microtubules via the neuronal microtubule-associated protein 2 (MAP-2) in the dendritic compartment of neurons (Farah et al. 2005). Interestingly, the non-neuronal microtubule-associated protein 4 (MAP-4), which has an identical microtubule-binding domain to MAP-2 was also detected in association with rough microsomes (Gilchrist et al. 2006). Whether MAP-4 and CLIMP-63 make a link between the rER and microtubules thus immobilizing rER structure in hepatocytes remains to be confirmed. Reticulons constitute a family of proteins implicated in the tubular structure of the smooth ER (Voeltz et al. 2006). Reticulon 3 was detected by mass spectrometry in smooth microsomes of rat liver by Gilchrist et al. (2006) and over-expression of this protein in Hela cells revealed production of fluorescent tubules, which co-distributed along microtubules as revealed by double-labeling immunofluorescence (Wakana et al. 2005). As proposed by Shibata et al. (2006) reticulons promote long unbranched ER tubules. Endoplasmic reticulum tubules have previously been shown to be highly dynamic structures capable of moving along tracks of microtubules (Waterman-Storer and Salmon 1998). Perhaps the tubular structure of the ER favors ER/motor/microtubule interaction? However if reticulons promote long unbranched ER tubules, how do they affect tritubular jonctions as observed in the large inter-connecting networks of tubules in sER in steroid cells or in hepatocytes? Proteins involved in signaling The ER is involved in many signaling pathways including the unfolded protein response (UPR) (Bernales et al. 2006), apoptosis (Chae et al. 2004; Nakajima et al. 2004), ubiquitination and proteasome degradation (Kostova and Wolf 2003) and some of the involved signaling molecules have been shown to associate in a transient manner with ER membranes. This includes Ras protein, which is involved in cell signaling (Sobering et al. 2004), the AAA ATPase p97, which is involved in ER assembly (Lavoie et al. 2000) and in proteasome degradation (Ye et al. 2003) and BAX, which is involved in apoptosis (Zong et al. 2003). Excluding proteins involved in ubiquitination and proteasome degradation (see above), few signaling proteins have yet to be detected in proteomics studies of ER fractions. This is true for a number of regulatory proteins such as SREBP-1, which regulates lipogenic enzymes (Bengoechea-Alonso and Ericsson 2007), Ire-1 which regulates the UPR (Bernales et al. 2006) and Bax, which is involved in apoptosis (Hetz et al. 2006). These signaling proteins probably represent low abundance proteins of the ER and require up-regulation following physiological activation to be detected by existing mass spectrometry techniques. Under conditions of accumulation of misfolded and unassembled proteins, the UPR acts to modify the ER by up-regulating chaperones so as to increase its folding capacity. Morand et al. (2005) used quantitative proteomics analysis and revealed a number of chaperones (GRP94 and PDI) to be up-regulated in ER microsomes from the liver of a fructose-fed, insulin-resistant hamster model and up-regulation was attributed to ER stress. Ras protein and p97 have been reported in association with ER fractions (Gilchrist et al. 2006; Galeva and Altermann 2002). Association of Ras with the ER is consistent with previous reports of the association of Ras with membranes of the secretory pathway including ER (Zheng et al. 2007). Association of p97 with ER is consistent with its role in ER assembly (Lavoie et al. 2000) and proteasome degradation (Ye et al. 2003). Phosphoproteins associated with the ER The ER is a membrane-bound compartment that functions by interacting with cytoplasmic and luminal-soluble proteins. Among the soluble proteins that interact with the ER membrane in a transient manner are signaling proteins. Reversible phosphorylation has been shown to control protein interaction at the ER of several signaling proteins including valosin-containing protein/p97 (Lavoie et al. 2000) BCL-2 (Lin et al. 2006) and Ire1 (Bernales et al. 2006). In addition, reversible phosphorylation has been shown to play a role in the control of ER structure. For example, phosphorylation of CLIMP-63 was shown to affect ER structure during interphase (Vedrenne et al. 2005) and phosphorylation of p47, a cofactor of p97, was shown to control ER structure at mitosis (Kano et al. 2005). Since the ER controls key metabolic events including events crucial to cell survival, it is expected that new phosphoproteins are yet to be identified, especially in relations to control of cell cycle events. A number of proteins have been detected in ER fractions, which have previously been shown to be phosphoproteins. Phosphoproteins were identified in transformed cells by analysis of tyrosine-phosphorylated peptides immunoprecipitated using anti-phosphotyrosine antibodies (Rush et al. 2005). A significant number of these previously identified tyrosine-phosphorylated proteins have in fact been detected in association with ER membranes by proteomics analysis (Foster et al. 2006; Gilchrist et al. 2006). Many of these proteins are not conventional ER proteins but rather are better known as cytosolic proteins; these include proteins involved in mRNA metabolism (PABP1, YB-1, ELAV-like 1, eEF-1-alpha), proteins involved in phospholipid binding (annexin I, annexin II), and proteins involved in de novo lipid synthesis (ATP citrate lyase). Some of these proteins are substrates of key signaling pathways including the Akt-signaling pathway (ex. ATP citrate lyase, Berwick et al. 2002) and some have been shown to have a phosphorylation status which has been associated with subcellular trafficking (Hibino et al. 2006). Thus proteins previously defined as tyrosine-phosphorylated proteins were observed in ER membranes by proteomics analysis and these may undergo cycles of tyrosine phosphorylation and dephosphorylation promoting specific ER-related functions in cells, which are yet to be better defined. Serine/threonine protein phosphorylation can also modify subcellular protein localization. Phosphorylation of the disc’s large tumor suppressor protein controls its membrane localization (Massimi et al. 2006). CLIMP-63 is serine phosphorylated during mitosis and regulates ER-microtubule binding (Vedrenne et al. 2005). Conclusions and future perspectives From available proteomics data parts of which are supported by electron microscope protein localization studies some tentative conclusions can be drawn about the relative segregation of proteins and molecular machines in the subcompartments of the ER. CLIMP-63 and ribosomes are enriched in rER. Reticulon, enzymes involved in ubiquitination, the proteasome, some cytoskeletal proteins, proteins involved in antigen processing, and coat proteins are enriched in sER. In contrast, proteins that appear equally distributed between rER and sER include proteins of the translocon, biosynthetic cargo, chaperones, proteins of detoxification, and proteins involved in lipid and glucose metabolism. Because proteomics of ER subcompartments is carried out using subcellular fractions, there are limitations that have to be considered when trying to interpret the results of the protein analysis. For example, the relative purity of the fractions will vary and proteins of fragments of contaminating organelles will be present in the fraction and be identified in the analysis. Moreover molecular dynamics at the cytosolic and luminal surfaces of the ER have to be taken into consideration. The composition of the ER is subject to change based on molecular interactions occurring on both cytosolic and luminal sides of the ER membrane. On the cytosolic side of the ER interaction with the cytoskeleton (to change the location, shape or size of the ER) or interaction with signaling proteins (to activate specific signaling pathways, e.g., apoptosis) leads to transient associations between the ER and cytosolic proteins. On the luminal side of the ER interactions between newly synthesized proteins and chaperones can vary under specific conditions (for example during ER stress) and will affect the overall molecular composition. Up-regulation of proteins involved in detoxification may occur under exposure to toxic chemicals and affect ER composition. Thus depending on the cell physiology protein associations with the ER may vary. Dynamics of protein interactions in organelles are often controlled by posttranslational modifications including phosphorylation. Understanding such modifications is key to understanding site-specific protein function. Proteomics studies of the subcompartments of the ER have lead to insights into the function of the different compartments of this organelle and new paradigms. However data obtained using proteomics analysis should be complimented by cytological techniques to confirm the localization of the proteins in the ER subcompartments and molecular biology should be used to modulate protein expression to examine the function. The combination of these approaches not only will yield new information about the proteins but they will also expand knowledge on the protein families to which they belong and/or protein complexes of which they are part of.

Matern_Child_Health_J-2-2-1592147

Treatment of Tobacco Use in Preconception Care

The preconceptional period provides an important opportunity to actively assess and treat tobacco use and to assist parents-to-be in quitting permanently to avoid smoking-related pregnancy and long-term health consequences. The use of tobacco prior to conception is associated with male impotence, conception delay, and primary as well as secondary infertility. During pregnancy, tobacco use increases the risk for spontaneous abortion, ectopic pregnancy, low birth weight, SIDS, premature birth, and other fetal and maternal complications [1]. Each year, almost one-half million babies in the United States are born to mothers who report smoking during pregnancy [2]. For smokers, tobacco use continues to be the leading cause of morbidity and mortality in the United States, resulting in approximately 440,000 deaths per year [3]. Women smokers are at increased risk for cancer, cardiovascular disease, and pulmonary disease. Since 1987, lung cancer has outpaced breast cancer as the leading cause of cancer death among women, and exposure to environmental tobacco smoke (ETS) is a cause of lung cancer and coronary heart disease among lifetime nonsmokers. Moreover, infants born to women who are exposed to ETS during pregnancy may also have increased health risks, including small decrements in birth weight and slightly increased risk for intrauterine growth retardation, compared with infants born to women who were not exposed [1]. Despite all that is known about the devastating health consequences of smoking, 20% of U.S. women smoked cigarettes in 2003 [4] and 11.4% of women giving birth reported smoking during their pregnancy [5]. It is encouraging that 70% of smokers want to quit, and about two-fifths quit for at least a day every year [4]. To avoid early pregnancy complications, women who smoke should be counseled to quit before they become pregnant. In addition, smokers who are partners of mothers-to-be should also be included in any treatment plans given the strong association with partner smoking and relapse [6] as well as concerns about ETS exposure. Treatment for tobacco dependence is safe, effective, available, and affordable [7]. The 2000 Public Health Service document, Treating Tobacco Use and Dependence: A Clinical Practice Guideline [7], as well as the 2001 Centers for Disease Control and Prevention publication, Guide to Community Preventive Services [8], both provide strong evidence-based guidelines for treating smokers using a comprehensive system that includes the 5A's (a brief 5-step counseling approach recommended by the 2000 PHS clinical practice guideline for all smokers). These documents also provide details of how reminder systems, telephone quitlines, decreased co-pays and costs of treatment, mass media campaigns, smoke free environments and increased prices for tobacco products support the cessation process [7, 8]. At the clinical level, every clinician providing preconception care should implement a tobacco treatment system based on the 5 A's and beginning with a systematic way to assess tobacco use. This evidence-based counseling approach includes asking every patient about tobacco use and, if they smoke, advising them to quit, assessing willingness to quit, assisting them in quitting, and arranging follow-up. Assisting may include the offer of pharmacotherapies or additional counseling, each of which doubles the quit rates in non-pregnant adult populations [7]. The effectiveness of counseling increases as the intensity (number and length of sessions) increases. At present, seven first-line FDA-approved medications are available: bupropion varenicline and five nicotine replacement therapies (gum, patch, lozenge, inhaler, and nasal spray) [7]. Because the safety and efficacy of pharmacotherapy during pregnancy has yet to be established, the preconception period provides an excellent interval before pregnancy to offer these effective adjuncts to quitting. Once abstinent, patients should be followed to help avoid relapse. For those smokers who are not willing to quit in the near future, the PHS clinical practice guidelines recommend the 5 R's (relevance, risks, rewards, roadblocks, repetition) to enhance patients’ motivation to quit smoking [7]. Clinical systems that provide tobacco-dependence treatment should also include a reminder system to alert providers to advise smokers in their practice [8]. This system might be an electronic medical record alert or simply a stamp or sticker on the patient's chart to indicate tobacco status - current, former, or never. The PHS guideline also recommends using the 305.1 ICD-10 code for tobacco dependence for billing purposes [7]. This code also may be used by health-care systems to monitor and improve the provision of effective treatment. State quitlines provide an effective vehicle for telephone counseling and can be accessed by calling 1-800-QUIT NOW, a national portal number to refer tobacco users directly to their state quit line based on their area code. A small number of states also provide over-the-counter medications to eligible populations in conjunction with telephone counseling. Another group of states (e.g., Maine, Massachusetts, Oklahoma, Oregon, and Wisconsin) encourages providers to fax referrals (with patient consent) to the state quitline, which in turn proactively calls the smoker to begin counseling. Most quitlines send a “quit kit” to each caller. With effective treatment, quit rates can be as high as 25%–30% in the general population [9]. Because many women spontaneously quit upon learning about their pregnancy—ranging from 11% to 28% in publicly insured pregnant smokers to 40% to 65% in privately insured pregnant smokers [10], it is reasonable to expect that effective preconception tobacco-dependence treatment could exceed these rates, particularly if partners who smoke are treated as well. Placing these cost-effective treatment systems into preconception practice should not be insurmountable, given the evidence, the affordability, the lessons learned from other clinical tobacco-dependence systems, and the availability of resources. Available Resources for learning more about tobacco dependence treatment:Treating Tobacco Use and Dependence. JAMA June, 28, 2000 (Summary article)Treating Tobacco Use and Dependence (full document). Available at www.surgeongeneral.gov/tobacco.Agency for Health Care Research and Quality, patient and provider materials. Available at www.ahrq.gov.The Guide to Community Preventive Services. Available at www.thecommunityguide.org .Telephone Quitlines: A Resource for Development, Implementation, and Evaluation. Available at www.cdc.gov/tobacco.Smoke-Free Families provider, patient, and system materials. Available at www.smokefreefamilies.org.Environmental Protection Agency, smokefree home ban materials. Available at www.epa.gov .March of Dimes at www.marchofdimes.com.Nicotine and Tobacco Research Journal supplement: Helping Pregnant Women Quit Smoking: Progress and Future Directions. Available at http://www.ntrjournal.org/pregnancy.html.

Intensive_Care_Med-3-1-1915615

Complete recovery from an unusual cause of coma

Case A 51-year-old man was admitted to the emergency room because of progressive lethargy, slurred speech, weakness of the right arm and low-grade fever. Physical examination showed no additional neurological deficits. He had a history of a tick bite 6 months previously which had been treated with antibiotics. Apart from leukocytosis (10.5 × 109/l) and raised C-reactive protein (62 mg/l), additional blood examination was normal. Cerebral contrast-enhanced computed tomography (CT) revealed no abnormalities. Cerebrospinal fluid (CSF) examination showed an elevated cell count of 66 × 106 cells/l (of which 97% were lymphocytes), slightly elevated protein concentration of 1.03 g/l, a normal glucose level of 3.7 mmol/l and an elevated pressure of 29 cmH2O. Case discussion The onset of progressive lethargy over a period of days, slurred speech and the weakness indicates a lesion of the central nervous system. Given the progression of lethargy, the fever and the absence of acute onset, an infectious origin seemed more likely. This was suggested by the CSF examination, showing an elevated cell count, especially lymphocytes. The differential diagnosis consisted of (viral) meningo-encephalitis – most likely caused by enteroviruses, arboviruses or herpesviruses – neuroborelliosis and vasculitis. Antibiotics and antiviral drugs were started. Case Within 2 days, the patient's condition deteriorated into a deep coma requiring mechanical ventilation. Initial treatment consisted of acyclovir and ceftriaxone, anticipating a possible neuroborelliosis and herpes simplex encephalitis. However, repeated blood and CSF cultures were normal, making an infective origin less likely. Serological tests for Lyme disease and repeated polymerase chain reaction (PCR) testing for herpes simplex virus (HSV) were negative. Systemic vasculitis was ruled out. The patient remained in an areactive coma without focal signs and abnormal brainstem reflexes. An electro-encephalogram (EEG) after 1 week showed non-specific diffuse slow activity. Three weeks after admission cerebral gadolinium-enhanced magnetic resonance imaging (MRI) was performed, showing numerous hyperintense lesions, on T2-weighted images, of the brainstem, the left cerebellar hemisphere, the basal ganglia on both sides and in periventricular locations (Fig. 1a–c). Fig. 1a–c T2-weighted MRI of the brain, demonstrating hyperintense lesions of a brainstem and left cerebellar peduncle, b basal ganglia and c left periventricular region. c–e T2-weighted MRI of the brain 2 months after therapy, demonstrating a decrease in white matter lesions Case discussion On ICU admission the main medical problems were (1) respiratory insufficiency requiring mechanical ventilation, (2) progressive loss of consciousness and (3) the unknown origin of the coma. Maintenance of an adequate airway and concomitant aspiration pneumonia necessitated mechanical ventilation. There are numerous possible causes of progressive loss of consciousness, including cerebrovascular accidents, cerebral infections (viral, bacterial, parasites, tuberculosis, Lyme disease), neoplastic and auto-immune diseases (sarcoidosis, vasculitis, SLE). Despite the elevated white cell count and the presence of protein in the CSF, no infectious origin was determined. Screening for HIV and coagulation disorders was negative. Repeated blood and CSF cultures were negative, and there was no response after 14 days of ceftriaxone and acyclovir. Nevertheless CSF analysis after treatment was interpreted cautiously. Many viruses responsible for meningo-encephalitis cannot be identified using common tests. MRI showed multiple hyperintense brain lesions, especially of the brainstem. Because of the massive involvement of the brainstem and persistence of coma for 2 weeks, the prognosis was considered poor. Case Acute disseminated encephalomyelitis (ADEM) could not be ruled out, so treatment was continued. Additionally, treatment with intravenous dexamethasone (4 mg four times a day) was started. Upon treatment with corticosteroids for 4 weeks the patient made a slow but full recovery within 2 months. After 2 months cerebral gadolinium-enhanced MRI was repeated, showing substantial amelioration of the intracerebral lesions (Fig. 1d–f). One year after treatment the patient still functions well, without relapse. Case discussion Elevated pressure, white cell count and protein level in the CSF in combination with the extensive white matter lesions confirmed the diagnosis of ADEM. ADEM is a rare cause of prolonged coma with complete recovery under specific treatment, and intensivists should be aware of this disease. Withdrawal or withholding of care in patients with prolonged coma can hardly be discussed as long as the cause of the coma remains unknown and the prognosis cannot be accurately established. Treatment with intravenous corticosteroids resulted in a full recovery. The absence of a previous infection is a peculiar aspect of our case. Comments This case illustrates the remarkable outcome after a long period of coma. Good recovery from a coma is observed in only 10% of reported cases and depends on the aetiology, the accompanying clinical signs, and the depth and duration of the coma [1]. ADEM is a rare monophasic illness that is thought to develop from antigenic mimicry, with antibodies having cross-reactivity to host epitopes in the nervous system. The disorder typically occurs following vaccination or a viral prodrome and is predominantly seen in children and in Japan, where viral encephalitis is more common [2, 3, 4, 5]. A preceding infection is reported in 50–75% of cases [3, 4, 5]. The estimated incidence is 0.8 per 100,000 population per year [3]. Some cases of ADEM among adults and the elderly have been reported, but the incidence is estimated to be considerably lower. The diagnosis of ADEM is reached on clinical grounds, evidence of white matter lesions on MRI and exclusion of other causes. These classic features were present in our case. However, diagnosis of ADEM may be difficult. Diagnostic overlap with multiple sclerosis (MS) may lead to underestimation of the prevalence [4, 5]. Some 0–33% of children and 35% of adults initially diagnosed with ADEM will eventually develop MS [4, 5, 6]. Therefore this diagnosis should be considered, especially in adults. Follow-up by repeated MRI, at intervals not shorter than 6 months, may anticipate this development [7]. The differential diagnosis consists of meningo-encephalitis, meningitis and cerebral involvement in auto-immune diseases [8]. In severe cases, ADEM may lead to coma. There is no standard treatment for ADEM. It is a rare disease, and no formal clinical trials of any therapeutic agent have been published. Thus the management of the disease rests on strategies that have appropriate effects on the plausible disease mechanisms. Present treatments rely on immunosuppression and immunomodulation. Intravenous corticosteroids (preferably methylprednisolone) is the first choice of treatment and usually leads to full recovery. Plasma exchange and intravenous immunoglobulin should be considered by deterioration on corticosteroids [9, 10]. In conclusion, treatment of coma should be continued when the cause of the coma is unclear, even if there are numerous lesions in the brain. ADEM should be considered in comatose patients with raised CSF pressure, protein and lymphocytes, negative CSF cultures, and multiple white matter lesions on cerebral MRI. ADEM is predominantly seen in children, but also occurs in adults. Recovery may last weeks.

J_Med_Internet_Res-4-2-1761933

Where Are They Now? A Case Study of Health-related Web Site Attrition

Background. When considering health-related Web sites, issues of quality generally focus on Web content. Little concern has been given to attrition of Web sites or the "fleeting" nature of health information on the World Wide Web. Since Web sites may be available for an uncertain period of time, a Web page may not be a sound reference. Introduction "We've all heard that a million monkeys banging on a million typewriters will eventually reproduce the entire works of Shakespeare. Now, thanks to the Internet, we know this is not true" [1]. Robert Wilensky The results of a recent major national survey found that about 110 million people in the U.S. — over half of the adult population — may be seeking health information online [2]. This compares with 54 million in 1998, 69 million in 1999 and 97 million in 2001. And according to the American Medical Association, on any given day, more people go online for medical advice than actually visit health professionals [3]. When considering health-related Web sites, issues of quality generally focus on Web content: how to find relevant information, and how to assess the credibility of the publisher as well as the accuracy and reliability of a document retrieved [4]. Little concern has been given to attrition of Web sites or the "fleeting" nature of health information on the Web. Scientist and scholar Sir Isaac Newton once said, "If I have seen farther than others, it is because I was standing on the shoulders of giants" [5]. Much, if not all, scholarship is based on relation to previous work, and when new scholarly work is produced, it is important that detailed and accurate information on sources consulted are cited. To facilitate referencing, scholarly works have been routinely collected and preserved in print by libraries and database producers [6,7]. But in terms of cataloging, storage and retrieval as it relates to the Web, the status quo does not apply. With the advent of the Web, libraries must now consider Web site information that may be created, change, move, expire and disappear; with no record of the information being preserved. Few libraries made the practice of collecting copies of Web pages [8]. Since Web sites may be available for an uncertain period of time, a Web page may not be a sound reference. If a Web page or link disappears, chances are almost nonexistent of locating the reference at a later time. As a safeguard, it has been recommended that individuals keep a personal copy of Web pages as evidence that the information existed [9]. To address the issue of attrition, a defined set of health-related Web sites was examined at two separate time intervals. Methods In an earlier study, a systematic survey was conducted to determine the validity of health claims on the World Wide Web for the herbal remedy Opuntia [10]. From December 1998 to May 1999, 184 Web sites were collected from which health claims were identified. Web sites were retrieved utilizing multiple search engines, and the Uniform Resource Locator (URL) for each Web site was recorded. In this study, to determine the degree of attrition, each of the 184 Web sites obtained and recorded from the previous study were revisited at a later period of time. During May 2002, the previously recorded URL for each Web site was entered into the address field of the browser Netscape Navigator (version 4.7, Netscape Communication Corporation, Mountain View, California.) It was documented whether the original Web site could not be found, moved to a different URL location, or the URL and site location was found unchanged from the original search. For A Web site whose URL remained unchanged, it was also noted whether the Web site had maintained currency, (i.e. updated) since the original posting. Since it is conceivable that inaccessibility of Web sites may be due to temporary server problems, another attempt was made to access the sites at different periods of time. For each "HTTP Error 404" or similar message obtained from the initial URL checks, an attempt to access these sites was made during June 2002 on various days and times of day in the manner described above. Results Results indicate that when each URL address from the original set of 184 Web sites was re-entered into the address field of the browser, 108 (59%) of the sites could not be found, 31 (17%) had moved to a new URL address, and 45 (24%) of the sites could be found from the original URLs obtained in the previous study. Of the Web sites that moved to a new URL address, only 7 sites provided a link from the original URL to redirect the viewer to the new location. Of the Web sites still in existence, 17 (38%) provided update information from the original posting. The information is summarized in Table 1. Table 1 Attrition of Health-related Web Sites for a Three-year Period ** Web Site Sponsor (No. of Sites) Not Found Moved To New URL URL Redirected URL to Site as Original Maintenance Update Provided Herbal Vendor (74) 46 14 1 14 7 Food/Recipes Products (7) 5 1 0 1 0 Educational Institution(24) 12 1 0 11 6 Government Institution (3) 1 2 1 0 1 Historical Essay (8) 1 1 1 6 0 Travel and Tourism (5) 1 2 0 2 1 Message Board (15) 15 0 0 0 0 Reference Guide (16) 8 6 3 2 1 Print Media* (24) 17 2 1 5 1 Expert (7) 2 2 0 3 0 Doomsday Group (1) 0 0 0 1 0 Totals (184) 108 (59%) 31(17%) 7(4%) 45 (24%) 17 (38%) * Includes book excerpts, newspaper and magazine articles, newsletters, a calendar reprint and a radio broadcast transcript ** Original Web site addresses and content are available on the World Wide Web at http://ismo.ama.ttuhsc.edu/users/~veronin/WebOpuntia.pdf In this study, attrition is defined as the unavailability of a Web site when known to be previously accessible based on a known URL address. This did not include sites that were redirected to a new URL. Approximately three years after initial posting, over two-thirds of the health-related Web sites reviewed could not be found or had moved with no forwarding URL, and about one-third of the remaining sites maintained currency of information. It appears that links are terminated as Web sites are moved or removed, or as servers close down. This supports the notion that it is difficult, if not impossible, to locate information that was previously found on the Web, and if a reference to an item is provided, there is no guarantee that viewers will be able to find the site at a later date. In this study, a comprehensive data set of Web sites on a specific health-related topic was obtained, and attrition was examined. Obviously an example from a single health-related topic is limited in what conclusions should be drawn. These findings cannot be generalized to other medical topics. But this raises the question that other health-related sites on the World Wide Web may vary in their degree of attrition, and warrants further research into methods of dealing with attrition with other medical topics. Discussion The average life of a Web page is about 77 days [11]. The perceived value of the Web lies in the immediate accessibility to a seemingly endless pool of information with no central controlling authority. This also makes the Web difficult to maintain. According to Chris Sherman, Associate Editor of SearchEngineWatch.com, (http://searchenginewatch.com),as automatic maintenance, most search engines remove missing URLs from their index when they recrawl and find that the pages are gone [12]. A different problem arises, though, when an organization has gone out of business but its site still exists. This is a much more difficult problem to handle, and to date, no search engine exists to locate or remove these sites. Enhancements in Web technologies hope to improve the problem of attrition. A prime example is the Internet Archive. The Internet Archive The Internet Archive (http://www.archive.org) is a digitallibrary of Web pages created with the lofty goal of cataloging all of the past and present publicly available material on the World Wide Web [11]. Accessible to the public for free, it contains more than 100 terabytes of data and is growing by 10 to 12 terabytes a month. Since 1996, the Internet Archive has been storing Web pages, including graphics files, from publicly accessible Web sites. A feature implemented October 2001 known as the "Wayback Machine" allows users to go back and view earlier versions of current Web sites or of Web sites that no longer exist. The Wayback Machine serves as a source to find Web pages when the page or host cannot be located [11]. When a user encounters a "File Not Found" or similar message on the Web, the Wayback Machine can be accessed to find a facsimile of the Web page. Though a significant accomplishment towards recovering lost Web pages, the Wayback Machine has limitations. It is not searchable by keywords or text in the manner of a general search engine. The user must know the precise URL of a particular Web page or site to access the Archive. Having entered a URL address, the viewer is presented with a list of dates that designates when a particular page was archived. Also, though the Internet Archive contains more than 100 terabytes of data, much is still missing. For example, it does not contain the older gopher content and other non-Web files prior to 1996, and a relatively small number of pages exists from 1996, with content increasing to recent times. Issues of Quality and Content The question may arise as to whether a relationship exists between Web site quality and attrition. Are poor quality sites more likely to disappear in time than sites of higher quality? A consensus has yet to be reached as to the properties a Web site needs to have to be considered "high quality." Wilson states that "quality remains an inherently subjective assessment, which depends on the type of information needed, the type of information searched for, and the particular qualities and prejudices of the consumer" [13]. Yet many organizations and individuals have identified standards of quality that should be applied to the Web [14]. A practical approach for assessment has been described by Risk that provides benchmarks of quality [15]. It includes assessing a site for information that is accurate, current, has a clear source, is referenced, has disclaimers and cautions if appropriate, clear, clean and pleasing design features and a well-defined purpose. These criteria were applied to the original sites in this study by this author to examine whether attrition may be influenced by quality. If a site possessed at least 5 of these attributes, it was considered "high" quality, 3 to 4 attributes, it was considered "moderate," and 2 or less it was considered "poor" quality. The results are summarized in Table 2. It appears that although the high quality sites make up only a small portion of the total number of sites retrieved (15%), half of the original high quality sites (14 of 28) could be located from the original URL or were redirected to a new URL. Conversely, only 10 of the 73 poor quality sites were accessible from the original URL entry, and only one poor quality site was redirected to another URL from the original site. This suggests that Web sites of higher quality may be less subject to attrition than those of poorer quality, and warrants further research on the relationship between Web site quality and attrition with other medical topics. Considering subject matter and attrition, it may be that certain topics (such as herbal remedies) can have periods of enthusiasm by the public then wane — which may be the case with these sites. Perhaps information on more mainstream topics (such as health risks and smoking) is less vulnerable to attrition. Table 2 Quality of Health-related Web Sites and Attrition Web Site Quality* (No. of Sites) Not Found Moved To New URL URL Redirected URL to Site as Original High (28) 10 7 3 11 Moderate (83) 38 21 3 24 Poor (73) 60 3 1 10 Totals (184) 108 (59%) 31 (17%) 7(4%) 45 (24%) * Quality assessed by author based on attributes described by Risk [15]: High = 5 or more, Moderate = 3 to 4, Poor = 2 or less Future Considerations It has yet to be determined with certainty the forces that influence the survival of Web sites. With the complex and dynamic nature of information flow on the Web, is there a form of "natural selection" at work in health Web site survival? If attrition is not related to the site's quality or subject matter, perhaps those with strong commercial backing may survive with greatest frequency. At this point we can only speculate what will endure. In some instances, Web site attrition may be desirable. A common complaint against search engines is that they return too many pages, and that many of the pages have low relevance to the query [16]. The most efficient search engines index only a fraction of the total number of documents on the Web, [17] and if sites of poorer quality go away, ideally this should help retrieval of documents of higher relevance to the user. Most quality issues with the Web focus on consumers, [18] however, a recent major poll revealed that physicians are using the Internet to increase their medical knowledge and improve the care they provide to patients [19]. Medical information can change rapidly with continuing breakthroughs and advances in medical knowledge. Availability of information through the Web would facilitate access to the most up-to-date information on current medical topics and scientific discoveries. Future research that is directed toward making sure Web site viewers always know the site will be accessible at a later time will enhance the Web as a valuable medical information resource.

J_Behav_Health_Serv_Res-4-1-2214828

Healthcare Utilization of Individuals with Opiate Use Disorders: An Analysis of Integrated Medicaid and State Mental Health/Substance Abuse Agency Data

Data from the Substance Abuse and Mental Health Services Administration’s Integrated Database (IDB) were used to examine the service use patterns of individuals with possible opiate use disorders in Washington State. Results indicate that regardless of Medicaid enrollment status, individuals who received mental health (MH) or substance abuse (SA) services only through state agencies received no inpatient substance abuse service. Furthermore, when compared with individuals who received at least one MH/SA service through Medicaid, those who received services only through the state agencies were less likely to have received any MH services and were more likely to have received residential SA services. This analysis highlights the importance of using integrated client data in providing a more comprehensive understanding of services to inform policy and raises significant questions about how regulatory requirements affecting different funding mechanisms might drive settings of care in ways not related to the care needed. Introduction The use of, abuse of, and dependence on opiates is a major public health concern.1,2 Although only about 0.1% of the U.S. population reported past-year heroin use in 1998,3 heroin and other opiate use accounted for about 30% of total spending for illicit drug use treatment and almost 18% of spending on drug-related crime.4 Psychological and physical health problems were also common among heroin and other opiate users in the mid- to late-1990s.5 As recently as 2003, heroin accounted for 23% of the mentions of substances used among emergency room patients and 41% of drug-related deaths recorded by medical examiners or coroners.6 The social costs of opiate abuse are not limited to illegal drugs. A recent study suggests that in 2001, the social cost of prescription opioid abuse in the United States was $8.6 billion.7 Despite the overwhelming societal costs associated with opiate use disorders, few studies have investigated the substance abuse (SA) or mental health (MH) treatment service patterns of individuals suffering from opiate abuse or dependence; rather, most studies focus almost exclusively on those in methadone maintenance treatment.8–10 These studies suggest that the co-occurrence of MH problems and other illicit drug dependence is quite high among individuals with an opiate use disorder.11–13 Yet, despite the increased incidence of MH conditions, individuals with a substance use disorder may not receive adequate levels of MH care beyond that directly related to their substance use disorder.10,11,14,15 Given these findings and the major public health concerns caused by opiate abuse and dependence, the lack of information about the broader MH/SA treatment service patterns of individuals with an opiate use disorder is a critical gap in the knowledge base informing policies affecting this population. This study used data from the Substance Abuse and Mental Health Services Administration’s (SAMHSA) Integrated Database (IDB) on individuals in Washington State with an indication of an opiate use disorder to determine what MH/SA services they received and through which auspice (i.e., Medicaid or state agency) they received them. Findings from this study offer two contributions to the current literature. First, the IDB presents a unique opportunity to study the behavioral health care utilization of individuals with opiate use disorders because it contains service use data linked at the client level from Washington Medicaid and from the Washington State Department of Social and Health Services’ (DSHS) Mental Health Division (MHD) and Division of Alcohol and Substance Abuse (DASA). The combination of Medicaid and DSHS data represented in the IDB provides a more comprehensive picture of service use patterns than might be obtained from studies that focus on only one data source. Second, the late 1990s is often characterized as a time of a heroin epidemic in the United States.16 Therefore, examining the MH/SA treatment service use patterns of individuals with opiate use disorders during this period can provide an especially relevant baseline for today’s policy makers as they attempt to address issues surrounding the emergence of new opiates. Data and Methods This paper uses IDB data on Washington State from three full calendar years (1996–1998). For a more detailed description of the IDB, see Coffey et al.,17 and for a detailed description of the methods used to link IDB service records across state organizations, see Whalen et al.18 In addition to service use information, the IDB contains information on patient demographics (e.g., age, gender, race/ethnicity), Medicaid enrollment status, MH/SA diagnosis and service codes, and limited provider information. Study population The study population for this analysis consists of individuals who have at least one record in the IDB indicating an opiate use disorder. An opiate use disorder could be indicated by one or more of the following: a diagnosis (either primary or secondary) of an opiate use disorder, a provider type indicative of opiate treatment, a service or procedure code indicative of opiate treatment (including methadone maintenance therapy but excluding methadone used for pain management), or a report of an opioid as the drug of choice. Opiate use disorder diagnoses were defined using the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM). ICD-9-CM codes of 304.0 or 305.5, including any subclassifications, were used to identify opiate dependence and abuse, respectively. State-specific provider codes were used to identify opiate treatment providers. Both standard [e.g., Healthcare Common Procedure Coding System (HCPCS)] and local procedure codes were used to identify opiate treatment services. Finally, drug-of-choice information was available from self-reports obtained by state agencies during client intake. In some cases, the drug-of-choice information was obtained more than 1 year before or after any SA or MH service provision, but these cases represent less than 3% of the sample. Preliminary analyses revealed that very few agency service records include diagnosis codes, but most Medicaid records do. Further investigation revealed that this is not an issue of Medicaid requiring a diagnosis for eligibility, but rather an issue of the data systems themselves. Agency services do not require a diagnosis code for reimbursement, so agency data systems do not track diagnosis. Conversely, Medicaid services often require a diagnosis for reimbursement, so Medicaid data systems track diagnosis. Because the presence or absence of a diagnosis is almost completely confounded by the use or nonuse of Medicaid services, information on service use differences associated with an opiate use disorder diagnosis is not presented. Client classification One of the primary objectives of this study was to examine the extent to which the MH/SA service use patterns of individuals with an opiate use disorder varied based on whether their MH/SA services were tracked by Medicaid or a state MH/SA agency. Importantly, service tracking may, or may not, be associated with full or partial financial coverage. Accordingly, for this study, individuals were classified based on the data source (Medicaid or state MH/SA agency) from which their IDB MH/SA service records were obtained. Based on this information, individuals were classified into three categories: Any Medicaid service: individuals who have at least one Medicaid MH/SA service record, regardless of whether they have MH/SA state agency service recordsAgency services only with Medicaid enrollment: individuals who have at least one MH/SA state agency service record and were Medicaid-enrolled at some point between their first and last observed MH/SA service record but have no record of receiving an MH/SA service through MedicaidAgency services only without Medicaid enrollment: individuals who have at least one MH/SA state agency service record and were not Medicaid-enrolled at any time between their first and last observed MH/SA service record Because the IDB integrates information from state Medicaid and state MH/SA agency data sources, overlapping records may occur if Medicaid reimburses a bill, but the state agency provides the service. To avoid overstating utilization rates, only one service date was counted for cases in which the same client, service type (MH or SA), modality/setting of service [i.e., inpatient (IP), residential/long-term care, or outpatient (OP)], and service date were reported on both the Medicaid and state agency databases. Individuals with these types of records were classified as any Medicaid service. Importantly, although individuals in the any Medicaid service group may have received any number, or even the majority, of their services through the state agency, preliminary analyses indicated that individuals who received both Medicaid and agency services were more similar to those who received only Medicaid services than they were to those who received only agency services. For this reason, individuals receiving any Medicaid service were combined into a single category. In addition to the client categorization described above, the standard IDB client classification was also used. The IDB client classification was used to identify individuals who received services for only MH conditions (MH-only), only SA conditions (SA-only), or co-occurring conditions (both MH and SA) during the study period. The IDB classifies individuals as having co-occurring conditions if they had any of the following within the 3-year study period: (1) both a primary MH and a SA diagnosis, (2) a primary MH and a secondary SA diagnosis, or (3) a primary SA and a secondary MH diagnosis. Individuals classified as having co-occurring conditions did not necessarily have MH and SA conditions concurrently. A client with an MH record at the beginning of the study period and an SA record at the end of the study period, for example, is classified by the IDB as having co-occurring conditions. In the absence of diagnosis information, MH-only and SA-only classifications were assigned based on the type of service received during the study period (see Coffey et al.17 and Bray et al.19 for a detailed definition of the primary MH/SA diagnosis category). The IDB client classification does not incorporate information on secondary diagnoses unless a primary MH/SA diagnosis is also present, nor does it incorporate drug-of-choice information from state agency intake records. Because both of these pieces of information were used to identify individuals with an opiate use disorder for this study, it is possible for individuals with an opiate use disorder to be classified as MH-only or having received no MH/SA service based on the IDB client classification. Service classification Standard IDB service type classifications were used to classify the MH/SA services received by individuals with an opiate use disorder. The IDB MH/SA service type classifies MH/SA service records as either MH or SA and within MH/SA as IP, residential, or OP. Many inpatient programs are part of psychiatric or general hospitals and generally use a medical model of substance disorders in which intensive medication and counseling are provided over a relatively short period of time.20 Residential programs typically are provided in a free-standing, designated residential treatment facility. Residential treatment is usually of longer duration than IP treatment and relies less on medical professionals. Residential treatment provides organized services by designated treatment personnel who provide a planned regimen of care in a 24-h setting and is intended to serve clients who need a safe and stable living environment to develop sufficient recovery skills.21 For a complete description of the criteria used to classify services, see Coffey et al.17 The vast majority of individuals with a possible opiate use disorder have at least 1 SA service record, but relatively few have MH service records; therefore, the IP, residential, and OP subclassifications were examined for SA service records but not for MH service records. Service encounters An individual’s total number of service encounter dates was defined as the count of unique dates within the MH/SA service window on which the individual had a record with at least one service of a given service category (MH, IP SA, residential SA, or OP SA). Within a single IP or residential stay, each daily service encounter date was counted separately. Using standard IDB definitions for the full SA population, this same information is presented for the broader SA population, excluding individuals with an indication of an opiate use disorder. Methods To characterize the level of contact individuals with an opiate use disorder have with the public treatment system, the analysis examined four key domains: (1) the proportion of clients using services, (2) the median length of the service window (i.e., the length of time between an individual’s first and last MH/SA service), (3) the number of days of Medicaid enrollment within the service window, and (4) the number of unique MH/SA encounter dates within the service window. Regression analyses were conducted to assess whether the service use patterns of individuals with an opiate use disorder differed significantly across the client classification categories after controlling for differences in the length of the MH/SA service window, the length of Medicaid enrollment, and demographics across the client categories. First, logistic regression models of the following form were estimated: where SERVi is a series of indicators for receipt of services of a given type. For the regression analyses, any MH services and OP and residential SA services were considered. Regression analyses for IP SA services were not conducted because those services were received solely by the any Medicaid service group. DEMOGi is a set of demographic characteristics (i.e., gender, age, and race/ethnicity); GROUPi is a vector of variables representing the data source categories of any Medicaid service (the referent), agency services only with Medicaid enrollment, and agency services only without Medicaid enrollment; WINDOWi is the MH/SA service window length in days; and ENROLLi is the months of Medicaid enrollment (including 0 months). The βs are vectors of coefficients to be estimated. Variables reflecting the primary diagnosis categories are not included because much of the information used to classify individuals as MH-only, SA-only, or co-occurring was also used to classify services as MH or SA. Next, regressions of the following form were run on days of service, conditional on service use: where DAYSi is a set of variables reflecting the unique days of care for each of the same types of care, and all other terms are as previously defined. Consistent with the recommendations of Manning and Mullahy,22 generalized linear model (GLM) estimation with a log link and a gamma distribution on the natural scale was used rather than ordinary least squares (OLS) on the log scale. Results Table 1 presents the demographic characteristics of the individuals identified as having an opiate use disorder in the IDB. For a point of comparison, Table 1 also presents the same information for the general IDB SA population, as defined in Coffey et al.,17 but excluding individuals with an opiate use disorder. Individuals with an opiate use disorder accounted for approximately 16% of the population with any SA disorder. In terms of demographics, when compared with individuals with a non-opiate SA disorder, individuals with an opiate use disorder are less likely to be male and more likely to be adults (as opposed to youth or elderly), but only minor differences in the distribution of clients across racial/ethnic categories are observed. Individuals with an opiate use disorder are less likely to be in the SA-only IDB client classification category. This is largely by construction because as discussed earlier, the current paper uses more inclusive data to identify individuals with a possible opiate use disorder than is used by the IDB client classification. Individuals with an opiate use disorder were more likely to have IP and residential SA services than the general SA population, but less likely to have any OP SA service. Individuals with an opiate use disorder were also more likely to have received any MH service compared to the general SA population. They also had longer MH/SA service windows, indicating longer periods of contact with the public MH/SA treatment system, and were more likely to be in the any Medicaid service and the agency services only with Medicaid enrollment groups. Table 1Characteristics of individuals with a possible opiate use disorder compared with the non-opiate SA populationPopulation characteristicNon-opiate SA populationOpiate usersAll users (N)83,79315,652Percentage of total SA population (%)84.315.7Gender (%) Male63.256.8 Female36.843.2 Unknown0.00.0Age (%) Youth (0–17)19.93.1 Adult (18–64)78.896.5 Elderly (65+)1.30.4 Unknown0.00.0Race/ethnicity (%) White70.077.6 Black9.010.6 Hispanic8.54.3 Native American7.84.5 Other3.62.6 Unknown1.00.3IDB client category (%) SA-only68.859.9 Co-occurring (MH+SA)31.234.7 MH-only0.01.9 Neither MH nor SA0.03.4MH/SA service type (%) SA services (%)  Any setting94.994.6  Inpatient3.06.2  Residential31.957.7  Outpatient80.971.4  No SA services5.15.4  Any MH service31.236.5 MH/SA service window length  25th percentile (days)38.078.0  Median (days)168.0348.0  75th percentile (days)446.0770.0 MH/SA service data source (%)  Any Medicaid46.051.0  Agency services only with Medicaid enrollment17.322.7  Agency services only without Medicaid enrollment36.826.3SA Substance abuse, MH mental health Figure 1 presents the median MH/SA service window and length of Medicaid enrollment across individuals with a possible opiate use disorder in each of the data source categories. The any Medicaid service group has the longest median MH/SA service window and the most median days of Medicaid enrollment within that window, followed by the agency services only with Medicaid enrollment group and then the agency services only without Medicaid enrollment group. Because the analysis only examines Medicaid enrollment within the MH/SA service window, the length of Medicaid enrollment is always less than the total service window length. The total Medicaid enrollment of an individual may be greater than that reported here, but by definition of the service window, no MH/SA service use occurred during days of Medicaid enrollment not captured by this measure. Importantly, the any Medicaid service group may have received substantial services from MH or SA state agencies, because individuals in this group are categorized as any Medicaid service only because they received at least one Medicaid service. Figure 1Median MH/SA service window length and days of medicaid enrollment among individuals with a possible opiate use disorder by data source category Figures 2 and 3 present the probability of service use and the median days of service conditional on service utilization for each of the data source categories. IP SA care was not included in Figures 2 and 3 because only individuals in the any Medicaid service group had IP SA utilization (the conditional median days of IP SA care for that group were 6). Figure 2 suggests that the two agency services only groups are more likely to receive residential SA services and are less likely to receive OP SA services or MH services. Conditional on receipt of services, Figure 3 suggests that the agency services only with Medicaid enrollment group received the most days of residential SA service, the agency services only without Medicaid enrollment group received the most days of OP SA care, and the any Medicaid services group received the most days of MH care. Figure 2Percentage of individuals with a possible opiate use disorder using MH/SA services by service type and data source categoryFigure 3Median days of care conditional on service use among individuals with a possible opiate use disorder by service type and data source category Table 2 presents results from the logistic regressions, including logit coefficients, their standard errors, and associated odds ratios (ORs) for the data source grouping variables. For all other covariates, only the estimated logit coefficients and their standard errors are presented. Table 2 shows that even after controlling for the longer service window and more months of Medicaid enrollment, the pattern observed in Figure 2 still holds. Compared with individuals in the any Medicaid service group, individuals in the agency services only with Medicaid enrollment and the agency services only without Medicaid enrollment groups had significantly higher odds of receiving at least residential SA service (OR = 2.941 and OR = 1.376, respectively) and lower odds of receiving any OP SA (OR = 0.311 and OR = 0.162, respectively) or any MH service (OR = 0.217 and OR = 0.110, respectively). Briefly examining the results for the control variables in the regression, females are significantly less likely to receive residential SA services. The racial/ethnic categories that are statistically significant suggest that racial/ethnic minorities are less likely than non-Hispanic whites to receive services of any type. Age categories reflecting individuals both younger and older than the referent of 36 to 40 were also, in general, less likely to receive services of any type when the associated coefficients were statistically significant. The exception is that individuals aged 21 to 35 with an opiate use disorder were more likely to receive residential SA services. Results with regard to demographic characteristics, especially race/ethnicity, should not be interpreted as evidence of disparities, however. Rather, they indicate the differential representation of demographic groups across the data and highlight the importance of controlling for those factors when assessing statistical significance. Unexpectedly, months of Medicaid enrollment are positively associated with a greater likelihood of service use only for MH services. For both types of SA care examined, longer Medicaid enrollment was negatively associated with the probability of service use, although the relationship was not significant for residential care. Table 2Logistic regression results for the probability of service useParametersProbability of residential SA service useProbability of OP SA service useProbability of any MH service useAgency services only with Medicaid enrollment1.079***−1.167***−1.526***Standard error0.0500.0610.052Odds ratio2.9410.3110.217Agency services only without Medicaid enrollment0.319***−1.819***−2.210***Standard error0.0520.0690.072Odds ratio1.3760.1620.110Control variablesIntercept0.446***1.091−0.274***Standard error0.0590.0780.065Female−0.519***0.344−0.064Standard error0.0360.0460.042Race/ethnicity (referent is non-Hispanic White)Black−0.269***0.371−0.493***Standard error0.0570.0750.069Hispanic−0.0810.036−0.402***Standard error0.0860.0970.116Native American0.342***−0.067−0.221**Standard error0.0870.1010.101Other−0.357***0.229−0.169Standard error0.1100.1450.128Unknown−1.656***−1.122**0.863*Standard error0.5070.4410.459Age (referent is 36 to 40)0 to 17−0.384***0.765−0.058Standard error0.1030.1460.12418 to 20−0.032−0.037−0.126Standard error0.1120.1320.13421 to 250.361***−0.0980.097Standard error0.0750.0860.08526 to 300.279***−0.0600.027Standard error0.0630.0760.07231 to 350.175***0.0340.093Standard error0.0580.0720.06641 to 45−0.205***−0.047−0.116*Standard error0.0550.0690.06546 to 50−0.465***−0.010−0.268***Standard error0.0650.0820.07851 to 55−0.537***0.001−0.425***Standard error0.0990.1290.12156 to 60−1.096***0.443**−0.718***Standard error0.1590.2200.18361 to 64−0.656***−0.713**−0.729**Standard error0.2410.2970.28865 plus−1.417***−0.285−0.675**Standard error0.3010.3440.322Service window length (days)0.0000.0030.001***Standard error0.00010.00010.0001Medicaid enrollment (months)−0.005−0.017**0.053***Standard error0.0040.0070.005IP Inpatient, SA substance abuse, OP outpatient, MH mental health*p < 0.10**p < 0.05***p < 0.01 Table 3 presents the regression results for days of care, conditional on having at least 1 day of service of that type of care. Because the natural logarithm of days of care is the dependent variable, the coefficient estimates, standard errors, and associated percentage changes for the data source categories are presented. For all other variables, only the estimated coefficients and their standard errors are presented. For residential SA care, the agency services only with Medicaid enrollment group is significantly associated with more days of residential SA care (28% increase), while the agency services only without Medicaid enrollment group is significantly associated with fewer days of care (56% decrease). Both agency services only groups are significantly and positively related to OP SA days of care: an estimated 18% increase for the agency services only with Medicaid enrollment group and an estimated 60% increase for the agency services only without Medicaid enrollment group. Finally, the agency services only without Medicaid enrollment group is associated with significantly more days of MH care than the any Medicaid service group (56% increase). Unlike in Table 2, no consistent pattern emerges with regard to the demographic control variables included in the linear regression. The length of the MH/SA service window is positively and significantly associated with days of care. Months of Medicaid enrollment are negatively and significantly associated with days of residential and OP SA care, but positively and significantly associated with days of MH care. Table 3Linear Regression Results for Unique Health Care Service Dates ParametersUnique dates of residential SA serviceUnique dates of OP SA serviceUnique dates of MH serviceAgency services only with Medicaid enrollment0.248***0.169***−0.009Standard error0.0300.0450.063Percentage change0.2820.185−0.009Agency services only without Medicaid enrollment−0.818***0.472***0.448***Standard error0.0380.0770.094Percentage change−0.5590.6040.564Control variablesIntercept3.310***2.552***2.337***Standard error0.0420.0650.067Female−0.0170.322***−0.045Standard error0.0250.0390.041Race/ethnicity (referent is non-Hispanic White)Black−0.0070.121**−0.010Standard error0.0400.0610.074Hispanic0.208***0.013−0.153Standard error0.0570.1060.133Native American0.254***−0.039−0.700***Standard error0.0520.0840.106Other−0.057−0.0160.211*Standard error0.0790.1230.128Unknown−1.268***0.000−1.340***Standard error0.4830.0000.443Age (referent is 36 to 40)0 to 170.549***0.374***0.224*Standard error0.0760.1120.12318 to 200.193***0.1350.366**Standard error0.0740.1230.14321 to 25−0.0280.188***0.062Standard error0.0450.0720.08526 to 300.0030.174***0.071Standard error0.0390.0610.07131 to 35−0.0260.0390.040Standard error0.0370.0570.06541 to 45−0.0310.137**0.115*Standard error0.0370.0580.06446 to 500.0370.1150.309***Standard error0.0460.0750.07851 to 550.0400.317***−0.080Standard error0.0730.1220.12356 to 60−0.229*0.264−0.037Standard error0.1340.2100.19661 to 64−0.618***0.283−0.093Standard error0.1900.3570.30865 plus0.701***−0.7000.116Standard error0.2711.3210.353Service window length (days)0.001***0.003***0.002***Standard error0.0000.0000.000Medicaid enrollment (months)−0.019***−0.024***0.013***Standard error0.0030.0040.003IP Inpatient, SA substance abuse, OP outpatient, MH mental health*p < 0.10**p < 0.05***p < 0.01 Implications for Behavioral Health This analysis examined the service use patterns of individuals with an indication of an opiate use disorder using IDB data from Washington for the period 1996 through 1998. Among individuals with opiate use disorders, the receipt of at least one MH/SA service through Medicaid appears to be positively associated with IP SA service use in that only individuals who met this condition had any record of an IP SA service. The receipt of at least one MH/SA service through Medicaid was also positively associated with OP SA service and with MH service use among individuals with opiate use disorders. The use of only state MH/SA agency services, on the other hand, was positively associated with the use of residential SA services. The findings suggest that the regulatory restrictions faced by state agencies and Medicaid may drive observed patterns of care for individuals with opiate use disorders. Specifically, the finding that agency-only MH/SA service use is associated with higher rates of residential service use and with no IP SA service use is likely tied to regulatory differences between Medicaid and the state agencies in Washington. For example, SA block grant funds, which are a key source of funding for both DASA and MHD, could not be used for IP hospital care during the period covered by this analysis. This restriction most likely induced state agencies to route patients for whom outpatient care is insufficient to residential care. Similarly, the Medicaid Institutions of Mental Disorders exclusion prohibits payment for psychiatric services received by adults in residential care facilities with more than 16 beds and so may induce providers to route more severe patients to IP care rather than to residential care. This explanation suggests that using administrative data to track service use patterns may be misleading because the collection of service data is driven by regulatory environments and billing systems that may not capture the actual intensity of care given. The results are subject to several limitations. First, although the IDB represents one of the most comprehensive cross-system databases used to examine this critical issue to date, it does not capture all possible services that could be used by individuals with possible opiate use disorders in Washington. Other possible sources include self-pay, private insurance, and the Department of Veterans Affairs (VA) system, among others. Another important limitation common to all administrative databases is that the actual treatment need of the population studied is unknown, so definitive statements cannot be made about the appropriateness of the services received. Furthermore, because of the limited time frame of the IDB, data are unavailable for individuals who used MH/SA services either before 1996 or after 1998. As a result, it is possible that this study has not captured the full service use history of some individuals who appear in the treatment system briefly at the beginning or end of the study period. A final limitation of this study is that information on prescription drug use is not available in the agency service records contained in the IDB and is therefore not considered in this study. Despite these limitations, the analysis of service use patterns of individuals with possible opiate use disorders during the late 1990s offers key implications for today’s policy makers as they attempt to address issues surrounding the emergence of new opiates. First and foremost, if policy makers are trying to track the service use of a small but important segment of the overall SA population, using integrated data is a necessity. When funding for treatment services is cut in an effort to contain costs, it is important to determine if those services have simply been shifted to other state programs, reflecting little net cost savings for states overall. The present study combines state MH/SA agency data with state Medicaid data, but including additional data sources (e.g., VA or criminal justice system) would provide an even more complete picture of service use patterns. Second, the utility of administrative data for analyses such as these is often limited by the influence of the regulatory environment, clinical practice patterns, and the institutional history of the data systems used. Given that state and federal policy makers increasingly rely on administrative data to assess the performance of the treatment system, the results clearly highlight the need to better track service provision. SAMHSA’s IDB, therefore, represents the vanguard of a new, expansive, cross-agency philosophy regarding administrative data sources and serves as a model for new and more comprehensive data integration efforts.

Virchows_Arch-3-1-2063564

Pancreatic intraductal papillary-mucinous neoplasms: a new and evolving entity

For a long time, intraductal tumors of the pancreas were neglected because they were misdiagnosed as mucinous cystadenocarcinoma, ordinary ductal adenocarcinoma, or chronic pancreatitis. Only in recent years have they been recognized as clinical and pathological entities. Most common are the intraductal papillary-mucinous neoplasms. Although they show an adenoma-carcinoma sequence, they have proved to have a more favorable prognosis than ductal adenocarcinoma, when resected in a preinvasive state. Recently, it has become clear that they constitute a heterogeneous group with at least four subtypes. Their stratification reveals that the various intraductal papillary-mucinous neoplasm subtypes have different biological properties with different prognostic implications. Historical notes and a rising incidence The pancreatic tumors that are characterized by an intraductal origin and growth pattern include intraductal papillary-mucinous neoplasms [3, 24, 41], intraductal tubular carcinomas [22, 45], intraductal tubular adenomas of the pyloric type [6], and intraductal acinar cell carcinomas [15]. Most common are the intraductal papillary-mucinous neoplasms (IPMNs). They are mucin-producing epithelial tumors that usually show a papillary architecture and are associated with dilatation of the ducts. Before 1990, these tumors were thought to be rare. They first started to be recognized approximately 20 years ago [31, 36, 39], when the many different names given to the tumor in the 1980s were replaced by the term IPMN [41]. This name was also introduced in the classification of exocrine pancreatic tumors propagated by the World Health Organization (WHO) [24] and the fascicles of the Armed Forces Institute of Pathology [44]. Since then, IPMNs have been reported with increasing frequency (Table 1) and currently account for about 7% of clinically diagnosed pancreatic neoplasms and up to 16% of surgically resected pancreatic neoplasms and for almost 50% of pancreatic cysts found incidentally [8, 17]. When only the cystic tumors of the pancreas are considered, IPMNs take first place, with a frequency of 24% [26]. Table 1Incidence data on intraductal papillary-mucinous neoplasmsAuthorPeriod 1Number of incidencePeriod 2Number of incidenceSohn et al. [43]1987–2001582001–200378Wada et al. [47]1988–2000632001–200337Our series1981–2000552001–200750 The new incidence data on IPMNs raise the question whether their increase in number is real or not. Of course, it is difficult to accept that IPMNs might have been overlooked in the past, not only clinically but also morphologically. There are good reasons, however, to believe that IPMNs did always exist and did not really increase in frequency. One reason is related to the rapid improvements in modern imaging techniques, which enable more precise recognition of cystic lesions, even if they are small and asymptomatic. Another is connected with the decreasing risk of pancreatic surgery. The most important fact, however, may be that until 1999, the distinction between IPMNs and mucinous cystic neoplasms (MCNs) was unclear, so that many IPMNs were classified as MCNs [50] or regarded as ductal adenocarcinomas or chronic pancreatitis. Adenoma-carcinoma sequence In IPMNs, the normal ductal epithelium is replaced by mucin-producing columnar cells showing papillary proliferations and variable degrees of cellular atypia, even within an individual neoplasm. They are graded according to the most atypical area as IPMN with low grade dysplasia (adenoma), IPMN with moderate dysplasia (borderline), and IPMN with high grade dysplasia (carcinoma in situ). An invasive component may be found in 38–50% of the cases [7, 26, 40, 43]. Progression from adenoma to carcinoma is estimated to occur at about 5 years [43]. IPMNs therefore provide a model of neoplastic progression from a benign intraductal neoplasm through increasing grades of dysplasia to invasive carcinoma. Prognosis after resection Between 80 and 90% of IPMNs are surgically resectable. For these IPMNs, a 5-year survival rate of 77–100% was reported, provided the tumors did not have an invasive component (Table 2). By contrast, IPMNs with an invasive component had a 3- to 5-year survival rate of only 36–46% [12, 16, 43] (Table 2). Interestingly, the survival rate did not appear to be dependent on the grade of dysplasia in the IPMN if there was no invasive component [12, 16, 43, 47]. These data imply that the overall outcome of IPMNs therefore largely depends on the presence of an invasive component. If the tumor is already invasive, criteria for a poor outcome are lymph node involvement, vascular invasion, and bilirubin elevation [13]. A comparison of the prognosis of all patients with invasive IPMNs with that of patients with ductal adenocarcinoma reveals that patients with IPMNs survive longer than those with ductal adenocarcinomas [29, 40, 43]. Table 2Five year survival rate and recurrence in 349a intraductal papillary-mucinous neoplasms NoninvasiveInvasive77–100%36–46%No recurrence93–98.7%52–70%Recurrence1.3–7%30–48%Localup to 6%8–48%Distant (metastases)1%12%Both0%10–48%a[12, 43, 47] Several studies have reported recurrences after resection of noninvasive IPMNs, some of which revealed only moderate dysplasia [12, 43, 47, 48]. The recurrences were either local or metastatic (Table 2). To explain the recurrences, particularly the local ones, it has to be assumed that either tumor tissue was overlooked at the pancreatic resection margin, or an invasive component remained undetected in the resected specimen, or there was multifocal disease. The last possibility has to be considered if the surgical margins were negative and the recurrence occurred in the pancreatic remnant. This has been observed only in a few cases [40]. When metastatic recurrences occur, it is most likely that they resulted from inadequate sampling that failed to detect an invasive component. Regarding the impact of a positive resection margin on IPMN recurrence, it is interesting to note that it has been reported that even IPMNs with positive margins did not recur during a median follow-up period varying from 19–40 months [13, 48]. The reason for this phenomenon might be that the growth of the remaining intraductal tumor tissue is so slow that clinical symptoms only appear after a follow-up period of 2 to 3 years. However, even if intraductal recurrences may take a long time to become clinically apparent, a positive margin in any IPMN case should lead to further tissue resection. Histological type and prognosis In 1991, it was reported that the invasive component of IPMNs corresponded either to an ordinary ductal adenocarcinoma or, more frequently, to that of a mucinous (colloid) carcinoma [49]. This observation suggested that IPMNs form a group of heterogeneous neoplasms. A further argument for the heterogeneity of IPMNs was the detection of IPMNs in branch ducts rather than in the main duct, where most of the IPMNs are found. Finally, it was recognized that IPMNs differ in their histological and cytological features and in their mucin profile [3, 4, 18, 28, 32, 33]. Currently, four subtypes of IPMN can be distinguished: an intestinal type, a pancreatobiliary type, an oncocytic type, and a gastric type [18]. The most common type of IPMN is the intestinal type (Table 3). It usually occurs in the main duct of the pancreatic head [9] and shows a villous growth pattern similar to that of villous adenoma in the colon. It also expresses MUC2 and CDX2 but not MUC1 (Fig. 1a,b). When this IPMN type becomes invasive, the invasive component resembles mucinous (colloid) carcinoma [5, 28], a tumor of which at least 80% is composed of pools of extracellular mucin containing single cells or strands of neoplastic glandular epithelium or even a small component of signet ring cells. Patients with colloid carcinoma have a 55% 5-year survival rate after resection [5]. These tumors therefore seem to be much less aggressive than ordinary ductal adenocarcinomas. Fig. 1Histological subtypes of intraductal papillary-mucinous neoplasms of the pancreas and their usual mucin pattern. a, b IPMN of the intestinal type positive for MUC2; c, d IPMN of the pancreatobiliary type positive for MUC1; e, f IPMN of the oncocytic type showing scattered positivity for MUC2; g, h IPMN of the gastric type, positive for MUC5Table 3Histopathological data on 105 intraductal papillary-mucinous neoplasms collected during a period of 26 yearsIPMNGastric (n = 27) (26%)Intestinal (n = 57) (54%)Pancreatobiliary (n = 7) (7%)Oncocytic (n = 14) (13%)Noninvasive (65%)n = 20 (74%)n = 35 (62%)n = 3 (43%)n = 10 (72%)Adenoma91215Borderline71821Carcinoma in situ4504Invasive (35%)n = 7 (26%)n = 22 (38%)n = 4 (57%)n = 4 (28%) The pancreatobiliary type of IPMN is much rarer than the intestinal type IPMN (Table 3). It shows complex arborizing papillae and expresses MUC1 only (Fig. 1c,d). Its invasive component usually corresponds to a conventional ductal adenocarcinoma. The prognosis of this type of IPMN, if invasive, seems to be similar to that of ductal adenocarcinoma and therefore poorer than that of the intestinal type of IPMN [4]. The oncocytic type of IPMN (also called intraductal oncocytic papillary neoplasm [2]) shows the same complex papillae as the pancreatobiliary type, but the lining cells reveal strongly eosinophilic cytoplasm. In addition, there are often numerous goblet cells. The tumor cells express MUC1 and MUC2 inconsistently (Fig. 1e,f). With fewer than 20 cases reported in the literature to date [2, 20, 34, 35, 37, 38, 42], the clinical and pathological behavior of this type is still unclear. Most of the cases (94%) were diagnosed as carcinoma, some of them with an invasive component or even distant metastases [37]. As the follow-up in this patient group is very short, no relevant data are available yet on survival and outcome. The gastric type of IPMN exhibits papillary projections lined by epithelial cells resembling gastric foveolar cells and shows pyloric gland-like structures at the base of the papillae. These cells express MUC5 (Fig. 1g,h), while MUC1, MUC2, or CDX2 positivity is only occasionally observed. The gastric type of IPMN corresponds to the branch duct type, which occurs in the periphery of the pancreatic parenchyma, most often in the uncinate process, where it usually presents as a multicystic lesion with cysts no larger than 3 cm [9]. The gastric type seems to be less aggressive, i.e., less invasive, than the other IPMN subtypes [10, 25, 33, 46] but may show severe cellular atypia in a few cases (up to 25%) [46] (Table 3, Fig. 2) The size of the lesion was unrelated to the grade of cellular atypia [46]. Fig. 2IPMN of the gastric type showing severe cellular atypia and MUC5 positivity Interestingly, pancreatic intraepithelial neoplasia (PanIN)-like complexes are frequently observed next to gastric type IPMNs. This raises the question whether IPMNs of the gastric type are a focal accentuation of a diffuse disease rather than a localized lesion. They might therefore also be related to the small peripheral cystic changes described by Kimura et al. [21] in non-neoplastic pancreata or the patchy lobular fibrosis associated with PanIN-1B lesions described by Detlefsen et al. [14] (Fig. 3). If this were the case, IPMNs of the gastric type would actually be large PanIN-1 lesions. An argument for this assumption is that both IPMNs of the gastric type and PanIN-1 lesions stain for MUC5 in the absence of MUC1 and MUC2 positivity. This assumption would also explain why it is difficult to distinguish PanIN lesions from some IPMNs [27] (Fig. 4), despite a consensus definition of both lesions [19]. Fig. 3PanIN-1 lesion associated with lobular fibrosisFig. 4Small multicystic duct-associated lesion in the periphery of pancreatic tissue suggestive of an IPMN of the gastric type but difficult to distinguish from a large PanIN-1 lesion Although the malignant potential of IPMNs of the gastric type seems to be rather low, it has to be pointed out that the fibrocystic changes that have been described in pancreata removed from patients with a strong family history of pancreatic cancer [11, 30] are similar, if not identical, to IPMNs of the gastric type and their associated PanIN lesions. This implies that IPMNs of the gastric type/PanIN-1 lesions are not innocuous lesions but have a malignant potential. Summary and perspectives The significance of IPMNs among the pancreatic tumors has increased greatly in recent years because of their improved recognition, both clinically and histopathologically and their much better prognosis than ordinary ductal adenocarcinomas. Moreover, they appear to fall into four subtypes that have special biological properties with prognostic implications. Of particular interest in relation to the development of ductal adenocarcinomas is the fact that the so-called gastric type IPMNs seems to occur in pancreata from patients with a strong family history of pancreatic cancer. Furthermore, it is of interest that the pancreatic IPMNs have their counterparts in IPMNs of the biliary duct system, where the same subtypes may occur [1, 23, 51, 52]. The treatment of choice is resection, but future trials may reveal that the extent of resection could depend on the IPMN subtype.

Eur_J_Pediatr-4-1-2413082

A 15-year-old girl with a large pericardial effusion

Pericarditis is a rare manifestation of tuberculosis and can be fatal. We describe a 15-year-old girl admitted for a large pericardial effusion. Subxiphoid pericardial biopsy was performed. Biopsy samples were positive for M. tuberculosis DNA by PCR, which confirmed the diagnosis of tuberculous pericarditis. A 15-year-old girl was admitted with chest pain, progressive shortness of breath, fever, nonproductive cough, decreased appetite, weight loss, and fatigue. The patient was born in Angola, but had lived in the Netherlands for 14 years. Seven months before admission she had traveled to the Democratic Republic of Congo in Western Africa. Her temperature was 39.7°C, her pulse 90 beats/min, respiratory rate 28 breaths/min, and blood pressure 96/58 mmHg. No enlarged lymph nodes were found. Soft cardiac tones and a friction rub were noticed. The liver was enlarged. Laboratory results showed normal white blood cells and platelets with a decreased hemoglobin (4.3 mmol/l). The erythrocyte sedimentation rate was 60 mm/h. A chest radiograph showed marked enlargement of the cardiac silhouette with no other abnormalities. Echocardiography revealed a large pericardial effusion containing exudative debris and fibrin strands (Fig. 1). Extensive laboratory studies for auto-immune diseases and infections, including HIV, did not reveal the cause. Fig. 1Echocardiography showing a large pericardial effusion with exudative debris and fibrin strands. RV right ventricle, LV left ventricle, LA left atrium, PE pericardial effusion Under general anesthesia the pericardial fluid was drained and a subxiphoid pericardial biopsy was performed. The PCR for M. tuberculosis DNA was positive on the pericardium, though not in the pericardial fluid. The patient was treated with four anti-tuberculous drugs (isoniazide, ethambutol, pyrazinamide, rifampicin) for 4 months and two anti-tuberculous drugs for an additional 4 months. Adjunctive treatment with predinisolone was tapered over a 12-week period. The patient completely recovered and did not develop any signs of constrictive pericarditis. She was followed up for more than 1 year and then discharged because of uneventful recovery. Discussion Pericarditis is a rare manifestation of tuberculosis that can be fatal and accounts for approximately 1–2% of all cases of tuberculosis [6]. Children in particular are at high risk of the development of extra-pulmonary disease [5]. Clinical manifestations of tuberculous pericarditis are nonspecific and cardiopulmonary complaints usually develop later. Accumulation of pericardial fluid may compromise venous return, leading to cardiac failure. Echocardiography plays a major role in confirming the presence of a pericardial effusion. In addition, several echocardiographic abnormalities including pericardial thickening, exudative coating and strands crossing the pericardial space are suggestive of tuberculous pericarditis. Thickened pericardium and fibrin strands are highly specific (94% and 88% respectively), and exudative coating has a high sensitivity (100%) in tuberculous pericarditis [4]. The diagnosis is confirmed with detection of Mycobacterium tuberculosis in either pericardial fluid or pericardial tissue. Pericardial fluid aspiration, however, has low diagnostic yield, both for PCR and culture, whereas pericardial biopsy provides the best chance of definitive diagnosis [2, 6]. A positive tuberculin skin test may increase the suspicion of tuberculous pericarditis, but a negative skin test does not exclude the diagnosis. A tuberculin skin test was not performed in this case because the girl had been immunized with BCG. There was no history of any family members being diagnosed with TBC. The spread of tuberculosis to the pericardium, resulting in an effusion, occurs most often from the mediastinal lymph glands. However, pericarditis may also represent reactivation of disease in the absence of an apparent primary focus of infection [3]. Current recommendations for treatment of tuberculous pericarditis include an initial four-drug regimen for a period of 8 weeks with a subsequent continuation phase of treatment for 4 months when the isolate is drug-susceptible [1]. Controversy exists with respect to the use of corticosteroids for tuberculous pericarditis. The American Thoracic Society’s consensus statement on tuberculosis recommends adjunctive therapy with corticosteroids (1 mg/kg of prednisolone with tapering over 11 weeks) for tuberculous pericarditis [1].

J_Headache_Pain-4-1-2276243

The primary headaches: genetics, epigenetics and a behavioural genetic model

The primary headaches, migraine with (MA) and without aura (MO) and cluster headache, all carry a substantial genetic liability. Familial hemiplegic migraine (FHM), an autosomal dominant mendelian disorder classified as a subtype of MA, is due to mutations in genes encoding neural channel subunits. MA/MO are considered multifactorial genetic disorders, and FHM has been proposed as a model for migraine aetiology. However, a review of the genetic studies suggests that the FHM genes are not involved in the typical migraines and that FHM should be considered as a syndromic migraine rather than a subtype of MA. Adopting the concept of syndromic migraine could be useful in understanding migraine pathogenesis. We hypothesise that epigenetic mechanisms play an important role in headache pathogenesis. A behavioural model is proposed, whereby the primary headaches are construed as behaviours, not symptoms, evolutionarily conserved for their adaptive value and engendered out of a genetic repertoire by a network of pattern generators present in the brain and signalling homeostatic imbalance. This behavioural model could be incorporated into migraine genetic research. Introduction The genetics of the primary headaches scored recent scientific successes due to the unravelling of the genetics of FHM. Deciphering of the patho-physiological mechanisms of these common diseases promises to bring the much needed knowledge for pharmacological treatments and therapeutic interventions. There are however also problems and controversies, some not solved by the genetic studies performed to date. The following is a brief subjective review of the available evidence, suggesting a role for epigenetic mechanisms and ending with the proposal of a behavioural model of the primary headaches possibly useful for the genetic studies. Primary and secondary headaches: symptoms, syndromes or diseases? Idiopathic and syndromic migraines Headaches/migraines are plagued by problems of definition: these terms describe symptoms (a feature which indicates a condition of disease, in particular one apparent to the patient, CED 2003), and at the same time, distinctive syndromes (a group of symptoms which consistently occur together) [1] or diseases (a disorder of structure or function in a human, animal, or plant, especially one that produces specific symptoms) [1] with recognisable diagnostic features, internationally defined [2]. The problems encountered with definitions become evident when dealing with primary or secondary headaches, and when considering idiopathic and syndromic migraines. Secondary headaches are those in which attacks occur due to a recognisable cause or disease, which itself represents the primary cause of the attacks. Syndromic migraines, contrasted with the idiopathic ones, are those in which attacks of migraine, clinically barely or not distinguishable from those occurring in the primary migraines, occur compounded with involvement of other systems. Syndromic migraines are often genetically determined. The concept of “syndromic,” potentially useful in the exploration of headache pathogenesis, has been applied to conditions such as deafness, visual loss and epilepsy, but has no place in the HCS classification that classifies headache attacks and not diseases, albeit distinguishing between primary and secondary headaches. These considerations may apply to the genetics of the primary headaches, since by adopting the HCS (2004), we consider symptoms, not diseases (much as if, studying the genetics of diabetes mellitus, we adopted a classification of the hyperglycemias). Genetic epidemiology of the typical migraines The typical primary migraines (MO and MA) all have a substantial risk of familial recurrence. When estimating the population relative risk of migraine in specified groups of relatives (i.e. the ratio between the probability that a relative versus a random member of the population is affected), first-degree relatives of migraine without aura probands have 1.9 times the risk of MO and 1.4 times the risk of MA, whereas first-degree relatives of MA probands have nearly four times the risk of MA and no increased risk of MO [3]. Since a family aggregation is implied when the risk ratio exceeds one, this confirms the familial liability for the migraines, even though familiarity is not yet heredity. A further analysis showed however that spouses of MO probands have 1.4 times the risk of MO, and spouses of MA probands have no increased risk of MA [3], thus backing a hereditary liability for MO and especially MA. Twin studies concur with this increased familial liability. Concordance rates for migraine are consistently higher among monozygotic (MZ) than dizygotic (DZ) twins. In particular, heritability estimates were around 52% in female twin pairs raised together or apart since infancy. In MZ Danish twin pairs, liability to MO resulted from additive genetic effects (61%) and from individual-specific environmental effects (39%), while in MA, correlation in liability was 0.68 in MZ and 0.22 in DZ, with heritability estimated at 0.65. Therefore, twin studies reveal that approximately one-half of the variation in migraine is attributable to additive genes, while the remainder is caused by unshared rather than shared environmental factors between twins [4, 5]. Several studies have analysed pedigrees with migraine, segregation analysis being performed to discover the genetic transmission pattern. Studies at first envisioned migraine as a simple mendelian disorder, inherited according to monogenic rules of transmission. Various modes of inheritance, autosomal dominant with female preponderance, possibly sex determined; autosomal recessive with 70% penetrance; polygenic; maternal and X-linked transmissions have been proposed, or rejected [6, 7]. Finally, based on complex segregation analysis, a multifactorial inheritance was considered the most likely pattern even in high-risk families with MA [8]. A single gene was considered unlikely, but, notably, in some families, a mendelian or mitochondrial inheritance could not be excluded [3]. Currently, migraine is widely considered a complex disease with multifactorial inheritance. This type of inheritance applies to many complex/quantitative traits, i.e. traits that vary continuously in a phenotypic range, and in which variation is quantitative, not qualitative. Examples of quantitative traits are height, body weight, etc. Such traits are influenced by multiple genes (each a quantitative trait locus QTL), each having a small quantitative effect and interacting with the environment. However, there is still no unequivocal evidence that migraine as a quantitative trait varies continuously in the general population, and moreover, genetic variation underlying a continuous character distribution can result from segregation at a single locus too. Therefore, considering migraine as a quantitative trait may still be unwarranted. The primary headaches also display considerable comorbidity, rarely incorporated into genetic studies. MA is comorbid with hypomania, depression and anxiety, and MO with phobia, panic and major depression. Other comorbidities are stroke, dyslipoproteinemias, essential tremor, paroxysmal dyskinesia and epilepsy. Merikangas et al. in a longitudinal genetic epidemiology study found that migraine was associated with mood disorders and drew attention to the fact that age at onset of anxiety disorders preceded, while onset of affective disorders followed that of migraine, findings consistent with a syndromic relationship between migraine and anxiety/depression [9]. These findings have been replicated, maternal depression being significantly associated with development of migraine in children [10]. Asthma, rhynitis and allergic bronchitis are also important comorbidities recurring in migraine families [11–13]. These comorbid clinical features should be properly incorporated in the genetic studies of the primary headaches. Mendelian migraines? The genetics of FHM and their putative relationship with the typical migraines MA/MO Migraines may be multifactorial, but mendelian migraines, i.e., migraines that conform to a mendelian type of genetic transmission, do exist. FHM is classified as a subtype of migraine with aura in the HCS (2004), and it conforms to an autosomal dominant pattern of hereditary transmission. Joutel et al. mapped FHM to chromosome 19, and in 1996 the first FHM gene, CACNL1A4, later termed CACNA1A, encoding the alpha1A subunit of the P/Q neural calcium channel, was discovered, accounting for both FHM 1 and episodic ataxia type 2 (EA2) phenotypes [14, 15]. Spinocerebellar atrophy type 6 (SCA6) was added to the FHM and EA2 phenotypes in 1997 [16]. Thus, FHM 1, EA2 and SCA6 are all allelic channelopathies, with missense mutations mostly accounting for FHM, mutations disrupting the reading frame for EA2 and polyglutamine expansions in the COOH gene terminal for SCA6. The phenotypic spectrum of the CACNA1A mutations was at first believed to consist either of pure FHM or of FHM associated with cerebellar atrophy. Why some mutations originate pure FHM while others elicit progressive or intermittent cerebellar features remains unclear [17]. Ictal coma after trivial trauma and essential tremor were associated clinical features in families harbouring particular CACNA1A mutations. The phenotypic spectrum of the CACNA1A mutations has further expanded to include ataxia induced by fever or high temperature [18], childhood epilepsy [19,20] and status epilepticus [21], paroxysmal paranoid psychosis with anxiety [22], benign paroxysmal torticollis of infancy, considered a migraine equivalent [23], and even myasthenic syndrome [24], since CACNA1A is also expressed on presynaptic neuromuscular junction terminals where it modulates transmitter release [25] even in the absence of any morphological changes in the junction or muscle weakness [26]. The paroxysmal clinical features of migraine, ataxia and epilepsy, together with the consideration that CACNA1A specifies for a calcium channel and that in the tottering and leaner mouse with epilepsy and ataxia, similar mutations are found in the mouse homologue of the calcium channel alpha1A subunit gene, led to the proposal that migraine be considered a calcium channelopathy [27]. The concept of migraine as a brain channelopathy fits well with the phenomenon of spreading depression [28], implicated in migraine attack pathophysiology. It is now accepted by the scientific community as an explicative model for migraine. However, the available genetic evidence is controversial or negative (see below). FHM was soon proved to be genetically heterogeneous, some families linking to chromosome 1 [29,30], and a second gene, ATP1A2, encoding the alpha2 subunit of the Na/K ATPase, was discovered in Italian families and accounting for a phenotype of pure FHM (FHM 2) [31]. New mutations were found in FHM 2 pedigrees [32], and soon the phenotypic spectrum of FHM 2, initially thought to be confined to pure FHM, broadened to include such features as coma, triggered by minor head trauma and angiography [33], recurrent comas [34] and epilepsy, namely benign familial infantile febrile convulsions [35]. Finally, cerebellar ataxia associated with epilepsy and mental retardation was described in an Italian FHM 2 family [36,37], findings later confirmed by Spadaro et al. and Vanmolkot et al. in other families [38, 39]. Phenotypes of alternating hemiplegia of childhood [40–42] and basilar migraine [43] described with ATP1A2 mutations further enlarged the clinical spectrum of FHM 2. Variability within the same family is notable, with FHM, cerebellar ataxia, recurrent paroxysmal dystonia and mental retardation all recurring together [42]. Lastly, mutations in the neuronal voltage-gated sodium channel SCN1A were reported by Dichgans et al. [44] to account for a phenotype of pure FHM (FHM 3), and there are still FHM families without mutations in any of the previously described genes, implying further genetic heterogeneity. Sporadic patients with HM, more common in clinical practice, also present problems, since mutations in the known FHM genes are only rarely encountered in this population [45]. An important corollary of the genetic discoveries obtained in the FHM was the proposal to consider FHM as a model for the typical migraines MO and MA [27]. This spurred the search for the involvement of FHM genes in MO/MA. Up to now the effort has been largely unrewarding. This in our opinion is also due to the misclassification of FHM as a subtype of MA [2], whereas FHM represents a syndromic migraine (see below). Some evidence in favour of linkage of typical migraines to the FHM locus on chromosome 19 was initially offered by May et al. [46], Nyholt et al. [47] and Terwindt et al. [48]. However, early negative studies [49–51] were later substantiated by systematic screening investigations of the CACNA1A in families with MO and/or MA [52–55], and to date, mutations in CACNA1A have never been demonstrated in kindreds without hemiplegic migraine, with or without aura. The same negative considerations apply to ATP1A2. Earlier evidence in favour of a role of the Chrlq3l locus or ATP1A2 gene in the typical migraines [56, 57] was superseded by negative findings and absent ATP1A2 mutations in typical migraine only pedigrees, even those displaying an apparently autosomal dominant mode of inheritance [58–61]. The FHM 3 SCN1A gene was discovered too recently for any conclusive study. Von Brevern et al. [62] however failed to find any CACNA1A, ATP1A2 or SCN1A mutations in patients with migrainous vertigo. Thus, there is no current evidence that the genes causing FHM represent major susceptibility loci for the typical migraines. Does such negative genetic evidence imply that FHM is not a useful model for migraine etiology? Several reviews of migraine pathogenesis apply the FHM model of neural channelopathy to the typical migraines. While such models are not justified genetically, it may be contended that FHM is nonetheless helpful in elucidating the pathophysiology of the migraine attacks. This consideration however is likely to apply to several clinical conditions all characterised by headache attacks of the migraine type. Migraine-like attacks indeed are found not only in the typical migraines, but also in other conditions, diseases or syndromes, in which they occur together with symptoms and signs of multisystem nervous or extra-nervous involvement. These “syndromic migraines” thus display bona fide migraine headache attacks at some times in their clinical course, and most of them have a genetic basis (Table 1). FHM is also characterised by multisystem neurologic involvement (migraine, hemiplegia, ictal recurrent comas, cerebellar atrophy, mental retardation, epilepsy, movement disorders, myasthenic syndrome, etc.), and therefore we make a plea for FHM to be considered more appropriately as a syndromic migraine and not a subtype of MA, as with the current HCS classification (2004). Table 1A list of proposed (and provisional) syndromic migrainesSyndromic migrainesGenes (chromosome) involvedMigrainous features (references)MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes)MTTL1, MTTQ, MTTH, MTTK, MTTS1, MTND1, MTND5, MTND6, and MTTS2 (mtDNA)Most frequent symptom: episodic sudden headache with vomiting and convulsions [63, 64]CADASIL (cerebral arteriopathy, autosomal dominant, with subcortical infarcts and leukoencephalopathy)NOTCH 3 (19p13.2–p13.1)MA in 22% [65]; migraine in 38% [66]HERNS (retinopathy, vascular, with cerebral and renal involvement and Raynaud and migraine phenomena)TREX1 (3p21.3–p21.2)Migraine in 70% [67]CCM (familial cerebral cavernous malformations)KRYT 1 (7q11.2–q21)Convulsions and migraine attacks [68, 69] Linkage and association studies in the typical migraines Several studies on the genetics of the typical migraines MO and MA applied genetic association, linkage and genome wide scanning methods. Most of these studies resulted in findings that either lack verification or are controversial. For MA, a genome wide scan on 50 multigenerational families in Finland identified a susceptibility locus on chromosome 4q24 [70]. Other loci for MA have been reported on chromosome 11q24 in Canadian families with an autosomal dominant transmission pattern [71], and on chromosome 15q11–q13 to a genomic region containing genes encoding for GABA-A receptors in ten Italian families again displaying an autosomal dominant transmission pattern [72]. For MO, or for pedigrees with MO mixed with MA, susceptibility loci have been reported on chromosome 6p12.2–p21.1 in Sweden [73], chromosome 5q21 [74], chromosome 14q21.2–q22.3 in an Italian family with MO [75], chromosome Xq24–28 in two large Australian pedigrees [76] and chromosome 19p13.3/2 to the insulin receptor gene INSR [77]. While many of such findings have still to be replicated, in some cases (the INSR gene) sequence studies have given negative results [78]. Other studies have examined candidate genes, implying that a pathogenetic (and a priori) hypothesis was formulated beforehand. This may be risky, considering that the pathogenesis of the migraine headaches is still imperfectly understood. Candidate genes explored were the mitochondrial DNA (mtDNA), or genes involved in prothrombotic or cardiovascular disease, or in the metabolism of biologic amines such as dopamine or serotonin, or in a variety of other metabolic systems. Several of the studies applied to mtDNA genes have yielded negative results, even though in some families migraine was reported to segregate with the Leber mtDNA 14484 mutation [79], and mtDNA mutations and haplotypes (haplotype U) have been associated with juvenile migraine stroke [80, 81] and with cyclic vomiting, considered a migraine equivalent in the pediatric population [82–84]. Contrasting results for genes involved in prothrombotic/cardiovascular risk, and for those involved in the metabolism of the biological amines serotonin and dopamine, or in several other metabolic pathways are summarised in Tables 2, 3, 4, and 5. Table 2Prothrombotic and cardiovascular risk genes and typical migraine geneticsProthrombotic/vascular risk genes or mutations examinedPhenotypesLDL receptor (19p13.2)Associated with MO [87]; not associated [88]Factor V R/Q 506 (Leiden mutation)Associated with MA [89]Not associated with migraine stroke [90]; not associated with MA/MO [91]; not associated with juvenile MA [92]Factor II 20210 G/ANot associated with MA/MO [91]Not associated with migraine stroke [93]Factor XIII Val 34 LeuNot associated with migraine [94]Decanucleotide insertion/deletion factor VII promoterNot associated with MA/MO [91]Alloantigenic platelet systems HPA-1 and HPA-2Not associated with MA/MO [91]Deficit of protein SAssociated with MA [89]Angiotensin converting enzyme (ACE)Allele D associated with MO and more frequent migraine attacks [95]Endothelial NO synthase inducible (NOS3; iNOS)Not associated with migraine [96, 97]Endothelin receptor A (ETA-231 A/G) polymorphismAllele G protecting from migraine [98]MTHFR (methylene-tetra-hydrofolate reductase) C677T/A1298CHomozygous mutation associated with MA [99], associated with MA [100]; risk for MA, modulated by thymidilate synthase gene [101]Table 3Serotonin metabolism genes and typical migraine geneticsSerotonin metabolism genes examined Phenotypes5-HTSERT (17q11.2–12) Allelic association with MO (increase of allele STin2.12 + decrease of allele STin2.10) and MA (same + increase of allele Stin2.9) [102]; 5HT-TLPR with MA [103]Allelic association with migraine (allele Stin2.10) [104]; borderline association with migraine [105]No association/linkage with migraine [106, 107]5-HT2A (13q14–21)Allelic (allele C) association with migraine aura [108]No association with migraine [105, 106, 109, 110]5-HT1B (6q13)No association with migraine [106, 110, 111]5-HT1D (1p36.3–34.3)No association with migraine [106, 110, 111]5-HT2B (2q36.3–q37.1)No association with migraine [106, 110, 111]5-HT2C (Xq22–25)No association with migraine [106, 110, 111]5-HT1B (6q13)No association with therapeutic response to triptans [112, 113]5-HT1F (3p12)No association with therapeutic response to triptans [112, 113]Table 4Dopamine metabolism genes and typical migraine geneticsGenes examinedPhenotypesDopamine receptor 2 (DRD2)Allelic association (allele NcoI) with MA comorbid with anxiety/depression [114]Allelic association (allele 1) with yawning/nausea during attack of MO [115]No allelic association (allele NcoI) with MA [116]No allelic association with MO/MA [107, 117, 118, 119]Dopamine receptors 1, 3, 4, 5 (DRD1, DRD3, DRD4, DRD5)No allelic association with migraine [115, 117, 120]Dopamine transporter (DAT)Association with chronic daily headache with drug abuse [86]COMT; MAO-A No association with migraine [117, 121]Dopamine-betahydroxylase (DBH)Association with migraine [107], especially males with MA [122]No association [123]Table 5Other genes implicated in typical migraine geneticsGenes examinedPhenotypesAndrogen/progesterone receptorsAndrogen receptor not associated; progesterone receptor associated with migraine [124]K channel KCNN3Allelic association (CAG repeats) with MO/MA [125]Not associated (CAG repeats) [126]Cytotoxic T lymphocyte antigen 4 (CTLA-4) Not associated with migraine [127]HLA-DRB1Allelic association with MA [128] Finally, a few studies have focused on the genetics of the chronic headaches, a major social problem, since these chronic headaches are often associated with drug abuse and afflict a remarkable percentage of the general population. Chronic tension-type headache displays a substantial familial recurrence, with lifetime relative risk estimated at 3.87 for parents and 3.53 for children of probands; the risk is greater for females (3.35) than for males (2.59) [85]. A genetic association study of chronic headache with drug abuse versus the dopamine metabolism genes by Cevoli et al. [86] found that allele 4 of the exon III VNTR polymorphism of the dopamine receptor 4 gene DRD4 was associated with chronic daily headache, and allele 9 of the dopamine transporter gene SLC6A3 was more common in chronic daily headache associated with drug abuse than in episodic migraine. Genetics of tension-type headache Remarkably, apart from an epidemiological genetic study that demonstrated a familial aggregation for chronic tension-type headache [85], there are no other genetic studies of this common disorder. Genetics of cluster headache Cluster headache (CH) has long been considered a sporadic disease. In recent decades, however, a familial recurrence has been appreciated, and the new HCS classification (2004) now states that CH may be transmitted as an autosomal dominant disease in about 5% of cases. Several CH cases have been reported among monozygotic twins and in family pedigrees [129–137], and family studies indicate that I-degree relatives of CH probands carry a 5- to 18-fold, and II-degree relatives a 1- to 3-fold increased relative risk of the disease [138–141]. CH has been considered a probable autosomal dominant disease with a penetrance of 0.3–0.34 in males and 0.17–0.21 in females [142]. The exact transmission pattern is however still debated [143, 144], and an autosomal recessive pattern has been advocated in certain families [137]. Several candidate genes have been analysed, in particular mtDNA mutations [145–148], HLA antigens [149–151] and CACNA1A polymorphisms [152, 153], usually with negative or controversial results. Other genes, such as the NO synthases NOS1, NOS2A and NOS3 [154], the elusive amine gene cluster [155], the CLOCK gene involved in the regulation of circadian rhythms [156,157] and the hemochromatosis gene [158], have been found not associated with CH. Recently, an association between CH and a polymorphism in the hypocretin receptor 2 gene HCRTR2 was reported by Rainero et al. [159], possibly accounting for the circadian recurrence of the CH attacks. Such an association, while confirmed by Schürks et al. [160], was rejected in a European multicentric study [161]. Recently, reports of CH associated with hemiparesis during the attacks suggested a relationship with FHM and ionic channelopathies [162]. Preliminary genic expression studies instead documented the activation of proinflammatory genes during the CH attack [163]. Genetics and epigenetics Epigenetics is the study of the changes in DNA and DNA-binding proteins that, albeit altering the structure of chromatin, do not modify the nucleotide sequence of DNA. The remarkable feature here is that some of these modifications may be associated with heritable changes in gene function. Commonly held concepts of heredity indeed pit environmental influences (nurture) against genetic background (nature) as totally separate causative factors. Genetic advances themselves have however demonstrated that the hereditary transmission of biological changes not encoded in the DNA sequence and dictated by environmental influences is possible. This part of genetics, called epigenetics, has received little or no attention in the genetic studies of the primary headaches. It is the contention of the author however that future epigenetic studies will account for several hereditary features of the primary headaches, in particular their comorbidities. All those (meiotic and mitotic) modifications in gene expression that are heritable but not encoded in the DNA sequence are defined as epigenetic. Molecular mechanisms implicated include (1) methylation of cytosine residues at C5 in dinucleotide CpG sites (localised especially in promoters of well over 40% of the genes and that, when methylated, cause silencing of the gene); (2) mechanisms of RNA interference, whereby microRNAs silence gene expression; (3) histone (DNA associated proteins) changes: activation or inactivation of genomic regions according to the “histone code”. All of these mechanisms result in the expression or silencing of genes, and underlie such phenomena as inactivation of the X chromosome and genomic imprinting. Several epigenetic diseases are already known that may be inherited through the somatic and the germinal line: fragile X syndrome, in which ATRX gene mutations modify the methylation pattern of ribosomial RNA and, by methylation of CGG expansions in the FMR1 gene, silence the gene; the Angelman and Prader-Willi, and Rett syndromes; also, many colonic cancers and leukemias. Important epigenetic differences that increase with age are found even between monochorial twins [164]. Notably, epigenetic modifications may increase with age and may also be prevented through interventions directed at DNA or histone methylation (with azanucleotides, antisense oligonucleotides, histone deacetylase). Even more remarkably, there is some evidence that lifestyles and even diet may play a role [165]. Epigenetic models for the primary headaches? There is consistent evidence that behavioural differences typical of specific inbred animal strains are the consequence of environmental influences acting especially during development rather than DNA changes. Mice strains with decreased environmental exploration behaviour (B6 strain) develop enhanced exploratory behaviour if nurtured in their first 3 months of life by BALB dams, a strain displaying intense exploratory behaviour; changes in behaviour appear to be linked to the type of maternal care, particularly licking of the pup by the mother, a behaviour demonstrated to affect the status of the endocrine stress system in mice [166]. Weaver et al. [167] showed how maternal care in the rat (licking and grooming the pup) modifies the methylation pattern of the promoter of the glucocorticoid receptor gene in the hippocampus; such epigenetic changes, evident from the first week of life, persist throughout the animal’s life but are reversible upon treatment with histone deacetylase inhibitors or upon intracerebral administration of methionine (an intervention that modifies the methylation pattern) [168]. Stress plays a remarkable role in the development of the nervous system: removal of rat pups from the mother causes reduced neurogenesis in the adult hippocampus through steroid-dependent mechanisms [169], and alters serotonergic transporter densities and serotonergic 1A receptors in the rat brain [170]. Administration of steroids to the mother before delivery causes changes in behavioural patterns in juvenile rats [171], and maternal deprivation in the immediate post-natal period modifies locomotor and steroid release patterns in the adult rat [172]. Epigenetic mechanisms also seem relevant for the formation of memory traces [173] and more generally for cognitive development [174]. Epigenetic mechanisms have been hypothesised for psychiatric disorders [175, 176] and many complex and multifactorial diseases affecting the brain or the inflammatory and immune systems [177–179]. There are still no studies of epigenetic mechanisms in the primary headaches. When considering, however, the important maternal influence in migraine genetics; the consistent and inherited co-morbidities especially for psychiatric and inflammatory-immune disorders; twin studies documenting that only about half of the variability is due to “genetic” factors; it is possible to envision that epigenetic mechanisms, especially those acting during nervous system development in early infancy and childhood, play a role in the heritability and pathophysiology of the primary headaches. Preliminary studies have already analysed attachment styles in adult migraineurs [180, 181], and a prospective investigation demonstrated correlations between events suffered during pregnancy and early life, and quality of adult life 31–33 years later [182]. It is reasonable to suggest that early life factors and attachment styles between mother and child represent determinants of epigenetic changes relevant in migraine pathogenesis. Such early pre- and post-natal environmental behavioural factors could be usefully analysed to define endophenotypes of adaptive behaviour useful in the genetic studies of the primary headaches. Final comments: a behavioural model of the primary headaches as fight-or-flight response and sickness behaviour to be incorporated into genetic research Consideration of epigenetic mechanisms may help in analysing behaviours during the headache attacks. Any genetic studies are ultimately dependent upon the definition of the phenomenon taken into consideration, and on how it is conceptualised. Therefore, studies have to rely upon conventional diagnostic criteria, in turn based on a priori interpretations. Most genetic studies have been performed within the frame of migraine interpreted simply as a “pain” trait with multifactorial inheritance. In a “harlequin” model, several genetic factors, each one having a small specific weight, interact with environmental factors to determine the migraine attack. Such a model should however be better tailored to suit phenomena such as the migraine attack and the migraine diseases that are really behavioural “processes” with an intrinsic logic of their own [183], one that is consistent within attacks, within patients and within populations. There is a need to conceive of the primary headaches along more useful scientific lines. Consistently lacking in the genetic studies is for instance any consideration of migraine as a behavioural response to environmental and/or endogenous triggers, a view that has scientific support [184] and that we recently revised to accomodate a Darwinian perspective [185]. According to our view, migraine and other primary headaches such as CH are behaviours, not symptoms, evolutionarily conserved for their adaptive value and engendered out of a genetic repertoire by networks of pattern generators present in the brain. These neural networks serve the homeostasis of the brain, with migraine pain considered a kind of visceral pain signalling homeostatic imbalance. The behavioural repertoire enacted during the migraine attack, complete with its full panoply of pain, cognitive, autonomic, motor, etc., symptoms and signs, is comparable to that defined as sickness behaviour and already known to develop in all mammals and other animals following challenge with infective and other pathogenic agents [186]. In contrast, behaviour during the CH attacks [187] resembles the fight-or-flight response of hypothalamic animals. These behaviours during the headache attacks really represent “healing” processes, and migraine may even be evolutionarily advantageous [188]. Thus, what is relevant in this new behavioural model is not the manifestations of the attack, but the factors triggering it, that, migraine being of the brain, must relate to still unknown disturbances of brain homeostasis [185]. Accordingly, it is these triggering factors rather than the manifestations during the attacks that may represent the features most relevant for a true dissection of the genetics of the primary headaches.

Psychopharmacologia-3-1-1915617

The PDE4 inhibitor rolipram reverses object memory impairment induced by acute tryptophan depletion in the rat

Rationale The selective type IV phosphodiesterase inhibitor, rolipram, has been shown to improve long-term memory and can reverse the cholinergic deficit caused by scopolamine. However, the underlying mechanisms of action of rolipram remain obscure. Introduction Inhibition of phosphodiesterase type 4 (PDE4) leads to an increase in intracellular cyclic adenosine monophosphate (cAMP) availability (Silvestre et al. 1999). cAMP is an important second messenger molecule in the process of intracellular signal transduction mechanisms (Bailey et al. 1996). Several studies have shown that rolipram, a selective PDE4 inhibitor, improves cognitive performance in young rats (Blokland et al. 2006; Imanishi et al. 1997; Rose et al. 2005; Zhang et al. 2000, 2004) and ameliorates scopolamine-induced memory deficits (Egawa et al. 1997; Imanishi et al. 1997; Rutten et al. 2006; Zhang and O’Donnell 2000). In a previous study, we have shown that rolipram reversed a scopolamine-induced deficit in an object recognition task (ORT) in male Wistar rats (Rutten et al. 2006). It was suggested that these effects may be related to the effects of elevated cAMP levels on neurotransmitter release. There is also evidence that there is a link between PDE4 inhibition and serotonergic neurotransmission (Schoffelmeer et al. 1985; West and Galloway 1996). The adenylate cyclase activator forskolin, as well as 8-bromo-cAMP, can enhance the electrically evoked release of 3H-5-hydroxytryptamine (Schoffelmeer et al. 1985). Thus, elevating cAMP levels by a selective PDE4 inhibitor could exert a general facilitatory effect on 5-HT release. Based on these findings, we argued that rolipram may also reverse memory deficits induced by a lowered 5-HT neurotransmission. Acute tryptophan depletion (ATD) is a well-established model to assess the role of serotonin in cognitive and affective functioning (Booij et al. 2003). The neurotransmitter serotonin is synthesized from its amino acid precursor tryptophan (TRP), which is obtained from our food. Free TRP is transported into the brain across the blood–brain barrier, but has to compete for entrance with five other large neutral amino acids (LNAAs: valine, leucine, isoleucine, phenylalanine, and tyrosine). The ratio of TRP and these other LNAAs (TRP/ΣLNAA ratio) is thought to be a more sensitive index of brain TRP availability (Fernstrom 1981; Wurtman et al. 1980). As 5-HT is synthesized from TRP, through the rate-limiting enzyme TRP-hydroxylase, the availability of TRP in the brain determines the amount of central 5-HT (Wurtman et al. 1980). Due to its reversible and non-intrusive effects, the method of ATD can be repeatedly used in animals and humans. In humans, acute tryptophan depletion has a negative effect on memory consolidation (Park et al. 1994; Riedel et al. 1999; Schmitt et al. 2000). Furthermore, memory impairments in the ORT have been observed in rats after ATD (Lieben et al. 2004b, 2005). Therefore, we hypothesized that rolipram could reverse an ATD-induced memory deficit in rats by elevating 5-HT release in the brain. In addition to this aim of the study, we further examined the relation between peripheral TRP and memory performance. We investigated the effects of different pretreatment times of the TRP-depleted mixture on object recognition memory. These data should provide more experimental support for a relation between plasma TRP and, consequently, central 5-HT and memory performance. Materials and methods Animals All experimental procedures were approved by the local ethical committee of the Maastricht University for animal experiments according to governmental guidelines. A total of 42 4-month-old male Wistar rats (Charles River, The Netherlands) were used (410–450 g). Rats were randomly assigned to either the biochemistry (n = 18) or the behavior group (n = 24). In the biochemistry groups, the animals were randomly subdivided over three treatment groups, i.e., saline, TRP+, or TRP− (n = 6/group). The animals were housed individually in standard type 3 Makrolon cages on sawdust bedding in an air-conditioned room (about 20°C). They were kept under a reversed 12/12-h light/dark cycle (lights on from 18:00 to 6:00 hours) and had free access to water. Rats were housed in the same room as where they were tested. A radio, which played softly, provided background noise in all rooms. All testing were done between 9:00 and 17:00 hours. Drugs and chemicals The gelatin hydrolysate (Solugel C) was obtained from PB Gelatins (Tessenderlo, Belgium). Glucodry 210 was obtained from the Amylumgroup (Koog aan de Zaan, The Netherlands). l-Tryptophan and rolipram were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Kaliumchloride (KCl), calciumchloride-dihydrate (CaCl2·2H2O), and 5-sulfosalicylic acid dihydrate were purchased from Merck (Darmstadt, Germany). Treatment During a period of 2 weeks preceding the experiment, the rats were handled and habituated to oral injections with normal tap water (10 ml/kg). On experimental days, the rats were not fed 14 h before treatment until the testing period was completed. This was done to minimize the availability of TRP from food, which would counteract the effects of the ATD treatment. The rats were orally treated with a protein–carbohydrate mixture containing TRP (TRP+ group; in which 0.28% TRP of the total protein was added to the mixture) or one lacking TRP (TRP− group; no TRP added to the mixture), or with saline. The composition of the nutritional mixture is shown in Table 1. Table 1Composition of mixture and determination of the amino acids in gelatin-based protein (mol) MixtureComposition Protein (Solugel) in 100 ml water100 g Aspartic acid4.8 Glutamic acid10.3 Hydroxyproline11.4 Serine3.4 Glycine23.2 Histidine0.8 Arginine10.0 Threonine2.0 Alanine10.4 Proline12.0 Tyrosine0.4 Valine2.2 Methionine0.7 Isoleucine1.0 Leucine2.5 Hydroxylysine0.9 Phenylalanine1.1 Lysine2.9Carbohydrate (Glucodry 210) in 80 ml water50KCl0.094CaCl2·2H2O2.32l-Tryptophan (TRP−group)0l-Tryptophan (TRP+ group)0.28The composition of the mixture (g) used in this experiment is described in italic. The amino acid spectrum (%) of the Solugel protein was obtained from PB Gelatins, Tessenderlo, Belgium. In every experiment, each dose contained Solugel C at 4.0 g/kg and Glucodry at 2.0 g/kg of the body weight and was given in a dose of 10 ml/kg between 8:30 and 12:30 hours. Blood samples were taken at baseline (20 min before treatment) and 60, 180, and 360 min after the first treatment. For the behavioral studies in the object recognition task, the mixture was administered at different time points (see below). Rolipram was freshly suspended in 5% ethanol, 1% tylose (methyl-cellulose), and 94% distilled water on every experimental day. The following doses of rolipram were tested: 0, 0.01, 0.03, or 0.1 mg/kg always in combination with the TRP− mixture (3 h before the first trial, T1). Rolipram was always administered 30 min before T1 (i.p. injection, dose 2 ml/kg). Biochemistry For the determination of plasma amino acid levels, blood samples were taken at resting values and repeated at several points in time. Blood sampling was done via a tail-incision method (Fluttert et al. 2000). Promptly after collection of blood in sodium heparin tubes (Microvette® CB 300, Sarstedt, Germany), the samples were kept on ice. After centrifugation of the blood samples (at 4°C for 15 min at 3,000×g in a Hettich EBA 12 centrifuge), plasma was deproteinized with cups containing dry 5-sulfosalicylic acid (6 mg/100 μl plasma), and the protein was spun down. Samples were frozen in liquid nitrogen and stored at −80°C. Before analysis, samples were thawed at 4°C, vortex-mixed vigorously, and centrifuged at 50,000×g in a Hereaus Model Biogufe Stratos for 10 min at 4°C. From the clear supernatant, 20 μl was mixed with 1,960 μl water and 20 μl norvaline and stored in the cooled (7°C) sample compartment until analysis. In addition to total plasma TRP, the concentrations of several other amino acids were determined with a fully automated high-performance liquid chromatography system after precolumn derivatization with ophthaldialdehyde (OPA; Van Eijk et al. 1993). OPA amino acids derivates were quantified with fluorescence detection. The concentrations of the total plasma amino acids were expressed as micromole per liter. Object recognition memory The object recognition test was performed as described elsewhere (Prickaerts et al. 2002).The apparatus consisted of a circular arena, 83 cm in diameter. Half of the 40-cm high wall was made of gray polyvinyl chloride, the other half of transparent polyvinyl chloride. A light bulb was switched on during testing only and provided a light intensity (20 lx) which was equal in the different parts of the apparatus. Two objects were placed in a symmetrical position about 10 cm away from the gray wall. We used four different sets of objects. The different objects were: (1) a cone consisting of a gray polyvinyl chloride base (maximal diameter 18 cm) with a collar on top made of brass (total height 16 cm), (2) a standard 1-l transparent glass bottle (diameter 10 cm, height 22 cm) filled with sand, (3) a massive metal cube (10.0 × 5.0 × 7.5 cm) with two holes (diameter 1.9 cm), and (4) a massive aluminum cube with a tapering top (13.0 × 8.0 × 8.0 cm). The objects could not be displaced by a rat. In the first week, the animals were handled daily and were adapted to the procedure in 2 days; that is, they were allowed to explore the apparatus (without any objects) twice for 3 min each day. In the two following weeks, the rats were adapted to the testing and i.p. administration procedure by a saline injection (0.4 ml) 30 min before the first trial until they showed a stable discrimination performance, i.e., good object discrimination (a d2 value of about 0.30, see below) at a 1-h interval on two successive sessions. Subsequently, testing of the drugs began. A testing session comprised two trials. The duration of each trial was 3 min. During the first trial (T1) the apparatus contained two identical objects (samples). A rat was always placed in the apparatus facing the wall in the center of the transparent front segment. After the first exploration period, the rat was put back in its home cage. Subsequently, after a delay interval, the rat was put back in the apparatus for the second trial (T2), but now with two dissimilar objects, a familiar one (the sample) and a new one. The times spent exploring each object during T1 and T2 were recorded manually with a personal computer. Exploration was defined as follows: directing the nose to the object at a distance of no more than 2 cm and/or touching the object with the nose. Sitting on the object was not considered exploratory behavior. To avoid the presence of olfactory trails, the objects were always thoroughly cleaned with alcohol (70%). Moreover, each object was available in triplicate so neither of the two objects from the first trial had to be used as the familiar object in the second trial. In addition, all combinations and locations of objects were used in a balanced manner to reduce potential biases due to preferences for particular locations or objects. As we expected ATD to impair memory performance, we needed a delay interval at which normal rats discriminate between the novel and familiar object. Therefore, we selected a delay interval of 1 h, as Wistar rats show good discrimination between the two objects after this interval (Rutten et al. 2006). Furthermore, we hypothesized that rolipram may reverse deficits induced by ATD in a 1-h delay ORT. In 1 week, three testing sessions were given, with a 48-h washout period in between. Tests were always conducted on Mondays, Wednesdays, and Fridays. In the first behavioral experiment, we investigated the effects of administration with TRP+ or TRP− mixture at different time points (i.e., 30 min, 1, 3, or 6 h) before testing in the ORT. Each rat always participated in each condition, and the order of test conditions was decided at random. The second behavioral experiment examined the effect of rolipram (30 min before T1) on object recognition performance in combination with the TRP− mixture at the most effective time point (i.e., 3 h before T1). Thus, five treatment conditions were tested, i.e., saline, vehicle and TRP−, rolipram 0.01 mg/kg and TRP−, rolipram 0.03 mg/kg and TRP−, and rolipram 0.1 mg/kg and TRP−. Each rat was tested in each condition, and the order of treatment conditions was decided at random. The same animals (n = 24) were used for both behavioral experiments; thus, control conditions (TRP+) from experiment 1 and saline from experiment 2 can be compared to each other. Statistical analysis Biochemistry The mean concentrations of plasma amino acids were determined for each treatment and time condition separately. The extent of reduction (expressed in absolute values and in percentage decline from resting values) was calculated for total plasma TRP concentrations and for the TRP/ΣLNAA ratio. Extreme values, as determined by an extremity test (see Dixon 1959), were excluded from statistical analysis. Differences in plasma amino acid concentrations were analyzed with a general linear model, with main factor ‘treatment’ and repeated measure factor ‘time’. Separate one-way analyses of variance (ANOVA) were performed to analyze differences between treatment conditions per time point. A post-hoc Bonferroni test was used to further characterize the differences between treatment conditions. Differences were regarded as statistically significant if P < 0.05. Behavior The basic measures were the times spent by rats exploring an object during T1 and T2. Table 2 shows the measures involved in the object recognition task (Prickaerts et al. 1997). e1 and e2 are measures of the total exploration time of both objects during T1 and T2, respectively. d2 was considered as index measures of discrimination between the new and the familiar objects. d2 is a relative measure of discrimination which corrects the difference between exploring the old and the novel object for exploration activity (e2) and appears to be independent of the total exploration times (see Şık et al. 2003). In the object recognition task, results are not reliable if an animal explores both objects in the second trial for less than 5 s (Şık et al. 2003). Therefore, in every session, animals that explored less than 5 s in the second trial (i.e., e2 < 5) were excluded from analysis. For all parameters, the effects of the different treatment, time, and interactions were analyzed with univariate ANOVA for the first experiment and one-way ANOVA for the second experiment. Significant effects were analyzed in more detail using the Bonferroni correction. To compare control conditions, i.e., the TRP+ (3 h before the first trial) condition of the first experiment with the saline condition of the second experiment, a t test was performed. Differences were regarded as statistically significant if P < 0.05. Table 2Measures involved in the object recognition testExplorationDiscriminatione1 is the measure of the time spent in exploring both identical objects (a1 and a2) in T1; e2 is the measure of the time spent in exploring both the familiar (a) and new object (b) in T2; d2 is the measure of discrimination between the new and familiar objects. Results Plasma TRP values The plasma TRP/ΣLNAA ratio is shown in Fig. 1. TRP/ΣLNAA levels changed over the 6 h [F(3,60) = 4.92; P < 0.01]. There was a treatment effect for TRP/ΣLNAA ratio [F(2,60) = 36.72, P < 0.01]. Post hoc analysis showed that the TRP− condition was significantly different than the TRP+ and the saline condition (Bonferroni, P < 0.05). In addition, the time × treatment interaction was also significant for the TRP/ΣLNAA ratio [F(6,60) = 7.80; P < 0.01]. Separate one-way ANOVA analyses for each treatment time point showed differences between treatment conditions at the 1-h time point [F(2,17) = 31.44; P < 0.01] and the 3-h time point [F(2,16) = 6.79; P < 0.01]. However, no differences between treatment conditions were observed at the baseline time point (−20 min) [F(2,15) = 2.03; n.s.] or the 6-h time point [F(2.17) = 3.33; n.s.] (see Fig. 1). Furthermore, post hoc analysis showed that the TRP/ΣLNAA ratio of the TRP-treated animals was lower than the TRP+- or saline-treated animals at the 1-h time point (Bonferroni; P < 0.01) and the 3-h time point (Bonferroni; P < 0.05). Fig. 1The effects of treatment time on the ratio plasma TRP/ΣLNAA (mean values and SEM). Percentages indicate the difference from baseline levels in the TRP− conditions. Asterisks indicate significant differences from baseline (*P < 0.05; **P < 0.01) Behavioral experiment 1 The effects of ATD on ORT performance are shown in Fig. 2 and Table 3. There was a general treatment effect on ORT performance [F(1,165) = 36.44; P < 0.01]. In addition, a time effect was observed on ORT performance [F(3,165)  =  2.86; P < 0.05]. The time × treatment interaction was also significant for the memory performance in the ORT [F(3,165) = 4.47; P < 0.01]. Separate analysis per treatment condition showed that there was a significant time effect for the TRP− condition [F(3,87) = 5.41; P < 0.01] in memory performance, but not for the TRP+ condition [F(3,84) = 1.77; P = n.s.]. Post hoc analysis showed that object recognition performance was significantly impaired when the TRP− mixture was administered 3 h before the first trial. Fig. 2The effects of treatment time of TRP+ or TRP− on the performance in object recognition task. Asterisks indicate a significant impairment in object recognition performance (*P < 0.05)Table 3Exploration times in the object recognition task at baseline and after ATD T1—30 minT1—1 hT1—3 hT1—6 hTRP+e126.62 (2.09)32.30 (1.37)27.82 (2.06)29.00 (2.50)e233.64 (2.40)49.78 (3.11)*28.61 (2.39)28.37 (2.15)TRP−e126.97 (2.11)30.39 (1.78)29.29 (1.66)24.98 (1.53)e239.26 (4.17)37.32 (2.40)30.06 (1.77)28.91 (2.69)*P < 0.05 (indicates significant differences to the baseline group) Behavioral experiment 2 The effects of ATD in combination with rolipram on object recognition performance are depicted in Fig. 3 and Table 4. A dose-dependent increase in discrimination performance was observed after treatment with rolipram [F(4,104) = 6.85; P < 0.01]. Post hoc analysis showed that a dose of 0.1 mg/kg rolipram reversed the effects of TRP− treatment (P < 0.05). Rolipram treatment decreased exploration times in the first trial [F(4,106) = 16.24; P < 0.01], but had no effect on exploration times in the second trial [F(4,106) = 1.53; n.s.; see Table 4). Post hoc analysis showed that for all of the rolipram doses the exploration in the first trial was lower than the saline control condition. Fig. 3The effects of rolipram treatment on ATD-induced deficits in the object recognition task. Asterisks indicate a significant difference in object recognition performance compared with the TRP− and vehicle condition (**P < 0.01). The 0.1-mg/kg dose of rolipram reverses the ATD effectTable 4Exploration times in the object recognition task after treatment with rolipram and TRP− mixture SalineVehicle0.01 mg/kg0.03 mg/kg0.1 mg/kgSalineTRP−TRP−TRP−TRP−e128.72 (2.52)26.18 (1.72)19.15 (1.66)*18.68 (1.35)*12.02 (1.53)*e228.73 (1.42)27.43 (1.80)21.68 (1.96)27.89 (2.63)27.29 (2.70)*P < 0.05 (indicates significant differences to the baseline group) A comparison between the d2 values of the control groups of the first and second experiment (TRP+ 3 h vs saline) showed that there was no difference between these two control groups [t(38) = 0.906; n.s.]. Discussion The present study shows that acute tryptophan depletion results in reliable lowering of plasma TRP and the TRP/ΣLNAA ratio. When rats were administered one dose (10 ml/kg) of the TRP− mixture, depletion was maximal (48%) 1 h after the treatment. The present data provide strong evidence that blood TRP levels predict object recognition performance and that ATD has only temporary effects on TRP levels and memory performance. When injected 3 h before T1, object recognition was maximally impaired. This is in support of the results from the blood TRP values (see above). Thus, TRP levels in the blood seem to be related with behavioral performance in rats. As previous studies have shown (Lieben et al. 2004a) a clear relation between plasma TRP and central 5-HT, these data provide further support that ATD can be used as a serotonergic-deficit model in the object recognition task. In the study by Lieben at al. (2004a,b), two doses of the TRP− mixture (10 ml/kg) were administered, which resulted in a 70% drop of the TRP/ΣLNAA ratio and in memory impairment in the ORT. In contrast, the present study showed that one dose of the TRP− mixture resulted in a 48% drop in the TRP/ΣLNAA ratio, which was found to induce memory impairment in the ORT. These findings suggest that a 50% reduction in plasma TRP levels is sufficient to impair object memory in rats. In the first behavioral experiment, no effects on exploration times in the ORT were observed, except from an increase in exploration time in the second trial of the TRP+ 1 h before T1 group. However, in the second experiment, a dose-dependent decrease in the exploration time of T1 was observed after treatment with rolipram. Previous work from our group has shown that as long as exploration in trial 1 is higher than 10 s, a reliable discrimination index (d2) can be calculated (Şık et al. 2003). Furthermore, as can be seen when exploration times are compared to d2 values, effects in exploration time are independent of effects in d2 values (Şık et al. 2003). Rolipram is known to have some sedative side effects (at higher dosages >0.1 mg/kg) (Griebel et al. 1991; Silvestre et al. 1999); nevertheless, the low dosages used in the present study improved memory performance. Rolipram has shown its pro-cognitive effects in several behavioral models. We have shown that rolipram treatment increases object memory in a time-dependent forgetting paradigm (Rutten et al. 2006). In addition, rolipram attenuated cholinergic deficits caused by scopolamine in several behavioral tasks (Imanishi et al. 1997; Rutten et al. 2006; Silvestre et al. 1999; Zhang and O’Donnell 2000). When rolipram (0.1 mg/kg) was administered in combination with the TRP− mixture (i.e., TRP− 3 h before T1 and rolipram 30 min before T1), we found that it reversed the effects of ATD. These are the first data suggesting that rolipram can reverse a serotonergic-induced memory deficit in rats. Our previous study indicated that rolipram can reverse the cholinergic deficit caused by scopolamine in the ORT (Rutten et al. 2006). Remarkably, a similar dose of 0.1 mg/kg rolipram was capable of reversing the serotonergic and cholinergic deficits in the object recognition task, which might be explained by a non-specific working mechanism of rolipram. Although the underlying mechanisms of action of the memory-enhancing effects of rolipram in these models remain to be determined, the present data could be explained via an enhanced cholinergic and serotonergic turnover by rolipram (Imanishi et al. 1997; Schoffelmeer et al. 1985). Alternatively, the effects of rolipram could also be explained in terms of only a cholinergic mechanism of action. Thus, previous studies have shown that the acetylcholinesterase inhibitor metrifonate ameliorates the effects of ATD as well and improves performance in scopolamine-deficit models of the ORT (Lieben et al. 2005). This suggests that the cholinergic and serotonergic systems act in a synergistic manner in memory performance (see Lieben et al. 2005 for a detailed discussion). Clearly, further research is needed to scrutinize the mechanism of action rolipram in the different animal models of memory. Another mechanism of action of rolipram that could explain the present data is related to the intracellular signaling in long-term potentiation (LTP). In the hippocampus, rolipram has shown to improve LTP through activation of the cAMP/PKA/CREB pathway (Frey et al. 1993; Impey et al. 1996). Moreover, these mechanisms have been linked to behavioral improvement in several behavioral tasks (Barad et al. 1998; Bernabeu et al. 1997; Blokland et al. 2006; Rutten et al. 2006). Consequently, it could be argued that the present data could be explained by the effects of rolipram on the cAMP/PKA/CREB pathway. However, a recent study by our group showed that the administration time of rolipram was of critical relevance. Rolipram was only effective in the 24-h interval ORT when administered 3 h after T1 (Rutten et al. 2006), and the mechanism of late LTP was proposed to explain these effects. However, in the present study, a 1-h interval in the ORT was examined, in which no gene transcription or protein synthesis is required. Therefore, we assume that the cAMP/PKA/CREB pathway cannot explain these effects. A recent PET study used radioactive rolipram as a measure for PDE4 binding and as an indirect index of cAMP signaling (Lourenco et al. 2006). Acute elevation of synaptic neurotransmitter levels (noradrenaline, 5-HT, and histamine) resulted in elevated cAMP levels that in turn elevated (R)-[11C] rolipram binding to PDE4 (Lourenco et al. 2006). According to this rationale, elevation of cAMP levels through inhibition of PDE4 can reverse cAMP-mediated deficits in behavior that occur due to a shortage of 5-HT, ie, reversing the ATD-induced object recognition impairment. As rolipram non-selectively facilitates several neurotransmitter systems, a broad mechanism of action can be expected. This can be favorable because influencing multiple systems increases the likelihood that several aspects of behavior could be enhanced. This may explain previous reports that rolipram was effective in depression models (Bobon et al. 1988; Norman et al. 1992; Weishaar et al. 1985). On the downside, the broad mechanism of action increases the risks for negative side effects, e.g., emesis, sedation, and nausea. In summary, the method of ATD can be used as a reliable method for inducing object memory impairment in rats. The selective PDE4 inhibitor rolipram was able to reverse this serotonergic deficit, which could be explained by different mechanisms of action. These data provide further support that PDE4 inhibition could be considered as a potential target to improve memory performance.

Plant_Mol_Biol-3-1-1876254

Tomato linalool synthase is induced in trichomes by jasmonic acid

Tomato (Lycopersicon esculentum) plants emit a blend of volatile organic compounds, which mainly consists of terpenes. Upon herbivory or wounding, the emission of several terpenes increases. We have identified and characterized the first two tomato monoterpene synthases, LeMTS1 and LeMTS2. Although these proteins were highly homologous, recombinant LeMTS1 protein produced (R)-linalool from geranyl diphosphate (GPP) and (E)-nerolidol from farnesyl diphosphate (FPP), while recombinant LeMTS2 produced β-phellandrene, β-myrcene, and sabinene from GPP. In addition, these genes were expressed in different tissues: LeMTS1 was expressed in flowers, young leaves, stems, and petioles, while LeMTS2 was strongest expressed in stems and roots. LeMTS1 expression in leaves was induced by spider mite-infestation, wounding and jasmonic acid (JA)-treatment, while LeMTS2 did not respond to these stimuli. The expression of LeMTS1 in stems and petioles was predominantly detected in trichomes and could be induced by JA. Because JA treatment strongly induced emission of linalool and overexpression of LeMTS1 in tomato resulted in increased production of linalool, we propose that LeMTS1 is a genuine linalool synthase. Our results underline the importance of trichomes in JA-induced terpene emission in tomato. Introduction Plants produce a wide variety of terpenoids that have primary functions as hormones (gibberellins, abscisic acid, brassinolide), sterols, pigments (carotenoids, phytol), and as parts of electron carrier moieties (ubiquinone, plastoquinone) (McGarvey and Croteau 1995). Most terpenoids, however, are secondary metabolites: over 20,000 different terpenoid structures from plants have been described (Sacchettini and Poulter 1997). Some terpenoids function in the direct defence strategy of plants as phytoalexins, which accumulate upon pathogen infection. Sesquiterpene phytoalexins (e.g., capsidiol) are characteristic for the Solanaceae (Chappell and Nable 1987; Egea et al. 1996). Volatile monoterpenes and sesquiterpenes can function in the interaction of plants with other organisms. They are present in floral scents that attract pollinators (Knudsen et al. 1993; Langenheim 1994) and are emitted by many plant species in response to herbivory by insects or spider mites. Induced terpene emission can attract predators or parasitoids of the herbivores, a mechanism commonly referred to as the indirect defense strategy of plants. This has been studied in, for instance, Arabidopsis (Arabidopsis thaliana) (Van Poecke et al. 2001), lima bean (Phaseolus lunatus) (Dicke 1994; Takabayashi and Dicke 1996), tobacco (Nicotiana tabacum; N. attenuata), maize (Zea mays), and cotton (Gossypium hirsutum) (De Moraes et al. 1998; Pare and Tumlinson 1999), and tomato (Kant et al. 2004; Takabayashi et al. 2000). These volatile terpenes are produced by sesqui- and monoterpene synthases of which several are induced by herbivory. For instance, nerolidol synthase activity is induced in lima bean, cucumber (Cucumis sativus), and maize (Bouwmeester et al. 1999; Degenhardt and Gershenzon 2000), whereas transcript levels of various other sesquiterpene synthases increase in, amongst others, maize (Shen et al. 2000), cucumber (Mercke et al. 2004), and wormwood (Artemisia annua) (Cai et al. 2002). In Arabidopsis, caterpillars (Pieris rapae) induce the myrcene/ocimene synthase AtTPS10 and the β-ocimene synthase AtTPS03, which coincides with increased myrcene emission (Van Poecke et al. 2001). Moreover, a β-ocimene synthase (LjEβOS) has been identified from Lotus (Lotus japonicus), which is induced by spider mite-feeding, resulting in increased emission of β-ocimene (Arimura et al. 2004b). However, artificial wounding or jasmonic acid (JA) treatment are often also sufficient to induce terpene synthases. The wound- and JA-induced synthesis of terpenes by coniferous plants is well described (Martin et al. 2002; Miller et al. 2005; Steele et al. 1995), and these treatments also mimic herbivore-induced responses in several angiosperms (Arimura et al. 2004a; Gomez et al. 2005; Schnee et al. 2002; Shen et al. 2000). Here we describe the identification and characterization of the first two monoterpene synthases from a solanaceous species (tomato), LeMTS1 and LeMTS2. We provide evidence that these genes are differentially regulated and more importantly, that expression of linalool synthase LeMTS1 is restricted to trichomes and induced by JA. Results Identification of tomato terpene synthases 1 and 2 (LeMTS1 and LeMTS2) In order to identify tomato monoterpene synthases that are induced upon spider mite herbivory, we first queried tomato EST databases (www.tigr.org and www.ncbi.nlm.nih.gov/BLAST/) with known monoterpene synthase sequences from other plant species. Retrieved tomato EST sequences were used to design a primer specific for monoterpene synthases (Fig. 1). We then used cDNA derived from spider mite-infested plants, to amplify a single 850 bp fragment with this primer and an oligo dT(18) primer. This fragment was subsequently used as a probe to screen available tomato cDNA libraries. Two different full-length cDNAs were identified; LeMTS1 (AY840091) was isolated from tomato leaves (sequence identical to the probe) and LeMTS2 (AY840092) from tomato roots and stems. The LeMTS1 and LeMTS2 open reading frames encode proteins of 609 and 590 amino acids (Fig. 1), which have predicted masses of 70.8 and 68.6 kDa, respectively, and are 71% identical. LeMTS1 and LeMTS2 contain amino acid motifs conserved in angiosperm (mono)terpene synthases. These are, based on LeMTS1 amino acid positions: R(45)R(46)W(55), required for the initial diphosphate migration (Williams et al. 1998); D(362)D(363)D(366), required for binding of a Mg2+ or Mn2+ ion, which is used as cofactor (Cane et al. 1996; Starks et al. 1997); D(505)T(509)E(513), required for binding of a second Mg2+ or Mn2+ cofactor; and D(586) in the active site cleft (Schwab et al. 2001; Whittington et al. 2002). LeMTS1 and LeMTS2 contain both putative (predicted with TargetP; Emanuelsson et al. 2000) plastid targeting signal peptides, which should be processed in the region upstream of the conserved R(45)R(46) arginine pair. Fig. 1Alignment of deduced amino acid sequences of LeMTS1 and LeMTS2 with three terpene synthases. A ClustalW alignment is shown of Mentha aquatica linalool synthase, a monoterpene synthase (C10), MaLin (AAL99381); Lycopersicon esculentum monoterpene synthase 1, LeMTS1 (AY840091); L. esculentum monoterpene synthase 2, LeMTS2 (AY840092); Mentha longifolia limonene synthase, MlLim (AAD50304), and L. esculentum germacrene C synthase, a typical sesquiterpene synthase (C15), LeGerC (AAC39432). Amino acids are shaded dark if identical in at least three of the five sequences and light if identical in LeMTS1 and LeMTS2 only. Conserved amino acid motifs typical for terpene synthases are boxed: RRx8W (Williams et al. 1998), DDxxD (Cane et al. 1996; Starks et al. 1997), Dx3Tx3E, and D (Schwab et al. 2001; Whittington et al. 2002). The presence of putative N-terminal plastid targeting peptides in the four monoterpene synthases and the primer used for amplifying the initial LeMTS fragment from cDNA of spider mite-infested leaves are indicated Remarkably, LeMTS1 contains an extra sequence stretch of 22 amino acids starting at N170 (Fig. 1). This sequence is not found in any other terpene synthase, nor does it have homology to any protein in the NCBI database (www.ncbi.nlm.nih.gov/BLAST/). Its presence can also not be explained by alternative splicing since the flanks do not contain canonical splice sites. This extra internal element is not an artefact of the isolated cDNA since it was present in four independent cDNAs isolated from different organs (data not shown). LeMTS1 and LeMTS2 belong to the TPSb subfamily of terpene synthases LeMTS1 and LeMTS2 are more similar to typical monoterpene synthases from, e.g., mint and citrus than to a typical tomato sesquiterpene synthase (Fig. 1). Based on protein sequence relatedness, LeMTS1 and LeMTS2 can be classified in the TPSb subfamily (Fig. 2), a distinct group of angiosperm monoterpene synthases, according to the original classification proposed by Bohlmann et al. (1997). So, unlike the snapdragon (Antirrhinum majus) monoterpene synthases from subfamily TPSg (Dudareva et al. 2003), including an Arabidopsis and a strawberry terpene synthase, the monoterpene synthases from the solanaceous family do not seem to form a new subfamily. Fig. 2LeMTS1 and LeMTS2 belong to the TPSb subfamily of terpene synthases. The TPS family is divided into subfamilies according to the nomenclature of Bohlmann et al. (1997). TPSa: angiosperm sesquiterpene synthases, TPSb: angiosperm monoterpene synthases, TPSc: angiosperm diterpene synthases, TPSd: gymnosperm mono-sesqui- and diterpene synthases, TPSe: a second group of angiosperm diterpene synthases, TPSf: a distinct group of (putative) linalool synthases mainly from Clarkia (Dudareva et al. 1996) and TPSg: a recently identified group of monoterpene synthases mainly from Antirrhinum (Dudareva et al. 2003). A limited amount of representative terpene synthases is shown. The bootstrapped phylogenetic tree was constructed using PAUP and TreeView software. A representative tree of 100 replicates is shown. Genbank protein accessions of terpene synthases shown are, top to bottom: TPSf: AAL24105, AAC49395; TPSe: BAC56714, AAC39443; TPSc: BAA84918, NP192187; TPSd: AAK83566, AAK39129, AAK39127, AAB71084; TPSg: NP176361, AAO42614, CAD57081; TPSa: AAU05951, AAK54279, AAC39432, AAG09949, Q40577; TPSb: NP189212, NP567511, AY840091, AY840092, AAC26018, AAV63791, AAL99381, AAD50304, AAG31435, AAM53943, AAM53945, AAF13356, AAK58723, CAC41012, AAS79351 Terpene synthase activity of recombinant LeMTS1 and LeMTS2 Recombinant LeMTS1 and LeMTS2 proteins were produced in Escherichia coli in order to characterize their enzymatic activities. The plastid targeting peptide was deleted up to one amino acid upstream of the RRX8W motif (Fig. 1) to produce a His-tagged ‘pseudo-mature’ form of the proteins, since it has been reported that this can improve protein expression without affecting activity and product specificity of the enzymes (Bohlmann et al. 1999; Williams et al. 1998). The soluble protein fractions were assayed for terpene synthase activity and products were analyzed on a GC coupled to a Time Of Flight-MS. Using the monoterpene precursor geranyl diphosphate (GPP) as substrate, LeMTS1 generated only linalool whereas LeMTS2 generated several monoterpene products: β-phellandrene, β-myrcene, and sabinene (Fig. 3a). Mass spectra and relative retention indices (Adams 2001) or authentic standards confirmed the identification of terpene reaction products. Extracts of E. coli cells expressing the empty vector control generated minor amounts of linalool (Fig. 3a) and occasionally geraniol. Thermally induced autoconversion or solvolysis of GPP to linalool has been observed before (Crowell et al. 2002; Jia et al. 1999), as well as conversion to geraniol by aspecific hydrolase or phosphatase activity in protein extracts (Crowell et al. 2002). However, LeMTS1 product levels are several hundred-fold higher than the background levels of the control (Fig. 3a). Fig. 3Enzymatic activity of recombinant LeMTS1 and LeMTS2 proteins. (A) GC/MS chromatograms of the LeMTS1 and LeMTS2 monoterpene products and the empty vector control with GPP as substrate. (B) GC/MS chromatogram of the LeMTS1 sesquiterpene product and the empty vector control with FPP as substrate. The chromatograms show detector responses for the terpene-specific ion mass 93. Products were identified as linalool (1), sabinene (2), β-myrcene (3), β-phellandrene (4), and (E)-nerolidol (5). Representative chromatograms are shown from assays of at least two independent experiments Enantiomer separation on an enantiomer-selective column showed that LeMTS1 produced only (R)-linalool (data not shown). LeMTS1 was also able to utilize the sesquiterpene precursor farnesyl diphosphate (FPP) as substrate to produce (E)-nerolidol (Fig. 3b), whereas LeMTS2 had no detectable sesquiterpene synthase activity. Both enzymes were unable to form terpene products from the diterpene precursor geranylgeranyl diphosphate. LeMTS1 and LeMTS2 are differentially expressed in various plant organs We investigated the organ specificity of LeMTS1 and LeMTS2 expression in a mature (13 weeks old) tomato plant (Fig. 4a). Transcripts of LeMTS1 were detected in young fruit, flower buds, petals, sepals, stems, petioles, and in young leaves. The expression pattern of LeMTS2 was very different: no transcripts were detected in petals, sepals, and leaves. However, LeMTS2 was expressed in roots. Fig. 4Spatial expression patterns of LeMTS1 and LeMTS2 in tomato. (A) Expression of LeMTS1 and LeMTS2 in different organs of an adult tomato plant. (B) Expression in trichomes or complementary tissue of petioles and stems. Expression was analyzed by RT-PCR. The data from one of two independent tissue sets that gave similar results is shown. RUB1 conjugating enzyme (RCE1, www.tigr.org: TC153679) was used as constitutive control. Ethidium bromide-stained agarose gels are shown For plants that contain glandular trichomes, monoterpene production is considered to be localized exclusively in these organs (Gershenzon et al. 1992; Iijima et al. 2004; Kutchan 2005; Turner et al. 1999; Turner and Croteau 2004). Tomato plants possess several types of trichomes including glandular trichomes that are present in high density on leaves, petioles, and stems and that contain terpenes (Maluf et al. 2001; Snyder and Carter 1985). Therefore, we investigated the location of LeMTS1 and LeMTS2 expression in trichomes of petioles and stems. This showed that LeMTS1 is expressed in trichomes, whereas LeMTS2 is expressed in the complementary tissue (Fig. 4b). LeMTS1 but not LeMTS2 is induced by spider mite feeding, artificial wounding, and jasmonic acid To study whether LeMTS1 and LeMTS2 were induced upon spider mite feeding, changes in transcript levels of intact plants were analyzed by RT-PCR since LeMTS gene-expression was too low to determine by RNA gel-blot analysis. Expression of LeMTS1 was indeed induced in leaves by spider mite-feeding, but LeMTS2, which expression was very low in leaves, was not (Fig. 5a). Spider mites damage plant tissue and induce JA- and SA-responsive genes such as a wound-induced proteinase inhibitor (WIPI-II) and a pathogenesis related protein (PRP6), respectively (Kant et al. 2004). Therefore, we investigated the effects of artificial wounding and exogenous application of JA and SA (Fig. 5a). Both artificial wounding and JA induced LeMTS1. In contrast, LeMTS2 was not induced by these treatments (Fig. 5a). SA-treatment induced the SA-marker PRP6 but neither LeMTS1 nor LeMTS2 was increased. Induction of LeMTS1 and WIPI-II was validated by real-time Q-RT-PCR. This showed consistent and clear induction of both genes by JA, artificial wounding and spider mites, although there was large variation in the level of induction between independent experiments (Fig. 5b). We routinely used the tomato cultivar Moneymaker for our experiments. However, in our earlier work (Ament et al. 2004; Kant et al. 2004) the cultivar Castlemart (CM) was used because it has the same genetic background as the defenseless1 (def-1) mutant (Howe et al. 1996). Here, we show that spider mite- and JA-induced LeMTS1 expression occurred both in CM and Moneymaker (Fig. 5b). Fig. 5Induction of LeMTS1 in tomato leaves in response to spider mites, wounding, and jasmonic acid (JA). (A) Gene expression analysis by semi-quantitative RT-PCR of LeMTS1 and LeMTS2 after various treatments, compared to the well known JA- and SA-marker genes WIPI-II (Graham et al. 1985) and PRP6 (van Kan et al. 1992), respectively. Treatments: JA, SA, and artificial wounding (W) after 24 h and spider mite-infestation (M) after 48 h. A representative result from three independent experiments is shown. RUB1 conjugating enzyme (RCE1) was used as constitutive control. Ethidium bromide-stained agarose gels are shown. (B) Validation of LeMTS1 and WIPI-II induction by real-time Q-RT-PCR. Two different tomato cultivars were used to verify induction by mites and JA. Values represent expression levels relative to controls, calculated after correction for expression of the control gene RCE1 (TC153679, www.tigr.org). Average values and standard errors are shown from three to five independent replicates To investigate whether spider mite-induced expression of LeMTS1 was dependent on JA or SA we made use of the def-1 mutant, which is impaired in induced JA-accumulation (Howe et al. 1996) and the SA-deficient, transgenic NahG tomato line (Brading et al. 2000). In plants with the def-1 mutation, LeMTS1 was not induced by spider mites, whereas the expression could be induced by exogenous JA (Fig. 6a). In contrast, the NahG plants showed higher and clearly induced LeMTS1 expression (Fig. 6b). This shows that induction of LeMTS1 expression is dependent on JA but not on SA. Fig. 6Spider mite-induced LeMTS1 expression is dependent on jasmonic acid (JA). (A) Expression of LeMTS1 in wild type (wt) or JA deficient def-1 mutant plants infested with spider mites (M) or treated with JA for 2 days. (B) LeMTS1 expression in wild type and SA deficient NahG plants in response to 2 days of spider mite-feeding. Gene expression was analyzed by RT-PCR, a representative result from one out of two independent experiments is shown. RUB1 conjugating enzyme (RCE1) was used as constitutive control. Ethidium bromide-stained agarose gels are shown JA induces expression of LeMTS1 in trichomes Since basal LeMTS1 expression in stems and petioles seemed to be restricted to trichomes (Fig. 4b), we addressed the question whether JA would induce expression of LeMTS1 solely in trichomes or also in other tissues. Indeed, JA-induced LeMTS1 expression was strongest in trichomes (Fig. 7). Interestingly, WIPI-II expression showed the opposite pattern. WIPI-II expression is very low in trichomes and basal as well as strongly induced WIPI-II expression occurred mainly in the complementary stem tissues. Fig. 7LeMTS1 is induced in trichomes by JA. Expression analysis of LeMTS1 and WIPI-II by real-time Q-RT-PCR. Intact plants were treated with JA, and subsequently trichomes were separated from remaining tissues of stems. The results from four independent experiments are shown as mean values and standard errors. Expression levels are indicated as expression relative to the control gene RCE1 Linalool accumulates in glandular trichomes and linalool emission is induced by wounding, jasmonic acid, and by ectopic LeMTS1 overexpression Figure 4b showed that, in petioles and stems, LeMTS1 was expressed specifically in the trichomes. Extraction of terpenes from the same tissues of that experiment demonstrated that linalool was produced predominantly in trichomes (Fig. 8). (E)-nerolidol and β-phellandrene also accumulated in trichomes. Conversely, the volatile benzenoid methyl-salicylate was not produced in trichomes and was also clearly detected in roots, in contrast to most terpenes. Fig. 8Linalool accumulation in trichomes. Relative levels of linalool, a selection of other terpenes and methyl salicylate in extracts of petioles and stems before and after removal of trichomes compared to total leaf and root extracts. Equal amounts of the different tissues were used (freshweight, 0.5 g), and trichome material was corrected for freshweight of the original petiole or stem. Averages of two measurements and min/max are shown Because LeMTS1 was induced by spider mite-infestation, wounding and JA, we investigated whether the emission of its in vitro products was induced concomitantly. Analysis of volatile production during 2 days of wounding or JA-treatment, conditions that induce LeMTS1 expression (Fig. 5), revealed that plants emitted more linalool after both treatments (Fig. 9a), while nerolidol emission was not induced. Fig. 9Increased linalool emission. (A) Linalool and (E)-nerolidol emission by intact tomato plants that were wounded (W) or treated with 1 mM jasmonic acid (JA) twice a day, for 2 days. The bar-graph shows the average (n = 3) emission over 2 days compared to the control and standard errors. Data (corrected for unequal variances) were analyzed by means of ANOVA followed by a Fishers-LSD post hoc-test. Linalool emission for both JA and wounding treatments was significantly increased compared to the control (P < 0.05), whereas (E)-nerolidol emission was not. (B) Linalool emission by a transgenic tomato line overexpressing LeMTS1. Headspaces were sampled from desiccators containing two plants each and compared to that of plants transformed with the empty vector. The graph expresses average values and standard deviations of four measurements. Emission of (E)-nerolidol, β-phellandrene, and methyl-salicylate did not significantly differ between plants. LeMTS1 overexpression in leaves was confirmed by RNA gel-blot analysis. The expression of RUB1 conjugating enzyme (RCE1) and ribosomal RNA levels (ethidium bromide-stained gel) are shown to indicate equal loading of the gel Next, we tried to overexpress the full length LeMTS1 cDNA (including the putative targeting peptide) in tomato plants, under control of the CaMV 35S promoter. Four transformation experiments yielded only two transformants of which one overexpressed LeMTS1. Compared to empty vector-transformed plants, LeMTS1 overexpressing plants emitted several hundred-fold more linalool into the plants headspace (Fig. 9b), while emission of other terpenes, including nerolidol, did not differ significantly from control plants. This suggests that LeMTS1 functions in planta as a true linalool synthase. Discussion Monoterpenes are abundantly emitted by tomato plants. For instance β-pinene, β-myrcene, 2-carene, β-phellandrene, limonene, and terpinolene are constitutively emitted, while linalool and β-ocimene emission is induced after spider mite feeding (Kant et al. 2004). In this paper, we describe the first two monoterpene synthases from tomato, LeMTS1 and LeMTS2 and show that LeMTS1 is induced by JA in trichomes. Identification of two tomato monoterpene synthases In vitro assays with the recombinant LeMTS1 and LeMTS2 proteins show that LeMTS1 has both (R)-linalool synthase and (E)-nerolidol synthase activity, and that LeMTS2 is a β-phellandrene/β-myrcene/sabinene synthase (Fig. 2a). LeMTS1 activity in planta seems restricted to linalool synthase activity since increased LeMTS1 expression by wounding, JA-treatment and ectopic overexpression led to increased linalool emission but did not affect nerolidol levels (Figs. 5, 9). Generally speaking, monoterpene synthases are plastid targeted and use GPP, while sesquiterpene synthases use FPP in the cytosol. There are however some exceptions to this rule. For instance, a cytosolic strawberry nerolidol synthase (FaNES1) has both (3S)(E)-nerolidol synthase and S-linalool synthase activity and is likely responsible for both linalool and nerolidol synthesis in planta (Aharoni et al. 2004). Vice versa, when FaNES1 was targeted to the plastid in Arabidopsis it also produced both linalool and nerolidol (Aharoni et al. 2003). The authors suggested that GPP and FPP substrate pools are not strictly separated between the cytosol and plastid, which has recently been supported by similar engineering experiments in tobacco (Wu et al. 2006). These observations might be explainable by for instance cytosolic as well as plastidial targeting of GPP synthase (GPS) in Arabidopsis and Litospermum erythrorizon (Bouvier et al. 2000; Sommer et al. 1995). In addition, FPP might be transported from the cytosol into plastids (Lichtenthaler 1999) and finally, the prenyltransferases GPS, FPS or GGPS might generate multiple products. However, since induced LeMTS1 expression does not correlate with increased nerolidol emission but only with increased linalool emission (Figs. 5, 9), we consider LeMTS1 to be a linalool synthase. Furthermore, we hypothesize that the plastidial FPP concentrations in tomato are negligible. Despite their high sequence homology (Fig. 1), LeMTS1 and LeMTS2 have different biochemical activities. It is commonly found that different terpene synthases within one plant species often have higher sequence similarity to one another than to functionally related terpene synthases of other plant species. For example, β-pinene synthase from citrus (Citrus limon) is more homologous to the citrus γ-terpinene synthase than to β-pinene synthase from wormwood (Fig. 2). It is also known from work with other terpene synthases that product specificity can depend on only a few amino acids (Kollner et al. 2004; Rising et al. 2000; Yoshikuni et al. 2006) and thus, product specificity of terpene synthases is not predictable on basis of protein sequence or specific amino acid residues. By comparing terpene synthase structures, attempts have been made to ascribe substrate binding and substrate modifications to certain residues. Modeling of the active sites of a Salvia (Salvia officinalis) sabinene synthase, a mint (Mentha citrata) limonene synthase and a mint linalool synthase led to the following observation (Crowell et al. 2002): compared to the other two enzymes, the linalool synthase has a three amino acid deletion in the active site pocket (two amino acids after the conserved D591, see Fig. 1), resulting in a more open structure of the so called J/K loop and providing an easier access of water during substrate ionization. Water access results in the premature release of the intermediate monoterpenol carbo-cation, before cyclization to a monoterpene olefin (non-hydrated) can occur. The tomato linalool synthase LeMTS1 also has a deletion in the active site pocket after the conserved D586 (Fig. 1). Although the deletion is eight instead of three amino acids, it indicates that LeMTS1 might utilize a similar structural mechanism to generate a monoterpene alcohol. LeMTS1 expression correlates with induced linalool emission The induction of LeMTS1 expression correlated with the increased emission of linalool by tomato plants. Wounding and JA application induced LeMTS1 (Fig. 5) as well as linalool emission (Fig. 9a). Induction of LeMTS1 upon spider mite-feeding (Fig. 5) coincided with linalool emission (Kant et al. 2004) in the tomato cultivar CM. Moreover, overexpression of LeMTS1 resulted in enhanced production of linalool (Fig. 9b) and no other volatile terpenes, providing evidence for in planta linalool synthase activity of LeMTS1. RNAi lines suppressing LeMTS1 expression in trichomes would provide additional evidence that LeMTS1 acts as a linalool synthase in planta. It is generally known that successful overexpression of terpene synthases can be problematic (reviewed by Chappell 2004). Substrate limitation and product conversions can explain low-terpene production levels (Aharoni et al. 2003; Lucker et al. 2001; Ohara et al. 2003). In addition, ectopic terpene synthase overexpression might lead to product toxicity (Aharoni et al. 2006; Besumbes et al. 2004) and selection for plant lines with low transgene expression or low terpene accumulation (Diemer et al. 2001; Krasnyanski et al. 1999). We managed to regenerate only one LeMTS1 overexpressing plant line after four successive, independent transformations. The empty vector and unrelated constructs gave at least 20-fold higher transformation frequencies. Similarly, LeMTS2 overexpression was unsuccessful, seven transgenic plant lines showed only expression of truncated transcripts (data not shown). LeMTS2 is expressed in roots, stems and petioles (Fig. 4a). In roots, only minute amounts of the main LeMTS2 product β-phellandrene were detected, hardly visible in Fig. 8 since levels are ∼300-fold lower than in leaves. The role of monoterpenes in roots remains unclear, although they might play a role in the defense response of plants. The release of the monoterpene 1,8-cineole from Arabidopsis roots is induced upon bacterial pathogen infection (Steeghs et al. 2004), although transcriptional regulation of the corresponding 1,8-cineole synthase (Chen et al. 2004) has not been shown yet. Although LeMTS2 is hardly expressed in tomato leaves (Fig. 4a), β-phellandrene and β-myrcene are constitutively emitted by tomato plants (Buttery et al. 1987; Kant et al. 2004). This suggests that the constitutive low expression of LeMTS2 might be sufficient to generate these monoterpenes. However, like the majority of volatile terpenes (data not shown), β-phellandrene is mostly located in trichomes (Fig. 8) where LeMTS2 is not expressed (Fig. 4b). This indicates that there are other β-phellandrene/β-myrcene synthases in tomato that synthesize these terpenes. JA-induced LeMTS1 expression occurs in trichomes Although the presence of terpene synthases in trichomes has been well documented (Bertea et al. 2006; Iijima et al. 2004; Lange et al. 2000), the regulation of their expression in trichomes remains obscure. Here we show that JA treatment of intact plants leads to higher transcript levels of LeMTS1 mainly in trichomes (Fig. 7). In contrast, WIPI-II expression is induced by JA in the complementary tissues, indicating that there is a tissue-specific differential activation of JA-regulated genes. Basal expression of WIPI-II was very low in tomato trichomes, although constitutive expression of a WIPI-II in glandular trichomes was recently demonstrated in nightshade (Solanum americanum) (Liu et al. 2006). It is unknown whether trichome-expressed WIPI-II plays a role in the defense against herbivores. Induction of WIPI-II in non-glandular tissue (mesophyll/parenchyma) meets the expectation that it should be present in those cells that suffer from herbivory. Previously, JA has been shown to increase trichome density on newly formed leaves of, for instance, Arabidopsis and tomato (Boughton et al. 2005; Traw and Bergelson 2003). Also, JA might induce trichome-based defenses directly. The production of acylsugars on the leaf surface (probably in trichomes) of Datura wrightii plants increased without affecting trichome density (Hare and Walling 2006). More clearly, nornicotin production on the Nicotiana repanda plant surface increased twofold within 6 h of JA treatment (Laue et al. 2000). Although it should be kept in mind that in the two above-mentioned studies, it was perhaps unjustly assumed that CHCl3 or CH2Cl2 extraction releases only trichome contents, it indicates that trichome-based secondary metabolite biosynthesis can be inducible. Results from our study show, for the first time, a specific transcriptional regulation of a gene involved in secondary metabolite biosynthesis in trichomes. Isolation of trichomes revealed that linalool synthase expression in tomato trichomes was induced more than twofold by JA after 24 h. In this paper, we report the identification of the first two monoterpene synthases from a plant of the solanaceae family. As it has recently been described that increased volatile terpene production in transgenic plants can benefit the indirect defense (Kappers et al. 2005; Schnee et al. 2006), it will be interesting to investigate the biological effect of LeMTS1 overexpression in tomato. Furthermore, the role of LeMTS2-derived terpenes in roots and stems remains to be investigated. Finally, the specific induction of LeMTS1 in trichomes by JA can provide an excellent opportunity for identification of novel, JA-related promoter elements, and transcription factors. Materials and methods Isolation of LeMTS1 and LeMTS2 cDNAs Tomato plants, Lycopersicon esculentum cv. Moneymaker (3–4 weeks old) were infested with 150 spider mites (Tetranychus urticae) as described by Kant et al. (2004). Leaves were collected and pooled after 2–5 days of infestation. RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA); cDNA was made using SuperscriptII RNAse H- (Invitrogen). A LeMTS fragment was amplified from this cDNA using the primer 5′-GATGACATTTATGATGTTTATGGC-3′ in combination with an oligo dT(18) primer. The primer was designed based on putative tomato monoterpene synthases (www.tigr.org: TC168035, TC160168, BG131411, and cLED9K13) as such that it should not anneal to sesquiterpene synthase cDNAs. The 850 bp PCR product was cloned into pGEM-T easy (Promega, Madison, WI, USA) and sequenced using the ABI PRISM BigDye terminator kit (Applied Biosystems, Foster city, CA, USA). cDNA was synthesized from leaves of 3-week-old Moneymaker plants and from hypocotyls and roots of 11-week-old Moneymaker plants with a ZAP-cDNA synthesis kit, cloned into the Lambda-ACT vector (Elledge et al. 1991) and packaged with a ZAP-cDNA Gigapack II gold cloning kit (Stratagene, La Jolla, CA, USA) 200,000 plaques of each library were screenend with the 850 bp RT-PCR product as probe, radioactively labeled by the ALL-IN-ONE Random prime labeling method (Sigma, Saint Louis, MI, USA). Filters were hybridized at 55°C in BLOTTO hybridization buffer (Sambrook and Russell 2001) and washed three times for 10 min in 1 ×  SSC, 0.1% SDS at 55°C. DNA from positive plaques was converted to pAct2 plasmids and their inserts were sequenced. LeMTS1 was obtained from the leaf cDNA library, LeMTS2 from the hypocotyl and root cDNA library. Sequences can be found in Genbank as AY840091 and AY840092, respectively. The presence of plastid targeting signals was predicted using PREDOTAR (http://genoplante-info.infobiogen.fr/predotar/predotar.html; Small et al. 2004) and TargetP (http://www.cbs.dtu.dk/services/TargetP/; Emanuelsson et al. 2000). Expression of recombinant proteins in E. coli, enzyme assays and product analysis LeMTS1 and LeMTS2 were subcloned into the pET32-a expression vector (Novagen, San Diego, CA, USA) after removal of the plastid targeting signal sequences up to one amino acid upstream of the conserved arginine pair (Fig. 1) by generating ‘truncated’ LeMTS1 and LeMTS2 PCR products with Pfu DNA polymerase (Stratagene). Forward primers contained NcoI- and reverse primers XhoI-restriction sites. LeMTS1 primers (5′–3′): GATCCATGGACACAAGGCGTTCAGGGAATTAC and GTACTCGAGCAAAGTAATAAAATGAAGCCTACG, LeMTS2 primers: GATCCATGGGTATCCGACGTTCAGGAAATTAC and GTACTCGAGAATAAAAGGTAATAATTCCTTGTC. pET32-LeMTS constructs were transformed into E. coli BL21(DE3) which were selected on Luria-Bertani (LB) plates containing 100 mg/l ampicillin. A single colony was grown overnight at 37°C on a plate, and transferred to 100 ml of LB with ampicillin. Cultures were grown at 37°C for 30–60 min to A600 of 0.6–1.0 before addition of 1 mM isopropyl-1-thio-β-d-galactopyranoside and grown at 20°C for an additional 8 h. Cells were harvested by centrifugation and resuspended in 5 ml monoterpene synthase assay buffer containing 50 mM HEPES pH 7.5, 10% glycerol, 5 mM DTT, 2 mM MnCl2, 10 mM MgCl2, proteinase inhibitor cocktail Complete (Roche, Basel, Switzerland), and 1 mM ascorbic acid. Cells were disrupted by addition of 1 mg/ml lysozym (Sigma), incubation on ice for 30 min and sonication. After centrifugation (10,000g) for 30 min at 4°C, 1 ml of the soluble enzyme fraction was assayed directly, or after purification of the His-tagged recombinant fusion protein on Ni-agarose beads (Novagen) for monoterpene, sesquiterpene or diterpene synthase activity using 10 μM of the substrates GPP, FPP or GGPP, respectively. The assay mix was incubated in a closed 20 ml vial at 30°C for 1 h under gentle shaking (150 rpm). Reaction products were sampled with a Solid Phase Micro Extraction fiber (SPME) for 10 min during which the vial was agitated and heated to 50°C. The SPME fiber was desorbed 1 min in an Optic injector port (ATAS GL Int., Zoeterwoude, The Netherlands) which was kept at 250°C. Compounds were separated on a DB-5 column (10 m × 180 μm, 0.18 μm film thickness; Hewlett Packard, Palo Alto CA, USA) in an 6,890 N gas chromatograph (Agilent, Amstelveen, The Netherlands) with a temperature program set to 40°C for 1.5 min, ramp to 250°C at 30°C/min and 250°C for an additional 2.5 min. Helium was used as carrier gas, the column flow was set to 3 ml/min for 2 min and to 1.5 ml/min thereafter. Mass spectra were generated with the ion source set to −70 V at 200°C and collected with a Time-of-Flight MS (Leco, Pegasus III, St. Joseph, MI, USA) at 1,671 V, with an acquisition rate of 20 scans/s. Terpene products were identified using authentic standards and comparison of ion specra and relative retention times (Adams 2001). For separation of terpene enantiomers, a Cyclosil-B column (30 m × 247 mm, 0.25 μm film thickness; Agilent) was used. The temperature program was isothermal at 115°C for 15 min, with a final ramp to 240°C at 120°C/min and 240°C for 5 min. Column flow was 0.7 ml/min. Plant treatments, plant headspace sampling, trichome isolation, and measurement of extracts For determining tissue specific gene expression, a 13 weeks old, untreated tomato plant was dissected to obtain material of each of the described tissues. For separation of trichomes from petioles and stems, 4-week-old plants were used. Trichomes were collected on the bottom of a 50 ml tube after vortexing several N2(l)-frozen petiole or stem segments, standing in upright position. The trichome fraction was used directly, whereas the remaining cleaned petiole or stem segments were thoroughly brushed to remove all remaining trichome material. Wounding-, spider mite-, and hormone-treatments (for both volatile sampling and gene expression analysis) were done using 3–4-week-old tomato plants. Wounding was inflicted by squeezing leaflets several times with a hemostat; a total of 150 spider mites were put on 3–4 leaflets per plant for 3 days; JA and salicylic acid were applied to plants by spraying 1 mM solutions made with tap water containing 0.05% SilwetL-77. Wounding- and hormone-treatments were done (cumulative) at 0 h and at 18 h and leaves were sampled at 24 h. Headspace sampling of plants was performed as described by Kant et al. (2004). Tenax sampling tubes were eluted with pentane : diethyl ether (4:1). The Eluted fractions were concentrated 20 times under a stream of N2 and 1 μl was injected into the GC injector port. For root-, leaf-, trichome-, stem- or petiole-extracts, 0.5 g tissue was used. The amount of trichome material was adjusted to be equivalent to the trichomes present on 0.5 g of the original stem or petiole. Tissues were ground in N2(l) and transferred to glass 20 ml vials containing 2 ml saturated CaCl2 (5 M) buffered in 100 mM sodium acetate (pH 4.5) which were capped immediately and kept at 5°C. Extracts were pre-incubated for 5 min at 60°C under agitation (500 rpm) and sampled for 10 min at 60°C on a 100 μM PDMS SPME fiber (Supelco, Zwijndrecht, The Netherlands). The SPME fiber was desorbed 1 min in the injector port of the GC/MS, which was kept at 250°C. GC/MS analysis was done as described earlier (Kant et al. 2004). Gene expression analysis by RT-PCR and real-time Q-RT-PCR For semi-quantitative RT-PCR, the tomato RUB1 conjugating enzyme (RCE1) was used as constitutively expressed control gene (www.tigr.org: TC153679). Initial PCR was performed with RCE1 primers, product levels were compared and individual cDNA samples were diluted accordingly, to ensure equal template concentrations. PCRs with primers of genes of interest were subsequently performed for non-saturating number of amplification cycles. RCE1 primers were always included in each experiment. Used primers (5′–3′) for RCE1: forward (F) GATTCTCTCTCATCAATCAATTCG; reverse (R) GCATCCAAACTTTACAGACTCTC, WIPI-II: F: GACAAGGTACTAGTAATCAATTATCC; R: CACATAACACACAACTTTGATGCC, PRP6: F: TCAGTCCGACTAGGTTGTGG; R: TAGATAAGTGCTTGATGTGCC, LeMTS1: F: GATGACATTTATGATGTTTATGGC; R: GGCCATCTCGAGACTTGAGAGCGAATGCAACATTAG, LeMTS2: F: GATGACATTTATGATGTTTATGGC; R: GGGTAATAATTCCTTGTCTTATTTC. Expression differences were validated by varying the amount of PCR cycles. For real-time Q-RT-PCR, total RNA was isolated using Trizol (Invitrogen) and DNA was subsequently removed with DNAse (Ambion, Huntingdon, UK). cDNA was synthesized from 5 μg RNA using SuperscriptII (Invitrogen) in 20 μl reaction volume that was diluted to 50 μl prior to using it for PCR. PCRs were performed in the ABI 7500 Real-Time PCR System (Applied Biosystems) using the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen). About 20 μl PCR reactions contained 0.25 μM of each primer, 0.1 μl ROX reference dye, and 1 μl template. The cycling program was set to 2 min 50°C, 5 min 95°C, 40 cycles of 15 s at 95°C and 1 min 60°C, and a melting curve analysis. Primer pairs were tested for specificity and for linearity with a standard cDNA dilution curve. Primers used: LeRCE1 QF: 5′-GATTCTCTCTCATCAATCAATTCG-3′ QR: 5′-GAACGTAAATGTGCCACCCATA-3′, LeWIPI2 (K03291) QF: 5′-GACAAGGTACTAGTAATCAATTATCC-3′ QR: 5′-GGGCATATCCCGAACCCAAGA-3′, LeMTS1 (AY840091) QF: 5′-TTTGGGGACATCTTCGGATGAA-3′ QR: 5′-CTACTCGAGTTACTTGAGAGCGAATGCAAC-3′. Expression levels were normalized using RCE1 mRNA levels. Generation of transgenic tomato plants The complete LeMTS1 cDNA, including the putative signal peptide, was cloned into the EcoRI-digested and Klenow-blunted binary vector pGreen1K (Brandwagt et al. 2002), resulting in a CaMV35S:MTS1:nos cassette. The construct was transferred to Agrobacterium tumefaciens strain EHA105 carrying the pSoup helper plasmid (Hellens et al. 2000). Tomato plants cv. Moneymaker were transformed essentially as described by Vanroekel et al. (1993). Heterozygous and homozygous LeMTS1 overexpressing plants were used to measure linalool production by sampling the headspace of intact plants (four sets of two plants for both LeMTS1 overexpressors and control plants).

Eur_Arch_Otorhinolaryngol-4-1-2217619

Morphology and function of Bast’s valve: additional insight in its functioning using 3D-reconstruction

The utriculo-endolymphatic valve was discovered by Bast in 1928. The function of Bast’s valve is still unclear. By means of orthogonal-plane fluorescence optical sectioning (OPFOS) microscopy 3D-reconstructions of the valve and its surrounding region are depicted. The shape of the duct at the utricular side is that of a flattened funnel. In the direction of the endolymphatic duct and sac this funnel runs into a very narrow duct. The valve itself has a rigid ‘arch-like’ configuration. The opposing thin, one cell-layer thick, utricular membrane is highly compliant. We propose that opening and closure of the valve occurs through movement of the flexible base/utricular membrane away from and toward the relatively rigid valve lip. Hypothesis The bottom or opposing wall of the uticulo-endolymphatic (Bast’s) valve is the moving and thus functional part of the valve. Background At the utricular end the utricular duct has a valve-like opening. This structure was discovered by Bast [3], who called it the utriculo-endolymphatic valve. It is since then also called Bast’s valve. The morphology of the valve has been researched in humans and several mammalians [1, 5, 10]. The function of Bast’s valve is still unclear. Bast himself suggested the closure of the utricular duct as the main function [3, 4]. Bachor and Karmody [2] follow Bast’s view, postulating that decreasing pressure in the whole endolymphatic system, secondary to collapse of the ductus reuniens, causes closure of the valve to prevent more loss of endolymph from the utricular system. Zechner [20] proposes that dysfunction of the valve causes endolymphatic hydrops. In this paper orthogonal-plane fluorescence optical sectioning (OPFOS) microscopy [6, 15–17] was used to obtain detailed three-dimensional reconstructions of Bast’s valve and the surrounding region in the intact guinea pig inner ear, as it is a technique capable of visualizing bone as well as soft tissue structures with high resolution, and is non-invasive. Material and methods Specimen preparation Two healthy female albino guinea pigs (Harlan Laboratories, UK), weighing 450 g, were used. Animal care and use were approved by the Experimental Animal Committee of Groningen University, protocol No. 2883, in accordance with the principles of the Declaration of Helsinki. The animals were terminated by lethal administration of sodium pentobarbital. After decapitation the bullas were dissected and fixated in a 10% formalin solution, neutrally buffered. Then the bullas were rinsed in aqua-dest. Decalcification in ethylenediaminetetraacetic acid 10% solution (EDTA; Sigma, ED5SS, pH 7.4) took place at a temperature of 50°C in a microwave oven (T/T MEGA microwave histoprocessor, Milestone) in eight sessions of 6 h. After decalcification the bullas were again rinsed with aqua-dest and dehydrated in a graded seven-step ethanol series (30%, 50%, 70%, 90%, 96%, 100%, and 100%). Spalteholz fluid, a 5:3 solution of methyl salicylate (Sigma, M-6752) and benzyl benzoate (Sigma, B-6630) [14], was thereafter used to achieve transparency of the specimen. The clearing process consisted of application of a succession of Spalteholz-ethanol solutions, 24 h each. The Spalteholz fluid fraction in the clearing session was 25%, 50%, 75%, 100%, 100%, respectively. Hereafter the specimen was dyed in a fluorescent dye bath of Rhodamine-B Isothiocyanate (RITC; Sigma, R-1755). RITC absorbs maximally at 570 nm and emits at 595 nm. The dye bath was prepared by dissolving 1.0 mg/ml RITC into ethanol, followed by dilution in Spalteholz fluid to a final dye concentration of 5 × 10−4 mg/ml [15, 16] (Voie 2003). The specimen was dyed for four days. OPFOS imaging system A schematic diagram of the optical setup of our custom-made OPFOS imaging device is shown in Fig. 1 [6]. Laser light, originating from a green frequency doubled Nd:YVO4 neodymium-laser (model DPGL-2050, Suwtech), with an emission wavelength of 532 nm and 52 mW maximal power, is expanded into a parallel beam of 28 mm diameter by a Galilean beam expander (BE, model 336, Spectra-Physics). The broadened and spatially filtered light falls onto a specially designed cylindrical lens (CL, custom-made by LiteTec Ltd., Essex, UK) of 30 × 30 mm and 80 mm focal length, which focuses the light along a single dimension (Z), thus creating a hyperbolic light pattern along the X-axis. Near the center of this focus, we can locally approximate the light intensity profile as a sheet of light in the X–Y plane, which will perform virtual slicing of a specimen object (O). In the plane where the object is sectioned by the sheet of light, the specimen emits fluorescence light, which is recorded by a CCD-camera (C, Firewire FO442BIC, Foculus) in the direction orthogonal to the light plane. In this way, a 2D image of a virtual section within the object is obtained. For the specimen to be compatible with the OPFOS method, the above described specimen preparation is necessary to make the object transparent, refraction index matched and fluorescent. In our setup the specimen is positioned inside a container with optical quality glass windows at the laser and CCD side, and filled with Spalteholz fluid, and can be positioned within the container by an object translation stage (OTS). Between recordings the object (and not the container) is moved along the Z-axis in small slicing steps with a high-precision DC-motor driven translation stage with position encoder (M112.1 High-Resolution Micro-Translation Stage with C862 Mercury II DC-Motor Controller, PI Polytec). A long working distance microscope objective lens (OL) with good numerical aperture (M Plan APO × 5, NA = 0.14, Mitutoyo) projects the fluorescence image on the CCD. A colour filter (Kenko R1 SR-60) placed before the objective lens blocks scattered laser light and transmits fluorescence light. Fig. 1Orthogonal-Plane Fluorescence Optional Sectioning (OPFOS) set-up. (BE: beam expander, FS: field stop, CL: cylindrical achromat, O: object, OTS: object translation stage, FTS: focusing translation stage, OL: objective lens with colour filter, CCD = charge coupled device camera) The virtual slicing is performed by a ‘plane’ with 3 μm 1/e²-thickness resolution in the center of the image and slightly thicker at the edges (less than 10 μm), with slicing steps of 2 μm, followed by storage in a personal computer. The two-dimensional stored images were processed with an IMOD (http://bio3d.colorado.edu/imod) software package for 3D-reconstruction. Input of relevant contours in each 2D-image was manually performed with a writing tablet (Wacom Cintiq 15X). Results Figure 2a shows an OPFOS image of a cross-section of part of the guinea pig inner ear. Figure 2b is an enlargement of the part of Fig. 2a inside the dashed box, in which Bast’s valve and its surrounding structures are clearly visible. Saccule, endolymphatic sinus, utricule and endolymphatic duct are filled with endolymph. Walls of the endolymphatic space are on the right side of Fig. 2b connected to bone (dense; non-compliant). The opposite walls are adjacent to perilymph (hypodense; compliant). Connective tissue (medium density; non-compliant) is present inside the lip of Bast’s valve and between the upper side of the utricule and bone. Figure 2c is another OPFOS image, showing Bast’s valve in detail. Fig. 2(a) 2D OPFOS-image of the region around Bast’s valve and the utricular duct, obtained with another OPFOS setup [7] than for (c) and following. The dashed box is shown in a larger magnification in (b), (b) Dashed box in (a) shown in a larger magnification, (c) 2D OPFOS-image of Bast’s valve. The arrows point toward very narrow passages Figure 3 shows a 3D-reconstruction of the entrance of Bast’s valve as seen from inside the utricle. The proximal end of the valve shows a rather rigid ‘arch-like’ configuration. Figure 4 shows a 3D-reconstruction of walls of Bast’s valve and the utricular duct. The shape of the utricular duct at the utricular side is that of a flattened funnel. This funnel quickly runs into a very narrow duct. Fig. 33D-Reconstruction of Bast’s valve as seen from inside the utriculeFig. 43D-Reconstruction of walls of Bast’s valve and the utricular duct. The shape of the utricular duct at the utricular side is that of a flattened funnel. This funnel runs into a very narrow duct Figure 5 is a 3D-reconstruction of part of the outer walls of the utricle and utricular duct. It again shows the small caliber of the utricular duct in relation to the size of surrounding structures. Fig. 53D-Reconstruction of part of the outer walls of the utricle and utricular duct. Note the small caliber of the utricular duct in relation to the size of surrounding structures Discussion In the 3D OPFOS-reconstruction of the entrance of Bast’s valve, shown in Fig. 3, the lip of the valve appears as a relatively rigid structure. In a comparable scanning electron microscopy (SEM) image the entrance of the valve is visible as a narrow slit in the utricular wall [9]. The valve lip is filled with connective tissue (Fig. 2c). This is even better visible in Fig. 6, which is a light microscopy (LM) image. This figure also clearly shows that the bottom of the valve has a thickness of only one cell-layer, and is as result highly compliant. Fig. 6Light microscopy image of lip and bottom of Bast’s valve Schuknecht and Belal [13] showed that the corium of the valve consists of fibroblasts and fibrocytes. Based on the study of 170 human temporal bones these authors propose that the valve is closed in normal ears and that its anatomical structure is ideally suited to permit the occasional egress of excessive accumulation of endolymph to be processed in the endolymphatic sac, while preventing an excessive loss of endolymph with the possible consequence of membrane distortions and interference with the motion mechanics of the vestibular sense organs. Opening of the valve, still according to Schuknecht and Belal [13], would then be accomplished by pressure displacement of the outer membraneous wall away from the more rigid inner valve lip. The arrows in Fig. 2c indicate positions were the valve appears to be closed. Also in Fig. 6 valve lip and bottom touch. Bast himself [5] was not sure about the valve being opened or closed in the normal situation. He writes: “As a matter of fact in many histological sections the epithelium of the valve is in contact with the opposing wall of the slit-like opening of the duct which suggests that such a closure may exist, at least at times, in the living ear.” He also was not certain about the mechanism that closes the valve [5]. On the one hand he supposed that a high perilymphatic pressure could close the valve by moving the opposing wall against the valve lip, as shown in Fig. 7. On the other hand he proposed closing of the valve lip against the opposing duct wall in the case of a relatively greater intra-utricular pressure. In this case he supposes that it is not the tip of the valve that moves, because it is bulky, but that the valve lip rotates at its base, where it is made up of loose perilymphatic tissue. Fig. 7Possible mechanism of valve closure (black arrows) or valve opening (grey arrows) In our opinion the latter mechanism is less likely, considering the observation that the valve opening in 3-D reconstruction is a rigid arch-like structure (Fig. 3), and not a flap hinged at its base, that could close an underlying opening. The latter mechanism may be inspired by 2-D pictures, like Fig. 2b, c and 6, where the valve tip appears as a flap-like structure. The rigidity of the valve tip was also noticed by Scheerer and Hildmann [12], who also thought it to be improbable that this structure can function as a valve. The walls of the endolymph filled spaces in the inner are highly compliant. As a result the pressure difference between endolymph and perilymph are negligible in the normal situation [19]. If the valve is open it seems, at first impression, easy to understand how an increase of perilymphatic pressure will close the valve by compression of its bottom, as is shown in Fig. 7. However, an increasing perilymphatic pressure will also increase the endolymphatic pressure inside the utricule and force endolymph out through the slit-like opening and prevent closure of the valve [11, 18]. As can be seen in Figs. 4 and 5 the utricular duct has a diameter in the order of 10 μm at its narrowest passage, preventing rapid flow of endolymph and possibly protecting the sensitive vestibular receptors against large fluid shifts in a short time. Konishi [8] found an open utriculo-endolymphatic valve in guinea pigs with an (experimental) endolymphatic hydrops. It is conceivable that an excess of endolymph will force the base of the valve opening away from the lip, as shown in Fig. 7. In future work, high-resolution OPFOS (HROPFOS) with slicing, a resolution of 2 μm [6] is feasible and may reveal even more detail. Conclusion The use of OPFOS imaging techniques and graphical 3D-reconstruction of the uticulo-endolymphatic (Bast’s) valve and its surroundings has given some additional insight in its functioning. It is most likely that opening or closure of the valve occurs through movement of the flexible base away from or toward the relatively rigid valve lip.

Eur_Radiol-3-1-1820761

Imaging oxygenation of human tumours

Tumour hypoxia represents a significant challenge to the curability of human tumours leading to treatment resistance and enhanced tumour progression. Tumour hypoxia can be detected by non-invasive and invasive techniques but the inter-relationships between these remains largely undefined. 18F-MISO and Cu-ATSM-PET, and BOLD-MRI are the lead contenders for human application based on their non-invasive nature, ease of use and robustness, measurement of hypoxia status, validity, ability to demonstrate heterogeneity and general availability, these techniques are the primary focus of this review. We discuss where developments are required for hypoxia imaging to become clinically useful and explore potential new uses for hypoxia imaging techniques including biological conformal radiotherapy. Introduction The suspicion that tumour hypoxia increased resistance to radiotherapy was first considered in the 1930’s but it was not until 1955 that Tomlinson and Gray showed that chronic hypoxia occurred in human bronchial carcinomas with necrosis occurring approximately 150 μm from blood vessels [1], which is a little larger than the currently known diffusion distance of soluble oxygen in tissues (approximately 70 μm). Decades of research in radiation therapy then followed, much of which focused on attempts to circumvent hypoxia-mediated radio-resistance but these efforts were only moderately successful. Over the last decade, it has become evident that hypoxia changes the patterns of gene expression in several ways that alters the malignant potential of tumours, leading to more aggressive survival traits. As a result, hypoxic cancers are difficult to treat, particularly by radiation and photodynamic therapy [2], but also by cytotoxic chemotherapy. Attempts at circumventing the cure-limiting impact of hypoxia have included the use of hyperbaric oxygen and radiation sensitizer drugs but these have, in general, not proved widely advantageous. However, attempts to take advantage of the presence of tumour hypoxia, such as hypoxia-specific cytotoxins, are more promising. As hypoxia-directed therapies enter into clinical trials, it has become important to non-invasively assess for the presence of hypoxia and to be able to follow how it is modulated by new therapies. Hypoxia imaging may help select the most appropriate population that would benefit from novel hypoxia-directed therapies. In this review we describe the causes for and the effects of tumour hypoxia, as well as summarise the lead contenders for human tumour imaging. We also assess where developments are required for them to become clinically useful imaging tests and explore potential new uses for hypoxia imaging techniques including biologically-directed conformal radiotherapy. Overview of tumour hypoxia & its importance For the majority of solid tumours hypoxia develops because of the inability of the vascular system to supply the growing tumour mass with adequate amounts of oxygen. Consequently, both low oxygen tensions and nutrient deprivation contribute to impaired tumour growth such that growth beyond 2 mm requires tumour neovascularisation. The major factors that play roles in the development of tumour cell hypoxia are the known abnormalities in structure and functioning of tumour microvessels [3], the increased diffusion distances between blood vessels (many of which may not even carry oxygenated red blood cells), the expanding tumour cell mass competing for oxygen and the reduced oxygen carrying capacity of blood due to disease- or treatment related anaemia. Thus, there are three distinct types of tumour hypoxia [4]: (1) Perfusion related (acute) hypoxia that results from inadequate blood flow in tumours that is generally the consequence of recognised structural and functional abnormalities of the tumour neovasculature. Such acute hypoxia is often transient, caused by temporary occlusions and temporary rises in interstitial pressure and can affect all cells right up to the vessel wall; (2) Diffusion related (chronic) hypoxia is caused by increased oxygen diffusion distances due to tumour expansion and affects cells greater than 70–100 μm from the nearest capillary, depending on where tumour cells lie in relation to the arterial or venous end of a capillary; (3) Anaemic hypoxia, which relates to reduced O2-carrying capacity of the blood and may be tumour associated or treatment related. The presence of hypoxia within human tumours before starting treatment has been observed in a variety of tumour types including squamous cell carcinomas, gliomas, adenocarcinomas (breast & pancreas) and in sarcomas. For example, in the normal cervix the pO2 is a median of 42 mmHg compared to a median of 10 mmHg in squamous carcinomas, and for cervix cancer the oxygenation status is independent of size, stage, histopathological type, and grade of malignancy [5]. Oxygen probes, that is, electrodes implanted directly into tumours to measure oxygen concentration by a polarographic technique [6–8] have shown (1) heterogeneity within and between the same tumour types of oxygen concentration and, (2) that hypoxia contributes to poor prognosis; pO2 < 10 mmHg results in poor local tumour control, disease-free survival and overall survival in squamous carcinomas of the head and neck and of the cervix [9, 10]. A large body of clinical evidence suggests that the hypoxia-mediated aggressive behavior of cancer cells and their resistance to therapy is orchestrated by the heterodimeric transcription factor, hypoxia inducible factor-1 alpha (HIF-1α), via a number of molecular events required for the adaptation of tumour cells to hypoxia (including unregulated glycolysis, angiogenesis and mutant p53) [11]. It is also important to realise that in some tumours including uterine lieomyomas, HIF expression is not always correlated with the presence of hypoxia suggesting that other factors including genetic events also contribute to activation of HIF, the most significant one of which is the loss of function of the Von Hippel-Lindau (VHL) tumour suppressor protein which results in constitutive activation of the HIF pathway. HIF-1 controls the expression of a variety of genes, the protein products of which play crucial roles in the acute and chronic adaptation of tumour cells to oxygen deficiency, including enhanced erythropoiesis & glycolysis, promotion of cell survival, inhibition of apoptosis, inhibition of cell differentiation, and angiogenesis. Thus, adaptive changes in the proteome and genome of neoplastic cells result in the emergence of more aggressive clones which are of cells that are more able to overcome nutrient deprivation or escape their hostile environments. Selection pressures by hypoxia and clonal expansion of the more aggressive cell types can result in exacerbations of regional hypoxia, further promoting the development of cell phenotypes that are treatment resistant (Fig. 1). Given the central role of HIF-1 in hypoxia mediated aggressive behaviour of cancer cells and their resistance to therapy, HIF-1 has become a target for the development of anti-cancer drugs [12]. Fig. 1Stylised diagram showing how hypoxia leads to therapy resistance and the development of an aggressive tumour cell phenotype. Figure adapted from [4] There is debate about whether there is a critical intratumoural pO2 below which detrimental changes begin to occur that is common across cell types. This occurs because experiments performed in cell cultures may not be applicable to in-vivo environments and some of the literature variation can be attributed to the tumour cell type chosen for experiments and the demands of host tissues. With these caveats in mind, the critical pO2 tensions below which cellular functions progressively cease or anticancer treatments are impaired are approximately as follows [13]: Effectiveness of immunotherapy becomes impaired (30–35 mmHg); Photodynamic therapy (15–35 mmHg); Cell death on exposure to radiation (25–30 mmHg); Binding of hypoxia immunohistochemical markers (10–20 mmHg); Proteome changes (1–15 mmHg) and Genome changes (0.2–1 mmHg). The differences in these numbers are smaller than the similarities so that, from a practical perspective, for solid tissue tumors in vivo, a value of between 5–15 mmHg is a good number to remember because of its impact on therapy. This number is in contrast to ischaemic hypoxia in the myocardium or stroke where detrimental effects are experienced at higher O2 [14]. In all these instances the critical oxygen level in tissues reflects the drive to match delivery with metabolic demand. As noted above, the presence of tumour hypoxia appears to impair the effectiveness of radiotherapy and radiosensitivity is progressively limited as tumour pO2 levels fall. Hypoxia-induced radioresistance is multifactorial with the presence of oxygen mediating DNA damage through the formation of oxygen free radicals which occurs after the interaction of radiation with intracellular water. The ratio of doses administered under well-oxygenated to hypoxic conditions needed to achieve the same biological effect (i.e., cell kill) is called the oxygen enhancement ratio (OER). For sparsely ionising radiations such as x- and gamma rays, the OER at therapeutic doses is between 2.5–3.5 [15]. That is, well oxygenated cells are about three times more sensitive to x- and gamma radiation than the same cells when they are hypoxic. Half maximal sensitivity to x- and gamma rays occurs at oxygen tensions of approximately 2–5 mmHg; above pO2 values of approximately 10–15 mmHg near maximal oxygen effects are seen. However, it should be recognised that sensitivity of cells to radiation is dependent on the phase of the cell cycle, with cells in the G1 phase having a lower OER (i.e., more radiosensitive) than cells in S-phase. As noted above, the oxygen effect is not the only mechanism for radioresistance in hypoxic tumour cells. Evidence is accumulating that the hypoxia-mediated proteomic and genomic changes may also contribute to radioresistance by increasing the levels of heat shock proteins (heat shock proteins (HSPs), are induced in response to environmental stresses like heat, cold and oxygen deprivation [16]) or by increasing the number of tumour cells that can resist apoptosis by mutating p53 (the slowing of cell division is dependent on a protein brake known as p53; the disruption of the functioning of this protein is associated with approximately 50–55% of human cancers). Clinical imaging of hypoxia As tumour hypoxia is an important biological characteristic and there is no good or easy clinical way to predict its presence, it has been suggested that imaging may be a good way of non-invasively selecting cancer patients who would benefit from treatments that overcome, circumvent or take advantage of the presence of hypoxia. Since tumour hypoxia is a key mechanism that leads to radioresistance, it has been repeatedly suggested that a hypoxia mapping technique could be integrated with conformal radiotherapy techniques to improve target delineation and dose delivery; this is discussed in more detail below. Imaging could also be used to document whether or not and the extent to which reoxygenation of tumours occurs during radiotherapy. Key requirements of any method that evaluates tumour hypoxia include non-invasive assessments that allow serial changes during treatment to be monitored and evaluation of heterogeneity between and within tumours. There are a number of ways in which tissue oxygenation status can be assessed in vivo (both invasive and non-invasive) or in vitro using material from biopsy. Non-imaging methods of assessing for the presence of hypoxia in tissues include histological appearance, immunohistochemical staining for intrinsic markers of hypoxia (e.g., carbonic anhydrase IX (CA-IX) and hypoxia inducible factor-1 (HIF-1)) and for the binding of externally administered nitroimidazoles [17, 18]. From an imaging perspective, an ideal test would: (1) distinguish normoxia / hypoxia /anoxia/necrosis, (2) distinguish between perfusion-related (acute) and diffusion-related (chronic) hypoxia if possible, (3) reflect cellular in preference to vascular pO2, (4) be applicable to any tumour site with complete loco-regional evaluation, (5) be simple to perform, non-toxic and allow repeated measurements, and (6) be sensitive at pO2 levels relevant to tumour therapy. Therefore, the challenge for hypoxia imaging is to measure low levels of tissue pO2 on a spatial scale similar to the O2-diffusion distance (70–100 μm); a much smaller dimension than can be achieved with human imaging techniques. Currently available MRI and PET methods were compared at a National Institute of Health/National Cancer Institute of the USA sponsored workshop in April 2004 and it was noted that only a few techniques have potential for in vivo assessment in humans particularly for repeated, sequential measurements. 18F-MISO and 60/64Cu-ATSM PET, and BOLD-MRI are the lead contenders for human application based on their non-invasive nature, ease of use and robustness, measurement of hypoxia status, validity, ability to demonstrate heterogeneity and general availability (Table 1). Table 1Comparison of techniques for evaluating human tumour hypoxiaTechnique and key referencesInvasive investigationRequires injectionMeasuresClinically Validated in RTGeneral availability (1–5; poor-wide)Monitors changes in pO218F-MISO PET [19, 24, 51–53]NoYesHypoxia±3No18F-AZA PET [54]NoYesHypoxiaNo2No18F-EF5 PET [55]NoYesHypoxia±2No64Cu ATSM [25, 26, 28, 34, 35]NoYesHypoxia±3NoBOLD-MRI [36]NoNo[dHb] in RBCsYes4YesPolorographic electrode [6–8]YesNopO2Yes2Yes 18F-MISO [18F]Fluoromisonidazole, 3-fluoro-1-(2′-nitro-1′-imidazolyl)-2-propanol or (18F-MISO), is the prototype hypoxia imaging agent whose uptake is homogeneous in most normal tissues, reflecting its high partition coefficient that nears unity, and whose delivery to tumours is not limited by perfusion [19]. The initial distribution of 18F-MISO is flow dependent, as with any freely diffusible tracer, but local oxygen tension is the major determinant of its retention above normal background in tissues after 2 hours (Fig. 2). 18F-MISO accumulates in tissues by binding to intracellular macromolecules when pO2 < 10 mmHg. Retention within tissues is dependent on nitroreductase activity (that is, on reduction status of a NO2 group on the imidazole ring) (Fig. 3) and accumulation in hypoxic tissues over a range of blood flows has been noted, including within the intestinal lumen where it is retained in anaerobes! Fig. 2The 18FDG-PET image (bottom left panel) shows increase uptake in both the oropharyngeal tumour (arrow) and in the left neck nodal metastasis (asterix). The 18F-MISO images (bottom right panel) were acquired in a dynamic mode and representative images after 1 minute, 30 minutes and 240 minutes are shown together with time-activity curves from the two regions of interest indicated in the FDG-PET image. The early distribution (1 minute) shows hyperperfusion in the region of the primary tumour and metastasis because of the high partition coefficient of 18F-MISO. After 2 hours, only the left neck nodal metastasis is shown to be hypoxicFig. 3The structure of [18F]-fluoromisonidazole, 18F-MISO, and its mechanism of retention in hypoxic tissues. The partition coefficient of 18F-MISO is near unity so the molecule diffuses freely into all cells. Once 18F-MISO is in an environment where electron transport is occurring (viable tissues), the –NO2 substituent (which has a high electron affinity) takes on an electron to form the radical anion reduction product. If O2 is also present, that electron is rapidly transferred to oxygen and 18F-MISO changes back to its original structure and can leave the cell. However, if a second electron from cellular metabolism reacts with the nitroimidazole to form the 2-electron reduction product, the molecule reacts non-discriminately with peptides and RNA within the cell and becomes trapped. Thus, retention of FMISO is inversely related to the intracellular partial pressure of O2 as shown in the lower left panel [45]. This mechanism is confirmed by the autoradiograph of a tumour spheroid (bottom right panel) with a radius of approximately 0.5 mm that shows no retention in the necrotic core or in the well-oxygenated outer sphere but intense uptake (white spots) in a donut like ring where cells are hypoxic Hypoxia can be imaged with 18F-MISO PET in a procedure that is well-tolerated by the patients. Imaging requires 20–30 min and starts anywhere from 75 to 150 min after injection, making it similar to the bone scan with which most cancer patients are familiar. Useful and well-validated images can be achieved with a modest dose of radiation, typically 250 MBq. No arterial sampling or metabolite analysis is required and synthesis is achieved through relatively simple modifications of nucleophilic displacement / deprotection synthesis boxes such as are used for fluoro-deoxyglucose (18F-FDG). In the USA, F-MISO has investigational new drug (IND) authorisation from the Food and Drug Administration (FDA) as an investigational product for use in humans. Unlike Eppendorf pO2 histography, 18F-MISO is only sensitive to the presence of hypoxia in viable cells; 18F-MISO is not retained in necrosis because the electron transport chain that reduces the nitroimidazole to a bioreductive alkylating agent is no longer active (Fig. 3). Limitations of 18F-MISO PET include the modest signal-to-noise ratio of raw 18F-MISO PET images but if a venous blood sample is acquired during the mid-course of the imaging procedure and used to calculate a Tumour:Blood (T/B) ratio image, then normoxic uptake (T/B < 1) can be electronically subtracted to increase image contrast. Several studies in a range of hypoxic tumours, stroke and hypoxic myocardium [14] have shown that a T/B of >1.2 reliably identifies the presence of hypoxia. The presence of high normal liver uptake impairs complete assessment of liver lesions and urinary excretion interferes with imaging near the bladder. 18F-MISO PET is able to monitor the changing hypoxia status of lung tumours during radiotherapy [20]. Studies in sarcoma [21] and head and neck cancer [22–24] have demonstrated a correlation of 18F-FMISO uptake with poor outcome to radiation and chemotherapy. Cu-ATSM Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) holds exceptional promise as an agent for delineating the extent of hypoxia within tumours with PET. Numerous pre-clinical studies have evaluated and validated its use for imaging of hypoxia in tumours and other tissues [25–32]. The mechanism of retention of the reagent in hypoxic tissues is largely attributed to the low oxygen tensions and the subsequent altered redox environment of hypoxic tumours (increased NADH levels) (Fig. 4). Clinical studies, well-tolerated by patients, involved 60Cu-ATSM imaging sessions of about 60 minutes with analysis of 30–60 minute summed-images. This time frame not only yields excellent data with good image quality (Fig. 4) in a very short time frame which opens up the opportunity with the shorter-lived 60Cu to perform multiple imaging sessions. A number of radioactive copper isotopes with longer half lives are available, e.g. 64Cu (t1/2 = 12.74 h) [33], enabling wide geographic distribution and the United States FDA recently approved an IND application for the study of 64Cu-ATSM for the imaging of hypoxia in human tumours. Fig. 4Retention mechanism of Cu(II)ATSM in hypoxic tissues. (a) Cu(II)ATSM is bioreduced (Cu(II) to Cu(I)) once entering the cell. The reduced intermediate species (likely to be [Cu-ATSM]-) is trapped within the cell because of its charge. This transient complex can then go through one of two competing pathways: reoxidation to the uncharged Cu(II) species (which can escape by diffusion), or proton-induced dissociation (which releases copper to be irreversibly sequestered by intracellular proteins). [Cu-ATSM]- favours the reoxidation route because it is easily oxidised but chemically more resistant to protonation. Copper from Cu(II) ATSM is trapped reversibly as [Cu-ATSM]- (if oxygen is absent), with the possibility of irreversible trapping by dissociation over a longer period. Cu(II)ATSM is thus hypoxia-selective. (b) High quality axial 60Cu-ATSM-PET image through the mid-upper thorax demonstrates heterogeneously increased uptake (arrow) within a known lung cancer in the aorto-pulmonary window and left suprahilar region. PET images representing summed data were obtained from 30 to 60 minutes after injection of 60Cu-ATSM In human studies of lung [34] and cervix cervical cancers [35], encouraging evidence has emerged that 60Cu-ATSM can act as a prognostic indicator for response to therapy. In the prospective study of 14 humans with non-small cell lung cancer, a semi-quantitative analysis of the 60Cu-ATSM muscle-to-tumour ratio was able to discriminate those likely to respond to therapy from non-responders [34]. A similar study in 14 women with cervical cancer demonstrated a similar predictive value in the tumour response to therapy [35]. BOLD-MRI Blood oxygenation level dependent (BOLD) and intrinsic susceptibility weighted MRI are interchangeable terms. As in any MR image, tissue contrast in BOLD images is affected by intrinsic tissue properties including spin-lattice and spin-spin relaxations. Additionally, BOLD MRI contrast is affected by blood flow and paramagnetic deoxyhaemoglobin within red blood cells (oxyhaemoglobin is not paramagnetic). Deoxyhaemoglobin increases the MR transverse relaxation rate (R2*) of water in blood and surrounding tissues thus BOLD-MRI is sensitive to pO2 within, and in tissues adjacent to perfused vessels [36]. Static tissue components include iron content (e.g. myoglobin found in muscle) and presence of fibrosis or ligamentous structures (e.g. in benign prostatic hyperplasia and the suspensory ligaments of the breasts) also affect the appearances of intrinsic susceptibility weighted images. In order to decouple the effects of flow from deoxyhaemoglobin and static components it is necessary to measure the T2* relaxation rate (R2* = 1/ T2*) which can be done by using a multi-echo GRE sequence (Fig. 5). Decoupling of flow from static effects on R2* images occurs because the flow component can be thought of as affecting individual T2* images of a multi-gradient echo sequence equally. It is important to remember that, although synthetic R2* images are free of the contribution of blood flow (that is, they mainly reflect deoxyhaemoglobin content and static tissue components), improving blood flow and vascular functioning will also increase tissue oxygenation, which can be seen by changes in R2* images. Fig. 5Data acquisition and quantification of BOLD-MRI in the prostate gland. Gradient recalled-echo MR images acquired at 1.5 T with a fixed repetition time and flip angle (TR ~100 msec; alpha 40 degrees) with lengthening echo-time (TE) are acquired through the prostate gland (bottom row of images). These images show increased susceptibility (T2*) effects with increasing TE. The rate of signal intensity decay in the dorsal aspect of the prostate is dependent on intrinsic T2* relaxation (local structure), deoxyhaemoglobin concentration [dHb] and local blood flow. Synthetic R2* (=1/T2*) images are created by plotting the natural logarithm of the signal intensity against the TE (top left panel). R2* map (top right panel) reflects on the structure of tissues and local [dHb] but inflow effects are minimised; however, R2* maps retain sensitivity to pO2 changes caused by alterations in blood flow The observations made in the previous paragraph imply two intuitive inferences. (1) BOLD-MRI images are more likely to reflect on acute (perfusion-related) tissue hypoxia which, as stated above, occurs because of transient occlusions of vessels, simply because hypoxic areas extend to the level of the blood vessels. In contradistinction, chronic hypoxia is less likely to be reflected by BOLD-MRI because the red blood cells in vessels are too distant from the area of hypoxia. (2) For BOLD-MRI to be able to inform on tissue oxygenation status, it is important for red blood cells to be delivered to the tissue in question. Human and xenograft studies have shown that tumour perfusion varies widely and that red blood cell perfusion is not simply related to the absence/presence of vessels; plenty of tumour vessels maybe present but perfusion by red blood cells may not occur [37]. This observation probably explains in part why no direct correlations between baseline R2* and tissue pO2 have been observed (that is, R2* does NOT measure tissue pO2). So it is necessary to know or to determine the distribution of blood volume in tissue in order to be able to correctly interpret R2* images in order to infer oxygenation status. Thus, if a tissue is perfused but has a high baseline R2* in one area/region compared to another area/region in the same tissue (i.e. the statistic components are the same), then one can infer that the high R2* region is relatively more hypoxic; this hypothesis is supported by recent preclinical and clinical data [38, 39]. As stated above, the use of BOLD-MRI for assessment of tissue hypoxia is predicated on the assumption that the oxygenation of haemoglobin is proportional to blood arterial pO2 which is in equilibrium with oxygenation of surrounding tissues. Many studies have shown that changes in R2* in response to vasomodulation with Carbogen (95% CO2:5% O2) inhalation, for example, are temporally correlated with changes in tissue pO2. Tumours differ in their responses to carbogen inhalation with only 50–60% of human tumours showing changes in R2* [40, 41]. The reasons for these limited and heterogeneous responses are complex but undoubtedly include the fact that tumours have adapted to widely different perfusion and that, even when vessels are present, red blood cell transport along these vessels may not be effective as demonstrated by Robinson et al. [37]. Thus, hypoxic tumours with high blood volume (due to high microvessel density coupled with large vessels) will not only have raised baseline R2* values but are more likely to respond to Carbogen. This will be reflected by large changes in R2*; and it is these hypoxic tumours that show positive radiosensitisation with Carbogen. On the other hand, hypoxic tumours with low blood volume (due to lower microvessel density, or due to small vessels) will have lower baseline R2* values and are thus less likely to respond to Carbogen. In this situation, there will be negligible changes in R2* and such hypoxic tumours do not show radiosensitisation with Carbogen [42]. Readers should also note that the BOLD response to Carbogen is also dependent on the ability of the underlying maturity of the vasculature with mature vessels able to respond actively to vasoconstrictory and vasodilatory stimuli [43]. The primary advantages of BOLD-MRI are that there is no need to administer exogenous radioactive contrast material and images at high temporal and with high spatial resolution can be obtained and repeated as needed. It is possible to decouple the effects of flow and deoxyhaemoglobin which are seen in native BOLD images and so to demonstrate changes in oxygenation independent of changes in blood flow. Major limitations of BOLD-MRI include the fact that they do not measure tissue pO2 directly (either in blood or tissues because of a non-linear relationship of R2* and tissue pO2), the images obtained have low signal to noise ratio and clinical studies with Carbogen vasomodulation are technically challenging (approximately 25–35% of patient examinations fail due to respiratory distress caused by an increased respiratory drive induced by Carbogen [40, 41]. BOLD-MRI appears most sensitive to oxygen levels adjacent to perfused vessels (that is, perfusion related or acute hypoxia) and BOLD-MRI sensitivity to more distant diffusion related or chronic hypoxia is an unknown. Hypoxia guided radiotherapy Recently introduced, technological improvements in radiotherapy delivery systems, including intensity-modulated radiotherapy (IMRT), have provided a means for shaping the dose distribution not only to the geometry of target volumes, and also to the differences in radiobiology across tumours [44]. Thus, it is now possible to define an additional “target within the target” as 3D pixel maps of the prescribed dose incorporating biological information derived from functional images; sometimes called dose painting by numbers [45, 46]. This allows treatment to the desired dose with escalation based on biologically relevant data, such as hypoxia which was discussed above, that is mechanistically related to therapeutic outcome. As an example of this, the spatial distribution of 60/64Cu-ATSM uptake on PET was successfully fused with CT radiotherapy planning images to show a proof of concept. This theoretical treatment planning would deliver higher doses of radiation via intensity modulated radiotherapy techniques (IMRT) to the most hypoxic regions of head and neck tumours [47]. In the case of hypoxia, it is important to remember that if the tumour stays hypoxic throughout the course of a treatment, more (escalating) radiation alone may not be sufficient to overcome the hypoxia-induced resistance. Hence, from a therapeutic perspective it is additionally important to know when and if reoxygenation occurs after initial radiotherapy (either by a reduction of oxygen consumption by the arrest of proliferation or by death of well oxygenated cells [15] (Fig. 6). However, data correlating treatment success with hypoxia and reoxygenation changes occurring during treatment are sparse; the extent and rapidity of reoxygenation is impossible to predict for individual tumours. The complexity of the processes leading to reoxygenation suggests the need for repeated imaging during the initial phase of treatment to determine the best time for dose adaptation. Koh et al. used 18F-MISO PET imaging to detect reoxygenation of some lung tumours after just a few treatments, whereas other patients exhibited no reduction in hypoxia even over the full course of radiotherapy [20]. Thus, dose escalation to target hypoxic areas in all patients at the beginning of the treatment could be a wasted effort because reoxygenation would change its distribution. Dose escalation is possibly best undertaken towards the end of treatment, at a time when hypoxic radioresistant cells are predominate. Fig. 6The re-oxygenation phenomenon. Tumours contain mixtures of aerated and hypoxic cells. Radiation is effective at eliminating well oxygenated cells because they are radiosensitive. Reoxygenation cause the preradiation pattern to return which can be eliminated by further radiation fractions but a progressive decrease in the tumour mass occurs after a series of fractions. Figure adapted from [15] Recently, Thorwarth et al. presented a study where dynamic (mapping of perfusion) and static (mapping of hypoxia) 18F-MISO scans were obtained in 15 patients with head-and-neck cancers who were subsequently treated with chemoradiation [48]. Their data suggested that subsequent treatment failure was related to both pretherapy hypoxia and concurrent poor perfusion. This result hints that reoxygenation did not occur as a consequence of the deficient vasculature of tumours [49]. Thus, it may be necessary not only to target hypoxic regions but also areas of poor blood flow. Dose escalation maps can be derived from dynamic 18F-MISO PET scans for targeting poor perfusion, while functional planning target volumes could be obtained from late static scans (that reflect hypoxia); both concepts lend themselves equally well to IMRT (Fig. 7). As a caution, simulated treatment plans also suggest that some patients may require such high radiation doses that will lead to a high risk of late complications in the vicinity of the tumour bed, such as necrosis, rupture of blood vessels or excessive fibrosis. Fig. 7Dose escalation map superimposed on CT with Planning Target Volume (red). Isodose lines show conformality to hypoxic lymph node with a maximum dose increase by 20%. This is the same patient illustrated in Fig. 2 Challenges for hypoxia imaging techniques As hypoxia imaging techniques move from academic research environments to routine clinical usage, it becomes important to recognise the unique challenges of clinical translation. For example, it is important that patient examination times are short to improve patient compliance particularly for repeated examinations. Thus, the need for doing dynamic scanning followed by several static scans over a prolonged period of time with PET techniques could prove a disincentive for patients. However, as noted above, not all patients undergoing hypoxia studies by PET need dynamic imaging although it may be useful in selected patients where the aim is to demonstrate both perfusion and hypoxia (for example to demonstrate reperfusion-reoxygenation). In-contradistinction, the interpretation of BOLD-MRI does require that the distribution of blood flow/volume is known and this can be done in clinical studies using dynamic contrast enhanced MRI (DCE-MRI) [50]. Whatever the chosen technique for clinical translation, there needs to be standardisation of imaging procedures and analysis methods in order to allow techniques to become more completely validated, for use in clinical trials. Amongst other issues that require addressing when clinical trials are being designed include the need for quantification, test-retest variability and data collection in body parts where there is a large degree of physiological movement such as the lungs and liver. A practical question often asked is whether it is necessary to quantify imaging data to answer important clinical questions. Subjective assessments work well enough in the clinic; however it is important to realise that subjective criteria cannot be applied simply from one centre to another particularly, when different equipment and imaging routines and human observers are used. Quantification techniques aim to minimise errors that can result from the use of different equipment and imaging protocols. Quantification techniques also enable the derivation of parameters that are based on some understanding of physiological processes and so can provide insights into tumour biology, for example the simple T/B and T/M ratios described for the two PET procedures. Quantification techniques are preferred when serial imaging studies are anticipated, for example when evaluating response to novel anticancer therapeutics. The reproducibility of the imaging technique should also be known in order to estimate the sample size required to evaluate therapy efficacy. Variation between measurements of the same quantity on the same individual can be caused either by measurement error or by physiological changes between measurements. Whilst it is possible (in theory) to reduce measurement error, physiological variation is inherent, and can cause difficulty in attempts to characterise disease or to monitor the effects of therapy. An estimate of measurement error enables us to decide whether a change in observation represents a real change. Data addressing the precision and measurement variability of hypoxia imaging techniques are urgently needed and should be an integral part of any prospective study that evaluates functional response to therapy to allow assessments of individual patients and group changes. It is intuitive that analysis and presentation of imaging data needs to take into account the heterogeneity of tumour hypoxia. The presence of motion can invalidate functional parameter estimates particularly for pixel-by-pixel analyses and this is especially true for high spatial resolution techniques such as BOLD-MRI. Motion is averaged in PET imaging because imaging times are long. In that case, pixel-analyses of the data and the issues of heterogeneity assessment can become less meaningful. The first step in heterogeneity analysis includes ROI definition which should be performed independent on the hypoxia imaging being assessed. For BOLD-MRI this could be done by anatomic MRI images and for PET studies could include the CT component of CT-PET studies although some groups have used ROIs defined in 18FDG-PET images. However, ROIs defined on 18FDG-PET images are know to be prone to error as far as tumour boundaries are concerned and are highly dependent on the level of threshold chosen. Whole tumour ROIs yield outputs with good signal-to-noise ratio, but lack spatial resolution and are prone to partial volume averaging errors and thus are unable to evaluate tumour heterogeneity. Pixel mapping has the advantages of an improved appreciation of heterogeneity of hypoxia and the risk of missing important diagnostic information and of creating ROIs that contain more than one tissue type is reduced. An important advantage of pixel mapping is being able to spatially map tumour characteristics such as hypoxia, glucose metabolism and blood flow and to be able to probe the spatial correlations between different kinetic parameters, providing unique insights into tumour structure, function and response to treatment. Conclusions To summarise, tumour hypoxia is common and its effects represents a significant challenge to the curability of human tumours, leading to treatment resistance and enhanced tumour progression. Tumour hypoxia can be detected by non-invasive and invasive techniques but the inter-relationship between these techniques needs to be better defined; human validation of the utility of hypoxia imaging is sparse at best. Anti-hypoxia therapies exist in the clinic and more are on their way. Either they don’t work very well or we don’t know how to use them optimally. Hypoxia imaging may allow better definition of a sub-population of cancer patients that would benefit for novel anti-hypoxia directed therapies.

Surg_Endosc-3-1-2169269

Comparison of three perioperative fluid regimes for laparoscopic donor nephrectomy

Background Pneumoperitoneum (PP), as used for laparoscopic procedures, impairs stroke volume, renal blood flow, glomerular filtration rate and urine output. This study investigated whether perioperative fluid management can abolish these negative effects of PP on hemodynamics. Laparoscopic donor nephrectomy (LDN) has become the method of choice to procure kidneys from living donors, mainly because of the reduced procedure-related morbidity and faster convalescence period [1–3]. Despite the benefits to the donor, there are concerns over the transient deterioration of renal function in the recipient of the kidney procured by the laparoscopic technique, compared with open donor nephrectomy (ODN) [2, 4–6]. The exact mechanism of delayed graft function after LDN is not fully understood. Pneumoperitoneum (PP) elevates intra-abdominal pressure (IAP), causing a decrease in renal blood flow (RBF) and glomerular filtration rate (GFR) resulting in oliguria [7–10]. In an animal model, London et al. have shown that PP resulted in a decrease in RBF during normal saline infusion, whereas RBF did not decrease if volume expansion was given [11]. From these results vigorous hydration up to 2 l/h of crystalloids during LDN in patients has nowadays been advocated [1, 12–15]. In 52 patients, Bergman et al. [16] found, however, no difference in graft function after LDN between aggressive (>10ml/kg/h) and conservative (<10 ml/kg/h) intraoperative fluid management. Volume loading after establishment of PP is perhaps too late to counterbalance the collapsed venal system. Biancofiore et al. [17] studied the effect of volume loading on graft function with a crystalloid infusion starting the night before surgery. Early graft function did not differ between ODN and LDN, although the serum creatinine declined earlier, but not significantly, in those receiving kidneys from ODN procedure. Fasting before operation and induction of anesthesia leads to relative hypovolemia and the goal is therefore to compensate this before PP is started. In this study, we compared three different fluid regimes in LDN patients, in which the effect of pre-hydration together with a bolus of colloids given just before induction of anesthesia and a second one just before inflation of PP on hemodynamics was of special interest. Methods Patients undergoing LDN from June 2001 to November 2001 (N = 21) were included in the study. The anesthetic procedure was performed according to a strict protocol for medication, ventilation and fluid regimen. In our hospital the donor patients are admitted the day before the operation, they are fasted during the night from 00:00 and operated on at 08.00 the next morning. Patients were randomized the day before operation by sealed envelopes by the responsible anesthetist to three different fluid regimens (Table 1): in group 1 fluid administration was started at 22:00 the day before operation with 3 ml/ideal body weight (IBW)/h Ringers lactate (RL) until operation. Before induction of anesthesia, the patients received 6 ml/IBW of colloid (6% HES 130/0.4); thereafter 13 ml/IBW/h RL was started until nephrectomy, before installation of PP another bolus of 6 ml/IBW colloid was given. Group 2 received overnight infusion in the same way as in group 1 with a bolus of 6 ml/IBW colloid just before induction. During operation, an infusion was started with 13 ml/IBW/h RL and 2 ml/IBW/h of colloid was given for three hours. Group 3 was fasted from 00:00 on the day of operation and received only an infusion during operation with 13 ml/IBW/h RL and 4 ml/IBW/h of colloid for three hours. After nephrectomy the infusion protocol was adjusted, so that exactly six hours after start of operation all the patients had received in total 9 ml/IBW/h RL. Patients were fitted with anti-thrombosis stockings. Table 1.Infusion protocols used in the three groupsGroupnml RL/IBW/h pre-hydrationaml RL/IBW/h preoperativebml HES/IBW before inductioncml HES/IBW before PPdml HES/IBW/h after PPeml HES/ IBW total1731366122721363 × 21237163 × 412IBW = ideal body weight n = number of patientsa Prehydration from 22:00 the day before laparoscopic donor nephrectomy, until operationb The amount of Ringer’s lactate given during operation, until nephrectomyc Amount of 6% HES 130/0.4 given before induction of anesthesiad Amount of 6% HES 130/0.4 given before installation of pneumoperitoneume Amount of 6% HES 130/0.4 given per hour after installation of pneumoperitoneumHES = 6% HES 130/0.4 Induction of anesthesia was performed with propofol (2 mg/kg) after a bolus of sufentanil (0.3 μg/kg). Muscle relaxation was achieved with rocuronium (0.8 mg/kg) and monitored by train-of-four (TOF) guard, a bolus of rocuronium (0.3 mg/kg) was given for three or more twitches. Anesthesia was maintained with propofol by continuous infusion (4–11 mg/kg/h), aiming at a bispectral index between 45 and 55 (BIS monitor; Aspect Medical Systems, Newton, MA, USA), and analgesia was achieved by continuous infusion of sufentanil (0.4 μg/kg/h) until nephrectomy. One hour after the start of operation 20 mg mannitol was given intravenously. After intubation all patients were ventilated in a pressure-controlled mode using a closed-loop ventilator (Physioflex®, Dräger, Lübeck, Germany) with the following initial settings: FiO2 of 0.4, positive end-expiratory pressure (PEEP) of 7 cm H2O and peak inspiratory pressure (PIP) of 22 cm H2O. Ventilation frequency was adjusted to keep PetCO2 between 4 and 5.5 kPa. After induction of anesthesia and before positioning of the patient, an esophageal Doppler probe (HemoSonicTM 100, Arrow International Inc., Reading, PA, USA) was positioned for measuring stroke volume (SV) and left ventricular ejection time, corrected for heart rate (LVETc) [18–20]. After positioning the patient in full lateral nephrectomy position, PP was installed with an IAP of 12 mmHg, which was constantly maintained at this level. All operations were done by the same team of anesthesiologists and surgeons. The surgical techniques have been described in detail elsewhere [21]. Mean arterial pressure (MAP) and SV (available after induction of anesthesia) were monitored noninvasively every five minutes. Urine output was measured from 22:00 the day before until the introduction of PP (T0), and was then measured every hour up to six hours thereafter (T1–6). Blood samples of the donors were collected to determine creatinine levels the day before operation, after induction of anesthesia, six hours after installation of PP, two days, one month, and one year after operation. Creatinine clearance (CrCl) was determined using the Cockcroft-Gault formula [22]. Statistical analysis Data analysis was performed using SPSS for Windows (version 14.0, SPSS Inc., Chicago, USA). Data are presented as means with standard deviation (SD). Differences between the groups were analyzed using the independent t-test, depending on Levene’s test, pooled or unpooled. Repeated measures with a general linear model from SPSS were used to assess significance for CrCl. A (two-sided) p-value < 0.05 was considered statistically significant. Results Baseline characteristics are shown in Table 2 and were comparable between the three groups. After induction of anesthesia, SV was significantly higher in both pre-hydrated groups compared to the control group (Fig. 1). After repositioning from supine to lateral, SV decreased significantly in the control group but not in groups 1 and 2 (Fig. 1). After installation of PP, SV remained stable in group 1 but not in groups 2 and 3 (Fig. 1). Table 2.Demographic data on the three groups, mean (SD)Group 1Group 2Group 3Age (yrs)56 (11)53 (9)55 (12)Weight (kg)72 (9)82 (8)74 (7)IBW (kg)70 (8)78 (8)71 (6)Male/female 2/54/33/4Operation time (min)237 (37)251 (46)226 (31)IBW = ideal body weightFig. 1.Stroke volume changes during laparoscopic donor nephrectomy, comparing three different fluid regimens: # symbol p < 0.05 for groups 1 and 2 versus control group 3; o symbol p < 0.05 for group 1 versus group 2; × symbol p < 0.05 versus supine position. Data are mean ± standard deviation. After induction, LVETc was higher in group 1 compared to the control group during the whole procedure and remained stable (Table 3). In all groups MAP decreased after induction of anesthesia; in the control group MAP decreased significantly more compared to group 1 (p = 0.03). HR was comparable between the three groups (Table 3). Table 3.Data on hemodynamic parameters: heart rate (HR), mean arterial pressure (MAP) and left ventricular ejection time (LVETc), mean (SD). Columns correspond to seven times: before anesthesia (preop); after induction of anesthesia, supine position (supine); full lateral position (lateral); and mean measurement for the first, second, third and fourth 30 minutes after installation of pneumoperitoneum (PP30, PP60, PP90 and PP120, respectively)PreopSupineLateralPP30PP60PP90PP120HR  Group 165 (6)65 (13)63 (20)60 (9)61 (8)60 (10)61 (9)  Group 263 (11)60 (9)61 (14)62 (11)61 (9)63 (6)63 (8)  Group 3 (control) 73 (9)69 (14)61 (6)65 (10)63 (6)60 (7)61 (6)MAP  Group 1102 (11)85 (13)* #81 (19)*93 (14)96 (15)94 (17)98 (20)  Group 2102 (14)73 (10)*81 (15)*107 (10)107 (18)104 (8)102 (12)  Group 3 (control) 105 (12)70 (11)*79 (7)*103 (9)98 (11)95 (12)95 (15)LVETc  Group 1336 (19)#339 (20)#355 (26)#345 (31)335 (30)336 (33)  Group 2313 (29)308 (39)295 (58)°311 (20)°308 (27)300 (28)  Group 3 (control) 294 (26)284 (31)292 (26)307 (18)309 (18)311 (25)* p < 0.05 versus preoperative values# p < 0.05 groups 1 and 2 versus control group° p < 0.05 group 1 versus group 2 Urine output, measured from the start of operation until the moment of kidney extraction, was 1.9 ml/kg/h (range 1.2–3.2) for group 1, 1.4 ml/kg/h (range 0.8–2.3) for group 2, and 1.1 ml/kg/h (range 0.6–1.6) for group 3. In controls, the urine production was significantly lower compared to group 1 (p = 0.01). CrCl decreased in the control group directly after PP, but not in the other groups (Table 4). From two days postoperative, CrCl was comparable between the three study groups (Table 4). Table 4.Creatinine clearance [mean (SD)] in the three groups at six measurement points: one day before operation; just after induction of anesthesia; at 14:30, six hours after installation of pneumoperitoneum T6; two days after operation; one month after operation; and one year after operationGroup 1Group 2Group 3 (control)CrCl preop (ml/min)89 (19)101 (25)102 (34)CrCl after induction (ml/min)104 (18)109 (20)96 (31)CrCl after the operation (ml/min)87 (17)94 (14)73 (23)ªCrCl D2 (ml/min)63 (10)*65 (11)*64 (19)*CrCl after 1 month (ml/min)63 (12)*67 (11)*63 (24)*CrCl after 1 year (ml/min)71 (13)71 (13)*66 (25)** p < 0.05 versus preoperative valuesª p < 0.05 difference between CrCl preop and CrCl after operation, control group versus groups 1 and 2 Discussion This study showed that during LDN preoperative hydration together with a bolus of colloid given before induction of anesthesia and before installation of PP resulted in higher SV and higher urine output compared to a fluid regimen with only an intraoperative aggressive fluid infusion. The second group, which received no bolus of colloid before PP in contrast to group 1, showed a significant reduction in SV after installation of PP. In the control group, LVETc and urine output at the moment of kidney extraction showed significantly lower values compared to both pre-hydration groups. CrCl values six hours after the start of the operation was significantly reduced in the control group compared to preoperative values but not in the two pre-hydrated groups; this difference was reduced two days postoperatively. Clinical studies yield conflicting data concerning the effect of LDN on recipient graft function compared to ODN. The largest study to date compared more than 5,000 kidney transplants from a database and found that LDN was associated with slower early graft function compared to ODN. However, renal function and graft survival at one year was similar between both groups. This was confirmed by retrospective analysis of 120 LDN and 100 ODN in our own institution in which serum creatinine in the recipients was significantly higher in the LDN group only in the first week after transplantation [4]. One very important discriminating factor between ODN and LDN is the pneumoperitoneum. From experimental studies it has become clear that PP decreases RBF and that the magnitude of this decrease is affected by the IAP used, the volume status, and positioning. To counterbalance the increased IAP, vigorous intravenous hydration during LDN is nowadays recommended in an attempt to optimize preload and promote diuresis, but randomized clinical data are missing. In a porcine model, Demyttenaere et al. [23] showed that the decrease in SV and renal cortical perfusion could be prevented by a simple hydration of 15 ml/kg/h saline combined with a bolus 20 ml/kg saline, in accordance with the findings of London et al. [11]. This was also seen in the present study in which pre-hydration with a normal infusion with crystalloids during operation combined with a bolus of colloids just before PP did not decrease SV but improved diuresis. Besides PP, the kidney lateral decubitus position, which is an anti-Trendelenburg position, contributes to hemodynamic alterations by decreasing preload through the effect of gravity on venous return [24]. Yokoyama et al. [25] found no significant change in hemodynamic values after postural change of their patients from supine to lateral but a significant reduction in SV after postural change to kidney position; these patients received a fluid regime of 20 ml/kg/h of crystalloids. This was confirmed by our study in which the control group showed a significant reduction in SV after postural change from supine to kidney position whereas there was no reduction in the two pre-hydrated groups, which received a bolus of colloid just before induction (Fig. 1). After pre-hydration with crystalloids we infused colloids to achieve optimal plasma expansion just before installation of PP [26]. In our hospital we use 6% HES 130/0.4 for fluid expansion, because the rate for anaphylactic reactions is considerably lower than for gelatin products [27]. However, there are concerns that infusion of certain HES types may influence kidney function [28]. As long as adequate hydration using sufficient amounts of crystalloids are used, the latest generation of HES products (6% HES 130/0.4) do not increase the risk for renal dysfunction even when used in large amounts [29, 30]. Lang et al. [31] even demonstrated that 6% HES 130/0.4 improved tissue oxygenation during and after major surgical procedures compared with a crystalloid-based volume strategy. In this study, we used the HemoSonicTM, a transoesophageal Doppler ultrasonography (TOD) device, to measure blood flow in the descending aorta. Several studies have confirmed good correlation with cardiac output measured by the thermodilution technique [18, 32]. It has been shown that the accuracy of the device is somewhat operator-dependent [20] and therefore the same two people did all the measurements with this device in the present study. Feldman et al. [33] used LVET to guide their fluid management in LDN patients. In the present study it was shown that LVETc was significantly lower in the control group that did not received pre-hydration and increased over time (Table 2). It should, however, be taken into account that the blood flow with this device is measured in the descending aorta, which is around 70% of the total cardiac output. This could influence our measurements if redistribution of flow away from the descending aorta occurs because of elevated IAP and this is more pronounced in hypovolemic patients. Some other limitations of this study should be noted. In only four patients a MAG3 scan was performed, which provides the distribution of the function from the two kidneys of the donor. In the four measured patients the harvested kidney contributed 43–48% of the total kidney function, these four patients were divided over all three study groups. However because we do not have the data on the other patients, this could have biased our data on postoperative CrCl. Prehydration of the donor patients conform our protocol, started the night before operation, which contradicts fast-track surgery where kidney donor patients are admitted to hospital on the day of surgery. Also these patients can receive adequate pre-hydration, but further research should be done. In this study we focused on intraoperative hemodynamic changes. Our data show that preoperative hydration together with a colloid bolus given before induction of anesthesia and before installation of PP resulted in higher SV and higher urine output during LDN, compared to controls that received only an aggressive intraoperative infusion. While under-hydration may contribute to renal dysfunction, perioperative fluid excess can also cause problems, such as pulmonary edema, ileus and increased risk of cardiopulmonary and wound healing complications, which might result in longer hospital stay [34]. However, there is a need to ensure adequate hydration status during PP without being overaggressive. First, our fluid regime will be tested in a large prospective study in order to prevent the negative effect of PP on early graft function in the recipient, and to study possible side-effects in the donor.

J_Occup_Rehabil-3-1-2039785

Loss of Productivity Due to Neck/Shoulder Symptoms and Hand/Arm Symptoms: Results from the PROMO-Study

Introduction The objective of the present study is to describe the extent of productivity loss among computer workers with neck/shoulder symptoms and hand/arm symptoms, and to examine associations between pain intensity, various physical and psychosocial factors and productivity loss in computer workers with neck/shoulder and hand/arm symptoms. Introduction Neck/shoulder and hand/arm symptoms are a common problem in society, in particular among the working population. In the European Union, in 2000 and 2001, 23% of the working population reported work-related pain in neck or shoulders. The percentage reporting work-related muscular pain in the upper limbs was about 13% in the old member states and 20% in the new member states [1]. Although it is not clear to what extent work-related factors contribute to their origin, their impact on working life is huge. Neck/shoulder and hand/arm symptoms can interfere with activities at work, and can cause sickness absence and chronic occupational disability. In the Netherlands in 2001, incident cases of chronic disability for work due to neck and upper limb symptoms added up to 0.1% of the working population, and 6% of the total number of new disability benefits [2]. Besides sickness absence and chronic disability, neck/shoulder and hand/arm symptoms could also lead to reduced work effectiveness. Many workers still go to work despite the feeling that, in the light of their health, they should have taken sick leave. This phenomenon is known as sickness presenteeism [3]. Although they are present at work, their productivity could be reduced due to functional limitations. The extent of productivity loss while present at work is uncertain, but it has been suggested that it accounts for the majority of lost productivity costs associated with chronic pain [4, 5]. Therefore, more knowledge is needed to estimate the magnitude of productivity loss associated with neck/shoulder symptoms and hand/arm symptoms. Not all musculoskeletal symptoms involve sickness absence or productivity loss. It would be interesting to know more about factors that might influence productivity loss in symptomatic workers. We are inclined to think that these factors might be similar to risk factors for the occurrence of symptoms or for sickness absence due to musculoskeletal symptoms. However, this is not necessarily true and these factors could easily diverge. Knowledge on both types of risk factors is important for primary, secondary and tertiary prevention. While knowledge on risk factors for the occurrence of symptoms is required to prevent them, knowledge on factors associated with productivity loss is needed in the process of retention, reintegration and rehabilitation of workers with symptoms. Previous studies have shown that pain characteristics, like pain intensity or severity, were predictive factors for a poor prognosis of musculoskeletal symptoms [6–9]. Therefore, it seems plausible that high pain intensity will also have an impairing effect on productivity. Besides pain characteristics, physical, psychosocial and personal factors might affect productivity. In previous studies these factors were already identified as risk factors for the occurrence of symptoms [10–14]. However, as mentioned earlier, this does not necessarily mean they also affect productivity loss in symptomatic workers. The present study is different from studies examining risk factors for sickness absence, as productivity is a broader outcome measure. Moreover, the study population of this study consists of symptomatic workers, and distinguishes between symptomatic workers with and without productivity loss. Studies examining risk factors for sickness absence are usually held in a mixed study population and usually distinguish healthy and symptomatic workers from workers with sickness absence. The objective of the present study is to describe the extent of productivity loss among computer workers with neck/shoulder symptoms and hand/arm symptoms, and to examine associations between pain intensity, various physical and psychosocial factors and productivity loss in computer workers with neck/shoulder and hand/arm symptoms. Methods Study Population Data were used from the baseline measurement of the PROMO-study: Prospective Research On Musculoskeletal Disorders in Office Workers [15]. The main purpose of this study was to determine risk factors for neck/shoulder symptoms and hand/arm symptoms among computer workers. The study design was approved by the Medical Ethics Committee of the VU University Medical Center. The study included workers from five different companies. The five participating companies comprised an insurance company, a department of a university, a public transport company, a brewery, and a financial consultancy firm. Employees from these companies had administrative, professional or management jobs. Altogether approximately 9,000 employees were working in these companies. All employees were invited to participate in the study. To maximize the participation rate, various activities were arranged, varying from the dissemination of brochures to visits at the worksite. Almost 2,500 employees subscribed to the project and signed informed consent. They were requested by e-mail to fill out an electronic questionnaire, accessible via the Internet. Participants who did not want to fill out an electronic questionnaire could fill out a paper version. Out of these employees, 1,951 filled out the questionnaire at baseline, resulting in a response rate of 79% of the subscribed employees. In the PROMO-study, productivity loss due to neck/shoulder and hand/arm symptoms was self-reported and only assessed in workers reporting symptoms. Therefore, analyses concerning associations between various factors and productivity were limited to respondents reporting work-related neck/shoulder symptoms or hand/arm symptoms during the past three months. This selection contained 654 computer workers. Productivity Loss Productivity loss was only assessed for those respondents that reported regular or prolonged neck/shoulder symptoms or hand/arm symptoms in the past three months. A dichotomous variable was constructed, based on the answers on the following questions: (1) ‘Have your symptoms slowed down your work pace?’; (2) ‘Have your symptoms decreased your working hours?’; or (3) ‘Have your symptoms caused disability to work for one or more days?’ These questions were adapted versions of items used in the Swedish questionnaire used in the epi-mouse study. In the epi-mouse study, questions on productivity loss were validated through interviews [16]. They were asked for neck/shoulder symptoms and for hand/arm symptoms separately and referred to the past three months. If one or more of these questions were answered affirmative, it was defined as productivity loss. It was assumed that respondents answering that their symptoms decreased their working hours (2), but did not cause disability for work (3), had not been on sick leave. Only if the question concerning disability for work was answered affirmatively, it was defined as sickness absence. Neck/Shoulder Symptoms and Arm/Hand Symptoms In the questionnaire subjects were separately asked about symptoms in the neck/shoulder region and in the arm/hand region. The reason to separate these regions is the expected difference in relationship between symptoms and computer usage; computer usage seems to have more effect on arm/hand symptoms than on neck/shoulder symptoms [17]. In the present study the distinction between the regions was maintained, because the different relationship with computer usage might result in different effects on productivity loss. Subjects were asked to rate the occurrence of pain or discomfort in the neck/shoulder region as well as in the hand/arm region in the previous 3 months on a four-point scale: ‘no, never’; ‘yes, sometimes’; ‘yes, regularly’; ‘yes, prolonged.’ Subsequently, subjects were asked to estimate whether these symptoms were related to their work, which they could answer with ‘yes, completely’; ‘yes, partly’; ‘possibly’ or ‘no.’ Also, 8 possible specific causes of these symptoms were summed: sport injuries, accidents, skin diseases, a twist or sprain, a cut or burn, a congenital defect, rheumatic disorders and a slipped disc. Neck/shoulder symptoms and arm/hand symptoms were defined as regular or prolonged pain, completely, partly or possibly related to work, and not caused by any listed specific cause. Independent Variables To examine which factors are associated with productivity loss in computer workers with neck/shoulder and hand/arm symptoms, analyses were carried out with the following variables: pain intensity, physical activity in leisure time (with BMI as a possible confounder), working hours, mouse position, psychosocial load and overcommitment. Intensity of Symptoms The intensity of symptoms was measured using Von Korff scales [18]. Respondents were asked to indicate the mean intensity of their symptoms in the past three months on a scale ranging from 0 (no pain) to 10 (worst pain ever). If they reported symptoms in both neck/shoulder and hand/arm region, the region with the highest score was used in the analyses. Physical Activity and BMI Concerning physical activity, the respondents were asked about the number of days per week they usually performed activities of at least moderate intensity, and about the times per week they usually performed activities of vigorous intensity. One variable with three categories was constructed measuring physical activity. The categories were ‘no significant physical activity,’ ‘moderate intensity physical activity, but no physical activity of vigorous intensity,’ and ‘vigorous intensity physical activity.’ Moderate intensity physical activity was defined as performing physical activity causing increased breathing for at least 30 min per day with a frequency of at least 5 days per week [19, 20].Vigorous intensity physical acitivity was defined as performing physical activity causing sweating for at least 20 min per session with a frequency of at least 3 days per week [21, 22]. BMI was computed by body weight (kg) divided by square of height (m2). Data on weight and height were self-reported. Working Hours Respondents were asked how many hours per week they worked according to their contract. A dichotomous variable was constructed that distinguished between full-time workers working 37–40 h and part-time workers working 4–36 h per week. Mouse Position Respondents were asked about the position of their mouse while using it. This question was illustrated with five possible positions for respondents to select, and a category ‘other position.’ A dichotomous variable was constructed that distinguished between a mouse position close to the keyboard, and positions further away from the keyboard. This dichotomization corresponds to the dichotomization in previous research of Hagberg et al., who defined nonoptimal computer mouse position as: “Those who marked their computer mouse position outside a rectangle close to the operator in a workplace layout figure in the questionnaire” [16]. Psychosocial Load To assess psychosocial load, two concepts were used, namely effort-reward imbalance and job satisfaction. For the first concept Siegrist’s Effort-Reward Imbalance model (ERI) was used [23, 24]. The theory of this model is that a combination of high effort and low reward could lead to adverse health effects. Effort and reward were measured with the recommended scales of the ERI-model [25]. A validated Dutch version of the questionnaire was used [26] with scores varying from 1 (‘agree’) to 5 (‘disagree, and I am very distressed’). Many different methods have been used to construct a variable indicating ERI [27]. In this study a variable in four categories was constructed: no high effort and no low reward, high effort (but no low reward), low reward (but no high effort) and both high effort and low reward. High effort was assigned when a respondent reported to be distressed or very distressed about one or more of the 5 effort items. Low reward was assigned when a respondent reported to be distressed or very distressed about one or more of the 11 reward items. Cronbachs α was 0.68 for the effort scale and 0.80 for the reward scale. To assess job satisfaction respondents were asked how they enjoyed their work. To answer this question four categories were presented: ‘never,’ ‘sometimes,’ ‘often,’ and ‘always.’ This variable was dichotomized, resulting in a positive score on job satisfaction containing the responses ‘often’ or ‘always,’ and a negative score containing the responses ‘never’ or ‘sometimes.’ Personal Factor—Overcommitment Concerning personal factors, the personality trait overcommitment was assessed. The concept “overcommitment” specifies those cognitive, emotional and motivational components within the global concept of Type A behavior that are important in coping with work demands. Overcommitted workers may expose themselves more often to high demands at work, or they may exaggerate their efforts beyond what is formally needed [25]. Overcommitment was assessed with the short version of a standard questionnaire [23–25]. Subjects were asked if they strongly disagreed, disagreed, agreed or strongly agreed on 6 items (e.g., I get easily overwhelmed by time pressures at work). Scores were dichotomized (agree versus disagree) and the added scores on these items resulted in an overcommitment score ranging from 0 to 6. Cronbachs α of the overcommitment scale was 0.74. Analysis First, descriptive statistics were used to examine how often symptoms resulted in productivity loss, and to describe the components of productivity loss in terms of sickness absence and decreased performance at work. The association between productivity loss and several determinants was examined with logistic regression analyses using productivity loss as the dichotomous outcome variable. Univariate and multivariate analyses were carried out. In all multivariate analyses, age, gender, level of education, and intensity of symptoms were included as covariates. For the analysis with physical activity, BMI was checked for confounding and for the analysis with the psychosocial work characteristics, the other psychosocial work characteristics were checked for confounding. If their inclusion in the model did not result in a change of more than 10% in the effect estimate, these covariates were not considered as a confounder and not included in the final model. Results Productivity Loss Figure 1 shows that neck/shoulder symptoms were reported more frequently than hand/arm symptoms. Of the total population 10% reported both symptoms. On average, in 26% of the cases reporting symptoms, productivity loss was involved. If both symptoms were reported, they were more often accompanied by productivity loss (36%). Fig. 1Distribution of neck/shoulder and hand/arm symptoms in a population of computer workers (n = 1,951), and the fraction involving productivity loss within workers reporting symptoms Figure 2 shows what part of productivity loss was caused by sickness absence and what part was caused by a decreased performance at work. A decreased performance at work means decreased speed or decreased working hours, but no sickness absence. In 32% of all cases reporting productivity loss, this productivity loss was coming from sickness absence. Sickness absence occurred more frequently in workers reporting both symptoms (43%). Among workers reporting arm/hand symptoms, and no neck/shoulder symptoms, productivity loss was composed mainly of decreased productivity at work. Only 11% of these workers reporting productivity loss have been on sick leave during the last 3 months. Fig. 2Distribution of productivity loss due to neck/shoulder symptoms, hand/arm symptoms or both in a symptomatic population of computer workers (n = 654) and the fraction involving sickness absence within symptomatic workers reporting productivity loss Associations with Productivity Loss Table 1 shows odds ratios resulting from the logistic regression analyses. A higher odds ratio represents a higher probability of productivity loss. Pain intensity and psychosocial load, i.e., high effort and low job satisfaction were associated with productivity loss in computer workers with neck/shoulder or arm/hand symptoms. In the analyses of the psychosocial work characteristics, the other psychosocial work characteristics were identified as confounders, while inclusion of physical activity in the model did not change the odds ratios for more than 10%. Table 1Associations with productivity loss in a symptomatic population of computer workers (n = 654)% (n)Crude OR (95%CI)Adjusted* OR (95% CI)R2N if item deletedFull model**.140Pain intensity    Continuous measure (0–10)1.24 (1.13–1.37) 1.26 (1.12–1.41) .104Physical activity in leisure time    No significant physical activity63 (408)1.001.00.131    Moderate intensity (5 × 30 min/week)22 (141)0.73 (0.46–1.17)0.74 (0.43–1.27)    Vigorous intensity (3 × 20 min/week)16 (99)1.48 (0.92–2.39)1.27 (0.70–2.27)Working hours    Part-time (4–36 h)61 (399)1.001.00.135    Full-time (37–40 h)39 (255)1.37 (0.96–1.96) 1.36 (0.87–2.11) Mouse position    Close to the keyboard35 (229)1.001.00.134    Other position65 (425)0.93 (0.64–1.34) 0.70 (0.45–1.08) Psychosocial loadEffort-Reward Imbalance    No high effort, no low reward40 (258)1.001.00.115    No high effort, low reward20 (130)1.81 (1.10–2.99)1.43 (0.76–2.67)    High effort, no low reward18 (119)1.89 (1.14–3.16)2.26 (1.24–4.12)    High effort, low reward21 (138)2.69 (1.68–4.32)1.95 (1.09–3.50)Job satisfaction/task enjoyment    Always21 (138)1.001.00.110    Often65 (423)1.29 (0.80–2.08)1.62 (0.89–2.92)    Never/sometimes14 (93)3.10 (1.72–5.58)3.10 (1.44–6.67)Overcommitment    Continuous measure (0–6)1.06 (0.96–1.16) 1.09 (0.97–1.22) .140Abbreviations: OR, odds ratio; CI, confidence interval; R2N: Nagelkerke R2 index of global model fit (0 = lack of fit, 1 = perfect fit)* Adjusted for gender, age, level of education and intensity of symptoms; the analyses with effort-reward imbalance were additionally adjusted for job satisfaction and the analyses with job satisfaction were additionally adjusted for effort-reward imbalance** The full model contained gender, age, level of education and all variables mentioned in the table Physical activity in leisure time, working hours, mouse position and overcommitment were not associated with productivity loss in computer workers with neck/shoulder symptoms or arm/hand symptoms. Additional adjustment for BMI in the analyses with physical activity did not result in a change in odds ratio for more than 10%. Discussion The purpose of this study was to describe the extent of productivity loss among computer workers with neck/shoulder symptoms and hand/arm symptoms and to examine associations between various physical, psychosocial and personal factors and productivity. The results show that in 26% of all cases reporting regular or prolonged symptoms in the past three months, productivity loss was involved. Most productivity loss was found in workers reporting both neck/shoulder symptoms and hand/arm symptoms. Overall, about 32% of the productivity loss was coming from sickness absence. Sickness absence occurred more frequently in workers reporting both symptoms (43%) and considerably less frequent in workers reporting only hand/arm symptoms (11%). Symptomatic workers reporting unfavorable psychosocial work characteristics reported more productivity loss. Comparison with Previous Research The study population of the present study consisted of office workers with neck/shoulder or hand/arm symptoms. So far, almost all studies using productivity loss as an outcome measure were studies in a mixed population, containing subjects with and without musculoskeletal symptoms. Moreover, most previous research on productivity loss concerned sickness absence, while in the present study a decreased productivity while working was also included. Therefore, the results of previous studies are hard to compare with the present study. In a mixed population, associations between productivity loss and potential risk factors partly reflect the risks for symptom occurrence. In the present study we wanted to examine which factors are related to decreased productivity once symptoms have occurred. These factors do not have to be similar. Nevertheless, we will mention the results of previous research in this section. In the present study, physical activity in leisure time was not significantly associated with productivity loss in workers with neck/shoulder or hand/arm symptoms. We did not find other studies in a symptomatic working population. A few studies have examined the relation between physical activity and productivity loss in a general population. These studies often found a favorable effect of physical activity. A favorable effect was found of sporting activity on sick leave in general [28], and in particular sick leave due to musculoskeletal disorders [29, 30]. Also an association was found between higher levels of physical activity and job performance [31]. It seems that physical activity has a preventive effect and is positively associated with productivity, but does not affect productivity loss in workers already having symptoms. The present study did not examine the association between physical load and productivity loss extensively. Our hypothesis was that computer workers with a high physical load (for example adverse ergonomic working conditions or sustained computer or mouse usage) might experience more productivity loss. Conversely, some studies in a general working population examining the effect of the improvement of ergonomic working conditions have shown beneficial effects on productivity [32, 33]. An observational study found a weak association between an adverse mouse position and productivity loss [16] in workers with musculoskeletal symptoms. The present study could not confirm the relation between an adverse mouse position and productivity loss. It might be more interesting to examine the relation between productivity loss and duration of computer use, since studies show that duration is more consistently associated with musculoskeletal symptoms than posture [10, 17]. However, the design of the present study is not suitable to examine this relationship. The results of the analyses would produce a biased view, because in a population of computer workers, productivity loss means almost automatically less computer usage. Preliminary analyses confirmed this assumption: in the subpopulation of symptomatic computer workers, those reporting productivity loss also reported less computer usage, in particular mouse usage. Therefore, analyses examining the association between computer usage and productivity loss would result in a negative association, i.e., more computer usage, less productivity loss. However, we did examine the association with part-time versus full-time work, because this variable is probably not biased. Respondents were asked how many hours they worked according to their contract. The distinction might indicate a form of physical load, assuming that full-time workers are exposed to a higher physical load. Nevertheless, no relation was found between productivity loss and part-time/full-time work. No previous studies were found examining this relationship. Psychosocial load, in this study defined as effort-reward imbalance and job satisfaction, was strongly associated with productivity loss. No studies were found that examined these psychosocial work characteristics in relation to productivity loss or sickness absence due to musculoskeletal symptoms. Also, no studies were found that examined the relation between psychosocial work characteristics in general and productivity loss, other than sickness absence. Finally, no studies were found that examined these relations in a symptomatic population. In mixed populations, containing subjects with and without musculoskeletal symptoms, the relation between psychosocial load and sickness absence in general was demonstrated by several studies [34–36]. Few studies have examined the relation with sickness absence due to musculoskeletal symptoms, and their results were conflicting [37, 38]. Limitations of the Study In the present study, self-reported data were used to assess productivity loss. Productivity loss in this study contains two components: absenteeism and presenteeism. Self-reported data on absenteeism have been found to be reliable and valid when the recall periods are short [39]. However, measuring presenteeism is more complex. For most types of employment, there is no objective account of productivity with which to assess an employee’s performance. In the present study computer usage was measured by a software program. Therefore, in this study population of computer workers it might be considered to use computer usage as a measure for productivity. We decided not to do so, because we assumed that differences in computer usage would be larger between jobs than between workers with and without productivity loss. For this reason, differences in computer usage would probably reflect different kinds of jobs instead of differences in productivity. The lack of objective instruments to measure productivity also hampers the validation of self-reported measures. One study, using a rather similar productivity measure, compared self-reported productivity loss assessed with a questionnaire with interviews of 50 computer users who had reported musculoskeletal symptoms [16]. The results seem to indicate that productivity loss assessed in the questionnaire could be a slight underestimation. Another limitation concerning the productivity measure is its dichotomy. No distinction could be made between a minimal and a major loss of productivity. It would be more accurate and it would provide more insight into the magnitude of the problem, if the precise size of the productivity loss was assessed. However, it is very hard for workers to estimate the size of their productivity loss. A recent review concluded that no study has shown that employees can accurately transform their perceived impairments into a quantitative measure [39]. Therefore, since objective instruments to measure productivity are lacking, we are forced to limit ourselves to a subjective dichotomous measure of productivity. Although in the PROMO-study follow-up measurements were available, we choose to use a cross-sectional design. The main reason is that data on productivity loss were only available for workers with symptoms. Since symptoms generally have an episodic nature the selection of workers with symptoms at baseline is not identical to the selection of workers with symptoms at follow-up. To select workers with data on productivity at baseline as well as at one or more of the follow-up measurements would mean a selection of workers with symptoms at all these measurements. Such a selection would result in a small study population of workers with chronic symptoms. If we assume that the associations in this study are causal, still the direction of causality cannot be established due to the cross-sectional design. It could be that either psychosocial working conditions caused productivity loss, or productivity loss caused the reporting of adverse psychosocial working conditions, or both. Intervention studies focusing on the prevention of productivity loss among symptomatic workers might shed more light on the direction(s) of causality. Implications for Practice The results of this study show that employers should be aware that the consequences of neck/shoulder and hand/arm symptoms are more extensive than the visible sickness absence due to these symptoms. Only one third of workers who experience productivity loss due to their symptoms actually take sick leave. For the other workers productivity loss expresses itself in decreased performance at work. The results concerning factors associated with productivity loss are more difficult to interpret, since there is still a lack of knowledge on how they relate. It seems that symptomatic workers perform better in favorable psychosocial working conditions. An advantageous psychosocial climate might prevent productivity loss in symptomatic workers. In conclusion, most workers with neck/shoulder symptoms or hand/arm symptoms experience productivity loss from a decreased performance at work and not from sickness absence. Favorable psychosocial work characteristics might prevent productivity loss in symptomatic workers.

Eur_Spine_J-2-2-1602191

Mycoplasma hominis deep wound infection after neuromuscular scoliosis surgery: the use of real-time polymerase chain reaction (PCR)

Mycoplasma hominis is a commensal of the genitourinary tract. It mostly causes infections to associated structures of this system; however, occasionally it is a pathogen in nongenitourinary tract infections. Since, M. hominis strains require special growth conditions and cannot be Gram stained, they may be missed or delay diagnosis. This report describes a deep wound infection caused by M. hominis after neuromuscular scoliosis surgery; M. hominis was recovered by real-time polymerase chain reaction (PCR). An awareness of the role of M. hominis as an extragenital pathogen in musculoskeletal infections, especially in neuromuscular scoliosis, being a high-risk group for postoperative wound infection, it is necessary to identify this pathogen. Real-time PCR for postoperative deep wound infection, in patients with a history of genitourinary infections, decreases the delay in diagnosis and treatment. In these cases rapid real-time PCR on deep cultures should be considered. Introduction Deep wound infections complicating spinal surgery are sources of major morbidity in neuromuscular scoliotic patients. Enterobacter, Enterococcus, Escherichia coli, Proteus and Staphylococci account for the majority of postoperative spinal wound infections in these patients [28]. Increasingly, previously uncommon pathogens are being identified in surgical infections. These organisms may be difficult to identify and/or resistant to most of the broad-spectrum antibiotics used for preoperative prophylaxis and for the treatment of postoperative wound infections. Among these pathogens, Mycoplasma hominis has been recognized as a cause of postoperative wound infections [25, 27, 31]. M. hominis is a commensal bacterium in humans and is distinguished phenotypically from other bacteria by the minute size and lack of a cell wall. As a result, M. hominis cannot be Gram stained and is resistant to penicillin and other antibiotics that interfere with the cell wall metabolism. In addition, it is a fastidious slow-growing organism, which may not be readily identified by using routine culture protocols. Early diagnosis, however, is of utmost importance for adequate institution of appropriate antimicrobial therapy. To our knowledge, only two cases of postoperative spinal wound infections due to M. hominis have previously been reported [18]. We describe a case report of a M. hominis postoperative deep wound infection in a patient with a progressive myelomeningocele scoliosis who underwent posterior scoliosis surgery with bone allograft. Real-time polymerase chain reaction (PCR) provided a fast and secure diagnosis, which prevented further complications. Case presentation An 11-year-old girl with a complete paraplegia at level L2–L3 caused by a myelomeningocele was admitted to our hospital for surgical correction of a progressive right convex scoliosis. Preoperative physical examination revealed a flexible right convex thoracic scoliosis with a left convex thoracolumbar curve. The unsupported sitting anteroposterior radiograph showed a right convex thoracic scoliosis of 55°and a left convex thoracolumbar curve of 42°with no pelvic obliquity. Her medical history revealed a shunted hydrocephalus with an Arnold Chiari type II malformation, chronic urinary tract infections, and an auto-augmentation of the bladder. The patient was treated with single stage scoliosis correction involving posterior instrumentation (Xia™ spinal system, Stryker Spine, Cestas, France) from T3 to L5. The spondylodesis was completed by applying allograft bone chips (Netherlands Bone Bank Foundation, Leiden, The Netherlands) over the laminae in the thoracolumbar region. In addition, Collagraft® (Neucoll, Campbell, CA, USA), a synthetic bone graft substitute composed of collagen and a composite mineral (hydroxyapatite and tricalcium phosphate), was applied posterolateral in the lumbosacral region. Prophylactic antibiotics, cefazolin (cefalosporin, Kefzol®) 1,000 mg IV, were administered at the induction of anaesthesia, and as a second and third dose 8 and 16 h postoperatively, respectively [6]. Because of a chronic urinary tract infection including a positive culture with E. coli, cefradin (cefalosporin, Velosef®) 500 mg orally was continued for 9 days at 8-h intervals. The postoperative course was uneventful. At day 8 the patient was discharged. At discharge the wound produced negligible clear fluid at the distal end without any signs of infection. However, at day 18 postoperatively, the patient developed fever (39°C) and was readmitted to our centre. On physical examination, an enlarged distal wound dehiscence was seen with increased fluid production. Infection parameters showed an increased C-reactive protein (CRP) (120 mg/l; normal <10 mg/l) and white blood cell (WBC) count (10.9 cells×109/l; normal 4–10 cells×109/l). A surgical intervention was performed including a thorough debridement of necrotic tissue and removal of the bone grafts. The instrumentation was left in place. According to the established guidelines, cultures were taken of various deep areas by fluid aspiration and from the applied bone graft [15, 28, 32]. After extensively irrigating with pulsatile lavage, the wound was closed leaving gentamicin collagen fleeces (Septocoll®, Biomed, Darmstadt, Germany) over the instrumentation. Adjuvant therapy with gentamicin (aminoglycoside, Garamycinl®) 210 mg IV and flucloxacillin (isoxazolylpenicillin, Floxapen®) 2,000 mg IV were initiated immediately after obtaining appropriate intraoperative cultures, and was continued postoperatively at 6-h intervals. Intraoperative cultures from both the fluid aspiration and the bone graft yielded Mycoplasma after 4 days incubation. Real-time PCR on the peri-operative biopsy material was only positive for Mycoplasma and the species was identified by 16S DNA amplification as M. hominis (Fig. 1). Separately a Collagraft® sample of the same batch was tested for Mycoplasma, however, the culture as well as real-time PCR remained negative. Gentamicin and flucloxacillin were stopped, and doxycycline (tetracycline, Vibra-S®) 100 mg was administered orally at 12-h intervals and continued for 3 weeks. The patient was discharged 5 days after surgery. Fig. 1Agarose gel analysis of duplo PCR amplification of Mycoplasma DNA. M Molecular weight marker, Lanes 1 and 2 positive PCR amplification of clinical sample, lane 3 negative control, lane 4 positive control. Subsequent sequencing results revealed M. hominis Wound healing and temperature were monitored at regular intervals at the outpatient clinic. After 3 months, the infection parameters were normalized and the wound was healed (Fig. 2). Four years after surgery there were no signs of infection. Radiographs at final follow-up demonstrated no instrumentation failure and no loss of correction of the scoliotic deformity (Fig. 3).Fig. 2Clinical appearance 3 months after surgical intervention for deep wound infection, located at the distal end of the woundFig. 3AP radiographs of the lumbar spine at the site of the deep wound infection immediately after surgical intervention and debridement of necrotic tissue including removal of the bone grafts (a) and at 4 years of follow-up (b) Discussion Mycoplasma hominis is a commensal bacterium in humans. The organism is commonly associated with infections of the genitourinary tract, particularly in females. Rates of colonization in the urogenital tract range from 21 to 54% among women and from 4 to 13% among men [20]. Reports on M. hominis infections outside the genitourinary tract are scarce. Most of them are case reports describing extragenital infections, such as septic arthritis [17, 21], septicemia [11, 12], prosthetic valve endocarditis [3, 10], postoperative wound infections [25, 31], peritonitis [13], neonatal encephalitis [30], meningitis [7, 9], brain abscess [33], thrombophlebitis [27], and mediastinitis [19]. Extragenital infections are mainly related to patients with immunosuppression [3, 7, 9, 17, 19, 21, 25, 27, 30, 33]. Osteomyelitis caused by M. hominis is predominantly reported in combination with hypogammaglobulinemia [5, 14, 16, 24]. In an extensive overview of nongenitourinary M. hominis infections, Madoff and Hooper [18] described only two cases of postoperative deep wound infections caused by M. hominis after orthopedic surgery. Both cases concerned with deep wound infections after scoliosis surgery, one of them with a history of pyelonefritis. Postoperative deep wound infections after scoliosis surgery are more common in neuromuscular patients than in patients with idiopathic scoliosis [28]. Risk factors associated with increased postoperative wound infection rates include a generalized decline in the immune status of neuromuscular patients, poor personal hygiene, and soiling of the wound. Sponseller et al. [28] reported 25 patients (12%) with a deep wound infection out of a series of 210 surgically treated patients with neuromuscular scoliosis in a 10-year retrospective study. From these 25 patients, 16 had a scoliosis related to myelomeningocele. Two risk factors were found to be significant: the degree of cognitive impairment and use of bone allograft. Furthermore, 52% of the infections were polymicrobial, which could point to contamination during or after surgery. However, the authors did not report any case of M. hominis wound infections in their series. Antibiotics are used routinely to prevent postoperative wound infection in patients undergoing spinal implant procedures. Currently, first or second-generation cephalosporin (e.g. cefazolin) are recommended as prophylaxis [6]. Prophylactic cefazolin was also routinely given in our case. Since, cephalosporins act on the cell wall of organisms in a manner similar to the penicillins, postoperative wound infection by M. hominis was not prevented. Antimicrobial treatment for M. hominis include protein-synthesis inhibitors such as tetracycline (e.g. doxycycline) [26] and doxycycline was administered accordingly. The postoperative deep wound infection resolved favorably after surgical debridement and appropriate antimicrobial treatment with doxycycline. The source of M. hominis deep wound infection in our case is unclear. M. hominis is commonly associated with infections of the genitourinary tract [20] and patients with meningomyelocele are known to have a high incidence of urinary tract infections [8]. In addition, it has been shown that children with myelomeningocele have a high incidence of urological complications after surgical treatment of scoliosis [4]. Possibly, in our case the M. hominis could have been present in the urinary tract infection and spread hematogenous or by contamination, either at the time of surgery or secondarily through the wound. Other possible sources are the materials that were used during surgery. Obviously, the instrumentation and the applied Collagraft®, were sterilized. In addition, cultures and PCR on the same batch of the Collagraft® were negative. Bone allograft, used to induce and facilitate spinal fusion, could also have been the source of the infection. The fact that the bone allograft was positive for M. hominis, proved that it was infected before retrieval, however, not that it was the source of the infection itself. Bone allograft has not been reported as a carrier of M. hominis in the literature, however, it must be noted that the standard screening at the Netherlands Bone Bank Foundation does not include screening on M. hominis. Unfortunately, no other specimen of the donor could be retrieved for testing. As a result, haematogenous spread or contamination from a colonized urogenital tract or contamination by the bone allograft as source of the infection could not be excluded. In the presented case, cultures taken during the reintervention proved to be positive for Mycoplasma. However, the diagnosis of a Mycoplasma infection was delayed due to the fastidious nature of mycoplasmas. To identify the Mycoplasma species, which in our case was cultured, 16S DNA amplification was used. Because isolation of M. hominis is difficult, time consuming, and not routinely done, a rapid specimen processing is required. Real-time PCR on deep cultures could provide a rapid alternative with a higher sensitivity and specificity than culture for the detection of M. hominis [1, 2]. In conclusion, M. hominis infections should be considered in postoperative deep wound infection after neuromuscular spinal surgery, especially in patients with genitourinary tract comorbidity. Since, M. hominis is not covered by routine prophylactic and therapeutic antibiotics, rapid real-time PCR is advised in these patients to initiate appropriate antibiotic treatment.

