Its function is unknown, but it is considered to be a major structural component of metazoan nuclear pores. Yet, our previous findings showed pronounced differences in expression levels in embryonic mouse tissues and cell lines. In order to identify factors regulating GP210, the genomic organization of human GP210 was analyzed The human gene was mapped to chromosome 3 and consists of 40 exons spread over 102 kb. The deduced 1887 amino acid showed a high degree of alignment homology to previously reported orthologues. Experimentally we defined two transcription initiation sites, 18 and 29 bp upstream of the ATG start codon. The promoter region is characterized by a CpG island and several consensus binding motifs for gene regulatory transcription factors, including clustered sites associated with Sp1 and the Wilms' tumor suppressor gene zinc finger protein (WT1). In addition, distal to the translation start we found a (GT)n repetitive sequence, an element known for its ability to bind WT1. Homologies for these motifs could be identified in the corresponding mouse genomic region. However, experimental tetracycline dependent induction of WT1 in SAOS osteosarcoma cells did not influence GP210 transcription.Although mouse GP210 was identified as an early response gene during induced metanephric kidney development, and WT1 binding sites were identified in the promoter region of the human GP210 gene, experimental modulation of WT1 expression did not influence expression of GP210. Therefore, WT1 is probably not regulating GP210 expression. Instead, we suggest that the identified Sp binding sites are involved. Nuclear pore complexes (NPCs) provide the only known gateway for transport of RNAs to the cytoplasm and bidirectional transport of proteins between the nucleus and the cytoplasm. The NPC in vertebrates has an estimated mass of approximately 125 Mda. Structural studies suggest an octagonal rotational symmetry framework, from which 50–100-nm long fibrils extend into the nucleoplasm and cytoplasm. A comprehensive inventory of all NPC constituents has been made for yeast .In the list of metazoan nucleoporins, there are only two integral membrane proteins, gp210 -6 and POIn contrast to POM121, gp210 has an inverted topology with its main bulk residing in the lumen of the NE and only a short 58 residue C-terminal portion facing the NPC . The amiThe primary sequence of gp210 is known for rat  and mouscis-acting elements in the human promoter and its mouse counterpart. Several putative Wilms tumor suppressor binding 1 sites were found. Nevertheless, experimental overexpression of WT1 in SAOS osteosarcoma cells did not influence GP210 mRNA expression.In the current study we present the genomic structure of the human integral membrane glycoprotein 210 gene (GP210), the open reading frame sequence and a promoter region analysis. This was done in silico by taking advantage of the available human genomic sequence. Transcription start sites were determined experimentally by RNA ligase mediated rapid amplification of cDNA ends. Computer-assisted searches of the promoter sequence indicated putative consensus binding sites for transcription factors involved in tissue specific gene regulation. We also identified of shared putative We initially assumed that mouse gpP210 was a member of a yet undiscovered large family of tissue-specific nuclear pore membrane proteins, and initially named it POM210 . Since cThe deduced amino acid sequence of human GP210 contains 1887 residues, predicting a molecular mass of 205 198 Da and a pI of 6,41 of the non-processed protein. Alignment to the corresponding mouse and rat sequences displayed a high degree of homology (91,8% similarity and 88,9% identity compared to the primary structure of the rat protein). One insertion, an alanine at position 1858, makes the GP210 one residue longer compared to rat and mouse GP210. A signal peptide cleavage consensus site could be defined between residues 25 and 26 using the SignalP algorithm  elements (consensus Py Py C A N T/A Py Py) . In ordeAnalysis of the sequence surrounding the translation start site with the GRAIL program predicted a 1236 bp long CpG island, with a GC content of 75.3% starting 434 bp upstream of ATG, covering the first exon and extending into the first intron. We used a variety of promoter and transcription factor binding site algorithms to analyse the region upstream the start site for GP210, including TESS and Matinspector. By selecting for perfect matching and human consensus motifs a restricted number of putative transcription factor binding sites were found. Using these criteria, 22 motifs recognized by 14 different factors were defined on the sense strand, evenly distributed within 1000 bp proximal to the translation start codon (See Table 2, A sequence of 9433 bp (c047302867. Contig1) was found in the Mouse Genome Sequencing Consortium (MGSC) database. This sequence mapped to mouse chromosome 6 and contained the first exon, part of the first intron, and 3 kb of the upstream promoter region of mouse GP210. Similar to human, a GPC island containing 644 bp and with a GC content of 74% was found starting 268 nt upstream of the start codon and extending into the gene. Pairwise ClustalW alignment of this genomic clone showed 54% homology to its human counterpart within the first 500 bp upstream of the translation start  suppressor protein binding sites, four Sp1 biding sites, and one ETS binding site in a range of 315 bases just upstream of the translation initiation site. Some of these were conserved in the mouse promoter region.Mouse GP210 was initially identified as an early response gene to induction of embryonic kidney tubule development , suggestWT1 is a zinc finger transcription factor known to exist in different isoforms due to alternative pre-mRNA splicing. DNA binding specificity is determined by insertion or removal of three amino acids between zinc finger III and IV (referred to as WT1(+KTS) and WT1(-KTS)). The -KTS isoform have been reported to repress or activate target genes containing variations of an EGR1 related, GC-rich motif (consensus GXGXGGGXG) in their promoter . Other bcis-elements widely distributed in different types of transcription control regions [Sp1 is a transcription factor included in a small protein family (Sp1, Sp2, Sp3, and Sp4), whose members are binding to  regions . Althoug regions  and hema regions  have beeWe also found a putative ETS-2 binding site in the mouse and human promoter for gp/GP210. Ets-2 is a widely distributed member of the ETS family of transcription factors characterized by a unique winged helix-turn-helix domain, which specifically interacts with DNA sequences containing the purine-rich core motif, GGAA/T. Since several ETS family members binds to the same core motif it has been difficult to determine specific target genes for each member, gene targeting in mice implicates ETS-2 as an activator of metalloproteinases in placenta (MMP-3, MMP-9 and MMP-13) and a regulator of hair development . ElevateOur promoter region analyses, which identify WT1, SP1 and Ets-2 as putative transcription factors regulating GP210 expression are descriptive. GP210 expression in the developing kidney resembles that of E-cadherin, which has been show to be a bone fide WT1 target gene . WT1 A i1880-Pro1881 motif could be detected as a target for cyklin-B-p34cdc2 kinase and MAP kinase in vitro [The amino acid sequence of human GP210 revealed potential sites for phosphorylation and glycosylation. The role for phosphorylation of nuclear envelope associated proteins is not well understood, but is presumed to have a function in mitotic events ,38. Non-in vitro . A compain vitro , but the7 cells using the RNAeasy midi kit (Qiagen) following the recommendations of the manufacturer, including a DNAse treatment step to avoid chromosomal DNA contaminations. Northern blotting of total RNA from cells was performed as described [32P] dCTP by random priming using Megaprime DNA labeling system kit (Pharmacia). Filters were hybridized in 20 mM Na2HPO4 (pH 7.2), 7% SDS at 65°C for 18 hours. After washing in 20 mM Na2HPO4 (pH 7.2), 5% SDS at 65°C for 2 × 60 min followed by 2 × 60 min in 20 mM Na2HPO4 (pH 7.2), 1% SDS at 65°C the filters were exposed to Hyperfilm-MP films (Amersham) for 5 days at -70°C in the presence of intensifying screens. Band intensities were quantified using a PhosphoImager 400S .WCCS-1 Wilms tumor cells  and HeLaescribed . To visuescribed . Probes 2 atmosphere. For the induction, cells (at 70% confluence) were washed twice with PBS and refed with fresh media in the absence or presence of 1 μg/mL of Tetracycline. After 18 hours, the cells were washed twice with PBS and total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacture's instructions. 4 μg of the RNA were resolved on formaldehyde-containing agarose gels and transferred to Nytran membranes (Schleicher and Schuell, Keene, NH). Hybridization was performed in ULTRAhyb buffer (Ambion, Austin, TX) at 42°C. Briefly, filters were prehybridized in ULTRAhyb buffer for 6 hours followed by an overnight hybridization at 42°C with the 935 bp hGP210cDNA probe. A WT1 cDNA probe (exons 5 to 10) and a cDNA probe for actin were used as controls. Membranes were exposed to BIOMAX MS films (Kodak, Rochester, NY) at -80°C in the presence of intensifying screens. Probes were stripped by boiling the membranes in 0.1% sodium dodecyl sulfate/ standard saline citrate solution for 10 minutes.WT1-A SAOS cells were constructed from a cell line harboring the tetracycline-repressor-VP16 fusion protein , transfeTo determine transcription start, the RNA Ligase Mediated Rapid Amplification of cDNA Ends (RML-RACE) kit (Ambion) was used according to the instructions of the manufacturer's. Briefly 5 μg of Hela cell RNA or 2–5 μg of poly-A+ RNA from partially differentiated human G6 satellite cells  was treaSequencing was performed using ABI PRISM and the Dye Terminator cycle sequencing kit according to the manufacturer's directions (Perkin Elmer) and analysed by an automated ABI-310 fluorescent-dy sequencer (Applied Biosystems).. Open reading frame finding and all sequence analyses were done using the MacVector 6.5.3 sequence analysing software (Oxford Molecular Group). The BLAST  server of the National Center of Biotechnology Information (NCBI)  and The Mouse Genome Sequencing Consortium (MGSC) database  was used for sequence alignments. Signal peptide prediction was done using the SignalP v1.1 at WWW Prediction Server (Center for Biological Sequence Analysis, and membrane topology predictions at the HMMTOP server using the TopPred2 program [ and Matinspector [. Prediction of GPC islands and detection of repeats in sequences analysed were done with the GRAIL program [Exon-intron boundary predictions were done manually and using the Genscan web server at MIT  program . Transcrnspector  search p program .Table 1. Organization of the human GP210 gene including exon/intron sizes, splice acceptor consensus sequences and intron phases.Click here for filecis-acting elements of the human GP210 promoter. The start sites of the elements are indicated as 5' end of consensus sequence and relative to translation start as +1. The compilation was made using the TESS and MatInspector analyze programs. Results are restricted to human species and perfect match except for four WT1 binding sites indicated as a boxed nucleotide. Some abbreviations: EGR2, Early growth response gene 2; WT1, Wilms' tumor zinc finger protein 1; PuF, c-myc purine-binding transcription factor; IL-6. RE-BP, IL-6 Response element-Binding protein; NF-1, Nuclear factor 1; Ets-2, proto-oncoprotein; USF2, upstream stimulating factor.Table 2. Predicted Click here for file


