The ACE gene contains a polymorphism consisting of either the presence (insertion, I) or absence (deletion, D) of a 287 bp alu repeat in intron 16. The D allele is associated with increased ACE activity in both tissue and plasma. The DD genotype is associated with risk of developing ARDS and mortality. The frequency of the D allele is higher in patients with pulmonary fibrosis, sarcoidosis and berylliosis. The role of this polymorphism has not been studied in the development of BPD in the premature newborn.ACE I/D genotype was determined in 245 (194 African-American, 47 Caucasian and 4 Hispanic) mechanically ventilated infants weighing less than 1250 grams at birth and compared to outcome (death and/or development of BPD).2 on 28 days or 36 weeks PCA). Secondary outcomes  similarly were not influenced by the ACE ID polymorphism.The incidence of the D allele in the study population was 0.58. Eighty-eight (35.9%) infants were homozygous DD, 107 (43.7%) were heterozygous ID and 50 (20.4%) were homozygous II. There were no significant differences between genotype groups with respect to ethnic origin, birth weight, gestation, or gender. There was no effect of the ACE I/D polymorphism on mortality or development of BPD (OThe ACE I/D polymorphism does not significantly influence the development of BPD in ventilated infants less than 1250 grams. Prematurely born infants who require mechanical ventilation (MV) frequently develop chronic lung disease known as bronchopulmonary dysplasia (BPD). Oxidant injury, mechanical disruption of the lung, inflammation and subsequent failure of lung development are considered the major mechanisms in the pathogenesis of BPD. The development of BPD is characterized by an initial acute inflammatory component followed by variable degrees of lung fibrosis and failure of alveolar septation, both of which ultimately impair the development of the immature lung -5. The iThere is increasing evidence to support the role for the activation of the renin-angiotensin system (RAS) system during ALI. In ARDS there is an increase in bronchoalveolar lavage and a concomitant decrease in circulating angiotensin converting enzyme (ACE) activity,7. This There is significant variation in circulating ACE activity among individuals, which may be genetically determined. The human ACE gene is located on chromosome 17 (17q23) and contains a polymorphism consisting of either the presence (insertion, I) or absence (deletion, D) of a 287 bp alu repeat in intron 16. The delBecause of the relationship between inflammation and activation of the RAS and the association with lung fibrosis and adverse outcome in ARDS, the ACE l/D polymorphism may modify risk for the development of chronic lung disease or death in mechanically ventilated VLBW infants. The purpose of this study was to determine if there is a relationship between the ACE I/D polymorphism and respiratory outcomes of death or the development of BPD, in mechanically ventilated very low birth weight (VLBW) infants.Genomic DNA used for this case controlled study was extracted from archival tracheal aspirate (TA) pellets 223 patients) or blood (22 patients) collected prospectively as part of an ongoing study of genetic factors in the development of complications of prematurity. The TAs that were used as a source of genomic DNA were originally collected as part of long term longitudinal studies examining cytokine concentrations and the development of CLD[ patientsCultures for genital mycoplasmas were performed on samples collected in the first few days of life. Clinical and outcome data were abstracted from the clinical record and included information on respiratory outcome, survival and development of complications of prematurity.Isolation of total DNA from blood or TA pellets was performed using the QIAmp DNA Mini kits™ (Qiagen Incorporated, Chatsworth, CA). Briefly, TA pellets were suspended in 200 μl of sterile phosphate buffered saline by vigorous vortexing, then digested in proteinase K and applied to silica gel spin columns. Columns were washed in the manufacturer's supplied buffers and the total DNA was eluted in 200 μl elution buffer. Blood (200 μl) was extracted similarly to the TA pellets.2 1.5 mM, KCl 50 mM, 5%DMSO, Triton X-100 0.1%, 200 micromolar each of dNTPs, 10 pmol each primer, 1–2 U of Taq polymerase, and 1 microliter of DNA. DNA was denatured at 95°C for 5–10 min, followed by 30 cycles of denaturation at 94° for 60 sec, annealing at 67 for 60 sec, and extension at 72° for 2 minutes, with a final extension at 72° for 7 min. PCR products were separated on 2% agarose gel containing 0.5-microgram/ml ethidium bromide. After gel electrophoresis the bands were visualized by UV-transillumination. The PCR product is a 190 bp fragment in the absence of the insertion (D genotype) and a 490 bp fragment in the presence of the insertion (I genotype).ACE I/D polymorphism was performed by microplate PCR method as described previously. . Tracheal aspirate cultures obtained during the first few days of life grew Ureaplasma urealyticum (Uu) on at least one occasion from 74 of 217 infants cultured (34%) and Mycoplasma hominis (Mh) from 29 (13%). One hundred and fifty one (67%) infants were oxygen dependent at 28 days and 55 (25%) were oxygen dependent at 36 weeks PCA. There were 39 (16%) patients who died  during their initial hospitalization (24 before 28 days of age and 15 after 28 days).Two hundred and forty five (245) patients had complete clinical information on respiratory outcome and genomic DNA available for genotyping. Mean gestational age and birth weight of the study population was 26.4 ± 0.1 weeks and 869 ± 12 grams respectively. One hundred and ninety-four (79%) infants were African-American, 47 (19 %) were Caucasian and 4 (2%) were Hispanic. Male: female ratio was 147:98. All patients required MV at birth and 228 (93%) infants were treated with exogenous surfactant therapy  and Caucasian infants (0.59) (p = 0.664). Fifty (20.4%) infants were homozygous II, 107 (43.7 %) were heterozygous ID and 88 (35.9%) were homozygous DD.Baseline clinical characteristics of birth weight, gestational age, race, gender, TA isolation of Mh, and the need for surfactant replacement were not different between genotype groups Table . IsolatiClinical characteristics of surviving infants who were or were not oxygen dependent at days are shown in Table Because the ACE I/D polymorphism may have different functional effects on plasma and tissue ACE activities in different ethnic groups, we analyzed the effects of ACE on the incidence of BPD separately for Caucasian and African-American infants. (The effects of the ACE I/D polymorphism on the incidence of BPD and other outcomes for the combined group can be found in the online data supplement). Table The effects of ACE genotype on outcome in ventilated African-American infants less than 1250 grams are shown in Table Since birth weight and gestation are the primary determinants of adverse outcome, analysis was repeated in both infants greater than 750 grams and infants ≤ 750 grams. There was no significant effect of ACE genotype when this subgroup analysis was performed (data not shown).The RAS is activated during lung injury and plays a role in several pathological processes. In addition to the pulmonary endothelium, respiratory epithelium also possesses significant ACE activity. Fas-induced alveolar epithelial cell apoptosis is dependent on local AT-II production and interaction with it receptor.,11 FurthThe frequency of the D allele in our study population was not different than reported in our local population or for other groups,24,31. TIn our study population, the ACE ID polymorphism was not associated with an altered risk for the development of BPD, oxygen dependency at 28 days, death  or composite outcomes (BPD or death) suggesting that factors other than genetic variation at this locus contribute to these outcomes. This is in contrast to that seen in adults with ARDS or other lung disorders such as pulmonary fibrosis, sarcoidosis and berylliosis where the D allele is associated with disease -21.1 +915 G/C SNPs on the development of BPD[Indeed genetic factors may not have a large influence in a disease (BPD) where maturity at birth has such an overwhelming influence. Our previous study in this population failed to find any association of the tumor necrosis factor-α-308 G/A, MCP-1-2518 A/G or transforming growth factor-βnt of BPD AdditionUreaplasma urealyticum colonization in these infants. Chorioamnionitis, which is highly correlated with isolation of Uu, is a risk factor for PVL.[The finding of increased incidence of PVL in Caucasian infants with the II genotype may be due more to the greatly increased incidence of  for PVL. Because Our study has several limitations. The ACE I/D polymorphism has only been demonstrated to be functional in Caucasians. Data linThe observations of this retrospective case controlled study are also limited somewhat by selection bias. Because only infants who were mechanically ventilated were included, the true impact of this polymorphism on the complications of prematurity may be underestimated. Since this was a retrospective study using stored material no attempt was made to correlate phenotype with genotype (ie TA ACE measurements).Lung injury and the subsequent maladaptive repair process that leads to the development of BPD is complex with a great many factors that interplay to determine outcome. It is very likely that other genetic play a role in determining the risk of poor outcome. Polymorphisms in cytokines, their receptors, bacterial pattern recognition molecules, surfactant proteins, and heme oxygenase-1 are known to alter the course of other pulmonary diseases and should be examined as to their potential role in the development of BPD in the premature infant.The ACE I/D polymorphism does not significantly influence the development of BPD in ventilated infants less than 1250 grams.The author(s) declare that they have no competing interests.RJB conceived and organized the study, prepared the manuscript and performed the statistical analyses. KY performed the genotyping, and assisted with manuscript preparation. JL oversaw collection tracheal aspirates, assisted with recruitment of subjects into the original studies, and assisted with editing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Contains Tables showing baseline clinical characteristics of African American and Caucasian infants separately by Genotype groupClick here for file