J_Mol_Biol-1-5-1885968

Global Changes in Local Protein Dynamics Reduce the Entropic Cost of Carbohydrate Binding in the Arabinose-binding Protein

Protein dynamics make important but poorly understood contributions to molecular recognition phenomena. To address this, we measure changes in fast protein dynamics that accompany the interaction of the arabinose-binding protein (ABP) with its ligand, d-galactose, using NMR relaxation and molecular dynamics simulation. These two approaches present an entirely consistent view of the dynamic changes that occur in the protein backbone upon ligand binding. Increases in the amplitude of motions are observed throughout the protein, with the exception of a few residues in the binding site, which show restriction of dynamics. These counter-intuitive results imply that a localised binding event causes a global increase in the extent of protein dynamics on the pico- to nanosecond timescale. This global dynamic change constitutes a substantial favourable entropic contribution to the free energy of ligand binding. These results suggest that the structure and dynamics of ABP may be adapted to exploit dynamic changes to reduce the entropic costs of binding. Introduction An important goal of structural biology is to predict and manipulate the interactions between proteins and small-molecule ligands. To this end, extensive research efforts have been aimed at relating the known structure of protein–ligand complexes to the thermodynamics of that interaction. The success of these attempts has been limited, in part because of their neglect of the role of protein and ligand conformational dynamics in determining ligand-binding thermodynamics. A common view of protein–ligand interactions sees them arising from the hydrophobic effect (the entropically favourable exclusion of water from hydrophobic surfaces) together with shape complementarity between the protein and ligand. In this view, protein–ligand interactions are expected to be driven by favourable changes in entropy. Recent results suggest closer scrutiny of this view is warranted, even for predominantly non-polar interactions.1,2 One can contrast this former view of protein–ligand interactions with one that considers the restriction in conformational degrees of freedom, which must necessarily be entailed by binding. From this perspective, an unfavourable change in entropy is expected to characterise these interactions. This requires a larger, favourable enthalpic contribution, which may arise directly from the protein–ligand interaction or from solvation effects. The thermodynamic signature of protein–ligand interactions in this second view is typical of protein–carbohydrate interactions: entropic contributions to the interaction are typically large and unfavourable, and favourable enthalpic contributions drive the interaction. An example of such a system is the arabinose-binding protein (ABP). ABP is a member of the bacterial periplasmic binding protein family and serves as the initial component of the active transport system for the monosaccharides l-arabinose, d-fucose and d-galactose. Several members of this family, including ABP, have served as models for structural analysis of ligand-binding interactions,3,4 and have recently emerged as templates for design of novel ligand-binding proteins.5 ABP binds its ligands in the interface of two protein domains. The binding site displays an extensive network of H-bond and CH–π interactions characteristic of protein–carbohydrate association. Both structural and thermodynamic aspects of this interaction have been studied extensively,4,6–9 yet there is no clear understanding of the basis of either the large favourable binding enthalpy or the unfavourable entropy change on binding. Results and Discussion Entropic cost of binding It is evident that formation of a stable complex between a protein and a ligand will involve the loss of entropy associated with the free diffusion of one component with respect to the other. The magnitude of these unfavourable contributions to the protein–ligand interaction may be only approximated; here, we take an estimate of the loss of ligand translational and rotational entropy from the work by Turnbull et al and Lundquist and Toone, which converge at approximately 25 kJ/mol for the equivalent free energy penalty.10,11 We note that this estimate is consistent with a number of others in the literature, for systems ranging from small polar molecules to folded and unfolded proteins.12–15 It is generally assumed that the bound ligand will adopt a single conformation, optimised to the structure of the binding site, and thus will experience a loss of entropy reflecting the loss of conformational flexibility of the ligand in solution. Likewise, the protein-binding site might be expected to undergo a loss of entropy as it adopts only that subset of available conformations that are conducive to binding. This loss of internal conformational entropy of the ligand is challenging to assess, particularly for a flexible ligand such as galactose, which displays complex conformational behaviour in solution. Nonetheless, on the assumption that ligand degrees of freedom are substantially “frozen” on binding, the entropic penalty arising from loss in degrees of freedom of the galactose hydroxyl rotors alone is likely to be ∼ 30 kJ/mol.11 The ABP binding site contains a significant number of tightly bound water molecules, which play a role in governing substrate specificity,9 and in maintaining the structure of the binding site. Examination of the structures of ABP in complex with arabinose (1ABE), fucose (1ABF) and galactose (5APB) reveals some 15 crystallographically resolved and structurally conserved water molecules within the binding site (within 10 Å of a ligand heavy atom and within 5 Å of heavy atoms of both protein domains; Figure 1). The entropic cost of confinement of water molecules has been estimated to be as much as 8 kJ/mol per water molecule.16 Even if the cost of confinement of the water molecules in the binding site of ABP is much less than this maximal value, it is clear that the overall cost of constraining these water molecules in the binding site will be substantial. The experimentally observed entropy of the ABP–galactose interaction amounts to a TΔS° of –61 kJ/mol at 308 K.6 Together, the entropic costs above exceed significantly this experimental entropy change on binding. Similar discrepancies, in which measured entropies of association are more positive (i.e. more favourable) than might be expected from first principles, have been known for at least 50 years.17–19 Most often, the discrepancy has been attributed to solvation effects, such as the hydrophobic effect or desolvation of charged groups.17,18 It has been noted, however, that these discrepancies might be explained by the introduction of new degrees of freedom in the complex, which did not exist in the interacting partners before interaction.19 We investigate this latter possibility by examining the contribution of protein dynamics to the entropy of interaction of ABP with its ligands. Assignment of apoABP We have reported the determination of near-complete backbone resonance assignments for ABP in complex with its ligand, d-galactose.20 Assignments for apoABP were determined from these results, using an approach that combined conventional triple-resonance assignment strategies and 1H-15N heteronuclear single quantum coherence (HSQC) titrations of ABP with the fast-exchanging ligand 1-deoxy-galactose. Approximately 80% of the expected backbone resonances of apoABP were assigned. From these assignments, a comparison was made of the chemical shifts of the backbone amide resonances of ABP in the apo state and in the complex (Figure 1). As expected, large chemical shift changes are seen in the binding site. In addition, large chemical shift changes are seen in the region linking the two domains of ABP, suggesting that ligand binding might be associated with a change in relative domain orientation in ABP. Domain reorientations on binding are observed in other members of the periplasmic-binding protein family, and have been proposed for ABP on the basis of the results of small-angle X-ray scattering and computational studies.21,22 Small chemical shift differences are seen at sites distal to the binding site and hinge region. These differences suggest that subtle changes in conformation or dynamics throughout the molecule occur on binding. Domain orientation of apoABP Analysis of NMR relaxation for anisotropic molecules depends on knowledge of the molecular structure. It is therefore necessary to address the possibility of ligand-induced domain reorientation in ABP. We have done this using residual dipolar couplings (RDCs), which are sensitive to the average molecular orientation with respect to molecular alignment induced by a liquid crystalline solution.23 1H-15N RDCs were measured for 127 backbone amides in regions of defined secondary structure of apoABP. The measured RDCs were initially compared to those predicted on the basis of the crystal structure of ABP in complex with galactose. Agreement was poor (RMSD = 13 Hz, R-factor = 65%), confirming that conformational change does indeed occur on ligand binding. The extent of domain reorientation in ABP was determined using a structure calculation protocol in which the structure of each domain was minimised individually against the measured RDCs, followed by a simulated annealing procedure in which each domain was held rigid in its minimised conformation. This protocol was repeated 100 times with data generated by a Monte Carlo re-sampling of the experimental RDC data to assess the robustness of the protocol and the precision of the resulting structures. The agreement between the RDCs and the calculated structure is excellent, with an RMSD of 1.2 Hz and an R-factor of 6.0%. The quality of the structure is good, with 97% of residues in the core and allowed regions of the Ramachandran plot, and all residues showing correct peptide bond geometry. The backbone RMSD over the 100 structures derived from Monte Carlo re-sampling of the RDC data is 0.67 Å. The resulting ensemble of structures (Figure 2) showed apoABP to be “opened” by a domain hinge motion of ∼ 20° with respect to the crystal structure of the ABP–galactose complex. This represents a conformational change similar to those seen in other members of the periplasmic-binding protein family. In addition, small changes to individual domain structures are required for optimal agreement with RDCs. This supports the inference from chemical shift changes, that ligand binding has subtle conformational consequences throughout the protein. Dynamics of ABP The contribution of protein dynamics to the ABP–galactose interaction was assessed by means of Lipari–Szabo analysis of nuclear magnetic relaxation.24,25 This approach yields information on the extent and timescale of molecular motions occurring faster than the rotational diffusion of the protein. The Lipari–Szabo formalism is relatively free of assumptions regarding the physical model describing the motion under investigation, requiring only that the dynamics be described by a Markov process and that the internal dynamics is uncorrelated with the global tumbling of the macromolecule. The results of the Lipari–Szabo analysis, in the form of an order parameter, can be interpreted in terms of the conformational entropy associated with the measured motions by means of a specific motional model. It has been shown that for a wide range of models, the functional dependence of entropy on the order parameter is similar, suggesting that changes in order parameter can be related to entropy change in a model-independent fashion.26 We have measured relaxation data for 148 backbone amides of apoABP at three magnetic fields, and for 156 backbone amides of the ABP-galactose complex at two magnetic fields. The analysis of these data in terms of the Lipari–Szabo formalism yields order parameters that measure the extent of angular motion of individual amide bond vectors (Figure 3(a) and (b)). In both apoABP and the complex we see almost exclusively the large order parameters typical of the relatively rigid backbone of globular proteins. Remarkably, order parameters for apoABP are generally larger than for the ABP–galactose complex, indicating that pico- to nanosecond motions are more extensive in the complex than in the apo protein. Owing to the size of ABP and the associated spectral complexity, many residues have been excluded from the analysis, because spectral overlap precludes accurate measurement of relaxation rates. For this reason, no experimental data are available for residues involved directly in binding. Thus, our observations reflect changes in dynamics remote from the binding site, yet caused by the protein–ligand interaction. Indeed, the observed changes are seen to be distributed throughout the protein, albeit with a slight bias towards the N domain (in the sense that larger dynamic changes are seen preferentially in this domain) (Figure 3(c)). To confirm and further explore the basis of this result, we have performed molecular dynamics simulations for ABP in the apo state and in complex with galactose. By several measures, we see significant increases in backbone dynamics in the complex as compared with the apo protein. RMS deviations from the average structure for both the N and C domains are significantly larger for the ABP–galactose complex than for apoABP. In addition, fluctuations of backbone heavy-atom positions across the trajectory are generally larger in the complex than in the apo protein (data not shown). Furthermore, these dynamic changes are seen to be more pronounced in the N domain than the C domain, consistent with the experimental observations. To make a direct comparison between the experimentally observed order parameters and the simulations, backbone amide order parameters have been calculated from the molecular dynamics trajectory.27 We see good agreement between measured and calculated order parameters, albeit with a small tendency for the simulation to underestimate the experimental values (Figure 3(a) and (b)). Underestimation of measured order parameters by molecular dynamics simulations has been a common observation in previous comparisons between molecular dynamics simulation and NMR relaxation measurements, particularly in loops and other flexible regions.27–30 This small systematic discrepancy is of little importance in this instance, as we are interested principally in the difference in order parameter between the two states. Indeed, the changes in order parameter upon ligand binding reproduce the measured changes excellently, showing an approximately uniform decrease in order parameter upon binding across much of the protein (Figure 3(c)). The average change in order parameter (S2apoABP–S2ABPgal) determined by the two methods is identical, at 0.038; the uncertainty on this average value, derived by propagation of experimental uncertainties in the individual order parameters, is 0.005. The correlation coefficient between the experimental and simulated order parameters is 0.83 for the complex protein and 0.47 for the apo-protein. As well as validating our experimental results, the molecular dynamics simulations reveal details of dynamic changes in regions that could not be measured experimentally. Notably, the simulations reveal complex changes in dynamics in the binding site (Figure 4(b)). Several residues in the binding site display increases in flexibility upon binding, consistent with the trend seen throughout the rest of the molecule. Other binding site loops display a decrease in flexibility, more in keeping with the intuitive expectation that ligand binding will reduce the conformational freedom of binding site residues. A significant assumption entailed by the Lipari–Szabo analysis performed here is that the rotational diffusion of the protein can be fully characterised by a single diffusive process uncorrelated with the internal motions under investigation.24 This assumption is generally robust for globular, single-domain proteins, but might be called into question in the case of ABP, where flexibility of the domain hinge may result in a complex interaction between local protein conformation and rotational diffusion. In the case of the ABP–galactose complex, we assume such flexibility to be insignificant, as the ligand binds in and stabilises the domain interface. No such assumption can be made for apo ABP, however. In an attempt to assess the influence of inter-domain flexibility in apoABP on our results, we have repeated the Lipari–Szabo analysis, assuming each domain undergoes independent rotational diffusion. The expectation is that the apparent rotational diffusion of each domain should differ from that of the molecule as a whole if significant inter-domain flexibility is present. The best-fit diffusion tensors arising from the analysis of the two individual domains are very similar, but are slightly different from the diffusion tensor derived from the whole protein (Table 1). This suggests that inter-domain flexibility has only a minor influence on the measured relaxation rates. The orientation of the diffusion tensors for each domain is consistent with the RDC-derived structure of apo ABP, highlighting a consistency between the RDC and relaxation data that would not be expected if the relaxation data were influenced substantially by domain motions. Importantly, the Lipari–Szabo dynamic parameters derived from this analysis are identical, within error, with those of the initial analysis. Furthermore, we note that inter-domain flexibility can, to a first approximation, be accounted for by the so-called extended Lipari–Szabo treatment.25 Here, inter-domain flexibility is treated by the slow dynamic component (S2s, τs), while internal motions are treated by the fast dynamic component (S2f).31,32 Because the reported overall order parameter is the product of the fast and slow order parameters (S2LZ = S2f S2s), the effect of inter-domain flexibility (S2s < 1) will always be the overestimation of the extent of internal motions. As such, if domain flexibility in apoABP does influence our analysis, its effect will be the underestimation of the increase in internal dynamics upon ligand binding. In this context, it is of interest to note that there are more residues that are best treated by the extended Lipari–Szabo model in apoABP than there are in the ABP–galactose complex (Table 2). Additional evidence that inter-domain motions have little impact on our overall results is obtained from the MD simulations. The contributions of rotational diffusion to the simulated dynamics are removed in the conventional way by alignment of the protein at each snapshot to a single reference structure. Alternatively, contributions of both rotational diffusion and inter-domain flexibility can be removed by separately aligning a single domain to a reference structure before analysis of the internal dynamics of that domain. There is no significant difference between these two approaches in terms of the calculated order parameters (data not shown). This reflects the small amplitude of the domain flexibility observable in the production phase of the apo ABP trajectory. On this basis, it is likely that domain flexibility is either very small or occurs on a timescale slower than is detectable in the 20 ns simulations analysed here. Given the anomalously low viscosity of the TIP3P water model used in these simulations, the rate of domain flexibility in the simulation is likely to significantly overestimate the in vitro rate. Thus, the failure to detect significant domain flexibility suggests that the relevant timescale is slower than the overall rotational diffusion of ABP (∼ 15 ns), and therefore is not expected to contribute to the measured relaxation data. A further caveat of the analysis of NMR relaxation data concerns its insensitivity to dynamics occurring on a timescale similar to or slower than the rate of rotational diffusion. Given this insensitivity, it is conceivable that the apparent increase in flexibility of ABP on ligand binding may in fact represent a shift in the dynamic timescale. This would imply that the dominant timescale for motions in apoABP is slower than ∼ 15 ns, but that this shifts to a much faster timescale on ligand binding. There are two reasons to discount this interpretation of our data. Firstly, we consider it most likely that a timescale shift of the type considered here would result in a shift from predominantly simple Lipari–Szabo models in the apo-protein to a preponderance of complex models in the ligand-bound form, as new observable motional modes are introduced by the change in timescale. In fact, precisely the opposite trend is observed (Table 2). Secondly, we note that there is no evidence of extensive slow timescale motions in our molecular dynamics studies. The presence of dynamics on this timescale would be reflected by inadequate sampling over the course of our 20 ns trajectories. In fact, using the convergence test proposed by Best et al.,29 the calculated order parameter is judged to have converged in all but nine residues in apo ABP, and in all but four residues in the ABP–galactose complex. All residues for which dynamics are not converged are in flexible loops in ABP. To understand the thermodynamic implications of the observed changes in dynamics, we exploit the relationship between Lipari–Szabo order parameter (S2LZ) and conformational entropy (Sconf) derived by Yang and Kay.26 Of the 306 residues of ABP, we determined an entropy change associated with the measured change in pico- to nanosecond dynamics for 84 residues. Entropy change for the remaining residues could not be determined either because S2LZ could not be determined reliably for both the apo and ligand-bound states (203 residues), or because S2LZ > 0.95 in one or both states (19 residues). These data are plotted in Figure 5. The average entropy change per residue is 2.0(± 0.3) J/mol K. This amounts to an overall entropy change of 170(± 30) J/ mol K for the measured residues (TΔSconf = 52(± 9) kJ/mol at 308 K). If it is assumed that this average entropy can be applied to all un-measured residues, and that dynamics changes for each residue are not correlated, the total entropy change from changes to pico- to nanosecond motion on ligand binding amounts to 610(± 120) J/mol K (TΔSconf = 188(± 37) kJ/mol at 308 K). Clearly, this latter value amounts to an overestimate, as the assumption of un-correlated motion is not likely to hold for all residues of the protein. It is evident, however, that the entropy change associated with the change in pico- to nanosecond dynamics contributes significantly to the favourable free energy of binding, and in effect amounts to protein dynamics paying some of the unfavourable entropic cost of ligand binding. The experimental data available for this system is limited to probes of backbone dynamics. Given the good agreement between experiment and the molecular dynamics simulations, it is perhaps reasonable to infer something of the side-chain dynamics from these simulations. We observe considerably more variability in side-chain order parameters than is evident for the backbone, consistent with findings in other proteins. Furthermore, there is much greater variability in the change in order parameter observed upon ligand binding. Despite this, the changes in side-chain dynamics are broadly similar to those seen for the backbone, with the majority of residues showing a small increase in flexibility on ligand binding. As fast side-chain dynamics are correlated with local backbone dynamics only weakly, this suggests an additional source of favourable entropy change accompanying binding. A number of residues around the binding site show larger changes in dynamics on binding, reflective of both increases and decreases in flexibility. These changes reflect similar heterogeneous dynamic changes seen in the protein backbone of the loops that comprise the binding site. One particularly surprising aspect of these results is the dispersed and approximately uniform nature of the change in dynamics. This is unexpected, because fast motions in proteins, such as the pico- to nanosecond dynamics under study here, are almost exclusively local in character, with few if any correlations over distances longer than a few ångström units.30 One plausible explanation for the uniform nature of the observed effect is that it is an artefact arising from a systematic error in the determined rotational diffusion tensor for the apo and/or holo protein. To exclude this possibility, we have repeated the analysis using the approach described by Schurr et al., which fits the relaxation data for each residue individually, with a so-called local τm term to account for rotational diffusion.33 In this analysis, the sets of dynamic parameters determined for each residue are statistically independent of one another, and are independent of any structural model for the protein. We find no significant difference between order parameters derived from this analysis and those obtained from the conventional analysis, where rotational diffusion is treated as a global parameter in the form of an anisotropic diffusion tensor. Furthermore, the local τm values determined in this analysis are fully consistent with the diffusion tensor determined conventionally. We conclude that the results described here are robust with respect to the models of protein structure and rotational diffusion used in the analysis. These results therefore indicate a coordinated global change in local dynamics, initiated by the localised event of ligand binding. The physical basis for such a change is not clear, but it is not without precedent in the literature: several studies have identified favourable changes in pico- to nanosecond backbone dynamics on ligand binding to be similarly dispersed throughout the protein.34–36 Other studies have identified broadly dispersed changes in dynamics of contrasting sign, such that favourable changes in one part of a protein appear to compensate for unfavourable dynamic changes elsewhere in the protein.37,38 It has been appreciated for some time that protein dynamics might potentially mediate aspects of protein function including allosteric regulation39 and catalysis.40 Experimental evidence for such a functional role, principally from NMR spectroscopy, has been accumulating.41,42 These results raise the possibility that proteins may be adapted evolutionarily to exploit internal dynamic processes to functional ends. The results described here, together with previous observations,34–38 suggest that proteins may have evolved finely tuned networks of dispersed dynamic interactions that regulate the thermodynamics of protein–ligand interactions. We are actively exploring the mechanism of this phenomenon in ABP, and the contribution of other degrees of freedom to the binding entropy, particularly ligand and protein side-chain dynamics. Materials and Methods Protein expression and purification ABP was expressed as essentially as described,6,20 except that the Escherichia coli host strain was BL21(DE3), and the growth medium was M-9 minimal medium, supplemented with BME vitamins (Sigma). For the production of 15N-labelled protein, 15NH4Cl was the sole nitrogen source, and for production of 50% 2H, 13C, 15N protein, the medium was 50% 2H2O and the carbon and nitrogen sources were [U-13C, 50% 2H]glucose and 15NH4Cl, respectively. Purification was as described.6,20 NMR spectroscopy All NMR samples contained approximately 1 mM ABP in 20 mM potassium phosphate (pH 7.0), 3 mM sodium azide, 0.1 mM EDTA, 10% 2H2O. For studies of the complex of ABP with galactose, the sample contained approximately 5 mM d-galactose. Except where noted, NMR experiments were performed at 308 K on Varian INOVA spectrometers operating at 500 MHz, 600 MHz or 750 MHz 1H frequency and equipped with room-temperature triple-resonance z-gradient probes. The transverse relaxation optimized spectroscopy (TROSY)-HNCA of apoABP was acquired at 600 MHz with 16 transients and 1024, 60 and 40 complex points and spectral windows of 7510, 2800 and 4000 Hz in the 1H, 15N, and 13C dimensions, respectively. RDCs were measured at 303 K and 750 MHz using 3.5% (w/v) C5E12 alkyl-poly(ethylene glycol)/hexanol (1:0.96 molar ratio)43 as an alignment medium. 1H-15N one-bond couplings in the presence and in the absence of the alignment medium were measured using the 1JNH modulated HSQC described by Tjandra et al.44 The dephasing delay was varied in 1 ms steps from 6 ms to 20 ms, with two points duplicated for error estimation. Each increment was acquired with 40 transients, 1024 × 128 complex points and spectral windows of 6500 × 3200 Hz. Residues for which fewer than two zero-crossings could be clearly identified were excluded from further analysis. Uncertainty in the measured couplings was estimated using multiple fits using a bootstrap resampling procedure.45 Backbone amide relaxation parameters (15N R1 and R2, and the 1H-15N heteronuclear NOE) were measured using the pulse sequences reported by Farrow et al.46 Typical relaxation delays were 10.7 ms, 53.3 ms, 107 ms, 213 ms, 426 ms, 640 ms, 906 ms, 1230 ms, 1600 ms and 2130 ms for R1 and 12.4 ms, 16.6 ms, 24.8 ms, 33.1 ms, 49.7 ms, 66.2 ms, 82.8 ms, 99.4 ms and 133 ms for R2. Duplicate measurements were made for at least two points in each series for estimation of experimental error. In the R2 experiment, dummy Carr–Purcell–Meiboom–Gill sequences were applied before the recovery delay to compensate for sample heating caused by radio-frequency pulses, and all experimental series were acquired fully interleaved. Typically, 32 transients were acquired for R1 and R2 measurements, and 64 for the NOE, giving a total experimental time of approximately 60 h for the three experiments at each field. Measurements were made at 500 MHz, 600 MHz and 750 MHz for apo ABP, and at 500 MHz and 600 MHz for the ABP–galactose complex. Spectral assignment Resonance assignments for the ABP–galactose complex have been determined.20 Many peaks in the 1H-15N HSQC show small chemical shift changes between the apo and complexed states, permitting partial apoABP assignments to be determined on the basis of those for the complex. For assignment of 1H-15N HSQC resonances that do move on binding ligand, a titration of ABP with 1-deoxygalactose was used. 1-deoxygalactose binds ABP with an affinity four orders of magnitude less than that of galactose, and is in the fast-exchange regime. Assignments for a number of resonances remained ambiguous at this stage, owing to substantial crowding of the 1H-15N HSQC spectrum. These remaining ambiguities were largely resolved by means of i to i–1 connectivities derived from an HNCA spectrum of apoABP. Domain orientation of apoABP ABP is composed of two structural domains, the N domain (residues 1–103 and 257–277), and the C domain (residues 110–253 and 286–306). The remaining residues form a three-stranded linker between the domains, which retains some flexibility in the absence of ligand.21,22 The relative orientation of the two domains of apoABP was determined using 1H-15N RDCs. An initial estimate of the tensor describing molecular alignment of apoABP in 3.5% (v/v) C5E12/hexanol was obtained from the histogram of experimental RDCs.47 A starting structure was derived from the structure of ABP in complex with galactose (PDB ID 5ABP) by Powell minimisation of each domain individually against the measured RDCs.9 From this structure, domain orientation is determined by rigid-body/torsion angle simulated annealing in which both domains are held rigid. All calculations were performed using the IVM module of Xplor-NIH.48 The annealing protocol comprised an initial 3 ps/1000 steps of dynamics at 2000 K, after which the temperature was reduced to 25 K in 12.5 K steps, with 0.3 ps/100 steps of dynamics performed at each temperature. Default Xplor-NIH parameters were used throughout, and force constants were ramped using default values. Square-well potentials were used for all RDC constraints. Lipari–Szabo analysis of relaxation data Backbone amide relaxation parameters were analysed in terms of the extended Lipari–Szabo formalism,24,25 using the software relax.49,50 The 15N CSA was set at −170 ppm and the effective N–H bond length was 1.02 Å. Initial estimates for the rotational diffusion tensor of both apoABP and the ABP–galactose complex were obtained from the ratio of longitudinal and transverse relaxation rates.51 Residues that experience complex internal motion or Rex contribution were identified as described and excluded from this diffusion tensor estimation.52 For ABP–galactose, an axially symmetric diffusion tensor produces an optimal fit, while the fit to the apo ABP data is improved significantly by the use of a fully anisotropic diffusion tensor. Similar results were obtained using data collected at each field strength. Using these estimates, parameters of the extended model-free formalism were optimised for each residue individually, and the best parameter set identified by AIC model selection.49 All parameters, including the diffusion tensor, were then optimised. This process was repeated until the solution converged. The final optimised diffusion tensors are presented in Table 1, and the final dynamic parameter sets in Table 2. Additional analyses were performed as described.33 Here, the contribution to relaxation due to overall rotational diffusion is treated as a mono-exponential component to the correlation function, with a time constant determined individually for each residue; the so-called local τm. In this analysis, the dynamic parameters and local τm for each residue are optimised individually, and the optimal parameter set selected by AIC.49 We did not include chemical exchange (Rex) contributions to R2 in this analysis, owing to the tendency for un-physically low local τm values to be compensated by artefactual Rex contributions and low order parameters, particularly in the case of the ABP–galactose complex, where only two R2 values are available for each residue. Changes in conformational entropy associated with the observed changes in Lipari–Szabo order parameters were determined using the relationship described by Yang and Kay.26 This relationship is essentially independent of motional model for S2LZ ≤ 0.95, so only residues consistent with this criterion in both apoABP and the complex have been included in considerations of entropy change. Uncertainty in the calculated entropy change deriving from experimental uncertainty in order parameters was determined by standard error propagation methods. Molecular dynamics The simulations were carried out using AMBER 8,53 with the Cornell et al. force field.54 Initial coordinates of both holo and apo forms were based on the crystal structure of the ABP–galactose complex (PDB code 5ABP).9 The structures were processed by the XleaP module of AMBER, and the hydrogen atoms were added to the system. The structure of β-d-galactopyranose was optimised ab initio using the Gaussian 98 program† with the HF/6-31G* basis set, and RESP charges were generated and fitted.55 The ligand molecule was parameterised using GLYCAM force field.56 The models were subjected to short (1000 cycles) energy minimisation. Protein models were then immersed in periodic TIP3P water boxes. Approximately 5500 water molecules were added to each system. Simulations were carried out under NPT conditions at 300 K, using the particle mesh Ewald technique with 12 Å non-bonded cutoff and 2 fs time-step.57 SHAKE constraints with a tolerance of 10− 8 Å were applied to all hydrogen atoms during MD simulations to eliminate the fastest X-H vibrations and allow a longer simulation time-step. Translational and rotational centre-of-mass motion was removed every 10 ps. Equilibration started by 20 000 cycles of energy minimisation, with the atomic positions of the solute molecule restrained. It was followed by 100 ps of MD simulations, where the system was heated gently to 300 K and the constraints were released gradually (from 100 kcal/(mol·Å2) applied initially). The further equilibration took 4.9 ns. The production period took 20 ns for both systems. The coordinates were saved every 2 ps of MD simulation. Generalised order parameters were calculated from the trajectory of individual back-bone amide bond vectors as:27where x, y and z are components of a unit vector along the amide bond, and angular brackets denote the time-average over the trajectory. Convergence of the dynamics of interest is tested using the approach described by Best et al.:29 a cumulative time function S2LZ(τ) is defined using equation (1), with the time averages taken from t = 0 to t = τ. This function was evaluated for 100 equally spaced time-points across the trajectory. The trajectory was deemed to have converged if the difference between the maximum and minimum values of this function over the final 50 time-points (i.e. the final 10 ns of the trajectory) was less than 0.05. Any residue judged not to have converged was excluded from the analysis.

Extremophiles-4-1-2262144

Metabolism of halophilic archaea

In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. The metabolic diversity of halophilic archaea was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species: Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi, and the haloalkaliphile Natronomonas pharaonis. The comparative study reveals different sets of enzyme genes amongst halophilic archaea, e.g. in glycerol degradation, pentose metabolism, and folate synthesis. The carefully assessed metabolic data represent a reliable resource for future system biology approaches as it also links to current experimental data on (halo)archaea from the literature. Introduction Extremely halophilic archaea are a diverse group of euryarchaeota that inhabit hypersaline environments (3–5 M) such as salt lakes, salt ponds, and marine salterns. They are often referred to as “halobacteria,” named after the model organism Halobacterium salinarum, whose proton pump bacteriorhodopsin is one of the best-studied membrane proteins. Although haloarchaea share certain features in order to adapt to their extreme environment, i.e. acidic protein machineries, respiratory chains and rhodopsins, their metabolism is considerably different from each other. Although there are carbohydrate-utilizing species such as Haloferax mediterranei, Haloarcula marismortui, and Halococcus saccharolyticus, which catabolize hexoses (glucose, fructose), pentoses (arabinose, xylulose), sucrose, and lactose (Rawal et al. 1988; Altekar and Rangaswamy 1992; Johnsen et al. 2001; Johnsen and Schonheit 2004), other haloarchaea like H. salinarum are not capable of sugar degradation (Rawal et al. 1988). Instead, non-carbohydrate-utilizing species thrive on amino acids and typical compounds of hypersaline habitats. Haloferax volcanii, for example, is able to grow on glycerol and organic acids (Kauri et al. 1990) excreted by primary halophilic producers Dunaliella salina (Elevi Bardavid et al. 2006) and Microcoleus chthonoplastes (Zviagintseva et al. 1995), respectively. Haloarchaea differ not only in their catabolic pathways but also in their nutritional requirements. While simple growth media were described for Haloferax volcanii (Kauri et al. 1990) and Natronomonas pharaonis (Falb et al. 2005), H. salinarum exhibits complex nutritional demands. Growth of Halobacterium cells is often limited in synthetic media (Oesterhelt and Krippahl 1973; Grey and Fitt 1976), in spite of rich amino acid (at least 10 amino acids) and cofactor supplements (folate, biotin, thiamine). The metabolic diversity of halophilic archaea has not yet been investigated at the genomic level by metabolic reconstruction and comparative analysis. The absence of enzyme genes for numerous metabolic reactions in archaeal genomes has limited reconstruction of metabolic pathways so far. However, many of these pathway gaps have been elucidated recently with the discovery of novel non-orthologous enzymes in archaea, e.g. for the de novo synthesis of mevalonate (Barkley et al. 2004; Grochowski et al. 2006b), purines (Graupner et al. 2002; Ownby et al. 2005), and cobamide (Woodson et al. 2003; Woodson and Escalante-Semerena 2004; Zayas et al. 2006). Archaea also employ novel enzymes and precursors for pentose formation (Grochowski et al. 2005) and aromatic amino acid synthesis (White 2004; Porat et al. 2006) circumventing absent enzymes of the classical pentose-phosphate pathway. For this detailed review of haloarchaeal metabolism, metabolic pathways of halophilic archaea were systematically reconstructed. Currently, genome sequences of four diverse haloarchaeal species are available for comparative analysis (Table 1), namely that of the model organism H. salinarum (Ng et al. 2000; Pfeiffer et al. 2008; http://www.halolex.mpg.de), the metabolic-versatile H. marismortui (Baliga et al. 2004), the haloalkaliphile N. pharaonis (Falb et al. 2005), and the square-shaped Haloquadratum walsbyi (Bolhuis et al. 2006). The presented metabolic data will be a valuable resource for future system biology approaches, as each metabolic reaction has been carefully assessed and linked to experimental data from the literature. Table 1Overview of the currently sequenced haloarchaeaH. salinarum strains NRC-1/R1aH. marismortuiH. walsbyiN. pharaonisGene identifierVNG/OErrn, pNGHQNPSalt optimum [M]4–5 M4.5 M3.3 M3.5 M (pH 8.5)IsolationSalted fishDead Sea (Israel)Solar saltern (Spain)Soda lake (Egypt)Main research interestsRhodopsins, signal transductionVersatile nitrogen metabolismSquare-shaped cells, halomucinHaloalkaliphilicity, respiratory chainGenome size [Mb]2.61/2.724.373.242.80# Plasmids2/48 (incl. CHRII)12%GC chromosome68.062.447.963.4rRNA operons1321Fla genes (motility)YesYesNoYes# Transducer genes1821 (18)019# Rhodopsin genes (bop, hop, sop)4 (1,1,2)6 (3,1,2)3 (2,1,0)2 (0,1,1)aTwo strains of H. salinarum have been sequenced, strains NRC-1 (Ng et al. 2000) and R1 (Pfeiffer et al. 2008; http://www.halolex.mpg.de). These are virtually identical and differ only in their distribution of insertion elements and their plasmid arrangements. Unless mentioned otherwise, the two strains are not distinguished in this review, because they exhibit analogous sets of enzyme encoding genes. Gene identifiers of H. salinarum strain R1 (e.g. OE1001F) will be used throughout the text Materials and methods Metabolic reconstruction procedure Metabolic pathways of H. salinarum were reconstructed in a two-step procedure. At first, relevant reactions that take part in a given metabolic pathway were chosen from the complete set of reference reactions downloaded from the KEGG ligand database (Kanehisa et al. 2004). In the second step, organism-specific metabolic pathways were reconstructed by flagging each reaction existent or non-existent in H. salinarum with a certain confidence value. The enzyme gene predicted to catalyze this reaction in H. salinarum was linked to the reaction. In case experimental data is available from the literature, a comment was linked to the reaction or enzyme gene, which includes the PubMed identifier of the external reference. Each reaction was manually assessed based on automatic enzyme assignments derived from various similarity searches (i.e. blast search with UniProt enzymes, COG assignments, Pfam search), genome data (http://www.halolex.mpg.de), and experimental literature data for H. salinarum. In case, a reaction or enzyme is confirmed by experiments but no genetic evidence was found in the genome (indicating novel yet unknown enzyme variants), the reaction was marked existent and the conflict was labeled. The metabolic data for H. salinarum given in Supplementary Material S1 was internally stored in a MySQL database and managed via a Web-interface. Comparative analysis of the complete haloarchaeal gene sets For comparison of haloarchaeal gene sets, protein databases (fasta format) of the four completely sequenced haloarchaeal genomes, H. salinarum strain R1, N. pharaonis, H. walsbyi, and H. marismortui, were blasted against each other and against the non-redundant (nr) database. For example, each predicted protein sequence of N. pharaonis (species 1) was searched against the H. salinarum database (species 2) and the nr database (all species), then the difference between the best blast score for the general database (nr) and for the related species H. salinarum was calculated. Listing N. pharaonis sequences by these differences in descending order detects genes present in N. pharaonis but absent in H. salinarum. In few cases, the N. pharaonis gene had a close homolog in the general database but only a more distant homolog in H. salinarum. Such a gene is considered present in H. salinarum when the blast hit is highly significant (E-value better than e−20) were considered. The differential blast analysis routine was applied to all combinations of the set of four haloarchaeal genomes. A list of genes present in only a subset of genomes was obtained and filtered for metabolic function (Supplementary Material S2). Results and discussion The metabolism of H. salinarum was reconstructed by evaluating metabolic reactions of biochemical reference pathways from KEGG (Supplementary Material S1). Enzyme annotations from an automatic assignment routine and from curated genome data (http://www.halolex.mpg.de) were carefully assessed in order to avoid misassignments due to domain rearrangements (e.g. purine biosynthesis, Fig. 2) or overannotation of the exact substrate specifity (e.g. 2-oxoacid dehydrogenase complex). Novel enzyme variants closing former pathway gaps in archaea as well as previously published experimental data of H. salinarum were also considered throughout the reconstruction process. Comparative analysis of the currently sequenced haloarchaea, H. marismortui, H. walsbyi, N. pharaonis, and H. salinarum, disclosed major metabolic differences, i.e. alternative enzymes and metabolic pathways employed by haloarchaea (Supplementary Material S2). In the following, reconstructed metabolic pathways of halophilic archaea will be reviewed in detail with emphasis on different enzyme genes and metabolic pathways, which lead to the diverse catabolic and anabolic capabilities of halophilic archaea. In case an enzyme gene is present in all four haloarchaeal species, only the N. pharaonis identifier (e.g. NP1002A) will be exemplarily mentioned. Glycolytic pathways Major differences in sugar degradation capabilities were identified amongst halophilic archaea that reflect the previous classification of halophilic archaea into carbohydrate- and non-carbohydrate utilizers (Fig. 1, Supplementary Material S2). H. salinarum, H. marismortui, and H. walsbyi possess all required enzyme genes for the semi-phosphorylated Entner-Douderoff (ED) pathway previously described for carbohydrate-utilizing haloarchaea (Rawal et al. 1988; Danson and Hough 1992; Verhees et al. 2003). However, only H. marismortui and H. walsbyi encode a classical KDPG aldolase, which is the key enzyme of the pathway, in addition to the novel archaeal KD(P)G aldolase recently described for thermophilic archaea (Ahmed et al. 2005). Thus, operation of different ED pathway variants between H. salinarum and the other two species is indicated. It should further be noted that neither the ED pathway nor growth on glucose as the sole carbon source has been established for Halobacterium so far (Gochnauer and Kushner 1969; Rawal et al. 1988). N. pharaonis is likely to be incapable of glucose degradation, because it completely lacks all enzyme genes of the ED pathway. Fig. 1The central intermediary metabolism in halophilic archaea. The reaction arrows depict the reconstructed metabolism of the reference species H. salinarum (green reaction exists, red reaction absent). The four geometric symbols illustrate differences in enzyme gene sets between the four sequenced haloarchaea (square: H. marismortui, circle: H. walsbyi, diamond: N. pharaonis, triangle: H. salinarum, green gene exists, red gene absent). Reactions that have been investigated experimentally through NMR studies or enzyme activity tests in H. salinarum are highlighted by bold arrows (green reaction exists, red reaction absent). For some of the experimentally verified reactions, there are currently no genetic evidences in the H. salinarum genome (green arrows with red border). Vice versa, some reactions have been experimentally excluded, but probable enzyme genes are present in the H. salinarum genome (red arrows with green border). Compounds that have been identified through labeling studies are marked by asterisks. Proposed essential amino acids for H. salinarum are indicated (E). Compounds: AraHex—d-arabino-3-hexulose-6P, Ery4P—erythrose-4P, Frc—fructose, GAP—glyceraldehyde-3P, Glc—glucose, Gluc—gluconate, Glyn—glycerone, Glyc—glycerol, Glyox—glyoxylate, Icit—isocitrate, KDPG—2-dehydro-3-deoxy-6-phosphogluconate, Mal—malate, OA—oxalacetate, 2-OG—2-oxoglutarate, PEP—phosphoenolpyruvate, PGA—3-phosphoglycerate, Pyr—pyruvate, Rib5P—ribose-5P, Ribul5P—ribulose-5P, Suc—succinate, Xyl5P—xylulose-5P, Sed7P—sedoheptulose 7-phosphate, AcCoA—acetyl-CoA The carbohydrate-utilizing H. marismortui has acquired a wide range of enzyme genes involved in the uptake and degradation for various sugars. Fructose and sucrose are likely degraded via the modified Embden-Meyerhof (EM) pathway, which has been described for haloarchaea (Altekar and Rangaswamy 1992). The pathway involves the key enzyme 1-phosphofructokinase (EC 2.7.1.56, rrnAC0342) and sucrose 6-phosphate hydrolase (EC 3.2.1.26, rrnAC1479). Haloarcula also contains probable gene clusters for maltose uptake (maltose ABC transporter, rrnAC2346-rrnAC2349) and metabolism (several α-glucosidases, e.g. rrnAC0224) as well as a gene cluster for D-xylulose oxidation (rrnAC3032–rrnAC3039). The latter encodes the previously characterized D-xylulose dehydrogenase (rrnAC3034) (Johnsen and Schonheit 2004). Embden-Meyerhof pathway and gluconeogenesis Consistent with the previous biochemical findings (Rawal et al. 1988; Altekar and Rangaswamy 1992), genes for 6-phosphofructokinase (EC 2.7.1.11), the key enzyme of the classical Embden-Meyerhof pathway, are absent in all four haloarchaeal genomes. Alternative archaeal types of 6-phosphofructokinases that depend on ADP (Thermococcus, Pyrococcus) or PP(i) (Thermoproteus) (Kengen et al. 1994; Selig et al. 1997) as co-substrates have not been found either. However, a 1-phosphofructokinase gene for fructose degradation via the haloarchaeal variant of the EM pathway (Altekar and Rangaswamy 1992; Rangaswamy and Altekar 1994a, b) is present in H. marismortui (rrnAC0342). Although the upper, hexose part of the classical EM pathway is missing in haloarchaea, the lower, triose part of the pathway that leads to pyruvate is likely to be functional in all species, as required enzyme genes are encoded in their genomes. In fact, several enzyme activities have already been proven in H. salinarum (Rawal et al. 1988). The final glycolysis step from phosphoenolpyruvate to pyruvate might be catalyzed by pyruvate kinase (EC 2.7.1.40, e.g. NP1746A) and pyruvate, water dikinase (EC 2.7.9.2, e.g. NP1196A) in haloarchaea. In a thermophilic archaeon, both enzymes participated in glycolysis but only deletion of the pyruvate, water dikinase gene completely omitted growth on sugars (Imanaka et al. 2006). Complete gene sets for the reverse EM pathway (gluconeogenesis) from pyruvate to phosphorylated glucose were identified in all four haloarchaeal genomes. Consistently, labeling experiments (Sonawat et al. 1990; Ghosh and Sonawat 1998) and enzyme activity studies (Rawal et al. 1988) have confirmed gluconeogenesis in H. salinarum, which is required to synthesize hexoses for membrane constituents. For example, glucose was found to be incorporated into different sugar moieties (glucose, mannose, galactose) of halobacterial glycolipids (Weik et al. 1998). Furthermore, saccharide units are attached to surface proteins of H. salinarum such as the S-layer protein and flagellins (Sumper 1987). Sugar moieties of lipids and proteins are likely synthesized via nucleotide sugars. In accordance with this, several nucleotide sugar enzymes such as UDP-glucose 4-epimerase (EC 5.1.3.2, e.g. NP4662A) and UDP-glucose 6-dehydrogenase (EC 1.1.1.22, e.g. NP2322A, NP4668A) are present in haloarchaeal genomes. Entner-Douderoff pathway Variants of the classical Entner-Douderoff pathway are common in the archaeal domain of life (Danson and Hough 1992). Halophilic archaea such as H. vallismortis (Altekar and Rangaswamy 1992) operate the semi-phosphorylated pathway, where glucose is converted to 2-dehydro-3-deoxygluconate (KDG). This intermediate is then phosphorylated to KDPG and subsequently split into pyruvate and glyceraldehyde 3-phosphate. In Sulfolobus and Thermoplasma, a non-phosphorylated ED pathway has been described (in addition to the semi-phophorylated pathway variant), where KDG is cleaved without prior phosphorylation by a novel bifunctional KD(P)G aldolase (same COG0329 as dihydrodipicolinate synthase) (Ahmed et al. 2005). Orthologs for all genes of the characterized Sulfolobus ED pathway cluster (Ahmed et al. 2005; Kim and Lee 2005) were found in H. salinarum, H. marismortui and H. walsbyi, namely genes encoding d-gluconate dehydratase (EC 4.2.1.39, gnaD, COG4948, e.g. OE1664R), KDG kinase (EC 2.7.1.45, kdgK, COG0524, e.g. OE1266R), and KD(P)G aldolase (kdgA, COG0329, e.g. OE1665R). The ED pathway genes, except kdgK, are located in one gene cluster in the H. salinarum genome, which additionally contains a glucose 1-dehydrogenase gene (EC 1.1.1.47, e.g. OE1669F) (Bonete et al. 1996) as well as two conserved genes (e.g. OE1668R, OE1672F), which are candidates for the yet missing gluconolactonase (EC 3.1.1.17). The non-carbohydrate-utilizing strain N. pharaonis lacks all enzyme genes for the ED pathway. In addition to their archaeal KD(P)G aldolase (rrnAC0960, HQ2365A), H. marismortui (rrnAC3121), H. walsbyi (HQ1495A), and Haloferax alicantei (AAB40121, co-located with kdgK, AAB40122) also encode the canonical bacterial-type KDPG aldolase (EC 4.1.2.14, kdgA, COG0800), which is absent in Halobacterium, Sulfolobus, and Thermoplasma species. This suggests operation of different ED pathway variants in the two (halo)archaeal groups, and, probably, a ‘reduced’ semi-phosphorylated ED pathway in H. salinarum, which needs to be investigated in future. So far, experiments with glucose-grown cells of H. salinarum have shown the conversion from glucose to gluconate (Sonawat et al. 1990; Bhaumik and Sonawat 1999), but have not detected subsequent reactions from gluconate to pyruvate and glyceraldehyde 3-phosphate when applying indirect enzyme activity tests (Rawal et al. 1988). Oxidative pentose phosphate pathway Through the oxidative pentose phosphate pathway, phosphorylated glucose is oxidized to gluconate 6-phosphate (C6) and then converted to ribulose 5-phosphate (C5) by oxidative decarboxylation. In the archaeal domain of life, the oxidative PP pathway seems not to exist, because enzyme genes for this pathway are absent in archaeal genomes. However, haloarchaea show glucose 6-phosphate dehydrogenase activity (EC 1.1.1.49) in spite of lacking the respective enzyme gene (Aitken and Brown 1969) and encode orthologs of 6-phosphogluconate dehydrogenase (EC 1.1.1.44, COG1023, e.g. NP0286A). Thus, an operative, albeit modified oxidative PP pathway is indicated for haloarchaea. Pentose metabolism In bacteria, pentoses are synthesized via the non-oxidative part of the PP pathway, where fructose 6-phosphate (C6) and glyceraldehyde 3-phosphate (C3) are converted to ribulose 5-phosphate (C5) in a complex five-step pathway. The enzyme gene set for this pathway, consisting of transaldolase 1 and 2 (EC 2.1.2.1), transketolase (EC 2.2.1.1), and ribulose-phosphate 3-epimerase (EC 5.1.3.1), is absent in most archaea except Methanococcus jannaschii and Thermoplasma spp. (Soderberg 2005). Individual genes encoding ribose 5-phosphate isomerase (EC 5.3.1.6, e.g. NP0786A) for the conversion of ribose 5-phosphate to ribulose 5-phosphate are present in archaeal genomes. Although Methanocaldococcus jannaschii encodes all enzyme genes for an operative non-oxidative PP pathway, relevant intermediates (i.e. erythrose 4-phosphate, xylose 5-phosphate, sedoheptulose 7-phosphate) are absent in its cells (Grochowski et al. 2005). Instead, ribulose 5-phosphate is exclusively produced through the ribulose monophosphate (RuMP) pathway in M. jannaschii. This pathway is commonly employed for formaldehyde fixation and detoxification in bacteria but operates in reverse mode in archaea and, thus, substitutes for the classical non-oxidative PP pathway (Orita et al. 2006; Grochowski et al. 2005). The RuMP pathway converts fructose 6-phosphate to d-arabino-3-hexulose 6-phosphate and then to formaldehyde and ribulose 5-phosphate involving 6-phospho-3-hexuloisomerase (COG0794) and 3-hexulose-6-phosphate synthase (COG0269). These enzymes are encoded in all archaea except Thermoplasma and haloarchaea, which presumably operate the non-oxidative and a modified oxidative PP pathway for pentose formation, respectively (Soderberg 2005). Absence of the non-oxidative branch of the PP pathway in most archaea also results in the absence of the pathway intermediate erythrose 4-phosphate (C4), which is the precursor of aromatic amino acids in bacteria. While a group of archaea (e.g. Pyrococcus abyssi) encodes transketolase required for erythrose 4-phosphate synthesis, many archaea, amongst them haloarchaea, lack transketolase. These archaea operate an alternative pathway for aromatic amino acid synthesis starting from different precursors, 6-deoxy-5-ketofructose-1-phosphate (DKFP) and l-aspartate semialdehyde (see below) (White 2004). The key enzyme of the reductive branch of the PP pathway for CO2 fixation is ribulose-bisphosphate carboxylase (EC 4.1.1.39, RuBisCO). RuBisCO activity was previously detected in haloarchaea such as H. mediterranei but not in H. salinarum (Rawal et al. 1988; Rajagopalan and Altekar 1994). The CO2 acceptor and substrate of RuBisCO, ribulose 1,5-bisphosphate is not directly synthesized from ribulose 5-phosphate in archaea, but from the purine precursor 5-phospho-d-ribosyl-1-pyrophosphate (PRPP) (Finn and Tabita 2004). The required gene for this conversion is present in all haloarchaea (e.g. NP5174A), while RuBisCO (NP2770A) is only encoded in the N. pharaonis genome. Recently, it has been shown that purine and pentose metabolism are connected in archaea, as archaeal type III RuBisCO is involved in an AMP recycling pathway that is present in N. pharaonis (see below) (Sato et al. 2007). This novel pathway might be part of a cyclic CO2 fixation pathway in archaea consisting of (i) pentose formation (PRPP) from fructose 6-phosphate (e.g. via the RuMP pathway), (ii) conversion of PRPP and adenine to AMP by adenine phosphoribosyltransferase (EC 2.4.2.7, e.g. NP1254A, NP1426A), (iii) AMP recycling releasing adenine and 3-phosphoglycerate (and involving the CO2 fixation step), and (iv) gluconeogenesis for the conversion of 3-phosphoglycerate back to fructose 6-phosphate (Sato et al. 2007). Glycerol metabolism In many hypersaline habitats, glycerol is a highly abundant carbon source that is produced by the halotolerant green algae Dunaliella to protect itself from osmotic pressure (Borowitzka et al. 1977; Phadwal and Singh 2003). Haloarchaea are able to catabolize the abundant glycerol through phosphorylation to glycerol 3-phosphate and subsequent formation of dihydroxyacetone phosphate (DHAP) (Rawal et al. 1988; Nishihara et al. 1999). Consistently, haloarchaeal genomes encode glycerol kinase (EC 2.7.1.30, e.g. OE3762R), the multi-subunit glycerol 3-phosphate dehydrogenase (EC 1.1.99.5, e.g. OE3763F-OE3765F) and a potential sn-glycerol-3-phosphate ABC transport system (e.g. OE5166F-OE5170F). The haloalkaliphile N. pharaonis lacks glycerol degrading enzymes, which might be due to its soda lake habitat, where Dunaliella is not a main primary producer. It should be noted that glycerol degradation and lipid formation occur through separate pathways in archaea, meaning that the intermediate glycerol 3-phosphate derived from glycerol catabolism is not used for the synthesis of the glycerophosphate backbone of archaeal lipids (see below) (Nishihara et al. 1999). In H. salinarum, glycerol can also be converted to DHA by glycerol dehydrogenase (Rawal et al. 1988). For this reaction, a plasmid-encoded glycerol dehydrogenase (EC 1.1.1.6, OE5160F) is employed, which has been characterized and structurally elucidated (Offermann 2003; Horn 2006). The produced DHA might be phosphorylated by DHA kinase (EC 2.7.1.29) and fed into the lower EM pathway. However, potential DHA kinases genes (HQ2672A, HQ2673A) are only encoded by H. walsbyi, where they probably depend on a cytosolic phosphoenolpyruvate-dependent phosphotransferase system (HQ2709A) (Bolhuis et al. 2006). Pyruvate metabolism and tricarboxylic acid cycle Pyruvate metabolism The central metabolite pyruvate is converted to acetyl-CoA by pyruvate-ferredoxin oxidoreductase (EC 1.2.7.1, porAB, OE2623R/OE2622R) (Kerscher and Oesterhelt 1981a, b) and subsequently fed into the tricarboxylic acid (TCA) cycle. NMR spectroscopy experiments for H. salinarum have shown that 90% of the flux is channelled into the TCA cycle via pyruvate-ferredoxin oxidoreductase (Ghosh and Sonawat 1998; Bhaumik and Sonawat 1994). The remaining 10% of the pyruvate is converted to the TCA intermediate oxaloacetate by pyruvate carboxylase (EC 6.4.1.1), in order to fill up the oxaloacetate pool of the TCA cycle, when its intermediates are drawn off for biosynthetic purposes (Ghosh and Sonawat 1998). However, haloarchaea do not encode archaeal-type pyruvate carboxylase (Mukhopadhyay et al. 2000), but biotin carboxylases that are more likely to be involved in fatty acid degradation (e.g. NP4250A/NP4252A located within a fatty acid degradation cluster NP4230A–NP4258A). H. marismortui and H. walsbyi encode a phosphoenolpyruvate carboxylase (EC 4.1.1.31, rrnAC0562, HQ3197A), which has been proposed to be involved with a novel cytosolic phosphotransferase system (pNG7387-pNG7391, HQ1667A, HQ2709A) (Bolhuis et al. 2006). During gluconeogenesis, phosphoenolpyruvate is synthesized from oxaloacetate through malic enzyme (EC 1.1.1.38/39/40, e.g. NP0132A, NP1772A) and pyruvate, water dikinase (EC 2.7.9.1, e.g. NP1196A) in haloarchaea. The former anaplerotic enzyme has been shown to be active in H. salinarum, while the anaplerotic reactions catalyzed by phosphoenolpyruvate carboxykinase (EC 4.1.1.32/38/49) and oxaloacetate decarboxylase (EC 4.1.1.3) are missing in haloarchaea (Bhaumik and Sonawat 1994; Ghosh and Sonawat 1998). Under anaerobic conditions, it has been shown for H. salinarum that pyruvate is primarly converted to alanine, presumably by an aspartate transaminase, and to a larger extent to lactate and acetate (Bhaumik and Sonawat 1994; Ghosh and Sonawat 1998). In spite of proven lactate dehydrogenase activity in H. salinarum cell extracts (Bhaumik and Sonawat 1994), no clear lactate dehydrogenase homolog has been identified in haloarchaeal genomes. In M. jannaschii, it has been shown that lactate is produced from lactaldehyde, which might be derived from methylglyoxal (Grochowski et al. 2006a). The M. jannaschii lactaldehyde dehydrogenase is similar to several probable aldehyde dehydrogenases encoded in haloarchaea (e.g. NP1686A, NP3020A). Tricarboxylic acid cycle Although TCA cycles are highly variable and often incomplete within the archaeal domain of life (Huynen et al. 1999), haloarchaeal genomes encode the complete set of enzymes. For H. salinarum, activity of all these enzymes has been proven (Aitken and Brown 1969; Hubbard and Miller 1972; Kerscher and Oesterhelt 1981a, b; Gradin et al. 1985) and an operative TCA cycle has been shown by NMR spectroscopy (Ghosh and Sonawat 1998). In haloarchaea, pyruvate and 2-oxoglutarate are not converted by classical 2-oxoacid dehydrogenase complexes but by 2-oxoacid-ferredoxin oxidoreductases encoded by porAB and korAB genes, respectively (Kerscher and Oesterhelt 1981a, b). However, haloarchaeal and other archaeal genomes further contain gene clusters encoding all components of a 2-oxoacid dehydrogenase complex. In Thermoplasma acidophilum, it has recently been shown that the E1 component of the encoded 2-oxoacid dehydrogenase complex accepts branched-chain 2-oxoacids (Heath et al. 2004). Most likely, the haloarchaeal 2-oxoacid dehydrogenase complex is also involved in branched-chain amino acid degradation. When grown on acetate, Haloferax volcanii operates a glyoxylate bypass operon involving isocitrate lyase (EC 4.1.3.1) and a new type of malate synthase (EC 4.1.3.2) (Serrano and Bonete 2001). Homologs of both enzymes are present in the N. pharaonis (NP4432A, NP4430A) and H. walsbyi (HQ1720A, HQ3094A) genomes, while only a probable malate synthase is encoded in H. marismortui (rrnAC1965). Halobacterium lacks both glyoxylate cycle genes, although activity of both glyoxylate cycle enzymes has been demonstrated previously in this haloarchaeon (Aitken and Brown 1969). Nucleotide metabolism De novo synthesis of nucleotides The complete gene sets that are required for de novo synthesis of purines from ribose 5-phosphate and for de novo synthesis of pyrimidines from carbamoylphosphate and ribose 5-phosphate are present in haloarchaeal genomes. Haloarchaea reveal an unusual domain fusion pattern of purine synthesis enzymes (Fig. 2), because they contain a unique fusion of GAR and AICAR transformylases (EC 2.1.2.2/EC 2.1.2.3, purN/purH, e.g. NP1662A, OE1620R). Haloarchaea do not encode the novel AICAR transformylase (purP) (Ownby et al. 2005) present in most archaea, but encode the novel archaeal variant of IMP cyclohydrolase (purO, e.g. NP0732A, OE4329F) (Graupner et al. 2002). The four haloarchaeal strains show only few differences in their nucleotide metabolism, namely in the occurrence of pyrimidine kinases (EC 2.7.1.21: OE3159R, HQ1795, EC 2.7.1.48: OE2749F) (Supplementary Material S2). Fig. 2Domain rearrangement of enzymes involved in the de novo synthesis of purines. The pathway comprises 10 steps from PRPP to IMP. Fusions of enzyme genes are indicated by linked boxes. Non-orthologous enzymes are known for steps 3, 9, and 10 (unfilled boxes) but further archaeal enzymes for purine synthesis are unknown (question marks) Archaeal type III RuBisCO functions in AMP metabolism Ribulose-bisphosphate carboxylase (EC 4.1.1.39, RuBisCO) is a key enzyme for CO2 fixation via the Calvin-Benson-Bassham cycle in plants. However, RuBisCO was found to be involved in bacterial methionine cleavage and in the AMP metabolism of archaea (Sato et al. 2007). The latter pathway recycles the intracellular pool of AMP produced by ADP-dependent (AMP-forming) sugar kinases, which are involved in glycolytic pathways of archaea (Kengen et al. 1994; Selig et al. 1997). N. pharaonis is the only haloarchaeal species encoding all enzyme genes for this novel AMP recycling pathway, i.e. AMP phosphorylase (NP3958A), ribose-1,5-biphosphate isomerase (NP3202A), and RuBisCO (NP2770A), while H. salinarum exhibits only the isomerase gene (OE3610R) (Sato et al. 2007). N. pharaonis and some other archaea do not possess ADP-dependent sugar kinases that would produce AMP though. Instead, AMP recycling might be part of a cyclic pathway for CO2 fixation as described earlier. Lipid metabolism Membrane lipids of archaea consist of glycerol diether lipids with prenyl side chains instead of diacylglycerol esters. Specifically, membranes of H. salinarum contain core lipids with two phytanyl side chains (C20), and to lesser extents also other isoprenoid constituents such as squalenes (C30), phytoenes (C40), menaquinones (C40), and dolichol (C60) (Oesterhelt 1976; Lechner et al. 1985; Kushwaha et al. 1976) (Fig. 3). Furthermore, H. salinarum synthesizes several carotenoids from prenyl precursors, preferentially bacterioruberins (C50) and photoactive retinal (C20) (Oesterhelt 1976; Oesterhelt and Stoeckenius 1973). Retinal is incorporated into bacteriorhodopsin and other retinal proteins, which are unique to haloarchaea within the archaeal domain of life. Although fatty acids are not part of archaeal membrane lipids, small amounts of fatty acids (C14, C16, C18) have been detected in membrane proteins of H. salinarum (Pugh and Kates, 1994). Other haloarchaeal species likely possess similar membrane constituents as H. salinarum because they have the same gene set for the de novo synthesis of isoprenoids. However, specific prenyl-based compounds might vary from species to species, as in the case of diether core lipids found in haloalkaliphiles, e.g. N. pharaonis (C20–C20 and C20–C25 prenyl chains) (Tindall et al. 1984). Fig. 3Biosynthesis of isoprenoids in halophilic archaea. The isoprenoid precursor IPP is synthesized via the mevalonate pathway as shown by labeling studies (green reaction exists, red reaction absent, bold experimental verification). Various isoprenoids detected in membranes of H. salinarum (listed in boxes) are synthesized by a series of condensation reactions with IPP, which is added in head–tail (HT) or head–head (HH) fashion, and through desaturase reactions ([2H]). Enzyme gene sets for isoprenoid synthesis differ only slightly between halorarchaea (square: H. marismortui, circle: H. walsbyi, diamond: N. pharaonis, triangle: H. salinarum, green gene exists, red gene absent). Bacterial- (B) or archaeal-type (A) enzyme variants are indicated. Superscript “a” indicates C5-prenyl units are synthesized via the mevalonate pathway starting from two acetyl-CoA molecules and a still unknown C2-unit arising from amino acid degradation (Ekiel et al. 1986) Prenyl side chains of membrane lipids and other isoprenoids are derived via the mevalonate pathway in haloarchaea (Ekiel et al. 1986), while the glycerophosphate backbone of membrane lipids is formed from glycerol 1-phosphate. This membrane precursor is derived from the glycolytic intermediate DHAP via glycerol-1-phosphate dehydrogenase (EC 1.1.1.261, e.g. NP4492A), which is ubiquitously found in archaea (Nishihara et al. 1999). De novo synthesis of isoprenoids (mevalonate pathway) Like other archaea, halophiles synthesize activated C5-units [dimethylallyl and isopentenyl diphosphate (IPP)] for polycondensation of prenyl chains via the mevalonate pathway (Fig. 3). A previous comparative analysis of the mevalonate pathway (Smit and Mushegian 2000) identified gaps for three pathway steps in archaea, namely the lack of bacterial-type phosphomevalonate kinase (EC 2.7.4.2, COG3890), diphosphomevalonate decarboxylase (EC 4.1.1.33, COG3407), and IPP isomerase (EC 5.3.3.2, COG1443). Recently, an alternative type II IPP isomerase has been identified in archaea, which belongs to the same COG1304 as lactate dehydrogenase (Barkley et al. 2004). Furthermore, it has been suggested that mevalonate phosphate is first decarboxylated and then phosphorylated to synthesize IPP, while phosphorylation of mevalonate phosphate precedes the decarboxylation step in bacteria (Grochowski et al. 2006b). The archaeal-specific reactions involve a predicted phosphomevalonate decarboxylase (COG1355) and a characterized isopentenyl phosphate kinase (COG1608). Interestingly, haloarchaea may operate a chimeric mevalonate pathway (Fig. 3). Like other archaea, they encode isopentenyl phosphate kinase (e.g. NP2852A) instead of a bacterial phosphomevalonate kinase. However, haloarchaea lack the proposed archaeal phosphomevalonate decarboxylase gene, and, instead, encode a bacterial-type diphospho-mevalonate decarboxylase (e.g. NP1580A). Thus, neither the classical bacterial nor the proposed archaeal mevalonate pathway is complete. For the last mevalonate pathway step, only N. pharaonis and H. salinarum encode an archaeal-type II IPP isomerase (e.g. NP0360A), while all four haloarchaea possess a bacterial-type IPP isomerase (e.g. NP4826A). Future investigations are needed in order to clarify whether archaeal and bacterial enzymes are employed simultaneously in haloarchaea. The acquired bacterial enzymes might possibly have a higher substrate specificity or turnover for covering increased isoprenoid demands for retinal and bacterioruberin biosynthesis. A functional mevalonate pathway has been verified for H. salinarum by labeling studies (Ekiel et al. 1986), which lead to the proposal of an unusual first step. Lipid labeling patterns indicated that mevalonate is not synthesized from three activated acetate precursors but rather from two acetyl-CoA molecules and an unknown C2-unit. The latter is not derived from acetate but from degraded amino acids such as lysine. C5-isoprenoid precursors derived via the mevalonate pathway are elongated to trans- and cis-polyprenyl chains in head-to-tail fashion by (E)- and (Z)-prenyltransferases, respectively (E: NP3696A, NP4556A, NP0604A, Z: NP4550A, NP4544A). Exact chain-specificity of prenyltransferase orthologs needs to be determined experimentally, but the biosynthesis of dolichol and menaquinones found in H. salinarum requires prenyltransferases with long-chain specifities. Potential enzymes for squalene and phytoene synthesis through head-to-head condensation are also encoded in haloarchaeal genomes. Synthesis of carotenoids and retinal For carotene biosynthesis, phytoene is reduced to lycopene by phytoene desaturase that is encoded in haloarchaeal genomes (e.g. NP4764A, NP0204A). Lycopene is the branching point for the synthesis of β-carotene (C40) and bacterioruberins (C50) (Oesterhelt 1976). The reactions that lead to bacterioruberins have not yet been elucidated in detail, but lycopene cyclase activity (e.g. NP0652A) converting lycopene to β-carotene has been shown for H. salinarum (Peck et al. 2002). The derived β-carotene is cleaved by β-carotene mono-oxygenase into two retinal molecules (C20), which are incorporated in haloarchaeal rhodopsins. H. walsbyi encodes two cyanobacterial-like (HQ2381A, HQ2020A) and one plant-like β-carotene mono-oxygenase homologs (HQ3007A), and H. marismortui acquired a cyanobacterial-like β-carotene mono-oxygenase gene (pNG7272) on one of its plasmids (Bolhuis et al. 2006). H. salinarum and N. pharaonis lack β-carotene mono-oxygenase homologs and must therefore possess a still unknown non-orthologous enzyme for the oxidative cleavage of β-carotene. Previously, brp and blh have been shown to play a role in regulation or synthesis of retinal (Peck et al. 2001). Fatty acid metabolism Even-numbered fatty acids like palmitic and stearic acid are part of membrane proteins but not of membrane lipids in archaea (Pugh and Kates 1994). For example, palmitic acid is associated with halorhodopsin as a free fatty acid in H. salinarum (Kolbe et al. 2000). The origin of these fatty acids is unclear because archaea do not encode components of a fatty acid synthase complex. In methanogenic archaea, biosynthesis of fatty-acid-like compounds from 2-oxoglutarate by repeated 2-oxoacid chain elongation has been reported (White 1989). For the degradation of activated fatty acids via the ß-oxidation pathway, gene candidates are present in haloarchaea and most other archaea. However, since chain-length specifity of these enzymes is currently unknown, fatty acid degradation might still be limited to short chain lengths (e.g. for derivatives of branched-chain amino acids). A series of genes were assigned for Natronomonas (37 genes) and Haloarcula (29 genes) for the repeated four-step reaction sequence of the ß-oxidation pathway indicating a versatile fatty-acid metabolism in these two species. In contrast, few ß-oxidation genes are present in H. salinarum (12 genes) and H. walsbyi (6 genes). In accordance to these findings, growth experiments showed that N. pharaonis is able to grow on fatty acids of various lengths as carbon source (especially C14), while fatty acids seem not to be utilized by H. salinarum, as growth is reduced or diminished by long-chain (C14–C18) and medium-chain fatty acids (<C14), respectively (Konigsmaier 2006). Haloarchaea likely degrade odd-numbered fatty acids, because they possess probable propionyl-CoA carboxylase (EC 6.4.1.3, e.g. NP4250A/NP4252A), methylmalonyl-CoA epimerase (EC 5.1.99.1, e.g. NP1228A) as well as methylmalonyl-CoA mutase (EC 5.4.99.2, e.g. NP1226A, NP2710A). Natronomonas further encodes enzymes that are probably involved in propionate catabolism (NP6212A, NP4432A, NP4820A). Amino acid synthesis While certain haloarchaea such as Haloarcula hispanica (Hochuli et al. 1999) do not require any amino acids for growth, H. salinarum is grown in synthetic media with 10 to 15 amino acids (Oesterhelt and Krippahl 1973; Grey and Fitt 1976). Through metabolic pathway reconstruction and comparison, it can be proposed that H. salinarum has indeed reduced capabilities to synthesize amino acids. Specifically, H. salinarum lacks gene clusters for valine, leucine, isoleucine, lysine, and arginine (Supplementary Material S2). Furthermore, because of reduced folate biosynthesis, methionine biosynthesis via folate-dependent methionine synthase (EC 2.1.1.14) might be affected in H. salinarum. The predicted set of essential amino acids fits well to the set of amino acids that can be sensed by H. salinarum (Oren 2002) except for lysine, which is not an attractant signal, and for cysteine, which is sensed by BasT (Kokoeva et al. 2002) in spite of the fact that all enzyme genes for cysteine biosynthesis are present in its genome. The synthesis of several amino acids (glutamate, glutamine, proline, aspartate, asparagine, alanine, serine, phenylalanine, tryptophan, histidine) by H. salinarum has been verified by NMR labeling studies (Ekiel et al. 1986; Bhaumik and Sonawat 1994; Ghosh and Sonawat 1998; Engelhard et al. 1989) (Fig. 1). For H. marismortui, N. pharaonis, and H. walsbyi, complete independence from supplemented amino acids can be concluded from comparative analysis (Supplementary Material S2). De novo synthesis of all amino acids has already been proven for N. pharaonis by the development of a synthetic medium without amino acid supplements (Falb et al. 2005; Oberwinkler 2006). The three haloarchaea that do not require amino acids are also able to utilize several nitrogen sources because they posess gene sets required for urea conversion as well as for nitrate and ammonia assimilation (Falb et al. 2005) (Supplementary Material S2). Amino acid biosynthesis pathways of the three haloarchaea show only few differences, i.e. for proline, lysine, serine, and glycine synthesis (see below). Glutamate family (glutamate, glutamine, proline, arginine) The biosynthesis of glutamate from the TCA cycle intermediate 2-oxoglutarate is a major metabolic conversion in haloarchaea as shown by NMR labeling studies in Halobacterium. Labeled pyruvate, alanine, acetate, and glycerol were mainly found to be incorporated into glutamate, and a considerable part of the flux through the TCA cycle was shown to be channelled to glutamate (Ghosh and Sonawat 1998; Ekiel et al. 1986). Three paralogous glutamate dehydrogenase genes were found in Natronomonas (NP1582A, NP1806A, NP6184A), Halobacterium (OE1270F, OE1943F, OE2728R), and Haloarcula (rrnAC0384, rrnAC0775, pNG7157), but only one gene in Haloquadratum (HQ1880A). The activity of two Halobacterium glutamate dehydrogenases with NADP+ (OE1943F) and NAD+ (OE1270F) cofactor specificity has been investigated in detail (Bonete et al. 1987, 1989, 1990; Perez-Pomares et al. 1999; Hayden et al. 2002). In N. pharaonis, H. walsbyi, and H. marismortui, glutamate is also derived by glutamate synthase (e.g. NP1794A) from 2-oxoglutarate and glutamine, a reaction that is part of a proposed ammonia assimilation pathway (e.g. NP4224-NP4228A) (Falb et al. 2005). It should be noted that haloarchaeal cells amass glutamate in high concentrations as a compatible osmolyte (Kokoeva et al. 2002; Desmarais et al. 1997). Glutamine is derived from glutamate by glutamate-ammonia ligase (EC 6.3.1.2, e.g. NP4376A) which is present in all haloarchaea. Glutamate might further be converted to l-glutamate 5-semialdehyde and subsequently to proline. The required enzymes encoded by the proCBA gene cluster are only found in the genomes of Natronomonas (NP3974A-NP3978A) and Haloquadratum (HQ1844A-HQ1846A), but are absent in Halobacterium and Haloarcula. Biosynthesis of proline by H. salinarum has been shown previously (Ghosh and Sonawat 1998) indicating an alternative pathway in the latter two haloarchaea. Proline might be synthesized via 1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.2) and proline dehydrogenase (EC 1.5.99.8, putA). This notion is supported by the fact that the phylogenetic profiles of putA is complementary to that of the proABC cluster, i.e. putA is only encoded in H. salinarum (OE3955F) and H. marismortui (rrnAC2471), but is missing in N. pharaonis and H. walsbyi. Proline might also be derived by the cyclisation of ornithine as shown for M. jannaschii (Graupner and White 2001). Homologs of ornithine cyclodeaminase (EC 4.3.1.12) are encoded in all haloarchaea (e.g. NP0448, NP3802A). Glutamate is also a precursor of ornithine, which is converted to arginine by urea cycle enzymes. The genomes of Natronomonas, Haloquadratum, and Haloarcula possess argXCBDEF clusters (e.g. NP5258A-NP5268A) containing all required enzymes (except the argA gene) for the de novo synthesis of ornithine as well as a probable transcription regulator ArgX. The complete gene set for arginine biosynthesis is absent in Halobacterium. Instead, halobacterial arginine requirements are covered by the uptake of external arginine via a verified arginine-ornithine antiporter (OE5204R) (J. Tittor, unpublished results) encoded next to the arginine deiminase pathway cluster on plasmid PHS3. Aspartate family (aspartate, alanine, asparagine, threonine, methionine, lysine) Aspartate is derived from the TCA cycle intermediate oxaloacetate by aspartate transaminase (EC 2.6.1.1) of which several paralogs are present in haloarchaeal genomes (e.g. NP0824A, NP1666A, NP4024A, and NP4410A). These seem to be also involved in pyruvate transamination to alanine since experiments showed that the transaminase, which is involved in alanine synthesis in Halobacterium, uses aspartate instead of glutamate as amino group donor (Bhaumik and Sonawat 1994). In agreement with this, no clear orthologs of glutamate-pyruvate transaminase (EC 2.6.1.2) have been found in haloarchaeal genomes. Labeling studies showed that aspartate is converted to asparagine inH. salinarum (Engelhard et al. 1989), and, consistently, all haloarchaeal genomes encode asparagine synthase (EC 6.3.5.4, e.g. NP2978A). In a pathway analogous to proline synthesis from glutamate, aspartate is likely to be converted to l-aspartate semialdehyde. This compound is not only a precursor of threonine, methionine, and lysine in archaea, but is also required for aromatic amino acid synthesis via a modified shikimate pathway (see below). Enzyme genes for threonine biosynthesis from l-aspartate semialdehyde via l-homoserine and O-phospho-l-homoserine are present in all haloarchaea (e.g. NP0302A, NP4524A, NP5280A). In M. jannaschii, O-phospho-l-homoserine is not only converted to threonine but also to homocysteine (White 2003). Additionally, haloarchaea might synthesize homocysteine from homoserine via O-acetyl-l-homoserine (but not via O-succinyl-l-homoserine). This pathway requires clustered metX and metY genes (e.g. NP0280A-NP0284A) in case of direct sulfhydrylation of O-acetyl-l-homoserine. Alternatively, O-acetyl-l-homoserine could be converted to cystathionine and subsequently to homocysteine by thiol-lyase/-synthase (e.g. NP4746A). In the final step, methionine is likely derived from homocysteine by cobalamine-independent methionine synthase (EC 2.1.1.14, metE), of which two to three paralogs are found in haloarchaea (NP3670A/NP3672A) except Halobacterium (OE2668R). Since methionine synthase depends on the availability of folate, reduced folate synthesis in H. salinarum might also affect its synthesis of methionine. The diaminopimelate (DAP) pathway for the synthesis of lysine has already been proven for H. hispanica (Hochuli et al. 1999). Enzymes for the DAP pathway are present in Natronomonas, Haloquadratum, and Haloarcula, i.e. the dapABD gene cluster (e.g. NP1490A-NP1494A, dapC is synonym with argD) as well as lysA (e.g. NP1646A) and argG genes (e.g. NP5252A). Gaps within the DAP pathway (dapE, dapF) occuring in Haloarcula and Natronomonas are likely to be bridged by still unknown non-orthologous enzymes. Halobacterium is not capable of lysine biosynthesis because it does not encode any of the dap genes. Serine family (serine, glycine, cysteine) Serine biosynthesis might occur via two different pathways. Within the phosphorylated pathway, glycerate 3-phosphate, a glycolytic intermediate, is oxidized to 3-phospho-hydroxy-pyruvate, which is further converted to phosphoserine and to serine by transaminase and phosphatase activity. Alternatively, non-phosphorylated glycerate is directly oxidized to hydroxypyruvate and then transaminated. All four haloarchaea possess genes for phosphoglycerate dehydrogenase (EC 1.1.1.95, serA, e.g. NP0272A) and phosphoserine phosphatase (EC 3.1.3.3, serB, e.g. NP0274A) of the phosphorylated synthesis pathway. However, the enzyme gene for the intermediate pathway step, phosphoserine transaminase (EC 2.6.1.52, serC), is missing. Nevertheless, studies in Methanococcales revealed that broad-specificity class V aspartate transaminases (e.g. NP0884A, NP2578A) catalyze this reaction (Helgadottir et al. 2007). Apart from the phosphorylated serine synthesis pathways, Natronomonas, Haloquadratum, and Haloarcula might also operate the non-phosphorylated pathway because these species encode a probable hydroxypyruvate reductase (EC 1.1.1.81, e.g. NP1162A) for the initial step from glycerate to hydroxypyruvate. One of the class V aspartate transaminases might then catalyze the transaminase reaction from hydroxypyruvate to serine. Glycine is potentially derived from serine by glycine hydroxymethyltransferase (EC 2.1.2.1, e.g. NP2050A) in all haloarchaea. Halobacterium might additionally synthesize glycine from threonine employing threonine aldolase (EC 4.1.2.5, OE4436R), which is unique to the archaeal domain of life. H. salinarum would thus be adapted to its reduced folate synthesis (see below) as the folate-dependent glycine hydroxymethyltransferase is circumvented. All haloarchaea except H. walsbyi encode components of the glycine cleavage system (EC 1.4.4.2) which is encoded in a single gene cluster in Halobacterium (OE3274R-OE3278R). Future investigations might elucidate whether metabolic fluxes differ for the serine metabolism of Halobacterium and the other haloarchaea. Similar to the conversion of homoserine to homocysteine via O-acetyl-l-homoserine, serine is likely to be converted in all halobacteria to O-acetyl-serine (cysE, e.g. NP4172A) and then to cysteine by the incorporation of sulfide (cysK, e.g. NP4748A). An alternative cysteine biosynthesis pathway employed by M. jannaschii, which lacks cysE and cysK genes, starts from homocysteine and phosphoserine and leads via cystathionine to cysteine (White 2003). This pathway requires thiol-lyases and might be also operative in haloarchaea. Recently, also a tRNA-dependent synthesis pathway has been reported for M. jannaschii that leads from phosphoserine, an intermediate of the phosphorylated serine pathway, to Cys-tRNA (Sauerwald et al. 2005). Similar tRNA-dependent pathways have been suggested for glutamine and asparagine synthesis from glutamate and aspartate (Di Giulio 2003). For cysteine biosynthesis, sulfide needs to be assimilated from sulfate via adenylylsulfate, 3P-adenylylsulfate, and sulfite. Although archaea encode a gene for the small subunit (cysD) of the sulfate adenylyltransferase (EC 2.7.7.4, e.g. NP4570A), they lack the GTPase subunit (cysN). Genes for the two subsequent sulfate assimilation steps (cysC, cysH) are present in some archaea (e.g. Aeropyrum pernix) but are not present in any of the haloarchaeal genomes. Alternatively, thiosulfate might be converted to sulfite by thiosulfate sulfurtransferase [EC 2.8.1.1, e.g. NP3186A, NP4004A (N-terminus)] and subsequently to sulfide by one of the potential sulfite reductases [e.g. NP4004A (C-terminus), not in H. salinarum]. Biosynthesis of branched chain amino acids (valine, leucine, isoleucine) All enzyme genes for the de novo synthesis of valine, leucine, and isoleucine are present in Natronomonas, Haloquadratum, and Haloarcula but not in Halobacterium. Threonine-ammonia lyase (EC 4.3.1.19, e.g. NP1076A), which is required to synthesize the isoleucine precursor 2-oxobutyrate, is encoded in all haloarchaea. The threonine pathway leading to isoleucine is employed by H. hispanica (56% flux) but it is simultaneuosly operated together with the pyruvate pathway leading via citramalate to isoleucine (44% flux) (Hochuli et al. 1999). However, no sequences are yet available in public repositories for enzymes of this pathway, e.g. citramalate lyase (EC 4.1.3.22). A third potential pathway from glutamate to isoleucine via methylaspartate (mesaconate pathway) is not employed by H. hispanica (Hochuli et al. 1999). Mesaconate pathway genes (mamABC) were found in the genomes of H. salinarum and H. marismortui, however (see below). In Halobacterium, 2-oxobutyrate may further be derived from methionine by methionine gamma-lyase (EC 4.4.1.11), a reaction that releases volatile methanthiol from the cells (Nordmann et al. 1994). The fact that Halobacterium encodes enzyme genes for 2-oxobutyrate synthesis but lacks other isoleucine synthesis enzymes implies that 2-oxobutyrate is a precursor for further metabolic pathways in haloarchaea. Biosynthesis of aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine) The complete gene set for the classical shikimate pathway, where 3-dehydroquinate is derived from erythrose 4-phosphate (C4) and phosphoenolpyruvate (C3), is only present in Thermoplasma spp. and some other archaea (Soderberg 2005). In methanogenic archaea, 3-dehydroquinate is synthesized via a novel pathway starting from l-aspartate semialdehyde (C4) and 6-deoxy-5-ketofructose-1-phosphate (DKFP, C6) (White 2004). The DKFP pathway is also employed by haloarchaea as shown by the incorporation of labeled aspartate into the C11-position of tryptophan in H. salinarum (Engelhard et al. 1989). Furthermore, the two novel DKFP pathway enzymes for alternative 3-dehydroquinate synthesis, 2-amino-3,7-dideoxy-d-threo-hept-6-ulosonate synthase (COG1830 together with DhnA-type fructose bisphosphate aldolase; e.g. NP3160A) and 3-dehydroquinate synthase type II (COG1465, e.g. NP2238A) (Porat et al 2006) are encoded in haloarchaeal genomes. The precursor of the modified shikimate pathway, DKFP, presumably derives from methylglyoxal rather than from nucleoside diphosphate sugars in archaea (White and Xu 2006). Methylglyoxal is not created by methylglyoxal synthase (EC 4.2.3.3), which is absent in archaea, but from glyceraldehyde 3-phosphate using triosephosphate isomerase (EC 5.3.1.1, e.g. NP2182A, NP3716A). Another glycolytic enzyme, a multifunctional class I fructose-biphosphate aldolase (e.g. NP1594A) catalyzes the subsequent transaldolase reaction of methylglyoxal with fructose 1,6-diphosphate (or fructose 1-phosphate) to DKFP. The remaining biosynthesis steps from 3-dehydroquinate to chorismate follow the classical shikimate pathway in haloarchaea. Enzyme genes for biosynthesis of tryptophan, tyrosine, and phenylalanine from chorismate are also present in haloarchaeal genomes. Consistent with this, de novo synthesis of phenylalanine and tryptophan has been proven for H. salinarum (Ekiel et al. 1986; Engelhard et al. 1989). A second synthesis pathway for aromatic amino acids in archaea has recently been described for M. maripaludis (Porat et al. 2004), where aromatic amino acids are synthesized through the incorporation of exogenous aryl acids catalyzed by a ferredoxin-dependent indolepyruvate oxidoreductase (ior). This pathway is presumably not present in halophilic archaea, because their only two ferredoxin-dependent oxidoreductases have been already assigned otherwise (porAB, korAB) (Kerscher and Osterhelt 1981a, b). Histidine biosynthesis genes are partly clustered within haloarchaeal genomes. All enzymes for histidine biosynthesis starting from ribose 5-phosphate are encoded except for histidinol-phosphatase (EC 3.1.3.15). An unknown alternative enzyme is indicated for the missing pathway step, especially since histidine biosynthesis has already been proven experimentally in H. salinarum (Ekiel et al. 1986). Amino acid degradation Glutamate and aspartate degradation Glutamate and aspartate are major carbon substrates for halophilic archaea that are fed into the TCA cycle and subsequently into the respiratory chain for ATP production. Several transaminases are encoded in haloarchaea which likely catalyze the conversion of aspartate, glutamate, and other amino acids to TCA cycle intermediates. In H. salinarum and H. marismortui, glutamate might further be degraded to mesaconate by methylaspartate mutase (EC 5.4.99.1) and methylaspartate ammonia-lyase (EC 4.3.1.2). Genes for the mesaconate pathway (mamABC, OE4204F-OE4207F, rrn0684-rrn0687) are only encoded in the two haloarchaea and very few bacteria, i.e. two E. coli strains as well as a Clostridium and Treponema species. In thermophilic anaerobic bacteria, conversion of mesaconate to citramalate and subsequently to pyruvate and acetate has been proven (Plugge et al. 2001). However, the respective enzymes for the established reactions are not yet known. Mesaconate is also a potential precursor of isoleucine in haloarchaea, although not for the haloarchaeon H. hispanica (Hochuli et al. 1999). Arginine metabolism Urea cycle genes for the conversion of ornithine to arginine were found in all halophilic strains, but arginase (EC 3.5.3.1, rrnAC0383, rrnAC0453) converting arginine back to ornithine is only present in H. marismortui. Instead, H. salinarum metabolizes arginine to ornithine with concomitant ATP production via the arginine deiminase pathway (Hartmann et al. 1980). This pathway is rare amongst prokaryotes, and H. salinarum is the only archaeon that has acquired an arginine deiminase gene cluster (arcRACB, OE5205R-OE5209R, plasmid PHS3) (Ruepp and Soppa 1996) encoding all required arginine deiminase pathway enzymes as well as a probable transcription regulator ArcR. The gene for an arginine-ornithine antiporter (OE5204R) (J. Tittor, unpublished results) is also located next to the arginine deiminase gene cluster. Archaea also utilize arginine for the biosynthesis of polyamines such as putrescine, spermidine, or spermine (Graham et al. 2002b), which seem to be required in archaea for nucleosome maintenance in high-temperature environments (Higashibata et al. 2000). Orthologs of a novel arginine decarboxylase (EC 4.1.1.19, e.g. NP4484A) (Graham et al. 2002b) and agmatinase (EC 3.5.3.11, e.g. NP3022A, NP4754A) for the conversion of arginine to putrescine are present in haloarchaeal genomes. For pathway steps leading from putrescine to spermidine, no enzyme genes are encoded in halophilic archaea. Branched-chain amino acid degradation For amino acids with a more complex carbon scaffold, i.e. branched-chain and aromatic amino acids, separate biosynthesis and degradation pathways, regulated independently from each other, have evolved. Catabolism of valine, leucine, and isoleucine first involves a transaminase (e.g. NP5036A) for the conversion of branched-chain amino acids to the respective 2-oxoacids. The following decarboxylation is likely catalyzed by a previously unassigned 2-oxoacid dehydrogenase multienzyme complex (e.g. OE4113F-OE4116F), whose E1 component has recently been shown to accept branched-chain 2-oxoacids in T. acidophilum (Heath et al. 2004). The derived activated fatty acids are likely to be further degraded to acetyl-CoA and propionyl-CoA. Aromatic amino acid degradation Histidine is likely to be degraded to glutamate via the urocanate pathway by H. salinarum and H. marismortui encoding hutUGIH gene clusters (e.g. OE2734F–OE2739F). The two haloarchaea (and A. pernix) also acquired the only archaeal tryptophanases (EC 4.1.99.1, OE4331R, rrnAC2439) and H. salinarum the only archaeal kynureninase (EC 3.7.1.3, OE2332F) for potential indole or anthranilate formation from tryptophan, respectively. Further, haloarchaea (except H. walsbyi) encode orthologs (e.g. NP1194A) of the recently described l-tyrosine decarboxylase (EC 4.1.1.25) (Kezmarsky et al. 2005), which is required for synthesis of methanofuran in M. jannaschii. Complete aromatic amino acid degradation pathways could not be reconstructed but probable aromatic compound dioxygenases (COG0346, e.g. NP2650A) are encoded in all haloarchaeal genomes. Cofactor metabolism Bacteria and archaea are commonly able to synthesize cofactors de novo. However, the relevant biosynthesis pathways are often not completely understood, i.e. early pathway steps leading to biotin and thiamine. In fact, many genes (e.g. thiI, moaA) which have been associated with coenzyme biosynthesis have not yet been assigned to specific metabolic reactions. Upon metabolic reconstruction, archaea reveal many gaps where enzyme genes are replaced by still unknown non-orthologous genes. Although some novel enzymes have been elucidated in recent years, e.g. novel classes of GTP cyclohydrolases (e.g. NP4142A, NP2514A) (Graham et al. 2002a; El Yacoubi et al. 2006; Grochowski et al. 2007), several pathway gaps in archaeal coenzyme metabolism remain to be filled. Taking these limitations into account, de novo synthesis pathways for common cofactors are likely in haloarchaea. The compared haloarchaeal genomes show surprisingly different gene sets for cofactor synthesis pathways, and might have differing synthetic capabilities for coenzymes (Supplementary Material S2). N. pharaonis has the most complete set of cofactor synthesis genes and has been shown to grow independently of cofactors (Falb et al. 2005). It is capable of synthesizing menaquinone, coenzyme A, tetrahydrofolate (THF), molybdopterin, hemes, cobamide, flavins, nicotinamide derivates (NAD+/NADP+), biotin, thiamine, and pyridoxal 5-phosphate. The latter is presumably synthesized by novel pyridoxal 5-phosphate synthesis enzymes (pdxS/pdxT, e.g. NP4528A, NP0464A) like in B. subtilis (Raschle et al. 2005). While most enzymes for biotin synthesis are encoded in N. pharaonis (bioA absent) and H. marismortui (bioA and bioD absent), H. salinarum and H. walsbyi lack the complete biotin synthesis gene set. Thiamine biosynthesis pathways might also differ amongst haloarchaea because a couple of probable thiamine biosynthesis genes found in N. pharaonis are partly missing in other haloarchaea, i.e. thiM (NP4052A), thiE (NP4054A), and tenA (NP4080A, NP4082A). For the latter two enzymes genes, there are non-orthologous variants in haloarchaea (including N. pharaonis), tenA is analogous to thiC (NP2210A) and thiN (NP5168A, NP0546A (fused to thiD)) to thiE (Morett et al. 2003). Finally, gene sets for folate metabolism differ greatly between various haloarchaeal strains (see below) leading to the reduced folate synthesis capability in H. salinarum (Levin et al. 2004). Consistent with these findings, biotin, thiamine, and folate are supplied to the synthetic medium of Halobacterium (Oesterhelt and Krippahl 1973). The utilization of several cofactors has been confirmed in haloarchaea such as the use of nicotinamide derivates by glutamate dehydrogenases (NAD+: OE1270F, NADP+: OE1943F) (Hayden et al. 2002), the association of coenzyme A and ferredoxin to pyruvate- and 2-oxoglutarate ferredoxin oxidoreductases (OE1710R, OE1711R, OE2622R, OE2623R) (Kerscher and Oesterhelt 1981a, b) and the incorporation of flavin in dodecin (OE3073R) (Bieger et al. 2003). Respiratory chains of haloarchaea further involve menaquinone and certain hemes (Oesterhelt 1976; Sreeramulu et al. 1998; Scharf et al. 1997; Mattar and Engelhard 1997; Falb et al. 2005). Several copies of proteinaceous cofactors, e.g. ferredoxin, thioredoxin, halocyanin (Mattar et al. 1994), and Fe-S proteins, were also found in haloarchaeal genomes. These are likely used in various redox reactions. Heme and cobamide synthesis Menaquinone (men) and heme (hem) biosynthesis gene clusters are present in all haloarchaeal genomes. All heme synthesis genes for steps leading from glutamate to uroporphyrinogen III have been found, but genes for successive modifications of the porphyrin system to protoheme (e.g. by hemE) are absent as in most other archaea (Supplementary Material S3). Thus, an alternative heme biosynthesis pathway via precorrin-2 as shown for M. barkeri seems likely (Buchenau et al. 2006). A novel archaeal variant of heme A synthase (COG1612) (Lewin and Hederstedt 2006) for the conversion of heme O to heme A is present in haloarchaea (e.g. NP1770A, OE3306R). Halobacterium assimilates corrinoid precursors and cobamides through a high-affinity transport system, but is also able to synthesize cobamide (coenzyme B12) de novo (Woodson et al. 2003). Cobamide synthesis starting from uroporphyrinogen III has mainly been studied in bacteria, e.g. in Pseudomonas denitrificans (cob genes) which uses an aerobic pathway and in Salmonella typhimurium (cbi/cob genes) and Bacillus megaterium which employ an anaerobic pathway (Rodionov et al. 2003). The two pathway variants differ in their cobalt integration step occurring early in the anaerobic (S. typhimurium cbiK, B. megaterium cbiX) but late within the aerobic pathway (P. denitrificans cobN). Haloarchaea encode both cobalt chelatases, the oxygen-dependent (cobN, e.g. NP1092A) and the oxygen-independent type (cbiX, e.g. NP1108A) (Supplementary Material S3). Experimental data showed, however, that cobamides are synthesized under aerobic conditions in Halobacterium (Woodson et al. 2003). Haloarchaeal genomes encode most known cob genes but homologs of P. denitrificanscobG, cobF, cobK (S. typhimirium cobJ), and cobP (S. typhimirium cobU) are absent. This indicates alternative enzymes and reactions in archaeal cobalamin synthesis. For example, the bifunctional bacterial cobP enzyme gene (EC 2.7.1.156/2.7.7.62) is replaced by two novel genes cbiZ (e.g. NP5300A) and cobY (e.g. NP5304A) in archaea (Woodson et al. 2003; Woodson and Escalante-Semerena 2004). The novel archaeal variant of α-ribazole-5′-phosphate phosphatase (EC 3.1.3.71, cobZ, COG1267) (Zayas et al. 2006) for the synthesis of the cobamide α-ligand is not present in haloarchaea, however. Their genomes (except H. salinarum) contain the bacterial-type phosphatase gene (cobC in Salmonella, COG0406, e.g. NP1332A) encoded next to the hemCXD cluster (NP1326A–NP1330A) in Natronomonas. The cobamide α-ligand is derived by cleavage of the ribityl tail from flavin mononucleotide cofactor by BluB (COG0778) (Campbell et al. 2006), of which distant homologs are present in haloarchaea (e.g. NP0176A). Folate synthesis Folic acid and its derivates are synthesized from p-aminobenzoate (PAB) and a pteridine intermediate (Fig. 4, Supplementary Material S4). The latter is derived from GTP by a series of reactions involving folE, folB, and folK genes; all of them are missing in the archaeal domain of life except for H. marismortui. This species has acquired an extensive folate metabolism gene cluster located on one of its plasmid, which contains canonical GTP cyclohydrolase I (EC 3.5.4.16, folE, pNG7382). Recently, a novel archaeal GTP cyclohydrolase (e.g. NP2514A) producing a cyclic phosphate intermediate has been reported in archaea (El Yacoubi et al. 2006; Grochowski et al. 2007), but novel enzyme genes replacing folB and folK remain to be elucidated. PAB is usually derived from chorismate by aminodeoxychorismate synthase (EC 2.6.1.85, pabAB) and aminodeoxychorismate lyase (EC 4.1.3.38, pabC) which are clustered in haloarchaeal genomes (e.g. NP0798A-NP0802A). These enzymes have previously been annotated as copies of anthranilate synthase (same COG0147/COG0512) and branched-chain amino acid aminotransferase (same COG0115), respectively. The pabA gene (OE1570F) of H. salinarum strain R1 (but not of strain NRC-1) is disrupted by an insertion element, though, so that de novo synthesis of folate is likely omitted in this strain. Proposed haloarchaeal pab genes have not yet been characterized, and it should be noted that labeling studies for M. maripaludis suggest the synthesis of PAB from 3-dehydroquinate and not from chorismate in this archaeon (Porat et al. 2006). Fig. 4Folate biosynthesis and metabolism in halophilic archaea. Sets of enzyme genes, which are involved in folate metabolism, differ considerably between haloarchaeal species (square: H. marismortui, circle: H. walsbyi, diamond: N. pharaonis, triangle: H. salinarum, hexagon: H. volcanii, green gene exists, red gene absent). For details see Supplementary Material S4 For folate synthesis, the pteridine intermediate and PAB are converted to 7,8-dihydropteroate and subsequently to 7,8-dihydrofolate by the gene products of folP and folC (Fig. 4). Dihydrofolate is then reduced to 5,6,7,8-tetrahydrofolate by dihydrofolate reductase (EC 1.5.1.3, folA), which is encoded in all haloarchaea (e.g. NP2922A) except H. salinarum. Tetrahydrofolate synthesis in H. salinarum occurs via a novel alternative pathway, instead, where 7,8-dihydropteroate is reduced to 5,6,7,8-tetrahydropteroate by a flavin-binding Prd linker domain of the FolC-Prd-FolP fusion protein (e.g. OE1615R, NP1478A) (Levin et al. 2004, 2007). Tetrahydropteroate is subsequently converted to tetrahydrofolate by the FolC domain. The canonical dihydrofolate reductase FolA is not only involved in de novo synthesis of folate but is also required for the regeneration of dihydrofolate produced by thymidylate synthase (EC 2.1.1.45, thyA, NP2924A, HQ2456A). This enzyme is replaced by an alternative folate-independent enzyme encoded by thyX (OE2898R, rrnAC1121) in case a chromosomal folA gene is absent like in H. salinarum and H. marismortui (Supplementary Material S4). Tetrahydrofolate is likely metabolized to formyl- and methyl-THF in haloarchaea. However, only H. walsbyi and the H. marismortui plasmid pNG700 encode all required enzyme genes involved in folate coenzyme metabolism (e.g. HQ1768A, HQ2790A, HQ1756A). Conclusions Comparative analysis of enzyme gene sets of four halophilic archaea reveals analogous metabolic routes for the biosynthesis of nucleotides and prenyl-based lipids. Biosynthesis pathways for all proteinogenic amino acids are present for H. marismortui, H. walsbyi, and N. pharaonis, while H. salinarum has no biosynthetic capabilities for five amino acids. The loss of amino acid synthesis genes and the acquisition of a catabolic gene clusters for arginine and glutamate fermentation in Halobacterium might have been driven by the constant availability of external amino acids under extreme salt conditions, where few other halophiles compete for nutrients. Several pathway variations were observed for folate-dependent enzymes such as the occurrence of different types of thymidylate synthases or the circumvention of glycine hydroxymethyltransferase by threonine aldolase. These differences might be due to varying gene sets for folate metabolism, in particular the replacement of the canonical dihydrofolate reductase by dihydropteroate reductase in H. salinarum (Levin et al. 2004, 2007). While some haloarchaea may require thiamine and biotin supplements, all halophilic archaea possess the required enzyme genes for the de novo synthesis of cobamide, hemes, flavin and nicotinamide nucleotides, folate, coenzyme A, menaquinone, molybdopterin, pyridoxal 5-phosphate, and several proteinaceous cofactors like halocyanin. The compared haloarchaeal species can best be distinguished by their catabolic and central intermediary pathways. While H. marismortui has a versatile sugar and nitrogen metabolism, H. walsbyi encodes a unique sugar phosphotransferase system and H. salinarum is characterized by a variable energy metabolism. Its plasmids acquired arginine deiminase and cytochrome d oxidase gene clusters (OE6185F/OR6186F) as well as the gene for glycerol dehydrogenase. N. pharaonis is not able to catabolize glycerol (that is not common in its alkaline environment) and is further incapable of sugar degradation. However, the haloalkaliphilic strain encodes an archaeal-type RuBisCO that is probably involved in a novel carbon dioxide-fixation pathway (Sato et al. 2007). It appears that halophilic archaea have adopted several strategies in order to adapt to the nutritional conditions of hypersaline environments, e.g. by gain/loss of metabolic pathways, acquirement of plasmid-encoded enzymes, and utilization of secretion enzymes (Supplementary Material S2). Interestingly, haloarchaeal genomes often encode bacterial-type enzymes instead of (or alongside) non-orthologous archaeal variants, e.g. bacterial-type α-ribazole-5′-phosphate phosphatase (cobC), folate reductase (folA), and β-carotene monooxygenases. Within the mevalonate pathway, two bacterial-type enzymes (IPP isomerase, diphosphomevalonate decarboxylase) exist, which might have been acquired to cover high isoprenoid demands of haloarchaea. Future studies might elucidate whether halophilic archaea employ different enzyme sets for the synthesis of their membrane lipids and carotenes, the latter of which are unique to the haloarchaeal branch of the archaeal domain. The compiled data on haloarchaeal metabolism present a valuable resource for future system biology approaches (Gonzalez et al. 2008), because current knowledge of enzyme variants, domain rearrangements, and enzyme substrate specifities has been included throughout. Further, many of the predicted reactions and enzymes have been linked to available experimental data from enzyme activity tests and labeling studies conducted in the model species H. salinarum. Some of these experimental results however disagree with genomic findings, e.g. for enzymes of the glyoxylate cycle (Aitken and Brown 1969) and the Entner-Douderoff pathway (Rawal et al. 1988) (Fig. 1). These inconsistencies might be due to still unknown non-orthologous enzymes or may result from investigating different strains of Halobacterium, which have been renamed several times within the past decades. Although many pathway gaps in archaeal metabolic networks have been closed in recent years, novel enzyme and pathway variants still remain to be discovered within the archaeal domain of life. In haloarchaea, pathways for ribose 5-phosphate synthesis and for retinal formation need to be investigated. A future in-depth analysis of abundant non-orthologous gene displacements in halophilic and other archaea might further give interesting insights into the evolution of metabolic pathways. Electronic supplementary material Below is the link to the electronic supplementary material. Supplementary Material S1 (DOC 1.71 MB) Supplementary Material S2 (DOC 116 KB) Supplementary Material S3 (DOC 95.5 KB) Supplementary Material S4 (DOC 66.5 KB)

Pediatr_Nephrol-4-1-2275777

The neurogenic bladder: medical treatment

Neurogenic bladder sphincter dysfunction (NBSD) can cause severe and irreversible renal damage and bladder-wall destruction years before incontinence becomes an issue. Therefore, the first step in adequate management is to recognize early the bladder at risk for upper- and lower-tract deterioration and to start adequate medical treatment proactively. Clean intermittent catheterization combined with anticholinergics (oral or intravesical) is the standard therapy for NBSD. Early institution of such treatment can prevent both renal damage and secondary bladder-wall changes, thereby potentially improving long-term outcomes. In children with severe side effects or with insufficient suppression of detrusor overactivity despite maximal dosage of oral oxybutynin, intravesical instillation is an effective alternative. Intravesical instillation eliminates systemic side effects by reducing the first-pass metabolism and, compared with oral oxybutynin, intravesical oxybutynin is a more potent and long-acting detrusor suppressor. There is growing evidence that with early adequate treatment, kidneys are saved and normal bladder growth can be achieved in children so they will no longer need surgical bladder augmentation to achieve safe urinary continence in adolescence and adulthood. Introduction Neurogenic bladder sphincter dysfunction (NBSD) can develop as a result of a lesion at any level in the nervous system, including the cerebral cortex, spinal cord, or peripheral nervous system. Neurologic conditions in children leading to neurogenic bladder dysfunction are predominantly congenital neural tube defects (including myelomeningocele, lipomeningocele, sacral agenesis, and occult lesions causing tethered cord). Acquired causes such as spinal cord tumors or trauma or sequelae of transverse myelitis are less frequent. Whereas from an etiologic standpoint neurogenic bladder dysfunction is a heterogeneous group, medical management will be similar irrespective of the underlying cause. The vast majority of knowledge about NBSD management comes from long-term experience with myelomeningocele (MMC), the most common neural tube defect. Following the institution of a general treatment policy with advances in neurosurgical and orthopedic treatments in previous decades, governing the associated NBSD has become crucial for improving quality of life and life expectancy in children with neural tube defects. In MMC patients, disordered innervation of the detrusor musculature and external sphincter adversely affects bladder function, which if untreated not only leads to incontinence but also will cause secondary damage and dysfunction of both the upper and lower urinary tracts. Key elements in optimal NBSD management are early diagnosis (including NBSD typology) and early (presymptomatic) institution of adequate medical treatment. There is indeed growing evidence that management decisions made during infancy, which prevent both renal damage and secondary bladder-wall changes, potentially impact long-term outcomes for renal function and safe urinary continence. After describing the pathophysiology of NBSD and its possible consequences, this paper focuses on diagnosis (including early identification of patients at risk), treatment goals, treatment tools, and practical management of NBSD. Historical evolution The management of NBSD in children has undergone major changes over the years. A first milestone was the introduction of clean intermittent catheterization (CIC) in 1972 [1]. CIC (combined with anticholinergics if required) has made “conservative” (medical) management a successful treatment option, with a good outcome for quality of life and kidney protection. Further important breakthroughs were the wider application of urodynamic testing in the evaluation of infants and young children with suspected NBSD [2–4] and the better pathophysiological understanding of the natural history of NBSD in patients with spina bifida. In spina bifida, the natural history of the urinary tract in untreated NBSD is one of progressive deterioration by the age of 3 years in up to 58% of patients [5]. Several reports have shown this deterioration to be directly related to increased intravesical pressure. In 1981, the bladder pressure at which urethral leakage occurred was found to be a useful predictor of unsafe bladder function [2]. The leak-point pressure, as it is now commonly referred to, has become accepted as one of the urodynamic parameters that allows clinicians to differentiate patients with relatively low or high risk for subsequent upper urinary tract deterioration. In 1984, detrusor external sphincter dyssynergia (DSD) was identified as an important factor leading to functional obstruction, and intravesical pressure was recognized as the pathophysiological mechanism of subsequent upper urinary tract deterioration [3]. Shortly thereafter, urodynamics in infants and children was shown to allow a functional classification of NBSD that correlated with clinical entities of incontinence and obstruction, an approach that has allowed the concept of individualized and presymptomatic therapy in high-risk patients [6]. Pathophysiology of the neurogenic bladder Under normal conditions, the detrusor muscle, bladder neck, and striated external sphincter function as a synergistic unit for adequate storage and complete evacuation of urine. In healthy bladders, the change in bladder-filling pressure between empty and full is normally less than 10–15 cm H2O. Normal voiding pressures for males and females are from 50 to 80 cm H2O and from 40 to 65 cm H2O, respectively [7]. In patients with NBSD, disordered innervation of the detrusor musculature and external sphincter adversely affects bladder function. In recent years, it has become clear that children with this condition can be categorized into high- and low-risk groups for secondary damage from a neurogenic bladder based on intravesical pressure. When the detrusor (filling) pressure exceeds 40 cm H2O, glomerular filtration rate decreases and pyelocaliceal and ureteral drainage deteriorates, leading to obstructive hydronephrosis and/or vesicoureteral reflux [2, 8–10]. Even in the absence of reflux or upper urinary tract dilatation, high intravesical pressure can impair drainage of urine into the bladder. Any pathophysiologic process that causes either intermittent or continuous elevation of bladder pressure above 40 cm H2O places the child at risk for upper urinary tract dysfunction, urinary tract infections, and ultimately renal failure. Intermittent elevation of bladder pressure may occur from detrusor hypertonia, hyperreflexia, or both. Hyperreflexia may cause intermittent elevation of bladder pressure, especially if the external sphincter acts reflexively and tightens rather than relaxes in an attempt to prevent micturition [detrusor sphincter dyssynergia (DSD)]. Over a long period of time, hyperreflexia with pressures greater than 40 cm H2O may result in detrusor decompensation (areflexia from myogenic failure) or in detrusor hypertrophy with associated sacculations and subsequent diverticula formation. These pathophysiologic changes affect the elastic and vesicoelastic properties of the bladder and also result in mechanical ureterovesical junction obstruction. Continuous elevation of bladder pressure above 40 cm H2O may occur from a hypertonic detrusor or a hypertrophic small-capacity bladder secondary to outflow obstruction [11]. Bladder outlet obstruction is caused by DSD, or by fibrosis of the external urethral sphincter secondary to partial or complete denervation [3, 12, 13]. Bladder outlet obstruction will lead to elevated (pathologic) voiding pressures, which will contribute to either detrusor decompensation or hypertrophy. Finally, recurrent urinary tract infections due to bladder residue may aggravate damage to the neurogenic bladder through processes of transmural inflammation and fibrosis. Together with high intravesical pressures and/or vesicoureteral reflux, these lower urinary tract infections will lead to episodes of acute pyelonephritis and irreversible renal damage. Management of the neurogenic bladder General principles and treatment goals The cornerstone of optimal NBSD management is early identification and characterization (typology) and the institution of proactive therapy. Crucial for long-term prognosis of patients with NBSD is the fact that the management must start before consequences of bladder dysfunction become apparent. From the outset, the goals of management are to prevent or minimize secondary damage to the upper urinary tracts and bladder from the primary neurogenic bladder dysfunction and to achieve safe social continence [14]. Thus, long before continence becomes an issue, starting from the first year of life, management is directed at creating a low-pressure reservoir and ensuring complete and safe bladder emptying. Clean intermittent catheterization (CIC) or self-catheterization (CISC) in combination with anticholinergics (oxybutynin) is the standard therapy for children with neurogenic bladder dysfunction with detrusor hyperactivity and/or DSD [11, 15, 16]. This treatment is also feasible and effective in developing countries, where untreated neuropathic bladder is an important cause of preventable chronic renal failure [17, 18]. CIC enables complete bladder emptying and thus avoids bladder residues and consequent risks for infections. In the high-risk bladder with DSD, CIC also allows bladder emptying before the occurrence of otherwise “spontaneous” high-pressure voiding, which is known to be detrimental for kidney function and drainage. Oxybutynin, a bladder smooth-muscle relaxant, is used to improve bladder dynamics through suppression of detrusor hypertonicity and hyperreflexia. By doing so, oxybutynin eliminates (high-pressure) uninhibited detrusor contractions (and thus urinary leakage) and prevents high-pressure bladder storage (due to detrusor hypertonicity or low bladder compliance) and high-pressure emptying (in case of DSD). Early management, including diagnosis and identification of the high-risk bladder At birth, the majority of patients with neurogenic bladder has normal upper urinary tracts. Without proper management, urinary tract infections and elevated bladder pressures with secondary bladder-wall changes may cause upper urinary tract deterioration within 3 years in up to 58% [5]. One third of children who develop impaired kidney drainage do so within the first year of life [19]. The specific abnormalities vary considerably and are not predicted by the level of the spinal cord defect. Furthermore, the dysfunctional pattern may be dynamic, influenced by spinal cord surgery, tethering, and denervation. In the initial baseline evaluation, clinical observations must be completed with urinalysis (microscopy and culture), renal/bladder ultrasound, and cystourethrogram. These allow the experienced clinician to suspect the type of NBSD and to identify the high-risk subgroup. The next consideration is when to perform urodynamic studies. Two different opinions exist in the literature on the use of urodynamic studies in the early evaluation and further follow-up. In one approach, urodynamic assessment has become an integral part of the initial evaluation and subsequent management, as it allows recognition of the different subtypes of NBSD (typology), proactive interventions, evaluation and guidance of therapy, and early detection of neurologic deterioration (such as symptomatic tethering of the spinal cord [20]). Advocates justify this approach of routine urodynamics to minimize the deleterious effects of high intravesical pressure by directly measuring it rather than indirectly suspecting it from the development of upper and lower urinary tract changes on serial radiologic imaging. Several studies have shown that early urodynamic evaluation of children with NBSD allows the prediction of which newborns are at risk for upper urinary tract deterioration. Urodynamic risk factors are low bladder compliance, intravesical pressure more than 40 cm H2O, and DSD [2, 3, 6, 21]. The alternative to urodynamic-based management is serial radiologic imaging to detect secondary evidence of high bladder pressure. Critics of newborn and early infancy urodynamics refer to a lack of standards for performance and interpretation, which might lead to unnecessary interventions [22, 23]. Those authors recommend careful history, physical examination, upper urinary tract imaging, and close follow-up during infancy and childhood, reserving urodynamic studies only for patients with evidence of urinary retention on physical examination, new-onset hydronephrosis or febrile urinary tract infection, or for evaluation to achieve continence. Proponents of this approach with selective urodynamics suggest that close monitoring with prompt intervention at first signs of deterioration is effective in protecting the upper urinary tracts (including preservation of nephrons and thus renal function in the long run). A remaining concern, however, could be that in this more expectant approach, high intravesical pressures may have already resulted in irreversible and avoidable damage to the bladder wall, resulting in small-capacity, low-compliance bladders later in life. Although many questions regarding optimal evaluation and management remain unanswered, the consensus on the need of close surveillance, especially in the first years of life, plus the possibility that proactive treatment may be better for the bladder in the very long term, emphasize the need for an integrated approach in which clinical observations, serial imaging, and urodynamics are the basis for early adequate treatment. Urodynamic studies: special considerations in children with NBSD If properly performed, even with possible shortcomings in newborns and infancy, urodynamic studies allow direct diagnosis of NBSD and recognition of dysfunction subtypes. This functional classification allows adequate treatment for the different types and early proactive treatment for the bladder at risk [6]. It is important for the practitioner to understand the complexities involved in performing urodynamic studies in newborns, infants, and children. Urodynamic assessment can provide reproducible results in newborns and infants, but it requires attention to mechanical factors and filling rates. The younger the child, the higher the risk that mechanical factors (such as bladder-outlet obstruction by the catheter used for the investigation) may produce artificial information (elevated leak pressure or inability to void). It has also been shown that using a bladder infusion rate as close as possible to the natural filling rate is important for correct assessment of detrusor properties [24]. It is presumed that fast infusion rates overcome vesicoelastic detrusor properties, falsely indicating detrusor hypertonicity [11]. On the other hand, in children who have apparent low-pressure cystograms and who leak during filling (due to sphincter hypoactivity), detrusor hypertonia may be unrecognized [25]. In these children, it is important to perform a provocative study (including bladder neck occlusion with a balloon catheter) to identify unrecognized detrusor hyperactivity prior to bladder-neck surgery for treatment of incontinence. Electromyographic (EMG) evaluation of the external urethral sphincter is required to identify DSD. The use of concentric EMG needles is preferred, as it gives more reliable information than patch electrodes [11]. The combination of X-ray cystography with cystometrogram and sphincter EMG (video urodynamics) allows accurate evaluation of the link between intravesical pressure and vesicoureteral reflux and gives direct visual information of (dys)synergia between detrusor and sphincter mechanisms [26]. Clean intermittent catheterisation In children with neurogenic bladder, CIC is the first-choice treatment to empty the bladder adequately (no residue, no infection) and safely (prior to high-pressure voiding), and it is a valuable tool for achieving continence. The wide variety of used materials and techniques for CIC does not seem to affect efficacy and safety as long as some basic principles are applied: proper education and training, clean and atraumatic application, and achievement of good patient compliance on a long-term basis. For education, training, and further guidance during follow-up, a dedicated continence nurse is invaluable. Patients and caregivers must understand what is wrong with the bladder/sphincter and why CIC is proposed for treatment, and they have to learn how to catheterize properly. CIC has been successfully used by parents even in newborns and infants, becoming a part of their everyday routine [27]. Some authors prefer early institution of CIC in all infants with NBSD, given the fact that by the age of 3 years, CIC will be required in all for achieving continence, and given the difficulties of starting CIC at toddler age [28]. Such early institution of CIC seems to improve family compliance and their ability to assist the child in coping with their disease and with CIC [29]. CISC can be successfully taught to boys and girls who are motivated and who have developed the required dexterity, mostly around the age of 6 years. The required frequency of catheterization depends on several factors: fluid intake, bladder capacity, and bladder filling/voiding pressures. In practice, it is recommended to catheterize six times a day in infants (linked with feeding time) and five times a day in school-aged children. Although reported incidences of CIC-related infection risks are variable, it is generally agreed that the risk is low as long as complete bladder emptying is achieved. Furthermore, reused supplies are not related to more urinary tract infections [30]. If symptomatic infections occur, these are mainly caused by incomplete bladder emptying, and CIC appliance by child or caregiver needs to be optimized. To prevent urethral strictures and false passage in boys, catheter lubrication and avoidance of forceful manipulation during catheter insertion are advocated. Nonreusable low-friction catheters are considered valuable in high-risk male patients with urethral false passage or very tense sphincters but are unnecessary in routine cases [31]. To maintain therapeutic compliance with CISC in adolescents, psychosocial support is often required. Neurogenic bowel dysfunction with constipation and fecal soiling can interfere with the institution of a successful CIC treatment. Retained stools may mechanically impair bladder filling, increase detrusor irritability, or contribute to urine retention. Stool incontinence increases the risk of bladder contamination and urinary tract infection. An effective bowel management program is therefore needed. Finally, given the high prevalence of latex allergy [32], in the spina bifida population, a strict latex-free approach is of extreme importance. Pharmacologic treatment: anticholinergics Of the anticholinergic agents available, oxybutynin hydrochloride is most commonly used, and long-term experience supports its safety also in newborns and infants [33]. Oxybutynin is a tertiary amine with a well-documented therapeutic effect on detrusor hyperactivity, and its effectiveness is attributed to a combination of anticholinergic (M3-selective receptor subtype antagonism), antispasmodic, local anesthetic and calcium-channel-blocking activity [34]. Several studies have shown its efficacy for decreasing the filling pressure, increasing the capacity of the neurogenic bladder, and preserving renal function [35–37]. The usual dose regimen of oral oxybutynin is 0.3–0.6 mg/kg per day in three doses. In children with insufficient response or significant systemic side effects to oral oxybutynin, intravesical instillation of oxybutynin has been shown to be a highly efficacious, reliable, and well-tolerated therapy for children who would otherwise require surgical therapy [38–43]. Because a solution suitable for intravesical instillation was not available, crushed oxybutynin tablets were used in the earlier trials, with consequent problems of inconvenience and impracticability, and it was the belief of several authors that poor patient compliance could be resolved by an optimized drug preparation [40, 44]. It was subsequently shown that, indeed, eliminating the complex crushing preparation by child or parent makes intravesical oxybutynin therapy easy to use and acceptable for long-term therapy [41]. The mechanisms underlying the more potent and longer-acting detrusor-suppressive effects of intravesical oxybutyinin, as well as its better tolerability, have been investigated by several groups. It was demonstrated that a reduced first-pass metabolism of oxybutynin after intravesical instillation, resulting in a reduced generation of the N-desethyl metabolite, may explain the clinically relevant reduction of systemic side effects that characterizes intravesical compared with oral oxybutynin therapy [45]. In addition, these pharmacokinetic studies provided first evidence for a direct local rather than a systemic effect of intravesical oxybutynin on detrusor muscle [45]. Further evidence for a local effect of intravesically administered oxybutynin was provided by studies showing local (urothelial) accumulation, suppression of muscarinic receptor-mediated detrusor muscle contractions, and blocking of muscarinic receptors in bladder-afferent pathways [46, 47]. In most reports, intravesical oxybutynin is used in dosages between 0.3 and 0.6 mg/kg per day in two or three doses. Given its better tolerability compared with oral treatment, if required, intravesical dosages can be further increased up to doses of 0.9 mg/kg per day [43]. To date, the vast majority (∼ 90%) of patients can be treated successfully with the gold standard treatment of oxybutynin (oral or intravesical) and CIC. Other bladder-relaxant drugs include propiverine (10–15 mg b.i.d. or t.i.d., adult dose), trospium (20 mg b.i.d., adult dose), extended-release oxybutynin, and tolterodine (children 0.25–1 mg b.i.d., adults 1–2 mg b.i.d.). The current experience with compounds other than oxybutynin is still limited in children with neurogenic bladder [48, 49]. Botulinum A toxin injections into the detrusor muscle have been shown to be a potentially valuable approach in the neurogenic overactive bladder [50]. Repeated botulinum A toxin injections (as an alternative for or an additive to anticholinergics) could be considered to postpone or avoid surgical procedures in the small minority of children not responding to standard therapy with CIC and anticholinergics [51]. However, further investigations are required, given remaining concerns about costs and long-term efficacy and safety of prolonged botulinum A toxin administration. Although some authors have advocated alfa-receptor stimulation of the bladder neck, no validated medical treatment is available to enhance the bladder outlet. Medical management of NBSD in clinical practice Optimal management involves first, early diagnosis, including recognition of high-risk subtypes, and second, proactive institution of adequate treatment. Early proactive treatment of high-pressure dyssynergic lower urinary tracts is important in the long term, not only to preserve renal function [52] but also to prevent poor bladder compliance and the subsequent need for bladder augmentation [35]. Urodynamic assessment is used in newborns and infants to come to a functional classification of NBSD, allowing presymptomatic interventions in the high-risk groups and individualized treatment planning according to the type of dysfunction [6, 29, 53]. In clinical practice, four major subtypes can be used to describe NBSD (Fig. 1): sphincter overactivity combined with detrusor underactivity (type A) or overactivity (type B), and sphincter underactivity combined with detrusor underactivity (type C) or with detrusor overactivity (type D). The easiest type to treat is type A. This bladder type requires early treatment because of urine retention with high filling pressure and continuous leaking. Here, CIC alone is effective and sufficient and will make the bladder safe and infection free, and the patient will be dry in between (social continence). Good care to empty the bladder totally is most important to avoid bladder infections caused by residual urine. Dysfunctional type B will have high filling and high voiding pressures, being very unsafe from birth onward due to DSD. Here, the act of voiding has to be prevented. With oxybutynin, the overactive detrusor can be “pharmacologically converted” to an inactive reservoir (situation similar to type A), which has to be emptied with CIC. In type C, CIC reduces the degree of incontinence and offers much better control over urinary tract infections. To achieve continence, this type will at a later age need surgical intervention on the sphincter (e.g. sling operation). An important caveat here is that detrusor instability may emerge only after surgical improvement of outlet resistance. If this detrusor instability would remain unrecognized and untreated (with oxybutynin), bladder-outlet surgery would have converted a “wet but safe” into a “dry but unsafe” bladder. In the last dysfunctional subtype (type D), the bladder leaks due to detrusor instability and gradually becomes unsafe due to secondary bladder-wall changes with detrusor hypertrophy and loss of bladder compliance. Therefore, treatment consists of CIC combined with oxybutynin and, at a later age, bladder-outlet surgery. Fig. 1Classification of the neurogenic bladder, with four subtypes (a–d) according to dysfunctional activities of sphincter and detrusor. For each subtype, clinical implications if untreated and principles of management are summarized Once appropriate therapy has been initiated, adequate follow-up is required, with adjustments if needed (CIC frequency, medication dosing and administration route). Treatment efficacy can be assessed using clinical parameters (including CIC frequency and volume charts), urinalysis, renal and bladder ultrasound, X-ray cystography, and video urodynamics. As long-term sequelae of insufficiently treated neurogenic bladders (renal scarring, noncompliant fibrotic bladder) already have their origin in the first years of life, the frequency of multidisciplinary follow-up visits must be age dependent (3× yearly up to age 3 years, 2× yearly in school-aged children, yearly in adults). Typically, urinalysis and ultrasound are performed at all visits, cystography to investigate unexpected upper urinary tract infections, and urodynamics periodically to verify that under treatment, the catheterized bladder volumes are age appropriate [54] and stored under safe pressure conditions (storage of expected bladder capacity at pressures below 30 cm H2O; see [55]). With early instituted and optimal treatment, the large majority of patients can be adequately controlled without antireflux surgery or surgical bladder augmentation (Fig. 2). Augmentation cystoplasty is limited to a small group of patients in whom medical treatment fails (persistence of high filling pressures). In patients with insufficient sphincter activity, continence achievement will require bladder-outlet surgery in addition to medical treatment. In female wheelchair users, surgical intervention to provide a continent stoma will facilitate self-catheterization. Fig. 2Suppression of detrusor hyperactivity with resolution of reflux by nonsurgical management. Illustrative patient with high-risk neurogenic bladder sphincter dysfunction (NBSD) (type B), urodynamically showing early unsafe filling pressures (A) with high-grade reflux (a) and urosepsis before treatment. Under clean intermittent catheterization (CIC) plus oxybutynin, the unsafe high-pressure bladder was converted into a safe low-pressure reservoir with good capacity and disappearance of the reflux at control cystography 3 months later (b). Severe systemic side effects, making continuation of oral oxybutynin impossible, disappeared after switching to intravesical oxybutynin. Further urodynamic evaluations (B: after first intravesical administration; C: after 4 months) documented adequate suppression of detrusor hyperactivity (modified from [40]). Long-term (currently 13 years) continuation of CIC and intravesical oxybutynin has resulted in a safe and adequate capacity bladder with social continence for the patient Long-term outcome evaluation and need for life-long follow-up Lifelong follow-up with further periodic investigations of upper urinary tract changes, renal function, and bladder status is extremely important. There are two reasons why long-term outcome evaluation in adulthood and life-long patient follow-up are indispensable. First, for the individual patient, therapy is a life-long requisite, and verifying preservation of the patient’s kidneys is only possible by repetitive assessment throughout adolescence and adulthood. Second, in general, detailed long-term follow-up data will show whether a treatment policy driven by long-term goals is sufficiently effective or requires further adaptations. The effectiveness of efforts preserving upper urinary tract function can only be judged by assessing the ultimate outcome once these patients have reached adolescence or adulthood [29]. In populations with NBSD, no consensus exists as to how renal status is ideally evaluated [56]. In clinical practice, upper urinary tract deterioration or protection is often monitored by radiographic images of hydronephrosis and vesicoureteral reflux. Modalities used to look at renal functions include nuclear imaging [dimercaptosuccinate acid (DMSA) renal scan], urinary concentrating ability, and glomerular filtration rate assessment. For the latter, creatinine (Cr) clearance can be used for patients who are socially continent; for others, inulin or Cr ethylenediaminetetraacetate (EDTA) clearance can be used. Which (combination) of these tests is best to evaluate renal function requires further investigation [56]. Conclusions Medical management with CIC and anticholinergics is effective in preserving renal function and providing safe urinary continence in more than 90% of patients with a neurogenic bladder. Early diagnosis and treatment institution, long before continence becomes an issue at toddler age, can prevent both renal damage and secondary bladder-wall changes, thereby improving long-term outcomes. Compared with oral oxybutynin, intravesical oxybutynin has more potent and longer-acting detrusor suppressive effects with good tolerance and should be used prior to considering surgical therapies. Therapeutic goals should no longer be restricted to prevention of secondary damage to both upper and lower urinary tracts. Instead, our goal should be to achieve normal renal and bladder growth at safe bladder pressure, with appliance-free continence.

J_Mammary_Gland_Biol_Neoplasia-3-1-1820839

Extracellular Proteolysis in Transgenic Mouse Models of Breast Cancer

Growth and invasion of breast cancer require extracellular proteolysis in order to physically restructure the tissue microenvironment of the mammary gland. This pathological tissue remodeling process depends on a collaboration of epithelial and stromal cells. In fact, the majority of extracellular proteases are provided by stromal cells rather than cancer cells. This distinct expression pattern is seen in human breast cancers and also in transgenic mouse models of breast cancer. The similar expression patterns suggest that transgenic mouse models are ideally suited to study the role of extracellular proteases in cancer progression. Here we give a status report on protease intervention studies in transgenic models. These studies demonstrate that proteases are involved in all stages of breast cancer progression from carcinogenesis to metastasis. Transgenic models are now beginning to provide vital mechanistic insight that will allow us to combat breast cancer invasion and metastasis with new protease-targeted drugs. Introduction Breast cancer progression is accompanied by increased expression of proteases that are capable of degrading the extracellular matrix (ECM) leading to cancer cell invasion and eventually to dissemination of cancer cells to other organs. The proteolytic capability of breast cancers is reminiscent of the inherent proteolytic capability of the healthy mammary gland. Extracellular proteases are necessary for the dynamic tissue remodeling that takes place during normal mammary gland development and pregnancy cycling (reviewed in [1]). During development, the invasive phenotype of terminal end buds is associated with a controlled proteolytic digestion of ECM in the fat pad, and side branching of the newly formed ducts requires a focal degradation of basement membrane. The proliferative response of the epithelium during pregnancy and lactation is accompanied by limited proteolytic activity. In contrast, the involution of the lactating gland to a resting gland after weaning restructures the entire gland and requires extensive degradation of ECM and basement membranes. Extracellular proteases are thus more abundant during involution than at any other stage during development and the pregnancy cycle. The similarity between the proteolytic repertoire of the normal gland and the proteolytic capability of mammary cancers is best illustrated by the fact that the cellular expression patterns of individual proteolytic components are very alike, especially when comparing involution and cancer [2]. In terms of extracellular proteolysis, breast cancer in many ways resembles a mammary gland in a state of continuous and uncontrolled tissue remodeling. Matrix Metalloproteases and Plasminogen Activators Two families of extracellular proteases have been extensively studied in relation to mammary gland morphogenesis and cancer. These are the matrix metalloproteases (MMPs) and the group of serine proteases that govern the activation of plasminogen (Plg). Figure 1 illustrates the connected extracellular proteolytic network of the MMPs and the Plg activation system (PA system). Figure 1The PA and MMP systems in the mouse. Plasmin and MMPs are two major sources of extracellular proteolysis. Plg is converted to the active protease plasmin by one of three Plg activators, uPA, tPA, and pKal. The generation of plasmin is enhanced by the binding of uPA to the cell surface receptor uPAR, and by the binding of tPA to coagulated fibrin or to cell surface proteins (annexin II [86] and CKAP4 [87]). The conversion of pro-uPA to active uPA may be initiated by several proteases, depending on the tissue in question. Additional pro-uPA is then activated by plasmin in a positive feed-back loop. The PA system is tightly controlled by several serine protease inhibitors (serpins); the primary and most specific inhibitors are the Plg activator inhibitor PAI-1 that targets uPA and tPA, and the plasmin inhibitor α2-antiplasmin (α2AP). Major substrates of plasmin include fibrin, fibronectin, laminin, and other ECM proteins, latent TGF-β and other growth factors, and pro-MMPs. The 22 different mouse MMPs are all extracellular, and are either soluble or membrane-anchored. Like the serine proteases of the PA system, the MMPs are also synthesized as inactive proforms that require proteolytic activation. Ten of the 22 pro-MMPs, including the membrane-anchored MMPs, have a furin cleavage site and may be activated by furin-like proprotein convertases before they are secreted. The remaining pro-MMPs are activated extracellularly, typically by plasmin or by other MMPs. The MMP system is counterbalanced by a group of four inhibitors, TIMPs, that have varying specificities for individual MMPs. In addition, the membrane-anchored MMP inhibitor RECK regulates a subgroup of MMPs including MMPs-2, -9, and -14 [88]. An excess of plasmin or MMP activity may ultimately be cleared by α2-macroglobulin (α2M) which is a non-specific protease scavenger. The MMPs are collectively able to degrade any component of the ECM. Important substrates for the MMPs as a group include the native fibrillar collagens and all four major components of the basement membrane: collagen type IV, laminin, nidogen/entactin, and heparan sulfate proteoglycans. The liver-derived proenzyme Plg is found in high concentration throughout the body. In contrast, expression of the specific urokinase- and tissue-type Plg activators (uPA and tPA) is induced locally and thus determines where and when Plg is converted to the active protease plasmin [3, 4]. Plasmin, activated by uPA, is important for a number of tissue remodeling processes that, in addition to pregnancy cycling of the mammary gland [5], include wound healing [6] and embryo implantation [7]. In contrast, tPA is acknowledged to be primarily involved in thrombolysis and neurobiology [3, 4] but is also active during wound healing [8]. A newly identified Plg activator, plasma kallikrein (pKal), is involved in adipocyte differentiation in the involuting mammary gland [9] and in wound healing [8]. The MMPs constitute a large family of 22 extracellular metalloproteinases in mice (24 in humans) [10, 11] (an updated list of human and mouse proteases is available at http://web.uniovi.es/degradome/). MMPs are essential for a number of physiological tissue remodeling processes during ontogenesis and adult life, including mammary gland development [12, 13], bone development [14], wound healing [15], and embryo implantation [7]. During mammary gland development MMP-2 is involved in ductal elongation, and MMPs-2 and -3 are involved in ductal branching [13]. MMP-3 is also rate limiting for adipocyte differentiation during post-lactational involution [16]. The ductal tree is eventually fully formed in mice that are deficient in MMP-2, -3 or several other MMPs, indicating that the remaining MMPs are able to replace their activity in a functional overlap. Several members of the MMP and PA systems are involved in breast cancer progression. We will focus on the role played by these extracellular protease families during the various stages of breast cancer progression, from the initial hyperplastic lesion to disseminated disease, with particular emphasis on the insight gained from transgenic mouse models. Mouse Models of Breast Cancer The mouse mammary gland is ideally suited to study cancer progression for a number of reasons. First of all, there are many similarities between the human and mouse mammary glands (for detailed reviews see [17, 18]). Human and mouse mammary glands contain the same epithelial cell types: the ductal epithelial cells, luminal (milk secreting) epithelial cells, and the myoepithelial cells. They also contain adipose tissue and stroma, although the composition of the stroma differs between the human and mouse glands. One of the few differences between the species is that the epithelium in the mouse gland is surrounded by adipose tissue and a relatively sparse stroma, whereas the human mammary epithelium is encompassed by a specialized fibroblastoid stroma and is not in direct contact with adipocytes. The epithelial structure in the mature nulliparous mouse gland is also much less developed compared to the human gland and consists only of a branching but otherwise naked ductal tree. In contrast, the mature ductal tree in non-pregnant women contains groups of acini at the end of terminal ducts, named terminal ductal lobular units (TDLU). The mouse epithelium develops similar lobuloalveolar structures transiently during the estrus cycle in certain mouse strains [19] and always during early pregnancy, and in this way the resting human gland is structurally more similar to the pregnant mouse gland. In some transgenic breast cancer models, the earliest hyperplastic lesions may also resemble TDLU-like structures [20]. A second ideal feature of the mouse mammary gland is that it is readily accessible for monitoring tumor growth and for other experimental procedures. This has lead to the development of numerous transplanted models, chemically induced models and transgenic models. We will limit our discussion to the transgenic and chemically induced models since there are inherent problems of transplanted models that may introduce bias. Depending on the study design, these obstacles can include: (1) a species barrier between proteolytic components supplied by the transplant and the host (as documented for human and mouse uPA and its receptor uPAR [21]); and (2) a cellular inconsistency with respect to protease expression in the sense that the majority of proteolytic components are supplied by stromal cells in human breast cancers while epithelial-based cancer cell lines often express the very same proteolytic components [22]. This is presumably due to genetic alterations that have occurred during in vitro culture. Furthermore, (3) when gene-deficient mice are used as hosts for syngeneic transplantation, it is possible that a tumor-suppressive phenotype in the protease-deficient host is due to an immune response directed at the protease-producing transplant. A third advantage of the mammary gland for cancer research is that there are more transgenic models of mammary cancer than for any other cancer form, primarily because relatively specific promoters are available for targeting oncogene expression to the mammary epithelium. The promoters most frequently used stem from the whey acidic protein (WAP) gene, and the mouse mammary tumor virus long terminal repeat (MMTV). The promoters are not equally active at all stages of mammary gland development and the pregnancy cycle [23]. The MMTV promoter is active during puberty but is greatly enhanced during pregnancy. The WAP promoter is primarily active from mid pregnancy and during lactation. Accordingly, cancer progression is accelerated by pregnancies in most transgenic models and some models only develop tumors after a number of pregnancies. Finally, the tumor histopathology of several transgenic models is very similar to human breast cancers [17]. The histopathology seen in a single mouse model is, however, relatively uniform and often characteristic of the inducing oncogene, and in this way no single model replicates the variability of tumor phenotypes seen among different breast cancer patients [17, 24]. This drawback is to be expected, considering the fact that transgenic models rely on a well-defined initiating oncogene in mice with a homogenous genetic background (inbred strains). In contrast, human breast cancers arise from combinations of multiple mutations in women with heterogenous genetic backgrounds. A notable similarity between mouse and human tumors is the development of a reactive stroma and the appearance of extracellular proteolysis in the tumor microenvironment. There are, however, also significant drawbacks to the use of transgenic models, such as the fact that they express oncogenes in the entire mammary epithelium (or at least a large part of it) often giving rise to multiple tumors and in some models a general hyperplasia of the entire gland. By contrast, human tumors derive from a single site, therefore resulting in a single tumor in the breast. A comprehensive list of transgenic breast cancer models is available at http://emice.nci.nih.gov/ and has recently been reviewed [23]. Here we will focus on models that have been studied in relation to extracellular proteolysis. Table 1 summarizes the studies that describe MMP or PA system intervention in transgenic and chemically induced breast cancer models. It is evident from Table 1 that the role of proteases has almost exclusively been studied in models, in which one of the four oncoproteins ErbB-2 (neu) [25], Ras [26], Wnt1 [27], or the polyomavirus middle T antigen (PymT) [28] are induced by the MMTV promoter. ErbB-2 is often overexpressed in human breast cancers. Ras and the downstream effector proteins of Wnt1 and PymT are also frequently activated in human breast cancer, although mutation/overexpression of Ras and Wnt1 are infrequent in breast cancer and there is no cellular counterpart of the PymT oncogene [23]. In all four transgenic models, tumor incidence is 100% or at least approaches 100% depending on the time allowed for observation [29–32]. Tumor incidence has only in a few cases [29, 32] been affected in protease intervention studies (Table 1). Tumor onset in the ErbB-2, Ras, and Wnt1 models can be accelerated by multiple pregnancies [25, 30, 32] and mice used for experiments are frequently mated continuously, at least in the Ras and Wnt1 models [30, 32]. The PymT model is exceptionally fast even in non-pregnant mice, where the average tumor latency is approximately 1.5 months [31] compared to 4–7 months for multiparous mice in the three other models [25, 30, 32]. In all four models the tumors metastasize primarily to the lungs although the models differ in aggressiveness. The PymT model is the most aggressive and the Wnt1 model the least aggressive [31, 32]. A notable exception to the use of transgenic models in protease studies is the chemically induced mammary tumors that are induced by orally administered 7,12-dimethylbenzanthracene (DMBA) with or without hormone supplementation [33, 34]. The DMBA model is primarily a carcinogenesis model, since the tumors generally do not progress to form metastases [33, 34]. Table 1PA system and MMP intervention studies in transgenic and chemically induced breast cancer models.Proteolytic interventionMouse br.ca. modelPhenotypic effect on mammary neoplasiaReferencetumor incidencetumor onsettumor growth ratefinal tumor sizelung metastasis incidencelung metastasis burdencomments and additional phenotypesPlg activation systemPlg nullMMTV-PymT↔↔ (↓ trend)↔↔↓↓reduced metastasis in 129:BSw:FVB mixed strain but not in FVB mice[44] and unpublished datauPA nullMMTV-PymT↔↔(↓ trend)↔↔↔↓+ reduced lymph node metastasis[31]PAI-1 nullMMTV-PymT↔↔↔↔↔↔ (↑ trend)tumor vascularization not affected[45]uPAR nullMMTV-PymT↔↔↔↔↔↔no phenotype in the FVB strainunpublished dataMMP protease family MMP overexpressionMMTV-MMP-3 transgenenoneyes**tumors develop in the CD1 mouse strain but not in mixed B6:DBA mice[33, 61], see comment in [90]WAP-MMP-3 transgenenoneyes**tumors develop in the CD1 mouse strain but not in FVB mice[62], see comment in [90]MMTV-MMP-7 transgenenonenohyperplasia in multiparous mice but no tumors develop spontaneously[29]MMTV-MMP-14 transgenenoneyestumors develop spontaneously[63]MMTV-MMP-3 transgeneDMBA gavage↓↓opposite expectation[33]MMTV-MMP-3 transgeneDMBA gavage + MMTV-TGFα↔↔[33]MMTV-MMP-7 transgeneMMTV-neu↑↑↔↔[29]MMTV-MMP-7 transgeneApcMin + ENU↔[91] MMP inhibitionMMP inhibitor-treatment (galardin/GM6001)MMTV-PymT↓↓↓unpublished dataMMP-2 nullMMTV-PymT↔↔↔↔↓↓Fingleton and Matrisian, pers. comm.MMP-7 nullMMTV-PymT↔↔↔↔↔↔Fingleton and Matrisian, pers. comm.MMP-7 nullApcMin + ENU↓transient reduction in number of mammary tumors[91]MMP-9 nullMMTV-PymT↔↔↔↔↓↓reduced metastasis in mixed B6:FVB mouse strain but not in FVB miceFingleton and Matrisian, pers. comm.MMP-9 nullMMTV-neu↑(meeting report [67])MMP-11 nullMMTV-ras↔↓↓↑bidirectional effect[30]MMP-11 nullDMBA gavage↓↓ovarian and mammary carcinomas[66]MMP-13 nullMMTV-PymT↔↔↔↔↔↔unpublished dataWAP-TIMP-1 transgeneWAP-MMP-3↓reduced incidence of hyperplasias, no tumors develop[62]Albumin-TIMP-1 transgeneDMBA gavage + MPA↓early hyperplasia not affected[34]Albumin-TIMP-1 transgeneMMTV-PymT↔↓↓↔[34]MMTV-TIMP-1 transgeneDMBA gavage + MPA↔[34]MMTV-TIMP-1 transgeneMMTV-PymT↔↔↔[34]MMTV-TIMP-2 transgeneMMTV-Wnt1↓↓↓↓reduced mean vessel size in tumors[32]TIMP-2 gene therapyMMTV-neu↓↓[92]Abbreviations: ApcMin multiple intestinal neoplasia model in mice with a mutated Apc allele; B6, C57BL/6J; BSw Black Swiss; DMBA 7,12-dimethylbenzanthracene; ENU N-ethyl-N-nitrosourea; MMP matrix metalloprotease; MMTV mouse mammary tumor virus long terminal repeat promoter; MPA medroxyprogesterone acetate; PAI-1 Plg activator inhibitor-1; Plg plasminogen; PymT polyomavirus middle T antigen; TIMP tissue inhibitor of metalloproteases; uPA urokinase plasminogen activator; uPAR uPA receptor; WAP whey acidic protein promoter. A feature of any transgenic model that is particularly relevant to extracellular proteolysis is the transition from pre-invasive (carcinoma in situ, CIS) to invasive breast carcinoma. Most human pre-invasive lesions are ductal CIS (DCIS), and in these lesions the carcinoma cells accumulate in the ducts confined by the myoepithelial cell layer and the basement membrane. Proteolytic degradation of the basement membrane leads to depolarization of the myoepithelial cells and offers the carcinoma cells access to the surrounding periductal stroma [35, 36]. In some human breast carcinomas the tumor suppressive myoepithelium surrounding an emerging tumor can persist for a relatively long period of time. In the MMTV-PymT model the late stage tumors resemble human invasive ductal breast cancer, but the myoepithelium is lost very early in tumor progression and it is therefore a poor model of the CIS stage ([20] and unpublished data). The lack of an intact myoepithelium, as seen in the MMTV-PymT model, does not necessarily make the neoplastic cells invasive. These lesions fall in the broad category of mammary intraepithelial neoplasia (MIN), which includes CIS-like lesions but is used more generally in mouse tumors to describe pre-malignant epithelial cells that have significant nuclear atypia and are surrounded by an intact basement membrane [37]. A few transgenic breast cancer models have been described to develop CIS lesions with morphologic similarities to human DCIS and later progress into invasive breast cancer. These include the C3(1)-SV40-T prostate cancer model that also develops mammary lesions [38], the WAP-SV40-T model [39], and the MMTV-neu models [17]. However, the CIS stage has not been the focus of protease intervention studies so far. Protease Expression in Transgenic Breast Cancer Models Extracellular proteases are generally absent in resting mammary glands but are present as the gland undergoes the pregnancy cycle. The levels of a number of extracellular proteolytic components are particularly upregulated during post-lactational mammary gland involution. These include uPA, tPA, MMPs-2, -3, -9, -11, and their corresponding inhibitors PAI-1 and TIMP-1 [5, 40–42]. The upregulated proteases and protease inhibitors are presumably required for the orderly restructuring of the lactating gland to a virgin-like state (discussed later). During breast cancer development, a similar proteolytic program is activated in the diseased gland and extracellular proteases are abundant in invasive breast cancers of both humans and mice. Figure 2 gives a schematic comparison of the expression patterns of MMP and PA system components in human breast cancer and in the MMTV-PymT transgenic breast cancer model, which has been used to analyze the expression of several extracellular proteases. One study used laser microdissection to isolate cancer cells and stromal cells followed by quantitative RT-PCR to examine the expression level of MMPs and the PA system [43]. The study concluded that stromal tissue adjacent to cancer cells expresses higher levels of uPA, PAI-1 and MMPs-2, -3, -11, -13, and -14 than the cancer tissue. The predominantly stromal expression patterns of all these components in the MMTV-PymT model has been confirmed by in situ hybridization [31, 43–46]. Using immunohistochemistry, uPAR expression was found primarily in fibroblasts, endothelial cells, and in some macrophages in the MMTV-PymT model (unpublished data). In situ hybridization was used in another study on MMTV-PymT tumors to determine the expression pattern of four of the membrane-type MMPs: the transmembrane MMPs-14, -15, and -16 and the GPI-linked MMP-17 [46]. MMPs-14 and -16 were detected in the stroma whereas MMP-15 was the only protease found predominantly in the epithelium. MMP-17 expression was not observed in the MMTV-PymT tumors. The majority of these findings reflect the mRNA expression data for human breast cancer, since uPA [47], PAI-1 [48], and MMPs-2 [49], -3 [50], -11 [49], -13 [35], and -14 [51] all are found predominantly in the stroma (see suppl. data to review by Egeblad and Werb 2002 [10]). Immunoreactivity for uPAR is also primarily found in the stroma [52]. Figure 3 provides two examples of this close correlation between human and transgenic mouse tumors: MMP-13 is focally expressed in stromal cells in patient material [35] and in MMTV-PymT transgenic tumors [43] (Fig. 3c–d). Similarly, the primary uPA inhibitor PAI-1 is expressed in stromal cells in MMTV-PymT mice [45] and in breast cancer patients [48] (Fig. 3e–f). The PAI-1-producing stromal cells were identified as myofibroblasts in the human samples. It is of note that this cell type is uncommon in the normal mouse and human glands but is abundant in tumors from both transgenic mice (unpublished data) and breast cancers patients [53]. Figure 2Expression of MMPs and PA components in breast tumors in humans and in MMTV-PymT transgenic mice. MMPs and PA components are expressed by cancer cells or stromal cells in breast tumors or are present as ubiquitous plasma-derived proteins. The predominant source of each mRNA/protein is illustrated for human ductal breast cancer and for the MMTV-PymT model in mice. Only those proteases and related components that have been analyzed in the MMTV-PymT model are included in the comparison. Only mRNA analyses are included except for uPAR, which was analyzed by immunohistochemistry in both species. The “Not detected” category includes proteases that were sporadically expressed in the tumors. The data for the MMTV-PymT model: uPA [31]; PAI-1 [45]; uPAR, MMPs-7, -9, -10 (unpublished data); MMPs-2, -3, -11, -13, -14, uPA, PAI-1 [43]; MMPs-14, -15, -16, -17 [46]. The data for invasive ductal breast cancer: uPA [47]; PAI-1 [48]; uPAR [52]; MMP-2 [49]; MMP-3 [50], MMP-7 [89]; MMP-9 [72]; MMP-10 [89]; MMP-11 [49]; MMP-13 [35]; MMP-14 [51].Figure 3Examples of protease expression in breast cancer from transgenic mice and humans. Expression of MMP-13 during early invasion in mouse C3(1)-SV40-T breast carcinoma (a) and in human ductal carcinoma in situ (DCIS) (b). MMP-13 is focally expressed in periductal fibroblast-like cells (arrows in a, a’, and b’) in areas with early cancer cell invasion (inv) and is not expressed in adjacent non-invasive areas with in situ carcinoma (CIS). Expression of MMP-13 in invasive MMTV-PymT carcinoma (c) and in human invasive ductal carcinoma (IDC) of the breast (d). Focal expression of MMP-13 is seen in fibroblast-like cells located in the stroma surrounding invasive cancer cells in both mouse and human breast cancers (arrows in c and d). Expression of PAI-1 in invasive MMTV-PymT carcinoma (e) and in human IDC of the breast (f). The expression of PAI-1 is seen in fibroblast-like cells located in the stroma surrounding invasive cancer cells in both the mouse and human breast cancers (arrows in e and f). All panels are in situ hybridization using 35S-labeled probes. Panels a, c–f are counterstained with haematoxylin and eosin, whereas b is combined with pan-cytokeratin immunohistochemistry and (b’) with CK14 immunohistochemistry for myoepithelial cells [35]. Bars: a,c, 200 μm; b, 100 μm; d–f, 150 μm. Recently, the MMTV-Wnt1 model has also been used to study the expression levels of a number of MMPs during mammary tumor formation by RT-PCR and in situ hybridization [32]. MMPs-2, -3, -9, -13, and -14 displayed increased expression levels in hyperplastic glands and mammary tumors. MMPs-2, -3, and -9 were exclusively expressed in the stroma, while MMPs-13 and -14 were found predominantly in the cancer cells and to a lesser extent also in stromal cells. Again, most of these MMPs have been reported with similar expression patterns in human breast cancer, although MMPs-13 and -14 are predominantly stromal in ductal breast cancer [35, 51]. It is noteworthy that the MMTV-PymT and MMTV-Wnt1 models have broadly similar but not identical localization and expression of MMPs. The initial expression of proteases in human breast cancer may be associated with the transition from DCIS to invasive ductal carcinoma. The transition to invasive cancer can be identified histopathologically in some DCIS lesions, particularly facilitated by immunohistochemical staining of myoepithelial cells and intraductal neoplastic cells. Expression studies of MMPs in human DCIS material have shown that MMP-13 is focally upregulated in microinvasive foci (Fig. 3b) in contrast to MMPs-2, -11, and -14, which are all more broadly expressed in the samples [35]. All four of these MMPs are expressed in periductal (myo)fibroblasts, indicating an intense stromal involvement during early invasion. Brummer et al. found that TIMP-1 is also upregulated in areas of early invasion [54]. Mimicking the human situation, the C3(1)-SV40-T model develops DCIS-like lesions, in which MMP-13 is upregulated specifically in those DCIS-like structures that show signs of invasion (Fig. 3a). In human samples, uPA and uPAR are also focally upregulated in areas of microinvasion together with MMP-13, suggesting that plasmin-directed proteolysis coincides with MMP activity in the transition to invasive cancer [36]. Thus, the extracellular proteases expressed in transgenic breast cancer models generally mirror those identified in human breast cancer both in terms of expression and localization. These findings suggest that transgenic models are useful tools for the study of protease involvement in breast cancer invasion and metastasis. Functional Studies of Proteases in Breast Cancer Progression Proteases in Post-lactational Mammary Gland Involution Mammary gland involution following weaning can to some extent be regarded as a surrogate model for the role of proteases in breast cancer invasion due to the extensive tissue remodeling and extracellular proteolysis taking place. The first, reversible stage of involution is prematurely activated in Plg-deficient mice likely due to increased milk stasis during lactation [55]. However, the second, protease-dependent and irreversible stage of involution seems to be delayed in Plg-deficient mice [5]. Involution is eventually accomplished in Plg-deficient mice probably due to compensating action of other proteases including MMPs. In support of this hypothesis, implantation of TIMP-1 slow release pellets into involuting mammary glands delays alveolar regression [40], and likewise TIMP-3 null mice exhibit accelerated involution [56]. In contrast, transgenic overexpression of TIMP-1 in luminal cells did not provoke an involution phenotype [16, 57], which may be a cell-of-origin effect since endogenous TIMP-1 is produced by stromal cells during involution [40, 58]. Similarly, the accelerated involution observed in mice that overexpress MMP-3 in luminal cells [57, 59] could be an effect of overexpressing a stromal protein in luminal cells, and is not supported by evidence from MMP-3 null mice that do not show a change in mammary apoptosis [16, 57]. The fact that the PA system and the MMPs are required for the controlled tissue remodeling of the involuting mammary gland, suggests that it is the inherent proteolytic repertoire of the mammary gland that is reactivated in breast cancers. Proteases in Hyperplasia and Carcinogenesis The earliest stages of mammary carcinogenesis including the initial genetic alterations in the epithelial cells were previously considered to be independent of the surrounding stroma and extracellular proteolysis. It now seems that the structural integrity of the stroma is an essential factor in preventing carcinogenesis [60]. In fact, disruption of the ECM and cellular microenvironment caused by overexpression of MMPs-3, -7 or -14 in the mammary gland is sufficient to initiate mammary epithelial hyperplasia [29, 61–63]. Transgenic mice overexpressing MMP-3 or MMP-14 even develop tumors in the absence of a transgene-supplied oncogene, and a fraction of the tumors undergo malignant transformation [62, 63]. Similar transgenic overexpression of Plg activators has not been reported for the mammary gland, but a K14-uPA/uPAR double transgene elicits extensive skin hyperplasia [64]. The MMP transgenes all direct MMP expression to the mammary epithelium, using either MMTV or WAP promoters. Only MMP-7 is expressed by normal mammary epithelium [65], while MMPs-3 and -14 are expressed by the mammary stroma during development and in the mature gland [46, 61]. Thus, tumor growth and malignancy have only been reported in cases where extracellular proteases were expressed by cells that would not normally produce them. Nevertheless, ectopically expressed proteases act as independent tumor promoters, presumably by compromising the growth-inhibitory role of the stroma, and eventually lead to genetic instability as if they were conventional oncogenes. In light of the independent tumorigenic ability of some MMP transgenes, it is not surprising that tumor onset in transgenic breast cancer models is promoted by increased MMP activity and delayed by decreased MMP activity in some studies (Table 1). Tumor onset is thus promoted by MMP-7 overexpression in the MMTV-neu model [29] and delayed by MMP-11 deficiency in the MMTV-ras model [30] or by TIMP-2 overexpression in the MMTV-Wnt1 model [32]. Similarly, the incidence of DMBA-induced mammary tumors is reduced in MMP-11-deficient [66] and in TIMP-1-overexpressing mice [34]. The results of two studies were inconsistent with the general finding: MMP-3 overexpression resulted in reduced rather than increased tumor incidence in the DMBA model [33], and MMP-9 deficiency led to increased tumorigenesis in the MMTV-neu model [67]. This suggests that some MMPs may have a protective function in mammary tumorigenesis, similar to the protective role of MMP-8 in chemical carcinogenesis of the skin [68]. In contrast to the documented influence of some of the MMPs on tumor initiation, the PA system (including genetic deficiency in Plg [44], uPA [31], PAI-1 [45], or uPAR [unpublished data]) does not affect tumor onset in the MMTV-PymT model. Neither does TIMP-1 overexpression [34] or genetic deficiencies of MMPs-2, -7, -9 (Fingleton and Matrisian, personal communication) or MMP-13 (unpublished data) in the MMTV-PymT model. At present it is difficult to determine whether these differences reflect the nature of the different proteases or perhaps that the very early tumor onset, which takes place during puberty in the MMTV-PymT model, is less sensitive to the proteolytic environment. Proteases in Tumor Growth and Vascularization Mammary hyperplasias and CIS lesions are generally supplied with oxygen and nutrients by the pre-existing vasculature of the mammary gland. Tumor expansion beyond a certain stage requires the formation of new blood vessels. Angiogenesis, the sprouting of new vessels from existing vasculature, depends on proteolytic dissection of the endothelial basement membrane and other ECM components. Endothelial cells penetrate a newly deposited fibrin-rich matrix and use it as a temporary scaffold for vessel structure [69]. Accordingly, the fibrinolytic capacity of the PA system is crucial for vascularization in certain experimental settings, and PAI-1 may play a dual role as a protease inhibitor and cell migration mediator [3]. In tumors from breast cancer patients, endothelial cells are a considerable source of uPA, PAI-1 and uPAR suggesting a possible role in vascularization [47, 48, 52]. This has not been experimentally confirmed in transgenic tumors thus far (Table 1). In the MMTV-PymT model, tumors grow equally fast in the genetic absence or presence of Plg [44], uPA [31], PAI-1 [45] or uPAR (unpublished data), indicating that vascularization is not rate limiting for tumor growth in these cases. It is likely that functional redundancy with other proteolytic components compensates for the absence of a single component in the intact tumor. Even in PAI-1-deficient tumors, vascular density was not significantly different from wild type tumors [45]. It is also possible that vascularization of spontaneous tumors can be partially accomplished by endothelial precursor cells in a process termed vasculogenesis that may be independent of proteases or depend on a different subset of proteases [11]. Deficiency of MMP-9 reduces tumor growth in transgenic models of skin or β-cell islet tumors, most likely due to a requirement for MMP-9 to release vascular endothelial growth factor (VEGF) from ECM deposits in order to activate the angiogenic response [70, 71]. In human breast tumors MMP-9 is expressed by a subset of inflammatory cells and vascular pericytes, which could suggest a similar role of MMP-9 in breast cancer vascularization [72]. However, the evidence is less clear in transgenic breast cancer models (Table 1). Final tumor size in MMTV-PymT-transgenic mice is not significantly affected by single gene deficiencies of MMPs-2, -7, -9 (Fingleton and Matrisian, personal communication), or MMP-13 (unpublished data). Deficiency of a single MMP may be compensated by the presence of other MMPs. In contrast, simultaneous inhibition of a number of MMPs through a liver-specific TIMP-1 transgene significantly reduced final tumor size (by 42%) in MMTV-PymT mice [34]. Transgenic overexpression of TIMP-2 also caused a significant reduction in tumor growth rate and reduced the mean blood vessel size in MMTV-Wnt1 mammary tumors, providing direct evidence for an effect on vascularization [32]. MMP-11 may be important at this stage, since MMP-11-deficient mice develop significantly smaller tumors in the DMBA [66] and MMTV-ras [30] models. Surprisingly, a broad spectrum MMP inhibitor (galardin) only resulted in a 50% reduction in final tumor size in the MMTV-PymT model (unpublished data). It is possible that some MMPs promote while others inhibit tumor growth and vascularization, and the net effect of inhibiting a whole range of MMPs is rather limited. A protective role against tumor growth and vascularization has been observed in non-mammary models for MMP-3 [73] and MMP-19 [74]. Proteases in Cancer Metastasis In order to successfully form metastases, cancer cells must enter the lymphatic system or the bloodstream, survive in circulation and arrest in the vasculature, extravasate and finally be able to proliferate in the new tissue. There are several metastasizing transgenic models, but the majority of models metastasize rather infrequently and only when the primary tumors are quite large. In addition to the MMTV-PymT [28] and MMTV-neu [25] models, very few other breast cancer models metastasize sufficiently to allow rigorous quantification (reviewed in [75]). Metastasis can be greatly accelerated by combining e.g. an MMTV-neu model with a second mammary-specific transgene for VEGF or transforming growth factor-β1 (TGFβ1) [75–77], but these models require more elaborate breeding. Proteases are intimately associated with metastasis. A classic example is the osteolytic bone metastases that are so frequent in breast cancer patients. Breast cancer cells and the resident osteoclasts stimulate each other in a feed-back activation loop. Cancer cells stimulate MMP-dependent degradation of the collagen type I rich bone matrix by osteoclasts [78, 79]. As a consequence of bone lysis, latent growth factors are released from their deposits within the bone ECM, and are available to stimulate further cancer cell proliferation. Unfortunately, transgenic models generally metastasize to the lungs and in this way the models do not exactly mimic the organ preference of metastatic breast cancer in humans. The PA system plays a unique role in metastasis at least in the MMTV-PymT model that metastasizes to the lungs [28] and lymph nodes [31] (Table 1). uPA [31], PAI-1 [45], and uPAR (unpublished data) are all expressed both in the primary tumor and in the lung metastases. Metastasis is significantly reduced in both Plg-deficient and uPA-deficient mice, while these gene deficiencies have no significant effect on the primary MMTV-PymT tumors [31, 44]. Absence of the specific uPA inhibitor PAI-1 has a slight metastasis-promoting effect, but the difference is not significant perhaps due to the presence of alternative uPA inhibitors [45]. Deficiency of uPAR does not significantly affect metastasis in the inbred FVB strain (unpublished data). Plg and uPA may promote metastasis due to their fibrinolytic ability. Fibrin-deficient mice have not been studied in a transgenic model, but they are less susceptible to spontaneous metastasis from transplanted tumors [80] and lung colonization by tail vein-injected cells [81], suggesting that fibrin is important for sustained adhesion of circulating cancer cells in the target organ. Thus, metastasis may depend on an intact and balanced system of fibrin formation and fibrinolysis. Alternatively, Plg and uPA may promote metastasis by releasing growth factors from the ECM. Overexpression of relevant ECM-sequestered growth factors promotes breast cancer progression and metastasis in transgenic models [76, 77, 82, 83]. These growth factors include the possible plasmin substrates VEGF, TGFβ, and FGF (fibroblast growth factor), as well as the possible uPA substrate HGF (hepatocyte growth factor). There is also considerable evidence linking the MMPs to invasion and metastasis (reviewed in [10, 84]). In MMTV-PymT mice treated with the broad spectrum MMP inhibitor galardin, the average lung metastasis volume is reduced by approximately 99%, in the context of primary tumors that are only reduced by half (unpublished data). Metastasis incidence is also reduced in MMTV-PymT mice that overexpress TIMP-1 through a liver-specific transgene, but the reduction is less pronounced and is absent with a mammary gland-specific TIMP-1 transgene [34]. It will take a considerable effort to define which MMPs are responsible for the metastasis reduction observed with these inhibitors. MMP-9 deficiency results in reduced metastasis in the MMTV-PymT model, although this phenotype was mouse strain dependent (Fingleton and Matrisian, personal communication). Deficiency of MMP-2, which is abundantly expressed in the stroma of MMTV-PymT tumors [43], also results in reduced metastasis in the same model (Fingleton and Matrisian, personal communication). MMPs-2 and -9 have previously been associated with invasion and metastasis in a number of studies [84]. In contrast, there is no effect on metastasis in MMTV-PymT mice that are deficient in either MMP-7 (Fingleton and Matrisian, personal communication) or MMP-13 (unpublished data). Similarly, overexpression of MMP-7 in mammary glands has no effect on lung metastasis in the MMTV-neu model [29]. MMP-11 may play an unexpected role in actually preventing metastasis. In the MMTV-ras model, MMP-11 deficiency results in a higher number of metastases despite a lower number and volume of primary tumors [30]. Apparently, the transition to invasive carcinoma is greatly accelerated in the MMP-11 null mice, indicative of a protective function of MMP-11. A similar protective role against metastasis has been reported in non-mammary models for MMP-3 [73] and MMP-12 [85]. Thus, while the general MMP inhibitor galardin has a large inhibitory effect on metastasis, several single MMP knock-out studies report no effect or even the opposite effect on metastasis in breast cancer models. This demonstrates the potential of MMP inhibitors in preventing spontaneous metastasis but also underlines the necessity of evaluating a second generation of MMP inhibitors for clinical trials that are more specific than previous compounds. Transgenic models with a well-characterized involvement of MMPs in metastasis could be a useful tool in testing such compounds. Conclusion and Future Perspectives Transgenic breast cancer models are invaluable research tools because they recapitulate the entire process from the initial genetic events in normal cells to metastatic disease. Furthermore, the very similar expression patterns of extracellular proteases in human and mouse breast cancer samples suggest that transgenic mouse models are ideally suited to study the role of proteases in carcinogenesis and the progression to invasion and metastasis. We have only begun to examine gene deficiencies and transgenic overexpression of the various PA and MMP components in transgenic models. The studies that have been performed so far suggest that no single MMP or PA system component is likely to be all-important for any stage in tumor progression. Practically all of the protease intervention studies report partial phenotypes such as delayed carcinogenesis, slower tumor growth or reduced metastasis, if they report any phenotype at all. The absence of any absolute phenotypes, such as prevention of metastasis, suggests that many of the proteases have mutually overlapping functions. The challenge for developing novel therapeutics in the future will be to identify those proteases that can be targeted simultaneously to obtain a synergistic tumor-suppressive effect. Another area that has so far only been thoroughly examined in one or two transgenic models is the expression patterns of proteases. Using protease array data, it may be possible to draw some more detailed parallels between individual mouse models and certain histopathological subtypes of human breast cancers. It is very possible that the collective of transgenic breast cancer models available, rather than any single transgenic, will be needed to adequately model the heterogenous human disease. Another open question is whether certain proteases or inhibitors are up- or downregulated in response to certain gene deficiencies. This may provide vital information on functional overlap between individual proteases. Transgenic breast cancer mice may prove to be very reliable models not only for basic research but also for testing experimental anti-proteolytic therapeutics, due to the high degree of similarity with human breast cancer. However, the transgenic models also present some significant challenges. A key issue is that the experimental drug has to cross-react with the mouse version of the protease. Therefore, it may be necessary in some cases to develop two drugs in parallel, one for each species. Due to the extracellular nature of the PA and MMP systems, these represent excellent targets for therapeutic monoclonal antibodies. An elegant method for targeting extracellular proteases in transgenic mice is the development of monoclonal anti-mouse antibodies by immunizing protease-deficient mice with the target protease. In conclusion, transgenic breast cancer models will further the identification of extracellular proteases critical to cancer invasion and metastasis, and aid the development of new agents against the spread of cancer.

J_Mol_Biol-1-5-1885970

Crystal Structure of an Active Form of Human MMP-1

The extracellular matrix is a dynamic environment that constantly undergoes remodelling and degradation during vital physiological processes such as angiogenesis, wound healing, and development. Unbalanced extracellular matrix breakdown is associated with many diseases such as arthritis, cancer and fibrosis. Interstitial collagen is degraded by matrix metalloproteinases with collagenolytic activity by MMP-1, MMP-8 and MMP-13, collectively known as the collagenases. Matrix metalloproteinase 1 (MMP-1) plays a pivotal role in degradation of interstitial collagen types I, II, and III. Here, we report the crystal structure of the active form of human MMP-1 at 2.67 Å resolution. This is the first MMP-1 structure that is free of inhibitor and a water molecule essential for peptide hydrolysis is observed coordinated with the active site zinc. Comparing this structure with the human proMMP-1 shows significant structural differences, mainly in the relative orientation of the hemopexin domain, between the pro form and active form of the human enzyme. Introduction Connective tissue remodeling is a complex process involving a plethora of cytokines, growth factors, and turnover of extracellular matrix (ECM). The main enzymes that degrade ECM molecules are matrix metalloproteinases (MMPs), which are also known as matrixins. Under normal physiological conditions, MMP activity can be regulated at various stages: during transcription, proteolytic processing of their inactive pro forms, zymogens, as well as by inhibition of enzyme activity by endogenous inhibitors such as tissue inhibitors of metalloproteinases or TIMPs.1,2 These enzymes have a similar domain structure: an N-terminal signal sequence to target for secretion, a pro-peptide domain to maintain latency, a catalytic domain containing the catalytic zinc, a linker region, and a C-terminal four-bladed propeller structure called the hemopexin domain. Some of the MMPs have additional domains, e.g. the fibronectin repeats in gelatinases. These domains are important in substrate recognition and in inhibitor binding.3 The human MMP family to date comprises of about 23 enzymes that are classified based on their preferred substrate and cellular localisation: collagenases, gelatinases, stromelysins, elastase, membrane-type MMPs and so forth.4 Collagenases (MMP-1, MMP-8 and MMP-13) are the key enzymes that are capable of cleaving interstitial fibrillar collagen. Apart from these enzymes MMP-2 (gelatinase A) and MMP-14 (MT1-MMP) are also able to initiate the breakdown of collagen fibrils.5,6 Interstitial collagens I, II and III are triple-helical proteins that are the essential structural components of all connective tissues such as the cartilage, bone, skin, tendons and ligaments. These extracellular glycoproteins provide scaffolding of the tissue and play an important role in cellular processes such as cell migration, proliferation and differentiation. Physiological collagenolysis is integral to several biological processes such as embryogenesis, tissue repair and remodeling, angiogenesis, organ morphogenesis and wound healing.7,9 The collagenases cleave the triple-helical collagen approximately three-quarters away from the N terminus of the substrate, resulting in three-quarters and one quarter length fragments that are unstable at body temperature and undergo denaturation, rendering them susceptible to other non-specific tissue proteinases. However, under aberrant circumstances degradation of collagen results in pathological conditions such as cancer, atherosclerosis, arthritis, aneurysm and fibrosis.8,10 The proteolytic activity of the enzyme resides in the catalytic domain but it requires the hemopexin domain in order to cleave the three chains of the triple-helical collagen.11 The crystal structure of porcine MMP-1,12 determined sometime ago, revealed details of the active site structure and the specificity pocket but this structure does not shed any light on how collagenolytic MMPs can cleave the triple-helical collagen. Recently, the X-ray structure of human proMMP-1 (MMP-1 zymogen) was elucidated.13 The structure revealed the interaction between the pro-peptide and the hemopexin domain of the enzyme, which results in a “closed” conformation of the zymogen in contrast to the “open” conformation of catalytic domain of the active MMP-1. The three-dimensional structure of the human MMP-1 (E200A), an active site mutant, reported here takes us a step closer towards a more complete understanding of the interaction of the collagenases with the triple-helical collagens. It reveals new features of the active protease and provides a platform for understanding the structural changes that accompany zymogen activation. Results Overall structure The structure of human MMP-1 (E200A) was determined at 2.67 Å resolution with two monomers (chains A and B) in the asymmetric unit of the trigonal space group, P3221 (see Table 1 for crystallographic statistics). The overall domain structure of human MMP-1 is similar to that of the previously solved full-length enzymes (Figure 1). The structure comprises of the N-terminal catalytic domain, the linker region and the C-terminal hemopexin domain. The catalytic domain of one monomer contacts the hemopexin domain of the other monomer. Interestingly, the contact site used by the two monomers in the asymmetric unit to form the dimer is not the same as the dimerisation site observed in the proMMP-1 structure13 or that for the MMP-9 hemopexin domain.14 This indicates that the dimerisation mechanism is perhaps not a general rule of thumb for the hemopexin domains and is most probably a crystallisation artefact. Monomers A and B deviate from each other with an overall r.m.s. deviation of 0.87 Å (for 367 Cα atoms). The dimer is stabilised by eight hydrogen bonds and 54 van der Waals contacts (Table 2). These contacts are facilitated by a total of 33 residues (18 residues from monomer A and 15 residues from monomer B). Only nine amino acid residues are common to both the monomers: Pro104, Asp105, Leu106, Arg183, Trp184, Thr185, Val300, Phe301 and Gln335. Apart from the asymmetry seen in the residues that participate from each monomer at the dimer interface, we also observe a non-symmetric interaction pattern. However, it is believed that this dimer is not physiologically relevant, as we found that human MMP-1 is a monomer in solution (R.V. & H. N., unpublished results). Catalytic domain The structure of MMP-1 catalytic domain is very similar to those of other MMPs. The metalloproteinase domain is about 160 amino acid residues in length with the catalytic zinc ion residing in the C-terminal segment of this domain. The catalytic fragment of the protease consists of three α-helices and a highly twisted five-stranded β-sheet. The active site zinc is bound in the sequence HELGHXXGXXH by the three His residues: His199, His203, His209 and a water molecule at the active site cleft (Figure 2). This is the first MMP-1 structure where a water molecule essential for peptide hydrolysis is observed at the active site because all the previous structures of the metalloproteinase domain of MMP-1 are in the inhibited state of the enzyme. Also observed in this domain is the salt bridge between the ammonium group of the N-terminal, Phe81 and the carboxylate group of the side-chain of Asp232. The generation of the correct N terminus (Phe81) in the activation process of proMMP-1 is crucial for the enzyme to have full activity against collagen.15 If the N terminus is either longer or shorter the activity against collagen drops to 30–40%.15 Only with the correct N terminus the formation of the salt bridge is possible, and this stabilises the structure of the N terminus as was originally shown for MMP-8.16,17 Alignment of the pro-enzyme (1SU3) structure with that of the mature enzyme (2CLT here) shows Phe81 to have moved some 17 Å from its original position in the zymogen form of collagenase-1 (Figure 3(a)). The structure of proMMP-3 (PDB code: 1SLM)18 was aligned as well to help calculate the displacement of Phe81. The first seven N-terminal residues were not observed in the proMMP-1 structure because of lack of proper visible electron density.13 This deviation gradually decreases and by residue Pro88 the two structures are in register and remain remarkably similar throughout the catalytic domain. There are only slight differences, if any, in the side-chain orientation of the N-terminal residues in the catalytic domain cleft (Figure 3(b)). The structural zinc exhibits tetrahedral coordination facilitated by residues His149, Asp151, His164 and His177. The catalytic domain of MMP-1 also contains three calcium-binding sites. Either four or five liganding residues coordinate all the calcium ions, except one in the catalytic domain of monomer A (Table 3). One calcium ion packs the S-loop (between strands s3 and s4) against the liganding residues from strand s5. The second calcium ion of the catalytic domain is sandwiched between strand s3 and the loop containing strands s4 and s5. The third calcium site is determined by the presence of the critical residue, Asp105 that comes from the loop following strand s1. The other residues (Glu180 and Glu182) that provide coordination to this calcium ion come from the loop following strand s5. One interesting feature observed in this crystal structure by virtue of dimerisation is that residue Gln335 from monomer B contributes the fourth coordinating ligand for this calcium ion. This feature, however, is not replicated in the corresponding calcium-binding site in monomer B. Details of the metal site geometries can be found in Table 3. Linker region In MMPs the catalytic domain is followed by a stretch of 15–65 amino acid residues referred to as the linker or the hinge region. This region is rich in proline residues and replacement of those with alanine drastically reduced the collagenolytic activity of MMP-8 (neutrophil collagenase),19 indicating that the presence of the correct linker structure is important for collagenolysis. This region in MMP-1 with 16 residues is well defined in the present structure unlike that in the full-length crystal structure of porcine collagenase. The conformation of the linker region is quite similar in both proMMP-113 and the present MMP-1 structures (Figure 4(a)). The residues of the linker region make extensive contacts (six hydrogen bonds and 73 van der Waals contacts; Table 4) with both the catalytic and the hemopexin domain of the enzyme. These interactions stabilise the domain arrangement in MMP-1, which is required for the concerted action of the two domains. Comparing the interactions of the linker region with the catalytic and the hemopexin domain in both the pro-enzyme and the active enzyme did not reveal many differences. Most of the interactions seen in proMMP-1 (seven hydrogen bonds and 71 van der Waals contacts) are still observed in the present structure, indicating that the activation of the enzyme does not result in structural changes and the overall structure of the areas around the linker region are mostly conserved. Interestingly though, the hydrogen-bonding interactions (Table 4) within this region are quite different between the two forms of the molecule. There are a total of seven hydrogen bonds within the linker region of active MMP-1 (Figure 4(b)) compared to the five observed in proMMP-1 (Figure 4(c)), with only two bonds common to both structures This difference in the number of hydrogen bonds is perhaps not of much significance when seen in the light of the extensive contacts this region makes with the other two domains. Hemopexin-like domain The hemopexin domain starts with Cys259 and forms a complete circle by joining to Cys447 in a disulphide bond that connects blade bI with blade bIV, giving this domain the characteristic four-bladed β-propeller structure. Each blade starts near the periphery with either the motif DAA or DAX, in which the Asp residues (Asp266, Asp359 and Asp408) coordinate the central calcium ion through their carbonyl oxygen atom. Glu310 provides the fourth coordination thus completing the acidic patch at the entrance of the central, solvent-accessible channel (Table 3). The side-chains of these residues form salt bridges to the neighbouring β-strands holding the entrance of the central channel together. Three water molecules are found trapped in the centre of this channel. These, however, are not involved in the coordination geometry of the calcium ion at the tunnel centre. Two of the water molecules are at positions corresponding to the sodium and chloride ion in the proMMP-1 structure.12 The water molecule corresponding to the sodium ion is at hydrogen-bonding distances to the carbonyl oxygen atom of Ile268, Ala312, Ala361 and Val410. The same cannot be said for the one corresponding to the chloride ion, which does not make any hydrogen bonds with the main-chain amides of the residues mentioned above. It is very likely that the presence of these ions is a consequence of the crystallisation conditions rather than a stability requirement for the hemopexin-like domain. Comparison with the porcine MMP-1 and the human proMMP-1 structures Full-length human MMP-1, human proMMP-113 and porcine MMP-112 were superimposed on the basis of the catalytic domain of the three structures. Alignments were also done for just the hemopexin domain of the molecules. As expected both types of alignments gave the same outcome whereby the active form of the enzyme (human and porcine) were closer in their structure as opposed to the proMMP-1 structure. The average r.m.s. deviation over 367 Cα atoms of the porcine and human MMP-1 is ∼1.4 Å, whereas the r.m.s. deviation when aligning the full-length active MMP-1 with the proMMP-1 structure is 1.6 Å. If, however, we take only the hemopexin-like domain into consideration, then the conformational differences that lie within this domain are brought to light (Figure 5(a)). The superimposed porcine and human MMP-1 hemopexin-like domains vary by 1.2 Å (maximum displacement of 5.2 Å). This value increases to 1.7 Å when superimposing the hemopexin-like domains of the active human MMP-1 and the human proMMP-1. A considerable change in conformation is reflected in the residues that interact with the pro-domain. Most of these differences are by virtue of the Phe289-Tyr290-Pro291 region of the hemopexin-like domain. Table 5 shows these residues, their interactions and the shift in Cα position observed when the pro and active forms of human MMP-1 were superimposed. The largest movement of 16 Å is observed for the residue, Phe289 (Figure 5(b)). Such a perturbation in structural conformation is not evident in the catalytic domain of the enzyme (Table 5). It seems like Arg281 acts as a pivot around which the hemopexin-like domain undergoes displacement upon activation as suggested by Jozic et al.13 One region in the catalytic domain that generates interest is the region between Tyr218 and Tyr221. The shift in the Cα position between the active and the proMMP-1 is not as dramatic as that observed for the Phe289-Tyr290-Pro291 region but they are involved in several interactions with the pro domain around the cysteine-switch region. As a result when the pro-enzyme undergoes activation, this region of the catalytic domain opens wider and further exposes the crucial cis-configured Glu190-Tyr191 peptide in MMP-1. Discussion Reconciliation of structural findings: pro-collagenase versus active collagenase MMPs are multi-domain enzymes that consist of a pro domain, catalytic domain, a linker region and a hemopexin-like domain. Cleavage of the pro domain leads to a substantial rearrangement of the N-terminal residues 81–88 (Figure 3(a)). The activation process swings Phe81 towards the proteinase domain and terminates with a salt link between the amine of Phe81 and the carboxylate side-chain of Asp232. This interaction imparts several-fold greater enzymatic activity to MMP-1 than those with either an extended or a shorter N terminus, which lack the salt linkage.17 Previously an inhibitor-free structure of the catalytic domain of MMP-1 was reported.20 This structure was, however, unique in the sense that the N-terminal Leu-Thr-Glu-Gly (83–86) residues of one molecule occupied the active site of the other molecule thus forming an unnatural inhibited complex, which should not really be considered as an inhibitor-free structure. The present structure on the other hand stands unique in being elucidated in the absence of an inhibitor. This is the first unliganded structure of the full-length MMP-1 where a water molecule is seen at the active site providing the fourth ligand for the catalytic zinc in the tetrahedral coordination sphere (Figure 2). It must be mentioned here that the present structure is an active site mutant where the catalytic glutamate residue has been mutated to an alanine. It is likely that the scissile peptide bond and the catalytic glutamate (Glu200) flank the active site water molecule on either side. The favourable hydrogen bond between Glu200 and the water molecule would make it more nucleophilic. The strongly polarised carbonyl group of the scissile bond (by virtue of its interaction with the catalytic zinc) would face a properly oriented water molecule. An unstable transition state results in the transfer of one water proton to the leaving nitrogen (the amino group of the scissile bond) via the Glu200 carboxylate shuttle. This is followed by the cleavage of the peptide bond and concomitant shuttle of another water proton to the amino group. This hypothesis will, however, need to be substantiated with a transition state structure. The linker region is considered to play an important role in collagenolysis,19,21 but its exact role is not clear. The structures of both proMMP-1 and active MMP-1 have revealed the residues of this region to be in close contact with the catalytic and the hemopexin-like domain. Comparative analysis of the extensively similar contacts made by the residues of the hinge region reveal that despite being highly exposed with no secondary structure, the conformation of the linker peptide is perhaps a signature of its sequence and does not depend on the crystal packing (Figure 4(a)). Mutagenesis studies of this region result in decreased collagenolytic activity of MMP-121 and MMP-8.19 This may be due to structural changes in this region affecting the interactions with the catalytic and hemopexin domains that are required for collagenolysis.22 The cleavage of the pro domain to form the active enzyme is accompanied by major conformational rearrangement in the residues that interact with the pro domain. Residues from both the catalytic domain and the hemopexin-like domain make interactions with the pro domain residues. The effects on the overall backbone structure of the catalytic domain is minimal; the effects on the hemopexin-like domain is, however, quite dramatic (Figure 5(a)). The Phe289-Tyr290-Pro291 region of the hemopexin-like domain undergoes the most significant conformational change (Figure 5(b)). In essence, the hemopexin-like domain undergoes a major displacement towards the catalytic domain, thus widening the cleft between the proteinase domain and the hemopexin-like domain on the active site face of the enzyme. This altered configuration makes the active site residues and the RWTNNFREY (residues 183–191) interface, a segment critical for collagenolysis,23 more accessible for native collagen. Implications for collagenolysis Despite growing awareness of the importance of collagen-recognising determinants it is still unclear as to which part of the collagenase molecule makes the first contact with the triple-helical collagen. Several hypotheses have been suggested to try and explain the steps involved in collagenolysis (the most recent by Jozic et al.13). Docking studies were performed using a single-stranded, collagen α1(I)-like 15mer peptide with the Gly-Ile cleavage site incorporated in an attempt to better understand the functioning of the active enzyme. The peptide was modelled on the known structure of one of the chains of the triple-helical collagen (PDB code: 1BKV).24 Several solutions were generated. We used localised interactions as our guide to help us pick one possible solution: (a) contact of the carbonyl group of the Gly-Ile bond of the peptide with the active site determinants, especially the catalytic zinc; and (b) contact with the critical cis-configured Glu190-Tyr191 peptide. This solution revealed the peptide to be aligned to the continuous bulge-edge strand (Gly160-Phe166) and the wall-forming segment (Pro219-Phe223) in an extended manner. This solution, however, upon energy minimisation moved outside of the coordination sphere of the catalytic zinc but it was still close enough to be polarised by the catalytic zinc. Since the peptide was only 15 amino acid residues long, we could not see any interactions with the C-terminal hemopexin-like domain. It has been proposed that the triple-helical collagen would run via the Glu190-Tyr191 cis-peptide exosite making extensive interactions with the residues of the hemopexin-like domain.22 The structure of the active form of human MMP-1 has provided us insights on the conformational changes that occur upon activation of the pro-enzyme and these are valuable clues towards understanding the mechanism of collagenolysis. A better understanding of the mechanism of collagen cleavage will assist towards design of inhibitors that would specifically interfere with collagenolysis without affecting (beneficial) the cleavage of other substrates. Experimental Procedures Cloning, protein expression and protein purification The catalytically inactive mutant proMMP-1 (E200A) was cloned, overproduced, refolded, and purified as described.22 A catalytic site mutant of this enzyme was chosen in order to prevent autocatalytic cleavage during crystallisation. The mutant zymogen was activated as described22 at a final concentration of 554 μM in the presence of 1:80 molar ratio of MMP-3 lacking the hemopexin-like domain (MMP-3ΔC) and 1 mM 4-aminophenylmercuric acetate for 90 min at 37 °C. The activation mixture was directly applied to a Sephadex S-200 gel filtration column (diameter 26 mm, length 900 mm) in 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10 mM CaCl2, 0.02% (w/v) sodium azide, to separate the “active” form of MMP-1 (E200A) from MMP-3ΔC, 4-aminophenylmercuric acetate, and remnants of the pro-peptide. Fractions containing MMP-1 (E200A) were pooled and concentrated using a Vivacell 250 ml with a 5 kDa cutoff membrane, followed by a Vivaspin 20 with a 5 kDa membrane (Vivascience). The protein was stored at room temperature as incubation at 4 °C resulted in precipitation. Crystallisation, data collection and processing MMP-1 (E200A) was crystallised using the hanging drop vapour-diffusion method. The protein (2 μl at a concentration of 21 mg/ml) was mixed with 2 μl of the reservoir solution containing 0.1 M Tris (pH 7.5), 1.5 M ammonium formate and 10% (w/v) polyethylene glycol (PEG) 8000. Crystals appeared and grew to their maximum size within two weeks at 16 °C. A cryoprotectant solution prepared by supplementing the reservoir with 25% (v/v) glycerol enabled the crystals to be flash-frozen in liquid nitrogen. Flash-cooled MMP-1 (E200A) crystals were used to collect diffraction data to a resolution of 2.67 Å on PX 14.1 at the Synchrotron Radiation Source, Daresbury (UK). The data were processed and scaled using HKL2000.25 The crystals belong to the space group P3221, with cell dimensions of a = b = 138.48 Å and c = 110.05 Å. There are two monomers in the asymmetric unit with solvent content of about 60%. Data reduction using the program TRUNCATE26 estimated an overall B-factor of 63.1 Å2/Da from the Wilson plot. Details of the data processing statistics are presented in Table 1. Structure determination The solution for the MMP-1 (E200A) structure was found using the program PHASER.26 Human proMMP-1 (PDB code: 1SU3)13 was used as the search model with the pro-peptide domain removed from the structure. Log-likelihood gain (LLG) of 161 for the first solution increased to an LLG value of 1091 when the second monomer in the asymmetric unit was located indicating the solutions were indeed the right ones. Refinement Crystallographic refinement was carried out using the program CNS27 at 2.67 Å resolution against 85.1% of the measured data. A test set of random reflections of 2.2% was excluded from the full data set for cross-validation purposes by calculating the free R-factor (Rfree) to monitor refinement trend.28 Initial round of refinement with the two monomers found by PHASER26 resulted in an Rcryst of 45.1% and an Rfree of 50.6%. Once the six N-terminal residues in both the monomers were built in and all the ions (two zinc ions and four calcium ions per monomer) were added and the model subjected to simulated annealing, the Rcryst and the Rfree dropped to 31% and 38%, respectively. Iterative cycles of refinement (energy minimisation, simulated annealing and individual temperature factor (B-factor) refinement) using CNS27 and model building with reference to 2Fo-Fc and Fo-Fc maps using the program Coot29 progressively improved the phases. In the final stages of refinement, water molecules with peaks greater than 3σ in the Fo-Fc maps and those within hydrogen bonding distances from appropriate atoms were incorporated into the structure. The final refined structure at 2.67 Å resolution has an Rcryst of 22.3% and an Rfree of ∼26%. Both monomers, chains A and B have the full complement of the amino acids: from Phe81 to Cys447. Residues 117, 356, 369, 385, 386 and 424 of chain A and residues 257, 272, 287, 424 and 446 of chain B have been modelled as alanine residues because of the lack of visible electron density beyond Cβ atom. Analysis of the Ramachandran plot using the program PROCHECK26 indicated that ∼81% of the residues are in the most favourable region of the ϕ-φ plot and about 17% lie in the additional allowed region. The structure also consists of 198 water molecules. Details of the refinement statistics are given in Table 1. Protein Data Bank accession codes The atomic coordinates and the structure factors of the human MMP-1 (E200A) have been deposited with the RCSB Protein Data Bank (accession codes 2CLT and R2CLTSF, respectively).

Plant_Mol_Biol-4-1-2268718

Comparative molecular biological analysis of membrane transport genes in organisms

Comparative analyses of membrane transport genes revealed many differences in the features of transport homeostasis in eight diverse organisms, ranging from bacteria to animals and plants. In bacteria, membrane-transport systems depend mainly on single genes encoding proteins involved in an ATP-dependent pump and secondary transport proteins that use H+ as a co-transport molecule. Animals are especially divergent in their channel genes, and plants have larger numbers of P-type ATPase and secondary active transporters than do other organisms. The secondary transporter genes have diverged evolutionarily in both animals and plants for different co-transporter molecules. Animals use Na+ ions for the formation of concentration gradients across plasma membranes, dependent on secondary active transporters and on membrane voltages that in turn are dependent on ion transport regulation systems. Plants use H+ ions pooled in vacuoles and the apoplast to transport various substances; these proton gradients are also dependent on secondary active transporters. We also compared the numbers of membrane transporter genes in Arabidopsis and rice. Although many transporter genes are similar in these plants, Arabidopsis has a more diverse array of genes for multi-efflux transport and for response to stress signals, and rice has more secondary transporter genes for carbohydrate and nutrient transport. Introduction Cells maintain their biological activities by importing and exporting various substances. Provision of energy and nutrients and efflux of salts, biochemicals, and ions are necessary to maintaining biological activity in prokaryotic and eukaryotic cells. Environmental situations within cells differ among organisms: unicellular organisms cannot control the ion concentrations outside the cell, but multicellular eukaryotes (especially animals) can precisely regulate the ion concentrations of their cellular environments within micromolar ranges. Therefore, we can expect organisms to differ in gene number, structure, and function according to their biological abilities and environmental situations. Recent sequence analyses of entire genomes have made it possible to confirm the existence of homologous genes by computer data analysis. It is also possible to reveal the overall patterns of gene networks. In plants, complete genomic sequences are available for Arabidopsis and rice, but gene annotation programs are not yet sufficiently accurate to determine the function of all genes Instead, full-length cDNA data are useful for precise analysis of genes. Because transport activities are required at distinct levels in most tissues, we expected that the transcripts of most transmembrane transporters would be represented in full-length cDNA libraries from plants at various developmental stages, various plant tissues, and plants exposed to various treatments. We searched for orthologs of known membrane transport genes by using the 35,180 full-length rice cDNA sequences (Rice Full-Length cDNA Consortium 2003; Satoh et al. 2007) and genomic sequence data from Arabidopsis (Arabidopsis Genome Initiative 2000) and japonica rice (Goff et al. 2002; International Rice Genome Sequencing Project 2005) and global functional gene annotation in Arabidopsis and rice (Munich Information Center for Protein Sequences (MIPS) data service (http://mips.gsf.de/proj/plant/jsf/) (Schoof et al. 2004; Karlowski et al. 2003)) (RAP-DB = Rice Annotation Project Data Base: http://rapdb.lab.nig.ac.jp/ (Rice Annotation Project 2007, 2008)); the TIGR Rice Genome Annotation: http://www.tigr.org/tdb/e2k1/osa1/index.shtml (Ouyang et al. 2007). Transmembrane proteins have a hydrophobic structure, a pore-forming sequence, and molecule-binding sites. Because of these specific structural features, the identification of membrane transport orthologs is clear from computer calculations. Previous reports have characterized individual transporter protein families but have not extended to whole transport systems in general (Eng et al. 1998; Pao et al. 1999; Mäser et al. 2001; Sánchez-Fernández et al. 2001). In a more general analysis of various organisms, the features of prokaryotes were contrasted with those of eukaryotes (Ren and Paulsen 2005). However, differences among eukaryotes—especially animals and plants—were not a focus of that report. We also searched for orthologs of membrane transport gene in various organisms database (The Human Gene Nomenclature Database Search Engine (http://www.genenames.org/cgi-bin/hgnc_search.pl) (Wain et al. 2004); Genomic comparison of membrane transport systems (TransportDB: http://www.membranetransport.org/index.html) (Ren et al. 2004)), the functional genomics of plant transporters (PlantsT: http://plantst.genomics.purdue.edu/ (Tchieu et al. 2003) and ARAMEMNON (http://aramemnon.botanik.uni-koeln.de/) (Schwacke et al. 2003)). Here, we compare total membrane transport systems from diverse organisms and conclude that membrane transport genes exemplify evolutionary diversity of homeostatic systems. Evolutionary changes in gene families indicate the dynamics of alterations in biological systems and gene networks. Therefore, analysis of large categories of gene families may reveal many basic concepts of biological systems. General comparisons of membrane transport genes Membrane transport protein carries various materials for homeostasis. There are many clear features of domains (ex. Transmembrane, pore-forming, ATP binding, molecular capture), and functional features in the transport proteins. According to the structure and its functional systems, the membrane transport proteins have divided to three categories-pump, channel and secondary transporter. The pump system is the slowest (1–103 molecules/s) but environmental-independent system which consume energy (mainly ATP) for transport. The channel is the most rapid (107–108 molecules/s) and non-energy consuming systems but its need concentration gradients previously (transport directions are only according to the gradients). The secondary transport system adapts co-transport molecules movement energy to carry molecules. Therefore, it needs co-transport molecules and the transport directions have depended on environmental conditions and the speeds (102–104 molecules/s) are the middle of pump and channel. We summarized all three categories (pump, channel and secondary transporter) of genes and compared the total numbers of membrane transport genes in Escherichia coli, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Neurospora crassa, and Saccharomyces cerevisiae. The genome sizes among these organisms were diverse (4.6–3150 megabases), and the numbers of transmembrane genes ranged from 300 to 350 in E. coli, fungi, and yeast to about 1000–1200 in Arabidopsis and rice (Table 2). This indicates that a minimum number of about 300 gene species may be required to retain cell homeostasis. The greater numbers of transmembrane transport genes in Arabidopsis and rice may indicate additional redundancy as well as the modification of genes for new roles (e.g. addition of new substances, adaptation of systems for regulating transport, divergence of stage- and tissue-specific material transport), specialization for the various tissues and cells of multicellular organisms, and increased complexity of cells, which in eukaryotes have many additional organelles. The greater relative increase (plants versus bacterium, fungus, and yeast) in the numbers of membrane transport genes was less than has been reported for other gene categories, such as transcription factor genes and metabolic enzyme genes, in higher eukaryotes (Wray et al. 2003). This may indicate that adaptations in membrane transport are critical for the survival of organisms during evolution. The total numbers of membrane transport genes in higher plants (Arabidopsis, about 1000; rice, 1200; Table 2) are 1.2–2.0 times those in animals (fly, 600; nematode, 650; human, 750; Table 2). These differences in numbers of transporter genes may be related to differences in the need for efflux and influx systems in restricted habitation environments. Because of their immobility and the simplicity of their uptake systems, plant cells have more opportunity to absorb inappropriate substances and greater amounts of substrates and to synthesize larger amounts of secondary products than do animals. In accordance with their structures and mechanisms of action, transport proteins are classified into three classes: pump, channel, and secondary transporter (transporter). The composition ratios of these classes of protein were also compared (Table 2, Fig. 1). The numbers of pump genes in animals (72–82) were almost the same as in bacteria (70). The numbers of secondary transporter (animals, 350; bacteria, 230) and channel (animals, 160–320; bacteria, 15) genes were increased in animals. In particular, vertebrates (humans) had more (322) channel gene species than plants (130–180). We considered that this gene diversity in the development of channel systems was caused by the acquisition of a nervous system. The electrical transmission systems in nervous systems supplying organs (e.g. muscles, kidneys) need precisely controlled ion concentrations and the ability to make immediate changes in gradients. The development of active transport systems in animals allowed the regulation of rapid movements of the body and organs. Therefore, animals presumably acquired genes for the fastest transport-system channels. Plants also had more channel gene species than bacteria, although fewer than animals. Because plants do not transmit signals for quick movement of their organs, they do not need to regulate membrane voltages as precisely as animals. Additionally, signal-transmitting systems with ligand molecules (e.g. neurotransmitters) are not specific, unlike in animals. Therefore, the numbers of voltage-gated ion channels (VICs) and ligand-dependent channels were smaller in plants than in higher animals (Table 1). On the other hand, higher plants had increased numbers of genes for pumps (170–250) and secondary active transporters (660–760). Plant cells have chloroplasts, which synthesize carbohydrates for many biological activities, including protein synthesis and functioning of the ATP-dependent pumps. Plants presumably use ATP-consuming systems more easily than animals, and the pumps transport the molecules that act as the driving forces of the secondary active transporters. Additionally, plant-specific organelles and vacuoles provide pools of ions and catabolite molecules. Co-transport molecules for secondary transport also are safely and stably stored in the vacuoles. Therefore, plants are presumably able to constantly supply co-transport molecules for secondary active transporters, independently of environmental conditions. The existence of vacuoles gives plant cells more self-sufficiency than animal cells and explains the evolution of membrane transport genes for individual cell homeostasis in plants. Therefore, pump and secondary transporter systems in plants are more divergent than in animals. Fig. 1Numbers of membrane transporter proteins of each class. Membrane transporter proteins were categorized into three classes (ATP-dependent [pump], channel, and secondary transporter) and compared among Escherichia coli K12-MG1655, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens NCBI, Neurospora crassa 74-OR23-IVA, and Saccharomyces cerevisiae S228CTable 1Comparative analysis of membrane transporter gene in many organismsGene familyE. coli K12A. thalianaO. sativaC. elegansD. melanogasterH. sapiens N. crassa 74S. cerevisiae MaterialPresent inEnergy-dependent (pump)ABC 671101534851473124VariousAllArsAB 00101111AnionAllF-ATPase (catalytic) 15621222H+, Na+AllH+-PPase 03900000H+P, a (vacuolar)IISP 03501329ProteinAllMPT 061411814818ProteinEukaryote (mitochondria)P-ATPase 446572219321916H+, Na+, Ca2+ etc.,AllChannelACC00000700Cation (Ca2+)A (neuron)Annexin 0810071310Ca2+EukaryoteBcl-2 000011200AnionA (mammal )Bestrophin 000214400Anion (Cl−)A, P, F, B (G−)CD2000000900Ca2+A (B-lymphocyte )ClC 3714631031Cl−AllConnexin 000001800VariousA (vertebrate)CSC 02100000VariousP (chloroplast)CytB 01970101319H+AllE-ClC 04400400Cl−A (mammal), P (distant homolog)ENaC0002025800Na+A (epithelial cell, brain)GIC 019179272000Metal (Ca2+, K+), GlutaminAllHsp70 017230141440Cation AllICC00000100Cl−A (mammal)ICln 01210100Anion (Cl−)A, P (distant homolog)Innexin 000228000VariousA (invertebrate)IRK-C000132200K+A, BLIC 00069234500VariousAMid100000001Cation (Ca2+)YMIP23838771114H2O, CO2, NH3AllMIT20000023Metal (Mg2+,Co2+, Ni2+)Y, a, BMscL 10000000VariousBMscS 68700000VariousP, BNSCC2 01111101CationA, Y, FO-ClC00001600Cl−APCC 00005600Cation (Na+, K+ , Ca2+) APLM00000700AnionA (mammal)RIR-CaC 00053600Ca2+ATic11001200000VariousPTRP-CC 000572301Ca2+A, Y, UT00000200UreaA (vertebrate), B VIC 1351863319022K+, Na+ or Ca2+AllPhosphotransferase System (PTS)GPTS 60000000CarbohydrateBSSPTS 230000000CarbohydrateBSecondary TransporterAAA 02300000ATPPlant (chloroplast)AAAP 0437711151347Amino acid, AuxinA, P, F, YAAE10000000Aspartate, Alanine BAbgT10000000p-Aminobenzoyl-glutamate BACR3 00000011Arsenite, Antimonite Y, BAE 073421021Na+, HCO3− , H+, Cl− , H3BO3 (P, Y)A, P, YAEC 18800000AuxinP, Y, BAGCS 10000000Alanine, GlycineB, aAmt161362443NH3, CO2AllAPC2212141111141524Amino acid, PolyamineAllArsB 20005100Arsenite, Antimonite All (with distant homolog)BASS 15602501Organic acidAllBCCT 30000000Betaine/Carnitine/CholineB, aBenE 10000000BenzoateBCaCA 21223811884Ca2+AllCCC 01265911Na+, K+, Cl−AllCDF 2811871085Zn2+ or Cd2+ etc.,AllCHR00000010SO42− , CrO42−F, BCNT30022310NucleosideA, Y, BCPA1287115332Na+, H+AllCPA2 3321600121Na+, K+AllDAACS30062700Dicarboxylate, Amino acidA, BDASS 54543503VariousA, P, Y, BDcu 20000000C4-Dicarboxylate B (G−)DcuC 20000000C4-Dicarboxylate B (G−)DMT 161215715141869Various AllENT 08453411NucleosideA, P, YESS 10000000GlutamateBFBT 09800000Folate, BiopterinP, B, aFNT 40000011Formate, NitrateY, B, aGntP70000000CarbohydrateB (E. coli, Bacillus) GPH 69811520SugarA, P, F, a, BGUP 00100012GlycerolA, P, Y, F, BHAAAP 81000000Amino acidB, P (distant homolog)KDGT 100000002-Keto-3-DeoxygluconateBKUP 1132100010K+P, F, BLCT00002211CystineA, P, FLctP20000000Lactate, Glycolate B, aLIV-E10000000Amino acid (L,I,V)B, aLIVCS 10000000Amino acid (L,I,V)BLysE 10000000LysineBMC 052663445443434Various EukaryoteMET 00000300Nucleoside, etc.AMFS 70901451371448214185Various AllMOP8564800213Various (Drugs etc.,)AllMTC 02262311Various (Anionic substrate )EukaryoteNCS1 210000310Nucleobase, Thiamine P, F, Y, B, aNCS211121151410Nucleobase,etc.AllNhaA 10000000Na+,H+ProkaryoteNhaB10000000Na+, H+AllNhaD 02100000Na+(Li+), H+P, BNiCoT 00000010Ni2+, Co2+AllNramp171021223Metals (Fe2+, Zn2+ etc.,)AllNSS 00012211800Neurotransmitters etc.,AOAT 022381100VariousA, P, F, YOPT0153400043OligopeptideP, B, aOST 00000200Organic compound EukaryoteOxa114501101ProteinAll (with distant homolog)PiT21351211HPO42−, SO42−AllPnaS 10010200Inorganic phosphate A (mammal)POT 4506133421OligopeptideAllRFC 00033400Folate, TiamineARhtB 50000000Amino acidB, aRND 821244721Various AllSSS 4113191121VariousAllSulP 11115791144SO42−AllTat 10100000ProteinB (G−)TDT 14900021Tellurite, C4-Dicarboxylate All (with distant homolog)ThrE 10000002Threonine, Serine All (with distant homolog)TRAP-T 10000000VariousB, aTrk 21100022K+P, Y, BZIP 0131865253Zn2+, Fe2+AllUnclassifiedCtr1 00000001Dipicolinic Acid Y, BCtr2 05743222Cu2+AllFeoB 10000000Fe2+B, aFeT 00000001Fe2+, (Co2+, Cd2+)YFP 00000100Fe2+A (mammal)LPI 00000500ProteinAOFeT 10000012Fe2+,Fe3+Y, BPnuC 10000000Nicotinamide mononucleotideBPPI 00006630ProteinA, F, BPUP 115400000Peptide, Fatty acidP, BNote: Present in: A = animal, P = plant, F = fungi, Y = yeast, B = bacteria, a = archea, G− = gramm minus bacteria Energy (ATP, pyrophosphate)-dependent (pump) system We compared the ATP-dependent transport genes in this diverse set of organisms (Table 1, Fig. 2, and Supplemental data-2). The main roles of the ATP-dependent (pump) proteins are (1) to transport molecules in specific directions independently of the environmental situation; and (2) to transport ions to form a concentration gradient between the areas outside and inside the membrane (active transport). Because bacteria cannot control the concentrations of ions or metabolites outside the cell, their pumps work mainly to transport molecules. In E. coli, most of the ATP-dependent genes (93%) encode ATP-binding cassette (ABC) proteins, and some of them are reported to encode channel proteins (Fig. 2, Table 1) (Holland et al. 2005). The ABC proteins transport various substances (e.g. ions, peptides, nucleosides, amino acids, carbohydrates, proteins) ATP-dependently in all organisms (Kolukisaoglu et al. 2002; Garcia et al. 2004). In eukaryotes, many functional units are present within one polypeptide, whereas many bacterial ABC subunits are encoded by individual genes. This results in an inverted relationship between prokaryotes (E. coli) (72) and primitive eukaryotes (yeast) (43). Many bacterial ABC proteins are located in the plasma membrane and serve as the main forces in energy-consuming transport for both import and export of substances. In higher eukaryotes, ABC proteins tend to export substances rather than function in import reactions.Fig. 2Comparison of numbers of pump genes among various organisms. Pump gene numbers were compared among Escherichia coli K12-MG1655, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens NCBI, Neurospora crassa 74-OR23-IVA, and Saccharomyces cerevisiae S228C. ABC: ATP-binding Cassette; ArsAB: Arsenite-antimonite Efflux; F-ATPase: H+ or Na+-translocating F-type, V-type and A-type ATPase; H+-Ppase: H+-translocating Pyrophosphatase; IISP: General Secretory Pathway (Sec); MPT: Mitochondrial Protein Translocase; P-ATPase: P-type ATPase Structural analyses revealed that ABC proteins could be classified as half size (homo- or heterodimer functional) or full size (monomer functional) in accordance with their construction. The number of genes encoding ABC proteins in animals is the same as, or slightly less than, in bacteria, most of them representing whole subunits of ABC proteins. In contrast, the number of ABC genes in plants is about twice that in animals and bacteria, primarily due to the encoding of half-size proteins (Table 1, Fig. 2, and Supplemental data-2) (Sanchez-Fernandez et al. 2001; Sugiyama et al. 2006). This increase in numbers of genes encoding half-size (e.g., WBC, ABC2) and full-size (PDR; pleiotropic drug resistance) proteins supposedly permits stage- and tissue-specific regulation of various substances (including plant-specific substances). Therefore, increases in the capacity of import and export transport systems by an increase in the number of half-size ABC proteins have allowed plants to fulfill their unique roles. In the import and efflux of ions and the creation of ion gradients for secondary transport, P-type ATPases transport many species of ion (e.g. H+, Na+, K+, Ca2+) in both directions in the cell (Table 1, Fig. 2, and Supplementary data-2) (Axelsen and Palmgren 2001; Baxter et al. 2003). Control of ion concentrations outside bacterial cells is not possible. Only a few of the bacterial genes involved in systems for the uptake or efflux of ions (uptake: K+, Mg2+; efflux: Ca2+, Ag+, Zn2+, Co2+, Pb2+, Ni2+, Cd2+; uptake or efflux, depending on the system: Cu2+) have been characterized, and each of the enzymes encoded comprises a distinct subfamily (Banci et al. 2006). Eukaryotes have diverged in terms of their transport substances and have also adapted genes for making the ion gradients for secondary transport. One of the most important ATPases in animals—Na+/K+ATPase—does not exist in plants. Na+/K+ATPase makes a Na+ ion gradient across the plasma membrane and forms the basis of membrane voltage and secondary active transport in animals. On the other hand, plants use H+ gradients for secondary transport, and more than 10 isoforms and vacuole-type H+ATPases are involved in stage- and tissue-specific control. This difference in adapted ion gradients reflects the ion concentrations in the cells and the demand for nutrient ions. A constant supply of Na+ ions is required to retain homeostasis of animal cells and rapid signal transmission systems (e.g. muscles, nerves). In contrast, plants use the more abundant H+ ion to make cation gradients and have special systems for transporting H+ ions into vacuolar pools. For transporting H+ into vacuoles, higher plants have a vacuole-type ATPase and an H+-translocating pyrophosphatase (H+-PPase) (Table 1, Fig. 2, and Supplementary data-2) that are comparable to bacterial ATPase and PPase, respectively (Sivula et al. 1999; Sze et al. 2002). Therefore, plants presumably adapted bacterial H+-ATPase systems to help in H+ ion concentration steps, whereas animals developed completely new systems to make Na+ ion gradients. Ion channel systems Ion channels are the “gates” in the membranes that open or close in response to signals such as mechanical or electrical stimulation and ligand binding. Therefore, ion channels are closely involved in determining whether or not ionic gradients are available. Multicellular organisms can control the ion concentrations in the tissues on both sides of the cell membrane, whereas monocellular organisms usually find it hard to control the ion gradient outside the cell. Thus, the use of ion channels becomes restrictive and unidirectional in these more primitive organisms. Unlike in eukaryotes, the channel system in prokaryotes is not well adapted to transport (Table 1, Fig. 3, and Supplemental data-3). Of the total number of genes encoding membrane transport proteins in prokaryotes, fewer than 5% are channel genes, and they mainly regulate osmotic homeostasis in the cell. Ions (e.g. Cl−, K+, metal), water, and osmolytes are imported or exported by the channels in their restricted role. In contrast, animals can make various ion gradients precisely and, in particular, can develop channel systems very well.Fig. 3Comparison of numbers of channel genes among various organisms. Channel gene numbers were compared among many organisms (E. coli K12-MG1655, A. thaliana, O. sativa, C. elegans, D. melanogaster, H. sapiens NCBI, N. crassa 74-OR23-IVA, and S. cerevisiae S228C). ACC: ATP-gated Cation Channel; Bcl-2: Bcl-2; Bestrophin: Anion Channel-forming Bestrophin; CD20: CD20 Ca2+ Channel; ClC: Chloride Channel; Connexin: Gap Junction-forming Connexin; CytB: gp91phox Phagocyte NADPH Oxidase-associated Cytochrome b558 (CytB) H+-channel; E-ClC: Epithelial Chloride Channel; EnaC: Epithelial Na+ Channel; GIC: Glutamate-gated Ion Channel; Hsp70: Cation Channel-forming Heat Shock Protein-70; ICC: Intracellular Chloride Channel; Icln: Nucleotide-sensitive Anion-selective Channel; Innexin: Gap Junction-forming Innexin; IRK-C: Inward Rectifier K+ Channel; LIC: Ligand-gated Ion Channel of Neurotransmitter Receptors; Mid1: Yeast Stretch-Activated, Cation-Selective Ca2+ Channel Mid1; MIP: Major Intrinsic Protein; MIT: CorA Metal Ion Transporter; MscL: Large Conductance Mechanosensitive Ion Channel; MscS: Small Conductance Mechanosensitive Ion Channel; NSCC2: Non-selective Cation Channel-2; O-ClC: Organellar Chloride Channel; PCC: Polycystin Cation Channel; PLM: Phospholemman; RIR-CaC; Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca2+ Channel; Tic110: Chloroplast Envelope Anion Channel-forming Tic110; TRP-CC: Transient Receptor Potential Ca2+ Channel; UT: Urea Transporter; VIC: Voltage-gated Ion Channel Compared with other systems, the channel system is the fastest at transporting molecules without consuming energy (i.e. when it is not necessary to transport against electrical gradients). Therefore, animals (especially vertebrates) have adapted these systems to nerve and muscle signal transmission. Nervous-system-specific channels such as neurotransmitter-responsible channels (connexin, ENaC, innexin, LIC, RIR-CaC, TRP-CC) are found only in animals. The numbers of channels that are membrane-voltage-dependent for Ca2+, K+, and Na+ ion transport are dramatically increased in both vertebrates and invertebrates (Table 1, Fig. 3; Sheng et al. 2000; Du et al. 2002; Clapham 2003; Hua et al. 2003; Miyazawa et al. 2003). Because animals form Na+ and K+ gradients with the Na+/K+ ATPase pump system, there is also an increase in the numbers of species of Na+ and K+ ion channels in animals. Thus, the total number of channel genes in humans (320) is about twice that in plants (130–180) (Table 2, Fig. 3). This difference is supposedly related to the ecological and physiological specificity of plants. Sometimes constant acquisition of unevenly distributed resources is difficult for plants. Additionally, the ion concentrations in the vascular systems and intercellular spaces of plants, unlike those in animals, are difficult to control precisely. Therefore, some of the ion channels and membrane-voltage-dependent channels are not as divergent as in animals. Although plants lack neurotransmission systems and have fewer membrane-voltage-dependent systems that use Na+ and K+ ion channels, the channel genes for ion homeostasis and signal transduction (e.g. ClC, GIC) are as well developed as in animals, and the numbers of some channel genes (CytB and MscS) are in fact specifically increased in plants (Table 1, Fig. 3).Table 2Comparison of the genome size, total and membrane transport gene numbers in many organismsE. coli K12A. thalianaO. sativaC. elegansD. melanogasterH. sapiens N. crassa 74S. cerevisiae Genome Size (Mb)4.61254309712031504013Total gene number4,290 26,000 37,000 20,621 13,489 30,000 10,082 5,804 Total Transporter Proteins3549841200654590754344300Transporters per Mb genome76.747.822.846.754.30.248.6325.38Transporters per whole gene0.0820.0380.0330.0330.0440.0250.0340.052ATP-dependent pumps72 (20%)173 (18%)249 (21%)72 (11%)72 (12%)82 (11%)53 (15%)43 (14%)Ion Channels15 (4%)131 (13%)178 (15%)229 (35%)158 (27%)322 (43%)12 (3%)24 (8%)Phosphotransferase Systems (PTS)30 (8%)0000000Secondary Transporters233 (66%)658 (67%)762 (63%)349 (53%)351 (59%)336 (44%)273 (79%)227 (75%)Unclassified3 (1%)22 (2%)11 (1%)4 (1%)9 (1%)14 (2%)6 (2%)6 (2%) Comparison of protein structures indicates that the fundamental structures of ion channels (numbers of transmembrane domains, pore-forming helices) are common in many organisms, but local similarities in individual regions among organisms are low (especially in the N and C terminus fragments). Some of the genes (e.g. for shaker-type K+ channel, SKOR-type K+ channel) have low levels of similarity among the whole structures of plants and animals (Supplemental data-3). In contrast, the major intrinsic protein (MIP) gene family, which encodes water-transport proteins, is specifically well developed in plants (Zardoya 2005). The numbers of these genes in tissues and at different stages are 3–5 times those in animals (38 vs. 7–11) (Table 1, Fig. 3) (Quigley et al. 2002; Sakurai et al. 2005). MIPs are abundant in the plasma membrane (15–20% of total membrane protein) and vacuoles (30–50% of total membrane protein) of plants; therefore, a high level of water transport is carried out at plant cell membranes. The importance of the acquisition of water has presumably resulted in the diversification and development of water channels in plants. Plants also use water pressure for regulation of movement (e.g. stomatal opening, leaf and petal angle changes) and for transmitting signals in various homeostatic functions. Therefore, plants have presumably developed signal transduction systems that rely on water molecules instead of the neurotransmitters and membrane voltage changes used in animals. Structural analyses indicate divergence in the level of conservation of MIP subfamilies (PIP, TIP, SIP, NIP, AQP, and GLP) (Supplemental data-3E). The plasma-membrane-intrinsic proteins (PIPs) are well conserved among organisms, but animal- (AQP) and plant-specific (SIP, NIP, TIP) genes have diverged, and closely related channel proteins have low levels of similarity within each organism (Supplementary data-3E). This also suggests a specific diversification of the water transport genes in plants. The structures of the channels in plants are simple compared with those in animals (Supplementary data-3). A decrease in the membrane spanning times and in the number of subunits of working systems can be detected in many channels (e.g. VIC [VDCC], CIC) (Supplementary data-3). Therefore, animals have developed channel systems especially for the exquisite control of ions, membrane voltages, and the signal transduction pathways of specific ligands (e.g. neurotransmitters). Phosphotransferase system (bacteria) Bacteria have specific membrane transport systems (phosphotransferase systems) for carbohydrates (sugar) and phosphates (Tables 1, 2) (Barabote and Saier 2005). These systems use phosphate phosphoenol pyruvate (PEP) as the energy source for phosphorylation and for transport of carbohydrates with the aid of enzyme complexes. Carbohydrates (e.g. glucose, fructose, mannitol, sorbitol) are transported against a concentration gradient, with concomitant phosphorylation. PEP is transferred via the soluble (and non-sugar-specific) enzymes EI and HPr to the enzyme complex EII. EII is made up of components A, B, and C, which, depending on the sugar specificity and bacterium involved, may be the domains of composite proteins; component/domain C is a permease and is anchored to the cytoplasmic membrane. Because the amount of phosphorylation of the enzymes influences other regulatory mechanisms in the cells (e.g. catabolite repression, chemotaxis), the whole cell needs a multiple-component, complex enzymatic control system. Therefore, the larger and more complicated cells of animals and plants have not adapted this system and instead use ATP-dependent and secondary transporter systems to transport carbohydrates. Secondary transporter (transporter) system The secondary transporter system works via the concentration gradient of co-transporter molecules. It is efficient to use a few abundant molecules as common co-transporter molecules for various transport substrates. Therefore, in accordance with the species of ion for which there is a gradient between the inside and outside of the cell, major co-transporter molecules were selected and secondary transporter systems developed for them. There are more than 100 species of gene families in the secondary transporter systems of all organisms, and various substances are transported. Compared with those in the pump and channel systems, the genes involved in the secondary transporter system are most divergent in plants and bacteria; in animals there are about 350 of these genes (44–59% of the total) (Tables 1, 2, Fig. 4).Fig. 4Comparison of numbers of secondary transport genes among organisms. Secondary transporter gene numbers were compared among Escherichia coli K12-MG1655, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens NCBI, Neurospora crassa 74-OR23-IVA, and Saccharomyces cerevisiae S228C. AAAP: Amino Acid/Auxin Permease; AE: Anion Exchanger; AEC: Auxin Efflux Carrier; Amt: Ammonium or Ammonia Transporter; APC: Amino Acid-Polyamine-Organocation; BASS: Bile Acid:Na+ Symporter; BCCT: Betaine/Carnitine/Choline Transporter; BenE: Benzoate:H+ Symporter; CaCA: Ca2+:Cation Antiporter; CCC: Cation-Chloride Cotransporter; CDF: Cation Diffusion Facilitator; CNT: Concentrative Nucleoside Transporter; CPA1: Monovalent Cation:Proton Antiporter-1; CPA2: Monovalent Cation:Proton Antiporter-2; DAACS: Dicarboxylate/Amino Acid:Cation (Na+ or H+) Symporter; DASS: Divalent Anion:Na+ Symporter; DcuC: C4-dicarboxylate Uptake C; DMT: Drug/Metabolite Transporter; ENT: Equilibrative Nucleoside Transporter; FBT: Folate-Biopterin Transporter; GntP: Gluconate:H+ Symporter; GPH: Glycoside-Pentoside-Hexuronide (GPH):Cation Symporter; HAAAP: Hydroxy/Aromatic Amino Acid Permease; KUP: K+ Uptake Permease; LCT: Lysosomal Cystine Transporter; MC: Mitochondrial Carrier; MET: 4 TMS Multidrug Endosomal Transporter; MFS: Major Facilitator Superfamily; MOP: Multidrug/Oligosaccharidyl-lipid/Polysaccharide Flippase Superfamily; NCS2: Nucleobase:Cation Symporter-2; Nramp: Metal Ion (Mn2+–iron) Transporter; NSS: Neurotransmitter:Sodium Symporter; OAT: Organo Anion Transporter; OPT: Oligopeptide Transporter; PnaS: Phosphate:Na+ Symporter; POT: Proton-dependent Oligopeptide Transporter; RFC: Reduced Folate Carrier; RhtB: Resistance to Homoserine/Threonine; RND: Resistance-Nodulation-Cell Division; SSS: Solute:Sodium Symporter; SulP: Sulfate Permease; TDT: Tellurite-resistance/Dicarboxylate Transporter; Trk: K+ Transporter; ZIP: Zinc (Zn2+)–Iron (Fe2+) Permease Organism-specific gene families in bacteria include those encoding proteins (e.g. APC, HAAAP) that transport many species of amino acid or carbohydrate (GntP, Dcu), or other substances (BCCT, BenE) (Neidle et al. 1991; Golby et al. 1998; Saier et al. 1999; Jack et al. 2000; Samsonov et al. 2002; Prakash et al. 2003). Gene families in animals include genes for specific transport proteins (NSS, MET) that carry neurotransmitters and hydrophobic compounds (Beckman and Quick 1998; Hogue et al. 1999; Yamashita et al. 2005). Those in plants include genes with dramatically increased expression, encoding proteins (AAAP, CPA2, DMT, MFS, OPT, POT) that transport such substances as amino acids, cations, and carbohydrates (Fig. 4) (Fischer et al. 1998, 2002; Pao et al. 1999; Saier et al. 1999; Jack et al. 2001; Koh et al. 2002; Wipf et al. 2002). The greater number of secondary active transporters in plants is supposedly due to the existence of vacuoles. Secondary active transporters need an ion gradient across the membranes, and vacuoles can pool many substances at high concentrations and supply co-transporter molecules instantly at any time. Therefore, the secondary transporter systems easily control transport and have diverged more in plants than in animals. The major facilitator superfamily (MFS) is the largest secondary transporter family in whole organisms; its members transport various substances (e.g. carbohydrates, phosphates, amino acids, cations) with Na+ or H+ ions (Pao et al. 1999; Burckhardt and Wolff 2000; Lemoine 2000). There are many modifications of these superfamily genes among organisms. Bacteria have amino acid and drug efflux families, animals have neurotransmitter transport families, and plants have diverse carbohydrate-transport genes (Tables 1, 2, Fig. 4, and Supplementary data-4). The proteins have structures typical of secondary active transporters: 10–14 transmembrane domains, co-transporter molecules, and material-binding domains. Comparative analyses indicate that their fundamental structure—membrane-spanning, pore-forming, and substrate-binding sites—is conserved, but similarities between individual domains are generally low among organisms (Tables 1, 2, Fig. 4 and Supplementary data-4). The transporter proteins are encoded by single transcription units and work as monomers. Therefore, this transport system is easy to adapt to new purposes than are the higher pump and ion channel systems. Although the secondary transporter system can adapt to many kinds of concentration gradients, generally few ion species are used. Most of the secondary active transporters depend on two ions: Na+ and H+. In the whole organism, more than 80% of the genes depend on these ions as co-transporter molecules (Table 3, Fig. 5). However, in many cases each ion can be substituted: animals use Na+ ions as co-transporter molecules and bacteria and plants use H+ ions. This has caused animals to make a Na+ ion gradient with Na+/K+ ATPase for membrane voltage and plants and bacteria to make H+ ion gradients with P-type ATPase and H+-PPase, respectively. The transporter genes of animals and plants have specifically adapted to Na+ and H+ co-transporter systems (Table 3, Fig. 5). Therefore, both animals and plants have developed transporter genes adapted to ion gradients.Table 3Comparison of secondary transporter genesE. coli A. thalianaO. sativaC. elegansD. melanogasterH. sapiens N. crassa S. cerevisiae Present inMaterialCotransporterDirectionAAA 02300000P (chloroplast)ATPH+ in (same)AAAP0437711151347A, P, F, YAmino acid, AuxinH+ in (same)AAE 10000000Bl-Aspartate, l-Alanine each otherboth (anti)AbgT10000000Bp-Aminobenzoyl-glutamate H+ in (same)ACR300000011B, YArsenite, Antimonite ?outAE 073421021A, P, YNa+, HCO3−, H+, Cr, H3BO3H+, Na+, Cl−both (anti)AEC 18800024P, Y, BAuxinH+ out (anti)AGCS10000000a, BAlanine, GycineNa+, H+in (same)Amt 161362443AllNH3, CO2?bothAPC 2212141111141524AllAmino acid, PolyamineH+, Solutein (same)ArAE311900000P, Y, B, aArmate acid (Malate etc.,)?outArsB 20005100All (with distant homolog)Arsenite, Antimonite ?outBASS 15602501AllOrganic acidNa+in (same)BCCT 30000000B, aBetaine/Carnitine/CholineH+ in (same)BenE 10000000BBenzoateH+ in (same)CaCA 21223811884AllCa2+H+, Na+both (anti) CCC 01265911AllNa+, K+, Cl−Na+, K+, Cl−both (same)CDF2811871085AllDivalent cation (Zn2+, Cd2+) H+, K+out (antit)CHR 00000010F, BSO42− , CrO42−H+both (anti)CNT30022310A, Y, BNucleosideH+, Na+in (same)CPA1 287115332AllNa+,H+H+, Na+, Cl−both (anti)CPA2 3321600121AllNa+, K+H+ (or itself)out (anti)DAACS 30062700A, BDicarboxylate, Amino acidH+, Na+in (same)DASS54543503A, P, Y, B (G−)Aminoacid, Sulfate, Phosphate etc.,H+, Na+in (same), both (anti)Dcu20000000B (G−)C4-Dicarboxylate Dicarboxylateboth (anti)DcuC20000000B (G−)C4-DicarboxylateH+, Dicarboxylatein (same = H+) , both (anti) DMT 161215715141869AllC3 Carbohydrate, Sugar, Nucleotide etc.,H+, Nucleotideout (anti)ENT08453411A, P, YNucleoside?bothESS 10000000BGlutamateNa+in (same)FBT 09800000P, a, BFolate, BiopterinH+ in (same)FNT 40000011Y, a, BFormate, NitrateH+ in (same)GntP 70000000B (E. coli, Bacillus)CarbohydrateH+ in (same)GPH69811520A, P, F, a, BSugarH+, Na+ , Li+in (same)GUP 00100012A, P, F, Y, BGlycerolH+ in (same)HAAAP 81000000B, P (distant homolog)Amino acidH+ in (same)KDGT 10000000B2-Keto-3-DeoxygluconateH+ in (same)KUP 1132100010P, F, BK+noneinLCT06302211A, P, FCystineH+ in (same)LctP 20000000a, BLactate, Glycolate H+ in (same)LIV-E10000000a, BAmino acid (L, I, V)H+ in (same)LIVCS10000000BAmino acid (L, I, V)H+, Na+in (same)LysE 10000000BLysineH+, OH−out (OH− = same; H+ = anti)MC 052663445443434EukaryoteC3 Carbohydrate, Sugar, Nucleotide etc.,Variousboth (anti)MET00000300ANucleoside, Hydrophobic compound H+ both (anti)MFS 71921451341368114185AllC3 Carbohydrate, Sugar etc.,H+, Na+, Variousboth (H+, Na+= same; others = both)MOP (MATE) 8564800213AllVarious (Drugs, Polysaccharides etc.,)Na+, (H+)out (anti)MTC 02262311EukaryoteVarious (Anionic substrate )H+ in (same)NCS1210000310P, F, Y, a, BNucleobase, Thiamine H+ in (same)NCS2 10121151410AllNucleobase, (Ascorbate = (mouse only)) H+, (Na+= (Ascotbate))in (same)NhaA 10000000ProkaryoteNa+, H+Na+, H+both (anti)NhaB 10000000AllNa+, H+Na+, H+both (anti)NhaD 02100000P, BNa+, (Li+), H+Na+, (Li+), H+both (anti)NiCoT 02000010AllNi2+, Co2+Ni2+, Co2+both (anti)Nramp 171021223AllFe2+, Zn2+, Mn2+, Co2+, Ca2+, Cu2+, Ni2+, Pb2+etc.,H+ in (same)NSS 00012211800ANeurotransmitters, Amino acids, Osmolytes, Cl− etc.,Na+in (same)OAT 022681100A, P, F, YVariousAnion both (anti)OPT 0153400043P, B, aOligopeptideH+ in (same)OST00000200EukaryoteOrganic compound (mostly anions)each otherboth (anti)Oxa1 14501101All (with distant homolog)Proteineach otherboth (anti)PiT 21351211AllHPO42−, SO42−H+, Na+in (same)PNaS 10010200A, B (distant homolog)Inorganic phosphate Na+in (same)POT 4506133421AllOligopeptideH+ both (same)RFC00033400AFolate, TiamineH+, OH−, anionboth (anti)RhtB50000000a, BAmino acidH+ out (anti)RND821244721AllHeavy metals, Drugs, Lipids etc.,H+ out (anti)SSS4113191121AllSugar, Amino acid, Organo cation, AnionNa+, (H+ can replace, but reduces affinity)in (same)SulP 11115791144AllSO42−H+ in (same)Tat10100000B (G−), P (distant homolog)ProteinnoneoutTDT 14900021All (with distant homolog)Tellurite, C4-Dicarboxylate H+ in (same)ThrE10000002All (with distant homolog)Threonine, Serine H+ both (anti).TRAP-T10000000a, BVariousH+ in (same)Trk 21100022P, Y, BK+H+ in (same)ZIP 0131865253AllZn2+, Fe2+noneinNote: Present in: A = animal, P = plant, F = fungi, Y = yeast, B = bacteria, a = archea, G− = gramm minus bacteria; Direction: “in” , “out”, and “both” indicate transport direction of materials through cell membrane, and “same” means material and cotranspoter move to the same dirction, “anti” means the different directionsFig. 5Comparison of co-transport molecules of secondary active transporters among various organisms. Numbers of co-transport molecules of secondary active transporters were compared among Escherichia coli K12-MG1655, Arabidopsis thaliana, Oryza sativa, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens NCBI, Neurospora crassa 74-OR23-IVA, and Saccharomyces cerevisiae S228C In animals, many molecules are transported with different co-transporter molecules. Examples are (1) molecules (e.g. neurotransmitters, organic anions, phosphates) transported with Na+ ion (NSS, BASS, CPA2, Pnas) (Beckman and Quick 1998; Saier et al. 1999; Kramer et al. 2001; Segawa et al. 2002; Radchenko et al. 2006); (2) molecules (e.g. amino acids, nucleosides) transported with H+ ions (e.g. AAAP, APC, DMT, LCT, MET) (Steiner et al. 1994; Jack et al. 2001; Zhai et al. 2001; Gasol et al. 2004); (3) molecules (e.g. anions, cations, sugars, nucleosides) transported with either Na+ or H+ ions (e.g. AE, CaCA, DAACS, GPH, CNT) (Reinders and Ward 2001; Ritzel et al. 2001; Zhu et al. 2003; Cai and Lytton 2004; Ryan et al. 2004); and (4) others (e.g. CCC, OAT, RFC) transported with other molecules (Russell 2000; Flintoff et al. 2003; Hagenbuch and Meier 2003). In contrast, in plants, almost all substances (K+: KUP; N (): Amt; phosphate: PiT, Pht [MFS], and TPT [DMT]; : SulP; sucrose: GPH; sugar alcohol: SAT [MFS]; monosaccharide: MST [MFS]; amino acid: APC and AAAP; nucleosides: NCS1), including many cations, anions, metals, and drugs, are co-transported with H+ ions. Only BASS (organic acid transporter) and NhaD (Na+: H+ antiporter = Na+: efflux system of vacuole) use Na+ ions as co-transporter molecules for efflux of ions from the cytoplasm, so plants do not require extensive sodium uptake (Table 3, Fig. 5) (Smith et al. 1995; Naderi and Saier 1996; Fu and Luan 1998; Nozaki et al. 1998; Pao et al. 1999; Daram et al. 1999; de Koning and Diallinas 2000; Jack et al. 2001; Khademi et al. 2004). Accumulation of H+ ions makes solutions acidic and prevents many enzymatic and biochemical reactions, so plants pool H+ ions in vacuoles inside the cytoplasm, thereby establishing a stable ion gradient for transport energy. Plants make H+ gradients not only inside cells, but also outside cells by the H+ pump, and they absorb many substances by secondary transporter systems. This H+-adapting system presumably has led to diversification of H+ co-transport in secondary transporter systems in plants. Comparative analysis of membrane transporter systems in Arabidopsis and rice We also compared the numbers of membrane transporter genes in Arabidopsis and rice. The rice genome size (430 Mb) is more than three times that of Arabidopsis (125 Mb), but the total number of membrane transporter proteins (1200) is only 1.20 times that in Arabidopsis (1000) (Table 2). The proportions of pump, channel, and secondary transporter proteins are almost the same, and the numbers of genes in many individual gene families are 1.1–1.3 times those in rice. However, Arabidopsis has more multi-efflux proteins and proteins involved in secondary active transport (CPA2, DMT, MOP, NCS2) and in channel-type signal transduction systems (CytB, GIC) (Fig. 6). Arabidopsis is a wild plant that lives in diverse soil conditions and under environmental stress, whereas rice is a cultivated crop plant grown under more stable environmental conditions. Therefore, Arabidopsis has diverged more than the rice cultivar Nipponbare to form transporter systems involved in multi-efflux and stress response signaling. On the other hand, rice has more pump and secondary transporter genes (ABC, P-type ATPase, MFS, POT) for carbohydrate and nutrient transport systems (Fig. 6). This divergence of carbohydrate transporters might have been influenced by artificial selection, during which individuals with larger seed size and numbers were chosen to be cultivated. Additionally, amino acid (AAAP), ammonia (Amt), sulfate (SulP), metal ion (ZIP), Ca2+ (CaCA), and K+ transport protein gene families (VIC, KUP) are specifically diverged in rice. This may have been caused by the need for nutrient supplements for rapid growth in subtropical plants. These differences may also result from dissimilarities between monocots and dicots. Although the cell structures are the same in both plants, there are many differences in the basic structure of tissues and organs. Differences in root and vascular bundle structure may be related to differences in uptake efficiency of nutrients. The levels of uptake and transport amounts of ions such as K+, Fe2+, and other nutrients differ between monocots and dicots; this might be related to differences in the numbers of membrane transport genes. Fig. 6Comparison of membrane transport genes in Arabidopsis and rice. Numbers of the membrane transporter gene families are compared in Arabidopsis and rice. ABC: ATP-binding Cassette; P-ATPase: P-type ATPase; CytB: gp91phox Phagocyte NADPH Oxidase-associated Cytochrome b558 (CytB) H+-channel; GIC: Glutamate-gated Ion Channel; MIP: Major Intrinsic Protein; VIC: Voltage-gated Ion Channel; AAAP: Amino Acid/Auxin Permease; Amt: Ammonium or Ammonia Transporter; CaCA: Ca2+:Cation Antiporter; CPA2: Monovalent Cation:Proton Antiporter-2; DMT: Drug/Metabolite Transporter; KUP: K+ Uptake Permease; MFS: Major Facilitator Superfamily; MOP: Multidrug/Oligosaccharidyl-lipid/Polysaccharide Flippase Superfamily; NCS2: Nucleobase:Cation Symporter-2; OPT: Oligopeptide Transporter; POT: Proton-dependent Oligopeptide Transporter; SulP: Sulfate Permease; ZIP: Zinc (Zn2+)–Iron (Fe2+) Permease; PUP: Peptide Uptake or Activated Fatty Acid Export Permease Comparison of the membrane transport genes in Arabidopsis and rice pointed to the directions of evolution of these plants in response to the selection pressures of their differing environmental situations. The level of conservation of gene families in Arabidopsis and rice varies, depending on the category. Rice membrane transporters were 72% orthologous with those of Arabidopsis; this is higher than the 50% ortholog found in transcription factors (Xiong et al. 2005). The levels of expression of membrane transport genes are limited precisely within a small range, and these genes are critical for survival. This may be the reason why the membrane transport genes were less divergent than the transcription factors. Therefore, the specifically divergent gene families in Arabidopsis and rice are related to the indispensable and unique systems in each plant. Divergence of gene patterns might indicate differences between wild weeds and crop cultivars that have been selected for growth in specific and possibly new environments. Comparison of the overall gene compositions of bacteria (E. coli), animals (H. sapiens), and plants In the eight diverse organisms that we compared, the number of membrane transporter genes (300–1200) varied less than that of genes in other categories. The minimum number of genes indispensable to retaining cell membrane transport homeostasis thus seems to be 300–350, as found in bacteria, yeasts, and fungi. Many of the additional newly diverged genes of higher animals and plants are channel transporter genes and secondary active transporters that facilitate adaptation to fluctuating concentration gradients present in their environments. Moreover, many newly acquired transporters are highly specifically stage- and tissue-regulated and transport special substrates such as neurotransmitters (ACC) in neural cells or carbohydrates (POT) in leaf tissue. Comparison of the overall gene compositions of bacteria (E. coli), animals (H. sapiens), and plants (A. thaliana and O. sativa) reveals the strategies for osmotic pressure adjustment and the features of the substance-transport systems in each organism (Fig. 7). Since a monad cannot control the ion environment of its external world and has no need to communicate with other cells, the role of its transporter proteins is restricted to the control of material transport into and out of the cell. Therefore, transport depends mainly on an energy-consuming system (pump) and an internal ion-gradient-dependent system (secondary transporter). Because the bacterium has a cell wall, osmotic pressure is opposed by cell wall pressure and there is no need to form a molecular concentration gradient to prevent excessive accumulation within the cell. Additionally, bacteria are small (1–5 μm, <1/10 of the size of an animal cell) and do not have many of the organelles and membrane structures possessed by higher organisms. Therefore, transport of substances is simple and it is easy to control their concentrations in the cell. The genes encoding the ATP-dependent pump (ABC), phosphotransferase (PTS), small conductance mechanosensitive ion channel (MscS), and secondary active transporters (e.g. APC, DMT, MFS, NCS2, RND) with H+ or Na+ as co-transporter molecules have diverged in bacteria (Fig. 7). Fig. 7Summaries of comparison of membrane transport genes in bacteria, animals, and plants. Bacteria-specific genes = blue; animal-specific genes = red; plant-specific genes = green; bacteria- and animal-specific genes = purple; bacteria- and plant-specific genes = brown; genes with divergent numbers in organisms = yellow. ABC: ATP-binding Cassette; ArsAB: Arsenite-Antimonite Efflux; F-ATPase: H+or Na+-translocating F-type, V-type and A-type ATPase; H+-Ppase: H+-translocating Pyrophosphatase; IISP: General Secretory Pathway (Sec); MPT: Mitochondrial Protein Translocase; P-ATPase: P-type ATPase; ACC: ATP-gated Cation Channel; Annexin: Annexin; Bcl-2: Bcl-2; Bestrophin: Anion Channel-forming Bestrophin; CD20: CD20 Ca2+ Channel; ClC: Chloride Channel; Connexin: Gap Junction-forming Connexin; CSC: Chloroplast Outer Envelope Solute Channel; CytB: gp91phox Phagocyte NADPH Oxidase-associated Cytochrome b558 (CytB) H+-channel; E-ClC: Epithelial Chloride Channel; EnaC: Epithelial Na+ Channel; GIC: Glutamate-gated Ion Channel; Hsp70: Cation Channel-forming Heat Shock Protein-70; ICC: Intracellular Chloride Channel; Ic ln: Nucleotide-sensitive Anion-selective Channel; Innexin: Gap Junction-forming Innexin; IRK-C: Inward Rectifier K+ Channel; LIC: Ligand-gated Ion Channel of Neurotransmitter Receptors; Mid1: Yeast Stretch-Activated, Cation-Selective Ca2+ Channel Mid1; MIP: Major Intrinsic Protein; MIT: CorA Metal Ion Transporter; MscL: Large Conductance Mechanosensitive Ion Channel; MscS: Small Conductance Mechanosensitive Ion Channel; NSCC2: Non-selective Cation Channel-2; O-ClC: Organellar Chloride Channel; PCC: Polycystin Cation Channel; PLM: Phospholemman; RIR-CaC; Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca2+ Channel; Tic110: Chloroplast Envelope Anion Channel-forming Tic110; TRP-CC: Transient Receptor Potential Ca2+ Channel; UT: Urea Transporter; VIC: Voltage-gated Ion Channel; AAA: ATP:ADP Antiporter; AAAP: Amino Acid/Auxin Permease; AAE: Aspartate:Alanine Exchanger; AbgT: p-Aminobenzoyl-glutamate Transporter; ACR3: Arsenical Resistance-3; AE: Anion Exchanger; AEC: Auxin Efflux Carrier; AGCS: Alanine or Glycine:Cation Symporter; Amt: Ammonium or Ammonia Transporter; APC: Amino Acid-Polyamine-Organocation; ArsB: Arsenite-Antimonite (ArsB) Efflux; BASS: Bile Acid:Na+ Symporter; BCCT: Betaine/Carnitine/Choline Transporter; BenE: Benzoate:H+ Symporter; CaCA: Ca2+:Cation Antiporter; CCC: Cation-Chloride Cotransporter; CDF: Cation Diffusion Facilitator; CHR: Chromate Ion Transporter; CNT: Concentrative Nucleoside Transporter; CPA1: Monovalent Cation:Proton Antiporter-1; CPA2: Monovalent Cation:Proton Antiporter-2; DAACS: Dicarboxylate/Amino Acid:Cation (Na+ or H+) Symporter; DASS: Divalent Anion:Na+ Symporter; Dcu: C4-Dicarboxylate Uptake; DcuC: C4-dicarboxylate Uptake C; DMT: Drug/Metabolite Transporter; ENT: Equilibrative Nucleoside Transporter; ESS: Glutamate:Na+ Symporter; FBT: Folate-Biopterin Transporter; FNT: Formate-Nitrite Transporter; GntP: Gluconate:H+ Symporter; GPH: Glycoside-Pentoside-Hexuronide (GPH):Cation Symporter; GUP: Glycerol Uptake; HAAAP: Hydroxy/Aromatic Amino Acid Permease; KDGT: 2-Keto-3-Deoxygluconate Transporter; KUP: K+ Uptake Permease; LCT: Lysosomal Cystine Transporter; LctP: Lactate Permease; LIV-E: Branched Chain Amino Acid Exporter; LIVCS: Branched Chain Amino Acid:Cation Symporter; LysE: L-Lysine Exporter; MC: Mitochondrial Carrier; MET: 4 TMS Multidrug Endosomal Transporter; MFS: Major Facilitator Superfamily; MOP: Multidrug/Oligosaccharidyl-lipid/Polysaccharide Flippase Superfamily; MTC: Mitochondrial Tricarboxylate Carrier; NCS1: Nucleobase:Cation Symporter-1; NCS2: Nucleobase:Cation Symporter-2; NhaA: Na+:H+ Antiporter A; NhaB: Na+:H+ Antiporter B; NhaD: Na+:H+ Antiporter D; NiCoT: Ni2+–Co2+ Transporter; Nramp: Metal Ion (Mn2+–iron) Transporter; NSS: Neurotransmitter:Sodium Symporter; OAT: Organo Anion Transporter; OPT: Oligopeptide Transporter; OST: Organic Solute Transporter; Oxa1: Cytochrome Oxidase Biogenesis; PiT: Inorganic Phosphate Transporter; PnaS: Phosphate:Na+ Symporter; POT: Proton-dependent Oligopeptide Transporter; RFC: Reduced Folate Carrier; RhtB: Resistance to Homoserine/Threonine; RND: Resistance-Nodulation-Cell Division; SSS: Solute:Sodium Symporter; SulP: Sulfate Permease; Tat: Twin Arginine Targeting; TDT: Tellurite-resistance/Dicarboxylate Transporter; ThrE: Threonine/Serine Exporter; TRAP-T: Tripartite ATP-independent Periplasmic Transporter; Trk: K+ Transporter; ZIP: Zinc (Zn2+)–Iron (Fe2+) Permease; GPTS: general carbohydrate phosphotransferase; SSPTS: sugar specific phosphotransferase; Ctr1: Dipicolinic Acid Transporter; Ctr2: Copper Transporter; FeoB: Ferrous Iron Uptake; FeT: Low Affinity Fe2+ Transporter; FP: Ferroportin; LPI: Lysosomal Protein Import; OfeT: Iron/Lead Transporter; PnuC: nicotinamide mononucleotide(NMN) uptake permease; PPI: Peroxisomal Protein Importer; PUP: Peptide Uptake or Activated Fatty Acid Export Permease Increases in the size and complexity of cells with the emergence of organelles and an internal membrane structure arose in the process of evolution from prokaryote to eukaryote. The eukaryote’s inclusion of the prokaryote’s transport genes in the mitochondrion or chloroplast allowed the transport of homeostatic substrate to organelles, as well as other materials and the products of specific biosynthesis (tricarboxylic acid cycle, electron-transport chain, and photosynthesis). Some of the genes are indistinguishable between eukaryotic transcript and organelle transporter, but there are 50–100 typical organelle transporters (e.g. MC, MCX, Tat) in eukaryotes, and they make a large group (7–12% of all transporters). Furthermore, with the development of multicellular eukaryotes there came a need for organisms to develop a system of transmission between cells. Animals and plants developed specific systems to solve the problem. Animals lost the cell wall structure and gained cellular mobility and flexibility, which also promoted integration of the whole body and specialization of tissues and cells. The loss of the cell wall also led to the need to control osmotic pressure against the plasma membrane. Animal cells regulate osmotic pressure by raising the Na+ concentration outside the plasma membrane by active transport with Na+/K+ ATPase. This Na+ gradient is used not only for homeostasis of the osmotic pressure of the membrane, but also as an electrical potential to control the voltage-dependent channel system and for co-transport of symport or antiport molecules of secondary transportation. Animals then developed the channel system (e.g. Bcl-2, connexin, E-ClC, EnaC, ICC, Ic ln, LIC, PCC, PLM, RIR-CaC, VIC) for rapid transmission in nerve and muscle tissues, together with an Na+ gradient dependent on the secondary transport system (e.g. NSS, PnaS, SSS). This Na+ ion gradient adaptation system is a feature of the newly added transporters and has evolved with animal specificities, i.e. the need for rapid movement and adaptation to environmental stimulation. In contrast to animals, plants have retained the cell wall instead of acquiring mobility. Because their cell structures and sizes became complicated and large, plants also evolved a means of controlling osmotic pressure; this means differed from the cell-wall-dependent system of bacteria. Because the plant cell controls osmotic pressure by the turgor pressure of the cell wall, it does not require a sodium or other ion gradient and lacks an Na+/K+ ATPase for control of osmotic pressure against the plasma membrane. On the other hand, an increase in permeability to water is necessary for smooth transmission of water pressure in the large and complicated structures of the cell. Therefore, water channels are abundant in both the plasma membrane and the vacuoles (20–50% of membrane proteins), and the diversity of water channels (MIP) is also four to five times higher than that in bacteria and animals. Compared with circulation systems in animals, whole-plant circulation systems are simple and less controlled. Plant cells also need to be more self-complementary in their homeostasis. Additionally, plants have less choice in their environmental conditions (e.g. soil, weather, stress) and must have more transport systems for cell homeostasis than animals. This is why plant cells include vacuoles to pool many substances and bioactive compounds. Because plants obtain energy through photosynthesis, there are more ATP-dependent (pump) transporters (ABC, P-type ATPase) in their cells than in animal cells. Unlike the case in animals, voltage-dependent systems and Na+-ion-dependent secondary transport systems have not developed in plants; systems that rely on H+ rather than Na+ have developed as part of plant secondary transporter systems. Because the increase in H+ brings acidification, plants maintain the neutrality of the cytoplasm by accumulating H+ in vacuoles, which are membrane-bound organelles separated from the other cytoplasmic contents. By having an internal ion pool, plant cells can control the ion gradient easily and safely. Therefore, secondary transport genes (e.g. CPA2, DMT, MFS, MOP, POT) that use H+ as a co-transporter molecule have diverged far in plants (Fig. 7). The inclusion of pooling and energy-synthesizing organelles in the cell may be why plant cells (10–100 μm) are larger than animal cells (10–30 μm). Conclusions Comparative analysis of membrane transporters among these eight diverse organisms indicates the type of cell homeostasis, as evidenced by the pattern of gene conservation and diversification. Evolutionary changes in gene families, in general, indicate the dynamics of alterations in biological systems and gene networks. Therefore, analysis of large categories of gene families may reveal many basic concepts of biological systems. In practice, analyses of the membrane transporter mechanism are useful in revealing changes in the absorption of molecules by, or their efflux from, cells and tissues. This information also is useful for examining changes in soil adaptability, nutritional demand, and stress tolerance in plants. It may also help to improve the harvest of crop cultivars or extend areas habitable by plant species. Gene networks are intricately related, and analysis of the whole genetic structure is needed to gain a full understanding of biological phenomena and systems of gene regulation. We are continuing to analyze whole categories of genes in an effort to develop an overview of total gene networks. Electronic supplementary material (DOC 21 kb) (XLS 189 kb) (PDF 151 kb) (PDF 192 kb) (PDF 165 kb)

Chiropr_Osteopat-13-_-1151650

Is obesity a risk factor for low back pain? An example of using the evidence to answer a clinical question

Background Obesity as a causal factor for low back pain has been controversial with no definitive answer to this date. The objective of this study was to determine whether obesity is associated with low back pain. In addition this paper aims to provide a step-by-step guide for chiropractors and osteopaths on how to ask and answer a clinical question using the literature. Introduction Obesity is a problem of epidemic proportion [1,2]. Despite record rates of non-physician supervised dieting and the availability of numerous weight loss programs, the problem is not abating [3]. Complicating this, is that most primary care physicians do not treat obesity, citing a lack of time, resources, insurance reimbursement, and knowledge of effective interventions as significant barriers [4]. Musculoskeletal disorders including low back pain (LBP) represent a considerable public health problem and a common diagnosis creating absenteeism and the need for disability pensions [5]. It is estimated that about 80% of the United States and Canadian population will experience LBP during adulthood [6]. Most low back pain is self-limiting and will ultimately resolve in two weeks (50% of those affected) to six weeks (90% of those affected), however it remains an intriguing clinical problem [6]. It is widely noted that the economic cost of obesity and its related disorders are staggering, with lifestyle related conditions such as diabetes mellitus and coronary heart disease placing a large economic burden on the health care system [1-4]. However, low back pain also has a significant socioeconomic impact. Cost estimates range from US$20 billion to $50 billion annually, with 10% of the patients accounting for 85 to 90% of the costs [6]. In Australia, Walker et al estimated the cost of low back pain in 2001 alone to be AUD$9.17 billion [7]. One question, which arises from the discussion concerning obesity, is whether obesity is a risk factor for low back pain. "Buckwalter et al contended that a number of medical conditions including obesity, along with diabetes and hypertension, may influence the pathophysiology of diseases of the tendons and ligaments during the process of aging thus potentially leading to low back pain [8]. Along with low back pain, the conventional wisdom is that overweight persons are at risk of osteoarthritis in weight-bearing joints such as the knee, the hips, and feet [9]. To date, literature reviews have given conflicting views based on the available data and method of data retrieval. The purpose of this review is to establish, from recent research, if there is a causal link between obesity and the affliction of low back pain. A secondary purpose of this review is to present the concepts of evidence-based practice to aid the chiropractor or osteopath in looking for health-related evidence for their patients who present with obesity. Methods A MEDLINE search, from the National Library of Medicine, was used to ascertain pertinent articles between the years 1990 and 2004. The use of keywords "obesity", "body mass index", "BMI" and "low back pain" was used to obtain relevant studies. The references of papers retrieved were also reviewed, as were key texts and references. This section is devoted to presenting the concepts of evidence-based practice (EBP) to demonstrate to the reader the discovery process for finding a possible link between obesity and low back pain. The EBP method used is shown in Table 1. Table 1 Steps to asking the answerable question using EBP principles Step 1: Asking an answerable question Step 2: Selecting an evidence resource Step 3: Executing the search strategy Step 4: Examining the evidence summary Step 5: Application of the evidence The first step in this process is "asking an answerable question." In this paper we assume a patient has asked whether being overweight can cause low back pain. Construction of an appropriate answerable question would possibly be "Does an increased BMI cause low back pain?" "Does being overweight create osteoarthritis?" In this way questions can be constructed to allow the practitioner to effectively answer a clinical concern. Once the answerable question has been constructed the next task is to find an adequate resource. Internet access to the U.S. National Library of Medicine's MEDLINE or PUBMED, these database systems are considered by many experts to be the most up to date data source on medically related topics. The next step is to determine keywords to place in the search engine. From the answerable question(s) it can be appreciated that the initial keywords will be "low back pain", "BMI" and "osteoarthritis." This search constitutes the third step. In initiating the search, one should look for the search engines "limits" area. In this area can one designate an age group (ex: 45 to 60 years), date span of the literature search, (ex: 1998 to 2003), and to select either English language or articles in foreign languages. Once the search has been completed, the articles, which may answer the question, are isolated and can be read. Step four involves collating the evidence to answer the question. In searches one may find answers that were not known to exist, and information that may challenge an already preconceived notion. The evidence summary should list the main author and year the paper was published. This is in order to retrieve the data should anyone wish to examine the source. As an example of an evidence summary see Table 2. Table 2 Recent evidence: Obesity and low back pain (chronological order) Author, Year (Ref) N BMI LBP Association Melissas, 2003 [14] 50 >40 58% direct Bener, 2003 [10] 802 (26.4 males/ 27.8 females) 56.1% males 73.8% females moderate Tsuritani, 2002 [16] 709 -- 40.3% none Bowerman, 2001 [4] 252 -- 29.2% none Kostova, 2001 [12] 898 -- -- increased risk Bayramoglu, 2001 [15] 25 -- -- direct Mortimer, 2001 [13] 475 30 (43.6%) 31–40 (28.8%) 40+ (1.3%) -- increased risk Han, 1997 [11] 7018 women 5887 men NR -- females increased risk N = number; BMI = body mass index; LBP = low back pain; NR = not reported It is at this time that the clinician is ready for the final step of applying the evidence. In our example clinical data from the experimental literature may or may not indicate that there is a link between overweight and low back pain. Results The literature search into obesity and joint pain revealed several studies pertinent to the debate. Table 2 reveals an overview of the studies selected. Several studies [4,10-13] had large populations to draw from yet the data from these studies were not in agreement as to a cause or association. In fact, only two studies [14,15] found a direct association for obesity as a risk factor while two [4,16] studies found no association. Several of the studies reviewed were unable to clarify BMI to the satisfaction of the authors. Discussion Interest in the association between obesity and low back pain has piqued researchers interest for many years. Intuitively, a burgeoning waistline and an increased lordotic lumbar spine led researchers to conclude that overweight people would be more prone to low back pain. Historically, Kellgren and Lawrence (1958) found that that the prevalence of disk degeneration with obesity was not significant [17]. However, it was not until the mid-1970's when several studies observed a possible association. Obesity was found to increase the prevalence of disk degeneration significantly in a study by Magora and Schwartz in 1976 [18]. Barton et al (1976), in a review of 144 cases, found that 70% of those who complained of low back pain had been classified as being overweight [19]. This basic research appeared to conclude what was already intuitively thought about low back pain and increased weight. Body mass index Before an in-depth discussion of low back pain and obesity can ensue, the concept of Body Mass Index (BMI) needs to be discussed. BMI is a measure of fatness and is calculated by dividing the patient's weight in kilograms by height in metres squared kg/M2 [20]. It is widely accepted, easily measured, and predicts morbidity and mortality in many populations [15]. Obesity is generally defined as a BMI of 30 kg/m2 and higher [20,21]. Overweight is defined as a BMI between 25 and 30 kg/m2 [19,20]. Overweight tends to be more common in men with obesity being more prevalent among women [21]. When body weight is increased 20% above average, mortality rises to 20% for men and 10% for women [22]. (Table 3) Overweight individuals demand more from their cardio-respiratory and musculoskeletal systems [22]. It is known that more than 50% of adult Americans have a BMI equal to or greater than 25 [23]. Although there are certain limitations to BMI i.e. large muscular athletes who are in good cardiovascular shape, the rationale behind these numbers is that, across large population groups, there is an increased prevalence of certain diseases in people with a BMI over 25, and a much greater risk of disease and death in those with a BMI over 30 [4]. Being overweight or obese substantially raises the risk of developing hypertension, coronary heart disease, type 2 diabetes, stroke, gallbladder disease, sleep apnea and other respiratory problems, prostate and colon cancers [4,23]. Yet, the evidence to date linking it to low back pain is not as clear cut as it is with the previously stated pathologies. Table 3 Clinically relevant differentiation between obesity and overweight Overweight Obesity BMI of 25.0 to 29.9 kg/m2 BMI greater than 30 kg/m2 BMI calculation without benefit of BMI charts Body Mass Index (BMI) charts and hand held scales are available for individual clinician use. It is, however, unknown to what degree chiropractors or osteopaths use such tools. The following section is designed to aid the clinician with calculating BMI without benefit of chart or hand held scales. As noted earlier, BMI is calculated as weight in kilograms divided by height in square metres [20,24]. This method is often too difficult to calculate for most people. A simpler method for those using the imperial system of measures is to take body weight in pounds × 703/height in inches squared. For example, a person weighing 150 pounds at 6 foot tall would correspond to a BMI of 20.3. TABLE 4 Table 4 Calculation of BMI 150 × 703 = 105450 divided by 72 inches (6 foot) squared. 105450 divided by 5184 (72 × 72) = 20.3 BMI. Additional research findings Leboeuf-Yde concluded from a review of the literature that due to lack of evidence, body weight should be considered a possible weak risk indicator and suggested that there is insufficient data to assess if it is a true cause of LBP [25]. Kostova found that in men over 40, overweight, obesity and number of pack years of smoking, estimated by duration of smoking and daily cigarette consumption (more than 20 years and more than 20 cigarettes per day), increased the risk of developing back disorders [12]. Despite these two studies, Garzillo et al and Leboeuf-Yde et al have given conflicting opinions [26,27]. Garzillo's review of the data revealed a possible association between obesity and low back pain only in the upper quintile of obesity, and no evidence of a temporal relationship between weight change and changes in low back pain [26]. Leboeuf-Yde concluded from a twin study that obesity is modestly positively associated with low back pain, in particular with chronic or recurrent low back pain [27]. What appears to be a main concern in linking obesity as a causal factor for low back pain is the numerous variables encountered in these subjects. For example, it is hypothesized that overweight adult females may have negative self-concepts and body images compounded by chronic low back pain and obesity, these may be confounding factors [28]. Other variables such as less activity and/or muscular weakness leading to obesity are also possible considerations. Obesity and low back pain-related conditions Not only is there controversy in obesity and low back pain, but there exists conflicting views of obesity and low back pain-related conditions such as spondylosis, decreased physical activities and discal herniation. The studies demonstrating a positive association are many. O'Neil et al noted that increasing BMI is associated with more frequent findings of osteophytes (bone spurs) at both the thoracic and lumbar spines [29]. The correlation of osteophytes and increased BMI is highest at the thoracic level [29]. Biering-Strenson et al noted absolute weight and BMI are significantly higher in persons 60 years of age with spondylosis [30]. Both men and women with BMI of 30 kg/m2 or higher were twice as likely to have difficulties in performing a range of basic daily physical activities [30]. Compared with women with BMI lower than 25 kg/m2, those with BMI of 30 kg/m2 or higher were 1.5 times more likely to have symptoms of intervertebral disk herniation [31]. Conversely, Luoma et al concluded that disc degeneration is not related to body height, overweight, smoking, or the frequency of physical activity [32]. In addition, studies by Riihimaki, Symmons, and Kang have shown no association between BMI and low back related problems [33-35]. Confounding the data is that the mechanism by which excess body weight causes osteoarthritis is poorly understood [9]. It is believed that contributions from both local increased force across the joint and systemic factors play a role [9]. A discriminating factor between fit and unfit patients with back pain may be the fact that fit persons more frequently are still employed, and as such may be involved more in physical activity [36]. Table 5 indicates where the research currently exists for the link between low back pain and obesity along with obesity and osteoarthritis. Table 5 BMI-related risk of osteoarthritis and low back pain If your BMI is then your risk based solely on BMI <25 minimal 25 – <27 minimal 27 – <30 minimal 30 – <35 moderate 35 – <40 moderate >40 moderate to high We conclude, based on the available evidence to date, that those individuals with a BMI of under 30 are at a minimal risk of developing low back pain while those persons whose BMI increases to over 30 are a moderate risk of developing low back pain. We also suggest, based on the findings of the Melissas study [14] of those patients who relieved their low back pain symptoms after obesity surgery, that patients with a BMI of greater than 40 are at a high risk of developing low back pain. Albeit controversial, Table 5 may lead to a further refinement of risk of osteoarthritis and low back pain based solely on BMI. Limitations of obesity as a risk factor for low back pain A significant difficulty in ascertaining cause and effect between obesity and low back pain is undoubtedly the term "low back pain" itself. Low back pain is a symptom not a diagnosis. A specific diagnosis, instead of the generalized form of "low back pain" may help separate out the association between LBP and obesity. The Agency for Health Care Policy and Research (AHCPR) in their 1995 Acute Low Back Problems in Adults noted common diagnoses used to explain back problems [37] (Table 6). Given these possible diagnoses one can readily appreciate the dilemma in attempting to link obesity with its specificity in measurement to a broad symptom such as low back pain. Table 6 Common diagnoses used to explain back symptoms Annular tear Adult spondylolysis Myofascitis Fibromyalgia Disc syndrome Strain Spondylosis Lumbar disc disease Facet syndrome Degenerative joint disease Sprain Spinal OA Disc derangement/disruption Dislocation *Other potential causes of low back pain symptomology Failed Back Surgery Syndrome* Osteoporosis* Urinary tract infection* Compression fracture* Somato-visceral mimicry syndrome* Organic pathology (tumor, rheumatoid, endometriosis, arthritic disorders)* Leg length inequity* Sacro-iliac dysfunction* Hip disorder* **Disagreement in research as cause of low back symptomology Morbid obesity?** OA = osteoarthritis Another problem is the hypothesis that a person who suffers with continuing bouts of low back pain may be predisposed, due to inactivity or inability to exercise, to gain weight thus increasing their BMI. This hypothesis to our knowledge, has yet to be fully discussed and investigated. Conclusion The data for a link between obesity and low back pain appears to be controversial. Yet, this does not adequately address the appropriate therapeutic approach to the obese patient with low back pain. The studies chosen for this review fail to document a definitive causal link between obesity and low back pain. Further research and epidemiologic data is needed to continue the search for a definitive answer. Competing interests The author(s) declare that they have no competing interests.

Evid_Based_Complement_Alternat_Med-4-4-2176145

A Review of Yoga Programs for Four Leading Risk Factors of Chronic Diseases

Yoga, a form of physical activity, is rapidly gaining in popularity and has many health benefits. Yet healthcare providers have been slow to recognize yoga for its ability to improve health conditions, and few interventions have been developed that take full advantage of its benefits. The purpose of this article is to review published studies using yoga programs and to determine the effect of yoga interventions on common risk factors of chronic diseases (overweight, hypertension, high glucose level and high cholesterol). A systematic search yielded 32 articles published between 1980 and April 2007. The studies found that yoga interventions are generally effective in reducing body weight, blood pressure, glucose level and high cholesterol, but only a few studies examined long-term adherence. Additionally, not enough studies included diverse populations at high risk for diabetes and its related common health problems. Chronic diseases such as heart disease, stroke and diabetes are leading causes of death in the US (1). Common conditions for these chronic diseases are overweight, high blood pressure (BP), high glucose and high cholesterol. These conditions are commonly associated with each other. According to a prospective cohort study focusing on men (2), there were positive relationships between body mass index (BMI) and hypertension incidence. Men with a higher BMI have a higher likelihood to have diabetes and high cholesterol. Another study with men and women showed that overweight is linked to type 2 diabetes (3). Even among patients newly diagnosed with type 2 diabetes, the Hypertension in Diabetes Study found that ∼40% had hypertension, and that hypertension considerably increased mortality in people with type 2 diabetes (4). Another recent study found a 79% rate of hypertension in patients who had received outpatient care for type 2 diabetes for at least 2 years (5). Individuals with impaired glucose tolerance also are more likely to have risk factors for cardiovascular disease such as elevated levels of triglycerides and low-density lipoprotein (LDL) and low levels of high-density lipoprotein (HDL)(6). Promising in this regard is appropriate physical activity because it can reduce body weight, BP, glucose level and cholesterol (7,8). Yoga, a form of physical activity consisting of various postures (Asana) and breathing and meditation techniques (Pranayama) (9), has been shown to have therapeutic benefits for individuals with a wide range of health conditions, including hypertension (10) and diabetes (11). Yoga also appears to be effective in reducing stress (12) and improving exercise tolerance as it is related to cardiovascular response (13). The number of people practicing yoga in the US increased significantly between 1997 and 2002 (14). According to the 2002 National Health Interview Survey (14), 5% of American adults practiced yoga in the month previous to the study. Yoga also is well received as a therapeutic intervention; for example, participants in a yoga intervention for insomnia found that it was easily learned and performed (15). Despite its popularity and positive physiologic effects, however, yoga has not been widely recognized in efforts to prevent and treat major chronic health conditions. The purpose of this article is to review published studies using yoga practice to determine the effects of yoga on common health problems, such as overweight, hypertension, high glucose level and high cholesterol. Methods Articles were retrieved from electronic databases (CINAHL, Ovid MEDLINE and PsychInfo) using yoga as a keyword. This initial retrieval, performed in April 2007, yielded 2349 articles, of which 861 have been published since 1980. A search within those 861 articles, using the keywords overweight, blood pressure, glucose and cholesterol, identified 56 research articles. After the author had read the full text of the 56 articles and identified those that met the purpose of the study, 24 articles were excluded, mainly because they described outcome variables and characteristics of yoga that were irrelevant. Poor quality was not a reason for exclusion, but studies were excluded if they focused only on meditation or relaxation (Pranayama) or if they were case studies. This process resulted in a final total of 32 articles to be reviewed. The review comprised studies involving yoga interventions and using an experimental or quasi-experimental design. In addition, observational studies were included because they often supply important information beyond the results of clinical trials (16). Studies that used yoga as a control, not as an intervention, were included as long as they provided evidence of the effectiveness of yoga on variables of interest (overweight, BP, glucose and cholesterol). Findings Of the 32 articles reviewed, 12 described experimental studies, 18 described quasi-experimental studies and two described observational studies. Only 2 of the 12 true experimental studies (17,18) described the randomization method. Even though risk factors for chronic health conditions were used as keywords in the search strategy, many of the studies used healthy adult samples; only half of the 32 studies actually focused on subjects with diabetes (19–23), hypertension (10,11,18,24–27), or cardiovascular disease (28–31). Seven of the studies were conducted in the US; the others, in India and other countries. Content of Yoga Programs Other than duration and frequency, most articles did not describe the yoga sessions in detail. Only a few articles (17,20,22,31,32) provided details of the yoga sequences used. Some listed the names of postures and breathing techniques. Of the few articles that identified the type of yoga studied, the most common was Hatha yoga, a popular form of yoga in the Western world. The Compendium of Physical Activities, (33) a coding scheme that classifies physical activity based on energy expenditure, does not define energy expenditure while performing various types of yoga. However, Hatha yoga is classified as a conditioning exercise, in the same category as stretching, which has a metabolic equivalent (MET) of 2.5 (1 MET equals the amount of energy used for resting). A recent study (34) found that when young adult women (19 to 40-years old) performed a 30-min session of Hatha yoga, the MET was 2.17. However, if the session was made more active using Sun Salutations, a flowing series of physical postures, the MET increased to 3.74 (SD = 0.70). Frequency and Duration of Yoga Sessions The total dose of yoga training, which depends on both the duration and frequency of yoga sessions, also needs to be considered in evaluating and comparing yoga studies. The most common duration and frequency of yoga sessions in the studies were 30–60 min per session and sessions meeting daily for 4–10 weeks, but many studies used sessions meeting 2–3 times per week for 8–12 weeks. Some yoga programs met more frequently than others but for a shorter time span. For example, in a study by Damodaran et al. (24), persons with essential hypertension received 1 h of yoga training daily for 3 months, which is counted as 84 h of training, whereas Ray et al. (32) studied healthy young adults who received 1 h of yoga training three times a week for 10 months, for a total of 120 h. Some findings can be useful in trying to determine effective durations of yoga sessions. For example, in one study, 1 h daily yoga practice was associated with significant reductions in body weight and cholesterol levels after 4 weeks, and those significant effects lasted for 14 weeks (28). In a study of 20 patients with essential hypertension, daily 30-min sessions of yoga led to a decrease in BP by the fifth day (27). Adherence to the Yoga Program Any persistent benefits from yoga would rely on long-term adherence, which was examined in only a few of the reviewed studies. In some studies, after yoga training, subjects were asked to continue their programs with daily home practice (28,35). One of these (35) compared adherence to yoga practice after a 10-week yoga intervention between white and black American adults (mean age: 69 years for whites and 70 years for blacks). Although the dropout rate did not differ significantly (16% for whites, 22% for blacks), the black participants did not engage in yoga at home as frequently as whites. A different study (36) found greater compliance with subsequent home practice among participants in a yoga class that met three times per week than in those who attended a weekly yoga class (86% versus 65%, P < 0.05). However, found no difference in compliance with home practice between groups engaged in yoga or aerobic exercise (36). Weight Decreased According to a retrospective observational study of 15,550 adults aged 53–57 years (37), regular yoga practice for 4 or more years was significantly associated with weight loss by overweight participants. Several intervention studies (10,26,29,31,38–41) also showed that yoga practice was effective in reducing body weight. After 4-day residential yoga practice followed by 14 weeks of 1 h daily home practice, one study (28) found a significant loss in mean body weight from 72.26 to 70.48 kg among subjects with risk factors for coronary artery disease (CAD). Other studies found that yoga was associated with significant weight loss by subjects with CAD (29–31) and subjects without CAD (30). Manchanda et al. (29) showed a 7% loss of body weight among adult men with CAD after 1 year of yoga practice, and in a study by Schmidt and colleagues, healthy adults lost an average of 5.7 kg after 3 months of yoga practice (39). All overweight adults studied by Yogendra et al. reached a normal weight within 1 year after initiating yoga-based lifestyle modifications (31). However, this article did not show the effect size of this change. Blood Glucose Level Lowered Of the six studies that examined blood glucose, all found that yoga was effective in reducing blood glucose level (11,19–22,31). For example, after 4 months of yoga practice, fasting glucose fell significantly (from 144 to 119 mg dl−1, P < 0.005) in a group of adults with type 2 diabetes, (19). Another sample of 24 adults with type 2 diabetes had significantly decreased fasting glucose (from 190.1 to 141.5 mg dl−1, P < 0.001) after 40 consecutive days of yoga practice, (20). Adults with normal blood glucose levels (11) also had significantly lower glucose levels after 3–4 h of yoga practice for 8 days (P < 0.001). BP Decreased This review found ample evidence that yoga was effective in reducing BP. In a group of low-income elderly people, effects on systolic BP did not differ between a yoga class and an aerobic exercise class, both held three times a week for 10 weeks (36). However, eight other studies found that yoga practice was effective in lowering BP in healthy samples, regardless of the type of yoga (13,32,38–44). Yoga practice also significantly improved BP among people with hypertension (10,11,18,24–27), cardiovascular disease (28–31) or type 2 diabetes (19–23). For example, in 13 patients, aged 41–60 years, with essential hypertension (25), BP dropped significantly during the third week of a 4-week yoga program (1 h per day, 6 days per week), and it fell further after the program. For example, systolic BP dropped from 141.7 to 127.9 mmHg by the third week and to 120.7 mmHg by the fourth week. Cholesterol Level Improved The practice of yoga was associated with significant decreases in cholesterol among subjects with cardiovascular disease (28,29), hypertension (11) or type 2 diabetes (19). One study (28) examined a regimen involving 4 days of a yoga program at a residential course, followed by 1 year of yoga practice at home. In both men with angina and asymptomatic participants with CAD risk factors, all lipid variables except HDL decreased beginning the fourth week of yoga practice (e.g. total cholesterol fell from 206.6 to 193.6 mg dl−1), and the level of total cholesterol continued falling to 176.06 mg dl−1 at 14 weeks. A study of subjects at risk for cardiovascular disease and diabetes (11) found significant improvements (P < 0.01) in total cholesterol, triglycerides, LDL, HDL and very-LDL (VLDL, defined as total cholesterol minus LDL minus HDL) after short-term intensive yoga practice (3–4 h per day for 8 days). Notably, for subjects whose baseline total cholesterol was 200 mg dl−1 or higher, the reduction in triglycerides (from 151.5 ± 48.9 to 132.7 ± 50.5 mg dl−1, P < 0.001) and VLDL (from 36.7 ± 13.8 to 30.2 ± 14.6 mg dl−1, P < 0.001) was significantly greater than in subjects with lower baseline total cholesterol (triglycerides falling from 113.6 ± 46.5 to 110.5 ± 38.1 mg dl−1, P > 0.05; VLDL from 23.7 ± 12.8 to 23.2 ± 12.5 mg dl−1, P > 0.05). Finally, a study of healthy adults over 40 years old found that 5 years of yoga practice reduced age-related deterioration in cardiovascular functions (45). Although the article describing this observational study did not detail the type of yoga performed nor the frequency or intensity of the yoga sessions, the data showed a long-term change indicating the effectiveness of yoga on cardiovascular functioning. Four studies used aerobic training as an intervention and yoga as a control (38,43,46,47). One of these, a study of healthy active people aged 65 years or older (38), found that aerobic exercise produced no significant reduction in weight or BP, whereas 4 weeks of yoga practice did lead to some reduction in weight or BP (for example, systolic BP decreased from 146 to 139 mmHg). DiPietro et al. (47) found no change in glucose and insulin responses in their yoga control group. However, the yoga control group was monitored to ensure that pulse rates did not exceed 90 beats per min during yoga practice (47). Therefore, this restriction should be considered in interpreting this result. Discussion The reviews showed that yoga had beneficial effects on body weight, BP, blood glucose level and cholesterol level (Fig. 1). Nonetheless, several shortcomings in research on this topic need to be addressed, in order for clinical programs to capitalize on these health benefits. Figure 1.The possible effects of yoga on behavioral, psychological and physiological measures are illustrated. This article focuses on current evidences for physiological changes. Of the articles describing interventions, only a few provided details regarding the yoga programs or the names and sequence of yoga postures. Asana and Pranayama provide different types of health benefits, and therapeutic yoga programs can involve various combinations of these two components. An optimal comparison of study results thus requires knowing the combination of Asana and Pranayama used. The sequence of yoga postures can be inferred from the list of posture names, but more straightforward information is essential to future studies seeking to replicate or generalize the results. A related issue that remains to be resolved is how to standardize yoga exercises for research purposes. The optimal duration and intensity required to maximize the effectiveness of yoga need to be determined, as does the need for a booster to provide long-term effects. Because the MET of yoga is low, increasing the frequency may increase the benefits. However, these factors cannot be judged from the reviews studies; many articles did not clearly identify the dosage of the yoga program studied, and they focused on the short-term health benefits of yoga. Only a few studies included follow-up data beyond 6 months. It also remains to be determined whether more intensive training, that is, a greater dosage, improves the likelihood of adopting and maintaining active behavior in the long run. The samples in the reviewed studies pose additional dilemmas. It was not a surprise that a large portion of the studies were conducted in India, where the philosophy and practice of yoga originated. However, this focus on one geographical region, where yoga is particularly ingrained in the culture, limits the generalizability of results. Few studies have addressed variables of interest specific to minorities in the US (Blacks, Hispanics and Asians), which are populations believed to be more vulnerable to type 2 diabetes (48,49) and physical inactivity (8). Yoga has beneficial effects on various health conditions. A large portion of the reviewed studies analyzed the effects of yoga with healthy samples. There is evidence that yoga practice was more effective in lowering triglycerides among people with higher cholesterol than those with a lower cholesterol level (11). Therefore, it is important to consider participants’ health conditions because there are possible differences in the effects of yoga by their health severity. Conclusions Analyses of yoga intervention should be designed and conducted to identify programs best suited for diverse populations and for specific populations with high-risk factors for chronic health conditions. Such studies could guide the development of more practical and effective interventions.