Detection of Loss of Heterozygosity (LOH) is one of the most common molecular applications in the study of human diseases, in particular cancer. The technique is commonly used to examine whether a known tumour suppressor gene is inactivated or to map unknown tumour suppressor gene(s). However, with the increasing number of samples analysed using different software, no tool is currently available to integrate and facilitate the extensive and efficient data retrieval and analyses, such as correlation of LOH data with various clinical data sets.An algorithm to identify prognostic disease markers is devised and implemented as novel software called LDMAS. LDMAS is a software suite designed for data retrieval, management and integrated analysis of the clinico-pathological data and molecular results from independent databases. LDMAS is used in stratification of disease stages according to clinical stage or histological features and correlation of various clinico-pathological features with molecular findings to obtain relevant prognostic markers such as those used in predicting the outcome of cervical intraepithelial neoplasia (CIN). This approach lead to the identification of novel prognostic cervical cancer markers and extraction of useful clinical information such as correlation of Human Papilloma Virus (HPV) status with CIN lesions.® GeneChip Mapping arrays.A novel software called LDMAS is implemented and used to extract and identify prognostic disease markers. The software is used to successfully identify 4 novel prognostic markers that can be used to predict the outcome of CIN. LDMAS provides an essential platform for the extraction of useful information from large amount of data generated by LOH studies. LDMAS provides three unique and novel features for LOH analysis : (1) automatic extraction of relevant data from patient records and reports (2) correlation of LOH data with clinico-pathological data and (3) storage of complex data in flexible format. The first feature automates the creation of database of clinically relevant information from huge amount of data, the second feature extracts useful biomedical information such as prognostic markers in CIN and the third feature simplifies the statistical analyses of the data and allows non-statisticians to carry out the analysis. Additionally, LDMAS can be used to extract clinically useful markers from other diseases and interface to high throughput genotyping analysis software such as GDAS used to generate LOH data from Affymetrix Detection of LOH is one of the most common molecular applications in the study of human diseases, in particular cancer. It is commonly used to examine whether a known tumour suppressor gene is inactivated or to map unknown tumour suppressor gene(s). Detection of LOH not only helps in understanding the molecular mechanisms underlying the development of cancer, but also provides important information useful for disease diagnosis and prognosis. LOH detection is commonly carried out by the analysis of microsatellite markers using an automated DNA sequencer. With the raw data from the sequencer being stored in one file per lane together with corresponding clinical information and patient follow up data, each LOH study ,2 generaLDMAS package is composed of three modules:(1) MRES  which parses patient report files, extracts the information of interest and organises it into a structured format, applicable to LDAS(2) LDAS  which obtains LOH data from Genotyper  or GDAS  software and correlates it to clinical data obtained from MRES(3) LDMS (LOH Data Management Software) which is used to gather patients' clinico-pathological data and extract significant relationship between the various data sets® Windows 2000 or Windows XP operating system. Figure LDAS and LDMS work synergistically to manage and analyse LOH data. The MRES source code for automatic parsing of patient reports is written in C++ using C++ Builder 5.0 , LDAS is written in Visual Basic for Application as an Excel 2000 add-in and LDMS is written as Visual Basic for Application modules embedded within Access 2000 as fully functional software. These modules can be run independently and used for applications other than LOH. LDMAS runs on MicrosoftThe MRES module takes its input from any patient report file containing clinical details such as diagnosis, stage of the disease, treatment and follow up results, parses and formats patient's data into a structured format that can be saved as Excel spreadsheet. In this case, data were taken from SunQuest patient record system and MRES converted and produced the data as:(1) Hospital Number(2) Hospital ID(3) Patient Name(4) Date of Birth(5) Pathological specimen Number(6) Date of Diagnosis(7) Histological diagnosis.The user can manually check the data and use it as template for analysis. Data analysis is carried out using LDAS which obtains LOH data from Genotyper and correlates it to the clinical data obtained from MRES. LDAS obtains data in plain text format and can thus be easily interfaced to any LOH platform generating software such as Genotyper and GDAS. Finally all data are entered into LDMS for storage and intelligent mining of data. Database query results can be exported back to LDAS allowing correlation between LOH and various clinico-pathological parameters such as age, histological grade, treatment modality and their responses, and HPV status as well as carry out multivariate analysis to determine the sensitivity and specificity of the markers involved in the LOH study. A more detailed description of all the modules is provided in LDMAS user's guide [Additional file 1] and the example below.LDMAS offers several advantages to users. It is user friendly and its architecture is modular allowing versatility of use. It enforces the standardisation of procedures for studies involving large cohorts of individuals. The data is well organized since LDMAS systematically assigns LOH results of each case to its corresponding clinical information. Additionally, LDAS standardizes LOH data analysis implicitly and allows the user to edit the data manually if needed. Microsoft Excel has been chosen to implement LDAS because of its wide use, versatility and convenient statistical analysis features facilitating the implementation of multivariate analysis and correlation testing between LOH and clinico-pathological parameters.We divided the CIN groups into disease free indicating cases that become CIN free after treatment, and disease persistence/progression indicating cases that develop show progression or persistence of CIN despite treatment. We used LDMAS to retrospectively examine the prognostic value of LOH at 12 microsatellite markers including 10 from 3p14, 3p22-21, 6p21 and 11q23 which are frequently deleted in cervical cancer [Initially MRES was used to automatically parse 4300 patient records and extract clinico-pathological data including age, diagnosis, method of treatment and treatment response during follow up. Out of those, 164 cases with follow up of 3 or more years were chosen for the study and their clinico-pathological information was imported into LDAS. Initially, 71 out of the 164 selected cases were examined for LOH using 12 fluorescent microsatellite markers ran on ABI377 DNA Sequencer. LDAS was then used to identify the microsatellite markers for which LOH was significantly associated with disease persistence/progression of CIN using two tailed student t-test. Figure To validate this finding, LOH at these four markers was investigated in a further series of 93 cases. Compatible results were obtained from these additional cases.The two sets of data were combined and further compared using LDMS. Methodologies included :2 (chi-squared) test of LOH at each of the four microsatellite markers with age, various methods of treatment, different subtypes of HPV infection and between CINs showing disease free or disease persistence/progression.1) comparison using χ2) correlation of LOH data with histological grade of CIN, treatment response and various HPV subtypes.Through such complex analysis, we showed that concurrent LOH at two of the four microsatellite markers could identify 47% of CINs that showed disease persistence/progression with 100% specificity . FurtherBased on the above example, an algorithm can be developed to extract prognostic markers for other diseases. The algorithm can be summarised in the following pseudocode :(1) Divide the disease in groups according to the pathology staging(2) Parse patient data from clinical records and use the groups defined in part (1)(3) FOR each microsatellite markercarry out a two tailed student t-test between the disease groups using LOH dataIF t-test p ≤ 0.05Marker is significant in prognosis of the diseaseELSEMarker is not significant in prognosis of the disease2 (chi-squared) test of LOH with clinico-pathological data and correlation of LOH data with histological grade of CIN, treatment response and various HPV subtypes.(4) Validate the prognostic markers using χLDMAS has been implemented using the above pseudocode.® GeneChip Mapping arrays and fingerprinting studies. Modules such as MRES can be used independently to parse medical records facilitating extraction of specific clinical information of interest. Additionally, LDMAS can be used to extract clinically useful markers for other diseases.We have devised an effective algorithm to identify and extract useful markers that can be used to predict the outcome of disease and used the algorithm to successfully identify 4 novel prognostic markers that can be used to predict the outcome of CIN. The algorithm was implemented in a novel software called LDMAS which provides an essential platform for the extraction of useful information from large amount of data generated by LOH studies. Furthermore, LDMAS is used to efficiently store, manage and track the data. Its flexible nature allows the easy manipulation of data facilitating complex analysis as demonstrated in the current study. The various modules of LDMAS can be easily adapted and used with other applications such as high throughput LOH and genotyping using SNPs on AffymetrixThe source code and executable files for LDMAS modules as well as user manual including examples from real study data are freely available and can downloaded from our website at : Additionally examples of input files are provided from our website for users to test the software and assess its functionality.RH designed and developed and implemented LDMAS software and the web site. AE and RH did the experimental work to generate the data necessary to test and validate LDMAS. MD supervised the study and designed the experimental work using CIN biopsies and smears. All authors read and approved the final manuscript.