It is not currently known what is the patient's viewpoint of a "good" physician. We set out to define patient's priorities regarding different physician's attributes in 3 domains important in medical care.Patients hospitalized or attending clinics at a large teaching hospital selected the 4 attributes that they considered most important out of 21 listed arbitrarily in a questionnaire. The questionnaire included 7 items each in the domains of patient autonomy, professional expertise and humanism.Participating patients (n = 445, mean age 57.5 ± 16 years) selected professional expertise (50%), physician's patience and attentiveness (38% and 30%, respectively), and informing the patient, representing the patient's interests, being truthful and respecting patient's preferences (25–36% each) as the most essential attributes. Patient's selections were not significantly influenced by different demographic or clinical background. Selections of attributes in the domain of patient's autonomy were significantly more frequent and this was the preferred domain for 31% and as important as another domain for 16% – significantly more than the domain of professional expertise (P = 0.008), and much more than the domain of humanism and support (P < 0.0005).Patients studied want their physicians to be highly professional and expert clinicians and show humaneness and support, but their first priority is for the physician to respect their autonomy. That can be seen as a prototype or role model of a first class physician. However, these laudable qualities, discussed in many texts -3, were most important and would like the best in their own physician. No grading was required. The questionnaire aimed at the patient's image of an excellent physician in general, and did not specify whether the physician was hospital-based or in primary care. The 21 attributes included 7 characteristics in each one of three domains: reflecting professional expertise and high-quality care; reflecting a humanitarian, patient-centered approach; and reflecting patient's autonomy and attentiveness to the patient's preferences and rights. Then patients supplied basic demographic data and the questionnaire was collected about 20 minutes later. The selection of the 21 different physician characteristics or behaviors started from collecting pertinent articles on patient autonomy; physician's humanism and patient support; and on physicians' expertise and professionalism using the author's (AS) collection and a Medline search. Some of the articles were based on patient-derived data. The next stage involved a series of meetings and discussions at the Department of Ambulatory Care and Prevention, Harvard Medical School, Boston. Three senior researchers  – RH Fletcher, T Peters and AS, selected and categorized the 21 items based on the literature and on personal experience. The third stage was a validation study among thirty residents in various stages of their training. They were presented with a mixed list of the 21 items and requested to categorize each one into one of the 3 domains. To rule out that patients categorized these characteristics differently, we performed an additional post-study validation on 30 outpatients that were representative of our study population. Results were similar to those obtained from the residents and confirmed that the vast majority of participants view each of the items as representative of the corresponding domain (Appendix 1) [see Patients hospitalized at our 600-bed teaching medical center or attending one of its day clinics were randomly approached and asked to fill in a one-page questionnaire. Over a period of one month, patients were approached by one of the authors (DR) and invited to participate. Every third patient on the specific day's ambulatory clinic lists and every fifth patient on the list of patients hospitalized at the Department of Medicine were approached. If the fifth patient was found to be too ill to participate (e.g. ventilated, unstable or confused) the next name on the list was selected. These numbers were arbitrarily chosen. Following a brief explanation they were handed the questionnaire. The questionnaire listed 21 physicians' characteristics or behaviors regarding the care of patients, presented in a varying order (different order for different patients) to prevent bias due to an item's position on the list. Patients were asked to select 4 attributes that they considered the x 1) see . No chanA total of 450 patients received the questionnaire and all but 5 consented to participate and returned the filled questionnaires (n = 445). Patient's ages varied from 18 to 89 years (mean 57.5 ± 16) and two thirds were aged 50–80 years. Other patient's characteristics are summarized in Table Our study population of 445 patients was heterogeneous Table , yet no at all by almost 30% of participating patients. An overview of the results reveals that patients studied want their physicians to show professional expertise and provide humane personal care (a preferred or important domain for 39% or 28% of the patients, respectively) . Not surprisingly, patients want their physicians to be experienced and highly professional. This was the physician's characteristic that was chosen by 50% of the patients Table , Top. HoOn the other hand, 'humane' qualities that are traditionally considered important, such as showing empathy or being friendly with the patient were surprisingly found to be among the least selected attributes , headed the list.None declared.AS initiated, designed and supervised the study, analyzed the results and wrote the manuscript. DR performed the actual study. NJ provided statistical advice and analysis. All authors read, discussed, contributed to and approved the final manuscript.The pre-publication history for this paper can be accessed here:The patient's questionnaire including the 21 attributes in the three domains.Click here for file


Competition experiments demonstrate that while hMutSα predictably binds the G:T mismatch to a much greater extent than G:A, hMutSα demonstrates a surprisingly equal ratio of competitive inhibition for both G:T and G:A mismatch binding reactions at the H-ras hot spot of mutation. Further, mismatch repair assays reveal almost 2-fold higher efficiency of overall G:A repair (5'-nick directed correct MMR to G:C and incorrect repair to T:A), as compared to G:T overall repair. Conversely, correct MMR of G:T → G:C is significantly higher (96%) than that of G:A → G:C (60%).Electrophoretic mobility shift (gel shift) experiments demonstrate that DNA containing mismatched bases are recognized and bound equally efficiently by hMutSα in both MMR proficient and MMR deficient  may contribute to the observed increased frequency of incorrect repair of G:A, resulting in the predominant GGC → GTC  ras-activating mutation found in a high percentage of human tumors.Combined, these results suggest that initiation of  Factors contributing to these and other hot spots of mutation are still largely unknown, despite much investigation, but now appear to have several different contributions, such as type of DNA damage, genomic location, surrounding sequence, cell cycle position, efficiency of the optimal DNA repair pathway, and involvement of alternate repair and other cellular pathways.Several different DNA repair systems have evolved within all living cells to correct mispaired or damaged nucleotide residues generated either by endogenous events or by exposure to exogenous mutagenic agents ,2. The fras-activating mutations, have been reported as frequent occurrences in HNPCC and other MSI tumors [DNA mismatch repair (MMR) is a repair system that corrects mispaired nucleotides and insertion/deletion loops (IDLs), resulting from replication, recombination, or repair errors. Consequences of defects in this DNA repair pathway are evidenced by microsatellite instability (MSI), elevated mutation frequency throughout the genome, enhanced recombination events, as well as tolerance to cytotoxic effects of alkylating agents as evidenced by decreased apoptosis. Deficient MMR is, in turn, associated with hereditary nonpolyposis colorectal cancer (HNPCC), as well as other types of sporadic tumors in humans and animal models -6. AlthoI tumors ,8.E. coli [in vitro model [DNA MMR is conserved amongst highly divergent species, reflecting the essential role of this DNA repair process ,10. The E. coli -13. SeveE. coli -19. The E. coli ,21. The E. coli ,21-24. TE. coli . MutL hoE. coli . SimilarE. coli ,25,27-32E. coli ,30,33-35ro model .ras codon 12 to better understand molecular events leading to activating mutations at this site. Our current results indicate that initial recognition and subsequent binding for repair signaling by hMutSα may be two separably measurable steps in the MMR pathway that can significantly affect downstream cellular events.We have previously demonstrated that the efficiency of correct mismatch repair within the cell can differ significantly, depending on exact type of mismatch, site-specific location, phase of cell cycle, or cell type ,24,37,38ras in HCT116 + Ch.3 nuclear extract is composed of hMutsα, DNA binding reactions were conducted using [32P]-G:T oligos incubated with nuclear extracts from these MMR proficient cells. In confirmation of hMutSα binding to a G:T mismatch at this oncogenic site, the hMutSα :DNA complex was efficiently interrupted by goat anti-hMSH6 and with rabbit anti-hMSH2  by incubation with unlabeled G:A-oligo, to determine if equal ratios of competitive binding might occur for G:T and G:A, despite the very different avidities of hMutSα for each mismatch. This would provide an indication of equal recognition of each type of mismatch by hMutSα, despite unequal binding avidity for these two mismatches. For these experiments, it was necessary to extend incubation periods from 30 min. to 2.5 hr, as sufficient concentrations of gel shifted bands can be detected with the G:A oligo only after extended incubation (unpublished observations). As expected, binding of hMutSα to [32P]-G:A-oligo is approximately 100 fold weaker in intensity than for [32P]-G:T-oligo after the extended incubation , as determined by comparison of the densitometric intensity of each band. All of the above experiments have been repeated with similar results.An alternate approach to more precisely define the extent of this phenomenon was to measure competitive inhibition of radioactively labeled G:A-oligo (rather than of the avidly bound [ras codon 12 sequence within the pUC19 vector is nick-directed, yielding a high degree of correct repair by MMR proficient E. coli (DH5α); G:T → G:C = 95%, G:A → G:C = 78%, in agreement with our previously published results using a different plasmid vector [ras 69 mer into a pUC18 vector to determine effects of reverse orientation of the oligo and 'nicked' strand resulted in an almost identical frequency of correct repair at codon 12; G:T → G:C = 100%, G:A → G:C = 78%. These results confirm that the mismatch is recognized and repaired by nick-directed bacterial MMR and that there is no strand bias for MMR during high copy number bacterial replication of this plasmid. Background repair after direct transformation into NR9161 E. coli (MMR deficient) was consistently between 41–44% for both mismatches (data not shown), and is favorably comparable to an average of 50 – 60% background repair efficiency by this and other strains of MMR deficient E. coli .Table d vector . Additioras codon 12 middle base pair location containing a 5' nick on the "T" or "A" strand (as described in methods) with MMR proficient (HCT116 + Ch. 3) or MMR deficient (HCT116) nuclear extracts. Efficiency of correct, incorrect, and total (correct + incorrect) MMR for each mismatch was subsequently calculated (as described in methods) [total repair of the G:A mismatch  was almost twice as high as for G:T . This phenomenon was consistently observed within the several different experiments required to obtain the total results depicted in Table correct (nick-directed) repair of G:A → G:C (60%) was significantly low when compared to correct (nick-directed) repair of G:T → G:C (96%) (p < 0.005). Inadvertent nicks in the "G" (correct) strand of the H-ras 69 mer or pUC plasmid during preparation could not have contributed to the increased total, or increased incorrect repair (G:A → T:A) results for the G:A mismatch because the efficiency of incorrect repair would then be similar for both the G:T and the G:A mismatch. Instead, MMR results within Table E. coli nick-directed repair results are consistently high, for both mismatch-containing plasmid preparation subsequently used for each nuclear extract MMR assay (data not shown). As well, MMR deficient E. coli repair ratios (correct to incorrect) are 1:1 after transformation of unmethylated plasmids . In combination, all of the above control experiments demonstrate a lack of any significant unintentional misrepair events on either strand of DNA, for either site-specific mismatch plasmid preparation. Therefore, the results within Table in vitro MMR assay might cause an artifactual decrease in the activity of other DNA repair pathways.Results within Table methods) . Surprisras, as compared to G:T → G:C repair at this location [ras oncogenic hot spot within the nuclear environment. Our results suggest that, firstly, the MMR pathway is the primary, if not the only, DNA repair pathway that can recognize and is subsequently responsible for the repair of both mismatches at this oncogenic location within the cell. Secondly, although 'recognition' of either G:T or G:A by hMutSα is likely equal, and is essential for initiation of MMR, the avidity – or strength – of hMutSα binding to either mismatch is not equal, and may play a significant role in the decision of whether to activate nick-directed correct MMR, or the activation of an alternate response pathway. In support of this concept, and in correlation with weak hMutSα binding to G:A, we have observed significantly decreased nick-directed (correct) MMR of G:A → G:C (60%), as compared to nick-directed MMR of G:T → G:C (96%). We have however, consistently measured almost twice the efficiency of total repair of G:A (to either G:C or T:A) as compared to total G:T repair (to G:C or A:T) at this oncogenic site. This phenomenon appears to be due to increased non nick-directed incorrect repair of G:A → T:A (rather than to replication without repair, which results in a mixture of G:C and T:A).Previous investigations of ours have revealed significantly decreased nick-directed G:A → G:C repair at codon 12 of H-location ,37. Decrcorrect MMR, or with alternate events other than nick-directed MMR. In support of this concept, Wang, et. al. has recently demonstrated by atomic force microscopy that E. coli MutS-DNA complexes exist in two conformations [E. coli MutS in the role of 'authorizing' different repair events [This raises the possibility that (a) initial recognition of a mismatch is essential but separable from (b) differential binding of MMR proteins to specific mismatches, which in turn is directly correlated either with nick-directed rmations . The inir events . Also, ir events ,12.E. coli MutS-DNA recognition complex [Figure  complex . Brieflyras codon 12 location is activating, the majority of human tumors containing mutations at this site are G:C → T:A transversions [overall MMR of G:A, combined with a high ratio of incorrect MMR of G:A → T:A at this oncogenic location correlates well with the predominant GGC → GTC  ras-activating mutation found in a high percentage of human tumors [ras codon 12 oncogenic hot spot of mutation, combined with a complete lack of rescue by other DNA repair pathways, is biologically relevant.These results agree with, and build upon our previous experimental results, and also agree with the presence of specific mutations predominantly found in human tumors ,24,37. Iversions -44. Our n tumors ,44,45. T2. HCT116 + Ch. 3 cell line also received 0.4 mg/ml Geneticin (G418). Both HCT cell lines were kind gifts of C. Richard Boland; UCSD. Nuclear extracts were prepared as described previously [AATTCACGGAATATAAGCTGGTGGTGGTGGGCGCCGGCG GTTGGGCAAGAGTGCGCTGACCATCCAGG-3', as well as complementary noncoding oligomers, were obtained from Operon (Alameda, CA). The above 69 mer oligo is a portion of the coding sequence of human H-ras DNA. The bolded underlined Grepresents H-ras codon 12 middle G position, plus an additional 30 bases of H-ras sequence both 5' and 3' of codon 12, with an Eco R1 site 5' and a Bam H1 site 3' . The wild type sequence (coding strand containing codon 12 middle G) was 5'-phosphorylated. Mismatch containing noncoding strand sequences (either T or A opposite codon 12 middle base G) were not phosphorylated, thus providing a 5' nick in the strand containing the incorrect base after ligation into the pUC19 plasmid. All other reagents were purchased from Sigma unless otherwise noted.Human colorectal carcinoma cell lines HCT116 and HCT116 + Ch. 3 were cultured in Iscove's Modified Dulbecco's Medium supplemented with 10% fetal bovine serum (FBS) at 37°C, 5% COeviously . Synthet2, pH 7.5). Ligation of annealed mismatched oligos to Eco R1/Bam H1 digested pUC19 DNA was accomplished at a 20:1 molar ratio of 69 mer to pUC19 DNA (~3–6 ng DNA per μl of ligation solution), using T4 ligase, per manufacturer's recommendations . The pUC19 vector used for ligation of mismatch-containing H-ras oligo for subsequent measurement of MMR within nuclear extracts was grown in GM2929 (E. coli dam-dcm-). The pUC19 and pUC18 plasmids used as positive controls for correct bacterial mismatch repair were grown in DH5α (E. coli dam+).Complementary 69 mer oligos containing the wild type sequence at codon 12; middle base (coding strand G) and one mismatched base (noncoding strand T or A), as described above, were annealed in an equimolar ratio at a final concentration of 0.2 μg DNA per μl of annealing buffer (1 mM Tris-HCL, 1 mM MgCl32P]-dATP-labeled-69 mer duplexes by a fill-in reaction, using [α-32P]-dATP and Klenow polymerase, per manufacturer's protocol (Invitrogen). Each nuclear protein-DNA binding assay was performed using 0.8 – 1.0 × 105 cpm homoduplex or heteroduplex 69 mer and 5–10 μg protein from nuclear extract solution in an equal volume of 2X gel shift reaction buffer, resulting in a final concentration of 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCL, pH 7.5, 0.1 mg/ml poly [dI:dC], 5 mg/ml BSA, 6% glycerol, in a final volume of 15 – 20 μl. In addition, each reaction contained 100-fold molar excess (100X) unlabeled homoduplex 69 mer, unless otherwise noted. Incubations were for 30 min. at 37°C, unless otherwise noted. Gel shifts of each reaction were electrophoresed at 20 mA within a 4.8% nondenaturing acrylamide gel in 0.5 × TBE buffer, at room temperature. Radioactively labeled oligomers were visualized by autoradiography of the dried gel.Gel shift assays were performed using nuclear extracts from either HCT116 or HCT116 + Ch. 3 cells and [in vitro mismatch repair assay was performed essentially as described by Thomas et. al. [ras 69 mer, with a nick on the same strand and 36 bases 5' of the incorrect base, was incubated with 50 μg of HCT116 + Ch. 3 or HCT116 nuclear extract in a final volume of 25 μl containing 30 mM HEPES buffer, pH 7.9, 100 μM each of dATP, dCTP, dGTP and dTTP, 200 μM each of CTP, GTP and UTP, 4 mM ATP, 40 mM creatine phosphate, 100 ng/μl creatine phosphokinase, 0.5 mM DTT, and 7 mM MgCl2. Negative control experiments were performed in the above solution without nuclear extract. Each reaction was incubated at 37°C for one hour, as described [ras insert was recovered using Wizard SV Miniprep System per manufacturer's directions , and subsequently used to transform E.coli strain NR9161 (MutL-). In addition, DH5α (MMR proficient E. coli) were transformed directly with the ligated and nicked plasmid as a positive control for correct mismatch repair [Nae I, which recognizes a single restriction digestion site unique to the wild type H-ras sequence at codon 12 middle base pair (G:C) [An  et. al. , except escribed . Plasmidir (G:C) . After eir (G:C) ,37,38.MMR efficiency of each human nuclear extract assay, above MMR deficient bacterial background repair, was determined by the following equations :Total repair efficiency = 100 × (1 - [fraction of unrepaired plasmids incubated with human nuclear extract results / fraction of unrepaired untreated plasmids from direct transformation of NR9161]).Correct repair efficiency = 100 × {(fraction of correctly repaired incubated with human nuclear extract) - [(fraction of correct repair by NR9161 direct transformation) × ]}.Incorrect repair efficiency = Total repair efficiency - Correct repair efficiency.Statistical comparisons of G:T and G:A results were conducted by a non-parametric equivalent to the Student's t-test for differences between proportions. Statistical significance was indicated for those comparisons with a P < 0.05 assuming a 2-tailed distribution .ME designed and carried out the MMR assay studies and helped draft the manuscript. HH designed and carried out all other experiments. AS performed all densitometry measurements and helped draft the manuscript. MF and SB helped carry out the MMR assays. KW conceived of the study, participated in its design and coordination and wrote the final manuscript.