J_Plant_Res-3-1-2039807

Promiscuous, non-catalytic, tandem carbohydrate-binding modules modulate the cell-wall structure and development of transgenic tobacco (Nicotiana tabacum) plants

We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase/hemicellulase complex, and the less promiscuous tandem CBM2b-1-2 from the Cellulomonas fimi xylanase 11A. CBM-labelling studies revealed that CBM29-1-2 binds indiscriminately to every tissue of the wild-type tobacco stem whereas binding of CBM2b-1-2 was restricted to vascular tissue. The promiscuous CBM29-1-2 had much more pronounced effects on transgenic tobacco plants than the less promiscuous CBM2b-1-2. Reduced stem elongation and prolonged juvenility, resulting in delayed flower development, were observed in transformants expressing CBM29-1-2 whereas such growth phenotypes were not observed for CBM2b-1-2 plants. Histological examination and electron microscopy revealed layers of collapsed cortical cells in the stems of CBM29-1-2 plants whereas cellular deformation in the stem cortical cells of CBM2b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2b-1-2 stem this was observed in the xylem cells only. The cellulose content of the transgenic plants was not altered. These results support the hypothesis that CBMs can modify cell wall structure leading to modulation of wall loosening and plant growth. Introduction Plant cell expansion depends on the interplay between internal driving forces and the controlled yielding of the cell wall. The ability of the wall to extend under this internal pressure is largely determined by wall loosening processes that modify the interaction of cellulose microfibrils with one another, and/or with hemicelluloses (Darley et al. 2001). Likewise, organ growth is brought about by differential rates of wall loosening, which causes differential tissue growth and, consequently, tissue tension within the organ (Peters and Tomos 1996, 2000). Thus, progressive modification of the cellulose–hemicellulose (mostly cellulose–xyloglucan) networks is important in plant development. In addition to their effect on plant growth, there are indications that interactions of hemicelluloses with cellulose can affect cellulose fibre properties. Using the Acetobacter model system, it was shown that cellulose–glucomannan and cellulose–galactomannan composites cause 57 and 32% loss of the crystallinity, respectively, of the native bacterial cellulose (Whitney et al. 1998). For cellulose–xyloglucan composites 29% loss of cellulose crystallinity was observed (Whitney et al. 1999). It was also shown that the composites were less stiff, leading to a dramatic reduction in mechanical strength, for example 80% reduction in composites with xyloglucan (Whitney et al. 1999, 2000). There are also reports which indicate that fibre properties depend on the side-chain substitution of the hemicelluloses. Studies on Arabidopsis thaliana mutants with mutations of the MUR2 and MUR3 genes, which encode xyloglucan-specific fucosyl and galactosyl transferases, respectively, revealed that the tensile strength of the fibre was enhanced by galactosylation of the xyloglucan (Pena et al. 2004; Ryden et al. 2003). There are indications that the interactions between mannan-based polysaccharides and cellulose can affect the structural properties of the cell wall (Carpita et al. 2001; Hosoo et al. 2002). An immuno-localisation study of A. thaliana revealed that mannans were present in all thickened cell walls of stems and leaves, including those of the xylem parenchyma and epidermis (Handford et al. 2003). Mannan transglycosylase was recently identified and characterized as cell wall enzyme acting on mannan-based plant polysaccharides in primary cell walls of higher plants (Schroder et al. 2004). This body of evidence thus supports an emerging idea that mannan-based polysaccharides in cell walls may have a role analogous to that of xyloglucans, introducing flexibility and forming a growth-restraining network with cellulose. Proteins, for example expansins, have been shown to be involved in modifying cellulose–hemicellulose interaction, thereby loosening the cell wall and ultimately regulating growth and developmental processes in which rearrangements in the cell wall are thought to be important (Cho and Cosgrove 2000; Choi et al. 2003; Pien et al. 2001; Zenoni et al. 2004). There is evidence that (heterologous) carbohydrate-binding modules (CBMs) could also affect these interactions (Levy et al. 2002; Shpigel et al. 1998), leading to altered structural morphology in transgenic plants (Kilburn et al. 2000; Quentin 2003; Shoseyov et al. 2001) or plant growth (Safra-Dassa et al. 2006). CBMs are non-catalytic polysaccharide-recognizing modules, appended to glycoside hydrolases, that often degrade water-insoluble polysaccharides and concentrate the enzymes on their target polysaccharides. CBMs are grouped into families based on amino acid sequence similarities; there are currently 47 families of CBMs in the database (http://www.afmb.cnrs-mrs.fr/CAZY/). The biochemical characteristics of members of most of the CBM families have been established, including those that target non-cell wall polysaccharides such as starch and glycogen (reviewed by Boraston et al. 2004). Although most CBMs are linked to the catalytic modules of carbohydrate-active enzymes, some non-catalytic carbohydrate-binding proteins are not appended to any catalytic module. Examples of such proteins include putative CBMs of the A. thaliana former X8 family, now CBM43 (GenBank accession no. AL161503) (Henrissat et al., 2001), with olive pollen allergen Ole e 10 (GenBank accession no. AY082335) being the first characterized member of this family (Barral et al. 2005). Another example is the non-catalytic protein (NCP1) (CBM29; GenBank accession no. AY026754), which is a component of the anaerobic fungus Piromyces equi cellulase–hemicellulose complex (Freelove et al. 2001). Because of the heterogeneous nature of the cell wall polysaccharides, several CBMs have evolved structures which enable them to recognize more than one wall polysaccharide (Boraston et al. 2004). Promiscuous recognition of carbohydrates by CBMs enables the associated enzyme (complex) to be more efficient in cell wall degradation. McCartney et al. (2006) showed that different xylan-specific CBM families can vary substantially in their binding to the cell wall. This indicates that the specificity of CBMs is so intricate that after expression in planta their effects on cell wall modification might be very local. To really affect wall architecture, it might be important to select CBMs that bind to different kinds of polysaccharide (promiscuous), so that the effect is not confined to one location or cell type. Most research activity relating to the potential use of CBMs for plant cell wall modification has been on cellulose-specific CBMs. In this investigation we have used different CBMs which not only recognise hemicellulose as opposed to CBM3 but are also to some extent promiscuous. The CBM29 modules are an excellent model of promiscuity in protein–carbohydrate recognition. The modules bind soluble glucomannan, galactomannan, β-glucan, and hydroxyethylcellulose (HEC), and insoluble forms of cellulose and mannan (Charnock et al. 2002; Freelove et al. 2001). For all the ligands tested two appended copies of CBM29 (CBM29-1-2) had affinity substantially higher than the additive value of the individual modules. This indicates the two CBMs act in synergy to bind all their target ligands, with CBM29-2 having more affinity than CBM29-1. The tandem CBM29-1-2 was, additionally, able to interact with xyloglucan and different forms of xylans, whereas the single CBM had little affinity for these polysaccharides (Freelove et al. 2001; McCartney et al. 2004). An ex-situ labelling study has revealed that the tandem CBM29-1-2 modules bind strongly to the cell walls of the maize coleoptile sheath and the enclosed developing leaves (McCartney et al. 2004). This is indicative of the great abundance of their interacting ligands in the primary cell walls of grasses. It was therefore of interest to investigate the heterologous expression of these promiscuous CBMs in plants, and, more importantly, to compare their effects with those of less promiscuous CBMs, whose characteristics are described below. Some ligand promiscuity is also observed for two consecutive family 2b CBMs (CBM2b-1 and CBM2b-2) from Cellulomonas fimi xylanase 11A. The tandem CBM2b-1-2 (note the underlining used throughout this report to distinguish it from CBM29-1-2) has very strong affinity for xylan and much lower affinity for insoluble acid-swollen cellulose (Bolam et al. 2001). It also has an 18 to 20-fold greater affinity for xylan than the individual modules. We have expressed two CBM29 constructs, the tandem CBM29-1-2 and the single derivative CBM29-2, and the tandem CBM2b-1-2 construct in tobacco (all proteins targeted the cell wall) under the control of 35S cauliflower mosaic virus (CaMV) promoter. It is shown that cell wall structural alterations in transgenic plants expressing CBM29-1-2 are more severe than those in CBM2b-1-2 transformants. Our results indicate that promiscuous carbohydrate binding modules can modulate cell wall structure and the development of transgenic tobacco plants. Materials and methods Preparation of constructs Three constructs were prepared for expression in tobacco plants, one for single CBM29-2, one for tandem CBM29-1-2, and one for tandem CBM2b-1-2. The CBM29 and CBM2b-1-2 constructs were prepared by amplifying their respective gene fragments from pET22b and pET28a, recombinant plasmid vectors used for cloning and expression in Escherichia coli (Bolam et al. 2001; Freelove et al. 2001). For the CBM29-1-2 fragment the polymerase chain reaction (PCR) was performed using primers that included BamHI and SmaI recognition sites (5′- cgggatccgttagtgctacttactctgttgtttat-3′ and 5′-tcccccgggccttttaatttattgggtcaacgaaa-3′; the BamHI and SmaI sites, respectively, are underlined). The three bases highlighted in bold type are the stop codon. The amplified 914-base-pair fragment of the tandem CBM29-1-2 was digested with BamHI and SmaI (Invitrogen, The Netherlands) and cloned into a similarly digested binary vector pGreen7k (Hellens et al. 2000). Similarly, amplification of the single CBM29-2 fragment were performed using primers with the same restriction sites as for the tandem CBM29-1-2 (5′-cgggatcccgtaatgtcagagccacttacactgt-3′ and 5′-tcccccgggccttttaatttattgggtcaacgaaa-3′; the BamHI and SmaI sites, respectively, are underlined). The amplified 559-base-pair fragment was digested with BamHI and SmaI (Invitrogen, The Netherlands) and cloned into pGreen7k binary vector. Similarly, amplification and cloning of the 635-base-pair CBM2b-1-2 fragment were performed using primers that included SmaI and EcoRI recognition sites at either end (5′-cccgggtgacacgggcggaggcggcggt-3′ and 5′-ggaattctcagcccgtggcgca-3′; the SmaI and EcoRI sites, respectively, are underlined). The cloning of the three fragments was in-frame with fusion peptides of two sequences, which were cloned upstream in the binary vector. The first sequence codes for a tobacco transit peptide for transporting a cellular glycoprotein NTP303 across the plasma membrane into the cell wall (Wittink et al. 2000) whereas the second sequence encodes a hexahistidine tag. The sequence of the transit peptide was obtained as a product of annealing two oligonucleotide primers, TP1 (5′- agcttatgggaagtggtaaagtaacatttgtggctttgctactttgcctctccgtaggggtgatagctt-3′) and TP2 (5′-ctagaagctatcacccctacggagaggcaaagtagcaaagccacaaatgttactttaccacttcccata-3′). The underlined nucleotides produced overhangs of HindIII and XbaI, respectively. This fragment was then cloned into the HindIII and XbaI cloning sites of the vector. Similarly, the sequence of the hexahistidine tag was obtained and ligated into XbaI and BamHI sites as annealing product of His1 (5′-ctagaagaggatcgcatcaccatcaccatcacg-3′) and His2 (5′-gatccgtgatggtgatggtgatgcgatcctctt-3′), with the underlined nucleotides producing overhangs of XbaI and BamHI, respectively. The purpose of using the hexahistidine tag was to facilitate affinity purification of the CBM29 proteins. The control construct did not contain any of the CBMs, the transit peptide, or the hexahistidine epitope tag. All constructs were sequenced to verify their integrity. Immunofluorescence detection of CBM29-1-2 and CBM2b-1-2 binding and microscopy Wild-type tobacco plants (Nicotiana tabacum L.) were grown at 24°C under 16 h light and 8 h dark for 6–7 weeks. Regions of stem were excised and immediately fixed in PEM buffer (50 mmol L−1 PIPES (piperazine-N,N′-bis[2-ethanesulfonic acid]), 5 mmol L−1 EGTA (ethylene glycol bis(β-aminoethyl ether)-N,N,N′N′-tetraacetic acid), and 5 mmol L−1 MgSO4, pH 6.9) containing 4% paraformaldehyde. Samples were then dehydrated in an ethanol series (30, 50, 70, 90, and 97%, each for 30 min at 4°C, ethanol and wax 1:1, 37°C, overnight) and embedded in Steedman’s wax. Sections were cut to a thickness of 12 μm and collected on polylysine-coated microscope slides (BDH Laboratory Supplies, Dorset, UK), de-waxed, and re-hydrated through a reverse ethanol series into phosphate-buffered saline (PBS) (97% for 3 × 10 min, 90%, 50% and water for 10 min each, and a final step of water 90 min). For CBM labelling, sections were incubated in milk protein/PBS (to reduce any non-specific binding of proteins) and 5 μg mL−1 CBM29-1-2 for 1.5 h. Immunofluorescence detection of binding of CBM29-1-2 and CBM2b-1-2 to sections and microscopy were performed as described elsewhere (McCartney et al. 2004). Tobacco transformation and regeneration In vitro leaf explants of N. tabacum cv. Samsun NN were used for Agrobacteriumtumefaciens-mediated transformation. Cloned binary vector pGreen7k was co-transformed with the helper plasmid pSoup (Hellens et al. 2000) into A.tumefaciens strain LBA4404 by electroporation. This was plated out on LB-agar plates containing kanamycin (100 μg mL−1) and rifampicin (30 μg mL−1), and incubated for three days at 28°C, to obtain single colonies. The integrity of the binary plasmids was tested by restriction analysis of plasmids isolated from A. tumefaciens cultures used for plant transformation. LB medium (20 mL) without selection was inoculated with a single colony and incubated overnight at 28°C. The grown culture (100 μL) was added to a Petri dish containing 10 mL Murashige–Skoog (MS30). Leaf explants, without major veins and edges, from young seedlings were transferred upside-down in to the MS30 medium. Agrobacterium infection was performed in the dark for 2 days at 24–25°C. Two controls were used for the procedure, untransformed wild-type control and empty pGreen7k control without an insert. Leaf explants were washed in three changes of liquid washing medium of MS30 with 250 mg L−1 carbenicillin. The washed explants were transferred upside-up to an MS30-phytagel plate containing 0.1 mg L−1 α-naphthalene acetic acid (NAA), 1 mg L−1 6-benzylaminopurine (BAP), 200 mg L−1 kanamycin and 250 mg L−1 carbenicillin, and incubated overnight in the dark at 28°C. Plants were transferred to a growth chamber (25°C), where they were gradually adapted to light and incubated for callus induction. After two weeks in culture, calli generated were transferred to shoot-inducing medium, MS20-phytagel, containing 0.2 mg L−1 NAA, 2 mg L−1 BAP, 200 mg L−1 kanamycin, and 250 mg L−1 carbenicillin. Well-formed shoots were harvested and transferred to root-inducing medium, MS15-phytagel plate containing 100 mg L−1 kanamycin, 250 mg L−1 carbenicillin, and 200 mg L−1 vancomycin. Transformed plantlets were transferred to the greenhouse to generate mature plants. Between fifteen to eighteen antibiotic-resistant tobacco transformants were generated for each of the three CBM transgenes. RNA gel blot analysis of transgenic plants Total RNA was isolated from 3 to 5 g transformed in-vitro shoots as described elsewhere (Kuipers et al. 1995). Aliquots of 20 μg per lane were separated on a 1% formaldehyde agarose gel and blotted onto a Hybond-N nylon membrane (Amersham) by vacuum transfer in 0.4 mol L−1 sodium hydroxide. The membranes were hybridized with probes consisting of 60 ng [α32P]dCTP-labelled restricted fragments of CBM29-1-2, CBM29-2, or CBM2b-1-2. The radioactive labelled blots were exposed to X-OMAT S and AR scientific imaging films (Kodak) at −80°C. The blots were re-hybridized, after stripping, with a 489 bp [α32P]dCTP-labelled fragment of a tobacco 18S ribosomal RNA gene (GenBank accession no. AJ236016), as a control. The ribosomal probe was amplified from tobacco genomic DNA using oligonucleotides 5′-gaaactgcgaatggctcatt-3′and 5′-attaccgcggctgctggc-3′ for PCR amplification. Protein extraction Protein extracts were prepared from transformed tobacco stems using 250 mmol L−1 sodium phosphate buffer (pH 6.0), 0.5 mol L−1 NaCl, 5 mmol L−1 EDTA, 0.1% (v/v) Tween 20, protease inhibitor (Complete, Roche), 37.5 mg mL−1 PVPP (adapted from Vincken et al. 1998). The protein samples were incubated for 2 h at 4°C with gentle shaking. After incubation the samples were centrifuged at 4,500g and 4°C for 20 min. The supernatant containing the soluble protein fraction was then collected. The protein samples were then concentrated with Centricon Plus-20 devices (Millipore) according to supplier’s procedure. Protein concentrations were determined with an ESL protein assay (Roche) using BSA as standard. Incubation of RGS·(HIS)6-tagged size marker in protein extract of wild-type tobacco Protein extracts were prepared from wild-type tobacco as described above, either lacking or supplemented with 5 mmol L−1 EDTA and protease inhibitor (Complete, Roche). RGS·(HIS)6-tagged size marker was incubated with the protein extracts as follows. A batch of RGS·(HIS)6-tagged size marker (Qiagen) was dissolved in 100 μL 50 mmol L−1 Tris, pH 7.5, such that the final concentration of the individual proteins varied from 50 to 75 ng μL−1. The epitope-tagged size marker (2 μL) was added to 18 μL tobacco extracts. The mixtures were incubated at room temperature for approximately 15 h. As a control, RGS·(HIS)6-tagged size marker was incubated in 50 mmol L−1 Tris, pH 7.5. Western blot analysis The protein samples were separated with SDS-PAGE on 12% SDS-polyacrylamide gels (Mini-Protean II apparatus, Bio-Rad). The separated proteins were electroblotted on to a nitrocellulose membrane in 192 mmol L−1 glycine, 25 mmol L−1 Tris base, 0.1% (w/v) SDS, 20% (v/v) ethanol, pH 8.3. Blocking against non-specific binding was performed for 2 h with 3% (w/v) BSA (Sigma) in TBS (10 mmol L−1 Tris, 150 mmol L−1 NaCl, pH 7.5). The membrane was then incubated with a 1:1000 dilution of the primary anti-RGS·HIS antibody (Qiagen) in TBS solution containing 3% (w/v) BSA for 2 h. The membrane was washed twice in TBS-TT buffer (20 mmol L−1 Tris base, 500 mmol L−1 NaCl, pH 7.5, 0.05% (v/v) Tween 20, 0.2% (v/v) Triton X-100), and once in TBS buffer, for 10 min each. The membrane was then washed with 1:2000 dilution horseradish peroxidase (HRP) conjugated sheep anti-mouse antibody (Amersham) in TBS buffer containing 10% (w/v) non-fat dried milk. The membrane was washed in TBS-TT buffer four times, 10 min each, and subsequently submerged in Supersignal ULTRA substrate working solution (Pierce), which is a mixture of equal parts of Ultra luminol/enhancer solution and Ultra stable peroxide solution. Finally, the membrane was exposed to X-OMAT S Scientific Imaging films (Kodak) at room temperature. Monitoring the growth of the transformed plants To monitor growth of the transformed plants with time, three replicates per line were grown in the greenhouse. The transformants were grown in two series, the CBM29s and CBM2b-1-2 series, with each series having its separate controls. Height measurements of the stems were taken weekly for nine weeks, starting from week two after transplant. The height measurements of six individual high expressers, each from transgenic lines containing CBM29-1-2 and CBM29-2 constructs, six transformed controls, and six untransformed controls from week 3 to week 9 were subjected to one-way analysis of variance (ANOVA), using an α of 0.05. To test whether the differential stem elongation was significant, a Fisher’s unprotected least significant difference test was used. The null hypothesis of no difference between plant heights was rejected at an error of 0.05. Light microscopy Three individual plants per transgenic tobacco line and three wild-type plants, as control, were used for microscopic examination. Three transgenic lines were used per construct (two high expressers and one low expresser). Stem samples were taken from the second internode from the top of the plant. Stem sections 1 mm-thick were fixed in 3% glutardialdehyde (Merck) and 3% paraformaldehyde (Merck) in 0.1 mol L−1 phosphate buffer containing 0.1% Triton X-100 for 2 h. The samples were then washed and dehydrated in an ethanol series. After dehydration they were embedded in Technovit 7100 resin (USA) (Kuroiwa et al. 1990). Sections 4 μm-thick were stained with 0.1% toluidine blue (Aldrich) and examined under a bright field microscope. Cryo-scanning electron microscopy and quantification of cell size and cell wall diameter One high expresser from each of the CBM29s and CBM2b-1-2 transgenic lines and one individual plant each from their respective controls were used for cryo-SEM examination. The plants that were analysed were chosen on the basis of the results obtained from the extensive light microscopic analysis that showed a consistent pattern of alterations in high-expressing transgenic plants. Stem sampling was the same as for the light microscopy. Stem samples 6 mm-thick were mounted in a brass cylindrical sample holder with TBS tissue freezing medium (EMS, Washington, PA, USA). The frozen samples were placed in a sample holder in a cryo-ultra microtome (Reichert Ultracut E/FC4D) and cut at a specimen temperature of 100°C. These samples were first planed with a glass knife, after which the surface was planed with a diamond knife (Histo no trough, 8 mm 45°C; Drukker International, The Netherlands). After planing, the samples were placed in a dedicated cryo-preparation chamber (CT 1500 HF, Oxford instruments, UK). All the samples in the cryo-preparation chamber were freeze dried for 3 min at −90°C and 8 × 10−4 Pa, and subsequently sputtered with a 10-nm layer of Pt. The samples were cryo-transferred into the field emission scanning microscope (Jeol 6300F, Japan) on the sample stage at −190°C. All images were recorded digitally (Orion, 6 E.L.I. sprl, Belgium) at a scan rate of 100 s (full frame) at the size of 2,528 × 2,030, 8 bit. The images were optimized and resized for publication by use of Adobe Photoshop CS. To quantify the micrographic images, files were opened with Image J software developed at the National Institute of Health, USA (http://www.rsb.info.nih.gov/ij/). The surface area of three hundred cortical and xylem cells was measured as shown by the insets in Figs. 7a and 7b. One hundred cells per field of view were measured in three replicates. Similarly, cell wall thickness of 150 cells was quantified as shown by the inset in Fig. 7c. Fifty cells per field of view were measured in three replicates. Standard deviations of three measurements were determined. Isolation of cell wall material and analysis of the cellulose content Stem samples from one high expresser of the CBM29 and CBM2b-1-2 series, one pGreen7k vector control, and one wild-type control plant were ground to a fine powder in liquid nitrogen. For each isolation, 1 g of this stem material was extracted in a 50 mmol L−1 Tris[HCl], pH 7.2 solution containing 1% SDS, for 3 h at room temperature (RT) with continuous shaking. The cell wall material CWM was spun down by centrifugation at 13,000 rpm for 15 min. Subsequently, the residue was washed with water, ethanol, and acetone, then air-dried. The different cell wall materials (10 mg) were hydrolysed in 1 mL 2 mol L−1 TFA. The TFA-insoluble cellulose was spun down at 13,000 rpm for 15 min, and the pellet was suspended in 67% sulfuric acid. The suspension was heated and diluted appropriately to determine the cellulose content colorimetrically, using anthrone as colouring agent (Updegraff 1969). The acid hydrolysis was performed in quadruplicate. Results Expression of bacterial CBM29 and CBM2b-1-2 genes in tobacco To detect expression of the introduced CBM genes in the tobacco plant, we performed Northern blot analysis with total RNA. Figure 1 shows representatives of three classes of transcript expression (high, low, and none) of the three CBM modules as revealed by the Northern analysis. Ten of the CBM29-1-2 plants were classified as high expressers, five as low expressers, and two as none expressers. For the CBM29-2 plants, ten lines were classified as high expressers, seven as low expressers, and two as none expressers (note that none expressers may include plants with very low RNA expression, which could not be detected). For the CBM2b-1-2 plants, six each were classified as high and low expressers whereas four plants were classified as none expressers. Preliminary attempts at purifying CBM proteins using affinity purification with the hexahistidine tag were not successful (data not shown). Similarly, Western detection of a hexahistidine epitope-tagged fungal elicitor protein ECP2 that was infiltrated into the apoplast of leaves did not succeed in the tomato plants but succeeded in Arabidopsis thaliana (van Esse et al. 2006). The results indicated that the histidine tag was cleaved in the tomato plants but not in A. thaliana. These observations have since led to the idea that the hexahistidine epitope tags have cleavage sites recognized by proteases, which are specific for the cell walls of the solanaceous species. Fig. 1Transcript analysis of CBM29 and CBM2b-1-2 genes in transgenic tobacco leaves. A differential transcript expression pattern is shown in the upper panel with the representative of each class in the three transgenic lines CBM29-2 (a), CBM29-1-2 (b) and CBM2b-1-2 (c). Line 14 in aline 5 in both b and c, represent high expressers. Lines 9, 11 and 1 represent low expressers. Line 17 in a, line 6 in b and line 14 in c represent none expressers. The lower panel shows RNA blots for the 18S ribosomal RNA internal control for each of the series We corroborated this notion by incubating a protein ladder, consisting of five different proteins of known sizes, each containing an N-terminal RGS·(HIS)6–tag, in protein extracts of wild-type tobacco (Compier 2005). The tagged proteins of the size marker that were incubated in tobacco extract without EDTA and protease inhibitor were not detectable on a Western blot whereas the markers incubated in extract supplemented with EDTA and protease inhibitor were detected. This suggested that extracts from tobacco contained proteases that could remove the hexahistidine epitope tag. Because grinding of the plant material and protein extraction were performed at 4°C, and the protein extraction buffer contained EDTA and protease inhibitor, removal of the RGS·(HIS)6-tag from the fusion proteins had probably occurred already in planta, most probably extracellularly. It should be noted that with the presence of the NTP303 signal peptide, the fusion proteins would be secreted into the apoplast. Cleavage of the RGS·(HIS)6-tag was, therefore, likely to occur after translocation into the extracellular space. CBM29-1-2 and CBM2b-1-2 have different binding patterns in the stem Immunofluorescence detection of CBM29-1-2 and CBM2b-1-2 binding to stems of the wild-type tobacco plant was performed order to determine, a priori, the particular tissues of the stem where the introduced CBM29-1-2 module would bind. Indirect immunofluorescence micrographs (Fig. 2) revealed that CBM2b-1-2 binding is restricted to the secondary cell walls of stem vascular tissue—xylem cells and phloem fibres (Fig. 2a)—whereas CBM29-1-2 indiscriminately binds to every tissue of the stem, from the epidermis through the cortex and the vascular tissue to the pith parenchyma in the innermost part (Fig. 2b). In the control (no CBM) weak autofluorescence was observed in the xylem vessel cell walls (Fig. 2c). This ex-situ evidence for unselective binding of the CBM29-1-2 to both the primary and the secondary cell walls of the mature stem of the tobacco plant thus complements earlier evidence for its binding to a predominantly primary-walled maize coleoptile (McCartney et al. 2004). It should also be noted that tobacco is a dicotyledonous plant whereas maize is monocotyledonous, and that there are many differences between the polysaccharide composition of the walls of these species. Both results thus confirm the binding promiscuity of the tandem CBM29-1-2, as previously characterized (Charnock et al. 2002; Freelove et al. 2001). We also conclude that the CBM2b-1-2 ligands are present in the vascular tissue of the stem. Fig. 2Micrographs, obtained by indirect immunofluorescence microscopy, showing binding of CBM2b-1-2 (a), CBM29-1-2 (b), and control (no CBM) (c) to transverse sections of wild-type tobacco stem. The CBM29-1-2 binds indiscriminately to every tissue of the stem, from the epidermis through the cortex and the vascular tissue to the pith parenchyma in the innermost part whereas CBM2b-1-2 binding is restricted to the vascular tissues. ct cortical parenchyma, xy xylem cells, ph pith. Arrow head indicates the epidermis. Arrow indicates the phloem fibres. Scale bar = 100 μm Reduced stem elongation and prolonged juvenility are observed for CBM29-1-2 transformants An indication of reduced stem elongation in the transgenic plants expressing the tandem CBM29-1-2 was first apparent after the third week of transfer to the greenhouse. The different stem elongation of the tandem CBM29-1-2-expressing plants and the control plants became significant (P < 0.05) at weeks seven and eight, when other plants had reached their final height (Table 1, Fig. 3). The tandem CBM29-1-2-expressing plants continued to increase in height until the eleventh week, however, when they reached their final height, an average of 58 cm. There was, as a result, no significant difference between the heights of the tandem CBM29-1-2 lines at maturity and the heights of the single CBM29-2 lines, the pGreen vector control, and the wild-type control plants. There was no significant difference, at any time, between stem elongation of transgenic plants expressing CBM29-2 and the control plants. Table 1Plant height (cm) of transgenic lines CBM 29-1-2, CBM 29-2, empty pGreen7k control, and wild-type controlPlant lineWeeks after transplant3456789CBM29-1-29.5 ± 1.316.1 ± 2.227.0 ± 3.837.7 ± 5.543.1 ± 5.3a50.5 ± 5.4a56.6 + 6.7CBM29-29.3 ± 1.316.9 ± 1.927.5 ± 3.240.9 ± 5.050.7 ± 4.157.8 ± 4.163.8 + 4.7PGreen7k9.5 ± 1.617.3 ± 2.330.1 ± 3.844.6 ± 5.257.6 ± 7.163.1 ± 5.865.4 + 5.6WT Ctrl9.7 ± 1.317.1 ± 2.331.5 ± 3.750.6 ± 7.363.6 ± 7.168.4 ± 5.568.8 + 5.6The values are means and standard deviations for six transgenic lines (high expressers) and six control plants. Each line has three individual plants (three replicates)aPlant height significantly different from that of controlsFig. 3Reduced stem elongation in time, and delayed development of CBM 29-1-2 transgenic tobacco plants. The picture was taken at week eight In addition to reduced stem elongation it was also observed that flower development was delayed in the tandem CBM29-1-2-expressing plants. We observed that at week eight, when most plants had already flowered (Fig. 3) and set fruit (data not shown), the tandem CBM29-1-2-expressing plants were just developing flower buds (Fig. 3). The flower buds eventually developed into normal flowers and also set fruits. Most plants, except the CBM29-1-2 plants, had also lost many leaves, because of faster ageing (Fig. 3). There was no morphological or developmental change in the transgenic plants expressing the tandem CBM2b-1-2 gene compared with the pGreen7k vector control. The average plant height of the tandem CBM2b-1-2 series at maturity was 71 cm, which was comparable with the 75 cm average height for their pGreen7k vector control plants. We also did not observe a particular trend in plant development with regard to stem elongation, leaf abscission, or flower formation. Cellular alterations are observed in transgenic plants The cellular basis for the CBM29-1-2 plant phenotypes was examined by histological examination of the transgenic tobacco stems. The examination revealed a prominent layer of collapsed cells in the cortex of the stems of the tandem CBM29-1-2-expressing plants (Figs. 4a, 5a). We also observed that this morphological alteration is consistently more severe in the high expressers than in the low expressers. This alteration was also observed in the high expressers of the single CBM29-2, although it was much less severe (Fig. 4c). For the CBM2b-1-2 stems, irregularly shaped cells were observed in the cortex of the high expressers (Fig. 5c). The micrographs of both the empty vector control and untransformed wild-type control looked the same; hence the pGreen7k micrograph was taken to represent the controls (Fig. 4b). Altered cell enlargement across the tissues in the CBM29s-expressing stems was also observed. This was particularly true for the tandem CBM29-1-2-expressing plants (compare, for example, Figs. 5a and 5d with 5b/c and 5e/f, respectively). The altered cell size phenotype is widespread in the high and low CBM29-1-2-expressing plants. This is not observed, however, in the single CBM29-2-expressing stems, in which only the high expressers had noticeably larger cells in the epidermal, cortex, and xylem tissues than those of the controls. Similarly, there was an indication of larger xylem cells in the outermost layer of the CBM2b-1-2 stems than in controls (compare Figs. 5e and 5f). There was, however, no clear indication of altered enlargement of the stem pith parenchyma cells of the transgenic plants. Fig. 4Cross sections of transgenic and control stems, showing collapsed cortical cells and enlarged cortical and xylem cells of the CBM29-1-2 stems. Sections of CBM29-1-2 (a), empty Pgreen7k control (b), CBM29-2 stem (c), and CBM2b-1-2 (d) were stained with toluidine blue. The boxed area indicates tissues shown with higher magnification in Fig. 5. ep epidermis, ct cortex, xy xylem, ph pithFig. 5 Magnification (10×) of cross sections of CBM29-1-2 stem (a, d), control stem (b, e), and CBM2b-1-2 stem (c, f). Micrographs show collapsed cortical cells (a), enlarged cortical and xylem cells of the CBM29-1-2 stems (a, d), and irregularly shaped stem cortical cells of the CBM2b-1-2 stems (c). Asterisks indicate irregular cortical cells in the CBM2b-1-2 transformants; in the CBM29-1-2 transformants these cells are more apparent and are not highlighted. ep epidermis, ct cortex, xy xylem, pf phloem fibre Cryo-scanning electron microscopy (cryo-SEM) of tobacco stems To establish that the transgenic stems indeed had larger cells than those of the controls we made cryo-SEM examinations of stem samples. Figure 6a shows the collapsed and enlarged cells of the cortex in the sections of transgenic line 5 of CBM29-1-2, as previously observed by light microscopic examination. The cortical cells of the control plant were smaller and the collapsed phenotype was not apparent (Fig. 6b). The micrographs also showed that xylem cells were indeed larger in the CBM29s-expressing stems than in the controls (compare transgenic line 5, Fig. 6c and the empty vector control, Fig. 6d). The micrographs also indicated that the cell walls of the xylem cells were thinner in the CBM29s-expressing stems (Fig. 6c) than in stems of controls (Fig. 6d). Similar micrographs were obtained for the CBM2b-1-2 series, which confirmed that the radial surface area and cell wall diameter of their stem xylem cells were larger and thinner, respectively, than those of the controls (data not shown). These notable observations were later substantiated by quantification. Figure 7a shows that almost 50% and 40% of the stem cortical cells of the wild-type and pGreen7k control plants, respectively, were grouped in the small class (50–100 μm2), whereas fewer than 20% of the stem cortical cells of the CBM29s-expressing plants were grouped in the small class. An opposite trend was observed in the large class (150–200 μm2)—fewer than 20% of the cortical cells of the two control stems were grouped compared with more than 30 and 40% of the stems expressing the single CBM29-2 and the tandem CBM29-1-2, respectively. We thus infer that the cortical cells of the CBM29-2-expressing stems are larger than those of the control stems. Similar quantitative data were obtained from the xylem cells depicted in Fig. 7b, which shows that only the xylem cells of the CBM29-1-2-expressing stems are larger than those of the control stems. There was no difference between the size of the xylem cells of the single CBM29-2 stems and those of the controls. Similarly, we quantified the thickness of the cell walls of the xylem cells of the CBM29s-expressing stems and the pGreen7k control stems as presented in Fig. 7c. Approximately 45% of the cell walls of the xylem cells of the CBM29-1-2-expressing stem were grouped in the thin class (1.2–2 μm) whereas fewer than 10% of the stem xylem cell walls of the single CBM29-2, the pGreen7k control, and the wild-type control plants were grouped in this class. More than 60% of the xylem cell walls of the two control stems were grouped in the thick class (2.8–3.6 μm) whereas approximately 22 and 0% of the stem xylem cell walls of the single CBM29-2 and the tandem CBM29-1-2, respectively, were grouped in this class. We can also infer from this result that the thickness of the cell walls of the xylem cells of the CBM29-2-expressing stems (particularly of the tandem CBM29-1-2) are thinner than those of the pGreen7k and the wild-type control stems. Fig. 6Scanning electron micrographs of transgenic tobacco stems to show details of larger cells and thinner cell walls of the CBM 29-1-2 stems compared with the pGreen7k control. CBM29-1-2 (a) and (c), and empty pGreen7k control (b) and (d). a and b show the cortex tissue whereas c and d show the xylem tissue. pf phloem fibre, xy xylem, ct cortex. Arrow heads indicate collapsed layer of cortical cellsFig. 7Cell size distribution in the cortex (a), cell size distribution in the xylem (b), and cell wall thickness of xylem cells (c) in the stems of transgenic and the wild-type tobacco plants. Insets show how the surface area and wall thickness measurements were taken. Surface areas of 300 cells were measured, 100 cells per field of view. Cell wall diameters of 150 cells were measured, 50 cells per field of view. Error bars represent standard deviation. Grey bars represent wild-type plants. Diagonal bars represent empty pGreen7k transformants. Open bars represent single CBM29-2 transformants. Horizontal brick bars represent double CBM29-1-2 transformants A similar trend was observed for the xylem cells of the CBM2b-1-2 stems, which were larger than those of the pGreen7k control plants (data not shown). Similarly, the cell walls of the xylem cells of the CBM2b-1-2-expressing stems were thinner than those of the pGreen7k control stems (data not shown). Analysis of cellulose content A colorimetric assay was performed on samples of crystalline cellulose obtained from four separate acid hydrolyses to determine the cellulose content in the stem cell walls of the CBM29s and CBM2b-1-2 transformants and the control plants. There were no substantial differences between levels of cellulose in the stem cell walls of the transgenic tobacco lines expressing the single CBM29-2, the tandem CBM29-1-2, the tandem CBM2b-1-2, the empty vector control, and the wild-type control plants (data not shown). Hence, it can be concluded that use of these CBMs for cell wall modification did not interfere with the extent of cellulose biosynthesis. Discussion It has been suggested that networks of plant cell wall polysaccharides can affect the properties of cellulose fibres. We hypothesized that expression of promiscuous CBMs in tobacco plants might lead to modification of these networks, although they might also affect plant development in more general terms. In this study, expression of two promiscuous Piromyces CBM29-containing proteins was targeted at tobacco cell walls to provide baseline information on the possibility of using CBMs for in-planta modification of the properties of cellulose fibre for industrial applications. In parallel, we also targeted the expression of the less promiscuous tandem CBM2b-1-2 protein of the C. fimi xylanase 11A, with a view to comparing its effect with that of the more promiscuous CBM29-1-2. Our results reveal that the tandem CBM29-1-2 modified the tobacco cell wall structure with consequent altered cellular and organ morphology. The tandem CBM29-1-2 modules had more effect than the single CBM29-2. Expression of the tandem CBM29-1-2 modules in the tobacco cell wall affected plant development, as reflected by reduced stem elongation rate, enlarged xylem, and collapsed cortical cells. The stem phenotype of the CBM29-1-2-expressing plants was more of delayed development of the whole plant rather than mere elongation defects of the stem. We suggest that the delayed development (prolonged juvenility) was as a result of reduced rate of anabolism in the tandem CBM29-1-2-expressing plant. It could be that structural remodelling of the cell wall has affected the normal developmental cues that signal the cells to synthesize new cell wall materials. It is conceivable that when the cell walls are held loosely continuously as a result of the continuous presence of CBM29-1-2, the rate of wall extension will outpace biosynthetic rate. Consequently, excessive thinning of the cell walls may occur (Kutschera 1990), which may have led to the eventual collapse of, particularly, the primary walled cells (and not so much of the secondary-walled xylem cells). Thus, because of the extensive loosening of their cell walls and consequent altered expansion, the cortical cells may have yielded to tissue pressure within the stem. The altered cell enlargement, which led to the collapse of the cortical cells of the tandem CBM29-1-2-expressing stems, is proposed as the cellular basis for the reduced stem elongation. We have not noticed that the increased size of the xylem cells led to thicker stems. It is possible the gain in the size of the xylem cells is neutralized by the collapse of the cortical cells. The explanation given above is based on the assumption that the CBMs do not bind to polymers until they have reached the wall. It should be noted, however, that by placing a signal peptide at the amino terminus of the CBMs we have targeted them through the secretory pathway, which includes the site of hemicellulose biosynthesis (the Golgi apparatus) (Denecke et al. 1990; Vitale and Denecke 1999). It is, therefore, possible the CBMs had already bound to hemicellulose before their deposition in the cell wall. As a result, the hemicellulose content, length, and/or branching pattern might have changed; this, in turn, could affect their interaction with cellulose microfibrils, leading to structural alteration of the cell wall. The expression of the CBM2b-1-2 modules in the tobacco cell wall altered the cellular morphology of the transgenic plants, as was apparent from the irregular shape of the cortical cells and altered cell enlargement of the xylem cells of the stems. The deformation of the cortical cells of the CBM2b-1-2 expressing plants is, however, less pronounced than that of the cortical cells of the CBM29-1-2 transformants. The more obvious phenotypic changes in the CBM29-1-2-expressing plants is consistent with the result of the immunolabelling study with the proteins, which revealed that CBM29-1-2 binds to polysaccharides that are integral to both primary and secondary cell wall structure. CBM2b-1-2, in contrast, binds only to xylan and non-crystalline cellulose. As xylan is mainly present in the secondary cell wall, it is not strange to see the effect of CBM2b-1-2 most pronounced in the secondary walled xylem. The finding that CBM2b-1-2 did not label the cortex (Fig. 2a) suggests that the deformation of the cortical cells is an indirect effect of CBM2b-1-2. The cortical parenchyma has no secondary walls, suggesting that the walls are much weaker here; consequently, this tissue might collapse when challenged by excessive expansion of flanking tissues. The expression of the CBM2b-1-2 only had effect on cellular morphology and not on plant morphology whereas the expression of the CBM29-1-2 in tobacco led to altered structural morphology and altered development of the transgenic plants. The reduced stem elongation phenotype of the CBM29-1-2 tobacco plants is, however, in contrast with the report by Safra-Dassa et al. (2006) of enhanced growth of transgenic potato plants expressing a bacterial CBM3. These contrasting results might be because of the different specificities of the CBMs, because we have used double CBMs in our investigation as opposed to the single CBM they used. That the experiments were performed on different plant species may also have contributed to the different observations. There are reports attributing cell wall phenotypes in mutant or transgenic plants to abnormal wall assembly in general, or depletion of the cellulose content in particular (Szyjanowicz et al. 2004; Taylor et al. 1999, 2000; Tsabary et al. 2003; Turner and Somerville 1997). In all these instances the plants had deformed walls in vascular cells and reduced growth as a result of cellulose depletion. Our results reveal that wall deformation (cortical cells) can also occur without a marked reduction in cellulose content. To summarise, the results of our investigation suggest that expression of the promiscuous tandem CBM29-1-2 can alter cell morphology to a larger extent than that of more specific CBM CBM2b-1-2, although the actual effect is unpredictable. Use of CBMs for in-planta cell wall modification thus still needs further investigation.

Evid_Based_Complement_Alternat_Med-5-1-2249747

The Treatment of Pulmonary Diseases and Respiratory-Related Conditions with Inhaled (Nebulized or Aerosolized) Glutathione

Reduced glutathione or simply glutathione (γ-glutamylcysteinylglycine; GSH) is found in the cytosol of most cells of the body. GSH in the epithelial lining fluid (ELF) of the lower respiratory tract is thought to be the first line of defense against oxidative stress. Inhalation (nebulized or aerosolized) is the only known method that increases GSH's levels in the ELF. A review of the literature was conducted to examine the clinical effectiveness of inhaled GSH as a treatment for various pulmonary diseases and respiratory-related conditions. This report also discusses clinical and theoretical indications for GSH inhalation, potential concerns with this treatment, its presumed mechanisms of action, optimal doses to be administered and other important details. Reasons for inhaled GSH's effectiveness include its role as a potent antioxidant, and possibly improved oxygenation and host defenses. Theoretical uses of this treatment include Farmer's lung, pre- and postexercise, multiple chemical sensitivity disorder and cigarette smoking. GSH inhalation should not be used as a treatment for primary lung cancer. Testing for sulfites in the urine is recommended prior to GSH inhalation. Minor side effects such as transient coughing and an unpleasant odor are common with this treatment. Major side effects such as bronchoconstriction have only occurred among asthma patients presumed to be sulfite-sensitive. The potential applications of inhaled GSH are numerous when one considers just how many pulmonary diseases and respiratory-related conditions are affected by deficient antioxidant status or an over production of oxidants, poor oxygenation and/or impaired host defenses. More studies are clearly warranted. Introduction Reduced glutathione or simply glutathione (γ-glutamylcysteinylglycine; GSH) is found in the cytosol of most cells of the body (1). It is a tripeptide consisting of glycine, cysteine and glutamate. GSH functions in several enzyme systems within the body that assist with the quenching of free radicals and the detoxification of fat-soluble compounds (Table 1) (2–5). It also plays a significant metabolic role in supporting many different biochemical processes (e.g. amino acid transport, deoxyribonucleic acid synthesis and immune system augmentation) considered to be important mediators of health status (6). Table 1.Enzyme systems involving glutathioneEnzyme systemFunctionGlutathione synthetaseGamma-glutamyl cycle.Riboflavin-containing glutathione reductaseCatalyzes the conversion of oxidized glutathione (glutathione disulfide; GSSG) to its reduced form.GSH transferase isoenzymesConjugation of GSH with fat-soluble substances for liver detoxification and the detoxification of environmental carcinogens, such as those found in tobacco smoke.Selenium-containing glutathione peroxidase (GPX)Protects cells from hydrogen peroxides and lipid hydroperoxides. If not neutralized, these peroxides will damage cellular membranes and other vital cellular components.Leukotriene C4 synthaseConjugation of leukotriene A4 with GSH, resulting in the generation of leukotrienes C4. Gamma-glutamyl transpeptidase then metabolizes leukotrienes C4 to leukotrienes D4. Glutathione in the epithelial lining fluid (ELF) of the lower respiratory tract is thought to be the first line of defense against oxidative stress (6). The ELF concentration of GSH is 140 times that of serum concentrations with a redox ratio of > 9 : 1 (7). In fact, alternations in alveolar and lung GSH metabolism are widely recognized as a central feature among many inflammatory lung diseases (8–14). In healthy lungs, the oxidant burden is balanced by local antioxidant defenses. However, in lung diseases cellular damage and injury is mediated by an increased oxidant burden and/or decreased antioxidant defenses. In inflammatory lung diseases, supplementation with exogenous sources of GSH would be necessary to reduce the oxidant load and/or correct for antioxidant deficiencies within the lungs. A few published clinical studies have shown the oral administration of GSH to be ineffective at increasing plasma levels when given to healthy subjects (15), or when used for the treatment of hepatic cirrhosis (16). If the oral administration of GSH cannot raise plasma levels in healthy and diseased patients, it is doubtful that this method of delivery would have any appreciable effects at increasing GSH concentrations within the lungs. Intravenous administration might be effective since it bypasses the gastrointestinal tract, immediately enters the blood stream, and presumably would saturate body tissues such as the lungs. Unfortunately, the results of a study did not show intravenous administration to be effective at increasing GSH levels within the ELF (17). When 600 mg of GSH was delivered intravenously to sheep, the levels in the venous plasma, lung lymph and ELF increased only for a very brief period of time. However, when the same amount of GSH was delivered through inhalation (nebulized or aerosolized), the baseline GSH level in the ELF (45.7 ± 10 μM) increased 7-fold at 30-min (337 ± 64 μM), remained above the baseline level 1 h later (P < 0.001) and returned toward baseline levels by 2 h. Despite this short-term increase in GSH concentrations within the ELF, the inhalation method did not significantly increase the amount of GSH in the lung lymph, venous plasma and urine during the 2 h study period. The authors of this report concluded that inhalation specifically increased GSH levels at the lung epithelial surface. Given that inhalation is the only known method that increases GSH levels in the ELF for a significant duration, a review of the literature was conducted to examine the clinical effectiveness of inhaled GSH as a treatment for various pulmonary diseases and respiratory-related conditions. Only reports involving human subjects were included in the analysis. The clinical and theoretical indications for GSH inhalation were summarized and potential concerns with this treatment reported. Other pertinent details such as its presumed mechanisms of action and optimal doses to be administered were compiled and evaluated. Methods Literature Search Computer searches were conducted of English and non-English language articles in the Biomedical Reference Collection (1984 to August 2006), CINAHL (1982 to August 2006), MEDLINE (1965 to August 2006) and Nursing and Allied Health Collection (1985 to August 2006) databases. Articles were searched with the key search terms ‘Nebulized Glutathione,’ and ‘Glutathione’ in combination with ‘Aerosol’ OR ‘Inhalation.’ These keywords were also searched with words related to pulmonary and/or respiratory disease. To supplement the search, references of the articles found from the initial search were reviewed. Hand searching of relevant journals was also completed as part of the search. Selection of Articles To be included in the final review, articles had to report on the use and administration of inhaled GSH for pulmonary diseases and respiratory-related conditions in human subjects. Only peer-reviewed articles were reviewed. Quality Assessment An evidence grade was determined for each article. These evidence grades were adapted from the hierarchy of evidence developed by the Oxford Centre of Evidence Based Medicine (Table 2) (18). Table 2.Grades of evidenceASystematic reviews of randomized controlled trials and/or randomized controlled trials with or without double-blind placebo control.BSystematic reviews of observational studies and/or high-quality observational studies including cohort and case-control studies and/or cohort ‘outcomes’ research and/or nonrandomized controlled trials.CCase-series, case-reports, and/or poor-quality cohort and case-control studies.DExpert opinion without explicit critical appraisal or based on physiology, bench research or ‘first principles.’ Results A total of 12 reports were screened (9, 10, 17, 19–27). Only one report was excluded because it involved the use of inhaled GSH in sheep (17). In total, 11 articles were found to meet the inclusion criteria and were included in this review (9, 10, 19–27). Table 3 displays the characteristics of the studies included in this review. Table 3.Summary of articles demonstrating the effectiveness of inhaled glutathione for the treatment of pulmonary diseases and respiratory-related conditionsReferenceConditionNDosages of inhaled GSHOutcomeEvidence grade(21)AsthmaEight asthma patients [mean age, 29 ± 7 (standard deviation; SD) years]600 mg once weekly for 3 monthsA subset of patients with clinically stable mild asthma experienced a bronchoconstrictor effect when treated with inhaled GSH.A: Randomized placebo-controlled trial(23)Chronic otitis media with effusion (chronic OME)30 patients (3–12 years of age; mean age, 5.8 years) and 30 controls (3–12 years of age; mean age, 6.1 years)600mg of GSH in 4 ml of saline subdivided into five 2-min sessions by nasal aerosol every 3–4 waking h for 2 weeksGSH should be considered for the nonsurgical management of chronic OME.A: Randomized placebo-controlled trial(24)Cystic fibrosis (CF)Nine patients [mean age, 16.1 ± 1.44 (SD) years] received the S-nitrosoglutathione (GSNO) and 11 patients [mean age, 19.9 ± 3.45 (SD) years] received the phosphate- buffered saline (PBS) solution0.05 ml/kg of 10 mM GSNOThe treatment group showed a modest improvement in oxygenation that was thought to be independent of the physiological effects of nitric oxide.A: Randomized placebo-controlled trial(27)CF19 patients (6–19 years of age) were randomized to treatment [mean age, 13.3 ± 4.1 (SD) years] or placebo groups [mean age, 12.9 ± 4.9 (SD) years]Total daily dose administered to the patients in the treatment group was 66 mg/kg of body weightGSH can improve clinical parameters in CF patients, and that effective treatment should include the correction of GSH deficiency.A: Randomized placebo-controlled trial(9)Idiopathic pulmonary fibrosis (IPF)10 patients with IPF [mean age, 46 ± 3 (SD) years] and 19 normal nonsmokers [mean age, 36 ± 3 (SD) years]600 mg twice daily for 3 daysInhaled GSH might be beneficial among IPF patients by reversing the oxidant–antioxidant imbalance.B: Nonrandomized controlled trial(19)Human immunodeficiency virus (HIV) seropositive individuals14 HIV seropositive individuals [mean age, 32 ± 2 (SD) years]600 mg twice daily for 3 daysIt is a reasonable therapeutic strategy to augment the deficient GSH levels of the lower respiratory tracts of HIV seropositive individuals.B: Cohort ‘outcomes’ research(20)Chronic rhinitis13 patients with chronic rhinitis and 13 healthy subjects (4–15 years of age for all subjects; mean age, 8.2 years)600 mg daily for 14 daysStatistically significant improvement in nasal obstruction, rhinorrhea and ear fullness.B: Nonrandomized controlled trial(10)CFSeven CF patients [mean age, 25 ± 1 (SD) years]600 mg of GSH for 3 daysInhalation therapy with GSH does normalize the respiratory epithelial surface oxidant–antioxidant balance in CF patients.B: Cohort ‘outcomes’ research(25)CF21 patients with CF (16–37 years of age for all subjects)300 or 450 mg three times daily for 14 daysInhaled GSH can permeate the lower airways of the lungs and improve important parameters of lung function in CF patients despite not having any effect upon markers of oxidative injury.B: Cohort ‘outcomes’ research(26)CF17 patients with CF (18–29 years of age for all subjects; mean age, 24 years)450 mg three times daily for 14 daysInhaled GSH did not affect the oxidative status of the patients who were tested, but it did favorably modulate their immune responses.B: Cohort ‘outcomes’ research(22)EmphysemaOne (95 year-old male)120 mg of GSH in office, then 120 mg twice daily for 3 days, and continuation of treatment (dose unknown) for 2 yearsWhen the patient returned for a follow-up visit, he no longer required the use of his wheelchair and oxygen. The striking results were unexpected and unlikely to be due to placebo alone.C: Case report Discussion Based exclusively on the published evidence included in this review, inhaled GSH is potentially indicated for the following clinical conditions: cystic fibrosis (CF), chronic otitis media with effusion (OME), HIV seropositive individuals, idiopathic pulmonary fibrosis (IPF) and chronic rhinitis. These conditions were chosen since the published studies were of good quality, received A and B evidence grades, and their respective results demonstrated benefits from the use of GSH inhalation. Inhaled GSH cannot be recommended as a potential treatment for emphysema since the quality of evidence is lacking at the present time. The emphysema case report had notable limitations since serial spirometry was not documented, and the placebo effect could not be ruled-out (22). However, this does not necessarily indicate that GSH inhalation would be of no benefit for emphysema patients. There is experimental and human data demonstrating a link between GSH, oxidant-derived damage and possible protection against the development of emphysema. An in vitro study demonstrated that GSH could retard the oxidant-mediated down-regulation of α-1-proteinase inhibitor activity in smokers’ emphysema (28). This finding is important since one of the principal pathophysiological mechanisms of emphysema is the down-regulation of this enzyme by means of oxidative damage (29). Moreover, in a recent review of lung GSH and cigarette smoke-induced airway disease, increased GSH in the ELF of chronic smokers was presumed to be a protective adaptive mechanism against the development of chronic obstructive pulmonary disease (COPD) (30). Considering that not all chronic smokers go on to develop COPD, the authors in that review pointed out that genetic variations in the molecular mechanisms that regulate GSH metabolism might explain why some individuals are better protected against the development of COPD. It thus appears that emphysema patients are subjected to progressive tissue damage due, in part, to the consequences of GSH deficiency and/or genetic variations in GSH metabolism. Since GSH inhalation would presumably offer both antioxidant protection and GSH replenishment, this method of treatment would potentially benefit emphysema patients. Asthma is another condition where inhaled GSH cannot be recommended since this treatment caused notable side effects (e.g. breathlessness, bronchoconstriction and cough) in the cited study (21). These side effects were linked primarily to the production of sulfites that occurred when GSH was in solution. GSH inhalation should continue to be explored as a potential treatment for asthma. None of the asthma patients in the study had their urine tested for sulfites. A positive test for sulfites would have eliminated these patients from entering the study. Accordingly, the results might have been much more favorable if patients without sulfite sensitivities were included. This issue of asthma and sulfite sensitivities is an important one for clinicians to be mindful of. Sulfites are found in beer, wine, restaurant salad bars, seafood, potatoes, processed foods and many pharmaceuticals (31). Many asthma patients report being sensitive to sulfites. In an Australian study, ∼30% of asthmatic patients reported being sensitive to sulfites in wine (32). A more recent and rigorous scientific study, however, demonstrated that asthma patients can tolerate varying amounts of sulfites in wines ranging from 20, 75 or 150 parts per million (ppm) (33). Only a small minority of patients in this study (4 of 24 self-reported wine-sensitive asthmatics) exhibited reactions when challenged with 300 ppm of sulfites. One report indicated that 4–8% of asthmatics are sensitive to sulfites (34). Other reports have estimated the incidence of sulfite sensitivity to be around 5–11% (35,36). Even though the exact percentage of sulfite-sensitive asthmatics is difficult to ascertain, sulfite sensitivity is an important factor to assess when using or evaluating research done on inhaled GSH. Future Research Directions There are additional clinical conditions that might benefit from this type of treatment, but further studies are necessary. One such condition is Farmer's lung (FL), which is a hypersensitivity pneumonitis caused by the inhalation of thermophilic actinomycetes and spores of Aspergillus specie (11). A study was undertaken to investigate the effect of pulmonary GSH levels after hay exposure in patients with FL and in asymptomatic farmers (AF) (11). Fifteen symptomatic patients with FL [mean age, 42 ± 1 (SD) year] were compared with 10 AF [mean age, 43 ± 1 (SD) year] serving as the control group. All patients had baseline lung function testing and testing at various time intervals following hay exposures. The authors of this study concluded that FL and AF patients have characteristically different intrapulmonary levels of GSH, and that the pathogenesis of FL is likely related to GSH regulatory mechanisms. They also speculated that AF patients have a better ability to upregulate their pulmonary GSH levels, which would protect them against active disease. Clinical testing of inhaled GSH in patients with FL is warranted. The administration of GSH inhalation before and/or immediately following exercise is another potential application of this novel treatment. Exercise is a known inducer of oxidative stress leading to free radical production, which can encourage lipid peroxidation and tissue damage among individuals with deficient and/or impaired antioxidant systems. As stated in the beginning of this report, selenium is a cofactor in the GPX enzyme that protects cells from hydrogen peroxides and lipid hydroperoxides. When under situations of oxidative stress, the GPX enzyme will markedly increase in the lungs as an antioxidant adaptive response (37). By supplying more GSH to the lung tissues, more of this enzyme might be available to help reduce the production of free radicals associated with exercise. Although these assumptions are very speculative, it does seem possible and even logical that GSH inhalation would benefit those who regularly exercise by increasing exercise tolerance, and by maintaining and/or replenishing the antioxidant systems within the lungs. Multiple chemical sensitivity disorder (MCSD) is another condition that might be clinically responsive to this treatment. Patients with this disorder are known to have bronchial hyperreactivity and even exhibit asthma-like symptoms (38). Unlike asthma, MCSD is not associated with atopy and immunoglobulin E (IgE)-mediated allergic mechanisms (39). The prevailing theory explaining the cause of MCSD is a fusion between two separate theories—the neural sensitization and nitric oxide/peroxynitrite theories (40). This fusion theory, proposed by Pall, links long term potentiation of N-methyl-d-aspartate (NMDA) receptors at the synapses of nerve cells by glutamate and aspartate to an increased production of nitric oxide and its oxidant product, peroxynitrite (40,41). Treatment with antioxidants may improve symptoms of MCSD by reducing the peroxynitrite elevations and other biochemical dysfunctions that are associated with such elevations (40,41). Glutathione inhalation may be ideal since the primary route by which patients with MCSD get triggered is through smelling and breathing. Sulfite sensitivity would have to be considered since inhaled GSH could provoke adverse events. This treatment might be capable of providing antioxidant protection to both the upper and lower respiratory airways, which would theoretically help to reduce the symptoms of MCSD and the production of peroxynitrite. More research studies are necessary. Two final conditions, cigarette smoking and lung cancer, are worth mentioning since they are intimately related to each other and are affected by GSH and its related enzymes. These conditions are influenced by the glutathione S-transferase (GST) group of enzymes that are found in significant quantities in the bronchioles and alveoli of the lungs (42), and in very high concentrations in the bronchial epithelium (43). Among smokers, a lack of the GST mu enzyme was thought to be associated with a greater risk of lung cancer, especially if there was a cancer and/or lung cancer history among the relatives of the patients in this study (44). Since the GST mu enzyme detoxify carcinogens in tobacco, any deficiency of this enzyme was presumed to be associated with an increased risk of lung cancer. However, a more recent study pertaining to the GST group of enzymes found no such association (45). In this meta-analysis, polymorphisms in the GST genes had no associations or weakly positive associations with risk factors for lung cancer. Despite the need for more research, GSH inhalation might be beneficial for smokers to augment their GST enzymes, which would help facilitate the detoxification of carcinogens. Even though the best intervention for these patients would be smoking cessation, many patients lack the necessary willpower to quit. For these patients, regular GSH inhalation might reduce oxidants generated from cigarette smoke (∼1014 free radicals/puff) (46), and the epithelial lung injury associated with smoking (47). For lung cancer patients, the use of GSH inhalation is not recommended. Cancer cells use multiple mechanisms (e.g. altered transport of a drug, inhibition of drug-induced apoptosis and elevation of cellular GSH) to circumvent the cytotoxic effects of chemotherapeutic agents (48). Early research studies showed that GSH was able to reduce cytotoxicity to chemotherapeutic compounds by boosting the metabolism of drugs to less active compounds, or by the detoxification of free radicals (49,50). More recently, research has revealed that the levels of a specific GST enzyme increases among cancer cells with higher differentiation grades, and that these drug-resistant gene products are found in lung carcinomas at the time of surgical resection (51). There is also speculation that GSH might be capable of repairing drug-induced injury at the DNA level (48). A recent review article has described the involvement of glutathione in the detoxification or inactivation of platinum drugs—the most commonly employed drugs for the treatment of advanced stage lung cancer patients (52). Based on this information, it would be unwise and illogical to use GSH inhalation while lung cancer patients are undergoing active chemotherapy treatment. Mechanism of Action Inhalation of GSH results in a mechanism of action confined to the upper airways and lungs (Fig. 1), and will not influence plasma levels to a significant degree. In the studies that measured both lung and plasma levels of GSH, the plasma levels remained essentially unchanged following GSH inhalation. Seven of the studies included in this review demonstrated that GSH inhalation exerts its effects upon the lower respiratory tract (9, 10, 19, 24–27). The upper respiratory tract also appears to benefit from GSH augmentation. Two studies involving patients with upper respiratory tract diseases showed clinical benefits from GSH inhalation treatment (20,23). The predominant mechanism responsible for GSH's therapeutic effects are probably related to its antioxidant properties that offer protection against oxidative injury, and/or assist with the normalization of the oxidant–antioxidant balance within the upper and lower respiratory tract. Even though the majority of these studies suggested that antioxidant protection was the principal reason for the favorable treatment responses, some of the studies were unable to demonstrate a change in markers of oxidation from this treatment. More data is necessary to confirm the precise nature of GSH's antioxidant properties within the upper and lower respiratory tract. Additional explanations for GSH's therapeutic effects might include an improvement in host defenses (e.g. increased cytotoxic lymphocytes), and better oxygenation (e.g. an increase in oxygen saturation). GSH inhalation produced clinically meaningful results in the majority of diseases that were studied. Specifically, GSH inhalation was shown to improve clinical markers of respiratory function that inevitably impact upon quality of life and disease progression. These improvements were the most important outcomes and features of this novel treatment. Figure 1.Inhaled GSH’s mechanism of action. GSH, reduced glutathione; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity. Considerations Prior to Initiating GSH Inhalation The urine should be tested for sulfite sensitivity. A special test strip can be dipped in the urine, and is known as the ‘EM-Quant 10013 Sulfite Test.’ It can be easily located through any search engine on the Internet (53). Even though instructions for sulfite testing have been published elsewhere (54), a brief description of the procedure is outlined below: A random (fresh) urine sample is suitable, but a first morning void may be preferable due to its higher concentration. Once the test strip is dipped in the urine (for 1 s), the reaction zone changes color to indicate the concentration of sulfites present. After 30 s, the color on the test strip is compared to a color scale on the bottle indicating the concentrations of sulfites in the urine (can detect 10, 40, 80, 180 and 400 ppm of sulfites). The resultant concentration should be multiplied by a factor of 1.5 to provide the amount of free sulfites in mg/l (ppm). The strip will not detect below 10 ppm. The urine samples should be preservative free, and the urinary pH should also be tested with pH paper. If the urine pH is below 6, then the amount of sulfites might be underestimated by the test. In such cases, consider adding sodium acetate or sodium hydroxide to raise the pH to at least 7–10 (should not exceed a pH of 12), and then repeat with a new test strip. If the urine test were positive for sulfites (normally they are absent), the use of inhaled GSH would be strictly contraindicated. Method of Delivery, Recommended Daily Dosages and Side Effects With a nebulizer, a solution of GSH is made into an aerosol and is delivered to the upper respiratory tract and the lungs through a mask that covers the nose and mouth, or is delivered directly into the lungs via a mouthpiece. Any compounding pharmacist would be able to prepare the solution of GSH at the desired concentrations. The typical dosages used in the studies cited in Table 3 were 600 mg once daily, 600 mg twice daily, 900 mg daily, 1350 mg daily or a daily dose of 66 mg/kg of body weight. Better results are more likely to be achieved with doses of at least 600 mg or more each day. One of the studies used much larger doses (66 mg/kg of body weight) since the authors speculated that these would be necessary to replace half of the amount of GSH that is produced each day (e.g. a 150 lb male synthesizes 10 g daily and would need 5 g as a replacement dose) (27). When patients are unresponsive to doses in the range of 600–1350 mg per day, it might be suitable to try doses that would replace half the estimated amount of GSH that is synthesized each day. These gram doses might yield better clinical results. In terms of side effects, GSH inhalation is very safe. Minor side effects such as mild coughing and an unpleasant odor were reported in some of the studies included in this review. These minor side effects, better described as mild nuisance problems, were not severe enough to cause any of the study participants to discontinue treatment with inhaled GSH. The only worrisome or potentially life-threatening side effect to note is bronchoconstriction, which would be more likely to occur among sulfite-sensitive asthma and MCSD patients. However, if proper precautions such as sulfite testing are done prior to treatment, this serious side effect should be avoidable. Monitoring the Clinical Response to Inhaled GSH For pulmonary diseases or respiratory-related conditions, baseline pulmonary function testing with a spirometer or a simple peak flow meter is recommended prior to the first treatment. After a prescribed period of treatment time, pulmonary function tests should be repeated. This will help to establish if there are any clinical improvements from regular GSH inhalation. Conclusions GSH inhalation is an effective treatment for a variety of pulmonary diseases and respiratory-related conditions. Even very serious and difficult-to-treat diseases (e.g., CF, IPF) yielded benefits from this novel treatment. GSH inhalation is very safe, and rarely causes major or life-threatening side effects. The potential applications are numerous when one considers just how many pulmonary diseases and respiratory-related conditions are affected by deficient antioxidant status, poor oxygenation and/or impaired host defenses. More studies are clearly warranted.

Purinergic_Signal-4-1-2245999

Internalization and desensitization of adenosine receptors

Until now, more than 800 distinct G protein-coupled receptors (GPCRs) have been identified in the human genome. The four subtypes of the adenosine receptor (A1, A2A, A2B and A3 receptor) belong to this large family of GPCRs that represent the most widely targeted pharmacological protein class. Since adenosine receptors are widespread throughout the body and involved in a variety of physiological processes and diseases, there is great interest in understanding how the different subtypes are regulated, as a basis for designing therapeutic drugs that either avoid or make use of this regulation. The major GPCR regulatory pathway involves phosphorylation of activated receptors by G protein-coupled receptor kinases (GRKs), a process that is followed by binding of arrestin proteins. This prevents receptors from activating downstream heterotrimeric G protein pathways, but at the same time allows activation of arrestin-dependent signalling pathways. Upon agonist treatment, adenosine receptor subtypes are differently regulated. For instance, the A1Rs are not (readily) phosphorylated and internalize slowly, showing a typical half-life of several hours, whereas the A2AR and A2BR undergo much faster downregulation, usually shorter than 1 h. The A3R is subject to even faster downregulation, often a matter of minutes. The fast desensitization of the A3R after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor. This review describes the process of desensitization and internalization of the different adenosine subtypes in cell systems, tissues and in vivo studies. In addition, molecular mechanisms involved in adenosine receptor desensitization are discussed. Introduction Adenosine is an important neuromodulator involved in a variety of brain activities and it also serves many different functions in the periphery. This nucleoside, when extracellular, exerts its action via specific G protein-coupled receptors (GPCRs) of the P1 class, divided into four subtypes: A1R, A2AR, A2BR and A3R [1]. GPCRs consist of a single polypeptide, containing seven α-helices which are oriented perpendicular to the membrane. The N terminus is located at the extracellular side of the cell and often contains one or more glycosylation sites. The C terminus is located intracellularly and contains phosphorylation and palmitoylation sites, which are involved in regulation of receptor desensitization and internalization [2]. All adenosine receptors, with the exception of the A2AR, contain a palmitoylation site near the C terminus. The A2AR is the only subtype with an extraordinary long C terminus, 122 amino acids versus 36 amino acids in e.g. the A1R [3]. All the adenosine receptors are glycosylated on the second extracellular loop, although glycosylation does not appear to influence ligand binding. The third intracellular loop and/or the C terminus are involved in coupling the adenosine receptors to G proteins. Phosphorylation of in particular intracellular loop 3 is involved in desensitization and internalization of adenosine receptors [4–6]. Adenosine and analogues Adenosine, consisting of a purine ring connected to a ribose group, is the endogenous ligand for the adenosine receptors (Fig. 1). Under normal conditions, adenosine is continuously formed extracellularly by dephosphorylation of ATP, ADP and/or AMP to adenosine by NTPDases (ectonucleoside triphosphate diphosphohydrolases). However, the A3R can also be activated by inosine, a breakdown product from adenosine [5]. Most adenosine receptor agonists are analogues of adenosine, modified by N6, C2 and C8 substitutions at the adenine base, and C5′ modifications of the ribose moiety [5, 6]. Antagonists lack the ribose group and usually possess a mono-, bi- or tricyclic core structure, e.g. caffeïne, which contains a xanthine as basic structure (Fig. 1). For extended reviews on high affinity agonists and antagonists for adenosine receptors and their structure-activity relationships, see Palmer and Stiles [1], Fredholm et al. [5], Jacobson and Gao [7], Beukers et al. [8], Müller [9] and Klotz [10]. Fig. 1Chemical structures of the endogenous ligand adenosine and the antagonist caffeine Occurrence and physiological functions of adenosine receptors The adenosine receptors are widespread throughout the body and exert many different functions both in the CNS and in the periphery. The A1R is particularly prevalent in the central nervous system, with high levels in the cerebral cortex, hippocampus, cerebellum, thalamus, brain stem and spinal cord. Numerous peripheral tissues also express the A1R, including vas deferens, testis, white adipose tissue, stomach, spleen, pituitary, adrenal gland, heart, aorta, liver, eye and bladder. Low levels are found in the lung, kidney and small intestine [1, 5, 6]. The A1R is involved in cardiovascular effects (e.g. reducing heart rate), inhibition of lipolysis and stimulation of glucose uptake in white adipocytes and the modulation of neurotransmitter release in the CNS [1]. The A1R also plays a role in anxiety, hyperalgesia, bronchoconstriction and the glomerular filtration rate and renin release in the kidney [5, 6, 11]. In the CNS, the A2AR is highly expressed in the striatum and olfactory tubercle [1]. In the periphery, it is highly expressed in the spleen, thymus, leucocytes and blood platelets, and intermediate levels are found in the heart, lung and blood vessels [5, 6]. The A2AR is involved in the onset of vasodilation, inhibition of platelet aggregation, exploratory activity, aggressiveness and hypoalgesia [1]. In addition, A2AR plays a role in Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, ischaemia, attenuation of inflammation and neuroprotection, particularly in peripheral tissues [5, 6]. A2A receptor antagonists slow the neurodegeneration which occurs in Parkinson’s and Huntington’s disease and also prevent toxicity induced by beta-amyloid in the development of Alzheimer’s disease [12–14]. The A2BR is widely expressed in the brain, but generally at very low levels. In the periphery, high levels of A2BR were detected in the caecum, large intestine and urinary bladder. Lower levels were observed in the lung, spinal cord, vas deferens, pituitary, adipose tissue, adrenal gland, kidney, liver and ovaries [5, 6]. Since there is a lack of specific agonists for the A2BR, little is known about the functional significance of this receptor. However, the A2BR plays a role in mediating vasodilation in a.o. the aorta, the renal artery and the coronary artery of different species. It is also involved in allergic and inflammatory disorders [5, 6]. The A3R is expressed in the CNS, but at relatively low levels, and only the hypothalamus and the thalamus have been reported to contain A3R [15]. The highest levels of adenosine A3R have been found in the lung and liver, and somewhat lower levels were found in the aorta [1]. In addition, the A3R was found in eosinophils, mast cells, testis, kidney, placenta, heart, spleen, uterus, bladder, jejunum, aorta, proximal colon and eye, although with pronounced differences in expression level between species [5, 6, 16]. The A3R has been implicated in mediating allergic responses, airway inflammation and apoptotic events; however, the latter is dependent on the cell type involved and/or the type of activation [5, 16]. Furthermore, the A3R is involved in the control of the cell cycle and inhibition of tumour growth both in vitro and in vivo [6]. In fact, adenosine A3 receptors have been demonstrated to be more highly expressed in tumours than in healthy cells, suggesting a role for A3R as a tumour marker [17]. Signal transduction of adenosine receptors G protein-coupling and second messengers Heterotrimeric G proteins are guanine-nucleotide regulatory protein complexes composed of α and βγ subunits. They are responsible for transmitting signals from G protein-coupled receptors to effectors, e.g. adenylyl cyclase. Until now, 16 α, 5 β and 14 γ isoforms have been reported [18]. G proteins are divided into several subclasses with a specific activity profile: Gs proteins stimulate adenylyl cyclase, Gi proteins inhibit adenylyl cyclase and stimulate GIRK channels, G0 proteins stimulate K+ ion channels, Gq/11 proteins activate phospholipase C, G12 proteins activate Rho guanine-nucleotide exchange factors (GEFs) and the olfactory G protein, Golf, stimulates adenylyl cyclase. Upon receptor activation, both the α subunit and the βγ subunit can signal, but to different effectors [19–21]. For a recent review on G proteins, see Milligan and Kostenis [18]. The A1R is usually coupled to a pertussis toxin-sensitive protein, which mediates inhibition of adenylyl cyclase and regulates calcium and potassium channels [1, 3, 5, 6, 11]. Both the third intracellular loop and the C-terminal tail of the A1R are involved in coupling [5]. In addition, it has been reported that under certain conditions the A1R couples to Gs to stimulate adenylyl cyclase, or to Gq/11 to stimulate inositol phosphate production. Apparently, the specific activity state of the receptor or the nature of the agonist determine which G protein class is activated by the A1R [22, 23]. The A2AR in the periphery is coupled to cholera toxin-sensitive proteins, which increase adenylyl cyclase activity upon receptor activation. The A2AR in the striatum is presumably coupled to Golf [5, 6]. The third intracellular loop, but not the C terminus of the A2AR, is involved in coupling [5]. The A2BR is coupled to proteins leading to stimulation of adenylyl cyclase upon receptor activation [6]. There is quite some evidence that A2BR can activate phospholipase C as well, via Gq/11 proteins [5]. The A3R is coupled to pertussis toxin-sensitive proteins, which mediate inhibition of adenylyl cyclase. In addition, A3R can stimulate phospholipase C via Gq/11 proteins [5, 6]. For an extensive overview of adenosine receptor-G protein coupling, see Fredholm et al. [24]. Desensitization and internalization—general principles and players Mechanisms to dampen GPCR signalling exist at every level in the cell. In this section attention will be paid to the underlying principles of desensitization and internalization and the protein partners involved in these processes. Receptor localization Depending on the localization signal, GPCRs in the plasma membrane can be targeted to lipid rafts1 or caveolae2. Different regions of GPCRs can influence not only the targeting to either lipid rafts or caveolae but may also enable an interaction of the receptor with constituents of these rafts and caveolae. For instance the extracellular part of the receptor might interact with GM1 gangliosides (glycosphingolipids) present in lipid rafts/caveolae. In addition, the C-terminal fatty acid acylation or palmitoylation may also affect targeting of GPCRs to either lipid rafts or caveolae. Finally, transmembrane regions may interact with cholesterol in the lipid rafts resulting in a change in conformation of the α-helices. Since the conformation of α-helices depends on the activation state of GPCRs, it may well be that agonist binding to the receptor may affect its localization in lipid rafts by means of molecular transitions leading to receptor activation [25, 26]. Desensitization Desensitization reduces receptor activity and plays a role in signal duration, intensity and quality. Desensitization is initiated by phosphorylation of serine and/or threonine residues in the third intracellular loop and C terminus of the receptor. Two types of desensitization occur, heterologous and homologous desensitization, and both are the result of receptor phosphorylation. Heterologous desensitization is induced by phosphorylation of the receptor by protein kinase A or C—sometimes even without agonist occupancy. On the other hand, homologous desensitization is specific for agonist-occupied receptors and consists in most cases of two steps. First, the receptor is phosphorylated by one of the G protein-coupled receptor kinases (GRKs 1-7); then it binds to β-arrestin, of which two subtypes exist, which exhibit high affinity for agonist-occupied, phosphorylated receptors. β-Arrestin serves to sterically inhibit G protein coupling, thereby terminating the G protein activation, and may also target the receptor to clathrin-coated pits3 for internalization (Fig. 2) [19, 25, 27, 28]. Fig. 2Homologous (a) and heterologous (b) desensitization and subsequent internalization of GPCRs. subunit, subunit, L ligand, GRK G protein-coupled receptor kinase, P phosphorylated amino acids, β-arr β-arrestin, AP2 adaptin, E effector, second messenger, PKA protein kinase A, PKC protein kinase C Internalization Receptor desensitization, initiated by phosphorylation of the receptor by different protein kinases (A or C) or GRKs, can be subsequently followed by receptor internalization. Upon phosphorylation, β-arrestin 1 or 2 is attracted to the receptor [31]. β-Arrestins not only interact with the phosphorylated receptor, but also bind to the heavy chain of clathrin, to the β2-adaptin subunit of the clathrin adaptor protein AP2, and to phosphoinositides. These interactions direct the phosphorylated receptor to punctate clathrin-coated pits in the cell membrane, which are internalized by action of the GTPase dynamin. Upon internalization, receptors can either be rapidly recycled to the plasma membrane, targeted to larger endosomes and slowly recycled, or degraded in lysosomes. The final destination of the internalized receptors largely depends on the β-arrestin subtype (1 or 2) that is recruited by the receptor upon phosphorylation and the duration of β-arrestin binding. In this way, internalization may regulate receptor resensitization and contributes to a positive regulation of receptor signalling [19, 25, 31]. Internalization pathways From internalization studies with several receptors, it appears that the internalization pathway is specific for receptor type, cell type, metabolic state of the cell, cell-specific factors etc. Receptor trafficking can be regulated in different ways (Fig. 3): (a) the receptor resides mainly in lipid rafts/caveolae and enters the cell via this pathway by default; (b) the receptor is in lipid rafts, but leaves these upon agonist binding to be internalized via clathrin-coated pits; (c) the receptor moves into lipid rafts upon agonist binding and is internalized via this pathway; (d) the receptor moves into lipid rafts after agonist binding to activate certain signalling events, but is eventually moved out of the lipid rafts to be internalized via clathrin-coated pits. Internalization can even be achieved via uncoated vesicles or by a combination of two or more of the aforementioned pathways. For example, β1-AR is internalized via both lipid rafts and clathrin-coated pits. PKA phosphorylation directs β1-AR to a clathrin-coated pit, whereas GRK phosphorylation directs the receptor to lipid raft-mediated internalization [19, 25, 26]. Fig. 3Different internalization pathways, adapted from Chini and Parenti, 2004 [25]. Internalization via (a) lipid rafts/caveolae, (b) upon agonist binding, the receptor moves to clathrin-coated pits to be internalized, (c) the receptor moves into lipid rafts upon agonist binding and is internalized, (d) the receptor moves into lipid rafts upon agonist binding to activate certain signalling events, but is eventually moved out to be internalized via clathrin-coated pits. Ligand (L, green triangle), clathrin-coated pits (dotted blue lines) and lipid rafts (solid pink lines) are indicated Function of lipid rafts/caveolae The existence of lipid rafts/caveolae serves different functions. First of all, lipid rafts act as ‘stations’ in which GPCRs accomplish specific signalling tasks by meeting a selected set of signalling molecules, e.g. G proteins and adenylyl cyclases. Another possible function of lipid rafts is the protection of receptors from rapid constitutive or agonist-induced internalization, thus allowing their coupling to specific signalling pathways. In addition, caveolin may regulate the constitutive activity of receptors [32]. Finally, the endocytic pathways that GPCRs choose may depend on cell-specific factors. Switching the internalization pathway from lipid rafts/caveolae to clathrin-coated pits may alter the final receptor destination [19, 25, 26]. Research on adenosine receptors probably provided the first account of receptor internalization via caveolae and lipid rafts, as an alternative to the well-described β-arrestin pathway [33]. G protein-coupled receptor kinases (GRKs) Upon agonist binding to GPCRs, not only G proteins are recruited to the activated receptor conformation, but also two other protein families, namely the G protein-coupled receptor kinases (GRKs) and the β-arrestins [31]. The GRK family consists of seven different genes. The GRKs have been divided into three protein subfamilies, based on sequence similarity. GRK1 and GRK7 belong together. The second subfamily consists of GRK2 and GRK3 since their membrane recruitment depends on interaction with Gβγ subunits and phosphatidylinositol 4,5-bisphosphate. GRK2 and GRK3 are primarily responsible for agonist-dependent receptor phosphorylation, β-arrestin recruitment and functional uncoupling of the receptor. GRK4, GRK5 and GRK6 form the third subfamily and are constitutively associated with the membrane. GRK1 and GRK7 are expressed mainly in the retinal rods and cones, whereas GRK4 expression is limited to the cerebellum, testis and kidney. In contrast, GRK2, GRK3, GRK5 and GRK6 are widely expressed in mammalian tissues [31]. β-Arrestins β-Arrestins belong to the arrestin family of which four members have been identified. Arrestin 1 and arrestin 4 are expressed in the visual system in retinal photoreceptor cells; arrestin 2 (also called β-arrestin 1) and arrestin 3 (also called β-arrestin 2) are more widely expressed and are involved in the regulation of nonvisual GPCRs. The β-arrestins were originally discovered as molecules that bind to and desensitize the activated and phosphorylated form of G protein-coupled receptors as described before [19, 25, 27, 28, 31]. The β-arrestin protein consists of an N and a C domain, which are almost entirely composed of antiparallel β sheets, connected by a linker of 12 amino acids. A ‘polar core’ is embedded between those two domains, and its disruption by the phosphorylated C terminus of the activated receptor leads to conformational changes in the β-arrestin. The C tail of the β-arrestin is then released, exposing both the clathrin and the AP2 binding domains. Recently, it appeared that β-arrestins not only are involved in the desensitization of G protein-coupled receptors, but also act as signal transducers on their own. As a consequence, they emerge as multifunctional adaptor/scaffold proteins which mediate cellular processes such as chemotaxis, apoptosis and metastasis besides receptor signalling and trafficking. For an extensive and recent review on the interactions of β-arrestins with other cellular proteins, see Lefkowitz et al. [34]. GRKs and β-arrestins orchestrate GPCR activities at three different levels: (1) silencing: the functional uncoupling of the receptor from its G protein by a mechanism known as ‘homologous desensitization’, (2) trafficking, which involves receptor internalization, resensitization and/or degradation, and (3) cross-signalling: activation or inhibition of intracellular signalling pathways, independent of heterotrimeric G proteins [31]. Dampening adenosine receptor signals In this section we will discuss the evidence for and mechanisms of desensitization and internalization of the four adenosine receptor subtypes. For each receptor we will first summarize results from in vitro, ex vivo and in vivo studies. This will be followed by a more biochemical approach in which we will focus on the molecular mechanisms of adenosine receptor desensitization and internalization by paying attention to the role of accessory proteins, the influence of receptor mutations, etc. We will refer to earlier seminal publications, but mostly focus on more recent evidence for adenosine receptor desensitization and internalization. A1 receptor Cellular and physiological studies The early evidence for adenosine A1 receptor desensitization was largely obtained from primary cells, cell lines, tissues or tissue slices, and intact animals that were exposed to varying concentrations of adenosine receptor agonists (either A1-selective or not), often examined over several time periods. Stiles and co-workers were among the first to study A1 receptor desensitization in detail (1991). Ramkumar et al. pretreated DDT1 MF-2 cells, a smooth muscle cell line expressing both A1 and A2A receptors, with R-phenylisopropyl adenosine (R-PIA) for up to 24 h, after which the adenylyl cyclase activity was reduced by approximately 50%. This was associated with a significant decrease in cell membrane-bound A1 receptors and a concomitant increase of receptor number in intracellular compartments. The authors also showed an increase in receptor phosphorylation, nicely paralleling the time course of adenylyl cyclase modulation [35]. In a later study, Nie et al. reported similar findings in the same cell line, although desensitization occurred at a somewhat faster pace (4 h) [36]. Interestingly, Palmer et al. were unable to demonstrate desensitization in CHO cells expressing the human A1R [37]. Klaasse et al., however, were able to show internalization of the human A1R tagged with a C-terminal yellow fluorescent protein (hA1YFP-R), stably expressed in CHO cells. Exposure of these cells for 16 h to 400 nM or 4 μM CPA resulted in 25 and 40% receptor internalization, respectively. Addition of 10 μM of the allosteric enhancer PD81,723 did not accelerate the internalization process, but lowered the threshold concentration at which internalization occurred. Under those conditions a small degree of internalization was observed already at a concentration of 40 nM CPA, and at 400 nM CPA, 59% of the receptors internalized [38]. Exposure of cerebellar granule cells, endogenously expressing A1R, to 100 nM R-PIA for 2–48 h led to a blunting of the inhibition of adenylyl cyclase. Along with this observation, a decrease of [3H]cyclohexyladenosine ([3H]CHA) binding to intact cerebellar granule cells and an increase of [3H]CHA binding in microsomes was detected. Simultaneously, a decrease in the steady-state level of in plasma membrane and microsomes was observed. These findings point not only to homologous desensitization, but also to subsequent internalization of the A1R in the microsomal fraction of cerebellar granule cells upon long-term agonist exposure. However, no change in mRNA level was observed, suggesting that post-transcriptional regulation underlies receptor desensitization [39]. In hippocampal neurons, A1Rs are present on both presynaptic and postsynaptic terminals, as well as on the cell body and dendrites where they exert different actions. To address the question of whether desensitization was influenced by subcellular localization, neurons were exposed to the agonist 2-chloroadenosine (CADO, 20 μM) from 2 up to 96 h. It was found that upon agonist exposure in cultured hippocampal neurons, the presynaptic A1Rs desensitize less quickly (>12 h) than the postsynaptic A1Rs (2 h). In accordance, the recovery of desensitized presynaptic A1Rs also requires more time (48 h vs 8 h for postsynaptic A1R). Desensitization of the postsynaptic A1Rs apparently occurs at the level of the receptor, because the other elements of the signal transduction machinery appeared to be fully functional upon receptor desensitization. All in all, these results suggest that the extent and the kinetics of agonist-induced desensitization of A1Rs depend on the subcellular localization of the receptors [40]. Using a more intact preparation, Coelho et al. found that the density of A1R in rat hippocampal slices was decreased by 30% upon 60 min of imposed hypoxia. This desensitization could be mimicked by adding the A1R agonist CADO (10 μM) for 60 min and was prevented by adding the A1R antagonist DPCPX. These results suggest that hypoxia leads to an increase in extracellular adenosine levels, and a subsequent, quite rapid (<90 min) desensitization, possibly followed by subsequent internalization of the A1R in nerve terminals [41]. Rat in vivo studies also demonstrated A1R desensitization. In a study by Parsons and Stiles rats were chronically (6 days) infused with R-PIA. Examination of adipocyte membranes of both treated and control rats revealed a 40% lower inhibition of adenylyl cyclase in the treated animals, coinciding with a reduced number of A1Rs (68% agonist-occupied receptors remaining) as determined by radioligand binding [42]. Upon further study it was noticed that the effects on adenylyl cyclase were not A1R specific but were controlled at the level of adipocyte G proteins. Gi levels appeared downregulated, whereas the amount of Gs in the preparation was increased, although not at the level of their mRNA, suggesting a heterologous form of desensitization [43]. Chronic exposure of rats to R-PIA (6 days) also led to A1R desensitization in the brain, and subsequent reduced inhibition of adenylyl cyclase. This loss of response was accompanied by a significant decrease in both total numbers of A1R and proteins in synaptic plasma membranes in the brain, paralleling the finding by Stiles and co-workers in adipocytes [44]. As a consequence, a significant increase of A1R was observed in microsomes and coated vesicles, which suggested a role for coated vesicles in the internalization of A1R. Similarly, chronic agonist exposure of rats to NECA (6 days) resulted in a significant decrease of A1R in the high-affinity state in the rat brain, however without changes in adenylyl cyclase activity or inhibition of the proteins [45]. Molecular mechanisms As discussed in the general introduction to this review, receptor posttranslational modifications, receptor phosphorylation, recruitment of arrestins and the formation of clathrin-coated pits are elements of the molecular machinery of desensitization and internalization. In addition, other potential protein partners in the two processes have been studied for the various adenosine receptor subtypes, which will also be discussed. Effect of receptor posttranslational modifications on desensitization and/or internalization Gao et al. studied the effects of preventing palmitoylation of the A1R. It appeared that the Cys309Ala mutation, thus removing the palmitoylation site of the human A1R, had no effect on internalization [46]. These findings were later confirmed by Ferguson et al. [47]. GRKs and arrestins A1R phosphorylation by GRKs has been subject to debate. Palmer et al. and Ferguson et al. failed to demonstrate GRK-2 phosphorylation of A1R; in the latter study it was found that nonphosphorylated A1R redistributes arrestin 3 from the cytoplasm into punctate clusters at the plasma membrane [37, 47]. Nie et al., however, reported that within 1 h of exposure of DDT1MF-2 cells to R-PIA, rapid translocation of GRKs was observed from the cytosol to the cytoplasm [36]. In a further biochemical approach with both purified receptor and GRK-2, a phosphorylated receptor was obtained that showed enhanced affinity for arrestins over G proteins. The same receptor kinase, upon overexpression in FRTL-5 cells, influenced A1R signalling, however, not via -mediated adenylyl cyclase but through -mediated MAP kinase activation [48]. It may be concluded that under physiological conditions A1R phosphorylation does not (readily) take place, which would be a rationale for the long time periods required for receptor internalization. Other protein partners The ectoenzyme adenosine deaminase (ADA) regulates the extracellular adenosine concentration by converting excess adenosine into inosine. However, ADA also plays a role in the desensitization and internalization of A1R in smooth muscle DDT1MF-2 cells [49, 50] and LLC-PK1 epithelial cells [51], acting as a receptor activity-modifying protein (RAMP). Upon agonist exposure (100 nM R-PIA, 2 h), ADA and A1R formed complexes on the cell surface, clustered and internalized together to intracellular compartments. Such clustering of adenosine A1 receptors prior to internalization was also reported by Ciruela et al. and Saura et al. [49, 52]. The intracellular vesicles contained the lipid raft marker protein caveolin. Filipin, an agent that disrupts rafts or caveolae, inhibited A1R internalization. In contrast, acidic treatment disrupting clathrin-coated vesicles did not inhibit agonist-induced internalization of A1R. These results indicate that ADA and A1R form a stable complex in the cell membrane of LLC-PK1 cells, internalizing upon agonist exposure via lipid rafts, in a clathrin-independent pathway. Furthermore, a direct interaction of the C terminus of A1R with caveolin was demonstrated [50]. These and other data suggest that the mode of receptor compartmentalization in response to agonist stimulation may be governed by both receptor subtype and cell type [51]. Another accessory protein, the heat shock cognate protein 73 (hsc73), a member of the hsp70 family, was identified as a cytosolic component able to interact with the third intracellular loop of the A1R. The interaction between hsc73 (30 nM) and A1R led to a marked reduction in affinity of ligands for the A1R and also prevented activation of the G proteins, even more so than the addition of GTP analogues or GTP itself (100 μM). These effects were completely prevented by the addition of 25 nM ADA. A high percentage of A1R was coupled to hsc73 in cell lysate, according to immunoprecipitation experiments. A remarkable feature upon internalization of the receptor in DDT1MF-2 cells was found; A1Rs internalized via two different vesicle types, one in which A1R and hsc73 are colocalized and another in which hsc73 was absent. These observations open the possibility that signalling via GPCRs is regulated at least to some extent by heat shock proteins [53]. Receptor-receptor interactions Dunwiddie et al. found that activation of A3R with a selective A3R agonist resulted in subsequent heterologous desensitization of the A1R, as observed in electrophysiological experiments in the CA1 region of rat hippocampus. Similar results were obtained after A3R occupancy via a brief superfusion with a high concentration of adenosine [54]. Lopes et al. investigated how activated A2AR influenced A1R function and whether this interaction was modified in aged rats. In hippocampal and cortical nerve terminals, the A2AR agonist CGS 21680 (30 nM) was able to lower the binding affinity of the A1R-selective agonist CPA, and this was taken as proof for A1R desensitization. The effect was counteracted by the addition of the A2AR antagonist ZM 241385 (20 nM). This reduction in A1R function could only be detected in young adult rats (6 weeks), but not in old rats (24 months). The addition of a PKC inhibitor, chelerythrine (6 μM), also prevented the desensitization of A1R [55]. Similarly, Ciruela et al. [56] demonstrated that A1R-A2AR heteromers were able to modulate the glutamatergic neurotransmission in the rat striatum. The main biochemical characteristic of this heteromer is the ability of the agonist-occupied A2AR to reduce the agonist affinity of the A1R [56]. By binding to cannabinoid CB1 receptors in Purkinje fibers in the cerebellum, Δ9-tetrahydrocannabinol inhibits adenylyl cyclase and consequently motor coordination. Long-term Δ9-tetrahydrocannabinol treatment resulted in CB1R downregulation, desensitization of the protein and desensitization of adenylyl cyclase. G protein activation by A1R, however, was unaffected. Surprisingly, heterologous attenuation of A1R-mediated inhibition of adenylyl cyclase was observed. These results indicate that long-term Δ9-tetrahydrocannabinol administration produces a disruption of inhibitory receptor control of cerebellar adenylyl cyclase and suggest a potential mechanism of cross-tolerance to the motor effects of cannabinoid and A1 agonists [57]. D1R and A1R have been shown to form functionally interacting heteromeric complexes in engineered cell lines and in cortical neurons in culture. Pretreatment with an A1R agonist caused complex formation of both receptors. Combined pretreatment with selective agonists for both receptors (but not one agonist alone) substantially reduced cAMP accumulation induced via the D1R, indicative for this receptor’s desensitization [58]. A2A receptor Cellular and physiological studies DDT1 MF-2 cells, expressing both A1R and A2AR, were exposed to an A2A-selective agonist, which resulted in a rapid loss (t1/2 = 45 min) of agonist-stimulated cAMP production in these cells. This receptor desensitization, however, did not involve a reduction in cell membrane receptor number or a change in ligand affinity [35]. Prolonged exposure of PC12 cells, expressing both A2AR and A2BR, to AR agonists led to a fast (30 min) and significant inhibition of A2AR stimulation by the A2AR-selective agonist CGS21680. This effect appeared to occur at the level of adenylyl cyclase, since no change was observed in receptor number or in CGS21680’s affinity for the receptor. This conclusion was corroborated by the finding of reduced activation of adenylyl cyclase by forskolin. Longer agonist exposure (12–20 h) led to a reduction of levels, whereas no changes occurred in the short-term protocol [59]. NG108-15 neuroblastoma x glioma hybrid cells express both A2AR and A2BR [60, 61]. Treatment of the cells with the non-selective agonist NECA followed by its washout led to a rapid (t1/2 = 20 min) and pronounced reduction in cAMP production by the A2AR-selective agonist CGS21680 when compared to vehicle-treated cells. Palmer et al. [62] generated CHO cells solely expressing the recombinant (canine) A2AR and examined the desensitization process of this receptor. Cells exposed to NECA showed a rapid desensitization of A2AR-stimulated adenylyl cyclase activity with no obvious difference between pretreatment of 30 min or 24 h. This was associated with a slightly reduced affinity of the receptor for the A2AR-selective radiolabelled agonist [3H]CGS21680. Cell surface receptor numbers only diminished significantly (up to 40%) upon longer-term pretreatment (t1/2 = 8 h) [62]. Using a tissue preparation, Conti et al. investigated whether prolonged exposure of A2AR to the non-selective agonist NECA, or to the selective A2AR agonists CGS21680 and 2HE-NECA, influenced A2AR desensitization. The authors used the porcine coronary artery as a sensitive vascular model, expressing among others A2ARs. The arteries were first precontracted by adding 3 μM PGFα. NECA, CGS21680 and 2HE-NECA showed high affinities for the A2AR (EC50’s of 72, 40 and 20 nM, respectively) inducing vasorelaxation. Next, coronary arteries were pretreated with 10 μM NECA for 30 min or 2 h. After a 2-h washout period, the functional response was assessed. It appeared that preincubation with NECA did not hamper the vasorelaxing effects of CGS21680 and 2HE-NECA. However, NECA response curves were shifted to the right after NECA pretreatment [63]. These results seem inconclusive, since NECA pretreatment might ‘hit’ other adenosine receptor subtypes. A2ARs, next to A1R, are present on presynaptic baroreceptor afferent terminals within the nucleus tractus solitarius (NTS) in the brain [64]. It was observed that these A2AR modulate 5-HT release as a mechanism of baroreflex control mediated by the NTS. Low concentrations of the non-selective agonist NECA (0.3–3 nM), briefly exposed (5 min) to NTS brain slices, induced the release of 5-HT caused by the activation of the A2AR present at presynaptic nerves. This effect could be blocked by the addition of the adenosine A2AR antagonist 8-(3-chlorostyryl)caffeine (CSC; 100 nM). Longer exposure of NECA (20 min) to the NTS slices resulted in inhibition of the 5-HT release, probably caused by quick desensitization of the A2AR (15 min) and subsequent involvement of the A1R [65]. These findings were corroborated in a similar study with CGS21680, an A2AR-selective agonist [66]. The effects on AR fate upon chronic agonist exposure were studied in vivo in rat brain [45]. After 6 days of NECA treatment, the adenylyl cyclase activity in synaptic plasma membranes was decreased, suggesting a desensitization of A2Rs, although the authors did not specify or study which A2R subtype was involved. Gs protein levels were decreased indicating Gs downregulation as the mechanism of desensitization. Interestingly, Rekik and Mustafa [67] showed that chronic antagonist treatment (3 days) of porcine coronary arteries with ZM241,385 led to a decreased agonist responsiveness. Although A2A receptor expression went up, it appeared that the levels of Gs had decreased, altogether leading to a functional desensitization of the relaxing response by e.g. CGS21680 [67]. It should be kept in mind that ZM241,385 is also a potent antagonist for the adenosine A2B receptor. Molecular mechanisms Effect of receptor C terminus on desensitization and/or internalization To investigate the importance of the (120 amino acid residues) long C terminus of the A2AR receptor in inducing desensitization and internalization, Palmer and Stiles introduced several mutations and deletions into the receptor tail. It appeared that deletion of the last 95 amino acids of the C terminus, containing 10 possible phosphorylation sites, did not have any effect on radioligand binding, adenylyl cyclase activity or desensitization kinetics compared to the wild-type A2AR. However, when two possible phosphorylation sites (Thr 298 and Ser 305) just upstream the 95 deleted amino acids were mutated to Ala, short-term (30 min) agonist-induced desensitization was attenuated, while the long-term (24 h) desensitization was not affected. Single mutations revealed that mutation of Thr 298 alone was sufficient to reduce receptor phosphorylation and agonist-induced short-term desensitization. This study also shows that short-term and long-term desensitization have distinct structural requirements and do not occur via the same mechanism [4]. GRKs and arrestins Which GRK isoforms are involved in the phosphorylation of the A2AR is not entirely clear yet. However, a putative role for GRK2 and/or GRK5 has been suggested [4]. The role of GRK2 in agonist-induced phosphorylation and subsequent desensitization of A2AR and A2BR was thoroughly investigated by Kelly and co-workers. Wild-type GRK2 was stably expressed in NG108-15 cells, which endogenously express A2AR and A2BR. The acute stimulation of adenylyl cyclase by activation of A2AR was markedly reduced in NG108-15 cells overexpressing wild-type GRK2. This was probably caused by GRK2-dependent pre-desensitization of the A2AR by extracellular adenosine. This effect could be reversed by pretreating the cells 24 h with 0.5 unit/ml ADA [68]. The same research group investigated the effect of a dominant-negative GRK2 mutant on the desensitization of the A2AR [60]. Stable transfection of a GRK2Lys220Arg mutant in NG108-15 cells reduced desensitization of the A2AR by 50% following a 30-min treatment with the adenosine agonist NECA. Tumour necrosis factor (TNF)-α treatment of human monocytoid THP-1 cells expressing the A2AR prevented desensitization of this receptor, occurring under control conditions upon pretreatment with CGS21680 or NECA [69]. It was discovered that the TNF-α treatment prevented GRK2 translocation to and decreased GRK2 association with the plasma membranes of these cells as a consequence of the activation of a sphingomyelinase-dependent pathway. In another study, Mundell and Kelly investigated the effect of inhibitors of receptor internalization on desensitization and resensitization of ARs in NG108-15 cells [70]. Before agonist exposure, cells were pretreated with hypertonic sucrose or concanavalin A (ConA), both inhibitors of internalization. This pretreatment did not affect the agonist-induced desensitization of the A2AR. However, the resensitization of the A2AR upon agonist removal was abolished in the presence of ConA or sucrose. Arrestins 2 and 3 have been implicated in the downstream desensitization process. CGS21680 stimulation of a tagged A2AR transiently transfected in HEK293 cells induced the translocation of GFP-tagged arrestins 2 and 3 towards the plasma membrane. A dominant-negative arrestin 2 mutant inhibited agonist-induced internalization [71]. Other protein partners The long C terminus of the A2AR has been coined a ‘coincidence detector’ as it recognizes quite a number of other proteins such as α-actinin and ARNO, the Arf nucleotide site opener (for a review see Gsandtner and Freissmuth [72]). α-Actinin may play a role in receptor internalization, not unlikely for a protein involved in cytoskeletal arrangements [71], however ARNO does not [73]. Receptor-receptor interactions The A2AR colocalizes with D2 dopamine receptors in the basal ganglia, and their interaction has been documented on several occasions [74]. For instance, the protein-protein interaction between A2AR-D2R was confirmed in HEK293T cells. The most likely mode of interaction is that helix 5 and/or helix 6 and the N-terminal portion of I3 in the D2R approach helix 4 and the C terminus of the A2AR [75]. Within the scope of this review fits the observation that activation of the D2 receptor actually sensitizes A2AR-mediated increases in cAMP production, in CHO cells expressing both receptors [76] as well as in CAD and NS20Y neuroblastoma cells in which the D2 receptor was expressed [77]. In SH-SY5Y neuroblastoma cells co-stimulation of A2AR and D2R accelerated D2R desensitization, probably by causing or enhancing D2R internalization [78]. Recently it was found that in these SH-SY5Y neuroblastoma cells the A2AR also formed heteromers with the endogenously expressed CB1R. The CB1R is negatively coupled to adenylyl cyclase and requires previous or simultaneous activation of A2AR to signal in these cells [79]. A2B receptor Cellular and physiological studies The adenosine A2B receptor (A2BR) is endogenously expressed on most artificial cell lines, such as COS and HEK293 cells. The exception is formed by CHO cells that lack this adenosine receptor subtype. The A2BR, like the other three adenosine receptor subtypes, is subject to agonist-induced desensitization. This was measured on the level of cAMP production by Peters et al. [80]. Pretreatment of COS7 cells endogenously expressing A2B receptors with the non-selective agonist NECA (1 μM) for 1 up to 17 h led to a significant reduction in cAMP production upon acute agonist challenge, already after 1 h of pretreatment. Maximal reduction of cAMP production was reached after 4 h of pretreatment. CHO cells stably expressing a 5′ FLAG epitope-tagged A2B receptor showed the same result, albeit that maximal reduction of cAMP response was already achieved after 1 h of pretreatment. Also mouse 3T3-L1 cells and human HEK293 cells, endogenously expressing the A2BR, showed a decreased cAMP response after 2 h and 5.5 h of pretreatment with 1 μM NECA, respectively [80]. In NG108-15 cells, expressing both A2AR and A2BR, the rate of desensitization for both receptor subtypes appeared similar, with a half-life of 15–20 min. Pretreatment with NECA of these cells (0.1 mM, 30 min) reduced stimulation of adenylyl cyclase by A2BR by approximately 50% [60, 61]. Rat pheochromocytoma PC12 cells also endogenously express both A2AR and A2BR. Prolonged exposure (14 h) to 100 nM NECA or 1 μM CGS21680 significantly inhibited the cAMP response of the cells to subsequent stimulation with the A2A-selective agonist CGS21680. Although a 100-fold higher NECA concentration is needed to stimulate the A2BR compared to A2AR, Chern et al. found that the A2B receptor cAMP response was also desensitized upon desensitization of the A2A receptor. This observation can be explained by the fact that the protein level and the adenylyl cyclase activity were diminished upon long-term desensitization of the A2AR by CGS21680 [59]. The domain in which the receptor resides is also important for desensitization events, according to Sitaraman et al. NECA (10 μM) added to either the apical or basolateral side of T84 intestinal cells resulted in desensitization of the A2BR on the corresponding side within 2–3 h. However, whereas applying NECA to the apical side of the membrane had no effect on the basolateral A2BR, basolateral NECA induced a complete desensitization of the apical receptor. Since receptor trafficking may play a role in this cross-desensitization process, this effect may contribute to the desensitization and subsequent downregulation of the A2BR [81]. Trincavelli et al. observed a very rapid desensitization (t1/2 = 5 min) of A2BR in a human astrocytoma cell line. Both G protein coupling efficiency and cAMP production were diminished after pretreatment of the cells with NECA in the presence of an A2AR-selective antagonist [82]. Mundell et al. reported that acute exposure of human airway smooth muscle (ASM) cells to adenosine receptor agonists resulted in a rapid accumulation of cAMP, most probably via A2BR. Treatment with adenosine deaminase (ADA) suggested that ASM cells produce adenosine which feeds back on the cells’ A2BRs, thereby regulating basal cAMP levels and inducing a small degree of A2BR desensitization. Chronic treatment with adenosine agonists had a dual effect; both A2BR desensitization and adenylyl cyclase sensitization (an increased responsiveness of adenylyl cyclase upon stimulation) were observed [83]. Haynes et al. [84] studied A2BR desensitization in both pulmonary artery smooth muscle cells and a more integrated preparation, the isolated perfused lung. Pretreatment of the smooth muscle cells with NECA for 45 min abrogated the increase in cAMP response otherwise observed for both NECA and isoproterenol, suggesting heterologous desensitization. Indeed, experiments with cholera toxin showed that the desensitization took place at the level of the Gs protein-adenylyl cyclase complex. In the lung preparation NECA, when dosed acutely, caused a rapid vasodilation, which was not observed when the tissue was treated again with the same compound 45 min after the first dose [84]. Taken together, these results suggest that desensitization of the A2B receptor is a robust phenomenon, rather independent from the cell type or tissue in which it is expressed. Molecular mechanisms Effects of receptor mutations on desensitization and/or internalization Matharu et al. studied the regions of the A2BR which are responsible for the desensitization and internalization of the A2BR [85]. For this purpose they introduced point mutations or deletions in the C terminus of the A2BR. Deleting the final two amino acids (Leu330-stop) did not affect the internalization properties of the A2BR. The Phe328-stop and Gln325-stop mutants, however, were resistant to agonist-induced desensitization and internalization. GFP-tagged arrestin 2 did not translocate from the cytosol to the plasma membrane upon agonist stimulation of these two truncated receptors. From a single point mutation in the C terminus, Ser329Gly, it became clear that this serine residue is responsible for the rapid agonist-induced desensitization and internalization. Surprisingly, a further deletion mutant Ser326-stop was able to undergo rapid agonist-induced desensitization and internalization; however, arrestin 2 was not attracted to the plasma membrane upon agonist stimulation. It appeared that internalization of this truncated receptor occurred via an arrestin- and clathrin-independent pathway, probably via caveolin-mediated internalization since the internalization of Ser326-stop was dynamin dependent. The destination of this truncated receptor was also different: a compartment close to the plasma membrane, instead of early endosomes. Whether the receptor will undergo rapid agonist-induced desensitization and internalization or influence the rate, extent and mechanism of internalization depends on specific sites in the C terminus of the A2BR and can be influenced by their mutation or deletion. GRKs and arrestins The A2BR, endogenously expressed in NG108-15 cells, is desensitized through phosphorylation by GRK2. This GRK2 effect was selective for A2AR and A2BR, since the other endogenously expressed receptors (secretin R and IP-prostanoid R) were not desensitized in this way [68]. In addition, stable transfection of a dominant negative GRK2(Lys220Arg) mutant in the NG108-15 cells reduced the NECA-induced (30 min, 100 μM) desensitization of the A2BR by 50% [60]. Peters et al. studied the role of phosphorylation by PKA and PKC rather than GRK2 in agonist-induced desensitization in COS-7 cells transiently expressing the 5′-FLAG A2BR. They concluded that these second messenger-dependent kinases are not involved in A2BR phosphorylation [80]. A similar conclusion for A2BR phosphorylation in NG108-15 cells was reached by Mundell and Kelly [61]. Trincavelli et al. found that treatment of astrocytoma cells with TNF-α increased the A2BR functional response, without changing receptor protein or mRNA levels. TNF-α treatment appeared to markedly reduce agonist-dependent receptor phosphorylation and also attenuated agonist-mediated A2BR desensitization. These effects could be inhibited by the addition of the A2BR antagonist MRS 1706 [82]. These findings resemble data obtained with the A2AR (vide supra). Mundell et al. found that within the arrestin family, both arrestin 2 and arrestin 3 are involved in the internalization process of the A2B receptor. Within 1 min of agonist exposure, both GFP-tagged arrestin isoforms were translocated to the plasma membrane. Longer agonist exposure (>10 min), however, revealed that only arrestin 2 colocalizes with the receptor in the early endosomes. One explanation for this may be a higher affinity of the A2BR for arrestin 2 compared to arrestin 3. Interestingly, arrestins are not only involved in the internalization of the A2B receptor, but also in its recycling. It appeared that the expression of stable arrestin anti-sense constructs, reducing the levels of endogenous arrestins, not only resulted in less internalization of the receptors, but also impaired the recycling process significantly. In contrast to the internalization process, arrestin 3 induced a significantly faster rate of A2BR recycling compared to arrestin 2 [86]. Penn et al. also studied the behaviour of GFP-tagged arrestins, now in human airway smooth muscle cells. Arrestin 2 and arrestin 3 were able to reduce the cAMP production upon stimulation of endogenous A2BR by NECA and stimulation of β2-AR by isoproterenol. In addition, a punctate clustering was observed in the membrane upon exposure to either NECA (100 μM) or isoproterenol (1 μM), indicating that arrestins play a role in receptor trafficking. On the contrary, signalling and trafficking of the prostaglandin E2-R, another endogenously expressed receptor, was not affected by arrestin 2 or arrestin 3 [27]. A3 receptor Adenosine A3 receptors (A3R) evoke considerable interest as novel drug targets to address cerebral/cardiac ischaemia, cancer, inflammation, asthma and chronic obstructive pulmonary disease. So far, potent and selective antagonists for the hA3R have been identified; the disadvantage however is that those antagonists show extremely low binding affinity for the rodent A3R, typically 1,000 times lower than in humans. Since rodent models are essential for the pharmacological evaluation of new therapeutic agents, this forms a serious drawback. In addition, the adenosine A3 receptor (A3R) sequence holds a nuclear localization signal in its C-terminal tail [87]. This typical stretch of four amino acids (KKFK) in helix 8 may direct the receptor to the cell nucleus, not necessarily as a consequence of desensitization or internalization. This should be kept in mind when appreciating the studies described below. Cellular and physiological studies Ramkumar et al. studied the characteristics of rat A3R, endogenously expressed on RBL-2H3 mast cells. In one of their experiments the authors pretreated the cells with NECA and found a partial desensitization of the initial Ca2+ response to NECA [88]. Trincavelli et al. studied the relationship between agonist-induced desensitization, internalization and resensitization of hA3R in transfected CHO cells. Agonist-induced endocytosis of hA3R was investigated by immunogold electron microscopy of plasma membranes and intracellular vesicles and shown to occur with a half-life of 17 min. Subsequent removal of the agonist led to recycling of 90% of the receptor population to the cell surface, with a half-life of 35 min. Short-term exposure to agonist caused rapid desensitization, as assessed in cAMP assays. Removal of the agonist led to resensitization of the cAMP signal to 90% of the original signal within 120 min. Internalization did not affect signal transduction, as was demonstrated after blockade of internalization and recycling. Internalization occurred via clathrin-coated pits. These results show that hA3R undergoes agonist-induced endocytosis, which is not responsible for desensitization. Moreover, in the case of hA3R, receptor sequestration rather than desensitization seems to be the first step in a cycle of internalization, dephosphorylation and recycling to the plasma membrane [89]. A similar cell line was used by Palmer et al. In stably transfected CHO cells prolonged treatment with NECA (10 μM, 20 h) induced uncoupling of recombinant hA3R from Gi proteins and a functional desensitization. Upon this A3R desensitization, an approximately twofold increase in adenylyl cyclase activity was found in the presence or absence of forskolin. This sensitization of adenylyl cyclase activity was not caused by an altered level of inhibitory or stimulatory G protein expression. The occurrence of the sensitization was also not due to new protein synthesis, but probably to an increased coupling efficiency between Gs and adenylyl cyclase. Compared to control cells, long-term exposure of stably transfected CHO cells to NECA caused an increase in phosphorylation of the cAMP-responsive element binding protein upon addition of suboptimal concentrations of forskolin. The sensitization of adenylyl cyclase activity upon long-term treatment might provide a molecular basis for the observation that for several adenosine receptor-mediated events, acute agonist exposure produces opposite effects to those after chronic agonist treatment [90]. Ferguson et al. studied the fate of the rat A3R expressed in CHO cells. The receptors internalized rapidly after treatment with NECA or R-PIA over a time frame (t½ = 10 min) that followed receptor phosphorylation (t½ = 1 min) [91]. The desensitization, internalization and downregulation of hA3R was also investigated in human astrocytoma cells, natively expressing hA3R. Short-term (15 min) exposure of the cells to 100 nM Cl-IBMECA, an A3R-selective agonist, caused rapid receptor desensitization, subsequently followed by receptor internalization within 30 min. With the help of immunogold electron microscopy, the localization of the A3R was revealed. After 10 min of exposure, the A3R was found in smooth-surfaced pits and in uncoated vesicles in the cytoplasm. After 30 min of exposure, the A3R was found in vesicular endosomes. Upon removal of the agonist, resensitization of the A3R occurred within 120 min through receptor recycling to the cell surface. Long-term agonist exposure (1–24 h) resulted in a marked downregulation of A3R to 22 ± 3% of control after 24 h. In conclusion, multiple, temporally distinct and sequential processes are involved in the regulation of hA3R upon short- and long-term exposure to agonists [92]. A3Rs were found to be highly expressed on murine B16-F10 melanoma cells [93]. The authors examined the association between A3R trafficking and receptor functionality and tumour growth inhibition upon activation with the A3R-selective agonist IB-MECA. Exposure to 10 nM IB-MECA (5 min) led to rapid internalization of A3R to the cytosol, upon which receptors were directed to the endosomes for recycling or to lysosomes for degradation. The addition of 100 nM MRS 1523 (5 min), an A3R antagonist, was able to counteract the internalization process as well as the modulation of the Wnt pathway leading to proliferation, thereby emphasizing the involvement of A3R in this process. When the melanoma cells were injected into nude mice, tumours rapidly developed. Tumour growth was significantly inhibited after administration of IB-MECA to the animals, paralleled by a decrease in A3R expression in tumour lesions. In hypoxic human A172 and U87MG glioblastoma cell lines it was found that adenosine upregulates the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) via the A3 receptor. HIF-1α is a key regulator in the development of tumours. The addition of the A3-antagonist MRE 3008F20 inhibited the adenosine-induced HIF-1α and VEGF accumulation in these hypoxic cells, thereby showing a putative role in inhibiting tumour growth [94]. To circumvent the species differences mentioned in the introduction to the A3R section, Yamano et al. generated A3R humanized (A3ARh/h) mice in which the A3R was replaced by its human counterpart. The expression level of hA3R in the humanized (A3ARh/h) mice was equal to the expression levels of A3R in wild-type mice. A3R agonists were able to elevate the intracellular Ca2+ concentration in bone marrow-derived mast cells from the humanized (A3ARh/h) mice. However, the rate of hA3R internalization was markedly reduced compared with that of mA3R in these mast cells [95]. Molecular mechanisms Receptor phosphorylation, GRKs and arrestins Palmer and Stiles as well as Ferguson et al. investigated which amino acid residues in the C terminus are responsible and crucial for the rapid desensitization of the A3R [96, 91]. A triple mutant (Thr307, Thr318 and Thr319 to Ala) exhibited dramatically reduced phosphorylation, desensitization and internalization of the rat A3AR. Individual mutation of each Thr residue showed that Thr318 and Thr319 were the most important sites for phosphorylation. In addition, phosphorylation of Thr318 was necessary to observe phosphorylation of Thr319, but not vice versa. Moreover, changing Thr318 for a negatively charged residue (Glu) was not sufficient to retain phosphorylation at Thr319. Mutating two predicted palmitoylation sites, Cys302,305 to Ala resulted in agonist-independent basal phosphorylation of the rat A3AR. Such findings strongly suggest that palmitoylation of these Cys residues is an important factor in controlling accessibility of the C terminus of the A3R in the process of recruiting GRKs. In fact, the palmitoylation sites are highly conserved between different species, e.g. rat, mouse, human, dog, sheep. Taking these results together, it appears that GRK-mediated phosphorylation of the A3R C terminus follows a sequential mechanism, with the receptor palmitoyl moieties in an important regulatory role, and phosphorylation of Thr318 being particularly crucial as an essential first step. In an earlier study Palmer et al. replaced the carboxyl terminus of the A1R by that of the A3R [37]. This chimaeric construct was able to undergo agonist-stimulated phosphorylation and functional desensitization, similar to A3R. It was also found that purified GRK2, GRK3 and GRK5 were all able to enhance agonist-dependent phosphorylation of A3R as well as the A1-A3 chimaera. Trincavelli et al. studied the involvement of extracellularly regulated kinases (ERK1 and 2), members of the mitogen-activated protein kinase (MAPK) family, in A3R phosphorylation. It was found that within 5 min of exposure to 10 μM NECA, ERK1 and 2 were already phosphorylated in CHO cells stably expressing hA3R. An inhibitor of MAPK activation (PD98059) also caused inhibition of A3R phosphorylation, desensitization and internalization, probably by preventing the membrane translocation of GRK2. These results indicate that the MAPK cascade is involved in A3R regulation by a feedback mechanism which controls GRK2 activity and probably involves direct receptor phosphorylation [97]. The rat basophilic leukaemia cell 2H3 cell line (RBL-2H3 cells) endogenously expresses equal levels of arrestin 2 and arrestin 3. Both arrestin isoforms also have a high and comparable affinity for clathrin, thereby promoting agonist-induced internalization. RBL-2H3 cells also endogenously express a high level of A3R, which, however, neither recruited arrestin 3 nor arrestin 2 upon stimulation with NECA. Also no changes in A3R distribution were observed. One explanation is that A3R follows an endocytic mechanism upon agonist stimulation that does not involve arrestin-mediated clathrin-coated pit internalization. Another possible explanation is that arrestin recruitment was below the limit of detection in the RBL-2H3 cells [98]. Ferguson et al. observed that upon phosphorylation of A3R by GRK arrestin 3 is redistributed into punctate vesicles both at the plasma membrane and within the cytoplasm. Nevertheless, these authors also noticed that there was no colocalization between A3R and arrestin 3 [47]. Conclusions The adenosine receptor subtypes are differentially regulated when exposed to agonist treatment. A1Rs are not (readily) phosphorylated and slowly internalize with a typical half-life of several hours. This feature may cause less than average tachyphylaxis upon chronic agonist administration, for instance in type II diabetes. A2AR and A2BR show much faster downregulation with similar kinetics, usually < 1 h for short-term desensitization. Agonists for the A2A receptor, currently profiled in wound healing, may thus suffer from declining efficacy upon chronic administration, in contrast to those for the A1 receptor. The A3R undergoes even faster downregulation, often a matter of minutes. The latter receptor also holds a nuclear localization signal in its carboxy terminal tail, possibly obscuring true agonist-induced downregulation. The fast desensitization of the A3R after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor.

Diabetologia-4-1-2170455

Retinal haemodynamics in individuals with well-controlled type 1 diabetes

Aims/hypothesis Abnormalities in retinal haemodynamics have been reported in patients with type 1 diabetes in advance of clinical retinopathy. These abnormalities could therefore be useful as early markers or surrogate endpoints for studying the microangiopathy. Since the DCCT, the increased focus on good glycaemic control is changing the natural history of diabetic retinopathy. Based on this, the aim of this study was to investigate whether patients with type 1 diabetes treated entirely or mostly in the post-DCCT era and tested in the absence of confounding factors show retinal haemodynamic abnormalities. The DCCT proved that good glycaemic control is a powerful means of preventing retinopathy or slowing its course in many patients [1]. It also proved that today’s means of achieving good control are imperfect, and that some patients may still progress to sight-threatening stages of retinopathy. With the latter finding, the DCCT encouraged continuation of the quest for therapies able to pre-empt the effects of the residual hyperglycaemia; however, by increasing the prevalence of good glycaemic control it made any trial of such therapies extremely long and expensive. For this reason, there is heightened interest in early markers of retinopathy to be used as surrogates to test adjunct therapies. Retinal haemodynamics have been investigated extensively in the quest for early functional markers of diabetic retinopathy [2]; some of the changes detected appear to be a function of the duration of diabetes and the severity of retinopathy [2–4]. In patients with type 1 diabetes and absent or minimal retinopathy, several investigators using different techniques have observed decreased retinal blood speed [3–7]. These studies, performed in the early and mid-1990s, were in patients with rather poor glycaemic control by today’s standards, yet the decreased retinal blood speed could not be attributed solely to elevated blood glucose levels at the time of measurement because hyperglycaemia actually increases blood speed [2, 3, 7]. We investigated whether patients with type 1 diabetes treated entirely or mostly in the post-DCCT era and tested in the absence of confounding factors show retinal haemodynamic abnormalities. Methods The institutional review boards of the Schepens Eye Research Institute and Massachusetts General Hospital (MGH) approved this study. We recruited, from the MGH Diabetes Center and the greater Boston area, type 1 diabetic patients with the clinical characteristics of patients previously reported [3–7] to show decreased retinal blood speed: 18–45 years of age, 1–15 years of insulin dependence, and absent or minimal retinopathy. The non-diabetic participants, matched for age and sex, had no history of diabetes and had HbA1c levels lower than 6%. Exclusion criteria were pregnancy, smoking, systemic diseases other than type 1 diabetes, other retinal diseases, hypertension and renal insufficiency; also excluded were users of angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, vitamin E at doses greater than 400 IU/day, aspirin or other antiplatelet drugs, and ibuprofen. After giving informed consent, all participants in the study underwent a complete eye examination. Diabetic individuals were excluded if they had diabetic retinopathy greater than level 20 in the Early Treatment Diabetic Retinopathy Study severity scale (microaneurysms only). HbA1c was measured by the MGH HbA1c laboratory; a level >10% was an exclusion criterion. The retinal haemodynamic parameters were measured when capillary blood glucose levels were between 3.8 and 11.1 mmol/l in order to avoid the confounding effects of acute hypo- or hyperglycaemia on retinal blood flow [7]. The investigator performing the laser Doppler measurements (G. T. Feke) was masked to the diabetes status of the participants. The study eye was chosen on the basis of vascular anatomy. The superior temporal retinal arteries are preferred because temporal arteries are larger than nasal arteries, and the lesions of diabetic retinopathy are more prevalent in the superior temporal than in the other quadrants. The arterial blood column diameter and centreline blood speed were measured using a Canon laser Doppler retinal blood flow instrument (CLBF 100; Canon, Tokyo, Japan) [8]. The blood flow rate in units of μl/min is calculated automatically as the product of the cross-sectional area of the artery at the laser Doppler measurement site and the average blood speed, assuming a circular arterial cross-section [8]. Data are presented as means±SD. Parameters were compared between diabetic and control participants using the non-paired t test. Results Table 1 summarises the characteristics of the study population. The diabetic and control groups differed only in the levels of HbA1c and blood glucose. The diabetic participants received insulin through multiple daily injections or an external pump. The mean HbA1c level was 7.5% and the range 5.2–9.3%, eight patients having HbA1c levels above 8%. The mean duration of type 1 diabetes was 8.8 years. Only three patients manifested retinopathy, two of them showing one microaneurysm and one several microaneurysms. Of note, no diabetic individual who sought participation in the study was excluded because of advanced retinopathy, and only one diabetic individual was not included because of HbA1c greater than 10% (10.8%). Table 1Characteristics of the study population Diabetic individuals (n = 33)Controls (n = 31)p valueAge (years)30 ± 7.030 ± 5.60.86Sex (% female)48580.44HbA1c (%)7.5 ± 1.25.0 ± 0.3<0.0001Diabetes duration (years)8.8 ± 4.6n/an/aBlood glucose level (mmol/l)a7.7 ± 2.85.4 ± 0.8<0.0001Systolic blood pressure (mmHg)106 ± 11109 ± 70.27Diastolic blood pressure (mmHg)65 ± 866 ± 70.47Retinopathyb (%)9n/aData are means±SD or percentagesaMeasured immediately before the retinal haemodynamic studiesbAssessed using the Early Treatment Diabetic Retinopathy Study severity scale. Individuals with a score of greater than 20 were excluded from the studyn/a, not applicable Figure 1 presents the retinal haemodynamic measurements in 27 diabetic and 26 control participants in whom the measurements could be performed at the major superior temporal artery. Arterial diameter and blood speed were not significantly different between the diabetic patients and the controls (diameter: patients 116 ± 12 μm, controls 112 ± 10 μm, p = 0.18; blood speed: patients 32.9 ± 6.0 mm/s, controls 34.6 ± 6.8 mm/s, p = 0.34). Accordingly, retinal blood flow was the same in the two groups (patients 10.4 ± 2.4 μl/min; controls 10.4 ± 3.2 μl/min, p = 0.98). In the remaining six diabetic and five control participants there was early bifurcation of the artery, and one of the branches was used for the measurements; again, no differences were noted between the two groups. The sample size provided 80% power (α = 0.05) to detect differences as low as 0.75 SD between diabetic and control participants. This translates into differences of 7, 15 and 24% for retinal artery diameter, blood speed and blood flow, respectively. Fig. 1Retinal haemodynamic parameters in type 1 diabetic individuals and matched non-diabetic controls. The box plots present the data for the 27 diabetic and 26 control participants in whom the measurements could be performed at the major superior temporal artery. Each box plot shows the 10th, 25th, 50th (median), 75th and 90th percentiles of the indicated parameter. Values above the 90th and below the 10th percentile are plotted as points None of the haemodynamic measurements showed a significant correlation with coincident blood glucose levels, HbA1c, age, diabetes duration, systolic or diastolic blood pressure in either the diabetic or the control participants (data not shown). Discussion The important finding of this study is that in young patients with relatively well-controlled type 1 diabetes and no complications the baseline retinal circulatory parameters are within the normal range, even after several years of diabetes. These results differ from those of studies completed before the publication and widespread application of the DCCT results. In these earlier studies [3–7] the average HbA1c levels were 8, 10 or 12% compared with 7.5% in our diabetic individuals. Although we did not observe a correlation of HbA1c with retinal blood speed or flow, most likely because of the narrow range of HbA1c values, it was previously shown that retinal blood flow decreased with increasing HbA1c in type 1 diabetic patients without retinopathy [4]. The relation between HbA1c and retinal circulatory parameters must be interpreted with the knowledge that acute elevations [2, 7] or coincident high levels [2, 3] of blood glucose increase retinal blood flow, by increasing blood speed. Thus, the decreased blood speed and blood flow found in type 1 patients in previous studies indicated the presence of chronic effects of hyperglycaemia on retinal vessels in advance of clinical retinopathy. Elevated plasma glucose levels at the time of haemodynamic measurements could actually have masked, to some extent, such chronic effects of hyperglycaemia, contributing to the inconsistency with which retinal circulatory abnormalities were detected in diabetic patients without retinopathy [2]. The design of our study, requiring relatively normal glucose levels prior to the laser Doppler measurements (Table 1), limited such potential confounding effects, and the normal retinal haemodynamics that we recorded in diabetic participants should be informative of the status of retinal vessels. Our results are empowering for patients with type 1 diabetes and those who care for them, for two reasons. First, it is apparent that good glycaemic control is attainable in a non-clinical trial setting, and is sustainable over time. Second, such good control not only delays or prevents retinopathy, as shown by the DCCT, but also delays or prevents abnormalities in the retinal circulation that may be prodromes to the development of retinopathy. Normal retinal haemodynamics were also reported recently in patients with type 2 diabetes with no retinopathy and mean HbA1c of 7.2% [9] or 7.7% [2]. From the standpoint of finding early markers of retinopathy, our observations indicate that measurements of retinal circulatory parameters in the unperturbed state may not be sufficiently sensitive in patients with absent or minimal retinopathy and well-controlled diabetes. Insofar as the HbA1c levels in such patients are often still greater than normal, the residual hyperglycaemia is likely to leave signs on the retinal vessels, but these may be uncovered only when a functional challenge is applied to the vessels. In response to hyperoxia, used as a stimulus to test vascular reactivity, patients with well-controlled type 2 diabetes showed normal retinal haemodynamics [9], but type 1 patients in another study did not [10]. Unfortunately, the latter study did not report levels of glycaemic control. The next step will be to compare in the same well-controlled type 1 diabetic individuals the behaviour of retinal haemodynamics at steady state and in response to a challenge. In this way we will begin to reconstruct systematically the history of retinal microangiopathy in the post-DCCT era.

Neurosurg_Rev-2-2-1705527

Surgical management of dural arteriovenous fistulas with transosseous arterial feeders involving the jugular bulb

Dural arteriovenous fistulas located in the vicinity of the jugular foramen are complex vascular malformations and belong to the most challenging skull base lesions to treat. The authors comprehensively analyze multiple features in a series of dural arteriovenous fistulas with transosseous arterial feeders involving the jugular bulb. Four patients who underwent surgery via the transcondylar approach to treat dural arteriovenous fistulas around the jugular foramen were retrospectively reviewed. Previously, endovascular treatment was attempted in all patients. The success of the surgical treatment was examined with postoperative angiography. Complete obliteration of the dural arteriovenous fistulas (DAVFs) was achieved in three patients, and significant flow reduction in one individual. All patients had a good postoperative outcome, and only one experienced mild hypoglossal nerve palsy. Despite extensive bone drilling, an occipitocervical fusion was necessary in only one patient with bilateral lesions. The use of an individually tailored transcondylar approach to treat dural arteriovenous fistulas at the region of the jugular foramen is most effective. This approach allows for complete obliteration of the connecting arterial feeders, and removal of bony structures containing pathological vessels. Introduction Ten to 15% of intracranial arteriovenous fistulas are dural lesions [27], and these are most commonly related to dural venous sinuses [7]. Only a few previous articles have reported the less common intraosseous dural arteriovenous fistulas occurring at the anterior or posterior skull base [8, 19, 22, 26, 30]. Although the pathogenesis of dural arteriovenous fistulas (DAVFs) is not yet well understood, angiogenic factors and local hypoxia might play the most important roles in angiogenesis of DAVFs [24, 36–39]. The known experience suggests that the natural history of DAVFs is sometimes progressive, and intervention aiming at closure of these fisulas should be considered in selected cases—either surgically, endovascularly, or radiosurgically [6, 11, 15, 21]. In some circumstances, successful obliteration may not be possible by endovascular means, or patients may refuse the radiosurgical treatment. Direct surgery may be the best treatment option in such cases. Surgical treatment of the DAVFs at the jugular foramen region aims at removal of the bony parts that contain the pathological vessels, and physical interruption or obliteration of the arterialised leptomeningeal venous connection. In the patients we have treated so far, the fistula complex was located within or in the vicinity of the occipital condyle, rendering the suboccipital transcondylar approach as the best access route for surgical treatment. Several previous studies related to fistulas with transosseous arterial feeders dealt with endovascular embolization or with a similar surgical technique, but in many aspects different from ours [14, 20, 21, 26]. It was the aim of the present study to detail our surgical technique for the treatment of DAVFs in the region of the jugular foramen, and to retrospectively review the clinical outcome of four patients treated at our institute during a 5-year period. Patients and methods Patients Between June 1999 and February 2004, four patients underwent surgery at the Department of Neurosurgery, Philipps University for removal of DAVFs involving the jugular foramen. Patients ranged in age from 58 to 72 years, with a mean of 67 years. There were three females and one male; one individual had bilateral lesions. Four lesions were located on the right, and one on the left side. All patients were symptomatic, the most common presenting symptom being pulsatile bruit. The clinical details are summarized in Table 1. Table 1Summary of clinical featuresCase no.Age/sexPresentationLocationEmbolizationAngiographyAttempts and intervalOutcome1. WP58Y/MPulsatile bruit, rt. hypoglossal nerve paresisRt. jugular process and bulbYesFeeders: rt. ascending pharyngeal and occipital arteries of ECA2, 2 daysNo additional deficits; symptom free2. KW72Y/FPulsatile bruit, swelling of rt. upper and lower eyelidsRt. jugular process and bulbYesFeeders: Both ascending pharyngeal and rt. occipital arteries of ECA4, 5 daysNo deficits; symptoms improved3. SH70Y/FPulsatile bruitDorsal rim of foramen magnum, both occipital condyles and jugular processesYesFeeders: Both internal maxillary and occipital arteries of ECA2, 1 monthNo deficits, symptom-free4. BE68Y/FPulsatile bruitRt. Jugular tubercle and bulbYesFeeders: rt. ascending pharyngeal and occipital arteries of ECA2, 4 daysMild hypoglossal nerve palsy; symptom-freeAbbreviations: Y: years; M: male; F: female; Rt: right; ECA: external carotid artery; Attempts and interval: attempts of endovascular embolization, and timing between the last endovascular and surgical treatment All patients underwent preoperative angiography and at least two attempts of endovascular embolization. For the occlusion of arterial feeders, Guglielmi electrolytically detachable platinum coils were used. The decision for surgical intervention was made after the endovascular approach proved unsuccessful in terms of complete obliteration, and the patient developed neurological deficits or intolerable symptoms of pulsatile bruit. Case histories Case 1 A 58-year-old male presented with a 1-year history of pulsatile tinnitus in the right ear. Neurological examination revealed a partial right-sided hypoglossal palsy. Superselective angiography disclosed multiple DAVFs between the distal right sigmoid sinus and transosseous arterial feeders from the right ascending pharyngeal and right occipital arteries. Although the endovascular intervention resulted in coil occlusion of the occipital artery, the DAVFs still persisted (Fig. 1). Microsurgical intervention was performed due to intolerable pulsatile tinnitus, with the patient in the sitting position. Only minimal bony involvement around the jugular foramen was detectable on the CT scan (Fig. 1). Fig. 1DAVFs with transosseous arterial feeders. Right external carotid angiogram, antero-posterior (AP) (a) and lateral (b) views, demonstrate arteriovenous fistulas (arrows) adjacent to the right internal jugular bulb. The fistulas are mainly fed by the right-sided ascending pharyngeal and occipital arteries. Symptoms were improved following transarterial embolization with coils (c), but the fistulas persisted. Intraoperative photograph with the patient in the sitting position showing the electrodes for lower cranial nerve monitoring (d). Postoperative angiograms (e and f) demonstrate cure of the DAVFs. The same patient’s axial CT scan reveals the preoperative intraosseous lesion near the jugular process (g). Postoperative CT scan shows minimal bony removal (h) Case 2 This 72-year-old female had suffered from pulsatile tinnitus in the right ear for more than 18 years. During this period, the patient was angiographically diagnosed as having DAVFs in the region of the right jugular bulb. Four sessions of endovascular intervention were performed without success. Several months before admission, the patient experienced a clinical deterioration in the form of right periorbital swelling. In addition, there was excessive lacrimation of the right eye, and progressive numbness occurred around her right ear. On the CT scan, a bony erosion of the medial portion of the right occipital condyle and jugular tubercle became evident. A recent angiography disclosed the typical early filling of the right jugular bulb, and right sigmoid and transverse sinuses through intraosseous DAVFs mainly supplied by both ascending pharyngeal arteries and both occipital arteries (Fig. 2). The patient subsequently underwent surgical intervention in the left park bench position (Fig. 2). Fig. 2Lateral (a) and AP (b) preoperative angiograms of the right common carotid artery (CCA), demonstrating the right intraosseous DAVFs (arrows). Postoperative right CCA angiogram revealing the remaining fistula (c). On the CT scan (d) bony erosion of the medial portion of the right jugular tubercle becomes evident (white arrows). Selective injection of preoperative right occipital artery reveals intraosseous fistula (e). The patient is positioned in the left lateral park bench position (f) Case 3 A 70-year-old female presented with a 10-month history of bilateral pulsatile tinnitus predominant in the right ear. Neurological examination was irrelevant, and superselective angiography revealed numerous bilateral DAVFs in the foramen magnum region affecting both occipital condyles and jugular processes (Fig. 3). The fistula drained into both jugular veins, respectively. Feeding arteries were branches from the internal maxillary and occipital arteries. After partial occlusion of feeders on the right side, the intensity of the tinnitus decreased but still persisted. The patient was severely affected by this symptom. Surgery was therefore carried out using a right transcondylar exposure, and post-operatively the patient reported further improvement, with minimal residual tinnitus. During a 6-month follow-up, the intensity of left pulsatile bruit increased. Additionally, the patient developed a movement-related neck pain which proved to be caused by a fracture of the right occipital condyle, with dislocation of a bony fragment and craniocervical instability (Fig. 3). She underwent a second surgical procedure via a left transcondylar approach to treat the left DAVFs, followed by an occipitocervical fusion performed in the same surgical setting (Fig. 4). Fig. 3Bilateral intraosseous DAVFs causing bilateral pulsatile tinnitus. Right lateral (a) and AP (b) external carotid angiograms, as well as right AP (c) common carotid angiogram demonstrate multiple arteriovenous shunts fed by the right internal maxillary and occipital arteries. MRI (d) and CT (e) studies show the abnormal vessels posterior to the right jugular tubercle and the enlarged jugular foramen with its surrounding bony erosion, respectively (arrows). The photograph shows the skin incision for the right transcondylar approach (f). Intraoperative screenshot of navigation, applied for localizing the exent of the arteriovenous fistulas (g)Fig. 4Postoperative CT scan of the same patient shown in Fig. 3 reveals a fracture of the right occipital condyle with dislocation of a bony fragment (a). Postoperative angiograms of the right external carotid artery (ECA), lateral (b) and AP (c) views showing no arteriovenous fistula after the surgical resection of right intraosseous DAVFs. Preoperative angiogram of the left ECA revealing arterial filling of the jugular vein and left-side DAVFs (d). The patient is placed in the prone position for the surgery of the left intraosseous DAVFs and the occipito-cervical fusion (e). Postoperative lateral plain radiogram (f) and a schematic drawing (g) demonstrating the instrumentation used for the occipitocervical fusion (g) Case 4 This 68-year-old female complained of progressive pulsatile right-sided tinnitus for 1 year. Neurological examination was irrelevant. An angiogram obtained at our institution furnished evidence of DAVFs at the right occipital condyle and jugular tubercle. The fistula was mainly fed by meningeal branches of the right occipital and ascending pharyngeal arteries. The patient underwent embolization of the right occipital artery, aimed at reducing the arterial supply. Due to persistent arteriovenous connection and impairment of the patient’s quality of life, surgery was subsequently performed. Surgical technique All surgical procedures were performed by the senior author (H.B.). The first aim of surgery was an adequate exposure of the dorsolateral suboccipital region, including the medial portion of the occipital condyle, the lateral rim of the foramen magnum, the arch of the atlas and the horizontal portion of the vertebral artery, as well as the medial mastoid and particularly the jugular process region (Fig. 5). The patient was placed either in the sitting or in the lateral park bench position with the head flexed, turned to the ipsilateral and slightly tilted to the contralateral side. It was considered important to reduce the venous congestion by avoiding jugular-vein compression or by elevating the head when the lateral park bench position was chosen. Arterial feeders were occasionally encountered during muscular dissection in the course of the occipital artery in those patients in whom the artery has not been completely obliterated endovascularly before surgery. Fig. 5The particular arteriovenous fistulas are located within the occipital condyle and the jugular foramen region. These fistulas are fed by dural branches of both internal and external carotid arteries (upper). The drawing demonstrates the intraosseous fistulas, arterialized diploic veins and draining vein (lower) First, a small dorso-lateral suboccipital craniectomy was performed, which basically corresponds to a dorso-lateral enlargement of the foramen magnum. Bone drilling began in the supracondylar fossa, and was extended both medially towards the rim of the foramen magnum and laterally towards the mastoid. By gradually exposing the distal sigmoid sinus and the dura mater of the suboccipital and upper cervical region, transosseous arterial feeders were encountered, which occasionally caused a brisk arterial bleeding (Fig. 6). To control this bony bleeding, drilling was temporarily performed using the diamond burrs without saline irrigation (Aesculap drills). Application of bone wax or bipolar coagulation to stop the bleeding was usually ineffective. In the patients with severe bony involvement, the consistency of the cancellous bone was remarkably altered. In these cases the bone marrow was packed with Surgicel and then slightly compressed with cottonoid to control the bleeding. In some cases, the arteriovenous fistulas involved the posterior condylar emissary vein located within/around the posterior condylar canal (Fig. 7). These bony portions were completely drilled away. While continuing drilling within the occipital condyle, the hypoglossal canal and the medial portion of the jugular bulb were gradually exposed. The medial wall of the jugular bulb, being the site of the arteriovenous fistulas, was coagulated to interrupt small arterial feeders of this area (Fig. 6). Fig. 6Artistic illustration showing the transcondylar approach as applied to interrupt the connecting arterial feeders; the extent of the bony removal is judged according to the extent of the arteriovenous fistulas. Transosseous arterial feeders are encountered, which may occasionally cause a brisk arterial bleedingFig. 7Schematic drawing of intraosseous dural arteriovenous fistulas in the right hypoglossal canal and in the vicinity of the jugular foramen region. The feeders are the dural branches of the ascending pharyngeal artery. These arterial feeders connect with the inferior petrosal sinus, the jugular bulb and the posterior condylar emissary vein. (1: right jugular bulb, 2: right posterior emissary vein, 3: right inferior petrosal sinus, 4: arterialized venous plexus within the right hypoglossal canal, 5: right ascending pharyngeal artery, a: right internal auditory canal, b: pars nervosa of the right jugular foramen, c: right hypoglossal canal, d: right occipital condyle, e: anterior rim of the foramen magnum) Intraosseous DAVFs were also encountered around the hypoglossal canal, with arteriovenous shunts flowing into the venous plexus surrounding the hypoglossal canal, and more anteriorly in the region of the inferior petrosal sinus. To reach this area, the bony resection was extended into a deeper region. Use of the intraoperative microdoppler probe gave a good impression of the arterialization of the jugular bulb. Thus, persistence of intraosseous arterial feeders indicated that bony resection had to be continued by drilling the jugular tubercle, the occipital condyle and the jugular process. The required extent of bone removal was also estimated from preoperative imaging, and was related to anatomical landmarks such as the hypoglossal canal, the posterior condylar emissary canal and the dural entrance of the vertebral artery. Drilling was stopped only when no more arterial feeders were encountered, indicating that all feeders had been interrupted. Normalization of blood flow within the jugular bulb could be documented by using the microdoppler probe. To adequately drill the dorsomedial portion of the occipital condyle, a small portion of the lateral atlantal mass had to be resected in some instances as well. In such cases, complete exposure of the horizontal portion of the vertebral artery was necessary. However, there was no need for complete C1 hemilaminectomy. The suboccipital dura mater was not opened, since an intradural inspection of the jugular foramen was unnecessary as there were no intradural arteriovenous shunts. After meticulous extradural hemostasis, wound closure was obtained in several layers. Results All four patients underwent surgical obliteration of their DAVFs via the transcondylar approach. This access route provided a good exposure of the target site, and allowed occlusion of the intraosseous pathological vessels. There were no intra-operative complications, and no permanent major postoperative deficit occurred. Despite a certain blood loss during surgery, no patient required intra-operative or postoperative blood transfusion. Although an extensive resection of the involved occipital condyle, jugular process and jugular tubercle was carried out in all patients, an occipito-cervical fusion was necessary only in patient 3 with bilateral lesions. There were no additional neurological deficits, with the exception of patient 4, who experienced mild hypoglossal nerve palsy postoperatively. Postoperative angiography was obtained in each patient to confirm obliteration of the DAVFs. Three patients showed radiological cures, with no further treatment necessary. In patient 2, the DAVFs were significantly reduced surgically, but still persisted. Therefore, a second surgical intervention was proposed to the patient, who eventually refused. While preoperative complaints disappeared in three patients, they were clearly improved in one individual. Discussion Definition, terminology, pathology and symptoms Dural arteriovenous fistulas [DAVFs] are defined as abnormal arteriovenous connections occurring within the leaflets of the dura mater, usually within or near the walls of a dural sinus [13]. Although the term “dural arteriovenous malformations” [DAVMs] has been used widely in the neuroscience literature, current nomenclatures include terms such as “DAVFs” and “dural arteriovenous shunts”, according to an accepted view that many of these lesions are acquired [7, 9, 10, 18]. Initially, DAVFs were described only in association with venous sinuses. Subsequently, they have been found in other locations such as the tentorium, the skull base and the intraorbital area as well [30, 31]. Piske and Lasjaunias hypothesized that DAVFs can develop wherever veins have their course [31]. This would explain why DAVFs can be located entirely within the bone, because emissary veins have transosseous courses. The particular group of dural arteriovenous fistulas located within the occipital condyle and jugular foramen region are intracavarial (intracranial) arteriovenous shunts fed by dural branches of both internal and external carotid arteries. Some authors have named this particular lesion “intraosseous DAVMs” [26] or “intraossous DAVFs” [22]. The fistulas are located within the bony structures surrounding the jugular bulb (the major draining vein), and may involve the adjacent draining veins [8, 26]. DAVFs involving the transverse and sigmoid sinuses always cause a pulsatile tinnitus [28, 29]. It seems that this symptom is related to venous turbulence in contact with the petrous bone, or to the high shunt flow into the petrosal sinus [22]. This might explain why all our patients presented with bruit as the predominant symptom. Imaging DAVFs are often invisible on CT and MR studies [17, 23]. Consequently, a normal contrast-enhanced CT or MR study does not exclude a DAVF. Contrary to the ordinary DAVFs, CT and MRI studies aid in giving the diagnosis of this particular DAVFs when the patient presents with pulsatile tinnitus due to demonstrable abnormal vessels in the soft tissue beneath the skull base [12, 32], or when bony erosion due to intraosseous abnormal vessels is present, as in the patients of our series. However, cerebral angiography remains the most important modality in evaluating DAVFs. General management and endovascular intervention According to current opinion, no single treatment is ideal for the obliteration of DAVFs, and management requires an interdisciplinary team approach which must be individualized for each patient. Malik et al. reported two patients with intraosseous dural arteriovenous fistulas located at the foramen magnum. He postulated that the intraosseous nature was related to the large number of emissary veins in this region, and recommended the exploration of the adjacent bone until the entire fistula is exposed [26]. Our observations and intraoperative findings confirm this operative experience, that considerable but controllable hemorrhage can occur during bone removal. As being less invasive than surgery, endovascular technique is given preference as the primary treatment. However, transvenous embolization or transarterial coil embolization of the ascending pharyngeal artery during treatment of hypoglossal DAVFs may sometimes be associated with complications [14, 22]. An alternative surgical therapy is warranted if the endovascular technique eventually fails, as it happened to all patients of this study. The reason for failure of the endovascular intervention is probably the great number of arterial feeders that traverse through the occipital condyle and jugular process to enter the jugular bulb. A transvenous embolization was not carried out in our patients, to avoid the risk of major venous occlusion. Although stereotactic radiosurgery either followed or not followed by transarterial particulate embolization of accessible external carotid feeding vessels became the primary mode of treatment at some institutions [6, 15, 25, 33], it may not always replace open surgery. Radiosurgery is expected to cause obliteration of DAVFs between 1 and 3 years after treatment [6], and it is not considered as a standard therapy for all types of DAVFs. Benign cranial DAVFs [6, 34], though, are a developing indication for radiosurgery [34]; stereotactic radiosurgery may be considered as an alternative treatment for this type of DAVFs that have failed other treatments [20]. Surgical technique and avoidance of complications In the majority of our previously treated patients, we have used the juxtacondylar/transcondylar approach to expose various intradural craniospinal lesions [1–5, 16]. To treat DAVFs, this approach was slightly modified, and primarily applied with the purpose of interrupting the pathological arterial feeders. The extent of bony resection at the skull base was, therefore, individually tailored according to the extent of arteriovenous fistulas under intraoperative microdoppler guidance. The use of this surgical procedure was most effective, as all patients in our study showed postoperative clinical improvement, and complete obliteration of the intraosseous DAVF in three patients. Only one patient (patient 3), whose angiogram revealed residual AV fistulas, experienced previously developed mild periorbital edema. We believe that this was due to residual pathological vessels anterior to the jugular bulb, an area very difficult to reach surgically. During the removal of pathological bone containing abnormal fistulas in this patient, the dilated thin wall of the jugular bulb was slightly injured. After packing this area of venous bleeding with muscle fascia, no space for further drilling was available. A second, slightly-modified surgical intervention was, therefore, proposed to the patient to obliterate the remaining fistulas, but she refused. Partial resection of the occipital condyle in DAVFs may be associated with the risk of injury to the vertebral artery and/or jugular bulb, or damage to the hypoglossal nerve. Since the arteriovenous fistulas may be located within and around the hypoglossal canal as in patient 4 of our series, the rootlets of the nerve may be injured by surgical manipulation, even in the hands of a very experienced neurosurgeon. Preoperative imaging with thin-slice CT scans or image fusion technique (which was applied in the last three patients) [35] can provide valuable information about the local topographical anatomy. This information comprises the variable anatomical configuration of the jugular tubercle, the course of the canal containing the posterior condylar emissary vein, and the size and exact location of arteriovenous fistulas. Usually, the partial resection of the occipital condyle with opening of the atlanto-occipital joint does not create atlanto-occipital instability [2–5]. Only in the case of extensive bony drilling within the condyle, necessary due to the large number of transosseous pathological feeders, a partial condylar fracture may occur as it happened in patient 3 of this series. However, an occipito-cervical fusion was undertaken in this patient only because she harbored bilateral intraosseous DAVFs, and a second procedure on the opposite side was required that would have significantly destabilized the atlanto-occipital region. Conclusion Dural arteriovenous fistulas located around the jugular foramen are a less common type of DAVFs. The suboccipital transcondylar approach is considered the best access route when surgery of this particular pathology is intended. The amount of pathological bone that must be removed by drilling depends upon the extent of transosseous arterial feeders. Surgical obliteration of this complex vascular malformation is possible, at least in the cases in which the arterial feeders do not extend too far anterior to the jugular bulb. With this surgical technique, an atlanto-occipital instability may occur in very large or bilateral lesions, requiring occipito-cervical fusion.

Virchows_Arch-4-1-2233709

Downregulated parafibromin expression is a promising marker for pathogenesis, invasion, metastasis and prognosis of gastric carcinomas

Parafibromin is a protein encoded by the hyperparathyroidism 2 oncosuppressor gene and its downregulated expression is involved in pathogenesis of parathyroid carcinomas. To clarify the roles of parafibromin expression in tumourigenesis and progression of gastric carcinomas, it was examined by immunohistochemistry (IHC) on tissue microarray containing gastric carcinomas (n = 508), adenomas (n = 45) and gastritis (n = 49) with a comparison of its expression with clinicopathological parametres of carcinomas. Gastric carcinoma cell lines (MKN28, AGS, MKN45, KATO-III and HGC-27) were studied for parafibromin expression by IHC and western blot. Parafibromin expression was localised in the nucleus of gastric epithelial cells, adenoma, carcinoma cells and cell lines. Its expression was gradually decreased from gastritis to gastric carcinoma, through gastric adenomas (p < 0.05) and inversely correlated with tumour size, depth of invasion, lymphatic invasion, lymph node metastasis and Union Internationale Contre le Cancer (UICC) staging (p < 0.05) but not with sex or venous invasion (p > 0.05). Parafibromin was strongly expressed in older carcinoma patients compared with younger ones (p < 0.05). There was stronger positivity of parafibromin in intestinal-type than diffuse-type carcinomas (p < 0.05). Univariate analysis indicated cumulative survival rate of patients with positive parafibromin expression to be higher than without its expression (p < 0.05). Multivariate analysis showed that age, tumour size, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging and Lauren’s classification but not sex, venous invasion or parafibromin expression were independent prognostic factors for carcinomas(p < 0.05). Downregulated parafibromin expression possibly contributed to pathogenesis, growth, invasion and metastasis of gastric carcinomas. It was considered as a promising marker to indicate the aggressive behaviours and prognosis of gastric carcinomas. Introduction Parafibromin is a protein encoded by the hyperparathyroidism 2 (HRPT2) oncosuppressor gene, whose mutation causes the hyperparathyroidism–jaw tumour syndrome. The disease is an autosomal dominant disorder characterised by the occurrence of parathyroid adenoma or carcinoma, fibro-osseous jaw tumours of the mandible or maxilla and renal neoplastic and non-neoplastic abnormalities, such as Wilms’ tumour, hamartoma or cystic renal disease [1, 16, 20]. HRPT2 gene is located in human chromosome 1q31.2, consists of 17 exons and spans 18.5 kb in the genome. It encodes a 2.7-kb transcript which is translated into a 531-amino-acid parafibromin protein with a molecular weight of 60 kd [3, 4, 23]. The 200-amino-acid C-terminal segment of parafibromin shares 32% identity and 54% homology with cell division cycle 73, a Saccharomyces cerevisiae protein forming the polymerase-associated factor 1 (Paf1) complex, which is associated with ribonucleic acid (RNA) polymerase II and involved in transcript site selection, transcriptional elongation, histone H2B ubiquitination, histone H3 methylation, poly (A) length control and coupling of transcriptional and posttranscriptional events [10, 17, 18, 24, 27]. Parafibromin overexpression was documented to inhibit colony formation and cellular proliferation and induce cell cycle arrest in the G1 phase, indicating that parafibromin has a critical role in cell growth [28]. Northern blot analysis showed HRPT2 expression in heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas [4]. Western blot study revealed parafibromin expression as a 60-kd band in the adrenal gland, heart, pancreas and kidney but 40-kd immunoreactive bands in the heart and skeletal muscle of human [25]. Immunohistochemically, higher expression of parafibromin was found widespread in glomerular mesangial cell, hepatocytes, cells of the base of gastric glands, renal cortex tubules and the pars intermedia of the hypophysis [17]. Subsequent investigations have revealed that mutations in HRPT2 are present in 66–100% of sporadic parathyroid carcinomas [7, 20]. Hyperparathyroidism–jaw-tumours-syndrome-related and sporadic parathyroid carcinomas are characterised by loss of parafibromin nuclear immunoreactivity [5, 22]. Selvarajan et al. [19] found that parafibromin expression was inversely linked to tumour size, pathologic stage and lymphovascular invasion of breast carcinomas using immunohistochemistry in a tissue microarray (TMA) study. These findings suggested the potential roles of parafibromin in pathogenesis and progression of malignancies. Gastric carcinoma ranks as the world's second leading cause of cancer mortality behind lung cancer despite a sharp worldwide decline in both its incidence and mortality since the second half of the 20th century [9]. Tumourigenesis and progression of gastric carcinoma is a multistage process with the involvement of a multifactorial aetiology, which mainly results from gene–environment interactions [32, 33]. Gastric carcinomas are classified into early and advanced ones on the basis of whether the carcinomas invade into the muscularis propria of the stomach [12]. In 1965, Lauren [13] classified gastric carcinomas into intestinal- and diffuse-type ones based on the morphological appearances. Intestinal-type carcinomas are characterised by cohesive carcinoma cells forming gland-like tubular structures with expanding or infiltrative growth pattern. However, the cell cohesion is less apparent or absent in diffuse-type carcinoma and cancer cells diffusely spread in the gastric wall [31]. Generally, there is a favorable prognosis for the patients with early or intestinal carcinoma compared with the other type. In our study, parafibromin expression was examined in gastric carcinoma, adenoma, gastritis and gastric carcinoma cell lines and compared with the clinicopathological parametres of carcinomas, as well as prognosis to explore the clinicopathological significance and molecular roles of parafibromin expression in stepwise development of gastric carcinoma. Materials and methods Subjects Gastric carcinomas (n = 508) were collected from the surgical resection, adenoma (n = 45) from endoscopic biopsy or polypectomy and gastritis (n = 49) from the endoscopic biopsy in our affiliated hospital, Himi Citizen Hospital and Kouseiren Takanoka Hospital between 1993 and 2006. All carcinomas were adenocarcinomas and the adenoma group was free from non-neoplastic polyp types, leiomyomas and benign gastrointestinal stromal tumours. The patients with gastric carcinoma were 354 men and 154 women (29~91 years, mean = 65.4 years). Among them, 191 cases have carcinomas accompanied with lymph node metastasis. None of the patients underwent chemotherapy or radiotherapy before surgery. They all provided consent for use of tumour tissue for clinical research and our University Ethical Committee approved the research protocol. We followed up all patients by consulting their case documents or through telephone. Pathology All tissues were fixed in 4% neutralised formaldehyde, embedded in paraffin and incised into 4-μm sections. These sections were stained by haematoxylin and eosin (HE) to confirm their histological diagnosis and other microscopic characteristics. The staging for each gastric carcinoma was evaluated according to the Union Internationale Contre le Cancer (UICC) system for the extent of tumour spread [21]. Histological architecture of gastric carcinoma was expressed in terms of Lauren’s [13, 31] classification. Furthermore, tumour size, depth of invasion, lymphatic and venous invasion were determined. Tissue microarray Representative areas of solid tumours were identified in HE-stained sections of the selected tumour cases and a 2-mm-in-diametre tissue core per donor block was punched out and transferred to a recipient block with a maximum of 48 cores using a Tissue Microarrayer (AZUMAYA KIN-1, Japan). Four-micrometre-thick sections were consecutively incised from the recipient block and transferred to polylysine-coated glass slides. HE staining was performed on TMA for confirmation of tumour tissue. Cell lines and culture Gastric carcinoma cell lines come from the Japanese Physical and Chemical Institute, including MKN28 (well-differentiated adenocarcinoma), AGS (moderately differentiated adenocarcinoma), MNK45 (poorly differentiated adenocarcinoma), KATO-III (poorly differentiated adenocarcinoma) and HGC-27 (undifferentiated adenocarcinoma). They were maintained in Roswell Park Memorial Institute 1640 (MKN28, MKN45 and KATO-III), minimum essential (HGC-27) or Ham’s F12 (AGS) medium supplemented with 10% foetal bovine serum, 100-units/ml penicillin, and 100-μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C. Total protein was prepared from all cells by cell disruption buffer according to Protein And RNA Isolation System manual (Arctiris Bioscience, USA). All cells were collected by centrifugation, rinsed with phosphate-buffered saline, fixed by 10% formalin and then embedded in paraffin as routinely processed. Immunohistochemistry Consecutive sections were deparaffinised with xylene, dehydrated with alcohol and subjected to antigen retrieval by irradiating in target retrieval solution citrate pH 6.0 (TRS, DAKO, Carpinteria, CA 93013, USA) for 15 min with microwave oven (Oriental Rotor Lmt. Co., Tokyo, Japan). Five percent bovine serum albumin was then applied for 1 min to prevent non-specific binding. The sections were incubated with mouse anti-parafibromin antibody (Clone 2H1, SC-33638, Santa Cruz, CA, USA; 1:40) for 15 min, then treated with the anti-mouse Envison-PO (DAKO, CA, USA) antibody for 15 min. Binding sites were visualised with 3, 3′-diaminobenzidine with the 5-min reaction. All the incubations were performed in a microwave oven to allow intermittent irradiation as described previously [11]. After each treatment, the slides were washed with Tris-buffered saline with Tween 20 (TBST; 10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) three times for 1 min. After being counterstained with Mayer’s haematoxylin, the sections were dehydrated, cleared and mounted. Omission of the primary antibody was used as a negative control. One hundred cells were randomly selected and counted from five representative fields of each section blindly by three independent observers (Takano Y, Li XH and Zheng HC). The positive percentage of counted cells was graded semi-quantitatively according to a four-tier scoring system: negative (−), 0~5%; weakly positive (+), 6~25%; moderately positive (++), 26~50%; and strongly positive (+++), 51~100%. Western blot Fifty-microgramme denatured protein was separated on an SDS-polyacrylamide gel (10% acrylamide) and transferred to Hybond membrane (Amersham, Germany), which was then blocked overnight in 5% milk in TBST. For immunoblotting, the membrane was incubated for 1 h with mouse anti-parafibromin antibody as described above. Then, it was rinsed by TBST and incubated with anti-mouse immunoglobulin G conjugated to horseradish peroxidase (DAKO, CA, USA, 1:1,000) for 1 h. Bands were visualised with X-ray film (Fujifilm, Japan) by ECL-Plus detection reagents (Amersham, Germany). After that, membrane was washed with WB Stripping Solution (pH 2–3, Nacalai, Tokyo, Japan) for 30 min and treated as described above except mouse anti-β-actin antibody (Sigma, MO, USA, 1:5,000) as an internal control. Statistical analysis Statistical evaluation was performed using Spearman correlation test to analyse the rank data. Kaplan–Meier survival plots were generated and comparisons between survival curves were made with the log–rank statistic. The Cox’s proportional hazards model was employed for multivariate analysis. P < 0.05 was considered as statistically significant. SPSS 10.0 software was employed to analyse all data. Results Parafibromin expression in gastric tumours and carcinoma cell lines As shown in Fig. 1, parafibromin was positively immunostained in the nucleus of MKN28, AGS, MKN45, KATO-III and HGC-27, and its expression level was consistent with the data of Western blot. Parafibromin was strongly expressed in the nucleus of gastric epithelial cells, adenomas and early carcinomas but not in given advanced carcinomas. Occasionally, it also appeared in stromal fibroblasts and lymphocytes but much weaker than epithelial cells or adenomas (Fig. 2).Generally, the stromal lymphocytes and fibroblasts were negative in cases where the tumour was negative. Overall, parafibromin expression was detected respectively in all gastritis (100.0%), 36 out of 45 adenoma patients (80.0%) and 233 out of total 508 gastric carcinoma patients (45.9%). Statistically, gradually reduced expression of parafibromin was seen from gastritis to gastric carcinoma through gastric adenoma (p < 0.05, Table 1). As summarised in Table 2, parafibromin expression was inversely correlated with tumour size, depth of invasion, lymphatic invasion, lymph node metastasis and UICC staging (p < 0.05) but not with sex or venous invasion (p > 0.05). Parafibromin was strongly expressed in older carcinoma patients compared with younger ones (p < 0.05). Intestinal-type carcinomas exhibited more frequent expression of parafibromin than diffuse-type ones (p < 0.05). Fig. 1Parafibromin expression in gastric carcinoma cell lines. a Parafibromin was positively immunostained in the nucleus of MKN28 (a), AGS (b), MKN45 (c), KATO-III (d) and HGC-27(e). b Cell lysate (50 µg) was loaded and probed with anti-human parafibromin antibody (60 kd) with β-actin (42 kd) as an internal control. Lane #1: MKN28; #2 AGS; #3 MKN45; #4 KATO-III; #5 HGC-27Fig. 2Immunohistochemical staining of parafibromin in gastritis, adenoma and carcinoma. Note parafibromin positivity was strongly observed in the nucleus of gastric superficial epithelium (a), and adenoma (c) and early gastric carcinoma (b), occasionally weaker in the stromal fibroblasts and lymphocytes(a, c), but not in given advanced gastric carcinomas (d), indicating that the internal positive control (stromal cells) was negative adjacent to the negative staining carcinoma cells but positive adjacent to the positive epithelial cellsTable 1Parafibromin expression in gastric tissue samplesGroupsNumberParafibromin expression−++++++PR (%)Gastritis4901741100.0aGastric adenoma459582380.0bGastric carcinoma508275556011845.9PR Positive rateaCompared with gastric adenoma or carcinoma, p < 0.001bCompared with gastric carcinoma, p < 0.001Table 2 Relationship between parafibromin expression and clinicopathological features of gastric carcinomasClinicopathological featuresNumberParafibromin expression−++++++PR (%)Rsp valueAge (years)  <6520912524243640.20.095<0.05  ≥6529915031368249.8Sex  Male35418837389146.90.054>0.05  Female1548718222743.5Tumour size (cm)  <426311626348555.9−0.237<0.001  ≥424515927263335.1Depth of invasion  Tis−126310230409161.2−0.344<0.001  T2-424517325202729.4Lymphatic invasion  −33115742458752.6−0.168<0.001  +17711813153133.3Venous invasion  −443236495110746.7−0.051>0.05  +6539691140.0Lymph node metastasis  −31713838449756.5−0.285<0.001  +19113717162128.3UICC staging  0–I29212336419257.9−0.292<0.001  II-IV21615219192629.6Lauren’s classification  Intestinal-type27310834389360.4−0.322<0.001  Diffuse-type22515721222530.2PR Positive rate, Tis carcinoma in situ, T1 lamina propria and submucosa, T2 muscularis propria and subserosa, T3 exposure to serosa, T4 invasion into serosa, UICC Union Internationale Contre le Cancer Univariate and multivariate survival analysis Follow-up information was available on 508 gastric carcinoma patients for periods ranging from 0.2 months to 12.2 years (median = 67.2 months). Figure 3 showed survival curves stratified according to parafibromin expression for gastric carcinomas. Univariate analysis using the Kaplan–Meier method indicated cumulative survival rate of patients with weak, moderate or strong parafibromin expression to be obviously higher than without its expression (p < 0.05; Fig. 3a). The significant difference disappeared if stratified according to the depth of invasion (Fig. 3b, c). Multivariate analysis using Cox’ s proportional hazard model indicated that age, tumour size, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging and Lauren’s classification (p < 0.05) but not sex, venous invasion or parafibromin expression were independent prognostic factors for overall gastric carcinomas (p > 0.05; Table 3). Fig. 3Correlation between parafibromin status and prognosis of the gastric carcinoma patients. Kaplan–Meier curves for cumulative survival rate of patients with gastric carcinomas according to the parafibromin expression in overall (a), early (b, EGC) and advanced (c, AGC) gastric carcinomasTable 3Multivariate analysis of clinicopathological variables for survival with gastric carcinomasNumberClinicopathological parametresRelative risk (95%CI)p valueAAge (≥65 years)1.929 (1.357–2.743)<0.001BSex (female)1.463 (0.983–2.179)>0.05CTumour size (≥4 cm)1.606 (1.013–2.547)<0.05DDepth of invasion (T2–4)6.530 (3.110–13.710)<0.001ELymphatic invasion (+)2.626 (1.516–3.374)<0.001FVenous invasion (+)0.959 (0.633–1.452)>0.05GLymph node metastasis (+)2.773 (1.525–5.043)<0.01HUICC staging (II-IV)0.294 (0.139–0.622)<0.01ILauren’s classification (diffuse-type)1.796 (1.212–2.661)<0.01JParafibromin expression (+~+++)0.792 (0.529–1.187)>0.05CI Confidence interval, UICC= Union Internationale Contre le Cancer Discussion HRPT2 has been isolated from complementary DNA libraries of parathyroid, kidney and bone tissue and encodes tumour suppressor protein parafibromin [4]. In the present study, the nuclear expression pattern was observed in the gastric epithelial cells, adenomas, adenocarcinomas and carcinoma cell lines consistent with previous reports in the gastric superficial mucosa, hepatocytes, kidney cortex tubules, adrenal gland, spleen lymphocytes, parathyroid tissue, adenoma and carcinomas, breast carcinoma [5, 8, 17, 19]. Although the result was in contrast with the paper of Porzionato et al. [17] possibly due to different incubation times of primary antibody, the great majority of immunohistochemical and cell transfection studies supported our observation of the nuclear staining [2, 5, 6, 8, 14, 19]. This study again demonstrates that parafibromin is nuclear and not cytoplasmic or nucleocytoplasmic in location as initially thought. The weaker expression of parafibromin in stromal cells than epithelial cells and adenoma might be due to the specificity of its cellular distribution as described previously [17]. It was found that the translocation of parafibromin to the nuclear compartment involved a function monopartite nuclear localisation signal at residues 136–139 [2, 6]. Parafibromin is a component of Paf1 complex in the nucleus, where it plays a role in cell cycle regulation, histone methylation, lipid and nucleic acid metabolism [10, 17]. The distribution pattern of parafibromin protein in gastric epithelial cells or tumour cells demonstrated its biological function in the nucleus. Statistically, parafibromin expression was gradually reduced from gastritis to carcinoma through adenoma in line with parathyroid carcinogenesis, suggesting that downregulated parafibromin expression might contribute to the malignant transformation of gastric epithelial cells as an early event. The positive rate of parafibromin expression was reduced to 80% in gastric adenoma and reached about 46% of gastric adenocarcinoma, supporting the involvement of parafibromin in the gastric adenoma–adenocarcinoma sequence. Actually, the adenoma can progress into and be incorporated with gastric well-differentiated carcinoma when it grows bigger and de novo carcinogenesis is well understood, especially in diffuse-type gastric carcinomas [34]. Higher parafibromin expression in adenoma and intestinal-type carcinoma indicated that decreased parafibromin expression might play an important role in de novo diffuse-type carcinogenesis but less in intestinal carcinogenic pathway. A body of evidences indicated that downregulation of tumour suppressor protein expression was due to genetic or epigenetic changes, like allelic loss, mutation, loss of heterozygosity (LOH), hypermethylation and microsatellite instability in malignancies [29, 30]. In the sporadic parathyroid carcinomas and hyperparathyroidism–jaw tumours, LOH or mutation of HRPT2 might cause the loss and inactivation of parafibromin protein [20, 25, 26]. Furthermore, the reduced expression of parafibromin was found to closely link to the tumour size, depth of invasion, lymphatic or venous invasion and UICC staging in line with the observation in breast carcinomas [19], indicating the inhibitory effects of parafibromin on tumour growth, invasion, metastasis and progression of gastric carcinomas. Drosophila Hyrax and its human orthologue, parafibromin, are required for nuclear transduction of the Wnt/Wg signal and bind directly to the C-terminal region of beta-catenin–Armadillo, thereby controlling transcriptional initiation and elongation by RNA polymerase II [15]. Parafibromin overexpression can inhibit colony formation, anchorage-dependent cell growth and cellular proliferation and induce cell cycle arrest in the G1 phase [28]. These findings demonstrated that loss of parafibromin expression had impact on the pathogenesis and progression of malignancies by promoting cellular proliferation. Additionally, parafibromin was expressed with a higher incidence in intestinal-type gastric cancer, which is presumed to arise from preceding dysplastic lesions, than diffuse-type one, which evolves without any precedent dysplastic changes. It is also demonstrated that distinct parafibromin expression underlies the molecular mechanisms for the differentiation of intestinal- and diffuse-type carcinomas. Until now, there is yet no paper describing the prognostic significance of parafibromin expression in malignancies. Here, for the first time, we analysed the relation of parafibromin expression with the survival rate of 508 patients with gastric carcinoma. The results revealed a close link between its loss and worse survival. If stratified according to the depth of invasion, the significant link disappeared, indicating that the relationship between parafibromin expression and prognosis depends on the depth of invasion. The multivariate analysis demonstrated that age, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging and Lauren’s classification but not parafibromin expression, venous invasion or sex were independent prognostic factors for carcinomas. These findings suggested that parafibromin expression is a promising indicator for the favorable prognosis of gastric carcinoma patients, albeit not independent. In the present study, a large number of gastric carcinoma cases were screened by TMA, which takes the advantages of high throughput, identical immunohistochemical conditions, and economy of samples, antibodies and time [33]. Although we used 2-mm-in-diametre needles, which are large enough to evaluate the morphological appearance and carefully selected representative regions with the reference of HE slides, it was difficult to avoid selection bias. Gill et al. [5] found that stronger parafibromin staining and more positive cells sometimes appeared at the edges of the parathyroid tumour than in the centre. This could be due to fixation methods, other processing issues or a biological phenomenon, for example tissue hypoxia in the centre of large tumours. Additionally, the collection of our samples (e.g. gastritis, adenoma and adenocarcinoma) respectively from the endoscopic biopsy, polypectomy, or surgical resection put forward their another possibility of selection bias because of different fixation and processing methods. Selvarajan et al. [19] found that parafibromin underexpression was correlated particularly with large tumour size which is in line with our finding. It was possible that weaker staining could be attributable to poor fixation properties in the centre of large tumours. Because tumour size is a key prognostic indicator, this artifact could explain the prognostic significance of parafibromin in gastric carcinomas. Therefore, it is a limitation of the present study not to separate the edge and centre of gastric carcinomas when establishing TMA. In the current study, the negatively staining carcinomas are associated with a negative internal control (stromal cells and lymphocytes) whereas the positive staining epithelium is adjacent to positive staining internal controls. Therefore, the negative staining of the carcinoma might be artificial, which should be considered as another limitation of the study. Our study might be mentioned as a preliminary experiment and the staining with original-size sections is an extensive work in the future using the gastric carcinoma samples, fixed and processed by the same approach. In summary, downregulated parafibromin expression might play an important role in malignant transformation of gastric epithelial cells. Its reduced expression was closely related to growth, invasion, metastasis and worse prognosis of gastric carcinomas. Its expression could be employed to differentiate the intestinal- and diffuse-type carcinomas and underlay the molecular mechanism about the differentiation of both carcinomas. It was considered as a promising marker to indicate the pathobiological behaviours and prognosis of gastric carcinomas.

Health_Care_Anal-4-1-2226192

The UNESCO Universal Declaration on Bioethics and Human Rights: Perspectives from Kenya and South Africa

In October 2005, UNESCO (the United Nations Educational, Scientific and Cultural Organization) adopted the Universal Declaration on Bioethics and Human Rights. This was the culmination of nearly 2 years of deliberations and negotiations. As a non-binding instrument, the declaration must be incorporated by UNESCO’s member states into their national laws, regulations or policies in order to take effect. Based on documentary evidence and data from interviews, this paper compares the declaration’s universal principles with national bioethics guidelines and practice in Kenya and South Africa. It concentrates on areas of particular relevance to developing countries, such as protection of vulnerable persons and social responsibility. The comparison demonstrates the need for universal principles to be contextualised before they can be applied in a meaningful sense at national level. The paper also assesses the ‘added value’ of the declaration in terms of biomedical research ethics, given that there are already well-established international instruments on bioethics, namely the World Medical Association Declaration of Helsinki and the CIOMS (Council for International Organizations of Medical Sciences) guidelines on biomedical research. It may be that the added value lies as much in the follow-up capacity building activities being initiated by UNESCO as in the document itself. Introduction In October 2005,1 the United Nations Educational, Scientific and Cultural Organization (UNESCO) adopted the Universal Declaration on Bioethics and Human Rights. The preamble states: “It is necessary and timely for the international community to state universal principles that will provide a foundation for humanity’s response to the ever-increasing dilemmas and controversies that science and technology present for humankind and the environment” [20, p. 3]. As the declaration is non-binding, this universal foundation will be implemented at the level of the nation-state. The onus is on UNESCO’s member states to incorporate the declaration’s provisions into their national laws, regulations or policies. This paper examines the synergies between the declaration and bioethics regulation in two countries, Kenya and South Africa. Before the UNESCO declaration was adopted, international instruments such as the World Medical Association (WMA) Declaration of Helsinki and the International Ethical Guidelines for Biomedical Research Involving Human Subjects of the Council for International Organizations of Medical Sciences (CIOMS) were already well-established in bioethics [5]. Indeed, the Helsinki declaration and the CIOMS guidelines are noted in the preamble of the UNESCO declaration [20]. These instruments have had a significant influence on the national bioethics policies of developing countries [1], including Kenya and South Africa. The objectives of this paper are twofold. The first is to assess the translation of universal principles into national practice. The comparison between the UNESCO declaration and Kenyan and South African bioethics forms the basis for this analysis. The second is to locate the ‘added value’ of the declaration, particularly to developing countries, given the pre-existence of international level bioethics documents. Empirically based, the paper draws on documentary evidence and interviews conducted in Kenya and South Africa in 2005 and 2006. Background The empirical research for this paper was carried out as part of a larger doctoral project on the global governance of human genomic and biomedical research and in particular the part developing countries play in this. The theoretical framework for this project is provided by international relations, a sub-discipline of political science. While international relations theory is not referred to overtly in this paper’s analytical sections, it provides the context for the understanding of universality contained therein. That is, universality is explored pragmatically, with regard to the relationship between broad principles negotiated at international level and their subsequent adaptation to national level policy and practice, rather than philosophically, in terms of universal versus pluralist moral reasoning. ‘Bioethics’ What exactly is meant by ‘bioethics’ is notoriously difficult to determine. This is illustrated by the fact that the UNESCO declaration contains no clarification of the term. While a definition appeared in earlier drafts, the impossibility of reaching a consensus on wording necessitated its being left out of the final version [18]. For the purposes of this paper, bioethics is understood specifically in terms of the regulation of biomedical research. The UNESCO declaration itself has a wider remit, covering medical practice and protection of biodiversity and the environment as well as research ethics [20]. Since the overall doctoral project is concerned chiefly with human subjects research, however, these broader considerations will not be discussed here. Methodology and Limitations Much of contemporary international relations theory concerns the roles of both state and non-state actors in global governance mechanisms. Reflecting this, the doctoral fieldwork consisted of 70 semi-structured interviews with a range of persons considered to be stakeholders in genomics and bioethics in France, the United Kingdom, Kenya and South Africa.2 The sample was chosen to reflect different societal perspectives on genomics and bioethics and thus consisted of those who formulate policy at international and national levels, those who must implement these policies in laboratories and ethics committees, those who claim to represent public concerns and those with commercial interests. The breakdown of sectors was as follows: policy-makers (20), scientists (17), ethicists3 (18), civil society representatives (12) and businesspersons (3).4 Of these 70 interviews, the data used in this paper draw on only 22 and come mostly from those with members of research ethics committees. Thus they are illustrative rather than representative. Studying a declaration as it evolves5 makes for exciting and contemporary research. It also carries limitations, however; in this instance, fieldwork may have been conducted too early to enable an assessment of the social and political impact of the UNESCO declaration in Kenya and South Africa. In the wider context of the doctoral research project, this limitation will be addressed by the inclusion of UNESCO’s previous declarations on genomics and genetics, which have had longer to become established within national policy frameworks.6 It is worth noting that the research process became by default an awareness-raising exercise, in that many of the people interviewed were previously unaware of the bioethics declaration, or indeed its predecessors. Previous Studies The UNESCO declaration features in several publications. Developing World Bioethics devoted a whole issue to the draft text in September 2005. The articles were largely critical, questioning the content of the draft declaration, how it had been put together and whether UNESCO was the right body to be taking on such an endeavour [8]. Professor Henk ten Have, Director of the Division of Ethics of Science and Technology at UNESCO, responded that the journal’s contributors were perhaps not au fait with how UN agencies work [14]. After the declaration was adopted, Herman Nys wrote an editorial for the European Journal of Health Law outlining its basic tenets and comparing it to the European Convention on Human Rights and Biomedicine. He emphasised the obligations of states to take on the declaration, despite its being legally non-binding [10]. Professor ten Have has himself written about the declaration, in the wider context of UNESCO’s activities in ethics. In his article in the Kennedy Institute of Ethics Journal he described how these activities fall into three areas, namely the adoption of normative instruments such as the declaration and their subsequent implementation through national level capacity building and awareness raising [6]. There has been a growing literature in recent years on research ethics in developing countries. A few examples, taken from the British Medical Journal, the Bulletin of the World Health Organization and PloS Medicine, will serve to illustrate that contextualisation of international instruments at national level and capacity for ethical review have been among the major concerns raised. Sylvester Chima has suggested that international guidelines need to be interpreted legislatively at local and regional levels and has thus called on the African Union to pass binding directives that are nevertheless adaptable to the laws of each state [3]. Kass et al., in a case study published in January 2007 reviewing the practices of research ethics committees in several African countries, including Kenya and South Africa, highlighted insufficient funding and training as the biggest challenges facing these committees and proposed that workshops be set up on how to apply ethical principles at local levels. They also called for more empirical investigation of ethics in African research [7]. Peter Singer and Solomon Benatar, in a 2001 article on the Helsinki declaration, contended that building capacity in research ethics will have far more impact on ethical standards than “revisions of this or any other research ethics code,” implying that declarations themselves are of limited use unless the capacity exists to implement them [15, p. 747). Zulfiqar Bhutta has also argued that strengthening local capacity in bioethics is key to promoting ethical health research in developing countries [1]. The UNESCO Declaration The Universal Declaration on Bioethics and Human Rights was adopted “by acclamation” at the UNESCO General Conference on 19 October 2005 [20, p. 5). This was the end of a process that began with an invitation by the 2001 General Conference to the UNESCO International Bioethics Committee to report on the possibility of elaborating a universal instrument on bioethics. The Committee recommended that this instrument be declaratory in nature (that is, non-binding) and the drafting process was launched in January 2004 [17, 19]. Thus the actual negotiation period lasted under 2 years. The declaration is aimed primarily at states, but can also be implemented by “individuals, groups, communities, institutions and corporations, public and private” where appropriate [20, p. 6]. It covers a wide range of bioethical principles, several of which had already become customary in bioethics and feature in documents such as the Helsinki declaration and the CIOMS guidelines (informed consent, for example). Some of the principles of particular relevance to developing countries will be elaborated further below. Bioethics in Kenya and South Africa Kenya and South Africa were chosen as fieldwork destinations because of their significant involvement in genomics and bioethics at local, national, regional and international levels. Both countries have recently adopted national guidelines on bioethics: in Kenya the 20047 National Council for Science and Technology (NCST) Guidelines for Ethical Conduct of Biomedical Research Involving Human Subjects in Kenya (‘human subjects guidelines’) and the 2005 Ministry of Health (MoH) Kenya National Guidelines for Research and Development of HIV/AIDS Vaccines (‘vaccines guidelines’) and in South Africa the Department of Health (DoH) Ethics in Health Research: Principles, Structures and Processes, which were drawn up by members of both the Department and the Interim National Health Research Ethics Committee, appointed under the National Health Act of 2003 [13]. Both Kenya and South Africa decided that national bioethics guidelines were necessary partly in order to protect poor and marginalised people from being exploited by unscrupulous researchers [9, 13]. Among other texts, the guidelines draw on the Helsinki declaration, the CIOMS guidelines8 and several documents from the World Health Organization and the United States, but are tailored to their national contexts, with specific provisions addressing the vulnerabilities that may have enabled past abuses [9, 11, 13]. Participants from ethics committees cited a similar assortment of guidelines and regulations—international, regional and national—as influential, including some from Europe, the United Kingdom and Australia. Synergies between the UNESCO Declaration and Bioethics in Kenya and South Africa As the UNESCO declaration is non-binding, if its principles are to be applied universally they will necessarily have to be reflected in national level documents and systems. This section compares the main tenets of the declaration that are of special relevance for developing countries with Kenyan and South African bioethics policy and practice. The comparison has two purposes. The first is to illustrate how internationally determined, universal principles might be implemented at national levels in developing countries. The second is to show to what extent these principles were already reflected in national systems, before the adoption of the declaration. Community Consent—Article 6 The UNESCO declaration states that for a research project on a group or community, agreement from representatives may be sought, in addition to that of the individual participants [20]. Community Advisory Boards facilitate this in some areas of Kenya (interviews, K_06:2005 and K_25:2005) and dialogue with community members through such boards is a requirement for HIV/AIDS vaccines research [11]. The South African guidelines stipulate community involvement and consultation for research involving ‘collectivities’, on issues such as ownership of data and distribution of benefits and harms [13].9 Vulnerability—Article 8 This article holds that “individuals and groups of special vulnerability should be protected and the personal integrity of such individuals respected” [20, p. 8]. The Kenyan human subjects guidelines give special instructions concerning research with underdeveloped communities, prisoners, married women in rural areas and pregnant or lactating women [9], while the vaccines guidelines state that the vulnerable and poor must be protected from exploitation [11]. The South African guidelines invite ethics committees to be “especially vigilant when considering research proposals involving vulnerable populations” [13, Preamble] and contain detailed provisions for research involving pregnant women, foetuses, prisoners and vulnerable communities [13]. Cultural Diversity and Pluralism—Article 9 Under the declaration these should be given “due regard” [20, p. 8]. Again, the national guidelines contain specific examples of what this might entail. The Kenyan human subjects guidelines, in the context of gaining informed consent from married women in rural communities, remind researchers that each of Kenya’s 42 tribes will have “unique sociocultural backgrounds” [9, p. 11], while the South African guidelines, in a section on indigenous medical systems, call on researchers to respect the cultures and traditional values of all communities [13]. The Kenyan vaccines guidelines are less detailed and, in a similar vein to the UNESCO declaration, simply require that research teams be sensitive to “sociocultural issues,” without specifying what these issues might be [11, p. 30]. Social Responsibility—Article 14 This article of the UNESCO declaration is considered particularly innovative [6]. It is perhaps not surprising, then, that it should prove more specific than any of the national guidelines as to how social responsibility could be implemented. Where the Kenyan and South African guidelines require research to be relevant to study populations and each country in general, by addressing either “health needs” (Kenya) [9, pp. 13, 16] or “broad health and development needs” (South Africa) [13, p. 3], the declaration states that scientific and technological progress should advance: access to healthcare and medicines; adequate nutrition and water; improved living conditions and environment; elimination of marginalisation and exclusion; and reductions in poverty and illiteracy [20]. Benefit Sharing—Article 15 The declaration is similarly specific with regard to the sharing of benefits, giving seven examples of what form this could take, including sustainable assistance to research participants and provision of new health products stemming from research [20]. It is the latter of these examples which features most prominently in the national guidelines. In Kenya, if research produces positive results, treatments should be available to participants [9]. Also, before HIV/AIDS vaccine research can take place, the “availability, affordability and accessibility” of its potential products must be considered [11, p. 30]. Research proposals in South Africa must indicate whether there is a reasonable likelihood that participants will benefit from the research and whether they will receive long-term therapy after the study [13]. More widely, research findings must “be translatable into mechanisms for improving the health status of South Africans” [13, p. 3]. In practice, ethics committees in both countries try to assess the extent to which participants will benefit from a research project (interviews, K_25:2005 and SA_19:2006), although they do not always find this a straightforward endeavour (interviews, SA_10:2006 and SA_35:2006). Transnational Practices—Article 21 This article calls on states to combat illicit trafficking of “organs, tissues, samples, genetic resources and genetic-related materials” [20, p. 10]. Transfer of tissues overseas has been a particular concern in both Kenya and South Africa, as reflected in their guidelines. The Kenyan vaccines guidelines aim to eliminate the unauthorised transfer of research materials and to this end contain a sample Biological Material Transfer Agreement [11]. Under chapter 810 of the South African National Health Act, an appendix to the DoH guidelines, the Health Minister may regulate “the importation and exportation of tissue, human cells, blood, blood products or gametes” [12, pp. 70, 62]. Ethics committees in both countries take tissue transfer equally seriously, examining closely any research protocol that involves movement of samples across borders. Generally they prefer a project to train people to analyse data in-country (interviews, K_17:2005, K_21:2005, K_25:2005, SA_19:2006, SA_21:2006 and SA_30:2006). Bioethics Education, Training and Information—Article 23 Under this article states are invited to foster bioethics training and education “at all levels” and to encourage information dissemination on bioethics [20, p. 11]. Such training and education is perhaps more widespread in South Africa than Kenya, although the UNESCO National Commission was in the process of setting up a bioethics centre to serve the East Africa region at the time of interview (interviews, K_01:2005 and K_16:2005). Several South African universities run courses in ethics or bioethics and two training initiatives, IRENSA and SARETI, serve sub-Saharan Africa as a whole (including Kenya).11 All three set of guidelines advocate bioethics training [9, 11, 13]. Universal Principles in the National Context The South African guidelines read: The challenge to international research ethics is the development of universal rules for research at a time when health care is being delivered within very different health care systems and in a multicultural world in which people live under radically different economic conditions. [13, p. 7]This paper asks whether the UNESCO declaration has met this challenge. Although promulgated with the expressed purpose of stating universal principles, the declaration acknowledges that such cultural and economic differences exist, through its articles on community consent, cultural diversity, vulnerability, social responsibility and benefit sharing. The Kenyan and South African guidelines demonstrate how the declaration might be applied at state level, with their prescriptions concerning, for example, how researchers should engage with communities and which particular members of society should receive special attention as vulnerable persons. That states may need to adopt particular interpretations of the declaration’s principles in order to realise them in national and local contexts is highlighted by ten Have in his paper on UNESCO’s ethical activities. He describes the adoption of the declaration as only the “start of a long process of detailed elaboration and consequent application” [6, p. 342] and in the context of the articles on consent and social responsibility states:As principles they are universally adopted, but in practice their application must be tailored in multiple ways to accommodate different types of research and health care, categories of patients and problems, and cultural settings and traditions. [6, pp. 342–343]The need for any national implementation of the UNESCO declaration to be adequately contextualised is brought out in the interview data from Kenya and South Africa, as are differences in opinion as to the usefulness of international guidelines in general. In Kenya, an academic scientist and advisor to both the government and UNESCO stated that it would be necessary to work with those government officers responsible for effecting international documents in law, in order to “translate it [the declaration] into what is happening locally” (interview, K_13:2005). Similarly, the head of a research institution attested: Something which became more and more legalistic in the detail and binding but which ignored local realities would be unhelpful. Something which tried to establish clear, agreed principles, which had been widely consulted, not just between member states, but with the kind of communities that are affected, would be very useful. (Interview, K_07:2005)In South Africa, one ethics committee member thought that, given different cultural contexts and sensitivities, declarations such as the UNESCO one would have to be formulated as generally as possible to enable universal applicability (interview, SA_08:2006). Another was of the view that national and local ethics guidelines are “the things to follow,” because what is applicable in one country may not be applicable in another (interview, SA_19:2006). Several saw at least some value in the UNESCO endeavour, but cautioned that universal principles must not be applied unthinkingly. Perhaps mirroring ten Have’s prediction that elaboration of the declaration will be a long process, they described how working out how to apply such principles in different contexts is often the most challenging aspect of implementing international instruments (interviews, SA_10:2006, SA_17:2006 and SA_24:2006). Added Value of the UNESCO Declaration The UNESCO declaration is considered to be of added value because it is the first intergovernmental instrument on bioethics. Ten Have describes the commitment by governments to an agreed set of principles on bioethics as the “innovative dimension” of the declaration [6, p. 342]. The Helsinki declaration and the CIOMS guidelines, by contrast, have been adopted by professional organisations (although CIOMS is in official relations with the World Health Organization). Where the former is officially directed at physicians or researchers, however, the latter, like the UNESCO declaration, are to be used in designing national policy on biomedical research ethics, particularly in developing countries [4, 21]. It may take time for the significance of the UNESCO declaration’s governmental backing to filter through to those who practise research ethics. An anonymous reviewer of this paper described the declaration as the “common denominator of global ethical thought,” but for many stakeholders the Declaration of Helsinki and the CIOMS guidelines fulfil this role, their professional origins notwithstanding. The Helsinki declaration is generally considered the foremost document globally on medical research ethics [2, 5] and, with the CIOMS guidelines, forms the bedrock of research ethics in many developing countries [1]. In Kenya and South Africa, where policy-makers and ethics committees have looked to these two documents for guidance at the global level, several of those interviewed were unconvinced that the UNESCO declaration, which they saw as simply another international instrument on bioethics, was necessary. As the declaration was adopted only relatively recently, such perceptions may change as both its contents and its intergovernmental status become more widely established (particularly as it construes bioethics in broader terms than only medical research ethics). If the UNESCO declaration is not to “remain paperwork,” as a non-binding instrument it must be effected by states [6, p. 343]. Kenya and South Africa already have national bioethics guidelines that complement the declaration to a large degree. They also have research ethics committees at institutional and national levels, or plans for constituting them. For those countries which have not already established bioethics systems, however, the UNESCO declaration could act as a catalyst to galvanise them into doing so. To this end, UNESCO’s ethics programme supports states in building bioethics capacity, through various activities. The first is to construct a database with information on ethics experts, institutions, teaching programmes and policies around the world. The second is to promote bioethics education, through teacher training and curriculum development. The third is to support the setting up of ethics committees and their subsequent operations [6]. These activities were broadly welcomed among those interviewed in both countries, with the caveat that they should not overlap too far with the initiatives of other organisations. UNESCO is in fact piloting an ethics teacher training course in Kenya in July 2007 [16], where there are fewer bioethics courses available than in South Africa. One area where ethics committees in Kenya and South Africa are in need of support is in the monitoring of research projects once they have been approved, as required by all three sets of national guidelines [9, 11, 13]. In practice, as found by Kass et al., funding can be an issue. One prominent Kenyan ethics committee had only recently carried out its first on-the-spot inspection at the time when interview data were obtained in November 2005. Prevented from conducting these inspections more often by financial constraints, the committee generally relies on reports from investigators and word of mouth (interviews, K_17:2005 and K_25:2005). The South African DoH guidelines require at a minimum that ethics committees request annual reports from principal investigators and establish a complaints procedure [13]; members of two different ethics committees described such measures as “passive monitoring”. As in Kenya, committees do not have the resources to carry out site visits (interviews, SA_10:2006 and SA_17:2006). The UNESCO declaration itself does not offer much by way of assistance, the only article that could possibly be taken to relate to post-approval monitoring stating, “Appropriate assessment and adequate management of risk related to medicine, life sciences and associated technologies should be promoted” [20, p. 10].12 Perhaps UNESCO’s ethics programme could help with training in these areas or encourage better funding, however. As highlighted by Singer and Benatar, capacity building is likely to advance research ethics further than are reformulations of bioethical principles. It may be, then, that the added value of the declaration will prove to lie more in UNESCO’s follow-up activities than in the document itself, at least in the medium term, the innovative sections on social responsibility and benefit sharing notwithstanding. Conclusion This paper has revealed something of a loose consensus, at least between UNESCO and those involved in bioethics in Kenya and South Africa, on two counts. With regard to universal principles, any attempt to implement them at national levels must be contextualised. Working out how to apply such principles in particular social and economic contexts is arguably as challenging as reaching agreement on how they should be constituted in the first place. With regard to the usefulness of the UNESCO declaration, the significance of its adoption as the first intergovernmental instrument on bioethics must be matched by action in the form of capacity building for it to be of added value in the realm of biomedical research ethics. The scope of this paper has been limited to a primarily pragmatic analysis of how universal principles can be applied at national level. The paper has not commented on whether the value of the universality that UNESCO has aimed towards in terms of a foundation for humanity is compromised if these applications are very different. Deeper reflection on the nature of universality in this context would require the input of trained bioethicists and philosophers. This paper highlights, then, the need for a cross-disciplinary approach to the analysis of international bioethics instruments. The scope of the study that engendered this paper was necessarily limited by the time and resource constraints of doctoral research. Further research could explore whether the experiences of other African countries have been similar to those of Kenya and South Africa. Francophone and lusophone states, in particular, may have quite different bioethical traditions and thus have had very different experiences in implementing bioethics policies.

J_Autism_Dev_Disord-4-1-2254472

Brief Report: Eye Movements During Visual Search Tasks Indicate Enhanced Stimulus Discriminability in Subjects with PDD

Subjects with PDD excel on certain visuo-spatial tasks, amongst which visual search tasks, and this has been attributed to enhanced perceptual discrimination. However, an alternative explanation is that subjects with PDD show a different, more effective search strategy. The present study aimed to test both hypotheses, by measuring eye movements during visual search tasks in high functioning adult men with PDD and a control group. Subjects with PDD were significantly faster than controls in these tasks, replicating earlier findings in children. Eye movement data showed that subjects with PDD made fewer eye movements than controls. No evidence was found for a different search strategy between the groups. The data indicate an enhanced ability to discriminate between stimulus elements in PDD. Introduction While most diagnostic criteria of Pervasive Developmental Disorder (PDD) are characterised by impairments, mostly with respect to social and communicational abilities, subjects with PDD excel on certain visuo-spatial tasks that supposedly reflect superior processing of fine detail (see Dakin & Frith, 2005, for a review). An especially robust finding seems to be the behavior of subjects with PDD on visual search tasks (Plaisted, O’Riordan, & Baron-Cohen, 1998; Plaisted, Saksida, Alcantara, & Weisblatt, 2003; O’Riordan, Plaisted, Driver, & Baron-Cohen, 2001). In such tasks, subjects are required to detect a target in a display containing a variable number of distracters. If the task difficulty is increased by adding distracters, usually RTs increase. In studies on PDD, two search task versions that differed in difficulty have been used. In both tasks children with PDD showed shorter RTs, as compared with normally developing controls (Plaisted et al., 1998; Plaisted et al., 2003; O’Riordan et al., 2001). The reason for this superior performance is not clear, but has been related to enhanced ability to discriminate between stimulus elements in subjects with PDD (O’Riordan & Plaisted, 2001), an explanation that has also been proposed for the superior performance of subjects with autism on other visuo-spatial tasks (Plaisted et al., 2003, see also Bertone, Mottron, Jelenic, & Faubert, 2001). So far, however, there have been no studies that have validated the claim for enhanced stimulus discrimination as an explanation for superior performance in search tasks in PDD more directly. It is well-known that stimulus discriminability affects eye movement parameters, especially number of fixations and fixation duration (Hooge & Erkelens, 1999). Therefore, studying eye movements during search tasks in subjects with PDD is a first step to gain more insight into the neurocognitive mechanisms of atypical visuo-spatial processing in this group. If increased stimulus discriminability is indeed the underlying factor for superior performance of subjects with PDD, it is expected that they need fewer and/or shorter fixations to identify the target. Also, eye movement parameters can be used to test alternative hypotheses for shorter reaction times in search tasks in PDD. More specifically, it has been argued that (healthy) subjects in a visual search task have a tendency to move their eyes even though in some situations it would be a better strategy not to do so, since longer fixations allow better extraction of (peripheral) information (Hooge & Erkelens, 1999; see also Rayner, 1998). It is well possible that individuals with PDD use a different search strategy in which they show longer, but less, fixations than controls. The first aim of the present study was to use eye movement parameters to test the two hypotheses described above for superior visual search in subjects with PDD. A second aim was to replicate the findings of Plaisted et al. (1998) and O’Riordan and Plaisted (2001) in high functioning adults with PDD. For these reasons we used the same search tasks as described in O’Riordan et al. (2001, second experiment) in a group of high functioning adults with PDD, matched on gender, IQ, and age to a control group. Methods Participants Two groups of young male adults participated, a group of eight high functioning individuals with PDD and a group of eight normal control individuals. The clinical subjects were recruited via the Department of Child and Adolescent Psychiatry at the Utrecht University Hospital. The study was described to the subjects and written informed consent was obtained. All subjects were administered the Wechsler Intelligence Scale, revised Dutch edition (WAIS). Diagnoses of either Autistic Disorder or Asperger Syndrome were made by a child psychiatrist and based on DSM-IV criteria. The parents of all subjects with PDD were administered the Autism Diagnostic Interview Revised (ADI-R) (Lord, Pickles, McLennan & Rutter, 1997; Lord, Rutter & LeCouteur, 1994). Due to technical problems, eye-movement data of one subject with PDD and one control subject were lost. The individual ADI scores of the remaining seven PDD subjects can be found in Table 1. Unpaired t-tests revealed that there were no significant differences between the remaining seven PDD and seven control subjects with respect to either age, TIQ, VIQ, or PIQ (see Table 2). None of the participants showed any visual or oculomotor pathology other than refraction anomalies. Subjects were allowed to wear their contact lenses or glasses. Table 1ADI scores on the four domains for individual PDD subjectsADI scores individual subjectsSocial interaction (cutoff 10)Communication (cutoff 8)Stereotypy (cutoff 3)Age of onset (cutoff 1)12218202181390324163542616855242410362120437281752Table 2Mean age and IQ characteristics of the control and PDD groupControlPDDAge21.222.1TIQ115.3121.9VIQ119.1124.0PIQ106.7114.0 Set up and Data Analysis Subjects sat in front of a LaCie Blue Electron lll 22′ Screen (0.394 m × 0.295 m, 1240 × 1024 pixels at 85 Hz,) at a distance of 0.64 m. Stimuli were generated by a Apple PowerMac G4/450 DP using a Matlab program based on routines taken from the Psychophysics Toolbox (Brainard, 1997; Pelli, 1997) and the EyeLink Toolbox (Cornelissen, Peters & Palmer, 2002). Movements of the left eye were measured at 250 Hz with the EyeLink 1 eye monitor. Head movements were prevented by the use of a chinrest. Data were stored on disk and were analysed off-line by a self-written Matlab program. The velocity signal of eye movements was searched for peak velocities above 20°/s. Each peak (in the velocity signal) was considered a potential indicator of the presence of a saccade. The exact onset of the saccade was determined by going backward in time to the point where the velocity signal dropped below the average velocity plus two standard deviations during the stable fixation period before the saccade. The exact offset of the saccade was determined by going forward in time to the point where the velocity signal dropped below the average velocity plus two standard deviations during the stable fixation period after the saccade. This method was adopted from Van der Steen and Bruno (1995). This procedure was followed by rejection/acceptation based on minimum saccade duration of 12 ms and minimum amplitude of 1°. When a saccade was removed, fixation time before and after this saccade and the duration of the saccade were added together. Stimulus and Task Two tasks were tested in separate sessions for each participant, with the order counterbalanced within participant groups. Both tasks contained tilted as well as vertical line elements. The tasks only differed in which of the two elements was target or distracter. Since an earlier study has shown a difference in task difficulty depending on target orientation (O’Riordan et al., 2001), the condition including a vertical target among tilted distracters will be referred to as the easy condition, and the condition with a tilted target (among vertical distracters) as the hard condition. Each task contained two crossed factors; set size (4, 16, or 25 items) and probe, target present or target absent. There were 30 trials at each unique combination of factors, yielding a session of 180 trials per task. The sequence of different tasks was randomised within each session. In each session, the participant was informed about the nature of the target (either vertical or tilted), but did not know when a target would be present or absent, or what the display size would be on any trial. The participants performed a binary choice RT task indicating whether a target was present or absent by a button press on each trial. Procedure The order in which the two conditions were presented was counterbalanced. Prior to each task participants were given 10 practice trials. Participants were instructed to respond as quickly and accurate as possible by pressing the five key on the numeric pad when the target was present and the four key when the target was absent. Thereafter, the eye link camera was attached to the head and calibrated. On each trial the sequence of events was as follows: a fixation cross was presented. The participant had to fixate at the cross and press the space bar subsequently. Fixation of the fixation cross was used for on-line drift correction of the eye tracker. Then, the search display appeared and remained visible until the participant responded. After the response the next trial started. Results To assure that a possible superior task performance of the individuals with PDD was not due to a difference in detection criteria (e.g., if speeded reaction time is accompanied by a decrease in number of correct answers), we performed an independent t-test on the error rates of both groups. No significant difference in error rate between the groups was found. One of the subjects in the PDD group showed a high number of errors and a deviant pattern in reaction time data, and was excluded from further analysis. The results described below are therefore based on six subjects with PDD, and seven controls. The mean RT, first fixation times, remaining fixation times, and number of fixations data were analysed using a repeated measures ANOVA, with one between subjects factor of group (control or autistic) and three within subject factors, condition (condition 1: tilted target or condition 2: vertical target), probe (target present or target absent) and set size (4, 16, or 25 items). Analyses of the first and remaining fixations were done separately, because there is evidence that they belong to different distributions (Hooge & Erkelens, 1996). Unless otherwise stated, a significance level of p < .05 was adopted for all statistical comparisons. Only main effects or interactions with Group will be considered. RT Analysis The analysis of variance revealed a main effect of Group, F(1,11) = 6.6, p < 0.05, reflecting the fact that individuals with PDD were significantly faster overall than the control individuals (737 vs. 1016 ms). A significant interaction Group × Display was found (F(2, 22) = 3.8, p < .05), as well as an interaction of Group × Display × Probe (F(2, 22) = 4.7, p < .05). Partial analyses showed that the Groups difference was significant at all set sizes, but that the effects were largest for set size 25 (all p < .05) (resp., F(1,11) 5.7, 5.4, 6.8). The interaction Group × Probe was marginally significant for set size 25 only (F(1,11) = 4.0, p = 0.72), indicating that at this set size, the Groups difference was largest if the target was absent (See Fig. 1). Fig. 1Means and standard errors of the reaction times for each group in trials with (left panel) and without a target (right panel), for the easy and hard condition Eye Movement Analysis The analysis of number of fixations revealed a significant effect of group (F(1,11) = 10.3, p < .01). The individuals with PDD made significantly less fixations than the control group (means, respectively, 1.3 and 2.1). In addition, there were significant interactions between Group × Probe, Group × Display and Group × Probe × Display. When tested per level of Display, subjects with PDD showed significantly fewer fixations for all display sizes (F(1,11) = 8.2, p = .015/9.4, p = .011/10.8, p = .007), but the effects were largest for the 25 set size. Interactions of Group × Probe effects were marginally significant for all set sizes (F(1,11) = 4.7, p = .052/4.0, p = .072/4.4, p = .06), indicating that group differences in number of fixations were largest in the target absent condition (see Fig. 2). Fig. 2Means and standard errors of the number of fixations for each group in trials with (left panel) and without a target (right panel), for the easy and hard condition No significant group differences were found for fixation times at either the first fixation or the remaining fixations. Discussion The present study was aimed to test two hypotheses on superior performance in visual search tasks in subjects with PDD. It has been suggested earlier that this superior performance reflects an enhanced ability to discriminate between stimulus elements (O’Riordan et al., 2001). We tested this hypothesis by measuring eye movements during search tasks in high functioning adult men with PDD and an age- and IQ-matched control group. In case of enhanced stimulus discriminability, shorter fixation times and/or fewer fixations were expected in the PDD group, as compared to the control group. The alternative explanation, that subjects with PDD use more efficient search strategies, was also tested. A more efficient search strategy would be reflected in a longer fixation time. The reaction time data showed that subjects with PDD were better than the control group for both easy and hard search tasks, especially in the largest set size and in trials in which the target was absent. These findings are in accordance with earlier studies on visual search in children with autism, indicating faster RT in the same type of tasks (O’Riordan et al., 2001; Plaisted et al., 1998). This indicates that superior search performance in subjects with PDD is a robust finding that can be demonstrated in both children and adults. Analyses of fixations during the search tasks showed that subjects with PDD made fewer fixations than controls. This difference in number of fixations was especially clear for the largest set size and for the condition in which the target was absent, analogous to the RT effects. In addition, it was noted that many subjects with PDD showed an absence of saccadic eye movements in target present trials, indicating that they were able to localize the target in a single glance. No differences between groups were found in fixation time. The absence of group differences in fixation time indicates that subjects with PDD followed the same search strategy as normal controls. However, the decreased number of fixations during the task in the PDD group is in agreement with the hypothesis of enhanced stimulus discriminability in subjects with PDD, as suggested by O’Riordan and Plaisted (2001). The finding that the pattern of effects for fixation times was strongly similar to the RT effect provides additional evidence that superior performance in this task is indeed related to enhanced discriminatory abilities in PDD. The question what the locus of this enhanced discriminability could be has been addressed by Cohen (1994), who tested a neural network model of processing in subjects with PDD. This model showed that an increase in processing units resulted in a strong ability to discriminate and learn stimulus patterns, along with a weak ability for generalization. Indeed, there is anatomical evidence for abnormal development of the cyto-architecture of the cerebral cortex of subjects with PDD, resulting in an increase in processing units. Recent post-mortem studies in subjects with PDD show an increased number of micro columns, albeit of reduced size (Casanova, Buxhoeveden, Switala, & Roy, 2002; Casanova et al., 2006). Concluding, the measurement of eye movements during search tasks shows that the superior behavior of subjects with PDD in these tasks cannot be attributed to strategy difference, but point indeed to increased stimulus discriminability in this group.

Mol_Biochem_Parasitol-1-5-2075530

The role of metacaspase 1 in Plasmodium berghei development and apoptosis

The malaria parasite encodes a wide range of proteases necessary to facilitate its many developmental transitions in vertebrate and insect hosts. Amongst these is a predicted cysteine protease structurally related to caspases, named Plasmodium metacaspase 1 (PxMC1). We have generated Plasmodium berghei parasites in which the PbMC1coding sequence is removed and replaced with a green fluorescent reporter gene to investigate the expression of PbMC1, its contribution to parasite development, and its involvement in previously reported apoptosis-like cell death of P. berghei ookinetes. Our results show that the pbmc1 gene is expressed in female gametocytes and all downstream mosquito stages including sporozoites, but not in asexual blood stages. We failed to detect an apparent loss-of-function phenotype, suggesting that PbMC1 constitutes a functionally redundant gene. We discuss these findings in the context of two other putative Plasmodium metacaspases that we describe here. 1 Introduction Malaria remains the most important parasitic human disease responsible for millions of deaths each year. The life cycle of malaria parasites is complex involving multiple life stages and requiring two hosts: mosquito and human. The uptake of gametocytes by the mosquito leads to rapid gametogenesis and fertilisation. Ookinetes are formed in the mosquito midgut lumen, which cross the midgut wall and transform into oocysts. Each oocyst can produce thousands of sporozoites, which after release traverse the mosquito hemolymph and infect the salivary gland tissues before they can enter the human host via the mosquito bite. Following inoculation, sporozoites infect hepatocytes and develop into liver schizonts. Liver schizont produce merozoites, which upon release into the blood stream initiate the erythrocytic cycle, the prime cause of clinical symptoms in malaria patients. Existing malaria control strategies are hampered by increasing parasite resistance in the field to established antimalarial drugs, as well as by widespread insecticide resistance in the mosquito vectors. Effective antimalarial vaccines are only at the preliminary stages of development. There is thus a great need to develop novel strategies for malaria control. Proteases are attractive antimalarial targets because of their indispensable roles in parasite development and infectivity. A wide variety of proteases of malaria parasites have been described [1]. Amongst these are cysteine proteases belonging to the Peptidase_C14 family (clan CD) which include caspases. Caspases belong to a distinct class of cysteine proteases with a so called Caspase Hemoglobinase Fold (CHF) [2]. Recently, two new families of predicted CHF proteases closely related to the caspases were identified, named paracaspases and metacaspases [3]. In metazoa, caspases are centrally involved with the molecular machinery of programmed cell death (apoptosis), and are responsible for many of the biochemical and morphological changes that accompany it. They cleave a variety of proteins ultimately leading to the disintegration of the cell [4]. Metacaspases have thus far only been identified in organisms that do not possess classic caspase genes: plants, fungi and the parasitic protists Trypanosoma, Leishmania and Plasmodium, and there is some evidence linking metacaspases to apoptosis in these organisms [5–10]. In Plasmodium falciparum a metacaspase gene (GenBank accession CAD52669, here named PfMC1) has been described possessing consensus histidine and cysteine residues that typically form the catalytic dyad in this family of proteases [1,3], suggesting that this parasite species may possess a mechanism of programmed cell death. Indeed, apoptosis-like DNA fragmentation has been reported to occur in blood stage parasites of P. falciparum in response to chloroquine treatment [11]. In addition, ookinetes of Plasmodium berghei were reported to undergo an apoptosis-like mechanism of cell death resulting in very substantial parasite losses [12]. In this study we investigate the role of PbMC1 in the reported programmed cell death in Plasmodium by constructing PbMC1-deficient parasites. We discuss our findings in the context of two additional Plasmodium metacaspase-like proteins that we describe here. 2 Materials and methods 2.1 Parasite maintenance, culture and purification P. berghei ANKA (clone 2.34) parasites were maintained in female TO mice by mechanical blood passage and regular transmission in Anopheles stephensi, as described [13]. Ookinete cultures were set-up as described [12,13] both with and without prior removal of white blood cells by CF11 columns. In general, 21–24 h ookinete cultures were centrifuged at 1500 × g for 5 min and the cell pellet incubated on ice in 20 volumes of 0.17 M NH4Cl to lyse the red blood cells. In some experiments ookinetes were further purified on Nycodenz cushens. Finally, parasites were concentrated by centrifugation at 1500 × g for 5 min, resuspended in RPMI 1640 or phosphate buffered saline, and immediately analysed. 2.2 Generation of PbMC1-KO parasites A 720 bp fragment was amplified by PCR from P. berghei genomic DNA with primers IF-MC3′-F (TAAACCATTGGTCATACCAAAAAATAATCAAAAAAATAACCAA) and IF-MC3′-R (CGGGCCGCTCTAGCATGACCAGGCTCAATAATTGAACA) and introduced into NdeI-digested pLP-DHFR2 via In-Fusion PCR cloning (BD Biosciences). The resulting plasmid, pLP-DHFR/MC, contains a loxP-prokaryotic promotor cassette (BD Biosciences), followed by a modified Toxoplasma gondii dihydrofolate reductase (dhfr) gene cassette [14], followed by the 3′UTR of pbmc1. A 680 bp fragment was amplified by PCR from P. berghei genomic DNA with primers pDNR-ΔMC-F (ACGAAGTTATCAGTCGACGGTACCCCATCATAAAGCAAAAAGC) and pDNR-ΔMC-R (ATGAGGGCCCCTAAGCTTATTTAACATAAATTTTGTCCATTT) and introduced into SalI/HindIII-digested pDNR-EGFP via In-Fusion PCR cloning. The resulting plasmid, pDNR-ΔMC/EGFP, contains two loxP sites flanking the 5′UTR plus first 11 codons of pbmc1 fused in-frame to the enhanced green fluorescent protein (egfp) gene, followed by the 3′UTR of P. berghei dhfr. The pbmc1-specific insert contained within pDNR-ΔMC/EGFP was introduced into pLP-DHFR/MC via Cre-loxP site-specific recombination (BD Biosciences) to give the transfection vector pLP-ΔMC/EGFP. Prior to performing transfections this plasmid was digested with KpnI and NotI to remove the vector backbone. Parasite transfection, pyrimethamine selection and dilution cloning were performed as previously described [15]. Genomic DNA extraction, and Southern blot were performed as previously described [14]. 2.3 Apoptosis assays Phosphatidylserine (PS) translocation was studied with fluorescein isothiocyanate (FITC)-conjugated Annexin V in conjunction with propidium iodide, by using the Annexin-FITC Apoptosis Detection Kit (Sigma–Aldrich) according to manufacturer's instructions. DNA condensation in the nucleus was assessed using acridine orange staining as described [12]. DNA fragmentation was assessed by in situ terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labelling (TUNEL), by using the ApopTag® Fluorescein In Situ Apoptosis Detection Kit (Chemicon International) according to manufacturer's instructions. Caspase activation was assessed with sulphorhodamine-VAD-FMK inhibitor of caspases, by using the CaspaTag™ Pan-Caspase In Situ Assay Kit (Chemicon International) according to manufacturer's instructions. 3 Results 3.1 Description of Plasmodium metacaspases Using BLAST searches of available Plasmodium genome sequences with the amino acid sequence of PfMC1 we identified orthologues in P. berghei and other Plasmodium species including P. chabaudi, P. yoelii, P. knowlesi, P. vivax and P. gallinaceum. Multiple alignment of the predicted PxMC1 proteins revealed conserved amino- and carboxy-terminal domains, separated by a region of variable length and sequence. These amino- and carboxy-terminal domains had, in Pfam and SMART searches, significant homologies with a C2 domain (a Ca2+-dependent membrane targeting module, Pfam identifier PF00168) and with the Peptidase_C14/Caspase domain (Pfam identifier PF00656), respectively (Fig. 1A). We also identified two paralogues of PxMC1 (named PxMC2 and PxMC3) in all Plasmodium species, each containing a region of significant homology to the Peptidase_C14/Caspase domain in Pfam and SMART searches. In contrast to the PxMC1 proteins however, sequence conservation in the much larger PxMC2 and PxMC3 proteins appeared to be limited to their C-terminal regions containing the putative caspase domains (Fig. 1A). Multiple alignment of the predicted caspase domains of PxMC1, PxMC2 and PxMC3 from P. berghei, P. falciparum and P. vivax, along with a metacaspase from yeast (YCA1) [6] shed further light on their structures (Fig. 1B). Like PfMC1, all PxMC1 orthologues possess histidine and cysteine residues predicted to form the catalytic dyad in this family of proteases. It is interesting to note that PbMC1 has the predicted catalytic cysteine located one residue upstream of the consensus position for this amino acid, with a proline in the consensus position (Fig. 1B). The same is true for PcMC1 and PyMC1 (data not shown). In contrast, PvMC1 has two adjacent cysteines, at the consensus position and the preceding residue (Fig. 1B). The same is found in PkMC1 and PgMC1 (data not shown). Catalytic dyad histidine and cysteine residues are absent in the consensus positions in the PxMC2 and PxMC3 proteins (Fig. 1B). However, PfMC3 and PvMC3 have a cysteine at the preceding amino acid position, similar to the situation in PxMC1 of rodent malaria species. 3.2 Generation and molecular analyses of PbMC1 knockout parasites Because PxMC1 proteins display the greatest sequence similarity with the consensus Peptidase_C14 domain and, of the three Plasmodium metacaspases described here, are therefore most likely to be active enzymes, we decided to investigate the expression of PbMC1 and its contribution to P. berghei parasite development. To this purpose we generated genetically modified parasites in which the PbMC1 coding sequence was removed and replaced with a reporter gene, enhanced green fluorescent protein (EGFP). In the strategy used, the 5′UTR and 3′UTR of the pbmc1 gene allow for double homologous recombination [15,16], introducing the EGFP coding sequence downstream of the first 11 codons of PbMC1 (coding for MSLQMDKIYVK), as well as inserting a modified T. gondii dihydrofolate reductase/thymidylate synthase (tgdhfr/ts) gene cassette conferring resistance to the antimalarial drug pyrimethamine into the pbmc1 locus (Fig. 2A). After transfection of purified P. berghei schizonts [15] we readily obtained pyrimethamine-resistant parasites. Diagnostic PCR amplification across the predicted integration sites showed that correct integration of the DHFR/TS cassette into the PbMC1 locus had occurred (data not shown). This was confirmed by assessing the integrity of a clonal population of the genetically modified parasite (named PbMC1-KO) by Southern blot analysis of HincII-digested genomic DNA. Accordingly, a probe corresponding to the 5′UTR plus PbMC1 coding region gave rise to expected bands of 4.5 and 2.1 kb in the parental wild-type (WT) parasites (Fig. 2B). In contrast, in the PbMC1-KO parasites expected bands of 4.5 and 0.9 kb were observed. The 0.9 kb fragment corresponds to 360 bp of the 5′UTR of pbmc1, its first 11 codons, plus 490 bp of the egfp gene (Fig. 2B). The 4.5 and 0.9 kb bands are more weakly labelled than the 2.1 kb band, because a smaller portion of the probe anneals to these fragments (Fig. 2A). A probe corresponding to the tgdhfr/ts gene gave rise to bands of 2.6 and 0.9 kb in the PbMC1-KO parasites, but no signal in the WT parasite sample as expected (Fig. 2B). These combined results are fully consistent with the structural removal of the PbMC1 coding sequence, and the integration of the reporter and selectable marker genes into the pbmc1 locus. 3.3 PbMC1 expression and loss-of-function phenotype PbMC1-KO parasites developed normally in mice and were morphologically indistinguishable from WT parasites in Giemsa-stained blood smears. Using UV microscopy EGFP fluorescence was readily observed in a subpopulation of parasitized erythrocytes. Upon gametocyte activation (by placing the infected blood in ookinete medium) the large majority (>90%) of fluorescent parasites emerged from the host cell (clearly visible by the rounding up of the parasite) by 15 min, typical of gametogenesis. The number of remaining intracellular fluorescent parasites was considerably lower than the percentage of trophozoites present in the sample as assessed by Giemsa staining. These are therefore likely to constitute immature gametocytes, or gametocytes that had failed to emerge. We were further able to distinguish between male and female gametocytes by observing exflagellation (i.e. the release of male gametes); this involved gametocytes that were not green. From these combined observations we conclude that the pbmc1 gene is expressed only in female gametocytes (Fig. 3A). As far as we could ascertain all females were positive and all males negative. Normal differentiation of gametocytes into ookinetes both in vitro and in vivo was observed. These ookinetes again displayed bright EGFP-based fluorescence under UV light (Fig. 3B). WT and PbMC1-KO parasite-infected A. stephensi mosquitoes developed comparable numbers of oocysts, indicating that the PbIMC1a-KO ookinetes are capable of normal midgut invasion and subsequent ookinete-to-oocyst transition on the hemocoel side of the midgut wall. Examination of oocysts on the midgut wall by UV microscopy displayed EGFP fluorescence in oocysts and the sporozoites contained within (Fig. 3C). Fluorescent sporozoites were observed in the mosquito salivary glands (Fig. 3D), which were infectious to mice upon infected mosquito bite. These combined results show that the pbmc1 gene is expressed in female gametocytes and all downstream mosquito stages including sporozoites, but not in the asexual blood stages. The apparently normal development of the PbMC1-KO parasite both in the vertebrate and insect hosts shows that pbmc1 is not an essential gene and may be functionally redundant. 3.4 Apoptosis in ookinetes It has been reported that, in P. berghei, a large proportion of ookinetes undergo cell death, displaying typical apoptosis-like features such as PS translocation, nuclear condensation and DNA fragmentation [12]. As caspases are well known to play a central role in apoptosis, we wanted to assess the effect of PbMC1 knockout on this process. Initially, we assessed the level of apoptosis-like cell death in WT ookinetes. Annexin V conjugated to FITC (staining green), which specifically binds to PS, was used to assess the proportion of ookinetes displaying PS translocation to the outer leaflet of the cell membrane. Propidium iodide (staining red) was used simultaneously to assess plasma membrane integrity (cell viability). Across 13 experiments conducted we observed an average 77% viable ookinetes (no staining), while an average 21% appeared dead (red only staining) (Fig. 4). The average proportion of ookinetes displaying PS translocation (green staining) was less than 3%. Only 0.5% of ookinetes examined showed PS translocation and were propidium iodide negative (green only staining) (Fig. 4). In addition to this assay, we used acridine orange staining to look for nuclear condensation, and TUNEL to assess DNA fragmentation in P. berghei ookinetes, as described [12]. We found no evidence for either of these processes occurring. Given the low numbers of positive cells in the above assays, we decided to look for the activation of caspases, a key event in caspase-dependent apoptosis, by using a fluorochrome-labelled inhibitor of caspases (CapaTag). In mammalian cells, these labelled inhibitors are permeant and covalently bind to the active centres of caspases with a 1:1 stoichiometry. In this assay, WT ookinetes displayed an average 3.8 ± 0.5% of positive cells (two experiments) after 21 h of culture, which, at 24 h had gone up to an average 14 ± 9% (four experiments). Under the same conditions, PbMC1-KO ookinetes showed an average 2.6 ± 0.3% positive cells (two experiments) at 21 h, and 13 ± 6% (five experiments) at 24 h of culture. We also used a green fluorescent P. berghei parasite with an intact pbmc1 gene (PbGFPCON [17]), that displayed an average 3.8% and 9.0% of positive ookinetes at 21 and 24 h, respectively. Thus, knockout of PbMC1 does not appear to significantly affect the percentage of ookinetes that bind CaspaTag. 4 Discussion In this paper we describe the generation of P. berghei parasites in which the caspase-like protein PbMC1 is replaced by the fluorescent reporter EGFP, allowing us to study both its expression profile and its loss-of-function phenotype using a single genetically modified parasite line. Our EGFP reporter data show the pbmc1 gene to be transcribed, and probably translated, in female gametocytes and all downstream mosquito stages including sporozoites, but not in asexual blood stages. However, we cannot rule out that the pbmc1 gene may be subject to translational repression. There are many examples of genes that are transcribed in the female gametocyte, but which have a translational block and are not translated until later in development, as was recently published [18]. Interestingly, pbmc1 is not one of the genes identified by these authors, supporting a scenario of direct translation. Knockout of PbMC1 expression does not seem to adversely affect any stage of parasite development, at least in vivo in the mouse and mosquito, or in the culture systems used in this study. This was surprising because pxmc1 is a highly conserved single copy Plasmodium gene, and because PxMC1 is predicted to possess consensus catalytic dyad residues and thus is likely to possess caspase activity. Although the two paralogues, PxMC2 and PxMC3, do not share the typical consensus catalytic dyad residues of the Peptidase_C14/Caspase family, it is possible that they constitute active zymogens by using alternate, functionally equivalent catalytic residues. Indeed, functional replacement between cysteine, serine or threonine residues has been reported for other cysteine and serine proteases [19–21]. If this is the case it is conceivable that one or both of the paralogues could carry out the role of PbMC1, hence leading to functional redundancy and resulting in an apparently normal mutant phenotype as observed. Interestingly, five metacaspase genes have been identified in Trypanosoma brucei, TbMCA1-TbMCA5, and two of these also lack a conserved histidine residue (replaced by a tyrosine, TbMCA1) and/or cysteine residue (replaced by a serine, TbMCA1 and TbMCA4) (reviewed in [22]). Whether any of these molecules are active enzymes remains to be determined. It has been previously shown in plants and fungi [6,9] that metacaspases can be involved in the regulation or the execution of programmed cell death. Because an apoptosis-like mechanism of cell death was reported to take place in ookinetes of P. berghei [12] we hypothesized that PbMC1 could be involved in this phenomenon. Of three typical apoptotic markers: PS translocation, nuclear condensation and DNA fragmentation, only PS translocation gave positive results in our hands. PS translocation does also occur when cells die from accidental cell death (oncosis), in which case membrane integrity is lost concomitantly. Accordingly, we cannot truly determine whether ookinetes that in our assay stained doubly positive for both Annexin V and propidium iodide were in the later stages of apoptosis, or whether they died from an unrelated death mechanism. The fact that a subset of ookinetes stained positive for Annexin V suggests that the low numbers of positive cells observed are not a result of a reduced PS content in the parasite plasma membrane. Overall, the results based on these three apoptotic markers indicate that apoptosis-like death in ookinetes, in our hands, is low. When we monitored for potential apoptotic ookinetes using CaspaTag, we obtained up to 14% positive cells. Surprisingly, no reduction of CaspaTag labelling was observed in PbMC1-KO parasites. Although PbMC1 is the most likely target of the CaspaTag inhibitor, it is possible that other targets of CaspaTag are present in ookinetes, which could mask any reduction in CaspaTag binding in the PbMC1-KO parasite. Clearly, the other two putative metacaspases are prime candidates. Indeed, P. falciparum transcriptome and proteome analyses indicate that PfMC2 is expressed in gametocytes and sporozoites [23–25]. Hence, its expression in other mosquito stages including ookinetes is likely. This scenario could also explain the normal development of the PbMC1 null mutant parasites by assuming a level of functional redundancy between the three Plasmodium metacaspases. Expression and knock out experiments with the other metacaspases are underway to test this hypothesis. Caspase activation is one of the earliest events in apoptotic mammalian cells. Hence, early apoptotic cells have activated caspases, but display no changes in plasma membrane integrity [26]. It was therefore notable that no EGFP-based fluorescence was observed in CaspaTag positive ookinetes neither in the PbMC1-KO nor PbGFPCON parasite lines, indicating that these cells had lost plasma membrane integrity before CaspaTag labelling occurred. This is consistent with results from Al-Olayan et al. [12] who reported that their caspase inhibitor-positive ookinetes were propidium iodide permeable. Moreover, we found that pre-treatment with the unlabelled inhibitor failed to reduce subsequent binding of CaspaTag (data not shown). In mammalian cells this has been reported to reduce CaspaTag binding by over 90% [26]. We should thus consider the possibility that these caspase inhibitors may have different specificities in Plasmodium than they do in mammalian cells. It is difficult to explain why in our hands the number of potentially apoptotic ookinetes detected was far below the numbers previously reported [12]. Despite numerous attempts we were unable to find conditions that led to increased numbers of ookinetes displaying apoptotic markers. We cannot rule out that small differences in the experimental set-ups might explain the conflicting findings. Alternately, the level of apoptosis detected in P. berghei ookinetes may depend on multiple factors that are still to be defined, for instance the exact conditions under which the gametocytes are generated in the mouse host. A not dissimilar discrepancy also exists for the reported apoptosis-like DNA fragmentation that occurs in blood stage P. falciparum parasites in response to chloroquine treatment [11], an observation that was not corroborated in a more recent study [27].

Atherosclerosis-2-1-2292239

Early changes in arterial structure and function following statin initiation: Quantification by magnetic resonance imaging

Effective LDL-cholesterol (LDL-C) reduction improves vascular function and can bring about regression of atherosclerosis. Alterations in endothelial function can occur rapidly, but changes in atherosclerosis are generally considered to occur more slowly. Vascular magnetic resonance imaging (MRI) is a powerful technique for accurate non-invasive assessment of central and peripheral arteries at multiple anatomical sites. We report the changes in atherosclerosis burden and arterial function in response to open label statin treatment, in 24 statin-naïve newly diagnosed stable coronary artery disease patients. Patients underwent MRI before, and 3 and 12 months after commencing treatment. Mean LDL-C fell by 37% to 70.8 mg/dL (P < 0.01). The plaque index (normalised vessel wall area) showed reductions in the aorta (2.3%, P < 0.05) and carotid (3.1%, P < 0.05) arteries at 3 months. Early reductions in atherosclerosis of aorta and carotid observed at 3 months were significantly correlated with later change at 12 months (R2 = 0.50, P < 0.001; R2 = 0.22, P < 0.05, respectively). Improvements in aortic distensibility and brachial endothelial function that were apparent after 3 months treatment were sustained at the 12-month time point. 1 Introduction HMG CoA reductase inhibitors or ‘statins’ reduce cardiovascular events by approximately 25–30% in patients with stable atherosclerotic disease [1,2]. Previous studies of ultrasound carotid intima media thickness (CIMT) have suggested relatively slow regression of atherosclerosis after 1–2 years of high dose statin treatment [3,4]. More recently, intravascular ultrasound (IVUS) of the coronary arteries has demonstrated coronary plaque regression following intensive LDL-C reduction in over 300 patients treated with 40 mg rosuvastatin for 2 years [5]. Corti et al. [6] were among the first to use serial magnetic resonance imaging (MRI), to demonstrate reduction in carotid and aortic atherosclerosis in 18 patients in response to 12 months of statin treatment. Subsequently, the same group demonstrated that more effective lipid lowering, to LDL-C < 100 mg/dL, was associated with a larger decrease in plaque size at 12 months [7]. Similarly, Saam et al. [8] have recently identified statin treatment as an independent predictor of reduced annual rate of carotid atheroma progression, measured using MRI. In the setting of acute coronary syndromes, early initiation of ‘intensive’ statin treatment can rapidly reduce cardiovascular risk within 4–16 weeks [9,10]. However, given the apparently slow changes in atheroma burden observed in previous studies [6], it has been hypothesised that the early clinical benefits of statins cannot reflect structural changes within arteries, but are due to ‘pleiotropic’ effects of statins such as improvement in endothelial function and reduction in inflammation and thrombosis [11]. Indeed, numerous studies have demonstrated rapid improvement in endothelial function in response to statin treatment [12–14]. However, there is also evidence that rapid changes in plaque size and composition might also be possible. Animal models of atherosclerosis indicate that potent correction of dyslipidaemia can result in prompt regression and favourable remodelling of plaques after only 9 weeks [15]. Furthermore, in humans, a recent study of treatment with intravenous apoAI-Milano induced modest but significant regression of coronary atherosclerosis after only 6 weeks [16]. We have previously shown that MRI can assess both central and peripheral vascular function, including measurements of arterial stiffness and endothelial function [17,18]. These parameters are of additional interest because they have been shown prospectively to predict cardiovascular risk [19,20]. By assessing both atherosclerosis and vascular function in the same patients we hoped to examine the extent to which changes in structural parameters might be anticipated by early functional changes. In this study, we used magnetic resonance imaging to evaluate, in vivo, the changes in structure and function in human aorta, carotid and brachial arteries at 0, 3 and 12 months in response to statin therapy. Notably, the mean post-treatment LDL-C achieved in this study reached the contemporary target of 70 mg/dL [21]. 2 Methods 2.1 Study population Newly diagnosed patients (n = 32) with coronary artery disease were recruited from the Cardiology Department of a single tertiary centre. Diagnosis was based on a history of typical symptoms of angina together with an exercise ECG that showed ischaemic-type ST segment changes or a stenosis of ≥50% in at least one coronary artery at angiography. No patients had taken statins prior to study enrolment. No other cholesterol modifying agents were permitted, but no further restrictions were placed on concomitant cardiovascular medications. Patients with acute coronary syndromes or contra-indication to MRI were excluded. MRI was performed at baseline, 3 and 12 months. At each time point, venous blood samples were obtained after a 12-h fast. Statin treatment was withheld until immediately after the first MRI scan, when a statin was started according to the preference of the managing clinician. Decisions regarding cholesterol treatment and management of cardiac risk factors were taken by the responsible clinicians. Statin dose titration, whilst allowed, was not protocol-driven. The study protocol was approved by the local Research Ethics Committee and all subjects gave informed written consent. Of the 32 subjects initially recruited, 8 did not complete all three study time points: 1 patient died from an out of hospital cardiac arrest, 5 were unwilling to return for follow-up scans due to claustrophobia and 2 were lost to follow-up. The 8 patients who did not complete the study were not significantly different from the 24 who did in terms of either their risk factors (age, cholesterol, diabetes, hypertension and smoking) or MRI measures of atheroma or vascular function at baseline. The data reported below refer to the 24 patients, who completed the study protocol. 2.2 Vascular MRI protocol Imaging was performed on a 1.5 T magnetic resonance scanner (Siemens Sonata, Erlangen, Germany) as previously described [17,18]. In brief, aortic imaging was performed using a combination of a two-element array surface coil placed on the chest and spine coil array. For carotid artery imaging, a two-element array surface coil was used (Machnet BV, Eelde, Netherlands) and for brachial artery imaging a surface coil was attached at the right elbow. Brachial artery blood pressure was monitored using a blood pressure cuff on the left arm. For quantification of aortic wall area, ECG-gated double-inversion recovery (black-blood) fast spin echo images were acquired during breath-hold (Figs. 1b and 2b). Typical parameters were FOV 200 mm, TR 750 ms, TE 11 ms, in plane resolution 0.8 mm × 0.8 mm, slice thickness 5 mm. Using an oblique sagittal image of the aorta as a pilot, 11 serial axial images were acquired with 5 mm interslice gap to cover the entire descending thoracic aorta. The midpoint of the right pulmonary artery in cross section was used as the anatomical reference for the first slice in baseline and follow-up scans. For the carotid arteries axial ECG-gated T2 weighted, black blood images of the neck were acquired during free breathing (Fig. 2b). Sequence parameters: FOV 150 mm, TR 2 R–R intervals, TE 81 ms, resolution 0.5 mm × 0.5 mm in plane, slice thickness 3 mm, no interslice gap. Nine images of the common carotid artery were acquired using the common carotid bifurcation as the anatomical reference position for baseline and follow-up scans. Care was taken to place aortic and carotid image slices perpendicular to the long axis of the vessel on the pilot images in order to limit partial volume effects. For analysis of aorta and carotid plaque images all identifying data were removed apart from a code number so that observers were blinded to both patient identity and study time point. The external vessel boundary and vessel lumen were contoured manually by one of two experienced observers (J.L., C.S.) using CMRtools software (Imperial College, London, UK). Vessel wall area was calculated from the difference between these two contours, and then normalised to external vessel area to yield a plaque index (PI), as previously described [22,23]. Plaque index for each patient was then expressed as the mean of all aortic or carotid slices. In keeping with previous studies of carotid atherosclerosis, a mean value was obtained for left and right carotid arteries combined [3,4]. A subset of images from four randomly selected patients (>100 images) were analysed by both observers to assess inter-observer variability. Steady state free precession (SSFP) acquisitions were used to determine aortic distensibility and brachial artery reactivity, as previously described [17,18]. Post processing of aortic and brachial images was performed using semi-automated edge detection methods developed with Matlab software (Mathworks Inc.) [24]. Maximum and minimum aortic cross-sectional areas over the cardiac cycle were measured, from which distensibility was calculated as the relative change in area divided by the pulse pressure. To assess brachial artery flow mediated dilatation (FMD), cross sectional images of the brachial artery were acquired at baseline and following release of a cuff inflated to 50 mmHg above systolic blood pressure on the forearm for 5 min. After 10 min, further brachial artery images were acquired following administration of 400 μg of sublingual glyceryl trinitrate to elicit maximal (endothelial-independent) dilatation. Maximum percentage change in cross sectional area was used to determine the response to each stimulus. 2.3 Serum and plasma assays Cholesterol and lipoprotein assays were performed using a Cobas-Mira Analyser (ABX Diagnostics, Shefford, UK). Total cholesterol was assayed using the enzymatic CHOD-PAP method and triglycerides were assayed using the enzymatic GPO-PAP method. HDL-cholesterol was assayed using a homogenous second generation PEGME method (Roche Diagnostics, Burgess Hill, UK). Apolipoprotein AI (apoAI) and apolipoprotein B (apoB) were assayed using immunoturbidimetric methods, using reagents supplied by ABX Diagnostics. C-reactive protein was analysed using ELISA (MP Biomedicals, UK) according to the manufacturer's instructions. 2.4 Statistical analyses Statistical analysis was performed using SPSS 12.0 (SPSS Inc., Chicago IL). The Kolmogorov–Smirnov test was used to assess whether data were normally distributed. Measurements at each time point were compared using repeated measures ANOVA for normally distributed data and Friedman analysis of variance by ranks for non-normally distributed data. Post-hoc analysis of paired time points was performed using a Bonferroni correction. Categorical data were analysed by the χ2 test. Data are presented as mean ± standard deviation or median and interquartile range as appropriate. Statistical significance was assigned at P < 0.05. 3 Results 3.1 Clinical and biochemical measures Baseline characteristics of the 24 patients who completed all three study time points are shown in Table 1. The most common statin dose used was simvastatin 40 mg daily (63% patients); other statin prescriptions are detailed in Table 1. Mean baseline total cholesterol was 187.9 mg/dL (LDL-C 112.7 mg/dL), however 3 months after commencing statins mean LDL-C was reduced by 37% to 70.8 mg/dL (P < 0.01) with a corresponding 24% (P < 0.01) reduction in apoB. Mean LDL-C at 12 months (79.3 g/dL) appeared slightly greater than at 3 months, but this was not statistically significant. Triglycerides and CRP did not change significantly at 3 or 12 months, though HDL cholesterol did show significant increase by 12 months – lipid and apolipoprotein data are shown in Table 2. Compared to baseline, at 3 months there were no significant changes in the proportion of patients taking either angiotensin converting enzyme inhibitors (12/24 versus 8/24: χ2 = 0.77; P = 0.38) or beta adrenoceptor blockers (22/24 versus 18/24: χ2 = 1.35; P = 0.25). By 12 months, the number of patients taking ACE inhibitors had increased significantly compared to baseline (17/24 versus 8/24: χ2 = 5.3; P < 0.05), though this would not have influenced the study findings at the early time point of 3 months. Beta blocker usage was not changed at 12 months (19/24 versus 18/24: χ2 = 0.11; P = 0.74). There was no significant change in either blood pressure or heart rate over the course of the study (Table 3). 3.2 Atherosclerosis regression All 24 patients had aortic plaque images of sufficient quality for analysis. Four patients had carotid plaque images at one or more time points of insufficient quality for analysis; therefore they were excluded from statistical analysis. Images (total > 100) from four randomly selected patients were analysed by both observers yielding inter-observer coefficients of variation for plaque index of 4.8% in the aorta, and 2.9% in the carotid. In both the aorta (Fig. 1a) and the carotid arteries (Fig. 2a), there were statistically significant reductions in plaque index after 3 months of statin treatment. Mean aortic plaque index decreased from 0.303 ± 0.024 at baseline to 0.296 ± 0.022 at 3 months (P < 0.05 versus baseline) and 0.288 ± 0.024 at 12 months (P < 0.01 versus baseline). Plaque regression in the aorta between 3 and 12 months was also significant (P < 0.05). In the carotid artery, plaque index fell from 0.446 ± 0.053 at baseline to 0.432 ± 0.046 at 3 months (P < 0.05 versus baseline) and 0.416 ± 0.032 at 12 months (P < 0.01 versus baseline). Plaque index for the carotid artery between 3 and 12 months did not show significant reduction (P = 0.09). The number of patients with early regression in the aorta at 3 months compared to baseline was 18/24, similar to that at 12 months where 20/24 showed regression (χ2 = 0.126, P = 0.72) (Fig. 1c). The number of patients with measurable early regression at 3 months in the carotid was 14/20, which was not significantly different to the 18/20 observed at 12 months (χ2 = 1.4, P = 0.24); see Fig. 2c. Furthermore within patients, the early (3 months) change in plaque index of aorta and carotid arteries showed significant correlation with the final change observed at 12 months (Figs. 1c and 2c). Mean aortic lumen area was 431 ± 77 mm2 at baseline, 434 ± 77 mm2 at 3 months (P = 1.0 versus baseline) and 442 ± 85 mm2 at 12 months (P = 0.09 versus baseline). Mean lumen area in the carotid arteries was 44 ± 9 mm2 at baseline, 44 ± 8 mm2 at 3 months (P = 1.0 versus baseline) and 43 ± 8 mm2 at 12 months (P = 0.55 versus baseline). 3.3 Physiological measures After 3 months of statin treatment, aortic distensibility increased by >20% at each of the three locations along its length. This effect was sustained but did not increase further at 12 months (Table 3). Flow-mediated dilatation of the brachial artery, a measure of endothelial function, also improved after 3 months by >30% (Table 3). Endothelial independent relaxation induced by GTN was not significantly changed after 3 months, but did show a significant increase by 12 months. 3.4 Relationship between variables Within individual patients there was no correlation between MRI quantification of atheroma burden in the aorta and carotid at baseline. Although at a group level, endothelial function and aortic compliance improved and atheroma burden diminished, there was no correlation of these changes within individual patients. Furthermore, there were no significant associations between measures of vascular function or atheroma burden and any of: attained LDL-C; change in LDL-C, HDL-C, apoB, apoA-I, or CRP. 4 Discussion In this study, we have observed that regression of atherosclerosis in response to statin treatment can occur earlier than previously appreciated in both the aorta and carotid arteries. The robustness of this observation is enhanced by the finding that, within individual patients, regression at the early time point of 3 months was closely related to the magnitude and direction of change at 12 months. Patients also showed early and sustained improvement in aortic distensibility and in flow mediated vasodilatation of the brachial artery. The magnitude of atheroma regression observed in our study after 3 months is consistent with previous longer-term studies of regression in response to statin treatment [6,7,25]. A lesser LDL-C reduction than that achieved in our study was not associated with early plaque regression after 6 months statin treatment [6]. Intensive LDL-C reduction has been reported to achieve greater regression, but this study did not include an early time point [7]. Thus, the early regression observed here is a new finding that accords with the analysis of Nissen et al. [5] in which atheroma regression, assessed by intravascular ultrasound in the coronary arteries, was predicted by attained mean LDL-C < 70 mg/dL. A recent observational study reported overall carotid wall area progression by 2.2% per year [8], although individual cases of regression of up to 7.9% were reported. Importantly, the population in that study differed by the inclusion of older patients with a higher prevalence of hypertension and selected only patients with carotid plaques of ≥50% stenosis on a prior duplex scan. Mean lumen size of the aorta and carotid appeared to show slight increase by 12 months, this did not reach statistical significance implying that regression was occurring predominantly by reversal of ‘positive’ vessel remodelling as suggested by Corti et al. [6]. There was significant change in plaque index between 3 and 12 months in the aorta, whilst the carotid artery showed a trend that did not reach significance (P = 0.09) over the same time period. This could reflect the smaller size of the carotid artery compared to the carotid, making a small change harder to detect. Alternatively the plaques in the aorta and carotid may differ in composition, with certain elements of the plaque likely to be more susceptible to removal than others. For instance, lowering plasma LDL-C could slow the rate of lipoprotein deposition in the arterial wall, allowing reverse cholesterol transport mechanisms to predominate, culminating in net regression [26]. Different effects on individual plaque components is suggested by MRI studies of advanced carotid atherosclerosis in which patients treated with aggressive lipid lowering therapy showed a reduction in size of lipid rich areas [27,28]. This study was not designed to examine plaque composition, though initiation of study to address this question has recently been reported [29]. New developments such as lipid selective contrast agents or higher field strength (3 T) imaging with improved resolution might further establish the degree to which plaque regression involves lipid removal [30,31]. Early clinical benefits of statins have also been ascribed to anti-inflammatory effects [32]. The degree of change in coronary atheroma volume measured using intravascular ultrasound has been related to the magnitude of change in both LDL and CRP [33]. However, in the present study, the early changes in plaque size occurred in the absence of significant change in CRP. Lack of measurable change in CRP probably reflects both the relatively small sample size, and the low baseline CRP level in this stable CAD population, comparable to the post treatment CRP levels in other studies [34]. We also found no relationship between the LDL-C attained and change in plaque size, which may again reflect the sample size and a clustering of LDL-C levels in the lower range precluding observation of a quantitative effect. However, the absence of changes in biochemical parameters despite plaque regression highlights the complementary role of MRI as an imaging biomarker. Rapid improvements in endothelial vasomotor function have previously been demonstrated in patients with atherosclerosis within weeks [12,14] or even days [13] of starting statin treatment. Thus our finding of increased flow mediated dilatation at 3 months was not surprising. By contrast, timing of statin effects on central arterial stiffness is less established. Improvements in large artery stiffness have been demonstrated after a year of statin treatment [35,36], although a shorter study of 8 weeks pravastatin treatment in patients with familial hypercholesterolemia did not show any improvement [37]. Hypertensive patients without coronary artery disease treated with high dose atorvastatin (mean LDL of <70 mg/dL) did show improvements in large artery stiffness after 3 months [38]. Our finding of rapid improvement in aortic stiffness was of similar magnitude and extends this potentially beneficial effect of statin treatment to a coronary artery disease population. We also observed that GTN mediated (endothelium independent) dilatation of the brachial artery was increased after 12 months statin treatment, as has been previously reported [39,40] and which might reflect downstream statin effects e.g. on smooth muscle cell sensitivity to nitric oxide. We initially hypothesised that changes in arterial function might predict changes in structural parameters, but in this study we found no correlation on an individual patient basis between changes is structure and function. Endothelial function, aortic stiffness and atheroma burden all represent different aspects and stages of disease. Although at a population level all these parameters may change in a favourable direction in response to treatment, individual patients may show variable response in each according to their stage of disease. As a result, it is likely that measures of vascular structure and function will provide complementary insights into vascular disease [18]. The capability of MRI to perform non-invasive assessment of changes in the arterial wall using relatively small numbers of patients is highlighted by this and other studies [6,22]. Accurate and reproducible assessment of the vessel wall is key to detection of atherosclerosis progression or regression [41]. The rationale for this approach has been illustrated by previous carotid intima-media thickness or coronary IVUS studies [42–44]. An early appreciation of effects on both structure and function, as provided by MRI, could be used to guide selection of novel agents prior to investment in major Phase III trials [45]. MRI is an emerging technique and unlike ultrasound measures of IMT and FMD, has not yet been validated as a means to predict future events in large studies. However, recent data shows correlation of aortic plaque burden by MRI with clinical risk scores [46]. As our findings suggest MRI potentially offers additional value through the assessment of arterial structure in multiple locations and complementary measures of vascular function. Therefore, it seems increasingly likely MRI will play a key role in evaluation of new and existing therapies and may even become part of individual patient risk assessment. 4.1 Study limitations A potential limitation of this study is the absence of a control group. However, given strong evidence of the benefits of early and intensive treatment with statins, a placebo controlled arm or even low intensity statin-treatment arm was not considered ethical. In common with several recent studies [5,6], we therefore conducted a longitudinal study of changes compared to baseline. As a result, it is not possible to exclude the possibility that the changes observed were due to an unknown factor, though in context, this is improbable. These patients were newly diagnosed with coronary artery disease, some of whom were taking vasoactive drugs such as ACE inhibitors and beta adrenoceptor blockers for hypertension prior to study enrolment. However, the increase in use of such medications between study baseline and the 3-month time point was small and not statistically significant and so the vascular changes observed over that period are not likely to be confounded by changes in these other medications. The 2–3% reductions in plaque index observed in this study at 3 months appear relatively modest when compared to decreases in lesion size of greater than 30% observed over a similar time frame in some animal studies. However, the interventions in animal models of atherosclerosis usually involve much more extreme changes in lipid levels than those achievable by statin treatment in humans. Thus, whilst statin treatment to achieve effective LDL-C reduction appears important for plaque regression, additional HDL-C based interventions in order to enhance reverse cholesterol transport may prove even more effective [26]. The patient population studied was overwhelmingly male and Caucasian therefore the findings may not be applicable across all population groups. 5 Conclusions This study shows that in a population of statin naïve, clinically stable but otherwise unselected coronary artery disease patients, cholesterol reduction using statins to mean LDL-C of approximately 70 mg/dL was associated with rapid regression of atheroma at 3 months. Early changes were highly correlated with changes after 12 months. These rapid structural changes were accompanied by early improvements in arterial stiffness and endothelial function that were sustained to 12 months. Use of multi-modal vascular MRI to detect early changes in atheroma and vascular function in small numbers of patients could prove to be an efficient strategy to screen novel anti-atherosclerotic agents.