Little is understood about the conceptual relationship of depression and quality of life (QoL). Judgments concerning both, implicitly or explicitly, involve a time perspective. The aim of this study was to test de Leval's theoretical model linking depression and QoL with a time perspective. The model predicts that changes in cognitions about one's past, present and future QoL, will be associated with changes in depressive symptomatology.Eighteen psychiatric in-patients with a clinically confirmed diagnosis of depression were assessed on commencing treatment and 12 weeks later. QoL was assessed by the Schedule for Evaluation of Individual Quality of Life (SEIQoL), depression by the Beck Depression Inventory (BDI-II) and hopelessness by the Beck Hopelessness Scale (BHS). Time perspective was incorporated by asking QoL questions about the past, present and future.Depression and hopelessness were associated with a poorer present QoL. Depression lowered present QoL but did not alter future QoL, as these remained consistently high whether participants were depressed or recovering. However, depressed individuals had a larger gap between their actual present QoL and future (aspired to) QoL. Changes in QoL were influenced by depression and hopelessness. Contrary to the model, perception of "past" QoL was not affected by depression or hopelessness.de Leval's model was largely confirmed. Thus depression and hopelessness influence a person's present and future QoL. The analysis of a temporal horizon was helpful in understanding the link between depression and QoL. Assessment of quality of life (QoL) has become increasingly important in health care, particularly as an evaluative method to measure outcomes of the impact of disease and interventions. To date it is unclear how research on QoL relates to other psychological constructs such as depression and anxiety. Many clinical studies assess a number of related psychosocial dimensions but without a theoretical basis for the unique contribution of each. On an intuitive level, QoL and depression can appear as opposing phenomena – crudely representing all the positive and negative aspects of well-being. Poor QoL is sometimes seen as a consequence of depression -3. On thA theoretical approach developed by de Leval ,5,8 trieThe "three-time-dimension" theory describes the dislocated temporal horizon of the depressed patient. It situates both depression and quality of life as part of a continuum in time rather than as independent phenomena. De Leval proposes that, for the depressed individual, time passes slowly, the present is dissociated from the past and the potential for the future is lost or viewed with hopelessness . During In de Leval's theory, QoL is perceived as the gap between actual experience and future aspirations. Whereas depression is placed on the past-present timeline, QoL is placed along the timeline using present and future. QoL according to this model is defined as being "the appropriateness of future aspirations to the present" or "the making present of the future". The larger the gap, the lower the QoL of the individual Figure . AlthougIn a cross-sectional study, de Leval  examinedpresent ill-being' in the case of depression) and anticipated status (i.e 'therapeutic future') and was measured by the discrepancy between present and future actual QoL scores. The level of phenomenological depression – i.e. the gap between a person's perceived past and present "ill-being" was measured by the discrepancy between the past and present actual QoL scores. These gaps are referred to as time comparisons gaps. The full model is displayed in Figure The present study sought to advance the understanding about the conceptual relationship of depression and QoL by empirically testing de Leval's model in a longitudinal study. It also sought to assess the model using a previously developed individualised system for assessing QoL. If de Leval's proposals concerning the centrality of the temporal assessment are correct, then any QoL assessment instrument which measures the present can be adapted to measure the past and the future as aspired to. In this study, the model was operationalised as follows: scores on standardised depression measures were taken to demonstrate an individual's level of depression, i.e., 'present ill-being'. QoL was the gap between an individual's present status  from time 1 to time 2 predicts the change in the gap between actual and aspirational QoL scores.Hypothesis 3: The change in hopelessness scores from time 1 to time 2 predicts the change in the gap between Hypothesis 4: A reduction in depression narrows the gap between the past and present, and enhances QoL by narrowing the gap between present and future QoL.Consecutive psychiatric in-patients N = 27) were approached to participate in a longitudinal study of depression. Study inclusion criteria were age ≥ 18 years, diagnosis of moderate to severe uni-polar depression (DSM-IV-criteria), a moderate or severe depression rating on the Beck Depression Inventory II [ were appQoL was assessed by the Schedule for the Evaluation of Individual Quality of Life (SEIQoL) , a well-The final assessment step in SEIQoL involves quantifying the relative importance (weight) of each cue to the judgment of participants' overall QoL. This is obtained by using a weighting disk, consisting of five interlocking coloured disks that can be rotated around a central point to form a pie chart. These disks are labelled with the five nominated cues and are adjusted by participants until they are satisfied that the proportion of the pie chart displayed by each life area accurately reflects the relative importance they attach to these life areas. The SEIQoL Index score is calculated by multiplying each cue weight with the relevant level of functioning. These five scores then are summed. The scale of the SEIQoL Index score ranges from worst possible (0) to best possible (100). This was done for each time anchor and point.To measure the temporal nature of the gap in terms of perceptions of their actual and aspirational past, present and future QoL according to the Leval's theory and Calman's definition of QoL, participants were asked to rate the status of each nominated SEIQoL cue (i.e. how were you doing?) based on the following questions:Past:- Where were you before you got depressed? - Where would you have liked to have been? Present: - Where are you now? - Where would you like to be? Future:- Where do you expect to be in a year’s time? - Where would you like to be in a year’s time? Thus scores were taken for actual present, aspirational present, actual past, aspirational past and actual future, aspirational future.The Beck Depression Inventory (BDI), a 21 item self-report instrument for measuring the severity of depression in adults, was developed for the assessment of symptoms corresponding to criteria for diagnosing depressive disorders based on the DSM-IV. In this study an updated version is used (BDI-II) [The Hopelessness Scale  is a 20 Of 27 patients approached, 24 gave written consent and took part in the study (89% response rate). Over the three month follow-up period, six patients dropped out (75% follow-up rate). Information on the complete sample of 18 patients was analysed. Sociodemographic and clinical details are presented in table Baseline scores of the depression measure (BDI-II) showed high levels of severity, which were reduced significantly after three months  was significantly higher at baseline and three months than actual past and this did not change over time table . Actual pd1 = .468, rfd1 = .475, both p < .05, rpd2 = .815, rfd2 = .730, both p < .01). In addition, correlations between the QoL preference comparison gaps for present, past and future assessment and hopelessness (h) were statistically significant at baseline for the present (p) gap and at three months (2) for present (p) and future (f) gaps . There was no difference between actual past and actual future QoL scores at baseline and three months (p = .475, p = .528). This confirms hypothesis 1a.The gap between actual and aspirational QoL is presented in table Multiple regression was conducted to investigate whether depression and hopelessness contribute to the change in the size of the gap of actual and aspirational QoL over time. Changes in depression and hopelessness scores over time were entered as independent variables in a multiple regression, while the change in gap scores was entered as the dependent variable. A significant amount of variance was explained by changes in the present and future gaps for actual and aspirational QoL (52.4%, 59,9% respectively both p < 0.01). Change in depression contributed to change (reduction) in the gap between present actual and aspirational Qol (β =.676). Change in hopelessness (β =.589) was the significant contributing factor for change (reduction) in the gap between actual and aspirational future QoL . This partly disconfirms hypothesis 4To investigate how depression and hopelessness may influence the change in the size of the gaps proposed by de Leval, multiple regression analysis was conducted. Change in depression and hopelessness scores over time were entered as independent variables in a multiple regression, while the change in the gap over time, was entered as the dependent variable. Changes in the size of the two time comparison gaps  were not influenced by change in depression or hopelessness scores . Secondly, the time comparison gap was assessed, i.e. where patients see themselves now in comparison to the past, and in comparison to the future. Both approaches were carried out on the basis of de Leval's model linking depression to all three time dimensions.Our findings are consistent with previous results, showing that people use their current affective state as a basis for making judgments of how happy and satisfied they are with their lives. A depressed person will usually see his or her well-being, social functioning and living conditions as worse than they appear to an independent observer or to patients themselves after recovery – the so called "affective fallacy" . ClinicaOur findings suggest the larger the present preference comparison gap, the greater the depression. While the preference comparison gap of the past did not change over time with the decrease of depression, there was a trend for the future preference comparison gap that might become statistically significant with a larger sample size. However, aspirations were not affected by depression, since they remained the same for past, present and future from baseline in hospital to three months later. Overall these findings may indicate that a reduction in the present preference gap should be the main goal for therapeutic actions for depressed patients. An achievable target may be to keep the same preference comparison gap between and over time points to maintain a healthy homeostasis and a good QoL figure . InteresSuccessful treatment of depression, indicated by a significant change of BDI-II and BHS scores, was accompanied by a significant reduction in the gap between present actual and aspirational QoL. A reduction in the gap between actual present QoL with the aspirational QoL ("where one would like to be right here and now") reflects a more positive view of the present and is accompanied by significant improvements in QoL. Depression was consistently correlated with the time comparison gap between present and future QoL assessments. However, depression was more highly associated with the present and future preference comparison gap at three months. This suggests, that depression may be more highly associated with the actual/aspirational gap when people are less depressed. In contrast, hopelessness was consistently correlated with the preference comparison future gaps and only at baseline with the gap of present actual/aspirational QoL. The gap between the actual past and aspirational past QoL was not explained by either depression or hopelessness. Change in depression or hopelessness was important in reducing the preference comparison gap for present and future QoL assessments. Change in depression was important in reducing the size of the present gap. Change in hopelessness significantly contributed to reducing the size of the future gap. Given that present "ill-being" is seen as depression, while hopelessness is future orientated and directed, this finding makes sense. This has implications for therapy: to improve the QoL of patients with depression, relief from depression must be provided. Hopelessness needs to be tackled in addition to achieving a realistic future QoL perspective.To a large extend this study confirms de Leval's theory. The assessment of de Leval's model: present and future QoL  and past and present  showed interesting results. The theoretical time comparison gap between a "healthy past" and present "ill-being" was confirmed by the significant difference between patients' perceptions of actual present and future QoL, at baseline. Over time, following appropriate treatment, this time comparison gap should close and QoL should improve, if the treatment is considered to be successful. The significant reduction of the past-present gap over time, and the loss in significance in size of this gap three months later, further confirms de Leval's model. This reduction of the time comparison gap between the present "ill-being" and a "healthy past" resulted in an improvement in QoL. The reduction was achieved by a change in the actual view of the present, leaving the view of the past unchanged figure .According to de Leval's model, recovery from depression is achieved when the appraisal of present and past QoL are very close, i.e. a very narrow gap. This means