TT virus is prevalent worldwide, but its prevalence and genotype distribution in Central and East-Europe has not been determined. The high prevalence of TTV in multiply-transfused patients points to the importance of a parenteral mode of transmission, but since more than half of the general population is infected other possible routes of transmission must be considered.In our study, we investigated the epidemiology, transmission and phylogeny of TTV in the Czech Republic. The following groups were selected: a control group of 196 blood donors, 20 patients with hemophilia, 49 intravenous drug users, 100 sex workers, 50 penitentiary prisoners, 208 healthy children aged 1 to 14 years, 54 cord blood samples, 52 patients with non-A-E hepatitis, 74 patients with hepatitis C, and 51 blood donors with increased ALT levels. Primers specific for the non-coding region were used. The genotype distribution was studied in 70 TTV-positive samples.The prevalence rate of TTV among the Czech population was 52.6%. We have shown that TTV is not transmitted prenatally. Children were infected after birth with two peaks: one at the age of two years and the other after the beginning of primary school. Adults have shown a further increase in the TTV prevalence with age. The highest TTV prevalence was found in the group of patients who had received multiple blood transfusions. The TTV prevalence rate in subjects at an increased risk of sexual transmission was not significantly higher than in the general population. Genotypes G2 and G1 were most prevalent among the Czech population, followed by G8 and G3. The subjects positive for markers of HBV and/or HCV infection tested significantly more often TTV DNA positive, which is suggestive of a common route of transmission of these three infections.This study on TTV prevalence, mode of transmission and age-specific prevalence is the most extensive study performed in Central and Eastern Europe. It showed insights into the epidemiology of TTV infection, but failed to associate TTV infection with clinical manifestations. TTV, a non-enveloped small circular single-stranded DNA virus, was recently placed in a novel virus family named Circinoviridae . In spitTTV has a worldwide distribution. The prevalence of the most common TTV genotypes G1 and G2 is similar all over the world, while the reports on the distribution of other genotypes are scarce and not conclusive. The rate of TTV DNA detection is influenced by the selection of the primer annealing sites. PCR using primers which target the ORF1 region can detect only TTV genotypes 1–6 of group 1, but PCR primers designed for the NCR can detect nearly all genotypes or genetic groups known so far [The prevalence of TT virus in the general population has ranged between 1.9 and 98%, with the highest rates detected in the African and South American countries ,6. The TThe mechanisms of TTV transmission have not yet been elucidated. Even though numerous studies have suggested that the parenteral transmission via transfusion of contaminated blood and blood products is the most common route of TTV infection , the detTTV was originally isolated from a blood-transfused patients in which increased alanine aminotransferase (ALT) levels were detected . TherefoTo investigate the possible routes of TTV transmission, age-specific prevalence and genotype distribution in the population of the Czech Republic, we analyzed sera of 854 subjects divided into 5 groups based on the type of risk of TTV transmission. The age-specific distribution of TTV and correlation with the anti-HBV and/or anti-HCV status were determined. TTV genotypes were determined in 70 patients selected from the different groups.The Human Subjects Committee of the Institutional Review Boards approved all experimental protocols, and all subjects enrolled in the study signed an informed consent form. Additional samples were obtained from the collection of the national reference laboratory for viral hepatitis (NRL-VH). Five groups of subjects were analyzed:Group 1  was selected from 778 healthy blood donors (mean age 29 years, age range 18–59 years). All donors had normal ALT levels, and were negative for anti-HCV, anti-HIV and HBsAg. Since the TTV DNA prevalence in the first 100 subjects tested was very high we decided to randomly select sera  to incluGroup 2  consisted of 20 hemophiliacs  were collected from 10 patients) (mean age 42 years, age range 21–74 years) and 49 IVDUs. Patients with hemophilia were screened for anti-HCV, anti-HIV, anti-HBc and HBsAg, IVDUs were tested for anti-HBc and anti-HCV.Group 3  included 85 sex workers and 15 promiscuous men (mean age 25 years, age range 18–43 years). All of them were screened for anti-HCV, anti-HBc, HbsAg and HIV.Fifty penitentiary prisoners (mean age 29, age range 16–58 years) were tested for a potential increased risk of parenteral and/or sexual transmission of the virus. They were screened for anti-HCV and HBsAg.Group 4 (at risk of transuterine and mother to child virus transmission) consisted of 54 cord blood samples and 208 sera of children selected by age .Group 5  included 52 patients with non-A-E hepatitis (mean age 40 years, age range 9–76 years), 51 blood donors with elevated ALT levels (mean age 39 years, age range 25–64 years) and 74 patients with hepatitis C (mean age 27 years, age range 2–56 years). All blood donors with elevated ALT levels and all patients with non-A-E hepatitis tested negative for anti-HCV and HBsAg. All blood donors were also negative for anti-HIV. All patients with hepatitis C were HBsAg negative.DNA was extracted from 200 μL of serum using the QIAamp Blood kit (QIAGEN Ltd., Crawley, UK) and dissolved in 100 μL of elution buffer (QIAGEN Ltd., Crawley, UK). Extracted DNA was stored at -20°C.PBMCs were separated by centrifugation from the whole blood on a Ficoll-Paque gradient (SIGMA, St. Louis, MO) according to the manufacturer's protocol. PBMCs were digested with proteinase K (100 μg/ml) (SIGMA, St. Louis, MO) in 1 ml of lysis buffer . Thereafter, proteinase K was inactivated for 10 min at 95°C and samples were stored at -20°C.Five microlitres of total DNA were analyzed in a PCR thermocycler PTC 200 . TTV DNA detection was performed with two sets of primers. A 271 bp long fragment of the ORF1 was amplified in a semi-nested PCR with a modified primer set designed by Okamoto . By nestFor the PCR with NCR specific primers the same conditions as for ORF1 specific PCR were used. In the first PCR we used 50 pmol of primer NG133 (3'GTAAGTGCACTTCCGAATGGCTGAG 5') and NG147 (3'GCCAGTCCCGAGCCCG AATTGCC 5'), for the second PCR 50 pmol of primers NG134 (3'AGTTTTCCA CGCCCGTCCGCAGC 5') and NG132 (3'AGCCCGAATTGCCCCTTG AC 5'). Each of the 35 cycles of the first PCR and 25 cycles of the second PCR consisted of 30 s of denaturation at 94°C, 30 s of annealing at 58°C and 45 s of elongation at 72°C, with a final extension for 7 min at 72°C. Ten microlitres of the PCR product were separated electrophoretically on a 3% agarose gel (NuSieve 3:1, FMC BioProduct, Rockland, ME).N) testing positive was used as a relative titer for determining the viral titer per 1 ml of TTV DNA in serum and PBMCs.DNA extracted from sera and PBMCs of 4 patients with hemophilia was serially diluted (in 10-fold steps) in distilled water and TTV DNA was determined by PCR with NCR primers. The highest dilution , which revealed a clear band on the agarose gel, was determined. PCR products were excised from a 3% agarose gel and purified with MinElute Gel Extraction kit (QIAGEN Ltd., Crawley, UK) according to the manufacturer's protocol. Both strands of the 271 bp long products generated by PCR with ORF1-specific primers were sequenced directly with the NG061 and NG063mod primers using the ABI Big Dye Sequencing kit (Applied Biosystems, Foster City, CA). The sequencing was performed on an ABI PRISM 310 automated DNA sequencer (Applied Biosystems, Foster City, CA).DNA sequences (of the following accession numbers: AY429576 – AY429589, AY433961 – AY434008, AY456097 – AY456103, AY484597) were aligned using the CLUSTAL X program  with theHBsAg (V2) AxSYM, CORE AxSYM, AUSAB AxSYM, HCV3.0 AxSYM (Abbott, Chicago, IL) tests were used for the detection of HBsAg, anti-HBc, anti-HBs and anti-HCV markers. HBsAg reactive samples were confirmed with the HBsAg Confirmatory AxSYM test.The statistical analysis was performed using the Fisher exact test. Odds ratios (OR) with 95% confidence intervals (CI) and two-tailed P values were calculated in 2 – 2 tables using the EPI INFO statistical package (version 2002) and GraphPad InStat (version 3.05) (GraphPad Software, San Diego, CA). In all tests, the basic significance level was P = 0.05.In total, 854 samples were screened for the presence of TTV DNA using a NCR-PCR. The TTV genotype was determined by sequencing part of the ORF1 region from 70 TTV isolates.The prevalence rates of TTV DNA in different groups are shown in Table As indicated in Figure As indicated in Table The results of our sequencing and phylogenetic analysis are summarized in Table Tree of four patients positive in both serum and PBMCs by the ORF1 PCR system yielded a PCR product adequate for sequencing analysis. Genotypes and sequence homologies are shown in Table Four groups of subjects were compared for correlation of past HBV and/or HCV infection with TTV DNA prevalence. We compared subjects at a higher risk of sexual transmission, IVDUs, patients with hemophilia and penitentiary prisoners. There was no evidence of present or past HCV or present HBV infection in blood donors. The prevalence of anti-HBc in blood donors in the Czech Republic is very low (1–2%, unpublished data). These data imply that our control group was at a low risk of sexually and parenterally acquired infections. Also all children were anti-HBc and anti-HCV negative. Both the groups of hemophiliacs and IVDUs presented evidence of frequent past or current HBV (50% and 22%) and HCV (82% and 72%) infection. Thirteen percent of sex workers and promiscuous men showed past or current HBV exposure. The study subjects positive for HBV and/or HCV markers had a significantly higher prevalence of TTV DNA regardless of the primer set used Table .The present study on TTV prevalence, mode of transmission and age-specific prevalence is the largest study performed in Central and East Europe. This study showed that TTV infection is quite common among the Czech population. The prevalence rates of TTV in blood donors (52.6%) were similar to those found in other developed countries (for review see ).The most interesting result was the lack of TTV DNA in 54 cord blood samples, suggestive of the absence of transuterine transmission of TTV. Furthermore, we have shown that the prevalence of TTV DNA was age dependent. In children, we observed a dramatic increase of TTV prevalence within the first two years of age. The prevalence of TTV further gradually increased in children aged 8 to 14 years. Similarly, Ohto et al. reported that the