Pediatr_Nephrol-3-1-1989764

The nervous system and chronic kidney disease in children

This paper provides a review of the literature on the nervous system involvement incurred by children and adolescents with chronic kidney disease (CKD), with a particular focus on neuropsychological functioning. In addition to an historical overview of earlier literature, published studies from the past 14 years that address both central and peripheral nervous system function in children with CKD are reviewed (1990–2003). These studies span work in neuroimaging, electrophysiology, and neuropsychology. A key focus for this review is on variables that might affect neurodevelopmental status in these children. The paper concludes with suggestions for achieving progress in the understanding of this complication of kidney disease in children. Introduction Associations between end-stage renal disease (ESRD) and neurocognitive dysfunction have been reported for over 25 years. Between 1977 and 1986, approximately 15 publications described dialysis in infants. In these early studies, approximately 65% of infants were reported to have developmental delay and 49% encephalopathy [1]. Between 1976 and 1984, the neurocognitive effects of aluminum-induced neurotoxicity secondary to then current treatments for chronic kidney disease (CKD) were reported [2, 3, 4]. These neurocognitive effects included seizures, speech disorders, dementia, and a slow electroencephalogram (EEG) pattern [2, 3]. These findings were important for the field in that they encouraged modifications to the treatment regimen for dialysis patients and highlighted a confounding factor in the relationship between CKD and neurocognitive integrity. By 1990 the use of aluminum was generally eliminated with improved dialysis water purification techniques and avoidance of aluminum-containing medications. In the early 1990s, erythropoietin was introduced into standard practice, resulting in improved anemia management for children and adults with ESRD and a diminution of anemia-related EEG and cognitive deficits reported in the adult ESRD population [5, 6, 7]. The purpose of this paper is to review the contemporary published literature that focuses on the neurological impact of CKD in children. Papers selected for this review have been published from 1990 to the present, and were selected to coincide with the improvements in treatment, including the diminution of aluminum exposure and availability of erythropoietin that occurred circa 1990. These studies span work in neuroimaging, electrophysiology, and neuropsychology to address central nervous system (CNS) function. We also present available studies addressing the integrity of the peripheral nervous system. Central nervous system studies Neuroimaging Imaging of the brain with computed tomography and magnetic resonance imaging (MRI) has been used to document the structural impact of ESRD in children. Contemporary estimates of the prevalence of cerebral atrophy are obtained from a report of 13 children with chronic renal failure from infancy. Brain atrophy was reported in 3 (23%) of these patients [8]. Publications with a focus on certain disease groups with a high risk for CNS disease, such as cystinosis, Lowe syndrome, and congenital nephrotic syndrome, estimate the prevalence of cerebral atrophy to range between 15% and 100% [9, 10, 11]. A recent study by Qvist et al. [11] described a cohort of 33 children, largely composed of children with ESRD from congenital nephrotic syndrome, tested an average of 6 years after renal transplant. MRI documented CNS infarcts or ischemic changes from both silent and clinically significant cerebrovascular events in 19 (58%). Although relevant for children with CKD, the frequency of brain structure abnormalities in these specific disorders may be greater than in a general pediatric CKD population with a variety of renal diseases. Electrophysiology findings Three reports of EEG findings in a cohort of children with CKD have been published since 1990. In one study focal and paroxysmal EEG abnormalities were observed in 12 (36%) of 33 children after renal transplant, with 5 (15%) receiving anticonvulsant therapy [11]. The second report of 14 patients documented unspecified EEG abnormalities in 6 (42%) [8]. This study also documented a normal auditory brain stem evoked response in 12 of 12 children tested [8]. Hurkx et al. [12] evaluated 22 children with chronic renal insufficiency (CRI) and dialysis dependence using somatosensory evoked potential of the right median nerve. They reported an increased interpeak latency (N13–N20) suggesting a delayed thalamocortical conduction. No differences in CRI versus dialysis subgroups were found. Investigations of adults with ESRD demonstrate that these tools are sensitive to kidney disease-related effects and may prove useful in future pediatric studies [5, 6, 13]. Neuropsychology findings Table 1 provides a brief description of 11 published studies that have been conducted over the past 15 years, with follow-up discussion on each of the studies within the broad domains of cognition listed below. These studies have employed samples ranging from 9 to 62 infants, children, and adolescents with CKD. The subjects experienced different types of treatment for their CKD (e.g., hemodialysis, peritoneal dialysis, kidney transplant, conservative). The research design varied (e.g., longitudinal, pre-post transplant, control groups) and different kinds of assessment methodology were used (e.g., infant development batteries, IQ batteries, specific measures of cognition, experimental tasks). In this regard, drawing specific conclusions for neuropsychological findings will be tentative, at best, but should provide guidance to clinicians and researchers as to how the field should move forward within this assessment domain.Table 1 Cognitive functioning in children and adolescents with end-stage renal disease (ESRD), 1990–2003 (CKD chronic kidney disease, SD standard deviation, CAPD continuous ambulatory peritoneal dialysis)Author/dateSample featuresComparison groupFindingsDavis et al. 1990 [28]n=37Pre-post transplant designMental development20 Dialysis at pre-transplant evaluation Improved from pre- (mean=77.0, range 50–116) to post-transplant (mean=91.4, range 50–117)17 Conservative at pre-transplant evaluationMean age at transplant=17.6 monthsMotor development Improved from pre- (mean=68.7, range 50–86) to post-transplant (mean=85.6, range 65–109)Cognition No change from pre- (mean=92.0) to post-transplant (mean=90.4)Overall development worse with early onset of ESRDFennell et al. 1990 [22]n=56n=56 healthy childrenCognitionMean age=13.6 yearsTesting at 6-month intervals Decreased verbal ability in CKDMean age of CKD onset=6.05 yearsVisual motor skills Decreased in CKDModality:Memory and learning Hemodialysis (7) Decreased in CKD and loss of function over time Peritoneal dialysis (12)Attention Kidney transplant (10) No differences between CKD and controls Conservative (27)Lawry et al. 1994 [18]n=24Transplant compared with dialysis plus conservativeCognitionModality: IQ: no differences between dialysis plus conservative (mean=92.91, SD=16.86) vs. transplant (mean=103.00, SD=11.97) Dialysis (9) Conservative (2) Kidney transplant (13) Correlation between age at onset of illness and IQ (r=0.49)Achievement Dialysis plus conservative less than transplant in maths (P=0.020), reading (P=0.028), and written language (P=0.028)Elzouki et al. 1994 [8]N=15NoneDevelopmental screeningModality: 3 of 15 with developmental delay Dialysis (6) Transplant (3) Conservative (6)Hulstijn-Dirkmaat et al. 1995 [14]n=31NoneDevelopmental indexMean age=2.5 yearsTesting every 6 months Conservative (mean=90.3, SD=14.3) greater than ESRD (mean=67.6, SD=17.3)Modality: Dialysis (16) Conservative (15)Verbal, perceptual performance, and quantitative scales No change over timeMendley and Zelko 1999 [19]n=9Pre-post transplant designCognition (baseline only) Full-scale IQ mean=91.6Mean age at pre-transplant testing=14.2 years Verbal IQ mean=91.4 Performance IQ mean=95.1Mean age at post-transplant testing=15.8 yearsAttention Improvement in sustained attention 1-year post-transplant (P=0.039)Pre-transplant modality:Executive functioning Peritoneal dialysis (5) Improvement in mental processing speed 1 year post transplant (P=0.008) Hemodialysis (3) Conservative (1)Mean age of onset of ESRD=11.9 yearsMemoryMean duration of ESRD prior to transplant=2.5 years Improvement in working memory 1 year post transplant (P=0.016)Warady et al. 1999 [16]n=28 infantsLongitudinal designDevelopment, generalModality: 6 of 28 children below the average range at 1 year of age CAPD at ≤3 months of ageCognitionTransplant at mean age=2.1±0.8 years Verbal IQ: 5 of 18 children below the average range at ≥4 years of ageMean age at follow-up=7.8±2.8 years (range 2.5–12.0 years) Non-verbal IQ: 8 of 18 children below the average range at ≥4 years of age 1 child within impaired range on both verbal and non-verbal IQLedermann et al. 2000 [15], Madden et al. 2003 [17]n=16 infants with ESRDLongitudinal designDevelopment, generalMean age at start of dialysis=0.38 years (range 0.02–1 year) 2 of 8 school-aged children had general delaysDuration of dialysis=17.3 months (range=1–59 months) 2 of 8 children <5 years of age had general delaysMean age at assessment=5.84 (range 1.58–12.00 years)Cognition IQ: 67% in average range, and 20% in low-average range (mean=86.5, range 50–102) Lower IQ scores for children with co-morbid diagnoses (mean=67.0) than for those with ESRD alone (mean=94.2)Attention 7 of 14 children with hyperactivity problemsSocial-behavioral 6 of 14 children displaying conduct problemsBrouhard et al. 2000 [21]n=62n=62 siblingsCognitionMean age: 13.8±0.4 years IQ: ESRD less than siblingsModality: 26 dialysis, 36 transplant No difference in dialysis vs. transplantAcademic achievement ESRD less than controls for all measures of spelling, reading, and mathematics No difference dialysis vs. transplantCorrelation between age of diagnosis and academic achievementCorrelation between parental education and academic achievementQvist et al. 2002 [11]n=33 transplant recipientsNoneCognitionMean age at assessment=8 years (range=7–12 years) Verbal IQ: mean=87.5 Non-verbal IQ: mean=87.5IQ range Low: 3/33 Low-average: 14/33 Average: 14/33 Above average: 2/33Neuropsychological battery No overall group deficits with attention, language, memory, or visuospatial abilities when compared with normative populationNeuropsychological deficits Attention: 8 of 33 children Language: 2 of 33 children Memory: 6 of 33 children Visuospatial: 8 of 33 childrenMotor function Hemiplegia: 3 of 33 children Bilateral infarction: 1 of 33 Cerebral palsy: 1 of 33Auditory function 2 of 33 children with moderate sensorineural hearing loss General neurocognitive function In infants and toddlers neurodevelopmental testing is conducted and analyzed in broad categories of overall development, mental development, and motor development. Hulstijn-Dirkmaat et al. [14] compared the general development in 15 toddlers receiving conservative therapy (CRI) with 16 dialysis-dependent children. The children with CRI had a better developmental index compared with the dialysis-dependent children (mean±standard deviation 90.3±14.3 vs. 67.6±17.3) [14]. Ledermann et al. [15] evaluated the long-term outcome of infants requiring peritoneal dialysis. In this study, 2 of 8 (25%) young children demonstrated developmental delay. Warady et al. [16] evaluated 28 infants at 1 year of age, all of whom were dialysis dependent. Of these 28 infants, 6 (21%) scored in the low-average or impaired range of general development. Based on these studies, approximately 20%–25% of children less than 5 years of age with CKD might be expected to show general developmental delays [8, 15, 16]. For older children, IQ is the typical measure of general cognitive function. In the sample of children studied with CKD, the distribution of IQ scores is shifted downward compared with the normal population, with low-average (IQ 80–89) and average (IQ 90–109) range scores predominating [11, 14, 15, 16, 17, 18, 19, 20]. In a study of 62 children with ESRD, dialysis and transplant combined, Brouhard et al. [21] described a significantly lower IQ in the children with kidney disease compared with their sibling controls. In 19 children with a mean age of 6.6±1.3 years who had ESRD from infancy, Warady et al. [16] reported a relatively intact IQ, with 15 of 19 (79%) in the average range. In this group, 13 of 18 (72%) achieved average verbal IQ scores, while only 10 (56%) scored in the average range in the nonverbal subtest [16]. When comparing transplanted patients with dialysis-dependent patients, variable results have been reported. Lawry et al. [18] compared 13 transplanted children with 11 dialysis-dependent children in a cross-sectional study and found a higher mean IQ in the transplant group (103.0±11.97 vs. 92.9±16.86). Conversely, Brouhard et al. [21] compared 36 transplant and 26 dialysis-dependent children in their cross-sectional study and found no significant difference between these patient groups. In aggregate it appears that general cognitive function is impaired in children with CKD, but the published literature is inadequate to fully characterize this effect with respect to CKD modality and differences between verbal and non-verbal IQ [11, 16, 19, 22]. Attention and executive function The findings in the pediatric CKD literature are mixed with respect to attention and executive function [11, 19, 22]. Fennell et al. [22] reported no differences in measures of sustained attention between children with all treatment modalities of CKD combined and matched controls. Conversely, Mendley and Zelko [19] documented longitudinal improvements in sustained attention (pre-transplant 2.19±1.29 vs. post-transplant 2.95±1.33, P=0.04) and mental processing speed (pre-transplant 2.28±0.72 vs. post-transplant 1.64±0.31 s, P=0.008) 1 year after transplant in 9 children. After renal transplant, Qvist et al. [11] reported no overall group deficits of attention in 33 children compared with the normative population [standard deviation score (SDS)=−0.2±0.4], although 24% of the sample showed generalized attention deficits. Language The prevalence of hearing loss among children with CKD is approximately 18%, and unrecognized or delayed diagnosis of hearing impairment may impede language development [11, 23]. Children with known hearing loss tend to be excluded from studies of cognitive function, and so their challenges are not likely to be fully reflected in the published literature [17]. Fennell et al. [22] documented deficits with verbal abstracting abilities in their matched control design for children with CKD. Qvist et al. [11] documented no overall group deficits of language compared with the normative population (SDS=−0.2±0.4), with only 6% of children showing evidence of generalized language deficits. More research needs to be conducted before determining whether language abilities will prove to be an area of concern for children with CKD, particularly with respect to potential delays secondary to hearing impairment. Visuospatial abilities Compared with a matched control population, early studies by Fennell et al. [22] documented deficits in visual-motor abilities in a cohort of 56 children with CKD. Qvist et al. [11] documented no overall group deficits with visuospatial abilities in 33 transplant recipients when compared with the normative population (SDS=−0.5±0.5); however, 24% of the cases did show generalized visuospatial deficits. Memory In a heterogeneous sample of children with CKD, Fennell et al. [22] reported lower memory scores for children with CKD compared with controls. In addition, a general loss of memory function over time was observed in 26 children with all treatment modalities of CKD over a 12-month testing period [24]. After kidney transplantation, Qvist et al. [11] reported no overall group deficits with memory compared with the normative population (SDS=−0.4±0.5), although 20% of the cases displayed generalized memory deficits. Conversely, Mendley and Zelko [19] documented longitudinal improvements in working memory in 9 children 1 year after transplant compared with the pre-transplant evaluation. Academic achievement Academic achievement in children with CKD is vulnerable to deficits in each cognitive domain noted above as well as an increased frequency of school absences. In a study by Lawry et al. [18], the combined dialysis and CRI group (n=11) with a mean chronological age of 14.9±3.3 years achieved at an age equivalent of 13.8±5.8 in mathematics, 15.6±7.7 in reading, and 11.2±7.2 in language. In comparison, the transplant group (n=13) with a mean chronological age of 13.8±3.2 years, achieved at an age equivalent of 16.2±7.8 in mathematics, 16.4±7.7 in reading, and 16.2±9.1 in language. This paper suggested that transplant recipients may have superior academic achievement compared with dialysis-dependent children [18]. Conversely, Brouhard et al. [21] compared the academic achievement of dialysis and transplant patients and normal sibling controls. They reported no difference in academic achievement between dialysis and transplant groups. However, the combined ESRD group of dialysis and transplant achieved below their sibling controls in spelling, arithmetic, and reading [21]. Observational studies of academic placement further indicate that regular education (with or without remedial tutoring) is used for 79%–94% of children with CKD, while 13%–15% receive special education services not related to hearing or visual impairments [11, 16]. This compares with national data in which approximately 10%–15% of students receive special education services [25]. Given the observed frequency of abnormalities across multiple areas of cognitive function, the frequency of special education services used by children with CKD appears low. Peripheral nervous system Peripheral neuropathy including diabetic neuropathy and dialysis-related amyloidosis has been reported in adults with ESRD [26, 27]. The frequency of peripheral neuropathy in children with CKD can only be grossly estimated based on the following study. Elzouki et al. [8] assessed nerve conduction velocity in 11 children with CKD and found 1 (9%) had diminished motor nerve conduction. Electromyograms performed in 9 of these patients revealed increased polyphagia of motor unit potentials in 4 (44%) [8]. At present, we are unable to find additional published studies examining peripheral neuropathies in children with CKD. Key variables affecting neurodevelopmental outcomes CKD is a complex disorder and, similar to other pediatric disorders, there are undoubtedly a variety of variables that contribute to the neurodevelopmental status of this population. Key variables presented here are postulates for the most part as the studies conducted since 1990 have not been adequately powered to assess multiple influential factors. The duration of CKD and the age at onset of CKD may affect cognition. Two separate studies have reported a poorer developmental prognosis for children with earlier-onset ESRD [18, 28]. It has been postulated that this may be the result of insults to the CNS at particularly vulnerable periods in development or the result of continuous insults over the majority of postnatal life among some children with long-standing kidney disease. The congenital disorders presenting with kidney disease early in life may influence the perceived relationship between duration of CKD and cognition, as these disorders may have other associated CNS abnormalities, such as pulmonary hypoplasia and hypoxia associated with obstructive uropathies, or structural anomalies with Joubert syndrome and Smith-Lemli-Opitz syndrome [29]. Hulstijn-Dirkmaat et al. [14] identified the presence of co-morbid conditions as a risk factor for cognitive impairment based on mean IQ scored (CKD alone 86.7 vs. CKD with co-morbidity 61.4, P=0.001). Conversely, Fennell et al. [24] found no correlation between age at onset of ESRD and cognition. The available data suggest that the duration of CKD and the age at onset of CKD negatively impact on cognitive functioning, but it remains unclear how these variables contribute to the type and severity of cognitive dysfunction. Having reached ESRD, the modality of renal replacement therapy may have an impact on neurodevelopment. Several studies have examined small groups of children either before and after renal transplant or as a cross-sectional study comparing renal replacement therapy modality groups [30, 31]. Fennell et al. [24] compared 10 transplant, 7 hemodialysis, and 12 peritoneal dialysis patients. Patients in both the transplant and peritoneal dialysis groups tended to have better attention and memory skills when compared with the performance of hemodialysis patients. Additional investigation is warranted to determine the differential effect of hemodialysis and peritoneal dialysis among children with ESRD with current dialysis adequacy standards [32, 33, 34]. From previous studies, a hypothesis can be formulated that motor skills, memory, attention, and mental processing may improve after renal transplantation [11, 16, 21]. Although existing literature suggests that a successful renal transplant may diminish the adverse developmental effects of ESRD, these same studies document the type and frequency of residual deficits in pediatric renal transplant recipients to be greater than in the normative American population [11, 16, 21]. Complications of CKD such as anemia, hypertension, and malnutrition likely affect neurodevelopment. Anemia has been shown to slow the cognitive event related potential in adults with CKD (mean hematocrit=23.7%) and impair cognitive function among otherwise healthy children aged 6–11 years with hemoglobin levels less than 11.8 g/dl [6, 35]. The optimal hemoglobin for cognitive function in children with CKD remains to be determined. The effects of hypertension on cognition may be related to the degree of blood pressure elevation, brain injury consequent to hypertensive or hypotensive episodes, and side effects of antihypertensive therapy. A recent study by Lande et al. [36] analyzed National Health and Nutrition Examination Survey III (NHANES III) data comparing 217 children with systolic blood pressure greater than or equal to the 90th percentile for age and gender with 4,860 normotensive children. The NHANES III study used subtests of the Wechsler Intelligence Scale for Children and reading and arithmetic subtests of a standardized academic achievement test. In this study, results suggested that hypertension was associated with lower scores in subtests representing memory, attention, and arithmetic. Furthermore, malnutrition in young infants without kidney or other chronic disease has been linked to impaired brain growth and developmental delay [37, 38, 39]. Significant effort has been applied to the nutritional support of children with CKD. However, in national registries of children with CKD, the mean weight z-score is 1.0–1.4 standard deviations below the American age-matched mean, and mean height z-scores are approximately 1.6 standard deviations below the American age-matched mean [40]. Anemia, hypertension, and malnutrition are likely key factors contributing to the cognitive deficits of children with CKD. Children with CKD are at risk for additional disorders that may accompany congenital kidney disease. Prematurity occurs with a greater frequency in children with congenital kidney disease and is associated with a greater frequency of CNS injuries and later developmental deficits [11, 41]. Socioeconomic status and parental education have a known impact on academic achievement among all children [42]. Although not unique to children with CKD, the identification of poor socioeconomic status or limited parental education may trigger additional educational evaluation and or services for children with CKD in these families. School absences are increased in children with CKD due to the need for outpatient medical visits, hospitalizations, hemodialysis, and acute illness [20]. Finally, the CKD population is at excess risk for sensory deficits, including congenital and acquired hearing impairment (9%-18%) and visual impairment, although the latter has not been examined thoroughly [11, 43]. All of these factors may contribute to the neurodevelopmental dysfunction of children with kidney disease. Conclusions Emergent findings appear to apply to three broad groups of children and adolescents: mild-to-moderate CKD, dialysis-dependent children, and transplant-dependent children. What is known about children with mild-to-moderate CKD is severely limited, with no focused studies addressing their neurodevelopmental needs. More is known about the pediatric dialysis population, with deficits in the areas of attention, language, visual-spatial abilities, and memory. However, the differentiation of problems that may relate to disease-specific variables, such as age at onset of kidney failure, anemia, and hypertension, remain relatively unknown and modestly examined at best. For the transplant-dependent group, cognitive deficits appear to persist, supporting the conclusion that the transplant does not result in complete neurocognitive “recovery.” The low frequency of peripheral neuropathy in children with CKD observed clinically and reported in a single study may be attributed to the low prevalence of diseases with microvascular complications such as diabetes mellitus, advanced atherosclerosis, and dialysis-related amyloidosis [8]. This pediatric advantage may be lost as patients survive into young adulthood. It is unlikely that purely pediatric follow-up studies will be able to characterize this potential late-term complication. Given the expected long-term patient survival of children with CKD, established guidelines for the provision of renal replacement therapy for children, and the data provided by studies among children with CKD, we believe that an organized, adequately powered approach toward the characterization of the neurodevelopmental impact of CKD is warranted [32, 33, 34, 44]. Ideally, this study or set of studies will include measures of neuroimaging, electrophysiology, and neuropsychology. Future research in this area should characterize potential risk factors, such as age at onset of disease, anemia management, and hypertension to advance our understanding of the magnitude of cognitive dysfunction and aid the identification of modifiable mediators of CNS deficits in children with CKD.

Arch_Microbiol-4-1-2270922

The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression

High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanellaoneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O2 deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research. Introduction The cultivation of microorganisms has been performed for more than a century beginning with Louis Pasteur (1879), Robert Koch (1881) and R. J. Petri (1882) (Sedgwick 1916; Gest 1987), with ever increasingly sophisticated methods becoming available over time. These methods range from using undefined medium (e.g., boiled meat extracts) in shake flask cultures where growth rate and substrate utilization (Narang et al. 1997) as well as proteomic profiles (Valentine et al. 2005; Wunschel et al. 2005) can vary widely, to defined culturing media combined with state-of-the-science bioreactors (Nethe-Jaenchen and Thauer 1984; Vasconcelos et al. 1994; Elias et al. 2005). A considerable amount of physiological (Keltjens et al. 1990; Dolla et al. 2000; Elias et al. 2004) and high-throughput functional genomic data (Beliaev et al. 2002; Thompson et al. 2002; Wan et al. 2004) has been generated from microbial samples produced using shake flask batch cultures. Although results may be statistically defensible, the inherent short-comings of this cultivation technique are that essential parameters directly impacting organism physiology are poorly described and difficult to control. These parameters include nutrient availability, metabolite production, specific growth rate, and poor mixing leading to cultural/environmental heterogeneity. Poor mass transfer kinetics can lead to unwanted limitations in dissolved gasses for use by the organism (such as oxygen or hydrogen) and the buildup of gaseous metabolic byproducts, such as CH4 and CO2 that can have secondary effects on cellular metabolism. For example, the accumulation of dissolved CO2 and the production of dissolved organic acids can significantly alter pH in poorly buffered systems (Ji et al. 1995; Narang et al. 1997; Mayville et al. 1999; Miller and Bassler 2001). Fluctuations of pH in bacterial cultures induce physiological responses in various organisms (Snoep et al. 1990; Blankenhorn et al. 1999; Stancik et al. 2002; Yohannes et al. 2004). Hence, the conditions of cultivation at the beginning of growth can be far different than those when the cells are harvested for analysis. Furthermore, the time of cell harvest during different “phases of growth” will also impact biochemical reaction rates (Wright and Holland 2003) and the complement of cellular proteins (Lipton et al. 2002; Wunschel et al. 2005). In actuality, the functional characteristics and relative abundance of proteins measured are derived from a heterogeneous cell population including cells in various stages of growth that are potentially expressing a range of metabolic states within relatively short time periods. We argue that the term “aerobic shake flask,” therefore, is more an operational description than an accurate description of the physiological growth conditions. In bioreactors, all relevant physiological parameters, including pH, dissolved oxygen (DO2), temperature, agitation, and the incoming gas flow rate and composition can be monitored and controlled. Because the nutrient medium can be of variable composition and added to the bioreactor at discreet rates, cells can be cultured with varying degrees of electron donor/carbon source or electron acceptor limitation and over a range of growth rates. The former has important ramifications given the effects of these limitations on cell physiology, while being able to address the issue of different growth rates enables studies that more closely mimic the variable and potentially long generation times typical of many habitats including sedimentary and aquatic environments. A complete mass (fermentation) balance can also be obtained by measuring biogenic gases produced during the cultivation by using in-line mass spectrometry (MS) in addition to the byproducts of carbon and energy source metabolism and the amount of produced biomass. The utilization of such advanced technologies are allowing for unprecedented insights into the metabolic complexities of microbiological systems. Controlled cultivation technologies, such as chemostats and turbidostats, minimize culture heterogeneity through continual and thorough agitation and by monitoring and controlling all culture parameters. This mixing substantially increases the mass transfer rates of both the influent gas stream and those gases resulting from microbial metabolism, thus creating a more homogenous gas profile throughout the culture (Pin et al. 2002) and reducing the gas effect on pH. Any change in pH that occurs during cell growth due to non-gaseous metabolic by-products is compensated for by monitoring probes and the addition of acid or base (typically HCl or NaOH) via computer controlled pumps. While there is a greater upfront capital investment for bioreactors, the investment is minor relative to the expense associated with high-throughput functional genomics techniques. Systems biology seeks to integrate high-throughput and comprehensive analytical techniques such as DNA and RNA microarrays, proteomics and protein interaction analyses, and metabolite measurements with computational biology (i.e., modeling) to describe the structure of the system and responses to individual perturbations. Such knowledge can be used, for example, to predict systems level responses to environmental changes/perturbations. In our opinion, a key element of systems microbiology research is the use of controlled cultivation techniques to generate samples under well-defined conditions where variations in intra- as well as inter-culture variability, are minimized. The work described herein was designed to quantitatively assess the relative suitability and limitations of various cultivation approaches for systems biology research. Cells cultivated in shake flasks, controlled batch bioreactors and chemostats were subjected to targeted as well as comprehensive proteomic analysis via two-dimensional gel electrophoresis (Beliaev et al. 2002; Abboud et al. 2005) and the MS based accurate mass and time (AMT) tag approach (Conrads et al. 2002; Smith et al. 2002), to determine the extent of biological variation. Recent reports have used controlled cultivation to determine the “metabolic flux” with different carbon, electron donor or nitrogen sources (Daran-Lapujade et al. 2004; Kolkman et al. 2005, 2006; Adams et al. 2006). However, this is the first report documenting the metabolic differences with varied electron-acceptor between cultures. Finally, while others have utilized steady state cultures and cite advantages similar to those mentioned above, this is, to our knowledge, the first report that assesses and quantifies the effect of cultivation methods on microbial physiology and cell proteome. Materials and methods Materials Acetonitrile and methanol were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Urea, dithiothreitol (DTT), and CaCl2 were obtained from Sigma-Aldrich (St Louis, MO, USA) while thiourea, trifluoroacetic acid, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate and ammonium bicarbonate were obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI, USA). Sequencing grade, modified trypsin was purchased from Promega (Madison, WI, USA) while ammonium formate was obtained from Fluka (St Louis, MO, USA). Water was purified using a Barnstead Nanopure Infinity water purification system (Dubuque, IA, USA). Cell cultivation and harvesting Shake flask cultures Shewanella oneidensis MR-1 (ATCC 700550) were cultivated in a defined medium as described elsewhere (Elias et al. 2005) except that 18 mM lactate was used instead of 6 mM and the PIPES concentration was increased to 30 mM since there was no pH control. Shake flask cultures (0.15 l) were grown aerobically at 30°C with constant shaking at 150 rpm. Duplicate cultures were inoculated at 2-h intervals over 20 h. After an additional 4 h of incubation the optical density (OD; A600), pH and DO2 were recorded and culture samples for organic acid analysis were taken via syringe, filtered (0.22 μm) and stored at −80°C until analyzed. The cultures that had grown for 24 h were in the late log phase of growth and were then harvested as previously described (Elias et al. 2005). First, 100 ml of culture was centrifuged and the resulting pellet dehydrated for dry weight biomass measurement (MA100 moisture analyzer Sartorius, Inc.; http://www.sartorius.com/). Then, the remaining cells were pelleted by centrifugation (27,000×g; 8 min; 4°C). The resulting pellets and approximately 2–3 ml of the supernatant were transferred to cryovials (1.0 ml) and centrifuged (9,000×g; 4 min; 4°C). After removal of the supernatant, the cryovials containing packed cell pellets were immediately stored at −80°C for later analysis by 2DE and AMT MS. Controlled batch cultures Cells were cultured in controlled batches using Bioflow model 110 reactors (New Brunswick Scientific, Edison, NJ, USA). For the direct proteome comparison, lactate was used at 18 mM as in the shake flasks but the PIPES concentration was lowered to 3 mM since pH control was present. Cells were grown with oxygen at 20% of dissolved saturation (DO2) using a combination of air and N2 gas while pH was constantly maintained at 7.00 ± 0.03 by the addition of 2 N HCl or 2 N NaOH. The temperature was a constant 30°C and the reactors were stirred at 400 rpm to minimize mass transfer limitations of O2 and CO2. Samples were taken for organic acid analysis as described above until the late log phase of growth at A600 ∼0.6. At this point cells were harvested for the analyses as described above for the shake flasks. Chemostat cultures Steady state cultures were attained using Bioflow model 3000 reactors (New Brunswick Scientific, Edison, NJ, USA) with the same medium and identical parameters and controls as described for the controlled batch cultures. Once the optical density (OD; A600) was approximately 0.6, fresh medium was pumped into the reactor at a dilution rate of 0.1/h and a constant 3 l volume maintained. Culturing continued until a constant OD and acid addition rate (i.e., steady state) was attained. Samples for organic acids were collected periodically and the cells harvested as above. Organic acid quantitation The organic acids lactate, acetate, pyruvate, formate and fumarate were measured using isocratic flow (0.5 ml/min) with both high performance liquid chromatography (HPLC) and ion chromatography (IC). The HPLC (Agilent 1100 series HPLC with a 300 × 78 mm Rezex organic acid column (Phenomenex, Torrance, CA, USA); 60°C; 35 min run time) used a dilute acid mobile phase of 0.005 N H2SO4. Quantification was accomplished by injecting the sample (25 μl) and using either a diode array detector or a fixed wavelength detector at 210 nm (4 nm bandwidth). Samples were prepared by filtering 1 ml (0.2 μm) and acidifying with 10 μl of 2.5 N H2SO4. Lactate had a retention time of 18.8 min and detection limit of ∼0.1 mM while pyruvate, acetate, and formate retention times were 13.4, 22.1, and 20.2 min, respectively with a detection limit each of ∼0.2 mM. For IC (Dionex DX-500) analysis was performed for the above organic acids as previously described (Senko et al. 2002; Elias et al. 2004). 2D-gel proteomic analysis Frozen cell pellets were mixed with 2 vols. of a solution containing 9 M urea, 2% 2-mercaptoethanol, 2% ampholytes (pH 8–10, BioRad), and 2% Nonidet P40 (a nonionic detergent). The cell lysate was then ultracentrifuged (435,000×g; 10 min) using a Beckman TL100 tabletop ultracentrifuge, and the soluble denatured proteins were recovered in supernatants from centrifugation. Protein concentrations were determined using a modification of the Bradford protein assay (Ramagli and Rodriguez 1985). Aliquots of sample containing 40 μg of protein were separated in the first dimension by isoelectric focusing using polyacrylamide gels containing 50% pH 5–7 with 50% pH 3–10 carrier ampholytes (Anderson and Anderson 1978a). After 14,000 V-h, the first-dimension gels were equilibrated with sodium dodecyl sulfate (SDS) and the proteins were separated by SDS-polyacrylamide gel electrophoresis as described (O’Farrell 1975) using a linear gradient of 10–17% acrylamide (Anderson and Anderson 1978b). Proteins were then detected by staining with silver nitrate (Giometti et al. 1991). The 2DE images were digitized using an Eikonix1412 scanner interfaced with a VAX 4000-90 workstation. The images were then transferred to a PC, converted to TIFF format, and then processed for spot detection and pattern matching using the Progenesis software (Nonlinear USA). One 2DE image was used as a reference pattern for the experiment. All patterns in the experiment were matched to the reference pattern so that the protein spots were given identification numbers. Statistical analysis of the relative abundance of each matched protein spot across the data set was done using a two-tailed Student’s t-test as previously described (Giometti and Taylor 1991). Proteins to be identified were cut from two to three replicate gels stained with Coomassie Blue R250 (approximately 200 μg of protein was loaded on each gel), and the proteins were digested in-gel with trypsin (Promega sequence-grade trypsin, 12.5 ng/μg). The resulting peptides were eluted from the gel pieces by extracting three times, first with equal parts of 25 mM ammonium bicarbonate and acetonitrile, then twice with equal parts of 5% (v/v) formic acid and acetonitrile. The eluted tryptic peptides were desalted and concentrated with a commercial ZipTip C18 pipette tip (Millipore). Peptide samples were then loaded onto a 365 × 100 μm fused silica capillary (FSC) column packed with 10 μm POROS 10 R2 packing material (PE Biosystem) at a length of 10–15 cm. Peptides were separated with a 30-min linear gradient of 0–60% solvent containing 80% acetonitrile and 0.5% acetic acid, and then entered into an LCQ ion trap mass spectrometer (Finnigan MAT). Tandem mass spectra were automatically collected under computer control during the 30-min LC–MS runs. MS/MS spectra were then directly subjected to SEQUEST database searches (Eng et al. 1994; Sadygov et al. 2002) by correlating experimental MS/MS spectra to predicted protein sequences in the S. oneidensis MR-1 open reading frame database. Mass spectrometry proteomic analysis Cell lysis and tryptic digestion Whole cell lysis was achieved by bead beating while tryptic digestion was achieved with sequencing grade-modified trypsin as previously described (Elias et al. 2005) and digested for 5 h at 37°C using a 1:50 (w/w) trypsin-to-protein ratio. Samples were quick frozen in liquid N2 and stored at −80°C until analyzed. Protein concentration was determined by the BCA assay kit (Pierce, Rockford, IL, USA). Mass spectrometric analysis The capillary LC system used was described previously (Elias et al. 2005) using 5,000 psi reversed-phase packed capillaries at ∼1.8 μl/min (150 μm i.d. × 360 μm o.d.; Polymicro Technologies) (Shen et al. 2001, 2002) with two mobile phase solvents consisting of 0.2% acetic acid and 0.05% TFA in water (A) and 0.1% TFA in 90% acetonitrile/10% water (B). The LTQ-FT data was processed using the PRISM data analysis system as described previously for LC-FTICR-MS data (Elias et al. 2005). Since the separation systems for both the LTQ-FT and the LCQ analyses were identical, peptide confirmation was based on both the calculated (from the mass tag database) and measured mass (from the FTICR analysis) of the peptide matching to within 6 ppm and the elution times matching to within 5%. All samples were analyzed as previously described (Lipton et al. 2002; Elias et al. 2005) using an LTQ-FT (Thermo-Finnigan, MI, USA). The mass spectrometer measurements were analyzed with SEQUEST (Eng et al. 1994) using our current database as previously described (Elias et al. 2005). Mass spectra were acquired with approximately 105 resolution. The peptide identifications were determined using the spatially localized confidence scoring (SLiC) algorithm (Norbeck et al. 2005) that incorporates a number of constraints and estimates the confidence of each peptide identification by yielding a score of 0–1. Recent work in our laboratory shows that a SLiC score of at least 0.7 is sufficiently rigorous (unpublished) and is used herein. Further, at least one high-confidence “unique” peptide (i.e., mapping to only one possible parent protein) and a total of two peptides was required for protein identification in each analysis. Results Effect of cultivation method on growth and metabolism Duplicate cultures were grown in; (1) shake flasks (uncontrolled batch) (2) controlled batch and (3) continuous flow reactors operating as steady state chemostats all with O2 as the sole terminal electron acceptor. Shake flask cultures exhibited an extended lag phase (approximately 13 h), which compared with the shorter lag phase ((5 h) in controlled batch cultures (Fig. 1a, b). The extended lag and slow growth rates ((0.15/h) in shake flasks correlated with elevated DO2 concentration that gradually decreased from nearly 100% of air saturation to approximately 20% of air saturation (Fig. 1a). Below 20% DO2 growth rates increased to approximately 0.275/h, which is close to the growth rate observed for controlled batch culture operated at DOT 20% of air saturation (Fig. 1a, b). By definition, chemostat cultures operating at steady state were maintained at a constant growth rate (0.1/h) and 20% DO2 throughout the course of the experiment (data not shown). Fig. 1A comparison of duplicate uncontrolled versus duplicate controlled (right) batch culturing of S. oneidensis MR-1. a The inconsistency in decreasing DO2 (dark filled diamond, open diamond) and growth (dark filled triangle, open triangle) in the uncontrolled cultures is apparent when compared to those for controlled batch cultures (b) using the same symbols. This variation is reflected in the (c, d) organic acid profiles. The lactate (dark filled diamond, open diamond), pyruvate (dark filled square, open square) and acetate (dark filled triangle, open triangle) all exhibited more similar profiles between culture replicates in the controlled batches Despite the similarity in growth rates between the duplicate flask cultures (Fig. 1a), the decrease in DO2, i.e., the O2 consumption rates, were dissimilar. These differences correlated with variations in the organic acid concentration profiles in shake flask cultures (Fig. 1c). Although the order of appearance and disappearance of the organic acids was the same between the flask culture replicates, the rate of lactate consumption varied significantly as did the transient concentrations of pyruvate and acetate. The differences in these profiles presumably reflect the metabolic variability between cultures at any given point along the growth curve, and may result from increased variability in the mass transfer kinetics in poorly mixed cultures compared to controlled batch. In comparison, maintaining a constant DO2 concentration in the controlled batch cultures resulted in a shorter lag phase with nearly identical growth rates (Fig. 1b). Cells in well-mixed controlled batch cultures were continuously exposed to a constant 20% DO2 concentration, as opposed to variable DO2 concentrations typical in “aerobic” flask cultures that in fact become O2-limited as cell densities increase due to poor mass transfer rates. This resulted in much greater consistency in organic acid concentration profiles between the replicates (Fig. 1d). Relating cultivation data to metabolic activity Not only does controlled cultivation result in more consistent metabolic activities within and between cultures as shown above, but the very nature of highly instrumented and controlled cultivation provides important characterization data that help to relate cellular metabolism with the environmental conditions experienced by the culture. In a controlled batch experiment designed to illustrate these relationships, cell growth (Fig. 2a) was correlated with the DO2 consumption and the amount of acid needed to maintain a constant pH (Fig. 2b), as well as the organic acid profiles (Fig. 2c). Figure 2a illustrates the increase in optical density measured during the course of the experiment. The solid circles indicate optical density of samples taken directly from the culture, while solid squares represent optical density measurements on the same samples after the addition of 10 μM Na-EDTA. This treatment destabilized cell aggregates (flocs) by chelating calcium, which can cross-link polysaccharides in extracellular polymeric substances. The ratio of flocculated:deflocculated cultures is termed the flocculation index (FCI) and is represented by the open triangles. Hence, flocculation indices above 1 indicate the presence of cell aggregates while values at or slightly below 1 indicate the absence of flocculated cells. The cultures lacked flocculated cells during the first 15 h of growth (Fig. 1a). Acid addition (Fig. 2b, red line) during this period directly tracked increases in optical density. Lactate served as the primary electron donor and carbon source, and decreased while pyruvate and acetate accumulated in the medium (Fig. 2c). Fig. 2Alignment of the (a) growth, (b) controlled parameters and (c) organic acid profile for S. oneidensis MR-1 cells grown in controlled batch to indicate consequent shifts in metabolism. a The growth of flocculated (dark filled square; after EDTA addition), and non-flocculated cells (dark filled circle) were initially compared to profile the degree of flocculation (dark filled triangle) during growth. When coordinated with changes in (b) pH, DO2 and acid addition and (c) the appearance and exhaustion of the organic acids lactate (dark filled diamond), pyruvate (dark filled square), acetate (dark filled triangle), changes in metabolism can be identified. These include the DO2 spike (solid arrow) signaling lactate exhaustion with the concomitant increase in cell floculation as well as the decreased acid addition rate (broken arrow) showing major carbon source exhaustion and the concomitant disappearance of flocculation The point at which lactate was completely consumed (18 h, solid gray arrow) corresponded with a transient spike in DO2 (Fig. 2b, blue line). This peak resulted from a rapid decrease in O2 consumption rate and a delayed response of the control loop used by the reactor to maintain constant DO2 values. The coordinated decrease in respiration rate and the complete consumption of lactate as the primary electron donor also correlated with the onset of flocculation, as indicated by an increased FCI. The organic acid analyses clearly indicated that pyruvate assumed the role of primary electron donor with no apparent change in acetate concentration. Cell flocculation peaked at ∼25 h, just as the last of detectable pyruvate was consumed. The subsequent decrease in the FCI was bimodal. The initial decrease in the FCI corresponded to a slow decline in alkalinity production as measured by decreasing acid addition to maintain pH. When the concentration of acetate fell to ∼15 mM and the FCI entered its second phase and decreased sharply, acid addition increased dramatically. This was likely a result of both the decrease in microbial metabolism and the aeration of the bioreactor. During active growth, lactate will be mineralized to both CO2 and bicarbonate with the CO2 being stripped from the culture via aeration and bicarbonate modestly increasing the alkalinity of the culture. As the metabolic rate of the culture decreases due to nutrient limitation, CO2 and bicarbonate generation will also decrease, but the CO2 will continue to be removed from the system while increasing amounts of bicarbonate will form carbonic acid and CO2. This will result in a net loss of protons from the system, thus increasing the pH and cause the increased addition of acid to the culture. Correlating protein expression with culture conditions Significant differences were observed between the proteomes of cells from uncontrolled “aerobic” flasks and controlled aerobic batch reactors when analyzed by 2DE and MS. In general, proteins predicted to be expressed in cells growing under low oxygen tensions or anaerobically [e.g., fumarate reductase (Myers and Myers 1992; Maier et al. 2003], OmpW, glutamine synthase, decaheme cytochrome c MtrA, MtrC, and OmcA (Beliaev et al. 2002, 2005; Fang et al. 2006) were exclusively expressed or detected with increased abundance in samples from shake flasks when compared to those from controlled batch and chemostat cultures. Fumarate reductase (spot 432, SO0970), an enzyme required for anaerobic growth with fumarate (Maier et al. 2003), was detected in high abundance in 2DE patterns of lysate proteins from cells grown in the flask cultures, but was not detected in samples from aerobic controlled batch reactors (Fig. 3). Similarly, the outer membrane protein OmpW (spot 1945, SO1673), and a hypothetical protein (spot 1275, SO3549) were present in 2DE patterns of proteins from flask culture lysates, but not detected in aerobic controlled batch reactors. A significant increase in the number of peptides corresponding to OmpW and fumarate reductase (8.1× and 6.3× higher, respectively, in the flask samples compared to controlled batch samples) was observed in the MS-based AMT tag analyses of these lysates (Table 1), confirming the 2DE results. It is presently unclear why OmpW and the hypothetical protein abundances increased in low DO2, but our laboratory has observed this trend in the former protein several times (unpublished). In addition, peptides representing the multiheme cytochromes MtrA, MtrC, and OmcA and outer membrane β-barrel protein MtrB, were observed in two- to fivefold higher abundance in the AMT tag analysis of samples from shake flasks versus those from controlled batch cultures. These observations strongly suggest the use of shake flask cultures to assess changes in protein expression patterns related to aerobic versus anaerobic metabolism could yield inaccurate results. Fig. 3Two-dimensional gel electrophoresis patterns of whole cell lysate proteins from S. oneidensis MR-1 grown in shake flasks or continuous batch cultures. The proteins indicated by spot number correspond to those identified as varying with statistical significance between the two different culture methods (see Table 1). The gel images are oriented with the basic proteins to the right, acidic proteins to the left, high molecular weight toward the top and low molecular weight toward the bottom. Flask: shake flask cultures (two replicate experiments); CB, controlled batch cultures (two replicate experiments)Table 1Differential protein detection between flask and controlled batch cultures as determined by 2DE and MSORFProtein nameSpot number2DEMSAvg flaskAvg CBP-valueFlask/CB ratioAvg flaskAvg CBFlask/CB ratioSO0970Fumarate reductase4324900ND––106 ± 3417 ± 2b6.3SO4349Ketol-acid reductoisomerase636114831500.0030.422 ± 6139 ± 260.2SO19312-Oxoglutarate dehydrogenase763495a9510.024a0.536 ± 146 ± 40.8SO3237Flagellin999271112239.9E-052.2108 ± 3673 ± 141.5–ND104811961968.8E-046.1–––SO3549Hypothetical protein1275648ND–––––SO3681Universal stress protein family138226007013.5E-063.751 ± 1213 ± 23.9–ND1426313ND–––––SO1673OmpW19452927ND––62 ± 248 ± 18.1SO4410Glutamine synthetase, type I1946100117471.8E-040.658 ± 1263 ± 30.9SO1776MtrB7 ± 14 ± 12.1SO1777Decaheme cytochrome c MtrA8 ± 12 ± 05.5SO1778Decaheme cytochrome c MtrC36 ± 618 ± 12.0SO1779Decaheme cytochrome c OmcA21 ± 311 ± 11.9ORF open reading frame (www.ncbi.nih.gov/), Protein name annotation for open reading frame (www.ncbi.nih.gov/), Spot number spot number assigned by Progenesis software using images shown in Figs. 3 and 5, Avg flask average integrated spot density in 2DE images (3–4 images per sample) from shake flask samples, Avg CB average integrated spot density in 2DE images (3–4 images per sample) from continuous batch culture samples, P-valueP-value from two-tailed Student’s t-test analysis of average integrated density values, Flask/CB ratio ratio of average integrated density values for flask and continuous batch culture samplesaNote that the difference in spot 763 is a function of the variability in the protein between the two flask cultures. In Flask 1, the average integrated density was 1020 with a coefficient of variation (CV) of 12.5% whereas in Flask 2, the average integrated density was 316 with a CV of 31.6%. The 495 represents the averaging of integrated densities from all of the patterns from these two flasks together with a resulting CV of 60%bThe average MS abundance data ± the standard deviation resulting from triplicate analysis of each biological sample Chemostats provide enhanced experimental consistency Chemostat cultures provide the highest level of control of any known cultivation technology (Monod 1950; Novick and Szilard 1950; Wimpenny 1985). To evaluate the reproducibility of chemostat cultures, duplicate bioreactors were operated in steady state with S. oneidensis MR-1 under 50% DO2 as above and the growth and carbon metabolism parameters were measured. Each of the aerobic cultures attained an initial maximum optical density of ∼0.70 by 20 h and eventually achieved a steady state cell density at 0.53 (A600 nm) (Fig. 4). The two reactors were highly similar in terms of growth and acid addition rates, as well as organic acid concentrations at the time of sampling of 100 h. Lactate, acetate, fumarate and succinate were all below detectable limits suggesting that the cultures were in fact carbon-limited during steady state growth. Pyruvate was present in both chemostatic cultures at 0.14 ± 0.02 mM. Dry weight biomass measurements confirmed the highly similar cell densities of 338.0 ± 4.3 mg/l with only a 1.3% variance between the reactors. In comparison, the duplicate controlled batch and shake flask cultures used in this study yielded similar biomass amounts but varied by 2.3 and 17.6%, respectively. These results support our contention of greater biological variability among replicate flask cultures relative to bioreactor cultures. Fig. 4Shewanella oneidensis MR-1 cultured in duplicate bioreactors at 20% DO2. The same inoculum culture was used for both bioreactors. Cells were harvested from each reactor when the optical density and organic acid concentrations were similar. The average dry weight biomass was 338 ± 4.3 mg/l Culture and proteome variability When the intercultural variability of the proteomes of cells from duplicate shake flasks, controlled batch bioreactors, and chemostat cultures was assessed using 2DE and MS, notable differences were identified. For 2DE analysis, digital images of the gels were processed for spot detection and pattern matching with one 2DE image as a reference pattern and all others matched to it to give the protein spots identification numbers. Statistical analysis of the relative abundance of each matched protein spot across the datasets employed a two-tailed Student’s t-test as previously described (Giometti and Taylor 1991). Differences in 2DE spot patterns were observed between the replicate flask cultures (Fig. 5a, d), including one unidentified protein (spot 771; P < 0.004). In contrast, there was relatively little variability (P < 0.05) in 2DE proteome profiles from the replicate gels from either the controlled batch or chemostat cultures. Fig. 52DE protein patterns from biological replicates of the three growth methods. The within method replicates show few significant differences. The chemostat and controlled batch replicates showed no quantitative differences with a statistical significance of P < 0.05 whereas the flask culture replicates showed one quantitative difference (Spot 771) with a difference of P < 0.004. SS steady state culture, CB controlled batch culture. The gel images are oriented with the basic proteins to the right, acidic proteins to the left, high molecular weight toward the top and low molecular weight toward the bottom Data from MS-proteome analysis was consistent with the 2DE results. Triplicate samples from duplicate cultures from shake flask, controlled batch, and chemostats were analyzed and Venn diagrams (Fig. 6) were constructed to illustrate the similarities and differences between biological and analytical replicates based on the cultivation method. A comparison of protein detection consistency between the biological duplicates showed that the flask grown cells (Fig. 5a) exhibit a higher degree of variability. Flask A averaged 972 proteins detected from triplicate analyses while flask B averaged 1,029 proteins with only 770 proteins (77%) in common. The consistency of detected proteins between biological replicates was higher in the controlled batch (84%, Fig. 5c) and chemostat cultures (88%, Fig. 5e). This was not due to a lack of consistency in the analytical replicates since protein detection consistency was between 88 and 89% for all three cultivation methods (Fig. 5d–f). Secondly, there was an overall decrease in the number of proteins detected from the flask grown (770) to the controlled batch (713) and to the chemostat (571) cultures. This can be explained by the presumed homogeneity of the culture increasing as the degree of control over pH and DO2 increased thus allowing the entire culture to experience similar environmental conditions. Fig. 6MS proteomic analysis of the same duplicate (left) shake flask, (middle) controlled batch and (right) chemostat grown cells that were analyzed by 2DE. The consistency in the proteins detected between biological duplicates from shake flask (a), controlled batch (c) and chemostat (e) cultures shows the increased similarity in proteomes with increased control over culturing parameters. This was not an effect of the instrumentation since analytical replicates from each of the shake flask (b), controlled batch (d) and chemostat (f) cultures consistently had (88% of the detected proteins in common. The values below each Venn diagram circle are the number of detected proteins for each biological (a, c, e) or analytical (b, d, f) replicate. The value in the overlap region is the number of proteins in common between the replicates with the corresponding percentage for ease of comparison. The percentage shown is the number of proteins in common divided by the average of the two replicates presented Discussion The advantages of controlled cultivation have been demonstrated for many different research applications in microbiology, including the determination of carbon, nitrogen or electron flow (Vasconcelos et al. 1994; Daran-Lapujade et al. 2004; Kolkman et al. 2005, 2006; Adams et al. 2006), kinetic constants for specific reactions (Nethe-Jaenchen and Thauer 1984), alternate roles for traditionally well-defined enzymes (Snoep et al. 1990), and whole cell protein fingerprinting (Wunschel et al. 2005). While there have been statements touting the advantages of controlled cultivation, an in depth investigation of the differences in metabolism between cultivation methods has not been performed. The closest exception to this reported that microarray results between three chemostatic cultures from three different laboratories were significantly more reproducible compared to a previous study that performed a similar comparison using shake flask cultures (Piper et al. 2002). However, the authors reported that there were differences in harvesting and other procedures which may well have skewed the findings. To this end, the experiments reported here were specifically directed toward determining whether or not controlled cultivation results in a more homogenous culture with regard to both targeted and comprehensive proteome profiles from three cultivation methods in our laboratory. Our results demonstrate that variations in DO2 concentration and consumption rates between replicate flask cultures in the late log phase of growth can impact metabolism. This was evidenced by the increased lag time in the uncontrolled flask cultures relative to the controlled cultivations. The data suggests that this may be an effect of oxygen toxicity when DO2 levels are above 20%. This has been recently tested in S. oneidensis MR-1 using sealed culturing vessels and levels above 21% headspace O2 were found to be inhibitory (McLean et al. 2007). Two possible explanations exist for this observation. It is possible that the extended lag and initial slow growth rate was the result of oxygen toxicity at DO2 values above 20%. However, the uncontrolled batch cultures contained 30 mM PIPES buffer to aid in the maintenance of pH during growth while the controlled batch and chemostatic cultures contained only 3 mM PIPES. Hence, there would have been a large difference in the ionic strength of the two media and so while DO2 toxicity may be the reason for the extended lag phase, other reasons cannot be ruled out. Separately, differences in acetate concentrations of the replicate shake flask cultures compared to controlled batch and chemostat cultures indicated that such differences may well have promoted additional biological variability. The difference in acetate concentrations between replicate shake flask cultures compared to the consistent acetate concentrations in controlled batch and chemostat cultures has particular implications for S. oneidensis strain MR-1. MR-1 can use lactate and pyruvate as an electron donor during anaerobic growth, but not acetate (Lovley et al. 1989; Nealson and Myers 1992). Thus, variations in the concentration of acetate and the variable DO2 uptake rates in shake flask cultures will cause a temporal shift in carbon source usage and electron flux. Such shifts can be expected to result in variable metabolic, transcriptomic and proteomic profiles between independent shake flask cultures if only a small number (1–3) of samples are taken, since one culture may have exhausted a carbon source and consequently shifted metabolism and electron flow while the “biological replicate” has not. Given the general metabolic flexibility of bacteria including S. oneidensis MR-1 and their ability to respond rapidly to changes in environmental conditions, changes in the expressed proteins (proteome) would be expected as a result of exposing cultures to different environmental conditions. Through the use of comprehensive proteome measurements, the results presented here demonstrate that chemostat cultures provide the most consistent source of cellular material for biochemical measurements involving microbial cells with a minimum contribution to those measurements from biological variation. Two independent methods of proteome measurement, 2DE and AMT MS, both showed that flask cultures yielded protein samples with more culture-to-culture variability than observed in either controlled batch cultures or continuous chemostat cultures, primarily among proteins thought to be associated with anaerobic or microaerobic metabolism. A similar effect was found with respect to differential regulation of genes via microarrays when the results of two different investigations were compared from yeast cells, showing less discrepancy between chemostatic cultures (Daran-Lapujade et al. 2004). Many of the proteins present in higher abundance in the aerobic shake flask cultures relative to the controlled batch or chemostat cultures in this study have previously been reported to be up-regulated in anaerobic compared to aerobic cultures (Beliaev et al. 2002, 2005; Fang et al. 2006). MtrA,B,C and OmcA are involved in metal-reduction in anaerobic cultures supplied with metals as terminal electron acceptors (Beliaev and Saffarini 1998; Beliaev et al. 2001; Myers and Myers 2002; Pitts et al. 2003) while the outer membrane protein OmpW and fumarate reductase have both been observed to be significantly more abundant in anaerobic cultures supplied with iron or fumarate as electron acceptor than in aerobic cultures (Fang et al. 2006). Thus, the shake flask cultures in the experiments described here must have included cells experiencing a lack of oxygen due to the nutrient depletion that is an inherent complication of such cultures, resulting in a greater range of expressed proteins. Achieving a systems-level understanding of microorganisms, including the underlying metabolic and regulatory networks that control cell physiology and the ability to predict responses to perturbations, is indeed an ambitious goal. Systems biology takes full advantage of the state-of-the-science technology and genomic information, with the scientific as well as the practical benefits expected to be plentiful. Our results emphasize that functional genomics can greatly benefit from well-defined and homogeneous cell cultures provided by controlled cultivation techniques. The use of controlled cultivation techniques ensures that the large and comprehensive datasets used in systems biology research are derived from well-characterized and homogeneous biological samples, thus reducing the inherent variability that often complicates drawing biological conclusions and will allow for more accurate and robust identifications of metabolic and regulatory networks.

Diabetologia-3-1-1914300

Automated measurement of brain and white matter lesion volume in type 2 diabetes mellitus

Aims/hypothesis Type 2 diabetes mellitus has been associated with brain atrophy and cognitive decline, but the association with ischaemic white matter lesions is unclear. Previous neuroimaging studies have mainly used semiquantitative rating scales to measure atrophy and white matter lesions (WMLs). In this study we used an automated segmentation technique to investigate the association of type 2 diabetes, several diabetes-related risk factors and cognition with cerebral tissue and WML volumes. Introduction Diabetes mellitus type 2 is associated with accelerated cognitive impairment and an increased incidence of dementia [1]. Recently, we have demonstrated that cognitive impairments in patients with type 2 diabetes are accompanied by brain atrophy and ischaemic white matter lesions (WMLs) [2]. Other neuroimaging studies have also demonstrated that type 2 diabetes is associated with a moderate degree of cerebral atrophy [3–5] and with an increased occurrence of cerebral infarcts [6]. These studies have used either semiquantitative rating scales [3, 4] or cerebrospinal fluid to brain ratios [2, 4] to assess atrophy. Quantitative assessment of volumes of grey matter, white matter, lateral ventricle, and cerebrospinal fluid in patients with type 2 diabetes has not been performed. The association between WMLs and type 2 diabetes is debated. Although several studies found an association between type 2 diabetes and WML severity or progression [2, 7–13], others did not [4, 5, 14–18]. These inconsistencies are probably due to the use of different, mostly semiquantitative, WML grading methods, and differences in study design and population selection. Furthermore, even within the population of healthy elderly the variance in WML severity is large [19]. The present study aimed to determine quantitatively the effects of type 2 diabetes on cerebral tissue volumes and WML severity by using an accurate magnetic resonance (MR) imaging-based automated segmentation algorithm [20] in a large, representative, cross-sectional, population-based sample of patients with type 2 diabetes and control participants. A second aim was to study the association of several diabetes-related variables and cognition with cerebral tissue volumes and WML severity. Subjects and methods Participants Participants were recruited between September 2002 and November 2004 as part of the Utrecht Diabetic Encephalopathy Study (UDES), a cross-sectional, population-based study on determinants of impaired cognition in type 2 diabetes [2]. The UDES study aimed to identify potential risk factors for cognitive impairment in type 2 diabetes. Therefore, patients were not selected for the presence or absence of diabetic complications, comorbid conditions (e.g. hypertension) or exposure to other risk factors (e.g. smoking). For inclusion in the present study, participants had to be 55–80 years of age, functionally independent, and Dutch-speaking. Type 2 diabetes patients (n = 122; 56–80 years of age) were recruited through their general practitioner and had a diabetes duration of at least 1 year. Controls (n = 57, 55–78 years of age) were recruited among the spouses and acquaintances of the patients. Exclusion criteria for all participants were a psychiatric or neurological disorder unrelated to diabetes that could influence cognitive functioning, a history of alcohol or substance abuse, and dementia. Controls with a fasting blood glucose ≥7.0 mol/l were also excluded. Twice as many patients as controls were included to increase the statistical power of within-group analyses in the type 2 diabetes group. The study was approved by the medical ethics committee of the University Medical Center Utrecht and each participant signed an informed consent form.In a standardised interview, participants were questioned about diabetes duration, height and weight, history of hypertension and smoking, level of education (seven categories, corresponding to years of education: <6, 6–7, 8, 9, 10–11, 12–18 and >18, respectively [21]), medication use and history of vascular disease. Furthermore, all participants measured their blood pressure at home at nine different time points during the day. These measurements were used to calculate the mean arterial pressure. Hypertension was defined as an average systolic blood pressure ≥160 mmHg and/or diastolic blood pressure ≥95 mmHg and/or self-reported use of blood pressure-lowering drugs. BMI, fasting glucose and HbA1c were also determined. Hypercholesterolaemia was defined as a cholesterol:HDL-cholesterol ratio greater than 5 [22] or the use of cholesterol-lowering drugs. All subjects underwent a neuropsychological evaluation, including 11 different tests addressing the cognitive domains of visuo-construction, attention and executive function, information processing speed, memory and abstract reasoning [2]. For further analysis, the raw scores of the different tests were standardised into z-scores per domain. For the present study, these z-scores were averaged into one composite cognitive z-score.Brain MR images were acquired as part of the study. In 23 participants with type 2 diabetes and 11 control participants no MR images were available for automated analysis: in 14 cases (five controls, nine diabetic patients) an MR image had not been performed because of MR contraindications and in 20 cases (six controls, 14 diabetic patients) the scan could not be analysed automatically because of technical problems, such as failure to retrieve the digital MR images or incompleteness of the series of images, or because image quality did not allow automated processing. Data for 99 patients with type 2 diabetes and 46 controls were analysed. Age, sex and duration of diabetes for the excluded subjects were similar to those for the included subjects (control subjects, average age 64.6 years, five men and six women; diabetic subjects, average age 66.5 years, 13 men, ten women, average diabetes duration 8.7 years). MR imaging Brain MR images were acquired on a Philips Gyroscan ACS-NT 15 whole-body system operating at 1.5 T (Philips Medical Systems, Best, The Netherlands) with a standardised MR protocol (slice thickness 4 mm, 38 contiguous slices, 230 × 230 mm field of view, 256 × 256 scan matrix). Axial T1, inversion recovery (IR), T2, proton density (PD) and fluid attenuated inversion recovery (FLAIR) scans were made: T1, 234/2 ms (repetition/echo time); IR, 2919/410/22 ms (repetition/inversion/echo time); T2, 2200/100 ms (repetition/echo time); PD, 2200/11 ms (repetition/echo time); FLAIR, 6000/2000/100 ms (repetition/inversion/echo time). Image processing Preprocessing consisted of intrasubject registration of the five MR sequences and extraction of a brain mask. Registration was performed using an affine nine-parameter mutual information-based algorithm [23] with the FLAIR image as reference.Brain masks were constructed to exclude the skull, skin and background during classification. The masks were extracted by k-means clustering of the T1, IR, T2, PD and FLAIR images using eight clusters. The clusters containing cerebrospinal fluid and brain were combined. Holes in the mask were filled and appending structures, such as eyes, were removed using morphological operators. Dilation of the brain mask by three voxels ensured the inclusion of all cerebrospinal fluid. One mask had to be edited manually because appending structures were included. The final brain masks contained the whole brain including the cerebellum and brainstem.Segmentation of the MR IR and FLAIR images into white matter, cortical and subcortical grey matter, lateral ventricles, cerebrospinal fluid not including the lateral ventricles (CSF) and WML was executed fully automatically by means of a probabilistic k-nearest neighbour (KNN)-based classification algorithm [20]. The algorithm was trained on expert manual segmentations of ten subjects who had varying degrees of WML, who were similar in age to the participants of this study and scanned using the same protocol, but who did not participate in this study. Manual segmentation of grey matter and white matter was performed on MR IR images, because these provide optimal tissue contrast for the assessment of the grey matter–white matter boundary, and manual segmentation of lateral ventricles, CSF and WML was performed on MR FLAIR images. All MR images were available to the expert for reference. Performance of the classification algorithm was validated previously using a leave-one-out procedure and resulted in similarity indexes of at least 0.808, indicating excellent agreement [20]. For each participant, classification produces five separate images of white matter, grey matter, lateral ventricles, CSF and WML probability per voxel. As an example, the result of the classification of the MR FLAIR and inversion recovery images of a diabetes patient is shown in Fig. 1. White matter, grey matter, CSF and lateral ventricle volumes were calculated by summing over the probability image and multiplying with the voxel dimensions. WML volume calculation deviated slightly. A threshold of 0.5 was applied on the WML probability image and all unconnected voxels were removed. Then, WML volume was calculated by summation over the image and multiplication with the voxel dimensions. The intracranial volume was calculated as the sum of white matter, grey matter, lateral ventricles, CSF and WML volumes; total brain as white matter plus grey matter volume; and total CSF as CSF plus lateral ventricle volume. Volumes of automatically segmented tissues for the male control participants of the present study did not differ from the tissue volumes found for the manually segmented men (manually segmented men, n = 8; women, n = 2 [not compared with female controls]). Furthermore, all segmentations were carefully reviewed by one of the authors (C. Jongen). During this process, the author was blinded to the diabetes mellitus status of the participants. Twelve WML segmentations were manually edited to be sure that infarcted tissue was excluded and ten were edited because of artefacts in the FLAIR image. Fig. 1MR FLAIR (a) and inversion recovery image (b) of a diabetes patient with relatively severe WMLs. On the MR FLAIR image, the WMLs are clearly visible as white areas, whereas on the inversion recovery image the boundary between grey and white matter is much better defined. (c) The result of segmentation using the automated KNN-based algorithm. The colours indicate the different tissue classes: grey matter (yellow), white matter (dark blue), lateral ventricles (green), CSF (red) and WML (light blue) Statistical analysis Differences in demographic data and risk factors between control participants and those with type 2 diabetes were analysed using a univariate general linear model or logistic regression adjusting for age and sex. A univariate general linear model was used to analyse differences in tissue volume between type 2 diabetes and control participants adjusting for age, sex, intracranial volume and level of education. Additionally, separate analyses for men and women were performed, because cerebral tissue volumes are different among men and women. Kolmogorov–Smirnov tests showed that WML volume was not normally distributed. Therefore, we used a natural log transformation of WML volumes in the analyses. Lateral ventricle volume distribution was slightly non-normal. Using natural log-transformed volumes gave similar results to using untransformed volumes. For ease of interpretation, results for untransformed volumes are reported. Additionally, the associations of age with tissue volumes were analysed. Furthermore, associations between the tissue volumes with the composite cognitive performance were analysed for type 2 diabetes and control participants separately, adjusting for age, sex, intracranial volume and level of education. Within the group of type 2 diabetes patients associations between MR volume measures and diabetes duration, HbA1c, hypertension, mean arterial pressure, history of macrovascular disease, and hypercholesterolaemia were determined, adjusting for age, sex, intracranial volume and level of education. All data were analysed using SPSS 12.0.1 (SPSS Inc., Chicago, IL, USA). Results Patient characteristics are shown in Table 1. Table 2 shows the brain tissue and WML volumes unadjusted for differences in age or intracranial volume. Table 3 shows the results of the analysis of the tissue volume differences between controls and patients with type 2 diabetes. Diabetes patients had significantly smaller grey matter volume (estimated volume difference −21.8 ml; 95% CI −34.2, −9.4; p = 0.001; F = 12.091) and significantly larger lateral ventricle volume than controls (estimated volume difference 7.1 ml; 95% CI 2.3, 12.0; p = 0.004; F = 8.441). Total CSF volume was also larger in diabetes patients, but not significantly so (estimated volume difference 9.5 ml, 95% CI −0.4, 19.5; p = 0.060; F = 3.594). White matter volume was unaffected (estimated volume difference 2.8 ml, 95% CI −3.2, 8.8; NS; F = 0.844). WML volume was significantly larger in type 2 diabetes patients (56.5%; 95% CI 4.0, 135.8; p = 0.032; F = 4.684). The cumulative distribution of WML volume is shown in Fig. 2. All participants had at least some WML. However, in type 2 diabetes patients a smaller proportion of the subjects had very small lesion volumes (<0.5 ml) (Pearson χ2 with continuity correction, p = 0.014). Such very small volumes mostly reflect pencil-thin lining and capping, which is often considered to be a normal finding in this age group [19]. The regression analyses were repeated for men and women separately (Table 3). Although the direction of the effect of type 2 diabetes was similar in the two sexes, significant associations of type 2 diabetes with tissue volume were only found in women. Table 1Demographics of participants and risk factors Control participantsType 2 diabetes patientsMean difference (95% CI)aOdds ratio (95% CI)aMen/women20/2649/50–1.3 (0.6, 2.6)Age (years)  Men66.4 (6.3)65.9 (6.0)−0.5 (−2.8, 3.7)–  Women63.8 (5.0)65.9 (5.2)2.1 (−0.4, 4.5)–Level of educationb4 (3–5)4 (3–5)––Diabetes duration (years)–8.7 (6.1)––HbA1c (%)5.5 (0.3)6.8 (1.2)1.4 (1.0, 1.7)***–Use of insulin (%)–29.3––Hypertension (%)c28.370.76.2 (2.8, 13.5)***Mean arterial pressure (mmHg)97.5 (10.6)102.6 (11.5)4.8 (0.9, 8.8)*–History of macrovascular disease (%)4.327.3–8.3 (1.8, 37.0)**Hypercholesterolaemia (%)42.269.4–3.7 (1.7, 8.1)**Smoking ever (%)47.866.3–2.2 (1.0, 4.7)*BMI (kg/m2)27.2 (4.4)28.0 (4.4)0.8 (−0.8, 2.4)–Cognition (composite z-score)0.13 (0.45)−0.10 (0.63)−0.22 (−0.40, −0.05)*d–Data in first two columns are mean (SD) except for level of education, which is given as median (interquartile range)*p < 0.05; **p < 0.01; ***p < 0.001aAdjusted for age and sexbSeven categories, corresponding to years of education: <6, 6–7, 8, 9, 10–11, 12–18 and >18, respectivelycAll controls with hypertension and 96% of diabetic patients with hypertension used antihypertensive drugsdAdjusted for age, sex and level of educationTable 2Volumes of cerebral compartments (ml) unadjusted for age or intracranial volume Control participantsType 2 diabetes patientsMenWomenMenWomenWhite matter688.0 (49.3)621.8 (46.9)687.2 (56.5)601.0 (56.5)Grey matter401.7 (47.8)404.4 (40.8)388.3 (48.8)360.1 (35.6)Total brain1,090 (81.0)1,026 (72.9)1,076 (91.0)961.1 (71.0)Lateral ventricle33.9 (18.7)22.1 (8.66)36.9 (17.4)29.8 (13.9)Lateral ventriclea30.1 (23.9–36.6)20.3 (16.6–28.0)33.8 (24.9–42.6)26.7 (20.6–35.3)CSF excluding lateral ventricles273.3 (27.1)222.1 (25.1)279.7 (37.8)229.4 (35.9)Total CSF307.2 (32.3)244.2 (26.3)316.5 (42.4)259.2 (41.2)WML3.89 (5.76)2.98 (4.70)3.66 (5.37)6.19 (14.2)WMLa1.81 (0.47–3.48)1.00 (0.48–3.20)2.16 (0.91–3.86)2.56 (0.86–4.46)Intracranial volume1,403 (90.7)1,275 (82.2)1,398 (104.2)1,228 (93.7)aLateral ventricle and WML volumes are median (interquartile range); other data are mean (SD)Between-group comparisons and statistical analyses are presented in Table 3Table 3Adjusted tissue volume differences between participants with type 2 diabetes and control participants Estimated volume difference (ml)F valueAll White matter2.8 (−3.2, 8.8)0.844 Grey matter−21.8 (−34.2, −9.4)**12.091 Total brain−19.0 (−29.9, −8.1)**11.863 Lateral ventricles7.1 (2.3, 12.0)**8.441 CSF not including lateral ventricles9.5 (−0.4, 19.5)3.594 Total CSF16.7 (6.8, 26.5)**11.247 LN WML0.45 (0.04, 0.86)a*4.684Men White matter1.8 (−7.4, 11.0)0.157 Grey matter−14.5 (−33.6, 4.6)2.315 Total brain−12.7 (−30.6, 5.2)2.026 Lateral ventricles4.9 (−3.7, 13.5)1.283 CSF not including lateral ventricles6.9 (−10.2, 24.0)0.650 Total CSF11.8 (−4.9, 28.5)1.993 LN WML0.37 (−0.19, 0.93)b1.781Women White matter7.0 (−1.5, 15.6)2.696 Grey matter−37.1 (−54.1, −20.1)***19.062 Total brain−30.1 (−44.9, 15.4)***16.695 Lateral ventricles9.0 (3.2, 14.8)**9.563 CSF not including lateral ventricles17.2 (4.9, 29.4)**7.802 Total CSF26.1 (13.6, 38.7)***17.215 LN WML0.54 (−0.11, 1.20)c2.745LN WML Natural log of WML volumeData are mean (95% CI) adjusted for age, sex, intracranial volume and level of education (data missing for one woman with type 2 diabetes)*p < 0.05; **p < 0.01; ***p < 0.001a56.5% (95% CI 4.0, 135.8)b44.9% (95% CI −17.0, 154.7)c72.4% (95% CI −10.6, 232.4)Fig. 2Cumulative distribution of WML volume (control men, closed squares; men with type 2 diabetes, closed triangles; control women, open squares; women with type 2 diabetes, open triangles). Very small lesion volumes (<0.5 ml) were significantly more frequent among controls (p = 0.014) than participants with type 2 diabetes Within the patients with type 2 diabetes, no significant associations between MR volumes and diabetes duration, hypertension, mean arterial pressure or hypercholesterolaemia were found. Higher HbA1c levels were associated with larger grey matter volume (6.479 ml per unit HbA1c (%); 95% CI 0.2, 12.8; p = 0.043; F = 4.203), but not with any other volume abnormalities. A history of macrovascular disease was associated with a larger total CSF volume (12.4 ml; 95% CI 2.6, 28.7; p = 0.020; F = 5.659) and a smaller total brain volume (−15.0 ml; 95% CI −29.8, −0.2; p = 0.047; F = 4.057). In the diabetic participants lower composite cognitive performance was associated with significantly smaller total brain volume (−15.4 ml per unit of the composite cognitive z-score; 95% CI −27.6, −3.1; p = 0.015; F = 6.199), larger WML volume (57.4%; 95% CI 2.2, 142.4; p = 0.040; F = 4.370) and non-significantly larger total CSF volume (11.0 ml; 95% CI −0.6, 22.1; p = 0.051; F = 3.911). In the control group exploratory analyses showed an association between higher BMI and larger total CSF volume (2.0 ml; 95% CI 0.34, 3.6; p = 0.017; F = 6.287). No other significant associations were found within the control group. Across the whole population, age was significantly associated with reduced grey matter and total brain volumes and larger lateral ventricle, CSF, total CSF and WML volumes (p < 0.001; F > 20.424; adjusted for diabetic status, sex, intracranial volume and level of education). Age was the most powerful predictor of WML and lateral ventricle volumes and it came second after intracranial volume for predicting grey matter, CSF, total brain and total CSF volumes. No significant interactions between age and type 2 diabetes were found (all p > 0.14). The interaction between age and sex showed a trend towards significance, men having more grey matter decrease (−1.8 ml/year; 95% CI −3.9, 0.3; p = 0.092; F = 2.888) and women having more white matter decrease (−0.9 ml/year; 95% CI −1.9, 0.1; p = 0.079; F = 3.134) with age (other volumes p > 0.26). Discussion Our MR image-based segmentation paradigm showed that type 2 diabetes was significantly associated with smaller grey matter volumes and larger lateral ventricle and WML volumes, whereas white matter volume was not affected. A smaller grey matter volume suggests cortical atrophy, whereas a larger lateral ventricle volume may indicate subcortical atrophy. Separate analysis for men and women showed significantly smaller grey matter and total brain volumes and significantly larger lateral ventricle, CSF and total CSF volumes in female but not in male diabetic patients. The apparent differential effects of diabetes on brain volumes in men and women were an unexpected finding. Previous studies on cognitive functioning or dementia in patients with diabetes have provided no clear indications that the effects of diabetes on the brain might be sex-specific [24], although it should be noted that the role of sex has not yet been studied systematically. In the general population, intracranial volume and relative and absolute grey matter and white matter volumes are known to differ between men and women [25]. Sex also influences the effects of ageing [26–28], men being more severely affected by age-related grey matter decrease than women [29, 30], which was also found in this study. WML volumes among male and female controls were similar, but in female type 2 diabetes patients we found significantly larger WML volumes than in male type 2 diabetes patients. In conditions other than diabetes, more severe WMLs in women than in men have been reported [15, 31–33], but similar WML severity in both sexes has also been reported [19]. Diabetic women had non-significantly smaller age-adjusted total intracranial volumes than control women. However, the expected effects of these intracranial volume differences on brain tissue volumes were smaller than the observed differences in effects on grey matter, CSF and lateral ventricle volumes. Age is a very important predictor of brain tissue and WML volumes and age effects may have confounded the effects of type 2 diabetes in men and women in this study. Hence, the implications of the effect of sex observed in the present study remain to be determined. No statistically significant associations between MR image measures and hypertension or mean arterial pressure within the group of type 2 diabetes patients were found. Similar results were obtained with manual measurements of atrophy and with WML grading in the same study population [2], and in a study that assessed the relation between type 2 diabetes, blood pressure and temporal lobe atrophy [5]. In contrast, another study reported that hypertension was an important determinant of cortical atrophy in patients with type 2 diabetes [4]. We found an association between glycosylated haemoglobin and a slight increase in grey matter volume in type 2 diabetes patients. This counterintuitive finding may be due to chance. Other studies in subjects without diabetes have linked increased glycosylated haemoglobin with cerebral atrophy [34] and WMLs [35]. Cognitive impairment was associated with smaller total brain volume and larger WML volumes in type 2 diabetes patients. This is in line with observations from another recent study that reported an association between WMLs and subcortical brain atrophy and cognitive performance, in particular the speed of information processing [36]. This suggests that atrophy as well as WMLs have a negative impact on cognition. Previous studies that analysed WML volume or progression in type 2 diabetes patients have reported inconsistent results [4, 5, 7–18]. Several methodological factors may have contributed to these inconsistencies. First, techniques to measure WML severity have varied widely, from noting lesion presence or absence [12] to volumetric measurements [13, 18]. The majority of studies applied semiquantitative ordinal grading scales. It has been shown that these scales can give variable results [37] and some scales are relatively insensitive to mild to moderate WMLs. Second, the examined populations and the study design varied considerably [38]. Only a small proportion of the studies had a true population-based setting, whereas the majority of studies were hospital-based, involving cohorts of patients with stroke, hypertension or other forms of cardiovascular disease. Furthermore, several studies involved fewer than 20 type 2 diabetes patients [8, 11–13, 18]. These small sample sizes may have reduced statistical power. The present study used volumetric measurements in a large population-based sample. The combination of increased atrophy and increased WML volume indicates that type 2 diabetes is associated with mixed pathology in the brain. The association with vascular lesions, such as WML, is not surprising because diabetes is a well-known risk factor for cerebrovascular disease [39]. Indeed, previous imaging studies [38], as well as autopsy studies [40], have reported an increased occurrence of vascular lesions, in particular infarcts, in diabetic patients relative to controls. The mechanisms underlying accelerated brain atrophy in diabetic patients are less clear. Several processes related to glucose toxicity, abnormalities in cerebral insulin homeostasis, and microvascular abnormalities have been implicated [41], but their exact contributions to abnormalities in cerebral function and structure in diabetes still need to be elucidated. A limitation of our quantitative automated volumetric segmentation algorithm is slight overestimation of the CSF and slight underestimation of the lateral ventricle volume in subjects with markedly enlarged occipital horns. Some misclassification of interhemispheric CSF as lateral ventricle might also occur. However, the misclassified volumes are small compared with ventricle (about 2% and 6%) and CSF (about 0.3% and 0.4%) volumes and total CSF volumes are not affected. The artefacts were proportionally distributed across men and women and between type 2 diabetes patients and controls. Therefore, it is unlikely that these inaccuracies affected the analyses. Furthermore, all segmentations were inspected visually, so results will not have been affected by other segmentation errors. Our algorithm did not classify WMLs into deep and periventricular lesions. Such automated subclassification may provide valuable information; however, it is hard to achieve. WMLs tend to extend smoothly from the ventricular wall [42] and periventricular WMLs are often connected to deep WMLs on MR images. Therefore, defining meaningful boundary criteria that can be consistently and reproducibly applied automatically is difficult. Other published methods have used T1 and T2 [25], PD and T2 [26], T1 only [27], or T1, PD and T2 [30] MR images for segmentation. However, in a previous study it was shown that the use of IR and FLAIR images is highly preferable in brain tissue segmentation and that optimal performance of the automated KNN classification algorithm is achieved with this combination of images [20]. Furthermore, the learning data were segmented manually on the basis of IR and FLAIR images with the T1, T2 and PD images available to the expert for reference. Thus, our KNN classifier retrieves its learning data from expert segmentations based on all available information and segments using the optimal image combination for tissue classification. Our automated segmentation method offers major advantages over manual methods by enabling precise, objective and reproducible volumetric measurements of cerebral tissues in large numbers of patients. In future studies, this method might be used to quantify the progression of abnormalities on MR images in patients with type 2 diabetes, as well as in patients with other conditions. Such studies using quantitative MR data should focus not only on the differences between groups of patients and controls, but also on differences within a patient group to identify determinants of structural brain changes. Electronic supplementary material Below is the link to the electronic supplementary material.

Cancer_Causes_Control-3-1-1914283

Alcohol consumption, cigarette smoking, and endometrial cancer risk: results from the Netherlands Cohort Study

Objective To examine the association between alcohol consumption, cigarette smoking, and endometrial cancer. Introduction Endometrial cancer is the most frequently diagnosed gynecologic cancer in Europe [1]. The development of endometrial cancer has been related to exposure to estrogens unopposed by progestagens [2]. Many studies have shown a positive association between alcohol ingestion and estrogen levels in postmenopausal women. For instance, cross-sectional data from the European Prospective Investigation into Cancer and Nutrition (EPIC) suggested that elevated blood levels of estrone are observed with increasing alcohol consumption in postmenopausal women [3]. Thus, alcohol could be expected to increase endometrial cancer risk by elevating estrogen levels. An important determinant of estrogen levels in women is use of unopposed hormone replacement therapy (HRT). Accordingly, use of unopposed HRT is consistently associated with an increased risk of endometrial cancer [4–6]. Furthermore, it has been suggested that alcohol consumption increases estradiol levels in particular in postmenopausal women who are on HRT [7, 8]. However, previous studies have indicated that alcohol consumption is either weakly or not associated with a reduced risk of endometrial cancer and no significant interaction with use of HRT has been found [9]. Although several studies have analyzed the association between alcohol consumption and endometrial cancer risk [10–22], only few studies have examined the risk associated with various measures of alcohol consumption (e.g., amount and type of alcohol) including only one comprehensive prospective cohort study [18]. In contrast to alcohol, smoking has been hypothesized to exert anti-estrogenic effects [23] and to lower the risk of endometrial cancer in this way. Also, an effect modification by use of HRT seems reasonable [24]. Furthermore, it has been suggested that body mass index (BMI) and age at menopause might mediate part of the inverse association between smoking and endometrial cancer [24]. Earlier prospective studies [22, 25–29] have generally suggested that smoking is associated with a slight to moderate protection against endometrial cancer. However, to date, only the largest and most recent prospective cohort study has reported a significantly reduced risk in both current and past smokers [29]. Moreover, this large study explored the relationship between HRT and smoking and found no effect-modification, and also found that the association between smoking and endometrial cancer was not confounded by alcohol use [29]. The evidence from other epidemiological studies with regard to an effect-modification by use of HRT is ambiguous [24]. As many case–control [24], but only a few cohort studies have reported on the association between smoking and endometrial cancer [22, 25–29], the International Agency for Research on Cancer concludes that prospective cohort studies, in which selection and recall bias are minimized, are scarce [30]. Only two cohort studies have explored the association between smoking and endometrial cancer comprehensively by examining the risk associated with all common quantitative smoking measures (e.g., smoking duration, time since cessation) [28, 29]. Since only few prospective cohort studies have investigated the association between alcohol consumption, cigarette smoking, and endometrial cancer comprehensively, important features of this relationship are under-explored. Hence, we aim to provide additional evidence based on prospective data. Moreover, we intend to elucidate the hormonal mechanisms underlying endometrial carcinogenesis by investigating, first, whether BMI and age at menopause might act as intermediary variables in the association between smoking and endometrial cancer and by examining, second, whether there is evidence regarding a potential effect modification by HRT use. Materials and methods The Netherlands Cohort Study (NLCS) started in September 1986 when 62,573 women aged 55–69 years were enrolled in the cohort. Ethical approval was obtained from the ethics committee of the University Hospital Maastricht. All women were presumed to be postmenopausal. At baseline, data on dietary habits and other risk factors (such as alcohol consumption, smoking history, reproductive history, and anthropometry) were collected by means of a self-administered questionnaire. Data analysis was conducted according to the case–cohort approach. In this approach, cases are derived from the cohort (providing numerator information for the incidence rates), while the accumulated person-years at risk of the cohort are estimated from a random sample from the cohort, i.e., the subcohort (providing denominator information for the incidence rates). Following this approach, a subcohort of 2,589 women was sampled after the baseline exposure measurement. The subcohort has been followed up biennially by mail for vital status information. The vital status of subcohort members, who did not respond was completed by contacting the municipal population registers. Incident cases occurring in the entire cohort were detected by annual record linkages to the Netherlands Cancer Registry and the nationwide network and registry of histopathology and cytopathology in the Netherlands (PALGA). Further details on the design of the study and methods of follow-up have been presented elsewhere [31, 32]. The present analysis is restricted to cancer incidence in the 11.3-year follow-up period from September 1986 to December 1997. The completeness of cancer follow up was estimated to be at least 96% [33], and no subcohort members were lost to follow up. Three hundred and twenty-seven incident, microscopically confirmed, invasive, primary endometrial carcinomas were detected after a follow-up period of 11.3 years. Cases were excluded from analysis if they had been diagnosed with non-epithelial tumors (n = 12), and if information on either alcohol consumption or cigarette smoking was incomplete (n = 35). Women were eligible for the subcohort if they did not report at baseline that they had undergone hysterectomy. Application of this inclusion criterion yielded a subcohort of 2,229 members. Individuals were excluded from the analysis if they had been diagnosed with cancer other than skin cancer at baseline (n = 151) and if information on either alcohol consumption or cigarette smoking was missing (n = 177). After these exclusions, 280 cases and 1,901 subcohort members remained available for analysis. Questionnaire data Consumption of alcoholic beverages during the year preceding the baseline interview was assessed by consumption frequency questions on beer, red wine, white wine, sherry, other fortified wine, liqueur, and liquor. Categories ranked from ‘never’ to ‘6–7 times per week’ and information on the number of glasses per consumption day was also requested. Questionnaire data of all cases and subcohort members were key entered twice and processed in a manner blinded with regard to case/subcohort status in order to minimize observer bias in the coding and interpretation of data. The questionnaire has been validated against a nine-day diet record [34, 35]. The Pearson correlation coefficient between the mean daily ethanol intake assessed by the questionnaire and that estimated by the nine-day record was 0.86 for all subjects and 0.78 for users of alcoholic beverages [34]. Respondents that reported to drink alcohol less than once per month were considered non-drinkers. Four items from the questionnaire (red wine, white wine, sherry, and liqueur) were combined into one single wine variable since these items were highly correlated and separate analysis would have resulted in small numbers of subjects within each stratum. Mean daily alcohol consumption was calculated using the Dutch food composition table [36]. Based on data from a pilot study, standard glasses were defined as follows: 200 ml for beer, 105 ml for wine, 80 ml for sherry, and 45 ml for both liqueur and liquor, corresponding to 8 g, 10 g, 11 g, 7 g, and 13 g of alcohol, respectively. Smoking was addressed at baseline by questions on age at first exposure to smoking, age at last exposure to smoking, smoking frequency, and smoking duration of cigarette, cigar, and pipe smokers. As the vast majority of smoking subcohort members was cigarette smokers, analyses were restricted to that particular group. Based on the questionnaire data, the following cigarette smoking variables were constructed: cigarette smoking status (never versus ever and never versus former or current), frequency (number of cigarettes per day), duration (years), age at first exposure (years), and time since cessation (years). Time since cessation was calculated as ‘age at baseline’ minus ‘age at smoking cessation’. Concerning the use of HRT, women were asked whether they had ever used HRT because of complaints related to the menopause. We can assume that all members of our cohort were postmenopausal in 1986, when the baseline questionnaires were completed, and that possible treatment with HRT took place prior to 1986 in most women. Based on information regarding HRT prescription in the Netherlands in the past [37–39], we assume that all HRT users enrolled in the NLCS were treated with unopposed oral estrogens. Data analysis A variable was considered a confounder if (I) it was associated with endometrial cancer risk, if (II) it was associated with alcohol consumption or cigarette smoking, and if (III) age-adjusted hazard ratios changed by more than 10% after adjustment for the potentially confounding factor. Based on the literature [40] and previous analyses, we considered the following variables as potential confounders: age (continuous), age at menarche (continuous), use of oral contraceptives (ever versus never), duration of oral contraceptive use (continuous), age at first child birth (continuous), parity (continuous), age at menopause (continuous), use of postmenopausal hormones (ever versus never), duration of postmenopausal hormone use (continuous), non-occupational physical activity (categorized), BMI (continuous), height (continuous), energy intake (continuous), total fat intake (continuous), intake of saturated fat (continuous), intake of carbohydrates (continuous), intake of dietary fiber (continuous), intake of vegetables (continuous), intake of fruits (continuous), coffee consumption (yes versus no), education (categorized), diagnosis of hypertension (yes versus no), diagnosis of diabetes mellitus (yes versus no), family history of endometrial cancer (yes versus no), and if applicable: total alcohol consumption per day (continuous), type of alcoholic beverage (categorized), current smoking (yes versus no), number of cigarettes smoked per day (continuous), and duration of smoking (continuous). Incidence rate ratios (RR) and corresponding 95 percent confidence intervals (95% CI) for endometrial cancer were estimated in the age-adjusted and multivariate case–cohort analyses with categorized and continuous alcohol and cigarette smoking variables, using the Cox proportional hazards model [41] processed with the Stata statistical software package [42]. Standard errors were estimated using the robust Hubert–White sandwich estimator to account for additional variance introduced by sampling from the cohort. This method is equivalent to the variance–covariance estimator by Barlow [43]. The proportional hazards assumption was tested using the scaled Schoenfeld residuals [44]. Tests for dose-response trends in risk of endometrial cancer were assessed by fitting ordinal exposure variables as continuous terms. Tests for interaction were performed by using the Wald test. Two-sided p values are reported throughout the paper. Results The percentage of women reporting alcohol consumption was similar among cases and subcohort members (67.5% and 66.9%, respectively), as was the mean alcohol consumption per day among users in both groups (7.7 g with standard deviation (sd) = 10.8, and 8.5 g (sd = 10.4), respectively). Current smoking was less prevalent among cases than among subcohort members (15.4% vs. 21.8%), but the number of cigarettes smoked per day did not differ considerably between smokers in both groups (13.6 (sd = 8.4) and 13.2 (sd = 8.1), respectively). Drinkers reported a slightly higher age at menopause and a higher prevalence of both oral contraceptive use and current cigarette smoking than non-drinkers (see Table 1). With regard to smoking status, former and current smokers were slightly leaner and had fewer children than never-smokers. On average, current smokers reported having reached menopause 1 year earlier than former-smokers and never-smokers. The prevalence of both oral contraceptive use and alcohol use was higher among smokers than among never-smokers. Also, average alcohol consumption was approximately twice as high among smokers as among never-smokers (see Table 1). Table 1Means (standard deviation) and distribution (n) of potential confounders according to alcohol consumption and cigarette smoking status among subcohort members, the Netherlands Cohort Study (1986–1995) CharacteristicUnitAlcohol consumption statusCigarette smoking statusNo (n = 630)Yes (n = 1,271)Never (n = 1,100)Former (n = 387)Current (n = 414)Mean (sd)Mean (sd)Mean (sd)Mean (sd)Mean (sd)AgeYears61.8 (4.3)61.4 (4.3)62.0 (4.3)61.1 (4.4)60.7 (4.1)Body Mass Indexkg/m225.4 (3.9)24.9 (3.4)25.3 (3.5)24.7 (3.3)24.6 (3.8)ParityNumber of children2.8 (2.4)2.7 (2.2)3.0 (2.4)2.5 (1.8)2.5 (2.1)Age at 1st child birthYears22.0 (11.2)21.9 (11.3)22.2 (11.2)22.3 (11.3)20.8 (11.3)Age at menopauseYears48.3 (4.8)49.2 (4.3)49.1 (4.4)49.2 (4.1)48.1 (4.8)Total energy intake (including alcohol)kcal1,628 (415)1,724 (386)1,694 (393)1,676 (406)1,705 (406)Alcohol consumption G/day0 (0)8.5 (10.4)5.7 (7.8)10.4 (11.2)12.5 (12.8)CigarettesNo./day13.0 (8.8)11.1 (8.1)0 (0)9.8 (8.0)13.2 (8.1)n (%)an (%)an (%)an (%)an (%)aOral contraceptive useEver124 (20.0)336 (26.7)223 (20.6)127 (32.9)110 (26.8)Physical activity>30 min/day425 (69.1)975 (77.8)796 (74.1)306 (79.5)298 (72.9)Diagnosis of hypertensionYes198 (31.4)351 (27.6)339 (30.8)107 (27.7)103 (24.9)Diagnosis of diabetesYes33 (5.2)40 (3.2)48 (4.4)13 (3.4)12 (2.9)Hormone replacement therapyEver62 (10.0)152 (12.1)107 (9.9)58 (15.1)49 (12.0)Family history of endometrial cancerYes20 (3.2)32 (2.5)23 (2.1)16 (4.1)13 (3.1)Alcohol usersYes––663 (60.3)312 (80.6)296 (71.5)Currently smoking cigarettesYes118 (18.7)296 (23.3)–––a The percentage reported for some variables does sometimes not correspond with the numbers per smoker stratum since part of the information for these variables was missing Based on the literature and based on the methodological criteria specified above, we found the following confounders: age, BMI, parity, oral contraceptive use, non-occupational physical activity, hypertension, age at first child birth, and age at menopause. Alcohol consumption and cigarette smoking status were found to confound each other’s association with endometrial cancer. We controlled for all these confounders in multivariate analyses. In additional analyses, we mutually controlled the age-adjusted risk estimates regarding qualitative smoking measures for the other smoking measures. The multivariate risk estimates did not change substantially when oral contraceptive use (ever/never) was replaced by duration of oral contraceptive use (data not shown). Accordingly, we considered it sufficient to control only for oral contraceptive use (ever/never). Table 2 shows the results for the association between alcohol consumption and endometrial cancer. The multivariate adjusted RR associated with alcohol consumption was 1.06 (95% CI = 0.78–1.43). The multivariate RRs of endometrial cancer for women who consumed up to 4, 5–14, 15–29, and 30 or more gram of alcohol per day versus non-drinkers were 1.09 (95% CI = 0.78–1.52), 0.95 (95% CI = 0.62–1.45), 0.94 (95% CI = 0.52–1.69), and 1.78 (95% CI = 0.88–3.60), respectively. No significant trend was observed (ptrend = 0.62). Table 2Rate ratios of endometrial cancer according to baseline alcohol consumption in the Netherlands Cohort Study, 1986–1997Alcohol consumption (g/ day)Age adjustedMultivariate adjustedCategorical medianCasesPerson-years in subcohortRR (95% CI)aCasesPerson-years in subcohortRR (95% CI)bTotal alcohol    No0916,6411 (ref.)825,8371 (ref.)    Yes4.018913,7461.01 (0.77–1.32)17212,1371.06 (0.78–1.43)    0.1–41.61147,5991.10 (0.82–1.48)1056,6431.09 (0.78–1.52)    5–149.1473,6400.94 (0.65–1.37)393,2790.95 (0.62–1.45)    15–2920.9171,8220.69 (0.40–1.18)171,5750.94 (0.52–1.69)    ≥3037.3116841.20 (0.62–2.34)116391.78 (0.88–3.60)    p trend0.490.62Alcohol from wine    Yes3.218213,0091.06 (0.81–1.37)16611,5071.13 (0.84–1.52)    0.1–41.51258,0721.17 (0.88–1.55)1127,1131.16 (0.84–1.59)    5–148.9383,1460.91 (0.61–1.36)352,7841.07 (0.68–1.67)    ≥1521.8191,7910.80 (0.48–1.35)191,6111.11 (0.64–1.93)    p trend0.430.64Alcohol from beer    Yes1.14291,8731.15 (0.76–1.74)261,6291.30 (0.82–2.07)Alcohol from liquor    Yes3.7342,6480.93 (0.64–1.37)312,3491.11 (0.73–1.68)aRR = rate ratios; CI = confidence interval; n.a. = not applicablebRate ratios adjusted for age (years), body mass index (kg/m2), parity (number of children), use of oral contraceptives (ever versus never), non-occupational physical activity (low, moderate, active, very active), hypertension (yes versus no), age at first child birth (years), age at menopause (years), and current cigarette smoking (yes versus no) The multivariate RR of endometrial cancer for wine-consumers versus non-consumers was slightly, but non-significantly, elevated (RR = 1.13, 95% CI = 0.84–1.52). The RRs were also slightly, but non-significantly elevated when calculated according to different levels of wine consumption and no significant trend was observed (ptrend = 0.64). The multivariate RR of endometrial cancer associated with drinking beer equaled 1.30 (95% CI = 0.82–2.07) and the RR for drinking liquor was 1.11 (95% CI = 0.73–1.68). Regarding smoking, age-adjusted analysis revealed an inverse association between ever-smoking and endometrial cancer risk (RR = 0.70, 95% CI = 0.54–0.92, see Table 3). We observed significant inverse trends of endometrial cancer risk with all quantitative smoking measures. However, these trends became non-significant when never-smokers were excluded. Some of these age-adjusted risk estimates changed substantially after additional adjustment for current smoking status, smoking frequency and smoking duration (see Table 3). Table 3Rate ratios of endometrial cancer according to baseline cigarette smoking features in the Netherlands Cohort Study, 1986–1997Cigarette smoking featuresCategorical medianAdjusted for ageAdjusted for age, current smoking status, frequency and duration of smokingAdjusted for all confoundersCasesPerson-years in subcohortRR (95% CI)aCasesPerson-years in subcohortRR (95% CI)bCasesPerson-years in subcohortRR (95% CI)cSmoking status    Neverdn.a.18711,8721 (ref.)–––16910,3301 (ref.)    Ever smokers938,5160.70 (0.54–0.92)–––857,6440.71 (0.53–0.95)    Neverd18711,8721 (ref.)–––16910,3301 (ref.)    Former smokersn.a504,1810.77 (0.55–1.07)–––473,7570.83 (0.58–1.20)    Current smokersn.a434,3340.64 (0.45–0.91)–––383,8880.59 (0.40–0.88)    p trend0.010.01Frequency (cigarettes/day)    Neverd018711,8721 (ref.)18711,8721 (ref.)16910,3301 (ref.)    0.1–9 4383,7190.65 (0.45–0.95)373,5690.86 (0.49–1.50)333,2231.07 (0.58–1.98)    10–19 12282,4380.74 (0.48–1.14)272,4151.02 (0.54–1.92)252,2141.28 (0.66–2.46)    20+20222,0140.70 (0.44–1.12)222,0141.03 (0.46–2.29)211,7981.31 (0.56–3.03)    p trend0.040.750.43Duration (years)    Neverd018711,8721 (ref.)18711,8721 (ref.)16910,3301.0 (ref.)    0.1–19 10.5232,0090.74 (0.47–1.17)221,9910.71 (0.42–1.18)211,8480.77 (0.43–1.39)    20–3930524,2740.79 (0.57–1.10)514,1200.80 (0.47–1.34)473,6900.89 (0.51–1.56)    40+411319700.42 (0.23–0.75131,8870.44 (0.19–1.02)111,6970.37 (0.15–0.90)    p trend0.000.100.13Age at first exposure (years)    Neverdn.a18711,8721 (ref.)18711,8721 (ref.)16910,3301 (ref.)    <1917343,0980.71 (0.48–1.05)322,9470.78 (0.34–1.80)282,7240.91 (0.37–2.23)    19–2420212,5240.53 (0.33–0.86)202,3710.60 (0.29–1.27)202,1020.88 (0.38–2.02)    25+30312,6910.73 (0.49–1.09)312,5960.82 (0.47–1.46)282,3251.00 (0.53–1.87)    p trend0.010.470.94Time since cessation (years)    Neverdn.a18711,8721 (ref.)18711,8721 (ref.)16910,3301 (ref.)    20+e26.5201,1421.11 (0.68–1.84)171,0410.80 (0.41–1.57)179431.12 (0.52–2.41)    10–19e14141,4590.62 (0.35–1.09)141,4160.38 (0.14–1.03)131,2700.50 (0.17–1.45)    0.1–9e5131,3920.60 (0.33–1.08)131,3470.32 (0.10–1.06)121,2570.50 (0.12–2.00)    p trend0.030.060.26aRR = rate ratios; CI = confidence interval; n.a. = not applicablebRRs were adjusted for age (years). In addition, frequency was adjusted for current smoking status (yes/no) and duration (years); duration was adjusted for current smoking status and frequency (cigarettes/day); age at first exposure was adjusted for current smoking status, frequency, and duration; time since cessation was adjusted for frequency and durationcRRs were adjusted as mentioned under footnote b and additionally for body mass index (kg/m2), parity (number of children), use of oral contraceptives (ever versus never), non-occupational physical activity (low, moderate, active, very active), hypertension (yes versus no), age at first child birth (years), age at menopause (years), and alcohol consumption (gram/ day)dNever smoked cigars, pipe, or cigaretteseEx-smokers only When we adjusted for all confounders, multivariate analysis showed a statistically significant 29% reduced risk of endometrial cancer for ever-smokers when contrasted with never-smokers. When considered separately, the risk reduction appeared to be stronger among current smokers (RR = 0.59, 95% CI = 0.40–0.88) than among former smokers (RR = 0.83, 95% CI = 0.58–1.20). Tests for trends were not significant for any of the quantitative smoking variables when these were adjusted for age and additional confounders. The strongest reduction in risk, which could be observed in both univariate and multivariate models, was associated with a smoking history of 40 or more years compared with having never smoked (RR = 0.37, 95% CI = 0.15–0.90). Moreover, we observed a non-significant 50% reduction in risk in women that quit smoking either nine or less years ago or that quit 10–19 years ago. The data indicated no association between age at first smoking exposure and endometrial cancer. Omitting age at menopause and BMI from the multivariate models either separately or simultaneously did not cause meaningful changes in the corresponding estimates. No interactions in determining endometrial cancer risk could be observed between alcohol consumption and HRT use (p = 0.43), BMI (p = 0.38), age at menopause (p = 0.39), or current cigarette smoking (p = 0.83). When we stratified multivariate alcohol analyses according to smoking status, we observed a non-significantly lower risk of endometrial cancer among alcohol consumers that have ever smoked (RR = 0.91, 95% CI = 0.52–1.58) than among alcohol consumers that have never smoked (RR = 1.16, 95% CI = 0.81–1.67). Concerning smoking and HRT use, the interaction term was not statistically significant (p = 0.11). In this subset analysis, current smoking was associated with a reduced risk of endometrial cancer in women not using HRT (RR = 0.54, 95% CI = 0.35–0.84). In current smokers that did use HRT, the RR was 1.32 (95% CI = 0.57–3.04). Numbers were very small however: only 28 women were current smokers and used HRT and only eight women were current smokers and did not use HRT. Discussion Our results do not suggest a meaningful association between alcohol consumption and endometrial cancer risk. Current smoking is associated with a reduced risk of endometrial cancer. This inverse relationship is neither mediated by BMI nor by age at menopause. Regarding the biological mechanism underlying endometrial carcinogenesis, the so-called “unopposed estrogen hypothesis” is widely accepted [2]. According to this hypothesis endometrial cancer develops when the endometrium is exposed to high levels of unopposed endogenous or exogenous estrogens for a long period of time. This exposure results in elevated mitotic proliferation of endometrial cells which, in turn, increases the risk of DNA replication errors and DNA mutations which can lead to endometrial cancer [2]. Although female alcohol consumers could be expected to be at increased risk of endometrial cancer due to their hormonal profile, we have not detected significant associations between alcohol consumption and endometrial cancer risk. This finding is consistent with the vast majority of previous studies [13, 15–18]. However, in the EPIC study, significantly elevated blood estrone levels were found only in postmenopausal women who consumed more than 25 g of alcohol per day compared to non-drinkers [3]. Thus, possibly, a marked increase in estrone concentrations, and ultimately in endometrial cancer risk, can only be observed in women who consume more than moderate amounts. This notion might be supported by our data as we have observed an (non-significantly) elevated risk of endometrial cancer in women who reported to drink more than 30 g of alcohol per day. Based on literature reviews [7, 8], we hypothesized that we might find a positive association between alcohol consumption and endometrial cancer risk in particular among HRT users. However, our findings do not support the hypothesis of an effect-modification by HRT use; neither did most of the previous epidemiological studies [13, 17, 18]. Concerning smoking, an anti-estrogenic effect has been suggested [23], which should lower the risk of endometrial cancer according to the unopposed estrogen hypothesis. Accordingly, our data indicated a significant risk reduction in current smokers, just like the Nurses’ Health Study did, which has reported a RR of 0.72 (95% CI = 0.57–0.90) for current smokers [29]. In contrast, other cohort studies have observed non-significant associations [25, 26, 28]. We found that the inverse association between smoking and endometrial cancer was more pronounced among current smokers than among former smokers. These findings are in line with the evidence from several case–control [17, 45–47] and two cohort studies [26, 28] and they suggest that the degree of protection might partly depend on the time since smoking cessation. Although not statistically significant, our prospective data support this notion, because we observed that endometrial cancer risk is possibly higher in women that quit smoking 20 or more years ago compared to the risk in women that quit 19 years ago or less. In the epidemiological literature, a few studies presented data on time since cessation [17, 28, 29, 48–50], but only one of them found a significantly lowered risk among women that quit smoking less than 10 years ago [48]. Considering the risk associated with duration of smoking, we have observed a reduction in risk with a long smoking history, just like previous studies [17, 29, 47, 48]. With regard to smoking intensity, we have found that smoking 20 or more cigarettes per day is associated with a non-significantly elevated risk of endometrial cancer. Though one needs to bear in mind that the corresponding confidence intervals were large, these point estimates are possibly not in line with the majority of previous studies, which generally indicated that a high smoking intensity (e.g., smoking 20 or more cigarettes per day) is associated with a decreased risk of endometrial cancer [17, 22, 28, 29, 45, 47, 48, 51]. This inconsistency could be explained by the lack of adjustment for other smoking variables in earlier studies: only one [29] out of six [22, 25–29] prospective cohort studies has adjusted its smoking frequency estimates for the potentially confounding effects of smoking duration. When we omitted smoking duration from our multivariate models, the RRs for smoking frequency also suggested an inverse association with endometrial cancer (data not shown). Our results do not indicate any important association between the age at starting smoking and endometrial cancer risk. Four previous studies [26, 28, 47, 49] found a (non-significant) risk reduction with young age at first exposure, that is, starting to smoke between age 15 [28] or age 20 or earlier [49], but none of these studies has reported a significant trend. It has been hypothesized that smoking might lower the levels of estrogens partly by reducing the amount of fat tissue or by decreasing the age at menopause [24]. In our cohort, smokers were slightly leaner than non-smokers (24.6 kg/m2 vs. 25.3 kg/m2). Moreover, on average, current smokers appeared to have reached menopause 1 year earlier than never-smokers and former smokers (48.1 years vs. 49.1 years). However, in conclusion, our analyses indicated that BMI and age at menopause are no mediating factors. In contrast to smoking, use of unopposed HRT increases endometrial cancer risk in postmenopausal women. Although an interaction between smoking and HRT use seems biologically plausible, the small numbers in our analysis did not allow to draw firm conclusions regarding a possible effect modification by HRT use. Moreover, we had no precise information on what type of HRT women in the NLCS have used. If we could have included such information in our analysis, results might differ according to type of HRT used. Another potential drawback of our study might be misclassification of self-reported alcohol consumption and/or self-reported cigarette smoking. However, these misclassifications might be non-differential owing to the prospective study design. Consequently, the risk estimates would probably be biased towards no effect. Moreover, the correlation between the alcohol consumption measured by the NLCS questionnaire and the measurement in a nine-day record was high due to the large variation in alcohol consumption [34]. An important strength of our study is that the exposures were assessed prior to the diagnosis of endometrial cancer. Therefore, our findings cannot be influenced by recall bias. Moreover, selection bias is unlikely as the follow-up of subcohort members and cases was almost complete [33, 52]. Another strength is the way alcohol consumption and cigarette smoking was assessed in the NLCS. The detailed assessment enabled us to evaluate associations between endometrial cancer and various measures of both exposures. Furthermore, we were able to control for confounding by the most important risk factors of endometrial cancer [40]. To sum up our major findings, we found that alcohol consumption is not associated with endometrial cancer. Current smoking was associated with a reduced risk of endometrial cancer in postmenopausal women. This association was probably not mediated by a decreased BMI or by an earlier age at menopause. Larger prospective studies with information on the type of HRT are needed in order to investigate possible effect modification by different types of HRT. Possibly, the incidence of endometrial cancer could be reduced if smoking was more common in female populations; however, such a reduction would be overshadowed by a dramatically increasing incidence of many other chronic diseases. Thus, individuals should still be encouraged to quit or not to start smoking.

Med_Biol_Eng_Comput-4-1-2329731

Decomposition cross-correlation for analysis of collagen matrix deformation by single smooth muscle cells

Microvascular remodeling is known to depend on cellular interactions with matrix tissue. However, it is difficult to study the role of specific cells or matrix elements in an in vivo setting. The aim of this study is to develop an automated technique that can be employed to obtain and analyze local collagen matrix remodeling by single smooth muscle cells. We combined a motorized microscopic setup and time-lapse video microscopy with a new cross-correlation based image analysis algorithm to enable automated recording of cell-induced matrix reorganization. This method rendered 60–90 single cell studies per experiment, for which collagen deformation over time could be automatically derived. Thus, the current setup offers a tool to systematically study different components active in matrix remodeling. Introduction The extracellular matrix (ECM) provides a biophysical and biochemical environment for cell mechanical behavior. In turn, cellular interactions with the ECM resulting from adhesive, proteolytic and migratory activity govern continuous matrix reorganization. One such example occurs in eutrophic inward remodeling of small arteries [34]. Here, the existing collagen matrix is rearranged around a smaller diameter. Such inward remodeling is a hallmark of many hypertensive disorders and has been shown to have predictive value for cardiovascular events [9]. In vitro setups of cell-seeded matrix scaffolds allow studying the specific components that are active in vascular remodeling [22]. Especially, matrix reorganization can be monitored in the presence of specific cells, matrix elements, and blockers or markers of remodeling enzymes such as the matrix metalloproteases and transglutaminases [1, 2]. Several approaches using multi-cellular preparations have been developed. Thus, free-floating collagen gels can be seeded with contractile cells, and the remodeling is monitored from the dimensions of these gels [8, 13, 16, 20]. In the so-called culture force monitor, collagen gels mixed with cells are allowed to polymerize between a fixed plate and a force transducer. Contractile force development is measured during subsequent culture of this preparation [12, 14, 22, 36]. However, interpretation of data derived from these multi-cellular approaches is troubled by variability in cellular mechanical activity, lack of information on cell density, and synergistic contractile effects [30]. Therefore, microscopic observations on matrix reorganization by single cells may provide more detailed and fundamental information on the mechanisms of remodeling. Several groups have studied the effect of single cell activity on local deformation of matrices [5, 28]. The most frequently used substrates are silicone rubber [3, 6, 18, 21] and polyacrylamide sheets. Local deformation is then monitored by tracing the texture, sometimes facilitated by using fluorescent beads or imprinted micropatterns. Although these inert scaffolds provide a geometrical framework to study processes like cell locomotion, they do not resemble the physiological ECM. This has been solved in some cases by coating the polyacrylamide membrane with a thin layer of collagen [11, 17, 26, 27, 37]. However, remaining issues are the orientation of the collagen fibrils in the coating as compared to native collagen, and the inability of the cells to chemically modify (e.g. by cross-linking) the artificial sheets. A possible way to overcome these issues is the use of collagen-based matrices. Cell-induced deformation patterns of these matrices and the degree of reversibility of such deformation provide information on physical remodeling and the underlying biochemical processes. Deformations have been assessed by manually tracing beads or landmarks in consecutive images [6, 28, 30]. However, so far, only a few algorithms were developed for automatic detection of substrate deformations. In particular, very few of such algorithms allow tracing in the absence of embedded beads [23, 33, 35], and more stable algorithms are required. While single cell observations provide a more fundamental insight into matrix remodeling than macroscopic studies, a concern is efficiency. Time-lapsed video microscopy of single cells forms a labor-intensive and time-consuming experiment. Meaningful experiments require the comparison of large data sets, including different cell types, various matrix compositions, or the use of biochemical or molecular interventions. A fully automated, motorized microscopy setup is required that scans series of individual cells and their surrounding matrix. In addition, each individual cell should automatically be kept in focus during remodeling experiments, which take up 24 h or more. Finally, stable algorithms are needed for the automated analysis of geometrical reorganization with minimal user input. The aim of this study is to develop an automated technique that can be employed to obtain and analyze local matrix remodeling by individual cells. The system that we present allows for monitoring of ∼75 cells in parallel, using time-lapse video microscopy and computer-controlled stage positioning. In addition, we present and evaluate a new algorithm for automated detection of collagen matrix deformation around these cells. Methods Cell culturing and collagen matrix preparation Smooth muscle cells, obtained from mesenteric small arteries, were cultured in Leibovitz medium with 10% (v/v) heat-inactivated fetal calf serum. Cells from passages three to nine were used in experiments. Matrix constructs were produced from calf skin collagen (MP Biomedicals) at a concentration of 1 mg/ml; pH was buffered by HEPES, and a mix of antibiotics (PSF and ciproxin) was added. Immediately after preparation at 4°C, the collagen mixture was poured into a 3.8 cm2 culture well and a 1.5-h polymerization period at 37°C was allowed. Then, SMCs were seeded at a concentration of about 1 cell per mm2 in the presence of 1 ml serum-free Leibovitz medium. The cells were maintained in an incubation chamber that was set to a temperature of 37°C throughout the experimental procedure. After a stabilization period of about 1 h, cell–matrix interactions were monitored by microscopic imaging for a period of 24 h of spontaneous cell contraction. Automated microscopic imaging In order to enable simultaneous monitoring of cell-induced matrix remodeling at multiple locations, microscopy was combined with a motorized stage. Individual cells and their surrounding matrix were studied by phase-contrast microscopy (Olympus IMT-2 with 10× objective and 2.5× projection lens). Images were captured by a Qimaging Retiga SRV camera. The calibration factor for these images (1,392 × 1,040 pixels) was 0.88 μm/pixel. The microscopic field of view was set by a motorized stage, controlled by custom written software (Matlab 7.0 with Image Acquisition Toolbox 2.0). After manually determining and storing a set of x, y, z-coordinates for about 60–90 appropriate cells, these positions were tracked through time at a 15-min interval and images were captured by an automated procedure. During the time-lapsed image acquisition, samples were kept in focus by means of implementation into the acquisition software of one of the general auto-focus algorithms. Image contrast is optimal when a histogram of intensity values shows a broad distribution over all bins. This characteristic feature can be approximated by the standard deviation of pixel intensity values. For each image, contrast was enhanced by histogram equalization, and standard deviation was calculated. A normalized focus index (FI) was defined by dividing the standard deviation of the original image (SDoriginal) by the standard deviation of the contrast enhanced image (SDenhanced): For each cell, a series of images was captured at five different heights. The image was then defined to be in focus at the vertical level of maximal normalized focus. This height (z) was used as the central level when capturing the z-series in the next time step, thus allowing gradual vertical shift during the time-lapsed image acquisition. Finally, a stack of time-lapsed images was constructed for each x, y-position and analyzed off-line. Gel dynamics analysis Cell–matrix interactions were quantified offline using in-house designed, automated image analysis software (Matlab 7.0 with Image Processing Toolbox 4.2). Matrix reorganization was assessed by calculation of the displacement field around a cell. This was achieved by performing a cross-correlation between each two successive images in an image stack; resolution of the displacement field was refined by correlation of subimages of decreasing size. This procedure is explained below and illustrated in Fig. 1. Fig. 1Graphical representation of parameters used in matrix deformation analysis. The image shows a single SMC in the center, surrounded with a collagen matrix of relatively smooth texture. The white box (768 × 768 pixels) indicates the area for the most coarse correlation analysis. This correlation between two successive images was applied over an area as indicated by the yellow box (960 × 960 pixels). The green boxes show the more refined cross-correlation windows (stages 2–5). Table 1 indicates the area expansion used for these correlation analyses First, gross displacement was defined at the point of maximal correlation between two parent images I 1 (at t = t 0) and I 2 (at t = t 0 + Δt), using a correlation threshold of 0.5 and a maximum tested displacement of 11 pixels. Then, I 1 was decomposed into four equal-sized square subimages, for which another cross-correlation was performed. The position of the correlation subwindow of I 2 was refined using the displacement as calculated for the parent image, with a safety measure to prevent I 2 crossing the borders of I 1. This procedure of accurately positioning a correlation window not only reduced the possibility of an accidental cross-correlation match between a subimage of I 1 and any random subimage of I 2, but also drastically reduced calculation time. Resolution of the displacement field was refined to the fifth decomposition stage in our experiments (see Fig. 1), using maximum tested displacements (n cc) as indicated in Table 1. When insufficient correlation (r < 0.5) was found in any small area, displacement field from the next larger images were used. The calculated displacements were then assigned to the corresponding subimage centers. Subsequently, a continuous displacement field was obtained by bicubic interpolation. Finally, matrix compaction for each stack was quantified by tracking circular areas centered around the cell (see Fig. 5). Table 1Settings for matrix compaction analysis by decomposition cross-correlationDecomposition stageCross-correlation windowSearch area expansion (n cc)1768 × 768962384 × 384483192 × 19224496 × 9624 5 48 × 48 24 624 × 2412712 × 126Dimensions of the cross-correlation window I 1 were reduced by 50% for each decomposition stage. The search area of I 2 for correlation with I 1 was obtained by increasing the cross-correlation window with n cc pixels on all image sides. The settings which were used for gel dynamics analysis and validation are given in bold Validation The method described above (decomposition CC) was validated on several test series against a straightforward cross-correlation analysis (direct CC), with settings according to decomposition stage 1. The first test case consisted of an image of a collagen-embedded cell, which was artificially resized by 3%, thereby simulating matrix compaction. Secondly, increasing amounts of white noise were added to the resized image in order to test the stability of both correlation methods. Relative dispersion (RD), which is defined as standard deviation divided by mean, was used as a noise level index. Finally, image resizing was followed by a horizontal translation of 60 pixels for a low and high noise example (see Table 2). Table 2Characteristics of validation images: CC was tested by addition of Gaussian white noise with mean 0.0 and increasing variance levels, in several cases the image was resized or translatedIndexScaling (%)Gaussian white noise varianceRD imageRD increase (%)RD noiseHorizontal shift (pixels)a10.00000.2500b0.970.00000.2510.50c0.970.00050.2604.10.1040d0.970.00100.2697.60.1460e0.970.00200.28514.10.2070f0.970.00300.30120.20.2530g0.970.00400.31626.30.2930h0.970.00500.32931.70.3270i0.970.00050.2594.10.10860j0.970.00300.30320.20.26560Relative dispersion (RD) in an image is defined as standard deviation divided by mean, RD increase is expressed as RD value compared to case “a”, RD noise is calculated as ratio of noise variance and image mean Results Parallel recording of matrix compaction movies We were able to record on average 66 movies on collagen compaction by single cells in parallel at a time resolution of 15 min (n = 10 experiments). Critical issues that limited this number were the need to avoid rapid acceleration and deceleration of the microscope stage, and the time-consuming autofocus algorithm. If cells were properly focused in the initialization stage, only a small fraction (<5%) ran out of focus during 24-h compaction experiments. Under the given incubation conditions, the majority of the cells adhered firmly to the collagen matrix and demonstrated little migratory activity. Typically, cell centroids remained within a 150 μm radius of their original xy position. z position decreased slowly in many cases, reflecting compaction of the matrix in the vertical direction (data not shown). Validation of the collagen compaction analysis Analysis based on cross-correlation of images at a series of decomposition stages was compared with straightforward cross-correlation. This was performed on images simulating matrix compaction, subsequently followed by a challenge of increasing amounts of image noise and artificial translation. Decomposition CC was more time-consuming than direct CC: respectively, 84 and 54 s per image pair. This was due to a larger number of cross-correlations that have to be performed, and more complex data storage and lookup operations. For low noise levels (Table 2, case c, d), both analysis methods render 100% correct displacement vectors (see Fig. 2). When noise increased, the number of vectors in the smallest decomposition stage that had insufficient cross-correlation (r < 0.5) rose. However, this effect was smaller in the decomposition CC versus direct CC. As an example, the percentage correct displacement vectors at 20.2% noise (Table 2, case f) was 77.7% in direct CC versus 89.5% in decomposition CC. Concurrently, the average displacement error at this noise level amounted to 2.1 and 0.7 pixels, respectively. Fig. 2Noise sensitivity of the cross-correlation methods. Top from left to right simulated displacement field of 3% compaction (Table 2, case b), displacement field as predicted by direct CC, displacement field as predicted by decomposition CC; both cross-correlations were performed at a RD increase of 20.2% (Table 2, case f); vectors were not scaled to absolute displacements, but to optimal visual illustration; red asterisks indicate vectors based on cross-correlation values below threshold (0.5) at the highest resolution. Middle the fraction of displacement vectors in the smallest decomposition stage that is calculated correctly (error in horizontal and vertical direction ≤1 pixel). This fraction decreased at higher noise levels (relative dispersion: Table 2, case c–h). Bottom the average error between calculated and simulated displacement vectors increased for higher noise levels (Table 2, case c–h). The decomposition method resulted in a slightly lower number of incorrect vectors, and a large decrease in displacement error Figure 3 shows estimates for matrix compaction for these simulated deformations. The simulated compaction over a disc with 350 pixels radius was 6%. For noise levels up to 7.6% (Table 2, case d), such compaction was indeed found by both correlation methods. At higher noise levels, direct CC underestimated compaction much more severely as compared to decomposition CC. As an example, after a noise increase of 20.2%, decomposition CC estimated a compaction value of 5.9% as compared to only 4.1% for direct CC. Fig. 3Calculated area at a radial distance of 350 pixels after a simulated 3% compaction. Increasing displacement errors, caused by higher noise levels, resulted in a mismatch between calculated area and simulated compacted area (94% of original). The decomposition method showed stable results at larger relative dispersion values (Table 2, case c–h) When an additional horizontal shift was imposed, both analysis methods correctly estimated compaction for 4.1% noise (Table 2, case i). For higher noise levels, decomposition CC succeeded in a correct area assessment. In contrast, at 20.2% noise and 60 pixels translation (Table 2, case j), direct CC predicted 1.2% rather than 6% compaction of the 350 pixels disc (see Fig. 4). This effect was not due to more incorrect displacement vectors, but could be attributed to a rise in average displacement error up to 14.6 pixels. Fig. 4Sensitivity of the correlation methods to translation of the image. A horizontal displacement of 60 pixels was imposed after application of a 3% compaction and noise addition (4.1, respectively, 20.2% RD increase). Top left displacement field for high noise example (RD increase: 20.2%), as calculated by direct CC. Red asterisks indicate displacements where cross-correlation was below threshold (0.5); at these positions, a large mismatch between calculated compaction and simulated compaction occurs. Top right visual representation of matrix deformation at a radial distance of 270 pixels. Bottom calculated area remaining after a simulated compaction. At low noise levels (Table 2, case i), a horizontal shift after image resizing did not affect the calculated area; for high noise (Table 2, case j), failing correlation in the direct method results in a high offset in calculated area Collagen compaction by individual smooth muscle cells Figure 5 shows an example of collagen matrix compaction by an individual smooth muscle cell. In this particular case, gross geometrical reorganization occurred in the first 6 h of the 22-h observation period. Compaction magnitude decreases with distance, and there was a time delay of around 1.5 h before compaction visible at a radius of 97 μm (110 pixels) became apparent at a distance of 308 μm (350 pixels). Fig. 5Typical example of collagen matrix compaction by individual smooth muscle cell as estimated by decomposition cross-correlation, obtained at a resolution of 1 frame per 15 min. Top left displacement field represented by vectors, refined in five decomposition stages. Vectors originating from a red asterisk were created at a larger correlation scale; for the smallest window, cross-correlation value at these positions was below threshold (0.5). Top right visual representation of matrix compaction at a radial distance of 350 pixels, as estimated from a series of displacement fields, showing an initial (yellow) circle and its deformed (green) state. Bottom estimation of compacted area after 22 h at distances of 110, 230 and 350 pixels (scaling: 0.88 μm/pixel) Discussion This study aimed at developing an automated technique for obtaining and analyzing matrix remodeling by individual cells. Emphasis was put on construction of an automatic, reliable algorithm for assessment of a detailed matrix displacement field. Especially, refinement of a cross-correlation based image analysis with a decomposition scheme was investigated. While “classic” direct CC sufficed for pairs of images with high correlation and low noise, this was no longer the case when substantial matrix remodeling occurred within the time frame between two consecutive images. This resulted in failing of CC at spots of high geometrical reorganization. However, when using decomposition CC, the gross displacements at these positions could be estimated by analysis of parent images with larger dimensions. This way, at a RD increase of 20.2% the average displacement error was lowered threefold in decomposition CC as compared to direct CC. The method of refining displacement field accuracy with each decomposition step becomes progressively more important with larger displacements. Therefore we investigated the effect of a horizontal shift superimposed on a simulated compaction. Such shift occurred in our in vitro experiments when a group of neighboring cells pulled strongly on the matrix adjacent to a cell of interest. The shift interfered with direct CC because zero deformation was assumed when no proper correlation could be found. The result was an irregular displacement field (see Fig. 4). When using decomposition CC, on the other hand, the gross displacement was already detected in the first decomposition stage. Displacement values were then adjusted when local compaction was detected in subsequent decomposition stages. Using this strategy, a lack of local correlation at the finest resolution resulted in only a minor displacement error, with an average of 0.9 as compared to 14.6 pixels for direct CC. Both cross-correlation methods differ not only in stability of displacement field estimation, but in efficiency of calculation time as well. Direct CC requires a large search area, i.e. the subwindows of I 2 have to extend considerably beyond the boundaries of their corresponding I 1 subwindow in order to perform a meaningful correlation. On the other hand, with decomposition CC n cc can decrease with each stage (see Table 1), since each subwindow of I 2 is repositioned according to the preliminary displacement as calculated for its parent window. Due to the increased number of calculations and data manipulations that have to be performed, analysis time was still about 50% larger for decomposition CC at the settings as stated in Table 1. We chose a correlation threshold of 0.5 for both techniques, as well as cross-correlation windows as indicated in Table 1. These values were empirically determined as an optimum in the trade-off between calculation time, false positive displacements and overlooking local deformations. Clearly, these choices depend on the contrast and texture of the images and the nature of the deformation, and will need to be optimized for specific future experiments. Cell traction is frequently assessed by quantification of deformations in a flexible substratum. Both 2D and 3D approaches have been used, resulting in different cellular morphologies [19, 20, 29]. While the latter seems more physiological, interpretation of the 3D experiments is more complex [35]. 2D substrates offer more straightforward tools for analyzing mechanical behavior of single cells. These materials can be enriched with fluorescent microbeads to increase image contrast. In order to achieve a high resolution in the vicinity of cells, which can change their morphology rapidly, manual tracking of specific landmarks was employed in several studies [6, 28, 30]. However, this labor-intensive method inherently limits the number of cells under study. In several studies particle tracking was performed on individual microbeads [11]. In this case, resolution depends on particle distribution, which is typically far from homogeneous in collagen substrates [23]. Several nested cross-correlation methods have been developed [35]. All of these algorithms are based on empirical determination of square size, distance for pattern search, and normalized CC threshold. In order to spatially limit the search region, a relative translational shift has been derived from image registration [23, 33]. Several schemes have been implemented to increase displacement field resolution in a step-by-step manner. In a single refinement cycle, Dembo and colleagues [26] decreased the subwindow size if significant displacement was observed. On the other hand, window size can be increased if correspondence failure occurs at high resolution [23]. The algorithm developed by Wang and coworkers [33] resembled our decomposition CC to a large extent. However, these authors aimed at construction of traction fields with a smooth nature by incorporation of filtering procedures at several stages. Geometrical matrix reorganization provides a qualitative index of the traction forces present in the underlying material. The actual forces can be derived from a displacement field series using material properties of the ECM construct. However, for collagen scaffolds these are highly heterogenous. Frequently, local stiffness is estimated by either microneedles [5, 10, 21, 30] or optical tweezers [4, 15, 32]. Tractions can then be calculated by application of stress–strain relationships, including appropriate boundary conditions [7, 24, 25, 31, 37, 38]. However, this translation to quantitative traction forces lies beyond the scope of this article. In conclusion, we presented integral methodology for the study of matrix remodeling. These techniques allow systematic screening of the role of matrix components such as collagen, elastin, fibronectin and laminin. Likewise, the function of stationary cells like smooth muscle cells, fibroblasts, and osteoblasts can be investigated. Our method can be applied under a wide variety of other experimental conditions. The single requirement for the image quality is a sufficiently high contrast in the material under investigation, without the neeed for laborious and potentially interfering micropatterning. Furthermore, our graphical user interface enables a flexible tuning of parameters such as number of decompositions, size of correlation window, search area and cross-correlation threshold. Using our motorized microscopy stage it is possible to patch individual images together in order to create one large field of view for the study of motile cells such as keratocytes. Finally, the method can be extended with fluorescence imaging of specific cell structures, cytokines, hormones and enzymes [15].

Osteoporos_Int-3-1-1894753

Gender differences in the ratio between humerus width and length are established prior to puberty

Summary On a sample of 1,317 children aged 9.9 years we developed a novel method of measuring humeral dimensions from total body dual-energy X-ray absorptiometry (DXA) scans and showed that gender differences in the ratio between humeral width and length are established prior to puberty. Introduction Traditionally, the increased risk of osteoporotic fracture in women compared to men has been attributed to their low bone mass as a consequence of reduced peak bone mass acquisition and increased rates of bone loss following the menopause. However, more recent studies indicate that several other factors influence fracture risk independently of bone mass, such as skeletal geometry and the material properties of bone [1]. Furthermore there are gender differences in skeletal geometry that may contribute to the greater fracture risk in women compared to men. For example, long bone cross-sectional area is greater in men, which is thought to reflect higher rates of periosteal apposition from the time of puberty onwards [2]. One study of 68 girls and 59 boys aged 11.9 years, who underwent prospective peripheral quantitative computed tomography (pQCT) measurements, reported that periosteal growth was more rapid in pubertal boys compared to girls [3]. Skeletal growth is coordinated to ensure that the ratio between different skeletal dimensions is maintained despite rapid changes in size [4]. Therefore, it is possible that the greater cross-sectional area in boys compared to girls is a reflection of their larger size. On the other hand, the ratio between periosteal growth and longitudinal bone growth may be different in boys compared to girls, leading to gender differences in bone shape. However, little is known of the inter-relationships between longitudinal and periosteal bone growth, since investigation of possible gender differences in skeletal geometry have generally been confined to analysis of cross-sectional area; in the absence of techniques capable of simultaneous measurement of long bone length, it has been difficult to accurately assess how the ratio between longitudinal and periosteal growth is affected by gender and other factors such as puberty. Recently, we developed a novel method for evaluating long bone geometry based on analysis of the humerus on total body dual-energy X-ray absorptiometry (DXA) scans. The humerus offers important advantages over other long bones in that its entire outline can readily be traced on total body DXA images, and its shape can be modelled as a cylinder with reasonable accuracy. To explore the utility of regional DXA analysis at the humerus, ‘volumetric’ bone density of the humerus was derived by dividing humeral bone mineral content (BMC) by estimated humeral cylindrical volume, and then analysed in relation to fracture risk. Interestingly, humeral volumetric bone density obtained in this way was indeed related to fracture risk, as analysed in a subgroup of 1,317 children from the Avon Longitudinal Study of Parents and Children (ALSPAC), in whom total body DXA scans were available at 9.9 years of age [5]. In the present study, we aimed to characterise the influence of gender and puberty on the ratio between longitudinal and periosteal growth, by exploiting this novel technique of humeral geometric analysis. In particular, we wished to determine whether, in analyses combining fracture and non-fracture controls from the study described above, gender differences exist in the ratio between width and length of the humerus, and if so whether these differences are established prior to puberty. Methods Study population The Avon Longitudinal Study of Parents and Children (ALSPAC) is a geographically based cohort that recruited pregnant women residing in Avon with an expected date of delivery between 1 April 1991 and 31 December 1992. A total of 14,541 pregnancies were initially enrolled, with 14,062 children born. This represented 80–90% of the eligible population–see http://www.alspac.bris.ac.uk for further details [6]. Of these births, 13,988 were alive at 12 months. The population from ALSPAC used for this study consisted of 1,290 children: those who had DXA scans performed at aged 9.9 years, had measures of humeral dimensions performed and had data on pubertal stage available. Ethical approval was obtained from the ALSPAC Law & Ethics Committee, and Local Research Ethics Committees. Measure of size at birth In the immediate post-partum stage, whilst mother and child were still in hospital, trained ALSPAC staff measured crown-heel length with the Harpenden Neonatometer (Holtain Ltd., Crosswell, UK). Alternatively, this measure was collected from clinical records for babies who were not captured by the ALSPAC staff. Measures of size at age 9.8 years Children were seen in a research clinic at age 9.8 years (± 0.33 years). Parental consent and child’s assent were obtained for all measurements made. Height was measured to the last complete millimeter (mm) using the Harpenden Stadiometer. Weight was measured to the nearest 50 grams (g) using the Tanita Body Fat Analyzer (model TBF 305, HealthCheck Systems, New York, USA). Total body less head (TBLH) bone area (cm2), TBLH bone mineral content (g), TBLH bone mineral density (g/cm2), total body (TB) fat mass (kg) and TB lean mass (kg) were measured using a Lunar Prodigy dual-energy X-ray absorptiometer (Lunar Corp., Madison, WI, USA) on 7,444 children, using the default mode for all scans. The child was positioned carefully on the scanner and asked to lay their hands flat on the bed, palms down. Measures of humeral dimensions The present investigation was based on a subgroup of 393 children reporting fractures, for whom DXA scans were available at age 9.8 years, and an additional randomly selected group of 897 children, giving a total of 1,290 children. These children had originally been chosen for a study investigating the relationships between bone mass and fractures [5]. The measurer (EC) was blinded to the fracture status of the children. Customised settings were available on the Lunar Prodigy software and these were applied to the total body DXA image on screen. (No measures were performed during the DXA scan.) A region of interest (ROI) was drawn around the right humerus where possible (in case of movement artifact, the left humerus was used) after enlargement of the image to maximum magnification. The bone edge was detected visually with ease for the shaft and head of the humerus. At the distal end a straight line was drawn across the joint space from medial to lateral epicondyle, with the head of the ulna included within the humeral ROI. Where arm positioning was not ideal (such as palms not flat on the bed) the ROI was fitted as accurately as possible. The area (cm2) of the humeral ROI was recorded (Fig. 1). Length of the humerus was obtained by use of an electronic ruler positioned between its upper and lower extremities. Average humeral width (cm) was calculated as area divided by length. The humeral aspect ratio (AR) was calculated as humerus width divided by length and then multiplied by 100, so the AR is the humerus width expressed as a percentage of humerus length. Fig. 1Total body DXA scan showing position of the region of interest (ROI) around the right humerus. a Total body scan image. b Image following maximal (i.e. fourfold) magnification, which was selected prior to placement of the ROI and measurement of length by electronic ruler The precision of measurements of humeral geometry was calculated as the coefficient of variation (CV), based on ten scans with the measures repeated five times. The CV was 2.9% [95% confidence interval (CI): 2.1–3.7] for width, 1.5% (95% CI: 1.2–1.7) for length, and 3.2% (95% CI: 2.4–4.0) for humeral AR. Other measures The mother’s, partner’s and grandparent’s race and ethnic group and mothers’ highest educational qualification were recorded at 32 weeks gestation as described elsewhere [7]. Gender was obtained from birth notifications. At the time of the DXA scan and measurement of the anthropometric variables, the child’s age was calculated from the date of birth and date of attendance at the research clinic. Puberty was assessed by self-completion questionnaires using diagrams based on Tanner staging of pubic hair distribution for boys and girls, which we have previously found to show expected relationships with DXA measures in this cohort [8]. Prepuberty was defined as Tanner stage 1, and early puberty as Tanner stage 2. Statistical analyses Results from children reporting previous fractures and the randomly selected subgroup were pooled. Statistical analyses were performed with STATA 8.0. A two-tailed unpaired t-test was used to test the null hypothesis of no difference in the means for boys and girls. Linear regression was used to assess the associations between gender and humerus dimensions, which were adjusted for age on the day of the DXA scan and pubertal status. Additional analyses were performed following adjustment for TB fat mass and for fracture status. Results No differences in gender, ethnicity, socio-economic status, body composition or humerus dimensions were found between the children with and without fractures (results not shown) so these results were pooled for all further analyses. Length at birth, age at DXA measurement, height, weight, TB fat and lean mass and TBLH bone area of the 1,290 children in whom humeral dimensions were measured are shown in Table 1 according to gender and pubertal stage. There was no difference in age, height or weight between prepubertal boys and girls. However, boys had been longer at birth (by 0.3 cm on average), even after adjustment for gestational age and birth weight (P < 0.001). Prepubertal girls had a greater TB fat mass (P < 0.001), whereas prepubertal boys had a greater TB lean mass (P < 0.001). Puberty-related differences in size measures were also seen. For example, girls in early puberty (Tanner stage 2) were on average 4.4 cm taller and 5 kg heavier than prepubertal girls (Tanner stage 1). Boys’ pubertal stage showed similar trends to those observed in girls, but differences in size measures were considerably smaller. Table 1Mean age, height, weight, and DXA-derived total body fat mass and lean mass, and total body less head bone area for 648 boys and 642 girls with measurements of humeral size and dimensionsa BoysGirls Prepubertal, N = 551Early pubertal,N = 97P value for difference between boys in pre- and early pubertyPrepubertal, N = 548Early puberty, N = 94P value for difference between girls in pre- and early pubertyP value for difference between prepubertal boys and girlsP value for difference between boys and girls in early pubertyMeanSDMeanSDMeanSDMeanSDBirth length (cm)b50.91.550.91.50.99550.61.550.71.50.658<0.0010.316Age at DXA (years)9.80.39.90.30.6159.80.39.80.30.9460.6310.602Height (cm)139.46.1140.76.10.041138.76.0143.16.0<0.0010.0810.011Weight (kg)34.17.134.97.10.30334.27.339.27.3<0.0010.795<0.001TB fat mass (kg)7.34.97.74.90.4719.34.912.15.0<0.001<0.001<0.001TB lean mass (kg)25.33.025.63.00.31423.32.825.22.8<0.001<0.0010.332TBLH bone area (cm2)1,1411541,1611550.2611,1131571,217158<0.0010.0020.002aResults are shown separately for boys and girls, who are further subdivided according to results of Tanner stage self-completion questionnaire. P values shown are for the difference between prepubertal boys (Tanner stage 1) and boys in early puberty (Tanner stage 2); for the difference between prepubertal girls (Tanner stage 1) and girls in early puberty (Tanner stage 2); for the difference between prepubertal boys and girls; and for the difference between boys and girls in early puberty, all calculated by an unpaired Student’s t-test. For TB fat mass, TB lean mass and TBLH bone area results are adjusted for age at DXA measurement, by linear regression.bBirth length adjusted for gestational age and birth weight by linear regression. We then examined relationships between dimensions of the humerus, age, height and puberty. As expected, height was positively related to humerus, width and length based on analyses in boys and girls combined (P < 0.001). In spite of the relatively narrow age range of our study population, a positive association was also observed between age and width and length of the humerus (P < 0.001). In contrast, age and height were not related to humeral AR. Similar results were seen when boys and girls were analysed separately. Girls in early puberty had greater humeral width, length and area compared to prepubertal girls, but humeral AR in these two groups was similar (Table 2). In contrast, no differences were observed in any measure of humeral geometry between pre- and early pubertal boys. Table 2Humeral dimensions in prepubertal boys, boys in early puberty, prepubertal girls and girls in early pubertya BoysGirls Prepubertal, N = 551Early pubertal, N = 97P value for difference between boys in pre- and early pubertyPrepubertal, N = 548Early puberty, N = 94P value for difference between girls in pre- and early pubertyP value for difference between prepubertal boys and girlsP value for difference between boys and girls in early pubertyMeanSDMeanSDMeanSDMeanSDLength (cm)24.71.425.01.40.15924.91.425.91.5<0.0010.026<0.001Width (cm)1.920.21.930.20.6091.880.21.930.20.002<0.0010.985AR (%)7.780.77.760.70.6997.530.67.470.60.390<0.0010.006Area (cm2)47.76.048.46.10.29746.965.950.265.9<0.0010.0230.052aResults show mean and standard deviation for humeral length, width, aspect ratio (AR) and area. P values shown are for the difference between prepubertal boys (Tanner stage 1) and boys in early puberty (Tanner stage 2); for the difference between prepubertal girls (Tanner stage 1) and girls in early puberty (Tanner stage 2); for the difference between prepubertal boys and girls; and for the difference between boys and girls in early puberty, all calculated by an unpaired Student’s t-test. All analyses are adjusted for age at DXA measurement by linear regression. We then investigated the effects of gender on measures of humeral geometry according to pubertal stage, following adjustment for age of DXA scan. The humerus of prepubertal boys was slightly shorter (on average 0.2 cm), but of greater width (average of 0.04 cm) and area (on average 0.8 cm2), compared to prepubertal girls, as a result of which humeral AR was greater in prepubertal boys (an average of 3.2% greater, P < 0.001) (Table 2 and Fig. 2). Boys in early puberty still had a shorter humeral length, but a similar humeral width and a smaller area than girls in early puberty, as a result of which humeral AR remained greater in early pubertal boys (an average of 3.7% greater, see Fig. 2). Similar gender differences were seen after adjustment for TB fat mass and fracture status (results not shown). Fig. 2Associations between humeral geometry, gender and puberty, as determined in 1,290 boys and girls. Figure shows mean ± SD a humerus width (cm), b humerus length (cm) and c humerus aspect ratio (AR) according to gender and Tanner stage of puberty. P values are for the difference between boys and girls. All analyses are adjusted for age Discussion Humeral width and length were positively related to age and height in boys and girls combined, and to pubertal status in girls, in this contemporary cohort of pre- and early pubertal children. These observations are similar to those previously reported for other DXA-derived measures of bone size in this cohort, such as TBLH and spinal bone area [7]. In contrast, age, height and pubertal status did not influence the ratio between humeral width and length, as reflected by humeral AR, presumably reflecting the action of mechanisms to ensure that skeletal shape remains constant as bones grow. In both pre- and early pubertal boys, humeral length was found to be shorter compared with girls, whereas humeral width was similar or greater, resulting in a greater humeral AR in boys. Taken together, these findings suggest that gender, but not puberty, affects the balance between periosteal and longitudinal growth. Hence, differences in overall skeletal shape between boys and girls appear to be established prior to puberty. However, from the present study we are unable to determine whether the AR changes during later pubertal stages, or during subsequent ageing. Our observation that Tanner stage was found to affect humeral geometry in girls but not boys presumably reflects the fact that boys and girls in Tanner stage 2 are not equivalent in terms of skeletal development. The finding that height and weight differences between Tanner stages 1 and 2 were considerably greater in girls compared to boys is consistent with this view. Therefore, analyses of differences in humeral geometry between Tanner stage 2 boys and girls may have limited validity, since these may not have fully accounted for gender differences in skeletal maturity that are likely to have been present. Nevertheless, since humeral AR was unaffected by Tanner stage in the age of children studied, these reservations are unlikely to affect the main conclusion from this study, namely that humeral AR is greater in boys compared to girls as assessed at age 9.9 years. The gender differences in skeletal shape shown in Table 2 (approximately 2–3%) were smaller than those observed in fat mass, lean mass and bone area as in Table 1 (ranging from 7 to 27%). The larger gender differences in TBLH bone area (6.5%) compared with humeral area (2.7%) suggest that greater gender differences in bone area are present at other skeletal sites. Consistent with this conclusion, vertebral body size has been reported to be 11% [8] and 15% [9] larger in boys compared to girls. Our observation of an 8% gender difference in lean mass and 27% difference in fat mass, compared to the 1% gender difference in humeral length and 2% difference in width, perhaps reflects the strength of association between fat or lean mass and bone area [10]. Our conclusion that gender differences in humeral shape are established prior to puberty is supported by a previous study in which greater humeral width was seen in prepubertal boys compared to girls, based on radiogrammetry [11]. In the present study, the humerus was selected as the most suitable site for providing an accurate measure of aspect ratio by analysis of total body DXA scans. Although other skeletal sites were not evaluated, we assume that equivalent gender differences in periosteal relative to longitudinal growth are established prior to puberty throughout the appendicular skeleton. Consistent with this suggestion, the metacarpals and proximal radius have been found to be wider in boys compared to girls at all stages of development, as assessed by analysis of radiographs [12, 13]. Furthermore, in a recent study of 128 boys and girls, boys had higher rates of periosteal expansion relative to girls, as measured prospectively over 20 months at the radial midshaft by pQCT, and this gender difference was similar in early, peri- and postpubertal children [3]. In a recent analysis of 18-year-old males and age-, height- and weight-matched females, long bone width was found to be greater at the hip and distal tibia as measured by DXA and pQCT, respectively, in boys compared to girls [14]. Although our results suggest that gender differences in long bone width are due to more rapid periosteal apposition relative to longitudinal growth in boys prior to puberty, the precise timing of this gender effect is currently unclear. Review of the literature shows no evidence of gender differences in forearm bone width at birth [15], but studies of preschool and older children show conflicting results: Specker et al. [16] report no gender differences in radius width in children aged 1–6 years (based on 89 children), whereas Tanner et al. [17] found that the humerus is wider in boys compared to girls from age 3 years until the time of pubertal growth acceleration in girls (based on 505 children aged 3–18 years). It is also possible that the method used to measure bone size influences whether gender differences are found. For example, studies described above that used radiographs or pQCT identified gender differences [3, 11–13, 17], whereas those that used single photon absorptiometry did not [15, 16]. Prepubertal gender differences in the relative rates of longitudinal and periosteal growth that we observed may be mediated by alterations in endocrine factors. For example, prepubertal girls have higher levels of insulin-like growth factor I, estradiol and testosterone concentrations compared to prepubertal boys [18], all of which are known to have effects on both longitudinal and periosteal bone growth. In terms of the potential influence of these differences on fracture risk, according to beam theory, columns with larger aspect ratios (i.e. ratio of width to length) have a reduced fracture risk than columns with smaller aspect ratios [19]. In addition, the ratio between periosteal diameter to long bone length provides an approximate estimate of critical buckling load, such that a lower aspect ratio results in a long bone which is more prone to failure by buckling [20]. Furthermore, in children, the majority of fractures occur at the distal forearm and can be divided into two main types: simple torus fractures and the ‘greenstick’ variety, both of which are associated with buckling or bulging on the side of the bone in compression [21]. Therefore, theoretically, measurement of the ratio between long bone width and length from total body DXA scans as described here may provide an in vivo method for evaluating biomechanical strength of the skeleton. However, against this suggestion, boys have a higher fracture risk than girls in childhood [22], whereas girls have a smaller humeral AR. Furthermore, we found no relationship between humeral AR as measured in the present study and fracture risk [5]. One possible explanation for this lack of association is that our assumption that the humerus is cylindrical ignores gender differences in shape at the epiphysis or metaphysis which might contribute to fracture risk. On the other hand, humeral AR may predict fracture risk in certain adult populations, in view of evidence that bone width has previously been reported to be related to stress fractures in soldiers [23]. In light of our results, which suggest that humeral AR can be evaluated with relatively good precision, further studies are justified to determine whether this parameter represents a novel bone mineral density (BMD)-independent risk factor for upper limb fracture. The measure of humeral length from which we derived humeral AR is likely to be relatively accurate, since the upper and lower ends of the humerus are generally clearly visible on total body DXA scans (see Fig. 1). Alternative measurement techniques, such as pQCT, offer advantages over the approach described here, by measuring bone diameter directly, but do not provide a measure of bone length. Another limitation of the present study is that unlike girls, age 9.9 years appeared to be too young to evaluate possible effects of early puberty on skeletal development in boys. In future studies, we plan to repeat these analyses in older boys to confirm that as in girls, puberty increases humeral width and length whilst having no effect on humeral AR. In conclusion, we have found that long bone shape, as reflected by humeral AR which we derived using a novel technique from total body DXA scans, is unaffected by age, height and puberty, as evaluated in a child cohort of relatively narrow age range and range of Tanner stages. This finding suggests that the ratio between longitudinal and periosteal growth is controlled to ensure it remains constant during rapid growth. However, humeral AR was related to gender, suggesting that the greater periosteal diameter of boys compared to girls, which is well recognised, is a consequence of gender differences in the balance between longitudinal and periosteal bone growth. Interestingly, these gender differences in humeral AR were present in prepubertal children, possibly resulting from prepubertal differences in sex hormone levels. Further studies are justified to determine whether humeral AR is an important determinant of biomechanical strength and fracture risk, particularly in adult populations.

Surg_Radiol_Anat-3-1-1820760

Scapular deformity in obstetric brachial plexus palsy: a new finding

While most obstetric brachial plexus palsy patients recover arm and hand function, the residual nerve weakness leads to muscle imbalances about the shoulder which may cause bony deformities. In this paper we describe abnormalities in the developing scapula and the glenohumeral joint. We introduce a classification for the deformity which we term Scapular Hypoplasia, Elevation and Rotation. Multiple anatomic parameters were measured in bilateral CT images and three-dimensional CT reconstruction of the shoulder girdle of 30 obstetric brachial plexus palsy patients (age range 10 months–10.6 years). The affected scapulae were found to be hypoplastic by an average of 14% while the ratio of the height to the width of the body of scapula (excluding acromion) were not significantly changed, the acromion was significantly elongated by an average of 19%. These parameters as well as subluxation of the humeral head (average 14%) and downward rotation in the scapular plane were found to correlate with the area of scapula visible over the clavicle. This finding provides a classification tool for diagnosis and objective evaluation of the bony deformity and its severity in obstetric brachial plexus palsy patients. Introduction The most commonly injured brachial plexus nerve root in obstetric injuries is the fifth cervical (C5) root. The supplied shoulder girdle musculature is therefore most commonly affected and indeed shoulder deformities comprise the most significant problems in this injury [2]. Within the context of shoulder deformity, the medial rotation contracture (MRC) deformity results in the greatest morbidity. The pathophysiology of deformity has been known for over 100 years: paralysis of the shoulder abductors and external rotators leads to relative dominance of already strong internal rotators of the shoulder, with eventual clinically observed changes of arm posture. The imbalance causes progressive deformity in the growing shoulder which may progress to a fixed medial rotation position of the humerus as well as posterior subluxation of the humeral head [3]. This constant position of the shoulder has a deleterious effect on glenohumeral development [9, 14, 17] and is accompanied by a characteristic flexion and MRC of the shoulder. Furthermore, scapular growth is impaired compared to the normal side [12]. The patients exhibit a persistent elbow-bent posture, pronation position of the forearm, and apparent shortening of the arm (Fig. 1). In movement, there is loss of supination due to the abnormally situated arm in medial rotation, obvious elbow flare when the biceps is flexed, the “bugler’s position”, and awkward external rotation. This secondary structural shoulder deformity develops early and may persist despite improvement in neurological status [14]. Fig. 1At rest the child is noted to exhibit an internal rotation posture at the shoulder with elbow flexion and pronation of the forearm. There is apparent shortening of the arm There have been previous radiologic demonstrations of scapular deformity as a sequela of obstetric brachial plexus palsy using plain radiographs or computed axial tomography were used, but attention was directed solely to the glenoid fossa and adjacent structures, without evaluating the body, spine and acromion process [8, 11, 12, 17]. In the current study, we describe dysplasia and malposition of the entire scapula in obstetric brachial plexus palsy patients. We introduce a classification of this deformity which we term collectively Scapular Hypoplasia, Elevation and Rotation (SHEAR). Methods Patients A total of 30 obstetric brachial plexus palsy patients with glenohumeral internal rotation were evaluated in the past 6 months. We reviewed the clinical data on these patients and diagnosed and classified the scapular elevation according to the results of clinical examination and analysis. There were 10 boys and 20 girls ranging in age from 10 months to 10.6 years. Three of the children had not undergone any surgery. Twenty-four of the children had undergone latissimus dorsi and teres major muscle transfer, subscapularis, pectoralis major and minor contracture releases and axillary nerve decompression and neurolysis for correction of abduction at our institute in the past. Twelve of the latter group and one that had not undergone the aforementioned procedure had undergone primary neurological repair surgery. Measurements Transverse CT section and three-dimensional reconstructions of bilateral computerized tomograms (3D-CT) were used in the evaluation process. Trunk anterior and trunk posterior views of the whole shoulder girdle as well as superior trunk outlet and scapular posterior and medial views were examined. The contralateral scapulae were assessed for comparison. The height (H) of the scapula was measured on the scapular medial view as the length of the medial border between the superior angle and the inferior angle. Width was measured along the spine of the scapula on the scapular posterior view. The total width (W) was measured from the lateral end of the acromion to the most medial part of the scapula. Width of the body of the scapula (w) was measured as the distance from the glenoid to the most medial part of the scapula (Fig. 2a). Fig. 2Measurement of the different scapular variables. a The width is assessed in the posterior scapular view (left) along the axis of the scapular spine. The total width (W) is calculated as the length from the end of the acromion to the most medial aspect of the scapula. The scapular body width (w) is calculated as the distance from the mid glenoid to the most medial aspect of the scapula. The acromion length (acr) is also calculated on this view. The height (H) is measured along the medial border of the scapula in the medial scapular view (right). b Lines are drawn from the center of the glenoid cavity perpendicular to the vertebral axis line on the posterior trunk view. Superior displacement = A/H. Downward/upward rotation around an axis perpendicular to the scapular plane is defined as the difference between the angles α defined by the vertebral axis and the extension of the lines connecting the center of the glenoid cavity and the medial end of the spine of the scapula. c In the anterior trunk view three lines are drawn: Line 1 is the line between the sternoclavicular joint and the center of the acromio-clavicular joint, line 2 is the line between the center of the acromio-clavicular joint and the inferior angle of the scapula, line 3 is the vertebral axis. The superior scapular angle γ is the angle defined between lines 1 and 2 and the inferior scapular angle δ is the angle between lines 2 and 3. d In the superior outlet view the anterior/posterior rotation is calculated as the angle ϕ between the line connecting the acromio-clavicular joint to the superomedial angle and the line between the sterno-clavicular joint and the center of the acromio-clavicular joint (clavicular axis). e Schematic drawing showing the method of calculating glenoscapular angle (glenoid version θ), posterior subluxation of the humeral head and spinoscapular angle (ζ). The scapular line that connects the medial aspect of the scapula and the mid glenoid is drawn. A second line is drawn connecting the posterior and anterior margins of the glenoid. 90° are subtracted from the angle of the posterior medial quadrant defined by these lines to determine the glenoid version θ. A line perpendicular to the scapular line is drawn and the percentage of posterior subluxation is defined as the ratio of the distance from the scapular line to the anterior portion of the head to the diameter of the humeral head (LM/LN × 100). The spinoscapular angle (ζ) is measured as the angle between the scapular line and the medial plane Vertical displacement was measured on the trunk posterior view with reference to the contralateral side. Lines were drawn from the center of the glenoid cavity perpendicular to the vertebral axis line. Vertical displacement is the percentage of the difference between the levels of the two glenoids (A) divided by the height of the contralateral scapula (H). Positive sign denotes superior displacement and negative sign inferior displacement (Fig. 2b). In order to define the rotation of the affected scapula on an axis perpendicular to the scapular plane (downward/upward) we calculated the angle (α) between the extension of the line connecting the mid glenoid to the base of the spine of the scapula and the vertebral axis line (Fig. 2b). We also calculated two angles between three lines forming a triangle: Line 1 was drawn between the sterno-clavicular joint and the center of the acromio-clavicular joint, and line 2 between the center of the acromio-clavicular joint and the inferior angle of the scapula. Line 3 was the vertebral axis line. The superior scapular angle (γ) is the angle defined between lines 1 and 2 and the inferior scapular angle (δ) is the angle between lines 2 and 3 (Fig. 2c). The rotation on the axis of the scapular spine (anterior/posterior) was assessed on the superior outlet view by measuring the angle (ϕ) of convergence of the longitudinal lines of the shaft of the clavicle and the line connecting the superomedial angle of the scapula to the acromio-clavicular joint (Fig. 2d). The total area of the scapula was calculated on the scapular posterior view. The area of the scapula visible on the anterior view above the clavicle with reference to the contralateral side was measured on the trunk anterior view. For distance and area measurements, graphic software (Universal Desktop Ruler, AVPSoft.com) was used. On a transverse CT image at the mid-glenoid level a scapular line was constructed that connected the medial margin of the scapula to the middle of the glenoid fossa. The glenoscapular angle (glenoid version) was measured according to Friedman et al. [5] as the angle between the scapular line (connecting the scapular medial margin to the middle of the glenoid) and the line connecting the base of the anterior labrum and posterior labrum. By definition 90° are subtracted from the posteromedial quadrant angle to define the glenoscapular angle (θ) (Fig. 2e). The same scapular line is used to determine the degree of humeral head subluxation. The greatest diameter of the head was measured (LN) as the distance of the scapular line to the anterior portion of the head (LM). The percentage of subluxation was calculated as the ratio of these distances multiplied by 100 (Fig. 2e). Rotation about an approximately vertical axis (internal/external) was assessed by measuring the spinoscapular angle (ζ) between the scapular line and the median plane. Both the affected and contralateral sides were assessed and the values discussed take into account comparison between sides unless specifically indicated. Statistics Descriptive statistics were calculated for each variable, including mean, standard deviation and range. Comparisons of means for continuous variables were performed by the Pearson product correlation coefficients and the paired Student t test with Microsoft Excel 2003 software (Microsoft, Redmond, WA, USA). P values were two-tailed, and P values of <0.05 were considered to be significant. Results The patients were noted to have unilateral scapular elevation as a result of downward rotation to various extents. The 3D-CT data for the patients as well as the significance value between affected and contralateral side and the correlation coefficient of each variable to the area of affected scapula visible above the clavicle are summarized in Table 1. The area of scapula visible above the clavicle significantly correlated with downward rotational deformity (r = −0.65), affected to contralateral height ratio (r = −0.55), ratio of affected acromion to total width (r = 65), ratio of affected acromion to height (r = 0.85), affected to contralateral acromion length (r = 0.56), affected to contralateral area (r = −0.57), superior scapular angle (r = −0.84), inferior scapular angle (r = 0.83) and subluxation (r = −0.80). Table 1Details of the computed tomography data on the patientsAffectedContralateralAffected to contralateralPraMeanRangeMeanRangeMeanRangeHypoplasiaAcromion as percentage of widthacr/W × 10035.6 ± 5.6%24.4 to 48.9%32.4 ± 2.4%27.8 to 37.5%1.10 ± 0.170.70 to 1.58<0.0000.57Acromion as percentage of heightacr/H × 10036.0 ± 6.6%22.4 to 55.6%30.5 ± 3.0%24.4 to 35.7%1.19 ± 0.240.79 to 1.73<0.0000.71Affected acromion/control acromionacr/acr1.04 ± 0.140.73 to 1.28NS0.56Percent scapular area visible above clavicle17.76 ± 14.35%0.61 to 53.98%Percent ratio affected to contralateral scapular area86.0 ± 6.4%73.1 to 103.3%−0.57Affected to contralateral height ratioH/H0.89 ± 0.100.69 to 1.11<0.05−0.55Affected to contralateral width ratioW/W0.95 ± 0.070.78 to 1.09NS−0.14Height to scapular body widthH/w1.38 ± 0.200.95 to 1.811.40 ± 0.151.03 to 1.701.00 ± 0.130.80 to 1.30NS−0.30Affected to contralateral scapular body widthw/w0.90 ± 0.070.76 to 1.00<0.05−0.35Height to total width ratioH/W1.00 ± 0.130.68 to 1.261.07 ± 0.100.91 to 1.300.94 ± 0.130.69 to 1.17<0.05−0.35ElevationPercent vertical displacementA/H−1.6 ± 9.33%−20.2 to 18.8%−0.03RotationDownward/Upward displacement^α59.7° ± 13.5435° to 83°83.9° ± 10.1267° to 111°−24.2 ± 10.98−41 to −6<0.000−0.65Internal/External displacement^ζ−35.2° ± 8.5018° to 55°40.4° ± 4.9330.5° to 49°−6.5° ± 7.92−20.5° to 17°NS−0.22Anterior/Posterior displacement^ϕ35.4° ± 7.9320° to 48°43.2° ± 8.0225° to 54°−8.1° ± 4.89−18° to 4°<0.050.29Superior scapular angle^γ45.6° ± 11.5020° to 67°58.3° ± 7.3644° to 81°−12.8° ± 13.09−42° to 11°<0.000−0.84Inferior scapular angle^δ31.1° ± 16.51−2° to 67°9.7° ± 7.70−3° to 27°21.4° ± 17.72−9° to 62°<0.0000.83Percent subluxationLM/LN × 10025.7 ± 20.75%−27.7 to 53.0%49.0 ± 3.51%41.0 to 56.2−23.3 ± 21.21−76.0 to 4.6<0.000−0.80Glenoscapular angle^θ−20.4° ± 11.34−45° to 0°−2.9° ± 3.74−11° to 5°−17.6° ± 10.88−43° to 1°<0.000−0.51The exact measurements (as abbreviated in the second column) are defined in Fig. 2aCorrelation between ratio or difference of affected to contralateral side value to scapular area visible over clavicle Age did not significantly correlate with any of the parameters measured. Vertical displacement was noted to be either superior (15 patients) or inferior (15 patients) and did not significantly correlate with any of the parameters measured. Downward rotational deformity was found to correlate with superior (r = 0.53) and inferior (r = −0.53) scapular angle. Internal/external rotation did not significantly correlate with any of the parameters measured although only three patients had external rotation and two no rotation. Anterior/posterior rotation did not correlate significantly with any of the parameters measured although only one patient had posterior rotation and one no rotation. Area of scapula was found to correlate with superior scapular angle (r = 0.60). Affected acromion to height ratio correlated with affected superior scapular angle (r = −0.81), inferior scapular angle (r = 0.83) and with subluxation (r = −0.80). Affected to contralateral acromion to height ratio correlated with superior scapular angle (r = −0.64), inferior scapular angle (r = 0.62) and with subluxation (r = −0.62). Superior and inferior scapular angles correlated with subluxation (r = 0.63 and r = −0.63, respectively). Subluxation correlated with glenoscapular angle (r = 0.70). A grading scale was devised for assessing scapular deformity in brachial plexus palsy, based on the 3D-CT of the observed patients. The different stages are illustrated in Fig. 3. Fig. 3The different SHEAR stages of scapular deformity as determined by three dimensional computer tomography. In SHEAR Grade 0 less than 2% of the scapula, less than 20% of the superior border and less than 6.5% of the medial border are visible above the clavicle. In SHEAR Grade 1 2–3.6% of the scapular area, 20–45% of the superior border and 6.5–16.5% of the medial border are visible over the clavicle. In SHEAR Grade 2 3.6–20% of the scapular area, 45–58% of the superior border and 16.5–28% of the medial border are visible above the clavicle. In SHEAR Grade 3 20–45% of the scapula, 58–68% of the superior border and 28–50% of the medial border are visible over the scapula. In SHEAR Grade 4 more than 42% of the area of the scapula, more than 68.5% of the superior border and more than 50% of the medial border are visible over the scapula Discussion The position of the affected scapula did not follow the symptoms and characteristics of Sprengel’s deformity with congenital origin of scapular elevation. The hypoplasia and positioning of the scapula result from the brachial plexus injury and the apparent elevation is the result of downward rotation about an axis perpendicular to the scapular plane. The lack of substantial forces applied to the scapula by the weakened scapula stabilizer muscles leads to a decreased rate of bone growth [8, 11]. The observed decreased height to total width is partly the result of enlargement of the acromion, which may be caused by traction through the deltoid [7]. The acromion was found to be always tapered in the presence of the mildest deformity and often beaked and its ratio to both the total scapular width and height were significantly larger than the contralateral side (both P < 0.000). The ratio of affected acromion to height correlated with the area of scapula visible over the clavicle (r = 0.85), with both affected superior scapular angle (r = −0.81) and inferior scapular angle (r = 0.83) and with subluxation (r = −0.80). It seems that while the growth of height and width of the body of the scapula are impaired, the acromion follows a different pattern, of growth at the normal rate or at an aberrant rate if the deformity is more severe (correlation coefficient of affected to contralateral acromion and area of scapula visible over clavicle r = 0.56) and often impinges on the humeral head. The vertical displacement which was either negative or positive did not significantly correlate with any of the parameters assessed implying that the apparent elevation of the scapula is the result of rotational displacement unlike congenital scapular elevation where the severity of the deformity correlates with the level of the shoulder joint [4]. The affected scapula is not only malpositioned, it is also hypoplastic. Previous radiological assessments have used oblique images of the scapula [8, 11] and no report has utilized 3D-CT and measured these characteristics. In fact, in posterior trunk views the hypoplasia looks more exaggerated than by measurement in the scapular posterior view because the view is affected by the internal rotation. The total area of the hypoplastic scapula averages 14% less than the contralateral scapula and correlates with the severity of the deformity (r = −0.57). While the rotation about a vertical axis was usually internal, in three instances it was external with no correlation to the severity of the deformity or to any other parameter assessed. Rotation about the axis of the scapular spine was in all but one instance anterior, forming a narrower scapuloclavicular space on the superior trunk outlet view. During the development of scapular dysplasia, both downward rotational deformity (angle of the spine of the scapula) and decreased height to width ratio contribute to change in superior scapular angle and inferior scapular angle and both indeed strongly correlated with area of scapula visible above clavicle (r = −0.75 and r = 0.83) and degree of subluxation (r = 0.63 and r = −0.63). The clinical prognostic relevance of the various measurement methods is still under discussion [1, 13]. Global abduction has been correlated with glenoid version [17]. In the literature there seems to be no significant correlation between Mallet functional parameters other than global abduction and either radiographic parameters or age [13, 17] because active shoulder function is affected by the glenohumeral relationship, shoulder capsule, strength and physical properties of the muscles and the nervous system. In many patients, different versions of muscle release of the contracted internal rotators with or without muscle transfer of latissimus dorsi and teres major to the teres minor is performed early in life so that the weakened deltoid and supraspinatus muscles can achieve active abduction. The success of this intervention is very impressive and the abduction is often significantly improved ([6, 10, 15] and our unpublished data). While glenohumeral incongruence is delayed after muscle contracture release ([15] and our unpublished data), internal rotation of the arm and winging scapula often develop. The lack of correlation between age and any of the parameters of the anatomical deformity in our study, unlike reports by others [17], may reflect the fact that most of our patients had undergone contracture releases of the latissimus dorsi, teres major, subscapularis, and pectoralis major and minor with tendon transfer of latissimus dorsi and teres major. While global abduction in our series was near normal, the forces exerted by the muscles on the two scapulas are not equivalent obviously upholding impaired growth to the affected scapula. The rate of progression of the deformity is individual to the child. The rotation of the scapula causes positional changes in the acromio-clavicular joint. This causes impingement of the distal acromion and clavicle with the humeral head, resulting in the MRC positioning described above. The clinical implications of the SHEAR and resulting MRC are clear and have been described by Birch in detail [2]. The current standard of practice for treatment of MRC is muscle transfer if the shoulder is congruent [2, 16], corresponding to SHEAR 0–1 and humeral derotational osteotomy if the humeral head is incongruent corresponding to SHEAR 2–4 [16]. While derotational osteotomy may restore external rotation by placing the arm in a more functional position it addresses neither the scapular nor the glenohumeral deformities. Our findings that SHEAR score based on the percentage of scapula visible over the clavicle strongly correlates with rotation of the scapula, hypoplasia and subluxation, provides a convenient diagnostic tool to assess the presence of the MRC deformity. With the pathophysiology being thus established, anatomical correction of the deformity can be considered and planned, hopefully with improvement in functional parameters. Conclusion Scapular deformities common to the population of obstetric brachial plexus palsy patients due to muscular imbalances resulting from residual neurological deficit can be diagnosed and classified using the SHEAR classification and enable objective evaluation of the bony deformity and its severity as guide for treatment.

Skeletal_Radiol-3-1-1934928

The spine in Paget’s disease

Paget’s disease (PD) is a chronic metabolically active bone disease, characterized by a disturbance in bone modelling and remodelling due to an increase in osteoblastic and osteoclastic activity. The vertebra is the second most commonly affected site. This article reviews the various spinal pathomechanisms and osseous dynamics involved in producing the varied imaging appearances and their clinical relevance. Advanced imaging of osseous, articular and bone marrow manifestations of PD in all the vertebral components are presented. Pagetic changes often result in clinical symptoms including back pain, spinal stenosis and neural dysfunction. Various pathological complications due to PD involvement result in these clinical symptoms. Recognition of the imaging manifestations of spinal PD and the potential complications that cause the clinical symptoms enables accurate assessment of patients prior to appropriate management. Introduction Paget’s disease (PD) or osteitis deformans is one of the commonest metabolically active bone diseases, second in prevalence only to osteoporosis, characterised by a disturbance in bone modelling and remodelling because of an increase in osteoblastic and osteoclastic activity. It is more common in people of Anglo Saxon origin and is less common in the Far East, India, Middle East and Africa. The overall prevalence of PD is 3–3.7% and increases with age [1–3]. By the age of 90 years, the prevalence increases to about 10% [2]. As the aetiology of PD continues to be the subject of debate, it is variously classified as an infection, metabolic disorder and a neoplastic process [1, 4–15]. However, strictly speaking, as the unaffected skeleton is metabolically normal, it does not fulfil the criteria for a true metabolic bone disorder. The epidemiology of PD shows some significant changing trends in the clinical profilometry. First, recent studies have demonstrated that the incidence and prevalence of PD is gradually declining [16–18]. Second, there is a general trend towards reduction in the severity of the disease as measured by serum alkaline phosphatase levels [19, 20]. Third, there is a steady increase in the age at presentation by about 4 years per decade [18] and last, the proportion of patients with monostotic disease is increasing [18]. This in turn means that we are likely to see a higher incidence of monostotic vertebral involvement in the future. The spine is the second most commonly affected site (53%) [6], after the pelvis (70%) [21–23]. The disease is polyostotic in 66% of cases and between 35% [24, 25] and 50% [7, 8] have spinal involvement. Using multimodal advanced imaging, this review article focuses on the pathological processes that underlie the varied spinal manifestations and complications of PD. Pathomechanisms In PD the loss of homeostatic control leads to increased osteoblastic and osteoclastic activity and constitutes the background for the main three phases. The initial lytic phase represents a mainly osteoclastic activity, the late osteoblastic phase is characterised by new bone formation, while the intervening mixed phase is seen when there is a combination of osteoblastic and osteoclastic activities [24, 26]. One more phase, “inactive sclerotic phase”, characterised by normal or decreased bone activity, has also been described [5], when the stimulation of new osteoblast and osteoclast formation ceases. Although the bone is metabolically inactive, it maintains a sclerotic coarsened architecture [27]. In bones with a low trabecular/cortex ratio like the skull, femur and humerus, the early lytic phase is radiologically depicted by a clear leading edge at the interface with normal bone. The lytic phase is usually not detected in bones with a high trabecular/cortex ratio like the vertebra, sacrum and pelvis [28]. Vertebral body involvement at radiological diagnosis is virtually always complete, and therefore the leading edge present in the other affected bones is not seen in the vertebra [29]. These phases can be evident in the same patient and at the same time in different bones including the vertebral column. Although progression of disease occurs within an affected bone, the sudden appearance of bone involvement at new skeletal sites years after the initial diagnosis is uncommon [30, 31]. The pathomechanisms and the dynamics involved in bone remodelling in PD of the appendicular and axial skeleton at the periosteal and endosteal surfaces have been previously described [3, 5, 32]. The enhanced abnormal osteoblastic activity results in periosteal and endosteal new bone formation (apposition). The abnormal osteoclastic activity on the endosteal surface results in bone resorption (absorption). The various combinations of these mechanisms give rise to four different patterns of bone remodelling at the periosteum/endosteal interface leading to bone enlargement: periosteal and endosteal apposition; periosteal apposition and endosteal absorption; periosteal apposition with normal endosteal surface; and focal periosteal apposition –“pumice stone” appearance (Figs. 1, 2). The mechanisms in the spine commonly responsible for the changes on the periosteal and endosteal surfaces of the vertebral body and posterior neural arches are varied (Figs. 1, 2, 3, 4). These various mechanisms are not exclusive of each other, but can occur in combination in the same vertebra at various borders. Usually, one of the pathomechanisms predominates in the involved vertebra. The most frequent mechanism of vertebral body expansion is periosteal apposition and endosteal absorption. The new bone formation predominates on the periosteal surface and it is responsible for the vertebral body enlargement, while the absorption on the endosteum results in an increased bone marrow space. Periosteal/endosteal apposition and periosteal apposition with normal endosteal surface are two less common remodelling mechanisms seen in the vertebral body. In both, the apposition on the periosteal side results in vertebral body enlargement, but the bone marrow space is decreased or normal in size respectively. The least common mechanism of vertebral body expansion is focal periosteal apposition giving the “pumice stone” appearance (Figs. 1, 2). Expansion of the vertebral bodies seen radiologically occurs in 63% of cases [25]. The commonest mechanisms in neural arch involvement are a combination involving periosteal and endosteal apposition or periosteal apposition and endosteal absorption (Figs. 3, 4). In both of the mechanisms, the periosteal apposition causes a decrease in the size of the spinal canal resulting in spinal stenosis. Fig. 1Diagram depicting the osseous mechanisms involved in vertebral body enlargement in Paget’s disease and its effect on the size of the marrow (dashed arrows) and cortex (solid arrows). A normal vertebra is depicted in the centre of the figure. a Periosteal apposition, normal endosteum resulting in thickened cortex, but with normal marrow size. b Periosteal apposition, endosteal resorption results in normal cortical thickness and an increased marrow size. c Periosteal apposition/endosteal apposition results in a thickened cortex and reduced marrow size. d Focal periosteal apposition results in a focal “pumice stone”-like enlargementFig. 2Axial CT sections in different patients showing the various mechanisms described in Fig. 1 and their effect on marrow size (long white arrow) and cortical thickness (short white arrow). a Periosteal apposition, normal endosteum. b Periosteal apposition, endosteal resorption. c Periosteal and endosteal apposition. d Pumice stone type (dashed arrow) of focal periosteal apposition. Similar focal periosteal apposition of the spinous process is seenFig. 3Diagram showing the periosteal and endosteal Pagetic osseous mechanisms involving the cortex of the spinal canal resulting in spinal canal narrowing. Normal cortical thickness (orange) of the spinal canal (white) is depicted at the top. a Expansion of bone due to periosteal apposition/endosteal resorption results in a thin cortical outline (solid black arrow) of the narrowed spinal canal (dashed arrow). b Bony expansion due to periosteal apposition/endosteal apposition results in a thickened cortical outline (solid black arrow) of the narrowed spinal canal (dashed arrow)Fig. 4Axial CT images demonstrating the mechanisms in the posterior neural arch described in Fig. 3 and their effect on cortical thickness (solid arrow) and marrow size (dashed arrow). a Periosteal apposition/endosteal resorption. b Periosteal apposition/endosteal apposition Imaging manifestations Paget’s disease of the spine can either involve a single level or more than one level. The lumbar spine and more commonly the L4 and L5 levels are the most frequently involved sites (58%) [33], more so than the thoracic (45%) and the cervical vertebrae (14%; Fig. 5) [8, 23]. Involvement of the atlanto-axial region is very rare [8, 34, 35]. The vertebral body is almost always involved together with a variable portion of the neural arch. Isolated involvement of either the neural arch or the vertebral body is evident in only a small percentage of cases [25]. Fig. 5Vertebral Paget’s disease (PD) without expansion in two different patients. a PD of all the cervical vertebrae except C6. Note the absence of vertebral enlargement. There is sclerosis and loss of cortico-medullary differentiation of the vertebral bodies and the neural arches. b antero-posterior, c lateral radiographs and d axial CT through the L1 vertebra demonstrating sclerotic vertebra with no enlargement. The axial CT demonstrates trabecular and endosteal apposition, but no periosteal apposition accounting for the absence of enlargement. Diagnosis can be difficult and a biopsy (c) may be necessary in these cases Osseous changes (vertebral body) The radiological appearance of vertebral body expansion is characterised on radiographs by an increase in the antero-posterior and lateral vertebral dimensions. However, the height of the vertebra is unchanged. The bony vertebral end-plates are subchondral condensations of trabecular bone and do not represent true bony cortex, i.e. there is no periosteum/endosteum interface at the caudal/cranial aspect of a normal vertebra. The sagittal and lateral enlargement is due to one of the previously described pathomechanisms in the corticated portions of the vertebral body. The pathomechanisms are best appreciated on CT (Fig. 2), where the periosteal contour and the endosteal interface due to apposition and absorption are easily seen. Whatever the mechanism causing vertebral body remodelling, bone expansion is a common denominator in PD. Usually, the earliest phase seen radiologically in the vertebra is the mixed phase. The apparently “early” radiographic appearance of vertebral body involvement in PD is thickening and hypertrophy of the trabecular bone [36, 37], parallel to the end plates (Fig. 6), which can appear similar to a thickened cortex. CT reconstruction images can show this thickening and hypertrophy optimally. The combination of trabecular bone hypertrophy and thickening at the end-plates with apposition/absorption on the periosteal/endosteal surfaces at the anterior and posterior vertebral borders leads to the “picture frame” sign [36]. Radiographs demonstrate increased density in the vertebral periphery and a relatively lucent centre in the vertebral body [36, 38, 39], best appreciated on lateral views and on sagittal CT reconstructions (Fig. 6). Fig. 6a Lateral and b antero-posterior radiographs demonstrate expansion of the vertebra with characteristic sclerotic lines parallel to the end-plates due to trabecular hypertrophy, an “early” sign of PD. c Lateral radiograph in a different patient demonstrates the “picture frame” vertebra due to thickening of the cortex and trabecular hypertrophy at the end-plates Progression of the sclerotic phase in the spine leads to “ivory vertebra”, due to an increase in the density of the vertebral body, denser than the normal vertebral bodies. There is no change in the density and size of the adjacent intervertebral disc [40, 41]. Differential diagnoses of “ivory vertebra” include metastases, osteosarcoma, carcinoid and Hodgkin’s lymphoma [29, 36]. The increased size of the vertebral body is a useful clue to the underlying diagnosis of PD. Biopsy may be necessary in some cases when there is no expansion (Fig. 5). In the exceptionally rare cases of vertebrae presenting in the lytic phase, there is marked osteopenia of the vertebra on radiographs, giving a “ghost vertebra” appearance, because of the almost complete involvement of the vertebral body by the osteolytic process [42]. The lytic phase of PD involving the axis [43] and a rare pathological collapse of a purely lytic L5 vertebra [44] have been previously reported. It can be difficult to differentiate the lytic phase of PD from other causes of osteolysis and collapse. By demonstrating the cortical thickening, trabecular hypertrophy and vertebral expansion, CT confirms the lytic phase in the vertebral components due to the higher contrast resolution, the feasibility of “bone window” settings and multiplanar reconstructions. The trabecular hypertrophy and cortical thickening caused by the osseous involvement by PD results in a variable degree of low signal on both T1- and T2-weighted images. In the severe cases of vertebral sclerosis (ivory vertebra appearance) the whole vertebra demonstrates a diffuse low signal on both T1- and T2-weighted sequences. More commonly however, the signal characteristics are heterogeneous on both T1- and T2-weighted sequences due to changes in the intervening marrow space. PD of the vertebra is easily missed or misinterpreted on MR images in the early stages. This is especially true when there is an absence of classic changes including vertebral enlargement and cortical thickening. This is mainly because PD is a disorder of bone and the vertebral marrow is only secondarily affected. MRI can show discrete areas of marrow signal alteration involving vertebral bodies with low non-specific signal on T1-weighted images and high signal on T2-weighted sequences. The marrow changes due to vertebral involvement by PD are described in detail later. Osseous changes (posterior elements) Neural arch involvement can be difficult to evaluate on radiographs alone. The best modality for neural arch assessment is CT with reconstructions. When periosteal and endosteal apposition occur, a markedly sclerotic cortex can be seen [5, 8]. However, when the basic mechanisms are periosteal apposition and endosteal absorption, CT demonstrates an enlarged marrow space of the neural arch delineated by a thin sclerotic line due to the periosteal apposition (Fig. 4). Both processes result in a decrease in the spinal canal diameter. The presumed epidural fat ossification as previously described in Paget’s disease [45], results in the loss of the normal MR signal of epidural fat. This is probably a consequence of expanded Pagetic bone growth out of the neural arch components towards the spinal canal, reducing its size, rather than real ossification of the epidural fat. This can be misinterpreted as epidural fat ossification on radiographs and MR imaging and is best assessed on axial CT images (Fig. 7). Sometimes, however, the fatty marrow changes in the expanded Pagetic posterior neural arch can be misinterpreted on MR imaging as epidural lipomatosis (Fig. 8). CT again helps in the correct interpretation of this situation. This emphasises the need for a combination of imaging modalities to complement each other for accurate interpretation. Fig. 7a Sagittal T1-weighted MR image demonstrates PD in the L3 vertebral body and the posterior neural arch. The low T1 signal intensity mass (arrow) replacing epidural fat can be confused with epidural ossification. b However, an axial CT image confirms this to be due to expansion of the pagetic neural arch and not to ossification of the epidural fat. There is preservation of the intra-osseous fat as seen on both the MR and CT images, a useful discriminant from malignant infiltrationFig. 8Paget’s disease of T11 and T12 showing an increased amount of high MR signal (solid white arrow) in the posterior epidural space at these levels on a sagittal T2-weighted, b sagittal T1-weighted and c axial T1-weighted images. This can be mistaken for epidural lipomatosis. However, the d CT sagittal and e axial images demonstrate this to be due to the fat density (solid white arrow) within the expanded posterior neural arch involved in PD. The axial images (c,e) were obtained at the level of the tip of the solid arrows on sagittal images. There is also fusion of the vertebrae (dashed arrow) across the intervertebral disc. The combination of anterior and posterior vertebral involvement in this case resulted in severe spinal canal narrowing and cord compression. Note the high T1 signal indicating a high fatty marrow content within the pagetic T11 and T12 vertebrae Bone scintigraphy using 99-Technetium substrates and SPECT can aid the diagnosis of PD, showing a typical distribution of the disease. Scintigraphy demonstrates increased uptake in all the affected vertebral components [46, 47] because of its sensitivity to osteoblastic activity and can be very useful in showing activity in the posterior neural arch. As a whole body imaging modality, it also provides information about the overall distribution of the disease. Moreover, the fourth sclerotic inactive phase described by Milgram is diagnosed essentially on a bone scan where radiographically affected bone does not demonstrate increased activity due to the metabolic inactivity of the lesion [27]. However, increased activity on bone scintigraphy is usually non-specific and requires comparison with radiographs/CT. Various patterns of uptake are described on bone scintigraphy in PD [21, 46, 48, 49], all of which are non-specific. Imaging techniques such as PET and PET-CT, can contribute to the differential diagnosis of PD from other disorders in the spine, especially when non-specific PD changes are incidentally discovered, and to assess disease activity after treatment. In general 18F-fluoride positron emission tomography (PET) allows visualisation of the regional skeletal metabolic activity on the basis of the fluoride uptake on the bone surface when new bone formation and blood flow are greater [50]. The advantages of PET-CT on conventional nuclear bone scintigraphy include superior spatial resolution and more accurate quantification of bone activity. In PD, a general increase in global skeletal blood flow, measured by fluoride deposition, is seen associated with a regional increased uptake in the affected bone. Increased activity in the pagetic bone is usually recorded almost immediately after the injection and it remains high throughout the duration of the study. However, this finding is not specific to pagetic bony changes, giving rise to some false-positives. For this reason, the combination of non-specific activity on the PET image, the findings on the CT scan (PET-CT) and the correlation with other radiological examinations increases specificity [51]. Extra-osseous changes Paget’s disease can sometimes involve the soft tissue attached to the affected vertebra including the articular cartilage, the ligaments and the intervertebral disc [26, 45]. The articular facets are commonly involved in association with vertebral body and neural arch involvement. The affected facets are enlarged and sclerotic (Fig. 9). The pagetic vertebral body involvement can interfere with the nutrition of the intervertebral disc leading to its degeneration due to sclerosis adjacent to the end-plates resulting in decreased diffusion [26]. Fig. 9Facet involvement. a Axial CT section demonstrates incongruity at the facet joint (solid white arrow) due to an involved enlarged facet of one vertebra articulating with an uninvolved non-expanded adjacent vertebral facet. b Axial CT section in a different patient demonstrates advanced facet joint arthropathy (dashed white arrows) across two pagetic facets with complete loss of joint space and new bone formation contributing to spinal canal stenosis. This can progress to fusion across the facet joints Occasionally pagetic involvement of the spine can also produce extra-osseous extension resulting in ossification of the anterior/posterior longitudinal ligaments and ligamenta flava. Lateral radiographs and CT sagittal reconstruction demonstrate the ossified anterior and posterior longitudinal ligaments. The ligamenta flava ossification are seen on the AP view as radio-opacities adjacent to the facet joints. Para-vertebral soft tissue swelling seen radiographically or by CT can be due to extra-medullary haematopoiesis. When PD of spine results in extra-medullary haematopoiesis [52], there is extra-osseous soft tissue with signal characteristics similar to the adjacent vertebral marrow. CT and MRI demonstrate the soft tissue mass adjacent to the vertebral body (see Pathological complications) in communication with the bone marrow within the vertebra [52]. Bone marrow Paget’s disease is primarily a disorder of bone and not of the bone marrow. Secondary bone marrow changes do, however, occur. The marrow signal changes in established Paget’s disease have been well described and vary with the stage of the disease [53]. Low signal on T1-weighted images and mild high signal on T2-weighted images (Fig. 8) in the mixed hypervascular phase are seen. The sclerotic phase of PD results in low signal on both T1- and T2-weighted images in the vertebra due to increased trabecular thickness, sclerosis and marrow fibrosis (Fig. 10). There is fatty transformation in the latter stages when there is high signal on both T1- and T2-weighted images (Fig. 11). In the presence of osteolysis in pagetic vertebrae, a fat signal within the lesion is a useful clinical determinant between conservative management and biopsy in cases in which malignancy is suspected radiographically [54]. Fig. 10a Initial scintigraphy for back pain demonstrates isolated increased uptake at a single vertebral level (T8). On initial inspection sagittal b T1-weighted and c T2-weighted MR images do not show any abnormality of the vertebral body. There is, though, some abnormal low signal from the posterior elements (black arrow). The diagnosis is still not clear. d However, a CT scan demonstrates the clear posterior vertebral (black arrow) sclerotic changes consistent with PD. Even on CT there are only minimal changes in the vertebral bodyFig. 11On initial examination, a sagittal and b parasagittal T1-weighted, c sagittal and d parasagittal T2-weighted MR images of the lumbar spine do not demonstrate any obvious abnormality. e, f The radiographs, however, show classic pagetic changes of the L1 vertebra (dashed arrows) including vertebral expansion, sclerosis and cortical thickening. Review of the MRI shows some minor increased signal in the expanded L1 vertebral body on both T1 and T2 parasagittal images, suggestive of fatty marrow change (white arrows) Whenever vertebral bone marrow signal changes are seen on MRI, PD has to be considered in the differential diagnosis because of its non-specific and varied appearance in the bone marrow, and subtle bony alterations suggestive of PD have to be sought. Paget’s disease can, however, also coexist with other disorders. When the bone marrow is not or only minimally involved (Fig. 12) in PD, MRI will not reveal an appreciably altered signal on either T1- or T2-weighted images. The seemingly normal MR appearance of the vertebra does not exclude PD, but affirms only the normal appearance of the bone marrow, while the intervening trabecular bone is affected. For this reason, vigilance in recognising subtle signs like minor antero-posterior vertebral expansion and minimal cortical thickening is necessary as the signal characteristics within the centre of the vertebra may be entirely normal. MRI should be complemented by radiographs and CT in these instances (Figs. 10, 11, 12). PD is fundamentally a bone disorder, where the bone marrow may or may not be involved, showing areas of sclerosis, fibrosis, increased vascularity, residual haematopoiesis and fatty marrow replacement, resulting in a wide spectrum of MR signal features. For this reason, all these described MR findings need the comparison of radiographs and CT images, because only these imaging modalities are actually capable of showing the specific and pathognomonic bony changes of PD. The aim of the integration of different techniques is to make a correct diagnosis and avoid unnecessary biopsy. Fig. 12a T1-weighted, b T2-weighted sagittal images of the lumbar spine demonstrate no marrow abnormality. There is only a subtle antero-posterior expansion of the L2 and L4. The diagnosis in these patients can be missed on initial MRI. c Lateral radiograph of the lumbar spine demonstrates the classic pagetic changes including vertebral expansion, trabecular hypertrophy and cortical thickening in L2 and L4. There is an incidental non-pagetic vertebral compression at L1. There is again preservation of the fat signal within the vertebrae involved in PD Pathological complications Pagetic bone is structurally weak. Despite this, the involved vertebral body and posterior neural arch still have to fulfil the biomechanical role they were designed for. The disease will alter the normal metabolic function in the bone while biomechanical forces in turn modify further the metabolic and structural changes that arise. Various clinical complications can occur secondary to vertebral PD resulting in back pain, spinal stenosis and neural dysfunction. Whilst each of these clinical symptoms can be caused by a variety of pagetic complications, it has to be stressed that PD and its complications can be entirely asymptomatic[21]. Moreover, the symptoms could be due to other coexisting spinal disorders. Back pain is the most common clinical symptom associated with PD of the spine (Table 1). However, not all patients with PD of the spine are symptomatic [21]. Back pain in PD is due either to the disease itself or to the occurrence of different complications (Table 1). Back pain due to the PD itself is caused by an increased blood flow and modelling/remodelling process associated with the vertebral involvement including periosteal stretching and micro-fractures. In general, pagetic pain is a deep, dull ache or pain in the back that is unrelated to activity and not relieved by rest or non-steroidal anti-inflammatory medication. This pain is different from mechanical pain, which improves with rest and worsens with activity or a stressful posture of the back. It is also different from arthritic pain, characterized by aching and stiffness that is relieved by walking or by anti-inflammatory medication [24]. Between 11 and 54% [5, 55] of pagetic patients present with pain. In 24% of cases, back pain is due to PD itself [5, 8, 24]. In 50% of cases, it is due mainly to other complicating factors including fracture, spinal stenosis and facet arthropathy; less commonly it is due to spondylolysis with or without spondylolisthesis and discal involvement [26]. Another study found 88% of cases with back pain related to coexisting PD and osteoarthritis of the spine versus 12% of patients with pain attributed only to PD [56]. No particular difference in pain has been seen in multilevel involvement compared with single level involvement [24]. Table 1Causes of back pain in Paget’s diseasePeriosteal stretchingVascular engorgementMicrofracturesFacet arthritisIntervertebral disc diseaseOvert fractures of vertebrae, sacrumSpondylolysis/-listhesisSarcoma—very rare Spinal stenosis in PD of the spine has a prevalence of 33% of cases (Table 2) [5]. It is graded as mild if it is just perceptible, moderate if the area of the spinal canal is reduced by less than 50% and severe if that area is reduced by more than 50% [24, 25]. Spinal stenosis can present clinically as back pain and/or neurological dysfunction, as a consequence of cord (Figs. 8, 13) or nerve root compression. In some cases, spinal stenosis is not associated with back pain. Patients with severe stenosis seen on imaging can present without clinical symptoms and patients with mild or moderate stenosis can present with back pain [24]. This is possibly explained by the adaptability of the thecal sac and its neural elements to severe spinal stenosis without significant loss of function [5, 24]. The proportion of symptomatic patients with spinal stenosis ranges from 26% [57] to 33% [5]. Seventy-five per cent of these cases present with pain, but without neurological dysfunction [25]. Expanded pagetic bone involving all vertebral components (vertebral body, neural arch and facet joints) is the commonest cause of spinal stenosis (Fig. 13). It was reported by Wyllie in 1923 [58]. It is a consequence of a disturbance in the bone remodelling process, leading to a subsequent decrease in the spinal canal size [5, 24]. Less frequently, the stenosis can be due solely to expansion of the neural arch associated with the growth of pagetic bone involving the facets [8]. Table 2Causes of neural dysfunction in Paget’s diseasePosterior expansion of the vertebral bodyPosterior neural arch expansionFacet joint arthritis/overgrowthLigament ossificationSpondylolisthesisFracture retropulsionExtra-osseous involvement—PD, haematopoiesis, “pseudosarcoma”, malignancy“Arterial steal” syndromeFig. 13Sagittal T2-weighted MR image demonstrates cauda equina compression at the L1 level due to pagetic enlargement of the whole vertebra. Note the stenosis caused by expansion of both the vertebral body and posterior elements. Degenerative spondylolisthesis and stenosis at L4/L5 is noted Spinal stenosis in Paget’s disease has the unique radiographic feature of widening of the interpediculate distance on the antero-posterior view, with expansion of the vertebral body on the lateral view. CT is very useful for assessing the contribution of the facets and neural arch to the stenosis, not seen well on radiographs (Fig. 14). CT also helps to assess the location of the expanded bone to determine if the stenosis is central or lateral and to assess the severity of the stenosis. In cases in which cord compression due to the spinal stenosis is suspected, the diagnostic tier has to be completed by performing an MRI scan (Figs. 8, 13) [8, 24, 59]. Axial and sagittal images demonstrate the bone abnormalities, but they can also show myelomalacia in the spinal cord. The sagittal T2-weighted images show the pagetic bone compressing the cord with no evidence of normal CSF surrounding the cord. The axial images show the asymmetry or deformation in shape of the cord compressed by the expanded bone. Areas of myelomalacia demonstrate low cord signal on T1-weighted images and high signal on T2-weighted and STIR images, at the same level as the expanded pagetic bone. Fig. 14Post-myelographic lateral lumbar spine a radiograph and b axial CT scan demonstrate spinal block at the L1 vertebral level, which is pagetic. Note the severe degree of spinal stenosis despite the apparent increase in the interpedicular distance on the axial CT image Neural dysfunction can be due to spinal stenosis (33%) [24, 60], more frequently with thoracic spine involvement and less commonly with cervical spine involvement [61]. The less frequent neural involvement in lumbar spine involvement is due to the relatively capacious spinal canal in the lumbar region [5, 57, 62]. Although the pagetic process itself, together with compressive effects from the expanding bone, can give rise to symptoms and signs, other complications may develop and contribute to the symptoms complex of neural dysfunction (Table 2). Patients presenting with neurogenic pain due to cord compression by expanded pagetic bone (Fig. 20) can also respond well to medical treatment with calcitonin and bisphosphonates [63]. Surgical decompression is rarely necessary in cord compression as stenosis and neural dysfunction in PD may respond to medical treatment alone [5]. The “arterial steal phenomenon” is another factor contributing to neural dysfunction. This is described as a deprivation of blood supply to the spinal cord due to the preferential blood flow of the pagetic vertebra (Fig. 15) [64]. It is classified as a non-compressive spinal cord dysfunction and not directly related to pagetic bone growth and it can respond very well to treatment with calcitonin [64–66]. Fig. 15Sagittal fat suppressed T1-weighted image after gadolinium-DTPA administration in the same patient as in Fig. 8 demonstrates intense enhancement at both the T12/L1 and the L3/L4 levels (arrows). The enhancement in part reflects increased blood supply to the vertebra Compression fracture Compression fracture of the vertebral body is the commonest complication in PD of the spine and usually presents with sudden onset of back pain (Fig. 16). It occurs more frequently in the lumbar spine [59], and rarely in the sacrum [59], the coccyx [67] and the odontoid peg. Fig. 16Lateral and anteroposterior radiograph of the lumbar spine demonstrates severe compression of a pagetic L3 vertebra with retropulsion into the spinal canal. Note the increased interpediculate distance as a hallmark of PD Para-vertebral swelling can be seen as an indirect sign of the vertebral fracture in the acute phase. The fracture line itself is best seen on CT with sagittal reconstructions. Vertebral fracture can also contribute to the pain related to spinal stenosis by two mechanisms: posterior vertebral body wall bulging and retropulsion (Fig. 16) of a bone fragment into the spinal canal [8, 68], and cord compression. This can be recognised on radiographs in the lateral view and is associated with a decrease in the spinal canal size, but CT demonstrates this better. The fractured vertebra in PD sometimes appears osteopenic on radiographs. This could be due to a fracture occurring in the lytic phase of PD. However, as described earlier, it is unusual to see the lytic phase of PD in the spine. The osteopenia is most likely due to fracture-induced osteolysis. MRI can also contribute to the diagnosis of a compression fracture, even if its findings are not specific. The fracture line is seen as a linear or curvilinear low signal on T1-weighted images and high signal on T2-weighted and STIR images. Oedema surrounding the fracture can be an indirect sign. It is seen as high signal on inversion recovery and T2-weighted images and low signal on T1-weighted images. When the fracture is not recent, the signal on the MRI scan will be low on both T1- and T2-weighted images because of the sclerosis of the bone. In general, diffusion weighted imaging can help to differentiate osteoporotic from malignant vertebral body collapse [69]. It is not yet known if benign fracture in PD can be mistaken for a malignant compression on diffusion weighted imaging given the associated underlying marrow changes prior to the occurrence of the fracture. Facet joint arthropathy Facet joint involvement can cause back pain, lateral spinal stenosis and neural dysfunction. Facet involvement can lead to facet joint arthropathy due to two mechanisms. One is the direct extension of PD into the articular cartilage similar to that seen in large synovial joints and the intervertebral disc [26], the other is the pagetic involvement of the facets leading to expansion of the facet and resultant incongruity with the apposing uninvolved facet (Fig. 9). Facet joint arthropathy was noted in 17 out of 21 symptomatic patients with back pain in one study [25]. This gives rise to mechanical stress and early degenerative changes in the facet joint. This is one of the most important factors in back pain and also contributes to spinal stenosis. The spinal canal stenosis associated with facet joint arthropathy is typically lateral and can result in nerve root compression [5]. Patients with severe arthropathy often have symptoms of spinal stenosis, but they can be asymptomatic [24, 25]. Compared with radiography, CT gives a clearer depiction of facet joint changes (Fig. 9), showing the loss of articular space, the destruction of cartilage associated with subchondral erosion and articular incongruity (Fig. 9) [5, 26, 70]. Facet joint arthropathy may eventually lead to ankylosis of the articular space [25]. MR imaging can underestimate the bony changes associated with the facet involvement. Spondylolysis and spondylolisthesis Back pain may be also due to unilateral or bilateral spondylolysis if the pars interarticularis is involved in PD. PD, due to the associated modelling/remodelling activity, results in an increased susceptibility to insufficiency fractures. A lateral radiograph may demonstrate the typical spondylolysis (Fig. 17). However, due to the associated PD and sclerosis, this can be difficult to appreciate. CT is the best modality to detect this. Underestimation of this complication on radiographs and the relatively limited use of cross-sectional imaging in PD may be the reason for the under-reporting of this condition. Fig. 17Lateral radiographs in two different patients with PD showing two different mechanisms of spondylolisthesis: a spondylolytic (arrow) spondylolisthesis; b degenerative spondylolisthesis Spondylolisthesis in PD can be due to either spondylolysis or facet joint degeneration (Fig. 17). It is important to differentiate between the two entities. The degenerative spondylolisthesis results in spinal stenosis and neural dysfunction. The lateral radiograph is quite useful for detecting spondylolisthesis and to establish its grade and severity. However, this can be underestimated if the vertebral alignment is not assessed carefully. An enlarged vertebra due to PD may slip anteriorly over a normal vertebra, but the posterior vertebral body alignment may still be preserved due to the posterior expansion. The assessment of the posterior neural arch alignment, however, will reveal the subtle slip in these cases. The standard lateral radiograph can be complemented by dynamic views in flexion and extension, which can reveal the presence of spondylolisthesis under stress. Spondylolisthesis, and particularly spondylolysis, can be confirmed on CT with sagittal reconstruction images. Intervertebral disc involvement Paget’s disease can involve the intervertebral disc and cause intervertebral disc degeneration. The incidence of direct intradiscal transgression is about 10.7% [26]. In this study, 67% of patients with disc involvement presented with pain. Twenty-two percent were asymptomatic. Therefore, disc involvement can remain asymptomatic. Some studies describe the presence of para-vertebral soft tissue involvement without specific comment on the discal invasion by PD [45, 68]. PD transgressing the intervertebral disc occurs more often in the lumbar spine than in the thoracic and cervical spine. The mechanism is not very clear. Intervertebral disc degeneration can be caused mainly by the mechanical stress induced by asymmetry at the end plate attachment of annulus fibrosis between a pagetic and a normal vertebra, and also by direct invasion of the disc space by the pagetic process. Aggressive pagetic invasion at the disco-vertebral junction leads to pagetic tissue replacement of the cartilage end-plate and subsequently of the intervertebral disc. The direct resorption of the disc tissue with subsequent pagetic bone replacement in the disc is the most likely explanation [26]. PD can then invade the adjoining vertebra across the disc space. This can also occur by direct extension of PD along large pre-existing degenerative bridging osteophytes [26]. Radiological investigations in disc involvement show progressive disc space narrowing, loss of definition of the adjacent end-plates, sometimes indistinguishable from spondylodiscitis, and large degenerative osteophytes involved in PD (Figs. 8, 18). Intervertebral disc involvement can eventually lead to pagetic vertebral ankylosis (PVA) with an incidence of 4.4% [26]. Ankylosis is commoner in men, affects the thoracic spine in over 50% of cases [71] and is usually asymptomatic. The fusion between the vertebral bodies is easily appreciated as being due to Pagetic changes. The mechanisms that promote intervertebral disc involvement in PD are also in play when extra-osseous involvement of the spinal ligaments and para-vertebral tissues is seen (Fig. 19). Fig. 18Disc involvement. Serial radiographs 2 years apart in the same patient demonstrate progressive involvement of the L4/L5 intervertebral disc in Paget’s disease of the L5Fig. 19Extra-osseous Paget’s disease. a Sagittal CT reconstruction and b T1-weighted MR sagittal image demonstrate anterior longitudinal ligament ossification (white arrows). c, d Axial CT sections demonstrate ossification in the ligamentum flavum due to extra-osseous PD (black arrows) in a different patient. e Antero-posterior radiograph in another patient demonstrates paravertebral soft tissue swelling (dashed arrows) producing the “pseudo-sarcoma” appearance Neoplastic transformation Neoplastic transformation in pagetic bone is very rare in the spine (0.7%) [5, 8, 24, 72] and represents only 7% [73] of all sarcomatous degeneration in PD. It can present with back pain. Benign and malignant bone neoplasms can complicate Paget’s disease. The majority of malignant transformations are osteosarcomas. They seem to be related to one or two genes on the chromosome 18q [5, 74, 75]. Radiologically, this is seen on radiographs and CT images as a lytic lesion characterised by an aggressive pattern on a background of typical PD. It can be very difficult to diagnose this lesion on the radiographs because of the overlapping pagetic changes in the bone. However, as described earlier, it is unusual to see the lytic phase of PD in the absence of other complicating factors like a fracture. A para-vertebral soft tissue mass should always be regarded as possible neoplastic transformation associated with vertebral Paget’s disease, but more frequently is due to benign causes (Table 3). On MR axial and sagittal images the sarcomatous degeneration appears as a mass with irregular edges showing low signal on T1- and inhomogeneous high signal on T2-weighted images [8]. The preservation of the medullary fat signal on T1-weighted images excludes malignant degeneration and can be an extremely useful sign [54]. It usually shows inhomogeneous contrast enhancement after gadolinium administration because of its solid structure with internal vascularity. The other differential diagnosis to be considered is extra-medullary haematopoiesis. Less common is the “pseudo-sarcoma” appearance characterised by extra-cortical periosteal bone expansion or by a bulky juxta-cortical soft tissue mass similar in its characteristics on imaging to sarcomatous degeneration (Fig. 19) [11, 76, 77]. Malignant transformation can contribute to spinal stenosis [5]. For this reason the radiological assessment is not only focussed on the presence of a malignant mass, but also on the presence of cord compression, well documented on sagittal T1- and T2-weighted MR images. The spine is a common site for metastatic disease and the secondary deposits may also involve bones previously involved in Paget’s disease (Fig. 20). A biopsy may be necessary to differentiate among these various entities. Table 3Causes of paravertebral swelling in Paget’s diseaseExtra-osseous extensionFracture haematomaExtra-medullary haematopoiesisPaget’s sarcoma/other tumoursPaget’s pseudosarcomaFig. 20Vertebral metastasis from colon carcinoma. Sagittal a T1- and b T2-weighted MR images demonstrate metastasis in L2 and L4 vertebral bodies seen as discrete lesions (white arrows) with low signal on T1- and high signal on T2-weighted images. A further epidural lesion (black arrow) is seen in the spinal canal posteriorly at L3. Note the pagetic changes with expansion of L2 and L3 vertebral bodies Conclusion Recognition of the imaging manifestations of spinal PD and the potential clinical complications enables accurate assessment of patients prior to appropriate management. This knowledge should allow subtle PD to be identified on imaging when this is not suspected. This is especially relevant to MRI, as it has become the imaging modality of choice for investigating the spine. Patients presenting with back pain and spinal stenosis have to be assessed for pagetic complications before attributing the symptomatology to the disease itself.

Anal_Bioanal_Chem-3-1-1820759

Certification of butyltins and phenyltins in marine sediment certified reference material by species-specific isotope-dilution mass spectrometric analysis using synthesized 118Sn-enriched organotin compounds

A new marine sediment certified reference material, NMIJ CRM 7306-a, for butyltin and phenyltin analysis has been prepared and certified by the National Metrological Institute of Japan at the National Institute of Advanced Industrial Science and Technology (NMIJ/AIST). Candidate sediment material was collected at a bay near industrial activity in Japan. After air-drying, sieving, and mixing the material was sterilized with γ-ray irradiation. The material was re-mixed and packaged into 250 glass bottles (15 g each) and these were stored in a freezer at −30 °C. Certification was performed by use of three different types of species-specific isotope-dilution mass spectrometry (SSID–MS)—SSID–GC–ICP–MS, SSID–GC–MS, and SSID–LC–ICP–MS, with 118Sn-enriched organotin compounds synthesized from 118Sn-enriched metal used as a spike. The 118Sn-enriched mono-butyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) were synthesized as a mixture whereas the 118Sn-enriched di-phenyltin (DPhT) and triphenyltin (TPhT) were synthesized individually. Four different extraction methods, mechanical shaking, ultrasonic, microwave-assisted, and pressurized liquid extraction, were adopted to avoid possible analytical bias caused by non-quantitative extraction and degradation or inter-conversion of analytes in sample preparations. Tropolone was used as chelating agent in all the extraction methods. Certified values are given for TBT 44±3 μg kg−1 as Sn, DBT 51 ± 2 μg kg−1 as Sn, MBT 67 ± 3 μg kg−1 as Sn, TPhT 6.9 ± 1.2 μg kg−1 as Sn, and DPhT 3.4 ± 1.2 μg kg−1 as Sn. These levels are lower than in other sediment CRMs currently available for analysis of organotin compounds. Introduction Organotin compounds have wide ranging chemical and toxicological properties. They are widely applied as stabilizers of plastics, as fungicides and pesticides, and as marine antifoulants [1]. Use of tributyltin (TBT) and triphenyltin (TPhT) as marine antifouling agents has led to their almost global dispersal. Although use of TBT and TPhT has been controlled in Japan since 1989, the compounds are still prominent in the coastal sea waters of Japan. Thus, these organotins and their degradation products, di- and mono-organotins, in sediment are frequently monitored to evaluate organotin pollution in water environment. Quantification of organotin species in environmental samples is very difficult, because of their instability and the low concentrations present. Because long-term and worldwide monitoring is necessary for these compounds, comparability of the analytical results is required [2]. The National Metrology Institute of Japan (NMIJ) has already developed, in 2001, a marine sediment certified reference material (NMIJ CRM 7301-a) for analysis of mono, di, and tributyltins (MBT, DBT, and TBT) [3]. Certification was performed using two different types of species-specific isotope-dilution mass spectrometric (SSID–MS) method—gas chromatography coupled with inductively coupled plasma mass spectrometry (SSID–GC–ICP–MS) [4] and SSID–GC–MS combined with a microwave-assisted extraction—in which a mixture of 118Sn-enriched butyltins synthesized in our laboratory was used as a spike. In 2005 we developed a new marine sediment certified reference material, NMIJ CRM 7306-a, certified for di and triphenyltins (DPhT and TPhT) and the butyltins by using three different types of SSID–MS method. In the SSID methods, 118Sn-enriched TPhT and DPhT newly synthesized from 118Sn-enriched metal, and a mixture of 118Sn-enriched butyltins previously synthesized from 118Sn-enriched metal [4] were used as spikes. The certification strategy for NMIJ CRM 7306-a was almost the same as that for NMIJ CRM7301-a [3] but with two improvements to ensure the reliability of the CRM. One was the sterilization of the material with γ-ray irradiation. NMIJ CRM7301-a was not irradiation-sterilized because of possible degradation of the organotin compounds. Yang et al. reported that significant losses of butyltins in methanol occurs during γ-ray irradiation whereas such degradation is minimal in a sediment matrix [5]. In our preliminary test, no substantial loss of organotins was observed on γ-ray irradiation, and thus irradiation-sterilization was performed for CRM7306-a to ensure long term stability. The other improvement was that a total of six combinations of four extraction methods and three SSID–MS methods were used for analysis of the organotin compounds to ensure the reliability of the certified values. The overview of the analysis for certification is shown in Fig. 1. Although SSID–MS methodology can correct most of the systematic errors that occur in the analysis, it can not compensate for non-quantitative extraction from the sample or for species re-conformation in the sample preparation process [6–11]. Indeed, quantitative extraction of MBT is not an easy task, because MBT is strongly adsorbed by sediment matrices, because of its polarity [12–14]. It has been reported that DBT is degraded during microwave-assisted extraction [6] and pressurized liquid extraction [7] when high microwave energy or high extraction temperatures are used, although species interconversion can be taken into account in SSID–MS techniques that furnish data for more than one enriched isotope, as has been described by Encinar and co-workers [6–9]. In this certification, therefore, the four different types of extraction method, mechanical shaking, ultrasonic, microwave-assisted, and pressurized-liquid extraction were adopted to avoid possible analytical biases that caused by non-quantitative extraction and degradation or interconversion of analytes. SSID–liquid chromatography (LC)–ICP–MS, was adopted as an SSID–MS method in addition to SSID–GC–ICP–MS and SSID–GC–MS, because organotin separation by LC does not need a derivatization step. Thus, use of SSID–LC–ICP–MS can avoid potential degradation or interconversion of analytes in the derivatization step. This paper mainly describes SSID–MS analysis for certification of NMIJ CRM 7306-a Marine Sediment for organotin compounds. Fig. 1Overview of analysis for certification Experimental Preparation of sediment material The starting material for the CRM was collected in a bay near industrial activity in the Kyushu area, Japan. Approximately 300 kg surface sediment was sampled and the water was removed by filtration. The sediment material was air-dried for 2 weeks at room temperature (25–28 °C). After removal of visible external materials (rock, shell, etc.) the sediment material was ground in a high-purity alumina ball-mill. Sediment powder that passed through a 104-μm sieve was homogenized using a pan-type mixer. The sediment powder was then bottled in glass bottles (60 g each) and irradiation sterilized (60Co, 20 kGy). The irradiated powder was re-mixed and homogenized by use of a rocking mixer RM-10S (Aichi Electric, Japan) as a precaution against an inhomogeneous degradation of organotin compounds during sterilization. Finally, the powder was placed in amber glass bottles (15 g each) and stored at −30 °C. Conversion to dry mass basis The concentrations of the constituents of this CRM are given on a dry-mass basis. A dry mass correction factor for sample humidity was determined by drying the sample at 110 °C. After 5 h the sediment sample reached constant weight, so it was decided the drying time would be 6 h in this experiment. The dry mass correction factor at the time of certification was 0.959±0.003 (average±standard deviation for ten different bottles). Chemicals Tributyltin (TBT) chloride, dibutyltin (DBT) chloride, and triphenyltin chloride (TPhT) were purchased from Wako Pure Chemicals (Osaka, Japan). Monobutyltin (MBT) chloride and diphenyltin chloride (DPhT) were purchased from Aldrich (Milwaukee, WI, USA). Tripropyltin (TPrT) chloride was purchased from Merck (Darmstadt, Germany). Standard solutions of each organotin compound except DPhT were separately prepared by dissolving each compound in methanol (pesticide analysis grade, Wako) to avoid any disproportionation reactions with other organotins. The standard solution of DPhT was prepared by dissolving DBTCl2 in 0.005 mol L−1 HCl in methanol, to prevent self-disproportionation. All standard solutions were stored at −20 °C and diluted working solutions were prepared daily before the analysis. Sodium tetraethylborate (NaBEt4) was purchased from Wako Pure Chemicals. A 5% m/v solution of NaBEt4 was prepared in a glove box that was purged with N2 gas. Other chemicals used were of analytical reagent grade. Pure water prepared by use of a Milli-Q water-purification system (resistivity 18 MΩ cm, Nihon Millipore Kogyo, Tokyo, Japan) was used throughout the experiments. Synthesis of the 118Sn-enriched organotin compounds The mixture of 118Sn-enriched butyltin compounds used as the spike for the ID methods were synthesized from 118Sn-enriched tin metal (92% enriched) purchased from Trace Science International (Ontario, Canada). The synthetic procedure has been described previously [4]. 118Sn-enriched DPhT and TPhT were individually synthesized from 118Sn-enriched tin metal (98% enriched) purchased from Nippon Sanso (Tokyo, Japan). The synthetic procedures were almost the same as those described elsewhere [15]. A mixture of ca. 0.5 g 118Sn metal and ca. 2 g iodine was introduced into a 50-mL round-bottomed flask containing acetic acid (10 mL) and acetic anhydride (10 mL). A small crystal of potassium iodide was added as catalyst and the mixture was gently heated to reflux. After cooling in an ice bath, orange crystals of 118SnI4 (1.6 g) were collected. 118SnI4 was placed in a 50-mLround-bottomed flask containing 30 mL diethyl ether and 1 mol L−1 phenyl magnesium bromide in THF was added dropwise. After heating to reflux for 3 h the white-pink precipitate was collected by filtration after the Grignard reagent had been hydrolyzed with water. The solid was dissolved in dichloromethane and the solution was filtered to remove insoluble impurities. The dichloromethane was removed to leave 118Sn-tetraphenyltin (TePhT) as a white solid; this was washed with ethanol. 118SnI4 (0.25 g) and 118Sn-TePhT (0.5 g) were mixed in a glass tube and heated to 200 °C. The reaction products were dissolved in ethanol and the insoluble products were removed by filtration. By the addition of 20% potassium fluoride solution, 118Sn-TPhT fluoride was obtained as an insoluble salt. The fluoride was isolated by filtration, washed with a minimum amount of ethanol, and then treated with conc. hydrochloric acid and extracted with pentane. The extracts were dried with MgSO4 and the pentane was removed to yield 118Sn-enriched TPhT chloride as a white powder. 118Sn-enriched DPhT dichloride was prepared by treating 18Sn-enriched TePhT with HCl. The product was extracted with pentane. The extracts were dried with MgSO4 and the pentane was removed to yield 118Sn-enriched DPhT dichloride. Extraction procedure Ultrasonic extraction The ultrasonic extraction procedure for GC–ICP–MS and GC–MS was as follows. The sediment sample (ca. 0.5 g) was placed in a PFA centrifuge tube and spiked with an appropriate amount of the spikes. Then 2 g NaCl, 12 mL toluene containing 0.1% tropolone, and 10 mL acetic acid–methanol (1:1) were added to the tubes. The resulting mixtures were extracted in an ultrasonic bath for 30 min at 60 °C. After addition of 10 mL water the tubes were again shaken, for good phase separation, and then centrifuged at 3000 rpm for 5 min. Finally, the upper toluene layer was collected as the extract. For LC–ICP–MS, the extraction solvent was replaced with 10 mL acetic acid–methanol (1:1) containing 0.1% tropolone, and the same extraction procedure was performed. Mechanical shaking extraction The sediment sample (ca. 0.5 g) was placed in a PFA centrifuge tube and spiked with an appropriate amount of the spikes. Then 2 g NaCl, 12 mL toluene containing 0.1% tropolone, and 10 mL 0.5 mol L−1 HCl in methanol were added to the tubes, and the resulting mixtures were mechanically shaken for 60 min. After addition of 10 mL water the tubes were again shaken, for good phase separation, and then were centrifuged at 3000 rpm for 5 min. Finally, the upper toluene layer was collected as the extract. Microwave-assisted extraction Closed-vessel microwave-assisted extraction (MAE) was performed. The microwave system used was Mars X (CEM, USA). The sediment sample (ca. 0.5 g) was placed in a PFA vessel and spiked with an appropriate amount of the spikes. Then 2 g NaCl, 12 mL toluene containing 0.1% tropolone, and 10 mL 1 mol L−1 acetic acid in methanol were added to the vessel. The microwave irradiation program was ramp to 120 °C in 10 min then hold for 10 min. The mixture was transferred to a PFA tube containing 10 mL water and the tube was shaken, for good phase separation, and then centrifuged at 3000 rpm for 5 min. Finally, the upper toluene layer was collected as the extract. Pressurized liquid extraction Pressurized liquid extraction (PLE) was performed with an ASE 200 instrument (Dionex, USA). The sediment sample (ca. 1 g) was placed in an 11-mL stainless extraction cell containing a glass filter and quartz sand and was spiked with an appropriate amount of the spikes. After filling the extraction vessel with quartz sand, the cell was placed in the PLE system. The extraction solvent used was 0.5 mol L−1 acetic acid in methanol containing 0.2% tropolone. The extraction conditions were: oven temp. 110 °C, pressure 10.3 MPa, static time and cycle was 5 min×4 times. The extract obtained was transferred to a PFA tube containing 10 mL water and 12 mL toluene, and the tube was shaken, for good phase separation, and then centrifuged at 3000 rpm for 5 min. Finally, the upper toluene layer was collected as the extract. Derivatization procedure for GC–ICP–MS and GC–MS The extracted solutions were transferred to PFA centrifuge tubes and 25 mL ammonium-acetate buffer (0.5 mol L−1, pH 5) and 0.2 mL 5% NaBEt4 solution were added. The tubes were mechanically shaken for 20 min, for ethylation and extraction, and then centrifuged to achieve phase separation. The toluene layers were also transferred to the tubes and mixed with 2 g anhydrous sodium sulfate to remove the water. Clean-up procedure Clean-up on a Presep-C Florisil cartridge (Wako, Japan) was performed after the derivatization, except for LC–ICP–MS measurement. The toluene layer was evaporated to ca. 2 mL under a stream of N2 gas and loaded on to a cartridge previously conditioned with hexane. The eluent from the cartridge was collected in a 15-mL glass centrifuge tube. Hexane (6 mL) containing 5% (v/v) diethyl ether was then also loaded on the cartidge and the eluent was collected in the glass tube. Finally, the collected eluent was evaporated to 0.2 mL under a stream of N2 gas and used as the measurement sample solution. For the LC–ICP–MS measurement, a clean-up procedure using Presep-C18 cartridge (Varian, Australia) was performed. The extract was loaded on to a cartridge previously conditioned with acetone. The eluent from the cartridge was collected in a 15-mL glass centrifuge tube, evaporated to 0.2 mL under a stream of N2 gas, and used as the measurement sample solution. Determination of organotin compounds by ID–GC–ICP–MS The GC–ICP–MS used was that the GC (Agilent 6890GC) was coupled with an ICP–MS (HP4500, Yokogawa Analytical Systems, Tokyo, Japan) by means of a manufactured transfer-line (Yokogawa Analytical Systems). The GC column was HP-1 ms (30 m×0.32 mm i.d., 0.32 μm film thickness). The measured masses were m/z 118 and 120. The operating conditions and procedures for GC–ICP–MS were similar to those described elsewhere [4]. The concentrations of organotin compounds were calculated by inserting each value into the ID equation Eq. (1), based on a double-ID method [16, 17]: where Cx is the analyte concentration in the sample (mol g−1), mx the mass of sample (g) used for the sample-spike mixture, my the mass of spike solution (g) used for the sample-spike mixture, the mass of spike solution (g) used for the standard-spike mixture, mz the mass of standard solution (g) used for standard-spike mixture; R is the 120Sn/118Sn ratio in the sample–spike mixed solution, R′ is the 120Sn/118Sn ratio in the standard–spike mixed solution, Rx is the 120Sn/118Sn ratio in the sample solution, Ry is the 120Sn/118Sn ratio from the spike solution, Rz is the 120Sn/118Sn ratio in the standard solution, w is the correction factor for dry mass; n is the number of replicate measurements, and k, Ky, and K′ are the mass discrimination correction factors for each isotope ratio, which were calculated from the area ratio of 120Sn/118Sn for TPrT in each chromatographic run [4]. P is the purity of each organotin chloride reagent used for preparing the standard solutions, D is the dilution factor for each organotin chloride in the gravimetric dilution of the standard solutions, Mw the molecular weight of each organotin chloride, B the procedure blank, and E the variation introduced by extraction to the analytical results. Determination of organotins by ID–GC–MS The GC–MS used was Agilent model 6890GC/5983MSD (Agilent Technologies, Wilmington, DE, USA) with an HP-5 ms column (30 m×0.32 mm i.d., 0.25 μm film thickness). The measured masses were m/z 231 and 233 for MBT, m/z 261 and 263 for DBT and TBT, m/z 301 and 303 for DPhT, and m/z 349 and 351 for TPhT. The other operating conditions and procedures for GC–ICP–MS analysis were similar to those described previously [3]. The concentrations of organotins were calculated by inserting each value into Eq. (2), below, in which it was assumed that the relationship between mass ratio and peak ratio was linear. where Cx is the analyte concentration in the sample (mol g−1), mx the mass of sample (g) used for the sample-spike mixture, my the mass of spike solution (g) used for the sample–spike mixture, RWL the ratio of the mass in the spike solution (g) to the mass in the standard solution (lower level), RWH the ratio of mass in the spike solution (g) to the mass in the standard solution (higher level), R the measured abundance ratio in the sample–spike mixed solution, RL the measured abundance ratio in the standard–spike mixed solution (lower level), RH the measured abundance ratio in the standard–spike mixed solution (higher level), w the correction factor for dry mass, P the purity of each organotin chloride reagent used for the preparing the standard solutions, D the dilution factor for each organotin chloride in gravimetric dilution of the standard solutions, Mw the molecular weight of each organotin chloride, B the procedure blank, and E the variation introduced by the extraction to the analytical results. The mass bias correction was not performed for GC–MS measurements. In this experiment we prepared several sample–spike mixed solutions of different mass ratios and chose the solutions having values of RL and RH slightly lower and higher than the R value for calculation. Determination of organotin compounds by ID–LC–ICP–MS The LC–ICP–MS system used consisted of a PU-712i HPLC pump (Inert model; GL Science, Tokyo, Japan), a Nanospace SI-2 auto-injection sampler (Siseido, Tokyo, Japan), and an Agilent 7500c ICP–MS (Yokogawa Analytical Systems, Japan). The LC column used was a Mitysil RP-18 GP (150 mm×2.0 mm i.d. 3 μm). All tubing used was 1/16 in (0.13 mm i.d.) PEEK. The mobile phase was acetonitrile–water–acetic acid–tetraethylammonium chloride–tropolone 65:30:5:0.1:0.075 (%, v/v). The mobile phase flow rate was 200 μL min−1 and the sample volume injected was 5 μL. The sample-introduction device for ICP–MS was modified from the default settings as follows: a PFA μflow of 50 μL min−1 was used as a nebulizer, the double-path Scott type spray chamber was cooled to −5 °C, a 1.0 mm dimmer injector torch was used, and additional O2 (0.1 L min−1) gas was mixed with the argon axial gas flow via a T-adaptor. The LC column was directly connected via a 1/16 PEEK adaptor. The concentrations of organotins were calculated by use of Eq. (1). Homogeneity study The between-bottle homogeneity of the CRM was determined by analyzing sub-samples taken from ten bottles selected from the lot of 250 bottles. All the organotin compounds were determined by ID–GC–ICP–MS after ultrasonic extraction. Analysis of variance (ANOVA) of the data was performed and mean squares within group (MSwithin) and among group (MSamong) were calculated. Then standard deviations between bottles (sbb) were calculated by use of Eq. (3): If the measurement method was insufficient repeatable, the effect of analytical variation on the standard deviation between units ubb was calculated and used to estimate the inhomogeneity [18]. The ubb was calculated by use of Eq. (4): where denotes the degrees of freedom of MSwithin. Results and discussion Homogeneity study In the homogeneity study, between-bottle inhomogeneity (sbb) was only observed for TPhT, so ubb was used as uncertainty-derived inhomogeneity for TPhT. A relatively large sbb value (7.1%) was obtained for DPhT, although this value was not very large compared with uncertainties from other sources, for example the uncertainty of analytical results and between-method variance, as is shown later. These results indicate that this material may be considered homogeneous for butyltin and phenyltin analysis. Stability of organotins in this material We have been monitoring the stability of the butyltin compounds in NMIJ CRM7301-a since 2001. All three butyltins were sufficiently stable during storage for five years at −30 °C even though CRM7301-a was not sterilized. The new CRM 7306-a contains almost the same butyltin concentrations as CRM7301-a, its sample composition was also similar, and it was sterilized with γ-ray-irradiation. Thus, the butyltins in CRM 7306-a will also be stable for at least five years. Both the phenyltin and butyltin compounds in BCR CRM646, which was pasteurized at 100 °C, have been shown to be stable when the material is stored at below −20 °C [19]. Therefore, we decided to store CRM7306-a at −30 °C and to assess the stability of the material by measuring each organotin by ID–GC–ICP–MS over a period of 1 year, and that for CRM7301-a. Characterization of the synthesized 118Sn-enriched organotins Characterization of the synthesized 118Sn-enriched organotins was performed by GC–ICP–MS after mixing of each, dilution with methanol, and ethylation with NaBEt4. Figure 2 shows the GC–ICP–MS chromatograms obtained at m/z 118 and 120 for the synthesized 118Sn-enriched organotins mixture. The 118Sn-enriched DPhT and TPhT could be synthesized individually. The molar ratio of the 118Sn-MBT, 118Sn-DBT, and 118Sn-TBT in the synthesized mixture measured by GC–ICP–MS was approximately 7:10:5. Fig. 2GC–ICP–MS chromatograms of 118Sn and 120Sn obtained for the mixture of 118Sn-enriched organotin compounds. The chromatogram of 118Sn was shifted 9 s and 7500 cps for clarity Ten replicate measurements of the ethylated 118Sn-enriched organotin mixture by GC–ICP–MS were performed to obtain the 120Sn/118Sn ratio for each compound. The mass bias was corrected with the in-run correction method with TPrT [4]. The 120Sn/118Sn isotope ratios for 118Sn-butyltins were 0.055±0.001 (mean±standard uncertainty, n=10), with no significant differences. The 120Sn/118Sn isotope ratios for 118Sn-enriched DPhT and TPhT were 0.0020±0.0002 and 0.00020±0.0003, respectively. Assay for the standard solution of the organotins To obtain a standard solution with a well-defined concentration, an assay was performed to determine the purity of the natural abundance organotin chloride reagents used to prepare the standard solutions. Details of the assay have been described elsewhere [4]. In brief, the amounts of organic and inorganic tin species impurities in the organotin chloride reagents were estimated by GC–ICP–MS. The inorganic impurities, except for Sn, were checked by ICP–MS. The non-organotin organic impurities were also checked by GC–FID. The water content of the organotin chloride reagents were evaluated with a Karl–Fisher coulometric titrator (Model CA-05; Mitsubishi, Japan). The main impurities in the organotin chloride reagents used are water and tin species. Small amounts of inorganic and organotin impurities were observed in both the ethylated MBT and ethylated DBT solutions, and significant amounts of several organotin impurities were observed in the ethylated TBT, ethylated TPhT, and ethylated DPhT solutions. Among the organotin impurities observed in each ethylated organotin solution, inorganic tin, MBT, DBT and tetrabutyltin (TeBT), MPhT, and DPhT were identified from their retention times in GC–ICP–MS measurement and isomers of TBT (iso-TBT) and dioctyldibutyltin (DOcDBT) were identified from the fragment-ion patterns obtained by GC–MS. Two organotin impurities that could not be identified from their retention time were also observed in the ethylated-MBT solution, but the amounts of those were small (<0.03%). The purities of the organotin chloride reagent obtained were 99.4±0.2% for MBT, 99.5±0.2% for DBT, 96.8±0.2% for TBT, 98.6±0.2% for DPhT, and 98.8±0.2% for TPhT. The stability of the standard solution during certification is also important. Van et al. reported that DPhT in methanol was not stable because of its disproportionation reaction, although solutions of the butyltins and TPhT were found to be stable [20]. DPhT also reacted with MBT in mixed solutions in methanol. In contrast, Arnold et al. reported that DPhT in methanol containing 0.01 mol L−1 HCl was stable for six months [21]. Hence, the stability test for DPhT in methanol with and without 0.005 mol L−1 HCl was performed by GC–ICP–MS after storage at −20 °C for one month. Redistribution of DPhT to MPhT and TPhT was observed for the methanol solution, as has been described elsewhere [20]. In contrast, no degradation or redistribution of DPhT was observed for the methanol solution containing 0.005 mol L−1 HCl. Plazzogna et al. reported that the redistribution reaction between monomethyltrialkyltin (R3SnMe) and dimethyltin dichloride (Me2SnCl2) in methanol is occurred with dissociation of Me2SnCl2 [22]. They also reported that the redistribution reaction can be prevented by addition of chloride ion, as NaCl, which inhibits the dissociation of Me2SnCl2 in methanol. For DPhT in methanol the disproportionation reaction occurred with dissociation of DPhTCl2, and was prevented by addition of HCl, which inhibited the dissociation. Consequently, the standard solution of DPhT was prepared by dissolving DBTCl2 in methanol containing 0.005 mol L−1 HCl to prevent the self-disproportionation reaction. Evaluation of the degradation of DBT and TPhT during extraction Because SSID–MS methodology cannot compensate for non-quantitative extraction from the sample, aggressive conditions are required for solid–liquid extraction of strongly adsorbed species, for example MBT. Degradation or interconversion of species can occur under the aggressive extraction conditions [6–11]. Indeed, it has been reported that degradation of DBT occurs during microwave assisted extraction [6] and pressurized liquid extraction [7] when high-energy microwaves or high extraction temperatures are used. Degradation of TPhT during sample pretreatment has also been reported [11, 12]. In this certification, therefore, degradation of DBT and TPhT during extraction was checked using an 117Sn-enriched DBT solution obtained from the LGC, UK [15] and the single solution of the synthesized 118Sn-enriched TPhT. Both 117Sn-enriched DBT and 118Sn-enriched TPhT solutions were used to spike the sediment sample and then the four extraction methods were performed. After derivatization and clean-up the extract was analyzed by GC–ICP–MS. Because the 118Sn-enriched TPhT solution does not contain any phenyltins, degradation of TPhT in each extraction was evaluated from the difference between the natural abundance isotope ratio and the measured ratios of 120Sn/118Sn for DPhT. On the other hand, the 117Sn-enriched DBT solution contains small amounts (ca. 0.7%) of 117Sn-enriched MBT as an impurity. Therefore, the degradation of DBT in each extraction was evaluated from the difference between the natural abundance isotope ratios and the ratios of 118Sn/117Sn for MBT, corrected for 117Sn-enriched MBT. In this experiment, the measured 120Sn/118Sn ratios for DPhT obtained for all four extraction methods closely matched the natural abundance, and the results suggest that no significant degradation of TPhT to DPhT occurred during extraction. The degradation of DBT to MBT was also not observed in ultrasonic extraction and mechanical shaking extraction. Slight degradation of DBT to MBT was observed in microwave-assisted extraction MAE (0.8%) and in pressurized liquid extraction PLE (0.5%); these degradation levels were lower than those reported by Encinar [6, 7] (2–3% in both extractions under their optimized conditions). These differences could be because of different extraction conditions. The sample size used in our experiments was two to four times that used by Encinar, and the concentrations of acetic acid we used were lower. In addition, tropolone was used as a chelating reagent in all the four extraction methods. Formation of the tropolone-chelating complex might prevent degradation. The slight degradation of DBT observed would not significantly affect the analytical results obtained by use of MAE and PLE because the typical uncertainty of isotope-ratio measurement by GC–ICP–MS in this study was in the range 0.5 to 1%, and the relative standard deviations of the analytical results for DBT obtained by use of MAE and PLE were approximately 3%. As is shown later, the analytical results obtained by use of the four different extraction methods were in good agreement within the range of uncertainties. These results indicate that analytical biases caused by degradation or inter-conversion of analytes in the extraction would be negligible in this certification. Analytical results obtained by each method Determination of the organotin compounds in the candidate material by the six analytical methods was performed for the certification. Figures 3, 4 to 5 show the GC–ICP–MS, GC–MS, and LC–ICP–MS chromatograms obtained for the sample extract spiked with the 118Sn-enriched organotin compounds. It is apparent from Figs. 3 and 4 that adequate sensitivity for all the organotins was obtained in GC–ICP–MS and GC–MS measurements. Adequate sensitivity for the organotin compounds, except for DPhT, was also obtained in LC–ICP–MS; the sensitivity for DPhT was insufficient to obtain a reliable isotope-ratio measurement. The relative standard deviation for DPhT in LC–ICP–MS measurement was approximately 10% in triplicate measurement, and thus variation of the analytical results obtained was quite large. Thus, the analytical results for DPhT obtained by ID–LC–ICP–MS were not adopted for the certification. The analytical results, with their uncertainties, obtained by both analytical techniques are summarized in Table 1. The values were calculated as mass fractions (based on dry mass). The combined standard uncertainties for the analytical results obtained by each method were calculated by use of Eqs. (1) or (2). The uncertainties related to the standard solutions (uncertainty of P and D in Eqs. (1) and (2)) were not combined into uc because the same reagents were used for both analyses. They were combined later when the uncertainties in the certified values were calculated. Fig. 3GC–ICP–MS chromatograms of 118Sn and 120Sn obtained for the sample extract spiked with 118Sn-enriched butyltin and phenyltin compounds. The chromatogram of 118Sn was shifted 7 s and 2000 cps for clarityFig. 4GC–MS chromatograms of each measured mass obtained for the sample extract spiked with 118Sn-enriched butyltin and phenyltin compounds. The chromatogram for 118Sn was shifted 9 s and 500 cps for clarityFig. 5LC–ICP–MS chromatograms of 118Sn and 120Sn obtained for the sample extract spiked with 118Sn-enriched butyltin and phenyltin compounds. The chromatogram of 118Sn was shifted 9 s and 2500 cps for clarityTable 1Analytical results obtained by use of six combinations of extraction and measurement methods  Result±combined standard ua (μg kg−1 as Sn)ExtractionbMeasurementTBTDBTMBTTPhTDPhTUSEGC–ICP–MS44.3±1.151.2±0.965.9±1.17.8±1.43.4±0.3USEGC–MS43.8±1.750.7±0.866.6±1.75.8±1.53.9±0.4USELC–ICP–MS44.2±1.750.7±0.865.6±1.77.4±1.9–cMAEGC–ICP–MS43.8±1.552.1±0.968.2±1.27.0±1.02.9±0.13PFEGC–ICP–MS42.9±1.250.1±1.067.1±0.86.3±0.63.7±0.2MWEGC–ICP–MS47.0±1.352.5±1.267.6±0.88.7±1.74.4±0.8aCalculated by use of Eqs. (1) or (2)bUSE: ultrasonic extraction, MSE: mechanical shaking extraction, PFE: pressurized fluid extraction, MAE: microwave assisted extractioncTechnically invalid The analytical results obtained by use of the six methods were in good agreement within the range of their uncertainties; this agreement may indicate there were no significant analytical biases between measurement and extraction techniques for all the analytes. Therefore, all the analytical results obtained were treated equally for calculation and evaluation of the certified values and their uncertainties. Establishing certified values The analytical results in Table 1 were combined to provide the certified values listed in Table 2. Certified values are the weighted means of these results from the six methods, where 1/ui (ui is the uncertainty of each result) was used as a weight. Certified values are available for the concentrations of MBT, DBT, TBT, DPhT, and TPhT, as tin. Table 2Certified values and their uncertainties for mass fractions of organotin compounds in NMIJ CRM 7306-a TBTDBTMBTTPhTDPhTCertified value (mass fraction, μg kg−1 as Sn)4451676.93.4Relative standard uncertainty (%) Calibration solution ucal0.3%0.3%0.3%0.5%1.0% Analytical results uanal1.3%0.7%0.7%7.0%3.3% Between method umethod1.6%1.0%0.2%–– In-homogeneity sbb or ubb1.9%1.9%2.0%5.8%7.1%Combined uncertainty Relative (%)2.8%2.3%2.1%9.1%7.9% Absolute (μg kg−1 as Sn)1.31.21.40.60.3Expanded uncertainty U (k=2) (mass fraction, μg kg−1as Sn)3231.30.5 Uncertainty of the certified values The uncertainties of the certified values included the combined effects of method imprecision, possible bias effects among methods, material inhomogeneity, and stability. Components of the uncertainty of each certified value are listed in Table 2. Because the same reagents for calibration solutions were used for all the measurements, uncertainty of the calibration solutions (ucal) was combined with the uncertainty of the certified values separately from the uncertainty of the analytical results, as is described above. The property value was the weighted mean of the two results, and so the combined uncertainty of each analytical result (uanal) was given by Eq. (5): where xi are the results obtained by use of the six methods and wi are their weights. The between-method variance (umethod) was calculated from ANOVA on the data from the three techniques. The uncertainties derived from material inhomogeneity (ubb) were the estimates in the homogeneity study. As is described above, the butyltin and phenyltin compounds would be stable at lest five years at our storage temperature (−30 °C); we did not include the uncertainty from stability. The expanded uncertainty in each certified value is equal to U=kuc, where uc is the combined standard uncertainty, with coverage factor k=2, corresponding to 95% confidence intervals. Comparison with other CRMs The certified values for CRM7301-a and the CRM7306-a are summarized in Table 3. In comparison with the CRM7301-a, the uncertainties of the certified values for DBT and MBT in CRM7306-a are significantly smaller, despite the similar concentrations of the compounds. The main contributors to the uncertainty in the certified values for DBT and MBT in CRM7301-a were ubb and uanal [3]. The homogeneity of CRM 7306-a was improved by remixing after X-ray sterilization, and the reliability of the analytical methods used for certification was also better than those used for CRM7301-a. Thus, these improvements may lead to the small uncertainties. Table 3Certified values for butyltin and phenyltin in MNIJ7306-a and other CRMs certified by use of SSIDMS (unit: µg kg-1 as Sn) TBTDBTMBTTPhTDPhTCRM7306-a44±351±267±36.9±1.23.4±0.5CRM7301-a44±456±656±6HIPA-178±9SOPH-1125±7174±9PACS-2890±1051047±64 Most recently, several sediment reference materials for butyltin analysis certified by use of SSID–MS have been issued by the National Research Council Canada (NRCC, Canada). The certified values of the CRMs are also summarized in Table 3. HIPA-1 and SOPH-1 were certified, and PACS-2 was recertified by NRCC. Their certified and re-certified values were based on results from the Comite Consultatif pour la Quantite de Matiere (CCQM) comparisons [23–25], in which we participated. These CRMs have certified values for DBT and TBT only, and their concentrations are higher than those in CRM7301-a and CRM7306-a. The BCR 646 freshwater sediment for butyltins and phenyltins are also available from the European Commission Joint Research Centre (IRMM, Belgium), although SSID–MS was not used for the certification. The certified concentrations for BCR CRM646 are higher than those for CRM7306-a. In Japan, typical butyltin and phenyltin concentrations observed in environmental monitoring are not as high as they used to be. Reported TBT concentrations in sediment were below 80 μg kg–1, as Sn, in environmental monitoring by the Ministry of the Environment Japan in 1998 [26]. By using these CRMs properly, a wide concentration range can be covered in routine laboratories.

Purinergic_Signal-3-1-2096771

Chemotactic activity of extracellular nucleotideson human immune cells.

Purinergic P2 receptors are a class of plasma membrane receptors that are express in many tissues and are ligated by extracellular nucleotides [such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), uridine 5–triphosphate (UTP) and uridine 5–diphosphate (UDP)], which are released as a consequence of cell damage, cell stress, bacterial infection or other noxious stimuli. According to the molecular structure, P2 receptors are divided into two subfamilies: P2X and P2Y receptors. The P2X receptors are ligand-gated channels, whereas P2Y receptors are G-protein-coupled seven-membrane-spanning receptors. Several studies indicate that nucleotides play an important role in immune response modulation through their action on multiple cell types, including monocytes, mast cells, dendritic cells, neutrophils, and eosinophils. Recent work by our group and others identified extracellular nucleotides as chemotaxins for various human immune cells, including eosinophils, neutrophils and dendritic cells. In this review, we summarise recent findings in this field and put forward a hypothesis on the role of P2 receptors in the early recruitment of human immune cells to the site of inflammation. Introduction Cell migration plays a key role in a wide variety of biological processes, such as embryogenesis, development, angiogenesis, haematopoiesis, immune response and inflammation. In inflammation and host defence, the targeted trafficking of immune cells to tissues and/or lymphoid organs is one of the essential steps. Migration of the different leukocytes is tightly controlled by chemokines. These chemotactic cytokines are secreted proteins with a molecular weight of 8–0 kDa, which direct cellular traffic along ingeniously regulated concentration gradient in the extracellular space. Based on amino acid alignments, chemokines are divided into four families. According to the position and the spacing of the first two conserved cysteines or the lack of them, these families are distinguished as either C, CC, CXC or CX3C. Until now, C and CX3C are composed of only one member each, lymphotactin and fractalkine, respectively, whereas CC and CXC each consist of numerous, well-characterised members [1, 2]. The chemotactic effects of these molecules are mediated due to their interactions with different specific serpentine receptors that span the plasma membrane seven times and belong to the G-protein-coupled receptor family. Besides chemokines, many constitutive molecules can regulate the function of leukocytes and thereby modulate immune responses, e.g. following tissue damage, intracellular localised substances can be released into the extracellular space. For this reason, an increase of the extracellular concentration of certain molecules can be a very simple sign of cell damage. However, for constitutive molecules to function as chemotaxins, it is essential that they are recognised by immune system migration cells. In the past few years, evidence has accumulated strongly suggesting that nucleotides fulfil these requirements. They are present at high concentrations (5–0 mM) in the cytoplasm of all cells, whereas in the extracellular compartment, their concentration is in the nanomolar range. Nucleotides can be released into the extracellular space via nonlytic mechanisms through regulated transport; e.g. adenosine triphosphate (ATP) has been reported to be secreted by different cell types in a broad variety of conditions, such as shear stress, endotoxin stimulation, or at sites of platelet aggregation [3, 4] Hence, in tissues, nucleotides are able to generate concentration-dependent gradients, which can serve as chemotactic signals for different immune cells, causing migration. P2 receptors P2 receptors are subdivided on the basis of pharmacological, functional and cloning data into two families: the P2YR and P2XR [5–8]. P2YR are seven-membrane-spanning, G-protein-coupled receptors, and eight different P2YR subtypes have been cloned so far (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14) [5, 9, 10]. Activation of P2YR induces phospholipase C activation, inositol triphosphate generation, Ca2+ release from intracellular stores, and/or stimulation/inhibition of adenylate cyclase. Extensive pharmacological studies performed in P2Y transfected cells revealed that P2Y1, P2Y11, P2Y12 and P2Y13 selectively interact with ATP and/or adenosine diphosphate (ADP), whereas uridine 5–triphosphate (UTP) and uridine 5–diphosphate (UDP) are inactive [11–15]. In contrast the P2Y2, P2Y4 and P2Y6 subtypes are responsive to uridine nucleotides [16–18]. Whereas ATP and UTP activate P2Y2 with similar efficiency, UTP and UDP are most the potent agonists at P2Y4 and P2Y6, respectively [16–18]. In addition, it has been shown that P2Y14 specifically responds to UDP glucose and related sugar nucleotides but not to ATP, ADP, UTP or UDP [5, 9, 19, 20]. P2YR have been shown to modulate multiple cell function of various human immune cells, including cytokine release from dendritic cells, reactive oxygen metabolite production from neutrophils and eosinophils or chemokine release from airway epithelial cells [20–23]. P2XR are multimeric ligand-gated plasma-membrane ion channels activated by extracellular ATP and selective for monovalent and divalent cations [8, 24, 25]. At this time, seven different monomers have been cloned: the P2X1–P2X7 subtypes. Activation of P2XR leads to increased plasma membrane permeability to ions (Na+, K+, and Ca2+) and induction of apoptosis in human immune cells [22, 26] . In contrast to P2YR, the only currently known physiological ligand for all P2XR subtypes is ATP. P2 receptors and migration of neutrophils The first report that extracellular nucleotides can modulate human neutrophil function was a paper by Ward et al. showing that ATP and ADP can induce superoxide anion formation [27, 28]. Furthermore, it has been shown that human neutrophils or human promyelocytic HL60 cells respond to ATP, ATPγS and UTP with an increase in intracellular Ca2+ concentration via a pertussis toxin-sensitive G-protein receptor that coupled to the inositol phospholipid signaling system, suggesting involvement of P2Y subtypes [27, 29–33]. Activation of these intracellular signal-transduction systems is of great interest in the light of neutrophil recruitment to the site of inflammation, as increase in Ca2+ concentration is an important step in human neutrophil migration [34–36]. Furthermore, it has been shown that ATP also increases membrane expression of CD11b/CD18 and adhesion to albumin-coated polystyrene latex beads [37].Up-regulation of these molecules enhances the adhesion of neutrophils to other cells, e.g. between neutrophils and pulmonary endothelial cells, and could be of relevance for neutrophil migration across the vessel wall. However, after earlier observations suggesting that nucleotides might be chemoattractant for human neutrophils, it was not until the end of the 1990s that ATP and UTP were shown, by Verghese et al. [103] to induce actin polymerisation and chemotaxis in human neutrophils via the activation of P2Y2 (formerly known as P2U) receptor. Surprisingly, the first reverse transcripaise polymerase chain reaction (RT-PCR) data revealed that human neutrophils express only P2Y4 and P2Y6 but not P2Y1 and P2Y2 receptors [38]. However, recent reports showed that human neutrophils also express messenger ribonucleic acid (mRNA) for the P2Y2 and P2Y11 receptor subtypes [39, 40]. Among the P2X receptors, so far, only the present of the P2X7 receptor has been shown by Northern blotting and immunocytochemistry [41]. Because increased extracellular nucleotide concentrations are associated with cell damage/injury and human neutrophils are the initial cell type found at tissue injury sites, the conclusion could be drawn that extracellular nucleotides among other mediators are involved in the recruitment of neutrophils to the site of inflammation. P2 receptors and recruitment of human eosinophils Besides neutrophils, eosinophils also express P2 receptors. Several studies showed that human eosinophils express mRNA for the following P2Y and P2X subtypes: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y14, P2X1, P2X4 and P2X7 [23, 42–44]. Stimulation of P2 receptors expressed by eosinophils induces multiple cell responses, including production of reactive oxygen metabolites and secretion of eosinophil cationic protein (ECP) [23, 26]. Burgers and colleagues [45] showed that ATP, secreted by thrombin-activated platelets, was able to raise eosinophil intracellular Ca2+ concentration and made the cells chemotact towards platelets. These seminal studies were later confirmed by the identification of eosinophil P2Y and P2X receptors [42, 44] and the observations that nucleotides also induce up-regulation of adhesion molecule CD11b and actin polymerisation to important features involved in blood eosinophil recruitment to tissue [23, 42, 44]. In addition to direct chemotactic influences, ATP might also have indirect effects on eosinophil recruitment. We recently showed that ATP and UDP induce secretion of interleukin (IL)-8 by eosinophils [26]. This chemokine is a potent attractor for eosinophils themselves (and neutrophils), i.e. they are able to recruit more cells to inflammation sties. Increased secretion of IL-8 has been described in eosinophils from patients with bronchial asthma or atopic dermatitis [46]. Moreover, IL-8 concentration in bronchoalveolar fluids from asthmatic patients is increased significantly in comparison with that of healthy subjects [46]; therefore, one can suggest the involvement of different nucleotides in the direct or IL-8-mediated recruitment of eosinophils and thus in the development and maintenance of allergic diseases. P2 receptors and mast cells Mast cells are situated around blood vessels and nerves, especially at interfaces with the external environment, emphasising their role in immunity. They express several mRNAs that encode P2XR and P2YR subtypes [47, 48]. Human mast cells express P2X1 and P2X4, whereas the P2X7 receptor subtype is only expressed by human-cord-blood-derived mast cells when activated with anti-immunoglobulin (Ig)E [47]. Among the P2Y receptors, the presence of P2Y1, P2Y2, P2Y11, P2Y12 and P2Y13 subtypes were shown by RT-PCR [48]. Moreover, ATP and UTP enhance histamine release by human lung mast cells stimulated by cross-linkage of the fragment crystallisable (Fc)εRI [49]. This effect is attributed to the P2Y2 receptor. Data on chemotactic effects of nucleotides on mast cells are rare, but there is evidence that they might effect the migration of these cells. For example, McCloskey and colleagues showed that the nucleotides ADP, ATP and UTP are effective chemoattractants for rat-bone-marrow-cultured mast cells [50]. However, whether nucleotides can also induce migration of human mast cells remains to be elucidated P2 receptors and lymphocytes The first evidence for a role of extracellular nucleotides in human lymphocyte responses has been present for some time [51–53], but a systemic analysis of the expression and function of P2 receptors in human lymphocytes was only started at the end of the 1980s [30, 54–57]. Human B lymphocytes express both P2X and P2Y receptors [58, 59]. The presence of different P2Y receptors is indicated by the ability of ATP and many other nucleotides to trigger Ca2+ release from intracellular stores [60, 61] and the finding of P2Y1, P2Y2, P2Y4 and P2Y6 receptor-specific mRNA in lymphocytes [38]. Different studies suggest that human B cells express at least P2X7 receptor [22], but the identification of other P2X receptor subtypes is limited by the absence of specific antibodies [62, 63]. Nevertheless, confocal microscopy studies using anti-P2X polyclonal antibodies suggest the presence of P2X1, P2X2, P2X4 and P2X7 subtypes on human B lymphocytes [59]. However, despite the presence of functional P2 receptors, data about chemotactic effects on B lymphocytes induced by nucleotides are still missing. Functional and pharmacologic studies revealed that human peripheral T lymphocytes express P2X-like ATP-activated channels, most likely P2X1, P2X4 and P2X7, [22, 64, 65]. Functional activity of the P2X receptors on T cells has been shown by a large influx of Na+ and Ca++ from the extracellular medium caused by ATP and 3–O-(4-benzoyl)benzoyl-ATP (Bz-ATP) [65]. The expression of P2Y receptors subtypes is still unclear, and whereas Baricordi and coworkers described a lack of functional P2Y receptors expression [65], recent studies using complete lymphocyte populations (T and B cells) could detected all the target genes for P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12 and P2Y13 [66]. However, a functional expression of different P2Y receptors on human T lymphocytes has not been proven. Studies showing that activation of the P2X7 receptor by ATP and Bz-ATP induced shedding of CD23 and L-selectin from B and T lymphocytes of a B-chronic lymphocytic leukaemia (B-CLL) patient and from normal subjects, a classic effect of chemoattractants [67–70], leads to the assumption that the P2X7 receptor might be involved in the transendothelial migration of lymphocytes [71, 72]. But elegant experiments from Chen and coworkers using P2X7 antagonist in in vitro migration assays indicated that ATP is neither a chemoattractant that stimulates transmigration of lymphocytes nor an agonist that mediates the global L-selectin loss during transendothelial migration [73]. In any case a direct effect of extracellular nucleotides on T and B lymphocyte migration has not yet been investigated and, therefore, the role of nucleotides in the recruitment of lymphocytes remains unclear. P2 receptor in monocyte/macrophages Although the first report on a potential role of exogenous nucleotides on mouse macrophage function was a paper by Cohn and Parks from 1967 [74], a systemic investigation on human monocytes/macrophages was only started in the 1990s [30, 38, 75–78]. Freshly isolated blood monocytes have been shown to express mRNA for the following P2Y and P2X subtypes: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 P2X1, P2X4 and P2X7 [38, 66, 79]. However, other investigations found a lack of functional P2X7 in these cells, whereas the receptor appears during maturation of monocytes to macrophages [22]. Besides P2X7 receptors, human macrophages or macrophage cell lines have been described to express P2Y2, P2Y4 and P2Y6 receptors [30, 80, 81]. Activation of P2 receptors expressed by human monocytes/macrophages induces multiple cell responses, including increase of intracellular calcium concentration, induction of apoptosis, generation of reactive oxygen intermediates, NO generation and secretion of IL-1β, tumour necrosis factor (TNF)-α or IL-18 [22, 30, 77, 79, 82, 83]. In the light of migration, it is of great interest that extracellular ADP causes increased surface expression of MAC-1 (alpha M beta 2 integrin, CD11b/CD18) on monocytes [84] and that nucleotides can induce the adherence of monocytes to surfaces [38], which could be of relevance for monocyte migration across the vessel wall. Accordingly, migration of the mouse macrophage line J774 towards ADP has been demonstrated [50]. Interestingly, Warny et al. demonstrated that UDP activates IL-8 gene expression and IL-8 release in human monocytic cells [85]. Because IL-8 is a central mediator in inflammation and an important chemotactic factor for various cells, including neutrophils, eosinophils and CD16+ natural killer (NK) cells [46, 86–89], UDP [through its action on dendritic cells (DC)] might be indirectly involved in the recruitment of these cells to the side of inflammation. However, at this time, no information on the chemotactic activity of nucleotides on human monocytes/macrophages is available. P2 receptors in dendritic cells DCs are powerful antigen-presenting cells that circulate in the bloodstream or reside in peripheral tissues. They are characterised by a high antigen uptake capacity, recognition of constitutive or inducible endogenous ‘danger signals–provided by surrounding cells and a high responsiveness to chemotactic signals. The migratory ability of DCs is one of the main features in the initiation of immune responses. After acquiring antigens in the peripheral tissue, DCs migrate to the draining mediastinal lymph nodes to activate naive T cells [90–92]. Besides this ‘classic–action, some evidence also suggests that tissue-resident DCs are able to uptake tissue antigens and to migrate to the afferent lymph nodes, even in the absence of inflammatory conditions, thus contributing to tolerance maintenance [93, 94]. In the last few years, DCs came into the focus of researchers of the purinergic field, and the role of P2 receptors in DC migration came to the fore. RT-PCR analysis revealed that human DCs express a broad variety mRNAs for at least four subtypes of the P2X receptor family (P2X1, P2X4, P2X5, P2X7) and eight subtypes of the P2Y receptor family (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 P2Y12 P2Y13, P2Y14) [95–99]. First, studies by Liu et al. revealed that at least activation of the P2Y1, P2Y2 and P2Y4 mediates calcium release from intracellular storage. Furthermore, the observation that DCs redirect their dendrites towards a nearby patch pipette leaking ATP suggested that P2YR might mediate DC chemotactic response [96]. Indeed, ATP and UTP, probably via activation of the P2Y2, as well as ADP (via P2Y1?) turned out to be potent chemotactic stimuli for immature but not for mature DCs [100] In contrast, P2X receptor activation had only marginal chemotactic activity in both immature and mature DCs. Chemotaxis was paralleled by other intracellular signalling events, such as actin polymerisation and intracellular Ca2+ mobilisation. Recently, UDP could be added to the list of chemoattractant nucleotides, as it has been shown that UDP via binding to the P2Y6 receptor increased intracellular calcium, induced actin polymerisation and migration of immature, but again not mature, DCs [101]. The discrepancy between the responsiveness to extracellular nucleotides of immature and mature DCs could be explained by functional studies. They revealed a selective down-regulation of the Gi/o protein-coupled chemotactic P2Y receptor responsiveness during maturation. Surprisingly, immature and mature DCs expressed similar amounts of mRNA for the purinergic receptor subtypes P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2X1, P2X4 and P2X7 [100]. DC maturation encompasses a coordinated down-regulation of inflammatory chemokine receptors (CCR1, CCR2, CCR5 and CXCR1) and induction of CCR7 and CXCR4 [2]. In addition, during maturation, functional down-regulation of chemotaxis-regulating P2Y receptors are uncoupled to chemotaxis-associated signal transduction pathways. As a result, DCs lose sensitivity to inflammatory chemokines as well as to the nucleotides ATP, ADP, UTP and UDP. In addition, nucleotides can modulate the migration of DCs to chemokines, as immature and matured DCs stimulated with ATP gain the ability to migrate in response to CXC ligand (L)12 and CCL12 [102]. However, in contrast, Schnurr et al. reported that ATP through P2Y11 signalling could inhibit CCL21, induce migration of immature and mature monocyte-derived DCs and CD1a+ dermal DCs but not of CD1c+ peripheral blood DCs or IL-3R+ plasmacytoid DCs [98]. This controversy could be due to differences in the blood donors or preparation of monocyte-derived DCs in the different laboratories. Besides direct chemotactic effects on DCs, extracellular nucleotides are also involved indirectly via their action on DCs in the trafficking of other leukocytes through the release of chemokines. For example, ATP up-regulates the constitutive production of CCL22 [macrophage-derived chemokine (MDC)] and inhibits the lipopolysaccharide (LPS)-induced secretion of CXCL10 (IP-10) and CCL5 (RANTES), resulting in selectively impaired recruitment of type 1 but not type 2 T cells, suggesting a nucleotide-mediated communication between DCs and T cells an important event during antigen presentation in vivo [102]. In accordance, UDP can enhance the LPS-mediated release of chemotactic factor IL-8 via P2Y6 from mature DCs [101], so again, UDP might be indirectly involved in the recruitment of neutrophils, eosinophils and CD16+ NK cells to the side of inflammation. In sum, activation of DCs by extracellular nucleotides leads to multiple cell responses, which results in a direct migration of DCs and also maybe indirect (DC-mediated) recruitment of other immune cells to the site of inflammation. Conclusion Over the last few years, several studies have implied that extracellular nucleotides which are actively released or diffuse out of mechanically stressed, infected or injured cells might be involved in the early recruitment of immune cells to the site of inflammation/cell damage. In these in vitro experiments, it has been shown that extracellular nucleotides (mainly by activating P2YR subtypes) are direct chemoattractants for human neutrophils, eosinophils and DCs and/or they can modulate the chemokine production of eosinophils, monocytes and DCs, which might then influence the migration capacity of other immune cells. However, whether nucleotides play a role in the migration of immune cells in vivo still remains to be elucidated.