that a reduction of the gap between present and future QoL should improve current QoL. Although this study showed a decrease in depression over time by standard measures of depression and the size of the time comparison gap was significantly reduced between the two time points, the size of the gap did remain significant between the present and future at three months follow up. This finding may either indicate that the patients were still depressed at three months, or this part of the model could not be confirmed figure . de LevaSince we tested the model in a small and specialised sample, the findings are not generalisable. However, our study did test de Leval's model in a clinically depressed sample which is the focus of the most likely benefit of understanding the interplay of the two phenomena of depression and quality of life. It is also a challenging group to recruit, engage with and maintain in a research project.This study showed that depression influenced individual QoL by lowering the person's actual QoL. Thus depression was associated with a larger gap between current reality  and patient aspirations and realistic expectations. Aspirations for past, present and future QoL remained the same over time. However, patients actual appraisal of their present QoL improved with successful treatment of depression, reflected by a closure of the present preference gap.de Leval's model was largely confirmed. Thus depression and hopelessness influence a person's present and future QoL. The analysis of a temporal horizon was very helpful in understanding the link between depression and QoL. Therapeutic interventions can be considered as closing the gap between a person's present QoL and their present aspirations and realistic expectations for the future. This could be achieved, according to the data presented here, by changing the view of the present and not necessarily by changing the level of expected future QoL. Knowing the persons aspirational QoL level may be a helpful guide for the therapist in that it provides important information about a depressed person's position in the progress towards what he or she considers to be recovery.Margaret Moore contributed to conception and design, acquisition, analysis and interpretation of the data and revised the article. Stefan Höfer drafted the article and contributed to the analysis and interpretation of the data. Hannah McGee contributed to the conception and design and interpretation of the data and revised the article. Lena Ring contributed to the interpretation and revised the article.All authors have given final approval of the version to be published


Plexiform neurofibromas (PNF) are benign tumors of the peripheral nerve which mostly develop in patients with neurofibromatosis type 1 (NF1). Surgical interventions are usually not applied to children with small tumors. These are rather restricted to debulking of larger tumors in adults that cause clinical complications or aesthetic disfigurement. In most cases, a total resection of PNF is not possible due to the network-like growth of the tumors.Early surgical intervention was carried out for 9 small PNFs in 7 NF1 children. Tumor resection was performed following the graphical delineation of the affected skin and according the MRI findings.Total resection was achieved for all 9 PNF without causing any neurological or organic deficit. Annual magnetic resonance tomography over a period of four years did not reveal any relapse of the tumors.Early surgical intervention for small superficial PNFs in NF1 children have various advantages and may especially be considered a strategy to prevent progression. NF1 on 17q [Plexiform neurofibromas (PNF) are benign tumors originating from nerve sheath cells, subcutaneous, or visceral peripheral nerves and can involve multiple fascicles . PNF occ1 on 17q -5. At le1 on 17q ,6,7, whi1 on 17q -10.Previously we reported that PNF can be distinguished into three growth categories using magnetic resonance imaging (MRI): superficial, displacing and invasive. Superficial PNF arise from subcutaneous or cutaneous nerves and may remain within the upper layer of the skin – usually not involving major nerves . SubtotaIn this study, we present the results of early intervention for 9 PNFs in 7 NF1 children aged 3 to 15 years.The 7 children were examined in our NF-Clinic in the Department of Maxillofacial Surgery, University Hospital Eppendorf, Hamburg. Diagnosis of NF1 was based on the NIH criteria . InformeSeven NF1 children, five boys and two girls, aged 3 to 15 years were included in this study. All of them met the NIH diagnostic criteria for NF1 . PNF wasIn 2001, all seven children underwent surgical intervention for their nine PNF. During the mobilization of the underlying skin layers, three patients showed enlarged nerves running from subcutis to superficial muscle layers, which were identified and additionally resected. These enlarged nerves were proven to be PNF in all cases. Primary wound closure was achieved following the mobilization of the marginal skin. Healing followed without complications in all cases. The scars are relatively small and no hypertrophy was observed. The intervention was well tolerated by all children and recovery occurred within a few days.Postoperative MRI was done for three children. Six children underwent follow-up examinations annually by MRI for three years. One child was only examined in 2003, two years after the operation. MRI revealed successful total resection for all 9 tumors Figs. , 5, 6, 7In this study we demonstrated that small superficial PNF can be completely resected, leaving scars without hypertrophy. Operation is relatively uncomplicated, allows healing by primary intention, requires only a few days of hospitalization and is thus no burden for even the youngsters. Within the clinical and radiological follow-up period of three years there was no re-growth of the tumors. However, it cannot be excluded that all tumor cells have been resected. Therefore those patients warrant further annual clinical examination.The scars in some children may seem more obvious and disfiguring after the operation. However, in comparison to the complications the tumors may cause later, they are only of minor concern. Furthermore, these scars may be corrected later surgically.Without resection, these small PNF are likely to grow continuously to a large or even very large size which often causes aesthetic disfigurement, functional deficits and pain. Resection of large tumors is much more difficult and total resection is usually not possible any more. Currently it is not clarified whether or not growth patterns of PNF might possibly change from superficial to displacing or to invasive types over time. Early surgical intervention of small superficial PNF may thus be considered as a preventive strategy for later disfigurement and functional deficits.Adequate diagnosis of NF1 and thus small PNF in the pediatric age group is challenging. Children usually do not complain about subtle pain or discomfort in the affected skin region. Even in the presence of some symptoms parents are often not aware of the origin of the tumor. Only small PNF of the face and neck causing disfigurement in children tend to be recognized early. On the other hand, small PNF of the trunk and extremities which present with subtle hyperpigmentation, hair excess and palpable tumor tissue are frequently not noticed by the physicians. These tumors are rarely diagnosed correctly as PNF. In our cohort only one child received the diagnosis of a PNF correctly by the referring family physician. For better management and adequate treatment, efforts should be made to diagnose NF1 and PNF in their early stage.Early surgical intervention of small superficial PNFs is uncomplicated, without burden for even the youngsters and enables total resection of the tumors. It may be considered as a preventive strategy for later disfigurement and functional deficits.The authors declare that they have no competing interests.REF and RS carried out the surgical intervention for the tumors and post-operational care of the patients.CF carried out the MRI and was involved in the diagnosis of PNF.MH and VFM did the diagnosis and were responsible for management and consulting of the patients.REF and VFM designed the study and prepared the manuscript.All authors read and approved the final version of the manuscript.


Microbes are arguably the most adaptable organisms on Earth, inhabiting nearly every crevice of nearly every corner of the globe. Some invade the cavities of a wide variety of insects and other invertebrates while others colonize the skin, blood, eyes, and internal organs of animals. Still others thrive in such inhospitable places as the hydrothermal vents of the ocean floor and the Dry Valleys of Antarctica. These “simple” single-celled organisms have evolved unique molecules and strategies over some 3.5 billion years that suit life on the edge. With the sequenced genomes of nearly 140 microbial species in hand, scientists are gaining valuable insights into the nature of this adaptive diversity.Adapting to such radically different niches, it appears, has produced genes with diverse functions that evolve at very different rates. Genes that code for molecules essential for fundamental cellular functions like maintaining cell metabolism and structure tend to evolve rather slowly, while genes that make proteins charged with mediating cellular responses to internal or external changes often evolve relatively quickly. Pathogenic microbes in particular rely on a flexible genome to keep a step ahead of their hosts' similarly evolving defenses in the never-ending struggle to gain adaptive advantage. This adaptability underlies the increasing antibiotic resistance of diseases like tuberculosis, as selective pressures favor the expansion of resistant bacterial populations.Combating such problems requires a molecular understanding of bacterial infections, yet function has been ascribed to only a fraction of the genes found in microbial genomes. One approach to improve functional analyses of genome sequences combines bioinformatics with experimental methods. With such collaborations in mind, Yu Zheng, Richard Roberts, and Simon Kasif have developed a computational approach to help filter out the genetic noise and home in on genomic regions likely to contain clues to gene function. Their method relies on a novel way of classifying genes that flags sequences likely to reward biochemical and genetic efforts to analyze gene function.Many comparative genomic studies have focused on looking for sequence “motifs” that correlate with well-characterized protein sequences (that is, the amino acid sequence) and predicting function based on their similarity to the known protein sequences. Zheng, Roberts, and Kasif took a different approach, classifying genes based on their sequence variation. The researchers analyzed 43 fully sequenced microbial genomes and, after determining the degree of conservation or divergence among similar genes in different species, divided the genes into three broad categories: rapidly evolving genes unique to a particular species; highly conserved genes; and “segmentally variable,” or mosaic, genes. Stipulating that the boundaries between the categories are somewhat blurred, Zheng et al. define segmentally variable genes as regions that show a mosaic pattern of one or more rapidly evolving, variable regions interspersed with conserved regions. Based on evidence suggesting that retained variable regions tend to serve a function, the researchers predicted that these mosaic genes, with their highly variable, fast-evolving regions, would shed light on the forces that shape genome diversity and adaptation.For most of the microbes analyzed, mosaic genes accounted for about 8–20% of their genomes. Selecting several large families of mosaic genes, Zheng et al. explored the relationship between genes with known function and the structure of their variable regions. Noting an overabundance of particular functional categories in different species—such as signaling proteins that come into either direct or indirect contact with the cell's environment—the researchers speculate that the variable regions may constitute an adaptive layer for the microbe, as they not only “play a key role in mediating interactions with other molecules” but also support a microbe's ability to adapt to its particular niche. Several bacteria species, for example, contain roughly 40% more mosaic sensor genes involved in cell motility, which the authors attribute to the microbes' “expanded ability to detect different chemical signals and find favorable environments.”This regional variability appears to reflect the influence of selective pressures that fuel diversity through ongoing interactions with other rapidly evolving molecules in the environment, adding another source of genetic adaptability as cells adjust to new environments and outmaneuver pathogenic threats. While many of the mosaic genes identified encode proteins involved in host-pathogen interactions, defense mechanisms, and intracellular responses to external changes, their function is only broadly understood. While Zheng et al. cannot say to what extent variability affects function—Is extreme variability required for diversity or can modest variation suffice?—they are refining their classification of segmentally variable genes to address such questions. Until then, the authors' “mosaic” approach to understanding gene function promises to improve efforts to annotate the volumes of sequenced genomes on hand, offering biologists a much-needed tool to sift through the mountains of genomic datasets and identify promising targets for further study.