TTV prevalence in children at the age of 2 years was comparable with that in mothers, while children younger than 3 months of age were infected only exceptionally . In the In adults, we have shown the increase of TTV prevalence with age irrespective of study group. Similar results have been reported by others ,30-33.We observed an increased prevalence of TTV in various groups of hepatitis patients. Since many hepatitis viruses share the same modes of transmission, multiple viral infections may occur in one patient . Our resIn agreement with other studies we detected the highest prevalence of TTV in hemophiliacs and IVDUs, which supports the importance of the parenteral route of transmission of TTV ,35-38. NAs for TTV sexual transmission, the results of our study suggest that if TTV is sexually transmitted, this mode of transmission is likely to be less important. Even though 13% subjects of the group at risk for sexual transmission of TTV showed past or current HBV exposure, indicative of high promiscuity, the prevalence of TTV was not significantly different from that of the control group (62% and 52.6%). To the best of our knowledge, so far only one study found a significant difference in the TTV prevalence in sex workers , while oIn the group of penitentiary prisoners the significantly higher prevalence of TTV seems to be most probably a consequence of intravenous drug abuse rather than sexual promiscuity since the prevalence of HCV was four-times higher than in the group at risk for sexual transmission (20% versus 5%).An increased prevalence of TTV in non-A-E hepatitis patients was observed in the present study in agreement with many previous studies. Additionally, our data showed an increased TTV prevalence in blood donors with increased ALT levels. Several studies have shown a correlation between TTV-titer and elevation of serum ALT levels ,30 but tThe heterogeneity of TTV is extreme. Because the NCR region is too conserved for evolutionary analyses, the ORF1 region of the TTV genome is most often used for genotyping. Out of the 70 sequenced isolates, G2 followed by G1 were the most frequent genotypes among the Czech population. The phylogeny analysis showed no evidence of association of particular TTV genotypes with any of the risk groups.Our results demonstrated a high prevalence of TTV in the Czech population. Our data show the absence of transuterine transmission of TTV, but postnatal route of transmission from mother to child and infection via frequent social contacts seem to be very important modes of transmision in children. The sexual mode of transmission is most likely to be low effective. No convincing evidence was found to support the involvement of TTV in the pathogenesis of hepatitis.Our data, as well as the results of other studies, show that optimization of the primer set for more standard TTV detection and genotyping is still needed. Improved serological approach to TTV detection could be of value. It is evident that more data are still needed for a better understanding of the natural history of TTV infection.The author(s) declare that they have no competing interests.RT and VN conceived of the study and participated in its design and coordination. MS carried out most of the experimental work and participated on the preparation of the initial draft of the manuscript. Part of the experimental work was done by JK. RT did all the statistical analysis and evaluation of the results and writing of the final version of the manuscript. All authors contributed to the preparation of the final manuscript.The pre-publication history for this paper can be accessed here:


Xylitol is a 5-carbon sugar that can lower the airway surface salt concentration, thus enhancing innate immunity. We tested the safety and tolerability of aerosolized iso-osmotic xylitol in mice and human volunteers.This was a prospective cohort study of C57Bl/6 mice in an animal laboratory and healthy human volunteers at the clinical research center of a university hospital. Mice underwent a baseline methacholine challenge, exposure to either aerosolized saline or xylitol (5% solution) for 150 minutes and then a follow-up methacholine challenge. The saline and xylitol exposures were repeated after eosinophilic airway inflammation was induced by sensitization and inhalational challenge to ovalbumin. Normal human volunteers underwent exposures to aerosolized saline (10 ml) and xylitol, with spirometry performed at baseline and after inhalation of 1, 5, and 10 ml. Serum osmolarity and electrolytes were measured at baseline and after the last exposure. A respiratory symptom questionnaire was administered at baseline, after the last exposure, and five days after exposure. In another group of normal volunteers, bronchoalveolar lavage (BAL) was done 20 minutes and 3 hours after aerosolized xylitol exposure for levels of inflammatory markers.In naïve mice, methacholine responsiveness was unchanged after exposures to xylitol compared to inhaled saline (p = 0.49). There was no significant increase in Penh in antigen-challenged mice after xylitol exposure (p = 0.38). There was no change in airway cellular response after xylitol exposure in naïve and antigen-challenged mice. In normal volunteers, there was no change in FEV1 after xylitol exposures compared with baseline as well as normal saline exposure (p = 0.19). Safety laboratory values were also unchanged. The only adverse effect reported was stuffy nose by half of the subjects during the 10 ml xylitol exposure, which promptly resolved after exposure completion. BAL cytokine levels were below the detection limits after xylitol exposure in normal volunteers.Inhalation of aerosolized iso-osmotic xylitol was well-tolerated by naïve and atopic mice, and by healthy human volunteers. Human airway surface is covered by a thin layer of liquid (airway surface liquid [ASL]) that contains many antimicrobial substances including lysozyme, lactoferrin, human β defensins, and the cathelicidin LL-37 -4. The ain vitro [The airway epithelium is water-permeable . When lain vitro . Xylitolin vitro ,15; it hin vitro . It has in vitro ; nasal ain vitro . There aOsmotic agents such as hypertonic saline, which is ionic, and nonionic mannitol, dextran and lactose, have been used in human subjects to increase mucus clearance -23. HoweIn subjects with bronchiectasis, inhalation of dry powdered mannitol can increase the clearance of mucus without affecting lung function . HoweverWe hypothesized that aerosolized iso-osmolar xylitol is safe and well-tolerated well by normal subjects. We compared the safety and tolerability of aerosolized xylitol with normal saline, and carried out additional exposure studies using mice.enh = ([Te /0.3 Tr ] - 1) × [2Pef/3Pif ], where Penh equals enhanced pause, Te equals expiratory time (in seconds), Tr equals relaxation time (in seconds), Pef equals peak expiratory flow (in milliliters per second), and Pif equals peak inspiratory flow (in milliliters per second).All experiments were reviewed and approved by the animal care and use committee of the University of Iowa. Except during exposures and evaluation, mice were allowed access to food and water ad libitum. C57bl/6 mice (Jackson Lab, Bar Harbor, MA) underwent baseline methacholine challenge test using a whole-body plethysmograph (Buxco Electronics, Troy, NY) as previously described . RespiraMice (6/group) were exposed to aerosolized saline (0.9 % NaCl) or aerosolized xylitol (5% solution in water, equimolar to the NaCl) for 150 minutes in an exposure chamber; all mice were evaluated for bronchial hyperreactivity to inhaled methacholine (using the Buxco whole body plethysmography system) before and after the exposures; other mice were monitored periodically during exposure by whole body plethysmography. All mice underwent whole lung lavage the next day for cell count and differential. After euthanasia, the trachea was cannulated, and the lungs were lavaged with 3.0 mL of sterile normal saline (0.9% NaCl). The lavage samples were immediately processed for total and differential  cell counts. In a separate group of naïve mice, whole body plethysmography was used to monitor Penh, respiratory rate, and tidal volume periodically during exposure to xylitol and saline for 10, 20, 40, and 80 minutes for a cumulative total dose of 150 minutes.We repeated the saline and xylitol exposure protocol to 2 more groups of six mice each after they were sensitized to and challenged with an antigen . Mice weGiven the concerns that have been raised about the reliability of airway resistance measurement by Buxco equipment, in a select number of mice we confirmed airway hyperresponsiveness using invasive measurement. Airway responsiveness was measured 24 hours after xylitol exposure in ova-challenged mice and compared to measurements made on naïve mice and ova-challenged mice without any exposure. Mice were anesthetized with Ketamine at 90 mg/kg and Pentobarbital at 50 mg/kg and attached to a small-animal ventilator (Flexivent, SCIREQ). Animals were ventilated at 150 breaths/min. Positive end-expiratory pressure (PEEP) was maintained between 2–3 cmH2O, with the computer setting the tidal volume from the entered weight of each animal. Central airway resistance (R) was measured at baseline and after 10 sec. of nebulized methacholine at doses of 12.5, 25 and 50 mg/ml.The study was approved by the University of Iowa Institutional Review Board as well as the Food and Drug Administration. Since this is a pilot study and would be the first time xylitol is being used as aerosol, there was no information available on expected complications. Ten subjects aged 18 or greater were studied. Pregnancy or any chronic medical conditions such asthma, atopy, and diabetes were grounds for exclusion. After giving written informed consent subjects underwent a screening spirometry . Baseline measurements of serum electrolytes, and serum and urine osmolarity were carried out. Baseline oxygen saturation was measured using a pulse oximeter. A brief questionnaire of respiratory symptoms that was developed using a visual analog scale (VAS) was administered at baseline ,32.Subjects received 10 ml of aerosolized saline (generated using a Pari LC Plus nebulizer with Proneb Ultra compressor system, Pari Inc, Monterey, CA) . The parThirty minutes after the exposures, subjects completed a follow-up questionnaire, and underwent spirometry and O2 saturation measurement. The procedure was repeated after exposure to 1, 5, and 10 ml of 5% xylitol (Danisco Cultor, USA). Xylitol was prepared by adding 5 gm of crystal sugar to every 100 ml of sterile water (Abbott Laboratories, IL). The solution was sterilized using FDA approved techniques and osmolarity confirmed to be 292 mOsm using a 5500 vapor pressure osmometer (Wescor, Inc., Logan, UT). After completing the exposures, repeat blood and urine tests for electrolytes and osmolarity were carried out. Finally, subjects repeated the symptom questionnaire five days after the first visit, over the telephone. The pre-established criterion for discontinuing study participation was a decline in FEV1 by greater than 20% from baseline.Spirometry was performed using a Vmax V6200 Autobox (Sensor Medics Corp., Yorba Linda, CA), according to guidelines published by the American Thoracic Society . The spiThe amount of symptoms was assessed at baseline and after each exposure. Subjects scored chest tightness, shortness of breath, cough, headache, chills, muscle soreness, phlegm, nausea, stuffy nose, sneezing, and fatigue on a visual analog scale from 0–10 cm (0 being symptom-free and 10 being extreme amount) ,32.