Histochem_Cell_Biol-4-1-2413111

Tight junctions and the modulation of barrier function in disease

Tight junctions create a paracellular barrier in epithelial and endothelial cells protecting them from the external environment. Two different classes of integral membrane proteins constitute the tight junction strands in epithelial cells and endothelial cells, occludin and members of the claudin protein family. In addition, cytoplasmic scaffolding molecules associated with these junctions regulate diverse physiological processes like proliferation, cell polarity and regulated diffusion. In many diseases, disruption of this regulated barrier occurs. This review will briefly describe the molecular composition of the tight junctions and then present evidence of the link between tight junction dysfunction and disease. Introduction Tight junctions (TJ) (zonulae occludentes) form a continuous, circumferential, beltlike structure at the boundary between the apical and the basolateral membrane domains in epithelial and endothelial cells. By constituting a regulated diffusion barrier for the paracellular pathway, TJs establish separate compartments in multicellular organisms and are also crucial for the exchange of substances between the internal and external cellular environment by the expression of tissue-specific transport proteins and channels. The transmembrane proteins constituting the TJs are attached to the cytoskeleton, thereby linking cell–cell and cell–substratum adhesion sites. In addition, cytoplasmic plaque proteins constitute scaffolds for TJ assembly or are involved in the regulation of processes like transcription, proliferation and differentiation into a tissue-specific regulated diffusion barrier in physiology and development. Many excellent reviews have summarised data regarding the molecular composition and function of TJs (Ebnet 2008; Gonzalez-Mariscal et al. 2003; Matter and Balda 2003a; Mitic and Anderson 1998; Schneeberger and Lynch 2004; Tsukita and Furuse 1999; Zahraoui et al. 2000), so that the scope of the current review is focused on disturbances of TJ function in human diseases. Structure of the tight junction As the apicalmost part of the junctional complex (Farquhar and Palade 1963), the TJ forms a continuous, circumferential belt separating apical and basolateral plasma membrane domains, working as a barrier within the intercellular space and as a fence within the plasma membrane. In recent years, information on the molecular composition of TJs, in particular their transmembrane molecules, has accumulated, forming the basis of our current understanding of the structure and function of TJs in molecular terms (Fig. 1). The morphology of TJs has been intensively analysed by freeze–fracture electron microscopy (Staehelin 1973; Wolburg et al. 2003), where they appear as a set of continuous, anastomosing transmembraneous particle strands on the inner leaflet of the plasma membrane (P-face) with complementary vacant grooves on the outer leaflet (E-face) (Fig. 2). The number and complexity of ramification of the network of TJ strands depends on the cell type, in sum yielding differences in permeability barrier function between different tissues (Staehelin 1973). However, since a direct linear relationship between the complexity of the TJ strand network and the measured transepithelial electrical resistance (TER) could not unambiguousy be established for all cell types analysed, it was predicted that the strands might contain pores that fluctuate between open and closed conformations, suggesting that the TJ strands appear to be remarkably dynamic (Claude 1978). Fig. 1Molecular composition of tight junctions. The transmembrane proteins occludin, the claudin(s) and junctional adhesion molecule-1 (JAM-1) constitute the barrier formed by TJs sealing the paracellular space. They appear to be interacting in a homophilic manner, and occludin seems to co-polymerase into claudin-based TJ strands. Claudins adhere with each other in a homotypic as well as a heterotypic manner. ZO-1, -2, and -3 bind the cytoplasmic tail of occludin and link the TJ to the actin cytoskeleton. Proteins of the ZO family can shuttle to the nucleus to influence transcriptional processes in cellular proliferation and differentiation. The ZO-proteins have also been shown to interact with claudins and provide molecular scaffolds for TJ assembly. Cingulin is a 140 kDa TJ plaque protein which assoicates with the actomyosin cytoskeleton. Its putative function is transduction of the mechanical force generated by the actomyosin cytoskeleton important for cellular differentiation. The Ras target AF-6 interacts with ZO-1 and serves as a. peripheral component of tight junctions in epithelial cells. Symplekin is a 126 kDa protein that occurs and probably functions in the nucleus as well as in the TJ plaques. Tyrosine phosphorylated Par3 regulates tight junction assembly and promotes cellular polarity by intracellular signalling. Localization of 7H6 TJ-associated antigen along the cell border of vascular endothelial cells has been shown to correlate with paracellular barrier function. Additional proteins have been localised to the TJs but a function has presently not been assigned. Moreover diverse signaling proteins are detected at the apical junctional complex but they are not uniquely confined to the TJFig. 2Freeze–fracture image of the rat intestinal epithelium. The freeze fracture electron micrograph showns the apical brush border, the intramembraneous particle strands of the TJs and the lateral cell surface. The replica shows a continous network of TJ strands. Adapted from (Achler et al. 1989) with permission from D. Drenckhahn, Universität Würzburg. Bar 0.5 μm Occludin Recent identification of the TJ-specific integral membrane proteins forwarded our understanding of TJ molecular composition in mammals: occludin (ca. 60 kDa) was identified as the first integral membrane protein localised at TJs in chicken (Furuse et al. 1993) and then also in mammals (Ando-Akatsuka et al. 1996). The occludin transmembrane domain spans the membrane four times with a short cytoplasmic N-terminus and an especially long corboxy-terminal cytoplasmic domain (Fig. 3a). Occludin localizes to the TJs, and its overexpression increases TER in mammalian epithelial cells (Balda et al. 1996). However, transfection of insect cells devoid of endogenous TJs with occludin cDNA surprisingly demonstrated that occludin is not by itself sufficient to form TJ strands but its expression merely produced focal homophilic adhesion sites (Furuse et al. 1996). Moreover, disruption of both occludin alleles in embryonic stem cells did not prevent the formation of an effective diffusion barrier and the polarisation of epithelial cells, as shown by freeze–fracture analysis and TER assessment (Saitou et al. 1998). It was thus concluded that occludin is not required for the formation of TJ strands. However, occludin appears to interact, directly or indirectly, with claudins and is recruited into the long strands formed by coexpression of claudin-1 and claudin-2 (Furuse et al. 1998b, 1999). Balda et al. (1996) however reported that overexpression of both, wild type and COOH-terminally truncated occludin overexpression in cultured MDCK epithelial cells induces not only a modest increase in TER but also significant increase in the flux of the non-charged compound FITC-dextran 4 kDa. Using freeze–fracture electron microscopy and immunocytochemistry, they were able to attribute this increase in paracellular permeability to an altered distribution and membrane topology between neighbouring cells. Fig. 3Transmembrane proteins of the tight junction. a Murine occludin is a 521 amino acid protein. The first extracellular domain may be involved in cell–cell interaction containing a high tyrosine and glyine domain (triangles), acidic amino acids (orange-red), basic amino acids (green), neutral amino acids (cream). A second functional domain was assigned to the carboxy terminal 150 amino acids which appears to be responsible for the association of occludin with ZO-1. The occludin amino acid sequence is highly conserved between species (Ando-Akatsuka et al. 1996). b Claudins are four transmembrane domain proteins, containing two extracellular and one intracellular loop and a N- and C-terminal cytoplasmic domain. Claudins are a multigene family with so far 24 members identified in diverse species. In the first extracellular loop, claudins have a conserved common motif, GLWxxC(8–10aa)C (blue). Claudins further contain a PDZ-binding motif at the C terminus (orange) capable of bining to TJ plaque proteins like ZO-1. The claudins show a isotype-specific tissue expression pattern. c JAM-1 is an integral membrane protein expressed in endothelial and epithelial cells. Its extracellular domain can dimerize and bind homophilically. The intracellular domain (and in particular a PDZ-binding motif) enable JAM-1 to interact with structural and signaling proteins. JAM-1 is localized at the tight junctions of epithelial and endothelial cells and is involved in the regulation of junctional integrity and permeability. The polypeptide sequence of 299 amino acids has the typical feature observed in a type I integral membrane protein. A putative signal peptide may be cleaved between Leu23 and Val24 (pink), leaving 215 residues in the extracellular domain of the mature protein. A stretch of 17 hydrophobic residues (Ile 239 → Phe 255) presents a potential transmembrane region, and there are 45 residues in the cytoplasmic domain. The extracellular portion contains two domains with intrachain disulfide bonds typical of immunoglobulin-like loops of the V-type. Numerous PKC (blue) and Casein kinase II (yellow) phophorylation sites have been detected involved in platelet activation processes (Sobocka et al. 2000). Cys-43, -111, 146, -214 form dimer containing disulfide bridges and are labeled in black The claudins Searching for the proteins forming the structural backbone of the TJ strands, the Tsukita group re-examined the junctional fraction prepared from chicken liver and discovered the first members of the claudin family: two 22-kDa proteins, claudin-1 and claudin-2 (Furuse et al. 1998a). The claudins also have four transmembrane domains, but do not show any sequence similarity to occludin (Fig. 3b). So far, 24 members of the claudin family have been identified in different species. Claudins appear to be expressed in a tissue-specific manner; some claudins are expressed only in specific cell types, e.g. claudin-5 is expressed primarily by vascular endothelial cells or claudin-11 appears to be expressed solely in oligodendrocytes and Sertoli cells (Morita et al. 1999). Moreover, most cell types express more than two claudin isoforms conferring different size and charge selectivity qualities by the amino acid sequence of their extracellular loops, and the combination of those isoforms results in cell- or tissue-type specific barrier function by “heteropolymerisation” into the resulting TJ strands (see below). As demonstrated by immunogold electron microscopy, there is accumulating evidence now that the claudins do constitute the TJ strands as observed by freeze–fracture electron microscopy (Tsukita et al. 2001). Exogenous expression of claudins in fibroblasts devoid of endogenous TJs resulted in the formation of TJ strands. Summarizing existing data, there is clear evidence that the claudins constitute the backbone of TJ strands, while occludin, which itself cannot reconstitute such TJ strands, seems to play a permeability regulating role by incorporating itself into the claudin-based strands, a process, the mechanism of which however is not yet understood. The function(s) of occludin thus still remain to be elucidated. Junction-associated adhesion molecules (JAMs) A third class of integral membrane proteins comprises members of the immunoglobulin superfamily (Fig. 3c), and can be subdivided into a group consisting of JAM proteins and a group consisting of CAR, CLMP, ESAM and JAM-4 (Ebnet 2008; Ebnet et al. 2004). To date, at least three JAM isoforms have been described, which are expressed differentially in epithelial and endothelial cells, but also in cells devoid of TJ strands like leukocytes (D’Atri and Citi 2002). Except for JAM-1, little is known about the role of these proteins in TJ assembly and function. JAM-1, formerly F11 receptor (Sobocka et al. 2000) (Fig. 3c), may be involved in immune cell transmigration or cell adhesion (Bazzoni et al. 2000) but has not been localized to the junction strands. A putative PDZ binding motif in the cytoplasmic domain of JAM-1 has been identified, which has been able to interact with PDZ domains of ZO-1, AF-6, PAR-3 and MUPP1 (D’Atri and Citi 2002; Itoh et al. 2001). Moreover, JAM-1 has been shown to interact with cingulin. Thus, even though JAM-1 is not part of the TJ strands, it may well be involved in the propagation of signal cascades resulting from homophilic and heterophilic adhesion. A role for JAM-1 in the complex process of adhesion and transmigration of monocytes through endothelial cells has been demonstrated (Martin-Padura et al. 1998). As another function for the JAM proteins, a role in the assembly of TJs and regulation of paracellular permeability could be deciphered by ectopic transfection experiments (Cohen et al. 2001). Interaction of JAM-1 with occludin, so far not determined whether direct or indirect in nature, has been shown (Liu et al. 2000). TJ plaque proteins and the coordination of signaling at the TJ In addition to their prime function as a regulated permeability barrier in the paracellular pathway and as fence in the plane of the membrane, TJs play a pivotal role in organizing such diverse processes as morphogenesis, cell polarity, cell proliferation, and differentiation, which require the coordination of signals impinging on and emanating from the plasma membrane. Mounting evidence suggests that the cytosolic TJ plaque is one of the sites in which such signaling is coordinated (Ebnet 2008; Gonzalez-Mariscal et al. 2003; Schneeberger and Lynch 2004). Since identification in 1986 of zonula occludens (ZO)-1 as the first TJ-associated protein (Stevenson et al. 1986), almost 30 additional proteins have been described associated with the cytoplasmic aspect of TJs (Schneeberger and Lynch 2004). They can be grouped into two major categories: The first are the peripherally associated scaffolding proteins like ZO-1 (ZO-2, ZO-3, AF6, and cingulin) that appear to organize the transmembrane proteins and couple them to other cytoplasmic proteins and to actin microfilaments. The second are numerous “signaling” proteins (ZONAB, RhoA, RalA, and Raf-1) proposed to be involved in junction assembly, barrier regulation, gene transcription, and perhaps other, presently undefined pathways. TJs as permeability barrier One of the key functions of tight epithelia is the creation of a diffusion barrier by tight control of the transcellular and paracellular pathways. This is acomplished by asymmetrical distribution of transporters and channels for the transcellular route, and on the other side by regulation of diffusion through the paracellular space via integral TJ proteins (Turner 2006). Hereby, certain claudins participate in the formation of ion-selective pores: available data suggest that TJs on the one hand are permeable to small noncharged solutes up to a size of 0.43–0.45 nm in radius as shown by the use of the membrane-impermeant polyethylene glycol as a tracer, but investigations also showed that pores within the TJs are capable of discriminating between ion charge and size, which both are additionally dependent on concentration and pH (Schneeberger and Lynch 2004; van Bree et al. 1988). The two extracellular loops of occludin consist solely of uncharched amino acid side chains (Fig. 3a) represented mainly by an unusually high content of tyrosine and glycine (Furuse et al. 1993) so that a direct role for occludin in conferring charge selectivity is doubtful. However, occludin could increase electrical resistance by interaction with the extracellular domains of the differently charged residues of the different claudins (see below), since ablation of occludin is not without consequence despite the fact that the protein does not appear to be required for TJ strand formation. Occludin-null mice express a complex phenotype, which is, however, not manifested by structural or functional TJ abnormalities: in occludin −/− mice, TJs were not affected morphologically, and electrophysiological barrier function of intestinal epithelium was normal. However, histopathological abnormalities were detected in several tissues, i.e., chronic inflammation and hyperplasia of the gastric epithelium, calcification in the brain, testicular atrophy, the loss of cytoplasmic granules in striated duct cells of the salivary gland as well as the thinning of the compact bone, pointing to complex functions of the occludin protein (Saitou et al. 2000). Role of claudin proteins in formation of a TJ permeability barrier The lack of charged amino acid residues within the extracellular loops of occludin segregates occludin from the candidates contributing directly to the formation of ion-selective pores. The different claudins whose extracellular loops span a wide range of pKion the contrary appear to be excellent candidates to mediate ion selectivity in TJ permeability (Fig. 3b). Major insight into the distinct charge selectivity of the claudins came from the work of Furuse et al. (2001), who could relate the different levels of resistance between MDCK I cells (TER > 10,000 Ωcm2) and MDCK II cells (TER <200 Ωcm2) to a differential claudin isoform expression, while MDCK I cells express claudin-1 and -4 but not claudin-2, MDCK II cells additionally express claudin-2, which seems to mediate higher ion permeability by pore formation/inducing strand breaks. Notably, transfection of the tight MDCK I with claudin-2 cDNA resulted in 20-fold decline in TER but not in the number of TJ strands. A wide range of further transfection and mutagenesis experiments introducing or ablating different claudins has been performed to date, and been partially helpful to elucidate the conductance conferring properties of the resulting pores (Matter and Balda 2003b). These informative observations must however be viewed with some caution because transfection and site-directed mutagenesis may lead to more than a change in net charge as for example an altered molecular conformation, or altogether the introduction of an unusual claudin protein could lead to disturbance of strand organisation within native strands. By the construction of chimera, Colegio et al. (2003) were moreover able to attribute ion selectivity of the claudins to distinct stretches within the primary amino acid sequence of the extracellular loops leading to alterations in TER and charge selectivity in transfection experiments. Taken together, available data strongly suggest a key role for the extracellular loops of the diverse claudins in the formation of ion selective pores within the TJ strands. Role of occludin in formation of a TJ permeability barrier The role of occludin in TJs and a putative contribution to building up the TJ’s ion selectivity (the ion pores) is unclear; it has been demonstrated that overexpression of occludin in MDCK cells reduces ion permeability but in the same time was shown to increase the transepithelial flux of the uncharged compound mannitol (Balda et al. 1996). Further observations then implicated that occludin may be a target for receptor-initiated signalling involving the Rho family of GTPases, and that by an additional not-yet-understood mechanism, occludin phosphorylation may regulate TJ permeability independently of cytoskeletal activity (Hirase et al. 2001). Some indirect evidence for a role of occludin in strengthening barrier properties arose from heterologous expression studies: when expressed in L-fibroblasts that lack endogenous cadherin-based adherens junctions and ZO-1, little adhesiveness was measured (Kubota et al. 1999). However, when overexpressed in fibroblasts, which do have well developed ZO-1-containing adherens-like junctions (NRK-fibroblasts), increased Ca2+-independent adhesion was observed, indicating that when interacting with ZO-1, occludin mediates adhesive functions (Van Itallie and Anderson 1997). Addition of neutralizing peptides of the peptide sequence of the second extracellular loop elicited competitive interference with cell–cell-adhesion in a dose dependent manner, implicating that this loop directly participates in adhesive interaction (Wong and Gumbiner 1997). Relating epithelial malignancy to the loss of cell–cell-adhesion, it was demonstrated that overexpression of oncogenic Raf 1 in rat salivary gland epithelial cells (Pa4–Raf1) disrupts TJs and induces an oncogenic phenotype by downregulating the expression of occludin (Wang et al. 2005). Raf1-mediated transformation of Pa4–Raf1 cells was subsequently shown to induce transcriptional downregulation of occludin and forced re-expression of occludin rescued the polarized phenotype of epithelial cells (Wang et al. 2007). Interestingly, forced expression of an occludin mutant lacking the second extracellular loop did not rescue the epithelial phenotype in vitro nor did it prevent tumor growth in vivo. These results demonstrate that the TJ protein occludin has a potent inhibitory effect on the Raf1-mediated tumorigenesis, and the second loop of occludin appears to be required for suppression of Raf1-induced tumor growth, potentially by mediating cell–cell-adhesion as presented above contributing to contact inhibition of cell division. TJs and human disease Modulation of barrier function by cytokines Many cytokines have been shown to influence epithelial and endothelial TJ function and the actin cytoskeleton both in vivo and in vitro. To name the most prominent, the interleukins IL-0, IL-1, IL-3, IL-4, as well as tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) could be demonstrated to influence TJ barrier function in epithelia and endothelia (Ahdieh et al. 2001; Oshima et al. 2001; Youakim and Ahdieh 1999). This review will emphasise the influence of a few selected cytokines on TJ structure/function and its adjoining actin cytoskeleton in diseases like inflammatory bowel disease and inflammatory disease of the CNS (Table 1). Table 1Modulation of TJ barrier function in human diseaseClassificationTight junction protein affectedReferenceCancer Breast cancer: invasive ductal cancerClaudin-1, ZO-1Itoh and Bissell (2003), detailed here Prostate cancer: prostatic adenocarcinomasClaudins-1, -3, -4 and claudin-7Sheehan et al. (2007) Thyroid neoplasma, follicular adenomaOccludin and claudin-1, -4 and -7Tzelepi et al. (2007) Gastroesophageal reflux disease: Barrett’s  esophagus (dysplasia)Claudin-18Jovov et al. (2007) Lung cancer: Basaloid squamous carcinomaUltrastructural junctional dedifferentiationGilcrease and Guzman-Paz (1998)Inflammation Inflammatory bowel disease: Morbus CrohnClaudins-2,-3, -5, and -8, ZO-1Zeissig et al. (2007), detailed here  Collagenous collitisOccludin, claudin-2 and -4Burgel et al. (2002) Multiple sclerosisOccludin, claudin-5Förster et al. (2007), detailed hereHereditary diseases Hereditäry deafnessClaudin-14Ben-Yosef et al. (2003), detailed here Familial hypomagnesemiaClaudin-16Simon et al. (1999), detailed here cystic fibrosisOccludin, claudin-1, -4, JAM, ZO-1Coyne et al. (2002)Vision loss Diabetic eye disease: diabetic retinopathyOccludin, ZO-1Felinski and Antonetti (2005), detailed hereViral infection Reoviral infection (hydrocephalus, encephalitis)JAM-1Forrest et al. (2003)Bacterial toxins Clostridium perfringens enterotoxinClaudin-3 and -4McClane (2001), detailed here Inflammatory bowel disease: Crohn’s disease Crohn’s disease ranks among the chronic inflammatory bowel diseases (IBD) with diarrhoea as a leading symptom. This is chiefly attributed to epithelial barrier dysfunction, leading to an increased loss of solutes in the form of “leak-flux diarrhoea”. Notably, the integrity of the intestinal barrier is not only impaired in patients with Crohn’s disease but also many first-degree relatives of Crohn’s disease patients present with increased intestinal permeability in the absence of clinical symptoms (Hollander 1993; Peeters et al. 1997; Yacyshyn and Meddings 1995). From this observation it was concluded that barrier dysfunction in Crohn’s disease precedes the inflammatory process in contrast to many other inflammatory diseases, probably arising from genetic defects or driven by environmental factors like intestinal luminal antigens or bacterial toxins. The epithelial barrier in the intestine is established by a single layer of epithelial cells. However, barrier dysfunction alone does not to seem be sufficient to cause Crohn’s disease, since unaffected relatives of patients with Crohn’s disease comparably present increased permeability, but do not show the abnormal mucosal inflammatory response reported of patients with Crohn’s disease (Weber and Turner 2007). In healthy individuals, the intestinal mucosal barrier is organised through tight junction strands connecting the adjacent epithelial cells (Weber and Turner 2007). These TJs seal the paracellular space and an intact intestinal mucosa. TJ permeability is rate limiting for epithelial permeability and defects thereof are an established source for intestinal barrier defects as observed in Crohn’s disease. The basic understanding of TJ function we developed is that they form an ion-selective and size-selective paracellular gate whose permeability varies throughout the gastrointestinal tract (see above). According to this, it is not surprising that the pattern of claudin isoforms expressed varies alongside the gastrointestinal tract (Nusrat et al. 2000). Recent studies now show that the expression pattern of characteristic claudins is altered in Crohn’s disease (Weber and Turner 2007) with a concomitant effect on the integrity of TJ strands: Zeissig et al. (2007) showed by freeze–fracture electron microscopy that the overall number of TJ strands sealing the epithelial layer is reduced while concomitantly an increased number of strand breaks was noted in tissue from patiens with Crohn’s disease (Zeissig et al. 2007). The expression of 12 claudin isoforms in the intestinal epithelium was investigated in healthy individuals and patients with Crohn’s disease, leading to the insight that the expression of the claudins-3, -5, and -8 was decreased in active disease. On the contrary, expression of barrier-reducing claudin-2 was increased in histological samples from patients, which was particularly observed in the crypt epithelium. This altered pattern of claudin isoform expression was thus concluded to be partly responsible for the observed mophological and functional disruption of TJs. However, claudin isoform expression patterns were unaltered in patients with inactive Crohn’s disease, rendering this observed change in expression patterns rather a consequence than a cause of active disease. It is has been acknowledged for a long time that chronic inflammation in Crohn’s disease is associated with the overproduction of proinflammatory cytokines. Searching for the nature of these pro-inflammatory mediators effecting the TJ disruption and dysfunction, investigators (Conyers et al. 1990; Heller et al. 2005; Prasad et al. 2005) had identified IL-13, IFNγ and TNFα as factors in patient’s serum lowering resistance and opening TJs. However, differential effects of the different cytokines crititical to IBD pathogenis have been reported from studies in cell culture models, indicating that the mechanisms by which IFNγ and TNFα inrease paracelluar permeability differ from those used by IL-13 (Prasad et al. 2005). IFNγ and TNFα exposure of model cell lines led to a reduction in claudin-2 expression, while IL-13 exposure induced increased claudin-2 expression. Those observed discrepancies, which might be based on the source of epithelial cell lines, the experimental design or the doses of cytokines applied are the focus of current investigation. Other lines of research are concentrated on gaining more information on the source of pro-inflammatory cytokines in IBD and the mode how those cytokines may work together to modify claudin expression pattern, either in vitro or in vivo (Weber and Turner 2007). At the cell biological level, recent investigation demonstrated that IFNγ exposure promoted the endocytosis of occludin, JAM-1 and claudin-1 into early and recycling endosomes (Bruewer et al. 2005), an effect which appears to be mediated by RhoA/Rho kinase, which further induced a myosin II-dependent formation of vacuoles in the apical cell moiety (Utech et al. 2005). At the gene expression level, other research groups could demonstrate that TNFα induced an increase in Caco-2 TJ permeability mediated by activation of the pro-inflammatory transcription factor NFκB. NFκB action leads to a downregulation of specifically ZO-1 gene expression and a redistribution of ZO-1 protein away from the intercellular junctions (Ma et al. 2004). A comparable effect could be described in the nonintestinal model epithelial cell line, MDCK, and in the blood brain barrier model cell line cEND, where also disruption of TJs following exposure of epithelial and endothelial cells to TNFα could be demonstrated (Förster et al. 2007; Poritz et al. 2004). Blood-brain barrier compromise in neuroinflammation–multiple sclerosis Homeostasis of the central nervous system (CNS) microenvironment is essential for its normal function. It is maintained by the blood-brain barrier (BBB) which regulates the transport of molecules from blood into brain and backwards. The function of this highly specialised barrier is to (1) protect the brain from blood-borne substances and (2) to provide nutrient supply to the brain parenchyme by specialised transport systems (Rubin and Staddon 1999). The main structures responsible for this barrier property are the TJ. TJ are strongly developed in endothelial cells of the BBB but only moderately formed between endothelial cells of the peripheral vasculature: leaky blood vessels in the body allow many molecules to cross through to tissue, but the tight construction of the vessels in the CNS guards against brain entry, leading to high electrical resistance in the range of 1,500—2,000 Ωcm2, depending on the species investigated. In BBB-forming brain capillary endothelial cells (BCECs) the TJ proteins occludin and claudin-5, claudin-3, claudin-1 and claudin-12 were demonstrated by immunofluorescence and western blot analysis (Förster et al. 2007; Wolburg and Lippoldt 2002). Under physiological conditions, brain microvascular endothelial cells form a barrier limiting the extravasation of cells of the immune system like monocytes, lymphocytes and other leukocytes. It has been shown in this context that leukocytes can transmigrate to the BBB without net changes in vascular permeability and cell–cell contacts (Cramer 1992). However, increased leukocyte migration as in multiple sclerosis (MS) has been reported to lead to a re-organisation of the actin cytoskeleton and breakdown of the TJ proteins occludin and ZO-1 (Couraud 1998; Förster et al. 2007). MS is characterised as a chronic and progressive CNS disease with a remitting-relapsing profile. In the course of the disease, both demyelination and microvascular inflammation have been described as central pathophysiological insults. From an animal model for MS, experimental allergic encephalomyelitis (EAE) (Korner and Sedgwick 1996), the inflammatory cytokines TNFα and IL-1 have been delineated to be the key mediators inducing alterations in BBB permeability. Clinically matching data have been collected from MS patients, who show an elevation in TNFα and/or IL-1 in cerebrospinal fluid (CSF) (Weber and Rieckmann 1995). Cytokine-promoted opening of the BBB has been shown to occur due to degradation and decreased synthesis of TJ proteins (Chang and Werb 2001; Harkness et al. 2000; Silwedel and Förster 2006; Yang et al. 2006), resulting in compromised junctional integrity. Particularly, the TJ proteins occludin and ZO-1 have been shown to be negatively affected by the presence of these pro-inflammatory cytokines. Searching for the mechanism of TJ protein degradation, consecutive studies showed, that in many neuroinflammatory conditions, including MS, the matrix metalloproteinases (MMP) play an important role in disrupting the BBB. Amongst these, it has been demonstrated that selectively, MMP-9 (gelatinase B) is increased in CSF levels in MS patients. Tissue inhibitors of metalloproteinases (TIMPs) form complexes with either activated MMPs or with their pro-forms after their secretion, thus regulating MMP activity under physiological conditions (Brew et al. 2000; Yong et al. 2001). Levels of TIMP-1 are however reduced in MS patients relative to control patients, suggesting an imbalance in MMP-9/TIMP-1 ratios in MS (Avolio et al. 2005). MMP-9-mediated opening of the BBB then allows amplification of the inflammation, as demonstrated by radioisotopes (Kermode et al. 1990). Looking for therapeutical agents restoring the unbalaced MMP-9/TIMP-1 ratios in MS, Förster et al. (2007) showed that glucocorticoid treatment reduces the levels of MMP-9 markedly in a cell culture model of the BBB, an effect which was shown to be based on glucocorticoid-mediated transcriptional activation of the TIMP-1 gene. This study further demonstrated that the administration of the glucocorticoids dexamethasone and hydrocortisone preserved the functional integrity of the TJs and adherens junctions under pro-inflammatory conditions, chiefly by maintaining the levels of the TJ components occludin, claudin-1, claudin-12, ZO-1 and VE-cadherin, while dexamethasone effects on claudin-5 were negligible (Blecharz et al. 2008; Förster et al. 2007). Another study demonstrated differential susceptibility of cerebral and cerebellar endothelial cell lines to BBB breakdown in response to inflammatory stimuli (Silwedel and Förster 2006): increased fragility of the cerebellar BBB had been reported, as the typical caudal to rostral lesion development of MS and EAE with a preference of the cerebellum indicates (Cross et al. 1993; Juhler et al. 1984; Namer et al. 1993). Matching those studies, the authors could demonstrate a faster and more pronounced increase in permeability in the cerebellar BBB manifested by reduced TER and reduced TJ protein expression using the cerebellar BBB model cell line cerebEND (Silwedel and Förster 2006). The stronger effects of TNFα on endothelial cells of the cerebellum as compared the the cerebrum could be explained on the cellular level, where the TJ protein levels of claudin-5 and occludin were shown to be differently affected in cerebellar and cerebral microvascular endothelial cells: effects on occludin were more pronounced in cerebellar endothelial cells, while cerebral endothelial cells of the BBB showed a stronger decrease in claudin-5. Since it was shown that the main protein buiding up the barrier appears to be occludin, while claudin-5 protein, which is also well detectable in non-BBB endothelial cells, does not seem to influence barrier properties to the same extent (Foerster et al. 2002, 2005, 2006; Wolburg and Lippoldt 2002). One may speculate that the lower overall occludin contents of endothelial cells of the cerebellar BBB compared to the cerebral BBB can partly explain the greater sensitivity of the cerebellar BBB to inflammatory stimuli. Hereditäry diseases Hereditäry deafness: nonsyndromic recessive deafness DFNB29 The cochlea of the inner ear is divided into two compartments of different ionic composition. Hereby, the perilymph of the scala vestibuli and scala tympani is high in Na+ but low in K+ concentration, while the endolymph of the scala media resembles the intracellular ionic composition, being high in K+ but low in Na+ (Ferrary and Sterkers 1998). This ion gradient leads to the establishment of an 80–100 mV endocochlear potential, necessary for the depolarisation of sensory hair cells, enhancing the sensitivity of mechano-activated channels at the top of stereocilia (Hudspeth 1989). Besides, hereditary defects in the gap junction proteins connexin-26 and connexin-43 (Sabag et al. 2005) an association between tight junctions and hearing loss was established. The importance of claudin-14 expression in the cochlea was demonstrated by the identification of two different CLDN14 mutations that cause nonsyndromic recessive deafness DFNB29 in two large consanguineous Pakistani families (Wilcox et al. 2001). By in situ hybridization and immunofluorescence labeling mouse claudin-14 expression in the sensory epithelium of the organ of Corti was observed. By the generation and characterisation of claudin-14 knockout mice as a model for autosomal-recessive deafness DFNB29 it was revealed that deafness occurred due to death of outer hair cells. Searching for the factors that promoted increased death of outer hair cells, morphology of the TJs of claudin-14 knockout mice was evaluated. It was demonstrated that TJ strands between supporting cells and between outer hair cells and supporting cells in the organ of Corti are still present and appear morphologically normal in freeze fracture replicas. The analysed TJ strands which are composed of multiple claudins were thus concluded to have rather a functional than an ultrastructural defect (Ben-Yosef et al. 2003). Following the reasoning that claudins create charge-selective channels, Ben-Yosef characterised the electrophysiologic properties of claudin 14 by ectopically expressing it in MDCK cells, which revealed a strong discrimination of transfected monolayers against Na+ and K+ ions. Ben-Yosuf and coworkers thus concluded that the absence of claudin 14 from TJs in the organ of Corti might lead to elevated K+ concentration in the space of Nuel, which is normally filled with the K+-poor cortilymph and increased death of outer hair cells. Long-term exposure of the basolateral membranes of outer hair cells to high K+ concentrations had been shown to be toxic and eventually to lead to cell death before (Zenner 1986). By the identification of two claudin-14 missense mutations that were unable to form TJs after ectopic expression of CLDN14 in L mouse fibroblastes, Wattenhofer et al. (2005) were then able to prove that the basis for the development of deafness is incorrect recruitment and TJ strand formation in cells expressing mutated CLDN14. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) Inherited and acquired defects in epithelial membrane transport are responsible for many human diseases. In the kidney, tubular claudins play a role in extracellular or paracellular permeability. In this context, it has been shown that mutations in claudin-16 (Paracellin-1) impair the function of TJs in the paracellular resorption of Mg2+ and Ca2+ but not of monovalent cations (Simon et al. 1999), probably founding the molecular basis for the development of familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), an autosomal recessive renal tubular disorder. In FHHNC patients, the impaired tubular reabsorption of Mg2+ and Ca2+ in the thick ascending limb of the loop of Henle with an eventual progression can lead to end-stage renal disease (Lee et al. 2006). Functional tubular reabsorbtion mechanisms are vitally important, since a normal adult absorbs only 200 mg Ca2+ from the diet each day, but loses 8,000 mg to filtration across the glomerulus. To achieve normal calcium balance, the majority of the filtered load must be reabsorbed by the tubule, to approximately 90%, or 7,000 mg/day and this occurs mainly via the paracellular pathway driven by a lumen positive potential across the tubular wall. The structural backbone of the paracellular pathway must provide for the significant substrate selectivity and functional heterogeneity between nephron segments and thus also plays a pathophysiologic role in renal diseases. Clinically, the symptoms and gradual kidney damage caused by FHHNC cannot be alleviated by intravenous or oral supplementation with Mg2+, and kidney transplantation is often the only option at later stages of the disease. Recent studies (Simon et al. 1999) have reported that FHHNC is caused by mutations in a gene, which encodes the TJ protein, paracellin-1. Paracellin-1 (PLCN-1) belongs to the claudin family (claudin-16) that allows the paracellular transport of Mg2+ and Ca2+. As a member of the claudin family of TJ proteins claudin-16 exhibits four transmembrane spans with two extracellular loops and both termini located in the cytoplasm. To date, more than 20 distinct mutations have been identified, which affect either the trafficking of PLCN-1/claudin-16, or its permeability properties (Kausalya et al. 2006). Interestingly, FHHNC patients do not exhibit the salt wasting or metabolic alkalosis that is found in Bartter’s syndrome (Wong and Goodenough 1999). Based on these findings, it is tempting to hypothesize that PCLN-1 might form a Mg2+- and Ca2+ -conducting pore. It is therefore likely that PCLN-1 selectively mediates the barrier to divalent but not monovalent cations. Thus, not only can the paracellular barrier discriminate between charged and uncharged solutes, but it may also exhibit an even higher level of specificity distinguishing charge and ionic radius and size of the hydrate shell. As to molecular investigations based on the use of diverse cell culture models for kidney epithelial cells, there is some discrepancy between results obtained upon overexpression of PLCN-1 in two different kidney epithelial cell lines used. In the canine MDCK cell line, another group found PCLN-1 to decrease Na+ permeability, to decrease TER and to enhance transepithelial Mg2+ transport (Ikari et al. 2006). Another recent study performed in the porcine proximal tubule LLC-PK1 cells suggests that paracellin-1 functions to modulate paracellular conductance and not transcellular transport. They hypothesise that PLCN-1 mediates mostly paracellular Na+ permeation to build up the positive lumen potential and that this lumen-positive potential would be the driving force for the reabsorption of Mg2+ (Hou et al. 2005). As described for other molecules relevant to Mg2+ homeostasis such as ACDP2, (ancient conserved domain protein), a divalent metal transporter, or the Mg2+ transporter MagT1 (Wong and Goodenough 1999), the human PLCN-1 promoter appears to be responsive to external Mg2+ concentrations, since the renal epithelial cell specific PLCN-1 gene expression was shown to positively correlate with the amount of Mg2+ offered in the growth medium (Efrati et al. 2005). Further, mutations in PLCN-1 can affect claudin-16 protein targeting at the TJs (Kausalya et al. 2006). The mutation T233R for example prevents interaction of claudin-16 with ZO-1 and results in lysosomal targeting of claudin-16 instead. Protein kinase A (PKA) mediates phosphorylation of claudin-16 at Ser217, failure of which equally leads to dissociation of claudin-16 from ZO-1 and its translocation into the lysosomes (Ikari et al. 2006). In conclusion, although the mechanism of PLCN-1/claudin-16-mediated Mg2+ and Ca2+ reabsorbtion is not yet fully understood, it is clear that these claudins play a crucial role in this process. Recently, Konrad et al. (2006) additionally unravelled a role for claudin-19 in the development of FHHNC. They had clinically characterized one Swiss and eight Spanish/Hispanic families affected with severe hypomagnesemia due to renal wasting, nephrocalcinosis, and progressive renal failure but had to recognize that the individuals did not have any mutations in CLDN16. They showed that the renal phenotype of the affected patients was virtually undistinguishable from that of patients who presented with FHHNC with proven CLDN16 mutations. However, the affected individuals in these families also have severe visual impairment, characterized by macular colobomata, significant myopia, and horizontal nystagmus (Konrad et al. 2006). Konrad et al. were able to map a new locus for recessive renal magnesium loss on chromosome 1p34.2 and identified mutations in CLDN19, yet another member of the claudin family in patients affected with FHHNC and additionally, with severe ocular abnormalities. CLDN19, which encodes the tight-junction protein claudin-19 was demonstrated to be highy expressed in renal tubules and the retina. In an effort to understand the mechanisms underlying the roles of the two claudins in mediating paracellular ion reabsorption in the kidney, Goodenough and co workers (Hou et al. 2008) showed using pig kidney epithelial cells, that CLDN19 functioned as a Cl(−) blocker, whereas CLDN16 functioned as a Na(+) channel. Mutant forms of CLDN19 were shown to be associated with FHHNC since they were unable to block Cl(−) permeation. Coexpression experiments further elucidated that CLDN16 interacts with CLDN19 and that their association confers cation selectivity, suggesting a role for mutant forms of CLDN16 and CLDN19 in the development of FHHNC. Vascular system Diabetic retinopathy The barrier between the vascular lumen and neural layers in the retina and the brain parenchyme maintains a characteristic microenvironment and is essential for proper neuronal function. This blood-retinal barrier (BRB) consists of two anatomical entities: an inner BRB formed by junctions between endothelial cells of the retinal capillaries, and an outer BRB composed of TJs between retinal pigment epithelial cells. Clinical evidence from fluorescein angiography has demonstrated that the inner BRB appears to be the primary site of vascular leakage leading to macular edema. The BRB is built up and maintained by TJ complexes that make up the blood vessels of the microvasculature (Mitic and Anderson 1998). The principal proteins found in the retinal endothelial TJs are occludin and claudin-5 (Dejana et al. 2001). Loss of the BRB integrity and vascular permeability leading to macular edema (Vitale et al. 1995) characterise diabetic retinopathy. Macular edema in turn is thought to be directly responsible for vision loss in diabetic retinopathy (Felinski and Antonetti 2005). Vascular endothelial growth factor (VEGF) is thought to be chiefly responsible for this increase in BRB permeability, albeit other permeabilizing factors like histamin or the cytokines IL-1 and TNFα have also been implicated (Felinski and Antonetti 2005). Levels of VEGF, originally identified as an angiongenic factor serving as a mitogen for vascular endothelial cells have been recently correlated with increased vascular permeability (Felinski and Antonetti 2005), compatible with increased VEGF levels detected in the vitreous of diabetic patients for decades. There is enough evidence to show that diabetes and VEGF induce retinal vascular permeability by altering TJ complexes, evidenced for instance by the decrease in occludin content in the retinas of diabetic rats (Felinski and Antonetti 2005). In cultured primary bovine retinal endothelial cells, this observation was mirrored by the observation of decreased occludin contents after VEGF administration (Antonetti et al. 1998). Follow-up investigation underlined this and showed down-regulation of occludin in retinal endothelial cells in culture after VEGF stimulation. VEGF further activates PKC, which leads to increased occludin and ZO-1 phosphorylation (Harhaj et al. 2006). Phosphorylation of occludin and ZO-1 likely contribute to dysregulated endothelial paracellular permeability (Abbott et al. 2006). Regulation of PKC activity and TJ protein modifications may thus have therapeutic implications for treatment of diabetic retinopathy. Recent investigation has further revealed that alterations of the BRB may involve the active proteolytic breakdown of the endothelial cell TJs by MMPs as also evidenced in the case of MS (compare above) (Leppert et al. 2001). Specifically, elevated levels of MMP-9 have been seen when retinal endothelial cells were exposed to high glucose conditions as a cell culture model of diabetes, leading to proteloytic degradation of specifically the TJ protein occludin followed by disruption of the overall TJ complex (Giebel et al. 2005), rendering the regulation of MMP secretion and activity another therapeutical target in diabetic retinopathy. Multiple sclerosis See above. Tight junction alterations in cancer Decreased expression of claudin-1 correlates with malignant potential in breast cancer Changes in TJ function have been shown to be an early and key aspect in cancer metastasis (Ehler et al. 2001). Recent studies showed a role for claudin-1 in invasion and metastasis in mammary glandular differentiation (Förster et al. 2002) and carcinogenesis (Morohashi et al. 2007). Interestingly, there is an absence or significant loss of claudin-1 expression in several established breast cancer cell lines. Claudin-1 expression in primary human mammary epithelial cells, in contrast to low or undetectable levels of expression in a number of breast tumors and breast cancer cell lines, points to claudin-1 as a possible tumor-suppressor gene. In sections from surgically resected breast specimens, a significant loss of claudin-1 protein in breast cancer cells could be shown by immunohistochemistry (Tokes et al. 2005). This finding suggests that claudin-1 may play a role in invasion and metastasis. The expression profile of claudin-1 in non-malignant versus tumor-derived cells has made this gene an interesting candidate for involvement in tumorigenesis, namely by acting as a suppressor of mammary epithelial proliferation. In order to evaluate the CLDN-1 gene in sporadic and hereditary breast cancer, Krämer et al. (2000) characterized its genomic organization and screened the four coding exons for somatic mutations in 96 sporadic breast carcinomas and for germline mutations in 93 breast cancer patients with a strong family history of breast cancer. In addition, they compared the 5′-upstream sequences of the human and murine CLDN1 genes to identify putative promoter sequences involved in down-regulation of the CLDN1 gene under malignant conditions and examined both the promoter and coding regions of the human gene in various breast cancer cell lines showing decreased claudin-1 expression. However, neither in sporadic or hereditary breast cancers, nor in breast cancer cell lines they found evidence for a role of aberrant claudin-1 in breast tumorigenesis and concluded that other regulatory or epigenetic factors could be involved in the down-regulation of the CLDN1 gene during breast cancer development. A correlation between TJ abnormalities and neoplasia can be supported by several observations: alterations in the number, appearance, and permeability of TJs have been demonstrated in various cancer types (Cochand-Priollet et al. 1998; Soler et al. 1999; Swift et al. 1983) and in pre-malignant mammary epithelial cells (Förster et al. 2002). At the molecular level, the Drosophila lethal(1) discs-large-1 gene (dlg) product, which localises to TJ-related insect septate junctions has been shown to regulate epithelial cell proliferation. Genetic loss of dlg leads to a neoplastic overgrowth phenotype (Woods and Bryant 1991). The loss of expression of claudin-1 itself has been demonstrated in several mammary carcinoma cell lines (Swisshelm et al. 1999). The expression of the TJ plaque protein ZO-1 has been shown to be reduced in breast carcinomas or breast cancer cells (Hoover et al. 1998). These findings support the hypothesis that claudin-1 might be involved in the development of breast cancer and possibly in other epithelial tumors too. The molecular pathways leading toward the loss of claudin-1 expression however, remain to be explained. Bacterial toxins Clostridium perfringens enterotoxin Food poisoning by Clostridium perfringens enterotoxin (CPE) leads to diarrhea symptoms classified as “C. perfringens type A food poisoning and antibiotic-associated diarrhea”. The enterotoxin protein has been elucidated to intrude the cells by interacting with epithelial TJ, including certain claudins. Toxin-induced cytolytic pore-formation is a prerequisite for this and involves residues in the N-terminal half of CPE protein, while residues near the C-terminus are required for binding to claudins. In detail, the single 35 kDa CPE polypeptide first causes cellular damage by altering plasma membrane permeability by the formation of an approximately 155 kDa CPE-containing pore complex necessary for the subsequent interaction with TJ proteins via its C-terminal receptor-binding region (McClane 2001). CPE was then perceived to affect TJ structure and function in a way that permeability properties of the epithelial layer are altered leading to diarrhea (McClane 2001). Further investigation has determined the isoforms of claudins targeted by CPE. Sonoda et al. (1999) were able to show that the COOH-terminal half fragment of CPE (C-CPE) bound effectively to claudin-3 and claudin-4 in MDCK I cells expressing the claudins-1, -2, -3 and -4. They were further able to show that in the presence of C-CPE, reconstituted TJ strands gradually disintegrated and disappeared from their cell surface in C3L cells. As a consequence, because diverse claudins are overexpressed on some human cancers, the toxin became interesting for targeting chemotherapy in the manner of a Trojan horse (Long et al. 2001; Michl et al. 2001). Recently, van Itallie et al. (2007) aimed to solve the structure of the CPE claudin-binding domain to advance its therapeutic applications. The structure was shown to be a nine-strand beta sandwich with previously unappreciated similarity to the receptor-binding domains of several other toxins of spore-forming bacteria, giving strong evidence for the presence of a common ancestor for several receptor-binding domains of bacterial toxins (Van Itallie et al. 2007). The very recent elucidation of the structure of a 14-kDa fragment containing residues 194 to the native COOH terminus at position 319 by X-ray diffraction to a resolution below 2 Å showed that the structure is a nine-strand β sandwich with previously unappreciated similarity to the receptor-binding domains of several other toxins of spore-forming bacteria, i.e., the collagen-binding domain of ColG from Clostridium histolyticum and the large Cry family of toxins (including Cry4Ba) of Bacillus thuringiensis (Van Itallie et al. 2008). The authors speculate that the claudin-4 binding site is on a large surface loop between strands β8 and β9 or includes both of these strands. They further were able to clarify that the sequence crystallized binds to purified human claudin-4 with a 1:1 stoichiometry. The binding affinity was determined to be in the submicromolar range similar to that observed for binding of native toxin to cells (Van Itallie et al. 2008). These results could now provide a structural framework to advance therapeutic applications of the toxin. Conclusion and perspectives The discovery of the existence of occludin- and claudin-based TJs constituting a permeability barrier in epithelial and endothelial cells has paved the way for the elucidation of the molecular background of diverse human diseases the pathogenesis of which had been unclear before. It became clear that the presence of TJs is indispensable for tissue compartmentalisation and cellular homeostasis. Disturbances in TJ function are reflected in many diseases, but on the other hand, awareness of their involvement has facilitated therapy enormously. For instance, antiinflammatory or antimetastatic drugs could be developed based on their ability to repair broken barrier function. These strategies have become indispensable in the treatment of disease like MS, inflammatory bowel disease or diabetic retinopathy. Following the strategy of pathogenic agents, which use TJ proteins as a docking station to invade cells, even strategies for drug delivery techniques could be implemented. Future attempts will have to address the role of occludin and the exact contribution of the diverse claudins to the formation of ion selective pores, which are still open questions. A more thorough understanding will greatly facilitate diagnosis and the development of specific treatment regimens for diseases originating from impaired TJ function.

Ann_Biomed_Eng-2-2-1705520

Cellular and Matrix Mechanics of Bioartificial Tissues During Continuous Cyclic Stretch

Bioartificial tissues are useful model systems for studying cell and extra-cellular matrix mechanics. These tissues provide a 3D environment for cells and allow tissue components to be easily modified and quantified. In this study, we fabricated bioartificial tissue rings from a 1 ml solution containing one million cardiac fibroblasts and 1 mg collagen. After 8 days, rings compacted to <1% of original volume and cell number increased 2.4 fold. We initiated continuous cyclic stretching of the rings after 2, 4, or 8 days of incubation, while monitoring the tissue forces. Peak tissue force during each cycle decreased rapidly after initiating stretch, followed by further slow decline. We added 2 μM Cytochalasin-D to some rings prior to initiation of stretch to determine the force contributed by the matrix. Cell force was estimated by subtracting matrix force from tissue force. After 12 h, matrix force-strain curves were highly nonlinear. Cell force-strain curves were linear during loading and showed hysteresis indicating viscoelastic behavior. Cell stiffness increased with stretching frequency from 0.001–0.25 Hz. Cell stiffness decreased with stretch amplitude (5–25%) at 0.1 Hz. The trends in cell stiffness do not fit simple viscoelastic models previously proposed, and suggest possible strain-amplitude related changes during cyclic stretch. INTRODUCTION Mechanical stimuli are important for the health and maintenance of many biological functions.14 This is especially true in the cardiovascular system where the tissues and organs are undergoing constant cyclic loading. It is the cellular response to these mechanical forces that can either maintain healthy tissue or lead to a pathologic state.1 Mechanical stimuli have been used to create more functionally viable engineered tissues.2–5,9,15–17 It has been shown that constant amplitude cyclic stretch (a form of mechanical conditioning) of engineered tissue constructs can cause cells to reorient, increase cell proliferation, and produce more extracellular matrix (ECM).3 However, these studies generally have looked at how the tissue responds to mechanical conditioning over extended time.9,15 In addition, the studies generally quantify the mechanics using only the ultimate strength and modulus4,5,16 along with quantification of such biological factors as cell number, gene expression, and ECM components.2,3,17 Since it is the cells that actively respond to mechanical loads, it is important to understand the effects of mechanical conditioning on the individual components of the tissue (cell and matrix) and how the mechanical properties change from the time that the conditioning is initiated. Our earlier work has shown that tissue forces drop with subsequent stretches during repeated cyclic stretching.19 However, whether this drop occurs in the cells and/or matrix components is not known. Furthermore, it is not known how these drops are affected by different amplitudes and frequencies over many hours of continuous cyclic stretch. The objective of this study is to measure the short-term (<24 h) mechanical response of collagen gels populated with cardiac fibroblasts to cyclic stretch at different amplitudes and frequencies. Although engineered tissue constructs may be composed of several cell and matrix components, we chose to use a simple system composed of a single cell type and a single defined ECM component to study the mechanics of tissue components during cyclic stretch. This system provides a 3D environment for the cells while allowing the components to be easily modified and quantified. The benefits of using collagen, the main structural component in cardiovascular tissue, are that its initial concentration can be easily modified and it is more stable than other ECM components such as fibrin.4,5,10 By using a single cell type we can avoid complicating effects due to mixed cell populations and clearly understand that cell type’s contribution to the tissue mechanics. Cardiac fibroblasts are one of the two main cell types in cardiac tissue and are responsible for ECM maintenance and remodeling.7 These cells, along with the cardiac myocytes, undergo continuous cyclic stretch with each heartbeat. Thus, it is particularly useful to understand their cellular mechanics during cyclic stretch. By measuring the cell forces under various cyclic stretch amplitudes and frequencies, we can better understand the relationship between the applied strains and the cells’ mechanical response. Then we can model this behavior and be able to predict cellular responses to dynamic physiologic and pathologic loading conditions. METHODS Tissue Ring Fabrication Chicken embryonic cardiac fibroblasts (CECF) were isolated from 10 day old chicken embryos (Charles River Laboratories, Wilmington, MA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 3% fetal bovine serum (FBS), 50 units/ml penicillin and 50 mg/ml streptomycin (P/S). Tissues were fabricated as described previously.20 Briefly, CECF from passage 2–8 were added to a collagen solution containing monomeric rat tail tendon collagen (Upstate Biotechnology Inc., Lake Placid, NY) neutralized with 0.1 N sodium hydroxide (NaOH) and 2× concentrated DMEM. The final solution contained 1 mg/ml collagen and one million cells/ml. Then 1 ml of this solution was poured into cylindrical molds containing an inner mandrel (diameter=9.55 mm). After one hour of incubation, the solution gelled and the molds were filled with incubation medium containing DMEM supplemented with 10% FBS and P/S. After five additional hours, the gels were removed from the molds and transferred to spacers (Fig. 1) placed in incubation medium. The spacers consisted of two fixed rods (2.07 mm diameter) separated by a distance to maintain the internal circumference of the rings at 30 mm. Medium was changed every other day. FIGURE 1.Cardiac fibroblast populated rings on spacers during static incubation (A) Day 0 [6 h] (B) Day 2 (C) Day 4 (D) Day 8. Mean ring width was 0.64 mm after 8 days incubation (see Fig. 4).FIGURE 2.Illustration of peak force versus time. The gray curve is sample data obtained for 10 stretch cycles. The black line plots the peak force for each cycle vs. time. This peak force line is used for many of the following figures. Mechanical Tests After 2, 4, or 8 days of static incubation (Fig. 1) the width of the rings was measured and then the rings were removed from the spacers. The rings were then mounted on a mechanical tester described previously20 that allows independent programmable stretching of four rings while monitoring applied force. The test medium contained DMEM HEPES modification supplemented with 10% FBS and P/S. All rings were placed on the tester at a baseline length that maintained the inner circumference of 30 mm. This was defined as 0% strain. In early tests, stretching began immediately after mounting the rings. This led to increases in peak force after initiation of stretch for low amplitudes at early incubation times. Upon further study, it was determined this was a result of the fresh media and FBS used in the test baths and not a result of a mechanical stretch induced response. In all further studies, the rings were mounted on the tester and held at baseline length for 3 h to allow them to reach equilibrium before testing began. Effect of Cyclic Stretch Amplitude Four rings were tested at each of the three days with each ring stretched either 5%, 10%, 20%, or 25% at 0.1 Hz using a triangular displacement waveform (constant strain rate during loading). The force was continuously monitored at 10 Hz over 14 h. For many of the figures, the peak force, defined as the maximum force during each cycle, is plotted vs. time (Fig. 2). We use the term stretch to refer to the amplitude of cyclic stretching, while we use the term strain to refer to the amount of deformation at a particular point in the loading curve. To separate out the individual tissue component forces (cell and matrix), 2 μM Cytochalasin D (CytoD) was added either 3 h before or 12 h after initiation of stretch. CytoD disrupts actin filaments inside the cell and thus eliminates the majority of the cell-contributed force.21 The remaining force measured 120 min after addition of CytoD is termed the matrix force. Then the estimated cell force is calculated by taking the matrix force and subtracting that from the total tissue force without CytoD. FIGURE 3.Cell force calculation. Matrix force was measured by adding 2μM CytoD to rings prior to initiation of cyclic stretch. Cell force is calculated by taking the normalized matrix force (see Eq. 1) and subtracting it from the tissue force. To estimate the cell forces during the initial 12 h of cyclic stretch, it is necessary to use two separate rings subject to identical stretching protocols. This is accomplished by subtracting the forces measured in a ring with CytoD added prior to cyclic stretching from the forces measured in a ring where CytoD was added after 12 h of cyclic stretch. We denote these forces FC0(t) and FC12(t) respectively and similarly, we denote the peak forces PC0(t) and PC12(t). However, the peak forces after 14 h of cyclic stretch, when both rings have been treated with CytoD, can still differ between rings subject to identical cyclic stretch protocols due to variations between rings. To account for this variation, we scale the forces in the ring to which CytoD was added before initiating stretch using the following formula: We found that the shape of the loading/unloading curve was similar whether CytoD was added before or after initiation of cyclic stretch. We then subtracted this scaled, or normalized, force curve from the force curve for the ring with CytoD added after 12 h to obtain an estimated cell force (Fig. 3): Single Step Stretch and Hold Effect To compare the effect of a single stretch to cyclic stretching, a step stretch (stretch to a given stretch magnitude in less than 0.1 s) of 5%, 10%, or 20% was applied and the rings were held at this stretched length for 8 h while the force was monitored. The tissue was then returned to its baseline length and the force was monitored for an additional 6 h. In another set of experiments, CytoD was added 3 h before or 12 h after either a 10% or 20% step stretch. Effect of Cyclic Stretch Frequency A similar procedure was followed to test frequency effects. Four rings were tested at each of the three days with each ring stretched at either 0.25 Hz, 0.1 Hz, 0.01 Hz, or 0.001 Hz at 10% stretch amplitude. The current tester’s upper frequency limit is 0.25 Hz and thus unable to stretch at higher frequencies that are closer to physiologic values. CytoD was added either 3 h before or 12 h after initiation of stretch to separate out the cell and matrix components as a result of different frequencies of cyclic stretch. Cell Number After the completion of testing, some rings were washed in phosphate buffered saline (PBS) and then placed in 1 ml lysis buffer (0.1% sodium dodecyl sulfate (SDS) in PBS). The samples were sonicated until the rings completely disintegrated to release the DNA from the cells. Finally, 30 μl of sample was placed in 3 ml of Hoechst working solution (30 nM Hoechst 33258 dye (Sigma, St. Louis, MO) in PBS). The fluorescence was then read by a spectrophotometer and cell number determined from a standard curve obtained from samples with known numbers of cells. Immunohistochemistry Rings were removed from spacers after 2, 4, or 8 days static incubation and immediately placed in a solution of 4% paraformaldehyde for 30 min, washed with PBS and then permeabilized for 45 min in 0.2% Triton X-100 in PBS. This was followed by 1 h incubation in a blocking solution (2% normal goat serum and 0.02% sodium azide in PBS). After blocking, the rings were incubated overnight at 4°C in blocking solution containing TRITC phalloidin. Finally, the rings were washed again in PBS and mounted for viewing on a confocal microscope (Zeiss Confocor). RESULTS Ring Width Ring width was used as a metric for monitoring the extent of compaction during incubation. The initial width of the rings was approximately 10 mm. This width decreased until day 8 (Fig. 4(b)) when the average width was reduced to 0.64 mm. At this time the cross-sectional area of the ring appeared circular and the volume could be approximately calculated using a circular cross-sectional area and the circumference of the ring. A ring diameter of 0.64 mm corresponds to a 99% reduction in the initial tissue volume. FIGURE 4.Cell number and ring width during static incubation. (A) Cell number increases from the initial 1 million cells to 2.4 million cells after 8 days of static incubation. (B) Ring width decreased from an initial 10 mm to 0.64 mm after 8 days of static incubation, with a large amount of compaction occurring prior to day 2. Cell Number Rings started with a cell density of one million cells/ml (one million cells per ring). This number increased steadily for 4 days to 2.3 million cells then increased only slightly to day 8 with a total cell number of 2.4 million (Fig. 4(a)). When combined with the 99% reduction in tissue volume, this translates into a final cell density of 234 million cells/ml or a 234 fold increase in cell density. Amplitude Effect The effect of increasing stretch amplitude on the peak tissue and matrix forces is shown in Fig. 5. At all amplitudes, the peak tissue forces immediately after initiating stretch (Fig. 5(a)) increased with static incubation time while the peak matrix forces were similar (Fig. 5(c)). After 12 h of cyclic stretch, both tissue (Fig. 5(b)) and matrix forces had decreased substantially, with a larger percentage decline in the matrix force (Fig. 5(d)). To examine the relationships between tissue, matrix, and cell forces more closely, figures in the remainder of this paper show data from rings statically incubated for 8 days prior to cyclic stretch unless otherwise noted. Similarities or differences to days 2 and 4 are discussed in the text. In addition, all curves are averages across repeated (n=2–7) experiments. Due to the large number of different conditions tested and the relatively low number of samples (exact numbers shown in Fig. 5(e), 10(e)) rigorous statistical analysis was not possible. Tissue Force Higher stretch amplitudes resulted in higher initial peak forces (Figs. 5(a), 6(a), 6(b)). With each subsequent stretch, the peak force decreased with large drops during the first several stretches and sustained decreases during the first two hours, but only small further decreases in peak force over the next 10 h (Fig. 6(a)). In addition, this decrease in peak force was greater at higher stretch amplitudes both in terms of absolute force drop and percentage drop from first stretch. At days 2 and 4, after 12 h of stretch, higher amplitudes still have higher peak forces but the differences were smaller between the amplitudes than the first stretch differences (Fig. 5(a,b)). FIGURE 5.Peak tissue and matrix forces for different cyclic stretch amplitudes at 0.1 Hz. (A) Peak tissue force of the first cyclic stretch after 2, 4, or 8 days static incubation. Peak tissue force increases with cyclic stretch amplitude and incubation time. (B) Peak tissue force after 12 h of cyclic stretch after 2, 4, or 8 days static incubation. At day 8 the peak tissue force is similar regardless of cyclic stretch amplitude. (C) Peak matrix force of the first stretch after 2, 4, or 8 days static incubation. Peak matrix force increases with cyclic stretch amplitude. (D) Peak matrix force after 12 h of cyclic stretch after 2, 4, or 8 days static incubation. Higher amplitudes result in higher peak forces. (E) Number of rings averaged for data shown in Figures 5-8 at each amplitude and static incubation day. (F) Individual measurements used in panel B [Peak tissue force after 12 h, 8 days of incubation]. This shows that the trend within an experiment, i.e. rings fabricated on a single day, is consistent but the experiment to experiment variation in forces is large. Y-axis scales are different in each panel to emphasize the trends with amplitude and incubation time.FIGURE 6.Tissue force versus cyclic stretch amplitude after 8 days of static incubation. (A) Peak tissue force over 12 h of cyclic stretch. Peak tissue force dropped quickly after initiation of cyclic stretch followed by a continued slow decline. (B) First 5 tissue stretches after initiation of cyclic stretch. A large decline in tissue force and hysteresis occurred between the first and second stretch. (C) Last 5 tissue stretches after 12 h of cyclic stretch. The peak tissue force is similar at the four amplitudes. By day 8, similar peak tissue forces were seen after 12 h of cyclic stretch regardless of stretch amplitude between 5% and 25% (Figs. 5(b), 6(a), 6(c)). The rings subject to lower stretch amplitudes (5% or 10%) were able maintain a positive baseline force (i.e. force at the baseline, or zero strain, length). At the highest stretch amplitude the rings had periods of zero force during both the loading and unloading of the ring, meaning that at low strains the rings were slack (Fig. 6(c)). After the first stretch (Fig. 6(b)), the loading curve remained nearly linear at 5% stretch amplitude, but became increasingly nonlinear at higher stretch amplitudes (Fig. 6(c)). FIGURE 7.Matrix force versus cyclic stretch amplitudes after 8 days of static incubation. (A) Peak matrix force over 12 h of cyclic stretch. Peak matrix force dropped quickly after initiation of cyclic stretch followed by a slow decline for the 12 h duration. (B) First 5 matrix stretches after initiation of cyclic stretch. A large decline in matrix force and hysteresis occurred between the first and second stretch. (C) Last 5 matrix stretches after 12 h of cyclic stretch. Matrix loading curve is highly nonlinear and peak force increased slightly with higher cyclic stretch amplitude.FIGURE 8.Cell force versus cyclic stretch amplitudes after 8 days of static incubation. (A) Peak cell force over 12 h of cyclic stretch. Peak cell force increased for 5% and 10% amplitudes while it decreased for 20% and 25% amplitudes. (B) First 5 cell stretches after initiation of cyclic stretch. (C) Last 5 cell stretches after 12 h of cyclic stretch. The peak cell force and cell stiffness decreased with increased amplitude. The loading curves are nearly linear for all amplitudes.FIGURE 9.Response to step stretch and hold after 8 days of static incubation. The tissue, matrix, and cell forces are shown after a step stretch of either 10% or 20%. The tissue is held at the stretched length for 12 h. The tissue and matrix force for a 20% step and hold is higher than that for a 10% step and hold. The cell force is identical for 10% and 20%. Matrix Force Except for the first stretch, the matrix loading curves were nonlinear for all stretch amplitudes tested. Similar to tissue force, higher stretch amplitudes led to higher initial peak forces (Figs. 5(c), 7(b)). For all amplitudes, the peak force decreased with repeated cyclic stretching and this drop in peak force was larger at higher stretch amplitudes (Fig. 7(a)) but higher stretch amplitudes still maintained higher peak forces (Fig. 7(c)). For the first stretch, the ratio of peak forces at day 8 for 25% to 5% was 5:1, but this ratio decreased to 2:1 after 12 h of stretching, due to the peak matrix force dropping by a greater percentage at the higher amplitudes over time. The other distinct feature was the shape of the loading/unloading curves. After several hours of stretching the shapes of the loading/unloading curves were very similar for all stretch amplitudes, but the strain value at which the matrix force began increasing from zero during each loading cycle was shifted toward larger strain values. The amount of this shift was greater for higher stretch amplitudes (Fig. 7(c)). This meant that the matrix contributed to the tissue force during the loading only when the strain had reached a value from 3 to 7% less than its maximum value. This shape and shift is evident at days 2, 4, and 8. For all stretch amplitudes, the matrix is slack at low strains. Cell Force Cell force increased with increased incubation time. The increase in cell force after 12 h of stretch from 2 to 4 days of incubation was due to an increase in cell number (per cell force remained the same). However, the increase in cell force from days 4 to 8 could not be explained by the increase in cell number alone, indicating an increase in per cell force. The shape of the first force-strain curve for the cell differed from all subsequent curves. The initial baseline force was followed by a rise and then a leveling off with further increases in force seen only at the higher amplitudes (Fig. 8(b)). During the second stretch, the cell force loading curve was nearly linear. Unlike the tissue and matrix, the cell force loading curve retained a nearly linear shape throughout the 12 h of cyclic stretch even at 25% amplitude (Fig. 8(b,c)). At day 2, the peak cell force initially drops for several cycles, but then gradually increases to the end of the test. The size of the initial force drop is dependent on the stretch amplitude. The trend is similar at day 4, except the cell force at 25% stretch remains fairly steady instead of increasing. At day 8, the peak cell force, after the initial drop, increases only for the 5% and 10% stretch, while it decreases for the 20% and 25% stretch (Fig. 8(a)). Single Step Stretch & Hold Effect Similar to cyclic stretching at different amplitudes, the larger magnitude step stretches led to higher initial forces (Fig. 9). The tissue force was higher than the matrix force immediately after the step at the same magnitude. However, both tissue and matrix force dropped quickly and to a greater extent for higher magnitudes. The matrix force was lower than the tissue force during the entire 12 h hold period at the same magnitude indicating that the cells contributed to the measured tissue force throughout the hold period (Fig. 9). The rate of decrease during the 12 h hold period is similar for both the tissue and matrix. This is evident in the fact that very quickly after the step stretch the cell force approaches a constant for the 12 h (Fig. 9). The cell force is approximately the same at both 10% and 20% step stretches during the 12 h held at the stretched length. For both step stretch magnitudes, the cell force at the end of the 12 h hold period is similar to the cell force at the baseline length (data not shown) immediately before the step stretch is applied. Rings that did not have CytoD added before or during the step stretch were returned to their baseline lengths after being held at the stretched length for 8 h. The force dropped to zero but then began to recover with lower magnitude step stretches increasing their forces faster than higher magnitude step stretches. The recovered forces approached their baseline forces prior to stretch. After 6 h of monitoring force at baseline, rings stretched to 5% magnitude and held recovered more of their baseline force (84%) than rings stretched to 20% magnitude (42%). The force decreases more rapidly in the step stretch and hold than the peak force drops in cyclic stretching at the same amplitude. Frequency Effect Figure 10 illustrates the trends in the peak tissue (Fig. 10(a, b)) and matrix force (Fig. 10(c, d)) either at the first stretch or after 12 h of cyclic stretch. It also compares the peak force trends over stretch frequencies and static incubation time. At all frequencies the peak tissue and matrix force increased with static incubation (Fig. 10). FIGURE 10.Peak tissue and matrix forces for different cyclic stretch frequencies at 10% amplitude. (A) Peak tissue force of the first cyclic stretch after 2, 4, or 8 days static incubation. Peak tissue force increased with cyclic stretch frequency and incubation time. (B) Peak tissue force after 12 h of cyclic stretch after 2, 4, or 8 days static incubation. At day 8 the peak tissue force increased slightly with increased frequency. (C) Peak matrix force of the first stretch after 2, 4, or 8 days static incubation. Peak matrix force increased slightly with increased cyclic stretch frequency. (D) Peak matrix force after 12 h of cyclic stretch after 2, 4, or 8 days static incubation. Peak matrix force similar regardless of cyclic stretch frequency. (E) Number of rings averaged for data shown in Figs. 10–13 at each frequency and static incubation day. (F) Individual measurements used in panel B [Peak tissue force after 12 h and 8 days of static incubation]. This shows that the trend within an experiment (E#) is consistent but the experiment to experiment variation is large. Y-axis scales are different in each panel to emphasize the trends with frequency and incubation time. Tissue Force Initially, higher frequencies led to higher peak tissue forces (Figs. 10(a), 11(a), 11(b)). After an initial drop, the peak force remained steady or increased at days 2 and 4. However, at day 8 after the initial drop the peak force continues to decline over the rest of the experiment (Fig. 11(a)). After 12 h, higher frequencies still resulted in higher peak forces (Figs. 10(b), 11(c)). Another difference is in the baseline forces, where lower frequencies had higher baseline forces than high frequencies. Thus, rings cyclically stretched at lower frequencies have lower measured tissue stiffness (slope of the loading curve). FIGURE 11.Tissue force versus cyclic stretch frequency at day 8. (A) Peak tissue force over 12 h of cyclic stretch. Peak tissue force dropped quickly after initiation of cyclic stretch followed by a continued slow decline. (B) First 5 tissue stretches after initiation of cyclic stretch. A large decline in tissue force occurred between the first and second stretch. (C) Last 5 tissue stretches after 12 h of cyclic stretch. The tissue stiffness increased with increasing cyclic frequency. Matrix Force Initially, higher frequencies led to higher peak matrix forces (Figs. 10(c), 12(a), 12(b)). These matrix forces quickly dropped over the first few hours and then continued a steady decline for the remainder of the cyclic stretch (Fig. 12(a)). At day 8, all the peak matrix forces quickly approached the same value about 30 min after the start of stretch (Fig. 12(a)). In addition, unlike the tissue and cell curves, there is little difference between the matrix curves at the different frequencies after 12 h except that lower frequencies contribute over a slightly larger range of strains but the peak force is still the same at all frequencies tested (Fig. 12(c)). As with the matrix force at different amplitudes, the loading curve was nonlinear and the matrix contributed only to the tissue force when the strain was within 4–6% of its maximum value. FIGURE 12.Matrix force versus cyclic stretch frequency at day 8. (A) Peak matrix force over 12 h of cyclic stretch. Peak matrix force dropped quickly after initiation of cyclic stretch followed by a slow decline for the 12 h duration. (B) First 5 matrix stretches after initiation of cyclic stretch. A large decline in matrix force occurred between the first and second stretch. (C) Last 5 matrix stretches after 12 h of cyclic stretch. Matrix loading curves were highly nonlinear and the peak forces was similar at all frequencies. Cell Force Similar to the first force-strain curves at different amplitudes, the cell force initially rose, then leveled off or even decreased at the highest frequency (Fig. 13(b)). At lower frequencies the slope of the loading curve was lower than that for higher frequencies (Fig. 13(c)). This is clear at day 8 when, for low frequency, the baseline cell force was higher and the peak cell force was lower than that of high frequencies (Fig. 13(c)). At the two lowest frequencies the cells were able to maintain a baseline force while at the two highest frequencies the baseline cell force was near zero. This leads to a cell stiffness that was higher at higher cyclic stretch frequencies. This was similar to the trend seen with the tissue stiffness. FIGURE 13.Cell force versus cyclic stretch frequencies at day 8. (A) Peak cell force over 12 h of cyclic stretch. (B) First 5 cell stretches after initiation of cyclic stretch. (C) Last 5 cell stretches after 12 h of cyclic stretch. Cell stiffness increased with increasing cyclic stretch frequency. Immunohistochemistry Rings stained with a dye for actin cytoskeleton showed a distinct morphology that developed during the 8 days of incubation. Initially the cells were sparse with few cells on the outer layer of the rings (Fig. 14(a)). The inner core of the rings initially showed a higher cell concentration that had some alignment along the circumferential direction (Fig. 14(b)). As the rings incubated, this outer layer that was approximately 25 μm thick increased in density but showed little preferential alignment (Fig. 14(c,e)). The inner core of the rings also had some increase in cell density but was noticeably less dense than the outer layer (Fig. 14(d,f)). However, these cells were highly aligned in the circumferential direction (Fig. 14(d,f)). FIGURE 14.Confocal images at 2, 4, and 8 days of static incubation. F-actin fibers visualized using a TRITC phalloidin stain. (A) Outer surface (projection from 4–14 μm depth) after 2 days static incubation. (B) Inner core (projection from 150–160 μm depth) after 2 days static incubation. (C) Outer surface (projection from 4–14 μm depth) after 4 days static incubation. (D) Inner core (projection from 61–71 μm depth) after 4 days static incubation. (E) Outer surface (projection from 10–15 μm depth) after 8 days static incubation. (F) Inner core (projection from 60–65 μm depth) after 8 days static incubation. Field of View: 230 μm × 230 μm. Circumferential direction is in the vertical direction. DISCUSSION We have shown that a few hours after the initiation of stretch, higher cyclic stretch amplitude does not lead to higher cell or tissue forces. Thus, for the majority of the cyclic stretch duration, the peak stress experienced by the cells is independent of the stretch amplitude applied, at least in the range of 5%–25%. This is accomplished by the cells becoming less stiff at higher stretch amplitudes. The exact mechanism for this change in cell stiffness is unknown. We have found that the magnitude of the drop in tissue force after the addition of 2 μm CytoD is similar to the force drop after adding detergent (e.g. deoxycholate). Since the detergent treatment actually removes the cells,18 it is likely the actin cytoskeleton is primarily responsible for the change in cell stiffness. Whether this is a result of cytoskeletal remodeling and/or reduction in active contraction/relaxation of the myosin is not known. While the initial extracellular matrix consists entirely of collagen it is reasonable to assume that over 8 days of incubation time that additional cell-contributed matrix may be produced. We have incubated rings for 20 days prior to cyclic stretching and found that the matrix force-strain curves are similar to those after 8 days of incubation. Peak forces increase only a small amount at 5% or 10% stretch amplitude after 14 h of cyclic stretch at 0.1 Hz. These results suggest that the matrix mechanics do not change substantially even in the presence of cells that can produce additional matrix. In addition, we treated some rings with 30 mM ribose after 10 days of incubation, and incubated them for an additional 10 days to non-enzymatically crosslink the collagen.9 This treatment increased the peak matrix force during cyclic stretch with a 3 to 4 fold increase after 14 h compared to the untreated rings incubated for 20 days. The shapes of the matrix force-strain curves changed somewhat, with a shorter period of slack during each force-strain curve. The increased peak force with glycation was similar to Isenberg and Tranquillo9 but comparing the shapes of the force-strain curves is difficult due to differences in experimental methods. We also found that increased matrix stiffness due to crosslinking had little effect on cell force for 5% or 10% cyclic stretch amplitude at 0.1 Hz. Cell force-strain curves were almost identical for treated and untreated rings after 12 h of cyclic stretch. In 2-D cultures, cell traction depends upon the stiffness of the substrate.6 The cellular mechanism for this response is not clear, but Discher and co-workers suggest that both “outside-in” and “inside-outside-in” pathways may be involved. Whether 3-D culture conditions or cyclic stretching reduce the influence of matrix stiffness on cellular mechanics remains to be investigated. Since it is the cells that are responding to the mechanical conditioning it is important to realize that while the cells are undergoing a higher strain they are not undergoing a higher stress. So if the cells respond to stress, higher stretch amplitudes will not increase this response. Since both the cells and the tissues at different stretch amplitudes maintain similar stresses, it is reasonable to assume the cells are responding to maintain a certain range of mechanical stresses. Some limitations to estimating the cell-contributed force should be noted. Unlike the tissue and matrix force we are not able to measure the cell force directly but instead have to estimate it based on the direct measurement of the tissue and matrix. In addition, to estimate the cell force at more than a single time point, we must use the forces from different rings. Due to the ring to ring variations, it is necessary to normalize one ring to another to get quantitative values for the cell force. However, because this normalization occurs at 12 h, it is unknown how well this single point normalization works in predicting the cell force 12 h earlier during the initial stretches. Furthermore, the assumption that the cell force is simply the subtraction of the matrix force from the tissue force is only valid after a critical cell density is reached.11 At days 2 and 4 it is unlikely that this critical cell density is reached but it is surpassed based on cell density by day 8. Unlike the cells that respond to increased stretch amplitude by decreasing their stiffness, the matrix force-strain curves shift so that the matrix contributes only when the strain is within 3%–7% of the maximum value with the matrix contributing over a slightly larger range of strains at higher stretch amplitudes. This response of the matrix does not appear to depend upon the cells having an actin cytoskeleton, since we found a similar response when cytoD is added either before stretch is initiated or after 12 h of stretch. After 12 h of cyclic stretch, the peak matrix force is only slightly higher with increased stretch amplitude. With different frequencies of cyclic stretch, the peak matrix force is similar but the matrix contributes over a larger range of strains at lower frequencies. For a 10% stretch, the matrix contributes to the last 6% of the stretch at 0.001 Hz while contributing only to the last 4% at 0.25 Hz. The higher baseline tissue and cell forces seen at lower frequencies (0.001 Hz and 0.01 Hz) are consistent with a simple viscoelastic model because the tissues and cells have more time to recover before being stretched again. The same is seen in the lower amplitudes at 0.1 Hz (5% and 10%) which are able to maintain a baseline force. These correspond to the lowest strain rates and may allow for recovery. For a linear viscoelastic material undergoing continuous cyclic stretch, both higher stretch amplitudes and higher strain rates will lead to higher peak forces. For cyclic stretch at constant frequency, the highest stretch amplitude also corresponds to the highest strain rate. However, we observed that the peak cell force decreased as the amplitude (and the strain rate) increased. We also note that the decrease in peak cell force is a function of both stretch amplitude and strain rate because similar strain rates (5%/s) at different amplitudes (e.g. 10% @ 0.25 Hz and 25% @ 0.1 Hz) show different tissue, matrix and cell responses In the case of the matrix at 25% @ 0.1 Hz, there is no positive matrix force until the strain increases beyond 10%; whereas there is matrix force below 10% strain during cyclic stretch at 10% @ 0.25 Hz. To explain this complex behavior a model of cellular mechanics that includes strain-dependent and strain rate-dependent damage may be required. It is reasonable to assume large and/or quick deformations may result in disruption of the actin cytoskeleton8 leading to damage, and thus a reduction in the cell force and stiffness. However, any damage model will be complicated by viscoelastic effects. One possible mechanism for a strain dependent change in stiffness is an alteration in the amount of cell activation through a stretch-activated pathway. Two of the pathways that exist to activate myosin-light chain kinase and thus force generation in fibroblasts are a calcium dependent pathway and a Rho dependent pathway.13 To test the possible involvement of a calcium dependent pathway, we tested some rings in the presence of 50 μM or 500 μM of gadolinium chloride (GdCl3) to block stretch activated calcium channels.12 In the presence of 50 μM of GdCl3, day 2 rings showed a decrease in peak tissue force at both 5% and 10% cyclic stretch compared to untreated controls. This peak force decrease continued for the first 5–6 h of cyclic stretch then began to increase for the remainder of the 12 h of cyclic stretching but still did not return to the peak tissue force of untreated controls. However, for day 8 rings neither 50 nor 500 μM GdCl3 had an effect on the tissue force compared to untreated controls. Since GdCl3 reduces the tissue and cell force during the first several hours of cyclic stretch at day 2, it suggests that the calcium dependent pathway plays a role in maintenance of cell force at least during early tissue development. It is unclear what causes the cell force to increase after the initial decrease but it could be due to an upregulation of calcium independent pathways or incomplete inhibition of new or existing stretch activated channels. The lack of an effect of GdCl3 at day 8 suggests a reduced role of the calcium dependent pathway. In addition, these results have important implications for the tissue engineering field, especially for those tissues that undergo continuous deformations such as in the cardiovascular system. By isolating the cell contribution to tissue force, we are able to provide a better understanding of the mechanical environment of the cell and aid in designing mechanical conditioning protocols that improve tissue properties. For example, the peak cell force is not constant during cyclic stretch and thus cell stimulation varies with time. Furthermore, if cell response is dependent on cell stress or force, increasing the amplitude of conditioning will not increase and may decrease the applied cell force. Due to the large changes in mechanical behavior after initiation of cyclic stretch, it is important to characterize engineered tissues’ response to these loading conditions over longer time periods. Current mechanical characterization relies mostly on a few cycles of preconditioning followed by a test to failure.4,5,9,16 While failure tests are necessary, the response of the tissue and cells under physiologic loading is also vital. We have shown that mechanical properties may change dramatically over hours after initiation of stretch. These changes have implications on tissue function in vivo after implantation and these changes cannot be captured in simple failure tests. CONCLUSIONS We have shown that bioartificial tissue constructs consisting of a single cell type and matrix component are a useful model system in which to explore tissue, matrix, and cell mechanics. The cell response to cyclic stretch is complex and depends on both the amplitude and frequency of cyclic stretch. The change in cell stiffness with amplitude behavior does not fit current cellular mechanics models. Future investigations may explore the microstructural basis for this behavior.

Sleep_Breath-4-1-2194800

Armodafinil improves wakefulness and long-term episodic memory in nCPAP-adherent patients with excessive sleepiness associated with obstructive sleep apnea

Residual excessive sleepiness (ES) and impaired cognition can occur despite effective and regular nasal continuous positive airway pressure (nCPAP) therapy in some patients with obstructive sleep apnea (OSA). A pooled analysis of two 12-week, randomized, double-blind studies in nCPAP-adherent patients with ES associated with OSA evaluated the effect of armodafinil on wakefulness and cognition. Three hundred and ninety-one patients received armodafinil (150 or 250 mg) and 260 patients received placebo once daily for 12 weeks. Efficacy assessments included the Maintenance of Wakefulness Test (MWT), Cognitive Drug Research cognitive performance battery, Epworth Sleepiness Scale, and Brief Fatigue Inventory. Adverse events were monitored. Armodafinil increased mean MWT sleep latency from baseline to final visit by 2.0 min vs a decrease of 1.5 min with placebo (P < 0.0001). Compared with placebo, armodafinil significantly improved quality of episodic secondary memory (P < 0.05) and patients’ ability to engage in activities of daily living (P < 0.0001) and reduced fatigue (P < 0.01). The most common adverse events were headache, nausea, and insomnia. Armodafinil did not adversely affect desired nighttime sleep, and nCPAP use remained high (approximately 7 h/night). Adjunct treatment with armodafinil significantly improved wakefulness, long-term memory, and patients’ ability to engage in activities of daily living in nCPAP-adherent individuals with ES associated with OSA. Armodafinil also reduced patient-reported fatigue and was well tolerated. Introduction Obstructive sleep apnea (OSA) syndrome includes excessive sleepiness (ES), fatigue, sleep fragmentation, impaired alertness, cognitive dysfunction, and mood disturbances [1, 2]. OSA is typically characterized by recurrent complete or partial airway collapse during sleep, resulting in frequent apnea and hypopneic events lasting at least 10 s [1]. Although the reported prevalence of OSA in the general population varies as a function of diagnostic criteria [2], OSA syndrome has been estimated to occur in 4 and 2% of middle-aged North American men and women, respectively [3]. OSA can lead to serious medical, social, and public safety consequences, and patients are at an increased risk for cardiovascular disease [4] and motor vehicle and occupational accidents [5, 6]. Quality of life and social functioning are also significantly impaired in this patient population [7, 8]. Fatigue, in particular, can contribute to reduced quality of life [1, 9]; thus, the assessment of patient-rated fatigue is an important outcome to evaluate and monitor in patients with OSA. Nasal continuous positive airway pressure (nCPAP) therapy is the recommended standard of care for treating patients with moderate to severe OSA [10]. In a comprehensive review of 36 clinical studies, nCPAP therapy was shown to effectively reduce objective and subjective measures of sleepiness and improve quality of life in patients with moderate and severe OSA [11]. However, despite receiving adequate nCPAP therapy, half of patients with OSA still experience residual ES as measured by objective assessments as well as impairments in mood or cognition [12–18]. Cognitive impairment in patients with OSA is characterized by deficits in attention, learning and memory, and executive function (planning and problem solving) and may persist despite nCPAP therapy [12, 15–17]. A review of the literature suggests that while nCPAP therapy can substantially improve quality of life for patients with OSA, it has a smaller effect on improving cognitive performance [7]. Modafinil, a racemic mixture consisting of equal amounts of R- and S-enantiomers, has been shown to improve wakefulness in patients with OSA who experience residual ES despite regular nCPAP use [19, 20]. Pharmacokinetic studies have shown that the half-life of R-modafinil (10–14 h) is significantly longer than the half-life of S-modafinil (3–4 h) [21–23]. Armodafinil has higher plasma concentrations later in the day compared to modafinil on a “milligram-to-milligram” basis and improves healthy subjects’ wakefulness and ability to sustain attention for a longer period of time compared with modafinil [24]. Armodafinil has also recently been shown to improve wakefulness in patients with ES associated with narcolepsy in a 12-week double-blind, placebo-controlled study [25]. Recent findings from two 12-week, double-blind, placebo-controlled studies of similar design indicate that armodafinil improved wakefulness and overall clinical condition in nCPAP-adherent patients with OSA and residual ES [26, 27]. In the present pooled analysis of the two OSA studies [26, 27], the effects on wakefulness, cognition, and fatigue and the safety and tolerability of armodafinil compared with placebo were evaluated in OSA patients adherent to nCPAP therapy. Materials and methods Study design and procedures Data from two 12-week, multicenter, double-blind, placebo-controlled, parallel-group clinical studies were pooled for analysis. The pooling of these data was based on similarities in patient population, study design, and treatment duration in the two studies [26, 27]. One study was conducted in 36 centers across the USA, Australia, Russia, Germany, and France [26], and the other study was conducted in 37 centers across the USA and Canada [27]. The study protocols were approved by an Independent Ethics Committee or Institutional Review Board at each center. Both studies were conducted in accordance with the Good Clinical Practice: Consolidated Guideline [28] and national and local laws and regulations. Patients were randomized to receive armodafinil 150 mg (n = 129) or placebo (n = 130) daily in the first study [26] and armodafinil 150 (n = 131) or 250 mg/day (n = 131), or placebo (n = 130) in the second study [27]. Study drugs were initiated with a dose of 50 mg/day on day 1 and then increased in increments of 50 mg starting on day 2 and every 2 days thereafter until the assigned dose was reached (i.e., 150 mg/day on day 4 for both studies or 250 mg/day on day 8 for the second study). The study drugs were taken once daily in the morning (before 08:00 and approximately 30 min before breakfast). Monitoring of patients’ compliance with study drug administration was the responsibility of the investigator at each center and was assessed by completed study drug accountability records and reviews of patient diaries. nCPAP use at home was also monitored objectively by investigators at each visit to the study center, using the REMstar® Auto CPAP System (Respironics, Murrysville, PA). The REMstar Auto CPAP System was used in CPAP mode. Patient selection criteria Methods for selecting patients in both studies have been described previously [26, 27]. Men and women 18 to 65 years of age with a current diagnosis of OSA as defined by the International Classification of Sleep Disorders [1] were included in both studies. Patients were at least moderately ill (Clinical Global Impression of Severity of Illness rating greater than or equal to 4) [29] and had a complaint of ES. Patients were eligible if ES was determined to be pathological, based on an Epworth Sleepiness Scale (ESS) score greater than or equal to 10 [30]. All patients were receiving stable (greater than or equal to 4 weeks) and effective nCPAP therapy on a regular basis (greater than or equal to 4 h per night on greater than or equal to 70% of nights) during the 2-week evaluation period. The CPAP use was monitored using the REMstar Auto CPAP System. Effectiveness of therapy was determined by an apnea–hypopnea index (AHI) less than or equal to ten events per hour on nighttime polysomnography (PSG). Patient exclusion criteria Patients were excluded if they had any of the following: a diagnosis of a sleep disorder other than OSA, symptoms of ES associated with a clinically significant uncontrolled medical or psychiatric condition as determined by the investigator, any disorder that might interfere with drug absorption, distribution, metabolism, or excretion, a history of drug or alcohol abuse, as determined by the Diagnostic and Statistical Manual of Mental Disorders IV criteria [31] or by a positive result from the urine drug screen given at screening and again at the final visit, caffeine consumption greater than 600 mg/day, or a clinically significant sensitivity to central nervous system stimulants or modafinil. Women who were pregnant or breast feeding were also excluded. The use of any substance that could affect wakefulness or sleepiness (e.g., modafinil, sodium oxybate, melatonin, lithium, St. John’s wort, methylphenidate, amphetamines, pemoline, antipsychotic agents, benzodiazepines, zolpidem, anticonvulsants, or barbiturates), use of other excluded agents (e.g., monoamine oxidase inhibitors, anticoagulants), use of clinically significant amounts of nonprescription drugs within 7 days of screening, and use of investigational drugs within 1 month of screening was prohibited. All patients provided written informed consent before participation in these studies and were compensated for their participation. Assessments Efficacy assessments Efficacy assessments were performed for both studies at baseline and weeks 4, 8, and 12. The primary efficacy variables in the two individual studies were the change from baseline to final visit (week 12 or last postbaseline measurement) in mean Maintenance of Wakefulness Test (MWT) [32, 33] sleep latency averaged across the first four tests and the proportion of patients with at least minimal improvement on the Clinical Global Impression of Change (CGI-C) [29]. The MWT was conducted as six separate 30-min sessions at 09:00, 11:00, 13:00, 15:00, 17:00, and 19:00. Sleep latency was defined as the time to onset of the first of three consecutive epochs of stage 1 sleep or the time to onset of any epoch of stages 2, 3, and 4 sleep or rapid eye movement sleep [34]. Sleep latencies were averaged across the first four tests (09:00, 11:00, 13:00, 15:00) and the last three tests (15:00, 17:00, 19:00) to distinguish between early- and late-day effects, respectively. The proportion of patients with at least a minimal improvement on the CGI-C was assessed to determine patients’ overall clinical improvement in the individual studies; however, these data were not poolable for this analysis because of a substantial difference in the percentage of responders to placebo between the two studies. The Cognitive Drug Research (CDR) battery of tests [35, 36] was administered by computer as six separate 25-min sessions conducted at 09:30, 11:30, 13:30, 15:30, 17:30, and 19:30. The CDR battery includes five memory tests (immediate word recall, delayed word recall, numeric working memory, word recognition, and picture recognition) and three attention tests (simple reaction time, choice reaction time, and digit vigilance). Two composite factors are derived from the memory tests in the CDR: quality of episodic secondary memory, a measure of long-term memory that measures the ability to recall verbal and visual information, and speed of memory, which assesses the time it takes to decide whether information is held in memory. Two composite factors are derived from the CDR for attention: power of attention, which measures the ability to focus attention and avoid distraction (concentration), and continuity of attention, which measures the ability to sustain attention (vigilance). Similar to the MWT, the CDR scores were averaged across the first four test sessions (09:30, 11:30, 13:30, 15:30) and the last three test sessions (15:30, 17:30, 19:30) to assess cognitive effects on early- and late-day measurements. The patients’ ability to engage in daily activities was assessed at each visit by the ESS [30]. ESS total scores range from 0 to 24, with higher scores indicating greater sleepiness. An ESS score greater than or equal to 10 indicates pathological sleepiness [30]. The severity and impact of patient-rated fatigue on daily functioning were assessed at each visit by the nine-item Brief Fatigue Inventory (BFI) [37] and were based on changes in global fatigue (average of all nine questions) and worst fatigue in the past 24 h (item 3). The rating scale for the BFI ranges from 0 (no fatigue) to 10 (as bad as you can imagine). A score of greater than or equal to 7 for either global fatigue or worst fatigue in the past 24 h is indicative of severe fatigue. Both the ESS and BFI were administered before the first MWT session (09:00) at each visit. Safety and tolerability assessments Adverse events were monitored and recorded by the study investigators at each center throughout both studies. Clinical laboratory tests (blood chemistry, hematology, urinalysis), vital signs (resting heart rate and systolic and diastolic blood pressure 3 and 13 h postadministration), and electrocardiograms were obtained at screening, baseline, and weeks 4, 8, and 12. Physical examinations were performed at baseline and week 12. Patients’ use of nCPAP was monitored at least 2 weeks before baseline and throughout both studies using the Respironics REMstar Auto CPAP system. Effect on nighttime sleep was determined by PSG, which was performed the night immediately after the measurement of daytime MWT during the second screening visit and the final visit. The PSG was conducted for 8 h, starting within 30 min of the patient’s usual bedtime but not earlier than 21:30. Clinically significant elevations in resting systolic and diastolic blood pressure were defined a priori as greater than or equal to 140 mmHg with an increase of greater than or equal to 10% and greater than or equal to 90 mmHg with an increase of greater than or equal to 10%, respectively. Patients with worsening hypertension included those who had a history of hypertension at baseline and who started new antihypertensive medication and/or increased the dose of previously used antihypertensive medication during the studies. Patients with newly diagnosed hypertension included those who had no history of hypertension at baseline and who started antihypertensive medication during the studies. Patients at risk for hypertension included those who had at least two clinically significant elevations in blood pressure readings between baseline and final visit. Statistical analysis Descriptive statistics were used to summarize continuous and categorical demographic variables. Efficacy assessments were analyzed at weeks 4, 8, and 12 using observed cases and at final visit (week 12 or last postbaseline measurement) using the last observation carried forward approach. Efficacy analyses included randomized patients who received at least one dose of study drug and had a baseline measurement with at least one postbaseline measurement on the MWT and CGI-C. Safety and tolerability analyses included all randomized patients who received at least one dose of study drug. All efficacy assessments were analyzed by analysis of variance (ANOVA) with treatment and study as factors. Tests of poolability for all continuous efficacy variables across the two studies were conducted using an ANOVA with treatment and study and treatment by study interaction as factors. Vital signs and data from nighttime PSG were analyzed by Wilcoxon rank-sum test. For nCPAP use, the change from baseline to on-treatment values was analyzed using one-way ANOVA. All statistical tests were two-tailed, and the 5% level of significance was used. Results Patient demographics and disease characteristics Of 658 patients randomized, 651 were included in the safety analysis, and 601 were evaluable for efficacy (Fig. 1). Patient demographics and disease characteristics for all patients in the safety analyses are summarized in Table 1. The mean body mass index was 36.6 kg/m2. Patients reported high nightly nCPAP usage (mean [SD] hours of nightly use, armodafinil 6.9 [1.2] h; placebo 6.9 [1.0] h). The nCPAP therapy was effective, as shown by mean (SD) AHI values of 1.5 (3.3) and 1.2 (2.1) events/h in the armodafinil and placebo groups, respectively. Overall, 41% of patients had a history of hypertension at baseline. Fig. 1Patient dispositionTable 1Patient demographics and baseline characteristicsCharacteristicArmodafinil (n = 391)Placebo (n = 260)Age (years)Mean (SD)49.7 (9.0)50.3 (9.1)Sex, n (%)Men283 (72)183 (70)Women108 (28)77 (30)Race, n (%)White327 (84)224 (86)Black36 (9)21 (8)Asian6 (2)3 (1)Other22 (6)11 (4)Missing01 (<1)Weight (kg)Mean (SD)110.4 (24.6)111.2 (23.7)BMI (kg/m2)Mean (SD)36.4 (8.0)36.9 (7.5)CGI-S, n (%)Moderately ill219 (56)138 (53)Markedly, severely, or extremely ill172 (44)122 (47)nCPAP (h)Mean (SD)6.9 (1.2)6.9 (1.0)AHIMean (SD)1.5 (3.3)1.2 (2.1)History of hypertensionn (%)159 (41)108 (42)AHI Apnea–hypopnea index, BMI body mass index, CGI-S Clinical Global Impression of Severity of Illness, nCPAP nasal continuous positive airway pressure Effects on wakefulness Mean (SD) sleep latency at baseline across the first four MWTs (09:00, 11:00, 13:00, 15:00) was 22.8 (8.4) and 23.2 (7.9) min for the armodafinil and placebo groups, respectively. At all study visits, armodafinil significantly improved mean sleep latency across the first four MWTs compared with placebo (Fig. 2a). The mean change from baseline in sleep latency at final visit was 2.0 min for the armodafinil group compared with −1.5 min for the placebo group (P < 0.0001). Fig. 2Mean (SEM) change from baseline in sleep latency from the Maintenance of Wakefulness Test (MWT). a Sleep latencies averaged across the first four tests (09:00–15:00). b Sleep latencies averaged across the last three tests (15:00–19:00) Armodafinil also improved patients’ ability to maintain late-day wakefulness (MWTs at 15:00, 17:00, 19:00) compared with placebo (Fig. 2b). Mean (SD) sleep latency at baseline across the last three MWTs was 24.4 (7.8) and 24.6 (7.4) min for the armodafinil and placebo groups, respectively. The mean change from baseline in sleep latency at final visit was 1.1 min for the armodafinil group compared with −0.3 min for the placebo group (P < 0.05). Effects on memory and attention Compared with placebo, armodafinil significantly improved the quality of episodic secondary memory across the first four tests (09:30, 11:30, 13:30, 15:30) at all study visits (P < 0.05; Fig. 3a). The mean (SD) change from baseline in quality of episodic secondary memory at final visit was 10.2 (31.7) U for the armodafinil group compared with −0.7 (46.3) U for the placebo group (P < 0.01). Differences between the treatment groups did not achieve statistical significance at the final visit for speed of memory, power of attention, or continuity of attention across the first four tests. Fig. 3Mean (SEM) change from baseline in quality of episodic secondary memory. a Scores averaged across the first four tests (09:30–15:30). b Scores averaged across last three tests (15:30–19:30) Across the later three tests of the day (15:30, 17:30, 19:30), the difference in the quality of episodic secondary memory was statistically significant at week 8 for armodafinil compared with placebo (P = 0.011; Fig. 3b), with a trend toward significance at week 12 (P = 0.081). The difference between treatment groups was not statistically significant at the final visit for the quality of episodic secondary memory. There were no significant changes from baseline to final visit between the armodafinil and placebo groups for speed of memory, power of attention, or continuity of attention across the last three tests. Effects on patients’ ability to engage in activities The mean (SD) ESS scores were high at baseline (15.4 [3.5] and 15.9 [3.5] for the armodafinil and placebo groups, respectively), despite the high nCPAP use as shown in Table 1. Compared with placebo, treatment with armodafinil significantly improved patients’ ability to engage in activities of daily living (ESS) at all visits (P < 0.0001; Fig. 4). Nearly half of all patients (49%) in the armodafinil group responded to treatment (total ESS score less than 10 at final visit) compared with 26% in the placebo group. Fig. 4Mean (SEM) change from baseline in Epworth Sleepiness Scale total score Effects on fatigue The mean (SD) scores for global fatigue from the BFI at baseline were 4.8 (1.9) and 4.9 (1.9) for the armodafinil and placebo groups, respectively. Armodafinil significantly improved global fatigue at all visits compared with placebo (P < 0.01; Fig. 5a). Mean scores for worst fatigue in the past 24 h from the BFI at baseline were nearly identical for the armodafinil and placebo groups (7.2 [2.0] and 7.3 [2.0], respectively) and indicated severe fatigue (score greater than or equal to 7). Armodafinil significantly improved scores for worst fatigue in the past 24 h at weeks 4 and 12 and at the final visit (P < 0.05 vs placebo; Fig. 5b), with a trend toward significance at week 8 (P = 0.056 vs placebo). Fig. 5Mean (SEM) change from baseline in fatigue. a Global fatigue. b Worst fatigue in the past 24 h. BFI Brief Fatigue Inventory Safety and tolerability Armodafinil was well tolerated, with a low incidence of adverse events, most of which were mild to moderate in nature. Headache was the most commonly reported adverse event, defined as occurring in greater than or equal to 5% in either group (Table 2). Serious adverse events were considered by the investigator unlikely to be or not related to armodafinil (ulcerative colitis, n = 1; migraine, n = 1; worsening of Axis II disorder and mood disorder, n = 1; duodenal ulcer hemorrhage, n = 1). One serious adverse event was reported in the placebo group (gastroesophageal reflux disease). The most frequently occurring adverse events that led to discontinuation among patients receiving armodafinil were headache (n = 5) and nausea (n = 4). Table 2Adverse events occurring in ≥5% of patientsAdverse event, n (%)Armodafinil (n = 391)Placebo (n = 260)Headache65 (17)20 (8)Nausea22 (6)10 (4)Insomnia22 (6)3 (1)Dizziness19 (5)4 (2)Anxiety20 (5)2 (<1) There were no clinically significant changes from baseline to final visit in either the armodafinil group or the placebo group for laboratory values, electrocardiogram parameters, or physical examinations. There were also no meaningful changes (clinical or statistical) from baseline to final visit for systolic blood pressure (0.2 [14.2] mmHg for armodafinil vs −1.0 [14.6] mmHg for placebo), diastolic blood pressure (0.3 [9.3] mmHg for armodafinil vs −1.0 [10.0] mmHg for placebo), and heart rate (2.3 [9.6] bpm for armodafinil vs 1.4 [9.6] bpm for placebo). The incidence of patients with newly diagnosed hypertension was less than 1% in the armodafinil group and less than 1% in the placebo group. The proportion of patients at risk for hypertension was similar for both treatment groups (18% of 391 patients, armodafinil group; 16% of 260 patients, placebo group). The incidence of patients with worsening hypertension was 3% of 391 patients in the armodafinil group and 2% of 260 patients in the placebo group. nCPAP use remained high (approximately 7 h/night) throughout both of the studies. The duration of nCPAP use at final visit was reduced from baseline by a mean (SD) of −0.3 (0.7) h with armodafinil compared with −0.1 (0.6) h with placebo (a between-group difference of about 12 min; P < 0.0001). At final visit, AHI values remained low and were comparable between the armodafinil and placebo groups (P > 0.05 vs placebo). In addition, there were no significant changes from baseline in nocturnal PSG sleep variables between the armodafinil and placebo groups (P > 0.05; Table 3). Table 3Nocturnal polysomnography parametersaVariable (units), mean (SD)Armodafinil (n = 391)Placebo (n = 260)BaselineFinal VisitBaselineFinal VisitLatency to persistent sleep (min)22.3 (26.9)19.6 (20.5)21.3 (24.0)20.8 (21.4)Number of arousals, n20.0 (11.3)18.5 (10.2)18.7 (9.7)18.4 (10.4)Number of awakenings, n8.8 (4.7)9.2 (5.3)8.7 (5.1)9.6 (5.4)Sleep efficiency (%)82.4 (10.9)82.0 (12.0)82.0 (12.1)81.4 (11.2)Wake after sleep onset (min)66.6 (43.9)69.1 (48.5)68.7 (50.3)70.2 (46.5)Stage 1 (%)11.2 (6.4)10.5 (5.5)10.9 (6.1)10.6 (6.2)Stage 2 (%)59.3 (9.8)58.8 (11.2)58.8 (10.1)57.7 (11.2)Stage 3/4 (%)10.6 (9.0)10.3 (9.0)10.8 (9.6)10.9 (10.2)REM (%)18.9 (6.9)19.8 (7.1)19.5 (7.2)20.7 (8.0)REM Rapid eye movementaNo significant differences between baseline and final visit were observed. Discussion Recognizing and effectively managing ES in patients with OSA is essential for clinicians because of the profound impact that ES has on the patients, their families and coworkers, and the general public (e.g., increased risk of traffic accidents) [4–6]. Without appropriate treatment, ES adversely affects cognitive [15, 17], occupational [6], and social functioning [8]. Several studies evaluated in a comprehensive review [11] have shown that there is general improvement in wakefulness with nCPAP therapy, especially in patients with more severe OSA. Nonetheless, residual ES can persist even after effective nCPAP treatment [18], suggesting that there is need for adjunctive therapy to treat ES in these patients. The present study re-evaluates the findings from two separate studies on the effects of armodafinil in patients with refractory ES associated with OSA and provides additional information regarding secondary analyses, for which the individual studies may not have had adequate statistical power. In the individual studies [26, 27], armodafinil was shown to significantly improve daytime mean sleep latency across the first four tests on the MWT and overall clinical condition as assessed by the CGI-C. In the pooled analysis, armodafinil significantly improved patients’ ability to sustain wakefulness as objectively assessed by the MWT. Improvements in mean sleep latency were observed at the first visit at week 4 and were maintained throughout the remainder of the study. The lack of a consistent effect of armodafinil on wakefulness across the last three MWTs (15:00, 17:00, 19:00) likely reflects the high mean sleep latency (approximately 24.5 min) at these times. Behavioral effects, causing poorer performance on the last test of the day, and high intertest variability in sleep latency may have also contributed to this finding [38]. The individual studies also included the clinician’s subjective assessment of treatment effect on ES, the CGI-C. Data for the CGI-C, however, were not poolable for this analysis because of substantial differences in the proportion of patients considered responders to placebo between the two studies. In the individual studies, the proportion of patients considered responders to armodafinil and placebo was 71 and 53%, respectively, in one study [26] and 72 and 37%, respectively, in the other study [27]. Cognitive impairment is common in patients with OSA [17]. Although the etiology has not been definitively determined, decreased cortical activity because of impaired arousal or neuronal damage because of chronic intermittent hypoxia are possible causes [16, 39–41]. It is not yet clear what additional interventions may be effective in restoring cognitive function in patients with OSA. Findings from studies evaluating the effects of nCPAP therapy on memory and attention have yielded inconsistent results [15, 16, 40, 42, 43]. Unlike the findings from the individual armodafinil OSA studies [26, 27], the pooled analysis showed that adjunct armodafinil significantly improves long-term memory (i.e., quality of episodic secondary memory) at all study visits compared with placebo, indicating that treatment was associated with a greater ability to recall verbal and visual information. Interestingly, the long-term memory benefit occurred independent of a consistent benefit on attention and concentration. It is important to note that the CDR composite factors in both of the individual armodafinil OSA studies were secondary efficacy variables. Thus, although results from the pooled analysis provide a more precise estimate of the effects on long-term memory, further studies are needed to determine the potential role of armodafinil in improving cognitive function in nCPAP-adherent patients with OSA and associated ES. Armodafinil significantly improved patients’ ability to engage in activities of daily living at all visits as measured by the ESS. The patient population studied in this pooled analysis had severe ES (mean ESS score greater than 15) despite effective and regular nCPAP therapy. At the final visit, 49 and 26% of patients receiving armodafinil and placebo, respectively, had ESS scores less than 10, indicating that nearly half of patients no longer had pathological sleepiness with adjunct armodafinil treatment. Fatigue may be the presenting complaint of patients with ES [44] and is a common associated symptom [45, 46]. Armodafinil significantly reduced fatigue, a result similar to findings from the individual studies [26, 27]. Armodafinil reduced global fatigue from a baseline value of 4.8 to a final visit value of 3.6. Similarly, worst fatigue in the past 24 h was reduced from a baseline of 7.2 to 5.8 at final visit. These findings indicate that armodafinil significantly reduced the severity of global fatigue and worst fatigue in the past 24 h to the mild-to-moderate range of fatigue (BFI score less than 7 [37]). This pooled analysis shows that armodafinil does not adversely affect nighttime sleep in patients with OSA even with its longer-lasting duration of action compared with modafinil on a milligram-to-milligram basis [24]. Armodafinil was well tolerated; in general, adverse events were rated as mild to moderate in nature, although some patients discontinued because of adverse events. There were some minor changes in vital signs; however, these effects were not considered to be clinically significant. The patient population selected for both studies was verified to be adherent to nCPAP therapy; thus, the primary pathology of OSA was being treated in an appropriate and effective manner throughout the course of the two studies. At the final visit, the duration of nCPAP use had decreased minimally from baseline compared with placebo. Average nCPAP use remained high (approximately 7 h/night) and was effective (i.e., low AHI values). Adjunct armodafinil treatment did not affect arousals. Findings from the present pooled analysis are limited to patients with OSA who have residual ES despite regular and effective nCPAP therapy and should not be generalized to patients with OSA who are not receiving adequate nCPAP therapy or are not using it regularly. Additionally, the 12-week duration of treatment in these studies limits the applicability of observed results to a longer period of treatment. It should be recognized that armodafinil does not treat the underlying airway obstruction and should not be considered a replacement for nCPAP therapy in patients with OSA. Further research is needed to determine the role armodafinil may have in improving cognitive function and whether the significant reduction in fatigue observed in the pooled analysis will contribute toward improved quality of life in this patient population. In conclusion, pooled data from two 12-week, double-blind, placebo-controlled studies showed that once-daily administration of armodafinil significantly improved wakefulness when used as adjunct therapy in nCPAP-adherent patients with residual ES associated with OSA. The effect on wakefulness with armodafinil was maintained throughout the day. Importantly, adjunctive treatment with armodafinil was associated with significant improvements in long-term memory and patients’ ability to engage in daily activities. Armodafinil significantly reduced fatigue in the studied population and was well tolerated.

Childs_Nerv_Syst-3-1-1849423

Paradigm shift in hydrocephalus research in legacy of Dandy’s pioneering work: rationale for third ventriculostomy in communicating hydrocephalus

Objective This study aims to question the generally accepted cerebrospinal fluid (CSF) bulk flow theory suggesting that the CSF is exclusively absorbed by the arachnoid villi and that the cause of hydrocephalus is a CSF absorption deficit. In addition, this study aims to briefly describe the new hydrodynamic concept of hydrocephalus and the rationale for endoscopic third ventriculostomy (ETV) in communicating hydrocephalus. Introduction The modern era of hydrocephalus research began with the experimental studies of Dandy and Blackfan [3] in 1914, which, until today constitutes an unsurpassed contribution to this field. By plugging the aqueduct, they produced hydrocephalus in dogs. The division of hydrocephalus into the obstructive and communicating type originates from their work. The bulk flow theory and the concept of cerebrospinal fluid (CSF) malabsorption as the cause of hydrocephalus are also based on their experiment [3]. However, Dandy strongly opposed the view that idiopathic communicating hydrocephalus is caused by an obstruction at the arachnoid villi. Such an obstruction cannot cause a higher pressure in the ventricles than in the subarachnoid space but would rather dilate the subarachnoid space. He also objected to the view that the CSF is absorbed by the arachnoid villi and instead proved that the CSF is absorbed by the capillaries [3, 4]. If this is true, the modern era of CSF and hydrocephalus research started and ended with Dandy, who left the pathophysiology of idiopathic communicating hydrocephalus unexplained for future exploration. By going back to Dandy and following his lead of a capillary CSF absorption, a new understanding of hydrocephalus may be found. O’Connel [19] and Bering [1] suggested that increased pulse pressure in the ventricles is the cause of communicating hydrocephalus. In 1978, Di Rocco and Pettorossi [5] verified that an intraventricular pulsating balloon causes communicating hydrocephalus in sheep. In 1993, Greitz re-valuated the physiology of the CSF circulation and hydrocephalus by using flow sensitive magnetic resonance imaging (MRI) and radionuclide cisternography [9]. He suggested that the capillaries absorb the CSF and that the distending force in the production of chronic hydrocephalus is an increased systolic pulse pressure in brain tissue [14]. The hydrodynamic concept of hydrocephalus Classification of hydrocephalus The classification is based on capillary absorption of the CSF [9–14]. Hydrocephalus is divided into two main groups, acute hydrocephalus and chronic hydrocephalus [14]. Acute hydrocephalus is caused by an intraventricular CSF obstruction in accordance with the conventional view. As opposed to that view, it is suggested that chronic hydrocephalus is caused by decreased intracranial compliance. Causes of decreased intracranial compliance Although the etiology of the decreased intracranial compliance such as CSF obstructions at the foramen magnum and adhesions in the subarachnoid space after intracranial bleedings, trauma, operations and infections is well known, the etiology remains unknown in many idiopathic and congenital cases of hydrocephalus. However, the final stage of chronic hydrocephalus, independent of its etiology, is uniform. The final stage is characterised by narrow capacitance vessels and reduced cerebral blood volume. This reduces cerebral blood flow, reduces intracranial compliance and enhances the intracerebral pulse pressure. Intracerebral pulse pressure theory of chronic hydrocephalus Chronic hydrocephalus consists of two subtypes, communicating hydrocephalus and chronic obstructive hydrocephalus [14]. The associated malabsorption of CSF is not involved as a causative factor in chronic hydrocephalus. The theory is based on one basic mechanism, i.e. that chronic hydrocephalus is caused by decreased intracranial compliance increasing the systolic pressure transmission into the brain parenchyma [9, 10, 12–14]. The increased systolic pressure in the brain is the cause of the distension of the brain and ventricles. The systolic expansion of the brain distends the brain towards the skull and simultaneously compresses the periventricular region of the brain. The rationale result is the predominant enlargement of the ventricles and a narrowing of the subarachnoid space. Rationale for ETV in communicating hydrocephalus As described above, communicating hydrocephalus is caused by decreased compliance increasing the systolic pressure transmission into the brain. The systolic force compresses the brain including the intracranial capacitance vessels, i.e. the cerebral veins and capillaries. This results in a vicious cycle with narrow capacitance vessels and significantly reduced cerebral blood volume that causes further decrease in intracranial compliance and further increase in intra-cranial pulse pressure. It is obvious that an endoscopic third ventriculostomy (ETV) may interrupt the vicious cycle and reduce the systolic pressure in the brain simply by venting ventricular CSF through the stoma. The patent aqueduct in communicating hydrocephalus is too narrow to vent the ventricular CSF sufficiently. By reducing the pulse pressure, the ETV also reduces the compression of the capacitance vessels thereby restoring some degree of venous compliance, which in turn reduces the intracerebral pulse pressure further. ETV versus shunting The proposed concept opens a new avenue in that ETV may be an effective treatment also in communicating hydrocephalus [8, 16–18]. It thus constitutes an interchangeable alternative to shunting. The primary aim of the treatment of chronic hydrocephalus is to restore intracranial compliance, which is achieved by both treatments [14]. Effective shunting is based on a slight over-drainage of CSF that ultimately causes a direct and forced dilation of the compressed veins [14]. Therefore, shunting is more effective in increasing intracranial compliance than ETV where the dilating effect on the veins works indirectly by reducing the systolic compressing force on the veins. Because ETV provides a more physiological treatment and has less late complications than shunting, it may be of benefit to increase the number of patients finally treated by ETV [2, 6–8, 15, 17, 18, 20]. In patients shunted under 1 year of age, ETV should be considered as an alternative treatment when they grow older. However, the final answer as to which is the optimal treatment must be based on randomized clinical studies. Discussion The aim of this paper is to briefly describe the hydrodynamic concept of hydrocephalus, which is based on Dandy’s observation of capillary absorption of the CSF. Oi and Di Rocco [20] recently suggested that a minor CSF pathway through the brain parenchyma is the dominant CSF absorption site in the embryo, foetus and infant. The theory described here suggests that the minor pathway in the developing immature brain as well as the major pathway in adults are through the brain capillaries. It is beyond the scope of this brief communication to present the numerous physiological (and self-evident) evidences that the brain capillaries have the capacity to absorb fluids such as the CSF and its constituencies [3, 4, 9, 11, 12, 14]. This contrasts to the total lack of physiological evidence that the arachnoid villi can absorb the CSF. The CSF dynamics has been described in earlier published papers [9, 11, 12, 14]. The proposed concept indicates a paradigm shift in our view on hydrocephalus. It is suggested that chronic hydrocephalus is caused by decreased intracranial compliance rather than a CSF absorption deficit [14]. The associated CSF malabsorption is a secondary phenomenon to the decreased intracranial compliance. The only residue of the conventional view is acute hydrocephalus, which is caused by a CSF absorption deficit (of the periventricular capillaries). Normal-pressure hydrocephalus, aqueductal stenosis as well as communicating hydrocephalus in infants and adults are typical manifestations within the spectrum of chronic hydrocephalus. It should be emphasised that chronic hydrocephalus is an unstable hemodynamic disorder occurring at normal or slightly increased mean intracranial pressure with superimposed vascular pressure waves that can be fatal. The theory may thus have identified decreased intra-cranial compliance as a new and potential universal cause of chronic hydrocephalus. Decreased compliance is common to all different types of chronic hydrocephalus whatever their etiology. If the theory is correct, there is hope for improved treatment of chronic hydrocephalus of unknown etiology. How come then that treatment failure rather is the rule than the exception of this devastating condition? The reason may be that shunting and ETV essentially represents symptomatic treatments of hydrocephalus. To improve therapy, we have to find treatments that are more effective in increasing compliance in cases with unknown etiology or are directed against the cause of the decreased compliance in cases with known etiology.

J_Biomol_NMR-4-1-2268728

BioMagResBank (BMRB) as a partner in the Worldwide Protein Data Bank (wwPDB): new policies affecting biomolecular NMR depositions

We describe the role of the BioMagResBank (BMRB) within the Worldwide Protein Data Bank (wwPDB) and recent policies affecting the deposition of biomolecular NMR data. All PDB depositions of structures based on NMR data must now be accompanied by experimental restraints. A scheme has been devised that allows depositors to specify a representative structure and to define residues within that structure found experimentally to be largely unstructured. The BMRB now accepts coordinate sets representing three-dimensional structural models based on experimental NMR data of molecules of biological interest that fall outside the guidelines of the Protein Data Bank (i.e., the molecule is a peptide with 23 or fewer residues, a polynucleotide with 3 or fewer residues, a polysaccharide with 3 or fewer sugar residues, or a natural product), provided that the coordinates are accompanied by representation of the covalent structure of the molecule (atom connectivity), assigned NMR chemical shifts, and the structural restraints used in generating model. The BMRB now contains an archive of NMR data for metabolites and other small molecules found in biological systems. Organization. The BioMagResBank (BMRB, http://www.bmrb.wisc.edu/), the repository for experimental and derived data gathered from NMR spectroscopic studies of biological molecules (Ulrich et al. 2008), has been a member of the Worldwide Protein Data Bank (wwPDB, http://www.wwpdb.org/) since 2006 (Berman et al. 2003). Other wwPDB partners include the Research Collaboratory for Structural Biology (RCSB PDB, http://www.pdb.org/), the Protein Data Bank Japan (PDBj, http://www.pdbj.org/), and the Macromolecular Structure Database at the European Bioinformatics Institute (MSD EBI, http://www.ebi.ac.uk/msd/). These four groups work closely together with the goal of maintaining a single Protein Data Bank archive of macromolecular structural data that is freely and publicly available to the global community. The wwPDB Advisory Committee (wwPDBAC), which provides oversight and advice, meets yearly and currently is chaired by Stephen K. Burley. This committee consists of the leaders of the four sites plus two scientists nominated by each of the four partner sites. Additional ex officio members include scientists representing various stakeholder communities (e.g., International Union of Crystallography, International Council on Magnetic Resonance in Biological Systems, electron microscopy, structural genomics) and representatives of the international funding agencies that support the wwPDB member sites. On matters pertaining to NMR spectroscopy, the wwPDB is advised by members of the wwPDB NMR Task Force (NMR TF), which is chaired by Robert Kaptein (a wwPDBAC member). Historically, members of this TF have been nominated by the wwPDB leadership. Additional interested persons are welcome to join and participate. The wwPDB NMR TF meets periodically (usually more than once each year) at international NMR conferences. Data remediation. 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Ann_Surg_Oncol-3-1-2039827

Preoperative Chemoradiotherapy with Capecitabine and Oxaliplatin in Locally Advanced Rectal Cancer. A Phase I–II Multicenter Study of the Dutch Colorectal Cancer Group

Background We studied the maximum tolerated dose (MTD) and efficacy of oxaliplatin added to capecitabine and radiotherapy (Capox-RT) as neoadjuvant therapy for rectal cancer. Colorectal cancer is a major public health problem in the Western world and ranks as the third leading cause of death in both males and females. In 2000, more than 9000 new colorectal cancer patients were registered in the Netherlands, of whom 25% had rectal cancer.1 Surgical resection is the only curative treatment. However, following potentially curative resection, local recurrence rate varies between 5 and 40%.2–4 Total mesorectal excision (TME) is now the standard technique for primary resectable rectal cancer and has significantly improved local control.4–6 The cornerstone of the TME technique is the complete removal of the rectum and mesorectum to realize free circumferential resection margins. Adam showed in 1994 that the incidence of local recurrence 5 years after resection will rise from 10–78% in case of circumferential margin (CRM) involvement.7 So downstaging before the TME procedure may decrease the incidence of CRM involvement and local recurrence. In the Dutch TME trial, no tumor downstaging was detected in the week after 5 × 5 Gy.8 A recent Polish trial demonstrated that a radiotherapy schedule of 50.4 Gy combined with chemotherapy (5-FU/Leucovorin) followed after 4–6 weeks by surgery resulted in a significant higher percentage of downstaging compared with short-term preoperative radiotherapy of 5 × 5 Gy followed by surgery within 7 days.9 So downstaging is dependent on both the total radiotherapy dose and the interval between the end of the radiotherapy and the surgery. In general, to achieve downstaging this interval needs to be at least 6 weeks and the dose needs to be at least 45 Gy.9 Although the obtained downstaging after neoadjuvant radiochemotherapy did result in a favorable prognosis,10 it was not clear until recently whether the addition of chemotherapy to preoperative radiotherapy could increase the amount of downstaging and thus improve local control. The evidence that the addition of chemotherapy to preoperative radiotherapy improves local control rates has recently been shown by two separate trials. The EORTC 22921 trial has a two by two factorial design and randomized between preoperative radiotherapy (45 Gy) versus preoperative chemoradiotherapy (45 Gy combined with 5-FU/Leucovorin). The results demonstrated an increased local control rate for the chemoradiation arm: 91% versus 83%.11,12 A similar result was found in the French FFCD 9203 study, which randomized between preoperative radiotherapy (45 Gy) and preoperative chemoradiotherapy (45 Gy and 5-FU/Leucovorin) and which showed local recurrence rates of 16.5% and 8%, respectively.13 Based on these studies, prolonged preoperative chemoradiation is considered the standard treatment for resectable locally advanced rectal cancers. The EORTC study and the French study employed 5FU bolus injection modulated with leucovorin. However 5FU administered by continuous infusion or orally (capecitabine/ UFT) may be more effective and less toxic than 5FU administered by bolus injection.14–16 In parallel, the results of palliative chemotherapy in advanced colorectal cancer have been substantially improved by the combination of 5FU with oxaliplatin or irinotecan (reviewed by Punt17). Both in vivo and in vitro, oxaliplatin has been shown to have at least an additive interaction with radiotherapy in the management of digestive tract tumors.18–20 Incorporation of those drugs in combined treatment strategies could substantially improve the results obtained with bolus 5FU alone in downstaging and R0 resection rates in patients with locally advanced rectal cancer. We investigated the combination of capecitabine and oxaliplatin given concomitantly with radiotherapy in patients with locally advanced rectal cancer. The optimal dose of oxaliplatin was first established in a phase I study, and in the following phase II study the efficacy of this regimen was tested. PATIENTS AND METHODS Objectives The objective of the phase I study was to determine the maximum tolerated dose (MTD) of oxaliplatin in combination with a fixed dose of capecitabine and radiotherapy, and the objective of the phase II study was to determine the R0 resection rate and pathological complete response rate (pCR). Eligibility Criteria Eligibility criteria included histologically documented adenocarcinoma of the rectum within 15 cm from the anal sphincter, locally advanced stage T3 (distance to the endopelvic fascia < 2 mm) or T4 tumors based on computed tomography (CT) or magnetic resonance imaging (MRI) findings, performance status (Eastern Cooperative Oncology Group) 0–2, age >18 years old, adequate hematological, liver function, and other laboratory parameters (white blood cells 3.0 × 109/L, platelets >100 × 109/L, creatinin clearance > 50 mL/min, bilirubin < 1.5 times the upper limit of the normal range (ULN), and written informed consent. Patients with reproductive potential should use adequate contraceptive measures. Patients were excluded in case of prior chemotherapy and/or pelvic radiotherapy, acute bowel obstruction without colostomy, uncontrolled ischemic heart disease, peripheral neuropathy, or any uncontrolled serious systemic disease. The protocol was approved by the local ethics committees of all four participating hospitals. The study was performed within the framework of the Dutch Colorectal Cancer Group (DCCG). TREATMENT Radiotherapy All patients received radiation delivered by an isocentric three- or four-field technique, using a linear accelerator of at least 10 MV. The patients were treated in either supine or prone position with a full bladder. The radiation field extended superiorly to the L5/S1 junction and covered inferiorly the obturator foramina. The minimal inferior border extended 4–5 cm below the tumor. In case the tumor was located in the lower third of the rectum, the perineum was encompassed in the treatment field. The width of the AP-PA portals had to cover the lateral pelvic inlet with a margin of 1.5 cm. The entire sacrum was included with a dorsal margin of 1.5 cm. Anteriorly, the lateral fields had to encompass the tumor as determined by barium enema (optional) and pelvic CT scan. If there was clinical evidence of involvement of the bladder, the prostate, the cervix or the uterine body, not only the internal iliac nodes but also the external iliac nodes were included in the radiation field. Computerized dosimetry was routinely performed. Irradiation was delivered 5 days per week at a dose of 1.8 Gy/day to a total dose of 45 Gy with a boost to the tumor in 3 fractions of 1.8 Gy to a total of 50.4 Gy. Chemotherapy Capecitabine was administered orally twice daily at 1000 mg/ m2 on days 1–14 and 25–38. Oxaliplatin was given twice by intravenous infusion over 2 h, on the first day of radiotherapy and on day 29. Oxaliplatin doses were planned at 85, 100, and 135 mg/m2. Each dose level was to be administered to at least three patients. If a dose-limiting toxicity (DLT) was observed in ≥1 patient, a total of 6 patients had to be treated at that dose level. The maximum-tolerated dose (MTD) was defined as the level at which ≤2 out of 6 patients experienced DLTs without compromizing radiotherapy and surgery and was the recommended dose in the phase II study. Surgery Surgical resection was performed 6–8 weeks after completion of the chemoradiation therapy. The resection was performed according to the principles of total mesorectal excision as described by Heald2 and Enker.21 These principles include sharp dissection under direct vision following the lipoma-like surface of the mesorectum. Proximal transection of the inferior mesenteric artery was in general performed distal to the collateral between the left colic artery and branches of the sigmoid artery. Anterior dissection in male patients was carried out in front of Denonvilliers’ fascia. If tumor extended into the prostate a total exenteration was performed. In female patients, resection of the posterior vaginal wall was performed if necessary. A distal mesorectal margin of 2 cm was considered adequate if bowel continuity was to be restored. Pathology Pathology evaluation was standardized according to national guideliness. The CRM was assessed according to the method of Quirke et al.,7 and a margin of <1 mm from the primary tumor to the endopelvic fascia is considered positive. Statistics Twenty patients were to be included at the MTD to detect with a power of 80% a R0 resectability rate of >80% (with a 95% CI of 56–94%). Monitoring and Management During Treatment Physical examination and evaluation of toxicity was performed weekly. Complete blood count, electrolytes, creatinine, and total protein were determined weekly. Standard antiemetic prophylaxis with 5HT3 antagonist prior to oxaliplatin infusion was used. Adverse events were classified according to the National Cancer Institute (NCI) Common Toxicity Criteria (CTC) Version 2.0. Global quality of life (QOL) assessment was evaluated weekly during the chemoradiotherapy using a visual analogue scale (VASQOL) in which in a single 10 cm line anchored on the left with “worst” and on the right with “best” for QOL.22 Dose-limiting toxicities were defined as white blood cell (WBC) and platelets < 3.0 and < 100 × 109/L, respectively, before start of the next treatment cycle with oxaliplatin after a maximum delay of one week; diarrhea grade > 2; oral mucositis grade >1; skin toxicity grade > 2 before start of next treatment cycle with capecitabine, despite a delay of dosing for 1 week; neurosensory toxicity grade > 2 despite a delay in dosing for 1 week; occurrence of any grade 4 toxicity; and any event that would compromise the administration of radiotherapy. RESULTS Patients Characteristics Between June 2003 and December 2004, 22 patients with T3/T4 rectal cancer from four different centers were enrolled in the study. Patient characteristics are shown in Table 1. All patients were evaluable for toxicity during chemoradiation. One patient was lost to follow-up after chemoradiation (surgery was performed abroad). Therefore, 21 patients were evaluable for clinical outcome after surgery. Twenty patients (91%) received the planned dose of chemotherapy. All patients received the planned dose of radiotherapy. The median follow-up was 14 months. TABLE 1.Patient characteristics (n = 22)CharacteristicsNo.Median age (years)58Range (years)45–70Men/Women12/10Performance status  03  119Distance of tumor to anal verge (cm)  0–511  5–107  10–153  Unknown1T status  T3a17  T4b5a T3 beyond the fascia recti.b T4: into pelvic organs (1 ureter, 2 vagina, 2 prostate) Toxicity At the first oxaliplatin dose level of 85 mg/m2, one patient experienced DLT (hospitalization for grade 3 nausea and grade 2 diarrhea). This dose level was then expanded to six patients, and DLT was observed in another patient (hospitalization for grade 3 diarrhea). Therefore, a dose of 85 mg/m2 oxaliplatin was considered the MTD. In the additional 16 patients, two more patients experienced grade 3 diarrhea. Hand-foot syndrome, myelosuppression, and neurotoxicity were observed only in a minority of patients and were of mild severity. The grade 3/4 toxicity data are shown in Table 2. TABLE 2.Toxicity in the study population (n = 22)StudyPhase IPhase IIDose oxaliplatin (number of patients)85 mg/m2 (3)85 mg/m2 (3)(22)Toxicity grade (NCI CTCV2.0)Toxicity grade (NCI CTCV2.0)Toxicity grade (NCI CTCV2.0)343434Leukopenia––––––Neutropenia––––––Thrombopenia––––––Anemia––––––Diarrhea––1–4–Abdominal pain––––––Nausea1–––––Mucositis––––––Neurotoxicity–––––– Surgery TME surgery was performed after a median of 47 days (range 4–8 weeks range in days) following the completion of chemoradiation. Mean hospitalization time was 14 days (range 9–95; 70% < 21 days). The main surgical complications were: major bleeding (1), rectal perforation (1), ureter lesion (1). Postsurgery treatment related complications were seen in 12 patients with mild wound infections (6), wound dehiscences (2), (sub)ileus (4), rhabdomyolysis (1), and in one patient a life-threatening multiorgan failure after a perforation in an abcess located near the anus praeter. Rhabdomyolysis is an unusual complication. In this patient it was probably caused by the operation procedure and not by this specific procedure as there were no other postoperative complications. Three patients (15%) needed reoperation for perforation (1) and pelvic abscesses (2). The 60-day mortality rate was nil. Efficacy Twenty-one patients with a T3–T4 tumor based on CT or MRI underwent surgery with 10 abdominoperineal resections (APRs) and 10 low anterior resections (LARs); in one patient the tumor was not resectable. A R0 resection was achieved in 17 patients (81%, 95% CI 58–95%). A pCR was observed in 2 patients (10%, 95% CI 1–30%), and in one patient (5%) the surgical specimen only showed minimal microscopic disease. The postradiotherapeutic pathologic staging yielded in seven patients (33%; 95% CI 15–57%) a downstaging to pT0–T2. After a median follow-up of 14 months, four patients have died due to metastatic disease and one patient has experienced a local recurrence. Quality of Life The VASQOL score was measured weekly during chemoradiotherapy. The QOL decreased after 4 weeks treatment from 80% in week 4 to 60% in week 6 (p < .002, student-t test). DISCUSSION Based on the EORTC 22921 and FFCD 9203 phase III studies,11,13,23 preoperative chemoradiation with 5FU and 45 Gy or higher is now considered to be the standard of care for locally advanced rectal cancer. In these studies, 5FU was given as a daily bolus infusion in week 1 and 5. To improve on these results, we tested the feasibility and efficacy of the addition of oxaliplatin to capecitabine in this combined treatment strategy. Our recommended dose for the phase II study was oxaliplatin 85 mg/m2 on day 1 and 29 plus capecitabine 1000 mg/m2 twice daily during 14 days starting on day 1 and 25 in combination with radiotherapy at a total dose of 50.4 Gy. In our phase I/II study, we found grade III diarrhea in 19%, a R0 resection in 81%, downstaging to T0–2 in 33%, and a pCR in 10% of patients. Table 3 summarizes the studies with the addition of oxaliplatin to a fluoropyrimidine (IV or orally) and radiotherapy. These studies show a toxicity and efficacy profile comparable to the results from our study. TABLE 3.Locally advanced rectal cancer, recent neoadjuvant studies with fluoropyrimidines and oxaliplatin in combination with radiotherapyStudyDesign (phase)StageNEBRT (Gy)CTR0 (%)pCR (%)Toxicity grade 3–4 (%)TotalDiarrheaMyelotoxicPreoperative radiotherapy vs. chemoradiotherapy (with 5 FU)EORTC 2292111,23IIIT3/T4 101145––538a17–1–455FU pre–1454–34–9–FFCD 920313IIIT3/T476245–9043––455FU pre911215––Bujko9IIIT3/T43165 × 5–8713––50.45FU pre961718––Preoperative CRT with capecitabineKim30IIT3/T4/N+4550.4 Cap 3–31174–Dunst31/Glynne- Jones32IIT3/T4/N+9850.4 + 5.4Cap 1–422410Chau33IIT3+, low T3, N27754Cap 1b99244 deathsYerushalmi16IIT3/T4 (T2 10%)4650.4 5FU vs Cap 2–17443–302Preoperative CRT with 5 FU based chemotherapy and oxaliplatinGambacorta34IIT33050Tomox–3013310Gerard26IIT2/T3/T44050.4FU + Ox 1–151853Rodel35IIT3–43250.4Capox 479191680Carraro36IIT3/T42250.4FU + Ox 2–14–2714Aschele25I/IIT3/T4/N+2550.4FU + Ox 3892824164Glynne-Jones32I/IIUnresectable9445Capox 278281310–Machiels24IIT3/T4/N+4045Capox 18314–30–Hospers (2006) This studyI/IIT3/T42250.4Capox8110–18–Cap 1: 825 mg/m22× daily, 7 daysCapox 1: Cap 825 2× daily, Monday–Friday, oxali 50 weeklyTomox: raltitrexed 3, oxali 130, day 1, 19, 38Cap 2: 825 mg/m2 2× daily, Monday–FridayCapox 2: Cap 650 2× daily, 7 days, oxali 130 per 2 weeksFU + Ox 1: 5FU 350 daily, Oxali 130 weekly, week 1 and 5Cap 3: 825 mg/m2 2× daily,14 days (2×)Capox 3: Cap 825 2× daily, 7 days, oxali 60 per 2 weeksFU + Ox 2: 5FU 375/4 days, Oxali 25/4 days, week 1 and 5Capox 4: Cap 825 2× daily, 2 × 14 days, oxali 50, day 1, 8, 22, 29FU + Ox 3: 5FU 225 daily, Oxali 60 weeklya ≥Grade 2 toxicity.b 12 weeks neoadjuvant CAPO. Diarrhea generally occurred during the last 2 weeks of chemoradiation, which was accompanied by a significant decrease in QOL. As shown in Table 2, the addition of oxaliplatin increases the incidence of toxicities when compared with the use of 5FU alone. The observed postsurgical morbidity (mild wound infections excluded), the reoperations and the median hospitalization time in our study were 30%, 15%, and 14 days, respectively, and are comparable with other studies.24–26 We found a similar efficacy in pCR and R0 compared to other studies (Table 3). However, comparing the efficacy of these regimes is hampered by a number of technicalities. These comprise differences in 1) staging techniques, 2) the determination of distance of the tumor to the endopelvic fascia, 3) pathology techniques, and 4) the difference in interval between neoadjuvant treatment and surgery. The staging techniques used to define the locally advanced stage of the primary tumor and lymph nodes are clinical examination, CT, MRI, endoultrasound or combinations, the use of ultrasmall particles of iron oxide (USPIOs).27 These different staging techniques may lead to variabilities in pretreatment staging and therefore compromise the quantitation of downstaging. Moreover, the tumor might be still visible on posttreatment imaging, but it may have become nonvital. The distance of the tumor to the endopelvic fascia will influence the local recurrence rate. The determination of a pCR and an R0 resection depends on the sophistication of the pathology techniques used, and the definition of a R0 resection (distance tumor to resection or to CRM) is not uniform. In the various clinical protocols, the time interval between chemoradiotherapy and surgery is not standardized, which may influence the degree of downstaging but also the ultimate results of local cure. Therefore, cross-study comparison should be interpreted with caution. The end points used in this study are known to have a different impact on clinical outcome: a pCR is associated with a low local recurrence rate, and prolonged DFS and is not significantly related to the cT and cN categories.28 R0 resection is known to be a highly relevant prognostic factor.29 Comparing our results on pCR and R0 to other oxaliplatin-containing studies, we found similar results; however, these were also observed using monotherapy with 5FU or capecitabine (Table 2). Therefore, it is yet unknown whether the increased toxicity of adding oxaliplatin to a fluoropyrimidine in neoadjuvant chemoradiation schedules results in a clinical benefit for patients with rectal cancer. In conclusion, this multicenter study demonstrated a neoadjuvant regimen with Capox-RT with an acceptable acute toxicity profile. Randomized phase III studies with in the standard arm 5FU will be necessary to show the true benefit of this approach. In such prospective studies, the standardization of the staging technique (i.e., the distance of the tumor to the endopelvic fascia), TME surgery, radiotherapy, and pathology should minimize the influence of these factors on the outcome.

Clin_Auton_Res-3-1-1858602

Reduced brain perfusion and cognitive performance due to constitutional hypotension

This review article includes a systematic evaluation of the empirical data concerning deficits in mental ability, brain perfusion, and cerebral functioning due to chronically low blood pressure. A number of studies have provided strong evidence for reduced cognitive performance in hypotension, particularly in the domains of attention and memory. EEG studies have demonstrated that the hypotension-related poorer mental ability is also reflected in diminished cortical activity. Contrary to convention, more recent research has suggested a deficient regulation of cerebral blood flow in persons with low blood pressure. In addition to reduced tonic brain perfusion, studies demonstrated insufficient adjustment of blood flow to cognitive requirements. Altogether, these findings suggest that more attention should be allocated to chronic hypotension in both research and clinical practice. Introduction Chronically low blood pressure is accompanied by a variety of complaints including fatigue, reduced drive, faintness, dizziness, headaches, palpitations, and increased pain sensitivity [1–4]. In addition, hypotensive individuals report cognitive impairment, above all deficits in attention and memory. Nevertheless, it is generally the case that in research, as well as in clinical practice, relatively little importance is ascribed to hypotension. One reason for this is that, despite mental symptoms, cerebral dysfunction generally is not taken into account [1]. This is a consequence of the current doctrine that low systemic blood pressure is compensated by autoregulatory processes which prevent reduced blood perfusion of the brain [5, 6]. Some recent findings challenge this doctrine: reduced cognitive performance in hypotension has been demonstrated by neuropsychological testing, and EEG studies have revealed diminished cortical activity. Moreover, the assumption of unimpaired brain perfusion in hypotension no longer holds. In the present review the necessity of a reappraisal concerning hypotension is discussed in light of the relationship between blood pressure and cerebral functioning. Constitutional hypotension Hypotension has been defined by the World Health Organization (WHO) [4] as a low blood pressure with a systolic reading below 110 mmHg in males and below 100 mmHg in females, regardless of diastolic blood pressure. The concept of constitutional hypotension refers to a chronic condition of inappropriately reduced blood pressure independent of the presence of further pathological factors. Both secondary hypotension (e.g. due to blood loss or medication) and orthostatic hypotension (caused by circulatory problems when assuming an upright position) are distinguished from the constitutional form [1, 6]. Constitutional hypotension is relatively widespread in the general population. In a representative German sample of more than 7,000 subjects approximately 3% fulfilled the criteria of the WHO [c.f. 1]. In contrast to elevated blood pressure, which constitutes a significant risk factor for cardiovascular disease, hypotension is commonly not regarded as a severe condition. Nevertheless, its impact on personal well-being and quality of life has been shown in several epidemiological studies. For instance, Wessely et al. [7], as well as Pilgrim et al. [3] demonstrated impaired physical and mental health in the case of low blood pressure. Reduced health-related quality of life [8] and a heightened degree of depressiveness [9] were also reported by hypotensive individuals. Chronic hypotension has been identified as a major risk factor in pregnancy [10]. Moreover, several studies focusing on the elderly population have reported associations between low blood pressure and the prevalence and incidence of Alzheimer’s and vascular dementia [c.f. 11–14]. Whilst a few longitudinal studies examining this relationship have been published [15, 16], the causal role of low blood pressure in degenerative brain disorders has thus far not been proven [e.g. 13].1 Concerning the etiology of hypotension, reduced liquid intake and low body weight may be considered [c.f. 1, 17]. A dysregulation of the autonomous nervous system may additionally be of significance. Various findings on reduced electrodermal activity [18] and increased heart rate variability [19] reflect diminished sympathetic tone and habitually heightened parasympathetic activity in persons with low blood pressure values. This is corroborated by findings on the effects of sympathomimetic drugs [20, 21]. In mixed samples of persons with constitutional and orthostatic hypotension, α and β adrenergic substances were shown to lead to blood pressure elevation accompanied by a reduction of subjective symptoms. A malfunction of the arterial baroreceptor system was postulated as a further etiological factor [22]. Responding to mechanical stretch of the vessel walls, the baroreceptors form part of a negative feedback loop (“baroreflex”) compensating phasic blood pressure fluctuations [23]. Increased sensitivity of the baroreceptor system may result in stabilizing blood pressure at a lower level. In addition to findings in animals [24], this hypothesis is supported by a still unpublished study of our own group which yielded increased baroreflex sensitivity in the case of low blood pressure. Cognitive deficits in constitutional hypotension Table 1 presents the most relevant studies devoted to cognitive functioning in hypotension. In addition to the main results, the table includes information concerning the respective samples and assessment instruments. Table 1Studies on cognitive performance in hypotension (SBP, systolic blood pressure; DBP, diastolic blood pressure)AuthorsMain focusSamplesAssessment instrumentsMain resultsRichter-Heinrich et al. [18]Attentional performance in hypotension and hypertension30 hypotensives (SBP < 106 mmHg); 40 hypertensives (SBP > 140 mmHg); 31 normotensive controls; age 16–40 yearsKonzentrations-Verlaufs-Test (course of concentration test) [25]Reduced attentional performance in the case of both lowered and elevated blood pressureStegagno et al. [26]Attentional, memory and arithmetic performance in hypotension17 female hypotensives (SBP < 100 mmHg); 19 normotensive controls; mean age 23 yearsReaction times to acoustic stimuli; digit span [27]; recall of word lists [28]; serial subtractionsProlonged reaction times, poorer performance on the verbal memory and arithmetic tasks in hypotension, no effect for digit spanCosta et al. [29]Attentional and memory performance in hypotension26 female hypotensives (SBP < 105, DBP < 65 mmHg), 22 normotensive controls; mean age 29 yearsAufmerksamkeits-Belastungs-Test (attentional strain test) [30]; Zahlen-Verbindungs-Test (trail making test) [31]; recall of word lists [28]Poorer attentional and memory performance on each of the three tests in hypotensionMorris et al. [32]Relationship between blood pressure and cognitive performance in elderly personsRepresentative population sample covering the total spectrum of blood pressure (n = 5,816; age over 64 years)East Boston Memory Test [33]; Symbol Digit Modalities Test [34]; Mini-Mental State Examination [35]Weak U-shaped relationship between blood pressure and cognitive performanceWeisz et al. [22]Attentional performance in hypotension25 female hypotensives (mean SBP = 102 mmHg); 25 normotensive controls; age 19–44 yearsAttentional and Cognitive Efficiency Battery [36]Reduced performance on a subtest assessing cognitive flexibility in hypotensionDuschek et al. [37]Attentional performance in moderate hypotension26 borderline hypotensives (mean SBP = 112 mmHg); 29 normotensive controls; mean age 26 yearsReaction times to acoustic stimuli; Aufmerksamkeits-Belastungs-Test (attentional strain test) [30]; Zahlen-Verbindungs-Test (trail making test) [31]Prolonged reaction times and poorer performance on the “Aufmerksamkeits-Belastungs-Test” in moderate hypotension, no effects for the “Zahlen-Verbindungs-Test”Duschek et al. [38]Attentional and working memory performance in hypotension with motor performance and mood controlled40 hypotensives (SBP < 105 mmHg in women, SBP < 110 mmHg in men); 40 normotensive controls; age 19–45 yearsTestbatterie zur Aufmerksamkeitsprüfung (battery for the assessment of attention) [39]; Motorische Leistungsserie (motor performance series) [40]; Befindlichkeitsskala (mood scale) [41]Poorer performance on six tests assessing tonic and phasic alertness, selective, divided and sustained attention as well as working memory in hypotensives with fine motor performance and mood controlled A pioneering study investigating the relationship between blood pressure and cognitive abilities was conducted by Richter-Heinrich et al. [18]. They showed reduced performance of individuals with both lowered and elevated blood pressure on a test of concentration. A more comprehensive assessment was carried out by Stegagno et al. [26], in which poorer performances of hypotensive subjects on a verbal memory test and on an arithmetic task, as well as prolonged reaction times to acoustic stimuli were observed. Costa et al. [29] found reduced scores on two standard German paper–pencil tests assessing selective attention and cognitive speed. Moreover, as in the Stegagno et al. [26] study, impaired verbal memory performance was documented. Weisz et al. [22] reported that female hypotensive subjects performed significantly worse than controls on a computer-based test measuring attentional flexibility [36]. Subjects with only a moderately decreased blood pressure were assessed by Duschek et al. [37]. In this sample, reduced attentional performance and prolonged reaction times were again found as compared to normotensive controls. Duschek et al. [38] investigated the relationship between low blood pressure and attentional abilities through the application of a multidimensional diagnostic approach. They presented their subjects with a battery of six computer-based tasks [39] focusing on tonic and phasic alertness, selective, divided and sustained attention, as well as working memory. Additionally, in order to control for possible confounders, a test battery examining fine motor abilities [40] and a mood questionnaire [41] were presented. Reduced performance of hypotensives was evident in each of the six cognitive tests. The significant differences between hypotensive and control subjects persisted even when the effects of motor performance and mood were controlled. Altogether, the existing data provide strong evidence for cognitive deficits related to constitutional hypotension, especially in the domains of attention and memory. The reduced performance seems to be a direct consequence of low blood pressure rather than an effect of impaired well-being related to this state. Up to now there have been no empirical data concerning specific effects of hypotension-related deficits on everyday life. Nonetheless, these findings raise concern regarding the impact of the deficits on attention-demanding activities (e.g. professional or academic), including public health-related functions (e.g. traffic safety) [42]. It would appear that both extremes of the blood pressure spectrum are accompanied by a decrease in cognitive ability. A number of studies concerned with elevated blood pressure showed reduced performance on various cognitive tests [43, 44]. This is in line with epidemiological studies reporting an inverted U-shaped relationship between blood pressure and cognitive performance in elderly persons [c.f. 11, 13, 32, 45, 46]. Therefore, it may further be hypothesized that there is a relatively small range of normal blood pressure in which the brain exerts its optimal function. Neuroelectrophysiological correlates of hypotension-related cognitive deficits Cognitive deficits in low blood pressure states are also reflected in decreased cortical activity. In accordance with earlier results [22, 29], Duschek et al. [47] found the amplitude of the contingent negative variation (CNV) to be reduced in a hypotensive sample defined according to the WHO criteria [4] (c.f. Fig. 1). Fig. 1The CNV occurs during the period between a warning signal (S1) and a second stimulus (S2) demanding a motor, verbal or cognitive response. As can be seen in the figure, its amplitude was found to be reduced in hypotensive subjects (N = 40) as compared to normotensive controls (N = 40); modified from [47] The CNV is an evoked potential which is generally viewed as a neuroelectrophysiological correlative of attentional processing [48]. This is supported by pharmacological studies showing that the effects of various stimulant and sedative drugs on attention involve an increase or decrease, respectively, of the CNV amplitude [49, 50]. Moreover, attentional deficits due to brain lesions and psychiatric diseases are associated with a reduced CNV [51, 52]. In the Duschek et al. [47] study, the amplitude of the CNV proved to be negatively correlated with reaction time, which in turn was prolonged in the hypotensive group. This highlights that the CNV constitutes a brain electrical correlate of an aspect of cognitive functioning which is affected in hypotension. The relationship between blood pressure and cortical activity is also reflected in a negative correlation between blood pressure and α wave activity in the spontaneous EEG [47]. This suggests that low blood pressure is associated with a reduced tonic cortical arousal [53]. On the behavioral level, higher degrees of α activity are known to be associated with generally diminished vigilance and preparedness to react [54]. Considering the physiological processes mediating the relationship between blood pressure and brain function, it must be taken into account that blood pressure can influence cortical activation processes via afferent projections [47, 55–57]. The brain continuously receives information about the state of the cardiovascular system by means of viscero-afferent fibers [23]. These afferent signals enter the brain via brainstem nuclei. From there, ascending pathways continue via hypothalamic and thalamic regions to cortical areas such as the anterior cingulate, the insula and the prefrontal lobe [58]. The latter areas, in particular the prefrontal cortex and the anterior cingulate are of crucial importance in the regulation of cortical arousal, as well as for attentional processes [59, 60]. Thus, these brain structures may represent functional interfaces between cardiovascular activity and attention [42]. Also neurochemical mediators should be regarded in the relationship between blood pressure and cortical activity. Noradrenaline is involved in the control of cortical arousal and attentional processes, as well as in the genesis of the CNV [59, 61]. Catecholamines are also of great influence in the regulation of blood pressure [62]. Bearing this in mind, one could speculate on the neurochemical level about a specific role of noradrenaline linking cardiovascular and cortical activation. Cerebral blood perfusion in constitutional hypotension It is generally assumed that in healthy individuals processes of autoregulation keep the cerebral blood flow constant within a wide range of arterial pressure. In order to ensure stable perfusion, cerebral resistance vessels constrict during increases and dilate during reductions in systemic blood pressure [5]. Autoregulation is considered to be a protective mechanism which prevents brain ischemia during blood pressure decrease, and guards against capillary damage and edema formation during periods of elevated blood pressure. Under normal conditions the limits within which cerebral blood flow is assumed to be constant are approximately between a mean arterial pressure (MAP) of 60 and 150 mmHg [5, 63, 64]. If blood pressure is outside of this range, cerebral blood flow rises or falls with respective increases or decreases in blood pressure. Slight reductions of blood pressure below the lower limit of autoregulation can be compensated by an increase of the extraction coefficient of oxygen from the blood. Further reductions are accompanied by symptoms such as pallor and dizziness and ultimately lead to irreversible brain damage [5]. MAP in individuals with constitutional hypotension usually does not decrease beyond the assumed lower MAP limit of autoregulation (60 mmHg). In light of this, low blood pressure in these subjects should be compensated, and cerebral blood flow should not be affected. This assumption was challenged by Duschek and Schandry [65]. They recorded blood flow velocities by means of transcranial Doppler sonography in both middle cerebral arteries (MCA) in hypotensive individuals, defined according to the WHO [4], and in normotensive control subjects. Contrary to the current doctrine, MCA blood flow at rest was found to be substantially bilaterally reduced in hypotensives (c.f. Fig. 2). Fig. 2Mean flow velocities in the left and right middle cerebral arteries under resting conditions in hypotensive (N = 40) and control subjects (N = 40); bars represent standard errors [65] The perfusion territory of the MCA includes subcortical areas, large fractions of the frontal and parietal lobes, as well as the temporal lobes [66]. Thus, the latter finding demonstrated that, despite autoregulation, blood pressure in subjects with constitutional hypotension is not sufficient in maintaining the perfusion of a large part of the brain at the level of normotensive individuals [see also 67]. In explaining this unexpected result, it must be noted that the determination of the lower limit of autoregulation was exclusively based on the experimental manipulation of blood pressure employing pharmacological methods, as well as head up-tilt [68–71]. However, such a transient reduction of blood pressure is most certainly not comparable to conditions of chronic hypotension. Additionally, in all of these studies global cerebral blood flow was assessed by means of the oxygen difference method according to Lennox and Gibbs [72], which may be less sensitive to perfusion changes than Doppler sonography. Nevertheless, more recent findings suggest values for the lower MAP limit of autoregulation to be considerably higher than 60 mmHg [73–75]. In a critical review of the literature, Drummond [76] postulated an average lower limit of no less than 70 mmHg. Moreover, the limit seems to vary strongly across individuals. Waldemar et al. [77], for instance, reported an inter-individual range between 53 and 103 mmHg. In accordance with Duschek and Schandry’s [65] data, the doctrine of stable cerebral perfusion down to the limit of a MAP of 60 mmHg can no longer be supported by the current state of research. A further important aspect of cerebral hemodynamics concerns the continuous adjustment of brain perfusion to current requirements. Due to the close coupling of neural activity and brain metabolism, cerebral activation processes are accompanied by changes in cerebral blood flow [78]. Neurovascular coupling is based on the contraction and dilation of small resistance vessels, resulting from the changing metabolic demands of neuron populations in the vicinity [64]. With regards to hypotension-related cognitive deficits, the extent of blood flow adjustment to mental activity was also tested in the Duschek and Schandry [65] study. Subjects were presented with a simple attentional task (motor reactions to visual stimuli cued by acoustic signals). During task execution blood flow velocities in the MCA of both hemispheres were recorded by means of functional transcranial Doppler sonography (for technical details see [66]). As expected, mental activity was accompanied by a substantial increase of MCA perfusion. In control subjects, however, this increase was approximately 70% stronger than in hypotensives (c.f. Fig. 3). It would seem that blood pressure in the latter group was not sufficient to enable adjustment of brain perfusion to cognitive demands, as seen in normotensives. Moreover, hypotensive participants showed prolonged reaction times, and a positive correlation was found between reaction speed and the extent of blood flow increase. This finding corroborates the significance of hemodynamic adjustment for optimal cognitive functioning. Fig. 3Changes of blood flow velocities in the left and right MCA during the execution of an attentional task in hypotensives (N = 40) and controls (N = 40). The subjects had to press a key in response to a visual imperative stimulus which was announced by a cuing tone. In both MCA the rise in flow velocity which occurred during the anticipation of the imperative stimulus was substantially less pronounced in hypotensives. A second flow velocity maximum visible after the motor reaction was also slightly reduced in hypotensives [65] Final comments There is strong evidence suggesting that chronically low blood pressure is accompanied by diminished cognitive performance, primarily involving attention and memory [26, 29, 38]. Recent research has provided an insight into the psychophysiological mechanisms of action underlying these deficits: EEG studies have demonstrated that the weaker cognitive performance is associated with reduced cortical activity [22, 29, 47]. Furthermore, deficient regulation of cerebral blood flow must be assumed in hypotension. In addition to diminished tonic brain perfusion, reduced adjustment of brain perfusion to cognitive demands was documented [65]. It stands to reason that, as a consequence of this situation, a diminished metabolic supply of the brain tissue accounts for the cognitive deficits. The cognitive deficits can widely affect the every day life of hypotensive persons, and more attention should therefore be allocated to this topic within basic and clinical research, as well as in clinical practice [1, 17]. This finally leads to the issue of the treatment of hypotension. Empirical knowledge concerning the effectiveness of antihypotensive therapy continues to be scant. A variety of treatment strategies such as physical training, increase of liquid intake and pharmacological measures have been suggested [1]. In two clinical trials the administration of sympathomimetics was found to result in a reduction of subjective hypotensive symptoms [20, 21]. Recent data of our own group suggest that cognitive performance may also be enhanced by pharmacological blood pressure elevation [79]. Based on a placebo controlled design, the vasopressor agent midodrine was shown to raise cerebral blood perfusion as well as performance on a test assessing selective attention [30] in hypotensive subjects, defined according to the WHO [4]. The results of these pioneering studies are promising. Further research would appear worthwhile and should aim to establish precise guidelines which enable chronic hypotension to be efficiently and effectively dealt with in practice.

Apoptosis-4-1-2311383

Protein kinase A-mediated CREB phosphorylation is an oxidant-induced survival pathway in alveolar type II cells

Oxidant stress plays a role in the pathogenesis of pulmonary diseases, including fibrotic lung disease and cancer. We previously found that hydrogen peroxide (H2O2) initiates an increase in Ca2+/cAMP-response element binding protein (CREB) phosphorylation in C10 alveolar type II cells that requires activation of extracellular regulated kinases 1/2 (ERK1/2). Here, we investigated the role of crosstalk between protein kinase A (PKA) and epidermal growth factor receptor (EGFR) in oxidant-induced signaling to ERK1/2 and CREB in C10 cells. Application of H2O2 increased nuclear accumulation of PKA, and inhibition of PKA with H89 reduced oxidant-mediated phosphorylation of both CREB and ERK1/2. Single cell measurements of cAMP and redox status, using a FRET-based biosensor and a redox-sensitive GFP, respectively, indicated that H2O2 increases production of cAMP that correlates with redox state. Inhibition of EGFR activity decreased both H2O2-induced CREB phosphorylation and translocation of PKA to the nucleus, suggesting that crosstalk between PKA and EGFR underlies the oxidant-induced CREB response. Furthermore, knockdown of CREB expression using siRNA led to a decrease in bcl-2 and an increase in oxidant-induced apoptosis. Together these data reveal a novel role for crosstalk between PKA, ERK1/2 and CREB that mediates cell survival during oxidant stress. Introduction Lung epithelial cells, a target cell of inhaled oxidants, are subjected to a variety of environmental stresses, including oxidizing gases, particulates, and airborne microorganisms. Although the formation of oxidants is normally well regulated, excessive production may cause inflammation and cellular injury. Numerous studies suggest a role for oxidant stress in the pathogenesis of pulmonary diseases, including asthma, pulmonary fibrosis, and cancer [1]. Reactive oxygen species (ROS) are involved in multiple physiological processes through their capacity to regulate the expression of several growth factor receptors, protein kinases and phosphatases. In alveolar type II lung epithelial cells, hydrogen peroxide (H2O2) induces tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) [2] and leads to activation of protein kinase C (PKC) [3] and the mitogen activated protein kinases (MAPK) p38, c-Jun amino-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) [4, 5]. H2O2 also activates the expression of transcription factors, including Ca2+/cyclic AMP-response element binding protein (CREB) [6] and members of the activator protein (AP-1) family, c-jun and c-fos [7], resulting in proliferation [4] and apoptosis [6, 8]. CREB is a 43 kDa transcription factor belonging to the basic-leucine zipper (bZIP) family and is regulated by phosphorylation at serine residue 133 [9, 10]. Phosphorylation of CREB initiates recruitment of co-factors to the Ca2+/cAMP-response element (CRE) that are necessary for transcriptional activation such as CREB-binding protein (CBP300) [11]. CREB activation is regulated by both Ca2+ and cAMP, which have been shown to regulate both ERK1/2- and protein kinase A (PKA)-mediated CREB phosphorylation [6, 12, 13]. Many genes important for regulation of proliferation and apoptosis, including c-fos and bcl-2, respectively, contain CREs in their promoter [11]. Our previous studies have shown that exposure of alveolar type II cells to bolus H2O2 or asbestos fibers results in CREB activation that is dependent on ERK1/2 activation [6, 14]. Furthermore, we found that preventing CREB activation promoted cell survival and enhanced bcl-2 transcription, revealing a potential role for CREB in H2O2-mediated apoptosis [6]. Because of known crosstalk between PKA, ERK1/2, and CREB in many cell types and the dose-dependent effects of H2O2, the goal of this study was to determine their relative importance in oxidant-induced signaling. The effects of transient bolus H2O2 as well as a peroxide generating system were tested to represent a range of oxidant concentrations that lead to cell proliferation and/or apoptosis. The data support the hypothesis that PKA and EGFR are central regulators of oxidant–induced ERK1/2 and CREB activation and demonstrate a link between PKA, EGFR, and CREB in cell survival following exposure to H2O2 in alveolar type II cells. Materials and methods Cell culture and treatments C10 cells, a contact-inhibited, non-transformed murine alveolar type II epithelial cell line [15], were grown in CMRL 1066 medium supplemented with L-glutamine, penicillin/streptomycin, and 10% fetal bovine serum (FBS) (GIBCO BRL, Rockville, MD). Cells were grown to 90% confluence, and then complete medium was replaced with CMRL 1066 medium supplemented with l-glutamine, penicillin/streptomycin, and 0.5% FBS for 48 h before exposure to agents. H2O2 (Sigma, St. Louis, MO) was added to the medium at concentrations from 100 μM to 300 μM. Recombinant glucose oxidase (GO; 5 mU/ml, 15 mU/ml, or 30 mU/ml) (Roche, Indianapolis, IN) was used to provide low level fluxes of H2O2 [16, 17]. Forskolin and epidermal growth factor (EGF) (Sigma, St. Louis, MO) (10 μM and 100 ng/ml, respectively) were used as positive controls for induction of phospho-CREB and phospho-EGFR, respectively. Tumor necrosis factor α (TNFα) (Calbiochem, LaJolla, CA) was used at 0.1 μg/ml to induce apoptosis. Control cultures received medium without agents and were treated identically. The ERK1/2 inhibitor, U0126 (10 μM for 30 min prior to treatment), the EGFR tyrosine kinase inhibitor, Tyrphostin AG1478 (10 μM for 1 h prior to treatment), and the antioxidant enzyme, catalase (1000 U/ml 1 h prior to treatment) were obtained from Calbiochem. The PKA inhibitor, H89 (10 μM for 1 h pre-treatment) was obtained from Biomol (Plymouth Meeting, PA). N-acetyl-l-cysteine (NAC; 10 mM 16 h prior to treatment) and DL-Buthionine-(S,R)-sulfoximine (BSO; 10 μM 16 h prior to treatment) were purchased from Sigma (St. Louis, MO). Western blot analysis After C10 cells were exposed to agents as described above, the cells were washed twice with cold PBS and collected in 4X sample buffer (200 μM Tris, pH 6.8, 4% SDS, 4 mg/ml bromophenol blue, 0.04% β-mercaptoethanol, 40% glycerol, 2 μM pyronin-Y). The amount of protein in each sample was determined using the RC/DC protein assay (Bio-Rad). About 30 μg of protein was separated by a 10% SDS-PAGE and transferred to nitrocellulose. Western blots were performed as described previously [6] using antibodies specific to total and phosphorylated CREB (1:1000; rabbit polyclonal anti-CREB, Cell Signaling Technologies, Danvers, MA; 1:500; rabbit polyclonal anti-phospho-CREB, Cell Signaling Technologies), total and phosphorylated ERK1/2 (1:1000; rabbit polyclonal anti-ERK1/2, Cell Signaling Technologies; 1:500; rabbit polyclonal anti-phospho-ERK1/2, Cell Signaling Technologies). Antibody binding was detected using horse radish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (1:5000; Jackson ImmunoResearch Laboratories, Inc., West grove, PA), followed by chemiluminescence (Kirkgaard and Perry Laboratories, Gaithersburg, MD). QuantityOne (Bio-Rad, Hercules, CA) was used to quantify band density, and intensity of phospho-CREB bands were normalized to the intensity of the corresponding total CREB bands. Live cell imaging of cAMP and redox potential C10 cells grown on glass coverslips in 6-well tissue culture dishes were transiently transfected with 8 μg of a plasmid encoding a unimolecular fluoresecent resonance energy transfer (FRET)-based cAMP biosensor, Epac1-camps (provided by Dr. Martin Lohse, University of Würtzburg) [18, 19], or 1 μg of a plasmid encoding a redox-sensitive green fluorescent protein (GFP), roGFP2 (a gift from Dr. James Remington, University of Oregon) [20], using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the manufacturer’s instructions. The transfection efficiency was approximately 50%. Imaging experiments were conducted after 48–72 h of transfection, and the growth medium removed and replaced with Krebs-Ringer bicarbonate solution (KRB; 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 1 mM KH2PO4, 25 mM NaHCO3 or 10 mM HEPES-NaOH (pH 7.40), and 2 mM glucose). Coverslips were placed into a heated microperfusion chamber mounted on the specimen stage of an inverted fluorescence microscope (Nikon TE-2000U) equipped with a CARV spinning disk confocal system (Atto Bioscience Inc., Rockville, MD). Cells were continuously superfused with Krebs-Ringer bicarbonate solution (2–5 ml/min) at 37°C. Dithiothreitol (DTT) (Calbiochem, LaJolla, CA) was used to calibrate the redox minimum. For imaging Epac1-camps FRET and roGFP2, cells were visualized with a Nikon Super Fluor 40× objective. MetaFluor/MetaMorph software was used for image acquisition and analysis (Universal Imaging). The Epac1-camps EYFP excitation wavelength was 440 nm. Dual emission ratio imaging at 485 nm and 535 nm for Epac1-camps and dual excitation ratio imaging at 400 nm and 490 nm for roGFP2 were accomplished using a computer-controlled high speed filter wheel (Lambda 10-2 optical filter changer with rotation every 60–80 ms, Sutter Instrument Co., Novato, CA). For roGFP2, a 505DRLP dichroic mirror and an emission filter, 535DF25, were used. Images (50–250 ms exposure) were captured every 10 s with a 16-bit Cascade 650 digital camera (Roper Instruments, Trenton, NJ) and background-corrected by manual selection of background regions [19]. The ratio values were normalized to the average baseline values measured 1-min prior to application H2O2. Epac1-camps FRET decreases with increasing cAMP concentration, thus increased [cAMP]c is correlated with an increase in the relative emission ratio of ECFP/EYFP (485/535) [18]. Data were thus expressed as relative ratio 485/535 for Epac1-camps and fold increase in relative ratio 400/490 for roGFP2. Immunofluorescence in C10 cells C10 cells were grown on glass coverslips for all experiments. After experimental exposures, immunofluorescence to detect the catalytic subunit of PKAα was performed as previously described [6, 21]. Briefly, cells were washed with phosphate buffered saline (PBS), fixed in 3.7% formaldehyde, permeablized with −20°C methanol and incubated with blocking solution containing 2% bovine serum albumin (BSA) in PBS. Cells were incubated with primary antibody (1:100; rabbit polyclonal PKAα catalytic subunit antibody, Santa Cruz, Santa Cruz, CA) diluted in 2% BSA plus 0.1% Triton X-100 in PBS (BSA/PBS-T) overnight at 4°C. Secondary antibody (1:400; AlexaFlour 568 goat-anti-rabbit IgG, Molecular Probes) diluted in BSA/PBS-T was applied for 1 h at room temperature (RT), and followed by incubation with nuclear counterstain, YOYO-1 iodide (Molecular Probes, Carlsbad, CA; 1:10,000), 1 unit/ml RNase, and 0.1% sodium azide in BSA/PBS-T for 30 min at RT. Coverslips were mounted onto slides with AquaPolyMount (Polysciences, Inc. Warrington, PA). For each sample, confocal images were collected in fluorescence modes using a Bio-Rad MRC1024ES confocal scanning laser microscope (Bio-Rad, Hercules, CA). For quantification, a nuclear mask was generated in Corel Photopaint using the YOYO-1 image template, and pixel intensities for PKAα were determined within the nuclear mask area as previously described [22]. Transient transfections with small interfering RNA (siRNA) The siCONTROL non-targeting siRNA #2 and SMARTpool mouse CREB siRNA (100 nM; Dharmacon, Lafayette, CO) were transfected into C10 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the manufacturer’s instructions. After 4 h in transfection media, FBS was added to 10% and cells were incubated for 24 h. Cells were then transferred to reduced serum media (0.5% FBS) for 24 h prior to treatments. Real time quantitative PCR (RT qPCR) Total RNA was extracted from C10 cells using the RNeasy™ PLUS protocol for total RNA isolation from animal cells (Qiagen,Valencia, CA). cDNA was reverse transcribed from 500 ng total RNA using an Omniscript™ Reverse Transcriptase RNase free DNase kit (Qiagen), with an oligo dT primer, according to the manufacturer’s protocol. RT qPCR primers and probes for B-cell lymphoma-2 (bcl-2) and c-fos were obtained as Assays-on-Demand™ kits from Applied Biosystems (Foster City, CA). PCR products were detected by TaqMan qPCR, as previously described [23]. Expression levels of target genes were determined using hypoxanthine-guanine phosphoribosyl transferase (hprt) as the internal standard. Samples were run in duplicate from 3 independent experiments and the comparative Ct (cycle threshold) method for relative quantity (RQ value) was used to calculate relative mRNA expression among samples. Detection and quantification of apoptosis Apoptosis was detected by measuring single-stranded DNA using Apostain™ as previously described [24]. Briefly, cell monolayers grown on glass coverslips were treated with 30 mU/ml GO for 24 h, then fixed in methanol for 24 h at −20°C, boiled for 5 min in PBS containing 5 mM MgCl2, and then immersed in ice-cold PBS for 10 min. Cells were blocked with 40% FBS and then incubated with Apostain F7-26 (Alexis Biochemicals, San Diego, CA, 10 μg/ml) followed by HRP-conjugated secondary antibody (goat anti-mouse IgM; Jackson Labortories, West Grove, PA, 1:400). To visualize secondary antibody binding, the peroxidase substrate DAB (Sigma) was used. Coverslips were mounted onto slides with AquaPolyMount (Polysciences, Inc. Warrington, PA) for subsequent examination using bright field light microscopy. To determine the numbers of apoptotic cells and total cell numbers per field, 5 random fields were evaluated per experimental condition at x200 total magnification. Statistical analysis Statistical analyses were performed using non-normalized data, and pair-wise comparisons between treatment groups were achieved using Student’s t-test or Mann–Whitney Rank Sum Test method for unequal variances. ANOVA (Holm–Sidak method) was used for multiple comparisons. Differences were considered statistically significant at P < 0.05. Results CREB is phosphorylated after exposure to H2O2 and glucose oxidase We have previously shown that H2O2 causes CREB phosphorylation in alveolar type II cells [6]. Here, we further show by Western blot that the CREB response to bolus addition of H2O2 (Fig. 1a) and H2O2 generated by glucose oxidase (GO) (Fig. 1b) is reduced when pretreated with the antioxidants NAC or catalase at effective concentrations documented previously in C10 cells [25]. Fig. 1Bolus and glucose oxidase generated H2O2 mediate CREB phosphorylation in an oxidant-dependent manner. (a) C10 cells, pre-incubated with 1,000 U/ml catalase (CAT) for 1 h, were treated with 10 μM forskolin or 100, 200, or 300 μM H2O2 for 10 min, and then analyzed by Western blot analysis for phospho-CREB (p-CREB). (b) C10 cells, pre-incubated with 10 mM NAC for 16 h or 1,000 U/ml catalase (CAT) for 1 h, were treated with 10 μM forskolin or 5, 15, or 30 mU/ml glucose oxidase (GO) for 4 h, and then analyzed by Western blot analysis for phospho-CREB (p-CREB). An antibody recognizing total CREB was used as a control for protein loading in A and B. Data are representative of 3 separate experiments H2O2-induced CREB phosphorylation is decreased after the reduction of PKA activity Because PKA is an important regulator of CREB [9], we investigated its role in H2O2-induced CREB phosphorylation. Cells were examined after exposure to lower concentrations previously associated with expression of cyclin D (100 μM) and higher concentrations known to induce apoptosis (300 μM) [6, 26]. Exposure to H2O2 led to CREB phosphorylation in a concentration-dependent manner that was significantly decreased after reduction of PKA activity using the specific inhibitor H89 (Fig. 2a, b). The pattern of CREB phosphorylation in response to H2O2 closely paralleled that seen in response to activation of PKA by forskolin. H89 also reduced H2O2-mediated ERK1/2 activation, suggesting that PKA may regulate CREB activation directly or indirectly through ERK1/2-mediated CREB activation. Fig. 2H2O2-mediated CREB activation is inhibited by the PKA inhibitor, H89. (a) C10 cells, pre-incubated with 10 μM H89 for 1 h, were treated with 10 μM forskolin or indicated concentrations of H2O2 for 10 min, and then analyzed by Western blot analysis for phospho-CREB (p-CREB), total CREB, phospho-ERK1/2 (p-ERK1/2) and total ERK1/2. (b) Quantification of p-CREB band intensities from A corrected using the corresponding CREB band intensity and normalized to the untreated control. Data represent 4 separate experiments; *P < 0.05 when compared with corresponding condition without H89. (c) C10 cells, pre-incubated with 10 μM H89 for 1 h, were treated with 10 μM forskolin or indicated concentrations of glucose oxidase (GO) for 4 h, and then analyzed by Western blot for phospho-CREB (p-CREB), total CREB, phospho-ERK1/2 (p-ERK1/2), and total ERK1/2. (d) Quantification of p-CREB band intensities from C corrected using the corresponding CREB band intensity and normalized to the untreated control. Data represent mean ± SEM from 4 separate experiments; *P < 0.05 when compared with corresponding condition without H89 Similar to results with bolus addition of H2O2, H2O2 generated by GO stimulated CREB phosphorylation in a dose-dependent manner and the phosphorylation was significantly reduced by PKA inhibition at higher concentrations of GO (Fig. 2c, d). However, unlike bolus addition, H2O2 generated by GO led to a dose-dependent increase in ERK1/2 phosphorylation that was marginally sensitive to inhibition of PKA activity, suggesting that the duration of the stimulus may cause differences in signaling patterns. H2O2 stimulates cAMP production in C10 cells Single cell imaging was utilized to determine whether H2O2 exerts a direct effect on upstream regulation of PKA through production of cAMP. Epac1-camps, a FRET-based cAMP biosensor, was expressed in C10 cells. Epac1-camps senses changes in cytoplasmic cAMP levels ([cAMP]c) through a cAMP-dependent conformational change resulting in reduced FRET between ECFP (485 emission) and EYFP (535 emission) [18, 19]. Exposure to H2O2 caused a monophasic increase in [cAMP]c (Fig. 3a) that was comparable to the response elicited by direct activation of adenylyl cyclase with forskolin, but with a slower rise time (Fig. 3c). H2O2 generated by GO stimulated a rise in [cAMP]c similar in magnitude to bolus addition of H2O2, but with a longer lag-time presumably due to the slower generation of oxidant (Fig. 3b). Fig. 3H2O2 stimulates an increase in cAMP levels. C10 cells were transiently transfected with the cAMP FRET indicator Epac1-camps and then exposed to 10 μM 250 μM H2O2 (a), 15 mU/ml glucose oxidase (GO) (b), or 10 μM forskolin (FSK) (c) for the times indicated. Data (means ± SEM) are expressed as the relative ratio (485/535) of the cAMP response from the live emission recording of a representative cell. Grey bars indicate time of addition and length of exposure. Results are representative of ≥9, 5, and 4 cells (A, B and C, respectively) Single cell imaging was also used to measure redox status and correlate cAMP levels with redox status. The redox sensitive GFP variant, roGFP2, was expressed in C10 cells. Oxidation is detected by roGFP through a change in GFP excitation from 400 nm and 490 nm [20]. The time course of H2O2-mediated cAMP production correlated well with the level of intracellular oxidation measured by roGFP2 (Fig. 4a). Upon removal of H2O2, the redox state returned to baseline approximately 36 min after the initial exposure (Fig. 4b). Fig. 4Redox status correlates with cAMP level in cells responding to H2O2. C10 cells were transiently transfected with the redox indicator, roGFP2 and then exposed to 250 μM H2O2 for 10 min followed by 1 mM DTT (a) or 15 mU/ml glucose oxidase (GO) for 65 min, followed by 200 μM H2O2, and then 10 mM DTT (b). Data (means ± SEM) are expressed as the fold change in the excitation ratio at 400/490 nm normalized to the average base-line ratio values measured 1 min before stimulation. Results are averages of ≥23 and 11 cells (A and B, respectively). Grey bars indicate time of addition and length of exposure, and WO indicates wash out with KREBs buffer EGFR tyrosine kinase activity is important for signaling from H2O2 to PKA and CREB It has been shown that the protein tyrosine activity of EGFR is important for EGF-mediated stimulation of adenylyl cyclase [27]. Thus to determine the contributions of EGFR in the observed H2O2-induced CREB phosphorylation, cells were evaluated with or without pretreatment with the specific EGFR tyrosine kinase inhibitor, AG1478. Application of AG1478 significantly reduced CREB phosphorylation in response to bolus addition H2O2 and to H2O2 generated by higher concentrations of GO (Fig. 5), suggesting that EGFR activity is involved in H2O2-induced CREB phosphorylation. As expected, reduction of EGFR activity also inhibited H2O2-induced ERK phosphorylation (Fig. 5). Fig. 5Reduction of EGFR tyrosine kinase activity leads to the inhibition of both H2O2-induced CREB and ERK1/2. C10 cells, pre-incubated with the EGFR tyrosine kinase inhibitor AG1478 (10 μM) for 1 h, were treated with 10 μM forskolin or 100 ng/ml EGF for 5 min, the indicated concentrations of H2O2 for 10 min (a) or glucose oxidase (GO) for 4 h (c), and then analyzed by Western blot analysis for phospho-CREB (p-CREB), total CREB, phospho-ERK1/2 (p-ERK1/2), and total ERK1/2. (b, d) Quantification of p-CREB band intensities from A and C respectively corrected using the corresponding CREB band intensity and normalized to the untreated control. Data represent mean ± SEM of 3 separate experiments; *P < 0.05 when compared with corresponding condition without AG1478 Immunofluorescence evaluation of activated PKA translocation to the nucleus was used to more directly test the effects of H2O2 on PKA activation and to further evaluate a role for EGFR. Exposure to H2O2 led to an increase in the PKA free catalytic subunit that was significant in the nucleus at 300 μM and similar to the response to forskolin. Inhibition of EGFR tyrosine kinase activity with AG1478 prevented the H2O2-induced increases at both 200 μM and 300 μM (Fig. 6). Fig. 6Reduction of EGFR tyrosine kinase activity inhibits the H2O2-induced nuclear translocation of activated PKA. C10 cells, pre-incubated with 10 μM AG1478 for 1 h, were exposed to 10 μM forskolin and H2O2 for the indicated concentrations for 10 min. (a) The PKA free catalytic subunit (red) was detected by immunofluorescence and nuclei were stained with YOYO-1 (green). (b) Quantification of PKA immunofluorescence pixel intensity within nuclei; *P < 0.05 compared to control, #P < 0.05 compared to treatment, n = 3 experiments, 5 fields/experiment Direct activation of CREB by PKA is a minor pathway in EGF-induced CREB phosphorylation Because of the known crosstalk between EGFR, ERK1/2, and PKA, we examined the role of PKA and ERK1/2 in forskolin- and EGF-induced CREB activation. Treatment with both forskolin and EGF resulted in a significant increase in CREB phosphorylation when compared to control cells (Fig. 7). Inhibition of PKA activity with H89 significantly decreased forskolin-induced CREB phosphorylation, but only blunted EGF-induced CREB phosphorylation. Inhibition of ERK1/2 phosphorylation with the MEK inhibitor, U0126, also considerably decreased CREB phosphorylation. The inhibitory effects of H89 and U0126 were not additive towards either forskolin- or EGF-induced CREB phosphorylation, suggesting that direct activation of CREB through PKA may play a minor role in the activation of CREB. Furthermore, both forskolin and EGF induced ERK1/2 phosphorylation, but only forskolin-mediated CREB phosphorylation was inhibited by H89, suggesting that PKA regulates CREB indirectly through the MAPK/ERK1/2 pathway. Fig. 7Crosstalk between PKA and ERK1/2 signaling is important in regulating CREB phosphorylation. (a) C10 cells, pre-incubated with 10 μM H89, U0126, or H89 and U0126 for 1 h, were treated with 10 μM forskolin for 10 min or 100 ng/ml EGF for 5 min, and then analyzed by Western blot analysis for phospho-CREB (p-CREB), total CREB, phospho-ERK1/2 (p-ERK1/2), and total ERK1/2. (b) Quantification of phospho-CREB in A corrected using CREB intensities and phospho-ERK/12 in A corrected using ERK1/2 intensities; *P < 0.05 for DMSO treatment compared to AG1478 treatment; #P < 0.05 for DMSO control compared to treatment. Data represent mean ± SEM of 3 separate experiments Knockdown of CREB using siRNA results in loss of oxidant-induced c-fos and bcl-2 transcription To more directly assess the role of CREB in oxidant-mediated responses, an siRNA approach was used to reduce levels of CREB within C10 cells. Transfection with siCREB resulted in greater than 80% knockdown of both CREB mRNA and protein levels (Fig. 8a, b). CRE-containing genes previously shown to be upregulated by H2O2 including bcl-2 and c-fos were found to be dose-dependently induced by GO. Induction was significantly reduced in cells transfected with siCREB (Fig. 8c, d), suggesting that CREB is essential for the transcriptional response of these genes to H2O2. Fig. 8Knockdown of CREB using siRNA results in loss of oxidant-induced c-fos and bcl-2 transcription. (a) C10 cells were transfected with 100 nM scrambled siRNA (siControl) or siCREB followed by Western blot analysis to detect CREB. β-actin was used as a loading control. (b) Cells were transfected as in A, followed by treatment with 5 or 15 mU/ml glucose oxidase (GO) for 6 h. Total RNA was extracted and cDNA was assessed by RT qPCR to quantify CREB, bcl-2 and c-fos mRNA levels. Shown are relative quantity (RQ) values normalized to the siControl sample. #P < 0.05 when compared to untreated siCon; *P < 0.05 when compared to treatment siCon Knockdown of CREB significantly increases the number of cells undergoing apoptosis following exposure to glucose oxidase Recently, we have shown that reduction of CREB activity, by introducing a phosphorylation-null CREB construct, led to a decrease in apoptosis [6]. To resolve whether CREB is essential for H2O2-induced apoptosis, C10 cells transfected with scrambled siRNA (siControl) or siCREB were treated with 30 mU/ml GO for 8 h and apoptotic cells were identified by Apostain (Fig. 9). H2O2 generated from GO induced a significant increase in the percentage of cells undergoing apoptosis compared to the control in both siControl and siCREB cells. Cells expressing siCREB had significantly higher levels of apoptosis than the siControl cells in both the untreated condition and after GO exposure, supporting a role for CREB in the regulation of apoptosis induced by H2O2. Interestingly, the apoptotic enhancing effect was not selective for H202-mediated apoptosis, as siCREB also enhanced the apoptotic effect of TNFα. Taken together, these results suggest that CREB plays an important role in the cellular response to oxidant stress including upregulation of survival genes and promoting cell survival. Fig. 9CREB is protective against oxidant-induced apoptosis. (a) C10 cells, transfected with siControl or siCREB, were exposed to 30 mU/ml glucose oxidase (GO) or 0.1 μg/ml tumor necrosis factor α (TNFα) for 8 h followed by identification of apoptotic cells using Apostain. (b) Quantification of % Apostain positive cells was determined by scoring the average of 5 fields/condition; *P < 0.05 when compared to untreated siCon; #P < 0.05 when compared to treatment siCon. Data represent mean ± SEM of 4 separate experiments. Bar = 100 μm Discussion Exposure to various environmental stresses, such as oxidative gases, metals, and particulates, initiates multiple physiological processes in alveolar type II cells. Oxidants have been implicated in the pathogenesis of lung cancer, pulmonary fibrosis, and asthma through their capacity to regulate an intricate network of protein kinases. Yet the relationship between oxidants and the pathogenesis of lung disease is unclear, primarily because of the lack of understanding of the mechanisms by which oxidants function in both normal physiological and disease states. In this study, we show for the first time that H2O2 exposure leads to an increase in cytoplasmic cAMP levels, followed by PKA-dependent CREB and ERK1/2 phosphorylation in alveolar type II cells. Furthermore, we show that EGFR activity is necessary for H2O2-stimulated nuclear accumulation of activated PKA and for PKA-mediated CREB phosphorylation. Together these data suggest that PKA is a central hub for interplay between EGFR, ERK1/2, and CREB signaling pathways activated by oxidant stress. Several studies have shown crosstalk between PKA and ERK1/2 signaling pathways. This crosstalk appears to be cell type specific, since an increase in cAMP-dependent PKA activation stimulates ERK1/2 in some cells, but suppresses it in others [28–30]. We have previously demonstrated that H2O2 exposure leads to ERK1/2-dependent CREB phosphorylation in C10 alveolar type II cells [6]. Studies here have dissected the underlying mechanisms to reveal that H2O2-induces cAMP formation and PKA activation that are important for both ERK1/2 and CREB phosphorylation. EGFR is overexpressed and activated in response to epithelial injury [31]. Its activation is believed to play an integral role in the cellular responses of lung epithelium to injury and oxidant stress, possibly by signal amplification through MAPK phosphorylation cascades. Oxidative modification of a reduced cysteine residue in the EGFR reversibly affects its activation [32, 33]. Moreover, Goldkorn and colleagues have shown that in alveolar type II cells, H2O2 induces EGFR phosphorylation on tyrosine residues [2]. These findings suggest that even though there is no specific receptor for oxidants, their signaling may be efficiently transduced through interactions with the EGFR. In the present study, we show that EGFR activity is necessary for both activation of PKA nuclear translocation and subsequent CREB phosphorylation in response to H2O2. Mitogenic signals transmitted through the EGFR have been shown to involve PKA-dependent signaling cascades. Tyrosine phosphorylation of the EGFR requires an increase in cAMP levels and PKA function in many, but not all cell types [34, 35]. In addition, tyrosine kinase activity of EGFR is required for growth factor stimulation of adenylyl cyclase activity, which leads to the activation of PKA [27, 36]. PKA activity has also been shown to promote downstream Raf-1 function and subsequent ERK activation [37]. Crosstalk has also been seen from EGFR to adenylyl cyclase activity and PKA in airway epithelial cells via beta adrenergic receptors [38, 39]. Thus, communication between EGFR and PKA is potentially bi-directional. The H2O2-mediated generation of cAMP and the CREB activation response to H2O2 increased with oxidant concentration but, with minor exception, were not dependent on whether oxidant was added by bolus or generated by glucose/glucose oxidase. Although reduction of the CREB response by PKA or EGFR inhibition was only significant for the higher oxidant concentrations, a trend was observed with lower concentrations. While it is difficult to accurately predict the physiological significance of these oxidant levels, when put in context with previous findings, these observations suggest that CREB signaling through PKA and EGFR could participate in signaling that affects both oxidant-mediated proliferation and apoptosis. Previously we demonstrated that expression of phosphorylation-incompetent CREB paradoxically increases bcl-2 transcription and reduces apoptosis following bolus H2O2 exposure [6]. Findings here show that siRNA-mediated knockdown of CREB results in a loss of bcl-2 mRNA and an increase in apoptosis in cells responding to an H2O2 generating system. The difference is likely due to the method of CREB inhibition, in that the dominant-negative CREB may have altered chromatin interactions with other transcription factors or caused quenching of the response. Putting these results in context with the findings of others, we propose that CREB activation through PKA signaling serves as a key survival pathway in cells responding to oxidant stress. PKA-and CREB-mediated gene expression may thus be important in regulating oxidant-mediated apoptosis resulting from lung pathologies, and future experiments will explore the importance of these signaling pathways in promoting changes in gene expression following lung epithelial cell injury or oxidant exposure.

Matern_Child_Health_J-2-2-1592145

The Importance of Preconception Care for Women With Disabilities

Overall estimates of the number of women with disabilities in the United States range from 16.8 to 28.6 million, or approximately one in every five women [1–3]. A large percentage of these women are in their reproductive years, and they often encounter greater obstacles to receiving health care than women without disabilities [4]. Some disabling conditions, such as spinal cord injury (SCI) and multiple sclerosis (MS), have low prevalence rates but are more common in the reproductive age group. Such conditions are often associated with a greater severity of disability and increased need for specialized services, including prenatal care [5]. Health care professionals need to be aware of the reproductive health care issues facing women with disabilities and take every opportunity to address them. Sexuality and reproductive health issues have received inadequate attention for women with disabilities. Many women with disabilities desire children and are capable of conceiving, but face considerable pressure not to reproduce [6, 7]. Although a growing number of women with disabilities are becoming pregnant, anecdotal evidence suggests that many of these women encounter negative attitudes towards their pregnancies and report difficulty receiving comprehensive prenatal care [6]. Women with disabilities who become pregnant face similar issues and concerns as pregnant women without disabilities. Although many pregnant women experience problems with weight gain, fatigue, fluid retention, bladder dysfunction, and urinary tract infections, these problems may be more serious in women with disabilities [8]. All women are concerned about the health of their unborn child. Like other women, women with disabilities express concern about the possibility of giving birth to a child with disabilities and may seek genetic counseling. On the other hand, not all disabled women embrace the idea of genetic testing. To these women the idea of genetic counseling raises psychosocial issues that may be difficult for them to consider [6]. Some disabling conditions pose unique problems during or after pregnancy. Complications associated with specific conditions, such as systemic lupus erythematosus (SLE), SCI, or MS, can affect pregnancy and should be evaluated prior to conception [8–12]. For example, an increased risk of fetal loss, growth retardation, premature placental aging with thrombosis, and preeclampsia are described in women with lupus [9]. Given the seriousness of these complications, ongoing monitoring and interventions to reduce them are critical. Pregnant women with SCI above the level of T6 also require special attention [7, 8, 10, 11]. Such women have an increased risk of autonomic dysreflexia during all stages of pregnancy and, if it is not recognized and treated, autonomic dysreflexia can be fatal [8, 11]. Although women with stable MS can safely carry a pregnancy to term, postpartum exacerbations of MS are quite common and occur in up to 30% of women within one month of delivery [11, 12]. Pregnancy management in women with disabilities should therefore include preconception counseling and evaluation to identify these and other potential problems that can occur throughout the course of the pregnancy. Health care professionals need to know that physical barriers, such as the lack of accessible scales or examination tables, present enormous and recurring obstacles to obtaining adequate prenatal care. Also, communication barriers, particularly for those who are deaf or hard of hearing, affect a women's ability to receive appropriate medical care. In addition, there are some aspects of gynecologic health care such as informed consent, sedation, and contraceptive issues that are unique for women with developmental disabilities. A recent publication from the American College of Obstetricians and Gynecologists highlights ways to improve care for this group of women [7]. Preconception care is associated with improved pregnancy outcomes. It is recommended that primary care providers assess all women of reproductive age for their preconception risk conditions and provide or refer individuals for interventions as appropriate [13]. When caring for a woman with a disability, a team approach involving the primary physician, obstetrician, anesthesiologist, neurologist, physiatrist, and other allied health professionals such as occupational and physical therapists, is recommended [6–8, 11]. This is particularly important, as many obstetricians are unfamiliar with disability-related complications. A multidisciplinary approach, which involves nursing and social work, can further improve pregnancy outcomes. This shared approach to pregnancy management can also increase the level of comfort for women with disabilities, by knowing that more than one health care provider is looking out for their well-being. Preconception counseling for women with disabilities should address the medical, psychological, and social impact of a pregnancy [10]. During preconception counseling, it is particularly important to evaluate the prenatal and postpartum social support systems available to women with disabilities. Body changes such as decreased mobility or bladder dysfunction increase the need for personal assistance with routine activities of daily living. Many women with disabilities also benefit from an occupational therapy evaluation to assess environmental barriers and explore adaptive equipment and techniques that facilitate breast feeding and infant care. Health care providers might want to recommend that women with disabilities explore creative parenting strategies such as those offered by Through the Looking Glass, a national resource center on parenting with a disability, located in Berkeley, California [6, 14]. An increasing number of women with disabilities are becoming pregnant. Despite this little is known about the reproductive experiences of these women. Population-based data describing the reproductive experiences of women with disabilities is virtually non-existent. Currently, disability is not regularly included in national or statewide surveys and surveillance tools related to pregnancy. Consequently, the true magnitude of health concerns, including reproductive issues, facing women with disabilities is largely unknown. To improve our understanding of these issues, disability should be included as a demographic variable in all national and state-level surveys addressing reproductive health issues. All women of childbearing age, including women with disabilities, should have access to preconception care that addresses the medical, psychological and social impacts of pregnancy. Women with disabilities can and do have healthy pregnancies. Accurate knowledge of disabling conditions and a shared multidisciplinary approach to pregnancy management are critical components for good pregnancy outcomes among women with disabilities.

Osteoporos_Int-2-2-1705543

Effectiveness of bisphosphonates on nonvertebral and hip fractures in the first year of therapy: The risedronate and alendronate (REAL) cohort study

Introduction Randomized clinical trials have shown that risedronate and alendronate reduce fractures among women with osteoporosis. The aim of this observational study was to observe, in clinical practice, the incidence of hip and nonvertebral fractures among women in the year following initiation of once-a-week dosing of either risedronate or alendronate. Introduction Osteoporosis, a common skeletal disease in older populations, leads to more than a million fractures annually in the United States [1]. Nonvertebral fractures represent 75% of osteoporotic fractures seen in clinical practice [2]. The incidence of nonvertebral fractures, especially at the hip, increases rapidly with age [3]. In order to prevent these fractures, US clinical guidelines recommend that candidates for osteoporosis therapy be identified by screening the bone mineral density of all woman ages 65 and over (age 60 for high risk populations) [4]. Oral bisphosphonates are currently the most common therapy for osteoporosis [5]. While the three most utilized bisphosphonates (alendronate, risedronate, ibandronate) approved by the Food and Drug Administration have been shown to reduce vertebral fractures in randomized clinical trials, it is unknown if these three are equally effective in reducing nonvertebral fractures of real-world patients in clinical practice. A comparison of the results from the randomized clinical trials of each bisphosphonate, though limited by methodological differences between trials, suggests potential differences in degree of fracture reduction across bisphosphonates. In the primary analyses of the trials that followed patients for at least 3 years, risedronate significantly reduced the incidence of nonvertebral fractures by up to 39% [6, 7]; alendronate reduced the incidence of nonvertebral fractures by up to 21% [8–10]; ibandronate did not reduce nonvertebral fractures [11]. Post-hoc analyses of these trial data suggest that there are differences in the onset of fracture reduction. In those analyses, reduction of nonvertebral fractures began at 6 months for 5 mg daily dosing of risedronate [12] and at either 12 months (when fractures were recorded as adverse events) for 10 mg daily dosing of alendronate [13] or 24 months for 5 mg daily dosing of alendronate [14]. These possible differences in both the amount and the onset of fracture reduction between the bisphosphonates could arise from their differences in structure, potency, and binding properties [15]. The only direct comparison of bisphosphonates in a randomized clinical trial is based on surrogate endpoints (e.g., changes in bone mineral density and markers of bone turnover) [16]. However, the association between changes in these surrogates and subsequent fracture reduction is not consistent across studies [17, 18]. Unlike randomized clinical trials based on surrogate endpoints, observational studies of large populations provide the opportunity to use major disease endpoints (e.g., hip fracture) as the outcome of interest. The limitation of observational studies can be misleading results from bias arising from non-randomized treatment groups. This bias can be accounted for in part by statistically adjusting for known risk differences between groups. Furthermore, when different therapies are available to be prescribed for the same indication, there is at least some expectation of similarity in prognostic factors between treatment groups occurring naturally [19]. For example, observational studies have compared both the many antihypertensive drug therapies and many statins for reducing the disease endpoint of myocardial infarctions [20, 21]. Since the once-a-week dosing regimens of both risedronate and alendronate have been available in the US since 2002, there is now an opportunity to observe their effect on reducing fractures in a large population of patients seen in clinical practice. Hence, we conducted an observational study across multiple US health plans to observe the incidence of hip and nonvertebral fractures among women ages 65 and over following initiation of therapy with once-a-week dosing of either risedronate or alendronate. Methods The RisedronatE and ALendronate (REAL) cohort study was a retrospective observation of bisphosphonate patients within healthcare utilization records in the United States. The analysis plan was based upon an earlier report [22]. All authors had access to the data. For assurance of reproducibility [23], the analyses were independently replicated by the respective organizations of the authors. The reporting of this study is consistent with the STROBE guidelines [24]. Data source The data source was commercially available datasets of healthcare utilization from the 1 health plan within Ingenix Lab/Rx (Eden Prairie, MN; data through June 2004) and the 100 health plans of employers within MedStat Marketscan (Ann Arbor, MI; data through September 2004). These datasets contain a longitudinal history of patient-specific data including demographic information (sex, age, dates of dataset inclusion), clinical encounters (inpatient and outpatient services by associated procedures and diagnoses specified by the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM)), and outpatient pharmaceutical dispensations (retail and mail order specified by the national drug code (NDC)). To maximize sample size, the two datasets were combined for all analyses. At the time of data extraction for the current study, the combined datasets contained 12 million persons across 34 states in the US. Study population Within the data source, candidates for study inclusion were all women aged 65 and older with any use of once-a-week dosing of risedronate (35 mg; Actonel, P&G Pharmaceuticals) or once-a-week dosing of alendronate (35 or 70 mg; Fosamax, MSD) after July 2002, a date corresponding with contemporaneous commercial availability of once-a-week versions of both therapies (Fig. 1). Patient exclusion criteria and rationale were: 1) less than 6 months (182 days) of health plan enrollment before their first bisphosphonate use after July 2002, a history period to measure baseline fracture risk; 2) less than 3 months (92 days) of health plan enrollment after their first bisphosphonate use, a minimum observation period with expectation of any fracture reduction (based on post-hoc analyses of clinical trials [12, 14]); 3) any bisphosphonate use (daily, weekly, or Paget's doses) during their 6 month history period, to include only patients who are new users of bisphosphonates; 4) diagnosis of malignant neoplasm, ICD-9-CM 140 - 208; or Paget's disease, ICD-9-CM 731.0 during either the 6 month history period or first 3 months of observation, to exclude patients with fracture risk not related to osteoporosis; and 5) discontinued therapy within the first 3 months, a minimum period of therapy adherence. Fig. 1Identification of the study population Length of observation Observation for a subject was censored at one of following end points, whichever occurred first: date of fracture, 12 months after date of first bisphosphonate prescription, end date of health plan enrollment, date of switch between bisphosphonate therapies or end date of therapy adherence. Adherence was measured as a function of the gaps between refills, which provides the best available measure within datasets of medical claims [25]. Once the gap between the completion of a 30-day supply and the start of a new prescription exceeded 15 days, the end date of therapy adherence was the prescription date before gap plus 45 days. For a 90-day supply, a gap of 45 days was allowed between completion and a new prescription, the end date of therapy adherence was the prescription date before gap plus 135 days. For the last prescription, the end date of therapy adherence was the date of last prescription plus 45 days for a 30-day supply and date of last prescription plus 135 days for a 90-day supply. Fracture outcomes From the data source, two outcomes were identified: subjects with nonvertebral fractures collectively (hip, wrist, humerus, clavicle, pelvis, leg - sites previously specified [12]) and subjects with a hip fracture. Vertebral fractures were not included because the majority of them do not come to clinical attention and thus not systematically captured in the data source. In an attempt to identify incident fractures more likely related to osteoporosis, we used a series of exclusion criteria (Table 1). A total of 109 hip and 507 nonvertebral fracture patients were available for analyses. Table 1Identification of fractures outcomes in the study population (n = 33,830) Hip fractureaNonvertebral fracturebSubjects with a medical claim for fracture during the observation period after initial bisphosphonate.135923Exclusion of medical claim if a fracture at the same site both before and after start of bisphosphonate therapy; in order to increase the likelihood of including only new fractures.−16−368Exclusion of medical claim if a fracture at an unspecified sitec before the start of bisphosphonate therapy; in order to increase the likelihood of including only new fractures.−2−6Exclusion of medical claim if an open fractured; in order to decrease the likelihood of including traumatic fractures. −3−11Exclusion of medical claim if documented cause (E-codes) of injury is other than an accidental falle; in order to decrease the likelihood of including traumatic fractures.−5−31Subjects with a fracture outcome109507aInpatient ICD-9-CM codes (820.x, 733.14)bIn addition to inpatient hip fractures, inpatient and outpatient ICD-9-CM codes for fracture of the wrist (813.x, 733.12), humerus (812.x, 733.11), clavicle (810.x), pelvis (808.x), and leg (821.x, 823.x, 733.15, 733.16)cICD-9-CM code (733.10 or 733.19)dICD-9-CM code that is not 733.1x or where the 4th digit is not = .1, .3, .5, .9eICD-9-CM code with “E” classification (E880 - E888) for accidental falls Statistical analyses To assess the comparability of baseline characteristics (see Table 2 for specific definitions of demographic characteristics and health history) between the risedronate and alendronate cohorts, the chi-square test was used for dichotomous variables and the Wilcoxon rank sum test was used for continuous variables. Table 2Comparison of baseline characteristics between cohorts in study CohortsCharacteristicRisedronateAlendronatep-valueNumber of women subjects12,21521,615Duration of observation period Days (mean)226238< 0.001Age at study entryYears & months (mean)74 & 1074 & 7< 0.001 Ages 65 – 74 (%)53.552.4 Ages 75 – 84 (%)36.836.7 Ages 85 and over (%)9.711.0Medications – 6 month historya Concomitant medications (mean)b4.03.6< 0.001 Gastrointestinal medication use (%)c26.220.1< 0.001 Estrogen use (%)d17.216.50.08 Other non-estrogen anti-osteoporotic use (%)e15.611.0< 0.001 Glucocorticosteroid use (%)f10.38.5< 0.001Medical encounters – 6 month historya Office visits (mean)5.65.1< 0.001 Hospitalization (%)8.28.20.87 Osteoporosis diagnosis (%)g37.733.8< 0.001 Osteopenia diagnosis (%)h12.510.5< 0.001 Bone densitometry procedure (%)i47.441.5< 0.001 Gastrointestinal diagnosis (%)j15.412.3< 0.001 Rheumatoid arthritis diagnosis (%)k2.72.30.01aSix months before and including date of starting first bisphosphonate prescriptionbBased on number of therapeutic classes with a prescription [34]cBased on NDC codes for at least one prescription for either: H2 antagonists (ranitidine, cimetidine, famotidine, nizatidine); Proton pump inhibitors (omeprazole, esomeprazole, lansoprazole, pantoprazole); Cytoprotectives (misoprostol, sucralfate) [35]dBased on NDC codes for at least one prescription of estradiol, conjugated estrogen, esterified estrogen, or estropipateeBased on NDC codes for at least one prescription of calcitonin or raloxifenef Based on NDC codes for at least one prescription for triamcinolone, prednisone, prednisolone, methylprednisolone, dexamethasone, budesonide, betamethasone, cortisone, or hydrocortisonegICD-9 733.0xhICD-9 733.90 and no record of 733.0xiCPT 76070, 76075, 76076, 78350, 78351, ICD-9 88.98jMultiple ICD-9 codes [36]kICD-9 714.0 For the primary analysis, the main outcome measures were the 6 and 12 month incidence of nonvertebral fractures and hip fractures. Cox proportional hazard modeling (PROC PHREG, SAS Institute, Cary, NC) was used to compare the incidence of fractures between risedronate and alendronate cohorts, adjusting for potential differences in measurable risk factors for fractures. A parsimonious model for each outcome was developed to enhance precision of the parameter estimates and interpretation of results. The selection of variables to be included in the model was based on forward selection. These models were checked against models based on backward selection. The appropriateness of the proportional hazard assumption was assessed by graphical and numerical methods (ASSESS statement, SAS Institute). In order to evaluate if the results of the primary analysis were dependent on methodology, sensitivity analysis were used to compare the incidence of fractures between risedronate and alendronate cohorts. These methods included: (1) an intent-to-treat analysis that observed all subjects for 12 months regardless of therapy adherence; (2) a proportional hazard model using the propensity score to adjust for differences in baseline fracture risk between cohorts; (3) use of different inclusion criteria for the study population; (4) use of different inclusion criteria for the study outcomes (see Fig. 5 for specifics). Results The risedronate cohort included 12,215 subjects on once-a-week dosing of 35 mg followed for a mean of 226 days on therapy. 37% of this cohort was censored before 12 months because of the end date of available data and 41% was censored for an end in therapy adherence. The alendronate cohort included 21,615 subjects on once-a-week dosing of 35 mg (8%) or 70 mg (92%) followed for a mean of 238 days on therapy. 33% of this cohort was censored before 12 months because of the end date of available data and 41% was censored for an end in therapy adherence. Upon start of bisphosphonate therapy, the two cohorts were different in several baseline characteristics (Table 2). Statistically, the risedronate cohort was older, had more concomitant medications, had more use of glucocorticoids, and had more patients with rheumatoid arthritis than the alendronate cohort - characteristics that may increase fracture risk. Conversely, the risedronate cohort also had greater past use of calcitonin or raloxifene - a characteristic that may decrease fracture risk. Within the 12 months prior to the initiation of bisphosphonate therapy, similar percentages of the two cohorts had a diagnosis for a nonvertebral fracture and a clinical vertebral fracture, while a statistically larger percentage of the risedronate cohort had a diagnosis for a hip fracture than the alendronate cohort (Fig. 2). Fig. 2Percent of patients with a clinical diagnosis of fracture before initiation of bisphosphonate therapy. 1ICD-9 codes 808.x, 810.x, 812.x, 813.x, 820.x, 821.x, 823.x, 733.10, -.12, -.14, -.19; 2ICD-9 codes 820.x, 733.14; 3ICD-9 codes 805.x, 806.x, 733.13; 4Subset (81%) of study population with available 12 month history. *Statistical difference (p<0.05) between cohorts During the 12 months of observation after the start of bisphosphonate therapy, 507 subjects had nonvertebral fractures. The site of nonvertebral fracture was wrist (30%), hip (21%), leg (17%), pelvis (15%), humerus (14%), and clavicle (3%). For the 109 women hospitalized with a hip fracture, the skeletal sites were intertrochanteric (46%), transcervical (28%), unspecified (20%), and trochanteric or subtrochanteric (6%). For the primary analysis of nonvertebral fractures, the fracture incidence was similar between the risedronate and alendronate cohorts over the first 3 months of therapy (Fig. 3). After 6 months of therapy, the risedronate cohort had a 19% lower (95% CI 0% – 35%, p-value = 0.05) incidence of nonvertebral fracture than the alendronate cohort. After 12 months of therapy, the risedronate cohort had an 18% lower (95% CI 2% – 32%, p-value = 0.03) incidence of nonvertebral fracture than the alendronate cohort (Table 3). Fig. 3Cumulative incidence of nonvertebral fractures in patients treated with alendronate or risedronate for up to 1 yearTable 3Cumulative incidence of fractures during therapyFracture typeCohort sizeNumber of women with a fracturePercent of women with a fractureaCrude rate ratiobAdjusted rate ratiob95% CIp-valueTime on therapy        Cohort       Nonvertebral6 Months Alendronate21,6152531.31–––– Risedronate12,2151231.140.870.810.65–1.000.0512 Months Alendronate21,6153432.30–––– Risedronate12,2151641.990.880.820.68–0.980.03Hip6 Months Alendronate21,615540.29–––– Risedronate12,215190.170.630.540.32–0.910.0212 Months Alendronate21,615800.58–––– Risedronate12,215290.370.680.570.37–0.870.01CI = confidence interval of adjusted rate ratioaProportion is based on Kaplan-Meier estimate of the survival function.bBased on Cox regression model.cBased on Cox regression model. Variables selected by forward stepwise selection where criteria for selection p < 0.1. Model for nonvertebral fractures included age, estrogen use, number of medications, rheumatoid arthritis diagnosis, and history of nonvertebral fractures. Model for hip fractures included age, estrogen use, number of medications, and history of hospitalization. For the primary analysis of hip fractures, the fracture incidence was similar between the risedronate and alendronate cohorts over the first 3 months of therapy (Fig. 4). After 6 months of therapy, the risedronate cohort had a 46% lower (95% CI 9% – 68%, p-value = 0.02) incidence of hip fracture than the alendronate cohort. After 12 months of therapy, the risedronate cohort had a 43% lower (95% CI 13% – 63%, p-value = 0.01) incidence of hip fracture than the alendronate cohort (Table 3). Fig. 4Cumulative incidence of hip fractures in patients treated with alendronate or risedronate for up to 1 year These differences between the risedronate and alendronate cohorts in the incidence of fractures were consistent across other methods of analysis (Fig. 5). Depending on the method, the risedronate cohort had an estimated 6% to 23% lower incidence of nonvertebral fractures and an estimated 30% to 50% lower incidence of hip fractures than the alendronate cohort through 12 months of therapy. All of these other estimates were within the confidence intervals of the primary analyses. Fig. 5Sensitivity analysis: Rate ratio for fracture in the first year of therapy between patients on risedronate and patients on alendronate; results of the primary analysis and 4 other methods of analyses Discussion In this observational study across multiple US health plans, we observed that patients on once-a-week dosing of risedronate had a lower incidence of hip and nonvertebral fractures than patients on once-a-week dosing of alendronate. Differences in fracture incidence between these two cohorts of patients were observed at 6 and 12 months after initiating therapy. As with all observational studies, systematic errors (e.g., selection bias, measurement misclassification) may be the basis for the observed results [26]. In this study, systematic errors may arise from differences in fracture risk between the 2 cohorts of patients at initiation of therapy. Between the two cohorts, there were statistical differences in measurable fracture risk characteristics (Table 2), there are likely to be differences in known fracture risk characteristics not available within medical claims data (e.g., bone mineral density, family history, smoking history), and there are likely to be differences in unknown fracture risk characteristics (i.e., those that are controlled through randomized trials). The differences in measurable fracture risk characteristics, for which a greater percentage of the risedronate cohort has risk factors for fracture than the alendronate cohort suggesting bias towards higher fracture rates in the risedronate cohort, are inconsistent with observed results. The near unity in fracture incidence between the two cohorts during the first 3 months of therapy (Figs. 3 and 4) - a period for which there is also unity in fracture incidence between bisphosphonate therapy and placebo in clinical trials [12, 14] - suggest that both cohorts had similar risk for fracture at initiation of therapy. However, differences in fracture risk at initiation of therapy between the two cohorts cannot be excluded. Within healthcare utilization data, which are collected for purposes other than research, misclassification of fracture events and of therapy use are inevitable. As a check on the data, the rate of fracture events and therapy use of these health utilization data are in agreement with other data sources. In the current study, the annual fracture rates following initiation of therapy (≈2.0% for nonvertebral fractures and ≈0.5% for hip fractures) are consistent with the annual rates in the treated population of clinical trials (between 2.0 and 2.3% for nonvertebral fractures and between 0.4% and 0.7% for hip fractures [6–10, 27]). In this study, risedronate patients constituted 25% [45,360/(45,360 + 137,412)] of the bisphosphonate users in the utilization data compared to 24% [13.6 million / (13.6 million + 44.1 million)] of all bisphosphonate prescriptions in the US during the same period [28]. A good method for evaluating misclassification within healthcare utilization data is through a medical chart review. In a prior study, the proportion of fracture claims confirmed by chart review to be a fracture was highest for the hip relative to other fracture sites [29]. Since the effect of misclassification at these other fracture sites is likely to be no different between cohorts (i.e., misclassified exposure does not depend on cohort status), the study results for nonvertebral outcomes are likely more attenuated by misclassification than results for the hip [30]. The strength of observational studies can be the generalizability of results. In contrast, the generalizability of results from randomized trials to a real world setting can be limited by differences between the two in relation to expertise of health care provider, quality of medical care, course of therapy, and types of patients [31]. For example, it has been observed that the majority of patients considered candidates for osteoporosis therapy by their physician would not meet the eligibility criteria for inclusion in the randomized trials [32]. Since the population within the current observational study is drawn from multiple health plans in many US states and consists of subjects with a mixture of health characteristics (e.g., prior gastrointestinal comorbidities), the results are likely to be generalizable. Furthermore, the length of observation of therapy adherence (≈232 days) in the current study was consistent to the previously reported average duration of adherence to bisphosphonate therapy (245 days) [33]. In conclusion, within this observational study of clinical practice, a cohort of patients receiving risedronate had lower rates of hip and nonvertebral fractures during their first year of therapy than a cohort of patients receiving alendronate. These results do not appear to be explained by baseline differences in fracture risk between cohorts. In addition, the observed rates of fracture were consistent with the fracture rates in clinical trials. Thus it appears, patients receiving risedronate are better protected from hip and nonvertebral fractures during their first year of therapy than patients receiving alendronate.

Apoptosis-4-1-2423419

Identification and characterization of the human inhibitor of caspase-activated DNase gene promoter

DNA fragmentation factor is a heterodimer complex of the nuclease CAD and its specific inhibitor ICAD, which can be activated during apoptosis to induce DNA fragmentation. Although ICAD expression levels have been quantified in a variety of human cancer cells, the mechanism of ICAD gene regulation remains unknown. In this study, we identified a 106-bp TATA-less region upstream of the transcription start site as a basal promoter of the human ICAD gene. An E-Box motif, which binds transcription factors of the basic helix-loop-helix/leucine zipper family, is responsible for transcriptional activity, as demonstrated using mutated promoter-reporters. A chromatin immunoprecipitation assay further demonstrated that Myc binds to an endogenous ICAD promoter. The functional importance of Myc in the regulation of ICAD transcription was also demonstrated by knock-down of c-Myc and N-Myc gene expression, as well as their ectopic expression. Structural analysis of the human ICAD promoter and identification of factors which regulate its activity might further our understanding of the biological role of ICAD with respect to regulation of apoptosis and cancer development. Introduction Apoptosis or programmed cell death ensures the elimination of unwanted cells during normal development and homeostasis [1]. This process is progressively inactivated during malignant development and loss of the capacity for apoptosis is a hallmark of malignant cells. Chromatin condensation and internucleasomal DNA fragmentation are typical nuclear features and well-recognized events in apoptosis. DNA fragmentation factor (DFF) is a heterodimer protein composed of a 40-kDa caspase-activated DNase (CAD) otherwise known as DFF40, and its cognate 45-kDa inhibitor (inhibitor of CAD: ICAD or DFF45 [2–8]). Both human genes map to 1p36 [9, 10]. CAD is thought to be responsible for the majority of nuclear activity resulting in chromosomal DNA fragmentation. When apoptosis is activated, ICAD is cleaved by executor caspases, mainly caspase-3, into three fragments, after which it dissociates from CAD, resulting in CAD activation. Thus, the DFF complex may play a role in malignant transformation, and up- or down-regulation of ICAD/CAD expression might correlate with cancer aggression. Expression levels of ICAD have been examined in a variety of human cancers. For example, ICAD expression is down-regulated during the exponential growth phase of human colon carcinoma cells [11]. In some neuroblastomas, preferential ICAD expression is observed in low-stage, but not in their high grade [12]. Other research suggests that down-regulation of ICAD may contribute to tumor growth and lymph node metastasis in esophageal carcinoma [13], and that ICAD expression might serve as a marker of aggressive tumor behavior with an associated poor prognosis in ovarian cancer [14]. We have previously demonstrated that the hepatitis C virus core protein, which not only encodes the viral nucleocapsid but has a number of properties enabling persistent viral infection, induces gene expression of ICAD, thereby increasing steady-state levels of the ICAD protein [15]. To date, the mechanism of transcriptional regulation of the ICAD gene is not well understood. Analysis of the gene structure of mouse ICAD showed that a 118-bp flanking region of the ICAD gene is required for promoter activity [16]. In this study, we identified a functional promoter of the human ICAD gene and investigated the role of c-Myc and N-Myc in regulation of the ICAD promoter. Materials and methods Cell cultures Human hepatoblastoma cells (Huh-7), human oral squamous carcinoma cells (HSC-2, HSC-3 and Ca22-9), mouse neuroblastoma cells (IMR-32 and GOTO) and mouse 3T3 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and 100 mg/ml of streptomycin and kept at 37°C in a 5% CO2 incubator. Cloning of the 5′-upstream region of the human ICAD gene Human genomic DNA was isolated from Huh-7 cell lines using the QIAamp DNA Mini kit according to the manufacturer’s instructions (QIAGEN). A 5′-upstream region of the ICAD gene was amplified by PCR using primers based on the genomic DNA sequence of the human ICAD gene (GenBank accession No. NM_004401). Primers containing EcoRI sites were used as follows: forward (5′-GAATTCAGGCTGGTCTCAAACTACTG-3′) and reverse (5′-GAATTCGATCTCGCCAGATTCTGGTA-3′). The PCR reaction was carried out at 94°C for 1 min, 55°C for 1.5 min, and 72°C for 2 min, for total of 35 cycles. Each PCR product was purified and subcloned into a pGEM-T Easy vector (Promega), followed by sequencing using an ABI PRISM 310 automated DNA sequencer (Applied Biosystems). 5′ rapid amplification of cDNA ends (5′-RACE) assay The 5′ ends of the ICAD transcript were cloned by 5′-RACE using a Gene Racer RACE-Ready cDNA kit in accordance with the manufacturer’s instructions (Invitrogen). Double-stranded cDNA molecules prepared from human liver poly(A)+ RNAs ligated by exposure to the adaptor were amplified in a primary PCR reaction using the adaptor primer 1 and the ICAD gene-specific antisense primer ICA1AS (5′-GTGCTGTTCGCGGCTGTAGTT-3′, nt −147 to −173), followed by a secondary PCR reaction using nested oligonucleotides, the adaptor primer 2, and the antisense ICAD specific primer ICA2AS (5′-CACGGTGACTGGTGTCAGGGACTTATC-3′, nt −228 to −254). PCR products were purified and cloned, followed by nucleotide sequencing as described above. Plasmid constructions The 1-kb sequence containing the human ICAD promoter was excised by digestion of the above-mentioned pGEM-T vector with SacI and HindIII, and then cloned into the firefly-luciferase-expressing reporter plasmid pGL3-Basic (Promega), resulting in pLUC1005. A series of constructs with 5′ end-deletions of the ICAD promoter were created by PCR amplification using the reverse primer (5′-CAAGCTTGCCTCCACAAGGTGGGACCTG-3′) and the following forward primers: 5′-GGCTAGCCAGTACCCATTTCTGAAGAAG-3′ (nt −936 to +71), 5′-GGCTAGCCCTCATTTGGGTCCATTTTCC-3′ (nt −622 to +71), 5′-GGCTAGCCAGCTTTTTCAGACAGAATGG-3′ (nt −272 to +71), 5′-GGCTAGCCAGCTTTTTCAGACAGAATGG-3′ (nt −205 to +71), 5′-GGCTAGCCAGCTTTTTCAGACAGAATGG-3′ (nt −145 to +71), 5′-GGCTAGCCAGCTTTTTCAGACAGAATGG-3′ (nt −106 to +71), 5′-GGCTAGCCTATTTAGTTTGGTTAGTAAT-3′ (nt −90 to +71), and 5′-GGCTAGCCCAGATGGTAAATATACACAA-3′ (nt −43 to +71). Each SacI/HindIII fragment was inserted into the pGL3-basic vector to yield pLUC(−936/+71), pLUC(−622/+71), pLUC(−272/+71), pLUC(−205/+71), pLUC(−145/+71), pLUC(−106/+71), pLUC(−90/+71) and pLUC(−43/+71). Transfection and reporter assay Huh-7 cells were seeded at 5 × 104 cells/well in 24-well plates and maintained at 37°C in a 5% CO2/95% atmosphere. DNA transfection of cells with each ICAD-promoter-luciferase construct (1 μg) with an internal control vector pRL-TK (0.1 μg) (Promega) was performed with Trans IT LT-1 (Mirus) during 6-h of incubation. Cells were then rinsed with phosphate-buffered saline (PBS) 48 h after transfection, and luciferase activity was measured in the cell lysate using dual luciferase assay reagents (Promega) [17]. Firefly luciferase activity was standardized according to Renilla luciferase activity. Chromatin immunoprecipitation Chromatin immunoprecipitation assays were performed using the ChIP assay kit (Upstate). Briefly, cells in 100-mm dishes were grown to 70% confluency over 48 h. The chromatin from formaldehyde-fixed cells was sonicated and immunoprecipitated using mouse monoclonal anti-c-Myc or anti-N-Myc antibodies (Santa Cruz). The chromatin immunoprecipitate was analysed by PCR with the following primer pairs: F1 (5′-CGAGCTCGGTATACATGCGTGTGCATCG-3′) and R1 (5′-CAAGCTTGCCTCCACAAGGTGGGACCTG-3′) for amplifying the region from nt −272 to −71 containing potential Myc-binding sites; and F2 (5′-GAGATCAAAACTGCAGTGAG-3′) and R2 (5′-CACTGTTGGAGATTGTTCAG-3′) for amplifying the region from nt −789 to −451 that does not contain Myc-binding sites. Western blotting Cells were washed with PBS and lysed in SDS sample buffer. Cell lysate samples were separated by 10% SDS-polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene difluoride membrane (Immobilion; Millipore). After blocking in nonfat milk solution (Blocking One; Nakaraitesk), the membranes were probed with monoclonal antibody against the ICAD protein (Santa Cruz), c-Myc (Sigma), or N-Myc (Santa Cruz), as the primary antibody for 1 h. After being washed, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse immunoglobulin as the secondary antibody, followed by visualization with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology). siRNA experiments siRNAs for human c-Myc, N-Myc and the negative control were purchased from B-Bridge International. Each siRNA consisted of three different target sequences as follows: 5′-GAGGAGACAUGGUGAACCA-3′, 5′-GAGAAUGUCAAGAGGCGAA-3′, and 5′-GAGAAUGUCAAGAGGCGAA-3′ for c-Myc siRNA; 5′-CGGAGAUGCUGCUUGAGAA-3′, 5′-CGGAGUUGGUAAAGAAUGA-3′, and 5′-CAGCAGUUGCUAAAGAAAA-3′ for N-Myc siRNA; and 5′-ATCCGCGCGATAGTACGTA-3′, 5′-TTACGCGTAGCGTAATACG-3′, and 5′-TATTCGCGCGTATAGCGGT-3′ for the negative control. Huh-7 cells were transfected with 100 pmol/ml siRNA using FuGENE6 (Roche) in Opti-MEM I (Invitrogen), after which the medium was replaced by standard DMEM after 6 h of transfection. RNA preparation and real-time quantitative RT-PCR Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. First-strand cDNAs were synthesized using the SuperScriptIII First-Strand Synthesis kit (Invitrogen) and then used as templates for real-time PCR. Quantitative PCR was performed using the ABI Prism 7700 sequence detection system using Taq-Man Gene Expression assays (Applied Biosystems). The standard curve was created using serially diluted total RNA obtained from Huh-7 cultures and β-actin was chosen as the internal standard to control for variability in amplification. Amplification was performed at 95°C for 10 min, followed by 40 cycles of amplification at 95°C for 15 s and 60°C for 60 s. Results and discussion Localization of transcription start sites of the human ICAD gene Precise localization of transcription start sites of the ICAD gene was examined in human hepatoma Huh-7 cells by 5′-RACE analysis. A fragment of approximately 100 bp was obtained (Fig. 1a) and cloned, after which sequencing analysis of several cDNA clones identified two of 5′ termini (Fig. 1b). These were located 71 and 68 nucleotides upstream from the translation start codon of the ICAD gene. From a database search of the DBTSS [18], we found that these are two of multiple sites for putative transcription initiation for the human ICAD gene. Although transcription may be initiated at both sites, the upper site was designated position +1 in this study. Fig. 1(a) Mapping of the transcription start sites by 5′ RACE analysis. Following nested PCR reaction, a PCR product of 100-bp was detected by 1.5% agarose gel electrophoresis (lane 2). Lane 1: no template control; lane M: 100-bp DNA ladder marker. (b) The 5′ flanking region of the human ICAD gene. The nucleotide sequence is numbered from the major transcription site, which is indicated by an arrow. Putative binding sites for transcription factors analyzed by the TRANSFAC database and search program were shown with underlines Characterization of the ICAD gene promoter To examine potential regulatory sequences involved in ICAD gene expression, a 1 kb 5′-upstream region of the ICAD gene was sequenced and analyzed. The overall GC content of the 1-kb genomic DNA fragment was 46%, while GC content among the 200 bp proximal to the ATG start codon was approximately 62%. A search of transcription consensus motifs using the TRANSFAC database [19] demonstrated that none of the major eukaryotic promoter elements, TATA-, CCAAT-, or GC box, were located within 200 bp upstream of the transcription start site, although a TATA-like element was found more than 400 bp upstream from the start sites, indicating that the human ICAD gene has a TATA-less promoter. No CpG island, which is frequently observed in the TATA-less promoter, was found by screening with the CpG island searcher (http://cpgislands.usc.edu/). Absence of a TATA-like sequence upstream of the transcription start site has also been reported for the mouse ICAD gene, suggesting that TATA-less promoters might be a common feature in transcriptional regulation of the ICAD gene. As shown in Fig. 1b, the 1-kb sequence contains potential binding sites for several transcription factors. The ability of the 5′-upstream region of the ICAD gene to function as a promoter was assessed by its capacity to drive the expression of a luciferase reporter gene. A series of constructs in which genomic DNA fragments were fused to a promoterless firefly luciferase gene of the pGL3-basic vector were generated with the 3′ end always terminating +71 bp from the transcription start site. The 5′ ends began at bases −936, −662, −272, −205, −145, −106, −90, and −43 (Fig. 2). Promoter activity was assessed by measuring luciferase activity in transiently transfected Huh-7 cells. Luciferase activity was normalized to Renilla luciferase activity. As shown in Fig. 2 on the right, the region extending from −936 to +71 had promoter activity since luciferase activity of the reporter gene was 45-fold that of the empty vector. A 5′ deletion of the promoter sequence to −662(pLuc(−662/+71)) enhanced promoter activity, and successive removal of nucleotides from −272 to −106 also enhanced promoter activity, suggesting the possibility of negative regulatory elements within the −936/−622 and −272/−106 regions. When the sequence from −106 to −90 was removed (pLuc(−90/+71)), promoter activity fell to parallel that of the pGL3 basic vector. This suggests the presence of a critical element in the region extending from −106 to −90, thus, we have identified the region 106 bp upstream of the transcription start site as the basal promoter of the human ICAD gene. Fig. 2Human ICAD promoter drives reporter gene expression in transiently transfected Huh-7 cells. Deletion constructs of the upstream region of the ICAD gene linked to the firefly luciferase reporter gene (LUC) are illustrated in the left. Huh-7 cells were cotransfected with a firefly luciferase reporter and a Renilla luciferase internal control (pRL-TK). The relative luciferase activity (RLU) was obtained by normalizing the firefly luciferase activity to the Renilla luciferase activity, and is expressed as a percentage of RLU of pLuc(−936/+71). The average values are shown with standard deviation (SD) (n = 4 per construct) Myc and USF enhance ICAD promoter activity As indicated in Fig. 1b, a computer-based sequence analysis revealed that the 106 bp region contains potential binding sites for GATA-1, deltaE, Myc, and various upstream stimulatory factors (USF). Within this region, there is a canonical E-box sequence (5′-CACGTG-3′) located between −103 and −98. E box motifs are known to bind to transcription factors of the basic helix-loop-helix/leucine zipper (bHLH LZ) family, including Myc and USF [20–23]. We examined the potential role of E-box-binding proteins, Myc and USF, in potentiating ICAD gene transcription. Myc is bound to a partner protein Max via a bHLH LZ domain and the Myc-Max heterodimer activates transcription by binding to an E-box sequence [24, 25]. Max is present in greater amounts than Myc since the Myc transcript and protein have shorter half-lives compared to Max [26, 27]. Therefore, it is highly likely that Myc is the limiting, regulated component of the heterodimer. To examine the potential role of E box binding proteins, Huh-7 cells were co-transfected with luciferase reporter plasmids and expression vectors for c-Myc, N-Myc, USF1, and USF2 (Fig. 3a). Luciferase activity of the −145/+71 promoter construct increased 2- to 5-fold, and 1.5- to 2-fold, when co-expressed with Myc or USF, respectively. The effect of c-Myc and N-Myc on activity of the −90/+71 construct was less than for the −145/+71 construct. USF expression did not enhance activity of the −90/+71 construct. Fig. 3(a) ICAD promoter activity after transient expression of c-Myc, N-Myc, USF1 and USF2. Huh-7 cells were cotransfected with either expression vector for c-Myc, N-Myc, USF1 or USF2 driven by the CMV promoter and pLuc(−145/+71), pLuc(−90/+71) or pGL3 basic. Each firefly luciferase reporter plasmid (n = 4) was cotransfected in cells with pRL-TK for normalization of the reporter activity. RLU is expressed as a percentage of that of pLuc(−145/+71) in the absence of expressing plasmids for Myc and USF. (b) Effect of substitution mutation in the E-box element on the ICAD promoter activity. Cells were cotransfected with pLuc(−106/±71) or an E-box-mutant, pLuc(−106/±71)mt and either expression vector for c-Myc or N-Myc. pRL-TK was also used for normalization of the activity. RLU is expressed as a percentage of that of pLuc(−106/±71) without over-expression of Myc To further investigate involvement of the E box sequence in ICAD promoter activity, a reporter construct with an E box mutation (CCCGCG) was constructed and luciferase activity was examined in transfected cells (Fig. 3b). The E box mutation resulted in marked down-regulation of reporter gene expression and the reporter activity expressed from the E-box mutant was little increased when co-expressed with Myc, suggesting a functionally important role of the −106/−90 region E box motif with regard to basal transcriptional activation of the ICAD promoter. These data indicates that E-box-binding proteins, especially Myc, actively participate in positive regulation of the human ICAD promoter. Myc binds to the ICAD promoter in vivo To obtain direct evidence of an interaction between Myc and the ICAD promoter in vivo, we next examined binding of c-Myc and N-Myc to the 5′-upstream region of the ICAD gene within the context of native chromatin in living cells by chromatin immunoprecipitation. An alignment of the sequence around putative Myc binding site of human ICAD promoter and the corresponding part of mouse ICAD sequence demonstrated conservation of Myc binding sequence in human and mouse (Fig. 4a) [16]. Proteins were cross-linked to genomic DNA isolated not only from human (Huh-7) but mouse (IMR-32 and GOTO) cells, followed by immunoprecipitation with normal rabbit IgG or polyclonal antibodies to either c-Myc or N-Myc. IMR-32 and GOTO are neuroblastoma cells with N-Myc gene amplification [28]. The precipitated DNA was then subjected to PCR utilizing primers designed to amplify a 343-bp fragment (−272/+71) or a 338 bp fragment (−789/−451) of the ICAD 5′ flanking region. As shown in Fig. 4b, the 343-bp DNA fragment was observed in the immunoprecipitate from Huh-7 cells following exposure to anti-c-Myc or anti-N-Myc, while amplification of the ICAD promoter fragment was not detected in the negative control immunoprecipitate. Under similar experimental conditions, we did not detect binding of c-Myc and N-Myc to the −789/−451 region, which is not thought to contain a Myc-binding site. Similar results were obtained from IMR-32 and GOTO cells, which were immunoprecipitated with anti-N-Myc antibody. These results demonstrate that Myc forms a complex with the human ICAD promoter in cells, presumably through binding to the E-box sequence. Fig. 4(a) An alignment of the sequence around putative Myc binding site of human ICAD promoter and the corresponding part of mouse ICAD sequence. (b) Binding of Myc proteins in the endogenous ICAD promoter. Crosslinked chromatins isolated from Huh-7, IMR-32 and GOTO cells were immunoprecipitated with indicated antibodies (c-Myc, N-Myc) or an equivalent amount of mouse IgG. Recovered DNAs were purified and PCR-amplified with primers for nt −272/±71 region or for nt −789/−451 region. Input represents 1% of chromatin sample applied for immunoprecipitation Myc-dependent expression of the ICAD protein The above data suggests that the ICAD promoter is regulated by endogenous Myc proteins. This assumption is supported by the loss-of-function studies shown in Fig. 5a. We employed siRNA (small interference RNA) transfection to knock-down the expression of c-Myc or N-Myc in cells, after which we analyzed the effect of reduced Myc expression on steady state levels of the ICAD protein by Western blotting. Treatment with siRNA specific for either c-Myc or N-Myc markedly reduced ICAD expression without affecting the expression of β-actin. In contrast, control siRNA did not reduce ICAD levels. We further investigated the effect of over-expression of Myc on ICAD protein levels (Fig. 5b). Transfection of Huh-7 and mouse 3T3 cells with a c-Myc or N-Myc expression vector enhanced ICAD protein levels with an effect that was less pronounced than in the loss-of-function studies, but reproducible nonetheless. These results suggest an important role of Myc proteins in positive regulation of ICAD expression. Fig. 5Myc-dependent expression of human ICAD. (a) Suppression of Myc expression decreases expression of ICAD protein. Huh-7 cells were transiently transfected with Myc siRNA (c-Myc, N-Myc) or the control siRNA (cont). Three days later, the cells were harvested and subjected to Western blotting. (b) Ectopic expression of Myc increases expression of ICAD protein. Cells (Huh-7 and mouse 3T3) were transfected with the expression vector for c-Myc or N-Myc, and after 3 days the cells were harvested for Western blotting. (c) Comparison of the mRNA expression of c-Myc and ICAD in Huh-7 cells and human oral squamous carcinoma cells (Ca22-9, HSC-2, and HSC-3). Levels of mRNA expression of ICAD and c-Myc were measured by quantitative real-time RT-PCR based on Taq Man chemistry. Results, relative to β-actin mRNA, are depicted as averages with SD (n = 3) Finally, we examined if there might be a correlation between ICAD and Myc expression levels in cancer cell lines. The mRNA expression of ICAD and c-Myc in human oral squamous cell carcinomas (Ca22-9, HSC-2, and HSC-3), in which ICAD expression has not been investigated to date, as well as in Huh-7 cells during their late exponential phase of growth, was analyzed by quantitative RT-PCR (Fig. 5c). ICAD mRNA was readily detected in all cancer cells but the expression level was not consistent. A relatively high level of expression was observed in Ca22-9 and HSC-3 cells, while HSC-2 cells demonstrated the lowest. Interestingly, expression pattern of c-Myc mRNA in these cell lines showed a tendency similar to that of ICAD, suggesting an important role of Myc in ICAD expression in human cancers. A large scale analysis for investigating correlation between ICAD and Myc expression in a variety of cancer tissues obtained from patients is ongoing. Several studies have shown up- or down-regulation of ICAD in a variety of human cancers [11–14, 29, 30]. Although transcriptional deregulation is presumably involved in this aberrant expression of ICAD, little is known about the transcriptional regulation of ICAD in human cells. This work was done to characterize the human ICAD promoter and to examine transcription factors which may be involved in its regulation. We experimentally determined putative transcription start sites by employing the 5′-RACE method, and identified a promoter region required for basal ICAD gene expression using promoter-reporter constructs with progressive deletions. A combined study of site-directed mutagenesis of a reporter construct and chromatin immunoprecipitation revealed the importance of an E box Myc-binding motif located at position −103 to −98 from the transcription start sites, as well as an in vivo interaction between c-Myc and N-Myc and the proximal ICAD promoter. Furthermore, we demonstrated the functional importance of Myc proteins with regard to transcriptional regulation of the ICAD gene from studies examining ectopic expression of c-Myc and N-Myc, as well as with RNAi technology. We showed that expression levels of ICAD and Myc correlate in some tumors. c-Myc and N-Myc are transcription factors of the bHLH LZ family that bind to the E box sequence within promoters to control proliferation, cell differentiation, and apoptosis [25, 31, 32]. c-myc and N-myc genes are deregulated in a number of human cancers and influence proliferation and growth. A link between Myc and cancer is well established both in vivo and in vitro, and oncogenic activation of Myc has been observed to promote the development of a number of clinically significant cancers [25, 33]. However, the molecular and cellular mechanisms of Myc-mediated transformation are not fully understood. Although a variety of Myc target genes were identified and recruitment of Myc to the target promoters such as prothymosin and telomerase were shown [34], there has been no direct evidence on involvement of Myc in regulation of ICAD expression. It is known that c-Myc activation usually occurs during the later stages of carcinoma in humans. Conversely, in premalignant cells, c-Myc is a robust stimulator of apoptosis and programmed cell death [32]. One area of investigation into cancer cell death mechanisms centers on the mechanism by which c-Myc stimulates or suppresses apoptosis. For instance, Myc has been reported to potentiate apoptosis through both p53-dependent and -independent mechanisms [35, 36]. Myc controls the balance between pro- and anti-apoptotic factors at the level of the mitochondria, thereby regulating cytochrome C release and activation of downstream caspases [37, 38]. Involvement of Myc in up-regulation of ICAD expression as demonstrated in this study might present a novel mechanism of Myc-dependent inhibition of apoptosis. It is possible that elevated levels of ICAD in cells inhibit activation of endonuclear activity, thereby increasing the threshold for apoptosis signaling. Kawane et al. have reported on the structure and analyzed the promoter of a murine ICAD gene, by which they demonstrated that a 118-bp flanking region of the ICAD gene is required for its transcription [16]. The mouse sequence shares approximately 82% homology with a corresponding upstream region of the human ICAD gene. The mouse ICAD promoter has a number of potential binding sites for transcription factors, such as Ikaros, c-Rel, Myc, and Gfi-1 [16]. Conservation of the Myc-binding motif among human and murine promoters suggests a functional significance of Myc in transcriptional regulation of ICAD expression. In conclusion, this is the first report to identify a functional promoter of the human ICAD gene, and to demonstrate that Myc proteins are able to positively regulate ICAD gene expression. Extensive apoptosis research to date has shown that tumor aggression depends on various defects in apoptosis signaling [39]. Further investigation into the molecular events linking Myc expression with ICAD gene regulation may provide insight into Myc’s role in cell proliferation, transformation and apoptosis.

J_Mol_Biol-2-1-2279226

Histone Modifications Influence the Action of Snf2 Family Remodelling Enzymes by Different Mechanisms

Alteration of chromatin structure by chromatin modifying and remodelling activities is a key stage in the regulation of many nuclear processes. These activities are frequently interlinked, and many chromatin remodelling enzymes contain motifs that recognise modified histones. Here we adopt a peptide ligation strategy to generate specifically modified chromatin templates and used these to study the interaction of the Chd1, Isw2 and RSC remodelling complexes with differentially acetylated nucleosomes. Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways. For example, histone H3 lysine 14 acetylation acts to increase recruitment of the RSC complex to nucleosomes. However, histone H4 tetra-acetylation alters the spectrum of remodelled products generated by increasing octamer transfer in trans. In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment. These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes. Introduction Packaging DNA into nucleosomes and higher-order chromatin structures restricts access to the underlying genetic material. Thus, the manipulation of chromatin is a key step in many nuclear processes such as transcription, replication and repair. One of the means by which eukaryotes achieve this is through the use of ATP-dependent chromatin remodelling enzymes that non-covalently alter its structure. These enzymes are members of a diverse group of proteins named after the archetypal Saccharomyces cerevisiae Snf2 protein; the Snf2 family. Multiple members of this family of proteins are present in the sequenced genomes of eukaryotes of which the chromatin remodelling enzymes form distinct sub-groupings.1 Recent crystal structures of the catalytic domain of two Snf2 related proteins2,3 highlight structural similarities with the RecA domain found in a broad range of helicase related proteins. Snf2 proteins use the energy from ATP hydrolysis to alter DNA–protein interactions. However, unlike bona fide helicases, the action of chromatin remodelling enzymes is not generally associated with separation of DNA strands. Instead they act to catalyse dynamic transitions in chromatin structure. This can result in a variety of different outcomes. In vitro, chromatin remodelling enzymes have been shown to cause alterations to DNA accessibility, effects on DNA and chromatin topology, partial or complete removal or exchange of histones from nucleosomes and translational changes in the position of the intact histone octamer along DNA.4 How these proteins interact with chromatin and the mechanism by which chromatin remodelling is achieved remains unclear. However, in many cases it is apparent that histone tail domains are important regulators of chromatin remodelling. Genetically, there are links between histone tails and remodelling. Deletion of the H2B tail bypasses the need for the SWI/SNF complex in the regulation of several genes,5 and deletion of the H2A tail affects transcription of some SWI/SNF dependent genes.6 Importantly, post-translational modifications of histone tails affect chromatin remodelling. The recruitment and activity of the SWI/SNF complex in vivo is widely linked to that of the SAGA histone acetyltransferase complex.7–9 In addition, many chromatin remodelling complexes contain domains shown to bind modified amino acids, such as bromo- and chromodomains, PHD and WD40 domains.10 In these cases modifications have been proposed to act as epitopes that facilitate recruitment of proteins containing the appropriate recognition domains. For example, histone acetylation has been found to facilitate retention of SWI/SNF on nucleosome arrays11 in a way that is dependent on bromodomains within the Snf2 protein.12 The majority of the best characterised modifications occur within the non-globular histone tail domains which protrude from the core of the nucleosome. These histone tails constitute up to 30% by mass of histones, but are not visible in the crystal structures of nucleosomes due to their high intrinsic flexibility and have been thought to be largely unstructured.13 Their importance is highlighted by the fact that deletion of histone tails has wide spread effects on chromatin structure and gene regulation.14–16 Histone modifications have been correlated with a variety of chromatin states. On a genome-wide basis, histone H3 K4 tri-methylation and H3 acetylation are strongly correlated with active transcriptional start sites, phosphorylated H2A.X foci mark sites of DNA damage, and H3 K9 tri-methylation promotes the formation of heterochromatin via interaction with HP1.10,17 This suggests that histone post-translational modifications are an important means by which cells spatially and temporally regulate specific loci independently of bulk chromatin. Rigorously studying the effect of histone tail modifications in vitro has been hampered by the difficulty in isolating histones with defined and homogeneous modifications. To circumvent this we have adopted an approach to chemically synthesise modified histones by means of native chemical ligation. This technology has previously been applied to generating chromatin with uniform histone N-terminal tail modification patterns.18–20 Using this approach we show that histone modifications can affect nucleosome remodelling via distinct pathways. Tetra-acetylation of histone H3 results in a modest increase in the rate of intrinsic nucleosome repositioning. The ATP-dependent remodelling enzyme RSC shows a striking preference for H3 but not H4 tetra-acetylated chromatin, remodelling this 16-fold faster than the control. By measuring the kinetic parameters of the remodelling reaction with a real-time ATPase assay, we show that this is due to a lower Km value for H3 acetylated nucleosomes. In contrast, tetra-acetylation of histone H4 affects nucleosome remodelling by the Isw2 enzyme via altering the Kcat rather than the Km of the ATPase reaction. We also show that the yeast Chd1 enzyme is dependent on the H4 tail for efficient nucleosome remodelling in a manner similar to Isw2. Remarkably, histone H4 tetra-acetylation affects the rate of RSC catalysed nucleosome transfer in trans but not nucleosome sliding in cis. Thus, nucleosome modifications can affect enzyme catalysed remodelling reactions by altering the outcome of the reaction, through allosteric effects on ATPase activity and by acting as binding epitopes for recruitment. Results Generation of modified chromatin Modified histones were generated by ligating a bacterially expressed Xenopus laevis globular histone containing an N-terminal cysteine and a tail peptide with the modification of choice synthesised chemically as a C-terminal thioester. The ligation was based on the protocol described by Dawson & Kent21,22 and used thiophenol as the catalyst (Figure 1(a)). A key requirement of the globular histone is that it has an uncapped N-terminal cysteine available for ligation. One strategy that has been used previously to ensure that the initiator methionine is removed is the introduction of a protease cleavage site just before the cysteine to expose it post-translationally.20 Mass spectrometry (Supplementary Data, Figure 1) and the work of other laboratories19,23 show that removal of the initiator methionine in vivo by Escherichia coli methionine aminopeptidases is efficient when cysteine is the adjacent amino acid. This alleviates the requirement for more complex systems for generation of N-terminal cysteine residues. Polyacrylamide gel electrophoresis reveals that under the conditions used, ligation does not proceed to completion (Figure 1(b), lane 1). However, the full-length modified histone can be separated from the unreacted globular domain and tail peptide via multiple rounds of ion exchange chromatography to obtain material that was greater than 95% pure (Figure 1(b)). The overall efficiency of ligation and subsequent purification is approximately 20% (data not shown). When modified histones were combined with other core histones to form histone octamers and purified by size exclusion chromatography, they displayed identical elution profiles to unmodified octamers (data not shown). Modified histone octamers could be reconstituted onto DNA fragments to form nucleosomes. Interestingly, acetylation of H3 and to a lesser extent H4 reduced the migration of nucleosomes on native polyacrylamide gels (Figure 1(c)). This has been observed previously in the Bradbury laboratory using hyperacetylated histones purified from HeLa cells24 and suggests that linker DNA conformation may be affected. This altered electrophoretic migration is not due to the ligation procedure as nucleosomes containing methylated lysine residues did not show this behaviour (not shown). Lysine acetylation can alter intrinsic nucleosome mobility Although nucleosomes are stable with respect to dissociation, they can undergo a range of dynamic fluctuations in their structure. For example, following thermal incubation nucleosomes are frequently observed to redistribute to thermodynamically favoured locations.25 This movement of nucleosomes from one translational position on DNA to another can be followed using native PAGE.26 Acetylated and control nucleosomes were reconstituted onto differentially labelled fluorescent DNA, mixed, incubated thermally and run on a native gel (Figure 2(a)). The different nucleosomes can then be visualised separately using selective excitation and emission filters. From this the proportion of nucleosomes at the initial and final position can be measured at each time point. The rate at which nucleosomes accumulate at their destination can then be plotted and a hyperbolic curve fitted to the data enabling the initial rate of redistribution to be estimated (Figure 2(b)–(e)). This approach has proved more reliable at directly comparing the behaviour of different nucleosomes.27 Although native thiol ligation results in a normal peptide bond without the introduction of unusual chemical moieties, it requires the presence of a cysteine residue at the point of ligation. For this reason, the unmodified control nucleosomes used as a reference are point mutants containing a cysteine at the equivalent position to the modified histones. The sites selected for ligation involved substitution of cysteine for amino acids with similar overall dimensions (e.g serine to cysteine). None the less it was important to show that introduction of these mutations did not affect the behaviour of nucleosomes. Supplementary Data, Figure 2 shows a comparison of the thermal mobility of wild-type and cystein point mutant nucleosomes showing that the cystein mutations themselves have no discernable effect. The effects of the cysteine point mutations were also compared to wild-type histone sequences in other assays described below and in all cases no significant effect was detected (Supplementary Data, Figures 2, 3, 4; and data not shown). As a histone bearing the relevant cysteine point mutation is most closely matched to the product of a peptide ligation reaction, we have generally presented data using the point mutant as a control rather than the wild-type histone sequence. However, we are not aware of any cases where the choice of control affects the conclusions drawn. H4 acetylated nucleosomes were observed to be repositioned at the same rate as control nucleosomes (Figure 2(b)). In contrast, nucleosomes containing acetylated H3 relocated twice as fast as the control (Figure 2(c) and (e)). To test to what extent this was due to the partial neutralisation of basic charge that occurs upon acetylation of lysine residues, a histone H3 construct with lysine to alanine mutations at the four acetylated positions (H3 K9, 14, 18, 23A) was compared to wild-type (Figure 2(d) and (e)). This nucleosome repositioned at the same rate as the control indicating that charge neutralisation is not the sole mechanism by which acetylation affects nucleosome mobility. The RSC complex preferentially repositions tetra-acetylated H3 but not H4 nucleosomes RSC is an abundant and essential yeast chromatin remodelling complex that is closely related to SWI/SNF.28 A notable feature of the RSC complex is that it contains almost half of the known bromodomains in the S. cerevisiae genome. As bromodomains within other proteins have been found to recognise specific patterns of histone acetylation,29,30 a distinct possibility is that histone acetylation will influence the action of the RSC complex. To test this, the ability of RSC to remodel either H3 or H4 acetylated chromatin relative to unmodified control chromatin was assayed. RSC showed a dramatic preference for H3 (K9, 14, 18, 23) tetra-acetylated chromatin compared to an unmodified control (Figure 3(a)). From the initial rates of remodelling derived from the plotted data, this was calculated to be 16(±1.5)-fold faster than control (Figure 3(d)). When the rate of H4 (K5, 8, 12, 16) tetra-acetylated nucleosomes was measured, this was found to be indistinguishable from the rate of an unmodified control (Figure 3(b) and (d)). It is also worth noting that although the rate at which the H3S28C control nucleosomes are shifted in Figure 3(a) is slower than the rate at which the H4V21C nucleosomes are shifted in Figure 3(b), this should not be interpreted as indicating that the S28C mutation reduces the rate at which nucleosomes are repositioned by RSC. The reason that S28C nucleosomes are moved slower is due to the preferential engagement of RSC with acetylated nucleosomes at the expense of non-acetylated nucleosomes in the reaction mixture, as they are essentially in competition with each other. Indeed, comparing the rate of acetylated nucleosomes to either wild-type or cysteine containing control nucleosomes shows the same effect (Supplementary Data, Figure 3) This illustrates the importance of only making direct comparisons between the two templates present in the same remodelling assay. In this case, our data show that H3 acetylation stimulates nucleosome slding whereas H4 acetylation does not. Thus, none of the seven bromodomains found within the RSC complex appear to interact with acetylated lysines within the H4 tail in a way that affects nucleosome sliding. This is consistent with the observation that the relative rate of remodelling of a H3 and H4 acetylated nucleosome is not faster than a nucleosome that is just acetylated on the H3 tail (Figure 3(c) and (d)). H3 tetra-acetylation influences remodelling by the RSC complex by affecting Km but not Kcat of ATP hydrolysis and acts largely via H3 K14 acetylation The increased rate with which RSC repositions H3 acetylated nucleosome could be due to the modified lysine residues acting to recruit RSC or by allosterically affecting the remodelling reaction. To differentiate between these two options, the kinetic parameters of the ATP hydrolysis reaction were measured using a real-time fluorescent ATPase assay.31 The assay hinges on using a phosphate binding protein (PBP) labelled with a coumarin-based fluorescent dye, 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC), as a sensor for the amount of inorganic phosphate (Pi). On binding Pi, the labelled protein (MDCC-PBP) undergoes a shift in its emission wavelength coupled with a fivefold increase in fluorescence. When performed in a fluorimeter, this assay has the advantage of measuring ATP hydrolysis in real-time, from which kinetic parameters such as Km and Vmax are determined by non-linear fitting to the Michaelis–Menton equation (Figure 4(a)–(d)). We find that RSC has approximately threefold lower Km (tighter binding) for H3 acetylated nucleosome compared to the unmodified control, without affecting the Kcat of ATP hydrolysis (Figure 4(e)). This is consistent with preferential recruitment of RSC to H3 acetylated chromatin and the preferential binding of RSC to H3 acetylated nucleosomes (Supplementary Data, Figure 5). In contrast, RSC does not preferentially bind H4 acetylated nucleosomes, as the Km for these is the same as for the unmodified control (Figure 4(e)). Consistent with this, the Km value of the doubly H3, H4 acetylated nucleosome is indistinguishable from that of the H3 acetylated nucleosome (Figure 4(e)). Previous work from the Cairns laboratory has suggested the tandem bromodomains of the RSC4p subunit of the RSC complex interact with acetylated H3 K14.32 To test whether the single acetylation of lysine 14 is responsible for the dramatic effect of H3 (K9,14,18,23) tetra-acetylation on RSC activity, the singly acetylated nucleosome was generated and put through the same ATPase assay described above. From Figure 4(e) it can be seen that as for H3 tetra-acetylated nucleosomes, the Kcat value remains unchanged. In contrast, the Km value was significantly lowered, almost to the levels of the tetra-acetylated construct, suggesting that acetylation at a single residue, K14, confers the majority of the effect observed upon tetra-acetylation of the H3 tail. The H4 tail and its acetylation influence remodelling by Isw2 by affecting the Kcat of ATP hydrolysis Subfamilies of Snf2 family chromatin remodelling proteins contain different motifs capable of interacting with histones raising the possibility that they may be regulated in different ways by histone modifications. One of the defining characteristics of the Iswi subfamily of remodelling enzymes is that they require a particular epitope within the H4 tail for efficient remodelling activity.33–36 One of the lysine residues adjacent to this motif, H4 K16, can be acetylated, and there is evidence that this reduces the activity of ISWI containing remodelling complexes.19,37 When we compared the relative rate of mobilisation of H4 tetra-acetylated nucleosomes relative to an unmodified control by the yeast Isw2 enzyme, we found that the rate is approximately 1.5-fold slower (Figure 5(b) and (c)). This reduction, whilst significant, is not as severe as deletion of the H4 tail (Figure 5(a) and (c)). However, its biological importance is underlined in studies on flies, where defects in the genetic interaction between H4 K16 acetylation and ISWI result in large scale chromosomal abnormalities.37 Applying the same kinetic analysis performed previously to RSC shows that deletion or acetylation of the H4 tail lowers the turnover rate of the hydrolysis reaction (Kcat) without affecting nucleosome binding (Km) (Figure 5(d)). We have confirmed this by performing gel shifts of either full length or gH4 nucleosomes with Isw2 and observe no difference in binding (Supplementary Data, Figure 6). This contrasts with the effect of histone acetylation on remodelling by the RSC complex and shows that histone modifications can affect chromatin-remodelling enzymes at different stages of the reaction cycle. The yeast Chd1 enzyme requires the H4 tail for efficient chromatin remodelling Chd1 belongs to a phylogenetic subfamily that is distinct from either of the previously studied enzymes1 and contains a chromodomain that has the potential to recognise histone modifications. To investigate the histone dependence of this enzyme, we first tested the effect of deleting individual histone tails. We found that Chd1 is unable to efficiently slide nucleosomes lacking the H4 amino terminal tail (approximately sixfold slower than control: Figure 6(a), compare lanes 1–6 with 7–12). This requirement for the H4 tail is similar to that seen with Iswi remodellers such as Isw2, although the magnitude of the effect is lower. Mutation of residues 16–19 to alanine caused a 4.4-fold reduction in Chd1 activity (Figure 6(b)). Although this effect is significant it does not fully match the effect of a H4 truncation raising the possibility that the amino acids recognised by Chd1 include residues in addition to those within the 16–19 region. Acetylation of residues within the H4 tail also affected Chd1's nucleosome remodelling activity. Indeed, when this was tested, there was a modest 1.4-fold reduction in the initial rate of remodelling of acetylated H4 versus unmodified nucleosomes (Figure 6(c) and (d)). This raised the question of whether, as in the case of Isw2, the H4 tail was an allosteric activator of ATP hydrolysis or whether it was required as a binding epitope to recruit Chd1 to nucleosomes. Comparing the ATP hydrolysis rates of Chd1 with full-length and gH4 nucleosomes revealed that binding (Km) was unaffected (see also Supplementary Data, Figure 6(b)) and it was again the catalytic turnover rate which decreased in the absence of the H4 tail (Figure 6(e)). H4 acetylation leads to loss of histones in trans Studies with the RSC and SWI/SNF complexes in vitro have shown that in addition to being able to redistribute nucleosomes along DNA fragments, they can transfer histone octamers from one DNA fragment to another at lower efficiency. This entails the displacement of the histone octamer from one molecule of DNA and subsequent transfer onto a separate one. The SWI/SNF complex has been implicated in the disassembly and subsequent reassembly of nucleosomes at the Pho5 promoter.38,39 This loss of nucleosomes at the promoter is correlated with spikes in histone acetylation levels, leading to the hypothesis that ATP-dependent remodellers may recognise this signal and promote transfer. To test whether there is a direct link between histone modification and octamer transfer, the efficiency of octamer transfer from different chromatin substrates by the RSC complex was measured. The octamer transfer reaction involves incubating a “cold” donor nucleosome with a “hot” 32P-labelled accepter DNA fragment in the presence of RSC and ATP. The reaction products were run on a native polyacrylamide gel and transfer is indicated by a shift in mobility of the “hot” accepter DNA. If the accepter DNA has a histone octamer transferred onto it, it should now have the same mobility as a control nucleosome reconstituted onto the same fragment (Figure 7(a)). This was quantified as the fraction of radiolabelled free DNA incorporated into nucleosomes, as measured by mobility on a native polyacrylamide gel (Figure 7(b) and (c)). The amount of octamer transfer catalysed increased over time and was dependent on both ATP (Figure 7(b), lanes 1–3) and RSC (data not shown). As expected, octamer transfer from H3 acetylated nucleosomes was greater compared to unmodified nucleosomes (Figure 7(b), compare lanes 1–3 and 4–6). This is consistent with RSC having a lower Km for H3 acetylated nucleosomes (Figure 4) and the preferential nucleosome sliding seen in Figure 3. Surprisingly, when we looked at H4 acetylated donor nucleosomes, these were transferred considerably more efficiently than unmodified nucleosomes. Indeed, the amount of transfer was almost as high as that of H3 acetylated nucleosomes (Figure 7(c)), despite RSC not showing any increased affinity towards H4 acetylated chromatin (Figure 4). A doubly H3 and H4 acetylated nucleosome was in turn transferred more efficiently than either of the single histone acetylations. This suggests that H4 acetylation does not act at the level of stimulating RSC directly but rather by predisposing the nucleosome to be remodelled in trans, perhaps by destabilising it. Consistent with this, we find that H4 acetylated nucleosomes are more prone to thermally induced histone H2A/H2B dimer loss than H3 acetylated nucleosomes (Supplementary Data, Figure 7). Discussion Histone acetylation alters the intrinsic dynamic properties of nucleosomes Since they were discovered in the 1960's histone modifications have been predicted to affect chromatin structure and gene regulation.40 Subsequent studies have revealed correlations between histone modifications and transcription. For example, the recent application of genome-wide microarrays to map histone modifications, reveals that modifications such as H3 acetylation are found at the promoters of active genes and that their levels correlate with that of transcription.41–46 There are two prevailing views as to how modifications such as acetylation affect transcription. The first is based on the recruitment of activators due to recognition via modification binding modules. The number of modifications and respective binding domains identified has resulted in the hypothesis that these may form a code, which is read to determine a specific response.47 Alternatively it has been suggested that lysine acetylation may have a structural effect on chromatin structure by neutralising the charge interactions between histone lysine residues and DNA, resulting in a more open conformation.48–50 Several lines of evidence suggest that this can destabilise chromatin fibres.19,51–53 Histone acetylation may also affect the structure of mononucleosomes. Several studies suggest that DNA within acetylated nucleosomes is more accessible.48,49,54 It has been shown that acetylated histone tails adopt a more α-helical conformation,55 and that acetylated nucleosomes have a reduced linking number,24 consistent with wrapping less DNA. However, in this view, the role of individual histone modifications has been less clear. Here we show that acetylation of the H3 tail increases the inherent thermal mobility of nucleosomes whereas acetylation of H4 does not (Figure 2(e)). This increase in mobility does not appear to be due to charge neutralisation alone, as mutation of the relevant lysine residues to alanine did not produce the same effect (Figure 2(d)). The mechanisms behind this are not immediately clear; however, it is interesting to note that acetylation of H3 but not H4 increased the distance between the arms of linker DNA within a mononucleosome, as measured by FRET.53 We have also recently observed that individual histone tails have non-redundant effects on nucleosome dynamics,27 suggesting that the relationship between histone modification and chromatin dynamics may be more complex than initially appreciated. It will be interesting to examine how these effects interact with each other to alter chromatin structure and function in vivo. Histone acetylation affects ATP-dependent chromatin remodelling through altering either the Km or Kcat of ATP hydrolysis As their names suggest, ATP-dependent remodelling enzymes use the energy of ATP hydrolysis to alter chromatin structure. Based on the helicase domain of the catalytic subunit, chromatin remodelling complexes can be classified into a number of different subfamilies.1 Here we look at examples from three distinct subfamilies of chromatin remodellers: Chd1, Isw2 and RSC. The ATPase activity of these enzyme classes is stimulated by DNA and nucleosomes. Whereas it had been noted that histone tails and modifications affected their remodelling activity, it was not clear in many cases whether this was due to altered binding of modified nucleosomes or due to altered rates of ATP hydrolysis after binding. Using a real-time ATPase assay we show that depending on the particular enzyme involved either may be the case. Analysis of the RSC complex confirmed that the improved remodelling of acetylated nucleosomes is due to higher substrate binding affinity and not due to increased ATP turnover (Figure 4(b)). This is consistent with previous observations of bromodomain containing remodelling enzymes, such as SWI/SNF, which show improved binding to acetylated chromatin templates.12,56 Remarkably, given the large number of bromodomains, the RSC complex shows no improved binding of H4 tetra-acetylated nucleosomes but rather, specifically H3 acetylated nucleosomes. By using chromatin with defined modification patterns we show that mono-acetylation at lysine 14 results in an increase in binding almost to the level seen with H3 tetra-acetylation. Consistent with this the Rsc4p subunit of RSC interacts genetically with H3 K14 but not H3 K9.32 Studies from the Workman laboratory have shown that SWI/SNF and RSC have increased affinity for nucleosomes that have been acetylated by the NuA4 HAT complex.12,56 We show here that RSC does not preferentially bind H4 K5,8,12,16 tetra-acetylated nucleosomes (Figure 4(b)); however, these observations can be reconciled by the fact that NuA4 shows HAT activity towards other histones besides H4, most notably H2A,57,58 and it may be this which results in the improved binding. For this reason, it would be interesting to test the affinity of RSC for nucleosomes containing acetylated H2A or H2B. Indeed, in high-resolution microarrays, H2A K7 acetylation but not H2B K16 acetylation is typically found in the same region of genes as H3 acetylation, and both are positively correlated with transcription.46 In the case of Isw2, we show that acetylation of H4 reduces nucleosome remodelling by lowering the Kcat of ATP hydrolysis and not by inhibiting nucleosome binding (Figure 5(c)). Thus, the unmodified H4 tail is required as an allosteric activator of ATP hydrolysis. This is consistent with the previous observation that acetylated H4 peptides have a reduced ability to stimulate ATP hydrolysis by dISWI in comparison to unmodified peptides.34 We also find that Chd1 activity is affected by modification and alteration to the H4 tail in a similar way to the Isw2 complex (Figure 6). It is notable that in the case of both Chd1 and Isw2, the effects of tail acetylation or truncation on ATPase activity are smaller than the effects on nucleosome sliding. It is possible that there is some amplification of the effect on ATPase activity due to multiple rounds of remodelling being required for repositioning. Alternatively, alterations to the H4 tail may reduce the efficiency with which these enzymes reposition nucleosomes in addition to altering their ability to hydrolyse ATP. Previously, the requirement for the H4 tail for efficient nucleosome remodelling had only been observed for members of the Iswi subfamily of remodelling enzymes. However, there is evidence that there may be a significant degree of overlap in the function of Chd1 and Iswi subfamilies in S. cerevisiae. Isw1, Isw2 and Chd1 are all involved in transcription termination59 and their combined deletion is synthetic lethal with cellular stress.60 These complexes also reposition nucleosomes onto the same subset of thermodynamically favourable positions in vitro.61 Interestingly, like members of the Iswi subfamily, Chd1 is efficient in the generation of regularly spaced chromatin arrays,62 suggesting that a tightly coupled functional interaction with the H4 tail may be important for this activity. Studies with the similar Drosophila Mi-2 chromatin remodelling complex have shown that while its ATPase activity is stimulated by the assembly of histones into nucleosomes, this does not require histone tails.63 Whereas Mi-2 has similarity to Chd1, it is a member of a phylogenetically distinct group,1 suggesting that of the CHD-like remodellers, dependence on the H4 tail may be restricted to the Chd1 subfamily. H4 acetylation promotes octamer transfer in trans by the RSC complex A distinct activity of the SWI/SNF and RSC complexes is their ability to remove nucleosomes and displace histone octamers in trans. The targeted removal of nucleosomes has been shown to be an important phenomenon in nuclear processes such as transcription64–66 and DNA repair67,68 and interestingly chromatin remodelling enzymes are involved in both. In testing the role of histone modifications on octamer transfer in trans by RSC, we found that H3 tetra-acetylation resulted in a large increase in transfer (Figure 7(b) and (c)). This effect of H3 acetylation in promoting octamer transfer can be explained through improved recruitment due to the increased affinity for the modified nucleosome. Indeed, octamer transfer by the homologous SWI/SNF complex has been shown to require its bromodomains to mediate octamer transfer of SAGA acetylated nucleosomes.69 An unexpected finding was that H4 tetra-acetylation also had a stimulatory effect (Figure 7(b) and (c)), in stark contrast to the results seen with nucleosome repositioning in cis (Figure 3(b) and (d)). However, the additional effect of H4 acetylation was confirmed by the fact that a doubly H3 and H4 acetylated nucleosome was transferred better still than a singly H3 acetylated nucleosome. Kinetic analysis of ATPase activity clearly shows that H4 tetra-acetylated nucleosomes are not bound any better than unmodified nucleosomes and they do not alter the rate of ATP hydrolysis by RSC (Figure 4(b)). Thus, the increase in octamer transfer seen with H4 acetylated nucleosomes is not due to the modification recruiting or stimulating RSC activity but rather by affecting the nucleosome in such a way as to make it easier to transfer. In agreement with this, we find that H4 acetylated nucleosomes show increased thermally induced H2A/H2B dimer loss (Supplementary Data, Figure 7), indicating that this modification can affect chromatin dynamics independently of external factors. Interestingly, previous studies have shown that acetylation reduces the thermal stability of nucleosomes and H4 tetra-acetylation in particular reduces the thermal stability of nucleosomes almost to the level of bulk acetylation.70,71 The finding that histone acetylation improves the efficiency of histone octamer transfer by remodelling enzymes helps to explain discrepancies between previous studies of transfer efficiency. Previous studies that used native histones bearing modifications observed octamer transfer at a higher efficiency than was detected in another study using recombinant unmodified histones.72 The idea that acetylation of different histone tails may not be functionally equivalent is not a new one. Genome-wide microarray studies in yeast have shown that not all acetylation is positively correlated with transcription,42,46 and H4 acetylation does not substitute for H3 acetylation at Adr1-dependent genes.73 We propose that histone modifications may provide a means to facilitate histone eviction by ATP-dependent chromatin remodelling enzymes. Thus, acetylation, particularly of H4, may act in parallel with recruitment by activation domains to promote the removal of nucleosomes by remodelling enzymes such as RSC and SWI/SNF.74 This may also have relevance to processes other than transcription such as DNA repair. Intriguingly, H4 acetylation is very rapidly targeted to these regions75,76 making it tempting to speculate that this modification also acts to promote nucleosome loss during DNA repair. Experimental Procedures Purification of remodelling enzymes TAP (tandem affinity purification) tagged yeast strains were either purchased from Euroscarf (Germany) for CHD1 and ISW2, or in the case of RSC the strain BCY211 was kindly supplied by Brad Cairns. Six litres of yeast were grown at 30 °C to an A600 of 2–2.5 in 3×yeast extract, peptone, adenine, d-glucose and frozen by dropwise addition into liquid nitrogen. Yeast cells were lysed by mechanical disruption using a blender (Waring) kept cold by addition of solid CO2. This was then thawed and purified using standard TAP protocols77 except using higher stringency wash buffers (20 mM Na-Hepes (pH 7.4), 350 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) Tween-20, 1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride, 2.6 mM aprotinin, 2 μg/ml leupeptin, 1 μM pepstatin). The purified eluate was concentrated using a Centricon YM-50 concentrator (Millipore) to 200–300 μl, dialysed against wash buffer without protease inhibitors and stored as 10 μl aliquots at −80 °C. The purity of Isw2 and Chd1 is indicated by Stockdale et al.,61 the purity of RSC complex is illustrated in Supplementary Data, Figure 8. Native peptide ligation H3 Δ1-27 S28C (NCBI: CAD89679) and H4 Δ1-20 V21C (NCBI: CAD89677) were generated by sited directed mutagenesis. To ensure that the N-terminal cysteine within the globular histone was available for ligation, up to 10 mg of lyophilised histone was dissolved in 1 ml 6 M guanidine chloride(GnCl) (pH 7), 10 mM DTT, and incubated at 50 °C for 30 min. This was then dialysed extensively against three changes of 4l of 10 mM sodium acetate (pH 5.2), 1 mM EDTA, allowing at least 3 h per step. The fully reduced histone was then lyophilised and stored. Modified tail peptide thioesters were purchased from CSS-Albachem (Scotland) and aliquots of peptide in use stored at 10 mM in MilliQ water at −80 °C. The ligation conditions used were similar to those used by Dawson & Kent.21,22 The 1–2 mM globular histone was dissolved in 6 M GnCl, 0.2 M phosphate (pH 7.3) together with 0.4–0.5 mM thioester peptide and 2% (v/v) thiophenol, typically to a final volume of 200–300 μl. This was vortexed, and the reaction left at room temperature for 16–24 h. The reaction was stopped by adding DTT to 100 mM and dialysing the reactants against three changes of 500 ml SAUD0 buffer (7 M urea, 20 mM sodium acetate (pH 5.2), 5 mM 2-mercaptoethanol, 1 mM EDTA pH 8.0, 0 mM NaCl) using a 12–14 kDa MWCO dialysis membrane. The dialysed ligation mixture in SAUD0 buffer was spun to remove protein precipitate, and the supernatant loaded onto a 1 ml SOURCE 15S (Pharmacia) ion exchange column running at 1.5 ml/min to separate unligated globular histone from the full length product. A stepwise elution using SAUD0 (buffer A) and SAUD2000 (buffer B) (7 M urea, 20 mM sodium acetate (pH 5.2), 5 mM 2-mercaptoethanol, 1 mM EDTA (pH 8.0), 2 M NaCl) was used to elute in as small a volume as possible. The H3 Δ1-27 S28C and H4 Δ1-20 V21C constructs elute at a conductivity of 14 milliSiemens (mS), equivalent to approximately 180 mM NaCl, whereas full length histones elute at less than 25mS, 500 mM NaCl. This is typically 9.2%, and 25% SAUD2000, but due to variations in buffer preparation, the concentration of the two buffers required to obtain this conductivity should be determined empirically. We found that histones can elute in multiple peaks even though they are identical by SDS–PAGE and mass spectrometry, presumably representing differentially folded sub-species. The globular histone was eluted over 15 ml and the full-length histone over 10 ml, before the column was washed in SAUD2000 for 6 min in-between runs to remove tightly bound proteins and then re-equilibrated in buffer A. The relevant fractions are collected, diluted with SAUD0 to a final salt concentration below 100 mM NaCl and reloaded onto the column. We found that two to three rounds of purification were required to obtain greater than 95% pure ligated protein as determined by SDS–PAGE (Gradipore, Australia). The final fractions are concentrated using a YM-10 centricon (Millipore) to approximately 200 μl and their concentration measured by absorbance at 276 nm. Nucleosome reconstitution Recombinant Xenopus laevis histone proteins were expressed and purified as described.78 Where necessary site-directed mutagenesis was carried out using the Stratagene Quickchange kit. Nucleosomes were assembled by mixing equimolar amounts of histone octamer and DNA in high salt and performing stepwise dialysis into low salt as described.79 DNA was generated by preparative PCR using primers obtained from Eurogentec (Belgium) fluorescently labelled where appropriate. Nucleosomes were assembled on DNA fragments based on the MMTV nucleosome A positioning sequence80 or the synthetically selected 601 sequence.81 We have adopted a nomenclature in which the lengths of DNA extensions on either side of a nucleosome are indicated as numbers on either side of a letter that defines the core positioning sequence used. So 54A18 designates a nucleosome positioned on the MMTV nuc A positioning sequence with a 54 bp extension to the upstream side and a 18 bp extension to the downstream side. The oligos used to generate the 54A18 fragment were 5′TATGTAAATGCTTATGTAAACCA and 5′TACATCTAGAAAAAGGAGC; for the 54A54 fragment 5′TATGTAAATGCTTATGTAAACCA and 5′ATCACATGTGAAAGTTAAAAAA; for the 0W0 fragment 5′CTGCAGAAGCTTGGTCCC and 5′ACAGGATGTATATATCTG; for the 54W0 fragment 5′TATGTCCATGCTCATGCC and 5′ACAGGATGTATATATCTG; for the 36W36 fragment 5′GGCGAATTCGAGCTCGGTAC and 5′AGGTCGACTCTAGAGAATCC. The PCR fragments were purified by ion exchange chromatography on a 1.8 ml SOURCE 15Q (Pharmacia) column. Radiolabelled DNA was prepared using T4 polynucleotide kinase (New England Biolabs) and [γ-32P]ATP (Molecular Bioproducts) according to the manufacturer's specifications. Nucleosome remodelling Thermal remodelling reactions were performed by incubating nucleosomes in a thin-walled 200 μl PCR tube (ABgene, UK) in 50 mM NaCl, 50 mM Tris (pH 7.5) at 47 °C in a PCR machine with a heated lid for the specified amount of time. At the end of the reaction, sucrose was added to 5% (w/v) and the reactions placed on ice. The samples were separated on 0.2× Tris–Borate EDTA, 5% native polyacrylamide gels for 3.5 h at 300 V at 4 °C with pump recirculation. Gels were scanned in a Fuji Phosphoimager FLA-5100 and the bands quantified with Aida software (Fujifilm). For each time point the fraction of nucleosomes remodelled was calculated as the intensity of the destination position divided by the sum of the start and destination intensities. These data were then corrected to set the zero time point to zero percentage remodelled. The data points were plotted onto a graph and a hyperbolic curve of the form y = x/(n+x) fitted non-linearly to the data using the Solver add-in for Excel over 1000 iterative cycles. The initial rate of remodelling was calculated by differentiation of the curve and solving for time equals zero. At this point the initial rates for unmodified and modified nucleosomes within the competition assay were divided by each other to describe the initial rate of remodelling relative to the unmodified control. The average and standard deviations were calculated from at least three independent repeats. ATP-dependent remodelling reactions were performed as for thermal repositioning except using a reaction buffer containing 50 mM NaCl, 50 mM Tris (pH 7.5), 3 mM MgCl2, 1 mM ATP. These were incubated at 30 °C with the amount of remodelling enzyme and for the time specified in the figure legends. The reactions were stopped using 500 ng of HindIII-digested bacteriophage lambda competitor DNA, adding sucrose to 5% (w/v) and placed on ice. Nucleosome binding assays were performed in 50 mM Tris–HCl (pH 7.5), 50 mM NaCl, 3 mM MgCl2 and 3% (w/v) Ficoll-400 unless otherwise stated. ATPase assay The ATPase assay was performed as described.31 The reaction was measured in solution in real-time on a Cary Eclipse fluorimeter (Varian, Australia). The excitation and emission wavelengths were set to 430 nm and 465 nm, respectively, with a 5 nm slit width and polarisers used to compensate for anisotropy. A calibration curve using MDCC–PBP at 5 nM and increasing amounts of inorganic phosphate was performed to determine the range across which the detection is linear. MDCC–PBP was diluted using 10 mM Pipes (pH 7) to 50 nM, this was mixed with nucleosome and enzyme in a final buffer concentration of 50 mM NaCl, 50 mM Tris (pH 7.5), 1 mM MgCl2 to a final concentration of 5 nM. Enzyme and nucleosome concentrations are as stated in the Figure legends. Measurements were performed with 1 mM ATP which had previously been treated to remove inorganic phosphate contamination.31 However, identical results were obtained using 100 μM ATP which had not been treated with the phosphate mop. The measurement of the rate of hydrolysis was performed using the Cary Eclipse software and non-linear fitting of the Michaelis–Menton equation to the data done within the Solver add-in for Excel over 1000 iterative cycles. Octamer transfer and dimer exchange Octamer transfer was performed under the same conditions as standard ATP-dependent remodelling. The 15 nM donor nucleosome (assembled on unlabelled 0W0 DNA) was incubated with 10 nM RSC and 1 nM radiolabelled acceptor 0W0 DNA for the specified amount of time in the presence of 1 mM ATP. The reactions were stopped by the addition of excess cold DNA and analysed by native PAGE. Thermal dimer exchange was performed as described.72 Briefly, this is performed as for nucleosome repositioning, except with a twofold molar excess of H3/H4 tetrasomes assembled onto 147 bp ‘601’ DNA.81

Pediatr_Surg_Int-_-Immediate_Access-2292794

Adults with corrected oesophageal atresia: is oesophageal function associated with complaints and/or quality of life?