Obesity, the most common cause of insulin resistance, is increasingly recognized as a low-grade inflammatory state. Adipocyte-derived resistin is a circulating protein implicated in insulin resistance in rodents, but the role of human resistin is uncertain because it is produced largely by macrophages.The effect of endotoxin and cytokines on resistin gene and protein expression was studied in human primary blood monocytes differentiated into macrophages and in healthy human participants.Inflammatory endotoxin induced resistin in primary human macrophages via a cascade involving the secretion of inflammatory cytokines that circulate at increased levels in individuals with obesity. Induction of resistin was attenuated by drugs with dual insulin-sensitizing and anti-inflammatory properties that converge on NF-κB. In human study participants, experimental endotoxemia, which produces an insulin-resistant state, causes a dramatic rise in circulating resistin levels. Moreover, in patients with type 2 diabetes, serum resistin levels are correlated with levels of soluble tumor necrosis factor α receptor, an inflammatory marker linked to obesity, insulin resistance, and atherosclerosis.Inflammation is a hyperresistinemic state in humans, and cytokine induction of resistin may contribute to insulin resistance in endotoxemia, obesity, and other inflammatory states. Inflammatory stimuli affect resistin expression in human macrophages and raise serum resistin levels in healthy volunteers Dietary and lifestyle changes during the last century have entailed an unprecedented epidemic of obesity and associated metabolic diseases, including type 2 diabetes and atherosclerosis . Many inWe originally identified and characterized resistin as a circulating mouse adipocyte gene product that is regulated by antidiabetic drugs . In rodeHere we show that the endotoxin lipopolysaccharide (LPS), a potent inflammatory stimulant, dramatically increases resistin production by inducing secretion of inflammatory cytokines such as TNFα. This increase in resistin production is blocked by both aspirin and rosiglitazone, drugs that have dual anti-inflammatory and insulin-sensitizing actions and have been shown to antagonize NF-κB. Indeed, activation of NF-κB is sufficient to induce resistin expression, and loss of NF-κB function abolishes LPS induction of resistin. Resistin serum levels are increased dramatically by endotoxemia in humans, and correlate with a marker of inflammation in patients with type 2 diabetes. Thus, systemic inflammation leads to increased resistin production and circulating levels in humans. The increased level of resistin in humans with obesity is likely an indirect result of elevated levels of inflammatory cytokines characteristic of states of increased adiposity. Hence, obesity and acute inflammation are both hyperresistinemic states associated with insulin resistance.6 cells per well, allowed to adhere for 4 h, then washed with Dulbecco's Modified Eagles Medium and further cultured in 10% FBS in Dulbecco's Modified Eagles Medium supplemented with 5 ng/ml GM-CSF (Sigma, St. Louis, Missouri, United States) to promote macrophage differentiation. All experiments were performed after overnight equilibration with macrophage serum-free medium  supplemented with 5 ng/ml GM-CSF. Cells were treated with LPS (Sigma), aspirin (Sigma), SN50, and/or control peptide (Biomol, Plymouth Meeting, Pennsylvania, United States), MG132, PD98059, SB20358 , and TNFα (R&D Systems, Minneapolis, Minnesota, United States). Neutralizing antibodies to TNFα, IL-6, and anti-IL-1β, as well as control IgG, were obtained from R&D Systems. Adenovirus expressing activated IKK in pAD easy with GFP and control vector was a generous gift from Steven Shoelson.Peripheral blood mononuclear cells were isolated from whole blood of healthy donors following apheresis and elutriation. Greater than 90% of these monocytes expressed CD14 and HLA-DR. Cells were plated in 24-well plates at a density of 10RNA was isolated using RNeasy Mini Kit , then subjected to DNase digestion followed by reverse transcription (Invitrogen). mRNA transcripts were quantified by the dual-labeled fluorogenic probe method for real-time PCR, using a Prism 7900 thermal cycler and sequence detector . Real-time PCR was performed using Taqman Universal Polymerase Master Mix (Applied Biosystems). The primers and probes used in the real-time PCR were the following: Sense-Resistin, 5′-AGCCATCAATGATAGGATCCA-3′; Antisense-Resistin, 5′-TCCAGGCCAATGCTGCTTAT-3′; Resistin Probe, 5′-Fam-AGGTCGCCGGCTCCCTAATATTTAGGG-TAMRA-3′; Sense human 36B4 sense, 5′-TCGTGGAAGTGACATCGTCTTT-3′; Antisense 36B4, 5′-CTGTCTTCCCTGGGCATCA-3′; and 36B4 Probe, 5′-FAM-TGGCAATCCCTGACGCACCG-TAMRA-3′.−deltadeltaCT, in which deltadeltaCT equals the deltaCT of the compound-treated group minus the deltaCT of the chosen control group, which was normalized to 1.Primer and probe for TNFα were obtained from Applied Biosystems. The cycle number at which the transcripts of the gene of interest were detectable (CT) was normalized to the cycle number of 36B4 detection, referred to as deltaCT. The fold change in expression of the gene of interest in the compound-treated group relative to that in the vehicle-treated group was expressed as 2p < 0.001), except that the Biovendor values were approximately 30% lower than those determined with the Linco assay. This appeared to be related to the standards used for calibration. Discrepant absolute values among different assays, including the Biovendor assay, were recently described by others [Resistin concentrations, in cell media and human plasma, were assessed with a commercially available ELISA (Linco Research, St. Charles, Missouri, United States) and normalized to cell protein. The average correlation coefficient for standards using a four-parameter fit was 0.99. Intra-assay and inter-assay coefficients of variance were 4.7% and 9.1%, respectively. Direct comparison of standard curves generated by the Linco kit with those yielded by another commercially available resistin ELISA (Biovendor Laboratory Medicine, Brno, Czech Republic) yielded high correlation , aged 18–45 y with BMI between 20 and 30 and on no medications, were studied. The University of Pennsylvania Institutional Review Board approved the study protocol, and all participants gave written informed consent. Following screening and exclusion of individuals with any clinical or laboratory abnormalities, participants were admitted to the General Clinical Research Center at the University of Pennsylvania for a 60 h stay. Serial blood samples were collected during the 24 h prior to and 24 h following the intravenous administration of human-research-grade endotoxin  at a dose of 3 ng/kg given at 6 AM. Plasma and whole blood RNA (PAX tube isolators, Qiagen) samples were isolated from blood, and stored under appropriate conditions for subsequent assays.Healthy volunteers , aged 35–75 y and free from clinical cardiovascular diseases, were recruited through the diabetes clinics at the University of Pennsylvania Medical Center and the Veterans Affairs Medical Center, Philadelphia, Pennsylvania, to an ongoing study of cardiovascular risk factors in type 2 diabetes. The sample was composed of 59% Caucasians and 35% African-Americans. All participants were evaluated at the University of Pennsylvania General Clinical Research Center in a fasting state at 8 AM. The University of Pennsylvania Institutional Review Board approved the study protocol, and all participants gave written informed consent. The patient population is described in more detail elsewhere [Participants with type 2 diabetes  for continuous variables. Because of baseline variation in cell populations between batches of primary human monocytes isolated from multiple donors, cell culture experiments were performed in triplicate and data from representative experiments are presented. For cell culture experiments with multiple treatments, analysis of variance (ANOVA) was used to test for differences in means across treatment groups. When significant global differences were found, post hoc The regulation of resistin expression was studied in primary cultures of human monocytic cells. Immediately upon plating of elutriated primary human monocytes, resistin gene expression was detectable but highly variable from experiment to experiment (data not shown). One day after plating, resistin gene expression remained detectable at low levels A. SubjecInduction of resistin gene expression by LPS exposure of human macrophages began between 6 and 24 h after treatment, with peak expression at 24 h A. This tResistin gene expression was also induced by TNFα treatment of primary human macrophages A 28], a, a28], aMouse resistin, produced exclusively by adipocytes, is down-regulated by antidiabetic thiazolidinediones, including rosiglitazone . ConsistNext, we asked whether our findings from ex vivo studies of human macrophages would translate into in vivo observations in humans. Six healthy volunteers were injected with LPS, using a protocol similar to that shown to produce insulin resistance . BaselinPatients with type 2 diabetes and insulin resistance, many of whom are obese, have elevated levels of several inflammatory markers, including IL-6, TNFα, and sTNFR2 . LPS admWe have demonstrated that, in human macrophages, an inflammatory cascade with secretion of cytokines, including TNFα and IL-6, is sufficient and necessary for the induction of resistin. Insulin sensitizers that have anti-inflammatory properties, including a synthetic PPARγ agonist as well as aspirin, suppress macrophage resistin expression, as does direct inhibition of NF-κB. Experimental endotoxemia in healthy volunteers, based on the well-established gram-negative bacterial inflammatory response in humans ,39,40, iIn rodents, resistin is produced exclusively by adipocytes, regulates normal glucose homeostasis, and causes insulin resistance at high circulating levels ,13. TranR = 0.31, p < 0.001) was found in a cohort of 879 non-diabetic individuals, in whom resistin levels independently correlated with coronary atherosclerotic disease (M. P. Reilly, M. Lehrke, M. L. Wolfe, A. Rohatgi, M. A. Lazar, and D. J. Rader, unpublished data).Clearly the relationship between obesity, inflammation, and resistin expression is complex, and needs to be systematically studied in larger and varied patient populations. Intriguingly, we found a strong correlation between plasma levels of resistin and sTNFR2, the soluble cleavage product of the activated TNFα receptor, in diabetic patients. A comparable correlation between resistin and sTNFR2  are linked to diabetes, one possibility is that obesity in humans, by being similar to inflammation, causes immune cells to make lots of resistin and hence promotes diabetes that way.More research is necessary to confirm these findings and to find out how important resistin is as a link between obesity and diabetes, and how resistin promotes diabetes.http://www.niddk.nih.gov/health/nutrit/pubs/unders.htmUnited States National Institute of Diabetes, Digestive, and Kidney Diseases (NIDDK) information on obesity: http://diabetes.niddk.nih.gov/NIDDK information on diabetes: http://www.idf.org/International Diabetes Federation:


Quercetin and quercetin glycosides are widely consumed flavonoids found in many fruits and vegetables. These compounds have a wide range of potential health benefits, and understanding the bioavailability of flavonoids from foods is becoming increasingly important.This study combined an in vitro digestion, a lactase treatment and the Caco-2 cell model to examine quercetin and quercetin glucoside uptake from shallot and apple homogenates.p < 0.05), but had no significant effect on quercetin-3-glucoside recovery (p > 0.05). Digestion increased the Caco-2 cell uptake of shallot quercetin-4'-glucoside by 2-fold when compared to the non-digested shallot. Despite the loss of quercetin from the digested shallot, the bioavailability of quercetin aglycone to the Caco-2 cells was the same in both the digested and non-digested shallot. Treatment with lactase increased quercetin recovery from the shallot digestate nearly 10-fold and decreased quercetin-4'-glucoside recovery by more than 100-fold (p < 0.05), but had no effect on quercetin recovery from apple digestates. Lactase treatment also increased shallot quercetin bioavailability to the Caco-2 cells approximately 14-fold, and decreased shallot quercetin-4'-glucoside bioavailability 23-fold (p < 0.05). These Caco-2 cells had lactase activity similar to that expressed by a lactose intolerant human.The in vitro digestion alone significantly decreased quercetin aglycone recovery from the shallot digestate (The increase in quercetin uptake following treatment with lactase suggests that dietary supplementation with lactase may increase quercetin bioavailability in lactose intolerant humans. Combining the digestion, the lactase treatment and the Caco-2 cell culture model may provide a reliable in vitro model for examining flavonoid glucoside bioavailability from foods. Cardiovascular disease and cancer are the two most common causes of death in the United States and most industrialized nations. A diet high in fruits and vegetables has been correlated with a reduced risk for both cancer and heart disease ,2. It isA phytochemical of particular interest is quercetin, a strong antioxidant that is widely consumed in many fruits and vegetables. Quercetin has potential protective effects against both cancer and heart disease. Briefly, quercetin has been found to down regulate expression of mutant p53 in breast cancer cells, arrest human leukemic T-cells in G1, inhibit tyrosine kinase, and inhibit heat shock proteins . QuercetMore work is needed to understand the bioavailability of quercetin and other flavonoids from foods. The Caco-2 cell culture model is a well-established in vitro technique, extensively used to study intestinal cell absorption of compounds such as pharmaceuticals and nutrients; it is an excellent in vitro tool to study bioavailability of specific compounds. We have previously used the Caco-2 cell culture model to examine the uptake of quercetin from apple and onion extracts . Using tin vitro digestion has been paired successfully with the Caco-2 cell culture model to study iron and carotenoid bioavailability [in vitro digestion with the Caco-2 cell culture model could be quite useful in more specifically analyzing quercetin bioavailability from foods. At this time there is little information available describing the effects of digestion on flavonoids from foods. Vallejo et al. [To further understand quercetin bioavailability, it is important to examine the effects of digestion on foods prior to intestinal uptake. An lability ,24. Use o et al.  found tho et al. .in vitro model would aid in evaluating bioavailability of phytochemicals from foods by offering a simple method to screen for factors that may affect intestinal absorption of quercetin and quercetin glucosides, such as the food matrix, food processing, digestion, and interactions with other foods. Human and animal models can be expensive and time consuming, while a cell culture model allows for rapid, inexpensive screenings. The Caco-2 model has the potential to be a good model to measure quercetin absorption, however there are some drawbacks. Caco-2 cells have been shown to express significantly less lactase phlorizin hydrolase (LPH) than the average human small intestine [A good ntestine . Since tntestine , this deCaco-2 cells used in our lab have expressed greater LPH activity (3 mU/mg protein) than other Caco-2 cells (0.3 mU/mg protein) , resultiThe objectives of this study were (1) to develop an optimized in vitro digestion method for examining quercetin and quercetin glucoside recovery from digestates using onions and apples; (2) to examine the effect of lactase on shallot digestates; and (3) to examine Caco-2 cellular uptake of quercetin and quercetin glucosides from digested and lactase treated shallot.Kluyveromyces lactis, activity of 3000 units/mL), quercetin, and quercetin-4'-glucoside were purchased from Sigma Chemical Company (St. Louis, MO). Quercetin-3-glucoside was purchased from Indofine Chemical Company, Inc (Hillsborough, NJ). Caco-2 cells were obtained from the American Type Culture Collection (Rockville, MD) and were cultured in Dulbecco's Modified Eagle Medium  supplemented with 5% fetal bovine serum (Gibco Life Technologies, NY), 10 mM HEPES, 50 units/mL penicillin, 50 μg/mL streptomycin, and 100 μg/mL gentamicin, and were maintained at 37°C in 5% CO2.Shallots and onions (Northern Yellow) were obtained from a local grocery store. Apples (Red Delicious and Cortland varieties) were obtained from the Cornell Orchards (Cornell University, Ithaca, NY). Porcine pepsin, bile extract, pancreatin, lactase  using a Waring blender, and then homogenized using a Virtis 45 homogenizer. The total homogenates were aliquotted in 15 mL centrifuge tubes and stored at -20°C until use.3. Porcine bile extract and pancreatin were added to a final concentration of 1.1 and 0.175 mg/mL, respectively. The pH was increased to 6.5 by drop-wise addition of 1M NaHCO3, and the samples were incubated for 1 hour in a water bath at 37°C. Following digestion the pH was decreased to 2 by addition of HCl and the digestates were stored at -80°C for further analysis.For the digestion treatment, 2 g aliquots of the food sample were placed in a centrifuge tube with an equal amount of saline. The pH was decreased to 2.0 by drop-wise addition of 1M HCl, and porcine pepsin was added to a final concentration of 1.3 mg/mL. The digestate was incubated in a shaking water bath at 37°C for 30 minutes. The pH of the digestate was then increased to 5.8 with the drop-wise addition of 1M NaHCOTo examine and optimize the effects of digestion time and pH on the recovery of compounds from the digestate, the above parameters were varied. To examine the effects of pepsin digestion time, the pepsin digestates were incubated for 0, 30, 60 or 90 minutes and then incubated with the intestinal digestion enzymes for 60 minutes. To examine the effects of intestinal digestion, the samples were incubated with pepsin for 30 minutes, then incubated with pancreatin and bile for 0, 30, 60, or 90 minutes.The effects of 100 μM ascorbic acid and a nitrogen environment on quercetin and quercetin glucoside recovery from onion and apple digestates were examined. Following homogenization and prior to digestion, the food samples were mixed 1:1 with saline containing 200 μM ascorbic acid, leaving a final sample concentration of 100 μM ascorbic acid. During the digestion procedure described above, the samples were flushed constantly with nitrogen.The effect of pH of either 6.5 or 7.0 during intestinal digestion on quercetin and quercetin-3-glucoside recovery was compared. The effect of the storage pH was examined by comparing digested samples having either a final pH of 2.0 or 6.5. The effect of storage pH on 20 μM pure quercetin and 20 μM quercetin-3-glucoside was examined by comparing recoveries from samples stored at pH = 2.0, 3.5, 5.0 or 7.0. All samples were stored overnight at -80°C. Prior to HPLC analysis samples were thawed and extracted 4 times with acidified ethyl acetate (pH 2.0), evaporated to dryness and reconstituted in 2 mL acidified methanol (pH 2.0).Doses of lactase (0.5 units to 3000 units per gram sample) were applied to 1 gram shallot extract and incubated for 15 minutes at 37°C. The final pH was brought to 2.0 and the samples were stored at -80°C. The time kinetics were examined by incubating 1 gram shallot extract with 100 units of lactase for 0, 15, 30, 60, 90, 120, 240, and 720 minutes.The effect of both lactase and digestion were examined by digesting the samples with pepsin and pancreatin as described above, then incubating the samples with 100 units of lactase for 30 minutes at 37°C. Samples were stored overnight at -80°C.Prior to HPLC analysis, all digestate samples were thawed and extracted 4 times with acidified ethyl acetate (pH 2.0), evaporated to dryness and reconstituted in 2 mL methanol.5 cells per well in a collagen coated 6-well, flat bottom plate and incubated at 37°C in a 5% CO2 environment. Caco-2 cells were used between passages #10–25, and the cells reached confluence approximately 5 days post seeding. Culture media was changed three times a week. On day 14 post seeding, the DMEM was removed and the cells were rinsed three times with phosphate buffered saline (PBS).Caco-2 cells were seeded at a density of 5 × 10Shallot homogenates were digested as previously described and were placed directly on the 14 day old Caco-2 cells, or the samples were diluted 1:2 or 1:4 in HBSS . Cells were also incubated with non-digested shallot homogenates for comparison. For each treatment, two wells of cells were used for each sample, and each treatment was repeated in triplicate.2. The shallot treatment and HBSS was removed and the cells were rinsed three times with 20% methanol in PBS. Cells were scraped in acidified methanol (pH = 2.0) and the wells were rinsed three times with methanol. The scraped cells were sonicated for 15 minutes, centrifuged at 1600 g for 5 minutes, and the methanol supernatant was collected. The cells were rinsed three more times with methanol, the supernatants were collected and the methanol extracts were evaporated to dryness under nitrogen and reconstituted in 400 μl acidified methanol for HPLC analysis.To examine the effect of lactase on quercetin uptake from shallots, shallots were digested then incubated with lactase  for 20 minutes at 37°C. The digested shallot homogenates and the digested plus lactase treated shallot homogenates were diluted 1:2 in HBSS and placed on the cells. In all experiments, Caco-2 cells were incubated with treatment for 30 minutes at 37°C in 5% CO5 cells per well in a 6-well flat bottom plate. The cells were cultured in DMEM spiked with different doses of either lactose (10, 50, 100, 500, and 1000 μM), or forskolin (1, 10, 50, 100, and 200 μM). Media was changed every two days, and cells were harvested and lactase activity was measured at 14 days post-seeding. Lactase activity of Caco-2 cells was measured using a method adapted from Dahlqvist [Caco-2 cells were seeded at a density of 5 × 10ahlqvist . Cells wahlqvist . The resQuercetin and quercetin-3-glucoside content of untreated homogenates, digestates, and Caco-2 cell extracts were determined using an RP-HPLC procedure with a Supelcosil LC-18-DB column (150 mm × 4.6 mm, and 3 μm pore size). Waters 515 HPLC pumps (Waters Corp., Milford, MA) and a Waters 2487 dual wavelength absorbance detector (Waters Corp., Milford, MA) set at 370 nm were used for all HPLC analysis. Quercetin, quercetin-3-glucoside, and quercetin-4'-glucoside were used as standards. For the analysis of quercetin, quercetin-3-glucoside, and quercetin-4'-glucoside in the apple peel extracts, shallot extracts, and digestate extracts, the solvent system used was (A) acidified water  and (B) acetonitrile. The gradient method was the following: 0.0 min, flow rate = 1.4, (A) 90% and (B) 10%; 53 min, flow rate = 1.5, (A) 80% and (B) 20%; 58 min, flow rate = 1.7, (A) 65% and (B) 35%; 64 min, flow rate = 1.4, (A) 90% and (B) 10%. Twenty μL injections were made for each sample. Quercetin, quercetin-3-glucoside, and quercetin-4'-glucoside concentrations in the apple peel extracts, shallot extracts, and in the digestates were extrapolated from the pure quercetin and quercetin-3-glucoside standard curves.All data were reported as means ± SD for three replicates of each treatment. An analysis of variance (ANOVA) was used to compare results between treatment groups, and pairwise multiple comparisons were performed using Fisher's LSD with an individual error rate of 0.05. The statistical analysis was completed using Minitab Release 12 software (State College, PA).The total pepsin digestion time and pancreatin/bile digestion time had little to no effect on recovery of both quercetin and quercetin-3-glucoside from apple and onion homogenates when treated for up to 60 minutes (data not shown). After 90 minutes of pepsin digestion and 90 minutes of pancreatic digestion, quercetin and quercetin 3-glucoside in both the apple and the onion decreased slightly. Based on these results we chose to use a 30-minute pepsin digestion and a 60-minute pancreatin/bile digestion. In past studies, quercetin from onion and quercetin from quercetin-4-glucoside supplements reached the plasma in less than an hour following consumption by human volunteers . TherefoThe presence of ascorbic acid and nitrogen had no effect on quercetin or quercetin-3-glucoside recoveries from the digestates (data not shown). Quercetin and quercetin-3-glucoside recoveries from digested samples treated with ascorbic acid, nitrogen or both ascorbic acid and nitrogen were not different from the recoveries from the untreated digested samples.p > 0.05). However, following overnight storage at -80°C, the samples stored at pH 2.0 had significantly greater quercetin and quercetin-3-glucoside recoveries than samples stored at pH 6.5 and 7.0 (p < 0.05). Pure quercetin and quercetin-3-glucoside were also more stable at lower storage pH following digestion . Quercetin-3-glucoside recoveries from the digested apple, onion, and pure compound were 87.7, 89.5, and 86.4%, respectively. Quercetin recovery was significantly reduced in digested pure quercetin and digested onion samples, when compared to non-digested samples (p < 0.05). Quercetin recovery was lower than quercetin-3-glucoside recovery and tended to vary more, depending on the food matrix: 52.5% and 74.3%, from the onion and pure compound, respectively. There was no significant difference in quercetin recovery between to the non-digested and digested apple homogenates. There was only trace or no quercetin in non-digested apple homogenates, so the appearance of any quercetin following digestion resulted in a net increase.Based on these results, optimal digestion conditions were decided to be: pepsin digestion at pH 2.0 for 30 minutes, pancreatin/bile digestion at pH 6.5 for 60 minutes, and a final storage at pH 2.0. Ascorbic acid and nitrogen treatments were not continued. Using these conditions, the effect of digestion on quercetin and quercetin-3-glucoside recoveries from apples, onions, and pure quercetin and quercetin-3-glucoside was examined . Caco-2 cells treated with shallot homogenate absorbed approximately 2.9 ± 0.65 nmol of quercetin-4-glucoside, and Caco-2 cells treated with digested shallot absorbed 5.4 ± 0.04 nmol. Quercetin aglycone recovery from the digested shallot extract was only 47% that of the non-digested homogenate . Caco-2 cells absorbed 2.8 ± 0.4 nmol and 2.7 ± 0.2 nmol quercetin from the non-digested and digested shallot homogenates, respectively. Absorption of both quercetin-4-glucoside and quercetin from digested shallot followed a dose response. The Caco-2 cells absorbed quercetin 4'-glucoside and quercetin incrementally less from the digested samples that were diluted 1:2 or 1:4 in HBSS.Quercetin-4'-glucoside and quercetin were absorbed by Caco-2 cells following treatment with both digested shallot and non-digested shallot homogenates Figure . Quercete Figure  insert, Addition of lactose and forskolin, a specific inducer of lactase , to Cacop < 0.05; Figure p > 0.05).Treatment with 100 units lactase/g sample had a significant effect on both quercetin and quercetin-3-glucoside recoveries from the shallot . These cells had approximate lactase activity of between 2–4 mU/mg protein, consistent with what we previously reported . LactoseSince lactase activity could not be increased in the Caco-2 cells, we combined a lactase treatment with digestion to provide an intestinal uptake model that is more comparable to a lactose tolerant human, or a lactose intolerant human ingesting a lactase digestive aid to help improve lactose digestion. Not only did lactase increase the amount of quercetin in digested shallot homogenates, but it also increased the amount of quercetin taken up by the Caco-2 cells from the digested shallot extracts. This suggests that a lactase containing digestive aid may increase absorption of quercetin from onions in lactose intolerant humans. Combining the Caco-2 cell model with an in vitro digestion, simulating stomach and small intestinal digestion, and a lactase digestion may provide a more useful model to examine and screen for bioavailability of flavonoid glucosides from common foods Figure .Following an in vitro stomach and small intestinal digestion, the recovery of quercetin and quercetin-3-glucoside is optimized at a storage pH at or below 3.5. Storage at pH above 3.5 results in loss of most of the compounds. The in vitro digestion increased the uptake of shallot quercetin-4'-glucoside by the Caco-2 cells. Quercetin uptake by the Caco-2 cells was similar between digested and non-digested samples, despite the fact that approximately 50% of shallot quercetin is lost during digestion. Treating shallot digestates with lactase increased the recovery of quercetin aglycone 10-fold and decreased the recovery of quercetin-4'-glucoside. Lactase treatment increased the bioavailability of quercetin aglycone 14-fold and decreased the bioavailability of quercetin-4'-glucoside to the Caco-2 cells. Combining pepsin, pancreatin/bile, and lactase digestions with the Caco-2 cell culture monolayer may results in a useful model for studying flavonoid bioavailability from foods.Many advances have been made in understanding flavonoid bioavailability, however many questions still remain unanswered. Food processing, interactions with other compounds as well as with other foods are all factors that may affect bioavailability of flavonoids. A simple and inexpensive screening model would be beneficial as a first step in examining many of these factors. The Caco-2 cell model has the potential to be a good model for analyzing intestinal uptake of quercetin, quercetin glucoside and other flavonoids. While the Caco-2 cell model will never be an exact replica of a human small intestine, it could provide a valuable tool for initial screenings of large quantities of samples relatively quickly and inexpensively. In this study, it was found that a simple digestive aid such as lactase might increase quercetin bioavailability. This may be of importance to lactose intolerant people. Such significant trends may be examined in more detail using human or animal models.


Mutations of the BRAF gene are the most common genetic alteration in melanoma. Moreover, BRAF mutations are already present in benign nevi. Being overexpressed and mutated, B-Raf is a potential target for the immune system and as this mutation seems to be an early event, a humoral immune response against this antigen might serve as a diagnostic tool for detection of high risk patients.372 sera of 148 stage IV melanoma patients and 119 sera of non-melanoma patients were screened for B-Raf, B-Raf V599E and C-Raf specific antibodies by an ELISA assay. Sera were screened for specific total Ig and for IgG. Serum titers were compared with a two tailed Mann-Whitney U test. Sera with titers of 1:300 or higher were termed positive and groups were compared with a two tailed Fisher's exact test.B-Raf specific antibodies recognizing both B-Raf and B-Raf V599E were detected in 8.9% of the sera of melanoma patients and in 2,5% of the control group. Raf specific IgG was detected in some patients at very low levels. B-Raf specific antibody responses did not correlate with clinical parameters but in some cases, B-Raf antibodies emerged during disease progression.These findings imply that B-Raf is immunogenic in melanoma patients and that it might serve as a potential target for immunotherapy. However, B-Raf specific antibodies emerge at rather late stages of melanoma progression and are present only with a low frequency indicating that spontaneous B-Raf specific antibodies are not an early marker for melanoma, but rather may serve as a therapeutic target. Cutaneous malignant melanoma is responsible for 1% of all malignant tumors with a rising incidence in the Caucasian population . InvasivOn the molecular level, melanoma is associated with several genetic changes, including mutations or transcriptional variations in tumor suppressors like p53, CDKN2A/p16, CDKN1A/p21 or in oncogenes like N-Ras . Recentl+ T-cell responses in melanoma patients [+ T-cells in melanoma patients reactive against an HLA B27 restricted B-Raf V599E epitope encompassing the mutation identified using computer assisted algorithms [+ or CD8+ T-cell responses is not suitable for screening larger numbers of patients to estimate responder frequencies.Promising results with new approaches for melanoma therapy have been obtained with active immunizations against tumor associated antigens (TAA) like MAGE-3 , MART-1 patients . Moreovegorithms . HoweverTo determine the frequency of B-Raf specific responses in melanoma patients and to evaluate whether B-Raf specific immune responses could serve as a melanoma marker we examined the Raf specific humoral response using an ELISA assay with purified recombinant B-Raf, B-Raf V599E or C-Raf protein.Patient sera were obtained from frozen stocks and were collected over a period of 5 years. All patients gave informed consent to use their sera for scientific analysis. Control sera were obtained from patients of the dermatology department without signs of melanoma. Control patients were fully anonymized and no further information is available.Recombinant wild type B-Raf, B-Raf V599E and C-Raf proteins were expressed in Sf9 cells and purified as GST fusions (B-Raf V599E, C-Raf) or His-tagged proteins (B-Raf) as described . Purity 405) in a TECAN Spectra Thermo microplate reader.372 sera of melanoma patients were analyzed for B-Raf V599E specific response, 271 sera were analyzed for B-Raf wt and C-Raf specific responses and 119 sera of non melanoma control patients of the dermatology department were used and analyzed for B-Raf V599E, B-Raf wt and C-Raf specific responses. For determination of serum levels of total Ig or IgG, an ELISA assay was developed. 1 μg/ml of purified B-Raf, B-Raf V599E or C-Raf protein in 100 μl coating-buffer or buffer alone as background control (carbonate buffer, pH 9,6) was coated on NUNC 96-Well MaxiSorp plates at 4°C overnight. Plates were washed twice with washing buffer (0,05% Tween (Sigma) in PBS) and blocked with 1% BSA (Sigma) in PBS. After washing twice, serial dilutions of human sera, starting at 1:100, in 100 μl conjugate buffer (1% BSA, 0,05% Tween in PBS) were incubated for 1.5 hours at 37°C. After four wash steps, alkaline phosphatase coupled goat anti human Ig (IgM + IgA + IgG, Dianova) or goat anti human IgG (Dianova) diluted 1:2000 in 100 μl conjugate buffer was added. After 1 h at 37°C and two washing steps, 50 μl of pNPP (Sigma) substrate in buffer was added. The reaction was incubated at room temperature and stopped after 20 min by 50 μl 1 M NaOH. Optical density was read at a wavelength of 405 nm (ODFor determination of specificity, a similar protocol was used, but plates were coated with different concentrations of antigen (0, 0.1, 1.0 and 2.0 μg/ml antigen in coating buffer). Each concentration was determined in duplicate. In addition, specificity was further confirmed using an unrelated antigen, prostate specific antigen (PSA, Sigma), at concentrations of 1 μg/ml for ELISA analysis. Two patients exhibited non-specific serum responses and were excluded from further analysis.For competition ELISA performed with some positive sera, a similar protocol was engaged, using a fixed dilution of the sera and B-Raf or B-Raf V599E antigen for coating. Competition was performed by adding different concentrations of B-Raf or B-Raf V599E (0.0, 0.1, 1 or 2 μg/ml) to the human sera at the corresponding incubation step.405 value exceeding the Cut Off 0.275, corresponding to the mean + 1.5 SD of the background values of positive sera at serum dilutions of 1:50. Raf specific titers were compared to the background values using a two tailed Mann-Whitney U test with a 95% confidence interval. The amount of positive sera within the two groups were defined as sera exceeding a given titer; the final definition of positive sera were sera with titers of 1:300 or higher. Frequencies of positive sera were compared using two sided Fisher's exact test with 95% confidence interval.For the analysis of the ELISA data, the titer of the sera was defined as the last serial dilution with an OD405 and titers were attributed as described using a cut off OD405 of 0.275 (fig. 372 sera of 148 melanoma patients and sera of 119 control patients were screened for Raf specific antibody responses by an ELISA assay using a secondary antibody directed against IgG, IgA and IgM. Serial dilutions of the sera were analysed for their OD275 fig.  and 1B. 275 fig. . Specifi275 fig.  and WEST275 fig. , but mos275 fig. . In all 275 fig.  showed nComparison between Raf specific antibody responses in sera from melanoma patients and control patients reveals apparent differences figure . Raf speAnother interesting point to note is the small, although not significant, number of control patients with detectable levels of Raf specific antibodies. At least two out of 119 control patients showed a very strong B-Raf specific response in the same range as we have observed for positive melanoma patients figure . Due to For different extracellular tumor antigens, including gangliosides, in melanoma, positive associations between the induction of IgM and IgG antibodies and survival time have been reported  and the + or CD8+ T-cells. This question still remains to be clarified as up to now only advanced stage patients have been examined. It is obvious that at this stage the occurrence of antibodies is not correlated with prognosis and that it is very unlikely that the antibody response itself will have an influence on the disease. However, as it is rather unlikely that the frequency of a B-Raf specific T-cell response exceeds the frequency of antigen responses, our data can help to set the upper limit for the expected frequency of B-Raf or B-Raf V599E specific T-cells as 8.5% which allows the rational design of search strategies especially for Raf specific CD8+ T-cells.As oncogenic mutation of B-Raf and even transformation by other oncogenic events is frequently accompanied by Raf overexpression, the induction of an antibody response is not necessarily due to the presence of the mutation. Even though a polyclonal IgM response is very unlikely to be specific for a point mutation, this notion would explain the lack of specificity for the mutational epitope. However, it is also possible that a cellular immune response prior to the induction of Raf specific antibodies has occurred. Such a T-cell response directed against Raf or other tumor antigens, would also result in the lysis of tumor cells and the liberation of intracellular antigens. It is important to mention that the lysis leading to the formation of antibodies against intracellular antigens can be caused by T-cells other than the already observed B-Raf V599E specific CD4Even though the detection of B-Raf specific humoral and T-cell responses suggest that B-Raf/B-Raf V599E is immunogenic and that a response could be induced in at least a part of the patients, this notion does not allow conclusions on its suitability as a target for immunotherapy per se. In theory, an optimal tumor antigen is exclusively expressed in the tumor tissue and essential for tumor cell growth and survival to avoid the emergence of escape mutants or antigenic loss, whereas the immunological attack of the tumor cell is independent of the function of the antigen . To date+ and our earlier demonstration of B-Raf V599E specific CD8+ T-cell responses this study makes the screening for novel B-Raf V599E MHC class I epitopes for vaccination approaches a promising task.Taken together we have demonstrated that B-Raf/B-Raf V599E specific antibodies are detectable in 8.9% of advanced stage melanoma patients and B-Raf V599E might therefore be a valuable target for immune therapy. Combined with the independently described B-Raf V599E specific CD4None declared.JF and JCB designed the study and performed the data and statistical analysis. JF set up and supervised the ELISA and wrote the report. JCB set up and supervised the sample collection and was involved in writing the report. TP and VH were performing the ELISA assays, CSV was responsible for serum collection, transport and maintenance. EBB and URR were involved in providing the conceptual framework for this study. All authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:Supplementary figure with WESTERN Blot analysis of positive sera confirming specificity.Click here for file