++ and Mg++ and suspended in complete medium, Roswell Park Memorial Institute (RPMI) tissue culture medium (Gibco/BRL, Gaithersberg, MD). Differential cell counts were determined with cytospin (Shandon, Pittsburgh, Pa) slide preparations by using Wright-Giemsa stain. The cell-free fluid was frozen at -70°C until required for cytokine assay.We also examined the effect of aerosolized xylitol on markers of inflammation in the airways. A separate group of subjects underwent bronchoscopy and bronchoalveolar lavage (BAL) according to American Thoracic Society standards at 30 minutes (n = 6), and 3 hours (n = 5) after exposure to 10 ml of aerosolized iso-osmolar xylitol . BAL wasCytokine measurements were performed using enzyme linked immunosorbent assays for IL-6 and LTC-4. IL-6 levels were determined by a Quantikine Human IL-6 ELISA kit . The limit of detection of IL-6 is 0.70 pg/ml. LTC-4 (leukotriene) levels were determined by a leukotriene C4 EIA kit . The limit of detection of LTC4 is 10 pg/ml. LTC4 of BALs were extracted and concentrated with Cysteinyl-Leukotriene Affinity Sorbent .We studied ten subjects with a gradual increase in exposure dose in the pilot safety study. Differences were analyzed using t-test, Wilcoxon signed rank test, and one way and two-way repeated measures analysis of variance (ANOVA) as indicated. Ninety-five percent confidence intervals were calculated where appropriate. All analyses were performed using SAS version 8.2 (SAS Institute, NC) and at a 5% significance level.Mice tolerated the exposures well without any visible distress. The corresponding volume of the 150-minute exposure was approximately 45 ml. Among naïve mice, exposure to xylitol resulted in no significant change in bronchial hyperresponsiveness compared to saline Figure . We carrWhole lung lavage showed no significant differences in lavage fluid cell count and differential due to xylitol exposure. Naïve mice exposed to saline or xylitol demonstrated, as expected, a macrophage-predominant response. In contrast, OVA-sensitized/-challenged mice were characterized by airway eosinophilia in both saline- and xylitol-exposed groups Table . In summTable As shown in Table An additional 11 subjects underwent bronchoscopy and bronchoalveolar lavage following xylitol inhalation. The mean cell count in the BAL fluid at 20 minutes (n = 6) and 3 hours (n = 5) after xylitol exposure was 1.2 ± 0.07 million cells/ml and 2.94 ± 1.48 million cells/ml respectively. All cell preparations had between 95–100% alveolar macrophages. BAL IL-6 and LTC-4 levels after xylitol exposure were below 0.70 pg/ml and 10 pg/ml respectively at all time points.Lower respiratory tract colonization is an important step in the pathogenesis of pulmonary manifestations of chronic diseases such as CF and dyskinetic cilia syndrome and certain acute clinical entities such as ventilator-associated pneumonia. There is a continuing need for simple, cost-effective, and safe intervention to decrease colonization of lower airways. Studies have shown that lowering the salt concentration of airway surface liquid can enhance innate immunity by increasing the potency of the natural antimicrobial peptides. In addition to increasing the activity of individual ASL factors, lowering the NaCl concentration also independently enhances synergistic interactions . Thus, lXylitol, when applied to airways as an iso-osmolar agent, can potentially lower airway salt concentration and therefore lower bacterial colonization in chronic infections. In addition to having low transepithelial permeability, it has the added advantage of being poorly metabolized by bacteria. In recent years, many osmotic agents have been aerosolized into human airways for mucus clearance. However, there are reports of bronchospasm associated with their use. This is the first study to our knowledge to use xylitol in an aerosolized form.The main adverse effect reported from oral xylitol use was diarrhea when the dose exceeded 40–50 gm/day . Intraveet al. [2. The depth of ASL may vary from the trachea to the small bronchioles; if an average depth of 10 μm is estimated, the total ASL volume would be ~2.5 mL. Thus, if we assume a uniform aerosol distribution, administration of a total volume of 2.5 mL of 300 mM xylitol to the airways would be expected to lower the salt concentration in half simply by a dilutional effect. If the mean ASL depth were 20 μm, then 5 mL of delivered solution would be required. Because the solution is iso-osmotic, immediate, major osmotic shifts of water across the epithelium should not occur, which leads to dilution of the salt concentration. Moreover, with time, the volume and salt concentration may decrease due to Na+-dependent salt absorption, the osmotic effects of which are counterbalanced by xylitol in the ASL [We made calculations of the amount of xylitol to be delivered to the airway surface of an adult. Mercer, et al.  measured the ASL .et al. [Our preliminary calculations for dosing for mice experiments were derived as follows; Mercer, et al.  also estet al. . Assuminet al. , which wThis study shows that aerosolization of iso-osmotic xylitol is likely to be safe and well tolerated by human volunteers. There was no change in spirometry, laboratory test results as well as BAL cytokine levels after xylitol exposure. Earlier studies have reported bronchial hyperresponsiveness with aerosolization of hypotonic and hypertonic solutions. Thus, aerosolization of iso-osmotic xylitol could be tested for prevention and treatment of airway colonization.There are several potential limitations with this study. The validity of body plethysmography as a measure of respiratory physiology in mice has been recently questioned ,49. HoweIn summary, our data indicate that iso-osmotic xylitol can be safely delivered by aerosol to normal volunteers. Studies of safety with long-term exposure to animals are required before human use can be attempted. This could lead to exciting interventions to enhance the innate immunity of airway epithelia.ANOVA Analysis of VarianceASL Airway Surface LiquidCF Cystic FibrosisFEV1 Forced Expiratory Volume in 1 secondGSD Geometric Standard DeviationPenh Enhanced PauseVAS Visual Analog ScaleBAL Bronchoalveolar Lavage


There is little information on the effectiveness of modern compared to traditional malaria treatment from the rural areas of Africa.Follow-up of 402 episodes of clinical malaria among pre-school children in Nouna Health District, northwestern Burkina Faso. The exposure of interest was the type of treatment ; the outcome was clinical response to treatment.Out of the 402 observed malaria episodes, 87% were treated with chloroquine and 13% with traditional remedies. Overall, community effectiveness was 67% with chloroquine and 54% with traditional treatment. Chloroquine effectiveness was associated with age and ethnicity. An additional interview survey demonstrated wide variations in the dosages of chloroquine given to young children in this community.The effectiveness of chloroquine, when used within the community, was significantly lower in this study than its known efficacy in the study area. This concerns, in particular, the very young children. These findings demonstrate the need for better education of parents about correct dosage of first-line malaria drugs, and for particular attention in the treatment of very young children. It has been estimated that at least one million annual malaria deaths occur among young children in rural sub-Saharan Africa (SSA). Most of these deaths are in areas with little access to health services -4. UnderThe situation is now complicated by the increasing resistance of Plasmodium falciparum to chloroquine in most SSA countries . MoreoveTraditional treatment for malaria is very common, particularly in the rural areas of SSA, but only few data exist regarding the efficacy of such treatments ,12. In rThis paper reports on the community effectiveness of chloroquine compared to traditional remedies in the treatment of uncomplicated falciparum malaria in young children of rural Burkina Faso.The study was conducted in the rural part of the research zone of the Centre de Recherche en Santé de Nouna (CRSN), in Nouna Health District, north-western Burkina Faso. The CRSN study zone comprises 41 villages and Nouna town, with a total population of 55,000. The Nouna area is a dry orchard savanna, populated by subsistance farmers of different ethnic groups. There is a short rainy season which usually lasts from June until October. Malaria is holoendemic but highly seasonal in the study area . Formal This is an observational study at the community level. Data for this study were extracted from the database of a trial on the effects of zinc supplementation on malaria morbidity conducted in the Nouna area in 1999 . During Field methods for data collection have already been described . In brieAll relevant signs and symptoms manifested by the children were recorded on a standard questionnaire during regular visits of field staff. Moreover, a comprehensive questionnaire was filled in for each disease episode which included information on specific signs and symptoms as well as place, time and type of treatment received. No specific information was recorded on the dosage of chloroquine and on the type of traditional remedies given to the study group.For this study, only children with a diagnosis of falciparum malaria, defined as an axillary temperature of ≥37.5°C together with a density of ≥5,000 P. falciparum parasites per μl of blood were considered. Inclusion criteria were falciparum malaria treated with chloroquine (with or without antipyretics) but without traditional remedies (group CQ), or with traditional remedies only (group TRAD). Exclusion criteria were a history of febrile illness during the previous four weeks, malaria treatment during the previous four weeks, any treatment with another antimalarial drug during follow up, and missing temperature measurements during the two-weeks follow up period. Multiple episodes of malaria in individual children were considered to be independent with regard to treatment choice .To evaluate treatment outcome, a modified definition of the WHO protocol for assessment of therapeutic efficacy of antimalarial drugs in areas with intense transmission was used . TreatmeTo retrospectively