The aim of this study was to evaluate oesophageal function after correction of oesophageal atresia in adults, and to investigate the association between complaints, oesophageal function and quality of life (QoL). Twenty-five adults were included who participated in previous follow-up studies, during which complaints of dysphagia and gastro-oesophageal reflux (GOR), results of upper gastrointestinal endoscopy, oesophageal biopsies and QoL had been collected. Manometry was performed in 20 patients, 24 h pH-measurements were performed in 21 patients. pH-values (sample time 5 s) were calculated using criteria of Johnson and DeMeester. Associations were tested with ANOVA and χ2-tests. Ten patients (48%) reported complaints of dysphagia, seven (33%) of GOR. The amplitude of oesophageal contractions was low (<15 mmHg) in four patients (20%). pH-measurements showed pathological reflux in three patients (14%). Patients reporting dysphagia more often had disturbed motility (P = 0.011), and lower scores on the domains “general health perceptions” (SF-36) (P = 0.026), “standardised physical component” (SF-36) (P = 0.013), and “physical well-being” (GIQLI) (0.047). No other associations were found. This study shows a high percentage of oesophageal motility disturbances and a moderate percentage of GOR after correction of oesophageal atresia. Patients reporting dysphagia, whom more often had disturbed motility, seemed to be affected by these symptoms in their QoL. Introduction At present, the survival rate of patients with oesophageal atresia (OA) is approximately 95% [1, 2]. With the decreased mortality, the interest in morbidity, especially the long-term results after correction of OA, has increased over the years. Several long-term follow-up studies have shown that long lasting gastro-oesophageal reflux (GOR) is a frequent problem after correction of OA, although intestinal metaplasia, as its theoretical consequence, is rare [3–6]. In these studies, GOR has been either diagnosed by upper gastrointestinal (GI) endoscopy with biopsies, by 24 h pH-measurements, or by both, showing varying degrees of GOR. Besides pH-measurements, oesophageal manometry has been performed in several studies, showing oesophageal motility disturbances in most patients [7–10]. The true impact of this finding on individual daily life is not clear. In our centre, we have performed several long-term follow-up studies in a relatively large group of patients after correction of OA [5, 6]. Patients underwent upper GI endoscopy with biopsies and quality of life (QoL) measurements [11]. The first aim of this study was to evaluate the presence of GOR and oesophageal motility problems in a group of our adult patients treated for OA. The second aim was to investigate if there was an association between complaints, oesophageal function, and QoL-measurements. This association has not been investigated before. Because of the influence of complaints of dysphagia and GOR on daily life, we hypothesised that patients with complaints have a poorer QoL than patients without complaints. Patients and methods Twenty-five patients over 18 years of age who participated in previous follow-up studies after correction of OA [5, 6, 11], and gave informed consent to participate, were included in this study. In these previous studies, data regarding the results of upper GI endoscopy, biopsies of the distal oesophagus, and QoL had been collected. From the QoL study, we used the results of the Medical Outcome Study 36-Item Short-Form Health Survey (SF-36) and of the gastro-intestinal quality of life index (GIQLI) [12, 13]. After approval of the study protocol by the Medical Ethical Committee, all patients received a written invitation to participate in the study. All patients who gave their informed consent, underwent manometry and pH-measurements and were asked if they experienced difficulties swallowing solid foods (dysphagia) or experienced heartburn or retrosternal pain (GOR-related complaints). Oesophageal manometry was performed using the UPS-2020 measurement stationary system (MMS, Enschede, The Netherlands) with software version 7. The pressure was measured with the Unisensor Microtip catheter type 8304-00-9980-D with three pressure transducers on a 5 cm distance from each other. The lower oesophageal sphincter basal or resting pressure (LOSP) and relaxation after swallowing, the motility in the oesophageal body after at least six wet swallows of 5 ml water, and the upper oesophageal sphincter pressure (UOSP) and relaxation were calculated. The amplitude of oesophageal body contractions was categorized as “low” (<15 mm Hg), “moderate” (15–35 mm Hg), or “normal” (>35 mm Hg). The encountered oesophageal motility disorders were classified according to the guidelines proposed by Spechler et al. [14]. Based on the basal LOSP, LOS relaxation, peristaltic wave progression, and distal wave amplitude, oesophageal motility disorders were classified into four categories: (1) “inadequate LOS relaxation”, (2) “uncoordinated contraction”, (3) “hypercontraction”, and (4) “hypocontraction” or “ineffective oesophageal motility”. pH-measurements were performed using the Comfortec dual channel pH probe (Sandhill Scientific), which was positioned with the pH measurement points 5 and 20 cm above the manometrically established upper border of the LOS. The position of the probe was checked by X-ray. Ambulatory pH measurement was performed during 24 h using the GORD pH-recorder (Sandhill Scientific) with a sample time of every 5 s. The pH values were calculated using the criteria of Johnson and DeMeester [15]. The data were entered into a database and analysis was performed using SPSS (Statistical Package of the Social Sciences) 10.0.1 for Windows. We tested for association between functional results and QoL by applying ANOVA and χ2-tests or Fisher’s exact tests. Definitions Before testing for association, the results of functional tests were dichotomized. If upper GI endoscopy showed a normal oesophagus or grade I oesophagitis (according to the modified system of Savary-Miller) [16], this was scored as “normal”. Grade II oesophagitis or worse was scored as “abnormal”. If the biopsies of the distal oesophagus showed normal oesophageal epithelium or mild reflux oesophagitis (according to Ismael-Beigi) [17], this was scored as “normal”. Moderate oesophagitis or worse was scored as “abnormal”. As all patients were diagnosed as having “ineffective oesophageal motility”, this variable could not be used. Instead, we used the amplitude of oesophageal body contractions as a measure of outcome of manometry, because decreased amplitude implies a defective peristaltic function of the oesophagus. The results were dichotomized as “normal” or “abnormal” (moderate or low amplitude). The results of pH-measurements were also dichotomized as “normal” or “abnormal” (minor or pathological reflux). Results Patient characteristics Patient characteristics are described in Table 1. All patients had undergone a primary end-to-end anastomosis for Gross’ type C OA. Two patients were using proton-pump inhibitors, they stopped taking their medication two days before the start of the study. Table 1Patient characteristics of 25 patients participating in the studyMean (range) or n (%)Age (years)28.5 (18–42)Gender  Male16 (64%) Female9 (36%)Concomitant congenital anomalies None17 (68%) Present8 (32%)  Anorectal malformations3 (12%)  Cardiac malformations4 (16%)  Vertebral malformations1 (4%)  Limb malformations2 (8%)  Other malformations2 (8%)Anti-reflux procedure in past No23 (92%) Yes2 (8%)Anastomotic stenosis in past No18 (72%) Yes7 (28%)Current educational status Primary school1 (4%) Basic high school9 (36%) Advanced high school7 (28%) University5 (20%) Unknown3 (12%) Unfortunately, it was impossible to perform manometry and pH-measurements in four patients. In one patient it was impossible to introduce the catheter through the nose due to resistance of the patient, who decided to withdraw from the study. In three out of four patients it was impossible to introduce the catheter due to oesophageal stricture. All of these three patients had been treated for anastomotic stricture in childhood, one patient had undergone anti-reflux surgery in childhood. Two of these three patients had complaints of dysphagia at the time of the study. The stricture of these three patients was treated with dilatation. Since dilatations may influence the results of manometry and pH-measurements, no measurements were performed in these patients. Due to a technical failure, the data of the manometry of one patient could not be retrieved. Dysphagia was reported by 10/21 patients (48%), GOR-related symptoms were reported by 7/21 patients (33%). Manometry The data of 20 patients could be analyzed. The upper oesophageal sphincter (UOS) responded normally to swallowing in all patients. Mean UOS pressure was 30.8 ± 15.5 mm Hg. Oesophageal contractions were observed in all patients. One or more propulsive contractions were observed in 14/20 patients. All patients showed one or more non-transmitted contractions. Retrograde contractions were observed in 7/20 patients. Mean minimum oesophageal body amplitude pressure was 20.7 ± 13.4 mm Hg. Mean maximum oesophageal body amplitude pressure was 32.0 ± 15.5 mm Hg. The amplitude of oesophageal body contractions was low (<15 mm Hg) in 4 patients (20%), moderate (15–35 mm Hg) in 10 (50%) and normal (>35 mm Hg) in 6 (30%). In all patients, the LOS showed complete relaxation upon swallowing. Mean LOSP was 13.1 ± 7.2 mm Hg. According to the guidelines of Spechler et al., all patients were classified as having “ineffective oesophageal motility” [16]. The LOS pressure was normal and LOS relaxation was complete in all patients. Wave progression varied from normal to absent progression, and the distal wave amplitude was low in ≥ 30% of wet swallows (data not shown). pH-measurements The data of 21 patients could be analyzed. The results are described in Table 2. pH-measurements showed a normal pattern in 17 patients (81%), minor reflux in 1 (5%), and pathological reflux in 3 (14%). In both of the patients who were taking proton-pump inhibitors, pH-measurements showed pathological reflux. Table 2 Results of 24 h pH-measurements in 21 patientsProximal mean % ± SDDistal mean % ± SDTotal time pH < 40.2 ± 0.41.5 ± 2.2Upright time pH < 40.2 ± 0.62.2 ± 3.3Supine time pH < 40.0 ± 0.30.4 ± 1.0SD standard deviation Associations The association between symptoms and results of endoscopy, oesophageal biopsies, manometry, pH-measurements and QoL is shown in Table 3. Patients reporting dysphagia, more often had disturbed motility (P = 0.011). These patients also had significantly lower scores on the domains “general health perceptions” (P = 0.026), and “standardised physical component” (P = 0.013) of the SF-36; and on the domain “physical well-being” (P = 0.047) of the GIQLI. Table 3Association between symptoms and results of endoscopy, oesophageal biopsies, manometry, pH-measurements, and quality of lifeComplaintsDysphagiaGORYesNoYesNoEndoscopy Normal99612 Abnormal1212Oesophageal biopsies Normal6547 Abnormal3526Manometry Normal0*6*15 Abnormal10*4*68pH-measurements Normal107512 Abnormal0422SF-36 scales Physical functioning86.5 (19.2)94.1 (8.3)96.7 (5.2) 88.1 (16.4) Physical role-functioning77.8 (38.4)100.0 (0.0)83.3 (40.8)92.9 (20.6) Emotional role-functioning77.8 (44.1)96.7 (10.1)83.3 (40.8)90.5 (27.5) Bodily pain78.2 (26.3)92.5 (13.9)85.5 (24.5)86.3 (20.5) General health63.6 (23.6)**84.6 (15.1)**86.7 (14.8)70.2 (22.7) Vitality50.0 (15.4)52.3 (12.9)58.3 (13.3)48.2 (13.2) Social functioning88.9 (22.0)93.2 (18.8)97.9 (5.1)88.4 (23.2) Mental health82.7 (15.781.5 (16.7)87.3 (12.5)79.7 (17.0)SF-36 component summary scales PCS49.5 (7.4)***56.4 (3.4)***55.6 (4.9)52.3 (6.9) MCS49.0 (10.9)49.5 (7.4)50.3 (7.9)48.8 (9.5)GIQLI scales Physical well-being21.2 (3.1)****24.5 (3.7)****23.3 (3.8)22.9 (3.9) GI symptoms64.1 (8.5)68.6 (7.8)68.0 (6.5)66.0 (9.0) Social well-being12.0 (1.7)12.2 (0.4)12.0 (0.6)12.1 (1.3) Emotional well-being15.1 (1.5)15.9 (0.5)15.5 (1.0)15.6 (1.2)Total score112.4 (13.3)121.3 (9.6)118.8 (10.3)116.6 (12.9)Data are shown as n, or mean (standard deviation)GOR gastro-oesophageal reflux, SF-36 Medical Outcome Study 36-Item Short Form Health Survey, PCS physical component summary, MCS mental component summary, GIQLI gastro-intestinal quality of life index*P = 0.011, **P = 0.026, ***P = 0.013, ****P = 0.047 No association was found between complaints of GOR and quality of life; nor between the results of endoscopy and the results of pH-measurement and/or manometry; nor between the results of oesophageal biopsies and the results of pH-measurement and/or manometry (data not shown). Problems with the initial surgical repair in childhood (i.e. anastomotic leak or stricture needing dilatation) did not influence the findings in the current study. Discussion The results of the present study confirm that oesophageal motility disturbances are frequently present after correction of OA. Low or moderate amplitude of oesophageal body contractions were found in 14/20 patients (70%), all patients showed one or more non-transmitted contractions, and retrograde contractions were observed in 7/20 patients (35%). All patients met the manometric features of “ineffective oesophageal motility”, as described by Spechler et al. [14]. The manometric findings in the present study are comparable to those described in other studies [7–10]. Based on pH-measurements, the prevalence of GOR in the current patient group is lower than expected. pH-measurements showed minor or pathological reflux in 4/20 patients (20%). None of these patients had undergone anti-reflux surgery in the past. In other studies, the prevalence of GOR based on pH-measurements varies from 17 to 54% [7–9]. Unfortunately, the criteria used for diagnosing GOR and the age of the patient groups studied also vary between studies. The question is, what the influence is of the disturbed oesophageal motility and GOR found in previous studies on the daily life of adults with corrected OA. This is the first study in which complaints and QoL after correction of OA have been combined with long-term studies of oesophageal function: endoscopy, oesophageal biopsies, manometry and pH-measurements. Patients reporting dysphagia more often had disturbed motility, and showed significantly lower scores on the domains “general health perceptions” and “standardised physical component” of the SF-36, and on the domain “physical well-being” of the GIQLI. However, GOR-related complaints were not associated with disturbed oesophageal function, and did not influence QoL. It is important to consider that this group of patients has grown up with these symptoms, and may probably have gotten used to it. The fact that complaints of dysphagia affect the QoL and GOR-related complaints do not affect QoL may be explained by the influence of these complaints on daily activities such as eating. Motility problems after correction of OA were first reported by Haight [18] in 1957. The main cause of the abnormal oesophageal motility after correction of OA is not clear. Some studies propose a congenital nervous abnormality as the cause of motility disturbances. In the foetal rat model for OA, abnormalities were found in the course and branching pattern of the vagal nerves [19]. However, an acquired cause is also suggested, i.e. surgical damage to vagal fibres that innervate the oesophagus [20]. Abnormal oesophageal motility can cause symptoms of dysphagia. It can also worsen the effects of GOR, since malfunction of the peristaltic pump will result in a delayed clearance with a longer period of stasis of refluxed material in the oesophagus. In conclusion, this study shows a high percentage of oesophageal motility disturbances and a moderate percentage of GOR after correction of OA. Only patients reporting dysphagia, who more often had disturbed motility, appeared to be affected by these symptoms in their QoL.

Eur_Arch_Psychiatry_Clin_Neurosci-3-1-1800370

Deficit of social cognition in subjects with surgically treated frontal lobe lesions and in subjects affected by schizophrenia

The ability of humans to predict and explain other people’s behaviour by attributing independent mental states such as desires and beliefs to them, is considered to be due to our ability to construct a “Theory of Mind”. Recently, several neuroimaging studies have implicated the medial frontal lobes as playing a critical role in a dedicated “mentalizing” or “Theory of Mind” network in the human brain. In this study we compare the performance of patients with right and left medial prefrontal lobe lesions in theory of mind and in social cognition tasks, with the performance of people with schizophrenia. Introduction Social cognition refers to the ability to perceive, interpret and provide an adequate response to affective and other interpersonal cues [1]. Pioneering studies have found that schizophrenic subjects show social cognitive impairments, in particular in modifying their behaviour when interacting with other people and in recognizing social information cues [2]. People with schizophrenia also show a great deal of social naivety in interpersonal situations [3]. The ability to engage in competent social relations and to understand social information depends on the adequate functioning of a mental mechanism termed Theory of Mind (ToM), that allows people to understand and interpret their own and other people’s mental states and hence to predict and explain their behaviour [4, 5]. Evidence from neuroimaging and neuropsychological studies has led researchers to conclude that ToM is subserved by dedicated brain systems, including the amygdala, the temporo-parietal junction, the orbital frontal cortex and, in particular, the medial frontal lobes [6, 7]. The results of these studies have been used to argue that the medial frontal lobes play a critical role in a dedicated mentalizing system [7, 8]. Neuropsychological literature specifically relevant to the medial frontal cortex is scarce. Actually, only three studies have included patients with relatively focal medial frontal lobe damage and have sought to investigate correlations between specific frontal brain areas and performance in ToM tasks [9–11]. However, there are important limitations to these investigations. Stuss et al. [10] reported that patients with right frontal and bilateral frontal lobe damage were impaired in their ability to infer visual experience in others. The authors conclude that acquired brain damage to the medial frontal lobes does impact on ToM ability, but aetiology of brain damage for each patient is not reported, the text implies that most of the bifrontal group had suffered head traumas. It is often very difficult to assess the extent of brain damage reliably after head trauma as widespread damage can often occur through axonal shearing and other effects (e.g. [12]). The second neuropsychological study [9] reports that patients with left and right-sided lesions were equally impaired, while lesion size was unrelated to performance and no effect of lesion location was found when comparing patients with focal dorsolateral, medial or orbital frontal lesions. Also, a recent group study of empathy in patients [11] included an assessment of performance on the Faux Pas Test, a probe of ToM ability. Patients with frontal lobe lesions were impaired in this task. Specifically, patients with ventromedial frontal lobe damage made significantly more errors than patients with posterior lesions or healthy controls in the Faux Pas Test. However, a detailed analysis of lesion sites associated with ToM deficits in these previous studies revealed a particularly important role for the right ventromedial prefrontal frontal lobe [11, 13]. Damage in these areas induces behavioural changes affecting personality (indifference), impaired social judgement, reduced affect and goal-directed behaviour, self-monitoring deficits [14–16]. More recent studies have provided evidence that is in disagreement with a single area hypothesis involved in ToM processing. In fact, the neural network involving the right and left temporo-parietal-junction (TPJ R and L); the posterior cingulated (PC), and medial prefrontal cortex (MPFC) seems to be crucial in processing the complex reasoning involved in mentalizing [17, 18]. Bird et al. [19] studied a patient affected by extensive damage of the medial frontal lobes reporting, interestingly, a dysexecutive syndrome with confabulation with preserved performance on some ToM tasks. Saxe and Wexler [20] in an fMRI study in normal subjects, described an equally selective profile of activation of the above-mentioned areas in a multicomponent pattern of activation fMRI methodology. Frontal lobe damage in patients has long been linked to impairments in social behaviour [21]; in fact, they have been described as presenting diminished social awareness and a lack of concern for social rules [15, 22]. An interesting approach to social competence deficits in schizophrenic people may be represented by the Machiavellian Intelligence Hypothesis (MIH, [23, 24]). According to this hypothesis, in the development of intelligence, social, rather than technical, efficiency represents the main selective pressure of human evolution [25]. Social efficiency is represented by the ability to understand the intentions and beliefs of others with the aim of deceiving and manipulating them to achieve relevant objectives, such as control of food sources or sexual partners [26]. Recent literature fosters that these abilities are localised in the ventromedial prefrontal cortex: selective damage to these areas causes a relevant impairment of interpersonal relationships and in regulating behaviour according to social rules [27–30]. Patients with lesions to the orbitofrontal cortex also have disinhibited/socially inappropriate behaviour. Grafman interpreted the patient’s impairment in terms of an inability to access “social schema knowledge” stored in the frontal lobes [31]. Social schema knowledge is thought to inhibit aberrant behaviour. Patients with orbitofrontal cortex lesions who cannot access social schema knowledge fail to inhibit aberrant behaviour, such as physical threats and aggression. The prediction of a similar cognitive profile in terms of ToM abilities and social competence between frontal lesion subjects and schizophrenic people has been investigated and confirmed [6, 7]. An extensive and careful review recently published [32], reported a general agreement about the nature and extension of ToM dysfunctions in people affected by schizophrenia. These dysfunctions are symptoms related [33], disease specific and state independent. The neural architecture of the social cognitive dysfunction of schizophrenia is of paradigmatic importance for the understanding of social cognitive dysfunction and, more importantly, for the understanding of the consequences at the behavioural level [34]. A previous seminal study provided evidence that structural orbitofrontal cortex abnormalities are related to social dysfunction in schizophrenic people [35]. Moreover this prefrontal area has been unequivocally involved in the social cognitive deficits associated with this disorder [36]. However, empirically controlled investigations in which the cognitive profile of brain damaged patients was compared with schizophrenic subjects with an appropriate set of ToM and social intelligence tasks are lacking, thus leaving several crucial questions largely unresolved. In the current study, we examined the performance of stabilized schizophrenic outpatients, inpatients with focal damage of left and right ventromedial prefrontal lobes and healthy controls, in ToM abilities, in social competence and tactical strategy (Machiavellian Intelligence), to clarify whether schizophrenic patients demonstrate impairment similar to ventromedial prefrontal lesion patients and whether their performance in these tasks can be differentiated from their performance in tasks sensitive to neuropsychological dysfunction, including “executive” functions. Our prediction is that an overlapping dysfunctional cognitive profile should emerge between brain damaged and schizophrenic subjects, when compared to healthy subjects. We also addressed several methodological issues raised by earlier studies using social cognition tasks created for adults and not for children. Methods Participants The subjects for this study included 18 adult neurosurgical patients with unilateral frontal lobe lesions who had undergone surgery at the Department of Neurosurgery of “S. Salvatore” Hospital, L’Aquila, Italy. The patients were consecutively enrolled in the study from January 2003 to September 2005. Only patients with exclusive frontal lesions were identified and brain damage was confirmed through neuroimaging, with pre- and postoperative CT scanning and MRIs. Nine subjects with left side medial prefrontal cortex (LMPFC) lesions and nine subjects with right side medial prefrontal cortex (RMPFC) lesions were studied and underwent neuropsychological examinations (Fig. 1A, B). Fig. 1(A) MRI T1 weight (axial view) with gadolinium showing low density abnormalities (meningioma excised, patients L.F.) orbitofrontal cortex on the left side. (B) CT scan image showing low density abnormalities (meningioma excised, patients L.G.) orbitofrontal cortex on right sides Thirteen subjects had intra- or extra-axial tumours (72%); 3 had spontaneous haemorrhage (17%) and 2 (11%) had intracerebral haemorrhage from ruptured aneurysms. In the RMPFC group seven patients had a tumour removed (four had a meningioma excised, 2 had a high grade glioma excised and 1 had an oligodendroglioma excised), one had a right anterior communicating artery (ACoA) aneurysm clipped, following rupture and one had spontaneous intracerebral haemorrhage with no evidence of arterial malformations. Of the LMPFC patients, six had a tumour removed (five had a meningioma excised and one had a high grade astrocytoma excised), two had spontaneous intracerebral haemorrhage with no evidence of arterial malformations and one had a left ACoA aneurysm clipped following rupture. The location of the experimental group’s lesions were defined anatomically as medial (Broadman area 9 and 46) and orbital (Broadman areas 10, 11, 12 and 25) and further classified according to the prefrontal sectors of functional significance into which the lesions encroached (Tables 1, 2). Table 1Socio-demographic details of subjects with ventromedial frontal lobe lesionsSubjectsSiteAgeEducationAetiologyM.DRight frontal lesion5613MeningiomaF.S.Right frontal lesion508MeningiomaL.GRight frontal lesion448MeningiomaP.G.Right frontal lesion635OligodendrogliomaT.RRight frontal lesion625Glioma grade IIIE.GRight frontal lesion698Spontaneous intracerebral haemorrhageS.MRight frontal lesion6413Glioma grade IVA.URight frontal lesion3518Intra-cerebral haemorrhage from aneurismal sac ruptureM.URight frontal lesion4418MeningiomaL.FLeft frontal lesion418MeningiomaL.PLeft frontal lesion305MeningiomaM.NLeft frontal lesion6813Spontaneous intra-cerebral haemorrhageM.M.Left frontal lesion5513Spontaneous intra-cerebral haemorrhageR.MLeft frontal lesion548MeningiomaD.SLeft frontal lesion628MeningiomaR.TLeft frontal lesion7313Astrocytoma grade IIIA.CLeft frontal lesion598MeningiomaR.GLeft frontal lesion388Intra-cerebral haemorrhage from aneurismal sac ruptureTable 2Classification of the RMPFC and LMPFC experimental group according to the prefrontal sectors of functional significance into which the lesions encroachedPatientSexAge (years)AetiologyLesion locationFronto-orbitalMedialRF1F56Meningioma+RF2F50Meningioma+RF3M44Meningioma+RF4F63Oligodendroglioma+RF5M62Glioma grade III+RF6M69SIH+RF7F64Glioma grade IV+RF8F35IhAsc+RF9M44Meningioma+LF1M41Meningioma+LF2M30Meningioma+LF3F68SIH+LF4M55SIH+LF5M54Meningioma+LF6M62Meningioma+LF7M73Astrocytoma grade III+LF8F59Meningioma+LF9M38IhAsc+SIH = spontaneous intracerebral haemorrhage; IhAsc = intracerebral haemorrhage from aneurismal sac rupture Subjects were assessed with neuropsychological test batteries 20–40 days after surgery. A control group of schizophrenic subjects and a control group of psychiatrically and neurologically healthy subjects were studied as well. Twenty male patients, all native Italian speakers, whose symptoms satisfied the DSM-IV criteria for schizophrenia [37], participated in the study. They were all patients of the Department of Psychiatry of the University of L’Aquila and diagnosis was re-confirmed on admission to (and 6 months after discharge from) the Day-Hospital (DH) using a non-structured interview conducted by two psychiatrists (M.C., R.R) referring to DSM-IV criteria. All the subjects, whose assessment took place when clinically stable within a month of admission to the DH and establishment/confirmation of diagnosis, were treated with maintenance antipsychotic drugs. The mean daily dose was 310.3 (SD 143.67) mg/equivalents of Chlorpromazine [38]; this dose-equivalence with a typical antipsychotic is necessary in order to compare the different antipsychotic drugs as their strength may be different when administered to patients. Twenty neurologically and psychiatrically healthy control subjects (matched for age and education) were included. Exclusion criteria were: history of neurological disease including epilepsy, head trauma or mental retardation. All subjects provided informed consent to participate in the study. Materials and procedure Clinical assessment Clinical assessment in the sample with MPFC lesions was performed by using a non-structured clinical interview and Brief Psychiatric Rating Scale (BPRS) version 4.0 translated into Italian by [39] and through the neuropsychiatric Inventory (NPI), a semistructured clinicians interview using the protocol described by [40]. For the schizophrenic sample, frequency and severity of current symptomatology was registered by using a non-structured clinical interview and Brief Psychiatric Rating Scale (BPRS) version 4.0 (Modified 24-item version, translated into Italian by [39] and Clinical Global Impression scale (CGI) [40]. The prevalent symptomatology was also investigated using the Scale for the assessment of Positive Symptoms (SAPS; [41]) and Scale for the assessment of Negative Symptoms (SANS; [42]). The SAPS consists of 34 items and is divided into four subscales: hallucinations, delusions, bizarre behaviour and formal thought disorder. The SANS consists of 25 items and is designed to measure five domains: affective flattening or blunting, alogia, apathy, asociality and impaired attention. We also evaluated social function with AD-Disability Assessment [40]. Socio-demographic and clinical data are reported in Table 3. Table 3Socio demographic, clinical details in the total sampleSchizophrenia (no. 20)Right medial prefrontal cortex lesion (no. 9)Left medial prefrontal cortex lesion (no. 9)Healthy controls (no. 20)Sex (M:F)7:135:42:715:6Age (years)42.7 (1.8)49.6 (10.3)46.4 (18.6)38.9 (2.8)Education (years)8.8 (0.8)9.2 (2.6)12.2 (3.97)13.4 (1.2)Day since surgery–91.4 (11.6)88.8 (13.7)–IQ level88.8 (5.7)93.1 (2.4)98.7 (3.6)–NPI total score6 (0)10 (0)9.8 (5.3)–Duration of illness (years)12.30 (4.13)–––BPRS total score44 (11.1)52.5 (4.9)55.5 (17.5)–C.G.I.4.5 (0.6)–––SANS50.42 (20.9)–––SAPS42.57 (19.1)–––Social function (AD)2.5 (1.1)–––The values are means; standard deviation are in parenthesis Neuropsychological assessment All the schizophrenic subjects, with the exception of two positive schizophrenics, were also administered three neuropsychological tests for assessment of executive functions: the WCST, the Tower of London Test and a Phonemic Verbal Fluency Test (Table 4). Table 4Neuropsychological detailed of total sampleSchizophrenia (no. 20)Right medial prefrontal cortex lesion (no. 9)Left medial prefrontal cortex lesion (no. 9)Healthy controls (no. 20)Verbal fluency33.1 (10.4)21.2 (8.39)11.3 (4.19)38.7 (7.5)Tower of London29.8 (4.4)26.3 (6.5)19.2 (4.3)34.6 (1.9)WCST no. of category3.46 (2.41)2.66 (1.7)1.87 (2.1)5.7 (0.3)WCST % of perseverative errors27.66 (15.12)42.9 (22.6)50.7 (21.3)2.9 (2.6)Verbal memory19.1 (8.7)28.6 (8.01)8.3 (2.25)29.6 (3.01)The values are means; standard deviation are in parenthesis The WCST (128 cards) was administered with standard instructions, as described by Spreen and Strauss [43], whereas scoring followed Heaton’s [44] rules. Scoring and administration instructions for the Tower of London Test were those described by Krikorian et al. [45]. Scoring and administration instructions of the Phonemic Verbal Fluency Test were those described by Novelli et al. [46]. Visuo-spatial intellectual level was assessed by means of Raven’s Progressive Matrices [47]. Theory of mind tasks Four stories were read to the participants individually to assess ToM competence. The stories were structured to assess the ability to understand first and second order false beliefs in adult subjects [9]. First order false beliefs require a subject to make an inference about the state of the world. To assess first order ToM two stories were used: The washing machine task [9] and The Cigarette Task [48]. Second order stories measure the capacity to understand other people’s false beliefs. To assess second order ToM two stories were used; The Burglar [49] and The Wallpaper Story [9]. These stories were presented to the subjects in a series of cartoons in which the various actions of the characters are depicted in sequences. All the subjects were asked a ToM question and three control questions: False belief test question. This was designed to elicit a response that demonstrated the ability to make inferences about another individual’s mental state, namely, that a character in the story holds a false belief.Fact question. This was posed to determine whether subjects understood the actual sequence of relevant events that had occurred in the story which is in contrast with the sequence as understood by one of the characters in the story and that leads to his coming to a false conclusion.Memory question. This was used to assess whether memory for story details was approximately intact. The stories were the same for all the subjects examined. Each subject obtained a score ranging from 0 to 1 in the case of a correct answer to a False-belief question, to the fact question and to the memory question, where 0 = incorrect answer; 1 = correct answer. If the subject gave a correct answer to both the first order stories, s/he had a global score for first order ToM equal to 1 (non-casual performance). For second order false belief stories we followed the same methods. Social cognition tasks The following tasks addressed two aspects of social cognition. The ability to process the appropriateness of behaviour in different social contexts [21]; and the ability to use tactical strategy (Machiavellian Intelligence). Social situations task This task investigates the capacity to judge the appropriateness of behaviour that may induce anger in observers. Procedure. Nine short stories describing social situations incorporating behaviour were read by the patient. At various points in each story, the patient was asked to comment on how appropriate the behaviour was, giving a score from A to D. “A” scores meant that he judged the situation as normative. “B” to “D” scores meant that he judged the situation as a norm violation and indexed the extent of the violation (“B” scores being mild and “D” being serious). Blair and Cipollotti [21] report that previous piloting on a large, independent sample of healthy controls had resulted in the identification of a set of consistently identified normative situations and violations. Two scores were obtained for this task: one referring to the number of normative situations and the other to the number of violations correctly identified. The third refers to the extent to which the patient judged the violations to be socially inappropriate. For each situation, the participant obtained a score between 0 and 3, matching their response of “A” to “D” (i.e. “A” = 0, “D” = 3). Mach IV Scale We used the Mach IV Scale to assess the “Machiavellian Intelligence” of participants [50]. This is a self-report Likert scale, with scores ranging from 1 to 7 (where 1 = I totally disagree; 4 = no opinion; 7 = I totally agree), composed of 20 items, each consisting of a statement. The Mach IV scale is a method for assessing awareness and social functioning in a social context characterised by interpersonal deception (“Machiavellianism”). From the 20 statements of the Mach IV Scale we extracted two groups of items: (1) 5 items describing duplicity tactics, e.g.: “It is wise to flatter important people” (“tactics+”) and (2) 4 items describing a disagreements with tactics duplicity, e.g.: “When you ask someone to do something for you, it is best to give the real reason” (“tactics−”). The items were labelled for coding as follows: Positive tactics (tactics+): subjects must agree with statements reporting the ability to manipulate other people’s intentions and actions, according to Niccolò Machiavelli’s beliefs. Negative tactics (tactics−): subjects must agree with statements reporting judgements of correct and honest behaviour; for this reason they cannot use “intentional deception” mechanisms involved in the Machiavellian Intelligence Hypothesis. These features depend on an accurate interpretation of even the most particular intention of respondents [32, 51]. Human nature components referring to “people’s knowledge”, in particular the degree of cynicism concerning other people’s intentions and decisions are strictly related to the ability to interpret other people’s mental states. Statistical analysis One-way ANOVA was used to compare demographic, clinical information and neuropsychological Assessment and social cognition tasks. The Kruskaal ± Wallis test was used to analyse the level of significance of patients’ scores on ToM tasks. Results No differences among groups emerged for age [F(3,40) = 0.271, P = 0.751], years of education [F(3,38) = 0.387, P = 0.51] and estimated IQ [F(3,38) = 0.44, P = 0.64] (Table 2). Clinical assessment Neither were differences found between the four subgroups in age [F(3,38) = 0.271, P = 0.751, sex ratio, standard of education [F(3,38) = 0.387, P = 0.51], IQ [F(3,38) = 0.387, P = 0.51], in BPRS total scores [F(3,38) = 0.345, P = 0.72] and in NPI total score [F(3,38) = 0.748, P = 0.508]. Executive function The three groups differed significantly in the planning Tower of London task [F(3,40) = 22.568, P = 0.000]; Verbal Fluency [F(3,40) = 40.023, P = 0.000], and No. of categories achieved in the WCST [F(3,40) = 9.578, P = 0.000] and perseverative errors [F(3,40) = 10.694, P = 0.000]. The performance data for the four groups on the tests of executive functioning are shown in Table 3. The LSD method was used for post hoc comparisons. This revealed that in Verbal Fluency both frontal groups (LMPFC and RMPFC) performed significantly worse than the schizophrenic group and control subjects. Bonferroni tests on the Tower of London task showed impaired performance for the LMPFC group only when compared to both the schizophrenic and healthy subjects control groups. The LMPFC group performed significantly worse than the RMPFC subjects. First-order false belief tasks False-belief test question: Groups differed significantly on the non-parametric Kruskall–Wallis test: χ2(3) = 14.664, df = 3, P < 0.002. Post hoc (Bonferroni methods) comparisons showed that the RMPFC group’s performance, differed significantly from that of the LMPFC (RMPFC vs. LMPFC mean differences = −0.665; P < 0.005) but not from schizophrenic subgroups, and that the overall percentage of correct scores for patient groups was significantly lower than those of the normal control group. Fact questions: The percentage of correct scores revealed no significant overall differences between groups. Memory questions: The percentage of correct scores showed no significant group differences (Fig. 2). Fig. 2Performance of study groups (schizophrenics, Right Medial Prefrontal Cortex—RMPFC Lesion Subjects, Left Medial Prefrontal Cortex—LMPFC Lesion Subjects and healthy controls) on First-order False Belief tasks Second order theory of mind False-belief test question: Groups differed significantly: Kruskall–Wallis χ2(3) = 11.72, df = 3, P = 0.008). Post hoc (Bonferroni methods) analyses revealed the most impaired performance for the RF and schizophrenia groups when compared to LMPFC (LMPFC vs. RMPFC mean differences = −0.598; P < 0.007; LMPFC vs. schizophrenics mean differences = −0.623; P < 0.000). However the overall percentage of correct scores for patient groups was significantly lower than those of the normal control group. Fact questions: The percentage of correct scores revealed no significant overall difference between the groups. Memory questions: The percentage of correct scores showed no significant group differences (Fig. 3). Fig. 3Performance of study groups (schizophrenics, Right Medial Prefrontal Cortex—RMPFC Lesion Subjects, Left Medial Prefrontal Cortex—LMPFC Lesion Subjects and healthy controls) on second-order False Belief tasks Social situation task The ANOVA comparison between RMPFC, LMPFC, schizophrenics and healthy controls showed statistically significant differences in the ability to identify normative situations [F(3,58) = 3.179, P = 6.073]. Post hoc (Bonferroni methods) comparisons showed that the RMPFC group’s performance, differed significantly from that of the LMPFC (RMPFC vs. LMPFC mean differences = −1.904; P < 0.001; LMPFC vs. schizophrenics mean differences = 1.623; P < 0.002) and healthy controls but not from schizophrenics. There were no significant differences between the four groups in the total score of the norm violations (Fig. 4). Fig. 4Performances on social situation task in all groups (schizophrenics, Right Medial Prefrontal Cortex—RMPFC Lesion Subjects and Left Medial Prefrontal Cortex—LMPFC Lesion Subjects). The figure reports the scores in correct identification of normative situation and the mean scores of norm violations Mach IV scale The ANOVA comparison between RMPFC, LMPFC, schizophrenics and healthy controls showed statistically significant differences on the following Mach IV items (tactics+) item “Trusting someone means getting into trouble” [F(3,58) = 3.179, P = 0.035]; (tactics+) “It’s hard to be successful without taking short-cuts” [F(3,58) = 3.959, P = 0.000]; (tactics−) “There is no need to deceive anyone” [F(3,58) = 7.759, P = 0.021]; (tactics−) “It is possible to be good in all situations” [F(3,58) = 3.406, P = 0.027]. Post hoc multiple comparison (Bonferroni methods) showed that RMPFC score lower for items indicating agreement in strategic thinking (tactics+); than LMPFC, schizophrenics and controls showed higher scores for items indicating disagreement with strategic thinking (Tactics). Results are displayed in Fig. 5. Fig. 5Performance on items of Mach IV Scale in the four groups for strategic thinking Correlation analyses No significant correlations were found between ToM first order and ToM second order questions and executive functions (verbal fluency, WCST number of categories and perseverative errors, Tower of London) in normal controls, and in subjects with left and right frontal lesions. No significant correlations were found between the Mach IV scales (good tactical strategy and negative tactical strategy) and executive functions (verbal fluency, WCST no. of categories and perseverative errors, Tower of London) in normal controls, and in subjects with left and right MPFC. There was a significant correlation between first order ToM scores and duration of illness (r = −0.375; P < 0.029); between first order ToM scores and SANS total scores (r = −0.562; P < 0.03); between first order and second order ToM scores and social functioning total scores (AD) (first order = −0.489; P < 0.036; second order r = −0.543; P < 0.029). The significant correlation was also found in the schizophrenic sample between ToM performances and verbal fluency (r = −0.527; P < 0.000). No significant correlations were found between the Mach IV scale and social cognition task and psychopathological and clinical variable (SANS, SAPS and CGI). Discussion One of the distinctive attributes of human social cognition is our propensity to build models of other people’s minds: to make inferences about the mental states of others. Several neuroimaging studies have attempted to elucidate the neural substrates that support this distinctively human ability that is impaired in people with schizophrenia. The main aim of the present paper is to establish whether patients affected by schizophrenia show an impairment in several social cognitive tasks as demonstrated in other researches [5, 52–54] and if this cognitive profile is comparable to patients with a unilateral brain lesion involving orbito-ventromedial areas of the frontal lobes. Our results are in line with other studies: in people with schizophrenia there was an impairment of social cognitive abilities and this deficit appears to be related to negative symptomatology [55] and to be a key determinant of functional outcome, including social outcome [56]. It has been suggested that theory of mind deficit make unable schizophrenic subjects to interact effectively with their social environment, but that a lack of certain aspects of social cognition will lead to social misperceptions. In addition to these clinical and outcome goals, there is increasing interest in identifying the neural substrates that underlie social cognitive deficits in schizophrenia. For all of these reasons we compared the performances on social cognition tasks of schizophrenic subjects with the performances of MPFC subjects. Among the several studies which investigated the effects of frontal lobe lesions (dorsolateral and ventromedial/orbital) on performance in ToM tasks [9–11, 57–59], some of these including patients with bilateral frontal lobe damage, are limited because of a lack of detailed anatomical specification of lesion location [10, 11, 57]. Moreover, most of patients with bilateral lesions had suffered head trauma, an aetiology associated with rather diffuse brain damage that is particularly likely to impinge on orbitofrontal brain areas. Thus, the present study strictly tests the hypothesis that the unilateral (right or left) medial frontal cortex is implicated in the neural network sub serving ToM [8] which is based on well established evidences suggesting the implication of the ventromedial frontal lobe areas in playing a critical role in a dedicated “mentalizing” or ToM network in human brains ([7, 28, 60] for a review). We found out that subjects with RMPFC lesion are impaired in ToM tasks of “false beliefs”, showing thus a very similar cognitive dysfunctional profile to people affected by schizophrenia in all Theory of Mind tasks and in all social cognition tasks. A normal performance on control questions indicates an unimpaired comprehension of stories and suggest that the task was sensitive in detecting TOM impairments. In addition, schizophrenics and subjects with RMPFC lesion also showed impairment in the social cognition tasks, in fact they both failed to discriminate in judging inappropriate behaviour likely to induce anger in observers. This was unlike patients with LMPFC who showed no impairment on any of these tasks. This is clear evidence that the medial frontal cortex plays a critical role in a dedicated “mentalizing” brain network that underpins ToM ability [7, 8]. Our findings are in agreement with previous “lesional” studies, showing the association between right medial area damage and more severe ToM deficits [11, 13]. Siegal et al. [13] reported that ToM impairments seem to be associated with right hemisphere damage. In the present study we report a dissociation in RMPFC damage patients who displayed a defective ToM performance in contrast to LMPFC patients. LMPCF subjects show lower performances than RMPCF subjects in other cognitive competences but have normal performance in ToM competences and our results confirm the results obtained by Siegal and Surian [61]. In addition, when a more sophisticated social ability is required in order to perform second order false belief tasks correctly, also LMPFC damaged subjects fail to perform at a normal level and show a statistically significant impairment, even though to a lesser degree, when compared to subjects with RMPFC and schizophrenic people. A possible explanation is that LMPFC is involved in more sophisticated mentalizing tasks and that an intact right hemisphere structure is nevertheless required [58]. Normal subjects and neurosurgical subjects with unilateral LMPFC lesions perform fairly well on tasks related to tactical strategy, showing correspondingly low scores on the ingenuity aspect of thinking. The present study also provides further data on the neural prefrontal areas involved in social cognition tasks and in strategic thinking [21]. Social efficiency is represented by the ability to understand the intentions and beliefs of others with the aim of deceiving and manipulating them to achieve relevant objectives, such as control of food sources or sexual partners [26]. Recent literature fosters that these abilities are localised in the frontal cortex [27, 28]; the ability to recognise and manipulate hierarchical states to achieve some advantage would be localised in the amygdala and right hemisphere [62]. Schizophrenic people and subjects with RMPFC lesion showed impaired performance on tactical strategy associated with relatively “high levels” of social ingenuity and have an impaired ability to access “social schema knowledge” which is stored in the frontal lobes [31]. Such patients cannot access social schema knowledge and fail to inhibit aberrant behaviour, such as physical threats and aggression [21, 63]. Interestingly a completely reversed pattern characterizes the performance of LMPFC lesion subjects and healthy controls, in fact we found out a complete disassociation of the neural prefrontal areas located in the medial part of the hemisphere in sub serving human ability to think strategically, indicating that the cortical organization related to tactical aspects of Machiavellian Intelligence is lateralized to the right hemisphere [64]. Our results are at slight variance with the pioneering study of Rowe et al. [9] who found a significantly impaired performance of both the RMPFC and LMPFC subjects in first and second order ToM tasks. In this study cortical lesions were non-exclusive medial hemisphere but includes subjects with dorsolateral prefrontal cortex (DLPC) damage. Studies of normal subjects have used a variety of imaging techniques, designs, and test materials, but especially PET and fMRI to define brain regions specifically activated during a ToM task [8, 65]. Such studies have consistently shown activation of the medial prefrontal gyrus (MPFG) and the Temporo Parietal Junction. Frith and Frith [7], reporting data on studies carried out in adults, have revealed an MPFC system of three components that are consistently activated during both implicit and explicit mentalizing tasks. This brain region is probably the basis of the decoupling mechanism that distinguishes mental state representations from physical state representations. We can speculate, according to the Edelman model [66, 67], that a hierarchical organization of mental operation, when disrupted at a specified level, impairs the integrity of final output via an interruption of the chain of events required to perform a task. This study is limited by the small number of patients with unilateral frontal lobe lesions due to the rarity of such lesion. Despite this limitation, this study provides further evidence that social competence is compromised in RMPFC subjects very closely to schizophrenics and these data seem to elucidate the possible neuroanatomic structure alteration present in schizophrenia. However, we are confident that there is a wide range of behavioural manifestations of frontal lobe dysfunction, and ToM impairments clearly cannot account for all of these, nor is it likely to be responsible for all reported difficulties in social cognition. In contrast, ToM tests are designed with the aim of isolating those aspects of social cognition associated with two-way reciprocal interactions that rely crucially on ToM ability and false belief tasks have facilitated the demonstration of a mentalizing impairment in subjects with lesions of the prefrontal cortex, which is independent of non-mental state inference. In conclusion, our findings provide evidence that lesions to the right MPCF determine an incapacity to understand ToM “false belief” stories and to use tactical strategy and understanding of social schema. Other authors have reported the same results: subjects with right hemisphere damage, but not subjects with left hemisphere damage, had difficulties in performing simple theory of mind tasks [13, 68]. The difficulties shared by subjects with right hemisphere damage and young children on ToM tasks may have a similar origin [61]. These may both derive from a pragmatic deficit that prevents subjects from interpreting the implicit questions correctly, rather than from a conceptual deficit concerning the ability to represent mental states. The response pattern of subjects with ventromedial prefrontal damage on ToM tasks adds new evidence to the growing literature on the effects of the right hemisphere on various pragmatic aspects of language production and comprehension [13]. The good performance of subjects with RMPCF lesions in executive function and verbal memory tasks suggests that their difficulties are due to a reduced sensitivity to the constraints that guide the interpretation and production of contextually appropriate utterances [61]. It may be concluded that the ToM disorder in RMPCF subjects does not stem from executive function or memory deficits and that deficits in ToM and executive functioning in subjects with frontal lobe lesions are not causally related, even though our findings are in disagreement with Channon and Crawford [69] who found a relationship between executive functioning and ToM ability in adults with damage to the frontal lobes [70]. We support two positions: first, that a specialized, discrete ToM module, or set of modules, is located in the frontal lobes, but is functionally independent and second that these deficits can co-occur, on the basis of the proximity of the respective underlying neural areas.

J_Gastrointest_Surg-4-1-2231408

Helicobacter Genotyping and Detection in Peroperative Lavage Fluid in Patients with Perforated Peptic Ulcer

Introduction and Objectives Certain Helicobacter pylori genotypes are associated with peptic ulcer disease; however, little is known about associations between the H. pylori genotype and perforated peptic ulcer (PPU). The primary aim of this study was to evaluate which genotypes are present in patients with PPU and which genotype is dominant in this population. The secondary aim was to study the possibility of determining the H. pylori status in a way other than by biopsy. Introduction Over the past decades the incidence of perforated peptic ulcer (PPU) has declined in the western world. However, with an incidence varying between two and 10 per 100,000, it still is a problem in modern society.1 Moreover, mortality rates caused by gastric and duodenal ulcer perforation vary between 10 and 40% and zero and 10% respectively, and is higher among elderly patients.2,3 Several risk factors for PPU have been described such as smoking, alcohol abuse, and history of peptic ulcer disease (PUD).2 However, the main pathogenic factors are considered to be the use of non-steroidal anti-inflammatory drugs (NSAID) and the presence of H. pylori.2 Helicobacter pylori are widespread bacteria, with a prevalence ranging from 25% in the industrialized world to more than 70% in developing countries.4,5 Most infected people remain asymptomatic; however, a small group of carriers will develop PUD. Of patients who have developed PPU, 70% will test positive for H. pylori,2 suggesting the pathogenesis of perforation is associated with the presence of H. pylori. In addition, it is shown that different genotypes of H. pylori are associated with different clinical manifestations like PUD and gastric cancer.6,7 Two well-known H. pylori genes that have been associated with PUD are the cytotoxin-associated gene (cagA) and the vacuolating cytotoxin gene (vacA).6,8–10 VacA is present in all H. pylori strains and is associated with gastritis, PUD, and gastric carcinoma.10–12 It encodes for a vacuolating cytotoxin that causes epithelial cell injury and interferes with the immune system.13,14VacA contains at least two variable regions, the signal peptide (s)-region and the middle (m)-region. The s-region contains two allelic types, s1 and s2. The s1 strain has several subtypes, being s1a, s1b, and s1c.15 Two allelic types exist for the m-region, m1 and m2. The latter has two subtypes, m2a and m2b.16 CagA is considered a marker for a genomic pathogenicity (cag) island that is associated with enhanced virulence.17 If PPU is associated with a specific H. pylori genotype it may be feasible to limit the patients undergoing antibiotic therapy to those who have this genotype. When this specific type is not present, another cause of PPU should be looked for and antibiotic therapy should not be started. This would mean cost reduction and, probably, a reduction in the speed of the development of antibiotic resistance. Currently, gastric biopsy during endoscopy is a generally accepted method to diagnose H. pylori infection. However, patients with PPU will not undergo endoscopy but will generally be operated upon immediately. Taking a biopsy intraoperatively implicates a higher risk of bleeding and more difficult closure of the defect. Therefore, surgeons are reluctant to take a biopsy. The primary aim of this study was to evaluate which genotypes are present in patients with PPU and if a genotype is dominant in this population. The secondary aim was to study the possibility of determining the H. pylori status in a manner other than by gastric tissue biopsy. Methods From 30 consecutive patients operated on for PPU serum samples, gastric tissue biopsies, lavage fluid, and fluid from the nasogastric tube were collected. These patients were treated in five different medical centers throughout the Netherlands. In each of these centers approval of the medical ethical committee was obtained. Immediately after collection, the materials were frozen at −20°C. One researcher performed the analysis and genotyping. For H. pylori genotyping, the presence of cytotoxin-associated gene (cagA) and the s- and m-region genotypes of the vacuolating cytotoxin gene (vacA) were determined. DNA was isolated according to Boom’s method as described previously.18 A guanidine thiocyanate (GuSCN) solution was added to the collected material to induce lysis of the bacteria, releasing their DNA. After addition of the silica particles (Celite) the suspension was centrifuged. The silica particles, with the attached DNA, were washed with subsequently GuSCN-containing washing buffer, ethanol 70% and acetone. After drying, the DNA was eluted in an aqueous low salt buffer. The isolated DNA was amplified by means of polymerase chain reaction (PCR) and subsequently the presence of cagA and different types of vacA were analyzed by means of reverse hybridization on a strip (32). This assay consists of a nitrocellulose strip that contains dT-tailed oligonucleotide probes immobilized as parallel lines. For each strain, 10 μl of each PCR product (containing biotin at the 5′ end of each primer) was denatured by the addition of an equal amount of 400 mM NaOH and 10 mM EDTA in a plastic trough. After 5 min, 1 ml of prewarmed hybridization solution (2× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 50 mM Tris–HCl [pH 7.5], 0.1% SDS) was added, and a strip was submerged and incubated in a shaking water bath at 50°C for 1 h. The strips were washed with 2 ml of 2× SSC-0.1% SDS for 30 min at 50°C. Subsequently, the strips were rinsed three times in phosphate buffer, and conjugate (streptavidin–alkaline phosphatase) was added. After incubation at room temperature for 30 min, the strips were rinsed again and 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate substrate was added. Hybrids are visible as purple probe lines. Interpretation of the hybridization patterns was performed visually. As a control, a β-globin PCR was performed. Patient related factors were obtained prospectively. Statistical analysis was performed with SPSS for Windows, version 11.0. Results A total of 30 patients were included of whom nine were women. The average age was 65 years, varying between 40 and 87. Ten patients (33.3%) were operated laparoscopically. The perforation was found prepyloric in 11 patients, at the site of the pylorus in eight patients and postpyloric in 11 patients. A total of five (16.7%) patients had a history of PUD. Ten patients (33.3%) used NSAID’s, two patients (6.7%) used steroids, three patients (10.0%) used acid reducers, and one patient (3.3%) used a proton pump inhibitor (PPI) before admission to the hospital. The average hospital stay was 11.9 days, varying between 3 and 37 days. Fluid from the nasogastric tube was obtained from 25 patients, lavage fluid from 26 patients, serum samples from 20 patients, and ulcer biopsies from 18 patients. The results of the genotyping are depicted in Table 1. Table 1Helicobacter pylori Status and GenotypeThe colors represent the β-globin and H. pylori status of the patient. The β-globin determination was performed as a control. In nine samples of nasogastric tube fluid and in two samples of lavage fluid it was negative, rendering these results as unreliable. Therefore, these results were excluded from further analysis. Table 2 represents the frequency of the individual genes and the allelic types found in the different samples by means of PCR and LiPA. Table 2Frequencies of Individual Genes and Allelic TypesGenotypeFluid from Naso-Gastric TubeLavage FluidGastric Tissue BiopsyControl Non-UlcerNo.%No.%No.%%VacA s11090.91477.8777.846.9VacA s20015.5111.138.4VacA multiple19.1316.7111.114.7Total11100181009100100VacA m1654.5950.0555.629.4VacA m2436.4844.4333.355.9VacA incomplete genotype19.115.6111.10 (14.7 % incomplete)Total11100181009100100CagA positive981.81477.8555.647.1Total11100181009100100“VacA multiple” means that more than one allelic type or subtype has been found in one sample.In each different type of sample one incomplete genotype occurred, which is indicated as “vacA incomplete”. The “Control non ulcer” column represents the frequencies, found by van Doorn et al., in a population without PUD and is added to allow easy comparison. These tables show that for vacA the allelic type s1 is predominantly present in all three types of samples. In the s1 positive strains, subtype s1a is predominant as depicted in Table 3. Table 3Distribution of the vacA s1 SubtypesVacA SubtypeFluid from Naso-Gastric TubeLavage FluidGastric Tissue BiopsyControl Non-UlcerNo.%No.%No.%%S1a880.01392.9571.481.3S1b220.017.1228.618.7S1c0000000Total10100141007100100The s1a subtype is predominant in all sample types.The “Control non ulcer” column represents the frequencies, found by van Doorn et al., in a population without PUD and is added to allow easy comparison. With regard to the middle region of vacA the incidence of m1 allelic type is slightly higher; however, the difference is less outspoken compared to s1. The m2a was the only subtype that was found in the samples. In three samples, the genotyping was incomplete (Tables 1 and 2), meaning that determination of the middle region was not possible. This was most likely caused by the small number of bacteria present in those samples. With regard to the secondary aim of this study, analyzing possibilities to diagnose H. pylori presence in another fashion than through biopsy, the H. pylori status found in each type of sample was compared. A correlation was found between the H. pylori presence in biopsy and its presence in lavage fluid (Fisher’s exact test, p = 0.015), indicating lavage fluid is a valid alternative for determination of H. pylori infection. The sensitivity and specificity of the lavage fluid analysis was calculated, considering biopsy as a golden standard. Fourteen patients, of which the lavage fluid as well as the biopsy was analyzed, were included into this calculation (patients 2, 4, 5, 6, 8, 10, 15, 23–25, 27–30, Table 1), which is shown in Table 4. Of the remaining patients, either the biopsy or the lavage fluid was missing; therefore, these data cannot be used in the sensitivity/specificity calculation. Table 4Calculation of Sensitivity and Specificity of Lavage Fluid Analysis BiopsyLavage fluid+–Total+8210–044Total8614Sens 8/8 = 1Spec 4/6 = 0.67 The sensitivity was 100%, which means that in case of the presence of H. pylori in the biopsy specimen, the lavage fluid analysis detected it in 100% of cases. The specificity of lavage fluid analysis was 66.7%, which means the chance for false-positives is over 30%. With regard to gender, age, BMI, history of PUD, location of perforation, complications after procedure, and use of steroids, PPI, or antihistaminic medication, no statistically significant correlation was found. Discussion Concerning the role of H. pylori in the pathogenesis of PPU, some studies have been reported comparing the prevalence of H. pylori infection in patients with PPU to the prevalence in controls. They appear to be similar, suggesting that other factors like NSAID use play a role.19–21 However, the substantial genetic heterogeneity of H. pylori that has been revealed over the years leads to the hypothesis of a specific genotype causing PPU.5 Controls might test positive for H. pylori, but not develop PPU because it would not be this specific genotype that is isolated. This study of a selected population of patients, all with PPU, shows a limited diversity of H. pylori genotypes as represented by Table 1. VacA s1 strains are predominantly present in the three sample types of which s1a is the predominant subtype. Concerning the vacA m-region, the m1 strains are found in a majority of cases; however, the difference is less convincing than for vacA s1. Except for the biopsy samples, the cagA positive strains were predominantly present is this population. In the biopsy samples, the frequency of cagA-positive strains seemed to be low; however, this number is distorted because in two of nine positive biopsies, a decent comparison with the other samples was not possible. In patient 5, genotyping of the lavage fluid and nasogastric tube fluid was incomplete, and for patient 15, the opposite was the case. This means that the actual incidence should be 71.4 % (5/7). Summarising, these results shows that the vacA s1, cagA-positive strains were predominant in this population of patients with PPU. This finding is in accordance with literature reporting correlations between the presence of vacA s1, cagA-positive strains and PUD.6,10 Therefore, detection of the genotype vacA s1 does not specifically predict PPU; nevertheless, clinicians should be aware of this association. In Tables 2 and 3 the genetic distribution in a Dutch population without PUD, as found by van Doorn et al., are added for comparison. The frequencies found in this study for vacA s1, m1, and cagA-positive strains are clearly higher than in the non-PUD group, confirming the aforementioned hypothesis. However, with regard to the subtypes, Table 3 shows an almost similar distribution of frequencies, suggesting that determination of the allelic subtype is of less importance. In only 60% of patients biopsies could be analyzed. The reason for missing 40% is the restraint of the surgeon to take a biopsy when risk of bleeding and more difficult closure of the defect was estimated to be too high, which emphasizes the importance of finding an alternative. To do so, the H. pylori status of the patient as determined by biopsy was compared to the status as determined by analysis of nasogastric tube fluid, lavage fluid, and serum. A statistically significant correlation was found between the H. pylori status in biopsy and its status in lavage fluid (Fisher’s exact test, p = 0.015). This finding suggests that determination of the H. pylori status can be done with lavage fluid as well, obviously without any risk of bleeding and closure related difficulties. The sensitivity is 100%, but the specificity is 66.7%. This could mean the chance for false-positives is over 30%, which is not optimal and could lead to therapy overshoot. However, considering the fact that with the lavage a larger area is sampled, rendering the chance of positive test results higher than in biopsy, it is more likely to find false negative biopsy results. This could lead to a therapy undershoot, which obviates the importance having an alternative for a biopsy. In only two samples, both nasogastric tube fluids, a H. pylori genotype was isolated, while β-globin tested negative. In nine samples (seven nasogastric tube fluid, two lavage fluid) both β-globin and H. pylori tested negative. This means that either no humane cells were present in the samples, which is unlikely, or that an error in the PCR procedure had occurred. Because this was unclear these results were considered unreliable. Therefore, it still could be possible that nasogastric tube fluid is a good alternative for determining the H. pylori status as well. Overall, these results are positive, however they should be confirmed in a larger population. Conclusion This study shows that in a population of 30 patients with PPU, vacA s1, cagA positive strains are predominant. This finding is in accordance with literature reporting correlations between the presence of vacA s1, cagA-positive strains, and PUD. Therefore, detection of this genotype does not specifically predict PPU. Nevertheless, clinicians should be aware of this association. This study shows as well that it is feasible to use intraoperative lavage fluid to determine the H. pylori status of the patient, implicating that biopsies, with a risk of bleeding and more difficult closure of the defect, are not necessary anymore. In addition, considering the fact that a larger area is sampled with lavaging, biopsies may result in more false negative results leading to insufficient therapy.

Int_J_Parasitol-1-5-1885961

Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference

The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis. 1 Introduction The cattle tick Boophilus microplus is an important pest of cattle in subtropical and tropical regions of the world (Estrada-Pena et al., 2006). Although all Boophilus species including B. microplus have been reclassified to the genus Rhipicephalus (Murrell and Barker, 2003), we maintain use of the previous genus assignment for the purpose of biological clarity. Besides causing direct production losses and leather damage, B. microplus transmits several cattle pathogens, including Babesia bovis, Babesia bigemina and Anaplasma marginale. Control of B. microplus depends primarily on the use of acaricides or genetically resistant animals. Both approaches have limitations, including development of acaricide resistance, environmental contamination, pesticide residues in food products, the expense of developing new pesticides and the difficulty of producing tick-resistant cattle while maintaining desirable production characteristics (Willadsen, 2004). Other tick control approaches which show promise are the use of biological control agents (reviewed by Samish et al., 2004) and anti-tick vaccines (reviewed by de la Fuente and Kocan, 2003; Willadsen, 2004). Two commercial vaccines have been developed for control of tick infestations on cattle, TickGARD Plus® in Australia and Gavac® in Cuba. Both are based on the same recombinant antigen named Bm86, a glycoprotein of unknown function which is located predominantly on the surface of midgut digest cells (Gough and Kemp, 1993). This ‘concealed’ antigen is not naturally exposed to the host’s immune system. Lysis of midgut digest cells occurs in ticks that feed on vaccinated cattle, resulting in leakage of blood meal into the tick hemocoel. The overall effect of the vaccine is on engorging female ticks and includes a decrease in the number and weight of replete ticks and oviposition. While Bm86-based vaccines were effective against several other tick species, including Boophilus annulatus (Fragoso et al., 1998; Pipano et al., 2003), Boophilus decoloratus, Hyalomma anatolicum anatolicum and Hyalomma dromedarii, they were not effective against Amblyomma variegatum, Amblyomma cajennense and Rhipicephalus appendiculatus (de Vos et al., 2001; Rodriquez and Jongejan, unpublished data). Another ‘concealed’ antigen, Bm91, was shown to increase the efficacy of the Bm86 vaccine for B. microplus when co-administered (Willadsen et al., 1996). Bm91 is a low-abundance glycoprotein located in the salivary glands and midgut of B. microplus (Riding et al., 1994). The protein, a homologue of carboxydipeptidase, shares many biochemical and enzymatic properties with mammalian angiotensin converting enzyme, but its natural substrate has not been identified (Jarmey et al., 1995). More recently, a protein first labeled 4D8 and now called subolesin was identified through Ixodes scapularis cDNA expression library immunisation as a potential tick-protective antigen. Immunisation trials using recombinant subolesin caused reductions of larval, nymphal and adult I. scapularis infestations (Almazan et al., 2003, 2005a,b). The protein was later found to be conserved among ixodid tick species. Characterisation of its function by RNA interference (RNAi) in I. scapularis, Amblyomma americanum, Rhipicephalus sanguineus, Dermacentor variabilis and Dermacentor marginatus suggested involvement of this protein in the modulation of blood ingestion and reproduction. Therefore, the generic name “subolesin” was introduced for the 4D8 proteins and “subA” for the subolesin-encoding gene (de la Fuente et al., 2006a). Gene silencing by RNAi of subA and Rs86, the homologue of Bm86, in R. sanguineus, revealed a synergistic effect in which the expression of both genes was silenced and resulted in decreased tick attachment, feeding and oviposition (de la Fuente et al., 2006c). RNAi is a conserved post-transcriptional gene-silencing mechanism present in ticks and a wide range of eukaryotes in which double-stranded RNA (dsRNA) triggers a sequence-specific degradation of cognate mRNAs. It has been an effective tool to study the function of tick proteins at the tick–pathogen interface in a number of three-host tick species such as in I. scapularis which transmits Anaplasma phagocytophilum and Borrelia burgdorferi (Pal et al., 2004; Ramamoorthi et al., 2005; Sukumaran et al., 2006). RNAi was used to study the function of several tick salivary gland proteins involved in feeding of A. americanum (Aljamali et al., 2003; Karim et al., 2004), Haemaphysalis longicornis (Miyoshi et al., 2004) and I. scapularis (Narasimhan et al., 2004). The inducer of RNAi, dsRNA, is injected into nymphal or adult ticks which are then allowed to feed normally. Capillary feeding of dsRNA (Soares et al., 2005) or incubation of isolated tick tissues with dsRNA (Aljamali et al., 2003; Karim et al., 2005) are other methods used successfully to silence genes in ticks. These studies suggest that RNAi is systemic and effects gene silencing throughout the tick. In one-host Boophilus ticks, with all life stages feeding and molting on the same host, alternative strategies are required to conduct gene silencing by RNAi as compared with three-host tick species which spend their non-parasitic life stages off-host. Herein, we examined two methods of dsRNA delivery and its effect on the one-host tick B. microplus: (i) injection of dsRNA into freshly molted females and (ii) injection of dsRNA into engorged females. The latter method caused gene-specific silencing in the oviposited eggs and larvae that hatched from these eggs. We believe this is the first report of the silencing of the expression of Bm86, Bm91 and subolesin in Boophilus ticks as quantified by real-time RT-PCR using two routes of dsRNA delivery. 2 Materials and methods 2.1 Experimental animals Three Holstein–Friesian calves, 5 months of age (#7793, #7794 and #7799), were used. All animals had no previous exposure to ticks. All tick feedings were approved by the Animal Experiments Committee (DEC) of the Faculty of Veterinary Medicine, Utrecht University (DEC No. 0111.0807). 2.2 Ticks and tick feeding Boophilus microplus ticks originating from Mozambique were provided by ClinVet International (Pty), Bloemfontein, South Africa. The ticks were subsequently maintained on cattle at our tick rearing facility. Larvae were kept off-host at 20 °C with 95% relative humidity. Patches used for tick feeding with inner dimensions of 60 × 85 mm and sewn to an open cotton bag were glued to the shaved back of calf #7793 and #7794 using Pattex® contact glue (Henkel Nederland, Nieuwegein, The Netherlands). A batch of larvae eclosed from 1500 mg of pooled eggs oviposited by 25 females (approximately 24,000 larvae), was divided on day 0 between two patches on calf #7794. Since males appear earlier from the nymphal stage than females, approximately 500 unfed males were collected on days 13 and 14 and 600 unfed females on days 14 and 15 and incubated at 27 °C with 95% relative humidity. Freshly molted females were subjected to injection of dsRNA on day 15 as described below. For gene silencing in engorged females and their progeny, 25 engorged females with an average weight of 261 mg (248–272 mg) fed on calf #7794 were collected on day 21. Larvae which hatched from eggs laid by mock-injected, Bm86- and Bm91-dsRNA injected engorged females were fed in three patches on calf #7799. 2.3 RNA extraction and synthesis of tick cDNA for dsRNA preparations The viscera of five partially fed B. microplus females were dissected in ice-cold PBS and immediately stored in 1 ml Tri reagent (Sigma–Aldrich, Zwijndrecht, The Netherlands) at −80 °C. Total RNA was isolated and subsequently purified using the Nucleospin RNA II kit (Macherey-Nagel, Düren, Germany) in accordance with the reagent and kit manufacturer’s directions. Total RNA concentration was determined spectrophotometrically and the material was stored at −80 °C before use. Complementary DNA was made with the Revertaid first strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany) in accordance with the manufacturer’s protocol using random hexamer primers. Control reactions were performed using the same procedures but without RT as a control for DNA contamination in the RNA preparations. 2.4 Cloning and sequencing of the B. microplus subolesin gene Cloning and sequencing of the subA gene from the Mozambiquan B. microplus strain was performed as described elsewhere (de la Fuente et al., 2006a). The sequence has been submitted to GenBank and can be retrieved under Accession No. DQ923495. 2.5 dsRNA synthesis Oligonucleotide primers containing T7 promotor sequences at the 5′-end for in vitro transcription and synthesis of dsRNA were used to PCR-amplify cDNA encoding B. microplus Bm86 (421 bp), Bm91 (417 bp) and subolesin (381 bp). All oligonucleotide primers used in this study were synthesised by Isogen Life Science, IJsselstein, The Netherlands and their sequences are shown in Table 1. PCR products were purified using the GfX PCR purification kit (Amersham) and used as templates to produce dsRNA using the T7 Ribomax Express RNAi system (Promega, Leiden, The Netherlands). dsRNA aliquots were stored at −80 °C until used. 2.6 Injection of ticks with dsRNA Freshly molted females were placed on double-sided sticky tape with the ventral sides upwards and injected into the anal aperture with 0.5 μl Bm86, Bm91 or subolesin dsRNA alone or a combination of Bm86 and subolesin dsRNA (6–9 × 1011 molecules/μl) using a 10 μl syringe with a 33 G needle (Hamilton, Bonaduz, Switzerland) mounted on a MM3301-M3 micromanipulator (World Precision Instruments (WPI), Berlin, Germany) and connected to an UMPII syringe pump (WPI). The tip of a 27 G needle was used to slightly pierce the anal aperture before the 33 G needle was inserted. The dsRNA was dissolved in injection buffer (10 mM Tris–HCl, pH 7 and 1 mM EDTA). A control group was injected with injection buffer alone. The ticks were placed in an incubator at 27 °C with 95% relative humidity for 3–10 h following injection, before they were examined for mortality and placed in five separate patches, one for each group, on calf #7793. One hundred male ticks were placed in each patch simultaneously with the injected females. The ticks were checked twice daily and collected when they dropped from the host. Ticks still attached 14 days after the dsRNA-injection (day 29 after application of the larvae) were removed manually. All ticks were weighed separately within 1 h after collection and stored individually in 1.5 ml Eppendorf tubes with pierced lids at 27 °C and 95% relative humidity for oviposition. For the second experiment, engorged B. microplus females were injected with 5 μl of Bm86, Bm91 or subolesin dsRNA (1–2 × 1012 molecules/μl) or injection buffer alone in the right spiracular plate within 6 h after dropping off the host, using the same methods as described above, or left uninjected. They were stored individually in 2 ml Eppendorf tubes with pierced lids in an incubator at 27 °C and 95% relative humidity. Eggs were removed daily and each daily egg batch was stored separately under the same conditions. 2.7 Analysis to confirm gene silencing by quantitative RT-PCR Viscera was dissected from five females of each dsRNA-injected or mock-injected group after 6 days of feeding. Total RNA was isolated from these samples using Tri reagent and subsequently purified using the Nucleospin RNA II kit in accordance with the reagent and kit manufacturer’s directions. Total RNA was isolated from 100 mg eggs (14 days after injection), 50 mg larvae (at 6 days and 5 weeks after hatching) laid by/eclosed from the dsRNA- and mock-injected engorged females, 50 mg larvae at 10 weeks after hatching laid by the dsRNA- and mock-injected unfed females and from the dissected viscera of five females and five males which developed from 7-week-old larvae fed on animal #7799 using the same methods. cDNA from 1 μg of RNA (adults, eggs and 6-day-old larvae) and 0.3 μg of RNA (5-week-old larvae) was prepared using the Revertaid first strand cDNA synthesis kit (Fermentas) using random hexamer primers in accordance with the manufacturer’s protocol. All samples were analyzed for transcription of target genes by quantitative real-time RT-PCR using primers Bm86h-F6 and Bm86h-R4, amplifying a 117 bp section of the Bm86 gene; Bm91-F2 and Bm91-R3, amplifying a 129 bp section of the Bm91 gene and Bm-subA-F2 and Bm-subA-R2, amplifying a 166 bp section of the subolesin gene. Tick β-actin was included as a control and used for normalisation. A 126 bp fragment was amplified using primers Actin-F2 and Actin-R. All primer combinations amplified a different part of the targeted genes than the sections which were used for dsRNA synthesis, circumventing re-amplification of any unprocessed dsRNA. Twenty-five microlitres of real-time PCRs were performed using the Quantitect SYBR green PCR kit in accordance with the manufacturer’s protocol (Qiagen, Venlo, The Netherlands) on an iCycler real-time detection system (Bio-Rad Laboratories, Veenendaal, The Netherlands). Real-time PCR data were analyzed by iCycler IQ software version 1.0. 2.8 Analysis to check for the presence of dsRNA in eggs cDNA from 1 μg of total RNA of eggs 14 days post-oviposition was screened by quantitative real-time RT-PCR for the presence of non-processed Bm86, Bm91 and subolesin dsRNA to see whether the injected dsRNA was incorporated into the eggs and could be re-amplified. Oligonucleotide primers located within the region used for dsRNA synthesis of the Bm86, Bm91 and subolesin genes were used for this purpose. The following primer combinations were used: Bm86-F7 and Bm86h-R3, amplifying a 121 bp section of the Bm86 gene, primers Bm91-F3 and Bm91-R1, amplifying a 128 bp region of the Bm91 gene, primers BmsubA-F2 and BmsubA-R1, amplifying a 121 bp section of the subolesin gene. Real-time RT-PCR conditions were identical to those used to confirm gene silencing. 2.9 Statistical analysis Statistical analysis of data from two quantitative RT-PCR experiments, the weights of ticks after feeding and oviposited egg masses, was performed using Microsoft Excel and consisted of an unpaired t-test with unequal variances. Tick mortality was compared between the dsRNA- and mock-injected ticks by χ2-test. P values of 0.05 or less were considered statistically significant. 3 Results 3.1 Cloning and sequencing of the subolesin gene from B. microplus The subolesin gene (subA) from the Mozambiquan B. microplus strain was cloned and sequenced. This gene was found to be 99–100% identical to subA from B. microplus strains from Mexico and Brazil (de la Fuente et al., 2006a; our unpublished data). 3.2 RNAi in freshly molted B. microplus females Five groups consisting of 120 freshly molted B. microplus females were each injected with 0.5 μl of injection buffer in the following groups: (i) injection buffer alone, (ii) Bm86, (iii) Bm91, (iv) subolesin and (v) both Bm86 and subolesin-dsRNA. An average of 81 (76–82; 32.8% overall mortality) females were alive in each group 3–10 h following injection. These ticks were subsequently fed together on a calf with an excess of B. microplus males until the females became replete or for a maximum of 14 days. Tick weight after engorgement or manual removal, mortality rate, egg mass and hatching rate is presented in Table 2. A significant decrease in tick weight and oviposited egg mass, together with a higher mortality rate, was observed in the subolesin dsRNA injected groups compared with the control group (P < 0.01). Hatching rates were uniformly constant in the control, Bm86- and Bm91-dsRNA-injected groups (>90%), while in the ticks injected with subolesin dsRNA the hatching rate was lower (<20%). Gene silencing was confirmed by quantitative real-time RT-PCR (Fig. 1). The normalised transcript level of Bm86 was reduced with 86% in the Bm86-dsRNA-injected ticks and with 79% in the combined Bm86/subolesin-dsRNA-injected ticks, compared with the normalised transcript level of the mock-injected group (Fig. 1A). For Bm91, the normalised transcript level was reduced with 90% in the Bm91 RNAi-silenced ticks compared with the control ticks. A significant decrease in the Bm91 transcript level of 58% was observed in the subolesin dsRNA-injected ticks (P < 0.01) as well, but this reduction was not observed in the combined Bm86/subolesin-dsRNA-injected ticks (Fig. 1B). Normalised transcript levels of subolesin in the subolesin dsRNA- and combined Bm86/subolesin dsRNA-injected ticks compared with the control group were reduced with 90% and 80%, respectively (Fig. 1C). No differences were observed in the normalised Bm86 and Bm91 transcript levels of 10-week-old larvae which hatched from the Bm86- and Bm91-dsRNA-injected ticks compared with the mock-injected ticks (results not shown). 3.3 RNAi in engorged B. microplus females Five groups of five engorged females each with an average weight of 261 mg (248–272 mg) were injected with 5 μl of Bm86 dsRNA, Bm91 dsRNA, subolesin dsRNA, injection buffer alone or left uninjected within 6 h after dropping from the host. No reflux of the injected solution or hemolymph was observed from the puncture when the needle was gently withdrawn. Oviposition began in all groups within 3 days, except for one tick from the control-injected group and another tick from the Bm91 dsRNA-injected group which did not lay any eggs. The course of oviposition was not significantly influenced by the injection of dsRNA (Table 3), and dried or shriveled eggs were not observed, indicating that all eggs were successfully coated with a secretion from Gené’s organ. Interestingly, nearly all (>99.4%) eggs oviposited by engorged females injected with subolesin dsRNA showed an aberrant phenotype compared with those from the other groups. A typical example is shown in Fig. 2. Development of embryos in these eggs was not observed while many undifferentiated cells with some yolk cells were seen in Giemsa-stained egg crush smears. Most eggs did not hatch and eventually dried up and shriveled after 6–7 weeks of incubation at 27 °C/95% relative humidity. The few eggs from this group which did develop and hatched normally (<0.6%) were all laid during the first day of oviposition. A 64% decrease of subolesin transcript levels was found in eggs from the subolesin dsRNA-injected engorged females (Fig. 3C). Interestingly, a 30-fold increase in the Bm86 transcript level and a 56% decrease in Bm91 transcript level were also found in these aberrant eggs (Figs. 3A and B). An 84% decrease in the number of Bm86 copies was observed in eggs laid by the engorged females injected with Bm86 dsRNA (Fig. 3A) and the transcript level of Bm91 was reduced by 97% in eggs from the Bm91 dsRNA-injected engorged females compared with the mock-injected control group (Fig. 3B), confirming gene-specific silencing in the eggs of dsRNA-injected engorged females. Injected dsRNA could be re-amplified from the eggs of dsRNA-injected engorged females when primers located within the dsRNA sections were used, instead of primers located downstream of the dsRNA region which were used to demonstrate gene silencing (Table 1). Results of quantitative real-time RT-PCR performed with these primers showed levels of Bm86, Bm91 and subolesin which were 10.5, 4.3 and 4.5 times higher, respectively, in the Bm86, Bm91 and subolesin injected groups than levels found in the mock-injected group (Fig. 4). Gene silencing was observed in larvae 6 days after hatching; a decrease of 86% in Bm86 transcript level and of 91% in Bm91 transcript level in the Bm86- and Bm91 dsRNA-injected groups, respectively (Figs. 5A and B, grey bars). Subolesin transcript levels were not measured in the few larvae which hatched from the subolesin dsRNA-injected females due to their small number. Quantitative real-time RT-PCR analysis of RNA extracted from larvae 5 weeks after hatching (9 weeks after the initial injection of engorged females with dsRNA) revealed that genes remained silenced in both Bm86- and Bm91-silenced groups (Figs. 5A and B, black bars). This effect diminished over time, in particular for the Bm86-silenced group, in which transcript levels were now 67% lower compared with the control group. Bm91 transcript levels were still 90% lower in the Bm91-silenced group compared with the control group. When 7-week-old larvae from these three groups were fed in separate patches on a calf and total RNA from the viscera of adults which had developed from these larvae was analyzed by quantitative real-time RT-PCR, gene silencing was not observed (results not shown). 4 Discussion The production characteristics of the subolesin-silenced female B. microplus ticks corresponded with previous results from subolesin RNAi studies in other ixodid tick species, and typically resulted in decreased tick and egg mass weights and high mortality (de la Fuente et al., 2005, 2006a,c). The synergistic effect of combined silencing of the Bm86 and subolesin gene reported previously in R. sanguineus (de la Fuente et al., 2006c) could not be confirmed for B. microplus (Table 2). Significant changes in the production characteristics of the Bm86- and Bm91-silenced females were not observed, suggesting that the deleterious effect on ticks feeding on Bm86- or Bm91-vaccinated cattle is not caused by a loss of function of the Bm86 or Bm91 protein alone. In fact, the protective effect of Bm86-based vaccines is through gut damage mediated by anti-Bm86 antibodies during tick feeding (Willadsen et al., 1989). These results suggest that the protection mechanisms of Bm86 and subolesin-based vaccines are different and may contribute to the increased efficacy of Bm86 and subolesin combined vaccines. Quantitative real-time RT-PCR performed on samples taken 6 days after dsRNA injection and feeding resulted in a decrease of 79–90% of the targeted gene transcript level compared with the mock-injected group. This result was comparable with previous semi-quantitative measurements of the gene silencing effect by dsRNA-injection in A. americanum. In this RNAi study, a decrease of ∼90% to 50% in cystatin transcript level was observed from 24 h to 9 days of feeding (Karim et al., 2005). In the present study, although gene silencing was specific in the Bm86 and Bm91 dsRNA-injected groups, subolesin dsRNA injection resulted in both significantly decreased subolesin as well as Bm91 expression levels. This effect was not observed in the combined Bm86/subolesin silenced group, which may be explained by a slight difference in midgut:salivary gland ratio in the dissected viscera between the groups. Since Bm91 is present in relatively high concentrations in salivary glands compared with midgut (Riding et al., 1994), a shift in the midgut:salivary gland ratio of the dissected viscera in favor of midgut tissue could result in the lower Bm91 expression levels we found in this specific sample. Alternatively, the effect of silencing subolesin expression may affect the expression of other genes. The pleiotropic effect on tick tissues in which subolesin expression has been silenced suggests that this gene may be involved in the regulation of multiple pathways in ticks (de la Fuente et al., 2006a). Injection of pilocarpine solution into the hemocoel via the spiracular plate of engorged ticks has been described for inducement of salivation in engorged B. microplus ticks (Bechara et al., 1988). These openings of the tracheae are sclerotised structures located posterior to the fourth pair of legs. Their rigid structure allows for puncturing and injection of small quantities of fluid by a fine needle without subsequent reflux of the injected solution, hemolymph or tissue. When we injected Bm86, Bm91 or subolesin dsRNA into the hemocoel of engorged females, the course of oviposition appeared to be unaffected. Only one Bm91 dsRNA-injected female and one mock-injected female tick died before ovipositing. Interestingly, embryogenesis was undisturbed in eggs oviposited by the Bm86 dsRNA-, Bm91 dsRNA- and mock-injected engorged females, but an aberrant development was seen in the majority of egg masses oviposited by the subolesin dsRNA-injected engorged females. This egg phenotype has not been described previously and suggests that subolesin plays a role in embryonic development. When total RNA isolated from these aberrant eggs was analyzed by real-time RT-PCR, significantly higher Bm86 levels and decreased Bm91 and subolesin levels were found. Again, these results suggest that subolesin may be a regulator of transcription in ticks. Injected dsRNA was detected in eggs from dsRNA-injected engorged females by real-time RT-PCR using primers located within the dsRNA section, indicating that unprocessed dsRNA is incorporated in the eggs. The suggested route of incorporation of exogenously produced yolk directly from the hemolymph into oocytes (Saito et al., 2005) may be followed for the incorporation of dsRNA into oocytes as well. Further experiments are needed to determine whether this dsRNA forms a reservoir of mobile silencing signals inducing gene-specific silencing in eggs, or whether small interfering RNAs are responsible for this effect. Other possible routes for the mobile silencing signal to enter the oocyte are through the pedicel in the ovary or, once the oocyte has ovulated, by contact with cells from the genital tract. Some eggs (<0.6%) found in the batches laid during the first day of oviposition by subolesin dsRNA-injected engorged females, hatched normally. It is likely that these eggs developed to a stage which was not accessible by the dsRNA prior to injection of the dsRNA. They may have been ovulated eggs which were not in direct contact with the hemolymph or ones in which the shell was hardened during the oocyte passage through the oviduct and thus became impermeable to dsRNA during this process (Diehl et al., 1982). After dsRNA was injected into the body cavity of B. microplus, the gene silencing effect spread throughout the organism and its progeny. This systemic RNAi has been associated with the sid-1 protein, a transmembrane protein which enables passive cellular uptake of dsRNA (Winston et al., 2002; Feinberg and Hunter, 2003). Unfortunately, our attempts to detect a B. microplus sid-1 homologue, as described previously in studies on presence of a sid-1 homologue in grasshopper species Schistocerca americana, were not successful (data not shown) (Dong and Friedrich, 2005). The only protein currently present in the B. microplus expressed sequence tag (EST) database (Guerrero et al., 2005) which is associated with the RNAi machinery is the nuclease Argonaute-2 (Ago-2), the central catalytic component of the RNA-induced silencing complex (RISC) in mammals and Drosophila (Liu et al., 2004; Miyoshi et al., 2005). Homologues of other RNAi-associated proteins such as Dicer, which is responsible for the cleavage of exogenous long dsRNA into short interfering RNA (siRNA), remain to be identified in B. microplus and other tick species as well. RNAi described herein provides an important tool to screen for tick-protective antigens in this one-host tick species, B. microplus (de la Fuente et al., 2005) and allowed for characterisation of the effect and function of tick protective antigens, as well as the role of genes involved in tick–host–pathogen interactions and the transmission of tick-borne pathogens (de la Fuente et al., 2006b). Initiation and completion of the B. microplus genome sequencing project would greatly enhance these kinds of studies (Guerrero et al., 2006), as well as the availability of the genomes from cattle and the pathogens transmitted by B. microplus, most notably: A. marginale (Brayton et al., 2005), B. bigemina and B. bovis, which are currently being sequenced. Although sequences of the dsRNAs used in this study do not contain any significant overlap with other known B. microplus genes, the possibility of off-target gene silencing effects cannot be excluded due to the limited amount of sequence data available. Availability of the complete B. microplus genome sequence data will facilitate screening for potential off-target effects. These can subsequently be minimised by avoiding the use of dsRNAs or siRNAs containing sequences which are present in multiple genes. The effect of silencing tick genes suggested to be involved in embryogenesis such as vitellin degrading cysteine endopeptidase (Seixas et al., 2003), in the transovarial transmission of Babesia spp. or genes associated with acaricide resistance, which is measured in larvae by the Larval Packet Test (Li et al., 2003), could be studied using our method to silence genes in B. microplus embryos and larvae by injecting engorged females with dsRNA. These experimental approaches are more efficient and less labour intensive than the three other dsRNA delivery approaches into oocytes and embryos using microinjection (Wargelius et al., 1999), transgenic RNAi (Tavernarakis et al., 2000) and electroporation (Grabarek et al., 2002).

Eur_Arch_Otorhinolaryngol-4-1-2217621

Recurrent respiratory papillomatosis: an overview of current thinking and treatment

Human papillomaviruses (HPV) infection in benign laryngeal papillomas is well established. The vast majority of recurrent respiratory papillomatosis lesions are due to HPV types 6 and 11. Human papillomaviruses are small non-enveloped viruses (>8 kb), that replicate within the nuclei of infected host cells. Infected host basal cell keratinocytes and papillomas arise from the disordered proliferation of these differentiating keratinocytes. Surgical debulking of papillomas is currently the treatment of choice; newer surgical approaches utilizing microdebriders are replacing laser ablation. Surgery aims to secure an adequate airway and improve and maintain an acceptable quality of voice. Adjuvant treatments currently used include cidofovir, indole-3-carbinol, ribavirin, mumps vaccine, and photodynamic therapy. The recent licensing of prophylactic HPV vaccines is a most interesting development. The low incidence of RRP does pose significant problems in recruitment of sufficient numbers to show statistical significance. Large multi-centre collaborative clinical trials are therefore required. Even so, sufficient clinical follow-up data would take several years. Introduction Sir Morrell Mackenzie (1837–1892) was the first to recognize papillomas as a lesion of the laryngo-pharyngeal system in children in the late 1800s. It is now apparent that these benign tumours may occur at other parts of the upper gastrointestinal and respiratory tracts, and in all age groups. It was not until the 1940s that Chevalier Jackson (1865–1958) first coined the term “juvenile laryngeal papillomatosis”. The prevalence of laryngeal papillomatosis has been estimated at between four to seven cases per million person-years in the Western World [4, 5, 25, 43]. Furthermore, the incidence of recurrent respiratory papillomatosis (RRP) has been estimated at about 2 per 100,000 in adults and 4 per 100,000 in children [9]. The disease can be categorized into adult onset and juvenile onset forms. Age of first presentation of disease is usually in the teens (50%) for the juvenile onset form but can be as early as the first year of life. Initial presentation in the adult form tends to peak in the third and fourth decades. It is now well established that human papillomaviruses (HPVs) are the aetiological agent of many benign and malignant tumours arising from epidermal tissues. They are a necessary cause of the second most common female cancer worldwide, cancer of the cervix [7, 45], and strongly associated with several other ano-genital cancers such anal, penile, vulval and vaginal carcinomas [17]. Furthermore, there is mounting evidence of at least some head and neck cancers associated with HPV infection [15, 17, 23]. These malignancies are associated with ∼15 high risk (HR) types, in particular HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82. Benign tumours such as common warts, flat warts and genital warts are caused by low risk types such as HPV 1, 2, 3, 4, 6, 10, 11 and others. HPV 6 and 11 have been described as the dominant types found in RRP [10]. Despite the benign nature of these lesions, there is significant morbidity and occasional mortality due to multiple recurrences which necessitate hospital admission for surgical removal. Dissemination or extension of the growths into the lower airways indicates a poorer prognosis. The clinical behaviour is variable and lesions can regress, persist and in rare instances, progress to carcinoma if other environmental factors such as smoking or irradiation are involved. Epidemiology Human papillomaviruses infection in benign laryngeal papillomas is well established. One Danish study showed that 95% of solitary laryngeal papillomas were positive for HPV DNA by in situ hybridization [26]. Another study from Hong Kong found that 59% of laryngeal papillomas showed the presence of HPV 6, 11, 16, 18 subtypes, with 6 and 11 the dominant types [10]. Malignant transformation of some lesions has been described in association with HPV 11 integration into the genome and mutation of p53 [32]. Infection with HPV 11 is more likely to be associated with the development of distal airways disease than HPV 6. Clinical manifestation of disease prior to three years of age is a further risk factor associated with distal spread of disease [1, 47]. The vast majority of RRP lesions are due to HPV types 6 and 11, and the reservoir for these types is the human ano-genital tract. Ano-genital HPV is the commonest sexually transmitted viral infection and the prevalence of clinically apparent genital warts is thought to be the “tip of the iceberg” of HPV infection. Koutsky et al. estimates that ∼10–20% of the US population between 15 and 49 years of age have molecular evidence of infection and that another 60% have had prior infection [22]. In the UK, there were well over 81,000 incident genital wart infections reported from genitourinary medicine clinics in 2005 [16] and this has been increasing every year. Such high background prevalence suggests that there is a risk of mother to child transmission at the time of delivery, especially if there are noticeable genital warts. Data supporting this hypothesis showed that a history of maternal condylomata during pregnancy was associated with a 200-fold risk of RRP in the child [37]. An uncomplicated vaginal delivery in a mother with HPV infection has been estimated to carry a risk of transmission of 1:80–1:1,500 (median of 1:400) [34]. In one group of children with juvenile laryngeal papillomatosis, 54% had a maternal history of vulval condylomata at the time of delivery [13]. Another study found that in 77 mothers with condylomata at delivery, 9 children (11.6%) were later diagnosed with juvenile laryngeal papillomatosis [21]. The route of transmission is likely to be different in the juvenile onset and adult onset forms of RRP. Evidence for this has been suggested by a case control study, in which the risk factors for both forms were compared [20]. The authors found that adult onset patients were more likely to have had more sexual partners and oral sex than their controls. Patients with the juvenile form were more likely to have been born to teenage mothers and first-born children compared to their controls. Aetiological and histopathological features Papillomatous lesions preferentially occur anatomically at the sites of “transformation zones”, where squamous epithelia abut ciliated columnar epithelia but can infect anywhere in the respiratory tract [28]. The classical sites for recurrent disease in the upper aero-digestive tract would be the nasopharyngeal area of the soft palate, limen vestibuli, midzone of laryngeal area of the epiglottis, upper and lower margins of the ventricle, vocal fold undersurface, carina and bronchial spurs. It is interesting to note that papillomata have been observed at tracheotomy sites and tracts where the iatrogenic induction of change of epithelialization also occurs [19]. Human papillomaviruses are small nonenveloped viruses (∼8 kb), with a double stranded circular DNA genome encapsulated within an icosahedral capsid that replicate within the nuclei of infected host cells. The genome codes for 8–10 genes (median of eight). The late L1 and L2 genes code for the viral capsid proteins, the early proteins E1 and E2 are responsible for viral replication and transcription, and E4 appears to aid virus release from infected cells. The early genes E6 and E7 have transforming ability in in vitro assays for HR types but LR types have little to no ability for this feature [2, 33, 42]. Electron microscopic analysis reveals the virion to be ∼55 nm in diameter and the capsid to be comprised of 72 pentameric capsomers. The predominant protein in the capsid consists of the L1 protein, with a smaller proportion of L2 embedded deep within the protein shell. It is this L1 protein which provides the dominant antigenic epitopes recognized by neutralizing antibodies and forms the basis for the bivalent (GlaxoSmithKline) and quadrivalent (Merck, Hohenbrumn, Germany) vaccines currently available. The virus is thought to bind to and gain entry to its host cell, the basal keratinocyte, by microtrauma or abrasions to the surface epithelium. The receptor has not been definitively identified but α6-integrin and heparin sulphate may play important roles in viral entry [11, 18, 30]. Following infection and uncoating, the virus is thought to maintain its genome as a low copy number episome in the basal cells. It has been suggested that expression of E1 and possibly E2, may be sufficient for basal maintenance of viral episomes [50]. Viral early proteins E6, E7, E1 and E2 are expressed at low level in early passage cell lines derived from naturally occurring low-grade cervical lesions and the viral genome is maintained at around 10–200 copies per cell [8, 41]. The viral genome is amplified in differentiating keratinocytes via rolling-circle amplification that will synthesize sufficient viral genome for packaging [3, 12]. This requirement for differentiating epithelial cells is a key part of the virus life cycle but the normal restraint on cell cycle progression appears to be abolished by the E6 and E7 proteins and normal terminal differentiation is retarded [36]. These E6 and E7 effects on key apoptotic proteins such as Rb and p53 have been demonstrated in HPV 16 and other high-risk subtypes in in vitro assays [24, 27, 28]. HPV 6 and 11 E6 and E7 proteins do not readily bind to or degrade the p53 or Rb proteins [14, 31, 46]. This suggests that alternative mechanisms of altered cellular growth and proliferation may exist for the low-risk subtypes of HPV. Furthermore, there are little data on HPV 6 and 11 life cycles, replication, maintenance and viral production in respiratory cells. It is not known if these are similar to or different to disease in the ano-genital region. Treatment Surgical Surgical debulking is currently the treatment of choice; newer surgical approaches utilizing microdebriders are replacing laser ablation. Surgical excision aims to secure an adequate airway and improve and maintain an acceptable quality of voice [49]. HPV is present in the normal macroscopically unaffected mucosa and it is currently not possible to distinguish infected cells with a normal appearance from uninfected epithelia. Repeated recurrences are frequent, however, repeated attempts to treat the papillomas may cause serious complications [40]. Current practice in the treatment of RRP was recently evaluated by a questionnaire in the UK [44]. Various lasers such as CO2, KTP, and pulsed dye were found to be the preferred method of surgical removal of RRP in children [49]. Spontaneous ventilation (65.3%) is the preferred method of anaesthesia. The frequent recurrence of papillomas has resulted in the use of different adjuvant treatments alongside surgical removal of macroscopically obvious in the attempt to improve outcomes. In the future, advances in the understanding of the immune response to HPV may improve our treatment modalities and prevention strategies. Adjuvant treatment Adjuvant treatments currently used include cidofovir, indole-3-carbinol, ribavirin, mumps vaccine, and photodynamic therapy. As with surgical management, viral persistence occurs following treatment with these adjuvant modalities. Intralesional cidofovir may help control papilloma regrowth and reduce disease severity in many children with RRP [39]. In most cases, cidofovir would appear to be less efficacious in producing disease eradication. There appears to be little evidence to support prolonged treatment regimes (i.e. more than eight treatments) [35]. Subcutaneously injected cidofovir has been tested on cartilage in a rabbit model [39]. There was a positive dose-response relationship which existed for gross skin changes; however, there was no dose-response relationship for severity of change in the epithelium. Higher doses of cidofovir than commonly are used in the treatment of RRP may be safe, although the effects of repeat application and long-term complications are not yet known. In animals, cidofovir is carcinogenic (mammary adenocarcinoma in rats), embryotoxic and teratogenic [48]. Care must be taken in humans that the possibility of pregnancy is excluded when usage is considered. In view of the severe nephrotoxicity shown when intravenous cidofovir is administered to animals and humans, caution would be advised for repeated intralesional or subcutaneous applications for RRP treatment. The less common complications of bone marrow toxicity, iritis and uveitis may also arise and vigilance is required from clinicians. Controlled trials failed to provide sufficient evidence to draw reliable conclusions about the effectiveness of antiviral agents as adjuvant therapy in the management of RRP. Further research is required before any specific antiviral adjuvant therapy can be recommended. Factors leading to virus activation in RRP have not been recognized, however, extra-oesophageal acid reflux disease (EERD) has been suggested as a possible factor, initially by Borkowski et al. [6], and then by a group from Harvard in 2005 [29]. There is clinical evidence suggesting a link between the presence of EERD and RRP. Inflammation induced by acid exposure may result in the expression of HPV in susceptible tissues. Therefore, treatment of EERD should be considered in all patients with difficult to control RRP with EERD. Despite currently available surgical and adjuvant management options, tracheotomy may become necessary in selected patients with extensive disease. Decannulation should be performed as early as possible to avoid further spread of viral infection and improve the quality of life. The primary cause of papilloma extension to the lower airways appears to be iatrogenic, i.e. the tracheotomies performed in children with laryngeal papillomatosis (92.5% of cases). This was reported in a case group of 448 children with RRP treated in St. Vladimir Moscow Children’s Hospital between 1988 and 2003 [38]. The recent licensing of prophylactic HPV vaccines is a most interesting development. In particular, the quadrivalent vaccine from Merck & Sanofi-Pasteur (Gardasil®) which shows efficacy against HPV 6, 11, 16, 18 subtypes, may be anticipated to impact upon the incidence of RRP. The low incidence of RRP does pose significant problems in recruitment of sufficient numbers to show statistical significance. Large multi-centre collaborative clinical trials are therefore required. Even so, sufficient clinical follow-up data would take several years.

Eur_Radiol-4-1-2373858

Contrast-enhanced magnetic resonance imaging of the breast: the value of pharmacokinetic parameters derived from fast dynamic imaging during initial enhancement in classifying lesions

The value of pharmacokinetic parameters derived from fast dynamic imaging during initial enhancement in characterizing breast lesions on magnetic resonance imaging (MRI) was evaluated. Sixty-eight malignant and 34 benign lesions were included. In the scanning protocol, high temporal resolution imaging was combined with high spatial resolution imaging. The high temporal resolution images were recorded every 4.1 s during initial enhancement (fast dynamic analysis). The high spatial resolution images were recorded at a temporal resolution of 86 s (slow dynamic analysis). In the fast dynamic evaluation pharmacokinetic parameters (Ktrans, Ve and kep) were evaluated. In the slow dynamic analysis, each lesion was scored according to the BI-RADS classification. Two readers evaluated all data prospectively. ROC and multivariate analysis were performed. The slow dynamic analysis resulted in an AUC of 0.85 and 0.83, respectively. The fast dynamic analysis resulted in an AUC of 0.83 in both readers. The combination of both the slow and fast dynamic analyses resulted in a significant improvement of diagnostic performance with an AUC of 0.93 and 0.90 (P = 0.02). The increased diagnostic performance found when combining both methods demonstrates the additional value of our method in further improving the diagnostic performance of breast MRI. Introduction Breast cancer is the most commonly diagnosed cancer in women and the most prevalent cancer worldwide [1]. In breast imaging, mammography is still the most commonly used imaging techniques both in screening for and staging of breast cancer. However, dynamic contrast-enhanced magnetic resonance imaging (MRI) is becoming an increasingly important imaging modality in the detection and staging of breast cancer. Because of its superior sensitivity for the detection of invasive breast cancer, MRI has become a very important modality in breast imaging [2–5]. However, the classification of a lesion detected on MRI as benign or malignant still remains a challenge. Reported specificities in clinical studies range between 20% and 100% [6–15]. The main characteristics used for classification of detected lesions on MRI are the lesion morphology and the enhancement dynamics [4]. Dynamic evaluation is often based on late dynamic characteristics of enhancing lesions. In this approach, the decrease of signal intensity, often referred to as a type 3 curve or washout, is highly suggestive for breast cancer with the likelihood for malignancy of 87% [12]. This dynamic evaluation makes use of high-resolution T1-weighted MRI images with a relatively low time resolution of 42 s or more [3, 12, 16–20]. The high spatial resolution of these sequences is necessary for accurate morphologic evaluation. Irregular lesion contour, inhomogeneous internal enhancement and rim enhancement have been described as features indicating a malignancy [21]. Schnall et al. [4] found focal mass margins and signal intensity to be a highly predictive imaging features. However, the combination of both dynamic and morphological parameters resulted in the highest diagnostic accuracy in multivariate analysis. The evaluation of early enhancement using a high temporal resolution has also been a subject of study in breast MRI. Boetes et al. [6] found in a group of 87 lesions a sensitivity of 95%, a specificity of 86% and an overall accuracy of 93% based on early enhancement characteristics. In this study, a temporal resolution of 2.3 s was achieved using a turboFLASH sequence. The value of a high temporal resolution during initial enhancement was confirmed by Sardanelli et al. [22] who used a temporal resolution of 15 s during the first minute of enhancement. The overlap between malignant and benign enhancement curves was only 9% using the fast dynamic evaluation compared with 50% using a lower temporal resolution of 1 min. The value of first pass high temporal resolution imaging for the differentiation of benign and malignant lesions was studied in a direct comparison of steady-state dynamic MRI (30 s temporal resolution) and first-pass imaging (2 s temporal resolution) of induced mammary tumors in female rats by Helbich et al. [23]. In their study, an estimate of first-pass perfusion using T2*-weighted imaging almost reached a significant difference between benign and malignant tumors. All other methods used, including T1-weighted first-pass imaging, failed to differentiate benign from malignant tumors. Gibbs et al. [24] also used a high temporal resolution (10.5–14.5 s) in the evaluation of small breast lesions and evaluated their data using a pharmacokinetic model. The incorporation of data from pharmacokinetic modeling in the evaluation of lesions improved diagnostic accuracy in their group. High temporal resolution sequences often cover a limited area of the breast [6, 24]. These imaging protocols are, therefore, less suitable in clinical MRI or screening. For this study, we adjusted the scanning protocol in order to obtain a high temporal resolution during initial enhancement while covering both breasts entirely. The aim of this study is to asses the value of pharmacokinetic parameters derived from fast dynamic contrast enhanced imaging during initial enhancement in differentiating between benign and malignant breast lesions on MRI. Materials and methods Patient selection All lesions detected on clinically performed breast MRI examinations in the period from January 2004 until June 2005 were initially included. All detected lesions were evaluated based on the following inclusion criteria: (1) histological confirmed diagnosis or (2) follow-up based on unchanged MRI morphology and enhancement characteristics during at least 24 months indicating a benign nature of the lesion [25]. Lesions that could not be classified as benign or malignant using these criteria were excluded. The protocol was approved by the institutional review board. Imaging protocol All patients were examined using a 1.5-Tesla MRI scanner (Sonata or Symphony, Siemens, Erlangen, Germany) in combination with a double breast coil. In premenopausal women, the MRI examination was performed in the second week of the menstrual cycle to minimize enhancement of normal glandular tissue [26]. Prior to the MRI examination, an intravenous catheter was inserted in the left or right arm. All patients were placed in the prone position with the breasts in the double breast coil and positioned at the isocenter of the magnet. After localizer images were obtained in three directions, low spatial resolution proton-density-weighted images were acquired in the transverse plane covering both breasts completely (TE 1.56, TR 800, FA 8, FOV 320, slices 24, TA 50 s, image resolution 3.9 mm × 1.3 mm × 4.0 mm). Subsequently, a coronally orientated high-resolution three-dimensional fast low-angle shot series (FLASH 3D) was acquired (TE 4, TR 7.5, FA 8, FOV 320, slices 120, TA 86 s, image resolution 1.3 mm × 1.3 mm × 1.3 mm). Thereafter, high temporal resolution T1-weighted images (turboFLASH) were recorded 22 times with identical spatial resolution and orientation as the proton-density-weighted images (TE 1.56, TR 66, FA 20, FOV 320, slices 24, TA 22 × 4.1 s) during an intravenous bolus injection of a paramagnetic gadolinium chelate—0.2 mmol of gadoterate meglumine (Dotarem; Guerbet, The Netherlands) per kilogram of body weight—which was administered with a power injector (Spectris; Medrad, Pittsburg, USA) at 2.5 ml/s and followed by a 15-ml saline flush. Following these series, the FLASH 3D series was repeated four times. Total scan time for this protocol was 9 min 42 s, including the time needed to record localizer images. Image evaluation For the evaluation, the MRI data were divided into two sets of dynamic data for each patient. The first dataset contained the high spatial resolution T1-weighted images (FLASH 3D) only. These were used for the evaluation of both lesion morphology as well as signal intensity versus time curves. This method will be further referred to as the ‘slow dynamic’ analysis. The second dataset contained the proton-density-weighted images, the high temporal resolution images as well as the precontrast high spatial resolution sequence. A high-resolution subtraction of the pre- and first postcontrast FLASH3D series prepared on the MRI scanner was also included in this dataset to aid in lesion detection. The proton-density-weighted sequence was necessary for an accurate estimation of the T1 value necessary for the quantitative analyses. The evaluation of this dataset will be further referred to as the ‘fast dynamic’ analysis. In this fast dynamic analysis, the last three postcontrast FLASH 3D series were not used. All cases were evaluated prospectively by two experienced breast MRI radiologists (reader 1 and reader 2). Both readers had over 5 years of experience in dynamic breast MRI. The evaluation on the two workstations was performed independently in different sessions with at least a 2-month time interval between both sessions. For the slow dynamic analysis, a dedicated breast MRI workstation was used (Dynacad, Invivo, Germany). This workstation creates subtraction images for all time points after contrast administration, of which the first is automatically displayed together with the precontrast T1 acquisition, both in a coronal orientation. Furthermore, axial reconstructions were displayed for both the subtracted and T1-weighted images with color overlays of wash-in/wash-out enhancement characteristics projected over the T1-weighted images [27]. A maximum intensity projection and signal intensity versus time curves were also displayed. This display protocol resembles the protocol used in the clinical workflow of dynamic breast MRI in our hospital. A BI-RADS classification was assigned for each lesion based on their morphology and enhancement dynamics [28]. No clinical information, mammography or prior MRI data were provided to the readers during the evaluation of the cases. In the fast dynamic analysis, a workstation, developed in-house for the evaluation of dynamic contrast enhanced MRI, was used [29, 30]. On this workstation, pharmacokinetic parameters derived from the high temporal resolution turboFLASH series were automatically calibrated, calculated and displayed using color overlays. Examples of the recorded high temporal resolution enhancement versus time curves are presented in Fig. 1. In the preparation of this high temporal resolution data, each MRI signal enhancement/time curve was first fitted to a general exponential signal enhancement model, as described previously [31]. This reduces a curve to model with the following five parameters: baseline (s0); start of signal enhancement (t0), which defines the onset of the exponential curve; time-to-peak (ttp), the exponential constant; peak enhancement (sp), the signal amplitude at which the exponential curve levels off; and late wash, defined as the slope of the late part of the exponential curve. The reduced signal enhancement/time curve was converted to a reduced tracer concentration (mmol/ml)/time curve [31, 32], effectively converting sp to concentration tracer after initial rapid wash-in (often at a peak or plateau level) (Cgd,p). The reduced plasma concentration time curve was estimated using the reference tissue method [33]. Deconvolution of the plasma profile and estimation of pharmacokinetic parameters conformed to the theoretical derivations [34], but was implemented in the reduced signal space as: Ve = Cgd,ptissue/Cgd,pplasma; kep = 1/(ttptissue – ttpplasma). Ktrans = Ve × Kep. Where Ve is an estimate of the extracellular volume [%], Ktrans, the volume transfer constant (1/min), and kep, the rate constant (1/min), between extracellular extravascular and plasma space. The subscript ‘tissue’ stands for a measurement in the tissue under investigation and the subscript ‘plasma’ for the reference tissue plasma estimate. The reference tissue was automatically determined by selecting a set of voxels in the whole image volume [relative enhancement, (sp-s0)/s0] larger than 0.2 and smaller than 2.0). This was most often the pectoral muscle, sometimes the liver or spleen. The additionally recorded proton density images were used to correct for the coil profile. The data were presented on the workstation with high-resolution precontrast T1-weighted images in an axial, coronal and sagital reconstruction (FLASH 3D) as background. Color overlays were projected over the images representing Ktrans, kep and Ve parameter values that were based on the high temporal resolution images (turboFLASH). A subtraction image based on the pre- and first postcontrast FLASH 3D series was presented to aid in lesion detection. No criteria for differentiating between benign and malignant lesions were derived from the subtracted images. In this evaluation, the readers selected a region of interest (ROI) within the enhancing lesion. The ROIs were sphere-shaped and placed in an area within the lesion where the parameter values of Ktrans, Ve and kep were highest, based on the color-overlays. The outer limit of the lesion was used as a boundary of the ROI to rule out partial volume effects [35]. This method of ROI selection has previously been referred to as a hotspot method [36]. Each reader placed only one ROI per lesion. From this ROI, the workstation calculated the mean values for each of the pharmacokinetic parameters. Again, no clinical information, mammography or prior MRI data were provided to the readers during the evaluation of the cases. In case of multifocality, the tumor was analyzed as a single lesion. Fig. 1Relative enhancement versus time curves of a benign (a) and malignant (b) lesion. Note that the slope of enhancement and the level of enhancement is higher for the malignant lesion compared with the benign lesion. These fast dynamic acquisitions were analyzed as described in the Materials and methods section and resulted in the color overlays as presented in Figs. 4 and 5. The data used in this figure were respectively derived from a histopathology proven fibroadenoma and an invasive ductal carcinoma. The same lesions as presented in Figs. 4 and 5 Statistical analysis Differences in pharmacokinetic parameter values between the malignant and benign group were evaluated using an independent sample t-test. The performance of both methods was compared using a receiver operator characteristic (ROC) analysis. From the slow dynamic analysis, the reader’s final BI-RADS classification of the lesion was used in the ROC evaluation; from the fast dynamic analysis, the mean parameter values calculated from the ROI selected by each reader were used. Multivariate analysis was performed using logistic regression in order to evaluate the possible additional value of both methods to one another. Since the differentiation between benign and malignant lesions is more difficult in smaller lesions a subgroup of all lesions of 2 cm and smaller were also separately evaluated. The comparison of the various results, including the interobserver variability, was done by using the area under the ROC curve (AUC) as an estimate of diagnostic accuracy. A pairwise comparison was performed to evaluate differences in the AUC. P values <0.05 were considered to indicate statistical significance. Results A total of 870 consecutive clinical breast MRI examinations in 787 patients were performed. In these studies a total of 188 lesions were detected. Eighty-six lesions could not be included due to lack of histological diagnosis or insufficient follow-up. This resulted in a total of 102 lesions in 96 patients; 34 benign and 68 malignant lesions. The mean age was 51 years (range 28–74 years). Ninety-four lesions were included based on histological evaluation, eight lesions based on follow-up. Mean lesion size on MRI for the malignant group was 32 mm (range 9–90 mm) and this was 15 mm (range 5–50 mm) for the benign lesions. A total of 52 lesions were 2 cm or smaller; 25 malignant (mean lesion size 14 mm, range 6–20 mm) and 27 benign (mean lesion size 11 mm , range 5–20 mm). The histological evaluation of the malignant lesions was in 14 cases based only on the core biopsy, in 14 cases based on an excision biopsy or breast saving surgery specimen and in 40 cases based on the mastectomy specimen. Histological proven benign lesions were in 19 cases based on a core biopsy and in seven cases based on an excision biopsy. Eight lesions were proven benign based on follow-up, mean follow-up was 37 months (range 24–52 months). The histological composition of the entire group is summarized in Table 1. Table 1Histological composition of the benign and malignant group of lesions (IDC invasive ductal carcinoma, DCIS ductal carcinoma in situ, ILC invasive lobular carcinoma)Benign (n = 34)Malignant (n = 68)Fibroadenoma11IDC47Fibrosis4DCIS14Adenosis3ILC7Inflammation2Ductal papilloma2Scar tissue1Hyperplasia1Hamartoma1Radial scar1Follow-up8 Imaging results In the slow dynamic analysis reader 1 classified 25 lesions as a BI-RADS 2 (benign:malignant = 22:3), seven as BI-RADS 3 (4:3), 50 as BI-RADS 4 (6:44) and 20 an BI-RADS 5 (2:18). This was respectively 33 (24:9), 12 (4:8), 41 (5:36) and 16 (1:15) for reader 2. The ROC analysis for the slow dynamic analysis resulted in an AUC of 0.85 (95% CI = 0.773–0.918) and 0.83 (95% CI = 0.74–0.89) for reader 1 and 2, respectively. The mean volume of the ROIs selected by the readers in the fast dynamic evaluation was 0.51 cm3 for reader 1 (range 0.15–1.94 cm3, SD 0.30) and 0.52 cm3 for reader 2 (range 0.15–1.94 cm3, SD 0.41). No significant difference was found for ROI size (P = 0.72). The pharmacokinetic parameters used in the fast dynamic analysis showed a significant difference between the benign and malignant group for both readers (Table 2). The diagnostic performance of the fast dynamic analysis resulted in an AUC for Ktrans of 0.82 (95% CI = 0.735–0.905) and 0.82 (95% CI = 0.739–0.909) for reader 1 and 2. For Ve the AUC was 0.78 (95% CI = 0.682–0.873) and 0.77 (95% CI = 0.670–0.866) and for the kep parameter 0.72 (95% CI = 0.609–0.828) and 0.74 (95% CI = 0.629–0.841) for reader 1 and 2, respectively. Scatter plots of Ktrans and V displaying the parameter values of benign and malignant lesions found in the two readers are provided in Fig. 2. The comparison of the diagnostic performance from the slow dynamic analysis with the single parameter fast dynamic analysis showed no significant differences for the Ktrans and V parameter. A significant difference was found for reader 1 between the slow dynamic analysis and the kep parameter (P = 0.02) , the slow dynamic analysis showing better results. This was not found for reader 2 (P = 0.08). Table 2Mean pharmacokinetic parameter values categorized for malignant and benign lesions per reader. All parameter values proved significantly higher in the malignant group compared to the benign group (P < 0.01) Benign (n = 34)95% CIMalignant (n = 68)95% CIReader 1Ktransa1.20.9–1.42.32.1–2.6Ve41.634.9–48.363.958.6–69.1kepa3.02.7–3.33.83.5–4.0Reader 2Ktransa1.31.0–1.52.52.2–2.8Ve44.637.2–52.067.162.0–72.3kepa3.02.6–3.33.93.7–4.2a1/min.Fig. 2Scatter plots from the extracellular volume (V) versus the transfer constant (Ktrans) for reader 1 (a) and reader 2 (b). Benign and malignant cases were clustered. Clusters were summarized with an iso-probability contour computed from the cluster mean and covariance at a squared normalized radius of 2. The continuous-line ellipsoid represents the benign subgroup, the dotted-line ellipsoid represents the malignant subgroup Combining the pharmacokinetic parameters (Ktrans, Kep and V) in a multivariate analyses resulted in an AUC of 0.83 (95% CI = 0.74–0.90) and 0.83 (95% CI = 0.74–0.90) for reader 1 and 2. No significant difference was found between the multivariate fast dynamic and the slow dynamic diagnostic performance (P = 0.49 and P = 0.85). The multivariate analysis from all pharmacokinetic parameters combined with the slow dynamic analysis (combined analysis) resulted in an AUC of 0.93 (95% CI = 0.85–0.96) and 0.90 (95% CI = 0.83–0.95) for reader 1 and 2, respectively. The results from the combined analysis were significantly higher when compared with the fast dynamic analysis for both readers (P = 0.01 and P = 0.02). This was also found for the slow dynamic analysis (P = 0.02 for both readers). The ROC curves are presented in Fig. 3. Fig. 3ROC curve for reader 1 (a) and reader 2 (b) displaying the fast dynamic, slow dynamic and combined analysis. No significant differences were found between the fast and slow dynamic analysis in both readers. A significant difference was found between the slow dynamic analysis and the combined analysis for both readers (P = 0.02 for both readers). The comparison between the fast dynamic analysis and the combined analysis also resulted in a significant difference for both readers (P = 0.01 and P = 0.02). No significant differences were found between the two readers In the group of lesions of 2 cm and smaller, the slow dynamic analyses resulted in an AUC of 0.87 (95% CI = 0.75–0.95) for reader 1 and 0.79 (95% CI = 0.67-0.91) for reader 2. Overall, the fast analyses resulted in this group in an AUC of 0.83 (95% CI = 0.70–0.92) and 0.85 (95% CI = 0.72–0.93), respectively. No significant difference was found between the slow and fast dynamic analyses for both readers (P = 0.54 and P = 0.41). The combined analysis resulted in an AUC of 0.97 (95% CI = 0.88–0.99) and 0.94 (95% CI= 0.84–0.99), respectively. The results from the combined analysis were significantly higher when compared with the fast dynamic analysis for both readers (P < 0.01 and P = 0.04). This was also found when compared to the slow dynamic analysis (P = 0.03 and P < 0.01). No significant differences were found between the two readers in any of the analyses. An example of a benign and a malignant lesion is presented in Figs. 4 and 5. Fig. 4a Transverse reconstruction of the high-resolution subtraction sequence of the right breast. b Time versus relative enhancement curve of the slow dynamic series. Ktrans (c) and V (d) color overlay images of the right breast, including a scalar bar, to demonstrate the parameter values. The subtraction image shows a rounded, mostly sharp delineated lesion. The time versus signal intensity curve demonstrates a type 1 curve, indicative for a benign lesions. The readers classified this lesion as benign (BI-RADS 2) or probably benign needing follow up (BI-RADS 3) based on the slow dynamic analysis. The Ktrans and V parameter color overlays demonstrate relatively low values for both parameters (see Table 2 for comparison) indicative for a benign lesion. Histopathology proved this lesion to be a fibroadenomaFig. 5a Transverse reconstruction of the high-resolution subtraction sequence of the right breast. b Time versus relative enhancement curve of the slow dynamic series. Ktrans (c) and V (d) color overlay images of the right breast, including a scalar bar, demonstrates the parameter values. The subtraction image shows a spiculated lesion retromammillar. The time versus signal intensity curve demonstrates a type 3 curve (wash-out) suggestive for a malignancy. Both readers classified this lesion as malignant (BI-RADS 4) based on the slow dynamic analysis. The Ktrans and V parameter color overlays demonstrate high values for both parameters (see Table 2 for comparison), indicative for a malignant lesion. Histopathology proved this lesion to be an invasive ductal carcinoma Discussion In this study, we demonstrated that the pharmacokinetic parameters derived from fast dynamic scanning during initial enhancement were a valuable additional tool for the differentiation between benign and malignant breast lesions on MRI. The pharmacokinetic parameters were significantly higher for the malignant group compared with the benign lesions (Table 2). The diagnostic performance of the pharmacokinetic parameters was, compared with the results of the slow dynamic analysis, not significantly different. The combination of both methods, however, did improve the diagnostic performance significantly for both readers. These results were also found in the subgroup analysis of smaller breast lesions. The slow dynamic analysis resembles the evaluation as routinely performed in the clinical workflow in our hospital. The performance of the workstation used in the slow dynamic evaluation has already been investigated and proven by Wiener et al. [27] in the evaluation of breast lesions prior to surgical treatment. Schnall et al. [4] evaluated the performance of both dynamic and morphological features in 854 women with 995 lesions. The results of their multivariate evaluation based on both morphological and relatively slow dynamic lesion characteristics resulted in a similar diagnostic accuracy (AUC values of 0.87 and 0.88) compared with the results obtained in the slow dynamic analyses of our study (0.85 and 0.83). Our results found in the slow dynamic analysis are, therefore, considered representative for the diagnostic performance of an experienced radiologist in this group of patients. In the fast dynamic analysis, both the morphologic characteristics and slow dynamic characteristics were not included in the evaluation; instead, a quantitative analysis of pharmacokinetic parameters was used based on manual ROI placement within the lesion. In the literature, both a “hot-spot” and “whole-tumor method for ROI placement are reported [36]. In this study, we used a hot-spot method. The importance of a consistent ROI placing strategy has been described by Liney et al. [36]. In our study, both readers were instructed with a simple ROI-placing strategy placing the ROI in an area with the highest parameter values guided by color overlays. Since no significant differences were found in the ROC analyses in any of the pharmacokinetic parameters used it is assumed that the performance of the fast dynamic analysis was not negatively affected by this manual ROI selection strategy. The optimal strategy of ROI selection within a breast lesion is a subject that still needs to be further investigated; this is beyond the scope of this study. Gibbs et al. [24] found the use of quantitative pharmacokinetic parameters in the evaluation of sub 1-cm breast lesions to be beneficial. In their study of 43 women, a diagnostic accuracy of 0.92 was found combining the postcontrast images with the dynamic data in a logistic regression analysis. The exchange rate constant was found to be the best individual parameter with a diagnostic accuracy of 0.74. The Ktrans was also found to be the best individual parameter in our study with a diagnostic accuracy of 0.82. Furman-Haran et al. [18] concluded in their study of 141 lesions that the quantitative evaluation of perfusion parameters should be able to improve breast cancer diagnosis on MRI. In their study the Ktrans was also found to be the best discriminating parameter. Their analysis showed results of invasive ductal carcinoma versus fibroadenomas or fibrocystic changes. Unlike our study, the pharmacokinetic parameters used by Furman-Haran and coworkers were derived from high-resolution images with relatively a low temporal resolution of 2 min. Although our analysis used a more diverse histological distribution (Table 1) compared with the results presented by Furman-Haran et al. [18], only a relatively small number of benign lesions could be included. The diverse histological distribution also resulted in the inclusion of benign lesions that do not necessarily cause a diagnostic dilemma in daily practice. This can be seen as a limitation of our study. In the subgroup analysis of smaller lesions, a more equal distribution between benign and malignant lesions was found. The analysis performed in this subgroup also proved the additional value of the fast dynamic analysis in classifying small breast lesions on MRI. The three-time-point method used by Kelcz et al. [37] provides the reader with a composite image showing contrast uptake and wash-out characteristics related to the product of microvessel surface area and permeability, as well as to the extracellular volume fraction. In their study, the observers correctly diagnosed 27 of 31 malignant and 31 of 37 benign lesions (sensitivity 87%; specificity 84%) using the three-time-point method. The evaluation based on wash-in and wash-out curves in combination with morphology resulted in a sensitivity of 93% and a specificity of 82%. Our results not only demonstrate a similar performance of the pharmacokinetic analysis compared with the evaluation based on morphology and slow dynamics but also demonstrate the potential gain if both methods are combined. The results presented by Kelcz et al. [37] are, like other authors, again derived from high spatial resolution images with a relatively low temporal resolution of 2 min compared with our fast dynamic scanning protocol. With a scanning protocol using only the fast dynamic evaluation and morphology the scantime could be reduced significantly when compared with a protocol including the evaluation of wash-out. This without loss of diagnostic performance when compared with the results of the slow dynamic analysis in our study and the results presented by other authors [4, 12, 18]. This reduction of scantime can in the future contribute to the cost-effectiveness of MRI screening. However, since the highest diagnostic performance was obtained by combining both the fast and slow dynamic analysis, further studies are needed before the scantime can be reduced. The results presented in this study are our initial results using this scanning protocol. Therefore, no cut of values for the differentiation between benign and malignant lesions from the pharmacokinetic parameters were used in the evaluation or can be provided at this point. The results presented only show the potential of our method in differentiation between benign and malignant lesions in this group of patients. The value of our method needs to be further studied in a larger group, preferably using a more even distribution between benign and malignant cases and with lesions that can be classified on imaging as a BI-RADS 3 or higher. Unfortunately, the study design used did not allow a multivariate analysis combining the fast dynamic data with morphological characteristics. Also, the possible trade-off between the pharmacokinetic parameters based on initial enhancement and the wash-out based on late dynamic characteristics cannot be derived from these data. Both analyses will need to be performed in future projects in order to evaluate the full potential of the fast dynamic analysis as used in our study.In conclusion, pharmacokinetic parameters derived from fast dynamic imaging during initial enhancement have great potential in classifying enhancing lesions in the breast. In this study, the diagnostic performance for the fast dynamic analysis proved to be equal to the results of experienced radiologists using more common evaluation methods based on morphologic characteristics and slow dynamic enhancement characteristics. An increased diagnostic performance was found in combining both methods. This shows the additional value of this method in further improving the diagnostic accuracy of breast MRI.

Int_J_Legal_Med-3-1-1794629

Variation of 52 new Y-STR loci in the Y Chromosome Consortium worldwide panel of 76 diverse individuals

We have established 16 small multiplex reactions of two–four loci to amplify 52 recently described single-copy simple Y-STRs and typed these loci in a worldwide panel of 74 diverse men and two women. Two Y-STRs were found to be commonly multicopy in this sample set and were excluded from the study. Of the remaining 50, four (DYS481, DYS570, DYS576 and DYS643) showed higher diversities than the commonly used loci and can potentially provide increased haplotype discrimination in both forensic and anthropological work. Ten loci showed occasional missing alleles, duplicated peaks or intermediate-sized alleles. Introduction Y-STRs have key roles in the fields of forensic genetics, anthropological genetics and genealogy because of their ability to discriminate between male lineages and provide information about the relationships between them [1, 2]. The Y chromosome haplotype reference database [3] provides a widely used compilation of haplotype information constructed from a “minimal haplotype” of nine loci or a “minHt + SWGDAM core set” of 11 loci (http://www.yhrd.org/index.html). Some applications, however, require more Y-STRs. For example, a study of ∼1,000 men from east Asia found that almost 3% (27/1,003) shared the same 16-STR haplotype [4] and thus would not be distinguished by standard analyses. Most of the STRs on the Y chromosome have now been identified [5], and a set of 52 was highlighted that seemed particularly useful because their unit size was ≥3, they were single-copy, had a simple structure and showed variation in a set of eight diverse men. These additional loci proved to be useful in the east Asian study where 46 of them allowed a male lineage characteristic of the Qing Dynasty to be defined [4], but they clearly varied considerably in their diversity [4, 5] and may vary in other properties that affect their usefulness as well. In addition, it may often be impractical or impossible to type such a large number of markers. Further studies of these loci are therefore needed to identify the most useful subset. US population data for 16 of them have been presented [6], but data from other loci and populations are lacking. We have therefore established multiplex typing procedures for all of them and examined their variation in the Y Chromosome Consortium (YCC) worldwide panel of men [7]. Materials and methods The YCC panel consists of 74 male and two female DNAs; the men may be broken down into 26 from Africa, 26 from Asia and the Americas and 22 from Europe or the Middle East. In addition, the haplogroup R individual previously typed with all of the new markers [5] was included in this study to facilitate consistent allele calling. DNA was amplified before use with the GenomiPhi whole genome amplification kit (Amersham Biosciences, Amersham, UK) according to the manufacturer’s recommendations. A total of 52 polymorphic simple single-copy Y-STRs [5] were included in the present study. The published primers had been designed to operate under a common set of conditions and were therefore used in this study, except that a G was added to the 5’ end of the unlabelled primer if it was not already present to facilitate non-templated addition of an A to the labelled product strand [8]. Loci were tested in silico for potential interactions between primers using the AutoDimer software [9], and suitable sets were assembled into small multiplexes for experimental assessment resulting in 16 multiplexes each consisting of 2–4 loci (Table S1). Polymerase chain reactions (PCRs) were set up in 20 μl volumes containing 1× PCR buffer (Invitrogen, Paisley, UK), 1.75 mM MgCl2, 200 μM deoxynucleotide triphosphates (dNTPs; Amersham Biosciences), 1.0 unit of Platinum Taq DNA polymerase (5 U/μl, Invitrogen) with 10 pg–2 ng whole-genome-amplified DNA and primer pairs at the concentrations shown in Table S1. Thermal cycling was carried out in an MJ Research (Genetic Research Instrumentation, Braintree, UK) DNA Engine Tetrad™ 2 starting with denaturation at 95°C for 15 min, followed by 20 cycles of touchdown PCR: 94°C for 30 s, 70°C for 45 s, 72°C for 1 min, with a 1°C decrease in annealing temperature every cycle and then 15 cycles of standard PCR (94°C for 30 s, 50°C for 45 s, 72°C for 1 min) and finishing with extension at 60°C for 45 min and storage at 4°C. Products were analysed by mixing 1 μl of PCR product with 15 μl Hi–Di formamide and 0.2 μl size marker (CXR 60–400 bases, Promega UK, Southampton, UK) and running on 36 cm × 50 μm capillaries containing POP-4 polymer (Applied Biosystems) on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Warrington, UK). Electrophoresis was carried out at 3 kV for 3 s followed by 15 kV for 45 min with a run temperature of 60°C. Allele sizes were measured using GeneMapper v3.0 (Applied Biosystems). Most loci were sequenced because of the lack of previous sequence data, to confirm previous results or to investigate the structure of intermediate-sized sizes. Such alleles were amplified using unlabelled primers and sequenced by the Wellcome Trust Sanger Institute small-scale sequencing facility using standard methods. Results The 52 Y-STRs were examined in the 76 YCC samples and haplogroup R control individual, but the analyses presented in this paper (Tables S2, Tables S3) are based only on the YCC data to facilitate comparisons with other YCC results [10]. As expected, no specific products were obtained from the two female YCC samples in the size range examined, and single peaks were seen in all males for 40 of the STRs. The other 12 loci showed more complex patterns (Table 1). Products from four loci were missing in one (DYS525, DYS589, DYS636) or two (DYS556) individuals. These findings were reproducible and occurred in multiplex reactions that successfully amplified other loci, so that they may represent null alleles, but their structural basis remains to be determined, and they were treated conservatively as missing data in our analyses. Table 1Loci showing multiple peaks, missing peaks or intermediate allelesLocusIntermediate alleleaCommentsDYF386S1Two peaks in many individuals, excluded from analysisDYF390S1Two peaks in many individuals, excluded from analysisDYS448Two peaks in two individualsDYS52210 (U2Ains)Intermediate-sized allele. This insertion converts the reference sequence, which can be written ATAG ATG (ATAG)10 into ATAG AT A G (ATAG)10, which therefore has 12 copies of the ATAG repeat, but differs from the regular allele 12DYS525No product in one individual, two peaks in one individualDYS53111 (D6Tins)Intermediate-sized alleleDYS549Two peaks in one individualDYS556No product in two individualsDYS567Two peaks in two individualsDYS576Two peaks in two individualsDYS589No product in one individualDYS636No product in one individualU upstream, D downstream, ins insertionaNomenclature based on the standard recommendations [1] Two peaks were observed in many individuals for DYF390S1 and DYF386S1, and we interpreted these as duplicated loci that happened to have the same sized alleles in the small number of individuals examined before [5]; these two STRs were excluded from subsequent analyses. Five loci also showed two peaks of similar height in one (DYS525, DYS549) or two (DYS488, DYS567, DYS576) individuals, which may reflect rare duplications or somatic mutations in the YCC cell lines. In addition, two loci showed fragment sizes that did not fall into the expected size classes: DYS522 in one individual and DYS531 in 11 individuals corresponding precisely to haplogroup Q [7] and thus representing a variant characteristic of this haplogroup. The structural basis of these variants was determined by sequencing and found to arise from insertion events in the flanking sequences between the STRs and the primers (Table 1). Null alleles, occasional duplications and intermediate alleles have been found in the standard Y-STRs [1], and so we concluded that 50 of the 52 new Y-STRs merited further consideration as loci for wider use. We next examined the variation of these 50 STRs. The number of alleles ranged from two to 11, the diversity from 0.05 to 0.90 and the variance from 0.04 to 7.89 (Table 2). All of these characteristics were correlated, probably because of their common dependence on the repeat count. To interpret the values obtained, we have compared them with published data on the standard single-copy loci in the YCC panel [10]. Of the new loci, four (DYS481, DYS570, DYS576 and DYS643) showed higher diversity than the most variable standard locus DYS390 (diversity = 0.79) and 15 showed higher diversity than DYS393 (diversity = 0.66; Table 2). The discrimination of haplotypes that are not distinguished by the commonly used markers is a particularly useful property. As reported [10], eight pairs of YCC individuals carry haplotypes that are identical when the standard minimal set of Y-STRs is used. Two of these are from different populations (Mbuti Pygmy/Bantu speaker; English/German) and these were distinguished by seven and nine of the new loci, respectively. The other six pairs are from isolated populations, and these were distinguished by 2, 1, 1, 0, 0 and 0, respectively, of the new markers (Table S4). Although a total of 15 loci contribute to this increased discrimination, all of the five distinguishable haplotypes could be separated using just two of the most variable loci, DYS570 and DYS576. Table 2Variation of 50 new Y-STR loci in the YCC panelLocusMean repeat countNumber of allelesDiversityVarianceDYS48123.3110.907.87DYS57017.6110.863.89DYS57617.370.822.16DYS64311.190.822.33DYS48515.970.781.92DYF406S110.670.751.26DYS52211.260.741.03DYS58912.360.731.07DYS53311.260.721.03DYS54912.150.720.92DYS50512.160.711.00DYS50811.480.711.44DYS5259.970.710.95DYS53111.040.690.39DYS55611.760.671.03DYS57210.350.640.61DYS56511.560.640.71DYS5949.570.630.87DYS54011.440.620.52DYS48713.260.621.38DYS51110.950.620.73DYS5739.940.620.73DYS61712.450.621.06DYS49515.350.610.76DYS56710.460.610.72DYS49714.240.600.51DYS48813.460.580.91DYS49211.650.560.45DYS49012.970.562.43DYS56811.170.560.92DYS63611.350.560.49DYS53710.940.530.43DYS61811.840.510.37DYS63810.940.490.34DYS49112.150.460.40DYS5788.140.410.40DYS64011.230.390.39DYS47611.240.360.29DYS4949.040.350.28DYS6419.640.330.93DYS5549.050.310.36DYS57510.040.250.22DYS5838.030.220.12DYS5907.930.220.23DYS4807.930.220.16DYS56911.030.180.11DYS5309.120.170.09DYS5809.140.110.19DYS4728.020.080.04DYS5799.020.050.89Loci are ordered according to their diversity. Discussion We have investigated the properties of 52 new Y-STRs in a diverse worldwide set of males. We found that two of the Y-STRs were multicopy and thus not well suited to some applications and that the remaining 50 loci differed substantially in their properties. Our measurements of allele numbers, diversity and variance were overall consistent with the previous report [5]; correlation coefficients (R2 values) were 0.47, 0.58 and 0.67, respectively, but differed for some individual loci. The most variable Y-STR, in all respects, was DYS481, and this was not previously considered in detail because sequence data were not available before. Several other loci (e.g., DYS570, DYS576 and DYS643) may be particularly useful for increasing discrimination in forensic work, and the simple structure and mutational properties of this set make them the markers of choice for many population genetic studies. This is illustrated by considering the correlation between mean repeat count and variance in repeat number of the 50 simple loci: it was far higher (R2 = 0.67) than the value reported for complex Y-STRs (R2 = 0.34, [5]), suggesting that the simple STRs have simpler mutational mechanisms and may lead to more precise dates of lineages. The data in Table 2 and Table S3 now provide a basis for choosing the best simple loci and assembling them into a high-level multiplex reaction for more extensive population screening. Electronic supplementary material Below is the link to the electronic supplementary material. Table S1 Multiplex organization and primer concentrations (DOC 102 kb) Table S2 PCR product size range and allele range (DOC 92 kb) Table S3 Haplotypes of the YCC DNAs (XLS 58 kb) (Note: this table is provided as an Excel file and is the table mentioned in CE7. We transformed it into text because some journals insist on this, but the text version is difficult to interpret as CE5 highlights. The loci are ordered according to their positions in multiplexes 1–16 in Table S1 and Table S2 , and it seems most consistent to keep the same order for Table S3 ). Table S4 Subdivision of minimal haplotypes by new Y-STRs (DOC 87 kb)

Purinergic_Signal-3-4-2072910

Structural and functional evolution of the P2Y12-like receptor group

Metabotropic pyrimidine and purine nucleotide receptors (P2Y receptors) belong to the superfamily of G protein-coupled receptors (GPCR). They are distinguishable from adenosine receptors (P1) as they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species, and as many as eight functional subtypes have been characterized. Most recently, several members of the P2Y12-like receptor group, which includes the clopidogrel-sensitive ADP receptor P2Y12, have been deorphanized. The P2Y12-like receptor group comprises several structurally related GPCR which, however, display heterogeneous agonist specificity including nucleotides, their derivatives, and lipids. Besides the established function of P2Y12 in platelet activation, expression in macrophages, neuronal and glial cells as well as recent results from functional studies implicate that several members of this group may have specific functions in neurotransmission, inflammation, chemotaxis, and response to tissue injury. This review focuses specifically on the structure-function relation and shortly summarizes some aspects of the physiological relevance of P2Y12-like receptor members. Introduction Most cellular effects of nucleotides are mediated through two main families of specific cell surface receptors, the ionotropic P2X and metabotropic P2Y nucleotide receptors. The metabotropic receptors belong to the superfamily of G protein-coupled receptors (GPCR). The GPCR superfamily comprises at least five structurally distinct families/subfamilies (GRAFS classification) which share little sequence homology among each other [1]. The rhodopsin-like receptors (also called family A or 1) form the largest family in vertebrates. Eight different mammalian P2Y (P2Y1, 2, 4, 6, 11–14) receptor subtypes, all belonging to the rhodopsin-like receptors (family A), have been identified and proven to function as receptors for extracellular nucleotides so far [2]. Based on their preferential agonists, P2Y receptors have been subclassified into adenine nucleotide-activated (P2Y1, P2Y11, P2Y12, P2Y13), pyrimidine nucleotide-activated (P2Y4, P2Y6), ATP/UTP-activated (P2Y2), and UDP-sugar-activated (P2Y14) receptors. Most structural and functional studies were performed with the classic P2Y receptors P2Y1 and P2Y2 [3]. However, in comparison to other GPCR, large-scale structure/function data are rare, mainly due to difficulties in finding suitable P2Y-free heterologous expression systems [4]. Pharmacological characterization on both native and recombinant P2Y receptors is further hampered by the release of endogenous nucleotides and the hydrolysis or conversion of exogenous and endogenous nucleotides by ectoenzymes (for review see [5]). ATP and AMP are metabolized by cell surface enzymes, the ecto-NTPDase 1 (nucleoside triphosphate diphosphohydrolase 1, ectoapyrase, CD39, metabolizes ATP to AMP) and the 5′-ectonucleotidase (CD73, metabolizes AMP to adenosine). In addition, a nucleoside diphosphokinase catalyzes the transfer of the gamma phosphate of nucleoside triphosphates to nucleoside diphosphates. For example, in the presence of ATP, nucleoside diphosphokinase catalyzes the conversion of UDP to UTP. These specific problems may have contributed to many false and controversial declarations of P2Y receptors being activated by nucleotides [6–9]. Recent success in expressing GPCR in yeast, which is discussed below, may circumvent these problems and may engage structure-function-relation studies. Although in-depth phylogenetic analysis of GPCR groups indicates clustering of P2Y receptors, when compared with other GPCR of family A [1, 10], the adenine nucleotide-activated P2Y1 and P2Y12, for example, display only minor structural relation at the protein level. Phylogenetic analysis even revealed that nucleotide specificity e.g. for ADP evolved independently in P2Y1 and P2Y12. So we suggest that grouping of P2Y receptors should be performed on phylogenetic aspects rather than on ligand specificity. Based on their amino acid sequences P2Y receptors can be subdivided into at least two groups (Fig. 1). One comprises P2Y1, 2, 4, 6, 11, and the second group contains P2Y12–14. Additionally, several other structurally related GPCR, such as CysLT1R, GPR91, GPR99, and GPR34, cluster into both groups (Fig. 1). These receptors are activated by structurally different ligands such as leukotrienes, organic acids, and phospholipids. Probably because of their structural relation several P2Y receptors display agonist promiscuity. It was shown that not only ADP but also leukotriene E4 (LTE4) activates P2Y12 at nanomolar concentrations [11]. Cysteinyl leukotriene receptors (CysLT1R and CysLT2R) have been described to be activated by UDP in addition to activation by cysteinyl leukotrienes [12, 13]. Further, there is evidence that CysLT1R antagonists, such as pranlukast and montelukast, inhibit activation of P2Y1 and P2Y6 by 2MeS-ADP (2-methylthio-ADP) and UDP, respectively [14]. Although P2Y12 and cysteinyl leukotriene receptors are clinically used targets for prevention of thrombocyte aggregation (clopidogrel) and treatment of asthma (montelukast), respectively, the structural basis and (patho)physiological relevance of dual/multiple agonist specificity is obscure. Close structural relations between P2Y and other non-nucleotide receptors as well as the mentioned examples of ligand promiscuity indicate that nucleotides are probably only one of many natural agonists on the genuine P2Y receptors. Fig. 1Phylogenetic relation of human P2Y receptors. To evaluate the structural relation of P2Y receptors and related GPCR, amino acid sequences of human orthologs were aligned and phylogenetic relations were estimated using CLUSTAL W. The derived tree was essentially identical to former analysis [2, 10]. The P2Y12-like receptors cluster into a group (framed) that is distinct from other nucleotide receptors such as P2Y1 and P2Y2 Structural evolution of P2Y12-like GPCR Discovery and deorphanization of group members The ADP-(P2Y12)-like receptor group is the most recently identified P2Y receptor group and includes P2Y12, P2Y13, and P2Y14 (see Fig. 1, Table 1). Based on high structural similarity, the lyso-phosphatidylserine receptor (GPR34) and several orphan GPCR (GPR34-like, GPR82, GPR87, GPR171) are considered members of the structural P2Y12-like receptor group (3,11). Most members of this group were deorphanized by the so-called reverse pharmacology approach. The orphan receptors predicted from sequence data are now often used to ascertain the ligands by testing them on tissue extracts and subsequent fractionation or on huge libraries of bioactive compounds, whereas, traditionally, the bioactive ligand was used to identify the receptor (classic approach). Table 1Members of the P2Y12-like receptor familyReceptorG protein couplingNatural agonistTissue expressionP2Y12GiADP, CysLT-E4, phosphoribosyl pyrophosphatePlatelets, brain (glia) [19]P2Y13GiADP, diadenosine triphosphateSpleen and adult brain, lower expression in placenta, lung, liver, spinal cord, thymus, spleen, small intestine, uterus, stomach, testis, fetal brain, adrenal gland, lymph node, bone marrow, peripheral blood mononuclear cells, leukocytes [22, 45]P2Y14GiUDP-glucose, UDP-galactose, UDP-glucuronic acid, UDP-N-acetylglucosamineBrain (astrocytes, glia), placenta, adipose tissue, stomach, intestine, spleen, lung (epithelium), heart [15, 16, 71]GPR87Gin.kPlacenta, thymus, small intestine, colon, prostate, uterus, testis, peripheral blood leukocytes [28, 29]GPR171n.kn.kn.kGPR34Gilyso-phosphatidylserineBrain, glia cells, mast cells, spleen, heart, kidney, liver [27, 31, 34, 72]GPR82n.kn.kTestes, epididymis (unpublished own results)Based on phylogenetic analyses and structural similarities the seven GPCR clusters in a group which is distinct from other P2Y receptors (see Fig. 1). Although they share structural features it appears from current studies that not all members are receptors for nucleotides. The table lists the currently known receptor agonists, the receptor’s G protein specificity and expression patternn.k not known As the first member of the P2Y12-like receptor group P2Y14 (synonyms KIAA0001, GPR105, VTR 15–20) was identified [15, 16] and then deorphanized in the year 2000 [17]. UDP-glucose was found to activate P2Y14 by screening multiple receptors, each expressed in individual yeast strains, against a large library of over 700 known and putative natural GPCR agonists. In 2001, P2Y12 (initially named SP1999) was discovered and identified as the platelet clopidogrel-sensitive ADP receptor by a number of groups [18–21]. The P2Y12 was deorphanized with both the classic and the reverse pharmacology approaches. Hollopeter et al. used subfractionated transcripts from platelets and isolated the cRNA (complementary RNA, encoding e.g. the P2Y12 receptor) which mediated ADP-induced increases in K+ current after injection into Xenopus oocytes [19]. In contrast, fractionated rat spinal cord extracts were assayed for Ca2+ mobilization in cells transiently transfected with P2Y12 and chimeric Gα subunit [21]. Almost in parallel, ADP was shown to be an agonist for P2Y13 (former GPR86), a very close structural neighbor of P2Y12 [22–24]. 2MeS-ADP is the most potent agonist at P2Y12 followed by ADP, adenosine 5′-O-2-thiodiphosphate, and ATP. (N)-methanocarba-2-methylthio-ADP, a highly potent agonist at P2Y1, exhibited no and only minor agonist activity at P2Y12 and P2Y13, respectively [25]. Based on structural relations GPR34 [26, 27], GPR82 [28], GPR87 [29], and GPR171 (synonym H963) [30] also belong to the P2Y12-like group (see Fig. 1). Except for GPR34, which was recently identified as a receptor for lyso-phosphatidylserine [31] by a reverse pharmacology approach, all the other P2Y12-like receptors are still orphan GPCR. The evolutionary origins Based on structural similarities P2Y12-like receptors cluster into a group distinct from other P2Y receptors (see Figs. 1 and 2). This P2Y12-like receptor group can be subdivided into two subgroups. One subgroup encompasses P2Y12, P2Y13, P2Y14, GPR87, and GPR171, the other subgroup GPR34, GPR34-like, and GPR82 (Fig. 2). This may suggest that both subgroups evolved from gene duplications starting from two related members. The grouping is further supported by shared genomic localization. Chromosomal clustering in the human genome is found for P2Y12, P2Y13, P2Y14, GPR87, and GPR171 at 3q24-3q25 as well as for GPR34 and GPR82 at Xp11.4. This clustering is evolutionary well preserved. In the zebrafish genome P2Y12, P2Y14/GPR87, and GPR171 cluster at chromosome 15 and GPR34 type 2 and GPR82 are tandemly arranged at chromosome 9. Gene clustering is not only found for vertebrate P2Y12-like receptors but also for several other GPCR genes. Multiple copies of related GPCR genes, such as the human protease-activated receptors on 5q13, trace amine-associated receptors (TAAR, human 6q23.2), and CC-chemokine receptors (human 3p21.3), are the result of intrachromosomal gene duplications. The most impressive example is found in odorant receptors, where in humans, chromosome 11 contains nearly half of the odorant receptor repertoire, including a single cluster of more than 100 odorant receptors [32]. Gene amplification can be viewed as a dynamic and reversible regulatory mechanism that facilitates adaptation to variable environments. Clustered genes may confer selective benefits via their ability to be co-regulated and co-amplified. Indeed, even transcripts encoding a fusion protein of GPR34 and GPR82 have been observed (own unpublished results). However, the biological relevance of P2Y12-like receptor clustering in respect to transcriptional activity at their respective genomic loci, co-regulation, and transcript diversity need to be determined in the future. Fig. 2Evolutionary origin of P2Y12-like receptors. To subclassify and evaluate the phylogenetic relations between the P2Y12-like receptors, the amino acid sequences of orthologs from one species of each vertebrate class (if available) were aligned using CLUSTAL W (identity matrices) and a phylogenetic tree was constructed (1,000 iterations). Bootstrap values >600 were considered as significant to support a new branch Since P2Y12, P2Y14/GPR87, GPR34, and GPR34-like are all found in sharks (Mustelus manazo, Carcharodon carcharias) and chimeras (Callorhinchus milii) one can assume that P2Y12-like receptors arose more than 450 million years (Myr) ago, before cartilaginous and bony fish split. Our own polymerase chain reaction (PCR) amplification attempts as well as the ongoing lamprey (Petromyzon marina) and sea urchin (Strongylocentrotus purpuratus) genome projects revealed no P2Y12-like sequences yet, suggesting their origin in the very early Gnathostomata or, less likely, a loss in agnate and all non-vertebrates. Gene duplication can be a primary source of the genetic material from which genes with new functions evolve. One copy of a duplicated gene may become mutated and acquire unique functionality without risking the fitness of the organism ensured by the homolog. On the other hand, if not advantageous, continuous accumulation of mutations (neutral drift) will eliminate one of the genes, a process named pseudogenization. In fish genomes, there is only one respective ortholog with relation to P2Y12/P2Y13 and to GPR87/P2Y14. In the case of P2Y12/P2Y13 the fish ortholog is more closely related to P2Y12 implicating that P2Y13 evolved later in tetrapod evolution probably by P2Y12 duplication. However, one cannot rule out that the gene was eliminated in the common ancestor of sharks and bony fishes. The evolutionary mechanism of P2Y14 and GPR87 evolvement remains unsolved since their structural relation to the fish orthologs is similar. The absence of P2Y14 in the African craw frog (Xenopus) genome but the presence of ortholog sequences in all other sequenced tetrapod genomes does not necessarily implicate that P2Y14 derived from GPR87 in early tetrapod evolution. Again, P2Y14 gene loss in amphibians must be considered. Orthologs of GPR34, GPR87/P2Y14, and P2Y12 are found in all tetrapods and bony and cartilaginous fishes investigated so far. However, not all members of the P2Y12-like group appear to be present in all vertebrate classes. GPR82 has an ortholog in zebrafish but is not yet found in the genomes of pufferfishes and the lizard (Anolis carolinensis) genome. Although GPR171 is found in reptiles, birds, mammals, zebrafish, and chimera it appears to be absent in African craw frog, stickleback, and pufferfishes. This may implicate specific functions of some P2Y12-like receptors which are less important in some species or are compensated by other receptors and mechanisms. Signatures of gene inactivation (pseudogenization) are rarely found for members of the P2Y12-like group. Following gene duplication in the common ancestor of evolutionary basic fishes, like eels and carps [33], pseudogenization of one GPR34 subtype was identified in a salmon species (Keta salmon) (own unpublished results). The GPR34-like receptor is an evolutionary old P2Y12-like receptor being present in cartilaginous and bony fishes, amphibians, and birds but not in mammals (Fig. 2). Sequences with residual relation to the GPR34-like receptor are found in the platypus genome but it is most likely a pseudogene. Genomic organization of P2Y12-like receptors Comparison of transcript and genomic sequences provides information on intron/exon structure of a GPCR gene. Since the gain or loss of spliceosomal introns are unique events in evolution, they can serve as markers for phylogenetic analysis. Further, such analyses may reveal splice variants and may be informative about the promoter structure and gene regulation. Introns are the basis of alternative splicing, exon skipping, and RNA editing events and, therefore, can contribute to receptor diversity at a supragenomic level. Most coding regions of human P2Y12-like receptors do not contain introns (Table 2). One exception is GPR87 where the genomic sequence encoding the receptor’s N terminus is interrupted by an intron in mammalian and avian orthologs. Another intron which is rarely removed (cryptic intron) has been identified in the N terminus-encoding sequence of GPR34 [34]. Table 2Genomic organization of P2Y12-like GPCR. n.a. not analyzed because of lack of mRNA informationMemberNumber of introns in the 5′ non-coding regionNumber of introns in the coding regionApprox. size of genomic regionaP2Y12 Human1–2−47 kbp (2 different transcript starts) Mouse4−46.5 kbp Chickenn.a.−bn.a. (>1 kbp) African clawed frog−1 (N terminus)4 kbp Zebrafishn.a.−bn.a. (>1 kbp) Stickleback11 (TMD2)2 kbp Pufferfishn.a.1 (TMD2)>2 kbpP2Y13 Human−1 (rare transcript, N terminus, NM_176894) 3.2 kp (2 different transcript starts) Mouse1−3 kbp Chickenn.a.−bn.a. (>1 kbp) African clawed frog−1 (N terminus)14–19 kbpP2Y14 Human2−66 kbp (2 different transcript starts) Mouse1–2−16 kbp Chickenn.a.−bn.a. (>1 kbp)GPR87 Human11 (N terminus)22.7 kbp Mouse11 (N terminus)16 kbp Chicken21 (N terminus)9.5 kbp African clawed frogn.a.−bn.a. (>1 kbp)P2Y14/GPR87 Zebrafishn.a.−bn.a. (>1 kbp) Pufferfishn.a.−bn.a. (>1 kbp)GPR171 Human2−5 kbp Mouse1−4.5 kbp Chicken1−2.3 kbp Zebrafishn.a.−bn.a. (>1 kbp)GPR82 Human2−4 kbp Mouse2–3−6 kbp Chickenn.a.−bn.a. (>1 kbp) African clawed frogn.a.−bn.a. (>1 kbp) Zebrafishn.a.−bn.a. (>1 kbp)GPR34 Human3–41 cryptic (N terminus)8.2 kbp Mouse3–4−9.1 kbp Chicken1−2.5 kbp African clawed frog2−5.2 kbp Zebrafish  Type 11−7.3 kbp  Type 2−−1.2 kbp Pufferfishn.a.−bn.a. (>1 kbp)Except for GPR87, human P2Y12-like receptor members contain no intron in the coding region. However, this intronless gene structure is not well preserved in all vertebrates. The table summarizes the number of introns within the 5′ and coding region of selected vertebrate genes and estimates the size of the geneaBased on the 5′ longest transcriptbComplete open reading frame within the genomic sequence but intron within the very N terminus cannot be ruled out because of lack of mRNA information The genomic organization of the individual P2Y12-like receptor genes is not well conserved during vertebrate evolution (see Table 2). For example, the P2Y12 gene gained an intron in bony fish evolution disrupting the open reading frame of the transmembrane domain 2 (TMD2)-encoding part. Similarly, one of the two GPR34 paralog genes acquired an intron in the more recent bony fish evolution [33]. The two long-standing alternative explanations for the origin of introns, the intron-early theory and intron-late theory, remain a matter of continuous debate not only for GPCR [35]. The intron-early theory suggests that introns are extremely ancient characteristics of genes and that early genes were created through the intron-mediated shuffling of exons. However, numerous gene and genome comparison studies provided evidence that at least some introns are more recently acquired (intron-late theory). The P2Y12-like receptor group is, therefore, a nice example where introns were acquired in the coding region in some species during more recent evolution. In contrast to the coding region, the 5′ region of most members of the P2Y12-like receptor group displays a distinct intron/exon organization with sometimes multiple transcription starts. Also, the gene sizes strongly differ between the individual and even closely related receptors. Introns in the 3′-untranslated region (UTR) of P2Y12-like receptors have not been found yet. Large and complex organized 5′ non-coding regions of a gene may provide the basis for multiple promoter regions, cis-acting elements, and a variable 5′ UTR of the mRNA. Alternative structures of the 5′ UTR can contribute to expression regulation and alternative translation start points. For example, 5′ UTRs often contain small open reading frames (ORF) which can be translated via leaky scanning at the ribosome. Such leaky scanning can reduce translation of the downstream main ORF as shown for several genes including GPCR [36, 37]. It is of interest to note that both genes, GPR34 and GPR82, are located in antisense orientation within a large intron of the CASK gene. This position is conserved during vertebrate evolution. The CASK gene encodes a calcium/calmodulin-dependent serine protein kinase that is a member of the membrane-associated guanylate kinase (MAGUK) protein family. Since GPR34 and GPR82 transcripts are antisense orientated to CASK one can speculate that transcripts may regulate expression of CASK or vice versa. Such hypotheses will be addressed in receptor-deficient mouse models (see below) in the future. Key residues defining the individual member Many attempts have been made to identify structural signatures which are helpful in annotation and grouping of P2Y receptors. On the basis of available sequence data for validated P2Y receptors, key residues were extracted to define P2Y12-like and P2Y1-like receptor groups [11, 38, 39]. In recent studies, we have shown for several GPCR that a significant number of orthologs is required for identification of functional motifs and key residues [33, 40–42]. By mining public databases and by amplifying and sequencing P2Y12-like orthologs we have acquired large sets of sequences of P2Y12 (74 orthologs), P2Y13 (31 orthologs), P2Y14 (38 orthologs), GPR87 (51 orthologs), GPR171 (41 orthologs), GPR82 (34 orthologs), and GPR34 (133 orthologs) to determine structural conservation of the members and to identify amino acid sequence motifs and key residues that are unique for the P2Y12-like receptor group and the individual members (Fig. 3). These data sets were obtained from vertebrate species ranging from evolutionary old bony and cartilaginous fishes to the more modern mammals representing 450 Myr of evolution. The overall identity between P2Y12-like members is rather low ranging from 19% amino acid identity (human GPR82 vs human GPR87) to 47% (human P2Y12 and human P2Y13). Between the respective fish and mammal receptor orthologs P2Y12 (~49%) shows the highest identity followed by GPR171 (~48%), GPR34 (~40%), and GPR82 (~35%). Since not all P2Y12-like GPCR have orthologs in all vertebrate classes, we analyzed the structural conservation within this receptor group by comparing Ka/Ks values (ratio of the number of nonsynonymous substitutions per nonsynonymous site and the number of synonymous—or silent—substitutions per synonymous—or silent—site) of the P2Y12-like receptor ortholog set from species containing all group members (Table 3). Although all P2Y12-like receptors display a purifying selection mode of evolution (Ka/Ks <<1 indicates high conservation and elimination of deleterious mutations), there are significant differences between the members of this receptor group. P2Y12 and GPR87 were kept most conserved during evolution in birds and mammals. By contrast, GPR82 appears to be less constrained as already indicated by a relatively low conservation at the amino acid level (see above). Fig. 3Conserved residues in P2Y12-like receptors. To identify conserved group and member-specific positions the amino acid sequences of P2Y12 (74 orthologs), P2Y13 (31 orthologs), P2Y14 (38 orthologs), GPR87 (51 orthologs), GPR171 (41 orthologs), GPR82 (34 orthologs), and GPR34 (133 orthologs) were aligned using CLUSTAL W. Residues that are 100% conserved among the respective orthologs were boxed. Only a few positions are almost fully conserved among all members of the P2Y12-like group (position number refers to the relative numbering system by Ballesteros and Weinstein [73]): TMD1: Phe/Tyr1.39, Phe1.57; TMD2: Leu2.43, Asn/Asp2.50, Pro/Ala2.58; TMD3: Tyr/His3.33, Arg/Gln3.50; TMD4: Trp4.50; TMD6: Cys/Ser6.47; Pro6.50; TMD7: Asp7.49, Pro7.50, and the two Cys residues bridging extracellular loops 1 and 2. The approximate positions of the seven transmembrane domains (TMD) are given above the sequencesTable 3Sequence conservation of P2Y12-like GPCRReceptorKa/KsPi(mean ± SD)(mean ± SD)P2Y120.049 ± 0.0180.145 ± 0.022P2Y130.141 ± 0.0700.187 ± 0.023P2Y140.103 ± 0.0360.190 ± 0.024GPR870.046 ± 0.0270.134 ± 0.018GPR1710.078 ± 0.0120.158 ± 0.021GPR340.082 ± 0.0370.137 ± 0.026GPR820.202 ± 0.0490.167 ± 0.034To compare the sequence conservation of P2Y12-like receptors, ortholog sequences [relative positions 1.48 (in TMD1) to 7.68 (in C-term)] from 18 species were aligned and DNA polymorphism analyses were performed using DnaSP (version 4.1). The Ka/Ks ratio is calculated from the number of nonsynonymous substitutions per nonsynonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks) for any pair of sequences. The nucleotide diversity (Pi) is the average number of nucleotide differences per site between two sequences. The orthologs of the following species were analyzed because their genome contained all members of the P2Y12-like group: Bos taurus, Equus caballus, Canis familiaris, Pteropus vampyrus, Ornithorhynchus anatinus, Monodelphis domestica, Callithrix jacchus, Pan troglodytes, Homo sapiens, Macaca mulatta, Microcebus murinus, Pongo pygmaeus, Tarsius syrichta, Cavia porcellus, Mus musculus, Rattus norvegicus, Tursiops truncates, Gallus gallus As shown in Fig. 3, members of the P2Y12-like group share only eight fully conserved residues (Phe1.57, Leu2.43, Trp4.50, Pro6.50, Asp7.49, Pro7.50, and both Cys residues bridging extracellular loops 1 and 2) when sequences of more than 400 receptors of this group are compared. Although the ADP receptors P2Y12 and P2Y13 share more than 50 fully conserved residues none of these conserved residues is exclusively found in P2Y12 and P2Y13 but rather present also in other P2Y12-like receptors (see Fig. 3). Further, only a few residues are member specifically conserved (GPR171: Gln3.53 and Asn5.59; P2Y13: Met7.48; GPR82: Leu6.44 and Asp7.57; GPR34: Met7.52) and have not been found in other P2Y12-like receptors so far. These facts suggest that ligand and signaling specificity is determined by a combination of many, more or less conserved determinants. It has been proposed that His6.52/Arg6.55 and Lys7.35/Glu7.36/Leu7.39 within TMD6 and the ECL3, respectively, may present such motifs required for nucleotide binding [38]. However, such residue combination is also present in some GPR87, GPR171, and GPR34 orthologs which are not activated by ADP. This does not rule out that these residues are involved in nucleotide binding of e.g. P2Y12 but it implicates additional positions which determine ligand specificity. In-depth structure-function analysis e.g. by mutagenesis studies are required to identify key positions and their structural properties. Physiological relevance of P2Y12-like receptors Functional characterization of P2Y12-like receptors in heterologous expression systems Signal transduction of the ADP receptor P2Y12 via pertussis toxin-sensitive Gi proteins and adenylyl cyclase inhibition is well established [22]. Similar Gi protein-coupling specificity was found for P2Y13 [22, 24], P2Y14 [43], and lyso-PS receptor GPR34 [31, 33]. Because adenylyl cyclase inhibition assays are usually less sensitive and robust several other experimental setups were established to measure function of P2Y12-like GPCR. It has been shown that Gα15 and Gα16 can be activated by a wide variety of GPCR [44]. The ability of Gα16 to bypass the selectivity of receptor/G protein interaction was also useful to measure activation of P2Y13 [45]. It has been demonstrated that replacement of the four or five C-terminal amino acids of Gαq with the corresponding Gαi residues (referred to as GΔ6qi4 [46]) confers the ability to stimulate the PLC-β pathway onto Gi-coupled receptors [47]. Successful heterologous expression and activation by applying chimeric Gαqi4 has been demonstrated for P2Y12–14 and lyso-PS receptor GPR34 [25, 33, 48]. We have previously shown that P2Y12 as well as the lyso-PS receptor GPR34 display increased basal activity in functional assays when compared with other GPCR [33]. This high basal activity can be either discussed as the natural ground state of the receptor activity equilibrium or as the effect of receptor agonists that are present in the cell culture medium or that are released from cells. Analyzing mutations in the highly conserved DRY motif of P2Y12 we recently showed that basal activity is abolished in the DHY mutant but agonist-induced activation remains intact (Fig. 4). We conclude that the basal activity of the ADP receptor is rather a genuine property of this receptor and not due to continuous stimulation by agonists. High basal activity appears to be a general feature of all members of the P2Y12-like receptor group. In our initial ortholog screen we identified two GPR87/P2Y14 receptors, carp GPR87/P2Y14 types 1a and 1b (AY241103, AY241102), which differ in only ten amino acid positions. Interestingly, carp GPR87/P2Y14 type 1b displays higher basal activity and studies are ongoing to identify residues that promote constitutive activity. Fig. 4High basal activity is a genuine property of P2Y12. Basal activity of the wild-type P2Y12 (DRY motif) and a mutant P2Y12 (DHR motif) was determined in transiently transfected COS-7 cells. Gi coupling of P2Y12 was rerouted to IP production by co-transfection of a chimeric G protein GΔ6qi4 [46] Because many standard mammalian expression systems endogenously express P2Y receptors clear-cut functional studies are difficult to perform. One exception is the 1321N1 human astrocytoma cell line which does not express P2Y receptors. However, transient expression of GPCR in 1321N1 cells is limited by low transfection efficiency and stable transfection of these cells is usually required. Heterologous expression of GPCR in yeast was initially established for large-scale purification of receptor proteins. These advances in the expression of heterologous GPCR in the yeast Saccharomyces cerevisiae have led to the development of sensitive and selective assays of ligand-induced GPCR activation (reviewed in [49]). To facilitate a more systematic genetic analysis of GPCR function, e.g., by saturating random mutagenesis, we took advantage of a yeast expression system in which parts of the mammalian GPCR signaling system (GPCR and chimeric G protein) are linked to a modified yeast pheromone pathway (Fig. 5) [50]. The coupling of receptor activity to the genetically engineered yeast pathway allows for rapid and economical screening of substance libraries and randomly modified receptor libraries. Functional studies in yeast may have an advantage especially for P2Y receptors because one can (at least partially) circumvent specific problems in working with nucleotide receptors (see above). Only two NTPDases (GDA1 and YND1/APY1) have been found within the entire yeast genome which are mainly expressed in the Golgi apparatus [51]. Although conversion of nucleotides by nucleotidases and ectoapyrases occur also in yeast and one cannot exclude conversion of extracellular nucleotides by yeast enzymes, there is no endogenous plasma membrane P2Y receptor in yeast which can mediate and, therefore, interfere with transmembrane signaling of heterologously expressed P2Y receptors in yeast. We and others have successfully used this system for expression and functional study of P2Y12 [52], P2Y14 [53], and lyso-PS receptor GPR34 (unpublished data). As shown in Fig. 6a, P2Y12-expressing yeast cells grow only in the presence of the agonist 2-methylthioadenosine 5′-diphosphate (2MeS-ADP). Fig. 5Functional expression and in vitro evolution of GPCR in yeast. Genetically modified yeast cells are transformed with a mammalian GPCR. Following agonist activation and constitutive receptor activity, the receptor couples to a chimeric G protein [backbone yeast G protein Gpa1 in which the C-terminal five amino acids were replaced by the respective mammalian G protein sequence (e.g., from the Gi protein)]. Activation of the chimeric G protein enables yeast cells to grow on histidine-free medium by utilizing parts of the yeast mating pathway [49]. Gpa yeast ortholog of the mammalian G protein alpha subunit, Ste4 yeast ortholog of the mammalian G protein beta subunit, Ste18 yeast ortholog of the mammalian G protein gamma subunit, Ste12 transcription factor that is activated by a MAP kinase signaling cascade, Far1 cell cycle regulator that directly inhibits the yeast cyclin-dependent kinase Cdc28-ClnFig. 6Functional expression and random mutagenesis of P2Y12 in yeast. a The human P2Y12 receptor was transformed into modified yeast (see Fig. 4). The agonist 2-methylthioadenosine 5′-diphosphate (2MeS-ADP) induces a robust yeast cell growth. b The entire coding region of the human P2Y12 was subjected to random mutagenesis and transformed yeast cells were selected for growth in agonist-free U−/H−medium. Colonies that grow under this condition contain a constitutively active P2Y12. Exemplarily, mutations of a triple mutant (L115Q/F177S/R224G) were individualized and tested separately for constitutive activity. The data indicate that L115Q mainly contributes to the constitutive activity of the triple mutant Constitutively active GPCR are useful tools in studying the action of inverse agonists and activation mechanisms in GPCR. A constitutively active P2Y12 was generated by replacing the endogenous C terminus with the corresponding part of the human P2Y1 receptor [54]. Pharmacological evaluation of several P2Y12 antagonists revealed AR-C78511 (an adenosine derivative) as a potent P2Y12 inverse agonist. Traditional mutagenesis approaches are limited to screen for activating mutations because the number of mutant proteins that can be investigated is usually relatively small, primarily because of technical reasons and the time and costs needed to generate and analyze large numbers of mutant receptors. To circumvent these limitations, yeast has emerged as a highly useful host for the in vivo reconstitution of mammalian GPCR. Therefore, we applied a random mutagenesis and screening approach in yeast to identify key residues in maintaining ground stage of the human P2Y12. PCR-based random mutagenesis was optimized to induce approximately four mutations in 1 kbp. Several dozen clones were selected and are now under in-depth investigation. For example, we identified a triple mutant P2Y12 (Leu115Gln/Phe177Ser/Arg224Gly), and individual characterization revealed Leu115Gln (TMD3) to be mainly responsible for constitutive activity in the yeast expression system (Fig. 6b). These promising results in applying the yeast expression system await further efforts to identify functionally relevant determinants in P2Y12-like receptors. Establishing in vivo function of P2Y12-like receptors using mouse models Selective receptor ligand and receptor-deficient animal models are suitable tools to evaluate the physiological relevance of distinct GPCR. Enormous efforts have been undertaken in the development of selective and clinically useful ADP and other nucleotide receptor ligands. But except for P2Y12, dissection of the physiological relevance of all other P2Y12-like GPCR is at the very early stage. Since the pharmacological properties of ligands have been reviewed in detail elsewhere [2] we only shortly summarize available data on receptor-deficient animal models. Based on the clinical success of irreversibly bound P2Y12 antagonists, such as clopidogrel, the pivotal role of ADP in arterial thrombogenesis is well established. Although the combined action of P2Y1 and P2Y12 is necessary for the full platelet aggregation response to ADP, mice deficient for P2Y12 already display reduced platelet adhesion/activation, thrombus growth, and stability [18, 55]. Except for the altered platelet functionality P2Y12-deficient mice appear normal under standard laboratory conditions. In consent with this finding, we occasionally identified a frameshift (T insertion at base pair position 667) in the P2Y12 coding sequence of several individuals of the Asian house mouse (inbred laboratory strain of Mus musculus castaneus from of the Laboratoire Génome Populations Interactions Adaptation at the Universite Montpellier, France) during ortholog screening which showed no obvious phenotype in standard laboratory captivity. The platelet function was, however, not tested. The identified P2Y12 inactivation in some Asian house mice may reflect either natural polymorphisms present in the wild population or a new polymorphism that has been introduced during captivity. To distinguish between these possibilities, we analyzed the position 667 in P2Y12 from 88 wild M. m. castaneus trapped in Taiwan [41]. All contained the intact P2Y12 allele indicating that the inactive P2Y12 allele is very rare or absent in this wild M. m. castaneus population and favors the hypothesis of its acquisition in captivity (unpublished observations). The fact that receptor deficiency is per se compatible with viability and fertility appears to be true also for other P2Y12-like receptor-deficient species and mouse models. For example, mice individually lacking GPR82 and GPR34 are viable, fertile, and produced viable offspring (own unpublished observation). Further, there are several vertebrate classes and species naturally deficient in distinct P2Y12-like receptors. For example, P2Y13 is absent in fish genomes sequenced so far, GPR82 is not present in pufferfish genomes, and GPR171 is absent in pufferfish and Xenopus genomes. It appears that many functions of P2Y12-like receptors are more distinct and their disclosure requires specific challenging conditions. Indeed, P2Y12-deficient mice revealed an unexpected phenotype when specifically challenged. CNS injury is accompanied by release of nucleotides, serving as signals for microglial activation or chemotaxis. Microglia cells express several purinoceptors, including P2Y12. Microglia in P2Y12-deficient mice showed significantly diminished directional branch extension toward sites of cortical damage in the living mouse. These results imply that P2Y12 is a primary site at which nucleotides act to induce microglial chemotaxis at early stages of the response to local CNS injury [56]. Variants of P2Y12-like receptor genes within human populations Activating and inactivating mutations in GPCR have been made responsible for more than 30 different human diseases [57]. As expected from its pivotal role in platelet activation inactivating mutations in P2Y12 can cause a congenital bleeding disorder. Only a few missense (Met1Arg, Pro258Thr, Arg256Gln, Arg265Trp) and frameshifting (frameshift at amino acid position 240) mutations have been reported [19, 58–60]. Unexceptionally, the missense mutations found are at highly conserved positions and cause an impairment of receptor function. There are no other human diseases identified yet which are associated with dysfunction of P2Y12-like receptors. The antithrombotic effect of clopidogrel is considerably variable and the P2Y12 gene was screened for possible sequence variants. Five nucleotide variations were found in the human P2Y12 gene, two of them silent substitutions in the coding region [61]. Several studies were initiated to investigate the impact of P2Y12 polymorphisms on atherosclerosis [62, 63] and clopidogrel efficiency in preventing neurological events [64] showing no or some association to one haplotype. In single nucleotide polymorphism (SNP) projects, such as HapMap [65], SeattleSNP etc., silent variations have been identified at amino acid position Val4, Asn6, Gly12, and Phe182 of P2Y12. For example the C/T variation at amino acid position Phe182 is only found in the African population whereas the G/T variation at position Gly12 is present only in European and Asian populations. One missense mutation (Glu330Gly; receptor C terminus) has been detected in SNP projects. This variant is absent in European and Asian populations but displays an allelic frequency of about 15% in the African population. The functional relevance of this missense mutation has not been studied yet but a Gly at position 330 is naturally found in many other P2Y12 orthologs including primate P2Y12 almost ruling out a specific input in receptor function. One silent variant (C/T at Ile59) and one substituting variant (T/C at Met158Thr) were identified in the coding region of P2Y13. The Thr158 variant is less frequent in African populations (~4%) when compared to European (~21%) and Asian (~18%) populations. Met158 is not conserved and a Thr at this position is found in the dolphin (Tursiops truncates). Only two silent substitutions (A/G at Ala35 and T/C at Phe240) have been identified in P2Y14 so far. The GPR171 gene contains two more frequent silent substitutions (T/C at Tyr19, A/C Thr58) and one missense variant (A/G at Ile283Val, frequency about 6%) which is located within the DPXXY motif of TMD7. Ile283 is quite conserved among vertebrate GPR171 orthologs. The functional relevance of Val283 has not been studied yet but it occurs naturally in the zebrafish ortholog. In the human GPR87 gene three silent polymorphisms (G/A at Pro46, G/A at Leu179, C/T at Tyr355) and one nonsynonymous polymorphism (C/T at Thr205Met) are present. The position 205 is not conserved and Met205 is found in several rodents. Only rare silent substitutions in the coding regions have been found in GPR82 (C/T at Asp313) and in GPR34 (A/G at Val296) [34]. SNP stochastically occur in individual genomes and can amount to a reasonable frequency in populations by drift but also by selection. There are several approaches and methods which are suitable to distinguish between drift and selection. Population genetic models predict that selection can leave “footprints” in closely linked genomic regions. Several methods were developed to detect signatures of selective sweeps in genomic sequences [66]. All methods require data on allele variation (mainly SNP) within populations. Large genome-wide analyses have scanned the human genome for signatures of positive selection on the basis of nonsynonymous and synonymous substitution ratios or single nucleotide polymorphism (SNP) data. Several loci which contain GPCR genes have been identified using such methods, but the P2Y12-like receptor-containing loci showed no strong signatures of recent positive selection in these studies [67–70]. Conclusion P2Y12-like receptors which are grouped mainly by phylogenetic relations have been an inherent part of the vertebrate GPCR repertoire since more than 450 Myr. Although sharing features in respect to structural determinants and signal transduction the activating ligands are heterogeneous such as nucleotides, nucleotide derivatives, leukotrienes, and phospholipids. We are at the very beginning of understanding the physiological importance of the individual members. The nature of the ligands, first functional data, and expression of several members in migrating cells point at functions in immunologic response and tissue damage response. Doubtless, upcoming receptor-deficient mouse models and selective receptor ligands will help to unveil the functions of P2Y12-like receptor members. Further, one should consider combined receptor-deficient mouse models within this group but also with other P2Y receptors to uncover phenotypes which are hidden by receptor redundancy, e.g., in the case of ADP receptors.

Breast_Cancer_Res_Treat-3-1-2001224

Letrozole in the extended adjuvant setting: MA.17

Relapse after completing adjuvant tamoxifen therapy is a persistent threat for women with hormone-responsive breast cancer. Third-generation aromatase inhibitors, such as letrozole, provide a new option for extended adjuvant hormonal therapy after 5 years of tamoxifen. MA.17 was conducted to determine whether letrozole improves outcome after discontinuation of tamoxifen. Postmenopausal women with hormone receptor-positive breast cancer (N = 5,187) were randomized to letrozole 2.5 mg or placebo once daily for 5 years. At a median follow-up of 30 months, letrozole significantly improved disease-free survival (DFS; P < 0.001), the primary end point, compared with placebo (hazard ratio [HR] for recurrence or contralateral breast cancer 0.58; 95% confidence interval [CI] 0.45, 0.76] P < 0.001). Furthermore, letrozole significantly improved distant DFS (HR = 0.60; 95% CI 0.43, 0.84; P = 0.002) and, in women with node-positive tumors, overall survival (HR = 0.61; 95% CI 0.38, 0.98; P = 0.04). Clinical benefits, including an overall survival advantage, were also seen in women who crossed over from placebo to letrozole after unblinding, indicating that tumors remain sensitive to hormone therapy despite a prolonged period since discontinuation of tamoxifen. The efficacy and safety of letrozole therapy beyond 5 years is being assessed in a re-randomization study, following the emergence of new data suggesting that clinical benefit correlates with the duration of letrozole. MA.17 showed that letrozole is extremely well-tolerated relative to placebo. Letrozole should be considered for all women completing tamoxifen; new results from the post-unblinding analysis suggest that letrozole treatment should also be considered for all disease-free women for periods up to 5 years following completion of adjuvant tamoxifen. Introduction and rationale There is a persistent risk of breast cancer recurrence following primary treatment [1–3]. Initially, patients with hormone receptor-positive (HR+) breast tumors have a lower risk of recurrence than those with HR− tumors, but with longer follow-up, the opposite may be the case [3, 4]. For example, Saphner showed that the significantly higher hazard of recurrence in HR− versus HR+ patients in the time period 0–12 years (P < 0.00001) could be explained by the higher risk of recurrence in years 0–5 for HR− patients (P < 0.0001). However, between years 3 and 4, the hazard of recurrence for HR− and HR+ patients crossed, and beyond 5 years was actually higher for HR+ patients (P = 0.00002) [4]. These data clearly indicate the need for continuous hormonal treatment for women with HR+ tumors. The benefits of adjuvant hormonal treatment with tamoxifen were first demonstrated in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14 trial [5]. This large randomized, double-blind, placebo-controlled trial involving patients with node-negative, HR+ breast cancer demonstrated a significant prolongation of disease-free survival (DFS) among women treated with tamoxifen for 5 years, as compared with those receiving placebo. Updated results with longer follow-up demonstrated that the 5-year benefit in DFS with tamoxifen persisted through at least 10 years of follow-up, and a statistically significant survival benefit was also observed [6]. However, since the optimal duration of tamoxifen therapy was not known, patients who had completed 5 years of tamoxifen therapy and were disease-free were re-randomized to receive placebo or tamoxifen. Results published with a follow-up of 7 years after reassignment demonstrated a disadvantage in patients who continued tamoxifen compared with those who discontinued: DFS was 78 vs. 82%, respectively (P = 0.03), and overall survival (OS) was 91 vs. 94% (P = 0.07). Thus, extending tamoxifen treatment beyond 5 years was not deemed beneficial [7] nor recommended [8] when the MA.17 trial was initiated. While clearly in the best interest of patients, discontinuation of tamoxifen after 5 years creates a therapeutic dilemma because of the persistent risk of breast cancer recurrence. Relapse or appearance of new tumors after completion of tamoxifen therapy is relatively common in patients with HR+ tumors [1, 2, 4, 7]. The Oxford meta-analysis found that more than half of breast cancer recurrences and two thirds of breast cancer deaths occur after 5 years of adjuvant tamoxifen [3]. In the NSABP B-14 trial, the average annual rate of breast cancer recurrences was 8.9 per 1,000 patients who discontinued tamoxifen at 5 years [7]. Patients in whom tamoxifen is discontinued therefore require an alternative treatment option to provide continuing protection from recurrence. The loss of efficacy seen with long-term tamoxifen therapy may result from the emergence of a hormone-independent tumor phenotype [9, 10] or the induction of hypersensitivity to estradiol [11, 12]. Tamoxifen is thought to be more susceptible than aromatase inhibitors (AIs) to this adaptive change because of its intrinsic agonist properties [12]. Furthermore, data from the NSABP B-14 trial suggest that residual tumor cells may become tamoxifen-resistant, and that continued use of tamoxifen might in fact stimulate their proliferation [6, 13]. The development of highly potent and selective third-generation AIs provided a new hormone therapy option for patients with HR+ breast cancer [14–16]. It is suggested in independent studies that Letrozole (Femara®) is the most potent of the AIs as determined by in vitro assays [17] and clinical studies of total body aromatization [18]. A recent study has demonstrated that the more complete inhibition of aromatase achieved by 2.5 mg of letrozole than by 1 mg of anastrozole results in a greater degree of suppression of estradiol [19]. The clinical efficacy of letrozole was initially demonstrated in patients with HR+ metastatic breast cancer. In this setting, first-line therapy with letrozole was shown to significantly improve outcome compared with tamoxifen therapy [20, 21]. While OS was not significantly improved (34 vs. 30 months, respectively), an exploratory analysis of the patients that did not cross over showed a longer survival benefit for letrozole-treated patients (35 vs. 20 months) [22]. Second-line therapy with letrozole has also demonstrated significant clinical benefits in patients with HR+ metastatic breast cancer in whom tamoxifen has failed [23], and in one randomized trial demonstrated a significantly higher response rate than anastrozole in this population [24]. Data from in vivo models using MCF-7 cells transfected with the aromatase gene have shown that letrozole is more effective than tamoxifen and devoid of the agonist action observed with the selective estrogen receptor modulator [25, 26]. Although the mechanisms of estrogen blockade are different for letrozole and tamoxifen, combining the two agents did not increase antitumor activity in the MCF-7 xenograft model [25]. The authors proposed that sequential administration of tamoxifen and letrozole would be a more effective strategy [25]. This hypothesis was recently confirmed in vivo [27]. Using the MCF-7 model, it was demonstrated that tumors progressing on tamoxifen responded to second-line letrozole therapy, but tumors that progressed on letrozole did not respond to second-line treatment with tamoxifen or fulvestrant [27]. The evident need to continue hormone therapy after discontinuation of tamoxifen in patients with HR+ primary breast cancer and the potential efficacy of AIs in tumors resistant to tamoxifen provided the rationale for testing adjuvant letrozole after 5 years of tamoxifen. This paper reviews the key results from the landmark MA.17 trial [28, 29] and discusses the advantages of letrozole treatment after the recommended 5 years of tamoxifen therapy have been completed. To date, letrozole is the only AI approved in the United States and Europe for extended adjuvant therapy. Trial design and patients MA.17 was a phase III, randomized, double-blind, placebo-controlled trial of letrozole as extended adjuvant therapy in postmenopausal women with primary breast cancer who had completed approximately 5 years of adjuvant tamoxifen therapy [28, 29]. The aim of the trial was to determine whether letrozole improves outcome after the discontinuation of adjuvant tamoxifen therapy. The MA.17 trial was led by the National Cancer Institute of Canada Clinical Trials Group and included the North American Breast Intergroup and the Breast International Group. Institutional review boards of participating institutions approved the study protocol, and all patients gave written informed consent. Patient population The trial enrolled 5,187 postmenopausal women with early-stage breast cancer in whom 5 years of tamoxifen (range 4.5–6 years) therapy had been completed less than 3 months before enrollment. Eligible women had to have histologically confirmed, HR+ primary breast cancer. HR+ tumors were defined as estrogen receptor-positive (ER+) or progesterone receptor-positive (PgR+) as determined by a level of 10 fmol/mg of protein or a positive immunohistochemical analysis. Women were defined as being postmenopausal if they were aged at least 50 years at the start of adjuvant tamoxifen therapy, were <50 years of age at the start of tamoxifen therapy but postmenopausal at the initiation of tamoxifen therapy, were <50 years at the start of tamoxifen therapy but had undergone bilateral oophorectomy, were premenopausal and <50 years at the start of tamoxifen therapy but became amenorrheic during chemotherapy or treatment with tamoxifen, or were any age but had postmenopausal levels of luteinising hormone or follicle-stimulating hormone prior to the study. All women had a good performance status and life expectancy of at least 5 years. Randomized trial design Eligible women were randomly assigned to receive treatment with letrozole (2.5 mg) or placebo orally daily for 5 years (see Fig. 1). Women were stratified according to the tumor HR status (positive or unknown), lymph-node status (negative, positive, or unknown), and receipt or nonreceipt of previous adjuvant chemotherapy. Exploratory sub-analyses were based on these stratification factors and two additional covariates (criteria for the definition of postmenopausal status at the start of tamoxifen treatment and duration of tamoxifen treatment). Fig. 1MA.17 randomized trial design End points and rules for interim analyses The primary end point of the trial was DFS, defined as the time from randomization to the earliest recurrence of the primary disease (in the breast, chest wall, or nodal or distant metastatic sites) or the development of a new primary breast cancer in the contralateral breast. Secondary cancer and death without a recurrence or a diagnosis of contralateral breast cancer were not included as events. The trial was powered to detect a 2.5% improvement in 4-year DFS with letrozole (from 88 to 90.5%). Two interim analyses were scheduled, and stopping rules were specified a priori for interim monitoring [30]. The secondary efficacy end points of the trial were OS (defined as the time from randomization to death from any cause), annual incidence rate of contralateral breast cancer, long-term safety and tolerability, and overall and menopause-specific quality of life (QOL). In addition, distant DFS (DDFS), defined as the time from random assignment until the first observation of distant metastasis, was included as a secondary efficacy end point in the final analysis [29]. QOL and long-term safety were assessed as secondary end points [31]. Adverse events were graded according to the Common Toxicity Criteria of the National Cancer Institute (version 2.0). QOL was assessed with the Medical Outcomes Study 36-Item Short Form General Health Survey (SF-36) and the Menopause-Specific Quality of Life questionnaire [32, 33]. The effects of letrozole on lipid profile and bone mineral density (BMD) were assessed annually in companion studies to MA.17 [34, 35]. Efficacy of letrozole as extended adjuvant therapy A total of 5,187 patients were randomized to either letrozole (n = 2,593) or placebo (n = 2,594). Because of noncompliance, 10 patients in the letrozole and seven in the placebo arm were excluded from all analyses, leaving 5,170 patients (2,583 on letrozole and 2,587 on placebo) in the time to event analysis (50 patients deemed ineligible for several reasons and 33 with major protocol violations were included in the analysis). The final safety analysis excluded 21 patients who never received study medication, yielding a final safety population of 5,149 patients, 2,572 receiving letrozole and 2,577 receiving placebo [29]. Based on the 43% reduction in recurrence risk (P = 0.00008) with letrozole seen in the first interim analysis at 2.4 years’ median follow-up [28], the data and safety monitoring committee recommended that the MA.17 trial be discontinued early, and the participants were informed of the results. The trial was unblinded in October 2003, and patients on placebo were given the opportunity to switch to letrozole. Updated efficacy results after a median follow-up of 30 months confirmed the significant clinical benefits of letrozole as extended adjuvant therapy [29]. The updated trial results and recent additional efficacy analyses of MA.17 are summarized below. Letrozole significantly improves outcome At 30 months’ follow-up, letrozole significantly improved DFS, the primary end point, compared with placebo (see Fig. 2) [29]. The 4-DFS for patients receiving letrozole was 94.4%, compared with 89.8% for patients receiving placebo. The hazard ratio for recurrence or contralateral breast cancer was 0.58 (95% confidence interval [CI] 0.45, 0.76; P ≤ 0.76), representing a 42% reduction in risk for letrozole relative to placebo. The updated analysis also showed that letrozole produced a statistically significant improvement in DDFS (hazard ratio = 0.60; 95% CI 0.43, 0.84; P = 0.002), which may be regarded as a more meaningful end point than overall DFS; women with distant metastases inevitably die of breast cancer, and an improvement in DDFS may therefore translate into longer overall survival [36, 37]. Letrozole treatment non-significantly prolonged time to contralateral breast cancer incidence, resulting in a 37.5% relative risk reduction compared with placebo [29]. Fig. 2Kaplan–Meier curves for disease-free survival in the updated analysis of MA.17. N, number at risk; S, survival percent, with 95% confidence intervals (CIs) in parentheses. Reprinted from ref. [29] with permission The prospectively planned subgroup analysis showed that letrozole significantly improved DFS in all patients, irrespective of nodal status. The reduction in risk of recurrence in node-positive tumors was 39% (hazard ratio = 0.61; 95% CI 0.45, 0.84), and 55% in those with node-negative tumors (hazard ratio = 0.45; 95% CI 0.27, 0.73). While OS was not significantly improved (hazard ratio = 0.82; 95% CI 0.57, 1.19; P = 0.3), letrozole significantly improved OS in patients with node-positive tumors (hazard ratio = 0.61; 95% CI 0.38, 0.98; P = 0.04) (see Fig. 3), and this was the first survival advantage demonstrated by an AI in early breast cancer. Fig. 3Forest plots of the treatment effect (letrozole vs. placebo), in terms of overall survival, in subgroups defined by hormone receptor status, lymph node status, previous chemotherapy, menopausal criteria, and duration of tamoxifen treatment. For each subgroup, the hazard ratio for death from any cause is plotted as a solid square, and the area of the square is proportional to the variance of the estimated effect. The length of the horizontal line through the square indicates the 95% confidence interval (CI). The arrow at the end of the horizontal line indicates that the confidence interval is larger than the scale of the figure. Reprinted from ref. [29] with permission Additional MA.17 analyses Optimal duration of extended adjuvant letrozole The final MA.17 database, including all events up to the date of unblinding, was analyzed to examine the relationship between duration of treatment and outcomes [38, 39]. Data from this analysis have provided further evidence to support an extended duration of letrozole, as this cohort analysis found that the longer patients are exposed to extended adjuvant letrozole (at least out to 48 months), the greater the benefit [39]. The risk of disease recurrence increased over time in the placebo group, whereas in patients receiving letrozole, risk appeared to peak at around 2 years of treatment and decrease thereafter. In the overall patient population, hazard ratios for events in DFS and DDFS progressively decreased over time, favoring letrozole, with the trend being significant (P < 0.0001 and P = 0.0013, respectively). The trend for OS was not significant but was always <1. In the 2,360 patients with node-positive status, hazard ratios for DFS, DDFS, and OS all decreased over time, with tests for trend all showing significance (P = 0.0004, 0.0005, and 0.038, respectively). Considering the 2,568 patients with node-negative status, the hazard ratios for DFS decreased over time, with the test for trend being significant (P = 0.027), whereas the hazard ratios for DDFS and OS showed no significant change over time (see Table 1). Table 1Analysis of the hazard ratios for disease recurrence over time between the letrozole and placebo arms of MA.17Month after randomizationNo. at risk (letrozole/placebo)Hazard rate (letrozole)Hazard rate (placebo)Hazard ratio (letrozole vs. placebo)a122,425/2,4090.000930.001800.52 (0.40–0.64)241,555/1,5300.001050.002360.45 (0.33–0.56)36768/7230.000900.002610.35 (0.21–0.48)48244/2310.000590.003060.19 (0.04, 0.34)Reprinted from ref. [38] with permission from ElsevieraHazard ratios <1 indicate values in favor of letrozole MA.17 ITT analysis The intent-to-treat (ITT) analysis at 54 months’ follow-up looked at all outcomes, including all events before and after the unblinding, based on the original randomization of letrozole versus placebo; it did not take into account whether or not patients in the placebo group switched to letrozole at the unblinding of the data. The results further showed that patients randomized initially to letrozole had fewer DFS events than those initially randomized to placebo [40]. Publication of the final analysis of this data are awaited, but these provisional results highlight the strong beneficial effect of extended adjuvant therapy with letrozole when started immediately after tamoxifen. Impact of HR status on clinical benefit A retrospective analysis was conducted to determine whether HR status had an effect on the outcome of letrozole in the extended adjuvant setting [41]. ER and PgR positivity were defined as ≥10 fmol/mg protein or positive by immunocytochemical analysis. Preliminary results from 4,635 patients, based on local testing of HR status, showed that the reduction in risk of recurrence with letrozole compared with placebo was greatest in women with the most hormone-dependent tumors. The final results of this analysis await publication. These results should be interpreted cautiously, as this was an unplanned, retrospective analysis, and receptor levels were measured locally. Furthermore, from the outset, the analysis of outcomes in all other subgroups besides the ER+/PgR+ (n = 3,809) patients was weakened by the low numbers of patients in these groups (e.g., ER+/PgR−, n = 636) [42]. MA.17 post-unblinding analysis The trial was unblinded in 2003 because DFS values were met (stopping boundary nominal significance, P = 0.0008) and patients were given the opportunity to cross over. Post-unblinding analysis of MA.17 has provided additional efficacy data on patients who had crossed over from placebo to letrozole (n = 1,655), comparing them with patients who elected no treatment at the time of the unblinding (n = 613) [43]. These patients had not received any hormonal therapy after discontinuing tamoxifen. Data were adjusted for baseline patient and disease variables including tumor size, nodal status, and prior adjuvant chemotherapy. A preliminary analysis suggests that letrozole has significant clinical benefits in patients in whom treatment with the AI is started after a prolonged period since the discontinuation of tamoxifen. The publication of these results is eagerly awaited as they may effect patient care worldwide. MA.17 re-randomization A re-randomization of all patients completing letrozole to receive a further 5 years of letrozole or placebo is under way to confirm this finding [40, 44]. An amendment to this protocol allows women completing 5 years of any AI to be re-randomized to a further 5 years of letrozole or not regardless of prior tamoxifen or its duration. The MA.17 re-randomization study should provide additional insights into the efficacy and safety of extending letrozole therapy beyond 5 years [45]. Safety of letrozole in the extended adjuvant setting The women included in this trial had been disease-free for approximately 5 years during treatment with tamoxifen and, therefore, the safety and tolerability of continued hormone treatment with letrozole was an important consideration when the MA.17 trial was designed. Furthermore, early unblinding of the trial has not prevented the collection of long-term safety data, and additional sub-studies are providing useful information on the safety profile of letrozole in the extended adjuvant setting. The safety of AIs is discussed in detail in the paper by Dr. Perez in this supplement. The MA.17 trial showed that letrozole is extremely well tolerated relative to placebo. The most common adverse events reported were secondary to estrogen suppression and included hot flashes, myalgia, arthralgia, alopecia, and newly diagnosed osteoporosis. The increase in newly diagnosed osteoporosis (8.1% for letrozole vs. 6% for placebo; P = 0.003) [29] was predictable from the potent suppression of estrogens by third-generation AIs [17] and the association between estrogen levels and bone turnover [46–48]. Of note, no significant difference in clinical fracture rate was seen between letrozole and placebo groups (5.3 vs. 4.6%; P = 0.25) [29]. MA.17B is a companion study designed to compare the effects of letrozole (n = 122) and placebo (n = 104) on BMD in the L2–L4 (posteroanterior) region of the spine and hip [35]. At 24 months, patients receiving letrozole had a significant decrease in total hip BMD (−3.6 vs −0.71%; P = 0.044) and lumbar spine BMD (−5.35 vs. −0.70%; P = 0.008). Further follow-up is necessary to evaluate the long-term clinical implications of this modest increase in bone resorption and reduction in BMD in the spine and hip with letrozole compared with placebo. Prophylactic use of the bisphosphonate zoledronic acid is being studied as a means to prevent BMD loss [49, 50]. Results from two clinical trials have indicated that early use of zoledronic acid effectively prevents BMD loss in women receiving adjuvant letrozole [49, 51]. Estrogen has a beneficial effect on lipid profiles, and it has been suggested that AIs may have a relatively unfavorable effect. However, MA.17L, a substudy of MA.17 (n = 347), demonstrated that letrozole does not significantly alter serum cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, or lipoprotein (a) compared with placebo [34]. This is in agreement with the results of the main MA.17 trial, which showed no difference in hypercholesterolemia rates between placebo and letrozole [28]. Importantly, in the updated analysis of the MA.17 dataset, there were no significant differences between the letrozole and placebo arms in the incidence of hypercholesterolemia (16% in each arm; P = 0.79) or cardiovascular events (5.8 vs. 5.6%; P = 0.76) [29]. The QOL substudy was conducted in 3,612 patients treated in MA.17 (1,813 letrozole and 1,799 placebo) [31]. The analysis demonstrated that letrozole did not have an adverse impact on overall QOL, as determined by SF-36, which is an important and reassuring finding, as the extension of letrozole treatment for up to 10 years in this setting is now being tested [44]. Conclusions In recent years, considerable progress has been made in developing more effective hormonal treatments for women with breast cancer and improving the efficacy demonstrated with tamoxifen [3]. The pioneering MA.17 trial has demonstrated the need for extended adjuvant therapy after 5 years of tamoxifen to reduce the risk of recurrence in postmenopausal women with HR+ breast cancer. On the basis of this trial, letrozole was approved as extended adjuvant therapy, and it is currently the only AI approved for this indication [22, 52]. MA.17 demonstrated that extended adjuvant therapy with letrozole provides women further protection against relapse after the completion of tamoxifen. These findings support the concept that distant micrometastases that have survived 5 years of tamoxifen therapy remain highly estrogen-sensitive and responsive to extended adjuvant letrozole treatment. This is an important clinical benefit in view of the persistent risk of relapse beyond 10 years in patients with HR+ tumors [4]. In fact, the cohort analysis showed that in the placebo group, there was actually an increasing risk of disease recurrence over time after discontinuing prior tamoxifen [40]. The preliminary MA.17 ITT data suggest that the strongest beneficial effect is still obtained when starting letrozole within 3 months of completing tamoxifen [38], while the post-unblinding results suggest that women with hormone-dependent breast cancer who are prescribed letrozole following a prolonged delay after completing tamoxifen may experience a significant improvement in outcome [43]. Therefore, if a patient misses the chance to start letrozole within 3 months post tamoxifen, these results suggest that there is still a benefit to initiating letrozole therapy for up to 5 years following the discontinuation of tamoxifen. Importantly, women in all risk categories benefited in terms of reduced risk for recurrence of their cancer. Thus, in both node-positive as well as node-negative women, there was a strong improvement in DFS, and “low risk” status of the primary tumor should not preclude consideration of extended adjuvant therapy with letrozole. Extended therapy with adjuvant letrozole should therefore be considered for all women currently completing tamoxifen. In addition, in women who have completed tamoxifen within the last 5 years, introduction of letrozole for 5 years can be discussed because although we do not have level 1 evidence for benefit in this setting as yet, our post-unblinding analysis of MA17 strongly supports the potential for benefit in these women. Among the clinical questions left unanswered are the optimal duration of letrozole and the long-term safety of extended adjuvant therapy in women leading a normal healthy lifestyle. Preliminary results of a cohort study analysis of MA.17 provide support for the use of extended adjuvant letrozole for at least up to 4 years [40, 53]. The MA-17 re-randomization study will assign patients completing 5 years of letrozole to a further 5 years of letrozole or placebo and should provide data on the efficacy and safety of extending letrozole therapy beyond 5 years [45]. In conclusion, HR+ breast cancer presents an unremitting threat that may require life-long hormone therapy. The optimal hormone treatment strategy is evolving based on the results of landmark clinical trials in the initial adjuvant [54, 55], sequential adjuvant [56], and extended adjuvant settings [28, 29]. The optimal agent, sequence of treatments, or combination of treatments will be able to provide the greatest improvement in OS with minimal acute and long-term toxicity. MA.17 has demonstrated that letrozole is highly effective and extremely well tolerated when given in the extended adjuvant setting. The results of the pivotal MA.17 trial have changed current clinical practice to extend letrozole protective therapy in thousands of breast cancer patients currently receiving 5 years of adjuvant tamoxifen. The benefits of long-term adjuvant letrozole treatment clearly outweigh any adverse events in postmenopausal women who have survived breast cancer after initial adjuvant therapy with tamoxifen. Long-term side effects and risks continue to be monitored and taken into account when any individual patient is being considered for extended adjuvant therapy.

Purinergic_Signal-4-1-2245998

P2 receptors in cardiovascular regulation and disease

The role of ATP as an extracellular signalling molecule is now well established and evidence is accumulating that ATP and other nucleotides (ADP, UTP and UDP) play important roles in cardiovascular physiology and pathophysiology, acting via P2X (ion channel) and P2Y (G protein-coupled) receptors. In this article we consider the dual role of ATP in regulation of vascular tone, released as a cotransmitter from sympathetic nerves or released in the vascular lumen in response to changes in blood flow and hypoxia. Further, purinergic long-term trophic and inflammatory signalling is described in cell proliferation, differentiation, migration and death in angiogenesis, vascular remodelling, restenosis and atherosclerosis. The effects on haemostasis and cardiac regulation is reviewed. The involvement of ATP in vascular diseases such as thrombosis, hypertension and diabetes will also be discussed, as well as various heart conditions. The purinergic system may be of similar importance as the sympathetic and renin-angiotensin-aldosterone systems in cardiovascular regulation and pathophysiology. The extracellular nucleotides and their cardiovascular P2 receptors are now entering the phase of clinical development. Introduction Ever since the first proposition of cell surface receptors for nucleotides [1, 2], it has become increasingly clear that, in addition to functioning as an intracellular energy source, the purines and pyrimidines ATP, adenosine diphosphate (ADP), uridine triphosphate (UTP) and uridine diphosphate (UDP) can serve as important extracellular signalling molecules [3, 4] acting on 13 P2X homo- and heteromultimer ionotropic and 8 P2Y metabotropic receptor subtypes [5, 6] (Table 1). To terminate signalling, ectonucleotidases are present in the circulation and on cell surfaces, rapidly degrading extracellular ATP into ADP, AMP and adenosine [7, 8]. Evidence is accumulating suggesting an important role for the purinergic system in cardiovascular regulation [9–15]. It stimulates vasoconstriction and vasodilatation, growth of vascular smooth muscle cells and endothelial cells, angiogenesis, is involved in vascular remodelling, stimulates platelet aggregation, regulates coagulation, inflammation and several aspects of cardiac function. It is involved in blood pressure regulation, development of myocardial infarction, heart failure and xenograft rejection. The physiological effects of the purinergic signalling system are dependent on the release of extracellular nucleotides, the degradation by ectonucleotides, the type of P2 receptors expressed on the cells, their desensitisation rates and their second messengers. P2 receptors have highly specific organ distributions and they can be rapidly up- or downregulated. The purinergic system may be of similar importance as the sympathetic and renin-angiotensin-aldosterone systems in cardiovascular regulation and pathophysiology. The involvement of purinergic signalling in a variety of different cardiovascular clinical conditions has been addressed to a limited extent previously [15–19] and the large number of new discoveries calls for a review to summarise the most important roles of the purinergic system in cardiovascular regulation and disease. Table 1Receptor classification, intracellular signalling, ligands and selective agonists and antagonistsP2 subtypeI.c. signallingLigandSelective agonistSelective antagonistNon-selective antagonistP2Y1↑IP3ADP (ATP)MRS2365MRS 2179, MRS2500P2Y2↑IP3UTP = ATPMRS2498, UTPγS, INS3717Suramin > RB2P2Y4↑IP3UTP (=ATP in rodents)UTPγS, INS3717–RB2 > SuraminP2Y6↑IP3UDPMRS2666, MRS2633, UDPβSMRS2578P2Y11↑IP3, ↑cAMPATPAR-C67085MX, NF546NF157Suramin > RB2P2Y12↓cAMPADP–Clopidogrel, prasugrel, AZD6140, INS50589, AR-C9931 (cangrelor)P2Y13↓cAMPADP–MRS2211P2Y14IP3UDP-glucose, UDP-galactoseUDP-glucose, UDP-galactose–P2X1Positive ion channelATPα,β-mATPNF023, NF449TNP-ATP, Ip5IP2X2Positive ion channelATP–NF770Suramin, isoPPADS, RB2P2X3Positive ion channelATPα,β-mATPA317491, NF110SuraminP2X4Positive ion channelATPIvermectin potentiates–TNP-ATPP2X5Positive ion channelATP––Suramin, PPADSP2X6Positive ion channelATP–––P2X7Positive ion channelATP–KN62, KN04, MRS2427Coomassie brilliant blue GEctonucleotidaseApyrase, human SolCD39ARC67156 The research field has grown rapidly since the term P2 receptor was coined [2]. In preparation of this review more than 2,700 references on P2 receptors and cardiovascular regulation were found in PubMed. Among the 15 receptor subtypes, one is the target for one of the most widely used medical drugs—the platelet inhibitor clopidogrel (Plavix). As will be reviewed here, several of the other 15 receptors are promising drug targets to prevent cardiovascular disease. Regulation of vascular tone Vasoconstriction produced by ATP released as a cotransmitter with noradrenaline (NA) from perivascular sympathetic nerves was recognised early [20, 21]. However, following the seminal discovery of endothelium-dependent vasodilatation by Furchgott in the early 1980s, it was shown that ATP acted on endothelial cells to release endothelial derived relaxing factor [later shown to be largely nitric oxide (NO)] resulting in vasodilatation [22] and dual purinergic neural and endothelial control of vascular tone established [23, 24]. Neuronal regulation of vascular tone ATP is, together with NA and neuropeptide Y (NPY), a cotransmitter in sympathetic neurons [11, 25, 26], and sensory-motor nerves during “axon reflex” activity release ATP to dilate or constrict vessels [27, 28] (Fig. 1). The contribution of ATP to sympathetic contraction varies between vascular beds [25]. For example, it may mediate up to 50% of the neurogenic vasoconstriction via P2X1 receptors as seen in mesenteric arteries [29], about 70% in rabbit saphenous arteries and 100% in rabbit jejunal artery, while NA acts as a prejunctional modulator [30]. In the renal vasculature both P2X1 and P2Y2 receptors are important for neurogenic contraction. P2X1 receptors mediate contraction in afferent but are absent in the efferent renal arterioles [31, 32]. The result is that extracellular nucleotides selectively influence preglomerular resistance without having an effect on postglomerular tone. ATP is also released locally in the kidney in response to increased perfusion pressure, and in P2X1 receptor knockout mice auto-regulatory responses in kidney afferent arterioles are abolished, indicating an important role in renal glomerular pressure regulation [32, 33]. Fig. 1P2 receptor-mediated regulation of the circulation. See text for details. Purines and pyrimidines are released on the luminal side from endothelial cells, platelets and red blood cells (RBC) in response to hypoxia, acidosis, adrenaline, shear stress and other stimuli. When the endothelial cell layer is intact, the response is vasodilatation by the endothelial release of nitric oxide (NO), endothelium-derived hyperpolarising factor (EDHF) and prostaglandins (Pgl). When the endothelium is damaged, platelets accumulate, release ATP and ADP and mediate vasoconstriction via P2 receptors on the vascular smooth muscle cell (VSMC). On the adventitial side sympathetic and sensory nerves mediate vasoconstriction. Extracellular nucleotides are rapidly degraded by NTPDase1 on endothelium reducing ATP to AMP, followed by conversion to adenosine by CD73 (not shown). In the subendothelium NTPDase2 is present, degrading ATP to ADP, maintaining the platelet activating and contractile effects. Ado adenosine, CGRP calcitonin gene-related peptide, SP substance P Sympathetic nerves express inhibitory P2Y and P1 (A1) receptors indicating a P2 receptor-mediated negative feedback loop both directly by ATP and its degradation product adenosine [34, 35]. Ectonucleotidases are released from sympathetic nerves together with its substrate ATP, as a termination mechanism for the signalling [36]. Another balancing mechanism is the sympathicolytic effect seen in exercising skeletal muscle, which is important for increased blood flow in the working skeletal muscle during exercise, despite sympathetic NA release that normally would reduce blood flow. This sympathicolytic effect is mimicked by injection in the arterial lumen of ATP and UTP, inducing a vasodilatation that overrides sympathetic vasoconstrictor activity in human skeletal muscle, an effect not obtained by injection of adenosine [37], suggesting that ATP and UTP may mediate this important physiological mechanism. The first evidence of the presence of P2 receptor subtypes suggested P2X receptors on vascular smooth muscle cells (VSMC) and P2Y receptors on endothelial cells [38]. Rat blood vessel immunohistochemistry and human mRNA quantification have shown the P2X1 receptor to be the highest expressed subtype in smooth muscle cells [39, 40]. It has been difficult to prove the importance of other contractile P2X receptors besides P2X1 due to the paucity of specific agonists and antagonists. However, contractile effects of UTP suggested that P2Y receptors were present on VSMCs and mediated contraction [41]. Using selective pyrimidines resistant to degradation, it has been possible to show that contractile effects are mediated by both UTP-sensitive P2Y2 and UDP-sensitive P2Y6 receptors [42]. Ectonucleotidases in the vessel wall rapidly degrade nucleotides and markedly reduce their contractile effects. Pyrimidine analogues more resistant to ectonucleotidases are powerful vasoconstrictors and up to 1,000-fold more potent than the endogenous ligands [42]. Similarly, a prominent more potent UDP contraction has been seen in NTPDase1 knockout mice, demonstrating the protective effect of ectonucleotidases against nucleotide contraction [43]. In human saphenous vein grafts, used during coronary bypass, a stable UDP analogue stimulates a strong contraction lasting for hours, which is not desensitised [44]. The reason why the P2Y6 receptor is not desensitised is the lack of serine residues in the C-terminal part of the P2Y6 receptor [45]. Neither α1-adrenoceptors, nor P2Y6 receptors, are present in human coronary arteries, possibly to avoid deleterious vasospasm during ischaemia [44]. VSMC express P2Y12 receptors that mediate contraction after stimulation with ADP [46]. At the mRNA level, the P2Y12 receptor is the highest expressed ADP receptor and the second highest expressed P2 receptor in human VSMC [46]. The contractions are not inhibited in patients medicated with clopidogrel. However, drugs with antagonistic effects on P2Y12 receptors that reach the peripheral circulation (AZD6140 and INS50589), affecting both platelets and VSMC, could be of double therapeutic benefit in their prevention of both thrombosis and vasospasm [46]. In conclusion, extracellular nucleotides mediate vasoconstriction when released from nerves on the adventitial side, or when released in the lumen when the endothelium is damaged (Fig. 1). The most important contractile receptors on the VSMC are the ATP P2X1 receptor, the ATP/UTP P2Y2 receptor, the UDP P2Y6 receptor and the ADP P2Y12 receptor (Fig. 1 and Table 2). To determine the relative physiological importance of these receptor subtypes in different human vascular beds is an important task for the future. Table 2P2 receptor expression in the cardiovascular systemP2 subtypeEndotheliumVSMCHeartPlateletsRed blood cellsInflammatory cellsPerivascular sensory nervesPerivascular sympathetic nervesP2Y1High––High(Turkey)MediumMedium (neg. feedback)P2Y2HighHighHigh––HighLowPossibleP2Y4–Possible–––P2Y6MediumMediumHigh––P2Y11Possible–High––HighP2Y12–Medium–High–LowP2Y13––––HighLowP2Y14–––––P2X1–HighMediumHigh–MediumP2X2–––––HighMedium (pos. feedback)P2X3–––––HighP2X4High–Low––HighP2X5–––––P2X6–––––P2X7–Possible––LowHighUnless specified data refer to human tissue. High, medium or low expression represents an effort to summarise the results of a large number of mRNA, protein and pharmacological studies. Dash (–) denotes lack of convincing evidence for expression. VSMC vascular smooth muscle cell Endothelial regulation Shear stress and hypoxia are important stimuli of both ATP and UTP release from endothelial cells [47] (Fig. 1). Extracellular nucleotides have several important effects mediated by activation of endothelial cells. Vasodilatation and decreased blood pressure by release of prostaglandins and NO has been demonstrated in several studies [5], but P2 receptors also mediate release of endothelium-derived hyperpolarising factor (EDHF), which relaxes VSMC by activation of potassium channels, with subsequent hyperpolarisation [48–50]. Both UTP and ATP reduce forearm vascular resistance in a prostaglandin and NO independent way [51], indicating an important role for EDHF in P2 receptor-mediated vasodilatation in man. The P2Y1 receptor seems to be of major importance in most vascular beds [11]. However, several other P2 receptors are important for endothelial regulation. Pharmacology of vasodilatation and mRNA quantification in man indicates that P2Y2 and to a lesser degree P2Y6 also are important endothelial P2Y receptors [40, 52]. Knockout mice experiments recently confirmed this picture and demonstrated that the P2Y4 receptor does not mediate dilatation [53]. UTP and ATP are equipotent as vasodilators when infused in the human forearm circulation, indicating a role for both purinergic and pyrimidinergic receptors. The P2X1 receptor is not expressed on the vascular endothelium and the evidence for other P2X receptors has been scarce, except for the P2X4 receptor which is the highest expressed P2 receptor in endothelium [40, 54]. Using antisense oligonucleotides the P2X4 receptor was shown to be important for shear stress-dependent Ca2+ influx via an ATP-dependent mechanism [55]. This indicates that ATP and P2 receptors may be of importance for shear stress-mediated effects, which is in agreement with the well-established release of ATP from endothelial cells during shear stress [56]. Vessel dilation induced by acute increases in blood flow is markedly suppressed in P2X4 receptor knockout mice [57]. Thus, endothelial P2X4 channels are crucial to flow-sensitive mechanisms that regulate blood pressure and vascular remodelling [57]. Extracellular ATP in the circulation is rapidly degraded into ADP, AMP and adenosine by ectonucleotidases. Vascular NTPDase1 (CD39) is an endothelial cell membrane protein with both ecto-ATPase and ecto-ADPase activities [8, 58]. Ectonucleotidases are also released by shear stress from endothelial cells [59]. Reactive hyperaemia is the massive increase in blood flow that starts when a blood vessel is opened after a period of ischaemia. It is well known that ATP is released during ischaemia [7, 60]. Adenosine only mediates the late phase of the reactive hyperaemia as has been shown by infusion of adenosine deaminase or a selective A2A antagonist [61, 62]; ADP has been shown to mediate the mid-portion and peak of coronary reactive hyperaemia via endothelial P2Y1 receptors [63]. It has been proposed that different purines may mediate three phases of the reactive hyperaemia [63]. According to this hypothesis, ATP would mediate the first part of the hyperaemia via endothelial P2Y2 or P2X4 receptors. ATP would then be degraded to ADP, which mediates peak hyperaemia via endothelial P2Y1 receptors, followed by degradation of ADP to adenosine resulting in late phase hyperaemia mediated via A2A receptors on smooth muscle cells. Red blood cells as regulators of vascular tone The matching of oxygen supply with demand requires a mechanism that increases blood flow in response to decreased tissue oxygen levels. Several reports suggest that the red blood cell (RBC) acts as a sensor for hypoxia and different mechanisms have been suggested by which the deoxygenated RBC stimulates vasodilatation [37, 64–66]. RBCs contain millimolar amounts of ATP and possess the membrane-bound glycolytic enzymes necessary for its production [67–69]. ATP is released in response to reductions in oxygen tension and pH [37, 64] (see Table 3). It has been shown in vitro that vessels dilate in response to low O2 levels only when blood vessels are perfused with RBCs [70]. ATP is released in working human skeletal muscle circulation depending on the number of unoccupied haemoglobin O2 binding sites [64, 71]. Similar results have been shown in the coronary circulation of dogs [72]. The released ATP then binds to P2Y receptors on the endothelium and stimulates vasodilatation. Thus, the RBC functions as an O2 sensor, contributing to the regulation of blood flow and O2 delivery, by releasing ATP depending on the oxygenation state of haemoglobin. Table 3Release of purines and pyrimidines in the cardiovascular systemSourceEndotheliumVSMCHeartPlateletsRed blood cellsInflammatory cellsVascular nervesStimuliShear stress, hypoxia, high glucoseHigh glucose, hypoxiaHypoxiaCollagen, thromboxane, other platelet activatorsHypoxia, adrenaline, deformationInfl. activatorsSympathetic nerves, sensory nerve collateralsMechanismVesicularUnknownUnknownVesicularUnknownUnknown or vesicularVesicularNucleotideATP, UTPATP, UTPATP, UTPADP > ATPATPATPATPTargetEndothelium VSMCVSMCCardiomyocytes, blood vessels, intrinsic cardiac neuronsPlatelets; endothelium or VSMCEndotheliumVSMC, endotheliumVSMCDegradationNTPDase1NTPDase2, NTPDase1NTPDase1NTPDase1NTPDase1NTPDase1NTPDase1 (co-released)Stimuli represents stimuli leading to release. Mechanism refers to mechanism of release at the cellular level. Target represents the cells that are regulated by the released purine or pyrimidine. Degradation refers to the degrading enzyme responsible for removing the purine or pyrimidine after the release. VSMC vascular smooth muscle cell ADP activates a negative feedback pathway for ATP release from human RBCs via P2Y13 receptors [73]. Since blood consists of approximately 40% RBCs that contain a 1,000-fold higher ATP concentration than plasma (mmol/l vs μmol/l), even a minor release of ATP from the high intracellular concentrations could have major circulatory effects. A negative feedback system may therefore be of great physiological importance to mitigate ATP release. Another negative feedback system is the mechanism by which NO inhibits ATP release from erythrocytes, illustrating how NO may turn off ATP release [74]. NO is then scavenged by haemoglobin. Furthermore, the previous view of the RBC as a “passive bag that transports oxygen” is challenged. It now turns out that it releases ATP in response to stimuli and, as with most important signalling systems, it has a negative feedback system to terminate its release. The described negative feedback pathway may be important to avoid high extracellular concentrations of ATP. At levels above 100 µmol/l, ATP concentrations may exceed the catalytic capacity of ectonucleotidases and could, in fact, stimulate ATP release by increasing permeability of the RBC [75], probably via P2X7 receptors [76]. ATP may even release ATP from endothelial cells [77]. At high concentrations of ATP, a self-sustaining process may thus be instigated which may contribute to the irreversible stage of circulatory shock that can develop rapidly in severely ill patients. Similar mechanisms may be of importance in malaria because induction of the osmolyte permeability in Plasmodium-infected erythrocytes involves purinoceptor signalling [78]. A mechanism of ATP release from RBCs, which has eluded researchers for many years, has recently been suggested by Locovei and co-workers as being mediated via the gap junction protein pannexin-1 [79]. Erythrocytes do not form gap junctions, instead pannexin-1 forms a mechanosensitive ATP-permeable channel that mediates osmotically induced ATP release. Hypertension Blood pressure regulation by purinergic signalling is the net result of balancing contractile and dilatory effects as described above. ATP and UTP released on the luminal side, from endothelial cells and erythrocytes, stimulate vasodilatation, in contrast to release from nerves on the adventitial side, which results in vasoconstriction. ATP may also regulate blood pressure via renal mechanisms or brain stem regulation [31, 32]. ATP plays a significantly greater role as a sympathetic cotransmitter in spontaneously hypertensive rats (SHR) [80, 81], and there is increased responsiveness of the renal vasculature of isolated perfused rat kidneys to α,β-methylene ATP in SHR [82], while mesenteric vascular contractile reactivity to ATP via P2X1 and P2Y2 receptors is not altered in deoxycorticosterone acetate (DOCA)-salt hypertension [83]. In the aorta of SHR, endothelium-dependent relaxation to ATP is impaired because of the concomitant generation of an endothelium-derived contracting factor. Contractions to ATP were significantly potentiated in SHR aorta [84]. In hypertensive mature stroke-prone rats, NPY modulation of release of NA and ATP from sympathetic perivascular nerves in kidney vessels is impaired, which may account for the increased nerve-mediated responses [85]. Thus, animal models of hypertension reveal several alterations in purinergic signalling that may contribute to the development of hypertension. Diadenosine polyphosphates such as Ap4A, Ap5A and Ap6A are combinations of two adenosine molecules connected with four to six phosphate groups. They have been identified as vasocontractile agents [86], probably via actions on P2X1 and P2Y2 receptors. Ap5A and Ap6A are stored at higher levels in platelets from patients with hypertension and may contribute to their increased peripheral vascular resistance [87]. Up4A is a novel endothelium-derived vasoconstrictive factor more potent than endothelin in renal vasoconstriction [88]. It is released upon stimulation of the endothelium by acetylcholine, thrombin and mechanical stress and can be cleaved into either ATP or UTP to stimulate both P2X1 and P2Y2 receptors on VSMC resulting in increased blood pressure [88]. As mentioned above, the P2X4 receptor is the most abundantly expressed P2 receptor in the endothelium and mediates shear stress-stimulated vasodilatation. P2X4 receptor knockout mice have higher blood pressures and excrete smaller amounts of NO products in their urine than do wild-type mice [57]. The P2X4 receptor protein is upregulated in the placenta in preeclampsia [89]. Thus, shear stress-stimulated release of ATP acting on P2X4 receptors may be important for blood pressure regulation. The importance of P2 receptors for hypertension will not be proven until tested in man. It would be of great interest to perform a clinical study on the effects of P2X1, P2Y2 or P2Y6 receptor antagonists in the treatment of hypertension. Pulmonary hypertension Erythrocyte release of ATP appears to regulate pulmonary resistance under some conditions [90], and patients with pulmonary hypertension have been found with impaired release of ATP from RBC [69]. Endothelium-dependent relaxation to ATP has been demonstrated in human pulmonary arteries [91]. On the other hand, ATP is a mitogen for pulmonary artery smooth muscle cells, which may be relevant for the pathophysiological basis of pulmonary hypertension [92]. Again, the balance between endothelial versus smooth muscle stimulation by ATP may regulate blood pressure in different directions and we are far from a full understanding of the role of the purinergic system in pulmonary hypertension. Migraine and vascular pain Classic migraine is associated with two distinct vascular changes: an initial vasoconstriction, followed by vasodilatation associated with pain. It has been proposed that purinergic signalling is involved in these changes. ATP released from perivascular sympathetic nerves may participate in producing the initial vasospasm mediated by P2 receptors in vascular smooth muscle. Cerebral arteries are strongly contracted by UTP and especially UDP via P2Y6 receptors. A UDP agonist might therefore have similar effects as the 5-HT1D agonists frequently used to treat migraine [93]. ATP released by endothelial cells acting on P2 receptors on endothelial cells mediating release of NO and producing vasodilatation may contribute to the phase of reactive hyperaemia following vasospasm [94]. It has been suggested that antagonists to P2X3 receptors, which are located on nociceptive sensory nerve endings in cerebral vessels, may be promising candidates for anti-migraine drug development [95]. A recent report demonstrates selective upregulation of nociceptive P2X3 receptors on trigeminal neurons by calcitonin gene-related peptide and suggests that this mechanism might contribute to pain sensitisation in migraine [96]. P2Y receptors on trigeminal sensory nerves have also been implicated in pain in migraine. It has been claimed that migraine attacks are characterised by a relative depletion of sympathetic NA stores in conjunction with an increase in release of ATP [97]. In a review in the Lancet in 1996, Burnstock proposed that vascular pain, as in angina, ischaemic muscle and pelvic pain in women, may be initiated by ATP released from microvascular endothelial cells during reactive hyperaemia, diffusing a short distance to activate P2X3 nociceptive receptors on sensory nerves in the adventitia [26]. Atherosclerosis Atherosclerosis is the main cause of ischaemic stroke and cardiovascular disease and is now considered to be an inflammatory disease [98]. The formation of a plaque starts with the accumulation of cholesterol followed by invasion of macrophages taking up cholesterol and becoming foam cells. The plaque can be stabilised by smooth muscle cell formation of a fibrous cap that covers the lipid-rich region. However, stimulation of inflammation by oxidised low-density lipoprotein activates macrophages and dendritic cells into antigen-presenting cells, activating T lymphocytes resulting in release of cytokines and metalloproteinases degrading the fibrous cap. The end result is a vulnerable plaque and, when it ruptures, its highly thrombogenic contents activates platelets and causes the formation of a local thrombus occluding the artery or embolising, resulting in ischaemic stroke or myocardial infarction. Evidence both from basic research and from clinical studies indicates important involvement on several levels for purinergic signalling in the atherosclerotic process (Fig. 2). Interestingly, fish oil components increase the release of ATP [99] and a high cholesterol diet decreases ATP release from arteries [100, 101]. Fig. 2Functional roles of P2 receptors in the atherosclerotic inflammatory plaque and during restenosis. See text for details. Purines and pyrimidines acting on P2 receptors stimulate vascular inflammation both by actions on the endothelial cell (EC) and by effects on inflammatory cells. Furthermore, they stimulate vascular smooth muscle cell (VSCM) proliferation, the conversion to synthetic phenotype and production of matrix proteins. Mitogenic P2 receptors are upregulated by growth factors and cytokines. IL interleukin, MCP-1 monocyte chemoattractant protein-1, ICAM-1 intercellular adhesion molecule-1, TSP thrombospondin, IDO indoleamine 2,3-dioxygenase Atherosclerosis—P2 receptor-mediated effects on inflammatory cells Inflammatory cells express a large number of P2 receptors with multiple effects [102]. The final result is difficult to predict depending on the subtype of receptor expressed by a particular cell type and on the differentiation stage of the cell. The most important inflammatory cells for atherosclerosis are monocytes that differentiate into macrophages or dendritic cells in the plaque, and the T-helper and suppressor lymphocytes that coordinate the inflammatory reaction in the plaque [98]. A large number of P2 receptors are expressed on T lymphocytes and macrophages [14, 103] and have been suggested to be important players in atherosclerosis [14]. Strong evidence for a functional role exists for P2X7, P2Y2 and P2Y11 receptors. The P2X7 receptor is mitogenic and anti-apoptotic for T lymphocytes [104, 105]. P2X7 is important for release of interleukin (IL)-1 [106], tumour necrosis factor [107] and L-selectin, an adhesion molecule important for lymphocyte binding to endothelium [108]. All of these effects are known to be important for atherosclerosis development. The severity of arthritis is reduced in P2X7 receptor knockout mice [109] and rheumatoid arthritis is coupled to increased incidence of myocardial infarction. It would be of great interest to examine the P2X7 receptor knockout mouse in an atherosclerosis model. ATP and UTP are chemotactic for dendritic cells probably via the P2Y2 receptor and may attract inflammatory cells to the vascular lesion [110]. The P2Y2 receptor enhances the oxidative burst in human macrophages [111]. P2Y1 receptor deletion in knockout mice reduces the atherosclerotic lesions and the plaque area occupied by macrophages in ApoE knockout mice [112]. Whether this is due to platelet inhibition, endothelial or an inflammatory mechanism requires further studies. ATP inhibits CD4+ T cell activation via an increase in cyclic AMP (cAMP) probably via a P2Y11 receptor [113]. ATP acting on P2Y11 receptors regulates the maturation of human monocyte-derived dendritic cells and induces immunosuppression by inhibiting T-helper 1 cytokines and promoting T-helper 2 cytokines [114, 115]. A polymorphism in the P2Y11 receptor has been shown to have clinical importance by increasing the risk of myocardial infarction [116]. The G-459-A polymorphism, carried by one fifth of the population, causes an Ala-87-Thr substitution in the P2Y11 ATP receptor and increases the risk of myocardial infarction by 21%. The odds ratio increased stepwise depending on the number of Thr-87 alleles, and in subgroups in which the genetic influence is known to be of increased importance, family history of acute myocardial infarction (AMI), early onset AMI or the combined group of early onset AMI with family history. The mechanism by which the polymorphism causes AMI seems to be coupled to increased inflammation because the Thr-87 variant of the P2Y11 receptor was coupled to elevated C-reactive protein (CRP) levels. CRP is a marker of inflammation and an independent prognostic risk factor for the development of AMI [98]. Thus, the P2Y11 receptor is important in the development of atherosclerosis via modulation of inflammation either via effects on T lymphocytes or macrophage cells. Proinflammatory effects on the endothelium Even though P2 receptor-mediated activation of the endothelium stimulates release of NO, which inhibits inflammatory cells, it is now well established that important proinflammatory endothelial effects may also be triggered. A proinflammatory dysfunctional endothelium is crucial in the recruitment of monocytes to the atherosclerotic plaque and in the general extravasation and loss of peripheral resistance seen in sepsis. ATP stimulates neutrophil adherence to cultured endothelial cells [117]. ATP stimulates release of IL-6, IL-8, monocyte chemoattractant protein-1, growth-regulated oncogene α and increased expression of intercellular adhesion molecule-1 in human microvascular endothelial cells [118]. UTP and ATP stimulate expression of proinflammatory vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells through activation of the P2Y2 receptor and increases the adherence of monocytic cells to human coronary endothelial cells, an effect that was inhibited by anti-VCAM-1 antibodies [119]. VCAM is important for the recruitment of monocytes and lymphocytes. The VCAM stimulation is caused by P2Y2 receptor-induced transactivation of the vascular endothelial growth factor receptor-2 [120]. These effects have been confirmed in an in vivo neointima model, in which perivascular infusion of UTP enhanced infiltration by macrophages [121]. In conclusion, ATP and UTP stimulate several inflammatory responses known to be important for atherosclerosis development (Fig. 2). Restenosis and vascular smooth muscle cell proliferative disease VSMC proliferation contributes to plaque development, but it is probably beneficial by stabilising the plaque and forming a stronger fibrinous cap. However, there are several situations in which VSMC proliferation is important in vascular disease development: restenosis after balloon angioplasty, diabetic microvascular disease, chronic allograft rejection, pulmonary hypertension and possibly systemic hypertension. The latter is suggested since sympathetic nerves exert a trophic effect on vascular smooth muscle [122]. ATP is a cotransmitter from sympathetic nerves [25] and has been shown to be a more potent mitogen than the other cotransmitters NA and NPY [123]. Extracellular ATP is a potent growth factor for VSMC by activation of G protein-coupled P2Y receptors [12, 123–125]. UTP is also mitogenic, acting on P2Y2 receptors [123], as is UDP acting on P2Y6 receptors [124, 126, 127]. ATP is synergistic with polypeptide growth factors (e.g. platelet-derived growth factor, basic fibroblast growth factor) and insulin [123, 124]. The signal transduction is mediated via Gq proteins, phospholipase C and D, diacylglycerol, protein kinase C, extracellular signal-regulated kinase, phosphatidylinositol-3 kinase, MAPK/ERK activity kinase, mitogen-activated protein kinases and Rho [12, 128, 129]. Several immediate early genes are activated and the cell is taken through different phases of the cell cycle [125, 130]. Sometimes, as with UDP acting on P2Y6 receptors, progression is stimulated to both the S and G2 phases, that is through the whole cell cycle [127]. Mitogenic effects have been demonstrated in rat, porcine, and bovine VSMC and cells from human coronary arteries, aorta, and subcutaneous arteries and veins [12, 13, 124, 131]. The trophic effects on VSMC and the abundant sources for extracellular ATP in the vessel wall make a pathophysiological role probable in the development of atherosclerosis, neointima formation after angioplasty, chronic allograft rejection, pulmonary hypertension, diabetic microvascular disease and possibly hypertension. In these processes interaction between the VSMC and the matrix is important. It is therefore interesting that the human P2Y2 receptor contains an integrin-binding domain (RGD) in its first extracellular loop and that interaction with integrins influences P2Y2 receptor-mediated activation of G proteins [132]. ATP stimulates release of matrix metalloproteinase-2 (MMP-2) from human aortic smooth muscle cells and MMP-2 has been implicated in aortic aneurysm pathogenesis [133]. MMPs open up the matrix for migrating VSMC. ATP and UTP are potent chemotactic agents stimulating VSMC migration via P2Y2 and P2Y6 receptors [134]. These mechanisms, together with growth-stimulating effects, explain why perivascular infusion of UTP in a neointima model enhanced neointimal development [121]. Overexpression of the ATP- and UTP-degrading enzyme NTPDase1 reduces neointima development in rat aorta [135]. Augmentation of NTPDase1 activity is an important adaptive response for cardiac allograft survival, with increased inflammation, platelet deposition and infarction rates in NTPDase-deficient mice [136]. The mechanism is dependent on increased expression of the atherosclerotic matrix protein osteopontin [134, 137]. Diabetic microvascular disease Sympathetic vascular dysfunction in early experimental juvenile diabetes was recognised many years ago. Two weeks after induction of streptozotocin diabetes in rats, there was prejunctional impairment of sympathetic neurotransmission and impaired ATP-mediated endothelial function in mesenteric arteries [138]. Altered relaxant responses to ATP in the corpus cavernosum of men and rats with diabetes have also been reported [139]. As mentioned above, red blood cells may stimulate vasodilatation via release of ATP, which may be important in maintaining perfusion in the microcirculation. Interestingly, in erythrocytes from humans with type 2 diabetes ATP release is impaired, which is consistent with the hypothesis that the defect in erythrocyte physiology could contribute to the vascular disease associated with this clinical condition [140]. High extracellular glucose releases ATP and/or UTP in endothelial cells and pancreatic β cells [141, 142]. An increase in glucose from 5 to 15 mmol/l results in a marked increase in the proatherogenic nuclear factor of activated T cells signalling pathway in VSMC [143]. The effect is mediated via glucose-induced release of ATP and UTP, which subsequently activate P2Y2 but also P2Y6 receptors (after degradation to UDP). Thus, nucleotide release is a potential metabolic sensor for the arterial smooth muscle response to high glucose. Diabetic patients experience microvascular disease characterised by increased wall-lumen ratio, mainly because of an increase in VSMC and have higher rates of restenosis after coronary angioplasty. High glucose-induced release of extracellular nucleotides, acting on P2Y receptors to stimulate VSMC growth via NFAT (nuclear factor of activated T cells) activation, may provide a link between diabetes and diabetic vascular disease [143]. In conclusion, ATP/UTP/UDP stimulate VSMC growth, migration, release of MMPs and osteopontin that may contribute to the development of restenosis, diabetic microvascular disease, chronic allograft rejection, pulmonary hypertension and possibly systemic hypertension (Fig. 2). Vascular remodelling and angiogenesis Vascular remodelling P2 receptors may regulate VSMC phenotype and vice versa, and P2 receptor expression is markedly altered during phenotype changes [13]. The shift from a specialised contractile VSMC phenotype into a proliferating, matrix-producing, synthetic phenotype is a prerequisite for VSMC pathogenesis in vascular disease. Pacaud and co-workers found that the Ca2+-mobilising effects of 2-methylthioATP increased in VSMC during culture in serum, indicating upregulation of P2Y1 receptors in the transition from contractile to synthetic phenotype [144]. This was confirmed at the mRNA level where P2Y2 receptors were found to be upregulated, while P2X1 receptors were downregulated [145]. Mitogenic P2Y receptors are upregulated while ion channel receptors, with only contractile effects, are downregulated in the synthetic phenotype [145]. Growth factors, cytokines and interestingly also ATP are potent stimulators of P2Y2 receptor expression [146, 147]. Thus, factors of importance in the development of vascular disease increase mitogenic P2Y2 receptors; this is further supported by their upregulation in neointima after balloon angioplasty [148, 149]. ATP has a dual concentration-dependent effect on VSMC phenotype. Low ATP concentrations stimulate expression of genes specific for the contractile phenotype, while high ATP concentrations cause a phenotypic shift from the contractile to the synthetic phenotype, and this shift is dependent on a transient activation of protein kinase A, which inhibits activation of a serum response factor [150]. This previously unrecognised mechanism also appears to be important for the profound mitogenic effect of ATP. In intact human blood vessels examined in vitro, shear stress decreased contractile P2X1 receptor expression, but increased the expression of mitogenic P2Y2 and P2Y6 receptors in VSMC [151]. This mechanism could promote vascular growth and remodelling induced by shear stress as an adaptive response to increased flow. P2X4 receptor knockout mice are incapable of adaptive vascular remodelling, that is, a decrease in vessel size in response to a chronic decrease in blood flow [57]. Angiogenesis Very little is known about the role of P2 receptors in angiogenesis, but there are some studies suggesting their involvement. Ischaemia stimulates release of ATP [7, 60] and it has been shown to be a growth factor for endothelial cells [13, 152]. ATP and UTP stimulate cytoskeletal rearrangements with consequent cell migration of human endothelial cells [153] and ATP stimulates vasa vasorum neovascularisation in pulmonary arteries [154]. Newly developed vascular endothelia express very high levels of NTPDase1, also seen under hypoxic conditions [155]. NTPDase1 knockout mice exhibit disordered cellular migration and angiogenesis [156]. Varicose veins It has been suggested that cell lysis, consequent to P2X7 receptor-induced pore formation, contributes to the disorganisation and decrease in contractile myocytes in the media of varicose veins [157]. Upregulation of P2Y1 and P2Y2 receptors and downregulation of P2X1 receptors on smooth muscle of varicose veins is associated with a shift from contractile to synthetic and/or proliferative roles; this phenotypic change in smooth muscle leads to weakening of vein walls and may be a causal factor in the development of varicose veins [158], an interesting parallel to the receptor changes seen in response to increased shear stress (see above) [151]. Heart ATP is released in the heart as a cotransmitter together with catecholamines from sympathetic nerves, but it may also be released from other sources in the heart such as endothelium, platelets, RBC and ischaemic myocardium (Table 3) [7, 60, 159]. P2 receptors are abundantly expressed in the foetal human heart [160] as well as in the adult human heart [60, 161]. Inotropy In cardiomyocytes, ATP stimulates a pronounced positive inotropic effect and may also act in synergy with β-adrenergic agonists to augment myocyte contractility [60, 162–165]. ATP stimulates an increase in cytosolic calcium and evidence for the involvement of inositol 1,4,5-trisphosphate (IP3)-coupled P2Y2 receptors and ion channel P2X receptors has been presented [162, 164–167]. Ap4A, probably after degradation to ATP, increases force of contraction in human ventricular trabeculae [168]. The inotropic effects of ATP are dependent on both IP3 and cAMP [169]. The inotropic effects of catecholamines acting on β-receptors are mediated by an increase of cAMP and antagonists of these receptors are important drugs for the treatment of hypertension and reduce mortality in congestive heart failure. Similarly, ATP stimulates an increase in cAMP in cardiac myocytes and may act in synergy with the β1-adrenergic agonist, isoproterenol, by differential activation of adenylyl cyclase isoforms [60, 170, 171]. However, the ATP receptor mediating this increase in cAMP has not been related to any particular P2 receptor subtype in cardiac cells [60]. It could be the P2Y11 receptor that is additionally coupled to Gs and activates adenylyl cyclase [60, 172–174]. mRNA for the P2Y11 receptor has been detected in the human heart at high levels [161, 175]. The unstable agonist, UTP, has been shown to induce a positive inotropic effect in rat atria and in rat and guinea pig ventricular cardiomyocytes [164, 166, 176, 177]. Stable UTP and UDP analogues induce a pronounced inotropic effect on mouse cardiomyocytes [161]. The P2Y2 receptor is the most abundantly expressed receptor with very low levels of the P2Y4 receptor in the human heart [161], suggesting that the inotropic effects of UTP are mediated via P2Y2 receptors, while the UDP effects are mediated via the P2Y6 receptor [161]. These mechanisms are mediated via IP3-mediated signalling. In conclusion, the inotropic effects in man are mediated via P2Y2 and P2Y6 receptors and a P2Y11-like receptor (Fig. 3). P2X receptors are probably also involved, since several subtypes are expressed [60, 160], but it has not been possible to perform a clear receptor characterisation due to lack of selective antagonists. Fig. 3Functional roles of purines and pyrimidines acting on P2 receptors in the regulation of the heart. See text for details. a ATP, UTP and UDP exert inotropic effects on cardiomyocytes leading to increased cardiac output. b UTP and UDP stimulate hypertrophy of cardiomyocytes, while ATP can have apoptotic effects. c UTP protects against ischaemic injury and cardiomyocyte cell death. ATP-degrading enzymes preserve endothelial integrity and protect against allograft rejection. NTPDase nucleoside triphosphate diphosphohydrolase Myocardial infarction Using microdialysis, ATP in the interstitial space has been estimated to be 40 nmol/l, but the levels may increase markedly during electrical stimulation, ischaemia, challenge with cardiotonic agents, increase in blood flow, mechanical stretch and increased work load [60]. ATP is released from cardiomyocytes during reduced oxygen tension [60]. UTP and ATP are released from the heart during cardiac ischaemia [178] and patients with myocardial infarction have higher plasma levels of both ATP and UTP [161, 179]. There is a significant increase in ectonucleotidase activity (NTPDase1) in the hearts of patients with ischaemic heart disease [180] that could represent a compensatory mechanism against increased nucleotide levels during chronic ischaemia. Ischaemia-reperfusion provokes barrier failure of the coronary microvasculature, leading to myocardial oedema, ATP may protect against reperfusion-induced coronary endothelial barrier damage and inhibition of ATP degradation enhances the stabilising effect of ATP on barrier function [181]. The common P2Y11 ATP receptor polymorphism, Ala-87-Thr, is associated with both increased CRP and increased risk of developing myocardial infarction [116]. Based on this association, we hypothesise that the P2Y11 receptor plays an important role in cardiovascular biology and in inflammatory disease (see above), causing myocardial infarction via a proinflammatory effect. However, increased inotropic effects may also contribute. An interesting yin and yang situation for ATP and UTP has been revealed regarding hypertrophic effects. UTP but not ATP causes hypertrophic growth in neonatal cardiomyocytes [182] (Fig. 3). In contrast, ATP inhibits hypertrophy and may even induce apoptosis and necrosis [163, 183]. The reason for the difference in effects could be due to activation of P2X receptors or cAMP stimulation by ATP [60, 161]. Both UTP and ATP transactivate epidermal growth factor receptors, but only ATP stimulates the hypertrophic marker genes atrial natriuretic peptide and myosin light chain 2 [184]. Similarly, UTP but not ATP protects cultured cardiomyocytes against hypoxic stress [185]. Since UTP is released during preconditioning [178], a role for UTP in the protective effects of preconditioning is plausible. Recently, Yitzhaki and co-workers were able to demonstrate prominent reductions in myocardial infarction size and improved rat heart function in vivo, by a single intravenous bolus dose of UTP before ischaemia [186] (Fig. 3). Congestive heart failure There are several reports of alterations or adaptations of purinergic signalling during congestive heart failure. The positive inotropic effect of ATP is impaired in heart failure, but reversed by the angiotensin-converting enzyme (ACE)inhibitor imidapril [187]. The contractile responses for the P2Y11 receptor agonist AR-C67085 are decreased in heart failure, suggesting a downregulation of this receptor function in cardiomyocytes in a similar manner as seen for β1-receptors in congestive heart failure [169]. In human hearts, only the P2X6 receptor was altered in congestive heart failure [188]. In congestive heart failure P2X1 receptors are downregulated in VSMC in resistance arteries, which could represent a protective response against the increased sympathetic nerve activity and peripheral resistance seen in congestive heart failure [189]. Several P2 receptors have been suggested as targets for pharmacological treatment of congestive heart failure. Overexpression of P2X4 receptors has a beneficial, life-prolonging effect in a heart failure model [190]. Both ATP and catecholamines are released from sympathetic nerves, acting through cAMP-stimulating receptors to mediate positive inotropic effects, stimulating the same intracellular mechanisms as adrenergic β-receptors. It is possible that agonists for the P2Y11 receptor could be used to improve cardiac output in patients with circulatory shock. However, an even more important drug candidate would be a P2Y11 receptor antagonist that may be beneficial in patients with congestive heart failure. The extracellular pyrimidines UTP and UDP may be inotropic factors in man, acting on P2Y2 and P2Y6 receptors stimulating the same intracellular pathways as angiotensin II. Synthetic agonists could thus be used as inotropic agents during circulatory shock and antagonists may have effects similar to angiotensin II receptor blockers, being beneficial in the treatment of hypertension and congestive heart failure. Chronotropy and arrhythmia It has been difficult to determine the chronotropic effects of ATP due to the dominating inhibitory effects of adenosine on the AV node. We know that ATP increases the contractile rate in neuron-myocyte co-cultures but the effect is markedly reduced in non-innervated myocyte cultures [191]. ATP could induce arrhythmia based on its increased automaticity and early after-depolarisations [60]. The P2X1 receptor is colocalised with connexin 43 in gap junctions that transmit the contractile stimulus between cardiomyocytes [192]. However, so far no firm evidence exists of an important role for P2 receptors for chronotropy or arrhythmia. Platelets and coagulation Platelets ADP, released from erythrocytes or produced after ectonucleotidase breakdown of ATP released from erythrocytes and endothelial cells, was found to cause platelet aggregation as early as 1961, before the concept of P2 receptors was conceived [193]. Later, the effects were attributed to a single receptor, designated P2T, but it was not until 1998 that the three receptors involved were characterised [194–196]. Two ADP receptors (P2Y12 and P2Y1) and one ion channel ATP receptor (P2X1) are expressed on platelets [197, 198] (Fig. 4). The P2Y12 receptor is coupled to inhibition of cAMP. The molecular identifications of the P2Y12 receptor and generation of knockout mice revealed highly prolonged bleeding times, and their platelets aggregate poorly in response to ADP and display a reduced sensitivity to thrombin and collagen [199]. The P2Y1 receptor is responsible for ADP-induced shape change and weak, transient aggregation [200–202], while the P2Y12 receptor is responsible for the completion and amplification of the response to ADP and to all platelet agonists, including thromboxane A2, thrombin and collagen. The platelets also express P2Y1 receptors that have been shown in knockout mice to be of similar importance as P2Y12 receptors [200, 203]. Fig. 4P2 receptor-mediated regulation of coagulation and platelet aggregation. See text for details. ADP is an important mediator and positive feedback mechanism for platelet aggregation. Activated platelets stimulate coagulation, i.e. formation of a fibrin network. In contrast, ATP, ADP and UTP release tissue plasminogen activator (t-PA) from endothelial cells, resulting in degradation of the fibrin network. At the same time plasminogen activator inhibitor (PAI-1) is also released from endothelial cells by ATP stimulation, in turn inhibiting t-PA. Thus, P2 receptors regulate several balancing factors in haemostasis. Ado adenosine The physiological importance of the P2X1 receptor is not fully elucidated. However, recent studies have demonstrated that P2X1 receptors can generate significant functional platelet responses, independently and in synergy with other receptor pathways. In addition, studies in transgenic animals indicate an important role for P2X1 receptors in platelet activation, particularly under conditions of shear stress and thus during arterial thrombosis [204]. Synergistic interaction between ATP and NA in stimulating platelet aggregation may have significant clinical implications and suggests a prothrombotic role for ATP in stress [205]. NTPDase1 is an important inhibitor of platelet activation. Unexpectedly, NTPDase1-deficient mice had prolonged bleeding times with minimally perturbed coagulation parameters. Platelet interactions with injured mesenteric vasculature were considerably reduced in vivo and purified mutant platelets failed to aggregate to standard agonists in vitro [206]. This platelet hypofunction was reversible and associated with P2Y1 receptor desensitisation. In keeping with deficient vascular protective mechanisms, fibrin deposition was found at multiple organ sites in NTPDase1-deficient mice and in transplanted cardiac grafts. Endothelial ADPase activity is lost following ischaemia-reperfusion injury, xenograft rejection and inflammation resulting in increased levels of ATP and ADP [58, 207]. This may cause platelet aggregation but also other effects, such as mitogenic effects on VSMC. Infusion of systemic apyrase inhibits platelet aggregation and prolongs xenograft survival [208]. This has also been shown with adenoviral transfer of NTPDase1 [209]. NTPDase1 is also lost in vascular cardiac grafts subjected to oxidant stress. NTPDase2 only degrades ATP to ADP and has a prothrombotic effect. It is present in the subendothelium and can shift the balance in a prothrombotic direction after endothelial injury [210] (Fig. 1). ADP acting on P2Y12 receptors is not only important for platelet activation, it also stimulates vasoconstriction. Stable drugs with antagonistic effects on P2Y12 receptors, affecting both platelets and VSMC, could be of double therapeutic benefit in their prevention of both thrombosis and vasospasm [46]. Coagulation The prothrombotic effect of ADP on the platelets is counteracted by the effects of UTP and ATP on the endothelium that stimulates a substantial release of tissue-type plasminogen activator (tPA) with fibrinolytic effects [51, 211] (Fig. 4). To make the picture even more complex, ATP also releases plasminogen activator inhibitor (PAI-1) which in turn inhibits tPA [212]. Thus, P2 receptors are involved at several stages of haemostasis, which is important for the development of atherosclerosis and thrombotic occlusions leading to myocardial infarction and stroke. Clinical importance of P2 receptor-mediated regulation of platelets The first ADP inhibitors, ticlopidine and clopidogrel, were developed before the cloning and identification of the platelet ADP receptors. Later, it was concluded that they were prodrugs, converted in the liver to a metabolite that binds irreversibly to the P2Y12 receptor resulting in non-competitive antagonism. Ticlodipine has the disadvantage of a small risk of neutropaenia, so that clinically so far the most important contribution of drug development aimed at P2 receptors has been the beneficial effects of the platelet ADP receptor antagonist clopidogrel in atherosclerotic disease. In the CAPRIE trial, clopidogrel was even more effective compared to aspirin in preventing myocardial infarctions [213]. Clopidogrel, as an addition to aspirin, turned out to be necessary to prevent acute in-stent thrombosis in percutaneous coronary interventions. The CURE trial demonstrated that the addition of clopidogrel to aspirin reduced clinical events by 20% in acute coronary syndromes [213, 214]. This has also been shown for ST-elevation myocardial infarctions (COMMIT, CLARITY). However, long-term secondary prevention in high-risk groups with clopidogrel added to aspirin did not have significant benefits (CHARISMA). Clopidogrel combined with aspirin reduces cerebral emboli in patients undergoing carotid endarterectomy [215]. Clopidogrel (Plavix®) is now a blockbuster drug and one of the most sold drugs worldwide to the benefit of atherosclerotic patients. Nevertheless, clopidogrel is a rather weak antagonist at P2Y12 receptors with variable effects, often referred to as clopidogrel resistance. Newer P2Y12 receptor antagonists, such as prasugrel and AZD6140, have a stronger more consistent effect and are currently being studied in major clinical trials (TRITON and PLATO). Clinical importance of purinergic signalling in cardiovascular disease: future directions The P2 receptor family provides opportunities for drug development (see Table 4). The large number of receptor subtypes and the increasing knowledge of their tissue distributions may make it possible to target a specific cardiovascular organ with limited side effects. So far, only P2Y12 receptor antagonists have been explored in patients, but with extraordinary success. Clopidogrel is one of the most prescribed drugs in the world for the treatment of thrombosis, stroke and myocardial infarctions in millions of patients. The following compounds, prasugrel and AZD6140, are even more potent P2Y12 receptor antagonists with clinical benefits currently being tested in phase III trials. Improved platelet inhibition can also be achieved with antagonists against the other two P2 receptors on platelets. Studies in P2Y1 and P2X1 receptor knockout mice and experimental thrombosis models using selective P2Y1 and P2X1 receptor antagonists have shown that, depending on the conditions, these receptors could also be potential targets for new antithrombotic drugs. Table 4Cardiovascular disease targetsReceptor targetAgonist/antagonist activity of therapeutic drugDiseaseVascular smooth muscle cellP2Y2AntagonistHypertension, restenosis, diabetic microvascular disease, subarachnoidal bleedingP2Y6AntagonistHypertension, restenosis, diabetic microvascular disease, subarachnoidal bleedingP2Y12AntagonistHypertension, vasospasmP2X1AntagonistHypertension, migraine, subarachnoidal bleedingP2Y6AgonistMigraineSolNTPDase1EctonucleotidaseRestenosis, xenograft rejectionEndothelial cellP2Y2AntagonistAtherosclerosis, inflammationP2X4AntagonistVascular remodelling (cancer?), preeclampsiaHeartP2Y2AntagonistCongestive heart failureP2Y6AntagonistCongestive heart failureP2Y11AntagonistCongestive heart failure, myocardial infarctionP2Y2AgonistShock, reduced cardiac output, cardiac protectionP2Y6AgonistShock, reduced cardiac outputP2Y11AgonistShock, reduced cardiac outputSolNTPDase1EctonucleotidaseHeart transplantation, myocardial infarctionInflammatory cellsP2Y2AntagonistAtherosclerosisP2Y11AntagonistAtherosclerosisP2X7AntagonistAtherosclerosisPlateletsP2Y1AntagonistMyocardial infarction, stroke, peripheral artery diseaseP2Y12AntagonistMyocardial infarction, stroke, peripheral artery diseaseP2X1AntagonistMyocardial infarction, stroke, peripheral artery diseaseSolNTPDase1EctonucleotidaseMyocardial infarction, stroke, peripheral artery diseaseRed blood cellP2Y13AntagonistImproved peripheral circulationP2X7AntagonistShockPerivascular nociceptive sensory nervesP2Y1AgonistMigraine, vascular painP2X3AntagonistMigraine, vascular painPerivascular sympathetic nervesP2Y1AgonistHypertensionP2X2AntagonistHypertensionThe table presents possible targets for the development of new pharmaceutical compounds for the treatment of cardiovascular diseases ATP, UTP and UDP, acting on P2Y2 and P2Y6 receptors, are potent stimulators of inflammation, VSMC proliferation and migration. Antagonists to these receptors may be explored to protect against atherosclerosis, restenosis after balloon angioplasty, chronic transplant rejection and diabetic microvascular disease. It is possible that hypertension and pulmonary hypertension could be treated with P2X1, P2Y2, P2Y6 or P2Y12 receptor antagonists. In the heart P2Y2, P2Y6 and P2Y11 receptor antagonists may have similar beneficial roles as angiotensin inhibition or β-blockers in the treatment of congestive heart failure. In the acute phase of a myocardial infarction, a UTP analogue could protect cardiomyocytes and limit infarct size. A polymorphism in P2Y11 receptors could be considered as a prognostic marker to identify patients at risk of myocardial infarction and drugs aimed at the receptor could be explored to prevent myocardial infarction. A promising strategy would be to use soluble NTPDase to prevent thrombosis, restenosis and xenograft transplantation. SolNTPDase1 protects against brain damage in a stroke model and a human genetically engineered solNTPDase has been developed that could be used in clinical trials. Possible clinical indications could be stroke, myocardial infarction and transplantation. P2X3 receptor antagonists could be used against migraine and vascular pain [see 19, 26].

Osteoporos_Int-3-1-1915640

Vitamin K2 supplementation improves hip bone geometry and bone strength indices in postmenopausal women

Summary Vitamin K mediates the synthesis of proteins regulating bone metabolism. We have tested whether high vitamin K2 intake promotes bone mineral density and bone strength. Results showed that K2 improved BMC and femoral neck width, but not DXA-BMD. Hence high vitamin K2 intake may contribute to preventing postmenopausal bone loss. Introduction Since the discovery of osteocalcin, the vitamin K-dependent protein synthesized by bone tissue, attempts have been made to correlate bone vitamin K status with bone strength. The way in which these variables were measured varied in different studies. Obviously, the best way to assess bone strength is monitoring fracture incidence in a large study cohort over a long period of time. Unfortunately, this method is expensive and time consuming. Therefore, most authors have used bone mineral density as measured by DXA (DXA-BMD) as a surrogate marker for bone strength. A disadvantage of this technique is that it gives the amount of calcium per area, but does not take into account the three dimensions of the bone [1]. Findingreliable markers for bone vitamin K status turned out to be even more difficult. Dietary vitamin K may occur in different forms: K1 (also known as phylloquinone) and K2, which is a group name for a series of related compounds also known as menaquinones [2]. The various menaquinones differ structurally in the length of their isoprenyl side chain, which may contain four isoprenoid residues in menaquinones-4 (MK-4) up to nine residues in menaquinones-9 (MK-9). Still higher menaquinones have been described, but are increasingly rare in the diet. Dietary K1 originates mainly from green, leafy vegetables, whereas menaquinones are produced by bacteria and are mostly found in fermented foods such as cheese and the Japanese food natto (fermented soy beans) [3, 4]. Synthetic forms of vitamin K available in food supplements or pharmaceutical preparations are K1 and MK-4, recently also the natural MK-7 has become available. The recommended daily intake of vitamin K (all forms) is between 100 and 120 μg/day, but this value is based on the hepatic vitamin K requirement for blood clotting factor synthesis. The requirements of other tissues have not yet been defined, but are probably higher. In 1984, Hart and coworkers described a case-control study in which patients with femur neck fractures had lower circulating vitamin K1 concentrations than age- and sex-matched controls [5]. In a second study from the same authors it was found that subjects with low serum vitamin K1 concentrations had a lower DXA-BMD than those with normal or high serum vitamin K1 [6]. A potential bias in these studies is that low circulating vitamin K may be a marker for general poor nutritional status and that low DXA-BMD and increased fracture risk are associated with the combined deficiency of essential minerals, vitamins and proteins rather than with vitamin K alone. Others have published population-based studies in which an inverse correlation was found between fracture risk and vitamin K1 intake [7, 8]. It was found that the association between vitamin K1 intake and fracture risk was much stronger than that between vitamin K1 intake and DXA-BMD [9]. A drawback of these studies is that no attempts were made to also monitor vitamin K2 intake. Although in the western society K2 forms only 10–20% of the total vitamin K intake [3, 4], it is absorbed from the food matrix much better than K1 and may cover 50% of the total vitamin K absorbed. Moreover, it has been demonstrated that vitamin K2 rather than K1 is preferentially taken up by extrahepatic tissues such as bone and arteries [10]. Hence also vitamin K1 intake seems to be a questionable marker for bone vitamin K status. Possibly the best marker available today is circulating undercarboxylated osteocalcin (ucOC). Either the absolute concentration, the ratio between ucOC and total osteocalcin, or the ratio between ucOC and carboxylated osteocalcin (cOC) are used in the literature. The group of Delmas [11–13] and Luukinen et al. [14] reported a number of independent studies suggesting a strongly increased fracture risk in populations with elevated serum ucOC; later studies by the French group [15] and others [9, 16] showed that ucOC was inversely associated with DXA-BMD. It is at least remarkable that in all approaches, associations between vitamin K status and fracture risk were demonstrated more readily than those between K status and DXA-BMD. In a supplementation study with vitamin K1 alone no effect on DXA-BMD was observed during a 2-year treatment with 10 mg/day [17]. In another study from the same group, however, a synergistic effect of 1 mg/day of K1 with minerals and vitamin D [18] was found. In this study the rate of bone loss in postmenopausal women was reported to be 35% lower in the presence of minerals and vitamins K1 and D as compared to the group receiving minerals and vitamin D without vitamin K. A number of Japanese studies reported variable effects of vitamin K2 alone, but substantial beneficial effects if combined with vitamin D [19]. It has been reported by many different authors that in the healthy population serum osteocalcin is incompletely carboxylated, which is indicative for poor vitamin K status of bone [11, 14, 16, 20]. Based on our present knowledge of vitamin K-dependent proteins, it must be expected that non-carboxylated osteocalcin is not functional. Since the molecular function of osteocalcin has remained unclear thus far, we do not know whether it contributes to bone strength at all. From transgenic osteocalcin-deficient mice, we know that osteocalcin contributes to regulating the form and the dimensions of bone [21]. This may also be important for humans. The importance of bone geometry for bone strength is rapidly gaining interest [22–26]. Although DXA-BMD is still the determinant most generally used in the clinical evaluation of hip fracture risk, it has been stipulated that its uncritical use may lead to size-related artifacts in the estimation of bone strength and in the identification of fracture risk [27]. As was mentioned by Heaney [28], the ultimate concern in studying bone status is bone strength. Holding other variables constant, strength will increase both as bone mass increases and as bone size increases. When estimating bone strength, two strategies to also compensate for the dimensions of bone have been proposed. First, it is encouraged that densitometric comparisons between groups are based on bone mineral content (BMC) rather than on DXA-BMD [28]. Second, bone dimensions are used as independent determinants for bone strength. Important geometric parameters are the hip axis length (HAL) and the femoral neck width (FNW). Patients with low DXA-BMD or after experiencing a hip fracture had an increased FNW, suggesting an attempt to compensate for the increased fracture risk at this critical site [26]. On the other hand, it seems obvious that at comparable DXA-BMD a larger FNW will positively contribute to bone strength. In this way it is understandable that also and increase of BMC may contribute to bone strength, although it should be reminded that it is not the mass per se but the distribution of mass that is crucial for bone strength. The importance of combining bone geometry with DXA-BMD for expressing bone strength and fracture risk was realized by Karlamangla et al., who developed mathematical equations for calculating indices for compression strength, bending strength, and impact strength of the femoral neck [29]. In these equations, DXA-BMD is combined with HAL, FNW, height and body weight. We have used the same equations to analyse the data from a placebo-controlled intervention trial in which we compared the effects of vitamin K2 on DXA-BMD and bone strength indices in postmenopausal women, who were recruited from the apparently healthy population and thus represent the large group of free living elderly subjects. Additional or synergistic effects of calcium and vitamin D were not included in the design of our study. Subjects and methods Subjects The study was designed for apparently healthy, non-osteoporotic women. Participants were recruited by local newspapers. Inclusion criteria were female gender, age between 55 and 75 years at intake, Caucasian race, apparently healthy, at least 2 years postmenopausal and willingness to sign informed consent. Exclusion criteria were: a history of metabolic bone disease(s) or recent bone fractures (less than one year), low bone density (T-score < −2.5), ovariectomy, hysterectomy, oral anticoagulant treatment, hormone replacement therapy, treatment with bisphosphonates, calcitonin, prednisone, heparin, or vitamin K-containing vitamin concentrates and food supplements. Also subjects who had received an investigational new drug within the last 12 months were excluded from the trial. In total 325 women met the criteria and were randomized into our study. The participants were pre-stratified according to age: 105 in the age of 55–65 years, and 220 in the age of 65–75 years. The study protocol was approved by the University Hospital medical ethics committee. Masking Participants were identified by a single randomization number according to the randomization schedule generated by the University Hospital Pharmacy using a computer-generated random permutation procedure in the software package SPSS. Participants were randomly assigned to treatment with either 45 mg/day of MK-4 (menatetrenone, EISAI Co, Tokyo, Japan) or placebo, and compliance with treatment was decided from pill counts during home visits. The daily dose of MK-4 was given in three capsules of 15 mg each, which had to be taken at three time points spread over the day, preferably after the meal. The participant randomization codes were allocated sequentially in the order in which the participants were enrolled. After completion of all analyses the randomization code was disclosed to the investigator. Measurements The clinical end points chosen to evaluate the effect of the study treatment on bone were bone mineral density (DXA-BMD), bone mineral content (BMC) and bone strength indices of the femoral neck. DXA-BMD and BMC were measured at baseline and after 1, 2 and 3 years of treatment by dual energy X-ray absorptiometry (DXA) at the site of the left total hip and femoral neck, as well as the vertebrae (L2-L4) using a Hologic QDR 4500-A (Waltham MA, USA). The in vivo precision (CV) after repeated measurements in 10 healthy adults (30–40 years old) was 0.85% for the spine and 0.78% for the femur neck in short term (one day interval) and 1.03% and 0.98% in long term (one-year interval) precision measurements. The long-term precision of FNW assessment was 1.53%. At each time point, standing height and body weight were determined with standardized equipment and the body mass index (BMI) was calculated as the weight in kilograms divided by the square of the height in meters. The daily calcium intake was assessed using a validated food frequency questionnaire. Indices for compression strength (CSI), bending strength (BSI) and impact strength (ISI) at the site of the femoral neck were calculated from the mean femoral neck width (FNW) and hip axis length (HAL), together with height, weight and femoral neck DXA-BMD as described by Karlamangla et al. [29]. The mean FNW was obtained from the 1.5 cm wide femoral neck region of interest (area (cm2)/1.5 (cm)). The HAL reflects the distance along the femoral neck axis from the lateral margin at the base of the greater trochanter to the inner pelvic brim. The equations used were: Sample collection Fasting blood was taken and 2-h fasting urine was collected at baseline and after 3, 6, 12 and 36 months of treatment. After blood was taken, it was allowed to clot at room temperature for half an hour. Serum was prepared after centrifugation at 3,000 rpm, during 15 minutes. It was subdivided in small aliquots and kept at −80°C until use. Urine was collected as the second morning void after an overnight fast. Biochemical measurements In serum the following markers were tested, all based on enzyme immuno assays (EIA): total osteocalcin (t-OC, BioSource Europe SA); under-carboxylated osteocalcin (uc-OC) and carboxylated osteocalcin (c-OC) both obtained from Takara Shuzo Co Ltd., Shiga, Japan; bone-specific alkaline phosphatase (BAP, Alkphase B, Quidel/Metra Biosystems San Diego, CA, USA); N-telopeptides of type I collagen (sNTX, Osteomark sNTx, Ostex International, Inc., Seattle, WA, USA) and 25-Hydroxy vitamin D (25-OH D, OCTEIA 25-hydroxy vitamin D, IDS, UK). In the urine we measured free deoxypyridinoline by using an enzyme immunoassay (DPD, Quidel/Metra Biosystems). Urinary calcium (Ca) and creatinine (creat) were assessed according to standard methods. Dpd and Ca were corrected for the urinary creat concentration. Statistics The required numbers of participants in both age groups were assessed using separate power analyses (power of 90% and a significance level of 0.05). In the younger age group it was assumed that in the placebo group the mean bone loss is approximately 1.5% (4.5% in 3 years). The lowest detectable effect was set at an improvement of 30% (=3.15% bone loss after 3 years). Taking into account a drop-out frequency of 12% per year this group should consist of at least 76 subjects. In the older age group the mean bone loss was assumed to amount 1% per year (3% in 3 years). With a lowest detectable effect of 30% (=2.1% bone loss in 3 years) and a drop-out frequency of 19% per year, the minimal size of this group is 218 subjects. Statistical analysis was performed using the statistical package SPSS (SPSS Corp, Chicago, IL). The calculations are based on the intention-to-treat principle. Drop-outs were included in the analyses, with the last data available in all subsequent measuring points. All follow-up measurements were related to the baseline values and the proportional changes were calculated for each subject. Differences between and within the different groups were assessed with the unpaired and paired Student’s t-test, respectively. All data are given as mean values ±SE. The linear regression model was used to investigate the effect of treatment after adjustment for potential confounders. Change of FNW, HAL and bone strength indices after 3 years of treatment were used as dependent variables and age and BMI as independent variables. Results Baseline measurements and follow-up Three hundred fifty-one women were recruited for the study, 26 of whom were excluded before randomization for different reasons. After randomization there were 161 women receiving MK-4 and 164 women receiving placebo. The MK-4 group consisted of 54 women younger than 65 years of age and 107 women older than 65 years of age; in the placebo group these numbers were 51 and 113, respectively. The baseline characteristics of all participants in the placebo and MK-4 group are given in Table 1. No significant differences were found between both groups. However, when comparing the two age groups there was an age-related increase in weight and BMI and an age-related decrease in height (Table 2). DXA-BMD and BMC in the older age group were significantly lower at the site of the femoral neck, but not at the total hip and spine. From all serum markers for bone metabolism only BAP was higher in the older age group (borderline significant). Although there was an age-related decrease in serum vitamin D concentration, all participants had levels which were within the normal range at baseline (Tables 1 and 2) and at all subsequent measurements (data not shown). Urinary DPD/creat was markedly lower in the older age group compared to the younger group. Table 1Baseline characteristics of all participants subdivided according to treatment Placebo n = 164MK-4 n = 161P-valueAnthropomorphic variables:Age (years)66.0 ± 0.565.9 ± 0.40.9Years since menopause17.7 ± 0.717.2 ± 0.60.6Weight (kg)71.8 ± 1.070.3 ± 0.90.3Height (cm)162 ± 0.5161 ± 0.50.2BMI (kg/m2)27.3 ± 0.327.1 ± 0.40.7Calcium intake (mg/day)811 ± 26870 ± 320.1Non-smoking (%)87871.0Bone density characteristics:DXA-BMD (g/cm2) of: femoral neck0.688 ± 0.0070.706 ± 0.0080.1 Total hip0.842 ± 0.0090.861 ± 0.0080.1 Lumbar L2-L40.931 ± 0.0120.929 ± 0.0130.9BMC (g) of: femoral neck3.54 ± 0.043.65 ± 0.040.06 Total hip30.6 ± 0.4331.4 ± 0.390.2 Lumbar L2-L443.1 ± 0.7242.4 ± 0.760.6Hip geometry:Mean FNW (cm)3.43 ± 0.023.45 ± 0.0170.4HAL (cm)11.6 ± 0.0711.7 ± 0.060.2Bone strength indices:CSI (g/kg.m)3.41 ± 0.0553.52 ± 0.0520.1BSI (g/kg.m)1.00 ± 0.0171.04 ± 0.0170.2ISI (g/kg.m)0.24 ± 0.0040.26 ± 0.0040.03Serum markers for bone metabolism:c-OC (ng/mL)6.1 ± 0.26.2 ± 0.21.0uc-OC (ng/mL)3.3 ± 0.23.3 ± 0.20.4t-OC (ng/mL)13.2 ± 0.413.2 ± 0.50.7BAP (ng/mL)23.1 ± 0.723.6 ± 0.80.2NTX (nM)10.8 ± 0.210.7 ± 0.30.525-OH D (nM)67.2 ± 2.372.3 ± 2.60.09Urine markers for bone metabolism:Creatinine (mmol/L)3.5 ± 0.33.6 ± 0.21.0DPD / creat (μmol/mol)7.4 ± 0.27.5 ± 0.20.7Calcium / creat (mol/mol)0.29 ± 0.020.29 ± 0.010.7Only the BMC and the impact strength index of the femoral neck in the MK-4 group were slightly higher than in the placebo one. This difference is accounted for by expressing MK-4-induced changes as a percentage of the baseline values. All data are given ± SE.Table 2Baseline characteristics of participants subdivided according to age Age 55–65 n = 105Age 65–75 n = 220P-valueAnthropomorphic variables:Age (years)59.0 ± 0.369.3 ± 0.2Years since menopause9.7 ± 0.521.2 ± 0.4Weight (kg)67.4 ± 1.072.8 ± 0.80.0001Height (cm)163 ± 0.6161 ± 0.40.003BMI (kg/m2)25.4 ± 0.428.1 ± 0.30.0001Calcium intake (mg/day)816 ± 34858 ± 270.4Non-smoking (%)88870.9Bone density characteristics:DXA-BMD (g/cm2) of: femoral neck0.716 ± 0.0100.689 ± 0.0060.02Total hip0.871 ± 0.0120.842 ± 0.0070.03Lumbar L2-L40.940 ± 0.0150.925 ± 0.0110.4BMC (g) of: femoral neck3.69 ± 0.063.55 ± 0.030.02Total hip31.4 ± 0.630.7 ± 0.30.3Lumbar L2-L444.0 ± 0.942.1 ± 0.60.1Hip geometry:Mean FNW (cm):3.44 ± 0.0223.44 ± 0.0160.9HAL (cm):11.8 ± 0.0811.6 ± 0.060.03Bone strength indices:CSI (g/kg m):3.75 ± 0.0683.31 ± 0.0390.0001BSI (g/kg m):1.09 ± 0.0220.98 ± 0.0130.0001ISI (g/kg m):0.27 ± 0.0050.24 ± 0.0030.0001Serum markers for bone metabolism:c-OC (ng/mL)5.9 ± 0.26.3 ± 0.20.2uc-OC (ng/mL)3.3 ± 0.23.2 ± 0.10.8t-OC (ng/mL)13.9 ± 0.612.8 ± 0.40.1BAP (ng/mL)21.8 ± 0.824.1 ± 0.70.05NTX (nM)11.0 ± 0.310.6 ± 0.20.225-OH D (nM)76.5 ± 2.766.3 ± 2.30.005Urine markers for bone metabolism:creatinine (mmol/L)3.5 ± 0.33.6 ± 0.20.8DPD / creat (nmol/mmol)8.2 ± 0.27.1 ± 0.20.0001Calcium / creat (mmol/mmol)0.29 ± 0.020.29 ± 0.010.9The differences in all age-related variables were highly significant. All data are given±SE. After 3 years 257 women had completed the study: 133 women in the MK-4 group and 124 women in the placebo group. In total 68 women stopped during the study. This was 21% of the total. The largest group of participants (n = 30) stopped during the first 3 months of the trial. The most important reasons for discontinuation in this time period were various health problems (n = 18), low DXA-BMD (n = 17), lack of motivation (n = 10) and complaints about the capsules (n = 9). Subjects who stopped because of low DXA-BMD did so after consultation during the trial with their general practitioner; there were two complaints about the capsules in the MK-4 group and seven in the placebo group. Two participants died during the trial: one brain tumor and one cerebrovascular accident. Both casualties were in the placebo group. In total, 36 negative or positive side effects were mentioned by the participants. In the MK-4 group 16 women experienced negative effects and four experienced positive effects of the capsules; in the placebo group the numbers were 15 and one, respectively. The most frequent negative effects were gain of weight and gastro-intestinal complaints. However, there was no difference in total numbers of complaints between the MK-4 and the placebo group. Effect of MK-4 supplementation Effects on serum markers (if detectable) were maximal after 12 months of treatment, with no further increase of the difference from placebo at 36 months. In Fig. 1 we have plotted the changes of bone markers in serum during the first year. As was to be expected, a statistically significant effect of vitamin K treatment was observed on the serum concentrations of cOC and ucOC. The markers for bone formation (tOC and BAP) were also significantly higher in the MK-4 group as compared to the placebo one. No effect was observed on markers for bone resorption, or on DXA-BMD at any of the sites measured (Fig. 2a). Also urinary calcium excretion was not affected by MK-4 treatment (data not shown). It turned out that in the MK-4 group the BMC of the femoral neck decreased at a significantly lower rate than in the placebo one (Fig. 2b), and that this effect was paralleled by a significant increase in the femoral neck width, suggesting bone accrual at this critical site (Fig. 2c). The hip axis length (HAL) seemed to slightly increase with age in an MK-4 independent manner. From the DXA-BMD, the FNW and the HAL we have calculated the various indices for strength of the femur neck, and it resulted that MK-4 had a significant effect on all three indices (Fig. 2d). Notably the bending strength remained almost constant after the three years of intervention. Fig.1EffectofMK-4 on markers for bone metabolism during the first year of treatment. Bone formation markers are presented in graphs a–d (cOC, ucOC, tOC and BAP, respectively) and bone resorption markers in graph E and F (NTX and DPD/creat respectively). Closed symbols: MK-4; open symbols: placebo. Error bars represent SE. Significance of differences MK-4 compared to placebo: #: p < 0.05; ##: p < 0.005Fig. 2Effects of MK-4 on bone after 3 years of treatment. All data are expressed as a percentage of the respective baseline values. Graph A shows the data of DXA-BMD of the femoral neck (DXA-BMDFN), total hip (DXA-BMDtotalhip) and lumbar spine (DXA-BMDLS); graph B shows the BMC of the femoral neck (BMCFN), total hip (BMCtotalhip) and lumbar spine (BMCLS); graph C shows the femoral neck width (FNW) and hip axis length (HAL) and graph D shows the indices of the compression strength (CSI), bending strength (BSI) and impact strength (ISI). Open bars: placebo; hatched bars: MK-4. Error bars represent SE. Significance of change from baseline: *: p < 0.05, **: p < 0.005; significance of difference MK-4 compared to placebo: #: p < 0.05, ##: p < 0.005 Using a linear regression model, we have tested the statistical significance of the MK-4 induced effects. As is shown in Table 3, the differences in FNW, CSI, BSI and ISI between the MK-4 and placebo group were significant, both before and after adjustment for age and BMI. The effects on BMC, FNW and bone strength indices were observed for both age groups. As is shown in Fig. 3 the loss of bone strength in the MK-4 supplemented group was almost negligible in the group of 65 years and older. Table 3Effect of MK-4 treatment on geometrical variables Unadjusted adjusted mean difference (SE)pMean difference(SE)pFNW (%)+1.30 (0.50)0.01+1.34 (0.50)0.009HAL (%)− 0.25 (0.26)0.3+0.23 (0.22)0.9CSI (%)+2.26 (0.77)0.004+2.03 (0.76)0.008BSI (%)+3.80 (1.2)0.002+3.83 (1.2)0.001ISI (%)+1.98 (0.82)0.02+1.72 (0.79)0.03The increase/decrease of each variable was calculated as a percentage from its baseline value. The differences between the change in MK-4 group and that in the placebo group are given. A positive value reflects a more pronounced effect in the MK-4, a negative value reflects a more pronounced effect in the placebo group. The values are given before and after adjustment for age and BMI, adjusted values are quoted at the mean values for age and BMI of the total group (age = 65.9 years; BMI = 27.2 kg/m2).Fig. 3Effect of MK-4 on bone strength indices in 2 different age groups (55–65 and 65–75 years of age). All data are expressed as the mean percent change relative to the respective baseline values after 3 years of treatment with either MK-4 or placebo. Graph A shows the compact strength index (CSI), graph B the bending strength index (BSI) and graph C the impact strength index (ISI). Open bars: placebo; hatched bars: MK-4. Error bars represent SE. Significance of change from baseline: *: p < 0.05, **: p < 0.005; significance of difference MK-4 compared to placebo: #: p < 0.05 Discussion In this paper we demonstrate that the main effect of vitamin K2 on bone in the hip is an increase of the femoral neck BMC and width, resulting in maintenance of the calculated bone strength even at decreasing DXA-BMD after the menopause. This makes K2 to an interesting compound for combination therapy with other food supplements (calcium, vitamin D) or drugs (bisphosphonates) with known effects on also DXA-BMD. The potential synergism between K2 and other supplements/drugs, however, requires further investigation. In the literature there are many indications that vitamin Kcontributes to the maintenance of optimal bone strength. Besides various population-based studies and a number of clinical intervention trials investigating the effects of increased vitamin K intake, it has also been reported that subjects receiving vitamin K antagonists (oral anticoagulant therapy) have lower DXA-BMD and increased fracture risk [30–32]. Also experimental animals developed osteopenia upon treatment with the vitamin K antagonist warfarin [33]. On the other hand we must conclude, however, that in intervention trials the reported effects on DXA-BMD of vitamin K alone were small; most studies published thus far indicate that only in combination with calcium and vitamin D, vitamin K has a synergistic effect on DXA-BMD [18, 19, 34]. Osteocalcin is the most abundant vitamin K-dependent protein in bone, and the phenotype of transgenic osteocalcin-deficient mice suggests that it is primarily involved in the correct arrangement of hydroxyapatite crystals and in structuring the correct dimensions of bone [21]. Both factors may contribute to the total bone strength. Hence studies in which DXA-BMD is the only clinical endpoint do not give conclusive evidence on bone strength. If, for instance, the femoral neck DXA-BMD remains constant but increases in width the bone will become stronger and more resistant to fracture. Therefore, we have used mathematical procedures for calculating three indices for bone strength at the site of the femoral neck. Despite the very high dose of MK-4 used in the present trial, no effect was found on the DXA-BMD of the femoral neck, the total hip or the lumbar spine. On the other hand, significant positive effects were observed both on the femoral neck BMC and width. The relation between DXA-BMD, BMC and FNW can be seen from the formula: DXA-BMD=BMC/area, where FNW is one of the dimensions of the surface area. In case FNW is increasing, the projected area of the DXA-BMD measurement will increase too. In case BMC remains constant, the DXA-BMD values will decrease. In case FNW increases and BMC increases adequately, DXA-BMD remains constant, with a concomitant increase of bone strength. The importance of the FNW for the calculated bone strength is obvious from the equations for all three bone strength indices. Our results showed that during the entire intervention period there was no or very little decrease in bone strength in the K2 treated group, whereas there was substantial loss of bone strength in the placebo one. The favourable effect was found both in the young postmenopausal group (55–65 years old) as well as in the older group (65–75 years old). Since together with vitamin D and calcium vitamin K was also reported to have a positive effect on DXA-BMD it might even be speculated that the combination of these supplements will result in an increase of bone strength. After we obtained these data, we re-analysed previous studies from our lab, and it turned out that vitamin K1 had only little or no effect on the FNW. It should be pointed out, however, that the doses used in these studies were 1 mg/day and 10 mg/day, which is considerably lower than the 45 mg/day used in the present study. The first question that may be raised is whether the very high dose of vitamin K used in our study is required to obtain the observed effect. The present recommendations for vitamin K intake vary between 90 and 120 micrograms per day, and in a dose of 45 milligrams per day the vitamin is used as a drug rather than as a food supplement. It should be realized, however, that MK-4 has a very short biological halflife time (1–2 h), which is the reason why it was given three times daily in a dose of 15 mg per helping. Even at this regimen its short halflife will result in fluctuating K2 serum levels, which may explain the requirement of a high dose. At this time no data are available on the efficacy of MK-4 at lower doses, for instance in the range between 100–500 micrograms per day. A possible alternative to MK-4 might be using MK-7, the K2 vitamin most abundantly found in the Japanese food natto (fermented soy beans). MK-7 has probably a comparable effect as MK-4 [35], but it has a halflife in the circulation of 3 days, resulting in more constant plasma levels and accumulation in the blood and various tissues. Therefore, MK-7 is the most logical choice for use as a low-dose food supplement, because even at low intake relatively high blood and tissue levels can be obtained. Clinical trials in which the efficacy of MK-7 is tested are, however, lacking at this time. From our study it is not clear whether the high MK-4 regimen will increase vertebral bone strength in a similar way as observed for the DXA-derived femoral neck bone strength. To our knowledge no reliable formulas are available for calculating vertebral compression strength, the most important strength index for the vertebrae. However, the substantial and significant effect of MK-4 on the lumbar spine BMC warrants more elaborate studies in which the effect on vertebral fracture risk is investigated. A substantial risk reduction for vertebral fractures by long-term MK-4 treatment has also been reported by a number of Japanese studies [36, 37]. Another question that needs to be answered is why K2 has a much more pronounced effect than K1. One reason may be the different doses used: 45 mg/day for MK-4 and 1–10 mg/day for K1. Based on our present knowledge, all three doses are an excess; it seems not unlikely, therefore, that there is a qualitative difference between K1 and K2. The only difference between both vitamins is the aliphatic side chain, which does not influence the coenzyme function, but which does affect the distribution of vitamin K over the lipoproteins in the blood, and the transport to and absorption by the various tissues. It has been demonstrated in human volunteers that both K1 and K2 are transported to the liver via triglycerides, but that only K2 vitamins (both MK-4 and MK-7) are incorporated into the LDL fraction for transport to extrahepatic tissues [39]. It is not surprising, therefore, that in animal experiments it was found that K1 mainly accumulates in the liver, whereas MK-4 is far more abundant in other tissues including pancreas, bone and vessel wall [10]. In that perspective, K2 seems to be the most logical choice for supplementation because notably bone and arteries (and not the liver) were reported to have a sub-optimal vitamin K status in the majority of the healthy population. A limitation of this study is that it was conducted in non-osteoporotic women, in whom we tried to decrease bone loss and to maintain bone strength. Therefore, this study does not allow conclusions regarding beneficial effects of vitamin K2 on bone quality in women who have been diagnosed with osteoporosis already. A further limitation is that the study was exclusively conducted on women, so that no conclusions can be drawn for bone loss in men. A further limitation of our study was that the DXA-BMD of the spine was determined only in the postero-anterior (PA) position. This measurement includes not only the trabecular bone of the vertebral body, but also a substantial amount of cortical bone from posterior elements. During ageing, degenerative changes in the spine such as osteoarthritis or sclerosis of the endplates may occur. In those cases, PA projection may result in incorrect interpretation of the DXA-BMD and BMC values because the observed increase is due to degenerative changes and is not based on treatment effect. Lateral DXA-BMD measurements evaluate exclusively the vertebral body, allowing more sensitive detection of postmenopausal bone loss or DXA-BMD changes during therapy [38]. At the start of this study lateral DXA-BMD monitoring, however, was not yet available for clinical trials in our hospital. In conclusion we have demonstrated that vitamin K2 is capable of improving both the BMC and bone geometry at critical sites, leading to maintenance of the DXA-derived femoral neck bone strength in postmenopausal women during a 3-year study period. Even in the very high doses used in our study, adverse side effects of MK-4 were minor and not different from the placebo group. Therefore, it seems desirable to evaluate the cost-benefits of supplementing low dose vitamin K2 to all postmenopausal women. The optimal dose, the preferred form (MK-4 or MK-7), and synergistic effects of complementary food supplements (calcium, vitamin D) or medication (bisphosphonates), however, require further research.

Eur_Arch_Psychiatry_Clin_Neurosci-2-2-1705499

Epidemiologic and clinical updates on impulse control disorders: a critical review

The article reviews the current knowledge about the impulse control disorders (ICDs) with specific emphasis on epidemiological and pharmacological advances. In addition to the traditional ICDs present in the DSM-IV—pathological gambling, trichotillomania, kleptomania, pyromania and intermittent explosive disorder—a brief description of the new proposed ICDs—compulsive–impulsive (C–I) Internet usage disorder, C–I sexual behaviors, C–I skin picking and C–I shopping—is provided. Specifically, the article summarizes the phenomenology, epidemiology and comorbidity of the ICDs. Particular attention is paid to the relationship between ICDs and obsessive–compulsive disorder (OCD). Finally, current pharmacological options for treating ICDs are presented and discussed. Introduction Since the early 1990s, some researchers have suggested that the impulse control disorders (ICDs) might be conceptualized as a part of an obsessive–compulsive spectrum based on their clinical characteristics, familial transmission, and response to both pharmacological and psychosocial treatment interventions [1–3]. Over a decade of study and scientific developments have led a DSM-V task force to consider two important changes: separating obsessive–compulsive disorder (OCD) from the anxiety disorders and placing it in an autonomous category—the obsessive–compulsive spectrum disorders (OCSD); and creating several new autonomous disorders from those currently subsumed under ICDs not otherwise specified (ICD-NOS) [4], specifically including four new impulsive disorders, compulsive–impulsive (C–I) Internet usage disorder C–I sexual behaviors, C–I skin picking and C–I shopping. They are called compulsive–impulsive disorders due to the impulsive features (arousal) that initiate the behavior, and the compulsive drive that causes the behaviors to persist over time. The relationship between OCD and the OC spectrum has been supported by studies over the past decade, although recent studies have also supported additional models. Recent neuroimaging (PET, fMRI etc.) and genetics studies have increased understanding of the biological and neuroanatomical characteristics of the ICDs and have supported both the OC spectrum model and suggested other models [5, 6]. The pharmacological options, moreover, have been expanded based on recent research; traditional treatment with the serotonin reuptake inhibitors (SRIs) supported the OC spectrum model, but recent research demonstrating the efficacy of different pharmacological interventions suggests that additional systems are involved and other models may be useful. For example, the efficacy of pharmacotherapies acting on different systems of neuromediators (opioid antagonists, mood stabilizers, dopamine reuptake inhibitors), support different theoretical models for the ICDs and make clear that it is valuable to look at the ICDs from different theoretical perspectives that suggest different mechanisms might be important and raise new research questions. ICDs’ phenomenology, epidemiology and relationship with OCD ICDs are characterized by repetitive behaviors and impaired inhibition of these behaviors. Important defining criteria for these disorders include:The failure to resist an impulse to perform some act that is harmful to the individual or others;An increasing sense of arousal or tension prior to committing or engaging in the act;An experience of either pleasure, gratification, or release of tension at the time of committing the act. In addition, there is usually a pattern of engaging in the abnormal behavior in spite of adverse consequences (e.g., criminal changes, impairment of normal functioning, etc.). To demonstrate that a relationship exists between ICDs and OCD, there should be evidence that OCD is overrepresented in patients with ICDs and/or that ICDs are overrepresented in patients with OCD. Studies examining rates of OCD in patients with ICDs have reported inconsistent results, with some ICDs showing relatively high rates of comorbidity with OCD (trichotillomania, CI-shopping), and others demonstrating low rates (intermittent explosive disorder, pathological gambling, and C–I sexual behaviors). Pathological gambling (PG) is an impulse control disorder not otherwise specified (ICD-NOS) [4] that is characterized by recurrent and maladaptive patterns of gambling behavior that significantly disrupts the patient’s functioning in the personal, familial, or vocational spheres. Recent studies suggest that the prevalence of PG is between 1% and 3% of the adult population [7, 8], and a meta-analysis [9] estimated that 86% of the population of the USA are recreational gamblers (Table 1). The disorder usually starts during adolescence with a prevalence of approximately 4–7% in this population. However, over the last decade, there has been an unprecedented expansion of legalized gambling throughout North America, and, as a result, the prevalence of PG can be expected to increase. The disorder is currently more common in men than in women. Recent national studies on PG prevalence have also been conducted in New Zealand [10–12], Sweden [13, 14], Switzerland [15], Australia [16] and Great Britain [17], and despite the use of different methodologies and variable technical quality, problem gambling prevalence studies have shown a high degree of consistency in their general findings. Table 1Prevalence estimates of impulse control disordersImpulse control disorderReferenceType of communityPrevalence reportedPathological GamblingGerstein et al. (1999)Adult population1–3%Welte et al. (2001)Adult populationTrichotillomaniaChristenson et al. (1991)College students1.5% males; 3.4 femalesPyromaniaKosky and Silburn (1984)Children and adolescents2.4–3.5%Kolko et al. (1988)Children and adolescentsJacobson (1995)Children and adolescentsIntermittent Explosive DisorderMonopolis and Lion (1983)Psychiatric surveys1–2%Coccaro et al. (2004)Adult populationLifetime 11.1%; 1 month 3.2%KleptomaniaGoldman (1991)Adult population0.6%C–I Internet Usage Disorder–––C–I ShoppingBlack et al. (2001)Adult population 2–8%C–I Skin PickingDoran et al. (1985)Dermatologic patients2%Gupta et al. (1986)Dermatologic patientsC–I Sexual BehaviorsShaffer and Zimmerman (1990)Adult population5–6%Coleman, 1991Adult population A crucial issue to consider is the high rate of comorbidity among pathological gamblers. Patients with PG, at least those seeking treatment, have been found to score significantly higher than control populations on measures of depression [18], and have high incidences of various psychiatric disorders, including bipolar, anxiety and substance use disorders [19]. This frequent comorbidity is not surprising given the psychopathological core features of PG: impulsivity, compulsive drive to gamble, addictive features such as withdrawal symptoms during gambling abstinence, and bipolar features such as urges, pleasure seeking and decreased judgment due to unrealistic appraisal of the individuals’ own abilities. Several authors have noted the link between various core features of PG and neurobiological characteristics or treatment-response, and have conceptualized PG according to different models, thus placing it on different spectrums with the main psychiatric disorders of reference being OCD [1], addictive disorders [20], and affective disorders [21]. These models provide the theoretical rationale for the use of specific pharmacological treatments in PG. In addition, the models and related research findings may also suggest the presence of specific subgroups of patients with similar core features, comorbidity profiles and treatment-response within the population of pathological gamblers [22, 23]. The relationship between PG and OCD has allowed PG to be conceptualized as an OC spectrum disorder, within the impulsive cluster [1]. Patients with OC spectrum disorders, in fact, experience unpleasant feelings and physiological activation that result in an intense desire to perform a specific behavior in order to relieve the unpleasant feelings [24, 25]; this is the case in PG. In addition, a reduced capacity to resist gambling thoughts and urges leads to excessive gambling, in particular in the advanced phases of the disorder [26]. However, these patients differ from patients with OCD in important ways. Gambling behavior and thoughts are often experienced by these patients as ego-syntonic, while OCD obsessions and compulsions are generally ego-dystonic. In addition, the excessive doubt frequently experienced by OCD patients [24, 27, 28], as well as their harm avoidance, risk aversion and anticipatory anxiety [29], are not characteristic of pathological gamblers. OC spectrum disorders differ along the dimension of risk aversion vs. risk taking; the compulsive disorders are characterized by an overestimation of harm and by risk aversion while the impulsive disorders are characterized by an underestimation of risk and by risk seeking. Recently, the rate of comorbid OCD in individuals with PG was found to range from 1% to 20% [30] (Table 2). Table 2OCD rates in impulse control disordersImpulse control disorderReferenceRates of OCDPathological GamblingArgo and Black (2004)1–20%TrichotillomaniaChristenson and Mansueto (1999)3–27%Pyromania––Intermittent Explosive DisorderMcElroy et al. (1998)22%KleptomaniaPresta et al. (2002)6.5–60%C–I Internet Usage DisorderBlack et al. (1999)0% current; 10% lifetimeShapira et al. (2000)15% current; 20% lifetimeC–I ShoppingChristenson et al. (1994)12.5–30%McElroy et al. (1998)C–I Skin PickingSimeon et al. (1997)6–52%Arnold et al. (1998)Wilhelm et al. (1999)C–I Sexual BehaviorsKafka and Prentky (1994)12–14%Black et al. (1997) Patients afflicted with trichotillomania (TTM) describe an overwhelming urge to pluck out specific hairs; when they do so, the anxiety is momentarily relieved but is quickly replaced by another compulsive urge to pluck and even greater anxiety [31]. The exact prevalence of TTM is unknown; however, estimates from university surveys suggest that 1.5% of males and 3.4% of females endorse clinically significant hair pulling, with .6% endorsing all diagnostic criteria of TTM [32] (Table 1). The prevalence of non-clinical hair pulling behavior is even higher, up to 15.3%, in university surveys [33] (Table 1). In describing the phenomenological similarities between OCD and TTM, Swedo [34] highlighted the egodystonic feeling and the resistance experienced by patients with TTM and OCD. In addition, patients with TTM recognize the behavior as senseless, undesirable and performed in response to increasing anxiety, with resultant tension relief. Furthermore, a higher than normal incidence of both OCD and TTM has been reported in first-degree relatives of patients with TTM [35], and comorbidity data also support a relationship between OCD and TTM [36, 37] (Table 2). However, recent investigations [38, 39] have also included TTM in a spectrum of self-injurious behaviors (SIBs), including C–I skin picking, and underscored the phenomenological link among these SIBs and the differences between TTM and OCD [39]. In pyromania there is impulsive, repetitive, deliberate fire setting without external reward (e.g., arson for money, revenge, as a political act). There are very few community sample studies of firesetting, which is understandable since it is illegal and thus likely to be kept secret. The majority of epidemiological studies have focused on pyromania in childhood and adolescence and have reported the prevalence to be between 2.4% [40] and 3.5% [41, 42] (Table 1). In addition, several lines of evidence indicate that adolescent boys may be at higher risk for firesetting than adolescent girls [43, 44]. Among juveniles, firesetting is more prevalent in males than females, peaking between 12 years and 14 years [45]. Sixty percent of all fires in large U.S. cities are lit by individuals between 11 years and 18 years [46]. Besides young age, features such as temperament, parental psychopathology, social and environmental factors, and possible neurochemical predispositions [47] have been hypothesized to cause childhood pyromania. Some authors have noted a close link between firesetting and aggression [48] and between firesetting and antisocial behavior [49]. In addition, published data have shown high rates of conduct disorder among young arsonists [50]. Recent findings, moreover, revealed associations between firesetting and shyness, aggression and peer rejection [51]. No published studies of the relationship between pyromania and OCD in terms of comorbidity or family history are available. Intermittent explosive disorder (IED) is characterized by recurrent episodes of aggressive behavior that is out of proportion to psychosocial stressors and/or provocation and that is not better accounted by another mental disorder, comorbid medical conditions, or the physiologic effects of a pharmacologic agent or other substance with psychotropic properties [4]. Despite its inclusion in DSM for more than two decades, there are few studies of the lifetime prevalence of IED in either psychiatric or community settings. Clinical surveys of psychiatric inpatients [52], and clinical treatment studies on IED [53] had found rates of IED in psychiatric settings ranging from 1% to 2%. Recently, however, Coccaro and colleagues reported much higher rates of IED, 11.1% lifetime prevalence and 3.2% 1-month prevalence, in a community sample of 253 individuals [54] (Table 1). Based on these data, the authors estimated there are 1.4 million individuals with current IED in the US and 10 million with lifetime IED. As the authors suggested, prevalence rates so much higher than prior findings may reflect the changes in diagnostic criteria of IED from DSM-III [55] to DSM-IV [4] as well as the changes recently proposed in the development of research criteria for IED [56, 57]. A study by McElroy and colleagues reported rates of OCD in individuals with IED around 22% [58] (Table 2); recent studies investigating the rates of IED in patients with OCD have given lower estimates [59–61]. Kleptomania is a disorder in which the individual impulsively steals even though there is need to do so (i.e., the individual has money to pay for the stolen items or does not need the stolen goods). Like other ICDs, kleptomania is characterized by an anxiety-driven urge to perform an act that is pleasurable in the moment but causes significant distress and dysfunction [62]. The prevalence of kleptomania in the U.S. is unknown but has been estimated at 6 per 1000 people. [63] (Table 1). In addition, given the embarrassment surrounding kleptomania, it is often kept secret and thus goes undiagnosed [62]. Kleptomania is thought to account for 5% of shoplifting in the U.S. [64]. Based on total shoplifting costs of $10 billion in 2002 [65], this 5% translates into a $500 million annual loss to the economy attributable to kleptomania. This loss does not include the costs associated with stealing from friends and acquaintances or costs incurred by the legal system. Kleptomanic behavior carries serious legal consequences: approximately 2 million Americans are charged with shoplifting annually [66]. If kleptomania accounts for 5% of these, this translates into 100,000 arrests. Recent studies assessing the rate of OCD in patients with kleptomania have given widely differing estimates, ranging from 6.5% to 60% [67, 68] (Table 2). C–I Internet usage disorder, also referred as Internet addiction or problematic Internet use, has been proposed as an explanation for uncontrollable and damaging use of the Internet, and has only recently begun to appear in the psychiatric literature [69, 70]. People with problematic Internet use often report increasing amounts of time-spent web surfing, gambling, shopping or exploring pornographic sites. Others report spending time in chat rooms or corresponding by email. Frequently these people develop a preoccupation with the Internet, a need for escape to the Internet and increasing irritability when trying to cut back their Internet use. Ultimately, their attempt to cut back is unsuccessful. Functional impairments as a result of problematic Internet use include marital or family strife, job loss or decreased job productivity, legal difficulties or school failure [72]. Although diagnostic criteria for this disorder have been proposed, methods of assessing C–I Internet usage disorder are limited. In addition, although increasing research is being conducted on the topic, several published articles contain information that has not been empirically researched [73]. For some individuals, their excessive Internet use may be entirely accounted for by another Axis I disorder such as PG or C–I sexual behaviors; thus the Internet is functioning simply as another outlet for that disorder rather than being an additional disorder. Problematic Internet use has been reported in any age, social, educational, and economic range [74]. However, while previous studies tended to stereotype the classical Internet addicted patient as a young introverted man [75, 76], recent investigations have showed increasing rates of this disorder among women [74], as a result of the increased availability of the Internet. The prevalence of C–I Internet usage disorder is not known. Most of the studies related to this condition have been conducted with small samples. People enrolled, moreover, frequently had comorbid psychiatric diagnoses. In a recent study [71], Shapira and colleagues found that all subjects with problematic Internet use also met DSM-IV criteria for ICD-NOS. Studies assessing comorbidity rates between OCD and C–I Internet use reported estimates ranging from 10% to 20% for lifetime OCD and up to 15% for current OCD in Internet addicted patients [71, 77, 78] (Table 2). Further investigations on the epidemiology of this disorder are needed to clarify the scale and demographic characteristics of C–I Internet use. C–I sexual behaviors (C–ISBs) include repetitive sexual acts and compulsive sexual thoughts. The individual feels compelled or driven to perform the behavior, which may or may not cause subjective distress. Although generally not ego-dystonic, the behavior may interfere with several aspects of the patient’s life, causing social or occupational impairment, or legal and financial consequences [79]. C–ISBs involve a broad range of paraphilic or non-paraphilic symptoms [80]. Paraphilic C–ISBs involve unconventional sexual behaviors in which there is a disturbance in the object of sexual gratification or in the expression of sexual gratification (e.g., exhibitionism, voyeurism). Non-paraphilic C–ISBs, on the other hand, involve conventional sexual behaviors that have become excessive or uncontrolled [80]. The true prevalence of C–ISBs remains unknown, given the hetereogeneity of these disorders as well as the secretiveness of the condition for the majority of the afflicted patients. Investigations conducted in the early 1990s reported prevalence estimates of C–ISBs ranging from 5% to 6% of the US population [80, 81] (Table 1). Male patients have been traditionally reported to be more afflicted than women by C–ISBs [82, 83]. However, it is not clear how large this sex difference is and the extent to which the difference is due to men coming to the attention of professionals with greater frequency. Studies assessing the rates of OCD in patients suffering from C–ISBs [79, 84] reported estimates around 12% and 14% (Table 2). C–I shopping, also referred as compulsive buying, is characterized by maladaptive preoccupations or impulses to buy or shop that are experienced as irresistible, intrusive and/or senseless, accompanied by frequent episodes of buying items that are not needed and/or that cost more than can be afforded. Frequently, these patients engage in these behaviors for longer periods of time than intended, and they experience distress and significant impairment in social and occupational performance. As specified for many other ICDs, the excessive buying or shopping behavior does not occur exclusively during periods of hypomania or mania [85, 86]. A recent study on C–I shopping disorder estimated the prevalence of this disorder to be between 2% and 8% of the general adult population in the US [87]; 80% to 95% of those affected are female (Table 1). Onset occurs in the late teens or early twenties, and the disorder is generally chronic. Previous studies investigating rates of OCD in patients with C–I shopping reported rates of 12.5% to 30% [86, 88] (Table 2); lower rates of compulsive buying have been found in patients with OCD (from 2.2% to 10.6%) [59–61], except for the study of Lejoyeux and colleagues (23.3%) [89]. Patients with C–I skin picking frequently present to dermatologists, and it has been estimated that about 2% of dermatology clinic patients may suffer from this condition [90, 91] (Table 1). Prevalence in the general population or in psychiatric clinics is unknown. Skin picking is often not a transient behavior but may persist with a waxing and waning lifetime course. It should be considered pathological when it becomes habitual, chronic and extensive, leading to significant distress, dysfunction or disfigurement [38]. As reported by two recent studies, the majority of patients with C–I skin picking are women and their condition is assumed to be chronic, with excoriations on both single or multiple sites [92, 93]; the face is the most common site of excoriation but picking can involve any area of the body. Both studies found the majority of patients experienced increasing tension before the act (79–81%), relief after the act (52–79%), or both (68–90%). Comorbid lifetime rates of skin picking in patients with trichotillomania were approximately 10% in both studies [92, 93], whereas comorbid lifetime OCD was present in rates ranging from 6% to 19%. Wilhelm and colleagues [94] reported rates of OCD around 52% in a sample of 31 patients with C–I skin picking (Table 2). As mentioned for trichotillomania, the inclusion of C–I skin picking within a spectrum of self-injurious behaviors is receiving increasing support from clinical and neuroimaging studies [38]. Treatment options for ICDs Treatment options for ICDs include both pharmacotherapy and psychotherapy. During the last decade, increasing research has been conducted on different pharmacological treatments across several ICDs; however, while the efficacy of various treatments has been investigated in double-blind studies for certain disorders (i.e., PG, IED, C–I shopping), systematic research of clinical treatment is still lacking for other disorders (see Table 3). In addition, a crucial issue to take into account when considering pharmacotherapy for patients with ICDs is the comorbidity with other psychiatric conditions such as affective and addictive disorders. The presence of bipolar or addictive comorbidity, in fact, will determine the most appropriate choice when different treatments have proven to be effective for a specific disorder. Table 3Treatment options for impulse control disorders as reported in blinded and unblinded studiesImpulse Control DisorderDouble-blind studies (references)OutcomesOther treatment options as reported in open-label trialsPathological GamblingFluvoxamine vs. PC (Hollander et al. 2000; Blanco et al. 2002)SSD for Fluvoxamine; No SSD between Fluvoxamine and PC. Nefazodone, Bupropion, Citalopram, Divalproex, TopiramateParoxetine vs. PC (Kim et al. 2002; Potenza et al. 2003)SSD for Paroxetine; No SSD between Paroxetine and PC. Lithium vs. PC (Hollander et al. 2005)SSD for Lithium;Naltrexone vs. PC (Kim et al. 2001)SSD for NaltrexoneTrichotillomaniaClomipramine vs. Desipramine (Swedo et al. 1989)SSD for Clomipramine;Fluvoxamine, Citalopram, Venlafaxine, Naltrexone, Lithium, CBTFluoxetine vs. PC (Christenson et al. 1991; Streichenvein and Thornby 1995)No SSD between Fluoxetine and PCPyromania––CBT and other psychotherapiesIntermittent Explosive Disorder*Lithium vs. PC (Campbell et al. 1984 and 1995; Malone et al. 1998 and 2000) SSD for Lithium (in the Campbell’ study of 1984, Lithium was associated to Haloperidol)Clonidine*Divalproex vs. PC (Hollander et al. 2003 and 2005) SSD for Divalproex*Fluoxetine vs. PC (Coccaro et al. 1997) SSD for Fluoxetine*Carbamazepine vs. PC (Foster et al. 1989) SSD for Carbamazepine*Phenytoin vs. PC (Barratt et al. 1997; Stanford et al. 2001) SSD for Phenytoin*BBlockers vs. PC (Greendyke et al. 1986a and 1986b) SSD for BBlockers*Risperidone vs. PC (Buitelaar et al. 2001; Findling et al. 2001) SS for Risperidone*CBT vs. PC (Alpert et al. 1997)SSD for CBTKleptomania––Fluoxetine, Paroxetine, Fluvoxamine, Divalproex, Lithium, BenzodiazepinesC–I Internet Usage DisorderEscitalopram vs. PC (Dell’Osso et al. 2006**)SSD for EscitalopramPsychotherapyC–I ShoppingFluvoxamine vs. PC (Black et al. 2000; Ninan et al. 2000)No SSD between Fluvoxamine and PC;Fluvoxamine, NaltrexoneCitalopram vs. PC (Koran et al. 2003)SSD for CitalopramC–I Skin PickingFluoxetine vs. PC (Simeon et al. 1997; Block et al. 2000)SSD for FluoxetineClomipramine, SertralineC–I Sexual Behaviors––Lithium, Tricyclics, Buspirone, Fluoxetine, Nefazodone, Sertraline, NaltrexoneSSD = statistically significant differences; CBT = cognitive behavioral therapy; PC = placebo* Studies on patients with impulsive aggression features, rather than with a proper DSM diagnosis of IED** Open-label study followed by double-blind discontinuation phase (Abstract) PG is a good example of the importance of comorbidity determining treatment. PG has demonstrated a good response to selective serotonin reuptake inhibitors (SSRIs), mood stabilizers and opioid antagonists in double-blind studies [22, 95–99] (Table 3). Among all the antidepressants assessed so far, fluvoxamine [100], paroxetine [97, 98], citalopram [101], nefazodone [102], bupropion [103], (although only fluvoxamine and paroxetine in double-blind studies), the most convincing evidence is for the efficacy of the SSRIs. However, a major issue for this class of medication is the presence of bipolar spectrum comorbidity in some gamblers. This possibility needs to be carefully evaluated and excluded before treating pathological gamblers with antidepressants in order to avoid the possible reemergence of manic symptoms. The opioid antagonist naltrexone was effective in a double-blind trial, however, the risk of hepatotoxicity of this drug limits its use. Of note, the opioid antagonist nalmefene has shown to be efficacious in preliminary findings with better tolerability than naltrexone [104]. Patients with other addictive disorders (alcohol and other substances) and intense urges and craving might particularly benefit from opioid antagonists. Mood stabilizers and anticonvulsants (lithium and divalproex assessed in double-blind controlled trials) have shown good results in recent studies without any specific contraindications for their use across the different subtypes of gamblers. In addition, gamblers with consistent affective instability may particularly benefit from these treatments. Pharmacological treatment of TTM is not well established and, although SSRIs seem to show the best efficacy and safety, double-blind controlled studies on their use have given mixed results (Table 3). Clomipramine was found to be more effective than desipramine in a 10-week crossover study [105] conducted in the late 1980s. While subsequent uncontrolled studies found fluoxetine, fluvoxamine and citalopram to be efficacious in patients with hair pulling [106–110], two controlled studies [111, 112] with fluoxetine could not replicate the positive findings reported with SSRIs in the open-label trials. Positive results have been also reported in uncontrolled studies with venlafaxine, lithium and naltrexone [113–116] as well as in open-label augmentation studies with SSRIs and pimozide [117, 118]. However, treatment response is often disrupted by significant relapse during ongoing pharmacological treatment [117]. In a recent controlled study [119] comparing cognitive behavioral therapy (CBT) to clomipramine and placebo, CBT had a dramatic effect in reducing symptoms of TTM and was significantly more effective than clomipramine or placebo, underscoring the efficacy of behavioral as well as pharmacological treatment in hair pulling. To our knowledge, no controlled pharmacological trial has been conducted in patients with pyromania. Non-pharmacological interventions for firesetters, including CBT [120], short-term counseling and day-treatment programs [121], have shown some efficacy. Undoubtedly, pyromania represents an ICD needing systematic pharmacotherapy research. Treatment options for IED include the use of mood stabilizers, phenytoin, SSRIs, β-blockers, α2-agonists and antipsychotics (Table 3). Actually the majority of trials with these compounds have been conducted on individuals with impulsive aggression rather than with a specific diagnosis of IED, and several authors still don’t consider the current criteria for the diagnosis of IED to be adequate [122]. Nevertheless, the presence of impulsive aggression within the core features of IED allows us to put aside this nosographic debate. Among mood stabilizers, the most convincing evidence comes from controlled studies with lithium (especially in children and adolescents) [123–127] and divalproex [128]. This last medication demonstrated significant efficacy in different populations of aggressive subjects [129, 130]. Carbamazepine has also shown some efficacy in a small double-blind study and in open-label trials [131, 132]. Phenytoin has showed positive results in two controlled double-blind studies [133, 134] at doses up to 300 mg/d. With regard to SSRIs, a double-blind placebo controlled trial of fluoxetine [135] in patients with personality disorder showed reduced scores on measures of irritability and aggression in patients taking the active medication. B-blockers propranolol and pindolol have also shown positive results in controlled studies [136, 137], reducing aggressive behaviors in patients with brain damage, although their concomitant diagnosis of IED might be arguable as the aggressive behaviors may have a different etiology. The α-agonist clonidine was reported to decrease aggression in an open-label trial [138] with adolescents at dosages of 0.4 mg/d, although the tolerability was a problem for some subjects. The atypical antipsychotic risperidone was also showed to be effective in treating aggression in controlled studies [139, 140]. Finally, controlled studies of behavioral interventions including CBT, group therapy, family therapy and social skill training have shown them to be valid treatments for aggressive patients [141, 142]. The pharmacological treatment of kleptomania includes SSRIs, mood stabilizers and opioid antagonists, although none of these medications have been tested in blinded, controlled trials so far (Table 3). Among SSRIs, fluoxetine, alone or in combination with lithium or tricyclics, was shown to be effective in several case-reports [64, 143, 144], as were fluvoxamine and paroxetine [145–148]. Mood stabilizer trials and reports in kleptomanic patients showed mixed results for lithium [64, 144, 145], valproic acid [64, 149] and carbamazepine [64]. The opioid antagonist naltrexone was reported to be effective in two different case reports [148, 150]. Finally the benzodiazepines clonazepam and alprazolam provided some evidence of efficacy in treating kleptomania [64, 147]. In conclusion, as discussed in a recent review [151], SSRIs seem to be the most promising treatment for kleptomania (19 of 30 cases of successful pharmacotherapy reported in the literature), either as monotherapy or in combination with other psychotropic drugs. Given its recent recognition as a psychiatric problem, understandably no controlled pharmacological trials have been published on the treatment of C–I Internet usage disorder so far. Recently, Sattar and Ramaswamy [152] reported the case of a 31-year-old man with severe Internet addiction successfully treated with escitalopram (10 mg/d). Most treatment strategies for problematic Internet use have involved behavioral therapy techniques, which limit the amount of time on the Internet rather than requiring abstinence, as is done with many other addictions such as substance abuse. Self-help groups (both on and offline) are also being formed to address the problem. Our group has recently completed an open-label trial of escitalopram followed by a double-blind discontinuation phase in a population of C–I Internet users with preliminary positive findings [153]. Given the increasing use of the Internet in the new generations, a growing prevalence and incidence of this disorder is arguable. Clinicians treating subjects with ICDs should always assess the presence of this disorder in these patients given the relationship between C–I Internet use and some specific ICDs, such as pathological gambling and C–I sexual behaviors [154, 155]. Finally, controlled studies are expected in order to investigate the treatment response of Internet addicted patients to pharmacotherapy and psychotherapy. Although C–I sexual behaviors seem relatively common, controlled trials on pharmacological treatments for these disorders are still lacking, and the available literature on this topic consists essentially of open-label trials and case-report series (Table 3). Positive findings have been reported with lithium and tricyclics [156–158], SRIs [159–162], buspirone [163, 164] and nefazodone [165]. As for other ICDs, the opioid antagonist naltrexone has recently shown to be efficacious in some case-reports [166]. Finally, different forms of psychotherapy have been shown to be effective for specific subtypes of C–I sexual behaviors [167]. There is some evidence that C–I shopping has been effectively treated with several different compounds (Table 3). McElroy’s group [86] reported on 20 patients that benefited from antidepressants, often in combination with mood stabilizers. Black [168] reported fluvoxamine to be effective in patients without comorbid major depression, suggesting that improvement was independent of the treatment of mood symptoms. Naltrexone was found to be effective in a case series [169]. Two double-blind placebo-controlled trials [170, 171] did not confirm the superiority of fluvoxamine over placebo. However, these studies had the patients in both conditions keep a log of their shopping; keeping logs is a therapeutic intervention in itself and may have led to the failure of the fluvoxamine and placebo groups to separate. An open-label trial of citalopram [172] and a subsequent open-label trial followed by double-blind discontinuation [173], neither of which using shopping logs, reported positive results. Studies comparing the efficacy of pharmacological treatment with psychotherapy have not been published yet. Patients suffering from C–I skin picking often meet criteria for other psychiatric disorders (BDD and OCD), and frequently, due to medical complications of their psychopathology such as infection and scarring, they are referred to clinicians other than psychiatrists (i.e. dermatologists). The first controlled trial conducted by our group [97] found fluoxetine, at a mean dose of 55 mg/d for 10 weeks, significantly superior to placebo in decreasing the behavior in 21 adults with chronic pathologic skin picking (Table 3). More recently, a combined open-label and double-blind trial [174] confirmed the efficacy of fluoxetine in subjects with C–I skin picking. Previously, a retrospective treatment review of BDD patients with skin picking indicated that SRIs were effective in about half of 33 patients, whereas other agents were not [175]. In a subsequent open-label study [176], sertraline (mean dose: 95 mg/d) showed clinically significant improvement in 68% of 30 patients with skin picking after one month of treatment. Finally, uncontrolled psychodynamically oriented treatments and behavioral interventions have given mixed results described elsewhere [177]. Conclusions Current knowledge on ICDs in terms of epidemiology and pharmacological treatment varies notably across these disorders, with recent and continuing advances for some (i.e. pathological gambling and C–I shopping), and anecdotal and obsolete data for others. Undoubtedly, given the high prevalence estimates of some ICDs (i.e. pathological gambling and C–I sexual behaviors) as well as their comorbidity with other major psychiatric disorders, this group of disorders represents a global problem. Nevertheless, certain ICDs (i.e, pyromania, C–I Internet usage disorder) still need systematic epidemiological and pharmacological research. Studying the relationships between specific ICDs and other major psychiatric conditions (i.e. OCD, bipolar disorders, addictive disorders) in terms of phenomenological issues and comorbidity patterns is not only of theoretical interest; indeed, it provides the rationale for the use of specific pharmacological treatments and behavioral interventions. From this perspective, more than one decade after its introduction, the conceptualization of ICDs as obsessive–compulsive related disorders is still valid and has been confirmed by numerous studies; however, there is also evidence supporting the relationship between ICDs and addictive and affective disorders. Not only are the different models of conceptualizing the ICDs not mutually exclusive, but they can contribute to recognize specific subtypes within the disorders. As a result, different models of conceptualization of ICDs have led new developments in pharmacologic treatment of these disorders, with positive results obtained with mood stabilizers and opioid antagonists in addition to the SSRIs.

Med_Oncol-4-1-2386844

The effects inhibiting the proliferation of cancer cells by far-infrared radiation (FIR) are controlled by the basal expression level of heat shock protein (HSP) 70A

We developed a tissue culture incubator that can continuously irradiate cells with far-infrared radiation (FIR) of wavelengths between 4 and 20 μm with a peak of 7–12 μm, and found that FIR caused different inhibiting effects to five human cancer cell lines, namely A431 (vulva), HSC3 (tongue), Sa3 (gingiva), A549 (lung), and MCF7 (breast). Then, in order to make clear the control system for the effect of FIR, the gene expression concerned to the inhibition effect by FIR were analyzed. In consequence, basal expression level of HSP70A mRNA was higher in A431 and MCF7 cells than in the FIR-sensitive HSC3, Sa3, and A549 cells. Also, the over expression of HSP70 inhibited FIR-induced growth arrest in HSC3 cells, and an HSP70 siRNA inhibited the proliferation of A431 cells by irradiation with FIR. These results indicate that the effect of a body temperature range of FIR suppressing the proliferation of some cancer cells is controlled by the basal expression level of heat shock protein (HSP) 70A. This finding suggested that FIR should be very effective medical treatment for some cancer cells which have a low level of HSP70. Still more, if the level of HSP70 in any cancer of a patient was measured, the effect of medical treatment by FIR can be foreseen for the cancer. Introduction Far-infrared radiation (FIR), which causes heating, includes electromagnetic waves with wavelengths between 4 and 1,000 μm. Recently, there have been many studies of the effects of FIR on health and in the preservation of food. The available evidence indicates that whole-body irradiation by FIR has many biological effects. For example, hyperthermia (body temperature of 39–41°C) induced by whole-body FIR has been reported to substantially inhibit spontaneous mammary tumor growth in mice [1–4]. At normal temperature ranges (approximately 25.5°C), tumor growth in SHN mice can be inhibited by FIR [5, 6]. Furthermore, whole-body FIR irradiation is believed to improve human health and sleep by enhancing blood circulation in the skin [7, 8]. This is likely due to the ability of organic matter to absorb FIR at wavelengths between 7 and 12 μm. The effects of FIR, and particularly whole-body FIR, remain unclear, because the experiments are easily affected by environmental changes in temperature and humidity and by the presence of bacteria. Therefore, we developed a chamber for raising animals that emits FIR upon heating and is capable of maintaining steady conditions. This system employs a sealed heater with a carbon/silica/aluminum oxide/titanium oxide ceramic coating produced using a polycarbonate printing technique [9]. Using this system, we found that FIR inhibits tumor growth in the A431 tumor genesis model mouse by inhibiting the expression of matrix metalloprotease-1, 9, 10, and 13. Recent in vitro studies by Teraoka et al. found that FIR at wavelengths between 4 and 16 μm inhibits the growth of HeLa cells in vitro at 37°C [10]. Despite these findings, the molecular mechanism by which FIR affects cellular gene expression remains unclear. The lack of data on the effects of FIR on cells is due to the difficulty in stably irradiating cells with FIR under ideal culture conditions (i.e., 100% humidity, 37.0 ± 0.5°C, 5% CO2) and examining the effects of FIR at the cellular level. Therefore, using a polycarbonate printing technique, we developed a CO2 incubator with a sealed heater that has a carbon/silica/aluminum oxide/titanium oxide ceramic coating and emits FIR upon heating [11]. This CO2 incubator can stably emit FIR at wavelengths between 4 and 20 μm (maximum at 7–12 μm) under conditions of 100% humidity, 37.0 ± 0.5°C, and 5% CO2. Our results using this incubator indicate that a body temperature range of FIR radiation suppresses the proliferation of HSC3, Sa3, and A549 cells. But, A431and MCF7 was not affected. These differences may be caused by any control system competing with FIR. Therefore, the gene expression concerning to the effect of inhibition by FIR are analyzed in the present study. Still more, the effect of over expressing and suppressing candidate FIR response genes HSP70 we examined. Materials and methods FIR incubator As previously reported [11], we fabricated an FIR radiant-panel incubator by coating a carbon/silica/aluminum oxide/titanium oxide ceramic (radiation efficiency > 97%) using a polycarbonate printing technique (Bloodissue Co. Ltd. Tokushima, Japan). The incubator has a stably irradiate system with FIR at wavelengths between 4 and 20 μm (maximum at 7–12 μm) under conditions of 100% humidity, 37.0 ± 0.5°C, and 5% CO2 in air. Calculation of FIR absorbed per 10-cm tissue culture dish in the FIR incubator Fourier Transform Infrared Spectroscopy (FTIR) analysis revealed that the ceramics coating inside the CO2 incubator emits FIR at 4 W m−1 str−1 μm−1 at wavelengths between 4.486 and 20.256 μm, with a maximal emission of 11.6 W m−1 str−1 μm−1 at 9 μm, which is >95% of the emission rate of an ideal black body. Since the ceramic coating was maintained at 40°C, the total generating energy, integrated over the entire range of wavelengths, was calculated to be 130.225 Wm−2 str−1. The total area of the FIR-emitting ceramic surface was 1.2385 (m2). Therefore, the total energy emitted into the incubator was 161.28366 W/str. Assuming that FIR is emitted in all directions, the total emission was 2026.7502 J/sec. Given that the volume of the CO2 incubator was 0.1257 m3 and the volume of culture medium was 6 ml, the amount of energy absorbed by each 10-cm culture dish was 0.09674 J/sec. The surface area of each 10-cm culture dish was 78.5 cm2, so that the energy reaching the base of the dish was 0.001232 J/sec/cm2. Thus, over a 1-h period 4.4352 J/h cm2 was absorbed by each 10-cm culture dish. Cell lines and cell culture A431 human epithelial vulva carcinoma cells and Sa3 human gingival squamous carcinoma cells were purchased from RIKEN Cell Bank (Tsukuba, Japan). A HSC3 human tongue squamous carcinoma cells, A549 human lung carcinoma cells, and MCF7 human breast carcinoma cells were purchased from Health Science Research Resources Bank (Sennan, Japan). A431, A549, and MCF7 cells were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 nutrient mixture (Sigma, St. Louis, MO, USA). HSC3 and Sa3 cells were cultured in Eagle’s basal medium (Sigma). All culture medium was supplemented with 10% heat-inactivated fetal bovine serum, 100 μg/ml penicillin G, 100 μg/ml streptomycin sulfate, and 250 ng/ml amphotericin B (Invitrogen, Carlsbad, CA, USA). Cells were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. The medium were replaced every 2 days. Measurement of cell number and growth Cells (5 × 104) were plated in triplicate in 24-well plates (Nunc, Roskilde, Denmark). The attached cell populations were measured on day 8 using 0.2% Trypan blue and a hemocytometer. Incorporation of 5-bromo-2′-deoxyuridine (BrdU) was used to determine the amount of DNA synthesis. DNA synthesis by proliferating cells was assessed using a BrdU labeling and detection kit III (Roche, Mannheim, Germany) according to the manufacturer’s protocol. Briefly, cells (5 × 103 per well) were seeded in 96-well tissue culture plates (Nunc) and then placed in the FIR incubator for 4 days, and BrdU incorporation was measured during the logarithmic growth phase (i.e., before the cells were confluent) by treating the cells for 4 h at 37°C with 10 μM BrdU. BrdU incorporation was quantified by measuring the absorbance of the substrate reaction (405 nm) and the absorbance at the reference wavelength (590 nm) using an ImmunoMini NJ-2300 (System Instruments, Tokyo, Japan). Absorbance values directly correlated with the amount of DNA synthesis and therefore the number of proliferating cells. Histochemistry Cells were grown on 22-mm2 glass coverslips in 6-well culture dishes (Nunc). After 4 days of FIR irradiation, the cells were observed with a CK40 phase contrast microscope (Olympus, Tokyo, Japan), fixed, and stained with hematoxylin and eosin. For immunofluorescent staining of heat shock protein (HSP) 70, cells were washed in Phosphate Buffered Saline (PBS), fixed for 20 min in 4% paraformaldehyde in PBS, washed three times for 5 min each in PBS, and blocked for 1 h at room temperature with 5% goat serum. Cells were incubated at 4°C overnight in 1:200 mouse monoclonal antibody to HSP70 (Stressgen, Victoria, Canada) in PBS containing 1 mg/ml bovine serum albumin. After washing, the cells were incubated with 1:400 FITC (Fluorescein isothiocyanate)-labeled goat anti mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The localization of intracellular HSP70 protein was identified using a BX51 confocal microscope (Olympus) and a Cool SNAP CF digital camera (Roper Scientific, Trenton, NJ, USA) and calibrated using RS Image Express software (Roper Scientific). Microarray studies and data analysis Four days after FIR irradiation, two control and two FIR-irradiated samples were prepared for microarray hybridization. Total RNA was extracted using a Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Agilent human 1A ver.2 microarray slides (Agilent Technologies, Palo Alto, CA, USA) were used for the hybridization. The quality of RNA samples was monitored using an Agilent 2100 bioanalyzer (200 ng each). To produce labeled cRNA (complementary RNA), high-quality RNA was amplified and labeled with Cy5-and Cy3-CTP (Amersham Biosciences, Buckinghamshire, UK) using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent) according to the manufacturer’s protocol. After the amplification and labeling, the dye incorporation ratio was determined using a Nanodrop spectrophotometer, and the ratios were within 10–20 pmol per μg cRNA, which is the range suggested by the manufacturer for hybridization. For hybridization, an Agilent 60-mer oligo microarray (Rev. 7, SSC Wash/6-screw hybridization chamber) was used according to the manufacturer’s protocol. Briefly, 750 ng Cy3-labeled control and 750 ng Cy5-labeled MPP+-treated sample were mixed and incubated for 17 h with an SSC-washed microarray slide from an Agilent In situ Hybridization Kit. Sample pairs were dye-swapped and processed at the same time. The washed slides were immediately dried under a stream of ultrapure N2 in an ozone-free atmosphere. After drying, the slides were scanned using an Agilent Technologies Microarray Scanner with the PMT setting at 770 for Cy5 and 670 for Cy3, and the raw data were normalized and analyzed using GeneSpring 7.0 software (Silicon Genetics, Santa Clara, CA, USA). For normalization, per spot and per chip intensity-dependent (LOWESS) normalization was used to correct for the intensity-dependent ratio bias12. In addition, the following filters were applied to improve the quality of the data: eliminate saturated signal, eliminate non uniformity of background, eliminate non uniformity of feature, Feature Population Outlier, eliminate low-signal feature of background signal + 2.6 × SD, and eliminate P-value < 0.01. Genes were further classified for process and function according to their GO term information (http://www.godatabase.org). Stable transfection of HSP70A The HSP70A expression vector pcDNA3-HSP70A containing the cDNA for full-length human HSP70A was a generous gift from Dr. Hector R. Wong (Children’s Hospital Medical Center, Cincinnati, OH, USA). HSP70A cDNA was subcloned into the Xba I and Bam HI sites of pcDNA3.1(−) (Invitrogen). Cells grown on 60-mm dishes were transfected with 8 μg of pcDNA3-HSP70A, or pcDNA3.1 (Invitrogen) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The transfected cells were selected with 400 μg/ml G418 (Sigma), and clones formed were collected and maintained separately in medium supplemented with 400 μg/ml G418. Reverse transcription (RT)-polymerase chain reaction (PCR) Total RNA was extracted using Trizol reagent (Invitrogen) following the manufacturer’s instructions. The concentration and purity of the RNA were determined spectrophotometrically. One microgram of total RNA was reverse-transcribed into first-strand cDNA. Next, oligo(dT12–18) primer (Invitrogen), 10 mM dNTP mix (Invitrogen), 25 mM MgCl2 (Promega, Madison WI, USA), and 0.1 M dithiothreitol (Invitrogen) were added, and the mixture was incubated for 2 min at 42°C. Next, the RT reaction was performed by adding SuperScript II RT (Invitrogen) and incubating at 42°C for 50 min, followed by 70°C for 15 min. The reaction was terminated by rapid cooling on ice, after which RNA in the sample was degraded by treatment with RNase H (Invitrogen) at 37°C for 20 min. Polymerase chain reaction (PCR) was carried out using a 1-μl sample of the RT reaction and Ready Mix PCR Master Mix (AB gene, Epsom, Surrey, UK). The analyzed genes and the respective primer sequences were as follows: HSP70A, 5′-TGTTCCGTTTCCAGCCCCCAA-3′ (sense) and 5′-GGGCTTGTCTCCGTCGTTGAT-3′ (antisense); HSP70B, 5′-CTCCAGCATCCGACAAGAAGC-3′ (sense) and 5′-ACGGTGTTGTGGGGGTTCAGG-3′ (antisense); HSP70C, 5′-TTGAGGAGGTGGATTAGGGGC-3′ (sense) and 5′-AGCCTTTGTAGTGTTTTCGCC-3′ (antisense); glyceraldehyde-3-phosphate dehydrogenase (G3PDH), 5′-ACCACAGTCCATGCCATCAC-3′ (sense) and 5′-TCCACCACCCTGTTGCTGTA-3′ (antisense). PCR was carried out using cycles (30 for HSPs and 23 for G3PDH) of 94°C for 45 s, 58°C for 30 s (all HSPs) or 52°C (G3PDH), and 72°C for 90 s. Finally, primer extension was performed for 10 min at 72°C. A 10-μl sample of each PCR product was separated by electrophoresis on a 1.5% polyacrylamide gel in Tris borate/EDTA buffer and stained with ethidium bromide. Quantitative real-time RT-PCR data analysis To determine the level of HSP70A mRNA, quantitative real-time RT-PCR was carried out using a LightCycler and the Fast Start DNA Master SYBR Green I Kit (Roche). The reaction contained 50 ng of cDNA and 100 pmol of each primer in a final volume of 10 μl. The gene-specific primers were as follows: HSP70A, 5′-TGTTCCGTTTCCAGCCCCCAA-3′ (sense) and 5′-GGGCTTGTCTCCGTCGTTGAT-3′ (antisense); and α-actin, 5′-ATAGCACAGCCTGGATAGCAACGTAC-3′ (sense) and 5′-CACCTTCTACAATGAGCTGCGTGTG-3′ (antisense). The concentration of Mg2+ was 3 mM. In all cases, a first phase of denaturation was performed at 95°C for 10 min. Amplification was carried out for cycles of denaturation at 95°C for 10 s, hybridization for 10 s (58°C for HSP70A or 60°C for α-actin), and elongation at 72°C (20 s for HSP70A or 10 s for α-actin). Product specificity was evaluated by melting curve analysis. Fluorescence data were analyzed using LightCycler Software Ver. 3.5 (Roche). Crossing points were established using the second derivative method. The relative amount of target transcript in the sample was calculated by dividing the amount of target by the amount of internal standard (α-actin). Results were expressed as the target/internal standard concentration ratio calculated from the calibration curve. Since the target and internal standard genes had different sequence and amplicon lengths, it was expected that they would show different PCR efficiencies. Therefore, the PCR efficiency (10−1/m, where m is the slope from the calibration curve) was first established for each pair of primers. All reactions were performed in triplicate. HSP70 Enzyme-Linked Immuno Sorbent Assay (ELISA) HSP70 protein was quantified in cell lysates using a commercially available ELISA kit for human HSP70 (Stressgen). Cells (105 per well) were seeded in 6-well plates (Nunc). After 4 days, the cells were lysed, and all samples were assayed at optimal dilutions according to the manufacturer’s instructions. Protein extraction and Western blotting Cells (1 × 106) were grown in 60-mm tissue culture dishes (Nunc). After removing the cell culture medium from the culture dishes (Nunc) and washing the cells twice with cold PBS(−), the cells were lysed in lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and 1 μg/mL leupeptin). Protein levels were measured by the Lowry method [13] using a DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Cell lysate containing 15 μg of protein for HSP70 was subjected to Sodium Dodesyl Sulfate (SDS)-polyacrylamide gel electrophoresis. Separated proteins were then transferred from the gel to a polyvinylidene difluoride membrane. After blocking with 5% skim milk in PBS-Tween, the membrane was incubated for 1 h at room temperature with primary antibody in PBS-T containing 5% skim milk, followed by three 10-min washes with PBS-T. Next, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-labeled secondary antibody and washed three times for 10 min with PBS-T. Immunoreactive protein was detected using an ECL plus kit (Amersham Biosciences) and visualization by exposure to Hyperfilm (Amersham Biosciences). The primary antibodies used were rabbit anti-HSP70 (Stressgen), anti α-actin (Sigma), and the secondary antibody was horseradish peroxidase-conjugated anti-mouse (Zymed Laboratories, South San Francisco, CA, USA) or anti-rabbit IgG (Amersham Biosciences). Small interfering RNA (siRNA) We designed 21-nucleotide siRNAs targeting human HSP70A and HSP70C according to the manufacturer’s instructions (Dharmacon, Lafayette, CO, USA) and corresponding to the sequence 5′-AAGAACCAGGUGGCGCUGAAC-3′. It was not possible to design a siRNA specific to HSP70A because its mRNA was highly homologous to the mRNA for HSP70C (see Table 1). We used siCONTROL Non-Targeting siRNA (Dharmacon; 5′-UAGCGACUAAACACAUCAAUU-3′) as a negative control because it does not match the sequence of any known human or mouse genes. Cells (1 × 104/well) were plated in 96-well culture dishes (Nunc) and cultured for 24 h at 37°C in a 5% CO2 atmosphere. When the cells reached 70–90% confluence, they were transfected with 100 nM siRNAs to HSP70A and HSP70C or control siRNAs complexed with Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. The growth medium was removed after 6 h, and the culture dish was transferred to the FIR incubator. After incubation for 48 h at 37°C in a 5% CO2 atmosphere, cell proliferation was assessed by BrdU incorporation. Table 1Characterization of Hsp70 (A, B, and C)GeneLocationAmino acid homology to HSP70a (%)mRNA homology to Hsp70A (%)Alternative namesGenBankHsp70An6p21.3100100HSPA1A, Hsp72, Hsp70-1aM11717Hsp70Blq238484HSPA6, Hsp70BX51757Hsp70C6p21.39998HSPA1B, Hsp72, Hsp70-1bM5980aPercent amino acid homology to the HSP70 protein coded by Hsp70A Statistical analysis Data are means ± SE of replicate samples in single experiments or replicate experiments as described in the figure legends. Student’s t-test was used for comparisons between two groups. Multiple group comparisons were performed by one-way ANOVA, followed by the Tukey–Kramer multiple group comparisons test. All statistical analyses were performed using Statcel 2 software (OMS publishing, Saitama, Japan). Results FIR irradiation selectively inhibits the growth of specific cancer cell lines To clarify the effect of FIR irradiation on the proliferation of cancer cell lines, we irradiated five cancer cell lines (A431, HSC3, Sa3, A549, and MCF7) with FIR and measured the number of live cells by Trypan blue dye exclusion. Although the proliferation of HSC3, Sa3, and A549 cells were significantly suppressed on day 8 of culture (45.75%, 74.63%, and 65.79%, respectively), FIR irradiation had little effect on the growth of A431, or MCF7 cells (Fig. 1). Fig. 1Effect of FIR irradiation on cell growth of five cancer cell lines. Cells (1 × 105) were plated in 24-well dishes and cultured for 8 days. Cell numbers were counted every other day. Although proliferation of HSC3, Sa3, and A549 cells was suppressed on day 8 of culture, FIR irradiation had little effect on the proliferation of A431 and MCF7 cells. *P < 0.05 vs. unirradiated control cells Microarray analysis and extraction of candidate gene for FIR control Several gene families showed high correlations between endogenous expression (signal in the microarray) and the growth rate, proteins involved in cell proliferation, cytoskeletal proteins, cell cycle components, and protein kinases. Especially, in the analysis of stress factor, some genes encoding the HSPs which were well known that they participate in the cellular resistance to stress were focused. Of the 35 HSP genes on the microarray, HSP70 showed the highest correlation with the growth rate (Fig. 2a, b). We examined this further by real time RT-PCR, and we found that HSP70 is most highly expressed in A431 cells at all stages of the cell cycle. Lower levels of expression were found in HSC3 and Sa3 cells (Fig. 2c). We did not find a statistically significant difference between the expression of this gene between control (unirradiated) and FIR-irradiated A431, HSC3, or Sa3 cells. An ELISA for HSP70 in untreated cells showed similar results, specifically, that the expression of HSP70A was higher in A431 cells than in the HSC3 or Sa3 cells (Fig. 2d). Fig. 2Relationship between the expression of HSP70 and the suppression of proliferation by FIR irradiation. (a) Correlation between the signal from the microarray for the HSP family members in the control group and the correlation coefficient. (b) Correlation between the signal from the microarray for HSP70 and the growth rate in the control and the FIR-treated cells. (c) Expression of HSP70A mRNA in A431, HSC3, and Sa3 cells after 0.5, 1, 2, 4, 8, and 12 days of culture. The level of these mRNAs was determined by quantitative real-time RT-PCR. Bars represent the means and error bars the SD from three independent experiments. (d) Level of HSP70 protein on day 4 of culture. The concentration of HSP70 protein was measured by ELISA. Bars represent the mean, and error bars the SD (n = 4). *P < 0.01 vs. to A431 cells Increased expression of HSP70A improves the survival of HSC3 cells after a limited exposure to FIR To directly determine whether HSP70 can protect cells from FIR-induced cell death, we developed A431 and HSC3 cell lines stably expressing human HSP70A (A431-HSP70A and HSC3-HSP70A cells, respectively). Control cells were transfected with empty pcDNA3.1 (A431-Neo and HSC3-Neo; Fig. 3a, b). In our initial experiments, we found that exposure of HSC3 and Sa3 cells but not A431 cells to limited FIR causes G2/M arrest and induces partial hypertrophy to necrosis (data not shown). To determine whether increased expression of HSP70A confers protection against FIR, cell survival was examined in FIR-irradiated A431-HSP70, A431-Neo, A431-wt, and HSC3-HSP70, HSC3-Neo, and HSC3-wt cells. We found that over expression of HSP70A increased cell proliferation in A431 and HSC3 cells. Furthermore, the proliferation of FIR-irradiated and control (unirradiated) A431-HSP70A cells was similar (Fig. 3c). The survival rate after 6 days of FIR irradiation was significantly higher in HSC3-HSP70A cells than in HSC3-Neo or HSC3-wt cells. In addition, the proliferation of FIR-treated HSC3-HSP70A cells was similar to that of control HSC3-HSP70A cells. BrdU incorporation was significantly higher in FIR-irradiated or control A431-HSP70A cells than in A431-Neo or A431-wt cells (Fig. 3d). Although BrdU incorporation of FIR-irradiated HSC3-wt and HSC3-Neo cells was lower than in unirradiated HSC3-wt and HSC3-Neo cells, it was similar in FIR-irradiated and unirradiated HSC3-HSP70A cells. Surprisingly, BrdU incorporation by HSC3-HSP70A cells was significantly higher in both FIR-irradiated and unirradiated cells. These data demonstrate that over expression of HSP70A in HSC3 and A431 cells did not affect their proliferation, and also their morphology, even when they were irradiated with FIR (Fig. 3e). Fig. 3Over expression of HSP70 prevents the suppression of cell growth and induction of cell hypertrophy by FIR. (a) Real-time RT-PCR of HSP70 expression. Cells overexpressing HSP70 were established. (b) Representative Western blots demonstrating increased expression of HSP70 in stably transfected A431 and HSC3 cells. Lane 1, wild-type cells; lane 2, cells transfected with empty pcDNA3.1(+); lane 3, cells transfected pcDNA3.1-HSP70A. (c) Cell proliferation of empty vector-transfected and HSP70-overexpressing cells. Cell proliferation was the same in FIR-irradiated and untreated A431 cells overexpressing HSP70. In contrast, in HSC3 cells, over expression of HSP70 prevented FIR inhibition of cell proliferation. (d) BrdU incorporation in wild-type, empty vector-transfected, and HSP70-overexpressing cells treated with FIR. (e) Hematoxylin and eosin staining of wild-type, empty vector-transfected, and HSP70A-overexpressing A431 and HSC3 cells after 5 days of transfection. Note that the morphology of HSP70A-transfected HSC3 cells was unaffected by FIR irradiation Knocking down HSP70A by using siRNA improves the deth of HSC3 cells after a limited exposure to FIR We next examined the effect of knocking down HSP70A and HSP70C mRNA and HSP70 protein expression using siRNA. Transfection with HSP70A/C siRNA effectively decreased HSP70A and HSP70C mRNA (Fig. 4a) and protein levels (Fig. 4b) in both A431 and HSC3 cells without affecting the level of HSP70B mRNA or protein. HSP70A/C siRNA did not suppress BrdU incorporation in unirradiated A431 cells, but it suppressed BrdU incorporation in cells irradiated with FIR (Fig. 4c). Similarly, the HSP70A/C siRNA enhanced the suppression of BrdU incorporation by FIR irradiation. FIR irradiation also significantly suppressed BrdU incorporation in HSC3 cells transfected with the negative control siRNA (Fig. 4c). These results indicate that a decrease in HSP70 protein mediates the ability of limited FIR to inhibit the proliferation of A431 and HSC3 cells. Fig. 4Inhibition of HSP70 expression and BrdU incorporation in A431 and HSC3 cells after 48 h of transfection with 100 nM HSP70 siRNA. (a) RT-PCR of HSP70A, HSP70B, and HSP70C in A431 and HSC3 cells after treatment with siRNAs. C, wild-type control cells; NC, cells transfected with negative control siRNA; siRNA, cells transfected with HSP70-siRNA. (b) Western blot analysis of HSP70 protein expression in A431 and HSC3 cells. The lanes are as shown in panel A. (c) Inhibition of HSP70 suppressed FIR-induced DNA synthesis in A431 cells. Cells were transfected with 100 nM HSP70 siRNA and grown for 48 h. Bars represent the mean and error bars the SD (n = 5). *P < 0.05). In HSC3 cells, the random siRNA slightly suppressed DNA synthesis, whereas the HSP70 siRNA strongly suppressed DNA synthesis Discussion In the present studies, we showed that FIR suppresses the proliferation of HSC3, Sa3, and A549 cell lines. Two other cell lines, A431 and MCF7, showed almost no growth arrest in response to FIR. The effect of FIR does not seem to be related to the cell type or source, because these three cell lines have different origins: HSC3 is from a tongue squamous cell carcinoma, Sa3 is from a gingival squamous cell carcinoma, and A549 is from a pulmonary adenocarcinoma. FIR was found to cause hypertrophy without apoptosis in all three sensitive cell lines, although there was a slight increase in necrosis in the Sa3 cells by histological observation. In addition, the expression of apoptosis-related genes was unchanged in the FIR-sensitive cells by microarray analysis. Overall, FIR seemed to cause changes in the cytoskeleton, suppress proliferation, and induce some necrosis without apoptosis. These results raise the question of why proliferation of A431 and MCF7 was not suppressed by FIR. Perhaps something in certain cancer cell lines is present for resistance to FIR. We then focused on genes encoding the HSPs, which are well known to participate in the cellular resistance to stress. We found that HSP70 showed the highest correlation with the growth rate of cancer cells affected by FIR in 35 HSP genes on the microarray system. The expression of HSP70A mRNA was higher in FIR-insensitive A431 and MCF7 cell lines than in FIR-sensitive HSC3, Sa3, and A549 cell lines, although the gene expressions were not induced by FIR. This finding was confirmed by analysis of the expression of HSP70 mRNA and protein with real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. These results suggested that the effect on body temperature range by FIR, suppressing the proliferation of some cancer cells, may be controlled by the basal expression level of HSP70A. To test this hypothesis, the effects of overexpression and suppression of the HSP70 gene were studied. As a result, the overexpression of HSP70 inhibited FIR-induced growth arrest in HSC3 cells and an HSP70 siRNA inhibited the proliferation of A431 cells by FIR. These results confirmed that the effect on body temperature range by FIR, suppressing the proliferation of some cancer cells, is controlled by the basal expression level of HSP70A. HSP70 appears to be present in a variety of normal cell types and its expression may be induced by several stressors, such as hyperthermia, cardiac ischemia, infection, UV radiation, endotoxin, and nitric oxide to suppress or denature any foreign protein and restore an injured protein from lethal effects [12]. HSP70 seems to be particularly important for cancer cells. In human breast cancer, the expression of HSP70 correlates with increased cell proliferation, poor differentiation, lymph node metastases, and poor therapeutic outcome [13]. In vivo animal studies and clinical trials have revealed that hyperthermia may serve as a powerful tool in the treatment of prostate cancer [14–19]; at the cellular level, hyperthermic stress induces HSPs. Moreover, chemotherapeutic agents such as cisplatin, adriamycin, and bleomycin, as well as γ-radiation induce HSPs. HSP70 participates in cytoprotection and is associated with cellular resistance to lethal external effects [17–20]. However, in the present study, HSP70 was never induced by FIR. These results suggested that FIR has anti-tumor activity without inducing HSP70 as an anti-stress factor. This characteristic indicates that FIR may be suitable for medical treatment. Neutralization of the function of HSP70 or inhibition of its expression may inhibit tumor growth and/or sensitize tumor cells to chemotherapeutic agents without affecting normal cells. Furthermore, inhibition of HSP70 expression reduces cell survival. Conversely, microinjection of an HSP70-neutralizing antibody increases the vulnerability of cells to damage by sub-lethal temperatures [21, 22]. This finding was consistent with our results for FIR and the suggestion of HSP70 as the acting mechanism. However, FIR may be regarded as a sub-lethal stress that avoids an anti-stress reaction in some cancer cells. This is the first report suggesting that HSP70 may not rescue cells from the effects of FIR, although HSP70 is known to rescue cells from tumor necrosis, factor-induced caspase-independent programed cell death, heat shock, serum starvation, and oxidative stress [23]. This finding suggests that the effect of FIR may be a sub-lethal stress for cancer cells protected by HSP70 as an anti-stress protein. In other words, FIR may be a very effective medical treatment for some cancer cell lines that have a low level of HSP70. Furthermore, if the level of HSP70 in any cancer of a patient is measured, the effect of medical treatment by FIR on the cancer may be predicted. Conclusion It was found here for the first time the effect on body temperature range by FIR in several cancer cell lines in vitro, which is controlled by endogenous HSP70 to protect cells from FIR-induced growth arrest. This finding suggests that FIR may be a very effective medical treatment for some cancer cell lines that have a low level of HSP70. Furthermore, if the level of HSP70 in any cancer of a patient is measured, the effect of medical treatment by FIR on the cancer may be predicted.

Support_Care_Cancer-3-1-2071950

Dying at home or in an institution: perspectives of Dutch physicians and bereaved relatives

Introduction Previous studies have shown that most people prefer to die at their own home. We investigated whether physicians or bereaved relatives in retrospect differently appreciate the dying of patients in an institution or at home. Introduction The two most important events in life, that is, birth and death, relatively often occur at home in The Netherlands. Over the period 1995–2000, around one third of all Dutch births were home deliveries [1]. Further, in 2001, about 40% of all deaths have been found to occur at home, 40% in hospital, and about 20% in nursing homes [2]. Medical care in The Netherlands is strongly founded on home-based general practice. General practitioners or family physicians provide all basic medical care to outpatients and serve as a gate door to specialized care for patients with more complex health problems. In 2003, around 45% of deaths due to cancer occurred at home in The Netherlands [3]. In the UK, the percentage of cancer deaths at home is lower and falling, from 27% in 1994 to 22% in 2003 [4]. In a study of the place of death of cancer patients in the Houston area, USA, 35% died at home [5]. End-of-life care is thus, especially in The Netherlands, rather often provided in the home situation by general practitioners, home-care nurses, and informal caregivers. Research has shown that dying at home is usually preferred over dying in an institution, in The Netherlands as well as in other countries [6]. Such a preference seems to be predominantly shaped by whether or not people have sufficient informal carer resources [7]. In contrast, people who are concerned about the medical management of their symptoms may appreciate the easy access to professional caregivers in an institutional setting. Concerns about burdening relatives have also been found to contribute to a wish to die in an institution [7]. The finding that people’s wish to die at home becomes less predominant when death is nearing may be related to such concerns, as heavily burdening relatives with care duties is one of the most commonly recognized threats to a peaceful dying process [6]. A gradual shift in preference may also be due to the growing awareness of seriously ill people that dying sometimes involves problems and symptoms that are best treated by professional caregivers in an institutional setting. Finally, differential experiences of services also influence people’s preferences [7]. Enabling people to make genuine choices about their end-of-life care and to die at the place they prefer is often seen as a major challenge to current end-of-life care [8–10]. Home-care patients have been reported to have more control over the effects of their illness, medical care, and treatment received than patients receiving institutional care [11]. However, it is unclear if the relatively high home death rate in The Netherlands is really beneficial to the quality of death and dying. Little is known about the experiences of dying patients and their caregivers in different health care settings. We investigated to what extent dying in an institution or at home involves differences in care and its appreciation by physicians and bereaved relatives. Materials and methods Patients This study concerns a sub-sample of a cohort of 128 patients with advanced breast cancer, colorectal cancer, ovary cancer, lung cancer, or prostate cancer, who were followed for a study on end-of-life care needs and practices during the last stage of their lives [12]. Of these patients, 103 died within the time frame during which we were able to approach attending clinical specialists, general practitioners, and bereaved relatives for an after-death data collection. In 102 cases, physicians were willing to fill out a written questionnaire on the medical treatment and dying process of these patients: Questionnaires were filled out by clinical specialists in 30 cases, by general practitioners in 17 cases, and by both in 55 cases. Relatives who had been closely involved with the patient could be contacted in 91 cases; 63 gave their written consent to be personally interviewed at their own home. The reason for not participating was most often that relatives expected participation to be too burdensome. The patients for whom bereaved relatives participated in an interview had on average a longer disease duration (33 vs 21 months; p = 0.03) and were more often women (59 vs 28%; p = 0.004) than other patients. The Medical Ethical Committee of the Erasmus MC, University Medical Center Rotterdam approved the research protocol. Questionnaire for attending physicians As soon as we were informed of the death of a patient, physicians received a self-developed written questionnaire addressing the end-of-life medical treatment and decision-making. The median time between the patient’s death and filling out the questionnaire was 12.5 weeks (range, 1 to 59 weeks). The questions on end-of-life decision-making were based upon questionnaires that have proven to yield valid information in previous studies [2, 13]. In cases where both a clinical specialist and a general practitioner filled out a questionnaire, the information about the patients’ anti-tumor treatment history, medical decision-making, and any ‘negative’ aspects of the dying process were considered to be additive. Interview with bereaved relatives Interviewers were trained to carry out interviews with vulnerable people during a 2-day course. The interviews were on average held 4.9 months after the patient’s death (range, 0.8–9.5 months). The interview schedule included the following topics: personal characteristics of the bereaved relatives, such as age, sex, and relationship with the patient; symptoms of the patient, that is, loss of appetite, pain, fatigue, dyspnoea, nausea, mouth or mucous problems, incontinence, bedsores, confusion, anxiety, and depression; and whether or not the patient was treated for these symptoms. The patient symptom list was based upon the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 [14, 15] and the Problems and Needs in Palliative Care questionnaire [16, 17] and completed with psychological symptoms. Further, contacts with health care professionals, admission to hospital or other care institutions, the actual and preferred place of death, and problems in end-of-life care during the last 3 days of life were assessed using scales from the Voices of Informal Carers-Evaluation of Services (VOICES) questionnaire [18]. Finally, we asked the bereaved relatives about the burden of care giving. We adapted several questions from the Caregiver Reaction Assessment to make them applicable for an after-death interview with bereaved relatives [19]. Statistical analyses We compared the characteristics of patients who died in an institution with the characteristics of patients who died at home. Student’s t tests and χ2 tests were used to assess the statistical significance of differences between both groups. All analyses were done using the Statistical Package for the Social Sciences version 11.0. Results The characteristics of all 103 deceased patients and of 63 patients for whom an after-death interview with a bereaved relative was available are presented in Table 1. The mean age at death of all patients was 65 years; 48 patients (47%) were women. The most common types of cancer were lung cancer (48 patients) and breast cancer (19 patients). Of all 103 patients, 49 died within an institution: 35 patients died at a hospital department, 6 patients died after having been admitted to a department within a hospital, nursing home, or home for the elderly that was specialized in care for dying patients, 4 patients died within a general department of a nursing home or home for the elderly, and 4 patients died in a hospice. The remaining 54 patients died at home or in a home-like situation: 52 patients died in their own homes, 1 patient died in the home of a son, and 1 patient died during a holiday on a boat. The only significant difference between patients dying in an institution and patients dying at home concerned the percentage who had lived with a partner, which was 63% for patients who died in an institution and 83% for patients who died at home. All other characteristics of patients and relatives that are listed in Table 1 were similar in both groups. The characteristics of the group of patients for whom a relative could be interviewed were also similar to the characteristics of the total group. The interviewed relative was the patient’s spouse in 39 out of the 63 cases; in 19 cases, it was a son or daughter, and in 5 cases, another relative. The majority of relatives were women, and their mean age was 55 years. Table 1Characteristics of patients and bereaved relativesCharacteristicsAll deceased patients (N = 103)Deceased patients for whom a relative was interviewed (N = 63)Patients Age at death, in years [mean (SD)]65 (11)64 (11) Sex [n/N (%)]  Female48/103 (47)37/63 (59) Living arrangement [n/N (%)]  With partner76/103 (74)47/63 (75) Education [n/N (%)]  Lowa71/102 (70)41/63 (65) Religion [n/N (%)]  Religious62/103 (60)40/63 (63) Urbanization [n/N (%)]  Living in urban areab72/103 (70)46/63 (73) Primary tumor site [n/N (%)]  Lung48/103 (47)31/63 (49)  Breast19/103 (18)14/63 (22)  Other36/103 (35)18/63 (29) History of anti-tumor treatment [n/N (%)]  Surgery45/102 (44)26/62 (42)  Chemotherapy/hormone therapy84/101 (83)51/61 (84)  Radiotherapy54/101 (54)36/61 (59) Place of death  Institution [n/N (%)]49/103 (48)29/63 (46)   Hospital35/4924/29   End-of-life care unit6/492/29   Hospice4/493/29   Nursing home/home for the elderly4/49–  At home or in a home-like situation [n/N (%)]54/103 (52)34/63 (54)   At patient’s own home52/5432/34   Elsewhere2/542/34Bereaved relatives Age at the time of dying of the patient, in years [mean (SD)]–55 (15) Sex [n/N (%)]  Female–40/63 (63) Relative was patient’s [n/N (%)]:  Spouse–39/63 (62)  Son or daughter–19/63 (30)  Other relationship–5/63 (8)aLow education: lower vocational, lower secondary general education, or primary schoolbLiving in an urban area: patients who were treated as an outpatient in a hospital inside the Rotterdam area Some aspects of medical care are shown in Table 2. In both groups, physicians reported that about two thirds of the patients had died peacefully. Patients who died in an institution had less often (37%) been ready to die than patients who died at home (71%). Agitation was a common problem during the dying process. Most patients in both settings had been unconscious before death. Physicians had discussed a number of end-of-life decisions each with about one third of their patients: This holds for decisions to forgo potentially life-prolonging treatment, intensive treatment of pain, and active euthanasia. Sedation was the only treatment option that was discussed slightly more often with patients who died in an institution. Life had actually been shortened due to the forgoing of potentially life-prolonging treatment or to the use of potentially life-shortening drugs in about one third of all cases in both settings. Such medical decisions were usually made with clear consent of the patient, and life was generally shortened by less than 1 week. Table 2Dying in an institution or at home: the physician’s perspective Place of deathP value χ2 testIn institution (N = 49)At home (N = 54)N (%)Physician’s evaluation of dying processa Patient died peacefully28/41 (68)29/42 (69)0.94 Patient was able to say goodbye to relatives26/40 (65)33/44 (75)0.32 Patient was ready to die15/41 (37)31/44 (71)0.002 Patient died suddenly and unexpectedly13/42 (31)14/44 (32)0.93 Patient’s dying was preceded by period of agitation25/40 (63)29/41 (71)0.43 Patient’s dying was preceded by period of unconsciousness30/39 (77)26/42 (62)0.14Physician discussed with patient medical decisions that could shorten life Forgoing treatment13/49 (27)15/50 (30)0.70 Intensified pain treatment16/49 (33)16/50 (32)0.95 Sedation15/49 (31)8/50 (16)0.09 Euthanasia18/49 (37)19/50 (38)0.90Life was possibly shortened due to Forgoing treatment15/47 (32)14/52 (27)0.59  With clear consent of the patient11/1212/13  Life was shortened by more than 1 week4/110/9 Use of potentially life-shortening drugs16/46 (35)13/45 (29)0.55  With clear consent of the patient12/149/10  With the explicit goal of shortening life1/164/13  Life was shortened by more than 1 week1/132/10Information as provided by clinical specialist (n = 85) and/or general practitioner (n = 72). In case of conflicting answers concerning history of treatment, the information as provided by the clinical specialist prevailed. In all other cases, both sources were considered valid.aStatement was considered true if neither the clinical specialist nor the general practitioner considered it untrue. Bereaved relatives reported in 25 out of 63 cases that the patient had preferred to die at home (Table 3). Two patients had preferred to die in the hospital, 3 in a hospice, 2 elsewhere, and for 31 patients, the relative indicated that the patient had no clear preference. Most patients who had a preference died at the place they preferred, except for five patients who preferred home but died in the hospital. Patients who died in an institution had stayed there for on average 7 days. The number of transfers during 3 months before death was larger for patients who died in an institution (mean 1.9) than for patients who died at home (mean 1.2), and the number of medical specialties that were involved with the patient was also larger in institutions (mean 2.2) than at home (mean 1.8). Care involved on average six disciplines (medical and non-medical), both in institutions and at home. Table 3Preferred and actual place of deathPreferred place of deathaActual place of deathTotal (N = 63)In institutionAt homeHospital (N = 24)Hospice/end-of-life care unit (N = 5)At patient’s own home (N = 32)Elsewhere (N = 2)Hospital22Hospice33At home52025Other place112No (clear) preference16112231aInformation as provided by bereaved relative During the last 3 days of life, a large proportion of patients in both settings suffered, according to the reports of bereaved relatives, from fatigue, loss of appetite, pain, dyspnoea, and mouth or mucus problems (Table 4). Patients dying in an institution were somewhat more often incontinent and more often suffered from anxiety. Patients dying at home slightly more often had bedsores. There were no statistically significant differences between the settings in the degree to which symptoms were addressed with some form of medical treatment, except for loss of appetite, for which 6 of 22 patients who died in an institution received treatment, but only 2 of 30 patients who died at home (p = 0.04). The bereaved relatives’ evaluation of end-of-life care was in general positive in both settings. Some relatives of patients who died in an institution felt they had not sufficiently been involved in the decision-making, whereas all relatives in the other group were satisfied about their level of involvement. Table 4Dying in an institution or at home: perspective of bereaved relative Place of deathP value t testP value χ2 testIn institution (N = 29)At home (N = 34)Number of days patient stayed at place of death [mean (SD)]7 (6)59 (35)0.000Number of transfers [mean (SD)]1.9 (1.7)1.2 (1.3)0.047Number of disciplines involved with patient [mean (SD)]6.1 (2.3)5.8 (1.9)0.64Number of clinical specialties involved with patient [mean (SD)]2.2 (1.2)1.8 (0.8)0.011Patient [n (%)] Was fatigued22 (79)29 (88)0.33 Had loss of appetite22 (76)30 (88)0.20 Had pain20 (69)29 (85)0.12 Had dyspnoea18 (62)17 (52)0.40 Had mouth or mucous problems18 (62)16 (49)0.28 Was incontinent20 (69)13 (38)0.015 Had nausea9 (32)10 (30)0.88 Had bedsores5 (17)13 (38)0.07 Was confused14 (48)10 (29)0.12 Suffered from anxiety12 (41)5 (15)0.021 Was depressed3 (10)2 (6)0.54Evaluation of care during the last 3 days of life [n (%)] Assistance with personal care was sufficient27 (93)32 (94)0.87 Nursing care was sufficienta26 (90)33 (97)0.23 Relative was involved in decision-making25 (89)33 (97)0.22 Relative was sufficiently involved in decision-making24 (89)33 (100)0.049 Patient might have disagreed with medical decision(s)4 (15)1 (3)0.10 Relative disagreed with medical decision(s)7 (24)7 (21)0.78 It had been clear that patient was dying18 (67)26 (79)0.29aIncluding patients who did not need professional nursing care Further, of the bereaved relatives who had cared for a patient who died in an institution, 76% reported that it had significantly affected their own social life; this percentage was 94% for relatives who had cared for a patient who died at home (Table 5). A substantial number of relatives had only received some or no support from other family members (38%), and the number of relatives who reported that their own health had been affected by caring for the patient was also quite large (38%). Caring for a dying relative rarely yielded financial problems. Nearly all relatives thought it very important that they had been involved in caring for the patient. Table 5Dying in an institution or at home: experiences of bereaved relatives Place of deathP value χ2 testIn institution (N = 29)At home (N = 34)n (%)How often did you see patient in the last months of life?0.78 Every day24 (83)29 (85) Less than daily5 (17)5 (15)Did caring for patient affect your own social life?0.068 Yes22 (76)32 (94) No7 (24)2 (6)Did caring for patient result in financial problems?0.17 Yes4 (14)1 (3) No25 (86)33 (97)Did you receive support from your family in caring for patient?1.0 Much support16 (55)23 (68) Some or no support13 (45)11 (32)Did caring for patient affect your own health?0.62 Yes12 (41)12 (35) No17 (59)22 (65)Did caring for patient cost you a lot of energy?0.96 Often or all the time15 (52)15 (44) Rarely or sometimes14 (48)19 (56)How important was being involved with care for patient for you?0.65 Very important26 (90)32 (94) Important3 (10)2 (6) Discussion Medical care for patients who are in the last stage of life has received much attention during the past decade in The Netherlands. The organization of health care services for terminally ill patients has greatly expanded during a 5-year period from 1998 through 2003, during which the government financially supported six university-based centers for the further development of end-of-life care. After this period, the government took the position that, whereas death and dying are common events, the provision of end-of-life care should be part of the professional skills of all physicians, including general practitioners providing end-of-life care to patients who are staying at home. For complex problems, general practitioners and other physicians can now, in many places, seek support from expert teams [20–22]. Of the 103 patients in this study, who were, at inclusion, all treated as outpatients by a clinical specialist, about half died in their own home. Death in an institution mostly concerned a hospital, which was probably not, for all patients, foreseen as the place of dying. Patient characteristics did not clearly determine the place of dying in our study. Whether or not patients lived with a partner was the only variable that was significantly associated with the place of dying. Having access to informal care support is invariably found to be a strong determinant of being able to die at home [4]. Other factors that have been found to affect rates of dying at home are the health status and emotional capacity of the main carer, the availability and use of home-based end-of-life care services, the need for specialist symptom control, tumor type, distance to inpatient services, gender and age of the patient, the patient’s socio-economic status, and strength and visibility of patient or carer preferences concerning the place and circumstances of dying [4, 23–26]. Obviously, the number of cases in our study was limited, and we did not assess all possibly relevant factors, which precludes firm conclusions on determinants of the place of dying. However, whereas access to the available public end-of-life care services, both institutional and home-based, is virtually unlimited in The Netherlands and financially covered by either private or public insurance, care needs of patients are probably a predominant factor. The availability of in- and outpatient end-of-life care services is probably another important determinant of place of death. The number of patients who died in a specialized end-of-life care service in our study was limited. National statistics on the place of dying does not include hospices or end-of-life care units as a separate category. However, the percentage of cancer deaths inside such services as found in our study is probably comparable to the percentage in the total Dutch population. Recently, the number of beds in specialized end-of-life care services in The Netherlands has been rapidly increasing [27]. It is, therefore, likely that the proportion of cancer deaths in end-of-life care services will further increase in the coming years. However, the extent to which dying in an end-of-life care service will substitute dying in a hospital or dying at home is hard to predict. Probably, institutional end-of-life care services address very diverse needs of dying patients and their caregivers. Further, financial and other incentives that are aimed at setting the course for the supply of end-of-life care services may affect developments concerning the place of dying and end-of-life care as well [27]. It is remarkable that a substantial number of patients in our study did not have a clear preference concerning their place of dying. Obviously, we only have information about the preferences that patients had discussed with their interviewed relative. Patients may also have had wishes that relatives were not aware of. Nevertheless, the data in our study suggest that the place of dying is not a great concern for a substantial number of terminally ill cancer patients in The Netherlands. In general, we found few differences in the evaluation by physicians and bereaved relatives of the dying phase of patients who died in an institution and patients who died at home. Patients who died in an institution were, according to their physicians, less often ready to die, which may be due to the fact that hospitalization is typically forgone in patients who are expected to die at short notice. A sudden and more rapid deterioration than expected may also have been among the reasons to admit patients to hospital shortly before dying in some cases. We did not find substantial differences between both settings in most end-of-life decision-making characteristics. Relatives in both settings quite often (21–24%) stated that they had not agreed with the decision-making. Relatives of patients who died in an institution somewhat less often than other relatives felt that they had been sufficiently involved in the decision-making. Dissatisfaction of relatives with end-of-life decision-making in an institutional setting has been described elsewhere too [28–30] and has been attributed to many factors, such as a lack of time of professional caregivers, lack of skills in communication, failure to recognize end-of-life decision-making as a subject that could be discussed, ethical barriers, and the lack of emotional support for relatives [28–32]. It is unclear if our finding that relatives of patients who died outside an institutional setting more often feel satisfied about their involvement in the decision-making process is due to better communication in the home situation. However, the general practitioner, who often has a longstanding relationship with patients and their families, typically plays a key role in end-of-life care at home and may be better able to adequately communicate with family than institutional caregivers [33]. Medical decision-making may also be less complex or controversial for patients who die at home. The possibly rather complex decision-making in institutions is not associated with a higher prevalence of most symptoms. Fatigue, pain, and dyspnoea were among the most common symptoms in both settings, as has been found elsewhere too [34]. Incontinence was more common among patients dying in an institution, and this also holds for anxiety, which was reported as a problem for almost half of all patients dying in an institution. Incontinence and mental status have elsewhere also been found to be associated with a need of nursing home care [35]. In contrast, bedsores were more common at home. Virtually all relatives in both settings were satisfied about the personal and nursing care that had been provided to their dying relative. This may, to some extent, be indicative of the relative quality of end-of-life care in institutions and at home, but using satisfaction as an absolute indicator of the quality of palliative care services is quite problematic [36]. Further, most relatives were closely involved and appreciated their personal involvement very much, both for patients who died in an institution and patients who died at home. Nevertheless, caring for a dying relative often affected their own social life, especially when dying occurred at home, and took a lot of energy. Our study has several limitations. Firstly, the patients’ perspectives on their own dying process is lacking. Bereaved family members’ assessments are known to sometimes disagree with those of the patients’, especially for subjective aspects such as psychological symptoms and pain [37]. This is also true for physicians’ assessment of their patients’ condition. Secondly, our group of patients is probably not representative for all patients dying from one of the five major types of cancer because patients with a very poor health status and patients who died shortly after the diagnosis of incurable cancer were not included in the cohort study upon which the data collection for this study was based. Moreover, data from bereaved relatives could only be obtained for 61% of all patients, which may have resulted in an overestimation of the degree of satisfaction of relatives. The fact that patients of whom no bereaved relative participated in the study had a shorter disease duration at inclusion than other patients suggests that a rapidly progressive disease process makes it more difficult for bereaved relatives to talk about the last phase of life of the patient. In summary, we did not find major differences in the process and quality of dying between institutional settings and the home setting. Most patients with clear preferences concerning their place of dying were able to die at their preferred place. In about half of all cases, relatives were not aware of any preference of the patient concerning the place of dying, which is apparently not a major concern for many people. We conclude that the current situation in The Netherlands, in which the place of dying is mainly determined by the availability of informal caregivers at home and the care needs of the patient, involves no major threats to the process and quality of dying.

Int_Urogynecol_J_Pelvic_Floor_Dysfunct-4-1-2335287

Functional outcome after sacrospinous hysteropexy for uterine descensus

The study aimed to evaluate urogenital symptoms, defecatory symptoms and quality of life before and after a sacrospinous hysteropexy for uterovaginal prolapse. Seventy-two women with symptomatic uterovaginal prolapse were treated with sacrospinous hysteropexy. Before and after surgery, urogenital and defecatory symptoms and quality of life were assessed with a validated questionnaire. Anatomical outcome was assessed by means of pelvic examination before and after surgery. The mean follow-up time was 12.7 months. Scores on all domains of urogenital symptoms and defecatory symptoms, except for the pain and fecal incontinence domain, improved significantly. Also, quality of life improved on all domains. No major complications were encountered. Introduction In the current debate on the optimal surgical treatment of a uterine descent, several vaginal and abdominal techniques have been described. In case of a vaginal vault prolapse, the sacrospinous ligament fixation has proven to be an effective treatment [1]. The sacrospinous ligament fixation can also be performed as primary treatment for a uterine descent, a technique that can be referred to as ‘sacrospinous hysteropexy’. This procedure has been described in women who wanted to preserve the uterus to retain fertility [2, 3]. Several studies have shown that the sacrospinous hysteropexy is anatomical efficient and safe and most women are highly satisfied about the procedure [4–10]. Outcome in these studies was mainly assessed in terms of anatomical results, and the majority of these studies did not evaluate urogenital symptoms and quality of life with validated questionnaires. So, although anatomical outcome of the sacrospinous hysteropexy appears to be good, we cannot conclude from current literature that this type of surgery is associated with a significant functional improvement of urogenital and defecatory symptoms. Measuring this functional outcome preoperatively and postoperatively was one of the recommendations for future research from a recent review on the subject [11]. The main objective of this study was to assess urogenital and defecatory symptoms and quality of life before and after sacrospinous hysteropexy. Secondary, we assessed anatomical outcome. Materials and methods Patients In the period between December 2001 and April 2005, 72 women underwent a sacrospinous hysteropexy at the University Medical Center Utrecht, The Netherlands. A woman was eligible for participation if she had a symptomatic pelvic organ prolapse and wanted surgical correction with preservation of the uterus. Exclusion criteria were abnormal uterus or ovaries on ultrasound examination, abnormal bleeding pattern and abnormal cervical cytology. All women answered a standardized questionnaire covering urogenital symptoms, defecatory symptoms and quality of life before and after surgery. Urodynamic assessment was performed in all women preoperatively. We did not try to diagnose occult stress incontinence. The study was approved by the local ethics committee. Surgery Surgery was performed by two experienced surgeons who at least had performed 20 sacrospinous hysteropexies before start of the study. The sacrospinous hysteropexy was performed unilaterally to the right ligament. A midline incision in the posterior vaginal wall is extended to the posterior part of the cervix. Through sharp and blunt dissection, the right sacrospinous ligament is made visible with the use of three Breisky retractors. Two non-absorbable Prolene® sutures (Ethicon, Sommerville, NJ, USA) are placed through the right sacrospinous ligament, approximately 2 cm median to the ischial spine, and subsequently placed through the posterior side of the cervix in the midline. An additional anterior and/or posterior colporrhaphy (fascia plication), with absorbable Vicryl® 2-0 interrupted sutures (Ethicon, Sommerville, NJ, USA), was performed when indicated by the judgment of the individual gynecologist. In case of stress urinary incontinence, confirmed by urodynamic tests, a surgical procedure of the Tension-free Vaginal Tape (TVT, Ethicon, Sommerville, NJ, USA) was performed as described by Ulmsten [12]. All women received peri-operative thrombosis prophylaxis (anti-Xa) and a single dose of intravenous prophylactic antibiotic (amoxicillin/clavunalic acid). Post-operatively, a 14-French Foley indwelling bladder catheter with a 5-ml balloon was placed in all women and removed after 1 day (in case of an additional anterior colporrhaphy after 3 days). Measurements Data collection took place in October/September 2005, at least 3 months after surgery of the last participating woman. The following data were collected: age at the time of surgery; medical history; hospital stay and perioperative and postoperative complications. The anatomical outcome of all women was assessed with the Pelvic Organ Prolapse Quantification score (POP-Q) [13], which is described by the International Continence Society as a reliable and specific method to measure the pelvic organ support. Before surgery, POP-Q was performed by one of the two surgeons. After surgery, POP-Q was performed by one of three independent investigators. In the analysis, we dichotomized the POP-Q stage of prolapse into stage 1 or less and stage 2 or higher. Although we know that women with pelvic organ prolapse experience symptoms that do not necessarily correlate with the severity of prolapse, we have chosen this approach to try to separate potential clinical irrelevant from clinical relevant recurrences [14]. Urogenital symptoms were measured before and after surgery with a standardized questionnaire, the Urogenital Distress Inventory (UDI), which has been validated for the Dutch population [15]. In this validation study on a large population-based sample, it was shown that the domain construction of the Dutch version was different from the original one. The following five domains were identified: urinary incontinence, overactive bladder, pain, obstructive micturition and prolapse. The scores of these domains vary between 0 and 100. A high score on a particular domain indicates more bothersome symptoms. The incidence of urinary incontinence before surgery was measured as follows: a woman was considered to have stress urinary incontinence if she replied positively to the question “Do you experience urinary leakage related to physical activity, coughing or sneezing?”. Urge urinary incontinence was scored if the question, “do you experience urinary leakage related to the feeling of urgency?” was answered positively. All patients completed a questionnaire, the Defecatory Distress Inventory (DDI) to assess the presence of defecation symptoms before and after surgery. This questionnaire was developed by our research group to assess the presence of defecation symptoms [16]. The DDI consists of 15 items about symptoms related to obstructive defecation, constipation, fecal incontinence and pain related to defecation. The questions were developed after studying the literature and international definitions, interviewing patients who presented with constipation or fecal incontinence, and by interviewing three experts in the field from the Department of Surgery and Department of Obstetrics and Gynecology from the University Medical Center Utrecht, The Netherlands. Eventually, a structured interview of the 15 selected items was held with 20 female patients. The DDI was used as, at present, there are no other Dutch validated questionnaires to measure quality of life related to defecation symptoms. The design on the questions is identical to those of the UDI with domain scores between 0 and 100. Again, a high score on a particular domain indicates more bothersome symptoms. The DDI was used in previous studies at our department [4, 9, 17]. Before and after surgery, disease-specific quality of life was measured with the Incontinence Impact Questionnaire (IIQ), validated for the Dutch population [15]. These questions cover the following five domains: physical functioning, mobility, emotional functioning, social functioning and embarrassment. The score ranged between 0 (best quality of life) and 100 (worst quality of life). Effect sizes were measured as a useful way to estimate whether an improvement on a particular domain was considered to have a small, moderate or large clinical relevance. Statistical analysis Descriptive statistics were used for the whole population. To compare scores on urogenital and defecatory symptoms before and after surgery a paired samples t test was used. The significance level was set at α of 0.05. The effect size was calculated by Cohen’s d test which is defined as the difference between two means divided by the pooled standard deviation for those means [18]. An effect size of 0.2 was considered to be small, 0.5 to be median and 0.8 or higher to be large [18]. Statistical analysis was performed in SPSS 12.0 for Windows. Results Table 1 shows the characteristics of the 72 women. The fast majority had a combination of sacrospinous hysteropexy with an anterior colporrhaphy (87.5%). Five women (6.9%) had a sacrospinous hysteropexy of a stage 1 uterine descent. In these women, the surgeon decided to perform a sacrospinous hysteropexy during surgery because the uterine descent under anaesthesia was stage 2. The mean follow-up time was 12.7 months (median 11 months). Table 1Patient characteristicsPatient characteristicsn = 72Age (years)a57.2 (11.9)Surgery for prolapse in medical history3 (4.2)Surgery for urinary incontinence in medical history4 (5.6)Urinary incontinence before surgeryb40 (55.6) Urge incontinence9 (12.5) Stress incontinence21 (29.2) Combination stress and urge incontinence10 (13.9)Gynecological examination before surgeryDescensus uteri stage 2 or more67 (93.2)Cystoceles stage 2 or more57 (79.2)Rectoceles stage 2 or more19 (26.4)Enterocele1 (1.4)SurgerySacrospinous hysteropexy8 (11.1)Sacrospinous hysteropexy + anterior colporrhaphy54 (75)Sacrospinous hysteropexy + anterior and posterior colporrhaphy9 (12.5)Sacrospinous hysteropexy + posterior colporrhaphy1 (1.4)Additional TVT15 (20.8)Hospital stay (days)c3.5 (1–8)Follow-up in monthsc12.7 (3–33)Data are numbers (%)aMean (standard deviation)bAssessed with Urogenital Distress InventorycRange Perioperative and postoperative complications are shown in Table 2. One woman needed repeated surgery for postoperative bleeding a couple of hours after the primary procedure. Total blood loss was estimated at 400 cc. There were no incidental bladder or rectal injuries. Of the 20 women (27.8%) who had urinary retention over 100 ml after removal of the indwelling bladder catheter, five women (6.9%) needed intermittent self-catheterisation more than 2 weeks after surgery. However, at 4 weeks, no women had significant urinary retention. This complication only occurred in women who underwent an anterior colporrhaphy. Seven women with cystitis received antibiotics and made an uneventful recovery. Five women (6.9%) developed right-sided buttock pain which persisted longer than 2 weeks. It resolved spontaneously within 6 weeks. No removal of the sacrospinal suture was required. Table 2Complications related to surgeryPatient characteristicsn = 72Complications during surgery0 (0)Complications after surgery32 (44.4) Second surgery because of bleeding1 (1.4) Buttock pain13 (18.1)  Buttock pain <2 weeks8 (11.1)  Buttock pain >2 weeks5 (6.9) Vaginal hematoma2 (2.8) Urinary tract infection7 (9.7) Retention bladder20 (27.8)  Bladder catheterisation <2 weeks15 (20.8)  Bladder catheterisation >2 weeks5 (6.9) Vaginal adhesion3 (4.2)Data are numbers (%) During the follow-up period, a total of 16 women (22.2%) had a recurrent prolapse of one of the compartments. Five women (6.9%) had a recurrent prolapse of the uterus (four women stage 2, one woman stage 3). Ten women (13.9%) had a cystocele stage 2 or more (eight women stage 2, two women stage 3) and two women (2.8%) had a prolapse of the posterior compartment stage 2. All the women with a recurrent cystocele had had surgery of the anterior compartment combined with the hysteropexy, so there were no de novo cystoceles. The two women with a recurrent rectocele did not have surgery of the posterior compartment combined with the sacrospinous hysteropexy, so these can be considered as de novo rectoceles. Table 3 shows the results of the UDI, DDI and IIQ domain scores before and after surgery. On all urogenital domains, there was significant improvement as well as on all quality of life domains. Symptoms on domain constipation and obstructive defecation also improved significantly. Large effect sizes were found on domain pain (effect size = 0.92) and genital prolapse (effect size = 2.0) of the UDI. The domain physical functioning and emotional health of the IIQ also showed a large effect size (0.82 and 0.79, respectively). Table 3Urinary Distress Inventory (UDI), Defecatory Distress Inventory (DDI) and Incontinence Impact Questionnaire (IIQ) Before surgerya (n = 72)After surgerya (n = 72)P valueEffect sizeUDIUrinary incontinence18.5 (24.2)10.5 (21.0)0.0120.35Overactive bladder29.5 (26.7)14.4 (18.6)0.0000.66Pain30.1 (26.7)9.5 (16.7)0.0000.92Obstructive micturition27.0 (28.3)10.8 (19.7)0.0000.66Genital prolapse56.6 (32.0)5.6 (17.4)0.0002.0DDIConstipation11.9 (18.8)6.3 (9.8)0.0210.37Obstructive defecation13.3 (19.7)8.3 (11.8)0.0160.22Pain4.5 (16.3)2.8 (8.3)0.4980.13Fecal incontinence8.6 (20.7)8.5 (14.4)0.9550.01IIQPhysical functioning26.0 (24.4)9.4 (15.1)0.0000.82Mobility25.0 (22.8)12.6 (18.4)0.0000.60Emotional health23.2 (23.3)8.0 (14.2)0.0000.79Social functioning14.8 (19.1)4.5 (11.8)0.0000.65Embarrassment11.2 (13.8)6.7 (11.1)0.0130.41Effect size: 0.2 = small effect, 0.5 = median effect, 0.8 = large effectaMean (standard deviation) In addition to the table we made a sub-analysis for the 15 women who had additional surgery for urinary incontinence (TVT). These women improved significantly on the urinary incontinence domain after surgery (mean score: 26.7→6.7, p = 0.009). This improvement was not significant for the women without TVT surgery (mean score: 16.4→11.5, p = 0.162). On the other hand, the women with TVT surgery did not improve significantly on the overactive bladder domain (mean score: 24.3→16.23, p = 0.079) where the group without TVT did (mean score: 30.8→14.3, p = 0.000). Discussion The objective of this study was to assess quality of life and urogenital and defecatory symptoms before and after sacrospinous hysteropexy. The results show that a sacrospinous hysteropexy significantly reduced all urogenital and several defecatory symptoms and significantly improved quality of life. Effect sizes were large on domain genital prolapse and pain (UDI), and on domain physical functioning and emotional health (IIQ). It also anatomically cured the uterine descent in 93.1% of women. The sacrospinous ligament fixation was first described by Sederl (1958) [19]. Later it became more popular by Richter and Albright [20] (Europe) and Randall and Nichols [21, 22] (USA). Several modifications of their techniques have been described since. The anatomical results of 2,256 women after a sacrospinous ligament fixation of the vaginal vault were recently reviewed [1]. Objective cure rates varied between 67 and 96.8%, and subjective cure rates varied between 70 and 98%. Our findings are in line with these results, although the review focused on the sacrospinous fixation of a vault prolapse. Subjective outcomes are underreported in most studies on the sacrospinous ligament fixation. We have shown prospectively that bladder and bowel function improves significantly after a sacrospinous hysteropexy. There are a variety of reasons why women want to preserve their uterus. Among those reasons are: keeping their fertility, personal identity, but also the possibility that this kind of surgery might reduce operation time, estimated blood loss and postoperative recovery time [4, 23]. There are signs that removing the uterus may increase the risk of pelvic neuropathy, new onset urinary incontinence, bladder dysfunction and prolapse [24, 25, 26]. Several studies on the sacrospinous hysteropexy, as a technique in which the uterus is preserved, are available [3–6, 8–10, 27]. Among these studies, three were of prospective design [6, 8, 27], five were of retrospective design [3–5, 9, 10] and there was one case report [28]. One report described a different surgical technique and therefore cannot be compared with our study [2]. One study assessed risk factors for failure of sacrospinous hysteropexy [29] and another study only assessed sexual functioning after sacrospinous hysteropexy [30]. Anatomic success rates in these studies varied between 74 and 93.5%, which is comparable with our results. The main problems when comparing studies on the sacrospinous ligament fixation were recently debated by Morgan et al. [11]. They showed that there is a variety in definition of failure of sacrospinous ligament fixation due to differences in how anatomical outcomes are evaluated and which compartment of the vagina is considered. In our group, recurrent postoperative cystoceles, stage 2 or higher, were seen in 10 women (13.9%). This percentage is slightly lower than the 21.3% reported in the recent review [11]. However, our follow-up in the current study was relatively short and some recurrences may not have been detected in this timeframe.. In a previous retrospective study on the anatomical outcome of the sacrospinous hysteropexy by our group, we found a higher rate of recurrent cystoceles stage 2 or higher (30%) [9]. The follow-up period in this study was a mean of 23 months. The high rate of recurrent cystoceles may be related to the primary damage of neuromuscular support or may be the result of the retroverted axis of the vagina after sacrospinous hysteropexy. This last aspect, being regarded as an overcorrection, is held responsible for the high rate of cystoceles [27, 31]. However, in a study by Smilen et al. [32] the sacrospinous hysteropexy did not independently increase the risk of recurrent cystocele as compared to other surgical techniques. Apart from true genital prolapse symptoms, urogenital symptoms and also bowel symptoms improved after the sacrospinous hysteropexy. Because the majority of women in our study had their sacrospinous hysteropexy combined with an anterior colporrhaphy, one may argue that it was this anterior repair that relieved symptoms, and not the sacrospinous hysteropexy. However, it was shown that pelvic organ prolapse and urogenital symptoms were only slightly correlated to the site and severity of the prolapse [14]. This lack of a clear correlation between the site of the pelvic organ prolapse and symptomatology makes it very difficult in combination surgery to contribute functional improvement to a certain intervention. All we can conclude from our results is that surgical procedures that involve a sacrospinous hysteropexy show good functional outcome. It was shown that overactive bladder symptoms disappear after anterior repair in 60–82% of women [33]. We also found a marked improvement of overactive bladder symptoms after surgery in our group. However, this significant improvement was confined to the women who did not have a combined TVT procedure with their sacrospinous hysteropexy. Women who did have a TVT combined procedure experienced more bother on overactive bladder domain after surgery as compared to women without TVT surgery. This finding is consistent with literature on the TVT in which the development of overactive bladder symptoms after TVT surgery is reported to occur in up to 15% of women [34]. We have to keep this in mind when placing a TVT (prophylactic) in case of occult stress incontinence. After sacrospinous hysteropexy, postoperative complications occur, but none of them was life threatening. Most complications were self-limiting. The majority of postoperative complications were related to the bladder function. These complications did not occur in women who only had a sacrospinous hysteropexy. Therefore, it is likely that complications related to the bladder are the consequence of additional surgery and not the result of sacrospinous hysteropexy. The prevalence of buttock pain is estimated at 10 to 15% [35]. This pain can be explained by injury to surrounding nerves of the sacral plexus and branches of the pudendal nerve. In an anatomical study, the relationship of the pudendal nerve to the sacrospinous ligament was found to be variable (one branch of the pudendal nerve piercing through the ligament was found in 11%) [36]. Barksdale et al. also showed that nerve tissue is present and widely distributed within the sacrospinous ligament [37]. Therefore, although the placement of the suture two centimetres medial to the ischial spine protects against major nerve injuries, the complications of buttock pain cannot be prevented in all women. Fortunately, this buttock pain was shown to resolve spontaneously in most cases, as we also demonstrated in our series [9]. The strength of our study is that we measured urogenital and defecatory symptoms and quality of life in a large group of women who underwent a sacrospinous hysteropexy, with a validated questionnaire before and after surgery. There are some potential drawbacks that need to be discussed. First, there might be an indication bias. In our country, a vaginal hysterectomy is the standard surgical technique for correcting a uterine descent. Therefore, women that came to our hospital may have chosen specifically for this operation. They might have had high expectations of this procedure which could have influenced their outcome with respect to quality of life. Second, in some patients, follow-up time was limited to 3 months. Possibly, some recurrences had not yet developed at that time. Third, the study was performed in a single university hospital. The sacrospinous ligament fixation has become rapidly popular in our center and is performed by two surgeons. They are highly trained in performing the procedure. This might have influenced the outcome. Fourth, we did not compare the sacrospinous hysteropexy with another surgical technique to correct a uterine descent. Therefore, we cannot conclude that the sacrospinous hysteropexy is superior to other procedures. However, it is a safe and effective operation for women who wish to preserve their uterus at time of genital prolapse surgery.

Exp_Brain_Res-3-1-1914235

Error correction in bimanual coordination benefits from bilateral muscle activity: evidence from kinesthetic tracking

Although previous studies indicated that the stability properties of interlimb coordination largely result from the integrated timing of efferent signals to both limbs, they also depend on afference-based interactions. In the present study, we examined contributions of afference-based error corrections to rhythmic bimanual coordination using a kinesthetic tracking task. Furthermore, since we found in previous research that subjects activated their muscles in the tracked (motor-driven) arm, we examined the functional significance of this activation to gain more insight into the processes underlying this phenomenon. To these aims, twelve subjects coordinated active movements of the right hand with motor-driven oscillatory movements of the left hand in two coordinative patterns: in-phase (relative phase 0°) and antiphase (relative phase 180°). They were either instructed to activate the muscles in the motor-driven arm as if moving along with the motor (active condition), or to keep these muscles as relaxed as possible (relaxed condition). We found that error corrections were more effective in in-phase than in antiphase coordination, resulting in more adequate adjustments of cycle durations to compensate for timing errors detected at the start of each cycle. In addition, error corrections were generally more pronounced in the active than in the relaxed condition. This activity-related difference was attributed to the associated bilateral neural control signals (as estimated using electromyography), which provided an additional reference (in terms of expected sensory consequences) for afference-based error corrections. An intimate relation was revealed between the (integrated) motor commands to both limbs and the processing of afferent feedback. Introduction Recently, the relative importance of perceptual processes for motor control in general, and bimanual coordination in particular, has been intensely debated [e.g., see target article of Mechsner (2004) and associated commentaries]. Despite differences in the conceptual interpretation of empirical findings, there is ample evidence that perceptual factors affect bimanual coordination. For example, beneficial effects of augmented visual feedback on rhythmic bimanual coordination have been observed (Swinnen et al. 1997; Byblow et al. 1999; Mechsner et al. 2001), in combination with changes in concurrent neural activity (Debaere et al. 2003; Carson et al. 2005). In addition, it has been shown that perturbations of proprioception using tendon vibration result in disruption of the temporal coordination between the limbs (Verschueren et al. 1999; Steyvers et al. 2001). On the other hand, however, recent studies have indicated that important characteristics of rhythmic bimanual coordination are not primarily attributable to somatosensory feedback (Ridderikhoff et al. 2005b; Spencer et al. 2005), suggesting a key role for afference-independent (i.e., open-loop) control processes. Collectively, these findings motivated the present study on kinesthetic tracking, which (1) examined the closed-loop control processes underlying rhythmic bimanual coordination, and (2) addressed the potential interplay between open-loop and closed-loop control in the production of rhythmic bimanual movements. In the present study, we focus on rhythmic bimanual movements performed at the same frequency. Such movements are characterized by the presence of only two stable coordination patterns that can be performed without training (Zanone and Kelso 1992), and that are defined in terms of the relative phase between the hands (Φ). In most cases, one pattern (in-phase coordination; Φ = 0°; simultaneous activation of homologous muscles) is more stable than the other pattern (antiphase coordination; Φ = 180°; alternated activation of homologous muscles) (Kelso 1984; Swinnen 2002). These characteristics imply that the functional consequences of various control processes can be appreciated directly in terms of their contribution to the stability difference of in-phase and antiphase coordination. In general, these control processes can be classified according to three sources of interlimb interactions. One of these sources is afference-independent, and refers to open-loop processes involved in the integrated timing of the feedforward signals to both limbs. The other two sources are associated with closed-loop processes: a reflex-like interaction resulting in phase entrainment by contralateral afference, and intentional corrections of the timing based on the perceived error in the relative phase. This conceptual framework is motivated and described in detail elsewhere (Ridderikhoff et al. 2005b). In previous studies we found that the stability difference between in-phase and antiphase coordination depended mainly on the integrated timing of the feedforward control signals (Ridderikhoff et al. 2005b), and that phase entrainment by contralateral afference did not affect this difference (Ridderikhoff et al. 2005b, 2006). As regards the second afference-based source of interlimb interactions, timing corrections based on the perceived relative phase, a more complex picture emerged, which motivated the present study as discussed in the following paragraphs. Kinesthetic tracking tasks have been used to assess the role of afference-based interactions between the limbs in rhythmic bimanual coordination, by examining the coordination of unilateral active rhythmic movements with motor-driven oscillations of the contralateral limb (Viviani et al. 1997; Stinear and Byblow 2001; Ridderikhoff et al. 2005b). In agreement with the stability characteristics of rhythmic bimanual coordination, two previous studies on kinesthetic tracking have demonstrated differences between in-phase and antiphase coordination. One study revealed a more variable relative phase and larger response times on a secondary task for antiphase coordination than for in-phase coordination (Stinear and Byblow 2001). The other study showed that the mean relative phase was more susceptible to an external stimulus (i.e., less stable) during antiphase coordination than during in-phase coordination (Ridderikhoff et al. 2005b). Thus, the study of kinesthetic tracking indicated that afference-based interlimb interactions may contribute to the stability difference between in-phase and antiphase coordination. In terms of the aforementioned (afference-based) sources of interlimb interaction these pattern-related stability differences should be attributed to corrections based on the perceived error in the relative phase, because phase entrainment by contralateral afference has been found to stabilize in-phase and antiphase coordination in equal measure (Ridderikhoff et al. 2005b; Ridderikhoff et al. 2006). In our previous work we found that muscle activation patterns during kinesthetic tracking exhibited a striking similarity to the patterns observed in normal bimanual coordination, even though muscular activity was neither required nor of any consequence for the movement pattern of the motor-driven hand (Ridderikhoff et al. 2005b). In contrast, if motor-driven movements were used to induce phase entrainment by contralateral afference (i.e., when one limb was moved passively, but no coordination between the limbs was required), such activation patterns were not observed (Ridderikhoff et al. 2005b, 2006). Thus, the spontaneously adopted tendency of subjects to activate their muscles as if they were moving along with the motor-driven movement appeared directly related to the requirement of achieving a particular coordination pattern between the limbs. In principle, such spontaneous muscle activation in the driven limb may serve several purposes. It may be, for instance, that it reflects coactivation of gamma-motoneurons to enhance the sensitivity of muscle spindles (Gandevia et al. 1992). However, this possibility is less likely because several studies reported an increase of detection thresholds (Wise et al. 1998) and attenuation of muscle sense (Collins et al. 1998) during voluntary contractions (see Proske 2006 for a review). Alternatively, it may be that the activation of muscles in the motor-driven arm reflects the use of an internal control signal specifying the bimanual movement pattern. Considering that our previous work has indicated a crucial contribution of afference-independent interlimb interactions to the stability of the normal bimanual coordination (Ridderikhoff et al. 2005b), this internal control signal may well be generated reliably in an open-loop fashion. Hence, in the context of kinesthetic tracking this open-loop control signal may provide a suitable reference against which the error in the relative phasing (based on afferent signals) could be determined. Such involvement of motor commands in limb kinesthesis was recently demonstrated empirically (Gandevia et al. 2006), and has been advocated as a fundamental principle of motor control involving predictions of the movement’s sensory consequences via efference copy (e.g., Wolpert and Ghahramani 2000; Scott 2004). Note that this second account of the EMG activity in the driven limb, which in fact served as a working hypothesis for the present study, implies an intimate relation between two of the aforementioned sources of interlimb interaction, viz. error correction based on the perceived relative phase, and the integrated timing of the feedforward signals (providing the reference pattern). The aim of the present study was to examine the afference-based error corrections in detail using a kinesthetic tracking task, with a specific focus on the role of the muscle activity in the motor-driven arm, to elucidate the interplay of closed-loop and open-loop control processes in rhythmic bimanual coordination. We compared the performance during in-phase and antiphase coordination to further our understanding of the potential contribution of closed-loop control processes to the differential stability of these patterns. In addition, we examined the stability-related effects of bilateral muscle activation during kinesthetic tracking on the resulting coordinative stability. To address the latter issue we compared two conditions in which subjects were either instructed to keep the motor-driven limb relaxed, or to activate their muscles as if moving along with the imposed motor-driven movement. For the latter condition, the phase relations at the level of neural control signals (based on electromyographic data) and at the behavioral level (kinematics) were compared. The neural control signals represent the reference signal that may be used for the prediction of sensory consequences of the ongoing movement, whereas the kinematic phase relation reflects the actual quality of the performance. In both conditions, we performed an extensive analysis of the correlations between various kinematic variables to uncover the underlying structure of the timing corrections based on the (perceived) errors in the relative phase. Materials and methods Subjects Twelve healthy subjects (6 male, 6 female; aged 19–31 years) volunteered to participate. All subjects were right-handed according to their scores on a common handedness questionnaire (Oldfield 1971). They had no previous experience with the task and reported no (history of) neurological disorders. The local ethics committee approved the experiment and all subjects gave their written informed consent before the experiment was conducted. Apparatus Subjects sat in a height-adjustable chair with their elbows slightly flexed and their feet supported. Each forearm was placed in the apparatus in a neutral position (thumbs up and palms facing inward), and its position was restrained (by the support surface on the medial and ventral side, by two vertical foam-coated supports on the dorsal side, and by one horizontal foam-coated support on the lateral side) to prevent movements about the elbow. Both hands were fixated against the flat manipulanda using two Velcro straps, with all fingers extended. The apparatus only permitted flexion–extension movements of the wrist in the horizontal plane. The right manipulandum was mounted on a potentiometer (Sakae, type FCP40A-5k, linearity 0.1%) to register wrist joint angles during active movement, while the left was connected to a servo-controlled motor that moved the hand passively. The potentiometer’s output voltage was digitized by a 12-bit ADC (Labmaster DMA) and stored on a microcomputer at a sampling frequency of 1,000 Hz. The active movements were recorded with a precision of about 0.1°. The passive movements were generated using a DC brush motor (PARVEX, type RS440GR) that was controlled by a PC-mounted servo controller (ACS-Tech80, type SB214). The maximum torque of the motor was such that subjects were unable to alter the trajectory of the applied movements, and the maximum error in the trajectory of the passive movements was 0.26°. Subjects wore earmuffs with built-in stereo earphones (Bilsom 787, Flex II), which provided a moderate level of ‘white’ background noise to eliminate any auditory feedback from the motion of the motor. A white opaque screen was used to eliminate visual feedback of the hand movements. Surface electromyograms (EMG) were obtained from M. flexor carpi radialis (FCR), and M. extensor carpi radialis (ECR) of both arms. A bipolar arrangement of disposable electrodes (Medicotest, Ag/AgCl-electrodes, square 5 × 5 mm pick-up area) was attached with a center-to-center distance of 2 cm after cleansing and abrasion of the skin. The electrodes were positioned in the center of the muscle belly on the line from origin to insertion as determined by palpation. EMG signals were sampled at 1,000 Hz (TMS International, type Porti5-16/ASD; 22 bits ADC) after band-pass filtering (0.5–400 Hz), and stored on a microcomputer. Procedure Subjects were instructed to perform smooth oscillatory movements about the right wrist in such a way that (1) peak flexion and peak extension of both wrists were attained simultaneously (in-phase pattern), or (2) peak flexion of one hand coincided with peak extension of the other hand (antiphase pattern). To achieve this, the timing of the active right wrist movements had to be coordinated with the motor-driven movements of the left wrist. Only subjects that were able to perform both movement patterns in at least one of two selection trials at the start of the experiment were included (one candidate subject failed to meet this criterion). After the selection trials the EMG electrodes were applied, and subsequently all subjects performed maximum voluntary contractions (MVCs) by generating an isometric flexion or extension torque with each arm for approximately 3 s. For the purpose of normalization of the EMG, the maximum root mean square (RMS) value of two separate MVC measurements was used in the analysis. Additional instruction was given to subjects with respect to the muscle activity in the left (driven) arm. Subjects were required either to keep the muscles of the left (driven) arm as relaxed as possible (relaxed condition) or to activate the muscles of the left arm as if they were moving along with the motor-driven manipulandum (active condition). The resulting 2 (Pattern) × 2 (Activity) = 4 conditions were performed in separate blocks of trials, the order of which was counterbalanced across subjects. Each block of trials started with at least four practice trials to familiarize the subjects with the task (if necessary, maximally four additional practice trials were allowed). Once the subject was able to perform the task properly, based on visual assessment by the experimenters, six experimental trials were performed that were used for the analysis. For the motor-driven movements of the left wrist, sinusoidal trajectories with an average movement frequency of 1.4 Hz and mean amplitude of 35° (i.e., a range of motion of 70° about the neutral position of the wrist) were used. To create a challenging tracking task the period length and the amplitudes were varied within a trial. Because pilot experiments had shown that too much variability induced high levels of (mainly tonic) muscle activity in the relaxed condition, a moderate level of variability was selected, i.e., intermediate between natural variability and perfectly sinusoidal trajectories as used in previous studies (Ridderikhoff et al. 2005b; Stinear and Byblow 2001, respectively). Subjects started with a low level of variability in the selection trials (SDfrequency = 0.02 Hz; SDamplitude = 2°), which was increased in a step-wise fashion during the practice trials to the level of variability used in the experimental trials (SDfrequency = 0.03 Hz; SDamplitude = 5°). To assure a smooth start and finish of the trial, the amplitude of the motor-driven movements was gradually increased (from 0°) and decreased (to 0°) during the first and last 3 s of a trial, respectively. The duration of a trial was 30 s. To avoid transient effects and to exclude the cycles in which the amplitude of the passive movement was adjusted, the first 7 and the last 3 s of the trial were discarded, leaving 20 s per trial for analysis. Data reduction (kinematics) Figure 1 illustrates and defines the features of the time evolution of the joint angles on which the analyses of the kinematics were based. Because systematic differences in coordination were present depending on whether peak flexion or peak extension was chosen as reference, the relative phase between the hands (Φ) was calculated for each cycle as for flexion and as for extension, where ty,i and tx,i indicate the time of the ith peak flexion or peak extension of the right and left hand, respectively (for a similar method see, e.g., Carson et al. 1995). A positive relative phase meant that the right hand was lagging the left hand. Circular statistics (Mardia 1972) was used to calculate the mean and the circular standard deviation of the relative phase (SDΦ). The absolute error of the relative phase (AEΦ) was defined as the absolute difference between the mean relative phase and the required relative phase (0° for in-phase; 180° for antiphase). Fig. 1Main kinematic features of one full cycle of both hands, illustrated for the in-phase coordination pattern. Moments of peak flexion and extension at the start of the ith cycle in each hand are designated by tj,ik, where k indicates flexion (F) or extension (E), and j refers to the right hand (y) or left hand (x). The durations of the ith full cycle between two moments of peak excursion are designated by Δtj,ik. The duration of the ith half cycle is designated by δtj,il, where l indicates the orientation of the hand at the end of the half cycle: flexion (F) or extention (E). The (signed) error at the start of the ith cycle is designated by εik. The definition of the (signed) errors depends on the coordination pattern and is defined in terms of the relative timing of corresponding peak excursions in both hands (flexion–flexion and extension–extension for in-phase; flexion–extension and extension–flexion for antiphase) In a previous study (Ridderikhoff et al. 2005b), we showed that kinesthetic tracking performance can also be meaningfully evaluated in terms of correlations (RFC; FC = full cycle) between the signed timing error at peak flexion or peak extension (εi) and the duration of the following full cycle of the right (actively moving) hand (Δty,i). The rationale behind this measure is that if an error in the relative phasing is detected at the start of the ith cycle, this error may be compensated for by adapting the duration of the next cycle, resulting in negative values of RFC. Motivated by the aforementioned differences between flexion and extension that were observed for the relative phase (see also “Results”), we also calculated the correlation between this error in the relative timing and the duration of the following half cycle of the right hand (RHC; HC = half cycle). RFC and RHC are intimately related in that the error correction made during a full cycle is the sum of the error corrections made during its two half cycles, provided that the latter two corrections are independent of each other. However, in general this proviso is not met, because part of the correction in the second half cycle may in fact compensate for errors that arose in the first half cycle, which is reflected by two additional temporal correlations. First, deviations in the duration of the first half cycle of the left (driven) hand (as a consequence of the variability of the imposed reference trajectory) may result in errors at the start of the second half cycle that lead to adaptation of the duration of the consecutive half-cycle of the right hand. This dependency is reflected in positive ‘between-hands’ correlations (Rxy) between the durations of a half-cycle of the left (driven) hand and the following half-cycle of the right hand. Second, if in each half-cycle the errors are overcompensated, a sequence of alternating longer and shorter half-cycles is obtained. Such a dependency is reflected by negative ‘within-hand’ correlations (Ryy) between the successive half-cycles of the right hand. Thus, positive values of Rxy and negative values of Ryy reflect dependencies between the error corrections in successive half cycles that reduce their contributions (RHC) to the full cycle error corrections RFC. These four temporal correlations (RFC, RHC, Rxy, and Ryy) provide information about the temporal structure of the performance in terms of the timing of peak flexion and peak extension of the wrist. As such, these measures are more closely related to the underlying control processes than the global performance measures based on the relative phase. Exact definitions of (the relations between) these measures can be found in the Appendix in terms of the underlying covariances. Because systematic differences were found between the actual timing of peak flexion and that of peak extension, the effects of these two orientations on the error correction measures (RFC and RHC)—as obtained for timing errors at peak flexion or peak extension of the right (actively moving) hand in relation to the duration of the following (full or half) cycle of this hand—were examined as well. In addition, the effects of orientation on Ryy and Rxy were evaluated by comparing the correlations between the durations of the two successive half-cycles following either peak flexion or peak extension of the right (actively moving) hand. In this way, the implications of Ryy and Rxy for the effects of orientation that were observed for RFC could be discerned (see Appendix and Fig. 4). Data reduction (EMG) EMG records were bandpass filtered (10–400 Hz) using a second-order bidirectional (zero-lag) Butterworth filter (Merletti et al. 1999). To visualize the average muscle activity within a cycle, eight bins were defined in relation to the continuous phase of the movement Θ = arctan[(dθ/dt)/(2πfθ)], where θ and (dθ/dt) are joint angle and joint angular velocity, respectively, and f is the movement frequency. Thus, each bin represented an equal part of the phase of the hand oscillation. The first bin was centered around Θ = 0° (i.e., peak extension) and the fifth bin was centered around Θ = 180° (i.e., peak flexion). For each bin the RMS of the EMG was calculated and normalized to that obtained for the MVC. In addition to the comparison of conditions in terms of the average (normalized) amplitudes, the similarity of the rEMGs of homologous muscles was assessed using the weighted coherence. Specifically, the weighted coherence reflects the degree of similarity (or phase locking) of the activity bursts in the homologous muscles in terms of a weighted average of the coherence in the vicinity of the movement frequency (i.e., in the frequency band in which these bursts occur), yielding values between 0 (no phase locking) and 1 (perfect phase locking). Thus, the weighted coherence is an estimate of the strength of the interlimb coupling at the level of the neural control signals (Ridderikhoff et al. 2005b). In the present study it was used to evaluate differences in this regard between the relaxed and active conditions and between flexor and extensor muscles. The weighted coherence (Porges et al. 1980) of the full-wave rectified EMG (rEMG) of homologous muscles (CW) was calculated aswhere Δf defines a bandwidth around the movement frequency f (Δf = 0.1 Hz). Py is the power spectrum of the rEMG of the right arm, and Cxy is the coherence of the rEMGs of the homologous muscles in the left and right arm. The power spectra and the coherence were estimated with Welch’s modified periodogram method (Oppenheim and Schafer 1975) using a Hanning window of three cycles. In addition, the phase relations between rEMGs and joint angles θ were studied to compare the temporal relations observed at the level of the neural control signals to those observed at the behavioral level. This is of particular importance for the analysis of the contribution of the bilateral motor commands to the performance in the active condition. The phase shift between rEMG and θ was obtained from the cross-spectrum of rEMG and θ estimated at the movement frequency (using the same parameters as for the weighted coherence). Because flexion corresponded to negative values of θ, the phase shift between the EMG of FCR and θ was adjusted by 180° (cf. Ridderikhoff et al. 2004). Likewise, the relative phases between the rEMGs of homologous muscles were determined from the cross-spectrum of rEMGs of muscles in the left and right arm. The constant error of these relative phases was defined as the signed difference between the mean relative phase and the required relative phase (0° for in-phase; 180° for antiphase), with negative values indicating a relative phase lead of the activity in the right arm. The calculation of these measures required that the signals in question were phase and frequency locked, which implied that both measures could only be obtained for the active condition because only in this condition the rEMG of the muscles in the left (motor-driven) arm satisfied this requirement. For both measures (i.e., the phase shifts between rEMG and θ, and the relative phases between rEMGs of homologous muscles) the values obtained for FCR and ECR were analyzed separately to examine differences in the relative timing of flexors and extensors in relation to effects of coordinative pattern (for both measures) and limb (for the phase shifts between rEMG and θ). In addition, the values obtained for FCR and ECR were averaged to obtain a global measure for the phasing of the neural control signals to the limbs (Viviani et al. 1976). To visualize the main temporal relations in the movement system, the relative phase between the neural control signals, the phase shifts between the neural control signals and the movements of the left and right hand, and the average relative phase between the hands were examined in conjunction. In the active condition, FCR of the left (driven) arm of two subjects showed substantial reactive activity comparable in magnitude to that observed in the relaxed condition (see Fig. 5c; peak at bin 5, open symbols) in addition to the normal timing of muscle activation that was required in this condition (see Fig. 5c; peak at bin 2, filled symbols). This reactive activity was to some extent present in most subjects (as indicated by the peaks in Fig. 5, which was created without using the data of these two excluded subjects), but typically much less pronounced. For the two excluded subjects the large amplitude of the reactive activity resulted in a shift of the dominant frequency of the power spectrum of left FCR in this condition to twice the movement frequency (i.e., two bursts per cycle). The EMG data of these subjects were excluded from the analyses, because their inclusion resulted in a number of additional significant effects that could all be attributed to this reactive activity, but were not representative for the EMG data of the group as a whole. It should be noted, however, that all phenomena mentioned in the Results section were also observed for the excluded subjects. Statistical analysis Statistical analyses of the kinematics were performed using a 2 (Pattern: in-phase vs. antiphase coordination) × 2 (Activity: active vs. relaxed) × 2 (Orientation: flexion vs. extension) repeated measures analysis of variance (ANOVA). Also EMG-related measures were analyzed with repeated measures ANOVAs involving various, question-specific designs, which are described in the corresponding subsections of the Results. To facilitate the interpretation of the results obtained with ANOVA, paired-sample t-tests were used for post hoc analysis of the significant results (P < 0.05), and effect sizes (f) were calculated in terms of the partial η2 (Cohen 1988). The correlations were transformed to normally distributed variables using the Fisher transform. The same transformation was applied to the weighted coherence (Rosenberg et al. 1989). Whereas the inferential analyses were based on the transformed values, the corresponding untransformed values (bounded on the interval [−1, 1] for the correlations and on the interval [0, 1] for the coherence) are presented for reasons of clarity. Results Adequate performance of a trial was determined using the following inclusion criteria: (1) the number of cycles performed by the right hand and the (driven) left hand should be the same (i.e., no phase wrapping); (2) AEΦ should be smaller than 60°, and (3) the within-trial fluctuations of the relative phase should be within a range of 90°. In total 25 trials (8.7%) were excluded from the analysis: 9 trials in the active condition (3 trials for in-phase coordination, and 6 trials for antiphase coordination); 16 trials in the relaxed condition (7 trials for in-phase coordination, and 9 trials for antiphase coordination). In each condition all subjects were capable of performing the task in at least three trials in an adequate fashion. Relative phase Statistical analysis of AEΦ (see Fig. 2a) revealed a significant effect of Activity (F(1, 11) = 5.38; P < 0.05; f = 0.70), indicating that AEΦ was larger for the active condition (mean ± between-subjects SD: 28.2° ± 11.1°) than for the relaxed condition (20.6° ± 12.5°). In addition, a significant Pattern × Orientation interaction was found (F(1, 11) = 6.44; P < 0.05; f = 0.77). Post hoc analysis demonstrated two significant differences underlying this interaction, viz. between in-phase and antiphase coordination at peak extension (21.8° ± 11.0° vs. 26.8° ± 11.1°, respectively) and between flexion and extension during antiphase coordination (24.0° ± 10.0 vs. 26.8° ± 11.1°, respectively). The same statistical results were obtained for the constant errors in relative phasing (not shown), because the errors were almost always in the same direction (i.e., negative), indicating that the right (actively moving) hand was leading in time. Fig. 2a Mean absolute error of the relative phase (AEΦ). b Standard deviation of the relative phase (SDΦ). Results obtained for the relative timing of peak flexion (black bars) and peak extension (gray bars) are shown for in-phase (IP) and antiphase (AP), both for the conditions in which the muscles of the left hand were active (AC) or relaxed (RE). Error bars indicate the standard error of the mean To examine the stability of the coordinative patterns statistical analysis of SDΦ (see Fig. 2b) was conducted, which revealed significant effects of Activity (F(1, 11) = 5.94; P < 0.05; f = 0.74) and Pattern (F(1, 11) = 14.61; P < 0.005; f = 1.15). These effects resulted from, respectively, a larger SDΦ in the relaxed condition than in the active condition (12.7° ± 1.4° vs. 11.7° ± 1.4°) and a larger SDΦ for antiphase coordination than for in-phase coordination (12.8° ± 1.5° vs. 11.6° ± 0.87°). Movement amplitude To examine whether the different activity levels of the muscles in the left (driven) arm had an effect on the amplitude of the movements about the right wrist, the range of motion (i.e., the difference in peak extension and peak flexion) was determined for each cycle, averaged per condition and subjected to a 2 (Pattern) × 2 (Activity) repeated measures ANOVA. The analysis solely revealed a significant effect of Activity (F(1, 11) = 37.00; P < 0.001; f = 1.83), resulting from a larger movement amplitude in the active condition than in the relaxed condition (range of motion: 85.5° ± 23.8° vs. 58.6° ± 14.4°, respectively). Temporal correlations between kinematic variables The average correlations between the signed error and the duration of the following full cycle (RFC) or half cycle (RHC) are shown in Fig. 3a, b, respectively. Statistical analysis of RFC revealed significant effects for Activity (F(1, 11) = 7.05; P < 0.05; f = 0.80), Pattern (F(1, 11) = 5.29; P < 0.05; f = 0.69) and Orientation (F(1, 11) = 13.35; P < 0.01; f = 1.10). RFC was more pronounced (larger absolute values) for the active condition than for the relaxed condition (−0.76 ± 0.14 vs. −0.69 ± 0.14), for the in-phase pattern than for the antiphase pattern (−0.76 ± 0.14 vs. −0.69 ± 0.13), and for errors at peak flexion than for errors at peak extension (−0.75 ± 0.14 vs. −0.70 ± 0.14). Fig. 3Mean correlations between the signed timing error and the duration of the following full cycle (RFC, a) or half cycle (RHC, b) of the right (actively moving) hand. Results are shown for in-phase (IP) and antiphase (AP), both for the conditions in which the muscles of the left hand were active (AC) or relaxed (RE). RFC was calculated for full cycles following the error at two different orientations of the right (actively moving) hand: peak flexion (black bars) and peak extension (gray bars). RHC was calculated for the flexion half cycles following the error at peak extension (black bars) and the extension half cycles following the error at peak flexion (gray bars). Error bars indicate the standard error of the mean Statistical analysis of RHC revealed significant effects for Activity (F(1, 11) = 13.33; P < 0.01; f = 1.10) and Pattern (F(1, 11) = 13.02; P < 0.01; f = 1.09), indicating larger absolute values for the active condition than for the relaxed condition (−0.61 ± 0.17 vs. −0.50 ± 0.17), and for the in-phase pattern than for the antiphase pattern (−0.59 ± 0.17 vs. −0.52 ± 0.17). In addition, for RHC significant Activity × Pattern (F(1, 11) = 6.14; P < 0.05; f = 0.75) and Pattern × Orientation (F(1, 11) = 9.12; P < 0.05; f = 0.91) interactions were found. Post hoc analysis revealed that the first interaction effect resulted from a larger absolute value of RHC for in-phase coordination in the active condition than for the three other combinations of Activity and Pattern (see Fig. 3b). The second interaction effect was due to a larger absolute value of RHC for the extension half-cycle in the in-phase pattern than for the three other combinations of Pattern and Orientation (see Fig. 3b). The main effects of Activity and Pattern were qualitatively the same for RHC and RFC, but the effect of Orientation was markedly different. An explanation of this difference was found in a formal analysis of the relation between the covariances1 underlying RHC and RFC, which demonstrated that (unlike differences related to Activity or Pattern) differences between flexion and extension in RHC are completely unrelated to those in RFC (see Appendix). Two additional factors are involved in the relation between RHC and RFC (see “Materials and methods” and Appendix), which were captured by the within-hand and between-hands correlations of the durations of successive half cycles (Ryy and Rxy, respectively). The effect of Orientation in RFC can be attributed completely to (the covariances underlying) Ryy and Rxy (see Appendix). However, also for the other factors (Activity and Pattern) the relation between RHC and RFC may be affected by Ryy and Rxy, given the potential dependencies between error corrections in successive half-cycles. Thus, the temporal correlations were examined further in terms of the within-hand correlation Ryy and the between-hands correlation Rxy. Analysis of the between-hands correlation Rxy (Fig. 4b) revealed no significant effects. In contrast, significant effects of Activity (F(1, 11) = 8.83; P < 0.05; f = 0.90) and Orientation (F(1, 11) = 8.19; P < 0.05; f = 0.86) were found for the within-hand correlation Ryy. The effect of Activity revealed that the values of Ryy for the active condition were more negative than for the relaxed condition (−0.16 ± 0.17 vs. −0.02 ± 0.07). The effect of Orientation indicated that the correlations between the extension half cycle and the following flexion half cycle were more negative (−0.14 ± 0.10; gray bars in Fig. 4a) than those between the flexion half cycle and the following extension half cycle (−0.02 ± 0.17; black bars in Fig. 4a). The consequences of Ryy and Rxy for RFC are illustrated in Fig. 4c, d, respectively, which show the contributions of the covariances underlying Ryy and Rxy to RFC, according to the relations derived in the Appendix. The temporal relations expressed by Ryy and Rxy both result in a reduction of RFC (i.e., they reduce the effects of the half cycle error corrections, see “Materials and methods”); the effect of the former being larger than that of the latter (compare Fig. 4c, d). Both correlations resulted in a larger reduction of RFC for errors at peak extension than for errors at peak flexion, which explains the aforementioned effect of Orientation obtained for RFC. In addition, the effect of Activity for Ryy revealed that the error corrections in successive half-cycles were more correlated for the active condition than for the relaxed condition. This implied that in particular in the active condition a part of the adjustments of the half-cycle durations (captured by RHC) compensated for overcorrection in the preceding half-cycle. Fig. 4Mean correlations of the durations of successive half-cycles obtained during in-phase (IP) and antiphase (AP) coordination, while the muscles of the left (driven) hand were active (AC) or relaxed (RE): a within-hand correlations (Ryy); b between-hands correlations (Rxy). c and d indicate the relative contributions of the covariances underlying the within-hand and between-hands factors (Cyy and Cxy, respectively) by expressing them as a fraction of the covariance underlying RFC (i.e., CFC): negative values indicate a reduction of the absolute value of RFC (see Appendix). To facilitate comparison the values of Ryy and Rxy are presented in the same order as the corresponding values of RFC in Fig. 3. Error bars indicate the standard error of the mean. The legends of a and b also apply to c and d, respectively. Legends: FHC = flexion half cycle; EHC = extension half cycle; the arrows specify the temporal order of the half cycles. Whereas the second half cycle is always performed by the right hand, the first half cycle is performed either by the right hand (Ryy and Cyy, a and c) or by the left hand (Rxy and Cxy, b and d) EMG The average amplitudes of the EMGs (normalized to MVC amplitude) during the movement cycle are presented in Fig. 5, which exposes five noteworthy characteristics of the EMG data. First, in the right arm the EMG amplitudes were smaller in the relaxed condition than in the active condition (compare open and filled symbols, respectively, in Fig. 5a, b), corresponding to the observed differences in range of motion between these conditions. Second, in line with the task requirements, the EMG amplitudes of the muscles in the left arm were in general much smaller in the relaxed condition than in the active condition (compare open and filled symbols, respectively, in Fig. 5c, d). Third, for left FCR in the relaxed condition a distinctive (reactive) peak in the EMG amplitude was observed at bin 5, i.e., the moment at which the lengthening of the muscle started (open symbols in Fig. 5c). Fourth, for left ECR in the relaxed condition the EMG amplitude showed a slight modulation during the movement cycle that mimicked that of normal activation patterns (compare open symbols in Fig. 5d with the curves in Fig. 5b). Fifth, both left FCR and ECR in the active condition (filled symbols in Fig. 5c, d, respectively) were shifted leftward with respect to the (motor-driven) movement compared to the timing of right FCR and ECR when compared to the (self-generated) movement (Fig. 5a, b, respectively). Note that this phase advance of the EMG in the left arm relative to the corresponding hand motion was possible because (in contrast to the right hand) the movements of the (motor-driven) left hand were completely independent of its muscle activity. Fig. 5Normalized EMG amplitudes (RMS values) of FCR (a right; c left) and ECR (b right; d left), averaged over subjects, at eight phases of the movement cycle. Each graph shows the muscle activity for the conditions in which the muscles of the left (driven) arm were either active (filled symbols) or relaxed (open symbols), for in-phase (triangles; solid lines) and antiphase coordination (circles; dashed lines) separately Weighted coherence The weighted coherence of the rEMG of homologous muscles is shown in Fig. 6. Statistical analysis using a 2 (Pattern) × 2 (Activity) × 2 (Muscle) repeated measures ANOVA revealed significant effects of Activity (F(1, 9) = 103.98; P < 0.001; f = 3.39) and Muscle (F(1, 9) = 17.70; P < 0.01; f = 1.40), as well as a significant Activity × Pattern interaction (F(1, 9) = 10.68; P < 0.05; f = 1.09). The effect of Activity revealed that the weighted coherence was larger in the active condition than in the relaxed condition (0.85 ± 0.25 vs. 0.54 ± 0.63). The effect of Muscle showed that the weighted coherence was larger for ECR than for FCR (0.77 ± 0.38 vs. 0.62 ± 0.54), indicating a greater similarity of the EMG activity for extensor than for flexor muscles. Post hoc analysis of the Activity × Pattern interaction revealed no significant differences between in-phase and antiphase coordination for either the active condition or the relaxed condition (or vice versa), although the weighted coherence was larger for in-phase than for antiphase in the active condition (0.86 ± 0.22 vs. 0.83 ± 0.32), but smaller for in-phase than for antiphase in the relaxed condition (0.51 ± 0.70 vs. 0.57 ± 0.60). Fig. 6Mean weighted coherence (CW) of the rectified EMGs of homologous muscles: FCR (black) and ECR (gray) during in-phase (IP) and antiphase (AP) coordination, both for the conditions in which the muscles of the left (motor-driven) hand were active (AC) or relaxed (RE). Error bars indicate the standard error of the mean Temporal relations in the active condition In the active condition, the relative phase was determined at two levels: at the behavioral level (kinematics) and at the level of the neural control signals (based on rEMG). This distinction is important because for the right hand the phase shift between EMG activity and kinematics was constrained by the effector dynamics of the wrist (Ridderikhoff et al. 2004), whereas these constraints did not affect the phase shift for the left (motor-driven) hand. Hence, the two levels were not tightly coupled in this kinesthetic tracking task. To examine the relations between these levels, four different phase relations were studied in conjunction: the phase shifts between neural control signals and behavior for (1) the right hand and (2) the left hand, and the relative phase between the limbs at (3) the behavioral level and (4) the level of neural control signals. The results regarding the relative phase at the behavioral level were presented in the preceding. In the next two subsections the other phase relations (i.e., the phase shifts between rEMG and kinematics and the relative phase between rEMGs of homologous muscles) are presented. The results are combined in the final subsection to provide an overall picture of the temporal relations in the active condition across these different levels. Phase shifts between EMG and kinematics In the active condition differences between the left and right hand were observed with respect to the timing of the muscle activity relative to the ongoing movement (see Fig. 5). To analyze these differences, the phase shifts between rEMG and kinematics (see Table 1; negative values indicate that the EMG is leading the kinematics) were examined using a 2 (Hand) × 2 (Pattern) × 2 (Muscle) repeated measures ANOVA. This revealed a significant effect of Hand (F(1, 9) = 11.79; P < 0.01; f = 1.14), indicating larger phase shifts for the left hand than for the right hand (−145.3° vs. −114.4°). In addition, significant Hand × Muscle (F(1, 9) = 14.21; P < 0.01; f = 1.26), Hand × Pattern (F(1, 9) = 6.24; P < 0.05; f = 0.83), and Hand × Pattern × Muscle (F(1, 9) = 6.08; P < 0.05; f = 0.82) interactions were found. Post hoc analysis demonstrated that the Hand × Muscle interaction occurred because the phase shifts for ECR and FCR differed significantly for the right hand (−101.5° ± 21.2° vs. −127.4° ± 14.9°), but not for the left (motor-driven) hand. The Hand × Pattern interaction post hoc analysis revealed that the phase shifts during in-phase and antiphase coordination differed significantly for the left (motor-driven) hand (−149.9° ± 23.7° vs. −140.6° ± 27.2°), but not for the right hand. Post hoc analysis of the three-way interaction revealed that the phase shift for the left FCR during antiphase coordination deviated from the overall pattern: it was significantly different from the phase shift during in-phase coordination (in the absence of significant differences between in-phase and antiphase for any of the other muscles), and it was not significantly different from the phase shift for right FCR during antiphase coordination (whereas all other left-right comparisons yielded significant differences for both coordinative patterns). Table 1Phase shifts between rectified EMG and joint angles in the active condition (mean ± between-subjects SD) as determined from the cross-spectrum of these variables at the movement frequencyHandMuscleIn-phaseAntiphaseLeftFCR−148.2° ± 22.1°−137.5° ± 22.5°ECR−151.7° ± 27.8°−143.7° ± 33.5°RightFCR−123.1° ± 14.2°−131.6° ± 17.7°ECR−103.5° ± 17.7°−99.5° ± 15.2° Relative phasing of EMG The relative phase between the rEMG of homologous muscles in the active condition was calculated for FCR and ECR. The constant errors with respect to the reference values for in-phase (0°) and antiphase (180°) coordination (see Table 2) were subjected to a 2 (Pattern) × 2 (Muscle) repeated measures ANOVA. The analysis revealed significant effects of Pattern (F(1, 9) = 12.19; P < 0.01; f = 1.16), Muscle (F(1, 9) = 16.20; P < 0.01; f = 1.32), and a significant Pattern × Muscle interaction (F(1, 9) = 5.23; P < 0.05; f = 0.76). The effect of Pattern resulted in a significantly larger constant error for in-phase than for antiphase coordination (12.5° ± 18.0° vs. 0.3° ± 24.0°; positive values indicate a relative phase lead of the activity in the left arm). The effect of Muscle indicated that the average constant errors for FCR and ECR were significantly different (−9.3° ± 26.9° vs. 22.2° ± 20.6°). Post hoc analyses of the Pattern × Muscle interaction revealed a significant difference between the constant errors for in-phase and antiphase coordination for FCR but not for ECR (see values in Table 2). Table 2Mean constant errors in the relative phase for kinematics and EMG in the active condition (mean ± between-subjects SD)LevelIn-phaseAntiphaseKinematics−22.7° ± 10.4°−24.8° ± 11.4°EMG FCR0.1° ± 26.2°−18.8° ± 28.5° ECR24.9° ± 19.0°19.4° ± 24.7° Mean12.5° ± 18.0°0.3° ± 24.0°For the EMG the obtained values are presented for homologous FCR and ECR separately. The mean of the constant errors for FCR and ECR was adopted as the constant error of the neural control signal. Negative (positive) values indicate that the right limb is leading (lagging) the left limb Relative phasing of neural control signals and behavior To gain insight into the neurobehavioral basis of error correction, we examined the phase relations between the neural control signals and the overt behavior in the active condition. For this purpose, the phasing of the neural control signals was estimated for each limb separately by taking the average of the values obtained for FCR and ECR (see Tables 1, 2). The resulting phase relations are displayed schematically for in-phase and antiphase coordination in Fig. 7, which illustrates the muscle-independent phase relations addressed in the previous two paragraphs, showing only the effects of Hand and Pattern. First, the phase shift between the neural control signal and the wrist movements was larger for the (motor-driven) left hand (dashed arrows) than for the right hand (solid arrows)—significant effect of Hand. Second, the constant error in relative phase between the EMGs of homologous muscles was larger for in-phase than antiphase coordination (compare the corresponding diagrams)—significant effect of Pattern. Third, it can be appreciated from the figure that the phase shift between EMG and kinematics (larger shifts in the left hand, i.e., longer dashed arrows, for in-phase than for antiphase coordination) depended on the combination of hand and pattern—significant Hand × Pattern interaction. Finally, the diagrams indicate that, on average, the error in the relative phase was smaller for the neural control signal than for the kinematics (respective values are presented in Table 2). Fig. 7Diagrams illustrating temporal relations in the active condition for in-phase (left panel) and antiphase (right panel) coordination. Circles represent signal sources: θ is the joint angle, and ξ is the neural control signal (derived from the EMG, averaged over FCR and ECR). The length of the arrows indicates the phase shifts between the neural control signal and the kinematics (arrows point in the direction of time). The error of the relative phase Φ is equal to the inclination angle of the line connecting the circles at either the kinematic (θ) or the control level (ξ). The target values of Φ are indicated by the gray horizontal lines, and the dotted lines represent the relative error in Φ in the relaxed condition (available for kinematics only) Discussion The goal of the present study was to gain more insight into the error correction processes as allegedly implicated in bimanual coordination (Ridderikhoff et al. 2005b). To this end, we examined the temporal adjustments of the actively moving (right) limb in a kinesthetic tracking task based on the perceived (error in the) relative phase between the limbs. In particular, subjects tracked their motor-driven left hand with the actively moving right hand to achieve in-phase or antiphase coordination, in conditions where the left hand musculature was activated (as if moving along with the reference movement) or kept as relaxed as possible. In addition, the relation between bimanual (between-limbs) and unimanual (within-limb) factors was examined. Unimanual and bimanual factors were dissociated by comparison of several (related) temporal correlations and by examining the phase shifts between neural control signals and kinematics both within and between limbs. In line with our research questions the analysis focused on two aspects of the measurements, which are discussed in detail in the next two subsections: (1) differences in error correction between in-phase and antiphase coordination, and (2) effects of muscle activity in the driven (left) limb on these error corrections and on the overall performance of the task. In the last two subsections, we will then address the observed differences between the left and right hand and between flexors and extensors in terms of the phase shifts between EMG and kinematics and the relative timing of flexion and extension. Before embarking on these discussions, it should be noted that the stability of the bimanual patterns was affected by the muscle activity in the motor-driven limb, as SDΦ was larger in the relaxed condition than in the active condition. Furthermore, the variability of the relative phase (SDΦ) was smaller for in-phase than for antiphase coordination. This basic observation confirmed the result of an earlier study on kinesthetic tracking (Stinear and Byblow 2001) and indicated that also in normal bimanual coordination the stability difference of in-phase and antiphase coordination may be partly dependent on afference-based error corrections. Error corrections We performed an in-depth analysis of the corrections in the timing of the movements of the right (actively moving) limb based on the perceived errors in the relative phase. Since our analyses of the kinematics concerned the timing of discrete events (i.e., peak extension and peak flexion), the shortest possible time scale to study error corrections was related to compensatory adjustments of the duration of the half-cycle following the detected error. The related measure (RHC) indicated that the error corrections were more pronounced for in-phase than for antiphase coordination and that they were characterized by larger absolute values for the active condition than for the relaxed condition, in line with the observed effects for SDΦ. Additional analysis of the compensatory adjustments of the duration of the full cycle following the detected error (RFC) showed that both error correction effects persisted on a longer time scale. Therefore, we conclude that corrections in the movement timing of the right hand based on the perceived errors in the relative phase underlie the observed stability effects (SDΦ) of the examined coordination patterns (in-phase vs. antiphase) and muscle activity. It is important to consider error corrections at different time scales, because the dependency of the corrections performed in successive half-cycles may vary over conditions (see “Materials and methods”). The results revealed that in the present study these dependencies between successive half-cycles were reflected mainly by the within-hand correlation Ryy, indicating an important role for the unimanual coordinative processes underlying the rhythmic movements of the right hand. This role is of particular importance for the interpretation of the significant Pattern × Activity interaction observed for RHC, suggesting more pronounced error corrections during in-phase coordination in the active condition. Because the results obtained for Ryy demonstrated larger dependencies between successive half-cycles in the active condition than in the relaxed condition, the effects related to muscle activity were reduced at a longer time scale (RFC). As a consequence, the Pattern × Activity interaction was not significant for RFC and, thus, appeared to be of little functional relevance in terms of the coordinative stability. This illustrates how unimanual coordination (the flexion–extension coupling in terms of Ryy) may impinge on the effects of interlimb interactions (the error corrections in terms of RHC) in bimanual coordination. Two additional discrepancies between RHC and RFC were found in relation to effects of the orientation of the hand (flexion or extension). This was not surprising, because RHC and RFC are unrelated by definition with respect to these effects. As shown both analytically (for the underlying covariances, see Appendix) and empirically all differences between flexion and extension in RFC are attributable to the correlations between the durations of successive half-cycles within the right hand (Ryy) and between hands (Rxy). Specifically, the effect of orientation of the hand obtained for RFC was not related to error corrections based on the perceived relative phase, but rather reflected an asymmetry in the coupling of the unimanual flexion and extension half-cycles as captured by Ryy. In contrast, differences in the strength of the error corrections during the flexion and extension half-cycles are indicated by effects of orientation observed for RHC. Analysis of RHC revealed a significant Pattern × Orientation interaction, which indicated more pronounced error corrections in the half-cycle following peak flexion of the left (motor-driven) hand, in particular in the extension half-cycle during in-phase coordination (Fig 3b). In line with this result, we observed the smallest errors in the mean relative phase at the end of the half-cycles with the most prominent error corrections (i.e., at peak extension during in-phase and at peak flexion during antiphase coordination; see Fig 2a). A tentative explanation of these findings is that (relative) timing errors were detected most accurately at peak flexion of the driven hand and that, as a consequence, the corrections in the following half-cycle were most effective, leading to the highest accuracy in the relative phase at the end of these half-cycles. With respect to the two adopted measures of error correction (RHC and RFC) these analyses demonstrated that both measures should be evaluated in conjunction to assess the net effect of error corrections in relation to the effects of movement pattern and muscle activity, whereas RHC offers additional insight into the differences between flexion and extension in this respect. The analyses of the temporal correlations revealed several important global characteristics of the afference-based error corrections underlying rhythmic bimanual coordination. Although the analyses demonstrated that error corrections occur during the next half-cycle, it is important to emphasize that the precise time course of the error corrections remains unknown. That is, these methods do not reveal how long it takes until the ongoing movements are adapted to compensate for perceived errors, except for showing that such changes occur within the next half-cycle. Although, in principle, it is possible that timing errors are detected throughout the cycle and not only at peak flexion and peak extension, there is ample evidence indicating that coordinated rhythmic movements are characterized by the presence of so-called anchor points (Beek 1989), that is, discrete points in the cycle that have particular significance for the control of timing. Furthermore, several studies have demonstrated that anchoring occurs when peak flexion and/or peak extension are coinciding with an external stimulus (Byblow et al. 1994; Fink et al. 2000). These findings provide a rationale for the use of the moments of peak flexion and peak extension in the analysis of the temporal structure of the bimanual coordination pattern, and the instructions to subjects in terms of the timing of peak flexion and peak extension (see "Materials and methods") may have helped to establish such anchor points in the present study. However, further research regarding the existence of anchor points in the processing of kinesthetic feedback and the time course of the adjustments is required, for which the analyses in the present study may serve as departure point. For our current purposes, RHC and RFC provided important insights into the average amount of corrective activity that occurs at two relevant time scales in terms of the same kinematic measures that were used to assess the performance in terms of the relative phase between the hands. The effects of muscle activity in the driven limb The second objective of this study was to examine the neurobehavioral basis of error correction. To this aim, we manipulated the muscle activity in the motor-driven limb by instructing subjects either to activate their muscles in accordance with the ongoing motion or to keep them as relaxed as possible. The average EMG amplitudes (Fig. 5) clearly showed that this manipulation of muscle activity was successful in the sense that subjects managed by and large to avoid activating the muscles in the motor-driven left hand in the relaxed condition. This was further underscored by the fact that the values of the weighted coherence of rEMGs of homologous muscles in the relaxed condition did not exceed those obtained in previous studies involving passive (i.e., motor-driven) hand movement (Ridderikhoff et al. 2005b; Ridderikhoff et al. 2006). In those studies subjects had been explicitly instructed to ignore the motor-driven movement (i.e., no coordination between the hands was required) in order to study phase entrainment by contralateral afference, implying that the resulting values of the weighted coherence can be regarded as a baseline for the effect of motor-driven movement per se. In contrast, in the active condition the changes in the EMG amplitude of the motor-driven hand resembled those observed for actively performed rhythmic wrist movements (as obtained for the right hand), and significantly larger coherence of the homologous muscle activity was observed. For a proper evaluation of the adopted methodology it is relevant to note that the experiment actually involved a dual task paradigm, because in all conditions subjects were instructed to perform the required bimanual pattern and at the same time control the muscle activity in the driven arm. The additional cognitive load that is associated with performance of a secondary task may have augmented the intrinsic stability differences between in-phase and antiphase coordination (Temprado et al. 1999). However, the results did not indicate a differential effect of the two secondary tasks (active vs. relaxed condition) on the stability difference between in-phase and antiphase (i.e., no Activity × Pattern interactions were observed for SDΦ). From this we conclude that the additional cognitive load imposed by the secondary task was the same for the active and relaxed condition, and that the dual task paradigm did not confound the comparison between these conditions. In other words, we are confident that the observed differences between these conditions can be ascribed to the differences in muscle activity associated with these conditions, rather than differences in cognitive load. On the other hand, the dual task character of the present manipulations may explain why the present results regarding the SDΦ of in-phase and antiphase coordination agreed with those reported in the study of Stinear and Byblow (1999), in which an additional amplitude matching of the movements was required, but not with our previous (single task) experiment on kinesthetic tracking (Ridderikhoff et al. 2005b). Muscle activity clearly had a beneficial effect on coordinative stability, as the variability of the relative phase was significantly smaller in the active condition than in the relaxed condition (Fig. 2b). In addition, the adopted measures related to the error correction processes underlying kinesthetic tracking, showing larger compensatory adjustments of the timing of the right hand in the active condition (RHC and RFC; Fig. 3), indicated that kinesthetic afference was processed more effectively in this condition. Consequently, it seems unlikely that the muscle activity during kinesthetic tracking that we observed in our previous study (Ridderikhoff et al. 2005b) was merely a byproduct due to neural cross-talk.2 However, in addition to these benefits of muscle activity, we also found that the absolute (and constant) errors in the mean relative phase were larger in the active condition than in the relaxed condition (Fig. 2a), indicating that the coordinative pattern was performed less accurately in the former condition. Thus, muscle activation during kinesthetic tracking resulted in more pronounced and proficient error corrections in bimanual timing, but these corrections appeared to be performed with respect to less accurate reference values. In explaining this reduced accuracy in the active condition it proved essential to dissociate the temporal relations between the limbs observed at the level of neural control signals and those observed at the behavioral level, because these levels may involve separate reference signals. In the relaxed condition only the relative phasing at the behavioral level (kinematics) is available as reference for the error corrections. In contrast, the presence of a bilateral neural control signal in the active condition implies that the relative phase between the neural control signals (or motor outflow) may also be used as reference (e.g., by means of anticipation of the sensory consequences of the ongoing movement based on efference copies). Comparison of the constant errors at the two levels (Table 2) suggested an attraction toward the proper timing of the neural control signals, resulting in larger errors in the relative phase at the behavioral level (Fig. 7). This observation also revealed a discrepancy between the relative phases at the two levels, which resulted from an inadequate timing of the neural control signals of the motor-driven hand as explained in the next subsection. This interpretation of the results underscores that bimanual coordination benefited from muscle activity during kinesthetic tracking because the bilateral motor commands were used (e.g., via efference copy) as a reference signal for movement-elicited afference. The presence of this reference signal, which presumably allowed for prediction of the sensory consequences of the bimanual movement, resulted in more prominent error corrections, leading to a smaller variability of the relative phase in the active condition. Thus, the evidence suggested that an intimate sensorimotor integration, which has been proposed in the context of discrete goal-directed movements (e.g., Wolpert and Ghahramani 2000; Scott 2004), also underlies the coordination of rhythmic bimanual movements. In the context of the present study this interpretation can account for the observed effects of muscle activation on the stability as well as the accuracy of the relative phase. Timing of the EMG activity in the motor-driven arm Unlike the phase shifts between EMG and kinematics of the left (motor-driven) arm observed in the present study, the muscle activity observed during kinesthetic tracking in our previous study (Ridderikhoff et al. 2005b) was adequately timed with respect to the ongoing motor-driven movement. A possible explanation for this unexpected discrepancy between these two studies may be related to the instructions given to the subjects. In the active condition of the present study subjects were explicitly instructed to activate their muscles, whereas subjects did this spontaneously (i.e., without instruction) in our previous study. Given the instruction to activate the muscles as if moving along with the passive movement, subjects seemed to have aimed at generating contact forces that gave the impression that they were pushing or pulling the manipulandum in the correct direction. Comparison of the EMG amplitudes in the left (motor-driven) arm and the right (actively moving) arm (Fig. 5) indicated that this was accomplished by an earlier activation of the muscles in the left arm (relative to the ongoing movement) and an increase of the EMG amplitude (i.e., an increase of the muscle torque). These changes substantially increased the forces acting between the hand and the manipulandum in the direction of the ongoing movement. Note also that the EMG activity was minimal at the moments of peak excursion, i.e., when the movement changed direction (see Fig. 5). In other words, the results suggested that subjects in effect sought after the expected sensory consequences of moving something. This may be regarded as a misinterpretation of the instruction, because truly moving along requires the elimination of contact forces altogether. Flexor–extensor differences during active rhythmic wrist movement In addition to the effects of afference-based interlimb interactions (as reflected in the error corrections) the flexion and extension phases were also affected by the unimanual control processes underlying the rhythmic movement of the (right) wrist itself. In this regard, an asymmetry was found in the correlations between successive flexion and extension half-cycles (Ryy), indicating that the durations of the flexion half-cycles were adapted more to changes in the duration of the extension half-cycle than vice versa (Fig. 4a). Studies on unilateral rhythmic movements have also reported an asymmetric relation between flexion and extension half-cycles in rhythmic finger movements (Balasubramaniam et al. 2004), and a more adequate temporal control of wrist flexion than wrist extension (Carson 1996; Carson and Riek 1998). An adaptive relation between the relative timing of FCR and ECR on the one hand and the resulting wrist movements on the other hand was also indicated by the analysis of the phase shifts between EMG and kinematics. Different phase shifts were obtained for FCR and ECR in the right hand but not in the left hand, in which timing of EMG and kinematics were unrelated. Such differences in the relative timing of FCR and ECR activity during rhythmic wrist movements have been reported before (Ridderikhoff et al. 2004), and may reflect adaptations to the different physiological properties of these muscles that would affect the movement trajectories unless compensated for. Furthermore, the EMG analysis revealed that the coherence between left and right ECR was larger than the coherence between left and right FCR, may be related to the finding that neural cross-talk effects were larger for extensor than for flexor muscles (Ridderikhoff et al. 2005a). These combined results point to a more independent control of the timing of FCR in the context of rhythmic wrist movement. Conclusion The present study revealed two characteristics of the error corrections based on the kinesthetically perceived relative phase that are relevant to the understanding of rhythmic bimanual coordination, and supplement the findings of our previous study on the sources of interlimb interaction involved in this type of task (Ridderikhoff et al. 2005b). First, the corrections were more effective during in-phase than during antiphase coordination, resulting in a smaller variability of the relative phase in the former condition. This effect may be attributable to differences in the kinesthetic perception of variability in in-phase and antiphase coordination (Wilson et al. 2003), and indicated that afference-based error corrections contribute to the well-known stability difference of these bimanual patterns (Kelso 1984; Swinnen 2002). Thus, although somatosensory feedback is not essential for the stability differences between in-phase and antiphase (Spencer et al. 2005), the present study showed that afference-based error corrections augment the stability difference resulting from open-loop processes underlying the integrated timing of the efferent signals to both limbs (Ridderikhoff et al. 2005b). Second, the corrections (and, as a consequence, pattern stability) were enhanced if the muscles in the motor-driven arm were activated as if the limb was moving along with the motor. On the other hand, the accuracy of the relative phasing between the limbs was decreased in this situation, indicating that the corrections were based on a different reference signal than in the relaxed condition. The most likely explanation for these findings was that the bilateral neural control signal provided a reference for the evaluation of the kinesthetic afference, in the sense that on the basis of this signal the sensory consequences (of the bimanual movement pattern) could be anticipated. A closed-loop control process that exploits this kind of prediction is apparently more effective in terms of the stabilization of the relative phase. This result suggests an intimate relation between the integrated timing of the feedforward signals (underlying the bilateral activation pattern if both hands are moving actively) and the use of kinesthetic feedback in rhythmic bimanual coordination

Anal_Bioanal_Chem-4-1-2259237

Discrimination of biofilm samples using pattern recognition techniques

Biofilms are complex aggregates formed by microorganisms such as bacteria, fungi and algae, which grow at the interfaces between water and natural or artificial materials. They are actively involved in processes of sorption and desorption of metal ions in water and reflect the environmental conditions in the recent past. Therefore, biofilms can be used as bioindicators of water quality. The goal of this study was to determine whether the biofilms, developed in different aquatic systems, could be successfully discriminated using data on their elemental compositions. Biofilms were grown on natural or polycarbonate materials in flowing water, standing water and seawater bodies. Using an unsupervised technique such as principal component analysis (PCA) and several supervised methods like classification and regression trees (CART), discriminant partial least squares regression (DPLS) and uninformative variable elimination–DPLS (UVE-DPLS), we could confirm the uniqueness of sea biofilms and make a distinction between flowing water and standing water biofilms. The CART, DPLS and UVE-DPLS discriminant models were validated with an independent test set selected either by the Kennard and Stone method or the duplex algorithm. The best model was obtained from CART with 100% correct classification rate for the test set designed by the Kennard and Stone algorithm. With CART, one variable describing the Mg content in the biofilm water phase was found to be important for the discrimination of flowing water and standing water biofilms. Introduction Water quality assessment requires monitoring of carefully selected parameters. Usually water and sediment samples are collected in the hope that their chemical compositions will help to understand the nature of a given local or global environmental event. The chemical analysis of water indicates the water quality at the time of sampling, while analysis of sediments provides information about long-term environmental changes in aquatic systems. Biofilms reflect the environmental conditions in the recent past [1]. They are complex communities composed of microorganisms, which grow at almost any water–substrate interface. Biofilms can accumulate metal ions and play an important role in the processes of sorption and desorption of chemical elements [2]; therefore, they can be very useful bioindicators of water quality. The aim of this work is to investigate whether the biofilms grown in different aquatic systems can be discriminated on the basis of their chemical compositions. If the discrimination is possible, the next interesting question will be which measured parameters are responsible for it. For the purpose of the study, biofilm samples originating from different water bodies such as flowing water, standing water or seawater were collected. The elemental concentrations of biofilms and the water phase extracted at the sampling locations were then analysed using inductively coupled plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. To obtain useful information on the data collected, several pattern recognition techniques are going to be applied. These are principal component analysis (PCA), classification and regression trees (CART), discriminant partial least squares regression (DPLS) and uninformative variable elimination–DPLS (UVE-DPLS). PCA is an unsupervised pattern recognition method that aims to compress and to visualise the data structure, which allows for an easy interpretation of relationships between samples and the measured parameters. The supervised pattern recognition methods like CART, DPLS and UVE-DPLS aim to develop classification or decision rule(s) using a set of samples with known group origin. Then the classification rule(s) determines the belongingness of unknown samples to the available groups [3]. The application of such approaches in our study will provide a better understanding of the accumulation behaviour of different biofilms. Experimental Description of the sampling procedure The biofilm samples investigated can be divided into two groups: a group of systematically sampled biofilms and a group of uniquely sampled biofilms. The samples of the group of systematically sampled biofilms can be further split into two subgroups, namely, biofilms grown on polycarbonate plates and biofilms developed on natural substrates. A detailed description of the samples collected is presented in Table 1. Table 1Description of the biofilm and water samples collectedGroup of biofilmsSubgroup of biofilmsCharacter of the water phaseNumber of samplesSystematically sampled biofilmsBiofilms grown on polycarbonate platesf—the Saale river11s—the Teich pond22Biofilms grown on natural substratesf—the Leutra river13Uniquely sampled biofilmsBiofilms grown on natural substratesf—Celle (a, b), Lauscha (a, b, c), Oberpöllnitz, Falken, London, Munich, New York, Geithain, Steinach, Juquitiba12s—Chemnitz, New Hampshire, Bossow, Metebach, Erfurt, Rippachtal6m—Travemünde (a, b), Punta Skala, Nin, Majorca, Damp, Steinbeck7a, b and c denote different sampling locations.f a body of flowing water, s a body of standing water, m a body of seawater The biofilm samples collected in the Saale river, in a pond and in the Leutra river were systematically sampled, i.e. the biofilm and water samples were gathered within a definite period of time. The Saale river was chosen as a body of flowing water. The river flows through highly populated regions, which are heavily industrialised and are subjects of typical geogenic and anthropogenic pollution [4]. The Saale water and biofilm samples were collected within a 2-year period (from September 2003 till October 2005). The sampling point was situated in the village of Kunitz located downstream of Jena (Thuringia, Germany). This place was preferred, because it reflects a characteristic pollution of the nearby town. The Saale river is there about 1.5 m deep and Kunitz is remote from civilisation. These conditions facilitated the sampling campaign. A small pond of 0.75-m depth was selected as a typical example of a standing water body. It was located in the city of Jena. The sampling campaign duration was the same as for the sampling campaign in the Saale river. At these two locations, the biofilms were artificially grown on polycarbonate plates (10 cm × 10 cm) exposed vertically to the water (in the Saale river, in a streaming direction). The plates were fixed into polypropylene boxes, approximately 10 cm under the water surface and 1 m away from the riverbank. After a definite time of exposure, the biofilm samples were immediately transferred into plastic boxes filled with the river or pond water and transported to the laboratory. The plates were then washed with bidistilled water and the biofilm samples were scraped off the whole surface of the polycarbonate plates using a Teflon spatula. The river and pond water samples were collected every 2 weeks. The Leutra river, located in the city of Jena, was chosen as the second example of a flowing water body. The stony bed of the river, its small depth and good accessibility facilitated the sampling campaign. The biofilm samples from the Leutra river were scraped off the riverbed stones using a plastic spatula. They were placed into polyethylene bottles and transported to the laboratory. The sampling campaign at the Leutra river was held in autumn 2005 and in spring 2006. Additionally, water samples were collected. The sampling procedure, for water and uniquely sampled biofilms, was the same as that carried out for the Leutra river. The locations of the sampling sites were selected according to the availability of a suitable sampling device. The samples collected were also placed into polyethylene bottles and transported to the laboratory. Analytical procedure The biofilm samples collected were air-dried at 105 °C. Then, the samples containing 10–50 mg biofilm powder were dissolved in 3 ml of 70–72% perchloric acid and were heated for 3 h at 50 °C. The remaining dry matter of each sample was further dissolved in water so that the resulting solution was up to 5 ml. A Fisons Instruments (Beverly, MA, USA) Maxim 112 inductively coupled plasma optical emission spectrometer was used to analyse Al, Ca, Fe, K, Mg, Mn, Na and Sr, while Cd, Co, Cr, Cu, Ni, P, Se and Zn were determined with a PerkinElmer (Wellesley, MA, USA) Elan 6000 inductively coupled plasma mass spectrometer. An external aqueous calibration was adopted for the analysis by ICP-OES, while a standard addition procedure was used for the element analysis by ICP-MS. All contents correspond to the sample dry weight. The trueness of the measurements was tested by analysing a certified reference algae material. The element contents were certified for an aqua regia digestion. Additionally, the element contents were determined after microwave digestion with nitric acid. No differences between these two digestion methods were obtained. All the measurements were done in triplicate and the relative standard deviation of the technique was 10–15% for all the biofilms, indicating good repeatability of the measurements. Theory Classification and regression trees The CART method was proposed by Breiman et al. [5], for data modelling and classification. Depending on the type of the response variable, y (categorical or continuous), either classification or regression trees are built. In the present study, we will focus on constructing classification trees only. The goal of CART is to form a set of mutually exclusive regions in the data space, containing as homogeneous groups of objects as possible. This is achieved by finding optimal splits of some suitable explanatory variables at a given threshold value, such that a defined impurity function is minimised. The impurity function measures the homogeneity of each node obtained from the split. It takes the lowest value for pure nodes [5]. The nodes are split while a specified number of objects are not present in the child nodes or the nodes are not pure. A node which cannot be split any further is called a terminal node. One of the most popular impurity functions is entropy [5] and it is the function used in our study. Owing to a binary data splitting, the results of CART can easily be visualised as a binary tree, which consists of a number of nodes symbolising subgroups of data objects. In order to ensure good prediction properties of the constructed tree, the number of the tree nodes should be optimal. Selection of the optimal number of nodes relies upon a deletion of some nodes from the tree, which is done by means of the so-called cost-complexity pruning [5]. Discriminant partial least squares The DPLS approach aims to relate a set of n explanatory variables (predictors), X (m × n), to a dependent variable, y. The dependent variable, y, is either a discrete variable, representing the belongingness of m objects to two defined groups denoted by −1 and 1, or a binary variable. The popularity of the DPLS method in chemometrics is due to its attractive properties. DPLS can successfully deal with multicollinearity in the data by constructing a few (f) latent factors, T (m × f), which maximise the covariance between X and y [6]. In order to obtain DPLS models with good prediction abilities, an optimal number of factors should be chosen. The optimal number of factors is usually found with the help of a cross-validation procedure [6]. The model with the smallest root mean square error of cross-validation (RMSCV) is to be selected. The goodness of model fit is indicated by a root mean square (RMS) error, whereas the success of the prediction is expressed by a root mean square error of the test set. Moreover, the performance of DPLS depends on the set of samples used for its construction. The model set has to cover all possible sources of data variance. Furthermore, DPLS is sensitive to the number of objects used to build the model. Its performance is optimal when the model set contains two groups with the same number of objects [7]. The predictive ability of the model built also depends on the quality of the variables measured. Uninformative variable elimination–discriminant partial least squares Usually, the samples collected are characterised by a large number of variables in order to ensure a detailed description of the event studied. However, some of the experimental variables may be irrelevant for the particular discriminant problem. Such uninformative variables, which have a high variance, but small covariance with y, lead to a DPLS model with unsatisfactory predictive ability. Therefore, finding an optimal set of variables by discarding the uninformative variables from the data can substantially improve the DPLS model by a decrease of the prediction error for test samples or/and a decrease of model complexity. The variable selection method used in our study is UVE-DPLS [8]. With UVE-DPLS, variables with unstable regression coefficients are removed. In order to estimate the stability of the regression coefficients, a matrix, N (m × p), containing at least p=300 random variables, is augmented with the matrix of experimental variables, X (m × n), which results in a matrix, Z, of dimension m × n+p. To keep the influence of the variables added negligible, their elements are generated from the normal distribution and are multiplied by a small constant with magnitude 1 × 10–10. The matrix of regression coefficients is constructed by the use of a leave-one-out cross-validation procedure, i.e. m PLS models are built and each one by using m-1 objects. The stability of a variable is determined by the ratio of the mean of m regression coefficients and their standard deviation. The variables with absolute stabilities of regression coefficients below a given cutoff value are uninformative and are deleted from the data. The cutoff value is defined as the largest absolute value of all stability values for the random variables added. The goodness of a discrimination model is characterised by the percentage of correct classification or the so-called correct classification rate. It is commonly agreed that the higher the correct classification rate, the better the model. Additionally, one should consider sensitivity and selectivity of the model. For a two-class problem for instance, sensitivity is defined as the percentage of correctly classified samples of class A, while selectivity is the percentage of correctly classified samples of class B. Results and discussion All the data collected were organised in a matrix, X, of dimension 71 × 34. All 71 biofilm samples (Table 1) were characterised by the contents of 17 chemical elements analysed in the biofilms and in the water phases extracted at the sampling locations. Three groups of samples were distinguished depending on the type of water body, namely flowing water, standing water and seawater. Firstly, PCA was used for an overall exploration of the data structure. PCA is an unsupervised approach, and is frequently employed for data compression and visualisation [9]. With PCA, the original data matrix, X (m × n), is decomposed into two matrices: a scores matrix, T (m × n), the columns of which contain principal components (PCs) and a loadings matrix, P (n × n). PCs are found as linear combinations of explanatory variables by maximising the variance of projected data. The loadings matrix, P, describes the contributions of each variable to the constructed PCs. Prior to the PCA analysis, the explanatory variables were autoscaled, because they had been measured in different units. Autoscaling is performed by subtracting the column mean from each data element and dividing it by the corresponding standard deviation. It gives variables the same importance in the PCA analysis. The results of PCA for autoscaled data are presented in Fig. 1. Fig. 1Principal component analysis of the data set containing the uniquely sampled biofilms and the systematically sampled biofilms: a scree plot of the cumulative percentage of data variance explained by the consecutive principal components (PC), b projection of biofilms on the plane defined by PC 1 and PC 2, c projection of biofilms on the plane defined by PC 1 and PC 3, d projection of variables on the plane defined by PC 1 and PC 2 and e projection of variables on the plane defined by PC 1 and PC 3 The first three PCs explain about 50% of the total data variance (Fig. 1a). Figure 1a indicates that the compression is not very effective, because the data variance is distributed over all PCs. However, some general trends in the data structure can be revealed. All the seawater samples are differentiated from the standing water and flowing water samples in the PC 1–PC 2 score plot (Fig. 1b). The sea samples can be divided into two subgroups. The first subgroup contains the samples from Steinbeck (Germany), Travemünde (Germany) and Damp (Germany), located in the Baltic Sea, while the second subgroup includes the samples from Punta Skala (Croatia), Nin (Croatia) and Majorca (Spain), situated in the Mediterranean Sea. The standing water and flowing water biofilm samples overlap. The flowing water biofilm sample from Munich can also be distinguished from all the other samples along PC 2. This distinction is even more evident along PC 3 (Fig. 1c). Looking at the loading plots, shown in Fig. 1d and e, one finds the reasons for the objects’ distributions observed in the score plots. PC 1 represents the Sr, Cu, Mg, Se, K and Na contents in the water phase (W-Sr, W-Cu, W-Mg, W-Se, W-K, W-Na). This factor can be related to the salt content of the water phase and is conditionally called the ‘salt’ factor. The second PC, PC 2, is mainly associated with Fe and Mg (Fig. 1d). These elements are basic components participating in the biofilm formation. PC 3 reflects the Mn, Zn, Cd, Pb, Fe, and Co contents in the water phase. The presence of Cd and Pb is usually a result of environmental contamination and that is why PC 3 is associated with the anthropogenic influence. From the information obtained from the score and loading plots, it follows that the water phase of the biofilm grown in seawater is indeed richer in dissolved salts than the water phase of the standing water and flowing water bodies. The levels of Fe and Mg in sea biofilms are also higher in comparison with those for the other biofilms. The salt content, pH and temperature of water vary at different sampling locations and they influence the biofilm formation. It was reported in [10] that Mg strongly influences attachment and biofilm structure. The surface colonisation and biofilm depth increase with the increasing Mg concentration. The biofilms collected in Punta Skala, Nin and Majorca contain higher levels of dissolved salts in the water phase and higher Fe and Mg contents in comparison with the biofilms collected in Travemünde, Damp and Steinbeck. The sample originating from Munich shows a high anthropogenic influence, i.e. it has higher Mn, Zn, Cd, Pb, Fe and Co contents in the water phase and lower Fe and Mg contents in the biofilm in comparison with the other samples. In order to see whether the biofilms developed in standing water could be distinguished from the biofilms grown in flowing water, supervised approaches such as CART, DPLS and UVE-DPLS were applied. Furthermore, it was important to determine if the models constructed could predict the origin of new biofilm samples and how well. Another question to be answered was what variables are responsible for an eventual discrimination of groups. Only seven biofilms were grown in seawater; therefore, they were excluded from the forthcoming analysis. To construct a reliable discriminant model and to test its predictive ability, the data were divided into two subsets (model and test) with the Kennard and Stone [11, 12] and duplex [13] algorithms enabling a uniform subset selection. In the Kennard and Stone method, objects in the model set are selected sequentially, starting with the object closest to the data mean. The next object included in the subset is the one situated furthest away from the first one. The third object selected is the most distant one from the objects selected in the model set. The selection of objects continues while a predefined number of objects are not assigned to the model set. The remaining objects form the test set. As a similarity measure, the Euclidian distance was used. With the duplex algorithm, the two most distant objects in the data are found and included in the model set. The next two most distant objects are assigned to the test set. The remaining objects are consecutively added to the subsets, switching over to the most distant unassigned object with respect to the model set and to the most distant unassigned object with respect to the test set. The Kennard and Stone algorithm ensures that the objects in the model set cover all possible sources of data variance, while the duplex method guarantees the representativeness of both subsets. Selection of model and test sets should be done for each group separately. When a preprocessing procedure is required, the selection of objects is applied to preprocessed data. In our study, the model and test sets were selected using autoscaled data in order to remove the scale differences among variables while evaluating the Euclidean distances among objects. It should be mentioned that the performance of CART is not influenced by autoscaling. In our study, the model set of dimension 42 × 34 contains 21 flowing water and 21 standing water biofilm samples, whereas the test set of dimension 22 × 34 includes 15 biofilms of flowing water and seven biofilms of standing water. Results of CART, DPLS and UVE-DPLS for model and test sets designed with the Kennard and Stone algorithm To trace the importance of variables responsible for the discrimination of both groups, a classification tree was built. After tenfold cross-validation, an optimal tree, containing two terminal nodes, was selected. The cross-validation error is 14%, indicating a relatively good predictive ability of the constructed tree shown in Fig. 2. Fig. 2Classification tree constructed for 42 biofilm model samples with target variable describing the type of the water (flowing, f, or standing, s), in which the biofilms were grown Since there is only one split in the tree, the discriminant problem is rather simple and the most discriminative variable describes the Mg content in the water phase (W-Mg). As mentioned before, Mg plays an important role during the biofilm formation [10]. All the model set samples, belonging to the group of standing water (17 samples), have Mg content in water phase below 37 mg g−1. The remaining samples (21 flowing water biofilms and four standing water biofilms) are placed in the left terminal node, which results in a misclassification error of 9.5% for the complete tree. Although four model standing water biofilms are recognised as flowing water biofilms, the constructed classification tree provides a correct classification of 100% for the test samples (Table 2). Therefore, the model yields fairly high sensitivity (percentage of correct classification of the test flowing water biofilms) and selectivity (percentage of correct classification of the test standing water biofilms). Table 2Correct classification rate (CCR), sensitivity and selectivity of the modelsSelection of model and test setsKennard and StoneDuplexTechniqueCARTaDPLSUVE-DPLSbCARTcDPLSUVE-DPLSdFlowing water vs. standing water samplesCCR (%)100.081.890.986.486.486.4Sensitivity (%)100.073.386.7100.0100.0100.0Selectivity (%)100.0100.0100.057.157.157.1CART classification and regression trees, DPLS discriminant partial least squares regression, UVE uninformative variable eliminationaSelected variable: W-MgbSelected variables: W-Mg, W-Ca and W-SrcSelected variable: W-MgdSelected variables: Fe, Mg, Al, W-Cr, W-Cu, W-Mg, W-Ca, W-Sr and W-K Additionally, good discrimination results can be obtained when the primary split is made on the variable describing the Ca content in the water phase (W-Ca). This variable is a competitive variable selected after removing W-Mg. The split on W-Ca leads to a total misclassification error of 14.3%. The presence of Ca has been shown to have an influence on mechanical properties of biofilms [14]. In the next step of the investigation, DPLS was considered, in order to check if a discrimination model using linear combinations of explanatory variables can perform better than CART. The DPLS model has complexity 1. The RMSCV is 0.95 and RMS error is 0.64. The DPLS model constructed allows for 81.8% correct classification of the test set samples. The analysis of the misclassified test samples indicates that four out of 15 (26.7%) flowing water biofilms collected in the Leutra river are incorrectly predicted as standing water biofilms; therefore, the model has a lower sensitivity (73.3%) than the CART model. All the test samples belonging to the group of standing water biofilms are well predicted, which again indicates the high selectivity (100%) of the model constructed (Table 2). An improved DPLS model was obtained by use of the UVE-DPLS approach, after discarding the uninformative variables. The one-factor UVE-DPLS model constructed with three informative variables (W-Mg, W-Ca, W-Sr), offers a total correct classification of 90.9% for the test set. It yields a selectivity of 100% and a better sensitivity (86.7%) in comparison with the DPLS model, because only two out of 15 (13.3%) biofilms grown in the flowing water of the Leutra river are now assigned to the group of standing water biofilms (Table 2). The best discrimination results are obtained from CART, even though this model shows a misclassification error of 9.5% for the complete tree. Since the splits are done in a univariate way, the correlation between variables is not taken into account. Therefore, CART provides unsatisfactory results when a linear combination of variables is responsible for discriminating the samples. This, however, cannot be verified unless multivariate approaches such as DPLS and UVE-DPLS are used. Although CART and UVE-DPLS have different objective functions, common variables are selected as essential for the discrimination. The primary variable, W-Mg, and two competitive variables, W-Ca and W-Sr, in CART are also selected by UVE-DPLS. Results of CART, DPLS and UVE-DPLS for model and test sets designed with the duplex algorithm Results of CART, DPLS and UVE-DPLS were obtained using data designed with the duplex algorithm, which ensures the representativeness of the model and test sets. The classification tree built has two terminal nodes and the primary split is again made on the variable representing the Mg content (W-Mg) in the water phase. The cross-validation error is 7.1%. Two out of 42 model set samples are wrongly classified, which leads to a misclassification error of 4.8% for the complete tree. Compared with the previous results, the constructed tree shows a better performance for the model set samples, but worse prediction rates (Table 2); therefore, the model has again a fairly high sensitivity (100%), but quite low selectivity (57.1%). The DPLS model constructed for the data designed by the duplex algorithm shows slightly better prediction ability (86.4%) than the model built for the data designed by the Kennard and Stone algorithm (81.6%). It presents a better sensitivity (100%), but a reduced selectivity, with only 57.1% of standing water samples being well recognised. A discriminant model characterised by relatively high sensitivity and selectivity parameters is to be preferred over a model with a high sensitivity and a low selectivity. Therefore, the UVE-DPLS model for data designed by the Kennard and Stone algorithm is to be favoured (Table 2). All the methods allow a correct prediction for 86.4% of samples. The samples collected at Chemnitz and White Dak Pond, Metebach, are improperly classified by all methods. In fact, this is not a striking observation though when the data contain some samples that are different in comparison with the majority of samples. These samples are always assigned to the model set using the Kennard and Stone method and then the test samples are correctly predicted. Using the duplex method, we assigned some atypical samples to the test set, which results in a construction of models with too pessimistic predictive abilities. Results of CART, DPLS and UVE-DPLS for biofilm samples grown on natural substrates Another important issue to be discussed is whether the biofilm samples grown on natural substrates (see the group of uniquely sampled biofilms in Table 1) can be used to derive similar conclusions as those drawn using the whole data. If this is possible, the sampling procedure will be carried out in a simpler way, which will be less time-consuming and relatively low in price. For an initial inspection of the data structure, PCA was considered. PCA was applied to autoscaled data (25 × 34) containing only uniquely sampled biofilms and the results are presented in Fig. 3Fig. 3Principal component analysis of the data set containing the uniquely sampled biofilms: a scree plot of the cumulative percentage of data variance explained by the consecutive PCs, b projection of biofilms on the plane defined by PC 1 and PC 2, c projection of biofilms on the plane defined by PC 1 and PC 3, d projection of variables on the plane defined by PC 1 and PC 2 and e projection of variables on the plane defined by PC 1 and PC 3 The first three PCs account for 59.1% of the total data variance (Fig. 3a). Similar to PCA of the whole data, the compression is not very effective. The biofilms grown in seawater can again be distinguished along PC 1 (Fig. 3b). Moreover, two subgroups of sea biofilms are distinguished along PC 1 (Fig. 3c). The content of the subgroups is the same as before. The biofilm sample collected in Munich is again found far away from all the other samples. Another extreme biofilm sample, collected in the Aller river (Celle, Germany), appears along PC 3. Regarding the variable loadings (Fig. 3d, e), PC 1 is again associated with the salt content of the biofilm water phases, while PC 2 probably is now linked to the contamination of the biofilm water phases, because the variables W-Zn, W-Cd, W-Ni, W-Fe, W-Co, W-Mn and W-Pb possess high loading values. PC 3 consists of Zn, Cd and Pb, which are usually associated with an anthropogenic influence and this factor is therefore associated with contaminants accumulated by the biofilm. Summarising the results of PCA, one can additionally point out that the biofilm samples collected in Majorca, Punta Skala and Nin are richer in Zn, Cd and Pb in comparison with the remaining sea biofilm samples. Moreover, the highest Zn, Cd and Pb contents are characteristic for the biofilm sample from Celle. In order to construct the CART, DPLS and UVE-DPLS models, only data of natural biofilms grown in flowing water and in standing water were considered. Since the number of samples in each group is small (Table 1), the models were used for an exploratory purpose only. Because of this, the predictive abilities of the models were not tested using an independent test set. The complete classification tree with three terminal nodes is shown in Fig. 4. The primary split is made on the variable describing the Pb content in the water phase (W-Pb). W-Pb is the most discriminant variable. The next split on variable Al corrects the improper assignment of one sample and it is of a lower importance. Owing to the small number of samples, the required tenfold cross-validation procedure could not be applied and, therefore, the cross-validation error was not reported. All the biofilms grown in standing water are well classified, but two biofilm samples grown in flowing water are wrongly classified, which results in a total classification rate of 88.9%. The incorrectly classified samples originate from Steinach (Germany) and Geithain (Germany). Fig. 4Classification tree constructed for 18 biofilm samples with target variable describing the type of the water (flowing, f, or standing, s), in which the biofilms were grown The DPLS model constructed has complexity 1. RMSCV is 1.78 and RMS error accounts for 0.59. Two samples are incorrectly classified. One of them belongs to the biofilms of standing water and originates from Chemnitz (Germany), while the other one is the biofilm collected in the flowing water body (São Lourenço) located in Juquitiba (Brazil). The DPLS model built yields a total classification rate of 88.9%. It should be emphasised that DPLS can lead to a too optimistic result when the number of variables outnumbers the number of samples [7]. A remedy for this problem is to reduce the number of variables by the use of a feature selection technique, e.g. UVE-DPLS. The UVE-DPLS model has RMSCV of 0.95. One variable, namely W-Pb, is selected. However, all biofilms grown in flowing water are correctly classified with the model constructed, but all biofilms grown in standing water are improperly classified. Conclusions Discrimination between sea biofilms and the remaining standing water and flowing water biofilms is straightforward by investigating the score plots obtained from PCA. The loading plots emphasise the expected higher salt content of the water phases extracted from the sea biofilms as well as their higher levels of Fe and Mg in comparison with the other biofilms. A further discrimination between flowing water and standing water biofilms is possible by means of supervised methods like CART, DPLS and UVE-DPLS. The best discriminant model is obtained from CART. One variable describing the Mg content in the water phase (W-Mg) is enough to build a model with 9.5% misclassification error. All test samples selected by the Kennard and Stone algorithm are correctly classified using the constructed CART model. The DPLS and UVE-DPLS methods do not outperform CART for the data set studied and, therefore, it can be pointed out that a linear combination of explanatory variables does not lead to a better prediction for new samples. Moreover, CART appears as a very simple and efficient discriminant technique leading to a straightforward data interpretation in terms of explanatory variables. Hence, CART can be considered as a pilot discriminant approach. When the CART model is not satisfactory, one can apply discriminant methods, such as DPLS and UVE-DPLS, or if necessary to use a nonlinear multivariate classifier like, e.g., support vector machines. All discriminant models, CART, DPLS and UVE-DPLS, lead to 86.4% correct classification for the test set designed by the duplex algorithm. However, CART uses only one variable (W-Mg), UVE-DPLS selects nine variables and DPLS uses all explanatory variables to build the model. Discrimination of flowing water and standing water biofilms that are uniquely sampled, using CART, DPLS and UVE-DPLS models, is done only for a better understanding of the data collected. For a definite conclusion whether these two groups of samples can be discriminated, more samples are required to properly validate the discriminant models.

J_Autism_Dev_Disord-4-1-2268724

Face and Emotion Recognition in MCDD Versus PDD-NOS

Previous studies indicate that Multiple Complex Developmental Disorder (MCDD) children differ from PDD-NOS and autistic children on a symptom level and on psychophysiological functioning. Children with MCDD (n = 21) and PDD-NOS (n = 62) were compared on two facets of social-cognitive functioning: identification of neutral faces and facial expressions. Few significant group differences emerged. Children with PDD-NOS demonstrated a more attention-demanding strategy of face processing, and processed neutral faces more similarly to complex patterns whereas children with MCDD showed an advantage for face recognition compared to complex patterns. Results further suggested that any disadvantage in face recognition was related more to the autistic features of the PDD-NOS group rather than characteristics specific to MCDD. No significant group differences emerged for identifying facial expressions. Introduction Social cognition is a crucial component of healthy adjustment, and deficits have been reported in a range of psychiatric disorders in children and adults (for review, see Blair 2003; Phillips et al. 2003a; Walker 1981). A core deficit in children with autism spectrum disorders, or Pervasive Developmental Disorder (PDD) (as defined in the DSM-IV; (APA 1994) relates to social cognition, important aspects of which are, for example, the processing of faces and facial expressions. Encompassed by the overarching category of PDD is the diagnosis of Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS). Children with PDD-NOS form a heterogeneous group characterized by autistic-like symptoms of varying severity such as marked impairments in social interaction, communication and/or rigid and stereotyped behavior patterns, but fail to meet full criteria for autistic disorder (APA 1994; Walker et al. 2004). Within this heterogeneous group of PDD-NOS children, Cohen and colleagues highlighted the existence of a number of children with disturbances in various areas of functioning such as the regulation of state and arousal (i.e. anxiety and fears), social relations (i.e. detached, aggressive, clingy), and thought disorders (i.e. magical thinking, unusual thoughts, and difficulties in separating fantasy from reality) (Cohen et al. 1986). These children have been described in the past as schizotypal, having borderline disorder, childhood schizophrenia, childhood onset PDD or atypical PDD (for review see Ad-Dab’bagh and Greenfield 2001). The description of this group was refined through a specific set of diagnostic criteria, and the term ‘Multiplex Developmental Disorders’ (MDD) was proposed (Cohen et al. 1986). Subsequently, the criteria were altered and the term was modified to ‘Multiple Complex Developmental Disorder’ (MCDD) (Towbin et al. 1993). Although Cohen and colleagues positioned MDD within the broader classification of Pervasive Developmental Disorder (PDD), they also recognized the overlap with several other DSM-III (APA 1980) disorders (i.e. Avoidant Disorder, Overanxious Disorder, Schizotypal Disorder) (Cohen et al. 1986). To date, there is little research on these children, however further study is warranted since MCDD children may be at risk for a poor outcome in adulthood including Axis II disorder (Lofgren et al. 1991), and schizophrenia spectrum disorders (van Engeland and van der Gaag 1994). This study aims to directly compare children with MCDD to those with PDD-NOS on two measures of social cognition: face recognition and identification of facial expressions. Both of these skills are important to examine given the difficulties in social functioning reported in children with MCDD. Such studies are valuable since any emerging differences in social-cognitive functioning between these two groups of children would further support the validity of MCDD as a separate diagnostic construct. Further validation of the concept of MCDD would emphasize the importance of investigating etiology and efficacy of treatments for MCDD separately from PDD-NOS. On a symptom level, children with MCDD can be distinguished from other developmental disorders. A study examining the medical charts of children with MCDD, children with autism, and children with externalizing and internalizing disorders, reported that MCDD children, compared to children with autism, were more aggressive, more anxious, showed more psychotic thinking, and suspiciousness. Autistic children were more disturbed in their social interaction and communication and displayed more stereotyped and rigid behavior than MCDD children (van der Gaag et al. 1995). There is also evidence that children with MCDD or autism have elevated levels of formal thought disorder compared to children with Attention Deficit Hyperactivity Disorder (ADHD) and anxiety disorders (van der Gaag et al. 2005). A recent study directly comparing the symptom profiles of children with MCDD and PDD-NOS, reported that the greatest group differences (i.e. largest effect sizes) were found for psychotic thought problems (de Bruin et al. 2007). MCDD children, in comparison to PDD-NOS children, experienced more paranoia, incoherent thoughts and delusions. Furthermore, children with MCDD also had a higher frequency of anxiety disorders and disruptive behavior disorders according to the Diagnostic Interview Schedule for Children (Version IV) (DISC-IV) (i.e. Oppositional Defiant Disorder, and Conduct Disorder) compared to children with PDD-NOS. De Bruin and colleagues (2007) applied research criteria to assess MCDD and PDD-NOS independently of one another, and found that a greater percentage of children with PDD-NOS met criteria for a diagnosis of autism or autism spectrum disorder (ASD) and scored higher on measures of reciprocal social interaction and communication deficits as assessed using the Autism Diagnostic Observation Schedule-Generic (ADOS-G) (Lord et al. 1999). Findings suggest that children with MCDD, compared directly to those with PDD-NOS, can be differentiated on various dimensions, one of which (i.e. psychotic thought problems) might resemble difficulties experienced by adults with schizophrenia. Children with MCDD also demonstrate psychophysiological differences compared to children with autism, ADHD, dyslexia, and normally developing children as assessed by Event-Related Potentials (ERP) obtained during performance on a visual oddball task (Kemner et al. 1999). There is evidence for a blunted cortisol response to psychosocial stress compared to normally developing children (Jansen et al. 2000a) and children with autism (Jansen et al. 2003). Although both MCDD and autistic children are characterized by abnormal reactions to their social environment, group differences in cortisol response to psychosocial stress suggests that the disorders may have different etiological backgrounds or perhaps may be different neurobiological conditions. A blunted response to psychosocial stress among children with MCDD could also be partly due to higher levels of comorbid conduct disorders. Similar blunted cortisol responses to psychosocial stress have also been reported in adults with schizophrenia (Jansen et al. 1998, 2000b, 2003). This suggests that children with MCDD may possess a biological vulnerability to this disorder that may not be evident among children with PDD’s (at least autism). Findings to date on the symptom and biological/psychophysiological profiles suggest that children with MCDD form a group who may not be well placed under the umbrella of the PDD’s, since there is evidence that they may be at risk for developing schizophrenic spectrum disorders later in life. Despite the differences seen among children with MCDD compared to other clinical groups on a symptom, psychophysiological, and neurobiological level, no studies to our knowledge have assessed social-cognitive functioning in these children. The only study to investigate a related area, examined formal thought disorder in MCDD children compared with other clinical groups (van der Gaag et al. 2005). Although children with MCDD may demonstrate similar core social deficits as seen in children with PDD-NOS, parallels between the symptoms and biological reactivity in children with MCDD, and adults with schizophrenia spectrum disorders, suggest that a different pattern of performance on social cognitive tasks might emerge compared to children with PDD-NOS. We aimed to examine whether children with MCDD could be differentiated from children with PDD-NOS on two important aspects of social cognition: face recognition and the identification of facial expressions. Face Recognition Serra and colleagues provided empirical evidence that a more time-consuming, controlled, attention-demanding strategy may characterize face processing in children with PDD-NOS by demonstrating that children with PDD-NOS were significantly slower in face recognition than age-matched normally developing children while the recognition of abstract visuo-spatial patterns did not discriminate between the groups (Serra et al. 2003). Klin and colleagues studied face recognition in children with autism, PDD-NOS, and non-PDD disorders (Klin et al. 1999). Findings revealed a specific deficit in face recognition in children with autism (which could not be attributed to general cognitive ability), but no specific face recognition deficit in children with PDD-NOS. However, these authors did not investigate processing times. To our knowledge, no studies have examined face processing in children with MCDD, nor have any directly compared children with MCDD to those with PDD-NOS. We can only speculate on the results for MCDD children. However, if children with MCDD are distinct from those with PDD-NOS, we might expect them to show a different pattern of face recognition compared to children with PDD-NOS only (Serra et al. 2003), which might be characterized by less controlled time-consuming processing. Identification of Facial Expression Facial expression recognition has not been previously examined in children specifically diagnosed with MCDD. Considering that children with MCDD have been previously described as schizotypal or having a diagnosis of childhood schizophrenia (see Ad-Dab’bagh and Greenfield 2001), a study that most closely approximates the investigation of facial expression recognition in MCDD is one examining emotion expression recognition in children with schizophrenia, aggression, anxiety/depression, and normally developing children (Walker 1981). Walker (1981) reported that children with schizophrenia were less accurate in recognizing emotional expressions compared to the other groups. Deficits in identifying facial expressions have been reported in children with PDD (mainly autism) (Castelli 2005; Celani et al. 1999; Hobson et al. 1988). Although a review by Blair (2003) on facial expression recognition in neuro-cognitive disorders noted that once participants with autism were matched to controls on mental age, impairments in emotion expression recognition disappeared. Few studies have probed emotion processing in children with PDD-NOS. One study comparing normally intelligent PDD-NOS children with school children on emotion recognition (i.e. face, posture, and gesture recognition), found no differences between the two groups (Serra et al. 1998). Based on the above findings, and given the fears and anxieties inherent to the classification of MCDD, one might predict specific differences, particularly in the identification of threatening expressions (i.e. fearful and angry expressions) compared to children with PDD-NOS. This is based on previous research indicating that adults and children with elevated levels of anxiety demonstrate biases in processing anger/threatening expressions (Hadwin et al. 2003; Mogg et al. 2004). Aims and Predictions of the Study If robust social cognitive differences would emerge, in light of the symptom profile and biological/psychophysiological differences between the groups, there would be further evidence for recognizing a subcategory of MCDD within the DSM-V as already suggested by others (van der Gaag et al. 1995). Considering MCDD as qualitatively distinct from PDD-NOS has implications for treatment. Viewing MCDD as a subgroup of PDD-NOS may focus treatment on the improvement of social skills, whereas if the emphasis in MCDD was on the thought disorder and its relation to psychotic development as well as on anxiety symptoms, a more medication-focused approach might be preferred. However, if social cognitive skills were quite the same, such a finding could also suggest that the diagnostic boundaries of the DSM-categories are weak: people with schizoid/psychotic symptoms might have a neurobiological condition different from that of people with only PDD-symptoms though sharing important PDD core features. Such a finding would stress the necessity for profiling patients along various dimensions including their psychophysiological responsiveness and (social) cognitive capacities. We aimed to address whether children with MCDD significantly differ from children with PDD-NOS on two facets of social-cognitive skills: face recognition and the identification of facial expressions. Children with MCDD and PDD-NOS were carefully selected using explicitly outlined research criteria. Based on previous research:We predicted that children with MCDD would differ from those with PDD-NOS on recognizing neutral faces in comparison to complex patterns. More specifically, if indeed children with MCDD are not well-placed under the PDD’s, we would expect them to be faster and more accurate on face recognition compared to children with PDD-NOS, but perform similarly on especially complex pattern recognition.Children with MCDD would show differences in the identification of facial expressions, particularly a bias toward processing fear and anger expressions, as compared to children with PDD-NOS. Any significant differences found should not be explained by differences in mental age. Methods Participants The study sample was selected from 503 children, aged 6–12 years old, who were consecutively referred to the outpatient department of child and adolescent psychiatry, between July 2002 and September 2004. Referrals were comprised of a large variety of child psychiatric disorders (externalizing disorders, internalizing disorders, PDDs). Research criteria for MCDD and PDD-NOS were rated incompletely for 12 (2.4%) children, who were excluded from further analyses. Complete MCDD and PDD-NOS criteria were rated for 491 children. Twenty-nine (5.9%) children met research criteria for a diagnosis of MCDD. Eleven children (44%) meeting MCDD research criteria also met research criteria for PDD-NOS. These children were placed in the MCDD group. The parents of four of these children refused to participate in the study. Seventy-nine children met research criteria for a diagnosis of PDD-NOS without meeting research criteria for MCDD (PDD-NOS group). These children did not meet DSM-IV criteria for autism or Asperger syndrome. Children with an IQ score of less than 70 were not administered the neuropsychological battery; two children in the MCDD group, and 13 children in the PDD-NOS group were not administered the neuropsychological tasks on this basis. A further two children meeting MCDD criteria had missing data for the face recognition (FR) task and identification of facial expressions task (IFE). Thus, the MCDD group included a total of 21 children. In addition to the children without neuropsychological data due to a low IQ, five children in the PDD-NOS group were missing data for the FR task, and four were missing data for the IFE task, resulting in a total of 61 PDD-NOS children with FR task data, and 62 children with IFE task data. Ethics Participation was voluntary, and informed consent was signed by all parents/caretakers prior to participation in the study. Children who were 12 years old also signed the consent forms themselves. The Medical Ethics Committee of the Erasmus Medical Center approved the study. MCDD and PDD-NOS Research Criteria Explicit research criteria for MCDD and PDD-NOS were used (see Table 1) (Buitelaar et al. 1999a). Nine different child and adolescent psychiatrists were involved in rating these research criteria. Ratings were based on semi-structured interviews with the parents/caretakers and individual psychiatric observation of the child. Assessment information spanned from early development to current level of social, communicative, and adaptive functioning. School, relevant medical, and psychological assessment information were obtained as well. Immediately after all diagnostic procedures had been completed, MCDD and PDD-NOS research criteria were ticked as present or absent, and subsequently an algorithm, of which the rater was unaware, was used to determine whether the thresholds for research diagnoses of MCDD and PDD-NOS were met. For a detailed review of the development of the MCDD and PDD-NOS research criteria see Buitelaar and van der Gaag (1998). Table 1Research criteria used to identify children with MCDD and PDD-NOS (from Buitelaar and van der Gaag 1998)MCDD1PDD-NOS2(1) Impaired regulation of affective states and anxietiesA2. (1) Qualitative impairment in social interaction(a) Unusual or peculiar fears and phobias, or frequent idiosyncratic or bizarre anxiety reactions(a) Marked impairment in the use of multiple nonverbal behaviors, such as eye-to-eye gaze, facial expression, body postures, and gestures to regulate social interaction(b) Recurrent panic episodes, or flooding with anxiety(b) Failure to develop peer relationships appropriate to developmental level(c) Episodes of behavioral disorganization punctuated by markedly immature, primitive, or violent behaviors(c) A lack of spontaneous seeking to share enjoyment, interests, or achievements with other people (e.g. by a lack of showing, bringing, or pointing out objects of interest)(d) Lack of social and emotional reciprocity(2) Impaired social behavior(2) Qualitative impairments in communication(a) Social disinterest, detachment, avoidance, or withdrawal(a) In individuals with adequate speech, marked impairment in the ability to initiate or sustain a conversation with others(b) Markedly disturbed and/or ambivalent attachments(b) Stereotyped and repetitive use of language or idiosyncratic language(3) The presence of thought disorder(3) Restricted repetitive and stereotyped patterns of behavior, interests, and activities(a) Irrationality, magical thinking, sudden intrusions on normal thought process, bizarre ideas, neologism, repetition of nonsense words(a) Stereotyped and repetitive motor mannerisms (e.g. hand or finger flapping or twisting, or complex whole-body movements)(b) Perplexity and easy confusability. overvalued ideas, including fantasies of omni-potence, paranoid preoccupations, overengagement with fantasy figures, referential ideationB. Does not meet criteria for autistic disorder or for other specific pervasive developmental disorder1A total of 5 or more items from 1, 2, and 3, with at least one item from (1), one item from (2), and one item from (3)2A total of 3 or more items from (1), (2), and (3), with at least one item from (1) An interrater reliability study was conducted for 30 randomly selected children (27%). Two clinicians independently rated all MCDD and PDD-NOS research criteria. Agreement between the raters on the presence or absence of a PDD-NOS diagnosis was good (κ = .62). Agreement for MCDD diagnosis could not be calculated, as MCDD did not occur once in this subsample. Materials Procedure Children were assessed on two occasions, separated by a week. Testing was conducted in a quiet room in the outpatient department of the hospital. On the first occasion, the full Weschsler Intelligence Scale for Children (WISC-R) was administered, taking on average, 2 h per child. The social-cognitive tasks were administered on the second visit the following week as part of a larger neuropsychological battery taking approximately one and a half hours. Children were always tested in the morning to minimize the effects of fatigue and to maximize concentration. Intelligence: Weschsler Intelligence Scale for Children (WISC-R) The full WISC-R (revised for use in the Netherlands) was administered to the children. Based on the full-scale IQ score, each child’s mental age was computed using the following formula: Mental age = (age * full scale IQ score)/100. Mental age (MA) was included as a covariate since it has been shown to be an important mediator for group differences in social cognition (see Buitelaar et al. 1999b; Happe 1995). Baseline Speed (BS) A simple reaction time task was employed to obtain a baseline measure for the speed (BS) of responding with the response key to ensure children understood how to respond using the response key. Children were required to press a key with the index finger of their dominant hand when a square was presented. Thirty-two trials were administered. This task was a subtest of the computerized Amsterdam Neuropsychological Tasks (ANT version 2.1; de Sonneville 1999). The total baseline speed and the standard deviation (SD) of the BS were calculated. The SD provides a measure of the variability of performance. A higher SD could indicate less attention to the task. Face Recognition The Face Recognition (FR) subtest of the ANT 2.1 battery (de Sonneville 1999) was selected to measure the speed and accuracy of recognizing neutral faces. This task has previously been administered in studies assessing face recognition in children with PDD-NOS (Serra et al. 2003) and in normally developing children (de Sonneville et al. 2002). In the FR task, a target (neutral) face was presented for 2.5 s. Following the presentation of the target face, a set of four photographs of individuals was presented and children were required to indicate (using a two-key response panel) whether or not the target individual appeared in the set of four (see Fig. 1a). The sex and age category of the target (i.e. boys, girls, men or women) matched those of the subsequently shown set of four faces to be judged. In half of the trials (i.e. 20), the target individual did appear in the set of four and participants were required to press a ‘yes’ key (‘target’ condition), and in 20 trials the target individual did not appear in the subsequent set of four, requiring participants to press the ‘no’ key (‘non-target’ condition). Reaction time (RT) data and accuracy (assessed by calculating the proportion of correct trials out of the maximum score) were calculated for target and non-target conditions. Fig. 1(a) Face Recognition (FR) task. A neutral face (target stimulus) is presented for 2,500 ms, followed by a 500 ms delay. A display set of four neutral faces is then presented. The child must indicate (using a two-button response key) whether the target face is present in the display set (target condition) or not (nontarget condition). Reaction time and accuracy (proportion correct, with a maximum value of 1) data were calculated for target and non-target conditions. (b) Pattern Recognition (PR) task. The children must indicate whether a target pattern is present in one of four patterns presented in a display set. Presentation time parameters and outcome variables are the same as in the FR task. Two PR tasks are presented: (1) patterns involving a similar level of complexity to the FR task (i.e. Complex PR), (2) patterns of a dissimilar (i.e. Easy PR) level of difficulty compared to the FR task Pattern Recognition A subtest of the ANT 2.1 battery assessing pattern recognition (PR) was administered. The task consisted of two conditions, one containing visuo-spatial patterns that are dissimilar and hence easily distinguishable (easy condition), and the other containing complex patterns that are quite similar and therefore hardly distinguishable (complex condition) (see Fig. 1b). The same manner of responding was used as in the FR task, and the same four outcome variables were generated: RT and accuracy for target and non-target conditions. As with the FR task, there were 20 trials for the target condition and 20 trials for the non-target condition, each for the easy condition and the complex condition. Easy and complex patterns as target and non-target trials were presented in a random manner. Identification of Facial Expressions The “Identification of Facial Expressions”(IFE) subtest of the ANT 2.1 was employed to probe emotion processing (see Fig. 2). Children were required to respond as to whether a face displayed a particular target emotion (by pressing a “yes” button) or not (by pressing the “no” button). Four conditions were administered, each corresponding to a target emotion (i.e. happy, sad, anger, and fear). For each condition, children were instructed to focus on a particular emotion, and to respond whether the face demonstrated that particular emotion or not (i.e. for the happy condition, children were to respond “yes” if the face was happy or to press the “no” button if the face displayed a different emotion). Each emotion condition consisted of 40 trials, 20 of which were the target emotion (requiring a “yes” response) and 20 of which were a random selection of other emotions (requiring a “no” response). The images were digitized photographs of four adult identities (two men and two women). Four outcome variables were calculated for each emotion category: (1) RT for target condition (when the target emotion is presented) (2) RT for non-target condition (when the target emotion is not presented) (3) Accuracy in the target condition (4) Accuracy in the non-target condition. Accuracy (calculated for target and non-target conditions separately) was assessed by calculating the proportion of correct trials out of the maximum score. Fig. 2Examples of different expressions in the Identification of Facial Expressions (IFE) task. Children are presented with four different tasks (each corresponding to one of four target emotions: happy, sad, anger, and fear). For each task, children are required to focus on a particular emotion, and to judge whether the face displays a specific target emotion. The target consists of an adult face expressing one of four emotions. When the face matches the emotion a ‘yes’ response is required, when the face does not match the emotion, a ‘no’ response is required. A total of 40 trials per emotion condition were presented, with half of those trials requiring a ‘yes’ response (target), and half requiring a ‘no’ response (nontarget). RT and accuracy (i.e. proportion correct) for target and non-target conditions were calculated Statistical Analysis A series of multivariate General Linear Models (GLM) was conducted to compare children with MCDD to those with PDD-NOS-only on the various outcome variables of BS, PR, FR, and IFE tasks. For each task (except for the BS task which yielded reaction times only), separate repeated-measures analyses of covariance (ANCOVAs) were performed on the measures for accuracy and speed of processing. Assumptions underlying the use of parametric statistics were examined for each outcome variable. Where assumptions of normality were violated, transformations were applied. Only variables representing accuracy (for target and non-target conditions) were transformed. For these, we applied the arc sin transformation, since this is appropriate for proportional data (Howell 1997). Following these transformations, data were appropriate for the use of parametric statistics. For the repeated-measures ANCOVA’s, Wilk’s lambda and corresponding F-statistics and significance are presented where Mauchly’s test of sphericity was not significant. Where this test was significant, the corrected Greenhouse-Geisser degrees of freedom and significance levels are presented. Two-tailed tests were used. Effect sizes (small: ≥0.02 and ≤.06 ; medium: >.06 and ≤0.13; large: ≥0.14) were estimated using partial Eta squared (ηp2) (Stevens 1992). If any significant group differences were found, we repeated the analyses while covarying for the effects of mental age (MA) to exclude the possibility that the differences could be explained by variations in general intelligence. Results Group Characteristics Children in the MCDD group did not significantly differ from the PDD-NOS group on age (F(1, 78) = 2.29, p = 0.14), sex distribution (Fisher’s Exact = 1.00), VIQ (F(1, 82) = 0.22, p = 0.64), PIQ (F(1, 82) = 0.59, p = 0.44), FIQ (F(1, 82) = 0.04, p = 0.84) or MA (F(1, 78) = 1.07, p = 0.30). Means (SD) are presented in Table 2. There were no significant group differences in baseline speed (BS) (F(1, 79) = 0.21, p = 0.65) or SD of BS (F(1, 79) = 0.59, p = 0.44). This indicates that children in the PDD-NOS group were not significantly different from those in the MCDD group in terms of their basic ability to use the response key or basic attention to the task. Table 2Frequencies, Means (SD) for the MCDD and PDD-NOS groups for age, sex, and IQPDD-NOS, N = 62MCDD (all with MCDD), N = 21 ‘Pure’ MCDD subsample, N = 13 Mean (SD)Chronological age (years)9.22 (1.82) 9.89 (1.49) 9.87 (1.47) Male/Female (n)54/818/310/3Mental age (years)8.83 (2.10) 9.35 (1.60) 9.43 (1.77)IQ: WISC-RVerbal IQ95.54 (14.26) 97.33 (17.75) 98.77 (21.31)Performance IQ97.57 (16.64) 94.52 (12.55) 94.46 (10.15)Total IQ95.97 (14.39) 95.24 (14.17) 96.00 (14.52) Face Recognition (FR) Compared with Pattern Recognition (PR) Our first hypothesis was that children with PDD-NOS would be less accurate and slower in processing neutral faces compared to children with MCDD. Such a group difference was not expected for the recognition of abstract visuo-spatial patterns. Means and standard deviations (SD) for accuracy and speed of performance on both the face recognition (FR) and pattern recognition (PR) task are presented in parts 1 and 2 of Table 3, and generally indicate a slower speed of processing in the PDD-NOS group (for both face recognition and pattern recognition) while differences in accuracy appear less pronounced. Table 3Means (SD) for face recognition, pattern recognition, and identification of facial expressions tasksTask variablesPDD-NOSMCDD-allPure MCDDRT (ms): Mean (SD) Proportion accurate: Mean (SD)Face recognition (FR)N = 62N = 21N = 13Targets2155.29 (625.54)1862.71 (469.08)1853.12 (468.23)0.76 (0.17)0.83 (0.17)0.86 (0.11)Non-targets2751.15 (730.77)2599.61 (697.01)2701.30 (742.33)0.84 (0.12)0.84 (0.14)0.82 (0.15)Pattern recognition (PR)N = 61N = 21N = 13Easy PR: Targets1826.3 (519.12)1689.90 (509.77)1785.92 (544.13)0.96 (0.07)0.97 (0.05)0.98 (0.02)Easy PR: Non-targets1449.47 (470.34)1332.83 (356.20)1364.01 (392.80)0.85 (0.23)0.89 (0.17)0.87 (0.20)Complex PR: Targets2627.03 (723.78)2536.89 (763.48)2656.76 (886.98)0.83 (0.23)0.80 (0.26)0.78 (0.29)Complex PR: Non-targets3318.54 (984.58)3084.97 (845.74)3129.71 (1016.1)0.76 (0.20)0.84 (0.13)0.83 (0.14)Identification of Facial Expressions (IFE)N = 62N = 21N = 13Happy: Targets1001.03 (358.12)977.91 (347.91)1103.95 (380.76)0.92 (0.09)0.95 (0.04)0.95 (0.05)Happy: Non-targets1322.07 (482.98)1145.38 (352.43)1231.97 (394.17)0.95 (0.06)0.94 (0.06)0.95 (0.06)Sad: Targets1345.73 (419.14)1217.74 (414.07)1311.15 (467.99)0.69 (0.25)0.77 (0.21)0.81 (0.15)Sad: Non-targets1691.06 (657.64)1482.22 (412.78)1569.99 (382.69)0.78 (0.19)0.86 (0.13)0.86 (0.14)Anger: Targets1221.14 (513.73)1115.36 (275.29)1172.91 (296.49)0.73 (0.20)0.75 (0.21)0.79 (0.19)Anger: Non-targets1538.94 (614.53)1417.10 (361.06)1535.36 (347.95)0.89 (0.15)0.89 (0.13)0.87 (0.15)Fear: Targets1351.81 (573.98)1301.85 (491.01)1327.11 (586.66)0.79 (0.18)0.78 (0.24)0.80 (0.26)Fear: Non-targets1476.66 (519.16)1321.81 (300.87)1377.08 (355.14)0.82 (0.20)0.87 (0.14)0.87 (0.15) To test our hypothesis, two types of repeated measures analyses were conducted, each on our measures of accuracy and speed of processing separately. In the first type we compared the easy condition of the PR task with the performance of the FR task, in the second type we compared the complex condition of the PR with the FR task. Each analysis included two within-subjects variables: (1) “task” (FR versus PR) and (2) “response type” (target versus non-target). The between-subjects variable was group. Significant group by task interactions would indicate that the groups differ in their manner of processing neutral faces as compared to abstract patterns. Concerning performance on the easy condition of pattern recognition as compared to face recognition, there were no significant interactions involving group (i.e. group*task*response type or group*task) nor a significant main effects of group for either accuracy or speed of processing. Irrespective of group, a significant interaction between task and response type for RT (Wilk’s lambda = 0.26, F(1, 80) = 231.10, p < 0.001; ηp2 = 0.74), indicated that children took substantially longer to perform the FR task compared to easy PR, and this effect was particularly strong for the non-target condition. Comparison of the complex condition of the PR task with face recognition revealed a significant interaction between group, task (PR versus FR), and response type (target versus non-target) (Wilk’s lambda = 0.94, F(1, 80) = 5.07, p = 0.03; ηp2 = 0.06). Children with PDD-NOS were less accurate in recognizing the presence of a target face in the response set compared to children with MCDD, whereas this difference was not evident when children had to recognize a target pattern amongst hardly distinguishable other patterns (see Fig. 3). This interaction bordered on significance after controlling for MA (p = 0.05; ηp2 = 0.05). Such results did not emerge for speed of processing: there were no significant interactions; group*task*target (p = 0.91; ηp2 < 0.01) or group*task (p = 0.73; ηp2 < 0.01), nor was there a significant main effect of group (p = 0.23; ηp2 = 0.02). Irrespective of group, children processed faces faster than complex patterns (Wilk’s lambda = 0.64, F(1, 80) = 44.85, p < 0.001; ηp2 = 0.36). Fig. 3Accuracy of PDD-NOS versus MCDD children for face recognition (FR) and complex pattern recognition (PR). This figure is based on raw (untransformed) error rates, without covarying for MA. Error bars represent standard error of the mean Our first hypothesis was therefore partially supported: children with PDD-NOS were less accurate than children with MCDD in recognizing that a neutral face was present in the response set, but this effect was not seen when they were asked to note whether an abstract pattern was present amongst similar ones (i.e. complex condition). There was, however, no group difference in task dependent RTs. Identification of Facial Expressions (IFE) Task Our second hypothesis was that children with MCDD would demonstrate differences in the identification of facial expressions compared to those with PDD-NOS, particularly in the identification of fear and anger expressions. Means and standard deviations (SD) for both measures of speed and accuracy are presented in the third part of Table 3 showing a generally slower speed of processing in the PDD-NOS group while differences in accuracy appear less pronounced. To examine group differences in children’s identification of facial expressions, two repeated-measures analyses were conducted (one for accuracy and the other for speed of processing). The within-subjects factors included (1) emotion-category (four levels: happy, sad, anger, fear) and (2) response type (target versus non-target). The between-subjects factor was group. Contrary to our expectations, children with MCDD did not significantly differ from those with PDD-NOS on emotion-processing; there were no significant main effects of group for accuracy (p = 0.22) or for RT (p = 0.18), nor any significant interactions between group*emotion-category for either accuracy (p = 0.16) or RT (p = 0.98) data. We therefore found no support for our second hypothesis that children with MCDD would process facial expressions of emotion (particularly anger and fear faces) differently compared to children with PDD-NOS. Reanalyses Including ‘Pure’ MCDD Versus PDD-NOS Children Because approximately 40% of the children with MCDD also fulfilled research criteria for PDD-NOS, it is plausible that MCDD status might have been confounded with PDD-NOS. To ensure that the results were not simply due to the overlap between children with MCDD, and PDD-NOS, we repeated the above analyses, however this time comparing a sub-sample of the MCDD children who fulfilled research criteria for MCDD but not PDD-NOS (‘pure MCDD’; n = 13), to children with PDD-NOS (n = 61). For emotion-processing (i.e. Identification of Facial Expression task), similar results were obtained as with the larger MCDD group; no significant results involving group (‘pure MCDD’ versus PDD-NOS) emerged. Yet, re-analysis of the face versus pattern recognition performances revealed a group difference not previously found. This difference relates to the comparison between complex pattern recognition and face recognition; the significant interaction between task*response type*group previously found for only the accuracy data now emerged for RT data as well (Wilk’s lambda = 0.94, F(1,72) = 4.77, p = 0.03; ηp2 = 0.06). Children with PDD-NOS compared to the ‘pure’ MCDD group were slower in recognizing faces in the target condition, whereas no such group difference was found for complex pattern recognition. The interaction remained significant after adjusting for the effect of MA (p = 0.04; ηp2 = 0.06). All other results were similar when reanalyzed using this more ‘pure’ MCDD group. Discussion Previous research suggests that children with MCDD may form a group of children that is, based on symptom, biological, and psychophysiological profiles, well-distinguishable from those with autism, externalizing, and internalizing disorders. This group, though possibly at risk for poor prognosis in adulthood, including schizophrenia spectrum disorders, is currently subsumed under the larger, heterogeneous category of PDD-NOS. Yet, there is debate as to whether this group should be considered a separate diagnostic category within DSM-V (van der Gaag et al. 1995). To our knowledge, only one other study has directly compared children with PDD-NOS to those with MCDD, examining symptom differences between the two groups of children (de Bruin et al. 2007). The present study contributes to literature on this under-studied and somewhat controversial diagnosis, by examining whether children with MCDD can be differentiated from children with PDD-NOS on two domains of social-cognitive functioning; face recognition and the identification of facial expressions. The ability to recognize individuals, and to process the emotional cues of others quickly and accurately is a crucial component of social functioning and development (for reviews, see Blair 2003; Herba and Phillips 2004). Face Recognition Our first hypothesis postulated that children with PDD-NOS would demonstrate poorer performance in face recognition than children with MCDD, but that the two groups would perform similarly on detecting complex patterns. To test this hypothesis we adopted an approach used by Serra and colleagues (2003) which provided evidence that children with PDD-NOS may use a more attention-demanding strategy of face processing compared to typically developing children who were suggested to process faces more automatically. In that study, children with PDD-NOS showed an only small discrepancy in speed of processing complex patterns and faces, whereas typically developing children showed a greater advantage for face recognition compared to pattern recognition. We therefore predicted that children with MCDD, if indeed distinct from those with PDD-NOS, would demonstrate a more specific advantage for face recognition compared to complex pattern recognition, whereas children with PDD-NOS would show a smaller difference between processing faces and complex patterns. Our results are somewhat consistent with this hypothesis. When comparing easy pattern recognition with the recognition of unfamiliar faces, no group differences emerged; overall, children were faster and more accurate to recognize easy patterns compared to faces. However, when comparing the recognition of complex patterns with the recognition of faces, children with PDD-NOS appeared to process faces more similarly to complex patterns especially in the target condition whereas children with MCDD demonstrated greater accuracy for processing faces compared to complex patterns. This effect remained significant after adjusting for MA. Moreover, when investigating the ‘pure MCDD’ group (children who met criteria for MCDD, but not PDD-NOS), the advantage for face recognition could be shown to also exist for their speed of processing. These results suggest that MCDD-specific characteristics on their own (see Table 1: impaired regulation of affective states, impaired social behavior, and thought disorder) might not be associated with a more attention-demanding strategy of face recognition. In contrast, children with PDD-NOS, consistent with their symptoms of impaired non-verbal behavior, lack of social and emotional reciprocity, and lack of spontaneous affiliation with other people, appeared to process facial information in a way quite similar to how they process complex patterns. De Bruin and colleagues (2007), using data from the same participants as in the present study, reported that children in the MCDD group (36.0%) fulfilled criteria for ADOS-G classifications of autism or autism spectrum to a lesser extent compared to children in the PDD-NOS group (62.2%). Hence, the more time-consuming and therefore attention-demanding strategy of face processing described by Serra and colleagues and seen in our group of children with PDD-NOS could be due to the more ‘autistic’ characteristics of our PDD-NOS group. Face recognition appeared to be less attention-demanding in our MCDD group that did not meet the diagnostic criteria for a PDD-NOS. Our results therefore suggest that MCDD symptoms are associated with disadvantages in especially the speed of recognizing unfamiliar faces only to the extent to which they are accompanied by social problems severe enough to meet the diagnostic criteria for PDD-NOS. Identification of Facial Expressions Given the extensive fears and anxieties inherent to the diagnosis of MCDD compared to autism or PDD-NOS, we expected significant group differences to be evident in emotion-processing. We predicted that these group differences would be particularly marked for identifying facial expressions of fear and anger expressions, since previous research has indicated that aberrant patterns in processing certain emotions are specific to the symptoms of the particular disorder (Phillips et al. 2003a), and that adults and children with high levels of anxiety demonstrate biases toward processing threatening facial expressions (Hadwin et al. 2003; Mogg et al. 2004). Yet, we found no support that children with MCDD differed significantly from those with PDD-NOS in identifying fear or anger expressions, nor for any of the other emotion-categories we investigated. Taking the model of emotion-processing by Phillips and colleagues proposing the following three main components (Phillips et al. 2003b): (1) identification of the emotional significance of a stimulus; (2) production of an affective state; and (3) regulation of an affective state, we must note that only the first component was tested in the current paper. Evidently, the notable differences between children with MCDD and those with PDD-NOS found on a symptom level (de Bruin et al. 2007) do not translate to a more basic level of emotion recognition. Differences between the two groups in the production and regulation of affective states are nevertheless quite likely, since especially children with MCDD appear to have substantial difficulties regulating their affective state. Future work should incorporate tasks that allow for the examination of each of the three of the above-mentioned components of emotion-processing. In order to provoke changes in affective state such a task should take into account real-life situations or experiences that may trigger strong emotional reactions in these children. Such a task might help to extract further information on how the symptoms associated with MCDD may or may not discriminate emotion-processing ability from those with PDD-NOS. Strengths and Limitations To our knowledge this is the first study to directly compare children with MCDD to those with PDD-NOS (selected from a range of child psychiatric disorders) on social-cognitive functioning using well-validated research criteria for MCDD and PDD-NOS. A strength of this study was the selection of children with MCDD and PDD-NOS on the basis of explicit research criteria. Earlier studies on the neuro-cognitive profiles of children resembling MCDD included ill-defined groups representative of broad categories of disorder. Other studies examining symptom profiles and thought disorder using clearly defined groups of MCDD children selected these children from a sample of children with PDD, implying that MCDD can only occur within the broader category of PDD (van der Gaag et al. 1995, 2005). We screened children from a larger sample of outpatients, and applied the research criteria for both MCDD and PDD-NOS independently from one another. Therefore, a diagnosis of MCDD could occur if the child did not meet criteria for PDD, consistent with earlier work suggesting that only approximately half of the children with MCDD also met criteria for PDD-NOS (Towbin et al. 1993). Furthermore, PDD-NOS has in many studies been assessed as a ‘default’ diagnosis of the DSM when children did not quite meet the diagnosis for any of the other PDD subtypes rather than being explicitly defined on its own (see Walker et al. 2004). Explicit research criteria and not a DSM default option were applied in this study to identify children with PDD-NOS. However, we were also faced with a number of limitations. Since MCDD is not currently a DSM-IV diagnosis, all MCDD children had been assigned other clinical diagnoses. These diagnoses, based on DSM-IV and the DISC, included PDD-NOS, anxiety disorders (including separation anxiety and obsessive compulsive disorder), disruptive behavior disorders, in addition to ratings of psychotic thought problems (rated by the Child and Adolescent Functional Assessment Scale (CAFAS) and the CBCL thought problems subscale) (see also de Bruin et al. 2007). Approximately 40% of the MCDD children also met research criteria for PDD-NOS. Thus, although the PDD-NOS children in this study did not meet criteria for MCDD, some of the children in the MCDD group also met the criteria for PDD-NOS. Our results of an advantage for the speed of recognizing neutral faces compared to complex patterns among children in the ‘pure MCDD’ group suggest that the more attention-demanding strategy of face recognition used by children with PDD-NOS may be dependent to a larger extent on their ‘autistic-like qualities’ rather than specific characteristics of MCDD. It is also noteworthy that these results emerged despite the small number of participants in this ‘pure MCDD’ group. Future studies could aim for a purer comparison by using groups of PDD-NOS (without MCDD) and MCDD (without PDD-NOS). A further limitation was the relatively small sample size for the MCDD group. This was mainly due to the low prevalence rate of MCDD. However, another study assessing formal thought disorder in children with MCDD included a similar sample size (van der Gaag et al. 2005) and found higher rates of formal thought disorder in children with MCDD compared to clinical or healthy control groups. Furthermore, de Bruin et al. (2007) directly compared children with MCDD to PDD-NOS, and reported differences in symptom profiles using similar-sized groups while standard deviations in the dependent measures relative to their means (i.e. the variance coefficients) were greater in that study than those of, for example, the reaction times in our study. This suggests that the hypothesized group differences could have been smaller in order to be detected as significant. We are therefore not inclined to interpret the few significant differences in our study as being due to a power problem. We cannot exclude the possibility that children with MCDD and PDD-NOS use different cognitive strategies to achieve the same end level of performance. The use of neuro-imaging technologies and ERP studies would help to rule out this possibility, and may yield greater insight into the mechanisms underlying social cognition in these children. Conclusion Despite the existence of thought disorder in children with MCDD, the symptom level differences between PDD-NOS and MCDD children, and the biological/psychophysiological differences between MCDD children and other comparison groups, we found little evidence that children with MCDD are clearly distinguishable from those with PDD-NOS on the identification of facial expressions. Surprisingly, the high rates of anxieties and fears clinically characteristic of children with MCDD did not translate to any significant effects on our emotion-processing task. Further work is needed to probe whether more subtle emotion-processing differences exist. Such studies should focus on examining children’s processing of emotional stimuli within a context more relevant to ‘real-life’ as well as detailed evaluation of children’s emotion regulation ability. The only significant difference to emerge was that children with MCDD not meeting the criteria for a PDD diagnosis demonstrated fewer errors and a faster processing of unfamiliar neutral faces compared to children with PDD-NOS who processed faces more similarly to how they processed complex patterns. This suggests a disadvantage in face processing being related to the autistic characteristics of the PDD-NOS. Based on these findings, it is recommended that the impact of autistic features (amount and severity) are carefully considered when evaluating a child with MCDD symptoms since such features may yield relevant information about the child’s social cognitive abilities. Ideally, future work should include prospective designs which follow up children with MCDD who do and do not demonstrate autistic characteristics.

Appl_Microbiol_Biotechnol-4-1-2266783

Degradation of 4-fluorophenol by Arthrobacter sp. strain IF1

A Gram-positive bacterial strain capable of aerobic biodegradation of 4-fluorophenol (4-FP) as the sole source of carbon and energy was isolated by selective enrichment from soil samples collected near an industrial site. The organism, designated strain IF1, was identified as a member of the genus Arthrobacter on the basis of 16S ribosomal RNA gene sequence analysis. Arthrobacter strain IF1 was able to mineralize 4-FP up to concentrations of 5 mM in batch culture. Stoichiometric release of fluoride ions was observed, suggesting that there is no formation of halogenated dead-end products during 4-FP metabolism. The degradative pathway of 4-FP was investigated using enzyme assays and identification of intermediates by gas chromatography (GC), GC–mass spectrometry (MS), high-performance liquid chromatography, and liquid chromatography–MS. Cell-free extracts of 4-FP-grown cells contained no activity for catechol 1,2-dioxygenase or catechol 2,3-dioxygenase, which indicates that the pathway does not proceed through a catechol intermediate. Cells grown on 4-FP oxidized 4-FP, hydroquinone, and hydroxyquinol but not 4-fluorocatechol. During 4-FP metabolism, hydroquinone accumulated as a product. Hydroquinone could be converted to hydroxyquinol, which was further transformed into maleylacetic acid and β-ketoadipic acid. These results indicate that the biodegradation of 4-FP starts with a 4-FP monooxygenase reaction that yields benzoquinone, which is reduced to hydroquinone and further metabolized via the β-ketoadipic acid pathway. Introduction During the past decades, widespread application of fluoroaromatic compounds as agrochemicals and pharmaceuticals has lead to an increased occurrence of environmental contaminants containing fluorine (Key et al. 1997). Fluorinated compounds are rare in nature (Harper and O’Hagan 1999). The stability of the carbon–fluorine bond (116 kcal/mol in CH3F, compared to 81 kcal/mol for the carbon-chlorine bond in CH3Cl) makes most fluorine-containing compounds much more resistant to biodegradation than their unsubstituted analogs (Key et al. 1997). Furthermore, the van der Waals radius of fluorine is small (1.47 Å, in between that of a hydrogen and an oxygen). Yet, the electronegativity of fluorine causes strong polarization of C–F bonds, and fluorine substituents may be involved in biological interactions (Howard et al. 1996). Regardless of the recalcitrance of most fluoroaromatics to biodegradation, several bacterial cultures have been described that aerobically degrade fluorobenzoic acids (Oltmanns et al. 1989; Engesser et al. 1980; Harper and Blakley 1971; Schlomann et al. 1990). Research on bacterial fluorophenol degradation has been limited to studies with whole cells, cell extracts, or purified enzymes from Rhodococcus species that were obtained by enrichment with other aromatic compounds as a growth substrate (Boersma et al. 1998, 2001; Bondar et al. 1998, 1999; Finkelstein et al. 2000). The fluorobenzene-degrading organism Rhizobiales F11 could grow on 4-fluorophenol (4-FP), but information on the pathway of 4-FP metabolism is lacking. Cometabolic degradation of difluorophenols and trifluorophenols by several Rhodococcus species is initiated by a phenol hydroxylase that catalyzes ortho-hydroxylation, resulting in the formation of the respective fluorocatechol, which is then cleaved by an intradiol dioxygenase to produce fluoromuconate (Bondar et al. 1998). Conversion of 4-FP by whole cells of the phenol-degrading organism Rhodococcus opacus 1cp resulted in the formation of 4-fluorocatechol, 1,2,3-trihydroxy-5-fluorobenzene, and fluoromuconates (Finkelstein et al. 2000). Yeasts and fungi that are able to cometabolically transform fluorinated phenols have also been described. Whole cells of Exophiala jeanselmei transformed 4-FP into 4-fluorocatechol and 3-fluoromuconate (Boersma et al. 1998). Penicillium frequentans metabolized monofluorophenols in the presence of glucose or phenol. The metabolism of meta-or para-fluorophenols yielded the corresponding catechol and 4-carboxymethylenebut-2-en-4-olide (Hofrichter and Schreibner 1993; Hofrichter et al. 1994). None of these organisms could utilize fluorophenols as a growth substrate. To our knowledge, studies on the metabolism of fluorophenols by a bacterial culture that is capable of using such as compound as a sole source of carbon and energy have not been reported. In the present paper, we describe the isolation and characterization of a bacterial strain growing on 4-FP as the sole source of carbon and energy. Based on the identification of several intermediates, a metabolic route for the degradation of 4-FP by this strain is proposed. Materials and methods Media and growth conditions NB medium contained 8 g of Nutrient Broth (Difco) per liter. Mineral salts medium (MM) contained per liter 5.3 g Na2HPO4 12H2O, 1.4 g KH2PO4, 0.2 g MgSO4 7H2O, 0.5 g (NH4)2SO4, 5 ml trace metals solution (Janssen et al. 1985), and 10 mg of yeast extract (Difco Laboratories). When required, the solid medium was obtained by adding 15 g/l of Difco agar. Strain IF1 was grown at 30°C on a rotary shaker (180 rpm). Cultures were grown in 100-ml flasks filled to 25% of their volume and were closed with Teflon-lined screw caps. E. coli cells were grown in Luria–Bertani medium (LB) at 37°C on a rotary shaker. Enrichment and isolation of 4-FP-degrading cultures A variety of soil samples, collected from different sites in The Netherlands that are contaminated with halogenated aliphatic compounds (such as monochlorobenzene, hexachlorobenzene, and trichloropropane), were used as the initial inoculum for the 4-FP enrichments. The soil samples were used to inoculate flasks containing 30 ml of sterile minimal salts medium and 1 mM of 4-FP, supplied in the liquid phase as the sole carbon and energy source. The cultures were incubated at room temperature on a rotary shaker (150 rpm), and 40% of the suspension was transferred to a new flask containing fresh medium every 15 days. During this time, growth (optical density at 600 nm) and liberation of fluoride were monitored. Samples of the enrichment culture were periodically spread onto minimal salts agar plates containing 1 mM 4-FP and onto NB plates as soon as growth on 4-FP was established. Pure cultures were obtained by repetitive streaking onto solid MM containing 4-FP and tested separately for growth on 1 mM 4-FP liquid medium. Growth and fluoride release were again monitored to verify 4-FP degradation. Strains capable of 4-FP degradation as a sole source of carbon and energy were used for further experiments. Strain IF1 was deposited at Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, under accession number NCCB 100218. Sequencing of the 16S rRNA gene For cloning of the 16S ribosomal ribonucleic acid (rRNA) gene, a single colony of strain IF1 was directly used for polymerase chain reaction (PCR) amplification. The primers 63f (5′-CAGGCCTAACACATGCAAGTC-3′) and 1387r (5′-GGGCGGWGTGTACAAGGC-3′; Marchesi et al. 1998) were used for PCR amplification. The PCR reaction mixture (50 μl) contained Taq PCR buffer, 2.5 mM MgCl2, 20 pmol of each appropriate primer, 200 mM of each deoxyribonucleotide triphosphate, 1 U Taq DNA polymerase, and biomass of strain IF1. The PCR conditions were 94°C for 10 min followed by 1 min at 95°C, 1 min at 55°C, 1.5 min at 72°C, and 5 min at 72°C. The resulting fragments were cloned into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA) and transformed into E. coli TOP10 cells. The transformed cells were plated on LB plates containing 0.5 mg/ml of ampicillin, and the positive colonies were used for plasmid isolation and sequencing. Phylogenetic analysis Alignments of the 16S rRNA gene were made using sequences downloaded from the Ribosomal database project II (RDP II; Cole et al. 2005), after searching for nearest neighbors using the sequence match tool. Further searches were conducted using BLAST and FASTA of the European Molecular Biology Laboratory (EMBL) database for 16S rRNA gene sequences that are closely related to the 16S rRNA gene of strain IF1 but not available from the RDP II. Alignments were subsequently made using the profile alignment option in ClustalX (Thompson et al. 1997), refined using BioEdit ver 7.0.5.2 (Hall 1999), and subsequently parsed through Gblock (Castresana 2000) to remove ambiguously aligned sections and increase the robustness of the data. Phylogenetic trees were determined using the neighbor-joining (NJ) method. Evolutionary distances for the global tree were calculated using the Kimura-2-parameter model with a transition/transversion ratio of 2 (Fig. 1). Further trees were constructed in Phylip version 3.6a3 (J. Felsenstein), using maximum parsimony and maximum likelihood methods. All NJ trees were tested statistically by means of bootstrap analysis. Fig. 1Phylogenetic tree of the 16S rRNA gene sequence of strain IF1. The scale bar represents 0.1 fixed mutation per site. Bootstrap values were derived from 1,000 analyses. The DNA sequences were aligned using ClustalX, and the tree was constructed by the neighbor-joining program from a similarity matrix of pairwise comparisons made by using the Kimura-2-parameter algorithm. Tree a shows the global position of the strain IF1 while tree b shows the Bayesian tree and the more precise relationship of strain IF1 to other members of the genus Arthrobacter. The values in parenthesis in this tree are the posterior probabilities for the nodes that show where IF1 resides Preparation of cell extracts Cells were grown in MM with 4 mM 4-FP, harvested by centrifugation at 5,000 × g for 15 min, washed with ice-cold TD buffer (0.1 M Tris–HCl, pH 7.5, and 0.1 M 1,4-dithiothreitol), and stored at −20°C until further use. The frozen cells were thawed, resuspended in TD buffer, and incubated with lysozyme (50 mg/ml) for 1 h at 30°C. A French press was used to disrupt the cells, and the crude extracts were centrifuged at 10,000 × g for 15 min to separate the soluble from the particulate fraction. The supernatant was used for further experiments. Protein concentrations were determined with the Biorad protein assay kit. Enzyme assays Catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities were measured spectrophotometrically at 260 and 375 nm, respectively. The reaction mixtures contained 0.1 mM catechol, TD buffer, and cell-free extract (0.1 mg of protein) in a final volume of 1 ml. 4-Fluorocatechol 1,2-dioxygenase was measured as catechol 1,2-dioxygenase but with 4-fluorocatechol instead of catechol as the substrate. The 4-FP monooxygenase activity was measured spectrophotometrically by following the consumption of NADH at 340 nm in a reaction mixture containing cell-free extract (0.1 mg protein), 0.1 mM 4-FP, 0.1 mM NADH, and buffer in a total volume of 1 ml. The observed rates were corrected for substrate-independent NADH oxidation. Hydroxymuconic semialdehyde dehydrogenase was measured at 375 nm. Reaction mixtures contained (in a final volume of 1 ml) about 0.05 mM freshly prepared hydroxymuconic semialdehyde, TD buffer, 0.1 mM NAD, and cell-free extract (0.1 mg of protein). Hydroxymuconic semialdehyde was obtained by incubation of catechol with the cell-free extract of Pseudomonas putida mt-2 as described previously (Mars et al. 1998). Hydroquinone dioxygenase was assayed spectrophotometrically by monitoring the change in absorbance between 230 and 330 nm in a reaction mixture of 1 ml final volume containing 0.1 mM hydroquinone, TD buffer, and cell-free extract (0.1 mg of protein). Hydroquinone hydroxylase and hydroxyquinol dioxygenase were assayed by using a fiber optic oxygen sensor. Reaction mixtures contained 1 mM of substrate, MM, and cell suspension (0.5 mg/ml protein) in a final volume of 1.25 ml. Oxygen uptake measurements Strain IF1 was grown with glucose or 4-FP as the sole carbon source and harvested by centrifugation at 6,000 × g for 15 min at 4°C. Cells were resuspended in MM, and O2 consumption was measured with a fiber optic oxygen sensor (MOPS-1, ProSense BV, Hannover, Germany). All reactions were performed in a stirred vessel at room temperature. The reaction mixtures contained 1 mM of substrate (4-FP, hydroquinone, 4-fluorocatechol, hydroxyquinol, or catechol), MM, and cell suspension (0.5 mg/ml protein) in a final volume of 1.25 ml. Analytical methods For capillary gas chromatography (GC), 4 ml samples were extracted with 1 ml of diethyl ether. A model 6890 gas chromatograph (Hewlett-Packard) equipped with a flame ionization detector and a HP-5 column (Agilent 19091J-413, 30 m × 0.25 mm × 0.25 μm) were used for the analysis. GC-mass spectrometry (MS) analysis was carried out with a model 5973 mass selective detector (Hewlett-Packard) coupled to a HP 6890 series injector and a HP1 column (Agilent 19091Z-433; 30 m × 0.25 mm × 0.25 μm). High-performance liquid chromatography (HPLC) was carried out using a Chrompack C18 column (10 cm × 5 mm) connected to a Jasco UV-1575 detector, which monitored absorbance at 214 and 254 nm, and operated with Jasco PU-980 pumps and a Jasco AS-1555 sampler. The mobile phase was water/acetonitrile (70:30), 5 mM potassium phosphate (pH = 3), and 340 mg/l sodium dodecyl sulfate, and the flow rate was 1 ml/min. Liquid chromatography (LC)-MS was carried out with a ZMD Micromass spectrometer, equipped with a XTerra MS, Symmetry Shield C8 column (4.6 × 150 mm), a Waters 996 photodiode array detector, and a Waters 2690 separations module. Samples of 20 μl were analyzed, and compounds were isocratically eluted at a flow rate of 1 ml/min with a solution of water/acetonitrile (80:20) and 10 mM formic acid. Concentrations of free fluoride in the culture supernatants were measured with a fluoride electrode (model 96-09, Thermo Russell, Scotland). Fresh sodium fluoride standards were prepared for calibration curves. Chemicals 4-FP (>98%) was obtained from Sigma-Aldrich (Steinheim, Germany). All chemicals were of the highest purity grade available (Sigma-Aldrich; Acros Organics, Geel, Belgium). The compound 4-fluorocatechol was kindly provided by Dr. Erik de Vries. Purity of 4-FP, 4-fluorocatechol, and hydroquinone was checked by HPLC. Nucleotide sequence accession numbers The 16S rRNA sequence of strain IF1 was deposited at GenBank with the accession no. DQ425093. Results Isolation of a 4-FP-degrading bacterium To obtain a bacterial culture that is able to use 4-FP as carbon and energy source for growth, enrichments were performed and followed over time. Two months of selective enrichment by repeated transfer of samples from a culture that displayed growth on 4-FP to fresh 4-FP containing media resulted in a microbial consortium that was capable of growth on 4-FP as a sole source of carbon and energy. Samples of the consortium were repeatedly plated onto NB agar plates and MM agar plates containing 4-FP. This procedure resulted in the isolation of three pure strains named IF1, IF2, and IF3. The strains were restreaked on MM supplemented with 4-FP and inoculated in liquid cultures with 1 mM 4-FP. Fluoride liberation was observed for all three strains but not in control incubations to which no bacterial inoculum was added. All the three strains showed growth, as monitored at 600 nm, and thus were able to use 4-FP as a sole carbon and energy source. After 5 days of incubation in liquid culture supplied with 1 mM 4-FP, strain IF1 reached 100% fluoride release and an optical density (OD) of 0.119 at 600 nm. Strain IF2 released 76% of the fluorine and reached an OD600 of 0.09, while strain IF3 reached an OD of 0.076 and released 54% of the theoretical amount of fluoride ions. When a mixed culture of the three strains in liquid media was used, an OD600 of 0.217 and 100% fluorine release were measured after 4 days. Apparently, the mixed culture contained, even after prolonged adaptation, organisms with varying efficiencies of 4-FP utilization. Because strain IF1 showed the highest degradation rates combined with stoichiometric release of fluoride, this organism was chosen for further studies. Microbiological characterization Strain IF1 is a Gram-positive motile bacterium with a rhodococcus lifecycle. The optimal temperature for growth is 30°C. The 16S rRNA gene sequence was determined. Initial searches against the RDP II and EMBL rRNA databases resulted in very close associations with members of the genus Arthrobacter, the closest of which were the quinaldine-degrading strain Arthrobacter sp. KA1-1 (Overhage et al. 2005) and the 4-nitroguaiacol-degrading actinobacterium Arthrobacter nitroguajacolicus sp. nov (Kotouckova et al. 2004). The 16S rRNA gene sequences of these organisms were greater than 99% identical to IF1’s. The phylogenetic analysis places the isolates in the phylum Actinobacteria and the genus Arthrobacter (Fig. 1). The subtree that were obtained shows the detailed relationship of isolate IF1 to other members of the genus Arthrobacter. The topologies of all the trees that were obtained during statistical analysis were in agreement and clearly placed this isolate in the genus Arthrobacter (data not shown). Furthermore, the change in form during the growth cycle between rod and coccus is typical of the lifecycle of the Arthrobacter genus. Catabolic activities of Arthrobacter sp. strain IF1 To study the degradation potential of strain IF1, a range of organic compounds were tested as growth substrates. Cells were inoculated into MM, and different organic substrates were added at a concentration of 1 mM. After 72-h incubation growth, substrate disappearance and halide release were measured. Growth and substrate removal were found when catechol, hydroquinone, hydroxyquinol, benzoate, phenol, 4-fluorocinnamic acid, and 4-nitrophenol were used as substrates. The organism did not grow on 2-fluorophenol, 3-fluorophenol, 4-chlorophenol, 4-bromophenol, 4-iodophenol, fluoroacetate, trifluoroacetate, fluoroacetamide, trifluoroethanol, or on 2-bromoethanol. The fact that strain IF1 is capable of growth on catechol, hydroquinone, and hydroxyquinol but not on 4-fluorocatechol indicates that 4-fluorocatechol is not the most likely intermediate in the 4-FP pathway, although toxic effects could also play a role. To test the range of 4-FP concentrations tolerated by strain IF1, experiments were conducted in sealed flasks with 4-FP at concentrations of 1 to 7 mM as a sole carbon and energy source. Control assays without 4-FP showed no growth or release of fluoride, and sterile controls showed no abiotic loss of 4-FP. Between 1 and 4 mM 4-FP, the substrate was completely consumed, stoichiometric release of fluoride was observed, and biomass increased linearly with the amount of 4-FP added (Fig. 2a). This indicates that the degradation of 4-FP by strain IF1 does not give large amounts of fluorinated dead-end products. Concentrations of 4-FP above 4 mM caused a toxic effect on the growth of IF1 (Fig. 2b). The use of 5 mM 4-FP promoted growth, but a longer lag time was observed. At 7 mM 4-FP, no growth occurred even after 15 days of incubation. Fig. 2Fluoride release and biomass formation by strain IF1 with different concentrations of 4-FP. a Stoichiometric fluoride release. Symbols: squares, optical density at 600 nm; circles, fluoride concentration. b Growth in the presence of different levels of 4-FP. The initial concentrations used were: triangles, 1 mM 4-FP; circles, 4 mM 4-FP; diamonds, 5 mM 4-FP; squares, 7 mM 4-FP Growth on 4-FP and formation of metabolites A batch culture of strain IF1 supplied with 1 mM 4-FP as the only source of carbon and energy was monitored in time. Growth was accompanied by an increase in biomass, decrease in 4-FP, and formation of fluoride (Fig. 3). After 120 h, there was complete conversion of 1 mM 4-FP, and 1 mM fluoride was formed, indicating that there was no transient accumulation of fluorinated intermediates over the whole growth period. When cells grown on 4-FP were incubated in the presence of the iron chelator 2,2′-dipyridyl, no 4-FP degradation occurred, and no fluoride was released in the medium. This indicates that initial or further enzymes involved in 4-FP metabolism require ferrous ions for activity. Fig. 3Biodegradation of 4-FP and fluoride liberation in a batch culture of strain IF1. The carbon source used is 1 mM 4-FP. Symbols: triangles, 4-FP concentration; diamonds, optical density; squares, fluoride concentration To isolate intermediates of the degradation of 4-FP, samples from a batch culture containing 4 mM 4-FP were taken at appropriate intervals and analyzed by GC, HPLC, and LC-MS. Metabolites that were detected were numbered in order of time of appearance in the culture. GC analysis indicated that at least four metabolites were formed and degraded over time (Table 1). Metabolite I, the earliest product that was observed, had the same retention time as an authentic hydroquinone standard and metabolite II had the same retention time as a standard of hydroxyquinol. GC-MS analysis showed that the mass spectrum of metabolite I was indeed similar to that of the hydroquinone standard, with a molecular ion peak at 110 and at 55 m/z. Metabolite II gave a molecular ion peak at 126 and 63 m/z, which is typical for hydroxyquinol, and the spectrum coincided with that of a standard. Metabolites V and VI were detected by GC but did not ionize in GC-MS under the conditions tested and were not identified. Table 1Retention times in GC and HPLC analyses of metabolic intermediates formed by cells of strain IF1 growing on 4-FP and of some authentic standardsCompoundGC retention time (min)HPLC retention time (min)am/z of observed fragment ions detected by GC-MSMetabolitesI20.71.3110, 81, 54II24.31.1126, 80, 52III–c0.75–IV–2.6–V16.5––VI19.4––VII–3.1b159, 80dAuthentic standards4-FP15.73112, 83, 574-Fluorocatechol14.42128, 82, 51Hydroquinone20.71.3110, 81, 54Hydroxyquinol24.31.1126, 80, 52Catechol19.1––aElution followed at 254 nmbAnalyzed after separation on a Symmetry Shield C8 column (4.6 mm × 150 mm) as described in “Materials and methods” for LC-MS analysisc– Not determineddLC-MS analysis When the supernatant of IF1 cells growing on 4 mM 4-FP were subjected to HPLC analysis, four peaks were detected (Table 1). Metabolite I had the same retention time as the hydroquinone standard, and metabolite II coeluted with an authentic standard of hydroxyquinol. Hydroquinone was detected in culture supernatants by HPLC between 7.5 and 51 h (Fig. 4). LC-MS analysis of the extracted samples of the supernatant revealed the presence of a compound (VII) of which the negative ion spectrum shows a molecular ion peak at 159 m/z and a peak at 80 m/z, which are expected for the negative ionization of 3-oxoadipate. Fig. 4Accumulation of hydroquinone during growth of strain IF1 on 4 mM 4-FP. The optical density (diamonds), 4-FP concentration (squares), and hydroquinone concentration (x marks) are indicated. Hydroquinone was identified by GC-MS and quantified by GC-FID The above results suggest that 4-FP is initially converted to hydroquinone. The most likely enzyme involved in such a conversion is a 4-FP monooxygenase. Enzyme activities To test inducibility of enzymes involved in 4-FP degradation, cells of Arthrobacter sp. strain IF1 were grown on glucose or 4-FP, washed, and tested for oxygen consumption in the presence of different substrates. Washed suspensions of cells grown on 4-FP rapidly oxidized 4-FP, hydroquinone, and 1,2,4-benzenetriol (hydroxyquinol) without a lag. Catechol and 4-fluorocatechol did not stimulate oxygen uptake (Table 2). This result indicates that 4-fluorocatechol and catechol are not likely intermediates and that hydroquinone and hydroxyquinol are. Glucose-grown cells did not oxidize any of the aromatic substrates tested with the exception of hydroxyquinol. This observation implies that hydroxyquinol may be converted by a constitutive oxygenase, whereas most other enzymes seem inducible. Cells grown with hydroxyquinol showed complete conversion of benzenetriol in the presence or absence of 2,2′-dipyridyl, indicating that the putative hydroxyquinol oxygenase does not require ferrous ions to be active and that oxidation beyond this compound is not inhibited by the chelator. The formation of hydroquinone, which can be readily oxidized by 4-FP-grown cells, is in agreement with the involvement in 4-FP degradation of a 4-FP monooxygenase that is induced by 4-FP. Table 2Substrates oxidized by 4-FP and glucose grown cells of Arthrobacter sp. strain IF1aAssay substrateO2 uptake (μmol/min/mg of protein) by whole cells after growth onFluorophenolGlucose4-Fluorophenol0.356<0.001Hydroquinone0.313<0.001Hydroxyquinol0.1950.1564-Fluorocatechol<0.001<0.001Catechol<0.001<0.001a Oxygen consumption was measured with an oxygen sensor as described in Materials and Methods. All substrate were used at a concentration of 1 mM To study in more detail the degradation pathway of 4-FP by strain IF1, cell-free extracts of IF1 grown on 4-FP were investigated for the presence of several enzyme activities (Table 3). When catechol 1,2- and 2,3-dioxygenases were tested for, no activity was found. Furthermore, no activity was detectable for 4-fluorocatechol dioxygenase. Consequently, the pathway does not proceed through catechol or a substituted catechol. When cell extracts were assayed for 4-FP monooxygenase, activity could be found in the presence of NADH but not in the presence of NADPH as the reducing cosubstrate. This observation indicates that the cells contain an NADH-dependent 4-FP monooxygenase activity. HPLC assays of incubations of cell extracts with 4-FP showed that substrate conversion was complete. The formation of the putative 4-FP monooxygenase was induced by 4-FP because no activity was found in extracts of cells grown in the presence of glucose. Table 3Specific activities of enzymes in crude extracts of strain IF1 pregrown in 4-FP or glucoseAssay substrateEnzyme testedSpecific activity (U/mg of protein) after growth on4-FluorophenolGlucoseCatechol1,2-Dioxygenase<0.01<0.012,3-Dioxygenase<0.01<0.014-FluorocatecholDioxygenase<0.01<0.014-FluorophenolMonooxygenase0.12<0.01HydroquinoneDioxygenase<0.01<0.01Monooxygenase0.313<0.01Hydroxymuconic semialdehydeDehydrogenase<0.01<0.01HydroxyquinolDioxygenase0.1950.156 When hydroquinone was used as an assay substrate, its typical spectrophotometric peak at 288 nm was not replaced by a peak absorbing at 290 to 320 nm, which would have pointed to formation of hydroxymuconic semialdehyde. Thus, no hydroquinone dioxygenase was induced, or its product was rapidly further converted. The latter was ruled out by the observation that added hydroxymuconic semialdehyde was not converted by cell extracts of strain IF1, indicating that no hydroxymuconic semialdehyde dehydrogenase was induced during growth in the presence of 4-FP. These results make it unlikely that hydroxymuconic semialdehyde is an intermediate in the 4-FP pathway by strain IF1. When cell extracts were assayed for hydroxyquinol degradation, complete conversion of the substrate was observed with HPLC. When the reaction was monitored spectrophotometrically, the peak at 287 nm, typical of hydroxyquinol, was substituted by a peak at 245 nm, typical of maleylacetate. This indicates the involvement of a hydroxyquinol oxygenase that converts hydroxyquinol into maleylacetate, which is further converted into 3-oxoadipate. Discussion This study reports the isolation and characterization of Arthrobacter strain IF1, an organism capable of growth with 4-FP as a sole source of carbon and energy. The biodegradation of 4-FP by strain IF1 was analyzed by GC, GC-MS, HPLC, LC-MS, oxygen uptake experiments, and measurements of enzymatic activities. A metabolic pathway is proposed on the basis of the results of this study (steps 1 and 2, ortho-cleavage) and by analogy with other systems (Fig. 5). We suggest that the degradation of 4-FP in strain IF1 starts with the conversion by a monooxygenase to benzoquinone, which is immediately reduced to hydroquinone. Hydroquinone then undergoes a further hydroxylation to form hydroxyquinol. This benzenetriol is the ring fission substrate, which is transformed by ortho-cleavage and yields maleylacetate, which is further converted to 3-oxoadipate. Fig. 5Proposed pathway for 4-fluorophenol degradation by Arthrobacter sp. strain IF1. Hydroquinone and hydroquinole were identified as intermediates by GC-MS Up to now, two main metabolic routes for the aerobic degradation of halogenated phenols have been described in the literature. In bacteria that degrade mono- and dichlorophenols, a degradation pathway is usually observed in which the substituted phenol is hydroxylated to the corresponding catechol, which is followed by ortho-cleavage of the aromatic ring (Haggblom 1992; Hollender et al. 1997; Wieser et al. 1994). On the other hand, biodegradation pathways in which the substituted phenol is converted via hydroquinone to maleylacetate have been found, mainly in organisms that grow on polyhalophenols or 4-nitrophenol (Kiyohara et al. 1992; Xun et al. 1992; Kadiyala and Spain 1998; Nordin et al. 2005; Perry and Zylstra 2007). Most studies have focused on the organisms and pathways that involve degradation via catechols. A clear indication for the first step in the 4-FP catabolic pathway of strain IF1 was obtained by mass spectroscopic analysis of culture supernatants, which indicated the formation of hydroquinone as an early nonfluorinated intermediate. This result, in combination with the enzyme assays and oxygen uptake experiments, suggests the involvement of an inducible 4-FP monooxygenase that is NADH dependent, although the first expected product of this reaction, i.e., benzoquinone, was not detected. Monooxygenation of an aromatic substrate carrying an electron-withdrawing halogen group or a nitro-substituent can result in simultaneous hydroxylation and dehalogenation or nitrite removal. This has, for example, been described for the conversion of tetrafluoro-p-hydroxybenzoate by para-hydroxybenzoate hydroxylase (Husain et al. 1980) and the oxidation of 2,4-6-trifluorophenol by a monooxygenase from Ralstonia eutropha JMP134 (Xun and Webster 2004). The quinone that is produced is chemically reduced by NADH to a hydroxyquinol, leading to a net stoichiometry of two NADH oxidized per halide or nitrite that is released (Fig. 5). As in our case, no transient accumulation of a benzoquinone is usually observed, indicating that benzoquinone reduction is rapid. Other examples of halophenol degradation with formation of hydroquinone derivatives are the degradation of trichlorophenol to 2,6-dichlorohydroquinone by a strain of Pseudomonas pickettii (Kiyohara et al. 1992), the degradation of pentachlorophenol by a Sphingobium sp. through hydroxylation by a flavoprotein monooxygenase (Xun and Orser 1991), and the degradation of 4-chlorophenol by an Arthrobacter sp. (Bae et al. 1996). Our results further indicate that hydroquinone is not the ring fission substrate because no enzymatic activities for hydroquinone dioxygenase or hydroxymuconic semialdehyde dehydrogenase were found. Instead, upon conversion of hydroquinone by strain IF1, we observed the formation of a transient metabolite that was identified as hydroxyquinol, indicating that a second hydroxylation takes place before ring fission. The production of hydroxyquinol could be due to the action of a separate hydroquinone monooxygenase, or it could be caused by a second hydroxylation step by the 4-FP monooxygenase. Conversion of hydroquinone to hydroxyquinol before ring fission was also suggested for strains Arthrobacter that degrade 4-nitrophenol (Perry and Zylstra 2007) and 4-chlorophenol (Nordin et al. 2005). Ring fission of hydroxyquinol would produce maleylacetate, which we did not detect as an intermediate in 4-FP degradation, but a transient metabolite was found with mass properties identical to those of 3-oxoadipate, which is expected to be the next intermediate in hydroxyquinol degradation. Most cometabolic transformations of fluorophenols described in the literature involve the initial action of a phenol hydroxylase that results in the formation of fluorocatechols, which are subsequently transformed into fluoromuconates through a catechol 1,2-dioxygenase. The conversion usually proceeds with lactonization and elimination of fluoride (Boersma et al. 2001; Bondar et al. 1999; Finkelstein et al. 2000; Boersma et al. 1998; Bondar et al. 1998). Our results suggest that Arthrobacter strain IF1, which grows on 4-FP, possesses a new pathway for the degradation of 4-FP that involves immediate defluorination by a monooxygenase. Strain IF1 avoids the accumulation of possible toxic metabolites such as 3- or 4-fluorocatechol by this initial dehalogenation.

Neurosurg_Rev-3-1-2077911

Foramen magnum meningiomas: detailed surgical approaches and technical aspects at Lariboisière Hospital and review of the literature

Foramen magnum meningiomas are challenging tumors, requiring special considerations because of the vicinity of the medulla oblongata, the lower cranial nerves, and the vertebral artery. After detailing the relevant anatomy of the foramen magnum area, we will explain our classification system based on the compartment of development, the dural insertion, and the relation to the vertebral artery. The compartment of development is most of the time intradural and less frequently extradural or both intraextradural. Intradurally, foramen magnum meningiomas are classified posterior, lateral, and anterior if their insertion is, respectively, posterior to the dentate ligament, anterior to the dentate ligament, and anterior to the dentate ligament with extension over the midline. This classification system helps to define the best surgical approach and the lateral extent of drilling needed and anticipate the relation with the lower cranial nerves. In our department, three basic surgical approaches were used: the posterior midline, the postero-lateral, and the antero-lateral approaches. We will explain in detail our surgical technique. Finally, a review of the literature is provided to allow comparison with the treatment options advocated by other skull base surgeons. Introduction Meningiomas are common neoplasms representing 14.3 to 19% of all intracranial tumors [63]. Among all the meningiomas, only 1.8 to 3.2% arises at the foramen magnum (FM) level [3]. Nevertheless, meningiomas are the most commonly observed FM tumors, representing 70% of all benign tumors [13, 15–19, 55, 64]. Most of the time, these are strictly intradural. Ten percent have an extradural extension: Most are intra- and extradural, and a few may be entirely extradural [9, 13, 15–19, 24, 25, 56, 60, 61]. The lesion is often large when discovered because of their slow-growing rate, their indolent development, the difficulty of the diagnosis leading to a long interval since the first symptom, and the wide subarachnoid space at this level [7]. The prerequisite for treating FM meningiomas (FMMs) is the perfect knowledge of the surgical anatomy. We will then first detail a comprehensive review of the relevant anatomy of the FM area with special emphasis on the vertebral artery (VA) V3 and V4 segments. Second, we will describe our classification system. This classification system helps for determining the best surgical approach and for anticipating the position of lower cranial nerves. The technical aspects of our surgical approaches will then be detailed extensively. Finally, we provide a summary of the relevant literature, detailing surgical results and surgical approaches advocated by other skull base surgeons and discussing the extent of bone resection and the need for fusion. Surgical anatomy Limits of the FM A meningioma is considered to be located into the FM region if its insertion zone is mainly situated into the FM area. According to the FM limits that we have previously used, the FM area is defined by these landmarks [13, 18, 19] (Fig. 1): Anterior border: lower third of the clivus and upper edge of the body of C2Lateral borders: jugular tubercles and upper aspect of C2 laminasPosterior border: anterior edge of the squamous occipital bone and C2 spinous processFig. 1Illustration of the foramen magnum anatomy through a postero-lateral approach. The skin incision (dotted line) extends on the midline just upper to the occipital protuberance and curves laterally toward the pathological side. The right vertebral artery has been elevated from the C1 posterior arch. The C1 posterior arch has been resected on the pathological side, and a suboccipital craniectomy has been performed. The dura matter has been opened. 1 CN IX–X–XI, 2 PICA, 3 CN XII, 4 vertebral artery V4 segment, 5 C1, 6 dentate ligament, 7 vertebral artery V3 segment The VA V3 and V4 segments The VA V3 and V4 segments are important vascular structures whose anatomy must be perfectly known when approaching FMMs. In fact, after piercing the lateral aspect of the occipito-atlantal dura mater, the VA V3 segment becomes the V4 segment and then joins the contralateral one to form the basilar artery [8]. The VA V3 segment anatomy and techniques of approach, exposure, and transposition have been extensively described [8]. In summary, the VA V3 segment, also called the “suboccipital segment,” extends from the C2 transverse process to the FM dura mater. Its course is divided in three portions: a vertical portion, between the transverse processes of C2 and C1, a horizontal portion in the groove of the posterior arch of the atlas, and an oblique portion on leaving this groove up to the dura mater [8]. Along its course, this VA V3 segment can be at the origin of small collateral branches: in the middle of the posterolateral aspect of the C1–C2 portion, corresponding to the C2 radicular artery, a small muscular branch coursing posteriorly at the upper exit of the C1 transverse foramen, the posterior meningeal artery, and the posterior spinal artery, just before its dural penetration. This last branch enters the dural foramen where the C1 nerve root exits the spinal canal [50] and can also be bound with the VA by fibrous dural bands. The posterior spinal artery may also arise from the initial intradural part of the VA V4 segment or from the posteroinferior cerebellar artery (PICA) [11, 33, 43, 50]. Then, this branch runs medially behind the most rostral attachment of the dentate ligament [50]. On reaching the lower medulla, it divides into ascending and descending branches, providing, respectively, the vascular supply to a part of the medulla oblongata and the spinal cord [50]. The VA V3 segment is surrounded by a periosteal sheath that invaginates into the dura when piercing the FM lateral dura, thus creating a double furrow for 3–4 mm. In fact, the periosteal sheath is in continuity with the outer layer of the dura. At this level, the VA is attached to the periosteal sheath, and the adventitia is adherent to the double furrow forming a sort of distal fibrous ring [8]. In such a way, despite being the most mobile segment, the VA V3 segment is fixed at its extremities. Anatomical relations of the VA are modified by head movements of rotation, as well as during surgical positioning. In neutral position, the vertical and horizontal portions of the V3 segment are perpendicular. On the contrary, after head rotation, as required during an anterolateral approach, both segments are stretched and run parallel, only separated by the posterior arch of the atlas, because the C1 transverse process is pushed anteriorly by this movement, away from the C2 transverse process [8]. Abnormalities of the VA V3 segment are important to know and to identify preoperatively: In 40% of the subjects, the VAs size is different: One side is dominant, and the other is hypoplastic or atretic [8].The VA V3 segment can end at the PICA or at the occipital artery [12]. In 20% of the cases, the PICA originates extracranially, arising from the VA above C1, between C1 and C2, or even in the V2 segment [29, 36].The VA V3 segment can have an intradural course that corresponds to a VA duplication: One atretic portion follows the normal course, but the main portion pierces the dura between C1 and C2 [23, 26, 30, 40, 53].A proatlantal artery consists of a persistent congenital anastomosis between the carotid artery and the VA; it is often associated with an atretic proximal VA and an extracranial origin of the PICA [12, 36, 48].Finally, the groove of the arch of the atlas can be turned into a tunnel if the occipitoatlantal membrane is calcified or ossified, raising some difficulties to expose the horizontal portion at this level [23, 47]. The VA V4 segment ascends from the dura up to the anterior aspect of the pontomedullary sulcus where it joins the controlateral one to form the basilar artery. Initially, the VA V4 segment faces posteriorly and medially the occipital condyle, the hypoglossal canal, and the jugular tubercle. Later, the VA V4 segment lies on the clivus and runs in front of the hypoglossal and the lower cranial nerves rootlets. At the FM level, the VA V4 segment is at the origin of several branches: the PICA, the anterior spinal artery, the anterior and posterior meningeal arteries. The PICA is the main VA branch and originates at, above, or below the FM level [56]. The anterior spinal artery starts near the vertebrobasilar junction. Most of the time, the anterior spinal arteries of both sides joins together above the FM level near the lower end of the olives, before descending through the FM and running on the anterior midline [56]. In some cases, one artery is dominant, or no fusion exists between both arteries, only one of these arteries forming the anterior spinal artery. The vascular supply of the FM dura originates from the anterior and posterior VA meningeal branches and the meningeal branches of the ascending pharyngeal and occipital arteries [11, 42, 50]. Infrequently, meningeal branches are coming from the PICA, the posterior spinal artery, and the VA V4 segment. The anterior meningeal branch arises from the VA at the level of the third interspace. The posterior meningeal artery originates from the VA posterosuperior aspect when turning around the lateral mass of the atlas, above the posterior arch of the atlas, just before penetrating the dura, or just at the beginning of the V4 segment [8, 11, 50]. The ascending pharyngeal artery, a branch of the external carotid artery, gives meningeal branches passing into the hypoglossal canal and the jugular foramen. The meningeal branch of the occipital artery is inconstant and runs through the mastoid emissary foramen [50]. Lower cranial nerves The glossopharyngeal (CN IX), vagus (CN X), and spinal accessory (CN XI) nerves arise from rootlets of the postolivary sulcus and join the jugular foramen, passing ventral to the choroid plexus protruding from the foramen of Luschka and dorsal to the VA [49]. The accessory nerve is composed of rootlets originating from the spinal cord and the medulla. Spinal rootlets join together to form the main trunk, which ascends through the FM running behind the dentate ligament and unites with the upper medullary rootlets. Anastomoses with the dorsal roots of the upper cervical nerves are frequent, that with the C1 nerve root being the most common and largest one [49]. The upper medullary rootlets penetrate directly the jugular foramen [49]. The hypoglossal nerve (CN XII) arises from rootlets of the preolivary sulcus. This nerve runs anterolateral through the subarachnoid space and pass behind the VA to reach the hypoglossal canal. Rarely, the VA separates the CN XII rootlets [49]. The dentate ligament The dentate ligament is a white fibrous sheet that extends from the pia mater, medially, to the dura mater, laterally. It forms arches leaving passage to the VA for the first one and the second cervical nerve root for the second one. Our classification system of foramen magnum meningiomas Meningiomas are considered to be located in the FM area if their base of insertion is mainly located within the FM limits. This definition excludes tumors invading secondarily the FM region (Figure 2). Fig. 2Classification of foramen magnum meningiomas. Foramen magnum meningiomas are classified according to their compartment of development, their dural insertion, and their relation to the vertebral artery. The relation to the vertebral artery permits to anticipate the displacement of the lower cranial nerves. Tumors growing below the vertebral artery push the lower cranial nerves at the superior aspect of the lesion. On the other hand, tumors developed above or on both sides of the vertebral artery displace the lower cranial nerves in all directions and their position can then not be anticipated. Three basic surgical approaches are used. The extent of bone removal is delimited by the dotted lines The definitive objective of a classification system is to define preoperatively the surgical strategy based on preoperative imaging characteristics of the lesion. The surgical strategy in cases of FMMs is the surgical approach but also the anticipation of modified vital structure position. In our classification system, FMMs can be classified according to their compartment of development, their dural insertion, and to their relation to the VA [18]. According to the compartment of development, FMMs can be subdivided in: IntraduralExtraduralIntra- and extradural Intradural meningiomas are the most commonly observed. Extradural meningiomas like at any other locations are very invasive, into the bone, the nerves and vessels sheaths, and soft tissues. The VA sheath and even its adventitia can also be infiltrated. This raises some difficulties and explains the higher incidence of incomplete removal as compared to intradural meningiomas [31, 32, 39, 56, 60, 62]. According to the insertion on the dura, FMMs can be defined in the antero-posterior plane as: Anterior, if insertion is on both sides of the anterior midlineLateral, if insertion is between the midline and the dentate ligamentPosterior, if insertion is posterior to the dentate ligament Anterior meningiomas push the spinal cord posteriorly. Therefore, the surgical opening between the neuraxis and the FM lateral wall is narrow, and the drilling must extend to the medial part of the FM lateral wall to improve the access. In almost every case, no drilling of the lateral mass of the atlas and occipital condyle is necessary. Exceptionally, anterior meningiomas of small size without anterior compartment enlargement need more bone resection but never includes more than one fifth of these elements. On the other hand, lateral meningiomas displace the neuraxis posterolaterally and widely open the surgical access; therefore, the drilling has never to be extended into the lateral mass of the atlas or the occipital condyle. Finally, surgical strategies vary according to the relation to the VA, FMM having the possibility to develop: Above the VABelow the VAOn both sides of the VA Meningiomas are more often located below the VA. In this case, the lower cranial nerves are always pushed cranially and posteriorly. There is no need to look for them. They will come into view on reaching the superior tumoral part. On the other hand, if the lesion grows above the VA, the position of the lower cranial nerves cannot be anticipated; the nerves may be displaced separately in any direction. After partial debulking of the tumor, one has to look for them so as to identify and protect them during the tumor resection. In case of tumoral development on both sides of the VA, a similar problem may exist with the position of the lower cranial nerves. Moreover, the dura around the VA penetration may be infiltrated by the tumor. As previously mentioned, the dura is normally adherent to the adventitia, and complete resection of the tumor at this level is hazardous. In this case, which is rarely observed, it may be safer to leave a cuff of infiltrated dura around the VA and to coagulate this zone. Surgical aspects Preoperative and perioperative considerations Standard preoperative workup includes magnetic resonance imaging (MRI), computed tomography (CT) scan, and sometimes angiography. On MRI, gadolinium-enhanced sequences help to precisely delimit the dural attachment zone, the tumor, and its relation to neural and vascular structures. On T2-weighted images, the presence of an arachnoid plane between the tumor and the neuraxis is sometimes visible. Bone windows CT scan is helpful in case of extradural extension to investigate bone erosion and to schedule preoperatively the need for fusion. Conventional angiography is generally useless. There are only two indications for preoperative angiography: If a highly vascularized tumor is suspected and embolization is contemplatedTo perform a balloon occlusion test in case of VA encasement (extradural or recurrent meningioma and meningioma inserted around the VA). In our experience, it has never been necessary to occlude the VA. Intraoperative neurophysiological monitorings have been used by several surgeons [3, 7] and includes somatosensory-evoked potentials, brainstem auditory-evoked potentials, and electromyographic monitoring of lower cranial nerves, by recordings through an endotracheal tube (CN X) and with a needle in the sterno-mastoid (SM) muscle (CN XI) and the tongue (CN XII). Surgical approaches and techniques at Lariboisière Hospital The midline posterior approach The midline posterior approach is indicated for posterior meningiomas, whatever their intra- and extradural extension, if they remain posterior to the plane of the dentate ligament and medial to the VA [15–17]. In such cases, the neuraxis is pushed anteriorly. The patient may be in the sitting, ventral, or lateral position. To decrease venous bleedings, the sitting position is preferred at Lariboisière Hospital as far as there is no contraindication such as a patent foramen ovale; air embolism is prevented by hypervolemia and G-suit. The skin is incised on the midline from the occipital protuberance down to the upper cervical region. The midline avascular plane is opened between the posterior muscles, up to the occipital protuberance and down to the spinous process of C2. Bone opening is performed using a drill and Kerrison rongeurs and is always limited to the lower part of the occipital bone and the posterior arch of the atlas. The dura is then incised in a T- or Y-shaped fashion and retracted with stitches. Postero-lateral approach The postero-lateral approach is preferred for any intradural process located laterally and/or anteriorly to the neuraxis and for extradural lesions developed on the posterior part of the lateral FM wall [15–17]. For tumors extending far beyond the anterior midline, the postero-lateral approach has the advantage to allow a bilateral approach in the same stage (Fig. 3, 4, and 5). Fig. 3a–c Preoperative MRI. A large lateral foramen magnum meningioma displaces the neuraxis. d, e Postoperative CT scan. The meningioma has been completely resected. The spinal cord has regained a normal shape. f Reconstructed 3D CT scan after contrast administration. The resection of the posterior arch of the atlas is visible on the right side. The lateral mass of the atlas (star) was left intact. The vertebral artery (arrow) has been elevated from the C1 posterior arch (compare with the left side) during the dissectionFig. 4a, b Preoperative MRI. A large anterior foramen magnum meningioma severely compresses the neuraxis, which is reduced to a crescent (star). c, d Postoperative MR images confirm the complete resection of the tumorFig. 5Surgical steps during a postero-lateral approach. a The left vertebral artery V3 segment (black arrow) has been elevated from the lateral part of the C1 posterior arch (white arrowhead). The medial portion has been resected up to the midline (black arrowhead). The dural entrance of the vertebral artery, where the V3 segment becomes the V4 one, is delineated by the dotted line. b The dura matter has been incised. The inferior contraincision extends inferiorly to the site of entrance of the vertebral artery into the dura matter. The C1 posterior rootlets are identified (black arrowhead). The inferior portion of the meningioma (white star) severely compresses the spinal cord (black star). c The vertebral artery V4 segment (white arrow), the spinal accessory nerve (black arrowheads), and the XIIth cranial nerve (white arrowhead) have been controlled. Of note, the dura matter (black star) is stretched over the left C1 lateral mass by a stitch to enlarge the lateral access and after the occipital craniotomy, the fall of the left cerebellum is prevented by a blade (white star). d After the complete removal of the meningioma, both vertebral arteries are visible. On the left side, we observe the section of a feeding vessel (white arrow). The right PICAs is visible (black arrow) as well as the Xth (black star) and the XIIth (black arrowhead) cranial nerves The postero-lateral approach is a lateral extension of the midline posterior approach. The patient must be carefully positioned in the same position as during a posterior midline approach. The head must be placed in neutral position. Any flexion must be avoided because it decreases the space in front of the neuraxis and therefore may worsen the compression and the neurological condition. The vertical midline skin incision is identical, but the incision is curved laterally on the tumoral side just below the occipital protuberance toward the mastoid process. The posterior muscles are divided along the occipital crest and retracted laterally to expose the occipital bone, the posterior arch of the atlas, and the C2 lamina, if required. The exposure may be extended on a limited way on the contralateral side. VA exposure At this step, the VA running above C1 needs to be exposed to safely resect the C1 posterior arch up to the C1 lateral mass. The exposure of the horizontal segment of the VA V3 segment progresses from the midline of the posterior arch of the atlas laterally toward the atlas groove. The medial border of the groove is clearly marked by a step with a decrease in the height of the posterior arch. The safest way to expose the VA is to dissect strictly in the subperiosteal plane. The periosteum of the C1 posterior arch needs to be elevated from medial to lateral and inferiorly to superiorly. Working in this manner permits to expose the posterior aspect of the posterior arch of the atlas, to bring into view the step at the medial end of the groove and then to elevate the VA from the atlas groove. In fact, the VA V3 segment is surrounded by a venous plexus; both artery and veins are enclosed in a periosteal sheath. Both provide protection against VA damage. If tearing occurs, venous bleedings can be controlled by direct bipolar coagulation on the VA periosteal sheath. Dissection at the superior aspect of the VA is more difficult because there is no good landmark and the periosteal sheath is in continuity with the ligaments covering the occipital condyle. Cutting of the ligaments must proceed a few millimeters above the VA on the occipital condyle by following the superior aspect of the VA sheath. The occipitoatlantal membrane covering the groove of the posterior arch of the atlas can be calcified or ossified, turning the groove into a tunnel, raising some difficulties to expose the horizontal portion at this level [23, 47]. After being fully exposed, the VA can be gently displaced superiorly to show the lateral mass of the atlas. Bone opening The posterior arch of the atlas is resected with rongeurs from the midline toward the transverse process. To obtain a decompression of the neuraxis before the tumoral resection, the posterior arch of the atlas must also be resected beyond the midline toward the controlateral side. By this way, hyperpressure induced even by gentle manipulation during resection are not transmitted to the neuraxis. The lower part of the occipital bone is drilled or resected with rongeurs laterally toward the sigmoid sinus and also beyond the midline. The lateral extension of the bone opening is established according to the position of the meningioma. In case of lateral meningiomas, the spinal cord is displaced toward the contralateral side, and then any resection of the FM lateral wall is not necessary. In case of anterior meningiomas, the spinal cord is pushed posteriorly, and the surgical corridor has to be enlarged laterally by drilling up to the medial side of the C1 lateral mass and/or the occipital condyle. Drilling further the lateral mass of the atlas or the occipital condyle is exceptionally necessary. In the exceptional case where the opening must be enlarged, no more than 20% of the medial part of the FM lateral wall has to be drilled. In such a way, stabilization is never required. The cranio-caudal bone resection is scheduled according to the position of the tumor regarding to the VA. The drilling has to be extended toward the lateral mass of the atlas if the tumor grows below the VA, to the jugular tubercle and the occipital condyle if it grows above, and toward both parts of the FM lateral wall, if it encircles the VA on both sides. Dura opening The most adequate dural incision is a curvilinear incision starting at the inferolateral corner then running vertically at a paramedian level (5 to 10 mm from the midline) and curving toward the superolateral corner. This type of incision permits to take the maximum benefit of the bone opening without contraincision and is generally easy to close. Keeping the dura over the neuraxis provides protection during the surgical dissection. Moreover, arachnoid connections between the dura and the medulla are left undissected to prevent an anterior fall of the neuraxis during tumor resection. Intradural step Lateral meningiomas are directly brought into view and their dural attachment directly accessible. Contrarily, anterior meningiomas are partially hidden by the neuraxis. The first step of the intradural stage, before beginning tumor resection, is to identify several important structures and to open the surgical access to the lesion by completely releasing the neuraxis. Important structures are the VA, the accessory nerve, and the dentate ligament. The VA is identified by following the course of the V3 segment where it pierces the dura matter. The first two digits of the dentate ligament have to be divided. To further improve the surgical access, the first cervical nerve root can be divided distal to its connections with the accessory nerve. At this step, the surgical technique must be individualized according to the tumor location: above, below, or on both sides of the VA. As previously mentioned, in cases of tumor developed below the VA, the lower cranial nerves are always pushed cranially and posteriorly by the tumor. These nerves will then be found at the superior pole of the lesion at the end of the surgery. The resection must start at the caudal aspect of the meningioma with the goal to release the dural attachment and to suppress the vascular supply first; then, the tumor is debulked in a dry surgical field with a sucker, an ultrasound aspirator, or a laser, according to the tumor consistency. When liberating the dural insertion, it is important to keep undetached a small part of the base, at the side of the neuraxis, to avoid free movement of the lesion, which can be responsible for inadvertent damage to the neuraxis during the remnant resection. When hollowing the tumor, a small layer is also kept with its capsule against the neuraxis. This part will be resected at the last surgical step and under better conditions when the meningioma is completely devascularized and the surgical field widely open. If the tumor is developed above the VA, two special points must be taken into consideration: the displacement of the lower cranial nerves and the dissection of the VA branches. Indeed, in such location, the displacement of the lower cranial nerves cannot be anticipated. To prevent damage, the rootlets must be under control on the side of the jugular foramen and then followed along their courses more or less adherent to the meningioma. With the lesion being progressively debulked, the nerve rootlets can be more easily mobilized, often inferiorly, to allow a more confident tumor resection at some distance from fragile nervous structures. The tumor dissection from the VA branches, especially the PICA, is another difficulty encountered only with tumors developed above the VA. Precise knowledge of the patient anatomy based on preoperative investigation is mandatory. If the meningioma encases the VA, the technique is identical as described above. Special consideration is nevertheless required if the meningioma has its insertion on the dura surrounding the VA penetration. The dural resection is better achieved by progressing from the extradural side toward the intradural aspect, along the VA because the VA invaginates into the dura with its periosteal sheath. This furrow can be resected as much as the VA adventitia is not invaded. Dural closure A watertight dural closure is required to prevent a postoperative cerebrospinal fluid (CSF) leakage. The closure is generally easy with the curvilinear incision. If necessary, a dural patch using the suboccipital aponeurosis achieves a perfect closure. In any case, the muscular and aponeurosis layers must be tightly closed. Antero-lateral approach The antero-lateral approach is rarely used in our department as we consider that it is a good choice only in some meningiomas with extradural extension through the bony structures [15–17]. The antero-lateral approach to the craniocervical junction has been extensively described in the article focusing on the surgical exposure of the VA V3 segment and will not be detailed again for this reason [8]. Some points required nevertheless special consideration. The head has to be slightly extended with a rotation of 60° toward the opposite side. Because of this rotation, VA segments above and below the C1 posterior arch run parallel, separated by this bone. Opening the C1 transverse foramen is mandatory for VA transposition. Bone opening When completely liberated, the VA can be pulled out and transposed, bringing into view the FM lateral wall as the occipital condyle, the anterior and posterior arches of the atlas, the lateral mass of the atlas, and the C1–C2 joint become visible. The exposure must now be adapted to each case, depending on the location and extension of the meningioma. The odontoid process can be reached by passing over the C1–C2 joint. The mastoid process can be resected and small bridging bone removed to open completely the jugular foramen from the end of the sigmoid sinus, to the jugular tubercle just underneath the junction with the jugular bulb, up to the beginning of the internal jugular vein. Dura opening The dural opening is most of the time out of concern because the antero-lateral approach is mainly indicated for extradural meningiomas. When lesions are developed in both extradural and intradural compartment, the dura is already opened by the lesion and must only be enlarged. Dural closure Dural closure can be a considerable problem in cases where the dural defect is anteriorly located at the level of the clivus, the anterior arch of the atlas, and the odontoid process. Postoperative complications The antero-lateral approach could induce a transient dysfunction of the accessory nerve by the nerve manipulation, responsible for pain along the trapezius muscle and weakness of the trapezius muscle and/or the SM muscle. During this approach, manipulation of the sympathetic chain could also be at the origin of a transient Horner’s syndrome. VA damage has never been observed. In case of tumoral encasement, a balloon occlusion test must be realized. Preservation of the lower CNs can be hazardous. If CN IX and X are damaged, postoperative swallowing problems must be anticipated. Review of the literature Published series Yasargil has reviewed series published from 1924 to 1976 and counted 114 cases of FMMs. Since then, more than 400 cases of FMMs have been reported in the literature [2–7, 10, 18–22, 27, 35, 37, 38, 41, 44–46, 52, 54, 55, 57, 58]. George et al. [16, 19] reported on a series in the French literature of 106 craniocervical meningiomas from 21 hospitals. The largest single-center series have been published by Meier et al. [38] in 1984, Samii et al. [55] in 1996, and George et al. [18] in 1997. Table 1 summarizes published series over the last 20 years in the English literature. Table 1Review of the literature of published series of FMMs over the last 20°years in the English literatureAuthorYearNb ptFMM location (%)Recurrence (%)Va encasement (%)ApproachVA transpResection JTPartial mastNb CR (%)Extent CRInstability (%)Outcome (%)Resection (%)Transient morbidity (%)Permanent morbidity (%)Mortality (%)FURecurr (%)AnteriorLateralPosteriorImprovedUnchangedWorsenedTotalSubtotalGilsbach19875100–FLY1/3100.00.0200.0Guidetti19881782.40100.01211.0Sen, Sekhar19905802080–ELYYY1001/3–1/2020206060.040.06020.0Crockard199131003333TONo00331000.066.0100.010066.020.633.0Kratimenos1993810012.50–FLY1/3087.512.5025.0––Babu19949100ELYYY1001/3–1/2088.811.278.05611.19.40.0Akalan1994812.5087.50PM008812100.000.0Bertalanffy199619100–FL SO TCY1001/30100.00.000.0––Samii199638955540PM, LSO17.501/3063.030.037.056.021.05.0George1997404552.502.5038100Partial0902.507.5087.510.00.007.557.60.0Pirotte19986100–Y1001/2–1/30100.00.01717.0Sharma1999105050PM, FL00Yes100.015.0Salas199924100–TC/ELTJYY1001/3066.033.0–0.014.8–Arnautovic20001810011.10–TCY1001/2–1/30891175.012.55511.116.640.05.5Roberti200121EL TC76.024.021.509.5Goel20011710059SO11.801/3–1/4010082.018.060.0Nanda20026100–FL00100100.00.000.043.00.0Marin Sanabria2002772.5028.50TO, SO, TC291/3–1/2080.0020100.00.072.5514.0Parlato20037–Y<1/2086.0%14.00.024.00.0Boulton2003106010300070201090.010.040.0100.0Pamir20042291940FL951/3095.54.5274.500.040.00.0Margalit200518100Lat50Partial (9/18)0.0Bassiouni200625325711443FL0096.04.040.084.073.20.0CR Condyle resection, EL extreme-lateral, FMM foramen magnum meningioma, FL far lateral/postero-lateral, FU follow-up, JT jugular tubercle, mast mastoidectomy, Lat lateral, Nb number, pt patient, Recurr recurrence, SO suboccipital, TC transcond dylar, TO transoral, transp transposition, VA vertebral artery, Y yes Heterogenicity of published series A heterogenous group of patients constituted the published studies. Several studies included not only FMMs but also other FM pathologies such as neurofibromas, schwannomas, as well as other various lesions leading result analysis precarious [2, 4, 10, 35, 38, 41, 45, 57, 58]. Table 1 summarizes also the high variability of tumor location, rate of VA encasement, rate of tumor recurrence, and surgical approaches. The proportion of anterior FMMs is comprised between 12.5 [2] and 100% [3, 27, 41]. The subdivision between anterior and lateral FMM is not always detailed in studies including 90 to 100% of antero-lateral FMMs [4, 6, 20, 21, 35, 44, 46, 54, 55, 57]. Posterior FMMs were always included in studies with anterior or antero-lateral lesions with a variable proportion from 2.5, 5, 9, 11, 28.5, 30, up to 50% [5, 7, 18, 37, 44, 55, 58]. The rate of VA encasement can be higher than 59% and the rate of tumor recurrence up to 80% [21, 57] (Table 1). Surgical approaches, extent of bone resection, and need for fusion FMMs are undoubtedly challenging tumors, requiring special considerations because of the vicinity of the brainstem, medulla oblongata, lower cranial nerves, and the VA. Several approaches have been advocated. The definite goals are to achieve the largest tumor removal and the lowest morbidity rate as possible. Minimizing the morbidity is obtained by choosing the appropriate exposure. The approach must allow adequate controls of important neurovascular structures, without exposing to unnecessary risks. There is no discussion about the best surgical approach of posterior FMMs. The posterior approach is the best option, is well known by neurosurgeons, and is associated with a low morbidity rate. The debate about the best surgical approach is more open for lateral and mainly anterior FMMs. The transoral approach has been reported sporadically [10, 39]. Despite providing access to the anterior part of the craniocervical junction, this approach has several drawbacks in case of intradural lesions: increased risk of CSF fistula and meningitis after crossing of the contaminated oral cavity, poor access to laterally extending tumors resulting in a low rate of complete resection, and increased risk of posteroperative instability and velopalatine insufficiency [10, 39]. The two main surgical approaches reported in the literature are the far-lateral approach [28], also called postero-lateral approach or lateral suboccipital approach, and the extreme-lateral approach, also named antero-lateral approach. As detailed by Rhoton [51], the far-lateral approach is a lateral suboccipital approach directed behind the sternocleidomastoid muscle and the VA and just medial to the occipital and atlantal condyles and the atlanto-occipital joint. The extreme lateral approach is a direct lateral approach deep to the anterior part of the sternocleidomastoid muscle and behind the internal jugular vein along the front of the VA. In fact, both approaches permit drilling of the occipital condyle but provide a different exposure because of the differences in the approach direction. The extreme-lateral transcondylar approach for FMMs was first reported by Sen and Sekhar [57] in 1990. Further publications of the same experienced skull base surgeons followed [4, 52, 54]. Salas et al. [54] reported in 1999 several variations of the extreme-lateral approach based on a series of 69 patients, including 24 meningiomas. The lesions were removed most of the time through a partial transcondylar approach and more rarely through transfacetal, retrocondylar, and extreme transjugular approaches. During the partial transcondylar approach, the posterior one third of occipital condyle and superior facet of C1 were drilled away. These authors also performed a partial mastoidectomy [4, 54, 57]. Arnautovic et al. [3] published their experience in 2000 on a series of 18 ventral FMMs. They used also the extreme-lateral transcondylar approach. The condyle drilling ranged approximately from one third to one half of the condyle, without causing craniocervical instability. The extreme-lateral trancondylar approach requires the VA transposition to reach and drill the occipital condyle [3, 4, 27, 45, 54, 57]. As others [5, 6, 20, 21, 27, 35, 41, 44, 55, 58], we advocate rather the postero-lateral approach, also called far-lateral approach, even for anterior intradural FMMs. During this approach, the VA is controlled in the horizontal portion of the V3 segment, above the C1 posterior arch. VA transposition is rarely performed and has only been reported in one series [46]. The extent of FM lateral wall drilling is variable, in fact directly proportional to the tumor extension to the contralateral side. In the literature, the occipital condyle resection varies between 0 and 66% [27, 41, 44, 46, 55]. In a recent publication, Bassiouni [5] classified judiciously surgical approaches in two groups: transcondylar or retrocondylar if the occipital condyle is not drilled. In our review of the literature (Table 1), we found nine series in which the retrocondylar approaches was used to reach anterior or anterolateral meningiomas [2, 5, 7, 21, 35, 37, 41, 55, 58]. In four of these series, a retrocondylar approach was not used in all cases but in 89.2, 82.5, 71.5, and 50% [21, 35, 37, 55]. The five other series are homogeneous and only included patients treated through a retrocondylar approach [2, 5, 7, 41, 58]. Of these, three studies reported complete resection in 100% of the cases [2, 41, 58]. In the two others, complete resection was noted in 90 and 96% of the cases, and the remaining lesions were resected subtotally [5, 7]. Surgical results were good, and surgical morbidity and mortality rates low [2, 5, 7, 41]. Extradural lesions can be treated either by posterolateral or anterolateral approach (Fig. 2). The extent of drilling during this procedure can be larger but is only dictated by the tumoral invasion and must be limited to the destroyed or invaded bone. In such a way, the question of instability is only a preoperative concern. In our experience of tumors located at the craniocervical junction, we have not observed any instability if less than half of the C0–C1 and C1–C2 joints were resected [15]. In our experience, VA transposition was merely performed in selected cases of FMMs with extradural extension and never for intradural lesions. Surgical results Morbidity–mortality–prognosis factors In the Yasargyl’s review of the literature of series published before 1976, the overall mortality rate was approximately 13% but could be as high as 45% in some series [34]. Over the last 20 years, the overall mortality is 6.2%. The mortality rate is comprised between 0 and 25% (Table 1). Mortality rates higher than 10% were mainly observed in small series [4, 27, 37, 46, 57, 58]. In the Yasargil’s review, a good outcome was noted in 69% and a fair and poor outcome, respectively, in 8 and 10%. In series larger than ten patients published over the last 20  years (Table 1), neurological improvement, stability, and worsening were noted, respectively, in 70–100, 2.5–20, and 7.5–10% of the cases. The permanent morbidity rate is comprised between 0 and 60%. The permanent morbidity rate is lower through a far-lateral approach (0–17%), either transcondylar or retrocondylar, than through an extreme-lateral transcondylar approach (21%–56%, considering series without recurrent tumor). Lower cranial nerves dysfunctions are the most frequently encountered preoperative deficits. These deficits have the propensity to recover even completely postoperatively [3–5], except in cases of en plaque meningiomas or recurrent tumor [55]. Several factors lead the surgical procedure still more difficult, then influencing negatively the morbidity rate: anterior tumor location [18, 55], tumor size (smaller lesions are more difficult to resect because the surgical corridor is small), tumor invasiveness, extradural extension [18], VA encasement [22], absence of arachnoidal sheath [5, 14, 65], and adherences in recurrent lesions [4, 5, 55, 57]. Rate of tumoral resection Based on a multicentric study from 21 hopitals, George et al. [19] reported 77, 16, and 7%, respectively, of complete, subtotal, and partial removals. Over the last 20 years, most of the studies reported complete or subtotal removal of the tumor [2, 4–7, 10, 18–22, 27, 35, 37, 41, 44–46, 52, 54, 57, 58]. Factors limiting the resection completeness are adherences of the lesion to vital structures, VA encasement and invasiveness of the lesion. Adherences are observed during repeated surgery and explain the lower rate of complete tumor resection (60–75% of Simpson grade 1) in surgical series in which a high rate of recurrent tumors are included [3, 55, 57]. Eventually, in recurrent tumors, some authors advocate leaving a small tumor remnant to preserve a low morbidity rate [5, 18]. On the other hand, Arnautovic et al. [3] favored radical removal of recurrent tumors with the goal of providing a relatively long and stable postoperative course, even at the price of frequent but transient morbidity induced by lower cranial nerves dysfunction. Arnautovic et al. [3] have also demonstrated that the rate of complete removal is higher at first surgery than when treating recurrence, advising then to be more aggressive at the first surgical presentation. VA encasement was noted in 38 to 59% in some series [5, 18, 21, 44, 55]. This factor was recognized as an independent factor of incomplete removal [55]. The location of the meningioma, either intraextradural or extradural, reflects the tumoral invasiveness. These tumors are less favorable to be completely resected than pure intradural lesions [19, 39]. In the French cooperative study [19], the rate of complete removal of intradural, extradural, and intraextradural meningiomas was, respectively, 83, 50, and 45%. Our experience Our experience has been published in 1997 after the treatment of 40 FMMs operated in the period 1980–1993 [18]. Twenty-four lesions were intradural, two were extradural, and four were intraextradural. Eighteen were considered anterior, 21 lateral, and one was posterior. The tumor was above the VA in four cases, below in 20 cases, and on both sides in 16 cases. The posterolateral approach was used in 31 cases, the anterolateral one in five cases, and the posterior midline in four cases. The rates of complete resection for intradural and extradural lesions were, respectively, 94 and 50%. Postoperatively, the clinical condition improves in 90% of patients, remains stable in 2.5%, and worsened in 7.5%. Our present experience over the last 25 years is now based on 97 FMMs (unpublished data). Complete removal was achieved in 86% and subtotal removal in 11%. Subtotal removals were due to extradural extension or to recurrent cases. The rate of complete removal increased up to 94% when selecting only intradural lesions treated at first presentation. We mainly use the far-lateral retrocondylar approach for intradural FMMs. In some cases, the drilling of the FM lateral wall has to be performed for intradural anterior meningiomas but remains limited at worst to the medial 20% of the FM lateral wall. Whatever, this extent of drilling has to be tailored according to the tumor characteristics. We consider nevertheless that more bone resection is never necessary because of the anatomical anterior position of the lateral mass of the atlas and the occipital condyle. Our attitude has been reinforced by cadaveric study, which has demonstrated that increasing the bone drilling is not associated with a significant widening of the surgical corridor [41]. In fact, resecting one third and one half of the occipital condyle increases the visibility respectively by 15.9 and 19.9°. Two anatomic reports have demonstrated that condyle resection allows a wider angle of exposure to gain the anterior foramen area [1, 59]. However, these studies did not consider the fact that in surgical approaches to anterior lesions, space-occupying lesions can enlarge the surgical corridor. We consider that the extreme-lateral approach could be associated with a higher morbidity rate than the far-lateral approach. The exposure allowed by the far-lateral retrocondylar or partial transcondylar approach is adequate for resecting even anterior intradural FMMs. The supposed benefit in term of exposure provided by the extreme transcondylar approach is counterbalanced by the risks associated with the CN XI dissection, the VA transposition, and the condyle drilling. Conclusions FMMs are challenging tumors in the vicinity of the brainstem, the VA, and lower cranial nerves. Several surgical approaches are possible, each one with specific indications. The drilling of the FM lateral wall required during the approaches is always limited and by itself should not be at the origin of any instability. Postoperative complications can be dramatic and must be anticipated.

J_Autism_Dev_Disord-4-1-2268726

Brief Report: The Use of WAIS-III in Adults with HFA and Asperger Syndrome

The WAIS III was administered to 16 adults with high functioning autism (HFA) and 27 adults with Asperger syndrome. Differences between Verbal Intelligence (VIQ) and Performance Intelligence (PIQ) were not found. Processing Speed problems in people with HFA appeared. At the subtest level, the Asperger syndrome group performed weak on Digit Span. Comprehension and Block Design were relative strengths. In the HFA group, performance on Digit-Symbol Coding and Symbol Search was relatively poor. Strengths were found on Information and Matrix Reasoning. The results suggest that the VIQ-PIQ difference cannot distinguish between HFA and Asperger syndrome. WAIS III Factor Scale and Subtest patterning provides a more valid indicator. Introduction Over the past few years, interest in HFA and Asperger syndrome in adults with normal intelligence has increased markedly. However, not much is known about the cognitive profiles of these groups. Only a few studies exist about adults who function relatively well in society and have been diagnosed late in life (Howlin 2004; Vermeulen 2002). The present study aims to assess the cognitive profiles of this relatively well-functioning subgroup by means of the Wechsler Intelligence Scale III (WAIS III, Wechsler 1997). In WAIS III, the intelligence pattern is described at three levels: The first level contains Performance Intelligence and Verbal Intelligence. The second level consists of the four factor scales: Verbal Comprehension, Perceptual Organization, Freedom from Distractibility and Processing Speed. The third level contains the subtests. The following paragraphs summarizes research on the intelligence profiles of adults with the autistic disorder or Asperger syndrome on the basis of these three levels. The Performance IQ (PIQ)––Verbal IQ (VIQ) dichotomy has been incorrectly used for years to underpin the diagnosis of autistic disorder or Asperger syndrome. It is questionable whether the two constructs should even be applied in general, because research did not support the construct validity of the VIQ-PIQ dichotomy (Taub 2001). For adults with HFA, studies on WAIS-R have yielded contradictory results (Minshew et al. 1992; Siegel et al. 1996; Vermeulen 2002), which may reflect the validity problems of the VIQ-PIQ dichotomy (Arnau and Thompson 2000; Taub 2001). The factor scale level is of great importance in assessing cognitive abilities because factor analytic studies indicate that the factor scales give the best estimates of the four factors underlying intelligence (Arnau and Thompson 2000; Ryan and Paolo 2001). No studies have been performed on WAIS-III profiles for adults with HFA or Asperger syndrome as far as we know. Therefore no information is available on the results of factor scales in these groups. This leads to the conclusion that the most important factors of the intelligence pattern for adults with HFA or Asperger syndrome are still unknown. On subtest level, some studies on WAIS or WAIS-R found low Comprehension versus high Block Design scores (Goldstein et al. 2001; Rumsey and Hamburger 1988). A relatively high variability between the subtests scores in adults with HFA has also been reported (Siegel et al. 1996). In summary, research shows that among adults with HFA or Asperger syndrome, results of VIQ-PIQ differences vary and may be influenced by the validity problems of the VIQ-PIQ dichotomy. The factor scale scores and the subtest patterns provide a better representation of the intelligence pattern. Aims of the Present Study The present study aims to acquire insight into the WAIS III profiles of normal intelligent adults with HFA and Asperger syndrome. Profiles in the total group and differences between the two diagnostic groups will be examined. Methods Procedure All participants were recruited from the GGZ (Mental Health Center) Eindhoven and Oost-Brabant. The participants met the criteria for Asperger syndrome or HFA. Participants with relevant neurodevelopmental conditions and genetic conditions were excluded, as were institutionalized patients and patients with a Full Scale IQ below 80. Subjects The mean Full Scale IQ of the participants was 110.16, individual scores varied between 83 and 145 (see Table 1). Table 1Characteristics of participantsIQ and ageMSDRange    Full scale IQ110.1616.0583–145    Mean age41.9310.6720–60Diagnosisn%    Autistic disorder1637.2     Asperger syndrome2762.8 Gender    Male3990.7     Female49.3 Education    Lower/middle education1841.9     Higher education2558.1 Employment status    Employed or retired3069.8     Studying12.3    Unemployed1227.9 Current living circumstances    Lives with partner2353.5     Lives independently 1227.8     Sheltered living24.7     Lives with parents 614.0 All individuals ranged in age from 18 to 60 years. The mean age was 41.93. Of all participants, 25 finished higher education and 30 individuals had work. 23 participants lived together with a partner. The relatively large number of participants who had a relationship, worked and were well educated emphasizes the relatively high level of functioning in this group. Assessment of Disorder Hetero-anamnestic information was gathered using the Dutch version of the Autistic Disorder Diagnostic Interview, revised version (ADI-R, Lord et al. 1994), administered by psychologists who were officially trained in the administration and scoring of the instrument. To gather anamnestic information, a semi-structured interview was used to assess presence of the DSM-IV criteria of HFA and Asperger syndrome (APA 1994). Because of the controversial nature of the DSM-IV criteria (Ghaziuddin et al. 1992; Mayes et al. 2001), additional questions were used to differentiate between HFA and Asperger syndrome, based on the diagnostic criteria of Gillberg and Gillberg (1989) and ICD-10 (WHO 1993). Assessment of Intelligence The intelligence profile was assessed using the Dutch translation of the WAIS III (Wechsler 1997). The WAIS-III has excellent psychometric properties (Sattler and Ryan 1999) and has been validated for the Dutch population (Wechsler 1997). Results Analyses were done at the three WAIS-III levels: VIQ versus PIQ, the four factor scales and all subtests. Preliminary analysis included checks for normality, linearity, influential data points and assumptions of repeated measures. No serious deviations were found. T-tests showed that both diagnosis groups were comparable in education and work status, as well as in gender distribution. Differences Between WAIS III VIQ and PIQ Differences between VIQ and PIQ for all participants and both diagnostic groups were analyzed by means of paired t-tests. No statistically significant effects were found for any of the investigated groups (see Table 2). Table 2VIQ and PIQ differences in the total group and in diagnostic groupsVIQPIQMean difference nMSDMSDTotal group110.3013.83108.4218.211.8843Asperger111.41 13.57112.5217.281.1127Autistic disorder108.4414.49101.50 18.136.9416 Differences Between Factor Scale Scores Factor Scale profiles were studied within the total group and between the two diagnostic subgroups by means of repeated measures analysis of variance. Mauchly’s test indicated that the assumption of sphericity was not met. Therefore the degrees of freedom were corrected using the Huynh–Feldt correction (ε = .89). Post-hoc comparisons using the Sidak adjustment for multiple comparisons showed that the main effect of the WAIS III Factor Scale was statistically significant (F(2.7,109.7) = 7.0, p < 0.001). An interaction effect of differences in Factor Scale mean by diagnostic group was also found (F(2.7, 109.7) = 2.7, p = 0.05). To find out which differences in WAIS III Factor Scale means added to the significant main effect, post hoc pairwise comparisons were done. This showed that the main effect in the total group can be attributed to Processing Speed being significantly lower than Verbal Comprehension (p < .01) and Perceptual Organization (p < .005). Post hoc pairwise comparisons were done for the two diagnostic groups to analyse the ‘within group’ effect. In the Asperger group, no significant differences in Factor Scale mean scores were found. The HFA group however, showed a significant lower Processing Speed compared to Verbal Comprehension (p < .01), Perceptual Organization (p < .01) and Freedom from Distractibility (p < .05) (see Table 3). Table 3Factor scale scores for the total group and the diagnostic groupsFactor scaleMSDNVerbal comprehensionAutistic disorder107.5*12.116Asperger syndrome110.811.927Total109.6*12.043Perceptual organizationAutistic disorder105.0*18.716Asperger syndrome111.813.027Total109.3*15.543Freedom from distractibilityAutistic disorder105.1*18.216Asperger syndrome107.215.427Total106.416.343Processing speedAutistic disorder91.8*17.416Asperger syndrome106.519.427Total101.0*19.843*p < .05. Differences Between WAIS III Subtest Scores The Subtest profiles were explored within the total group and between the two diagnostic subgroups by means of a repeated measures analysis of variance. The assumption of sphericity was not met. Therefore the degrees of freedom were corrected using the Huynh–Feldt correction (ε = .82). Post-hoc comparisons were performed using the Sidak adjustment for multiple comparisons. The results (see Table 4) showed a significant main effect of the type of Subtest (F(10.7,438.7) = 4.8, p < 0.001). Table 4Mean standardized subtest scores for the total groupSubtest scores MSDnVocabulary11.63*2.56443Similarities11.422.49043Arithmetic11.77*3.04643Digit span10.723.26843Information12.42*2.77943Comprehension12.53*2.77243Letter-number sequencing10.982.95643Picture completion10.883.25343Digit-Symbol Coding9.81*3.43843Block design12.02*3.56243Matrix reasoning11.98*2.45443Picture arrangement11.533.73143Symbol search10.37*3.97043Object assembly11.163.08643*p < .05. An interaction effect of Subtest by diagnosis was also found (F(10.7, 438.7) = 2.1, p < 0.05), indicating that the patterning of the WAIS III subtest mean scores for the two diagnostic groups differs. Table 5 and 6 show the mean Subtest scores and standard deviations for the HFA group and the Asperger syndrome group. Table 5Mean standardized subtest scores for the autistic disorder groupSubtest Scores MSDNVocabulary11.312.49616Similarities10.941.76916Arithmetic11.443.70516Digit span11.313.40016Information12.13*3.28416Comprehension11.752.17616Letter-number sequencing10.253.15216Picture completion10.814.07016Digit-Symbol Coding8.38*3.03016Block design10.563.44416Matrix reasoning11.44*2.82816Picture arrangement10.193.67416Symbol search8.44*3.48316Object assembly9.883.32416*p < .05.Table 6Mean standardized subtest scores for the Asperger syndrome groupSubtest scoresMSDnVocabulary11.812.63227Similarities11.702.82627Arithmetic11.962.63827Digit Span10.37*3.20027Information12.592.48527Comprehension13.00*3.01327Letter-number sequencing11.412.80527Picture completion10.932.74527Digit-Symbol Coding10.673.43127Block design12.89*3.40127Matrix reasoning12.302.19827Picture arrangement12.333.59527Symbol search11.523.84727Object assembly11.932.71627*p < .05. Post hoc pair wise comparisons showed that the main effect in the total group can be attributed to the fact that Digit-Symbol Coding was significantly lower than Vocabulary (p < .05), Arithmetic (p < .05), Information (p < .005), Comprehension (p < .005), Block Design (p < .05) and Matrix Reasoning (p < .005). Furthermore, Symbol Search was lower than Information (p < .05) and Comprehension (p < .05). Post hoc pair-wise comparisons were also performed for the two diagnostic groups to analyze the ‘within group’ effect. The two groups showed significant differences in Subtest scores. In the Asperger syndrome group, Digit Span was lower than Comprehension (p = .005) and Block Design (p < .05). In the HFA group performance was significantly higher in Information compared to Digit-symbol Coding (p < .05) and Symbol Search (p < .05). Furthermore, Digit-Symbol Coding was lower than Matrix Reasoning (p < .05). Discussion WAIS VIQ Versus PIQ No significant differences were found between VIQ and PIQ in the total group nor in the two diagnostic subgroups. The results are in line with factor analytic studies showing that the VIQ-PIQ dichotomy is not valid for general populations (Arnau and Thompson 2000; Taub 2001). WAIS III Factor Scale Level The Asperger syndrome group was characterized by a flat Factor Scale profile in the Asperger syndrome group, while the HFA group performed significant low in Processing Speed. A low Processing Speed indicates problems in speed of processing visual information (Wechsler 1997). Adults with HFA apparently need more time than other people to process and integrate visual information and to act on this information. The Processing Speed performance of the HFA group might be influenced by problems with top-down processing and ignoring irrelevant details, which are characteristic of people with HFA (Happé 2005; Shah and Frith 1993). In order to maintain an overview of what they are doing, they work slowly. WAIS III Subtest Level Analyses showed different Subtest patterns in the HFA and the Asperger syndrome groups. The HFA group performed significantly low in Digit-Symbol Coding and Symbol Search. These two subtests together form the Processing Speed Factor. The low scores for these subtests represent the problems in speed of processing visual information as described in the preceding paragraph. The HFA group showed significantly high performance in Information and Matrix Reasoning. High scores for Information are in line with the fact that people with autism usually acquire much factual knowledge (Happé 1999). Matrix Reasoning taps nonverbal perceptual reasoning. Matrix Reasoning is the only Perceptual Organization subtest without a time limit and is possibly not influenced by low Processing Speed performance scores. The good performance of the HFA group can probably be attributed to their visual-spatial strengths (Lincoln et al. 1995; Tsatsanis 2005) and to the absence of a time limit for this subtest. In the Asperger group, scores for Digit Span were relatively low. Digit Span taps working memory capabilities (Wechsler 1997), which can been defined as ‘the ability to hold in mind past states of the environment and past actions while currently performing an action’ (Russell 1997). People with autism or Asperger syndrome tend to store information in details instead of using strategies, which often leads to problems in retaining information (Happé 2005; Minshew et al. 1992; Tsatsanis 2005). Low Digit Span scores in the Asperger group may reflect problems in applying strategies to retain information. The Asperger syndrome group performed significantly well on Comprehension. High scores on Comprehension in this group seem to contradict former research results (Klin et al. 2005; Mayes and Calhoun 2003; Siegel et al. 1996). However, people with Asperger syndrome often try to function in society by analyzing social situations at a cognitive level, which has been described as using an ‘explicit theory of mind’ (Frith and Happé 1999). A extremely well developed explicit theory of mind may have caused the Asperger syndrome group to have such high scores on Comprehension. The Asperger Syndrome group also performed significantly well on Block Design. Strengths in Block Design have often been reported in studies of people with HFA or Asperger syndrome (Happé 2005; Shah and Frith 1993). This has been attributed to strengths in processing unconnected stimuli outside a meaningful context, which go together with the central coherence problems seen in people with autistic impairment (Shah and Frith 1993). Conclusions The present study found people with Asperger syndrome to differ significantly from people with HFA in WAIS III Factor Scale profiles and WAIS III Subtest patterning. In people with HFA Processing Speed problems were found. Further, the HFA and Asperger syndrome group showed different subtest patterns. The present study supports the idea that HFA and Asperger syndrome can be differentiated empirically at the level of intellectual functioning. This lends support to the hypothesis that HFA and the Asperger syndrome are two separate disorders.

Eur_Arch_Otorhinolaryngol-3-1-2099165

Severe delayed posttonsillectomy haemorrhage due to a pseudoaneurysm of the lingual artery

A 3.5-year-old child is presented with severe, recurrent haemorrhages after a tonsillectomy. The haemorrhages were caused by a pseudoaneurysm of the lingual artery, which was visualised more than 2 weeks postoperatively by angiography. The pseudoaneurysm was successfully embolised by coils. Coiling is an adequate therapeutic option for severe posttonsillectomy haemorrhage due to arterial damage. Introduction Postoperative haemorrhage is the most common serious complication of adenotonsillectomy and its incidence is around 3% [1]. Intraoperative bleeding (<24 h) may be related to the surgical technique or a bleeding diathesis. Delayed postoperative haemorrhage (>24 h) predominantly occurs on the fifth to seventh-day postoperatively when the slough separates from the granulating fossa. The most critical posttonsillectomy haemorrhages are due to arterial dissections and aneurysms. In this report we present a boy with a severe, delayed posttonsillectomy haemorrhage due to a pseudoaneurysm of the lingual artery. Case report An otherwise healthy 3.5-year-old boy was brought in with severe oral bleeding. Nine days prior to the presentation he had undergone an adenotonsillectomy for chronic adenotonsillitis in another hospital. The tonsils had been removed with cold dissection and sutures had been placed in both tonsil areas. The procedure and the immediate postoperative period went uncomplicated. On the fifth postoperative day he had been admitted for a one-night observation because of haemorrhage from the mouth. During the admission no active bleeding was observed and his haemoglobin level of 4.9 mmol/l (normal values: 6.5–8.4 mmol/l) was treated conservatively. The second postoperative bleeding started 9 days postoperatively, late at night when the patient was lying in bed. There were no provoking events. The bleeding lasted around 10 min but had already stopped at arrival in our emergency room. We saw a pale and restless boy. Intra-oral examination in general anaesthesia showed lacerated pharyngeal muscles in the left tonsillar fossa and a loose suture was removed. The right tonsillar fossa revealed diffuse bleeding from the pharyngeal muscles without pulsating masses. The nasopharynx and tongue base showed no abnormalities. The haemoglobin was increased from 2.9 to 8.2 mmol/l by transfusion with two units of packed red blood cells. The coagulation values were within the normal range. The postoperative period was uncomplicated, so he was discharged after 5 days, 15 days after the primary intervention, with dietary advices. One day later the boy arrived by ambulance with heavy bleeding from the mouth. He was pale, shivering and drowsy. Again, the severe bleeding started without a provoking moment and persisted for about 15 min. Immediately, after arrival at our emergency department, he was re-explored intra-orally under general anaesthetics. We saw a bluish, pulsating mass of 5 by 12 mm low in the right tonsillar fossa without active bleeding. The faucial pillars on the right side were approximated without tearing the mucosa or induration of the aneurysm. However, during extubation a massive bleeding occurred. The boy was re-intubated, and with digital oral pressure during 15 min haemostasis was achieved. The patient was stabilised and an arteriography was performed. A pseudoaneurysm of the right lingual artery was visualized with selective catheterisation using a vertebral catheter (Fig. 1). The pseudoaneurysm was coiled distally and proximally using a microcatheter (Excelsior 10, Boston scientific, Natick, MA, USA) and microcoils (GDC 10, Boston Scientific, Natick, MA, USA). The close relation of the proximal part of the pseudoaneurysm to the entrance of the facial artery made coiling of the facial artery inevitable. The pseudoaneurysm was coiled successfully and no bleeding focus was seen on the control arteriography (Figs. 2, 3). During the procedures blood transfusion was needed to restore the low haemoglobin level. Fig. 1Pseudoaneurysm of the right lingual artery seen from right-anterior side (3D-surface rendering)Fig. 2Arteriography of pseudoaneursym of the right lingual artery seen from right-anterior sideFig. 3After coiling of the pseudoaneurysm, including the facial artery; compare to Fig. 2 The boy was extubated without any bleeding and 4 days after the embolisation he was discharged in a good clinical condition. At a follow-up examination, he did not report any intra-oral or neurological abnormalities. Discussion True aneurysms develop through congenital or acquired weakness of all three layers of the vascular wall. False or pseudoaneurysms may arise due to localised arterial wall laceration caused by blunt or penetrating trauma. The intima or adventitia layer of the vessel wall is dissected, which creates a periarterial haematoma. Pseudoaneurysms after tonsillectomy may be triggered by blunt or direct trauma during dissection or due to placing ligation sutures. Haemorrhages from posttonsillectomy pseudoaneurysms of the lingual, facial and internal carotid artery have been observed within 5 h postoperatively to the eighth postoperative day in a few cases [2, 3]. In those reports no intra-oral pulsating masses were observed. The pseudoaneurysms were shown with angiography and treated with embolisation. Mitchell et al. reported a case of fulminant bleeding during the tonsillectomy procedure from an aberrant lingual artery with a pseudoaneurysm, which was treated with embolisation [4]. A pulsating neck mass can also be the first symptom of a pseudoaneurysm, which has been seen in two cases without any bleeding [5, 6]. It is striking that the above-mentioned cases of posttonsillectomy pseudoaneurysms concerned children under the age of 10 years. No cases of posttonsillectomy haemorrhage due to pseudoaneurysms in adults have been reported. This suggestive higher incidence of pseudoaneurysms in children might result from the smaller anatomy and thinner pharyngeal muscles, and a subsequent higher risk of damaging the large vessels. Since the angiography of our patient did not show any aberrant courses of arteries, trauma to the artery during dissection or placing the suture in the tonsillar fossa could have caused the pseudoaneurysm in our patient. In the two first oral inspections no masses were observed, only after the third bleeding (16th day postoperatively) a pulsating mass was seen. This suggests a gradual development of the aneurysm. False aneurysms are known to expand when the periarterial clot dissolves and more blood flows into the periarterial space [5]. The size of the lesion to the vessel wall might determine the amount of blood and whether the pseudoaneurysm is large enough to be observed. Maybe the vessel lesion in our patient was small, which made the pseudoaneurysm grow slowly. Although no real provoking events seem to have initiated the bleedings, lying down in bed might have raised the blood pressure in the carotid artery, which made the aneurysm leak. A new clot made the severe short lasting bleeding stop. Severe posttonsillectomy haemorrhage is treated with external ligation of the internal or external carotid artery or endovascular coiling of the injured artery. Although a randomized controlled trial has not been performed, embolisation seems to have advantages over external ligation [7]. Firstly, the diagnostic evaluation can be combined with direct therapeutic intervention. Furthermore, coiling is more selective. This is especially convenient in posttonsillectomy haemorrhages, where several branches of the external or internal carotid artery could be damaged. The third advantage is that embolisation is less mutilating and has less risk of damaging the vagal and accessory nerves. The potential complications of embolisation are perforation of the vessel with leakage of coils, ischemia of mucosa or nerves, or vasospasm. External ligation seems to be the only quick option in massive bleeding and unstable patients. Conclusion Pseudoaneurysms are life-threatening and should be considered in severe posttonsillectomy haemorrhage. Pseudoaneurysms do not always present as intra-oral or cervical pulsating masses, and can develop gradually. If the technique and technical crew are available, interventional arteriography is strongly recommended in severe posttonsillectomy haemorrhage. It is diagnostic and therapeutic at the same time and more selective than surgical ligation.

J_Med_Internet_Res-5-4-1550579

How do Consumers Search for and Appraise Information on Medicines on the Internet? A Qualitative Study Using Focus Groups

Background Many consumers use the Internet to find information about their medicines. It is widely acknowledged that health information on the Internet is of variable quality and therefore the search and appraisal skills of consumers are important for selecting and assessing this information. The way consumers choose and evaluate information on medicines on the Internet is important because it has been shown that written information on medicines can influence consumer attitudes to and use of medicines. Introduction Consumers frequently use the Internet as an information source and it has been reported that 80% of adult Internet-users have accessed it for general health information [1]. More specifically, 36% of Internet-using consumers have used the Internet as a source of information on medicines [1]. It is broadly acknowledged that health information on the Internet is of variable quality as evidenced by the large number of studies that have explored the quality of consumer health information on the Internet [2]. This is to be expected because the Internet is a free medium. It has also been widely postulated that consumers searching for health information are in danger of being harmed by poor-quality information even though there is little evidence of this [3]. A consumer's risk for encountering poor-quality health information is purportedly related to the proportion of poor-quality information on the Internet and the consumer's ability to filter out this information [2]. As the quality of information on the Internet cannot be controlled, the more-imperative issue is the ability of consumers to search through information and assess its quality so they are able to avoid untrustworthy information [4]. An Australian study suggested that consumers found it difficult to describe how they distinguished good-quality information on medicines from poor-quality information on medicines on the Internet [5]. However, this study was limited by a small (N = 9), select sample and did not explore in-depth the way consumers searched for and selected information on medicines. There is little information concerning consumer Internet-search behavior for health information. One study reported that participants mainly select Web sites that looked and read professionally and preferred understandable Web sites from official sources that used scientific references [6]. When participants were observed while searching for health information on the Internet, it was found that they mainly used search engines and were described as having "suboptimal" search skills [6]. This study reported that participants did not find blatantly-incorrect health information in their searches [6]. This indicates that they had used selection criteria to decide on the Web sites, though the criteria were not fully described in this paper. Consumer use of information on medicines is an important issue because written information on medicines has been shown to influence consumer attitudes towards their medicines, and affect their medicine-taking behavior [7]. Furthermore, medicines, unlike general health issues, have overtly-commercial imperatives, which may influence the information available. Since the Internet has become a common source of information on medicines, it is important to identify the way consumers are using it. Therefore the aim of this study was to explore consumer use of Internet-based information on medicines. In particular, the objectives were to: examine consumer attitudes to the availability and quality of Internet-based information on medicines; explore consumer reasons for using this information; explore consumer experiences in searching for and appraising information on medicines; investigate the self-reported impact and application of this information. This paper will present results from the broader study on consumer experiences in searching for and appraising Internet-based information on medicines. Methods Selection of Method Focus groups were selected to address the study aims because they are useful for time-efficient, in-depth exploration of issues surrounding topics where there is little information [8- 10]. Since there is little known about how consumers use Internet-based information on medicines, focus groups were an ideal method for exploring this issue. The results of focus groups are not intended to be statistically generalizable, but are used to reveal the range of consumer opinions and attitudes. Research Instrument An interview guide consisting of general themes constructed from the literature was prepared (Table 1). This paper focuses on results ensuing from the exploration of themes 4, 5, and 6. The interview guide was composed of open-ended questions that addressed various issues pertaining to consumer use of Internet-based information on medicines; the questioning route was designed to stimulate discussion [11- 13]. Table 1 Themes for focus group interview guide* General opinions about the Internet as a source of information on medicines. Experiences in using the Internet to seek information on medicines. Reasons for seeking information on medicines. The methods and process of searching for information on medicines. Opinions and critique of the information found. Experiences in the evaluation of the quality of Internet-based information on medicines. Feelings after reading the information. Actions taken as a result of reading the information. Perceived benefits and drawbacks of the Internet as a source of information on medicines * This paper focuses on results ensuing from the exploration of themes 4, 5, and 6. The interview guide and questioning route was pretested with a convenience sample of consumers (N = 13) to test for interpretation, appropriateness, and comprehensiveness, and to establish face and content validity. No significant changes were made to the interview guide or questioning route as a consequence of this pretest. Participant Recruitment After approval was granted by the Human Research Ethics Committee of The University of Sydney, participants were enlisted for the focus groups by a recruitment agency. Participants were recruited from the agency's database of consumers across metropolitan Sydney, Australia via telephone using a screening questionnaire. Consumers were deemed to be eligible for this study if they had sought Internet-based information on medicines in the preceding 12 months. This bounded reference period was applied to allow for a suitable recall of past events [14] while also allowing enough time for consumers to have used the Internet for this purpose. Inclusion criteria required that participants were 18 years of age or over, did not require a translator to take part in focus group discussions, did not have training as a health professional, and did not have specialist Internet training. Participants were financially reimbursed for their time and travel expenses. Study Design Six focus groups were conducted in a number of locations around metropolitan Sydney in March to May 2003. To approximate a representative cross section of consumers, participants were recruited with the intention of including subjects from both genders and across different age groups. Focus groups were age stratified to achieve a level of homogeneity within each group. The use of stratification may increase congruency between participants, thereby allowing a more comfortable discussion [11,15]. Eight persons were recruited for each focus group to ensure that groups were large enough to motivate a discussion, yet small enough allow for all opinions to be heard [11]. The number of groups needed was not determined beforehand because data was collected until saturation occurred (the point where no new themes emerged) [10]. In this study, saturation occurred by the sixth focus group. The focus groups were facilitated by a skilled moderator while 2 assistant moderators observed and took notes. The group discussions were 1 to 1.5 hours in duration and were digitally sound recorded after permission was obtained from all participants. The recordings were transcribed verbatim. Participants also completed a demographics questionnaire that collected data on Internet usage. Data Analysis The verbatim transcripts were entered into NVivo qualitative software [16] and thematically content analyzed using a grounded theory approach. The grounded theory approach is an inductive approach to analyzing qualitative data, where ideas and emerging themes are systematically coded to generate theory [17]. Results This paper presents participants' responses to themes 4, 5, and 6 (Table 1). Responses to other themes are currently unpublished. Demographics Forty-six consumers participated in this study. The age of the participants ranged from 18 to 67 years, with a median of 41 years (interquartile range, 21 years) and a mean of 41.7 years (standard deviation, 12.7 years). Fifty-seven percent of the participants were female. The majority of the participants were employed full-time (58.7%) and about a fifth were either retired or full-time homemakers. Almost half the sample (47.8%) had occupations that could be classified as managers, professionals, or associate professionals [18]. A high proportion of the sample (65.2%) had completed further educational qualifications beyond high school, and 23.9% of the sample had a bachelors or postgraduate degree. Data on participant usage of the Internet is presented in Table 2. The majority of participants had a few years experience in using the Internet and over half had accessed it from both their home and workplace. In addition to using the Internet for information on medicines, most participants also used it for general health information and for services such as e-mail. Data on participant usage of the Internet for information on medicines is presented in Table 3. In addition to using the Internet, many participants also reported using other media such as magazines for information on medicines. This variety of information sources has also been seen in another Australian study on consumer use of Internet-based general health information [19]. Even though most participants (82.6%) were seeking information for themselves, many reported also searching for other family members. This was also reflected in the aforementioned Australian study that showed that 63% of Internet-using consumers sought health information mainly for themselves [19]. Table 3 Participant usage of the Internet for information on medicines (N = 46 participants) Characteristic Usage Frequency, Number of Participants Relative Frequency, (% of Participants) Media sources of information on medicines (more than one category could be selected) Internet Magazines Television Books Radio 46 31 23 22 9 100.0 67.4 50.0 47.8 19.6 Person that Internet medicine information was used for (more than one category could be selected) Self Spouse/partner Child Parent Another relative Friend 38 24 19 17 13 6 82.6 52.2 41.3 37.0 28.3 13.0 Health categories for which information on medicines had been sought for (more than one category could be selected) Allergies Arthritis/joint pain Asthma Cancer Skin disorders Hormones Other miscellaneous Child health Diabetes High cholesterol Immunization Pain and injury High blood pressure Mental health Digestion/stomach disorders Infections Migraine Osteoporosis Alzheimer's disease Dementia 20 14 14 13 12 11 11 10 10 9 9 9 8 8 6 6 6 6 5 5 43.5 30.4 30.4 28.3 26.1 23.9 23.9 21.7 21.7 19.6 19.6 19.6 17.4 17.4 13.0 13.0 13.0 13.0 10.9 10.9 Table 2 Participant usage of the Internet (N = 46 participants) Characteristic Usage Frequency, Number of Participants Relative Frequency, % of Participants Length of experience in the use of the Internet More than 5 years 4 to less than 5 years 3 to less than 4 years 2 to less than 3 years 1 to less than 2 years Less than 1 year 17 13 7 4 4 1 37.0 28.3 15.2 8.7 8.7 2.2 Location of Internet access Home and work Home only Work only 25 16 5 54.3 34.8 10.9 Activities that the Internet is used for (more than one category could be selected) Information on medicines E-mail Health information Travel information/booking Banking/financial services News, weather, sport Job or study related research Real estate Shopping—product research Games and hobbies Chat or instant messaging Shopping—purchasing Purchasing medicines 46 45 43 40 36 34 34 33 33 26 22 19 7 100.0 97.8 93.5 87.0 78.3 73.9 73.9 71.7 71.7 56.5 47.8 41.3 15.2 Searching for Internet-Based Information on Medicines Search Engines All participants had used a search engine to find information on medicines. Most participants had a single favorite search engine that they would always use, but a few reported using more than one search engine to find the information they required. The choice of search engines was determined by many different factors ranging from the default search engine on their browser to active selection based on self-developed criteria. Numerous participants were influenced by the search engine that was used by coworkers, for example: Group 4, Participant 8 I saw it on this guy's computer and . . . I thought 'Oh, I'm going to use this'. That's how I started it at work. Some participants also reported that their browser automatically defaulted to a certain search engine and a few participants were unable to identify the search engine they used, for example: Group 3, Participant 6 Couldn't tell you [the search engine] really. I just log on and use whatever comes on. Many participants used search engines recommended by family and friends. There were certain determinants that led some participants to actively choose a specific search engine. These included perceptions of the credibility of the search engine, ease of use, relation with services such as e-mail, and a lack of advertising. These determinants did not necessarily include perceived quality of the information on medicines obtained through their use. A few participants reported using AltaVista [20] because they thought it had an educational advantage, for example: Group 1, Participant 1 It's got an educational edge, that's my experience. When I was at university doing my second degree, that was one that was sort of promoted as credible I suppose. Some participants preferred to use Ask Jeeves [21] because they could enter the searches in a question or statement format rather than using search terms. Many participants reported using Yahoo! [22] because it appeared as a default homepage, was used as a personal e-mail account, or was advertised through other media. Yahoo! and Google [23] were also said to be useful for Australian-only searches. Google was undoubtedly the search engine the majority of participants used most and preferred. This was especially true of the younger participants. The common perception was that Google appeared to be straightforward and did not focus on advertising, for example: Group 4, Participant 6 It's just got less [rubbish]. It seems to be direct to what you want. I think that other [search engines] always have these categories and they always have suggestions for buying things and stuff like that but Google's pretty much straight to the point. It's simple. Participants also commented that this search engine was useful for suggesting spelling corrections when errors were made, as medicine names were sometimes difficult to spell. A few participants reported preferring Google as their search engine of choice specifically for health-related searches but were unable to explain reasons for their preference. Other search engines used by participants were metasearch engine Dogpile [24], Australian metasearch engine Search66 [25], Australian-based search engine Web Wombat [26], and ninemsn [27], the Australian-based access to search engine MSN Search [28]. Many participants who used metasearch engines were unaware of the difference between these and normal search engines. Generally, although a variety of search engines were used by participants when seeking information on medicines, the majority of participants used the same few dominant search engines. Participants generally preferred search engines with less advertising, and would continue to use the same search engine if they were successful in their searches. Most participants used the same search engine that they used for nonhealth information, and were usually influenced by what was used by friends, family, and colleagues. Search Processes Participants displayed a large variation in the process of searching for information on medicines. Most participants found information by typing the name of the medicine (drug name or brand name) into the search engine. A few participants felt this was the only way of finding information on a medicine, for example: Interviewer How do you put in your searches? Group 6, Participant 2 Medicines are really specific to just the name. Other participants reported looking for broader information, for example: Group 1, Participant 2 I often use a more general [search]. I might use something like 'women's health' or something. And I like to see a whole range of things . . . rather than targeting specifically . . . and then I choose within that. The information found through this type of search was said to be less specific to one medicine and had more general or comparative information. Some participants used more-advanced search techniques such as quotation marks, phrases, and extra words to narrow down their searches. They displayed an understanding of how these techniques helped to focus their searches, for example: Group 4, Participant 6 If you type it in with quotation marks, it'll search for those words together whereas if you type them separately, it'll just search for them anywhere. Participants reporting advanced skills were generally observed to be those who were younger or those who had greater experience of the Internet through work or study. However, it was clear that search skills varied significantly. The following interchange illustrates the mixed levels of understanding as to how search engines work: Group 5, Participant 4 [You need to] ask a specific question . . . 'What are the side effects?' rather than typing in 'penicillin'. Participant 6 Yeah, you really have to do a whole sentence. A whole statement. Participant 3 I would type in 'penicillin side effects'. Participant 4 'Then it could hit on 'penicillin' or it could hit on 'side effects'. The uninformed way in which some participants agreed upon what they considered to be optimal search skills was obvious in the group discussions. The majority of participants in this study who reported searching using less-than-optimal techniques—such as typing in whole questions—tended to be nonworkers, for example, full-time homemakers or retirees. The search skills of participants varied widely and these differences may affect the resulting information that participants encounter. Searching via a search engine however, was not the only way of finding information on the Internet on medicines. Other Methods of Finding Internet-Based Information on Medicines Some participants mentioned ways of finding information on medicines in addition to using search engines. A few participants said that they guessed the Web sites of medicines by typing the name of the medicine in the address bar in the format of www.[brand name or drug name].com. Several participants found information on medicines from Web sites recommended by family and friends, and from seeing advertisements in seniors' and health publications. Some reported bookmarking favorite Web sites for future reference and a few subscribed to mailing lists at health-related Web sites. One participant described searching for information on medicines using online journals. Although aware that the information was not aimed at consumers, this participant still chose to use this means to search for pertinent information on medicines: Group 2, Participant 2 I actually searched via . . . the professional journals . . . And I guess that was a little bit harder to do it that way because . . . reading through the journals was quite difficult. I tend to just go to the abstracts. Participants reported using a variety of search skills to obtain information on medicines. However, the important issue was how they selected and appraised the information. Appraising Internet-Based Information on Medicines Selecting Internet-based Information on Medicines Participants described different ways of choosing which Web site to visit when selecting from the numerous results obtained from using a search engine. Some worked down the list of results from the first one while others looked for keywords in the Web site descriptions or for the Web site's recency. Often participants made a judgment based on the URL (Web page address) of the result, for example: Group 4, Participant 1 I actually like looking at the actual web address, just seeing how professional it is. Like if it's some silly thing, I won't bother going into it. Many participants also reported looking for indicators in the Web site address to determine whether it belonged to a government, a university, an official organization, or a pharmaceutical company. Even though most participants said they would not go beyond the first page of the search results, one expressed the opinion that the best information was in the middle of the results and not on the first few pages. This participant had the erroneous opinion that the first pages of results are older and that results appeared mainly in the order in which the information had been created. Many participants reported looking for the country of origin of the information and preferred information generated from their country of residence, for example: Group 4, Participant 2 If I'm searching for a medication . . . and it brought up some things and I noticed it was in Australia, I click on that. These participants felt that Australian information would be more applicable to them and professed an awareness of health-setting issues such as differences in the brand names and availability of medicines in different countries. However, others had more confidence in United States-based information because they believed that this was where most new research was undertaken. It was clear that most participants did not pay conscious attention to how they selected Internet-based information on medicines, with one referring to the process as "a vibe" that you obtain through experience. Another described this as a feeling that "things have a look of credibility." Similarly, many participants had trouble in articulating their selection process, for example: Group 5, Participant 3 I find that sometimes I get to a site and I think 'Gee, this is a good site, but I don't know how I got there.' You know what I mean? You fluke it. Despite the inability of many participants to express how they selected information on medicines, many were able to express what they would not select. Participants reported quickly rejecting sites that were slow to load, sites that contained too many graphics, and sites that had pop-up advertisements. The process of selecting information on medicines varied among the participants. It appeared that all participants had their own criteria for selecting and rejecting information which may or may not appear logical to others. Credibility of the source, however, appeared to be a common determinant in the criteria of all participants. Credibility of the Source of Internet-Based Information on Medicines Participants expressed conflicting opinions about the credibility of the source of Internet-based information on medicines. Many participants regarded information produced by pharmaceutical companies to be the "official" information on a medicine and therefore trusted this the most, while many others were suspicious of a possible information bias, for example: Group 1, Participant 7 If you're looking at [a pharmaceutical company website], they've got factories throughout the world, they're a pretty good company so . . . you know that they've done so much research it's credible information. Group 1, Participant 6 If it's a pharmaceutical company, they're gonna put a good stance on their drug. Many other participants preferred information that originated from what they considered to be impartial and reputable sources such as government, professional, or disease-focused organizations, or university Web sites. A few participants also reported looking for credentials such as the author's qualifications when assessing the credibility of the information provider. A small number of participants preferred information written by other consumers who had personal experiences in taking the medicine. However, most participants expressed that they would be less likely to trust information on medicines generated by other consumers, for example: Group 1, Participant 6 There are chat rooms . . . if you've ever been prescribed such and such a medication; you'll get people from all around the world . . . Participant 2 Do you not find that a bit dangerous because everything is rather specific to each person's body? Participant 6 Oh yeah, but it would be comparable to having a chat with some of your friends. Some participants felt that the authorship of Internet-based information on medicines should be regulated and feared the reliability of the information because there was "no watchdog" for the information published on the Internet while others regarded it as analogous to the way they would trust information given in common conversation and therefore felt comfortable using information in this context. The credibility of the source of information on medicines was a strong determinant in the selection process. However, in addition to the source participants evaluated information using criteria described in the next section. Evaluating Internet-Based Information on Medicines Participants evaluated information on medicines using criteria such as the motive for the information, the language used, and the applicability to their needs. Almost all participants were skeptical to some degree of Internet-based information on medicines. Many participants professed a universal need for consumers to inherently distrust this information, and to interpret it accordingly. One participant stated that it is important to also consider why the information is on the Internet: Group 1, Participant 6 What are the motives? Are they conflicting, credible? Whoever has posted it, are they trying to make a profit? Other participants described the obviously difficult-to-believe nature of some of this information and looked for signs of conspiratorial or misleading language when deciding whether to trust the information, for example: Group 2, Participant 8 If it says 'hazard free' and 'completely no side effects', for example, I'm more likely to disbelieve than believe that In addition to this awareness of unreliable information on medicines, many participants also expressed an understanding that the information they find may not necessarily be applicable to them and that the information should not be used at face value, for example: Group 2, Participant 7 The thing with medicines is there's no sort of right or wrong . . . Everyone's different, everyone's going to have a different reaction. Group 2, Participant 6 When you ask the doctor, they tell you 'well, [the side effects] happen but it's not like that', I think what happens is that the information is not tailored for myself. It's general information. Pertinent to this appraisal was the information-filtering process described by participants: Group 2, Participant 4 It's always better to try and take as much information and try and sift out what's useless Group 1, Participant 6 When they're talking about people using this medicine, 'ninety-eight percent will die within five years' . . . you have to take that and filter it through a whole bunch of other variables . . . and whether [the information] is not terribly well informed or completely informed. One common way in which some participants were able to filter information on medicines was to use other Web sites for comparison and cross-checking, for example: Group 4, Participant 1 I always go to two or three sites. Although participants reported methods of evaluating information, many expressed a difficulty in their evaluation, for example: Group 2, Participant 4 How do you [figure] out what's useful? Group 3, Participant 7 How do you know what's reliable and what's not? Ultimately, despite an awareness of the shortcomings and difficulties in evaluating the quality of information on medicines, all participants saw the Internet as an important resource for this information, for example: Group 1, Participant 1 I think as patients you expect immediate information and the Internet, whether it's credible or not, it's the fact that people can get it. Discussion The issue of consumer use of Internet-based information on medicines is important because it has been shown that written information on medicines can be interpreted by consumers in ways that may lead to anxiety or apprehension [7,29- 32], and a refusal of prescribed medicines [33]. Conversely, it has been shown that written medicine information increases consumer knowledge about their medicines [29,34- 36] and that well-informed consumers with an increased understanding of the purpose of their medicines may have improved compliance and satisfaction with their therapy [29,31,37- 40]. However, studies on consumer use of written information on medicines have evaluated standardized information on medicines such as that produced by pharmaceutical companies, government or professional bodies, or health care practitioners [7]. In contrast, this study explored Internet-based information, which is neither standardized nor subject to universal quality control. Furthermore, medicines in particular are subject to commercial considerations that may have an impact on the motives for and quality of information. Therefore, the impact of Internet-based information on consumer use of medicines may differ from that reported from consumer use of standardized written information on medicines. The reported search skills of these participants were comparable to those of participants observed while searching for general health information [6] in that they mainly searched using simple strategies in a search engine and chose results primarily from the first page of search results. Although this similarity is not surprising, it does illustrate the overlap between appraising general health information and specifically medicines-related information. Indeed, it was not always possible for consumers in this study to speak on issues surrounding searching for and appraising information on medicines without speaking about other health-related issues. Participants in this study searched for information on medicines using a range of search techniques from simple 1-word searches and advanced techniques to suboptimal techniques. However, although some participants had little understanding of how search engines worked and possessed suboptimal search skills, a few participants described proficient search skills. Contrary to findings where consumers were observed to use information not applicable to their health setting [6], participants generally reported a strong awareness of the limitations of non-Australian information due to health-setting limitations pertinent to medicines use. Participants were conscious that there was an abundance of poor-quality information on medicines on the Internet. They were also predominantly aware that information on the use of medicines and on the incidence of side effects is often based on individual factors that should not be seen as applicable to everyone. Therefore, while consumer evaluation skills have been referred to as "meager" [41], the assumption that consumers believe everything they read does not take into account those participants who are savvy about issues such as bias, commercialism, and the lack of regulation of Internet-based information on medicines. However, the fact that many participants searched for information on a medicine by typing the brand name into a search engine would indicate that it was highly likely that they encountered the Web site of a pharmaceutical company on the first page of results [42], which raises the matter of consumer ability to interpret information on medicines that may not be comparative and unbiased in nature and not aimed at an Australian audience. Even though results from this study would indicate that many participants were aware of these limitations, others still viewed a pharmaceutical company Web site as the official, and therefore exclusive, information on a medicine; this indicates that some consumers may be unaware of or uninterested in information on medicines produced by alternate sources. Nevertheless, it has been suggested that consumers are more likely to search for alternate sources, rather than relying on product brands, as they become more experienced using the Internet [43]. It is clear that there was a variety of skills among participants. Many had not been conscious of some of the issues surrounding the process of searching for and appraising information on medicines and did not undertake this process in the most-constructive way. Furthermore, there have been few studies in the literature that have sought to educate consumers on strategies for effective use of the Internet for health information [44- 47]. Limitations in This Study There are several important limitations in this study. First, as this information is self-reported, consumers may not actually search for and appraise information in the same way as they describe. Such a discrepancy was demonstrated when participants in an observational study were reported to be less likely to look for the sources of the information than was apparent from claims in focus groups [6]. However, participants in that observational study were not searching for information that they would personally use; this may have meant that they were less concerned about the quality of the information. Second, the bounded period of 12 months in the inclusion criteria may be too long for consumers to correctly remember details of how they searched for and chose information. It might have been beneficial to actually perform a search as an activity to stimulate the participants' memories. Third, participants in group situations may feel compelled to provide socially-desirable answers that are not necessarily accurate. In this study, we sought to minimize this by informing participants that their results would be confidential and that they were welcome to speak about anything they felt even if they disagreed with someone else. However, this does not negate the problem. Although the use of individual interviews may help to minimize this discrepancy, this method is more time-consuming and cannot use group interaction for the generation of ideas. Last, certain actions are intuitive and therefore difficult to articulate. Most participants were not able to adequately describe their search and appraisal processes, which suggests that this process may largely be a form of tacit or implied knowledge. Therefore, future research needs to take into account actual observed (rather than reported) search and appraisal skills of consumers who are seeking information on medicines for their own use. Conclusion and Future Research The results of this study show that consumers may benefit from greater awareness and education on the significance of good search and appraisal skills for information on medicines so that this process is deliberate and conducted with thought rather than being random and tacit. Furthermore, there is evidence that consumers may support education that shows them how to search for information on medicines on the Internet [48]. However, health promotion and education needs to take into account the variety of consumer skills in both searching for and critically evaluating information. Pharmacists are in an ideal position to provide consumer training as they frequently counsel consumers on medicines [49] and have consumers present them with information from the Internet [19]. However, to successfully deliver this program, pharmacists need to be trained in these skills . Furthermore, the impact of pharmacist education on consumers' searches for Internet-based information on medicines and appraisal of that information needs to be evaluated. Therefore, future research by this team will be on the development of a health-promotion program for pharmacists to train consumers to search for and appraise Internet-based information on medicines.

Immunogenetics-4-1-2206249

Variation analysis and gene annotation of eight MHC haplotypes: The MHC Haplotype Project

The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine. Introduction The MHC has long been believed to be the most important region in the human genome with respect to infection, inflammation, autoimmunity and transplant medicine (Lechler and Warrens 2000). This was recently confirmed by the largest genome-wide association study carried out to date for seven common diseases, including two autoimmune diseases (type 1 diabetes and rheumatoid arthritis) and one inflammatory disease (Crohn’s disease). The highest associations were found between the MHC and these two autoimmune diseases (The Wellcome Trust Case Control Consortium 2007). The complex aetiology of MHC-associated disease coupled with high density, polymorphism, linkage disequilibrium (LD) and frequent non-Mendelian inheritance of gene loci have made it challenging to identify variations that cause or contribute to disease phenotypes. Additional limiting factors have been our incomplete knowledge of the allelic variation of genes and regions flanking the nine classical human leukocyte antigen (HLA) loci and the lack of a single haplotype reference sequence, the original reference sequence being a composite of multiple MHC haplotypes (Mungall et al. 2003; The MHC Sequencing Consortium 1999). Recognizing that the future identification of variants conferring susceptibility to common disease is critically dependent on fully informative polymorphism and haplotype maps, the MHC Haplotype Consortium formed in 2000 with the aim to generate these critical data and to make them publicly available as a general resource for MHC-linked disease studies. Similar efforts, but with different experimental approaches, were also carried out in Japan (Shiina et al. 2006) and the USA (Smith et al. 2006). To develop the resource, eight HLA-homozygous MHC haplotypes were selected on the basis of conferring either protection against or susceptibility to two autoimmune diseases, type 1 diabetes and multiple sclerosis, and that represented common haplotypes in European populations. In the subsequent years, incremental data, materials and tools comprising this resource have been released (Allcock et al. 2002; Horton et al. 2004; Stewart et al. 2004; Traherne et al. 2006) and have contributed towards the construction of a high-resolution LD map and a first generation of HLA tag single nucleotide polymorphisms (SNPs; de Bakker et al. 2006; Miretti et al. 2005) and the identification of a second MHC susceptibility locus for multiple sclerosis (The International Multiple Sclerosis Genetics Consortium; Yeo et al. 2007). In this paper, we report the final account of this international effort, including, analysis of the last four of the eight haplotypes, up-to-date variation statistics, gene annotation, population-specific aspects and a detailed description of the databases and tools for viewing and accessing the data in the context of existing genome annotation. Materials and methods Variation analysis The method previously reported for comparison of MHC haplotype sequences (Stewart et al. 2004; Traherne et al. 2006) was extended to cover all eight haplotypes. Briefly, the most suitable method proved to be a clone by clone comparison using the discrepancy-list option of the cross_match program (Green, unpublished; http://www.phrap.org/), an implementation of the Smith–Waterman sequence alignment algorithm (Smith and Waterman 1981), using the alignment of a haplotype clone sequence with the appropriate overlapping reference sequence from a PGF clone or clones. All variations were submitted to dbSNP using the submitter handle SI_MHC_SNP and user identifiers of the form [PGF BAC clone sequence version]_[position in PGF BAC clone sequence]_[variation change]. Thus, AL662890.3_6645_TC indicates a substitution in which the base T at base position 6645 in AL662890.3 (PGF BAC 308K3) was substituted by C in the other haplotype. In the case of indels, the ‘variation change’ consists of ‘i’ or ‘d’ (for insertion or deletion), followed by a numerical value for the length of the indel, in turn followed by the inserted or deleted sequence if this were of 12 or fewer bases. For longer indels, an X value is given, which refers to a look-up table (http://www.sanger.ac.uk/HGP/Chr6/MHC/Xfile). Thus, AL662890.3_7470_d8TACACACA indicates a deletion in AL662890.3 after base 7470 of the eight bases ‘TACACACA’. Further, AL662890.3_10559_i5ATATT indicates an insertion in AL662890.3 starting after base 10559 of the five bases ‘ATATT’. AL662890.3_7475_d14X1 indicates a 14-base deletion after base 7475 in AL662890.3 of a sequence coded as X1 which is ‘ATACACACACACAC’. Major indel sequences, appearing as breaks in the cross_match discrepancy lists between two clones from difference haplotypes, were extracted and subjected to analysis by RepeatMasker to detect the presence of retrotransposible elements. Gene annotation The finished genomic sequence for each of the eight haplotypes was analysed using a modified Ensembl pipeline (Searle et al. 2004). CpG islands were predicted on unmasked sequence. Interspersed and tandem repeats were masked out by RepeatMasker (Smit, AFA, Hubley, R & Green, P. RepeatMasker Open-3.0. 1996–2004, http://www.repeatmasker.org) and Tandem Repeats Finder (TRF; Benson 1999), respectively. The sequence was then BLAST searched (BLAST, basic local alignment search tool; Altschul et al. 1990) using a vertebrate set of complementary DNAs (cDNAs) and expressed sequence tags (ESTs) from the European Molecular Biology Laboratory (EMBL) nucleotide database (Kulikova et al. 2007), followed by the re-alignment of significant hits. Non-redundant proteins were aligned similarly. Protein domain matches were provided through alignment of Pfam to the genomic sequence using Genewise (Birney et al. 2004), thereby providing protein domain data to the annotator. Ab initio gene predictions were performed by Genscan (Burge and Karlin 1997) and Fgenesh (Salamov and Solovyev 2000), and potential transcriptional start sites were predicted by Eponine (Down and Hubbard 2002). Analysis results were displayed, and annotation was performed through an in-house annotation software system. Genes were manually annotated according to the human and vertebrate analysis and annotation (HAVANA) guidelines (http://www.sanger.ac.uk/HGP/havana/) using evidence based on comparison with external databases as of August 2005. All gene structures are supported by transcriptional evidence, either from cDNA, EST, or protein. In general, annotations are supported by best-in-genome evidence. Haplotype-specific evidence is assigned where possible. As with previous MHC annotation (Stewart et al. 2004; Traherne et al. 2006), some olfactory receptors have been built upon protein homology alone because of their restricted expression. Locus and variant types were annotated according to established standards (Harrow et al. 2006), with the modification that, within the MHC region, the artefact locus has been used to tag historically annotated structures that are no longer deemed valid. HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQA1, and HLA-DQB1 allele types were assessed by comparison against the IMGT/HLA database (http://www.ebi.ac.uk/imgt/hla/; Marsh et al. 2005). Annotation status of haplotypes The PGF, COX, and QBL haplotypes have already been annotated in detail (Stewart et al. 2004; Traherne et al. 2006). It was decided, however, to re-annotate and update this annotation to maintain consistency between all eight haplotypes with the current supporting evidence and pipeline analyses. The SSTO haplotype was manually annotated de novo. The new annotation from the PGF haplotype was projected through a DNA–DNA alignment to each of the remaining haplotypes (APD, DBB, MANN and MCF) where possible. This projection was checked thoroughly and non-alignable regions were manually adjusted (including the C4 and HLA-DRB1 hypervariable regions). Polyadenylation sites and signals were not annotated for haplotypes APD, DBB, MANN and MCF because of time constraints. In the main, however, these features may be assumed to correspond to the same positions as in the first four haplotypes. Combination of variation and annotation data By employing a series of Perl scripts, the array of haplotype variation was combined with the annotation of gene loci, repeat elements and microsatellites, extracted from the Vertebrate Genome Annotation (VEGA) database in general feature format (GFF; http://www.sanger.ac.uk/Software/formats/GFF/), to determine the variation status of all loci. Distribution of sequenced HLA haplotypes in Europeans To assess the distribution of sequenced haplotypes at the population level, 180 founder haplotypes were reconstructed using genotypic data from Centre d’Etude Polymorphisme Humain (CEPH) trios (de Bakker et al. 2006). A ~214 kb segment spanning the HLA–DRB1–DQB1 genes was selected for the analyses. This segment, represented by 54 SNPs, is delimited by rs2187823 and rs2856691, with NCBI build 36 chromosome 6 coordinates 32547486 and 32761413, respectively. Phased haplotypes with known HLA–DRB1–DQB1 alleles were then used to construct a neighbor-joining tree (Kumar et al. 2001) and a phylogenetic network (Bandelt et al. 1999). Resources All sequences presented in this paper have been submitted to the EMBL/GenBank/DNA Data Bank of Japan (DDBJ) database and allocated accession numbers. For clarity, all bacterial artificial chromosome (BAC) clones are referred to using their accession numbers. The annotation of each haplotype has been entered in the VEGA database and is accessible through its browser (http://www.VEGA.sanger.ac.uk). All variations from the study were submitted to dbSNP (http://www.ncbi.nlm.nih.gov/SNP) using the submitter handle SI_MHC_SNP. BAC clones from the CHORI-501 (PGF) and CHORI-502 (COX) libraries can be requested from BACPAC resources (http://www.bacpac.chori.org/). Clones from the other libraries can be requested from [email protected]. The web site for the MHC Haplotype Project provides links to various data resources (http://www.sanger.ac.uk/HGP/Chr6/MHC/). DAS sources for all substitutions and indels are available from http://www.das.ensembl.org/das as follows: ens_35_COX_SNP ens_35_COX_DIPens_35_QBL_SNP ens_35_QBL_DIPens_35_SSTO_SNP ens_35_SSTO_DIPens_35_APD_SNP ens_35_APD_DIPens_35_DBB_SNP ens_35_DBB_DIPens_35_MANN_SNP ens_35_MANN_DIPens_35_MCF_SNP ens_35_MCF_DIP These can be accessed via the VEGA browser. Results and discussion Variation analysis One of the main aims of the MHC Haplotype Project was to generate a comprehensive variation map of this most variable region of the human genome. To achieve this, eight haplotypes were sequenced and subjected to variation analysis. Table 1 details the lengths of the sequence contigs, the number of sequence gaps and the allelic types of major HLA loci for each haplotype. Of the eight haplotypes sequenced, three have already been described: PGF and Cox (Stewart et al. 2004) both of which formed single contigs of approximately 4.7 Mb, and QBL (Traherne et al. 2006), of approximately 4.2 Mb but with five gaps. The remaining haplotypes sequenced all contained gaps, their coverage ranging from 2.33 Mb (DBB with 28 gaps) to 4.19 Mb (MANN with 10 gaps). Table 1Haplotype sequence contig length, number of gaps and HLA allele typesHaplotypeLength (bp)GapsHLA-AHLA-BHLA-CHLA-DQA1HLA-DQB1HLA-DRB1PGF47548290A*03010101B*070201Cw*07020103DQA1*010201DQB1*0602DRB1*150101COX47318780A*01010101B*080101Cw*070101DQA1*050101DQB1*020101DRB1*030101QBL42492725A*260101B*180101Cw*050101DQA1*050101DQB1*020101DRB1*030101APD416096516A*01010101––––−DBB233010128A*02010101–Cw*06020101DQA1*0201DQB1*030302DRB1*070101MANN419101410A*290201B*440301Cw*160101DQA1*0201DQB1*0202DRB1*070101MCF408741315[A*020101]B*15010101Cw*030401DQA1*0303DQB1*030101–SSTO370424922A*320101B*44020101Cw*050101DQA1*030101DQB1*030501DRB1*040301Sequence length (bp) and number of gaps in each haplotype sequence, together with the HLA gene types obtained by BLAST against the IMGT/HLA database. Dashes or data in square brackets indicate the absence or the partial presence, respectively, of a gene owing to a sequence gap. For the variation analysis, each of the above haplotypes was compared with the PGF reference sequence, resulting in the identification of 44,544 variations (37,451 substitutions and 7,093 indels, Table 2), which have all been submitted to dbSNP. The success of this exercise is illustrated by the fact that examination of this public database (NCBI dbSNP build 127, March 2007) showed that there were only a further 19,598 variations, submitted by other laboratories, in this region which were not identified by this project. In accordance with the annotation that we also generated for each haplotype (see below), the variations shown in Table 2 were further classified as untranslated region (UTR), exonic, intronic, intergenic and eight more sub-categories (Table 3). Coding substitutions, which are of particular interest with respect to altered functionality, were further classified as synonymous, non-synonymous conservative, or non-synonymous non-conservative and grouped depending on whether they affected HLA or other genes (Table 4). The actual variations and affected amino acids can be viewed using the VEGA browser as illustrated in Fig. 1 and described in the corresponding section later on. In addition, we have analysed all haplotype sequences for inversions, which represent another important variation category that has been linked to genomic disorders (Shaw and Lupski 2004). Using Ssaha2 (Ning et al. 2001), we found no evidence of any inversion polymorphism within the generated sequences but could not exclude large-scale (e.g. involving entire MHC) inversions with breakpoints outside the MHC regions sequenced here. Fig. 1Annotation and variation data in VEGA. VEGA ‘overview’ (a), ‘detailed view’ (b) and ‘basepair view’ (c) example of the variation in the OR2J1 locus in which a STOP codon is present in all haplotypes except MCFTable 2Distribution of substitutions and indels amongst haplotypesHaplotypeSubstitutionsIndelsALLCOX15,9672,39318,360QBL15,2822,36017,642SSTO14,9822,30017,282APD4,2306834,913DBB14,2551,97516,230MANN12,1021,65413,756MCF10,7901,54512,335Overall37,4517,09344,544Number of variations found by comparing the PGF haplotype sequence with each of the other haplotype sequences in turn.Table 3Distribution of substitutions and indels within different sequence regions amongst haplotypesSequence regionBase pairsCOXQBLSSTOAPDDBBMANNMCFSIDSIDSIDSIDSIDSIDSIDCoding247,5053538503193802740351640193482UTR155,960382344385933135389326393033530931Intronic1,283,4723,1415713,1355902,6585056021472,8975092,1853932,126404Total intragenic1,686,9373,8766134,0766683,3695427141563,5745542,8894372,783437Pseudogenic57,223235152262122719101819110109611310Pseudogenic intron63,1085075422027215181582025822981317913Transcript exon78,0921903020733119227181361788167015Transcript intron332,7051,2431971,1862161,05315585291,2451921,08116126853REPEATS:LINEs608,4292,1102212,0152402,388255755932,0972172,0841931,530164SINEs428,5671,3814281,3164011,3113853461341,229318928241936271Other repeats487,8632,6052072,5182292,514207925562,7481992,1981772,170169Total in repeats1,524,8596,0968565,8498706,2138472,0262836,0747345,2106114,636604Microsatellite15,18518616895852221981429607661719068All above3,297,59012,3331,93311,8591,92011,4181,8013,16953311,5381,6059,5361,3158,1391,200Other intergenic996,7203,6344603,4234403,5644991,0611502,7173702,5663392,651345Total4,754,82915,9672,39315,2822,36014,9822,3004,23068314,2551,97512,1021,65410,7901,545Variations shown in Table 2 ascribed to sequence regions identified during annotation. These included exonic, UTR and intronic regions of coding; pseudogenic and transcript loci; repeat elements, microsatellites and other intergenic regionsS Substitution, ID indelTable 4Codon variation caused by substitutions in HLA and other gene lociCodons variation by virtue of substitutionsCOXQBLSSTOAPDDBBMANNMCFHLAOtherTotalHLAOtherTotalHLAOtherTotalHLAOtherTotalHLAOtherTotalHLAOtherTotalHLAOtherTotalSynonymous498113071106177725712912425666913559791388052132Non-synonymousTotal Conservative12576201184121305164722361927461207619614491235147562036842110102721749239131111829674010777601378235117Non-conservative573491824913172331058917533689673198652186Total174157331255227482236129365205171186145331203170373227108335Coding substitutions analysed for their effects on protein sequences and listed in by haplotype for HLA genes (HLA-A HLA-B HLA-C HLA-DRB1 HLA-DRA HLA-DQA1 HLA-DQB1 HLA-DPA1 HLA-DPB1) and for all other genes according to the changes they induced in codons as either synonymous, non-synonymous conservative, or non-synonymous non-conservative changes. Gene annotation There have been several previous annotations of the gene content of the MHC (Horton et al. 2004; Mungall et al. 2003; Stewart et al. 2004; The MHC Sequencing Consortium 1999; Traherne et al. 2006). The maximum region annotated in this study extends from the telomeric ZNF452 gene in the MHC extended class I region (COX haplotype) to the centromeric ZBTB9 gene just telomeric of the MHC extended class II region (PGF and SSTO haplotypes). The PGF haplotype (Stewart et al. 2004) remains the longest complete MHC haplotype, encompassing 320 annotated loci with 1,267 variants. The number of variants ascribed to each locus-type is listed in Table 5. A comparison of the statistics for loci in each haplotype is shown in Table 6. Table 5Splice-variant statistics for PGF annotationTypeNo.Total splice variants1,267Coding523Unprocessed_pseudogene50Processed_pseudogene41Expressed_pseudogene7Transcript271Putative71Retained_intron263Nonsense_mediated_decay30Artefact11Total loci320Splice variants annotated in the PGF haplotype.Table 6Gene annotation statistics for eight MHC haplotypesLocus typePGFCOXQBLSSTOAPDDBBMANNMCFCoding16515915013182146129150Transcript2828262619262722Putative181815156161214Pseudogenes total9895939859929575Unprocessed5048485336525342Processed4142403919343728Expressed75564655Artefact111110110000Total loci320311294281166281264261Total variants1,2671,1911,1551,0585681,1389601,115Annotation statistics for loci in each haplotype. For definitions of locus types see “Materials and methods”. VEGA database and browser The VEGA database provides access to gene annotation of the eight MHC haplotype sequences, a valuable public resource and a means of integrating annotation and variation data. The VEGA database also provides the facility to download nucleotide or peptide sequences for genes of interest, by selecting ‘export cDNA’ or ‘export peptide’ from the menu obtained by clicking on gene cartoons in the VEGA ‘detailed view’ or ‘basepair view’ window. From these, any desired alignments can be made. Variation data may be viewed in the browser linked to a distributed annotation system (DAS) source of any given variation (see “Materials and methods”). This is illustrated An example of the use of this browser to view a C to T substitution is illustrated for the OR2J1 locus (Fig. 1). An overview of the genomic environment is given in Fig. 1a, showing the gene within a cluster of olfactory gene loci on chromosome 6. The detailed view (Fig. 1b) shows OR2J1 with associated variations in all haplotypes. The basepair view (Fig. 1c) illustrates the presence of the C/T substitution in all haplotypes except MCF, and its positioning above the translated sequence, at the first position of a CAG codon, indicating the presence of a stop codon instead of glutamine. Annotation changes In addition to loci annotated in the previous studies, newly recognised with official Hugo Gene Nomenclature Committee (HGNC) symbols have also been annotated. These have included the mitochondrial coiled–coil domain protein 1 gene MCCD1 (Semple et al. 2003) and the related unprocessed pseudogenes MCCD1P1 and MCCD1P2, as well as the zinc-finger and BTB domain-containing protein gene ZBTB9, annotated at the very centromeric boundary of the sequenced region. The C6orf21 gene (De Vet et al. 2003; XXbac–BPG32J3.17-001) of the MHC class III region was annotated as a separate locus from the adjacent centromeric locus LY6G6D (splice variants XXbac–BPG32J3.4-001 and XXbac–BPG32J3.4-002). There was, however, a further coding splice variant of LY6G6D (XXbac–BPG32J3.4-004), which spanned not only the other LY6G6D splice variants but also C6orf21, suggesting that this is a possible so-called chimeric transcript (Parra et al. 2006). HLA-DRB1 hypervariable region Of the five newly annotated MHC haplotypes, APD alone exhibited the HLA–DRBDR52 antigenic specificity found on DRB1*3, DRB1*05 (DRB1*11 and DRB1*012) and DR6 (DRB1*13 and DRB1*14) haplotypes and encoded by HLA–DRB3, whereas the remainder (SSTO, DBB, MANN and MCF) exhibited the DR53 specificity, encoded by HLA–DRB4, here annotated for the first time in genomic sequence. The HLA–DRB53 sequences included three known loci (HLA–DRB4, HLA–DRB7 and HLA–DRB8), as well as three novel pseudogenes (DASS–218M11.1, DASS–23B5.1 and DASS–23B5.2). DASS–23B5.1 corresponds to a pseudogene derived from the gene for the protein kinase, interferon-inducible double-stranded RNA dependent activator (Chida et al. 2001) for which the symbol PRKRAP1 has now been recognised. A further processed pseudogene, FAM8A5P (Jamain et al. 2001), was also annotated in the DR53 specificity. HLA-V and HLA-P Our analysis showed that the two unprocessed class I pseudogenes HLA-V and HLA-P ( previously HLA-75 and HLA-90, Geraghty et al. 1992) should in fact be merged together; individually they merely represented the 5′ and 3′ portions of a single unprocessed pseudogene, separated by repeat elements. According to our annotation guidelines (see “Materials and methods”), the newly merged locus was assigned the symbol from the 3′ component, in this case, HLA-P. Best-in-genome nucleotide evidence was found to support five transcript variants at the 5′ end, which, together with evidence for continued locus-transcription, led us to designate the locus as a transcribed pseudogene. Because transcription appears to still occur at this locus, it was, therefore, designated as a transcribed pseudogene. A further six expressed pseudogenes were identified in the MHC region (HLA–DPB2, HLA-J, CYP21A1P, HLA–DRB6, HLA–L and PPP1R2P1). RCCX hypervariable region This module within the MHC class III region, named for its gene content (RP-C4A/B-CYP21-TNXB), may be duplicated or triplicated (Chung et al. 2002), and the pseudogenes CYP21A1P, TNXA and STK19P contain the complement component gene, C4, in either or both of the two versions, C4A and C4B (Awdeh and Alper 1980). This gene may also be present in either long (C4AL, C4BL) or short (C4AS, C4BS) forms depending on the presence or absence of an inserted HERVC4 element in intron 9. Contrary to our previous annotation (Stewart et al. 2004) see also legend to (Fig. 2), the PGF haplotype now appears to possess an arrangement in which C4AL precedes C4BL, whereas COX has a single module with C4BS and QBL has a single module with C4AS (Traherne et al. 2006). For the new haplotype sequences reported in this paper, SSTO was bimodular with two copies of C4BL, whereas DBB was bimodular with C4AL followed by C4BS. Although a sequence gap was present in MCF, this haplotype appeared to be bimodular in that, although the telomeric copy of the C4 gene could not be identified, there was evidence for the pseudogenes CYP21A1P, TNXA and STK19P in a telomeric module. The second centromeric module in MCF contained C4AL. The RCCX region in the APD and MANN haplotypes was incomplete because of sequence gaps. C6orf205 Variability in the C6orf205 gene has been reported to consist of extension of the minisatellite in exon 2 from 27 copies in PGF and COX to 31 copies in QBL (Traherne et al. 2006). In the newly annotated haplotypes, we found the minisatellite to extend to 29 in MANN. The APD, DBB and MCF possessed 27 copies. There was a sequence gap in this region in the SSTO haplotype. MICA The known allelic polymorphism of MICA reported for the DRB1*03 QBL cell line sequence, in which a four-base insertion (GCGT) extended the open reading frame in coding exon 5 haplotype (Traherne et al. 2006), was also present in the DRB1*07 MANN haplotype. The insertion was absent from PGF, COX and SSTO. No sequence was available in APD, DBB and MCF for this gene. PPP1R2P1 The intronless pseudogene PPP1R2P1 reported to have a full-length open reading frame in the PGF, COX and QBL haplotypes (Stewart et al. 2004; Traherne et al. 2006) was found to have a similar open reading frame in the DBB and MANN haplotypes but to have the frameshift mutation seen in the original chromosome reference sequence (Mungall et al. 2003) in the SSTO, APD and MCF haplotypes. PSORS1C1 The QBL haplotype remains the only one in which there was a single nucleotide deletion in a polyC tract of exon 5 (Traherne et al. 2006). DBB, MANN and MCF resembled PGF and COX. No sequence was available for this gene in SSTO or APD. POU5F1 The PGF haplotype has been reported to have a disrupted start codon for alternative splice variant of POU5F1 (Traherne et al. 2006). This disruption was not present in COX or QBL nor was it present in the further haplotypes reported in this paper, namely SSTO, DBB, MANN and MCF. APD had no sequence in this region. OR2J1 This olfactory receptor OR2J1 has been reported to have both functional and non-functional alleles (Ehlers et al. 2000), the latter the result of a premature stop codon at amino acid position 194 introduced by a substitution in the coding sequence. In our annotation, we found the PGF and MCF haplotypes to contain the full-coding sequence, whereas the COX, QBL SSTO, APD, DBB and MANN haplotypes to contain the truncated sequence as an unprocessed pseudogene (see above and Fig. 1). Other annotation differences Other loci included in the current but not the previous PGF annotation were HCG4P11, HCG4P8, HCG4P7, HCG4P5, HCG4P3 and the loci without symbols listed in Table 7. Previously annotated loci not annotated in this study or considered artefacts because they did not reach our current standards of annotation included HLA-X, C6orf215, HCG2P7, HCG8, HCP5P2, HCP5P3, HCP5P6, HCP5P12, HCP5P13, HCP5P14, HCP5P15, HCG8 and HCG26. Table 7Other newly annotated lociLocusLocus typeXXbac-BCX196D17.5TranscriptXXbac-BPG116M5.14PutativeXXbac-BPG116M5.15PutativeXXbac-BPG116M5.16PutativeXXbac-BPG118E17.9PutativeXXbac-BPG126D10.10Processed pseudogeneXXbac-BPG126D10.11Processed pseudogeneXXbac-BPG13B8.10TranscriptXXbac-BPG13B8.9Unprocessed pseudogeneXXbac-BPG154L12.4PutativeXXbac-BPG181B23.4TranscriptXXbac-BPG181M17.4PutativeXXbac-BPG246D15.8TranscriptXXbac-BPG248L24.10Unprocessed pseudogeneXXbac-BPG248L24.9Processed pseudogeneXXbac-BPG249D20.9PutativeXXbac-BPG250I8.13TranscriptXXbac-BPG254F23.5PutativeXXbac-BPG254F23.6PutativeXXbac-BPG254F23.7TranscriptXXbac-BPG254F23.7PutativeXXbac-BPG27H4.7TranscriptXXbac-BPG27H4.8TranscriptXXbac-BPG294E21.7Processed pseudogeneXXbac-BPG296P20.14PutativeXXbac-BPG296P20.15PutativeXXbac-BPG299F13.14PutativeXXbac-BPG308J9.3TranscriptXXbac-BPG308K3.5PutativeXXbac-BPG308K3.6TranscriptXXbac-BPG309N1.15Unprocessed pseudogeneXXbac-BPG32J3.18PutativeXXbac-BPG8G10.2Unprocessed pseudogeneDAQB-12N14.5TranscriptDAQB-331I12.5PutativeDAQB-335A13.8TranscriptNewly annotated loci without HGNC symbols. Non-canonical splice sites Eight variants within six loci were shown to exhibit haplotypic variation at their splice sites (canonical to non-canonical motif; Table 8). These variations may affect the gene expression at the post-transcriptional level. Hoarau et al. (2004, 2005) have already described the differential splicing within the HLA–DQA1 locus, and this can clearly be seen by comparing the new HLA–DQA1 annotation through the VEGA genome browser. Table 8Haplotype variation at splice sitesGeneVariantAffected exonsDonor*Acceptor*dbSNP cluster IDBest evidencePGFQBLCOXSSTODBBAPDMANNMCFTRIM3123/4ggttggrs28400887cDNANCNCNCCNDNCNCCTRIM3152/3ggttggrs28400887ESTNCNCNCCNDNCNCCC4B73/4ggtcgg–ESTNCNDNCCNCNDNDNDC4A73/4ggtcgg–ESTNCNCNDCNCNDNDNCHLA-DQA144/5ggtcggrs707947cDNACCCNCNCNDNCNCHLA-DQA154/5ggttaa/caars3667cDNANCNCNCCCNDCCHLA-DRB122/3gatcagrs9271083ESTNCCCCCNDCNDGene loci and variants that are affected by disruptive variations at splice sites. C Canonical splice site (donor = ngt; acceptor = nag), NC non-canonical, and ND no data (gene absent or gap). Donor and acceptor variable nucleotides in bold with equivalent dbSNP cluster ID number given in column to right. The C4A and C4B genes are, for these purposes, effective duplicates of each other. The two TRIM31 variants share the same splice site (but differ elsewhere in structure). The two HLA–DQA variants share the same donor but have alternative acceptors. Note the mutually exclusivity of these variants amongst the haplotypes (Hoarau et al. 2004; Hoarau et al. 2005). Combination of variation and annotation data The data for sequence contig length, gaps, variation rate within haplotypes and PGF coding gene annotation have been combined in the map in Fig. 2. This illustrates the concentration of variation around the HLA gene loci, specifically in 3 areas: around HLA-F, HLA-G and HLA-A; around HLA-C and HLA-B; and around HLA-DRB1, HLA-DQA1, HAL-DQB1, HLA-DQA2 and HLA-DQB2. The variation status of genes of the PGF haplotype is shown in Table 9. Fig. 2Variation and annotation map of eight MHC haplotypes. The map represents the complete reference sequence (orange bar split into three 1.6 Mb sections) labelled PGF and marked with a scale (Mb) and approximate megabase positions on the NCBI36 build of chromosome 6 (grey milestones). Below the reference sequence are arrows representing gene positions and orientations colour-coded for variation status (invariable, black; with synonymous variation only, green; with non-synonymous, conservative variation, red; with non-synonymous, non-conservative variation, purple; see Table 8) and their symbols on a band denoting MHC class (extended class I, green; class I, yellow; class III, pale orange; class II, light blue; extended class II, pink; outside MHC, pale grey). Above the reference sequence, coloured bands represent the sequences of the other seven haplotypes (COX, orange; QBL, mauve; APD, yellow; DBB, green; MANN, light blue; SSTO, dark blue; MCF, purple) with sequence gaps in dark grey; the RCCX hyper-variable region shown with green (C4A block) and/or red (C4B block) or black (block absent), and the HLA–DRB hyper-variable region in shades of blue-green. Above each haplotype bar, a bar-graph represents total variation between the haplotype and the reference sequence (total variations/10 kb) in dark red. Re-examination of the sequence AL645922 from the PGF haplotype, which contains the RCCX region, has shown that the original assembly was erroneous. Correction of these errors leads us now to the conclusion that the C4A gene precedes the C4B gene in this clone sequence. This new gene order is reflected in Fig. 2Table 9Variation status of the main coding variant of each gene in the PGF haplotype annotationInvariableSynonymous variation onlyNon-synonymous variationConservative variationNon-conservative variationABCF1BAT1aAGERBAT2AGPAT1BAT5BRD2aBAT3AIF1C2BTNL2BAT4APOMCREBL1C6orf21C4AATP6V1G2DAXXC6orf27C4BB3GALT4DDR1aCFBC6orf10C6orf134GNL1DOM3ZC6orf100C6orf136aGPSM3DPCR1C6orf15C6orf26GTF2H4EGFL8C6orf205C6orf48HLA-DOAaEHMT2C6orf25CLIC1HSPA1BFKBPLC6orf47CSNK2BLY6G6CGABBR1CCHCR1CUTAMSH5HLA-DMACDSNCYP21A2PBX2HLA-DOBCOL11A2DDAH2POU5F1HLA-DQB2DHX16FLOT1PPP1R11HLA-DRAHLA-AHLA-DPA1PRR3HSPA1AHLA-BHLA-DRB5RING1LY6G6DHLA-CHSD17B8RNF5MCCD1HLA-DMBKIFC1aRXRBMOGaHLA-DPB1LSM2SYNGAP1OR11A1HLA-DPB2LST1TRIM10OR2H2HLA-DQA1LTBTRIM26OR2J1HLA-DQA2LY6G5CTRIM27OR2J2HLA-DQB1LY6G6ETRIM39aOR2J3HLA-DRB1MAS1LVPS52PHF1HLA-EMRPS18BZBTB12PSMB9HLA-FNCR3ZBTB9RPP21HLA-GNEU1ZNRD1SFTPGHSPA1LNRMSKIV2LIER3OR2B3SLC44A4KIAA1949OR2H1TAP2LTAOR2W1TRIM15LY6G5BPFDN6WDR46MDC1PPP1R10ZBTB22MICAPRRT1ZNF311MICBPSMB8bNFKBIL1RDBPNOTCH4RGL2OR10C1RPS18OR12D2SLC39A7OR12D3STK19OR5U1TNFOR5V1TUBBPPT2ZFP57PSORS1C1PSORS1C2RNF39TAP1TAPBPTCF19TNXBTRIM31TRIM40UBDVARSVARSLGene coding sequences may be invariable (no recorded variation), have synonymous variation only (variation at the nucleotide but not the peptide level) or have non-synonymous variation (variation at both the nucleotide and peptide level), which in turn, may be conservative or non-conservative variation according to the criteria of positive or negative values in the BLOSUM62 matrix. The main coding variant is that numbered 001 in the VEGA database except for LY6G6E and HLA-DPB2 where the main variant is not coding. C4A and C4B were excluded from calculation of variation because the order of these genes in the PGF sequence precluded alignment with other haplotype sequences. Nevertheless, alignment of the coding sequences for each gene separately showed that there were non-synonymous, non-conservative variations. HLA-DRB5 is present in this study only in the PGF haplotype and, therefore, here appears invariableaCoding genes where the main variant does not harbour non-conservative, non-synonymous variation but other variants do (BAT1 BRD2 DDR1 C6orf136 HLA-DOA MOG KIFC1 and TRIM39).bSimilarly, coding genes where the main variant does not harbour conservative non-synonymous variation but other variants do (PSMB8). As well as the variations reported above, major indels revealed as breaks in cross_match discrepancy lists and analysed by RepeatMasker are given in Table 10. Many of these have been previously reported (Dangel et al. 1994; Dunn et al. 2003; Dunn et al. 2002; Gaudieri et al. 1999; Horton et al. 1998; Kulski and Dunn 2005; Stewart et al. 2004). These indels were most frequently but not exclusively associated with AluY elements. Table 10Major indels in the form of retrotransposible elementsChr6 pos’nFlanking lociPresence in haplotypeDetailsPGFCOXQBLSSTOAPDDBBMANNMCF29002370TRIM27:C6orf100CCCC??CCComplex region (A)29440424OR5V1:OR12D3✓✓?✓??XXAluYa529784097C6orf40:HCP5P15✓X✓✓?XXXAluYa5/8 175..30429788451Within HCP5P15XX✓X?✓✓XAluYa5/8 176..31029794763HCP5P15:HLA-F✓XX✓?XXXSVA_E plus simple rpt.s29922942HLA-G:MICF✓X✓✓✓✓✓✓L1ME3B 5940..616529954495MICF:HLA-H✓XXXXXXXHERVK9 inserted in MER930008633HLA-K:HLA-21✓XX✓XX✓?SVA E/F plus simple rpt.30106475HCG8:ETF1P1X✓XX✓✓XXAluYb830547387SUCLA2P:RANP1XXX✓?XX?AluJb 1..283 and parts of MLT1D/L1PBa31079582C6orf205:HCG22XX✓XXX?XAluYb8 37..29731117638C6orf205:HCG22✓XX✓✓X✓✓AluY (whole & part) and MER63 1017..106231301931HCG27:HLA-C✓✓X✓?✓✓✓HERV3 part (6489...7339)31320352HCG27:HLA-C✓XXX?XXXSVA_F 349..850 plus GC rich rpt.31358220RPL3P2:WASF5PXX✓X?XXXAluY 35..30631400900WASF5P:HLA-B✓✓✓✓?XXXAluSp plus L1PREC2 part (3205...4617)31405648WASF5P:HLA-B✓X✓✓?XxxHERVIP10F (part) and AluSg (only cf CX DB)31418854WASF5P:HLA-B✓✓✓✓?✓✓XL1PA5 part (5503..5876)31530995MICA:HCP5✓X?✓??X?SVA B/F plus simple rpt.s32421915within C6orf10✓XX✓XX✓XAluYb832486228BTNL2:HLA-DRA✓✓✓✓✓XXXL1P1/L1HS parts32655545HLA-DRB1 intron 5✓xxX?✓✓?AluYa5 within more or less partial LTR1232660731HLA-DRB1 intron 1X/X✓/XX/X✓/✓?✓/✓✓/✓?Tigger4/AluSx32661119HLA-DRB1 intron 1CCCC?CC?Complex region (B)32663167HLA-DRB1 intron 1X/✓✓/✓✓/✓✓/X?✓/X✓/X?AluSq/AluY32669534HLA-DRB1:HLA-DQA1CCCC?CC?Complex region (C)32679461HLA-DRB1:HLA-DQA1✓XXX?XX?AluY32693271HLA-DRB1:HLA-DQA1✓✓✓✓?X✓?L1PA4 (parts)32697545HLA-DRB1:HLA-DQA1XXXX?✓✓?L1HS 7..603232701428HLA-DRB1:HLA-DQA1✓X✓✓?xXxL1PA2 part and from CX: MER2B and AluY32728179HLA-DQA1: HLA-DQB1CCCC?CCCComplex region (D)32739664within HLA-DQB1XX✓X?X✓XAluY32743646HLA-DQB1: MTCO3P1XXXX?✓XXLTR1332746780HLA-DQB1: MTCO3P1XXXX?✓X✓L1PA4 (parts)32751442HLA-DQB1: MTCO3P1XXXX?X✓XLTR5_Hs32753489HLA-DQB1: MTCO3P1✓✓✓✓?X✓XL1PA10 268..4888 around L1PA4 (part)32756020HLA-DQB1: MTCO3P1XXXX?X✓XLTR5_Hs32764047HLA-DQB1: MTCO3P1✓✓✓✓?X✓XAluSx32765930HLA-DQB1: MTCO3P1XXXX?X✓XAluYa532785062MTCO3P1:HLA-DQB3✓✓✓✓?XXXTigger4 (Zombi)/L1HS (parts) and T-rich32795150MTCO3P1:HLA-DQB3XXXXX✓X✓AluY32796573MTCO3P1:HLA-DQB3XXXXX✓X✓AluY32815974HLA-DQB3: HLA-DQA2X✓XX✓XXXAluYa532857369HLA-DQB2:HLA-DOB✓X✓✓XX✓XAluYg632881426HLA-DQB2:HLA-DOBXX?✓✓XX?AluYa532887265HLA-DQB2:HLA-DOB✓X?XXX✓✓LTR42 and parts of L1MC5 and AluSc 3..10533201559within HLA-DPB2✓XXX✓?X?AluYb833234360HCG24:COL11A2✓✓✓??✓✓XAluY (1..293) AluJb (26..306)Where there was a break in the cross_match discrepancy list match between two clones, the inserted sequence was extracted and subjected to analysis by RepeatMasker to assess the number of major indels that were a result of retrotransposible elements. Chromosome 6 position (NCBI35/36) of the inserted sequence was that of the midpoint where the sequence was an insertion in PGF or the position before the deletion in PGF. Flanking loci were retrieved from the annotation. Insertion in a haplotypes is indicated by ‘✓’, deletion by ‘X’, complex regions by ‘C’. Where there is a sequence gap in a haplotype corresponding to the indel, this is shown by ‘?’. Four complex deletion/insertion events are listed: A, B, C and D; for details, see text. Four of these major indels were complex and designated as complex regions A, B, C and D in Table 10. They include three known regions from the comparison of the PGF and COX haplotypes (Stewart et al. 2004). Complex region A (involving MIR, MER41B, MER115, AluSx, Flam_C, AluSg, AluY, AluSx, L2 and MER38 elements) maps between TRIM27 and C6orf100 and was found to be deleted in COX but present in PGF, QBL, SSTO, MANN and MCF. Complex region B (involving L2 and AluY elements) maps to intron 1 of HLA–DRB1 and was also found by comparing PGF with COX, QBL, DBB, MANN and SSTO. Complex region C (SVA and low-complexity repeat elements) maps between HLA–DRB1 and HLA–DQA1 and was noted in COX as a deletion of the SVA and low-complexity repeats. Whereas, DBB, MANN and SSTO displayed the same deletion, as well as a telomeric deletion of AluSx/MIRb, QBL had both deletions plus that of an intervening 2.5 kb sequence containing Alu, L3 and MLT1A1 elements. Complex region D maps between HLA−DQA1 and HLA−DQB1 and is more complicated than previously reported. At the telomeric end, PGF lacks an L1PA4 fragment of >300 bp that is present in COX, QBL, SSTO and MCF and is also absent in DBB and MANN where it is interrupted by about 1.3 kb of SVA sequence. Centromeric to this PGF contains an AluSx, an AluY and an AluYd2, flanked by long interspersed nuclear element repeats, all deleted in the other haplotypes. Further towards the centromere there is an L1MA7 fragment, into which in PGF alone there are insertions of an AluSx followed by an AluY; a subsequent AluSg present in all haplotypes contains an insertion of 795 bp of SVA sequence in just COX and QBL. Finally, at the centromeric end of this region, PGF uniquely contains intact MER11C and LTR5 elements. Representation of haplotypes within European populations The eight haplotypes analysed in this study were selected on the basis of their association with type 1 diabetes and multiple sclerosis and their high population frequencies. To determine how representative these haplotypes are with respect to SNP haplotypic diversity in a population, we determined their distribution in the haplotypic tree space in the European population. For this analysis, we selected a segment of ~214 kb, spanning the HLA–DRB1 and HLA–DQB1 genes in a population of European ancestry with known HLA allelic data (de Bakker et al. 2006). Phylogenetic analysis of 180 founder haplotypes derived from genotypic data (54 substitutions) shows that the eight haplotypes selected as part of the MHC Haplotype Project share identical HLA alleles over most of the tree space (Fig. 3a), representing almost the entire variation observed in the population assayed with the exception of two branches (DRB1*1103–DQB1*0301 and DRB1*0101–DQB1*0501). Fig. 3Clusters of haplotypes in the European haplotypic diversity. Phylogenetic relationship of 180 founder SNP haplotypes from CEPH trios spanning a 214-kb segment of the MHC class II region, including the HLA-DRB1 and HLA-DQB1 genes (54 substitutions from rs2187823 to rs2856691). a Sequenced haplotypes are widely distributed in this NJ tree and represent the vast majority of the variation in the population sampled. Four-digit alleles are indicated for the corresponding DRB1 and DQB1 genes in each haplotype ID label to highlight the HLA haplotypic distribution based on the underlying nucleotide variation. The NJ tree was constructed using pairwise genetic distances considering the Kimura 2-parameters model without correction for rate variation among sites as implemented in the MEGA2 software (Kumar et al. 2001). b Each haplotype sequenced is associated to a single haplotype cluster. This phylogenetic network (Bandelt et al. 1999) also shows that clusters (shaded area) are constituted by one central haplotype and its derivatives. Circles represent individual haplotypes, and the size of the circle is proportional to the haplotype frequency. The length of the lines connecting nodes is relative to the distance between them, e.g. distances within shaded areas (clusters) never exceed three mutation steps. Cluster of haplotypes sharing HLA alleles with sequenced cell lines are named accordingly: COX and QBL: DRB1*0301 DQB1*0201–PGF: DRB1*1501 DQB1*0602–APD: DRB1*1301 DQB1*0603–MCF: DRB1*0401 DQB1*0301–DBB: DRB1*0701 DQB1*0303–SSTO: DRB1*0403 DQB1*0302–MANN: DRB1*0701 DQB1*0202. HLA haplotypes DRB1*1103–DQB1*0301 and DRB1*0101–DQB1*0501 indicate the two major haplotype clusters not represented in the MHC haplotype project data Haplotype diversity in this sub-population is restricted to relatively few haplotype clusters (Fig. 3b). Each cluster consists of a founder haplotype, depicted by the most frequent and centrally located haplotype within the cluster. Recently derived haplotypes show lower frequencies and are connected to the central haplotype by relatively few mutation steps (in this case, up to three). This phylogenetic network clearly shows that all the sequenced haplotypes occupy central positions in their respective haplotypic groups. Inferences about phylogenetic relationships between haplotype clusters are, however, only approximate as a consequence of recombination events. It should also be noted that SNP haploptypes derived from CEPH pedigrees of European ancestry by no means represent an exhaustive sampling of European diversity. Nevertheless, the sampling has been shown to represent the European population in the UK reasonably well (Ke et al. 2005). In conclusion, our analysis demonstrates that the HLA haplotypes selected for the MHC Haplotype Project are ancestral haplotypes, representative of MHC diversity in the European population. Conclusion and outlook The MHC Haplotype Project has succeeded in providing a new public resource for immune-linked disease and population genetic studies. First reports from studies using the resource indicate that it adds significant power to the identification and fine-mapping of disease-associated variations (Yeo et al. 2007). The data have also contributed to the recent identification of a first set of HLA tag SNPs, which hold great promise for future applications in clinical settings, e.g. to complement or replace classical HLA-typing in transplant medicine (de Bakker et al. 2006). While costs and other limitations of the current (capillary) sequencing technology have restricted our study to only few (eight) MHC haplotypes, the number of new variations found, combined with the fact that no variation plateau has yet been reached, indicates that there are many more variations to be discovered. The recent introduction of several new and massively parallel sequencing platforms (for review, see Bentley 2006) has created the opportunity to do just that by re-sequencing haplotypes and, eventually, entire genomes at the population level and as integral part of case control studies. Because of its wide-ranging medical importance, the MHC can be expected to be among the first regions of the human genome to be sequenced in this way. Such sequencing will provide the critical, and until now missing, data to identify causal variations and their underlying mechanisms on an unprecedented scale.

Acta_Neuropathol_(Berl)-2-2-1705477

The muscle protein dysferlin accumulates in the Alzheimer brain

Dysferlin is a transmembrane protein that is highly expressed in muscle. Dysferlin mutations cause limb-girdle dystrophy type 2B, Miyoshi myopathy and distal anterior compartment myopathy. Dysferlin has also been described in neural tissue. We studied dysferlin distribution in the brains of patients with Alzheimer disease (AD) and controls. Twelve brains, staged using the Clinical Dementia Rating were examined: 9 AD cases (mean age: 85.9 years and mean disease duration: 8.9 years), and 3 age-matched controls (mean age: 87.5 years). Dysferlin is a cytoplasmic protein in the pyramidal neurons of normal and AD brains. In addition, there were dysferlin-positive dystrophic neurites within Aβ plaques in the AD brain, distinct from tau-positive neurites. Western blots of total brain protein (RIPA) and sequential extraction buffers (high salt, high salt/Triton X-100, SDS and formic acid) of increasing protein extraction strength were performed to examine solubility state. In RIPA fractions, dysferlin was seen as 230–272 kDa bands in normal and AD brains. In serial extractions, there was a shift of dysferlin from soluble phase in high salt/Triton X-100 to the more insoluble SDS fraction in AD. Dysferlin is a new protein described in the AD brain that accumulates in association with neuritic plaques. In muscle, dysferlin plays a role in the repair of muscle membrane damage. The accumulation of dysferlin in the AD brain may be related to the inability of neurons to repair damage due to Aβ deposits accumulating in the AD brain. Introduction Aggregation of neuronal proteins is a common mechanistic theme in neurodegenerative diseases [13]. Whether due to point mutations or post-translational modification, normally soluble proteins are altered and form insoluble fibrillar aggregates [18]. Alzheimer’s disease (AD), the most common form of dementia, is characterized pathologically by abundant diffuse and neuritic plaques throughout most cortical regions. Amyloid β-protein (Aβ) is a 39–43 amino acid peptide cleaved from a larger precursor protein (amyloid precursor protein or APP) that deposits extracellularly as senile plaques either as loose, nonfibrillar diffuse plaques, or as a more compacted, fibrillar form, with dystrophic neurites coursing through the plaque [25]. Other aggregated proteins found in the AD brain include the microtubule-associated protein tau [11] as neurofibrillary tangles and the presynaptic protein α-synuclein [13] in the form of Lewy bodies. The alteration, deposition and aggregation of amyloid proteins may be an ill-fated response to the disease process or themselves lead to a cascade of cellular responses ultimately resulting in neuronal dysfunction and death. Nonbrain protein aggregates have been described in muscle disease. For example, amyloid-like deposits have been described in inclusion body myositis [2]. Other protein aggregates found in muscle disease include desmin-related myopathies, actinopathies and myosinopathies [30]. Aggregates containing these proteins have not been described in brain disease. Another muscle protein associated with myopathy is dysferlin. Dysferlin is a plasma membrane and cytoplasmic vesicle-associated protein implicated in three adult-onset muscle diseases (limb-girdle dystrophy, type 2B, Miyoshi myopathy and distal anterior compartment myopathy)[4, 9, 22, 24]. Although described in peripheral nerve [30] and demonstrated by Western blot in human and mouse brain [21], there is little known about the function of dysferlin in the brain or its role in neurodegenerative disease. We describe the pattern of dysferlin expression in the normal and AD brain and examine dysferlin accumulation in association with amyloid deposition in the brains of AD patients. Materials and methods Case materials Case materials were derived from research participants in a longitudinal study of healthy aging and dementia who were studied postmortem. The diagnostic criteria are consistent with probable AD reported by the National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer’s Disease and Related Disorders Association [26] and confirmed by autopsy to meet high probability of AD according to National Institute of Aging-Reagan criteria [28]. The clinical dementia rating (CDR) was used to determine the presence or absence of dementia and stage its severity [27]. The global CDR is derived by a synthesis of individual ratings in six cognitive and functional categories where CDR = 0 indicates no dementia and CDR = 0.5, 1, 2, or 3 corresponds to very mild, mild, moderate, or severe dementia [27]. The CDR is closely correlated with the presence of dementia pathology at autopsy [14]. The Washington University Human Studies Committee approved all procedures. Twelve brains were examined. Three cases were rated as nondemented (CDR 0) with a mean age of 87.5 years. Nine AD brains were examined; three each of mild (CDR 1), moderate (CDR 2) and severe (CDR 3). The mean age of the demented cases was 85.9 years with mean disease duration of 8.9 years (Table 1). Control cases were all Braak stage I or II [6]. Sampling during postmortem exam was done from 29 different cortical, subcortical and brainstem regions. Paraffin-embedded sample for this study were taken from midfrontal, cingulate and entorhinal regions; three sections were stained for each antibody with appropriate controls [15]. Biochemistry was performed on midfrontal gyrus adjacent to the section taken for immunohistochemistry for each case. Table 1Sample characteristics and findings at postmortem examinationCaseAge (years)SexCDRDisease duration (years)Brain wt (g) Braak scoresCERAD criteriaNIA–Reagan criteriaNeurofibrillaryAmyloid192F0–1,120IIA––285F0–1,100IA––392F0–1,210IA––485F181,200IVCDefiniteHigh599M121,230VBDefiniteHigh695M1141,360VCDefiniteHigh781M216790VICDefiniteHigh891M2111,580VBDefiniteHigh990M271,170IVBDefiniteHigh1086M3101,310VICDefiniteHigh1179F38770VICDefiniteHigh1288M3111,130VICDefiniteHigh Immunohistochemistry Immunohistochemistry was done as previously described [14, 15]. Briefly, serial 6 μm sections of paraformaldehyde-fixed, paraffin-embedded tissue were prepared and incubated overnight at 4°C with antibodies against dysferlin (Novacastra Laboratories), tau (17026, gift from J. Trojanowski), and amyloid β-protein (6E10, Signet). Sections were developed by avidin–biotin complex method (Vector Laboratories) for immunohistochemistry with DAB (brown), Vector Red (pink) and Vector SG (blue) as chromogens. Alexa-Red and Green (Jackson Laboratories) antibodies were used for immunofluorescence. Sections were examined with a Nikon E800 Research microscope, and images were digitized and viewed with AnalySIS analytic software (Soft Imaging Systems, GmbH). Western blots Western blotting and biochemical fractionation were performed as described previously [16]. Briefly, two sections of gray matter (0.3 g) from the midfrontal cortex of AD and normal age-matched control brains were dissected from underlying white matter. After dissection, gray matter was divided into two samples (Fig. 1). One sample was homogenized in RIPA (150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, 50 mM Tris, pH 8, 20 mM, NaF, 2 mM EGTA, 0.5% levamisole, 1 mM NaVO4) to estimate total dysferlin. The other sample was homogenized and subjected to sequential extraction buffers of increasing protein extraction strength. For each buffer, pellets were washed twice and supernatants pooled. Samples were first homogenized in 2 ml/gram of tissue of high-salt (HS) buffer (50 mmol/l Tris, pH 7.4, 750 mmol/l NaCl, 10 mmol/l NaF, 5 mmol/l ethylenediaminetetraacetic acid with protease inhibitors) and centrifuged at 100,000g for 30 min. The pellets were re-extracted and the supernatants were pooled. The pellets were sequentially extracted twice with 2 ml/gram of HS buffer/1% Triton X-100 (HS/T) and once with 1 ml/gram sodium dodecyl sulfate (SDS) sample buffer (1% SDS, 10% sucrose, 10 mmol/l Tris, pH 6.8, 1 mmol/l ethylenediaminetetraacetic acid, 40 mmol/l dithiothreitol). The pellets were extracted with 0.67 ml/g 70% formic acid (FA) and disrupted with two sequential 2-s sonication bursts. FA was evaporated in a vacuum centrifuge (Eppendorf). SDS sample buffer (0.67 ml/g) was added to the dried pellets, followed by vigorous vortex, and the pH was adjusted with NaOH. SDS sample buffer was added to the HS and HS/Triton fraction and all of the samples were boiled for 5 min. The FA samples were centrifuged at 13,000g for 5 min to remove insoluble debris. Five microliters of each fraction was loaded in separate lanes for SDS-polyacrylamide gel electrophoresis followed by Western blot analysis with antibodies again dysferlin. The immune complexes were visualized with the use of the ECL Plus kit (Amersham) according to the manufacturer’s protocol. The autoradiographs obtained were scanned and the band intensity quantified utilizing TotalLab software (Nonlinear Dynamics, Newcastle on Tyne, UK). Statistical analyses were performed by analysis of variance using SPSS statistical software. Fig. 1Schematic representation of sequential protein extraction buffers. Samples are divided into equal portions. The first sample is homogenized in RIPA buffer and blotted to estimate total dysferlin levels. The other sample is homogenized in sequential extraction buffers of increasing protein extraction strength. The first extraction is in high-salt (HS) buffer. The supernatants are saved and the pellet is extracted with HS buffer/1% Triton X-100 (HS/T) buffer. After saving the supernatants, the resultant pellet is homogenized in sodium dodecyl sulfate (SDS) sample buffer. Resulting pellets are finally extracted with 70% formic acid (FA). Supernatants are blotted to estimate differing levels of dysferlin solubility with the HS fraction representing the most soluble phase and the FA fraction representing the most insoluble phase Results Localization of dysferlin in normal (CDR 0) brain Microscopic evaluation of the dysferlin staining in the normal (CDR 0) brain localized expression to the cytoplasm of large pyramidal neurons of layers III, V and VI in the neocortex (Fig. 2a), layer II neurons of the entorhinal cortex, CA 1–3 neurons in the hippocampus and large projection neurons in the amygdala (not shown). Dysferlin was not localized to astrocytes, endothelial cells or microglia (arrowheads in Fig. 2b). Little to no dysferlin neuritic staining was seen in control cases (Braak stage I or II). Fig. 2Localization of dysferlin in brains of nondemented subjects. a, b Demonstrates the localization of dysferlin immunoreactivity in the brain. There is a neuronal expression of dysferlin localized in cytoplasm of large pyramidal (layers III, V, VI) neurons in the frontal cortex at low power (a). Little to no neuritic staining was detected. Dysferlin expression is confined to neurons (arrow, DAB brown in b) and is not seen in glial cells stained with GFAP (small arrowheads, Vector Red pink in b). Magnification of (a) is 100× and the scale bar in (a) = 50 μm. Magnification of (b) is 400× and the scale bar in (b) = 20 μm Dysferlin in the AD brain Dysferlin neuritic accumulation is found throughout the AD brain and in double-label studies it is localized in Aβ neuritic plaques in the neocortex (Fig. 3a). Double-labeled studies demonstrate co-localization of dysferlin (brown in Fig. 3b) and Aβ (pink in Fig. 3b) in the hippocampus. Fig. 3Localization of dysferlin with amyloid pathology in the AD brain. a, b Demonstrates the location of the dysferlin accumulation in the AD brain with double-labeled studies. a Diffuse dysferlin accumulation (DAB, brown) and Aβ (DAB-nickel, black) in the frontal cortex. Using VectorRed (pink, Vector Laboratories) as the chromogen for Aβ and DAB (brown, Vector Laboratories) as the chromogen for dysferlin, the pathologic deposition is localized as neuritic pathology in Aβ neuritic plaques in the neocortex hippocampus (b). Magnification of (a) is 100× and the scale bar in (a) = 20 μm. Magnification of (b) is 200× and the scale bar in (b) = 20 μm Dysferlin neuritic pathology occurs in conjunction with tau neuritic pathology In Fig. 4, we demonstrate that neurites in plaques may comprise tau, dysferlin or both. Triple-labeled studies of midfrontal (Fig. 4a), cingulate (Fig. 4b) and entorhinal (Fig. 4c) cortex illustrate the relationship of tau (pink, arrowheads) and dysferlin (brown, arrows) within an Aβ plaque (blue). Double-labeled immunofluorescent studies (Fig. 4d–f) demonstrate that tau (green in 4d) and dysferlin (red in Fig. 4e) have both distinct and overlapping (yellow in Fig. 4f) epitopes in neuritic plaques. Dysferlin does not appear to co-localize with the more abundant tau dystrophic neurites. Fig. 4Localization of dysferlin with tau pathology in the AD brain. a–f Demonstrates the location of the dysferlin and tau accumulation in the AD brain with triple-labeled immunohistochemistry studies (a–c) and double-labeled immunofluorescence studies (d–f). a Midfrontal, b cingulate and c entorhinal demonstrate tau (Vector Red, pink, arrowheads), dysferlin (DAB, brown, arrows) and Aβ (Vector SG, blue). Note the distinction between tau-positive and dysferlin-positive structures. Double-labeled immunofluorescence studies demonstrated neurites within amyloid plaques. Neurites may comprise tau (green, d), dysferlin (red, e) or overlapping epitopes (yellow in merged image of f). Magnification of (a–f) is 600× and the scale bar in (d) = 20 μm Biochemistry Western blot analysis following RIPA extraction demonstrated the presence of dysferlin as 230–272 kDa bands in all brains examined (Fig. 5a); however, there was a decrease in the total dysferlin content in CDR 3 (severe AD) brains compared to CDR 0 (nondemented). There is a progressive decrease in total dysferlin with progressive disease (r = 0.998, P = 0.001). Western blot analysis following serial extraction of proteins demonstrated a change in the solubility state of dysferlin across the spectrum of dementia (Fig. 5b). No dysferlin could be demonstrated in the HS fraction. In contrast to the control CDR 0 brains, however, there was a significant reduction of dysferlin in the HS/T fraction in the demented cases (CDR 1 and 2) and was undetectable at the CDR 3 level of severity (F = 2,991, P < 0.001). In the SDS-soluble fraction from the demented cases, there was an accumulation of 230–272 kDa bands with dementia progression, that is a more insoluble form of dysferlin was found in severe CDR 3 brains compared to milder stages of dementia (CDR 1) or in nondemented individuals (F = 3,183, P < 0.001). Dysferlin did not aggregate or accumulate in the FA-soluble fractions of the control or AD brains. Fig. 5Western Blot analysis of dysferlin in nondemented and AD brains. a Nondemented (CDR 0) and AD brains (mild = CDR 1, moderate = CDR 2, and severe = CDR 3) were extracted in RIPA as described in the Materials and methods. Five microliters of each extraction were loaded into separate lanes of a 12% SDS-polyacrylamide gel electrophoresis and after transfer to nitrocellulose membranes were probed with antibodies against dysferlin. The blot demonstrates the presence of dysferlin in both normal and AD brains with declining levels of protein across disease progression. Band intensities were quantified and compared in a bar graph. There is a linear decrease in total dysferlin associated with disease progression (r = 0.998, P = 0.001). b The same brains were sequentially extracted with HS, HS/T, SDS and FA as described in the Materials and methods section as loaded onto 12% SDS-polyacrylamide gel electrophoresis as described earlier. Dysferlin was not seen in either the HS or FA fractions. Dysferlin was demonstrated prominently in HS/T fraction in the CDR 0 brains, with a significant reduction (F = 2,991, P < 0.001) of dysferlin in the HS/T fraction in the demented cases (CDR1 and 2) and was nearly undetectable in severe AD (CDR 3) In contrast, the SDS fraction representing insoluble phases of dysferlin demonstrated significant increases (F = 3138, P < 0.001) in the 230–272 kDa bands as dementia progressed from mild (CDR 1) to severe (CDR 3) Discussion Dysferlin is a new protein described in the AD brain associated with neuritic plaques. Dysferlin is widely expressed in the brain and is found in intracytoplasmic compartments in pyramidal neurons in normal brains. Dysferlin begins to accumulate in dystrophic neurites in the AD brain at mild stages of dementia (CDR 1). Dysferlin is found at all stages of disease with greater accumulation in more advanced disease (CDR 3). Significantly, our Western blot analysis demonstrated dysferlin in RIPA-extracted fraction in all cases; however, in the serial extracted fractions an alteration in the solubility state of dysferlin was found in demented cases (CDR > 1) compared with nondemented cases. This coincides with the finding of neuritic pathology by immunohistochemistry. The HS/T fraction represents the transmembrane cytoskeletal fraction of normal dysferlin and decreases with advanced stages of dementia. This may represent the loss of membrane integrity and alteration in cytoskeleton structure. In contrast, the SDS-soluble fraction represents more insoluble forms of the protein suggesting aggregation of dysferlin may be related to disease progression. This alteration of solubility and accumulation is seen in other neurodegenerative diseases (e.g. Parkinson’s disease, multiple system atrophy, dementia with Lewy bodies) [15, 31]. Dysferlin is a member of ferlin family, homologous to fer1 protein in C. elegans [5]. It is highly expressed in muscle but little is known about the role of the protein in the brain. Several members of the ferlin family have been mapped. Dysferlin is found on chromosome 2p13 [1] and deficiency is associated with three clinical phenotypes: Limb-Girdle muscular dystrophy, type 2B, Miyoshi myopathy and distal anterior compartment myopathy [20, 22, 24]. Another member of the ferlin family, Otoferlin is mapped to chromosome 2p23 and is associated with autosomal recessive nonsyndromic deafness [31]. Two other members of the ferlin family have been described: Myoferlin (10q24) [7] and FerlL4 (20q11) [12]. Myoferlin is a type II transmembrane protein upregulated in Duchenne muscular dystrophy and may have a role in muscle regeneration and repair [10]. The function of Fer1L4 is unknown. Dysferlin is thought to play a role in muscle membrane maintenance and repair [3, 4, 9, 12, 19]. In C. elegans, the Fer1 protein plays a role in vesicle trafficking and membrane fusion and can bind to phospholipids in a calcium-dependent manner [22]. In dysferlin null-mice, muscle cells are defective in repairing membrane disruptions leading to degeneration [4, 21, 23]. If dysferlin has a similar membrane repair function in neurons, then it is possible that the deposition of dysferlin in AD may be related to the inability of neurons to repair damage due to accumulating Aβ pathology throughout the progression of AD. The findings reported suggest that there may be common mechanisms of membrane repair in degenerative diseases of muscle (e.g. dystrophinopathies and sarcoglycanopathies) and brain (e.g. AD). The aggregation of normally soluble proteins in fibrillar lesions is the neuropathologic hallmark of many neurodegenerative diseases. Whether the aggregates or their precursors are the actual toxic species is still under debate [8]; however, it is likely that the conversion of proteins from a soluble to insoluble state impairs the long-term viability of neurons. In addition, because dysferlin appears to accumulate in conjunction with Aβ deposition and disease progression it may be able to serve as a marker of disease progression. Continued efforts aimed at understanding abnormalities in the misfolding of protein leading to neurodegenerative disease such as Aβ and tau, or proteins associated with aggregation such as dysferlin will provide insights into disease mechanisms underlying neurological disorders characterized by abundant filamentous lesions.

Eur_J_Pediatr-4-1-2190787

Concordance between school outcomes and developmental follow-up results of very preterm and/or low birth weight children at the age of 5 years

Introduction Long-term follow-up studies have revealed a high frequency of developmental disturbances in preterm survivors of neonatal intensive care who were formerly considered to be non-disabled. These developmental disturbances interfere with the acquisition of everyday skills and, in particular, with normal school functioning. Introduction The survival rate of very preterm (VPT < 32 completed weeks of gestation) and very low birth weight (VLBW <1500 g) children has steadily improved during the last decades, but the prevalence of major disabilities or handicaps in these children has remained stable. Follow-up studies have shown that VPT and VLBW infants are at risk with regard to neurological dysfunctioning such as cerebral palsy and mental retardation [6, 17, 31, 33]. Even more children of these groups experience difficulties in intellectual, speech and language skills as well as clumsiness and attention and behaviour problems, all of which can affect school functioning [4, 12, 13, 16, 19, 24, 26]. Significant/severe neurosensory dysfunctioning is usually detected early, with the result that children with major handicaps often attend a school for special education from school entry. Minor developmental impairments and disabilities, however, frequently go undetected until school age (4 years in The Netherlands). These children start their school career in mainstream education. Once at school, however, certain learning problems may become apparent during the first 2 years (the kindergarten years), but more often these appear only when the children start formal academic training [15, 22, 34]. Several reasons may explain this phenomenon of 'growing into educational deficits' [2, 9, 18, 20, 27]. Mild developmental disturbances usually pose no problem for the child until greater demands have to be met at school or when more detailed evaluations are made. In addition, the cumulative effect of the failure to acquire basic skills and the resulting declining motivation may play an important role. In The Netherlands, schools are focused strongly on the early detection of children who need special assistance. After 1 year of education, at the age of 5, teachers usually have a good impression of both the cognitive and language development of their children and of their motor skills and behaviour. Standardized tests are used to assess children who are not doing well at school, and the data from these tests are used to underpin the need for special help for these children. Additionally, many neonatal intensive care units (NICUs) have follow-up programmes for premature children in which standardized assessments of different domains enable an overall evaluation of the child's development. Within the framework of follow-up research we assessed a group of VPT and VLBW children at the age of 5 years in different developmental domains. Perinatal, social and economic data as well as information on school functioning were available. This provided the opportunity to compare school outcomes and developmental follow-up outcomes, assuming that developmental skills mediate the effects of biological and social risk factors on school performance [35]. We studied the degree of agreement between follow-up assessments and school outcomes as well as the most important characteristics of the children (perinatal, developmental and socio-economic) in this context. Our aim was to obtain a understanding of the (potential) developmental disturbances in order to facilitate an adequate and timely signalling of children who need special help to meet their developmental capacities. Methods Study population The study population consisted of 768 infants of less than 32 weeks of gestation and/or weighing less than 1500 g who were born between October 1992 and December 1994 and treated in three Dutch neonatal intensive care units: the Radboud University Nijmegen Medical Centre, the Academic Medical Centre, Amsterdam, and the Máxima Medical Centre, Veldhoven. Mortality before the age of 5 years was 131 (17%). Forty-six children (6%) were excluded because they had participated in another study. Thirty-three children (4%) with severe or moderate cerebral palsy, blindness, mental retardation, chromosomal abnormalities, inborn errors of metabolism, personality developmental disorders and/or attendance at special schools or institutions were excluded. Sixty children (8%) of non-Dutch parents were excluded from the analyses since school problems could be caused by language and cultural problems. As a result, 498 children (65%) qualified for the study. School performance and socio-economic situation A questionnaire was mailed to the parents of the participating children 1 month before follow-up assessments were to be carried out; these were returned when the family attended the outpatient clinic for the assessment. The paediatrician initiated the follow-up assessment by checking the questionnaire and exploring the answers with the family in more depth. Within the framework of the present study we analysed questions addressing school performance and educational level of the parents (low, middle, high). School outcome was defined in two categories: (1) normal (mainstream education without problems, which means no remedial teaching or other forms of extra help) or (2) problematic (mainstream education with remedial teaching or special mainstream education; i.e. schools for children with mild learning, behavioural or cognitive problems). The parents were asked to fill in the school items together with the schoolteacher. The educational level of the parents was used in this study as a measure of the social and economic situation (SES) because it is the best indicator in predicting school outcomes in The Netherlands [5]. Perinatal data Perinatal data were prospectively collected during admission in the NICU, stored in the NICU databases and retrieved for data analysis. The following individual characteristics and perinatal variables were considered: gender, multiple birth, gestational age, birth weight, small for gestational age (SGA: birth weight below the 10th centile), low 5-min Apgar score (<7), intracranial haemorrhage grade 3 and 4 (ICH) [32], periventricular leucomalacia (PVL), bronchopulmonary dysplasia (BPD; oxygen dependency at 36 weeks postconceptional age or at 28 days of life), ante- or postnatal use of steroids and length of stay in NICU. Assessment at the age of 5 years Clinical assessments were carried out on the health and neurological functioning of the children (not reported in this article) and four developmental domains: cognition, language, motor functioning and behaviour. A paediatrician, a child psychologist and a paediatric physical therapist assessed the children. Appointments were scheduled at random. Cognitive development was assessed with the Revised Amsterdam Children’s Intelligence Test (RAKIT) for children aged 4–11 years (short version). This version has a correlation of 0.93 with the full-scale test. The concurrent validity with the Wechsler Intelligence Scale for Children-Revised (WISC-R) is 0.86 for total IQ (Intelligent Quotient) [3]. This test included logical reasoning, word knowledge, visual-motor integration and word fluency. It also included visual synthesis for children younger than 5.2 years and visual analysis and memory for children aged 5.2 years or older. The norm score (IQ score) of the test is 100 with a standard deviation (SD) of 15. All scores higher or equal to 85 are classified as normal, while scores below 1 or 2 SD’s are classified as mild or severe cognitive problems, respectively. The Dutch Language Screening Test assessed language and speech. This test consists of 39 items covering the use of vocabulary, comprehension, memory and production of language, use of plurals, prepositions and pronunciations. The total score varies from 0 to 52, with a score ≤18 considered to be normal, that of 19–25 considered to indicate mild problems and that of >25 to indicate severe problems [7]. The Movement Assessment Battery for Children (Movement ABC) was used to assess motor skills. A total motor impairment score (range: 0–40) was computed by summing the scores on all motor tasks. A total score ≤10.5 (15th centile) was considered to be normal, from 11.0 through to 17.0 (5th centile) considered to indicate children at risk and >17.0 to indicate abnormal motor development [11, 23]. To assess behavioural outcomes, we used the Child Behaviour Checklist for children aged 4–18 years (CBCL). This is a standardized parental questionnaire used to describe the skills and behavioural problems of children. It comprises 113 descriptions of behavioural problems, with each description scored as: 0 (not true), 1 (somewhat or sometimes true) or 2 (very true or often true). A total problem score is obtained by summing all items. The severity of the behaviour problems can be the borderline and clinical cut-off point, corresponding with standardized norm scores of 60 and 63, respectively [1, 30]. A normal score is <60, a mild problematic score is ≥60 and ≤63, and a severe problem score is >63. Statistical analysis In order to compare the results of the follow-up assessments and school outcomes, we cross-tabulated categorical data originating from the individual developmental test results of the children with school outcomes (normal or problematic). Normal follow-up outcomes were defined as four normal developmental scores or only one mild developmental problem score. Problematic follow-up outcomes were defined as two mild developmental problem scores, one severe developmental problem score or two or more severe developmental problem scores. Cross-tabulation was also used to describe the relation between the follow-up assessment outcomes and the school outcomes and the nominal variables (chi-square for testing). A one-way analysis of variance was used to assess within each of the follow-up outcomes the differences between the two types of school outcomes on the interval variables (F test for testing). The general linear model (GLM) was used for multivariate testing of the differences between the two school outcome groups within each of the two follow-up outcome groups. Results Of the 498 children included in this follow-up study, 143 (29%) were not assessed for various reasons (Table 1). Consequently, all outcome data, including developmental data, were collected on 355 children at the age of 5 years. No significant differences were found in perinatal data between assessed, partly assessed and unassessed children, with the exception of multiple births in that they were overrepresented in the assessed group [14]. Table 1Eligible and assessed children nPercentagenPercentageCohort 1992–1994768100  Died13117  Excluded because of participation in an other study466  Excluded because of severe handicap and/or in Special Schools334  Children of non-Dutch parents608Eligible49865498100  Address unknown255  Moved outside the country51  Treated in another hospital61  Impossibility to make a convenient appointment296  Refusal by the parents388  Assessment not fully performed408Assessed35571 Mutual correlations between child characteristics and perinatal variables were present but rather low. The percentage of children with all follow-up assessment results being normal or with only one mild developmental problem score was 64% (n=228); 36% (n=127) of the children had at least two mild problems or one or more severe problems according to the four developmental tests. No school problems were reported for 222 children (63%), while 133 children (37%) were reported to school problems as reflected in the need for special help (remedial teaching or other forms of extra help or specialized mainstream education). One hundred and seventy-five children (49% of the study group) had normal school outcomes and normal follow-up outcomes and 80 children (23% of the study group) had problematic follow-up outcomes and problematic school outcomes. These results show that for 72% (n=255) of the children there was a positive relation between school outcomes and the results of the follow-up assessments. Fifty-three children (15% of the study group) had school problems, while their developmental test scores were normal, and 47 children (13% of the study group) did not receive any extra help at school, although they had test scores that fell in the problematic range. However, for 100 children (28%) there was no concordance between the school outcomes and follow-up assessment results (Table 2). A significant difference (χ2=66.44, p=0.00) was found between the two school outcome groups, with more developmental problems in the school problem group. Children who received special help at school although they had obtained normal follow-up assessment results, differed significantly from the children with normal follow-up results without special assistance. This subgroup comprised more boys and SGA children who had lower Movement ABC scores (motor skills) and, in particular, lower language screening test scores. Table 2Follow-up outcomes versus school outcomesSchool outcomes:Normal: n=222 (100%)Problematic: n=133 (100%)Total: n=355 (100%)Follow-up outcomes:  Normal: n=228 (64%)   Four normal developmental scores124 (56%)26 (20%)   One mild developmental problem score51 (23%)27 (20%)  Subtotal175 →49%53 →15%  Problematic: n=127 (36%)   Two mild developmental problem scores8 (4%)10 (7%)   One severe developmental problem score36 (16%)54 (41%)   Two or more severe developmental problem scores3 (1%)16 (12%)  Subtotal47 →13%80 →23% For children who did not receive any extra help at school, even though problematic follow-up assessments were found, the results differed significantly from those who did receive extra help. This subgroup was less preterm, had higher Apgar scores, had had BPD less often and was treated less frequently with steroids during the neonatal period. Their scores on the Movement ABC, the language screening test and, in particular on the RAKIT (intelligence test) were better (Tables 3 and 4). Within each of the follow-up assessment groups (normal or problematic assessment results) the MANOVA showed significant differences between the two school outcome groups (F=3.47, p<0.01, df=6.22 and F=3.29, p<0.01, df=6.12, respectively). Table 3Means, standard deviations (SD) and one-way analyses of variance for normal and problematic school outcomes Follow-up outcomesSchool outcomesNormalProblematicFMeanSDMeanSDGestational ageNormal30.081.8530.501.542.33Problematic30.692.0129.902.303.82*Birth weightNormal1286.24319.201314.28307.09.32Problematic1225.09306.431241.22364.36.06NICU stayNormal27.2022.3325.0716.53.42Problematic26.9120.8236.9437.172.89RAKIT IQNormal104.8211.77102.3910.641.83Problematic95.1712.1986.7412.9213.19***Movement ABCNormal6.114.317.814.236.44*Problematic15.307.7118.389.103.80*Language scoreNormal8.294.6810.785.7410.48***Problematic11.557.8615.488.486.73*CBCL Total problem scoreNormal47.228.8348.068.25.38Problematic55.3210.7055.0512.06.02*p<0.05, ***p<0.001Table 4Comparison of child's characteristics, perinatal data and parents’ education with normal and problematic school outcomes Follow-up outcomesSchool outcomesχ2NormalProblematicPercentagePercentageMaleNormal42574.10*Problematic6463.01Multiple birthNormal35441.43Problematic3236.20SGANormal23374.29*Problematic4253.25Apgar scoreNormal1572.27Problematic47318.73*ICHNormal502.55Problematic46.21PVLNormal22.04Problematic261.09BPDNormal46.24Problematic0168.40**Neonatal steroidsNormal10.31Problematic0115.62*Parents education-highNormal4236.73Problematic39262.15Parents education-middleNormal4751.11Problematic3848.90Parents education-lowNormal1113.18Problematic2326.10*p<0.05, **p<0.01 Discussion The developmental skills of children are largely expressed in school functioning [25]. Therefore, it would be expected that there is a great concordance between the assessments of the NICU follow-up teams and school teams with respect to the identification of children who need special assistance at school. The results of this study support this expectation with respect to children with good developmental outcome assessments and those with obvious problematic developmental outcomes. While the latter group mainly consisted of children with very low gestational ages and/or serious perinatal problems, some were being faced with the direct adverse consequences of the use of neonatal steroids [36]. It is likely that the problems of these children and the genuine concern of parents and teachers has offered them the extra attention and assistance they required. Children with relatively normal developmental outcomes who, notwithstanding this, received extra help at their schools, still had language and/or motor problems that apparently interfered with school functioning, thereby causing schoolteachers to provide them with extra attention and help. The overrepresentation of boys in this group, as in both groups with problematic developmental outcomes, is a frequent finding [8] and because their behavior is often more demonstrative there is a stronger need to deal with. More children who were SGA were also found in this group. A number of these were still small at the age of 5 years, which may have led to overprotection or the conviction that the child needs more time or help to be ready for reading, writing or arithmetic [10, 21]. Further research should reveal if the problems of this latter group of children are attributable to not being ready for learning (pre) academic skills or to still being too playful, or whether it concerns serious learning and developmental problems. The limited number of children with developmental problems who were not signalled by the school did not have special characteristics with the exception of having slightly better developmental outcomes and/or having not been born extremely premature, having not been subjected to neonatal steroids and/or having had a somewhat less complicated neonatal period. It is possible that their developmental problems did not interfere with school functioning or they were able to compensate for them, but it is also possible that their parents and/or teachers did not signal their problems adequately. Parents are sometimes very relieved that the first difficult and worrisome years are over and unconsciously cut themselves off from new problems or both parents and teachers lower their expectations and demands because of overprotection. We did not find that less well-educated parents had more difficulties in mobilizing extra help for their children than relatively well-educated parents. Children of the former displayed developmental problems more often, but they received extra help and assistance in the same ratio as children of relatively more highly educated parents. Based on this line of reasoning a possible overrepresentation of children of less well-educated parents in the unassessed group would not change the degree of concordance between the follow-up results and school outcomes. One may question whether our developmental tests were sensitive enough to register minor neurological signs. In the overall study the paediatrician did a neurological examination (the modified Touwen examination) [28]. This examination classified 120 (34%) of the children participating in this study with neurological problems (disabling cerebral palsy, non-disabling cerebral palsy, minimal neurological dysfunction and gross motor retardation). Ninety-five (93%) of these children were also classified as problematic by one or more of the other instruments. While these neurological data provide a more complete picture, they do not improve the degree of concordance between school outcome and follow-up outcome (70%). Of the preterm children tested at age 5 years, one-half were found to have problems in the developmental assessments or in school functioning. This outcome confirms the findings of others that follow-up research in more than one developmental domain in combination with school outcomes shows a more problematic outcome picture [29]. Schools are doing quite well in identifying children with and without developmental problems, but the need for longitudinal multidisciplinary follow-up programmes in different developmental domains remains. Information that promotes the understanding of the development of these children during their school career provides schools and parents with more tools for early detection and will facilitate the design and evaluation of intervention programmes.

Clin_Auton_Res-3-1-1797060

The association of heart rate recovery immediately after exercise with coronary artery calcium: the coronary artery risk development in young adults study

We tested whether slower heart rate recovery (HRR) following graded exercise treadmill testing (GXT) was associated with the presence of coronary artery calcium (CAC). Participants (n = 2,648) ages 18–30 years at baseline examination underwent GXT, followed by CAC screening 15 years later. Slow HRR was not associated with higher odds of testing positive (yes/no) for CAC at year 15 (OR = 0.99, p = 0.91 per standard deviation change in HRR). Slow HRR in young adulthood is not associated with the presence of CAC at middle age. Introduction The prognostic value of slow heart rate recovery (HRR) after exercise in predicting cardiovascular disease (CVD) events has been established [5, 11, 12, 13, 15]. Initial increases in heart rate with exercise are due to parasympathetic withdrawal while sympathetic activation is responsible for heart rates greater than a 100 beats/minute. In the first two minutes following cessation of exercise, the rapid decrease in heart rate is principally determined by parasympathetic reactivation [1, 8]. Although slow HRR is associated with less autonomic nervous system responsiveness, the underlying mechanisms linking slow HRR to increased cardiovascular morbidity are not well understood. It is possible that slow HRR is associated with a higher susceptibility for atherosclerosis. Prior studies of patients referred for cardiac angiography for suspected ischemic heart disease (IHD) suggest an association between slow HRR and higher atherosclerotic burden [15]. Further, slow HRR has been observed to be associated with several risk factors for atherosclerosis [2, 3, 10]. However, the relationship between HRR and atherosclerosis in the general population has not been studied using a prospective study design. In a cohort of healthy young adults, we investigated whether slow HRR following a graded exercise treadmill test (GXT) was associated with the presence of coronary artery calcium (CAC), a marker of subclinical atherosclerosis, when assessed 15 years later. Methods and statistical analysis The Coronary Artery Risk Development in Young Adults study (CARDIA) is a longitudinal study designed to investigate the origins of cardiovascular disease in young adulthood [6]. Beginning in 1985, 5,115 African-American and Caucasian individuals [(African-American (52%) and women (54%)] ages 18–30 were recruited at sites in Birmingham, Alabama; Chicago, Illinois; Minneapolis, Minnesota; and Oakland, California. All participants gave informed consents prior to enrollment. At baseline, a symptom-limited maximal GXT was administered using the Balke protocol [14]. The test included nine 2-minute stages of increasing difficulty with participants encouraged to exercise to exhaustion, followed by a recovery period at a speed of 3.2 km/hour at 0% grade. HRR was defined as the difference between the maximum HR and HR at 2-minutes into recovery [10]. Participants were ineligible for exercise testing if they were on cardioactive medications, had a resting systolic or diastolic pressure >160 or >100 mmHg, or were febrile at time of examination. The rate of energy expenditure for the completion GXT was estimated and reported in metabolic equivalents (METs), as previously described [14]. Information on physical activity was collected by interview using a standardized questionnaire. At the year 15 follow-up examination, returning participants (n = 3,043) underwent coronary artery computed tomography (CT) scanning for the measurement of CAC. Mean HRR did not differ between those who did and did not return to the year 15 examination (42.8 versus 42.5 bpm, respectively; p = 0.55). Details of the scanning procedures have been described elsewhere [4]. Briefly, using standardized protocols, two scans were obtained for each participant (1–2 minutes apart) using electron beam CT scanners at the Chicago and Oakland sites and multidetector-row CT scanners at the Birmingham and Minneapolis sites. Calcium scores were calculated across each coronary artery and then summed across all the arteries. The final CAC score of positive scans was calculated as the mean of the two CAC scores obtained from each of the scans. Participants were sequentially excluded from this analysis for the following reasons: use of medications that affect heart rate (HR) (n = 27), unavailable GXT or CAC data (n = 661), missing data on blood pressure, lipids, glucose, or smoking (n = 353), pregnancy (n = 27) or those absent at the year 15 exam (n = 1,399). Following exclusions, 2,648 participants remained. Baseline characteristics were compared across sex-specific HRR tertiles. For continuous variables, test for linear trend was performed with HRR as a continuous variable using linear regression models. The Cochran-Armitage test was used to check for linear trend in binomial proportions across the HRR categories. Next, logistic regression was used to estimate the odds of the presence of CAC (defined as a CAC score >0) in relation to year 0 HRR (independent variable). HRR was modeled both as a continuous variable and in tertiles (fastest HRR tertile as the reference). Statistical significance was determined at P < 0.05. All analyses were conducted using SAS version 9.1 (SAS Institute Inc, Cary, NC). Results Demographic characteristics of the study sample, by 2-minute HRR tertiles, are presented in Table 1. Mean 2-minute HRR (standard deviation) for men and women were 44.3 (11.4) and 41.7 (11.5) bpm, respectively. Participants with slower HRR had less favorable GXT performance characteristics, a higher resting heart rate (both at year 0 and year 15) and reported less physically activity (both at year 0 and year 15). Table 1Baseline characteristics according to tertiles of 2-minute heart rate recovery (N = 2,648)aBaselineBaseline sex-specific HRR tertiles1 (slow)23 (fast)P trendNumber of women479463521NANumber of men392391402NAHRR (median [min, max])Women31.0 (1.0, 36.0)41.0 (37.0, 45.0)51.0 (46.0, 116.0)NAMen34.0 (−6.0, 39.0)44.0 (40.0, 48.0)54.0 (49.0, 97.0)NAAge (years)25.4 (3.5)25.1 (3.6)25.0 (3.6)0.003African-American, N (%)360 (41.3)383 (44.9)435 (47.1)0.014Body Mass Index kg/m224.9 (5.2)24.1 (4.2)23.8 (4.0)<0.001Resting heart rate (bpm) at year 071.5 (11.4)69.3 (10.2)66.1 (9.7)<0.001Resting heart rate (bpm) at year 1568.3 (10.8)68.2 (12.1)66.5 (11.3)<0.001Maximum heart rate (bpm)180.1 (15.8)180.0 (14.6)181.0 (13.9)0.001Estimated METs at peak exercise11.8 (3.0)12.1 (2.7)12.5 (2.7)<0.001Total physical activity score (exercise units) at year 0375.8 (273.0)428.6 (317.5)459.7 (307.3)<0.001Total physical activity score (exercise units) at year 15325.4 (278.1)346.9 (288.0)382.4 (296.6)<0.001aValues are expressed as mean (standard deviation) unless otherwise indicated. HRR = heart rate recovery. NA = not applicable. METs = metabolic equivalents The prevalence of CAC in the study sample was 9.0% (n = 239). Mean HRR at year 0 did not differ between those who had positive CAC scores versus those who had a CAC score of 0 at year 15 (42.8 bpm, for both). The unadjusted odds ratio (OR) for having a CAC score >0 for those in the slowest HRR tertile compared to those in the fastest HRR tertile was not significantly greater than 1.00 (Table 2). Similar findings were observed when HRR was studied as a continuous variable. Table 2Unadjusted odds ratio (95% confidence interval) for the presence of coronary artery calcium (score >0) at year 15 by baseline 2-minute heart rate recoveryTotal sampleCaucasian menCaucasian womenAfrican-American menAfrican-American womenOdds Ratio (95% CI) for threshold models based on HRR tertilesTertile 1 (slow)1.13 (0.81–1.57)0.90 (0.54–1.50)1.12 (0.53–2.33)1.11 (0.52–2.36)1.44 (0.60–3.49)Tertile 21.20 (0.87–1.66)0.90 (0.54–1.50)0.90 (0.41–2.01)1.55 (0.78–3.09)1.37 (0.57–3.32)Tertile 3 (fast)1.00 (Ref)1.00 (Ref)1.00 (Ref)1.00 (Ref)1.00 (Ref)Odds Ratio (95% CI) for HRR as continuous variablePer SD change in HRRa0.99 (0.87–1.13)1.04 (0.84–1.28)0.86 (0.65–1.15)0.95 (0.70–1.30)0.88 (0.60–1.27)HRR = heart rate recovery. CAC = coronary artery calcium. CI = confidence interval. SD = standard deviation aSD of HRR was 11.5 bpm In secondary analysis, similar findings were observed when the presence of higher CAC burden (defined as a score >100 [n = 34]) was studied, as well as when quartiles or quintiles of 2-minute HRR were analyzed. Lastly defining HRR at 1-minute into recovery also did not result in any association between HRR and CAC. Conclusion Slower HRR in young adulthood is not associated with the presence of CAC when assessed 15 years later in middle age (average age 40 years). Moreover, mean HRR at baseline did not differ between those with and without measurable CAC at year 15. While previous studies have examined the relationship of HRR with CAD events this study is first to investigate the relationship of HRR with a measure of subclinical atherosclerosis. Slow HRR has been associated with higher incident all-cause mortality, sudden cardiac death (SCD), and CVD events; however, the underlying mechanisms that link these relationships together are not known [5]. Morshedi-Meibodi et al. [12] using data from the Framingham Heart Study (FHS) observed slow HRR to be associated with coronary heart disease events (defined as acute coronary syndromes or SCD), suggesting a possible relationship between slow HRR and ischemic processes. Similarly, slow HRR has been observed to be associated with several risk factors for atherosclerosis [2, 3, 10]. In contrast to findings from the FHS, Jouven et al. [9] observed slow HRR to be related only to SCD and not death from myocardial infarction. Due to over lapping and discrepancy in clinical end points used in prior HRR studies, it cannot be determined whether slow HRR is associated with atherosclerosis or an increased susceptibility to lethal cardiac arrhythmias. The ability of coronary CT to assess the global burden of atherosclerosis in young adults is uncertain, and CAC is only a subset of atherosclerosis, representing calcified plaques which tend to be more stable than their lipid-rich counterparts [7]. Despite these limitations, our study suggests that slow HRR in young adulthood is not related to subclinical atherosclerosis at middle age, which supports the hypothesis that slow HRR is associated with mechanisms related to cardiac arrhythmia rather than atherosclerosis.

Biodegradation-3-1-2151777

Substrate specificity of a long-chain alkylamine-degrading Pseudomonas sp isolated from activated sludge

A bacterium strain BERT, which utilizes primary long-chain alkylamines as nitrogen, carbon and energy source, was isolated from activated sludge. This rod-shaped motile, Gram-negative strain was identified as a Pseudomonas sp. The substrate spectrum of this Pseudomonas strain BERT includes primary alkylamines with alkyl chains ranging from C3 to C18, and dodecyl-1,3-diaminopropane. Amines with alkyl chains ranging from 8 to 14 carbons were the preferred substrates. Growth on dodecanal, dodecanoic acid and acetic acid and simultaneous adaptation studies indicated that this bacterium initiates degradation through a Calkyl–N cleavage. The cleavage of alkylamines to the respective alkanals in Pseudomonas strain BERT is mediated by a PMS-dependent alkylamine dehydrogenase. This alkylamine dehydrogenase produces stoichiometric amounts of ammonium from octylamine. The PMS-dependent alkylamine was found to oxidize a broad range of long-chain alkylamines. PMS-dependent long-chain aldehyde dehydrogenase activity was also detected in cell-free extract of Pseudomonas strain BERT grown on octylamine. The proposed pathway for the oxidation of alkylamine in strain BERT proceeds from alkylamine to alkanal, and then to the fatty acid. Introduction Primary fatty amines contain a nitrogen atom attached to one long alkyl chain. Commercial primary alkylamines are usually mixtures of homologs because the sources of the hydrophobic groups are fatty acids derived from palm oil, coconut oil or tallow. The alkyl chains therefore vary in both chain length and degree of unsaturation. Alkylamines are primarily introduced into the environment through emissions during industrial production. The alkylamines produced in high volumes are toxic to aquatic organisms (Newsome et al. 1991; Schultz et al. 1991; Finlay and Callow 1997). Microbial degradation of primary fatty amines is therefore important for removal in biological wastewater treatment systems and to maintain low environmental concentrations. Yoshimura et al. (1980) studied the biodegradability of various fatty amines in the MITI test. The MITI test assesses the ready biodegradability by determining the biological oxygen demand (OECD 1992). Ratios of the biological oxygen demand and theoretical oxygen demand >0.6 were achieved in MITI tests, demonstrating the susceptibility of alkylamines (C8 to C18) to biodegradation (Yoshimura et al. 1980). Dodecylamine was also demonstrated to be biodegradable in another ready biodegradability test, i.e. Closed Bottle test (OECD 1992). The biodegradation of dodecylamine started immediately and reached ∼80% within a week (van Ginkel et al. 1995). The results obtained in ready biodegradability tests are far more satisfactory when supported by pure culture studies. Until now two pure cultures of bacteria have been shown to degrade primary fatty amines (Yoshimura et al. 1980; Selig et al. 1999). Possible intermediates of alkylamine degradation were not identified in these studies. Therefore, much remains to be learned about the substrate specificity of long-chain alkylamine degrading micro-organisms and the biodegradation pathway. The aim of this study was to investigate how alkylamines are degraded by micro-organisms using a pure culture capable of utilizing alkylamines as sole carbon and energy source. Substrate specificities of the pure culture and cell-free extracts catalysing the initial degradation steps are reported. Materials and methods Chemicals Samples of octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, octadecylamine cocoamine and tallowamine (Armeen®) were provided by Akzo Nobel Surfactants, Stenungsund, Sweden. All other chemicals were of reagent-grade quality and obtained from Sigma Aldrich, Zwijndrecht, The Netherlands or Akcross Organics, Geel, Belgium. The biochemicals were purchased from Boehringer, Mannheim, Germany. Activated sludge Activated sludge used as inoculum was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This activated sludge plant treats predominantly domestic wastewater. Media The mineral salts medium used for isolation and growth experiments contained the following in 1 l of deionized water; 1.55 g K2HPO4, 0.85 g NaH2PO4, 0.5 g NH4Cl, 0.1 g MgSO4 · 7H2O and 0.1 ml trace solution described by Vishniac and Santer (1957). The medium was sterilized by autoclaving for 20 min, together with any added growth substrate (1 g l−1). The volatile primary amines, i.e. octylamine and decylamine were added after autoclaving the mineral salt medium. When these volatile primary amines were used as growth substrate, silica gel was added to give a final concentration of 32 g l−1. Silica gel was added to reduce the concentration of alkylamines in the water phase. Enrichment, isolation and growth Aerobic long-chain alkylamine degrading micro-organisms were isolated from an enrichment culture developed from activated sludge. The enrichment culture was obtained in batch culture flasks containing 200 ml mineral salts medium and 1.0 g l−1 dodecylamine inoculated with 5 ml of activated sludge. After five subcultures a bacterium in this culture was streaked to purity on agar plates containing mineral salts medium, 1.0 g l−1 dodecylamine and 15 g l−1 agar. The cultures and agar plates were incubated at 30°C. The growth rate of strain BERT was recorded by measuring the increase in turbidity in culture flasks with a Hach Ratio XR turbidimeter (Hach, Loveland, CO, USA). The ability of the strain to grow on other substrates was tested using similar media in which dodecylamine was substituted with possible intermediates or other fatty amine derivatives. Culture conditions and preparation of washed cell suspensions and cell-free extracts Cells were grown on octylamine as sole nitrogen, carbon and energy source in a continuous culture. This continuous culture was run at 30°C in a 2-l fermentor (Applikon, Schiedam, The Netherlands) with a working volume of 1.0 l. The impeller speed was 500 rpm. The mineral salt medium was pumped continuously into the fermentor at a rate of 1.0 l day−1 by means of a peristaltic pump. Octylamine was introduced separately to the fermentor with a syringe pump and teflon tube giving a final concentration of 1.0 g l−1. This set-up was required because of transport of the alkylamine through silicone tubes. Cells were also cultivated in the fermentor with acetate, octanal and octanoate under the same conditions but with a mineral salts medium containing the respective organic compounds. The pH of the reactor was maintained at 7. Experiments on the nitrogen balance were also conducted in the fermentor fed with a mineral salts medium without ammonium chloride. The nitrogen recovery was estimated from the amount of nitrogen leaving the fermentor as biomass, ammonium and total nitrogen. The total nitrogen represents the residual substrate and/or water-soluble nitrogen-containing compound formed during the biodegradation process. Cells were harvested from the continuous cultures by centrifugation at 25,000g for 10 min and washed three times with 15 mM phosphate buffer, pH 7.0. Washed cells (10 ml) were disrupted by a French pressure cell at 35,000 psi (Thermo IEC, Needham Heights, MA, USA) After one pass the insoluble matter was precipitated at 50,000g for 20 min. Oxygen consumption The endogenous and substrate-dependent oxygen uptake rates by washed cell suspensions were determined in a polarographic oxygen monitor (Yellow Springs Instruments, Yellow Springs, OH, USA). The polarographic oxygen monitor consisted of a thermostated vessel with a magnetic stirrer, and an oxygen electrode to measure oxygen depletion. Cells were resuspended in phosphate buffer at 30°C in a final volume of 5 ml. After 5 min necessary to determine the endogenous respiration, the substrate-dependent respiration was measured by injection of 0.1 ml of a 1.0 g l−1 substrate solution into the vessel. Suspensions of alkylamines with more than ten carbons and octanal were heated prior to injection. Enzyme assays All enzyme assays were performed at 30°C. Spectrophotometric enzyme assays were carried out in a Shimadzu UV 160A spectrophotometer (Shimadzu, Kyoto, Japan) in 1 cm light-path cuvettes. The oxygen uptake was measured with a polarographic oxygen monitor (Yellow Springs Instruments). Alkylamine dehydrogenase (phenazinium methyl sulfate (PMS)-dependent) was assayed by recording the oxygen uptake in a polarographic oxygen monitor. The reaction mixture (volume 5 ml) contained 0.8 mM PMS and 0.7 g l−1 protein in a phosphate buffer (15 mM; pH 7). The reaction was started by injecting alkylamine in the reaction vessel at a concentration of 4.0 mM. Alkylamine dehydrogenase (PMS-dependent) activity was also determined by colorimetric detection of ammonium in a reaction vessel containing 8.0 mM octylamine, 0.8 mM PMS and 0.5 g l−1 protein in a phosphate buffer (15 mM; pH 7). The enzyme assays were carried out at least twice and the difference in specific activity was less than 20%. Alkylamine dehydrogenase (NAD(P)-dependent) activity was spectrophotometrically assayed by measuring the increase of NAD(P)H at 340 nm. The reaction mixture consisted of 1.3 mM NAD(P), a phosphate buffer (15 mM; pH 7) and cell-free extract (1.4 g l−1 protein) in a total volume of 3 ml. The alkylamine dehydrogenase (NAD(P)-dependent) activity was measured under anaerobic conditions to minimize NADH oxidase activity. For that purpose the contents of the cuvettes were stoppered with Suba Seal® septa (Sigma Aldrich, Zwijndrecht, The Netherlands) and flushed with nitrogen gas. Aldehyde dehydrogenase activity was also measured by spectrophotometric and a respirometric assays. Aldehyde dehydrogenase (PMS-dependent) activity was assayed respirometrically. The reaction mixture (volume 5.0 ml) contained a phosphate buffer (15 mM; pH 7), 0.8 mM PMS, cell-free extract (0.7 g l−1 protein). The reaction was started by injecting octanal in the reaction vessel giving 4.0 mM. Aldehyde dehydrogenase (NAD(P)-dependent) activity was determined by measuring the increase in absorbance at 340 nm. Reaction mixtures contained phosphate buffer (15 mM; pH 7), 0.15 μM aldehyde, 1.0 mM NAD and 0.1 mM KCN and enzyme solution in a volume of 3 ml. Analyses Ammonium was determined colorimetrically by forming indophenol blue with hypochlorite and salicylate in the presence of sodium nitroferricyanide as catalyst (Verdouw et al. 1978). Protein was quantified by bicinchoninic acid method. Cells were first lysed by incubating at 95°C with 1.0 M NaOH. The protein concentration was estimated by using the Bio-Rad Protein assay kit with bovine serum albumin as standard protein. Dissolved organic carbon and total nitrogen were quantified with a Shimadzu TOC apparatus (Shimadzu). Octylamine was analysed by ion chromatography with a Dionex DX-120 (Dionex, Sunnyvale, CA, USA). The Dionex DX-102 was equipped with an IonPac CS14 (4 mm) analytical column, a 25 μl sample loop, a CSRS-I in the external water mode and a CDM-3 flow-through conductivity cell with a DS4 detection stabilizer. The DX-120 was operated at a column temperature of 20°C and a detector temperature of 35°C. The eluent was deionized water with 5.0 mM methylsulphonic acid and 2.5% acetonitrile. The eluent flow rate was 1.0 ml min−1. The dry weight of the micro-organisms was determined by washing and concentrating a known volume from the continuous culture through centrifugation (25,000 g for 30 min). The concentrated biomass on a preweighed watch glass was dried for 1.5 h at 104°C. Results Isolation and characterization A culture capable of growth on dodecylamine was readily enriched from activated sludge through repeated transfers of cells to fresh mineral medium containing dodecylamine. Dodecylamine plates streaked with dilutions of this enrichment culture enabled the isolation of bacteria. The predominant isolate BERT was a Gram-negative, motile, non-spore-forming rod, 2.0–3.5 μm long and 0.6–0.8 μm wide. Oxidase, catalase, urease and alcohol dehydrogenase were present. The strain was able to utilize the following substrates: glucose, phenylacetate, citrate, malate, mannose mannitole and gluconate. It did not grow on maltose, trehalose, m-inositol, citraconate, erytritol, sorbitol, d-xylose, d-tartrate and l-arabinose. The strain was not capable of reducing nitrate. The identity of the primary alkylamine degrading bacterium was determined by the profile of cellular fatty acids and partial 16S rRNA gene sequencing. The profile of the cellular fatty acids is typical for the RNA group I of the genus Pseudomonas. The partial sequences have shown a similarity of 99.3% to Pseudomonas putida, P. plecoglosscida and P. alcaligenes. These characteristics only allow to place strain BERT within the RNA group I of the genus Pseudomonas. Growth A number of fatty amine derivatives were tested for their ability to support growth of Pseudomonas strain BERT. The strain was found to be able to grow on a wide range of long-chain primary amines, i.e. octylamine (in the presence of silica gel), decylamine (in the presence of silica gel), dodecylamine, tetradecylamine, octadecylamine, cocoamine oleylamine and tallowamine as sole source of carbon. Coco-1,3-diaminopropane and dodecyl-1,3-diaminopropane—both in the presence of silica gel—were also used as carbon and energy source by Pseudomonas strain BERT whereas dodecyldimethylamine, didodecylamine, didodecymethylamine, dodecyltrimethylammonium chloride did not support growth. Mineral salts media amended with possible intermediates of dodecylamine degradation such as dodecanal, dodecanoic acid and acetate supported growth of Pseudomonas strain BERT. Depletion of dodecylamine to non-detectable concentrations from the nitrogen-free mineral salts medium in batch cultures and the concurrent growth of the isolate clearly demonstrate that this isolate also used alkylamines as its nitrogen source (data not shown). Octylamine was used as growth substrate in a number of experiments because this amine is soluble in water at a concentration of 1.0 g l−1. Use of octylamine allowed, for instance, accurate measurement of the increase in turbidity due to growth. From the growth curve, a doubling time of 4 h was estimated (data not shown). Growth yield obtained with Pseudomonas strain BERT was 0.4 g dry weight of cells per g of octylamine utilized. The fate of the nitrogen of octylamine in terms of ammonium, biomass-nitrogen, nitrogen-containing soluble microbial products and residual octylamine was assessed in a continuous culture. Total nitrogen, ammonium and biomass-nitrogen were measured in triplicate. Approximately 25 ± 3% of the nitrogen was recovered as biomass-nitrogen. Octylamine-nitrogen was converted for 60 ± 4% into ammonium. Less than 1% of the nitrogen in the effluent was present as octylamine. Approximately 10 ± 2% of the nitrogen was recovered as water-soluble nitrogen-containing products. Respiration experiments The alkylamine-degrading isolate was grown on a number of different substrates as sole source of carbon after which respiration rates of washed cell suspensions were examined with a variety of substrates in order to identify possible intermediates of the degradation pathway (Table 1). Washed cell suspensions of Pseudomonas strain BERT grown with octylamine were capable of oxidizing octylamine, octanoate octanal and acetate. Whole cells of Pseudomonas strain BERT also showed octylamine-dependent oxygen uptake when grown with octanal, and octanoate. However, strain BERT grown with acetate did not respire octylamine. Finally, Pseudomonas strain BERT grown on octanal, octanoate and actate displayed significant activities with the alkanal and alkanoate tested (Table 1). Table 1Oxidation of various potential intermediates of alkylamine degradation by washed cell suspensions of strain BERT grown on octylamine, octanal, octanoate and acetateSubstrateGrowth substrateOctylamineOctanalOctanoateAcetatenmol min−1 mg−1 proteinOctylamine8525440Octanal6243660Octanoate84539041Acetate39354150Rates of oxygen uptake are expressed as nmoles O2 min−1 mg−1 protein after correction for endogenous respiration. The endogenous respiration of the strain grown on octylamine, octanal, octanoate and acetate were 8, 4, 8 and 6 nmol O2 min−1 mg protein−1, respectively The ability of octylamine-grown strain BERT to oxidize alkylamines was also evaluated (Table 2). Octylamine-grown cells were capable of oxidizing all alkylamines with alkyl chains ranging from 3 to 18 carbon atoms. The highest activity was observed when hexylamine, octylamine and decylamine were added to the reaction vessel. The oxidation rate of nonylamine was comparable to the activity found with octylamine. Propylamine was respired at a low rate. Low activities were also detected with alkylamines with more than 14 carbon atoms. All other fatty amine derivatives except for dodecyl-1,3-diaminopropane were not oxidized by strain BERT grown on octylamine (Table 2). Table 2Oxidation of various alkylamines by washed cell suspensions and alkylamine dehydrogenase (PMS-dependent)SubstrateWashed cell suspensionCell-free extractnmol min−1 mg−1 proteinButylamine145Hexylamine8830Octylamine8533Decylamine7634Dodecylamine6731Tetradecylamine5735Hexadecylamine3118Octadecylamine125Nonylamine8535Propylamine73Methylamine00Didecylamine00Decyldimethyamine00Decyltrimethylammonium 00Dodecyl-1,3-diaminopropane165Rates of oxygen uptake are expressed as nmoles O2 min−1 mg−1 protein. The rates for the washed cell suspensions are corrected for the endogenous respiration of octylamine-grown cells, i.e. 8 nmol O2 min−1 mg protein−1 Enzymatic activities Activities of enzymes that may be involved in alkylamine metabolism were tested in cell-free extracts prepared from cells grown on octylamine. Extracts of octylamine–grown cells released ammonium in stoichiometric amounts from octylamine in the presence of PMS (Fig. 1). In another experiment the presence of PMS induced the consumption of 0.08 mM oxygen upon the addition of 0.17 mM octylamine. The activity of the PMS-dependent alkylamine dehydrogenase with dodecylamine was 31 nmol min−1 mg−1 protein. NAD and NADP could not replace the artificial electron acceptor. The substrate specificity of cell-free extracts catalysing the conversion of alkylamines was studied by measuring the oxygen consumption in the presence of PMS. The enzyme was active towards alkylamines with alkyl chains with 8–18 carbons. Propylamine, nonylamine and dodecyl-1,3-diaminopropane also acted as substrate for the enzyme. Among the primary alkylamines examined, the most preferred were alkylamines with alkyl chains length ranging from 8 to 12 carbons. Methylamine secondary, tertiary and quaternary fatty amine derivatives were no adequate substrates (Table 2). Demonstration of alkanal dehydrogenase activity in cell extracts was achieved by incubating cell-free extracts with octanal and PMS and measuring the oxygen consumption. An activity of 19 nmol O2 min−1 mg−1 protein was measured. Alkanal dehydrogenase activity was not detected with other electron acceptors, i.e. NAD and NADP, despite repeated attempts. Fig. 1Formation of ammonium from 0.78 mM octylamine by cell-free extract of octylamine-grown cells in the presence (open square) and absence (filled square) of PMS. The protein concentration in the assay was 0.5 g l−1 Discussion The fate of long-chain alkylamines in the environment is largely dependent on the ability of micro-organisms to metabolize these compounds. Although degradation of alkylamines in OECD ready biodegradability tests has been observed (Yoshimura et al. 1980; OECD 1992; van Ginkel et al. 1995), little is known about the intermediates in the biodegradation process and the enzymes involved in the biodegradation. In this paper, we describe the properties of the newly isolated Pseudomonas strain BERT, which is capable of degrading long-chain alkylamines. Yoshimura et al. (1980) also isolated a Pseudomonas strain. Both Pseudomonas strains degraded primary alkylamines with varying alkyl chain lengths. Pseudomonas species are noted for their metabolic diversity and are often isolated from enrichments designed to identify bacteria that degrade anthropogenic organic compounds. Another species known to utilize a long-chain alkylamine as sole source of carbon and energy is from the genus Rhodococcus isolated from top soil (Selig et al. 1999). A nitrogen mass balance obtained in a continuous culture already, strongly indicated that octylamine a water soluble long-chain alkylamine is completely (ultimately) biodegradable because almost all of the octylamine-nitrogen was converted into biomass-nitrogen and ammonium. Other evidence of complete degradation of alkylamines has been obtained through the proposed biodegradation pathway. The biodegradation of alkylamines was assumed to proceed either via a Calkyl–N cleavage or oxidation at the far-end of the alkyl chain. Based on the induction pattern, and PMS-dependent dehydrogenase activity with alkylamines, the biodegradation of alkylamines is suspected to involve a cleavage of the Calkyl–N bond, which yield the respective alkanals and ammonium. The alkanals are oxidized by a PMS-dependent alkanal dehydrogenase to the respective fatty acids. The induction of long-chain alkanal dehydrogenase activity by growth of the bacterium on octylamine demonstrates that alkanals are significant intermediates. A PMS-dependent long-chain alkanal dehydrogenase was also detected in Acinetobacter calcoaceticus HO1-N grown on hexadecane and hexadecanol (Fox et al. 1992). At present it is unknown if both the alkanal and the alkylamine are dehydrogenated by the same enzyme in Pseudomonas strain BERT. Fatty acids produced by the alkanal dehydrogenase are channelled into the β-oxidation cycle (Ratledge 1994). The degradative pathway shown in Fig. 2 is inducible because octylamine is not oxidized by acetate-grown cells (Table 1). The degradation pathway is quite similar to those described for alkyltrimethylammonium salts, alkyldimethylamines and alkylbis(2-hydroxyethyl)amines (van Ginkel 1996, 2003). This biodegradation pathway provides evidence of total mineralization of alkylamines as indicated by the nitrogen mass balance and the ready biodegradability test results (Yoshimura et al. 1980; van Ginkel et al. 1995). Fig. 2Proposed pathway for the degradation of alkylamines in Pseudomonas strain BERT; (1) alkylamine dehydrogenase, (2) aldehyde dehydrogenase. The fatty acids formed are channelled into the β-oxidation cycle PMS, an artificial electron acceptor is generally used to assay enzymes known as quinoprotein enzymes. Physiological electron acceptors are cytochrome c, protein-bound haem and ubiquinone. Methylamine dehydrogenases (de Beer et al. 1980; Duine et al. 1990), short-chain alkylamine dehydrogenases (Shinagawa et al. 1988) and a long-chain alkanal dehydrogenase (Fox et al. 1992) have been identified as quinoproteins. Methylamine dehydrogenases catalyse the oxidative deamination of methylamine to methanal and ammonium (McIntire et al. 1990). The PMS dependent removal of ammonium from alkylamines in strain BERT may be catalysed by an enzyme similar to methylamine dehydrogenases although no activity towards methylamine was detected (Table 2). Zhu et al. (2000) demonstrated that the conversion of one amino acid of methylamine dehydrogenase altered the substrate preference of methylamine dehydrogenase to a large extent. The preferred substrates of the mutated enzyme are alkylamines with at least seven carbons. Long-chain alkylamines are probably also oxidized by an alkylamine dehydrogenase isolated from a butylamine-grown Pseudomonas sp. although the highest activity was found with butylamine (Shinagawa et al. 1988). A quinoprotein was also found in a P. putida catalysing the deamination of propylamine and butylamine both naturally occurring alkylamines (Adachi et al. 1998). Only a long-chain alkylamine dehydrogenase is required to convert anthropogenic alkylamines into compounds, which can enter an existing pathway. This enzyme may have evolved from methylamine dehydrogenases or short-chain alkylamine dehydrogenases involved in the degradation of naturally occurring amines. A major feature of Pseudomonas strain BERT is its broad substrate specificity with respect to primary alkylamines. Activities of washed cell suspensions and alkylamine dehydrogenase activities in cell-free extracts demonstrate that alkylamines with alkyl chain lengths ranging from 6 to 10 carbons are preferred. Odd alkyl chain amines were also respired by Pseudomonas strain BERT. Water-insoluble long-chain alkylamines are oxidized at a lower rate by washed cell suspensions and alkylamine dehydrogenase of strain BERT. This probably reflects the low bioavailability of the long-chain alkylamines. Limited bioavailability of long-chain alkylamines (C12 to C18) can also be concluded from the non-toxicity of the alkylamines to strain BERT at a concentration of 1.0 g l−1. In batch cultures growth of Pseudomonas strain BERT on octylamine and decylamine was only possible in the presence of silica gel. Silica gel reduces the toxicity of fatty amine derivatives through adsorption onto the silica gel (van Ginkel et al. 1992; Selig et al. 1999). The ease with which Pseudomonas strain BERT was isolated and the high growth rate of strain BERT are consistent with studies showing ready biodegradability of many primary amines (Yoshimura et al. 1980; van Ginkel et al. 1995). Based on the biodegradation pathway and the broad substrate specificity, significant differences in the biodegradability of alkylamines with varying chain lengths are not expected. However, degradation of primary alkylamines does differ in ready biodegradability tests (Yoshimura et al. 1980). This can be explained by biocidal effects and limited bioavailability. Reading across of the ready biodegradability test results enabled by this study reveals that all long-chain alkylamines—both saturated and unsaturated—are readily biodegradable. Future research should focus on identifying the intermediates and purifying the enzymes involved in the degradation of alkylamines.

J_Struct_Funct_Genomics-3-1-1769342

Coverage of whole proteome by structural genomics observed through protein homology modeling database

We have been developing FAMSBASE, a protein homology-modeling database of whole ORFs predicted from genome sequences. The latest update of FAMSBASE (http://daisy.nagahama-i-bio.ac.jp/Famsbase/), which is based on the protein three-dimensional (3D) structures released by November 2003, contains modeled 3D structures for 368,724 open reading frames (ORFs) derived from genomes of 276 species, namely 17 archaebacterial, 130 eubacterial, 18 eukaryotic and 111 phage genomes. Those 276 genomes are predicted to have 734,193 ORFs in total and the current FAMSBASE contains protein 3D structure of approximately 50% of the ORF products. However, cases that a modeled 3D structure covers the whole part of an ORF product are rare. When portion of an ORF with 3D structure is compared in three kingdoms of life, in archaebacteria and eubacteria, approximately 60% of the ORFs have modeled 3D structures covering almost the entire amino acid sequences, however, the percentage falls to about 30% in eukaryotes. When annual differences in the number of ORFs with modeled 3D structure are calculated, the fraction of modeled 3D structures of soluble protein for archaebacteria is increased by 5%, and that for eubacteria by 7% in the last 3 years. Assuming that this rate would be maintained and that determination of 3D structures for predicted disordered regions is unattainable, whole soluble protein model structures of prokaryotes without the putative disordered regions will be in hand within 15 years. For eukaryotic proteins, they will be in hand within 25 years. The 3D structures we will have at those times are not the 3D structure of the entire proteins encoded in single ORFs, but the 3D structures of separate structural domains. Measuring or predicting spatial arrangements of structural domains in an ORF will then be a coming issue of structural genomics. Introduction Genome sequencing projects provided a huge number of amino acid sequences without functional information (Stein 2001). To discover biological functions of those proteins, both computational predictions and biochemical experiments are necessary (Tsoka and Ouzounis 2000). Most of the proteins perform functions after forming specific 3D structures, and therefore protein 3D structure is one of the most valuable sources of information to predict protein function (Domingues et al. 2000; Xie and Bourne 2005). Protein function prediction based on 3D structures, especially protein surface structures, with evolutionary and/or physicochemical characteristics have been extensively studied (Lichtarge and Sowa 2002; Campbell et al. 2003; Kinoshita and Nakamura 2003; Laskowski et al. 2003; Ota et al. 2003; Pieper et al. 2006). However, determining protein structures of all the function-unknown proteins for applying these types of study is not practical. Proteins are classified into a large number of ‘families’ based on the amino acid sequence similarity (Dayhoff 1972), and proteins with similar amino acid sequences are known to have similar 3D structures (Chothia and Lesk 1986), all because the proteins in a family are evolutionary related (Doolittle 1995). Once we have 3D structure of at least one of the proteins in a family, then 3D structures of other proteins in the same family can be computationally deduced by ‘homology modeling’ (Burley 2000; Baker and Sali 2001). Based on this logic, structural genomics (SG) projects, which are to determine protein 3D structures of representatives for each family have been proposed and launched (Vitkup et al. 2001; Brenner 2000; Burley and Bonnano 2002). In homology modeling, corresponding residues between an amino acid sequence of structure unknown protein (target) and that of 3D structure known protein (template) in the same family are determined by sequence alignment and every residue in a template protein is replaced by that in a target protein (Marti-Renoma et al. 2000). SG projects have been providing new protein structures (Todd et al. 2005; Xie and Bourne 2005; Chandonia and Brenner 2006). Protein Data Bank (PDB) (Berman et al. 2000) now contains more than 390 3D structures for function unknown or hypothetical proteins (Stark et al. 2004). Protein function predictions based on 3D structures determined by SG projects are also in progress (Goldsmith-Fischman and Honig 2003; Liu et al. 2005; Petrey and Honig 2005). There are some projects that focus on a specific species and try to determine the 3D structures of whole proteins encoded in the genome of the species (Kim 2000; Yokoyama et al. 2000; Kim et al. 2003). Those projects provide a considerable number of 3D structures in a single protein family. This results in providing multiple templates for a single protein family and it can improve quality of homology modeling (Contreras-Moreira et al. 2003). We have developed FAMSBASE; a database for homology modeling 3D structures of whole proteins predicted on whole genome sequences, since 2001 (Yamaguchi et al. 2003; http://daisy.nagahama-i-bio.ac.jp/Famsbase/). FAMSBASE contains results of homology modeling by FAMS, a full automatic modeling software (Ogata and Umeyama 2000). Sequence alignments between whole ORFs and proteins in PDB are based on GTOP (Kawabata et al. 2002). We report here the update of the database including differences in the amount of structural data from the previous version, estimation of the time that whole ORFs predicted out of genome sequences are covered by homology modeling 3D structures and upcoming issues for utilizing those modeled structures. Methods Data update of FAMSBASE Correspondence between ORFs derived from whole genome sequences and protein amino acid sequences whose 3D structures are known is provided by GTOP database (Kawabata et al. 2002). The update in May 2005 of FAMSBASE is based on February 2004 version of GTOP. Protein 3D structures in PDB by November 2003 are used for homology modeling templates. FAMS (Ogata and Umeyama 2000) is applied by Umeyama et al. to pair-wise alignments between a predicted ORF sequence and an amino acid sequence with known 3D structure, and a 3D structure is modeled. All the results are stored in FAMSBASE. Assessing annual difference of data in FASBASE Based on the amount of data in FAMSBASE in 2001 and the amount of increase in the following years, a due year for whole proteome 3D structure models is estimated. Estimation is done residue-wise, not ORF-wise, since modeled structures in FAMSBASE are often limited to structural domains. In this report, structural domains refer to SCOP domains (Andreeva et al. 2004). All ORFs predicted out of genome sequences are divided into soluble and membrane proteins. The division is carried out by SOSUI (Hirokawa et al. 1998), and a protein with one or more transmembrane regions is classified into a membrane protein. The number of residues of whole soluble proteins encoded in the genome sequence (G) of species i is denoted as S, and the number of residues of whole membrane proteins is denoted as M. The number of residues included in modeled 3D structures of soluble and membrane proteins are denoted as S3 and M3, respectively. For a certain genome Gi, the coverage of modeled 3D structures in whole soluble proteins is then S3/S and the coverage for whole membrane proteins is M3/M. The coverage is summarized in different kingdoms of life as in the following equations; Both figures are calculated based on the data at the different times of FAMSBASE update, gradients in figures are then calculated, and the figures are extrapolated up to the year that coverage reaches to 100. It is getting to be known that not all ORFs assume stable 3D structures. Some parts of ORFs are considered to be natively disordered (Oldfield et al. 2005; Dyson and Wright 2005). Hence it is unlikely that coverage by homology modeling reaches to 100. We, therefore, estimate disordered regions in whole ORFs by DisEMBL (Linding et al. 2003) and omit these disordered regions from the calculation. Non-overlap multiple model structures in single ORFs Modeled 3D structures in FAMSBASE are often limited to structural domains. To find an ORF of which most of the entire 3D structure is modeled in pieces of structural domains, an ORF covered by non-overlapping three or more modeled 3D structures in eukaryotic genome is surveyed based on the following criteria; (1) 70% or more residues in the ORF are included in one of the modeled 3D structures, (2) the ORF contains three or more non-overlapping modeled structures, and (3) the sequence identity between a template protein and a target domain is no less than 25%. At the time of FAMSBASE building, five model structures are at most built for each ORF (Yamaguchi et al. 2003). Therefore, the expected number of modeled structures in the above criteria is between three and five. Prediction of domain interfaces The 3D structure in pieces for a single ORF needs to be assembled to model the entire 3D structure. For this procedure, a prediction of domain interfaces of each 3D structure is needed. A hydrophobicity index based on protein 3D structures is built for domain interface prediction. Hydrophobicity of amino acid residue is measured by buriedness of a residue inside the protein 3D structures. A representative 4,529 chains in PDB among which sequence identities are less than 30% were selected and solvent accessibility of each residue is calculated on a monomer state. For each amino acid residue type i (i = 1,...,20), the number of residue with accessibility no less than b (=0.0 − 1.0) is counted (Sb,i). Database derived hydrophobicity index (Ib,i) is obtained by; b is set to 0.15 to maximize the difference of Ib,i among different residues. The index has good correlation with Kyte and Doolittle hydrophobicity index (Kyte and Doolittle 1982). The index I0.15,i is assigned to every residue on the surface (accessibility no less than 0.15) of a modeled 3D structure. The hydrophobicity of each residue on a surface of a protein is then obtained by averaging the assigned values of residues within 7.0 Å from the residue in concern. A hydrophobic patch on the surface of the modeled structure is found as a cluster of surface residues with the hydrophobicity no less than 0.0. Results and discussion Coverage of whole protein space by homology modeling The latest update of FAMSBASE at May 2005 uses protein 3D structures deposited to PDB by the end of Nov. 2003 and ORFs predicted from genome sequences deposited by February 2004 (http://daisy.nagahama-i-bio.ac.jp/ Famsbase/). The latest FAMSBASE contains 1,396,272 modeled 3D structures of 368,724 ORFs derived from 17 archaebacterial, 130 eubacterial, 18 eukaryotic and 111 phage genomes; in total 276 genomes. Five models at maximum are built for each ORF in FAMSBASE. Those five models are the structure for the same or different regions in the ORF. When multiple models are built for the same region of ORF, we can evaluate the reliability of the model. When the model based on different templates have the similar 3D structures, then the 3D structure would be reliable. When the structures are different, the modeled structure would be less reliable. We further test the quality of modeled 3D structure by ProsaII (Sippl 1993) and find that about 72% of the modeled 3D structures are energetically ranked as number one and comparable to experimentally determined 3D structures. Some of the structures that fail the test are structures of a part of a large protein, mostly structural domains of large proteins. It is difficult to assess the quality of this type of domain structures, because interfaces of the domain for other parts of the protein are exposed in the modeled structures. Tendency of amino acid residue appearance in the interface is supposed to be different from that at the surface as we discuss down below. In the genome of 276 species, 734,193 ORFs are predicted. Therefore, in FAMSBASE, 3D structure of 50% (368,724/734,193) of ORFs have been built and stored (Table 1). These are about 47% of ORFs in archaebacterial genomes, about 52% in eubacterial genomes and about 49% of eukaryotic genomes.Table 1Number of ORFs and those with modeled 3D structures in 276 genomesSpeciesORFModel%ArchaeaArchaeoglobus fulgidus DSM43042,4071,23351.2Aeropyrum pernix K12,69478929.3Halobacterium sp. NRC-12,6051,19545.9Methanosarcina acetivorans C2A4,5442,12446.7Methanocaldococcus jannaschii DSM26611,77087549.4Methanopyrus kandleri AV191,68778446.5Methanosarcina mazei Goe13,3711,63448.5Methanothermobacter thermautotrophicus1,86999853.4Nanoarchaeum equitans Kin4-M53626449.3Pyrococcus abyssi Orsay1,78494252.8Pyrobaculum aerophilum IM22,6051,04740.2Pyrococcus furiosus DSM 36382,0651,03550.1Pyrococcus horikoshii OT32,06187942.6Sulfolobus solfataricus P22,9941,36545.6Sulfolobus tokodaii 72,8261,22843.5Thermoplasma acidophilum DSM17281,47884457.1Thermoplasma volcaniumGSS11,52683955.0sum38,82218,07546.6EubacteriaAquifex aeolicus VF51,55392959.8Nostoc sp. PCC 71206,1322,76545.1Agrobacterium tumefaciens C585,3013,01756.9A. tumefaciens C58 (Dupont)5,4023,02856.1Bacillus anthracis str. Ames5,3112,46346.4Buchnera aphidicola Sg55241074.3B. aphidicola50738575.9Bordetella bronchiseptica RB504,9942,93458.8Borrelia burgdorferi1,63953532.6Bacillus cereus ATCC 145795,2552,53448.2Candidatus Blochmannia floridanus58344776.7Bacillus halodurans C-1254,0662,12752.3Bradyrhizobium japonicum8,3174,44953.5Bifidobacterium longum NCC27051,73198556.9Brucella melitensis 16M3,1981,80156.3Bordetella parapertussis4,1852,52560.3B. pertussis Tohama I3,4472,17963.2Bacillus subtilis 1684,1062,15352.4Brucella suis 13303,2641,67751.4Bacteroides thetaiotaomicron VPI-54824,8162,46251.1Buchnera sp. APS57443676.0Clostridium acetobutylicum ATCC8243,8482,05353.4Coxiella burnetii RSA 4932,04592545.2Chlamydophila caviae GPIC1,00550550.2Caulobacter crescentus3,7372,08455.8Corynebacterium diphtheriae NCTC131292,2721,16551.3Corynebacterium efficiens YS-3142,9981,51350.5Corynebacterium glutamicum ATCC 130323,0991,55450.1Campylobacter jejuni1,63489354.7Chlamydia muridarum Nigg91148353.0Clostridium perfringens 132,7231,47054.0Chlamydophila pneumoniae AR391,11649544.4Chlamydophila pneumoniae CWL0291,05249647.1Chlamydophila pneumoniae J1381,06950146.9Chlamydophila pneumoniae TW-1831,11350145.0Chlorobium tepidum TLS2,2521,16651.8Clostridium tetani E882,4321,30653.7Chlamydia trachomatis D/UW-3/CX89448554.3Chromobacterium violaceum ATCC 124724,3852,34353.4Deinococcus radiodurans R13,1021,57950.9Escherichia coli K-12 MG16554,2842,39856.0E. coli O157:H75,4472,60747.9E. coli O157:H7 EDL9335,4492,62948.2E. coli CFT0735,3792,55847.6Enterococcus faecalis V5833,2651,56848.0Fusobacterium nucleatum ATCC 255862,0671,01148.9Geobacter sulfurreducens PCA3,4451,90255.2Gloeobacter violaceus PCC 74214,4302,20849.8Haemophilus ducreyi 35000HP1,71786550.4Helicobacter hepaticus ATCC 514491,87590248.1Haemophilus influenzae Rd1,7091,03860.7Helicobacter pylori 266951,56674147.3Helicobacter pylori J991,49174750.1Listeria innocua Clip112623,0431,64153.9Leptospira interrogans serovar4,7251,71936.4Lactococcus lactis IL14032,2661,25455.3Listeria monocytogenes EGD-e2,8461,65358.1Lactobacillus plantarum WCFS13,0091,64754.7Mycobacterium bovis subsp.3,9202,01851.5Mycoplasma gallisepticum R72637151.1Mycoplasma genitalium G3748030563.5Mycobacterium leprae TN1,60591857.2Mesorhizobium loti MAFF3030997,2813,82952.6Mycoplasma penetrans1,03747245.5Mycoplasma pneumoniae M12968833348.4Mycoplasma pulmonis UAB CTIP78239850.9Mycobacterium tuberculosis H37Rv3,9182,03652.0Mycobacterium tuberculosis CDC15514,1871,99047.5Nitrosomonas europaea ATCC 197182,4611,36655.5Neisseria meningitidis MC582,0251,01650.2Neisseria meningitidis Z24912,0651,02549.6Oceanobacillus iheyensis HTE8313,4961,89254.1Phytoplasma asteris, OY strain75442356.1Pseudomonas aeruginosa PAO15,5663,20657.6Porphyromonas gingivalis W831,90994449.4Photorhabdus luminescens laumondii4,6832,28648.8Prochlorococcus marinus MED41,71293354.5Prochlorococcus marinus MIT93132,2651,12249.5Prochlorococcus marinus marinus1,88293949.9Pasteurella multocida PM702,0141,23761.4Pseudomonas putida KT24405,3502,96855.5Pseudomonas syringae pv. tomato str.5,6082,93852.4Pirellula sp. 17,3252,58835.3Rickettsia conorii Malish 71,37457241.6Rhodopseudomonas palustris4,8142,73956.9Rickettsia prowazekii Madrid E83449859.7Ralstonia solanacearum GMI10005,1162,69852.7Streptococcus agalactiae2,1241,15954.6Streptococcus agalactiae NEM3162,0941,17456.1Staphylococcus aureus Mu502,7481,45152.8Staphylococcus aureus N3152,6241,44755.1Staphylococcus aureus MW22,6591,41053.0Streptomyces avermitilis7,6714,00152.2Streptomyces coelicolor A3(2)8,1544,19551.4Staphylococcus epidermidis ATCC 122282,4851,30352.4Shigella flexneri 2a 3014,4522,30651.8Shigella flexneri 2a str. 2457T4,0682,15953.1Sinorhizobium meliloti 10216,2053,49956.4Streptococcus mutans UA1591,9601,13658.0Shewanella oneidensis MR-14,7782,29147.9Streptococcus pneumoniae R62,0941,10152.6Streptococcus pneumoniae TIGR42,0431,13555.6Streptococcus pyogenes SF3701,69695656.4Streptococcus pyogenes MGAS82321,84599654.0Streptococcus pyogenes MGAS3151,86598652.9Streptococcus pyogenes SSI-11,86197652.4Salmonella typhi CT184,7672,34749.2Salmonella typhimurium LT24,5542,45754.0Salmonella enterica subsp. enterica4,3232,26352.3Synechocystis sp. PCC 68033,1671,67953.0Synechococcus sp. WH 81022,5171,24349.4Thermosynechococcus elongatus BP-12,4751,30352.6Thermotoga maritima MSB81,8461,05156.9Treponema pallidum subsp.1,03151750.1Thermoanaerobacter tengcongensis MB4T2,5881,40354.2Tropheryma whipplei TW08/2778349463.1Tropheryma whipplei str. Twist80849961.8Ureaplasma urealyticum61130349.6Vibrio cholerae N169613,8281,97151.5Vibrio parahaemolyticus RIMD 22106334,8322,46150.9Vibrio vulnificus CMCP64,5372,46154.2Vibrio vulnificus YJ0165,0282,49949.7Wigglesworthia brevipalpis61144172.2Wolinella succinogenes DSMZ 17402,0441,20859.1Xanthomonas axonopodis pv. citri 3064,4272,37453.6Xanthomonas campestris pv. campestris4,1812,28754.7Xylella fastidiosa 9a5c2,8321,15840.9Xylella fastidiosa Temecula12,0361,06652.4Yersinia pestis CO924,0832,11651.8Yersinia pestis KIM4,2812,12349.6sum396,126206,31152.1EukaryotesArabidopsis thaliana28,72314,39450.1Caenorhabditis briggsae14,7137,06348.0Caenorhabditis elegans22,2208,84139.8Ciona intestinalis15,8657,99450.4Drosophila melanogaster18,3029,54152.1Danio rerio26,58716,44361.8Encephalitozoon cuniculi1,99688744.4Guillardia theta Nucleomorph63230748.6Homo sapiens (ENSEMBLE)28,06315,46755.1Leishmania major Friedlin1736235.8Mus musculus24,92814,38257.7Neurospora crassa10,0883,80037.7Oryza sativa16,7244,51727.0Plasmodium falciparum 3D75,2681,90536.2Rattus norvegicus28,68216,74058.4Saccharomyces cerevisiae5,8692,91349.6Schizosaccharomyces pombe5,2612,80753.4Takifugu rubripes rubripes37,45215,20240.6sum291,546143,26549.1Phages/Viruses18646817.444AHJD2114.844RR2.8t2525120.2933W80911.3A11872912.5A5111100.0Aeh13315115.4APSE-154611.1B11119.1B10317423.5Bcep7816158.2BF238112.5bIL1706423.1bIL2856258.1bIL28661711.5bIL30956610.7bIL31029413.8bIL31122627.3bIL31227311.1BK5-T6369.5Bxb1861214.0C23925.1Cp-12827.1ϕCTX4748.5D29791519.0D3941111.7Rb1549612.2ϕg1e49612.2GA-13538.6Gh-1421228.6H-19B22418.2HF2114119.6HK02257814.0HK62058610.3HK97611016.4HP14137.3HP23638.3K1394449.1KVP403815715.02,38957712.3L-413C40410.0L5851214.1λ661827.3A261813.1Mu53611.3N15601321.7Mycoplasma virus P11100.0Enterobacteria phage P11100.0P242511.9P2236925.0P2758915.5P33549612.2P412216.7P60801316.3PA0134514.7PaP369811.6ϕKZ306258.2ϕCh19899.2ϕYeO3-12591322.0ϕ10551815.7ϕC3155814.5ϕ3626501020.0ϕE125711216.9ϕETA66812.1ϕNIH1.155610.9ϕPV8365913.8ϕSLT621219.4ϕadh63812.7ϕBT155916.4ϕA1122501020.0P682229.1ϕKMV481122.9PM22214.5PRD122418.2ΨM23113.2ΨM10037410.8PY54671014.9PZA27414.8R1t50612.0RB692565621.9RB492724918.0Rd47612.8RM3781461711.6PVL62812.9Sfi112514.0V53713.2SIO134617.6Sk15411.9SP620630.0SP βc21853317.8SPP110676.6MM153611.3ST64B56814.3ST64T65913.8720146817.4DT147714.9O12055747.0Sfi1945613.3Sfi2150918.0Stx2165116.7T3441022.7T42785820.9T7581017.2TM48955.6TP901-156712.5Tuc200956712.5Ul365858.6VHML57814.0VpV2626746.0VT2-Sa821113.4Wϕ4449.1Sum7,699107313.9Total734,193368,72450.2 When a modeled 3D structure is counted based on the number of amino acid residues, not on the number of ORFs, a different aspect emerges. Figure 1 shows the percentage of amino acid residues per ORF included in the modeled structures. ORFs without a modeled structure are omitted. Of archaebacterial and eubacterial genomes, in 60% of ORFs, more than 80% of the residues are included in modeled 3D structures, however, of eukaryotic genomes, only in 30% of ORFs, more than 80% of the residues are included (red and blue sections in Fig. 1). The proportion of residues in modeled 3D structure can be measured by the number of residues in a typical structural domain as shown in SCOP (Andreeva et al. 2004). The average size of protein domain is around 100–150 residues (Copley et al. 2002). In ORFs with modeled structures, a continuous region of residues with one domain or more remains as structure unknown in only about 18% of ORFs of archaebacterial and eubacterial genomes, whereas in about 60% of ORFs of eukaryotic genomes, the regions with one domain or more remain as structure unknown.Fig. 1Percentage of amino acid residues included in modeled 3D structures in each ORF is classified by 10% bins and shown in pie charts. ORFs without a modeled structure are not included. A number of ORFs with modeled structures and an average length of the ORFs are shown at the center of each pie chart. Sections bordered by thick black lines indicate that the unmodeled region in the ORF is no less than the size of a domain (about 150 residues) Annual difference of model structures In FAMSBASE of 2001, 38% of amino acid residues in all ORFs of archaebacterial and 40% of eubacterial genomes were included in modeled 3D structures (Yamaguchi et al. 2003). In the current update of FAMSBASE based on data by around 2004, 42% of amino acid residues in all ORFs in archaebacterial and 46% of eubacterial genomes are included in modeled structures. In eukaryotic genomes, 24% of amino acid residues in 2003, and 26% in 2004 are included in modeled 3D structures. Those figures can be used to estimate the time when modeled 3D structures of whole proteins predicted from genomes are obtained. The estimation for the time obtaining the whole soluble and membrane proteins are treated separately, because the speed of structure determination for soluble and membrane proteins seems to differ. The assumption for the estimation is that the speed for structure determination would stay the same and no new protein family would appear. For eubacterial genomes, 72.6% of residues in whole ORFs are predicted by SOSUI (Hirokawa et al. 1998) to encode soluble proteins and 27.4% to encode membrane proteins. This ratio is not so different from the previous prediction by Krogh et al. (2001). Of about 40% of whole eubacterial ORF that were with modeled 3D structures in 2001, approximately 90% were soluble proteins and 10% were membrane proteins. Therefore, about 50% (=0.40 × 0.90/0.726) of the whole soluble proteins were modeled. Of the whole membrane proteins in eubacterial genome, about 15% (=0.40 × 0.10/0.274) were modeled. In 2004, those figures are grown to 57% and 19%, respectively. In eubacterial whole ORFs, about 19.9% of amino acid residues are predicted to be included in disordered region by DisEMBL (Linding et al. 2003). Some of these regions are included in the modeled structures. These regions are either incorrectly predicted regions or incorrectly modeled regions. Assuming that the disordered regions without modeled 3D structures are correctly predicted, 10.8% of amino acid residues in soluble proteins were disordered and we would never obtain 3D structures of those regions. Then, by extrapolating the coverage of soluble proteins up to 89.2% (100–10.8) with the current growth rate, we can estimate that, by the year 2017, whole soluble proteins encoded in eubacterial genomes can be modeled (Fig. 2). Whole soluble proteins of archaebacterial genome can be modeled by 2021 and those of eukaryotic genomes, by 2031.Fig. 2Annual differences of modeled structures classified by kingdoms of life. The percentage is the number of amino acid residues included in modeled structures over the whole number of residues in predicted sequences for soluble and membrane proteins in each kingdom. (S) stands for soluble proteins and (M) stands for membrane proteins. Some of the residues are predicted to be in a disordered region. The percentage of residues in disordered regions is shown at the top Orengo et al. (1999) showed percentage of ORFs with protein 3D structures as between 30 and 46% in 1999. The genome sequences known by 1999 were mostly derived from prokaryotic species and the known protein 3D structures were mostly soluble proteins. Therefore, the figures they presented in 1999 should correspond to the figures of archaebacterial and eubacterial soluble proteins. When we extrapolate the figures of archaebacterial and eubacterial soluble proteins to the past in Fig. 2, the figures are around 40% in 1999, indicating that their figures approximately lie on the extrapolated lines. The current estimation indicates that we will obtain 3D structures of whole soluble proteins of eubacteria in 11 years and archaebacteria in 15 years. This estimation does not take into account the acceleration of structure determination speed by automation (McPherson 2004; DeLucas et al. 2005), which makes the due days closer to the present. For membrane proteins, speed of structure determination has been drastically accelerated by recent technical innovations (Kyogoku et al. 2003; Lundstrom 2004; Walian et al. 2004; Dobrovetsky et al. 2005), and therefore we will not linearly extrapolate the present status to estimate the due day for membrane proteins. Frequency of template structure in use When the template 3D structures used in FAMSBASE are classified by SCOP superfamily, which is a group of proteins that have low sequence identities but whose structural and functional features suggest that a common evolutionary origin is probable (Lo Conte et al. 2002), and frequencies of superfamilies in use are counted, ‘P-loop containing nucleoside triphosphate hydrolases’ superfamily is found to be the most frequent one; 7,532 times (about 12%) in whole archaebacterial model structures, 77,806 (about 10%) in eubacterial structures and 35,468 times (about 6%) in eukaryotic structures. The templates that follow in frequency in archaebacterial and eubacterial protein structures are ‘NAD(P)-binding Rossmann fold domains’, ‘4Fe–4S ferredxin’, and ‘PLP-dependent transferases’ superfamilies. In eukaryotic protein structures, ‘protein-kinase’, ‘immunoglobulin’ and ‘C2H2 and C2HC zinc fingers’ superfamilies, which appear specifically in eukaryotic genomes, follow the top. Differences in distribution of frequency of templates in different kingdoms of life are evident, when frequencies in use of template are plotted in descending order (Fig. 3). In any kingdoms of life, the frequencies of the most and the second most used templates exceed those of the remaining templates. The frequencies of templates in use drops first in archaebacterial protein structures and then in eubacterial protein structures. The descending curve of eukaryotic template frequency is less steep compared with the others, indicating that one template can produce a large number of domain 3D structures in eukaryotic ORFs. In other words, a significant number of proteins encoded in eukaryotic genomes are originated by domain duplication, as Koonin et al. (2000) demonstrated. Superfamilies with the 3D structures and with many copies in eukaryotic genomes, but seldom in prokaryotic genomes are ‘protein kinase-like’, ‘immunoglobin’, ‘RNA-binding domain’, ‘C2H2 and C2HC zinc fingers’, ‘WD40-repeat’, ‘glucocorticoid receptor-like’, ‘homeodomain-like’, ‘PH domain-like’, ‘RING-box’, ‘L domain’, ‘ankyrin repeat’, ‘ARM repeat’, ‘cytochrome P-450’ and ‘EF-hand’ superfamilies. These superfamilies are transcription factors, protein–protein interaction mediators and response factor for toxic substances, mostly known to be unique to eukaryotes.Fig. 3Frequency of template usage in descending order. Horizontal axis is a template and the vertical axis is a frequency of templates in use. Red line is a template usage in archaebacteria, blue line is eubacteria and green line is eukaryotes The ‘P-loop containing nucleoside triphosphate hydrolases’ superfamily outnumbering other superfamily in template frequency corresponds to the previous finding that the enzyme is highly frequently used in every kingdom of life (Leipe et al. 2003). When biological functions of these ORFs with the 3D structure of ‘P-loop containing nucleoside triphosphate hydrolases’ superfamily are classified, about half of the proteins are ABC transporters in archaebacterial and eubacterial proteomes, but numbers of G-proteins and motor proteins in eukaryotic proteomes are noticeable (Fig. 4).Fig. 4Protein family distribution of ‘P-loop containing nucleoside triphosphate hydrolases’ superfamily in each kingdom. In the three pie charts, the section with the same color is a category of the same family except for the white section In the last two years, new protein structures were determined and contributed to an increase in the number of templates for homology modeling. A part of those template structures are listed in Table 2. A part of those top 15 templates contributed a lot for the growth of modeled 3D structure database. In Table 2, 3D structure derived from SG projects is rare. The ratio of SG products in Table 2 is the same as that in PDB (Editorial Board, Nature Structural & Molecular Biology 2004). As the SG projects in US and Europe have proceeded to phase 2 (Service 2005), SG products are expected to contribute to increase in the number of templates in the near future. The qualities of protein 3D structures, namely, size, resolution, R-factors and so forth, derived from SG projects were compared with those in PDB and no obvious compromise in quality of SG products were found (Todd et al. 2005). The quality of homology modeling based on products of SG projects in the future, therefore, will be expected to be no less than the current quality.Table 2Top 15 modeling templates in the newly determined 3D structures between 2002 and 2003PDBIDChainNumber of uses as a templateSGaProtein name1q12A7,031NMaltose/maltodextrin transport ATP-binding protein MalK1l2tA6,529NHypothetical ABC transporter ATP-binding protein Mj07961oxxK3,948NABC transporter ATP-binding protein GlcV1pf4A3,202NTransport ATP-Binding Protein MsbA1nr0A2,640YActin interacting protein 1 Aip11ixcA2,495NLysR-type regulatory protein CbnR1ld8A2,410NFarnesyltransferase α subunit1ji0A2,331YABC transporter1oywA2,251NATP-dependent DNA helicase; RecQ helicase1kt1A2,198NFk506-binding protein FKBP511mt0A1,961NHaemolysin secretion ATP-binding protein; ATP-binding domain1mdbA1,745N2,3-dihydroxybenzoate-AMP ligase DhbE1nnmA1,730NAcetyl-CoA synthetase1gxrA1,715NTransducin-like enhancer protein 1 Esg11uohA1,706N26S proteasome non-ATPase regulatory subunit 10a PDB entry seemingly derived from the SG projects judged by description in PDB file is tagged Y, and the remaining entry is tagged N Whole structure and function of proteins from homology modeling of domain structures Protein function prediction, especially studies on enzyme specificity, based on homology modeling structures is intensively carried out in the field of drug design and related fields (Goldsmith-Fischman and Honig 2003; Kopp and Schwede 2004). Those studies are mostly based on homology modeling of domain structures. As mentioned above, most of the eukaryotic protein structures in FAMSBASE are 3D structures of structural domains, not the entire coding regions (Fig. 1). Protein functional sites are often located at a cleft of domains (Laskowski et al. 1996), and therefore understanding relative location of domains will be a critical issue. Xie and Bourne (2005) and O’Toole et al. (2003) also pointed out this problem and mentioned, “even if all the domains of a multiple-domain query sequence have determined structures, the individual structures will not enable accurate modeling of how they associate together in the structure of the entire proteins (O’Toole et al. 2003).” Figure 5 shows all eukaryotic ORFs whose 3D structures are mostly modeled in pieces. There are three types of enzymes and four types of cell surface receptors. A protein structure of ENSP00000264705 which is an ORF found in human genome can be modeled based on Escherichia coli carbamoylphosphate synthetase (CPS) and Pyrococcus abyssi asparatate transcarbamoylase (ATC). E. coli CPS is composed of a large subunit and a small subunit. CPS and ATC are the first and the second enzymes, respectively, in pyrimidine biosynthesis pathway. In mammalian genomes, those proteins are coded by a single gene and active in a hexamer form (Serre et al. 2004). Interactions between the large subunit domain and the small subunit domain of human CPS are conjectured to be the same as those between the large and the small subunits of E. coli CPS. N-terminal residues of the large subunit and the C-terminal residues of the small subunit are spatially located close in E. coli CPS, which permits the two chains to be chemically connected without disrupting subunit interfaces. To be active, human CPS should form a hexamer supramolecule and the interfaces for the supramolecule formation should be predicted from the modeled 3D structures. At the moment, the interfaces are unknown.Fig. 5Eukaryotic ORFs with multiple model structures covering more than 70% of entire protein. In each of the bar representation of proteins, a black box is a region with 3D structure. A name and PDB ID of a template structure and amino acid sequence identity between template and target domains are given below the black box. A yellow box is a putative signal peptide and green box is a putative transmembrane region. Template and modeled structures of ENSMUSP00000019416 were shown on the right side of the figure. Each domain is colored by hydrophobicity. A hydrophilic residue is in green and a hydrophobic residue is in red. A buried residue is in deep blue ENSMUSP00000019416 is an ORF found in mouse genome and encodes a putative cell surface receptor. The protein is predicted to consist of six consecutive Ig-fold domains. There is a putative transmembrane helix at the C-terminal region of the protein. Two consecutive Ig-fold domains are modeled without overlap, and no pieces of information for relative orientation of three modeled structures have been found. Information of interaction sites of those domains is required to build the entire structure of the protein and to predict a target molecule of this receptor. Computational analyses of domain interfaces and of protein–protein interfaces have been targets for extensive study for a long time, and some general characteristics have been found. One of them is the hydrophobicity of the interfaces (Wodak and Janin 2002). Hydrophobic clusters on the surface of modeled structures of ENSMUSP00000019416 are shown in right side of Fig. 5. One of the template structures, Nkp46 ectodomain, has hydrophilic surface (green) around the C-terminal residues of the domain, however the modeled structure has a hydrophobic surface (orange) at the corresponding area. The other template structure, LIR-1 D1D2, has a hydrophilic surface around the N-terminal residues of the domain, however the modeled structure has a hydrophobic surface at the corresponding area. The surfaces uniquely turned into hydrophobic in modeled structures are close to the residues that are chemically bonded in the target protein, and therefore both of the areas likely form interfaces of the two domains. The modeled structure based on LIR-1 D1D2 domain has another hydrophobic surface around the C-terminal residues, which may interact with CD158j-like domain located at the C-terminal side of the domain. Accuracy of homology modeling There are at least three major issues that affect accuracy in homology modeling; the best template selection, accuracy of an amino acid sequence alignment between template and target protein sequences and the accuracy of structure building procedure itself (Contreras-Moreira et al. 2005). Accuracy of the alignment is high, when sequence identity of template and target proteins is higher than 30%, and alignment of proteins with identity less than 30% is known to be less reliable, thereby accuracy of homology modeling deteriorates (Kopp and Schwede 2004). FAMS has been shown to construct relatively accurate model structures, even with low sequence identity between template and target sequences in CAFASP2, the homology modeling competition (Iwadate et al. 2001; Yamaguchi et al. 2003). A distribution of sequence identity between amino acid sequences of template and target proteins in FAMSBASE is shown in Fig. 6. Half of the model structures in FAMSBASE rely on alignments of sequence identity less than 20%. Figure 6 suggests that the current 3D structure database does not contain good enough structures for high quality homology modeling. SG projects will eventually provide better template structures, and improvement in target selection, alignment and modeling methods are also in pursuit to overcome the difficulties in homology modeling (John and Sali 2003; Wallace et al. 2005).Fig. 6Distribution of sequence identity between template and target amino acid sequences in FAMSBASE Conclusion Construction of database of whole genome homology modeling clarified that protein 3D structures of about 50% of the protein coding regions in whole genome can now be modeled. Maintaining the current speed of 3D structure determination, it will take, at most, 11 years to have enough templates to cover whole soluble proteins of eubacterial genomes, and 25 years to cover those of eukaryotic genomes. The current advancement in technologies of protein structure determination is expected to make these due times closer to the present. What we obtain at those times are not the 3D structures of entire proteins, but domain structures in pieces. A homology modeled domain structure is now in use of predicting domain functions, but predicting spatial arrangement of domains in a protein will be an important issue for function prediction.

Cancer_Causes_Control-2-2-1705485

Hypothesis: hair cover can protect against invasive melanoma on the head and neck (Australia)

The anatomic distribution of cutaneous melanoma reflects people’s levels and patterns of sun exposure. While examining trends of incident invasive melanomas by site in recent decades in Australia we noted significant increases in incidence on the ears but not the face or any other site in women younger than 40 years, by 6% (95% confidence interval [CI] 2–10%) per year, and 40–59 years by 7% (95% CI 4–10%) per year. Men of the same age showed no corresponding changes in ear melanoma. However incidence rates of ear melanoma in general were fourfold higher in males than females in Australia. Further, using data from the National Cancer Institute’s Surveillance, Epidemiology and End Results (SEER) Program, rates of invasive melanoma on the ear were found to be sevenfold higher in males than females in the US population in the same period. Higher rates of scalp and neck melanomas were also seen in men and women in both populations. We therefore speculated that the isolated rises of ear melanoma in younger women in Australia, and the higher overall rates of ear, scalp and neck melanoma in men compared with women, reflect differences in hair coverage. We tested the specific hypothesis that hair cover reduces sun exposure of the ears using experimental manikins and found that hair cover of the ear reduced solar ultraviolet-B exposure by 81% [SE ±8] compared with uncovered ears. We conclude that hair cover can protect against invasive melanoma on the ear and may similarly protect on the scalp and neck. When discretionary, hair may be an important additional factor to be considered for melanoma prevention. Introduction Sun exposure is the major environmental cause of melanoma [1] and so the anatomic distribution of melanoma in Caucasian populations largely reflects relative levels of sun exposure of different body sites [2]. Similarly in a given population, changes in site distribution of melanoma have long been known to offer clues about change in people’s sun-related behavior [3]. For example Magnus [4] analyzed melanoma incidence rates by age cohort and tumor site in Norway 1955–1977 and found that trunk and lower limb melanomas had increased much more than face and neck melanomas among younger generations, consistent with changes in clothing and sun-tanning habits in the first half of the twentieth century. Here we describe an isolated rise in the incidence of melanoma of the ear in women under 60 years of age in Australia. This distinct increase of invasive melanoma on a discrete site indicated some diminution in covering of the ear in young women in preceding decades. While a change in hat wear was one possible explanation, we speculated that a change in hair cover may also explain the observed rise, and that less hair coverage of the head among men in general may explain their higher relative incidence of melanoma on the ears and scalp compared with women [2]. When we searched for published data regarding the sun protection factor of hair we found none. Hence we set out to test the specific hypothesis that hair cover protects the ear from sun exposure using experimental manikins. Recent trends in melanoma in Australia Based on data for 109,062 invasive melanomas assembled by the Australian Institute of Health and Welfare from cancer registries in all Australian states and territories, trends in incidence of cutaneous melanoma in Australia 1982–1999 by sex and site were examined. Sites of invasive melanoma were pre-classified by cancer registries according to a standard protocol. For each site annual notifications were provided with age in 5-year bands. Population estimates by age and sex were obtained for each year from the Australian Bureau of Statistics. Annual sex-specific incidence rates of melanoma age-standardized to the WHO World Population were computed for each site and relative rates of increase were calculated for three age bands, less than 40 years, 40–59 years and 60 years and over, by regressing the logarithms of these rates on years. The average relative change in melanoma incidence rates over all sites each year was estimated as 3.2% (99% CI 3.0–3.4%) in males and 1.5% (99% CI 1.3–1.8%) in females but increases varied by sex and age. Highest increases were seen in men 60 years and over (4.4% per year, 95% CI 4.2–4.6%), followed by women 60 and over and men 40–59 years (2.9% per year), and then by women aged 40–59 years (1.5% per year) (Table 1). In contrast persons under 40 years showed little to no relative increase in invasive melanoma in the last two decades: young men showed a 1.4% annual increase of borderline significance and women under 40 years showed virtually no change in melanoma incidence (Table 1). Table 1Age-standardized incidence of melanoma on head and neck sites and all sites in Australia 1982–1986 and 1995–1999 by age and sexSiteAge (years)No. of melanomasIncidence rates per 100,000% Annual relative increase (95% CI)1982–19861995–1999MalesEar<401790.150.222.0 (−0.9–5.0)40–59 4260.951.31 1.9 (0.0– 3.8)≥60 1,1153.296.464.2 (3.0– 5.4)Face<40 3920.340.502.6 (−0.7–4.5)40–59 1,1252.483.85 3.2 (2.1–4.4)≥60 3,54611.0620.303.7 (3.0–4.4)All sites<40 10,7318.9111.981.4 (1.0–1.8)40–59 19,89843.3864.652.8 (2.5–3.1)≥60 27,95785.34166.534.4 (4.2–4.6)FemalesEar<40 1060.080.176.1 (2.4–9.9)40–59 1300.230.537.0 (3.6–10.4)≥60 2260.580.921.9 (−0.8–4.6)Face<404510.400.540.9 (−0.9–0.7)40–598402.482.340.0 (−1.3–1.4) ≥603,5089.7315.282.4 (1.8–3.1)All sites<4013,49413.4915.290.3 (0.1–0.7)40–5917,32844.2454.691.5 (1.2–1.8)≥6019,65455.7487.052.9 (2.6–3.2) Recent site-specific trends in melanoma on the head and neck in Australia Site-specific trends in melanoma for males and females within each age group reflected the above trends for all sites combined, such that men 60 and over generally had the highest relative increases on each anatomic site and women under 40, the least (data not shown for trunk and limbs). The striking exception was a significant 6.1% increase (95% CI 2.4–9.9%) in incidence rates of melanoma on the ears in women under 40 years based on 106 invasive tumors diagnosed in the study period. A similar rise of 7% (95% CI 3.6–10.4%) was seen for ear melanomas in women 40–59 years based on 130 invasive tumors (Table 1). The incidence rates of melanoma on the face in young women and those under 40–59 years showed little change (0.9% and 0.0% respectively), and similarly on the scalp and neck (data not shown). In contrast, young men showed no significant relative increases of melanoma on the ear or any other sites on the head and neck in the same period. Men aged 40–59 years also showed no significant relative increases of melanoma on the ear but modest relative increases in melanoma on the face of around 3% per year (Table 1) and also on the scalp and neck. Sex-specific differences in incidence rates of melanoma on the head and neck Between 1982 and 1999 in Australia the overall age-standardized incidence rate of ear melanoma in males was 0.91 (99% CI 0.85–0.97) per 100,000, 4 times higher than the corresponding rate of 0.24 (99% CI 0.21–0.26) per 100,000 in females. The male excess was seen in all age groups though the difference in rates of ear melanoma between young men and young women was small by the end of the study period, compared with the sevenfold sex-specific difference seen in older men and women over 60. The incidence rate of scalp and neck melanoma (sites pre-combined) in males was 2 times higher than in females (2.18, 99% CI 2.09–2.27 and 1.08, 99% CI 1.02–1.14 per 100,000 respectively) though again the differences were narrowed in the youngest age group. Incidence rates of melanoma on the face were slightly higher overall in males than females (2.62, 99% CI 2.52–2.72 and 2.06, 99% CI 1.98–2.14 per 100,000 respectively) but were no different in men and women under 40 years (Table 1). Differences in incidence rates of melanoma on the ear, scalp, face and neck were seen even more clearly in an earlier unrelated study of the Queensland data for 1987 [2] where site-specific incidence rates of melanoma had been calculated and further adjusted for relative body surface area. Incidence of invasive melanoma on the ear in Queensland males was 209 per unit area of skin per 100,000 per year compared with 50 per unit area of skin per 100,000 per year in females, again a fourfold difference. On the scalp there was a 20-fold greater incidence in males than females and on the neck a fourfold difference. Again rates on the face in males and females showed very little difference in Queensland in 1987 [2]. To assess whether comparable patterns were seen in other populations, age-standardized incidence rates of melanoma per 100,000 for 1982–1999 were calculated for sub-sites of the head and neck for the regions covered by the National Cancer Institute’s Surveillance, Epidemiology and End Results (SEER) Program of population-based cancer registries [5]. In males compared with females there was a sevenfold higher incidence rate of ear melanoma and a threefold higher rate of scalp and neck melanoma compared with a less than twofold higher incidence rate of melanoma on the face. Hypothesis The systematically higher incidence of invasive melanoma on the ear, scalp and neck compared with the face in males and females is consistent with the general hypothesis that hair cover can offer long-term protection of the skin from sun exposure. We tested the specific hypothesis that hair cover of the ear protects it from solar ultraviolet (UV) radiation by conducting an experiment using manikin head-forms in which the difference in solar UV radiation to ears with and without hair cover was measured. We used four manikin head-forms: three wearing human hairpieces (blonde, brown and black) covering the ears and one uncovered (Fig. 1). These were set facing north on a concrete floor and exposed to natural sunlight in Brisbane (27°S) from 11 am to 1 pm Australian Eastern Standard Time daily for five consecutive days in May 2005. Cloud cover was zero and surface reflectivity less than 7%. Sun exposure was measured by UV-sensitive polysulfone dosimeters placed on the ears and nose of each manikin. Fig. 1Measurement of solar ultraviolet radiation on ears in relation to hair cover using head-forms and polysulfone dosimeters Ears covered by hair received on average 81% [SE ±8] less solar UV radiation than bare ears when exposed to the sun. As a control site on the face, the corresponding solar UV exposures to the noses of the manikins were monitored (Fig. 1) and showed a slight variation during the experiment in the reverse direction, namely a 5% [SE ±3.4] increase in UV radiation to the wigged manikins compared with the bare manikin. Discussion These experimental data support our hypothesis that hair cover offers substantial sun protection of the skin of the ear and by extrapolation, the skin of the scalp and back of the neck as well if hair is worn long. Taken together with the recently observed change in incidence of invasive melanoma on the ear in women in Australia and the consistent pattern in Australia and the United States of lower incidence of melanoma in females on the scalp, ear and neck – sites that are more frequently covered or screened by hair than in males, the evidence suggests that hair cover can indeed protect against invasive melanoma on these sites. Other explanations for the observed site- and sex-specific patterns can be considered. Change in diagnostic trends was unlikely to explain the observed rise in ear melanomas in younger women since in situ melanomas were not included in the analyses. One possibility is that the distinct increase of ear melanomas in Australian women under 60 was due to the loss of some other form of physical protection. For example the rise in popularity of caps in the 1980s resulted in a decrease in cover of the ear but not the face in young women who wore brimmed hats in preceding decades [6]. If this were the sole explanation, some analogous rise in the rate of ear melanomas in men might have been expected as an aftermath as well, but has been observed neither in Australia nor the US. Another cause of increasing sun exposure of the ears in young women may have been the upsurge in women’s participation in outdoor sport that also occurred in the 1970s and 1980s [7] if caps rather than hats were worn. However even if change from hats to caps did explain the recent rise in incidence of ear melanomas in Australian women, difference in hat wear between the sexes does not explain the systematically higher rates of invasive melanoma in men than in women across the various sub-sites of the head and neck with less discrepant rates on the face. (Indeed facial hair cover in men with beards may mitigate the effect of overall higher outdoor exposure of men than women for facial melanomas). We also speculated that younger women’s hair styles may have changed over time and become shorter leading to greater exposure of the ears and neck, but there is little evidence available to address this question. Finally in view of the known sex-specific differences in melanoma incidence on sites such as the scalp, it is surprising that the sun-protection factor of human hair has never been measured before. This absence of quantitative data contrasts with the extensive information available about the sun-protection provided by clothing and sunscreens [8, 9]. If our general hypothesis is confirmed, hair cover of the ears in particular would add another potential means of melanoma prevention, especially in high-risk populations, among individuals in whom amount of hair cover is discretionary.