estimate the chloroquine dosages usually given to febrile young children in the study area, 55 individual interviews with randomly selected mothers of young children from three villages in the study area were conducted during a malaria survey at the end of the 2001 rainy season. The villages were purposely selected to represent the rural study population in its socio-cultural, demographic and geographical diversity. Questions concerned the kind of treatment  and the dosages of western drugs used during treatment of the last febrile episode of survey children, the current availability of chloroquine in the household and the origin of chloroquine drugs. The weight of children was measured with a Salter hanging spring scale.Blood films were kept in closed slide boxes until they were transported to Nouna (two to three times per week). They were Giemsa-stained at the Nouna hospital laboratory and transported afterwards to the Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Ouagadougou for reading. All films were examined by two experienced laboratory technicians using a ×100 oil immersion lens and ×10 eyepieces. In case of significant discrepancy between the results of the two technicians, blood slides were read by a third investigator. Blood films were analysed for the species-specific parasite density per μl by counting against 500 white blood cells and multiplying by sixteen (assuming 8,000 white blood cells per μl of blood). Slides were declared negative if no parasites were seen in 400 fields on the thick film.Data were entered at the data management department of the CRSN into a data bank (Microsoft Access, version 97). All questionnaires were checked by supervisors before computer entry. Parasitological data were entered into EpiInfo (version 6.0) at the CNRFP, and the data were transferred to the CRSN. All data were checked for range and consistency before data entry.The outcome of malaria episodes (AR vs. TF) was assessed dependent on the treatment given by the caretakers. Episodes in which the child had received at least one dose of chloroquine were grouped in the treatment group CQ, and episodes in which caretakers had given only traditional treatment in the treatment group TRAD. The hypothesis was tested that the samples in the CQ and the TRAD treatment groups were from populations with the same distribution of baseline variables using the Wilcoxon ranksum test for continuous variables and the chi-squared test for categorical variables. Community effectiveness of treatment with chloroquine was assessed both by intention to treat (i.e. adherence to advice by the field workers to give chloroquine) and considering only those cases who actually complied. A stratified analysis by age bands was started, assuming that age would be an important determinant of treatment outcome. Logistic regression modelling was then used to control for differences in the distribution of baseline characteristics such as age, sex and parasite density in the two treatment groups. Stata 7.0 statistical software was used for all calculations.Approval for the RCT from which the data used were drawn was granted by the Ethical Committee of the Heidelberg University Medical School and the Ministry of Health in Burkina Faso.A total of 330 children (175 boys and 155 girls), who contributed 402 episodes of falciparum malaria, were followed up. Most of the episodes (65.7%) occurred between August and October (Table In 350 (87.1%) of the episodes, the child had received at least one dose of chloroquine (treatment group CQ); in 52 episodes (12.9%), the caretakers had given only traditional treatment (treatment group TRAD). Table The caretakers had been advised to give chloroquine to all children participating in this study. Compliance with this advice, stratified by age group, is shown in Table The crude odds ratio for TF in the CQ group vs. the TRAD group was 0.59 with a 95% confidence interval that included unity . During the most recent fever episodes of respective children, home treatment with chloroquine was reported in 32/55 (58%) , treatment at the local health centre in 15/55 (27%), traditional treatment in 6/55 (11%), and only paracetamol treatment in 2/55 (4%). The mean weight of chloroquine-treated children was 8.5 kg (range 3.2 – 12.0 kg). The mean (median) total dose of chloroquine used was 34 mg/kg and 25 mg/kg respectively (range 5–110 mg/kg) (figure 1). Only 9/55 (13%) mothers reported current availability of chloroquine tablets in their household (median 3 tablets, range 1–10). Most chloroquine drugs were purchased from local shops and drug sellers.The main finding of this study is a rather low community effectiveness of chloroquine in the treatment of malaria episodes among young children in rural Burkina Faso. Overall, a failure rate of 35% was observed which declined only marginally to 33% when malaria episodes in which caretakers did not comply with the advice to give chloroquine were excluded. This is considerably higher than the 10% treatment failure rate observed in a study on chloroquine efficacy in young children of the area . These fThe effectiveness of treatment with chloroquine increased significantly and almost linearly with age, even after controlling for parasite density. The most likely explanation is a rapid development of immunity within the age range of our study children, as has been observed in other studies ,17,18. TOnly a small number of episodes were treated solely with traditional treatment, possibly a consequence of the view of the local population that western malaria treatment is more effective than traditional treatment . This maThis study has two limitations. First, information on the dosage of chloroquine and on the nature of traditional remedies given to children in the study was lacking. However, data from the interview survey provide a good estimate of chloroquine dosages used. Although these data demonstrate a wide variation of chloroquine dosages, it is reassuring that the reported median dosage complies with the national recommendations of a total dose of 25 mg/kg chloroquine for uncomplicated malaria. So why is there such a difference between efficacy and effectiveness? The reason may well be a combination of partly chloroquine underdosing, poor quality of drugs purchased from local shops and drug sellers and high frequency of vomiting in febrile young children -23. MoreIn conclusion, this study provides clear evidence for a rather low and age-dependant community effectiveness of chloroquine in the treatment of P. falciparum malaria in an area of rural Burkina Faso where the efficacy of chloroquine is still sufficiently high in use as first-line treatment. The observed discrepancy between efficacy and community effectiveness implies that many children received insufficient doses of chloroquine. Our findings thus point to the importance of (1) better education of parents on correct dosage of first-line malaria drugs, as well as on danger signs requiring a visit at a health centre, (2) quality-control of all malaria drugs used in the community, and (3) particular care in the treatment of immunologically naïve very young children. Finally, more studies on the types and effects of traditional treatments used in different population groups are needed.


Brugia malayi is endemic in several Asian countries with the highest prevalence in Indonesia. Determination of prevalence of lymphatic filariasis by serology has been performed by various investigators using different kinds of antigen (either soluble worm antigen preparations or recombinant antigens). This investigation compared the data obtained from IgG4 assays using two different kinds of antigen in a study on prevalence of antibodies to B. malayi.Serum samples from a transmigrant population and life long residents previously tested with IgG4 assay using soluble worm antigen (SWA-ELISA), were retested with an IgG4 assay that employs BmR1 recombinant antigen (BmR1 dipstick [Brugia Rapid™]). The results obtained with the two antigens were compared, using Pearson chi-square and McNemar test.There were similarities and differences in the results obtained using the two kinds of antigen (SWA and BmR1). Similarities included the observation that assays using both antigens demonstrated an increasing prevalence of IgG4 antibodies in the transmigrant population with increasing exposure to the infection, and by six years living in the area, antibody prevalence was similar to that of life-long residents. With regards to differences, of significance is the demonstration of similar antibody prevalence in adults and children by BmR1 dipstick whereas by SWA-ELISA the antibody prevalence in adults was higher than in children.Results and conclusions made from investigations of prevalence of anti-filarial IgG4 antibody in a population would be affected by the assay employed in the study. Brugia malayi and Brugia timori [Brugia endemic countries of Malaysia and Indonesia [Lymphatic filariasis affects approximately 120 million people worldwide. Ten percent of these infections are attributed to ndonesia . This dindonesia  and diffConversely, serological diagnostic methods exhibit better sensitivity than detection of microfilaria by thick blood smear, allow the detection of amicrofilaraemic infections among "endemic normals" and afford daytime finger-prick blood sampling  .Brugia infection, anti-filarial IgG4 assay may be the next best alternative for detection of brugian filariasis [However, reports on antigen detection test for brugian filariasis have not demonstrated high levels of sensitivity ,7. Thus lariasis . Anti-filariasis -13 and dlariasis -17. Detelariasis ,19.Studies assessing antibody prevalence of lymphatic filariasis have employed assays that use either soluble worm antigens or recombinant antigens ,13,18-21Previously, an ELISA employing soluble adult worm antigen (SWA-ELISA) had been performed to determine prevalence of anti-filarial IgG4 antibodies on a set of serum samples from Indonesia. These samples were collected from:1. A transmigrant population that migrated from a non-filarial endemic region to an area endemic for Brugian filariasis and;2. Life-long residents of the Brugian endemic area .B. malayi recombinant antigen (BmR1 dipstick [Brugia Rapid™]) and detection of IgG4 antibodies were evaluated using the same set of serum samples. The BmR1 dipstick test has previously been shown to be highly specific and sensitive for the detection of brugian filariasis. In a study involving four international laboratories, the BmR1 dipstick was found to be 93% sensitive and 100% specific when tested with 535 serum samples from patients with various infections and healthy controls [In the present study a rapid test based on controls . In anotcontrols .The present study demonstrated that interpretations of some aspects of the seroepidemiology of filarial infection are affected by the kind of antigen employed in the assay. Thus this study, which utilized the same set of serum samples on assays using two kinds of antigens, highlights the role of the kind of antigen employed in the comparison of results of prevalence studies.B. malayi [The details on the sera and study population are as described previously . Briefly. malayi ,25. The B. malayi worms were purchased from TRS labs, Athens, Georgia, USA. Female worms were freeze dried, ground to powder, dissolved in phosphate buffered saline (PBS), homogenized and slowly stirred overnight at 4°C. The protein concentration was determined by 2,2'-biquinoline-4,4'-dicarboxylic acid disodium salt hydrate (BCA) method before storage at -20°C.Adult Bm17DIII DNA sequence, GenBank accession no. AF225296. Southern blot hybridization assays performed on cDNA libraries of L3, L4, mf, adult male and adult female B. malayi demonstrated that the DNA sequence is present in all of the five kinds of libraries . Preliminary immunohistological studies suggest that the expressed antigen is found in the epithelial membranes of the adult female uterus .This BmR1 dipstick (Brugia Rapid™) was performed as described previously and according to the instructions of the manufacturer [16, Malaysian Bio Diagnostics Research Sdn. Bhd., Bangi, Selangor, Malaysia]. The BmR1 recombinant antigen was expressed from The results obtained with BmR1 dipstick were compared to the results obtained previously with SWA-ELISA. The similarities and differences in the results were analyzed by comparing proportion of related samples using McNemar test; and comparing proportions of unrelated samples by using Pearson Chi-square (if indicated Fischer exact test was used instead).B. malayi, as determined by BmR1 dipstick assay and SWA-ELISA, in the transmigrant population who had resided for various lengths of time in the endemic area and the antibody prevalence in the LLR population. The first detection of IgG4 antibody by the BmR1 dipstick was recorded at 3 years post-exposure, the antibody prevalence increased from 0% in the new arrivals (≤ 1 month and 2–4 months) to 7.4%, 11.1%, 39.1% and 42 % in populations exposed to the infection for 3, 4, 5 and 6 years respectively. Comparison of IgG4 assays using the two kinds of antigen demonstrated similarities in three areas. First, using both SWA-ELISA and BmR1 dipstick, the total prevalence of specific IgG4 in the transmigrant population was found to increase with increasing length of residence in the endemic area. Thus the BmR1 dipstick test confirmed the previously reported finding which demonstrated that the development of anti-filarial IgG4 correlated with the duration of exposure in previously unexposed population [Figure pulation . Second,pulation . Thus thpulation . Third, Differences between the two kinds of antigen were also observed Figure . First, Figure Strongyloides antibody responses was found to be associated with higher antifilarial IgG4 responses in assay that uses crude filaria extract as compared to assay that uses B. malayi Bm14 recombinant antigen [Thus the BmR1 dipstick test demonstrated that the establishment of anti-filarial IgG4 in the children and adults of the transmigrant population occur at comparable rates; this is not in agreement with the previous finding, using SWA-ELISA, that demonstrated IgG4 was established more rapidly in the adult population than in children . The abo antigen .The discrepancy between the results of SWA-ELISA and BmR1 dipstick may also be partly due to the greater sensitivity of the former as compared to the latter. Out of 120 of 381) discrepant results, 111 were positive by SWA-ELISA but negative by BmR1 dipstick. Out of these 111 samples, 38 had low ELISA titers . If the stringency of the cut-off value of the SWA-ELISA is increased from 4.02 to 4.5, then the discrepancy can be considerably reduced to 88 (of 381), with 73 positive by SWA-ELISA but negative by BmR1 dipstick. On the other hand, since BmR1 dipstick test has been reported to be highly specific 1 discrep, it is ualbeit equally specific) than BmR1 dipstick test [B. malayi infection. Thus in this study if the ELISA format had been used to determine prevalence of IgG4 antibodies to BmR1 instead of the dipstick format, the SWA-ELISA would still be detecting significantly more positives than the BmR1-ELISA.Due to the problem of maintaining the antigenicity of the BmR1 antigen when shipped from Malaysia to Netherlands, the dipstick (immunochromatography) assay, which can be transported at room temperature, was employed to test the BmR1 antigen. However the difference in the assay formats is unlikely to be the reason for the lower overall antibody prevalence levels seen with the latter. This is because we have previously shown that ELISA using BmR1 was less sensitive . In the adult population, except for the earlier initial detection, a similar pattern was observed with the results of the BmR1 dipstick i.e. increasing rate of antibody prevalence in the transmigrants with exposure; and the IgG4 prevalence at year 6 is not significantly different (p = 0.430) than the antibody prevalence in the LLR adult population.This study highlights that assays using both BmR1 and SWA antigens demonstrate an increasing prevalence of specific IgG4 antibodies in the transmigrant population with increasing length of residence in an area endemic for brugian filariasis, and, by six years residency that the antibody prevalence was similar to that observed in the LLR population. This study also documented three main differences in results derived from assays using two antigens i.e.1. Earlier detection and higher rate of antibody prevalence by SWA-ELISA as compared to the results demonstrated by BmR1 dipstick;2. Similar rate of acquisition of antibody prevalence in children and adults by BmR1 dipstick; whereas by SWA-ELISA adults were found to become antibody positive faster than in children;3. By BmR1 dipstick the increasing total prevalence of IgG4 with exposure was primarily due to the adult population, whereas by SWA-ELISA this was attributed to both children and adult populations.Brugia malayi infection may vary with the kind of antigen used in the assay. Thus comparison of results of different studies must take into account the kind of antigen employed, especially if one study uses native antigen and another uses recombinant antigen. It would be interesting to compare seroepidemiological data of IgG4 assays using two different recombinant antigens on the same population.This study demonstrates that some aspects of seroepidemiology of Rahmah Noordin is the inventor of the commercialized BmR1 dipstick test (Brugia Rapid™)RN drafted the paper and supplied the BmR1 dipstick test; SW & AM supplied the sera and edited the paper; LBH performed most of the statistical analysis; ES and MY conceived the study and contributed significantly in editing of the paper: In addition ES performed the BmR1 dipstick test and some statistical analysis. All authors read and approved the final manuscript.


Osteoid osteoma is a benign bone neoplasm which is seen in the long bones of appendicular skeleton. It is rarely seen in the cranium. Skull base osteoid osteoma is extremely rare and has been anecdotally reported.The authors report a case of a large osteoid osteoma of the ethmoid with intraorbital and intracranial extension in a 33 year old male patient. He presented with loss of vision in the left eye. The intra-cranial extension was excised through a single burr-hole fronto-orbitotomy. The ethmoid and orbital portions were approached and excised through a Weber-Ferguson incision and inferior orbitotomy. Radical excision of the tumor could thus be achieved through a craniofacial approach.Although benign and rare, skull base osteoid osteoma can present with neurological deficit due to its mass effect and involvement of vital structures. A multispeciality team approach is advisable in such cases if radical excision is to be achieved. A craniofacial approach made radical single stage excision of this multicompartmental osteoid osteoma possible with an uneventful postoperative period. Osteoid osteoma is a rare benign osteoblastic lesion usually involving the long bones of the lower limbs. Cranial involvement has been mainly localised to the skull vault. Osteoid osteoma of the skull base is a rare entity . SurgicaA 33 year old male presented with decreased vision in the left eye and left sided headache of 3 months duration. Examination revealed left eye blindness with primary optic atrophy and no other neurological deficit. Imaging revealed a bony tumor in the left ethmoid sinus invading the left orbit and compressing the left optic nerve. Intracranial extension into the anterior cranial fossa on the left side was noted Figure . Core biA multispeciality team approach was devised to achieve radical excision of the tumor. Bicoronal scalp flap and pericranial flaps were raised separately. A single burr hole left fronto orbital bone flap was raised including the orbital roof and left zygomaticofrontal process Figure . Dura waPost-operatively patient had a frozen left eye  with no improvement in the left eye vision in the immediate postoperative period. His postoperative period was otherwise uneventful. Histopathological examination revealed an osteoid osteoma Figure . On follOsteoid osteoma is a benign osteoblastic lesion and constitutes 1% of all bone tumors and 11% of benign bone lesions . CranialThe radiological diagnosis rests on Computerised Tomography and isotope bone scan . RadiogrThe treatment generally consists of en bloc resection or curettage of the tumor. Recurrence rate after incomplete resection may be upto 10% . Our patHistologically the nidus is sharply delineated from the surrounding variably thick layer of dense bone. The nidus is composed of more or less calcified osteoid lined by plump osteoblasts within a highly vascularised connective tissue stroma  Figure . It doesOsteoid osteoma of the skull base is rare and anecdotally reported. Radical excision is difficult especially if the tumor involves major blood vessels and cranial nerves. The surgical team constituted the neurosurgeon, surgical oncologist and plastic surgeon. A craniofacial approach made radical single stage excision of this multicompartmental osteoid osteoma possible with an uneventful postoperative period.All the authors of the article "ETHMOIDAL OSTEOID OSTEOMA WITH ORBITAL AND INTRACRANIAL EXTENSION -CASE REPORT" hereby declare that there are no competing interests – financial and non financial.SBP performed the craniotomy, excised the intracranial extension of the tumor, assisted in excising the orbital portion, drilled the ethmoidal portion and drafted the manuscript.KH excised the orbital portion and helped draft the manuscript.MSV and US performed the orbitotomy, helped in tumor excision and closure.DJ did the histopathological examination and helped in drafting the manuscript.All authors have read and approved of the manuscript.The pre-publication history for this paper can be accessed here: