This paper compiles data from different sources to get a first comprehensive picture of psychometric and other methodological characteristics of the Menopause Rating Scale (MRS) scale. The scale was designed and standardized as a self-administered scale to (a) to assess symptoms/complaints of aging women under different conditions, (b) to evaluate the severity of symptoms over time, and (c) to measure changes pre- and postmenopause replacement therapy. The scale became widespread used (available in 10 languages).A large multinational survey (9 countries in 4 continents) from 2001/ 2002 is the basis for in depth analyses on reliability and validity of the MRS. Additional small convenience samples were used to get first impressions about test-retest reliability. The data were centrally analyzed. Data from a postmarketing HRT study were used to estimate discriminative validity.Reliability measures (consistency and test-retest stability) were found to be good across countries, although the sample size for test-retest reliability was small.Validity: The internal structure of the MRS across countries was astonishingly similar to conclude that the scale really measures the same phenomenon in symptomatic women. The sub-scores and total score correlations were high (0.7–0.9) but lower among the sub-scales (0.5–0.7). This however suggests that the subscales are not fully independent.Norm values from different populations were presented showing that a direct comparison between Europe and North America is possible, but caution recommended with comparisons of data from Latin America and Indonesia. But this will not affect intra-individual comparisons within clinical trials.The comparison with the Kupperman Index showed sufficiently good correlations, illustrating an adept criterion-oriented validity. The same is true for the comparison with the generic quality-of-life scale SF-36 where also a sufficiently close association has been shown.The currently available methodological evidence points towards a high quality of the MRS scale to measure and to compare HRQoL of aging women in different regions and over time, it suggests a high reliability and high validity as far as the process of construct validation could be completed yet. The interest of clinical research in aging women and males increased in recent years and thereby the interest to measure health-related quality of life and symptoms. Women, as do men, experience an age-related decline of physical and mental capacity. They observe symptoms such as periodic sweating or hot flushes, impaired memory, lack of concentration, nervousness, depression, insomnia, and bone – joint complaints.women, not by their physician [The Menopause Rating Scale (MRS) is a health-related quality of life scale (HRQoL) and was developed in response to the lack of standardized scales to measure the severity of aging-symptoms and their impact on the HRQoL in the early 1990s. Actually, the first version of the MRS was to be filled out by the treating physician but methodological critics lead to a new scale which can easily be completed by hysician .The validation of the MRS started some years ago -6 aimingThe scoring scheme is simple, i.e. the score increases point by point with increasing severity of subjectively perceived symptoms in each of the 11 items (severity 0 [no complaints]...4 scoring points [very severe symptoms]). The respondent provides her personal perception by checking one of 5 possible boxes of "severity" for each of the items. This can be seen in the questionnaires in the additional files linked to this publication. The composite scores for each of the dimensions  is based on adding up the scores of each item of the respective dimensions. The composite score  is the sum of the dimension scores. The three dimensions, their corresponding questions and the evaluation are detailed and summarized in an attached file linked to this publication [see Additional file 1].).The MRS scale became internationally well accepted. The first translation was into English . Other tLike in other QoL scales, it is a challenge to satisfy the demands of a clinical utility and outcomes sensitivity, and this in addition to the conventional psychometric requirements of test reliability and validity.The aim of this paper is to present additional psychometric data to discuss the methodologically relevant characteristics of the MRS scale.The development of the scale, instrument characteristics , and norms and standardized scores have been published elsewhere -5. This Europe (Germany, France, Spain, Sweden), North America (USA), Latin America (Mexico, Argentine, Brazil), and as example for Asia – Indonesia. Study participants were accrued as a random sample of females aged 40 to 70 years from existing population panels. The sample size in each of the countries was about 1000 females aged 40–70 years, with exception of USA (n = 1500). The participation rates ranged between 46 and 94% across countries. The demographic details of the sample are: On average, about tertiles of the respondents were under 50 years, between 50–59, and over 60 years old in most of the countries, however, about 50% were less than 50 years in Indonesia and in Brazil. The majority of respondents reported a Christian religion in Europe (range: 74% (Germany) to 96% (Spain), 85% in USA, and in Latin America (range: 95% (Argentina) to 97% (Mexico). The use of the MRS was part of this survey, i.e. multinational data became available to reconsider methodological issues more thoroughly such as internal structure of the scale, reliability , and reference values for different population.During the last two years a number of smaller and larger investigations were made from different groups to further check methodological features of the scale. We performed recently a large, multinational survey to represent the situation across nine countries and cultures using existing and for the respective countries representative panels between November 2001 and February 2002 to get information about knowledge, attitudes and behaviour related to hormonal treatment in women aged 40–70 years: For the purposes of reliability assessment we performed a few preliminary studies with a test-retest approach. These small, descriptive studies of community samples of women aged 40–70 were done in summer and fall 2002 by local collaborators in the respective countries, but they were done separately and independent from the main study. These studies were done just for orientation with convenience samples – not representative for the respective population.There is only one intervention study  available to our knowledge. This study has been published  but not With these data available, we were able to scrutinize many methodological characteristics of the MRS scale to review most fundamental psychometric characteristics as well validity parameters.The assessment of scientific measurements depends first of all on the evidence of replicability (consistency) and test-retest reliability. In contrast to systematic and random variation, reliability gives an estimate of method-related measurement error which should be low not to hide or dilute intended systematic changes – due to treatment for example.Table The test-retest correlation coefficients (Pearson's correlation) support the suggestion of a good temporal stability of the total scale and its three sub-scales Table , althougThe test-retest coefficients of the total score range between 0.8 and 0.96 across Europe, North and Latin America, and Asia. When it comes to the subscales with much fewer items, the variation increased and some of the coefficients went down to 0.5 . Altogether, the test-retest stability over a time period of two weeks aggregated at the international level supports the notion of a very acceptable test-retest reliability of the total scale and their three sub-scales.Although there is an impressive set of information currently available concerning the reliability of the MRS scale, there are also limitations: Small sample sizes prevent a final conclusion regarding test-retest reliability in some of the languages the scale has been translated in.Similar to reliability which assesses the consistency of measurement, the validity estimates if a scale measures what it intends to measure. But whereas reliability can be determined straight forward with very few indicators, the validity is almost always a continuous process . It is a process of accumulating evidence for a valid measurement of what is purposed. Therefore, the currently available data are already fairly comprehensive and do pave the way for a focussed and continuing validation process.The first step of validation is usually to multivariately demonstrate a similar internal structure ("dimensions") of a given scale through factor analysis.The first factorial analysis in 1996 was applied to identify the dimensions of the scale. Three dimensions of symptoms/complaints were identified : a psychThe recent large, multinational survey in nine countries of four continents provided data to compare with the initial standardisation sample of the MRS. The question was: Is the internal structure of the MRS results comparable among different countries or cultures. Astonishingly similar factor loadings of the 11 items of the 3 domains of the MRS were observed , and sleep disturbances both 0.5 in somatic and psychological domain (Latin America). These two items had similar problems in Spain, Mexico, and Brazil but not in other countries  will be the main criterion which might not be affected by potential slight differences in the patient reported outcome structure. Therefore the general agreement in the internal structure of the MRS scale across country groups, even accepting the possibility of slight differences in two items  would be closer to 0 than the correlations with the construct of the aggregate total score to which all sub-scales should significantly contribute. But that is theory; Table There are different categories of severity of complaints or problems with QoL. For the comparison of these categories across countries or cultures it is important to understand the prevalence of complaints. Currently, there is only one table with reference values and definitions published – for the German population from the initial standardization of the MRS . Are theThe data of our large multicultural survey permitted such comparisons. The mean values (SD) of the MRS total score and the three domains are depicted in Table However, the total, psychological and somatic scores were systematically higher in Latin America, and systematically (significantly) lower in Asia (Indonesia) than in Europe/North America. The urogenital scores were significantly lower in Latin America/Indonesia than in Europe/US. Obviously the perception of the prevalent symptoms depends on cultural factors – or the symptoms show real differences in prevalence. Thus, direct comparisons of MRS scores between Europe/North America on the one side and regions in Latin America and Asia cannot are not recommended. This does not effect intra-individual comparisons (e.g., pre/ post therapy) within these countries and it may also very little affect the comparison of relative changes (pre/post treatment) among different countries. The latter is a hypothesis and needs further evidence form research/experience.Similar mean values could still have a different distribution across the proposed four categories of "severity of complaints": no/little symptoms, mild, moderate, and severe complaints, i.e. for the total scale and the three domains. The prevalence of these categories across the four regions studied is seen in Table In fact, the comparison with other scales of similar purpose is important. It is known from other quality of life scales that comparisons with scales with similar purposes are much more important than comparisons with so-called objective parameters such as exercise tests, physiological or chemical parameters – in our case with hormones.Health related quality of life should be validated against quality of life measured with other generic QoL scales (e.g., SF-36), and against specific instruments to measure symptoms in aging women (e.g. Kupperman index). These data were published elsewhere  but willAlthough the Kupperman Index was not validated according to psychometric standards it is still in use in the medical practice to monitor menopausal symptoms. Therefore a comparison with the fully standardized MRS seems to be reasonable. If one divides the distribution of both scales into quartiles and compares the frequencies, both instruments were found to be closely associated: Kendall's tau-b coefficient 0.75 (95% CI 0.71–0.80) . SimilarTwo sub-scales of the multi-domain quality of life scale SF-36 was compared with the MRS: the somatic sum score (with somatic domain of MRS) and the psychologic sub-scales of both instruments. Both somatic domains were sufficiently well and significantly associated: Kendall's tau-b = 0.43 (95% CI 0.52–0.35); Pearson correlation coefficient r= 0.48 (95% CI 0.58–0.37). That means, the higher the score in the somatic dimension of the MRS, the lower the quality of life according to the somatic sum-score of the SF-36 . Similari.e., the ability of the scale to accurately measure treatment effects and to predict the clinically based assessment of physicians, was not analysed so far. At present, there is one post-marketing study that can be used to preliminary assess discriminative validity. The results will be published soon elsewhere. To this end, many clinicians understand the term "validity" and mean high utility for clinical work or research.The MRS scale is a standardized HRQoL scale with good psychometric characteristics. The use in many countries offered the possibility to compare the test characteristics across countries. Reliability measures (consistency and test-retest stability) were found to be good in all countries where data were obtained – however, some samples were very small and therefore considered as preliminary information.The validity was measured in its various forms: The internal structure of the MRS across countries was sufficiently similar to conclude that the scale really measures the same phenomenon in women with complaints. The sub-sores and total score correlations showed high coefficients with the total score and less among the sub-scales. This however indicates that the subscales are not fully independent in practice.Comparisons of reference values from different populations showed that the MRS scores can easily be compared between Europe and North America/US. Direct comparisons between Europe/North America and Latin American countries and Asia (Indonesia) should be considered with caution because the severity of reported symptoms seems to differ. The reasons are not clear, further research is needed.The comparison with other scales for menopausal symptoms (Kupperman Index) showed a sufficiently close association and correlation coefficients, i.e. illustrating a good criterion-oriented validity. The same is true for the comparison with the generic QoL scale SF-36 where also high correlation coefficients have been shown.Thus, the currently available methodological evidence points towards a high quality of the MRS scale to measure and to compare HRQoL of aging males over time or intervention. It suggests a high reliability and high validity as far as the process of construct validation could be completed.KH: responsible for drafting the manuscript and running analyses. AR: responsible for designing and overseeing the multinational survey (2001/2002), contributed to writing and revising of the paper. PP: co-ordination of the field work of the multinational survey, setting up the initial database, and contributed to writing of the paper, responsibility in developing/validating the MRS scale. HPGS: Major responsibility in developing the MRS scale, contributed to writing/revision of the manuscript. FS: Provided data of a clinical study, contributed to writing of the manuscript. LAJH: responsible for the collection and evaluation of the data, and involved in writing/revising the paper. DMT: responsible for checking the integrated database, responsible for several analyses regarding validity, and contributed to writing of the paper.


Poxviridae, was used extensively in the past as the Smallpox vaccine, and is currently considered as a candidate vector for new recombinant vaccines. Vaccinia virus has a wide host range, and is known to infect cultures of a variety of cell lines of mammalian origin. However, little is known about the virus tropism in human leukocyte populations. We report here that various cell types within leukocyte populations have widely different susceptibility to infection with vaccinia virus.Vaccinia virus, the prototype member of the family We have investigated the ability of vaccinia virus to infect human PBLs by using virus recombinants expressing green fluorescent protein (GFP), and monoclonal antibodies specific for PBL subpopulations. Flow cytometry allowed the identification of infected cells within the PBL mixture 1–5 hours after infection. Antibody labeling revealed that different cell populations had very different infection rates. Monocytes showed the highest percentage of infected cells, followed by B lymphocytes and NK cells. In contrast to those cell types, the rate of infection of T lymphocytes was low. Comparison of vaccinia virus strains WR and MVA showed that both strains infected efficiently the monocyte population, although producing different expression levels. Our results suggest that MVA was less efficient than WR in infecting NK cells and B lymphocytes. Overall, both WR and MVA consistently showed a strong preference for the infection of non-T cells.When infecting fresh human PBL preparations, vaccinia virus showed a strong bias towards the infection of monocytes, followed by B lymphocytes and NK cells. In contrast, very poor infection of T lymphocytes was detected. These finding may have important implications both in our understanding of poxvirus pathogenesis and in the development of improved smallpox vaccines. Poxviridae, is a large DNA virus whose replication takes place in the cytoplasm of the infected cell [in vitro, little is known about the ability of vaccinia virus to infect different cell types in vivo. Vaccinia virus host range in cell culture is known to be determined by several genes. The importance of host restriction has been highlighted in recent years by the growing use of the Modified Vaccinia Ankara (MVA) virus strain, whose replication is severely impaired in human cells [Vaccinia virus, the prototype of the ted cell . Althougan cells -4. Genesan cells .In addition to host range genes, there are a number of factors that might influence the infection rate of a given cell type, such as the accessibility and amount of receptors, the ability to internalize the virus, and the metabolic state of the cell. In addition to cellular factors, genetic differences in the virus might influence the efficiency and fate of the infection. For instance, cellular nucleotide pools can be one of the factors that, in conjunction with the expression of viral thymidine kinase (TK), may influence the rate of infection.in vivo may be determined by many complex factors that may be dependent on the cell type and metabolic state.The above considerations led us to hypothesize that, although receptors for vaccinia seem to be ubiquitous, and virus replication is relatively independent from the host cell, virus tropism We have focused here on the differences between two widely used strains of vaccinia virus (Western Reserve-WR and MVA), and also to their respective TK(-) mutants, in their ability to infect different cell types in fresh human PBLs.+, up to 20%) and NK cells (CD56+, up to 9%).Previously, we have shown that GFP expression from a vaccinia virus recombinant can be used to monitor infection by flow cytometry . Where aVaccinia virus MVA and TK-deficient viruses have been proposed as improved recombinant vaccines. In particular, the highly attenuated MVA strain has elicited much interest as a safer vaccine vector. We studied the influence of the virus strain and the TK phenotype in the infection of human PBLs. We thus constructed GFP expressing viruses from vaccinia virus MVA strain, by inserting the GFP cassette downstream of the F13L gene, using an intergenic region for the insertion. Additionally, thymidine kinase-deficient virus recombinants WR-TK(-) and MVA-TK(-) were constructed by inserting the GFP cassette within the viral TK locus. Those viruses grew to high titers and produced, upon infection of cell lines, bright GFP fluorescence (not shown).+ and CD8+ cells were poorly infected, although there was indication of an increased infection of low-CD8 T lymphocytes in comparison with high-CD8 cells. Notably, this experiment confirmed that most of the monocytes (CD14+) was infected in our experimental conditions, and showed a high level of GFP fluorescence. It was of interest to directly compare the ability of vaccinia MVA to infect PBLs with that of the standard laboratory strain WR. A side-by-side comparison of WR-GFP and MVA-GFP showed that both viruses infected a high percentage of CD14+ monocytes (83 and 70%, respectively), and a low percentage of T lymphocytes (0.46 and 0.2%, respectively). No significant differences were noted in the percentage of CD4 cells infected with both viruses. Although both virus strains were able to infect the majority of monocytes, MVA-GFP produced a lower level of GFP fluorescence than WR-GFP in the infected monocytes. Those differences could be the result of a lower expression level, or a delay in the course of infection, by the MVA strain.The four GFP-expressing viruses were used to infect fresh human PBLs from a different individual, and subjected to flow cytometry analysis at 7 h.p.i. Fig. . The res+ B lymphocytes (7.1% vs 3.5%) and CD56+/CD16+ NK cells(7.6% vs 4%).In addition to increased GFP expression levels, WR-GFP was also more efficient than MVA-GFP for the infection of CD19As stated above, we constructed recombinant viruses from both WR and MVA by insertion of GFP into the TK locus. Infection of different populations in human PBLs with these viruses was again monitored in paralell using specific antibodies Fig. . InfectiDetection of cells infected by GFP-expressing vaccinia viruses provide a fast and sensitive method to measure virus infection . In thisIt is to note that the approach used in this work only allows the detection of viral gene expression derived from the infection, but does not address whether the infection results in the production of progeny virus. Early reports indicated that vaccinia virus cause cythopathic effect in human leukocytes, although only replicated in mitogen-stimulated cell populations, indicating that active cell replication is required for virus replication ,8. In thOur results point to a significant preference of vaccinia virus for certain cell types. In particular, monocytes were the most susceptible cells, followed by B cells and NK cells. In contrast, T cells were infected at very low rates. These observations are in broad agreement with previous studies, where different infection rates have been noted between monocytes and lymphocytes  and betwComparison of the patterns obtained with the two virus strains and their TK(-) mutants indicate that both the virus strain and the TK phenotype may determine the amount of gene expression, as was revealed by the intensity of GFP fluorescence in infected monocytes. In addition, the ability of the virus to infect certain cell types (CD19) seems to be affected to a certain extent by disruption of the TK gene. While this may be derived from our inability to detect those infected cells because of decreased gene expression, we cannot rule out a more direct requirement of TK activity in those cells.MVA vaccinia virus strain has elicited much interest recently because of its safety record. Because clinical complications and side effects of smallpox vaccination are a critical issue in the event of mass vaccination, understanding the basis of MVA attenuation may lead to the development of better vaccine vectors. In this study, a number of differences were noted between the rates of infection obtained with WR and MVA virus strains. While both viruses were able to infect the monocyte population, WR infected B cells and the NK population (CD56, CD16 positive cells) more efficiently than MVA. Whether these observations have implications on the pathogenicity or immunogenicity of MVA will require further studies.The fact that both WR and MVA showed a strong preference for certain cell populations indicate that, in addition to host range genes, there are other factors that might influence the infection rate of PBL cells. Those might include a variety of such as the accessibility and amount of receptors, ability to internalize the virus, and the metabolic state of the cell.Monocytes (CD14+ cells) were the cells in the PBL population that showed a greater susceptibility to vaccinia virus infection, as measured by viral gene expression. On the other hand, T lymphocytes (CD3+ cells) were infected with low efficiency. An intermediate susceptibility was detected in B lymphocytes (CD19+ cells) and NK (CD56+ cells). Both the use of a highly attenuated virus strain (MVA) or the disruption of the thymidine kinase gene lead to decreased gene expression in the infected cells. Those observations highlight the existence of a different degree of susceptibility to infection if PBL subpopulations, a fact that may have important implications in understanding virus pathogenicity and immunogenicity.2 atmosphere at 37°C. Plasmid pRB21 [Vaccinia virus strain WR was grown and titrated in BSC-1 or CV-1 cells, grown in minimal essential medium (EMEM) supplemented with 5% fetal bovine serum (FBS) and 2 mM L-Glutamine. MVA virus and recombinants were grown in BHK-21 cells (ATCC CCL10) cultured in BHK medium containing 5% FBS, 3 g/ml tryptose phosphate broth and 0.01 M hepes. All cells were maintained in a 5% COid pRB21  containsGCTAGCAAAGGAGAAGAA) and GFPH3 (TTTAAAGCTTTACTAGTGGATCCTCAG), that include NheI and HindIII restriction sites, respectively. After digestion with NheI and HindIII, the gene was inserted into the corresponding sites in plasmid pRB21 [Plasmid pRBrsGFP, designed to mediate the insertion of the gene coding an enhanced version of the green fluorescent protein gene, rsGFP, (Quantum Biotechnologies, Inc.) was constructed as follows. rsGFP gene in plasmid pQBI25 was amplified using oligonucleotides GFP 5' (AATATAAATGid pRB21 , downstrXhoI and BamHI.Plasmid prsGTK, containing the above GFP expression cassette located between recombination flanks for the TK locus, was obtained by cloning the rsGFP cassette from plasmid pRBrsGFP in plasmid pGPTK (Sanchez-Puig and Blasco, unpublished) after digestion with Viruses WR-GFP and MVA-GFP were obtained by transfection of plasmid pRBrsGFP in cells infected with WR mutant vRB12  or MVA, Recombinant virus VVrsGFPTK, was isolated after transfection of plasmid prsGTK in cells infected with virus WR. Recombinant virus plaques were isolated by plaquing on 143B TK(-) cells in the presence of 25 μg/ml bromodoxyuridine. GFP positive plaques were identified under the microscope, and plaque-purified three times before amplification.Recombinant virus MVA-GFPTK was isolated by transfection of plasmid prsGTK in MVA-infected BHK-21 cells. GFP-positive virus were identified under the microscope, isolated by three consecutive rounds of plaque purification in BHK-21 cells and amplified in BHK-21 cell cultures.Finally, virus recombinants were analyzed by Southern Blot, using digoxigenin-labelled GFP gene sequence as the probe. The analysis demonstrated that the recombinants contained the GFP expression cassette in the desired genome position and that they were stable, double recombinants.Peripheral blood mononuclear cells from healthy subjects were obtained by density gradient centrifugation of heparinized blood on Ficoll-Paque . Cells obtained from the interface were washed three times in saline solution and then resuspended in complete medium (CM) consisting of RPMI 1640 (Gibco, Life Technologies, Germany) supplemented with 10% FBS (Gibco), 2 mM L-glutamine (ICN, USA), 100 U/ml each of penicillin and streptomycin (Laboratorios Normon, Spain). Viability of the isolated cells always exceeded 95% as determined by trypan blue exclusion.5 PBLs were infected with virus recombinants VV-rsGFP, VVrsGFPTK, MVA-GFP, and MVA-GFPTK, at 10 p.f.u./cell, in 0.7 ml of RPMI medium containing 2% FBS. After 1 h adsorption, cells were pelleted and resuspended in 2 ml of fresh RPMI medium containing 2% FBS. At different infection times, the cells were sedimented by low-speed centrifugation, resuspended in 100 μl FACS-FLOW, and labeled with the appropriate conjugated monoclonal antibodies (mAb) for flow cytometric analysis (FCM) .Infection of human PBLs was performed as follows: 2 × 10Cells were incubated with the antibodies for 30 min at 4°C in the dark, washed twice with saline solution and finally resuspended in 200 μl Cytofix/Cytoperm (BD Pharmingen). Cells were analyzed in a FACSCalibur (BD Biosciences, San Diego, CA) and data were processed with Cell Quest software (BD).The author(s) declare that they have no competing interests.JMS carried out the isolation of virus recombinants and performed viral infections and participated in the drafting of the manuscript. LS and GR performed the preparation of PBLs, carried out the flow cytometry and elaborated the data. GR participated in the interpretation of the data and helped in the elaboration of the manuscript. RB conceived the study, designed the virus recombinant constructs, supervised the experimental work and drafted the manuscript. All authors read and approved the final manuscript.


Most patients with non-small cell lung cancer who initially respond to gefitinib or erlotinib (tyrosine kinase inhibitors) ultimately develop resistance and disease relapse. What is the mechanism for this resistance? Uncontrolled proliferation of tumor cells is a hallmark of cancer. In many types of cancer, mutations in genes that activate cellular signal transduction pathways contribute to enhanced proliferation and survival of cancer cells. One well-characterized example is mutation in tyrosine kinases, enzymes that regulate the growth and survival of cells. Tyrosine kinase activity is tightly regulated in normal cells, but is dysregulated due to mutation in some cancers, including lung cancer, resulting in enhanced proliferation and survival of cancer cells. The tyrosine kinases are attractive candidates for molecularly targeted therapy in cancer, because cancers become dependent on growth signals from the mutant tyrosine kinases. Tyrosine kinases require ATP for their enzymic activity, and thus small molecules that mimic ATP can bind to mutant kinases and inactivate them.BCR-ABL gene rearrangement [The paradigm for tyrosine kinase inhibition as treatment for cancer using small-molecule inhibitors was first established in the context of chronic myelogenous leukemia (CML) associated with the angement . Imatiniangement , and alsangement .PDGFRB [PDGFRA [KIT [Imatinib is also a potent inhibitor of the ARG, KIT, PDGFRA, and PDGFRB tyrosine kinases. As a consequence, there have been additional dividends from the United States Federal Drug Administration approval of imatinib for treatment of BCR-ABL-positive CML. For example, imatinib is effective in treatment of chronic myelomonocytic leukemia with gene rearrangements that constitutively activate PDGFRB , of hype [PDGFRA , and of FRA [KIT  .epidermal growth factor receptor (EGFR) in patients with NSCLC that are associated with clinical response to the small-molecule EGFR inhibitors gefitinib (Iressa) or erlotinib (Tarceva) [More recently, this paradigm has been extended to treatment of non-small cell lung cancer (NSCLC). Several mutations have been identified in the context of Tarceva) ,8,9, incTarceva) ,8,9. TheBCR-ABL kinase domain itself, including T315I [As might have been anticipated in treatment of cancer with any single agent, resistance to small-molecule tyrosine kinase inhibitors has emerged as a significant clinical problem. This was first appreciated in patients with CML treated with imatinib whose tumors developed resistance, and has been most extensively studied in that context. Although there are many potential mechanisms for development of clinical resistance, most cases of imatinib-resistant CML are due to point mutations in the ng T315I ,11. Siming T315I ,12. Thesng T315I ,14.PLoS Medicine, Pao and colleagues have identified acquired mutations in patients with NSCLC that appear to explain clinical resistance to gefitinib or erlotinib [In an elegant new study in rlotinib . The mecStructural models of EGFR provide structural insights into these biological data. A ribbon structure of erlotinib bound to the EGFR kinase domain  shows thKRAS alleles that have previously been shown by these same authors to confer resistance to these inhibitors [These observations provide convincing evidence that, at least in some patients with NSCLC, resistance to gefitinib or erlotinib can be attributed to acquisition of a T790M mutation in the context of EGFR. However, three additional patients with clinical resistance to gefitinib or erlotinib did not have the T790M mutation, nor did they have mutant It will be important to identify alternative small-molecule inhibitors for the T790M resistance mutation. Structural data suggest that one compound, lapatinib, may subserve this purpose , but it Pao and colleagues' superb study also highlights several important points that may guide development of kinase-targeted therapies in the future. It is clear that, to the extent that small-molecule kinase inhibitors are effective as single agents in treatment of cancer, resistance will develop. Furthermore, based on previous experience, some of these patients are likely to harbor acquired point mutations in the target kinase that confer resistance. Resistance mutations identified via in vitro screens have shown a high degree of correlation with those that develop in vivo, as shown in screens for imatinib-resistant BCR-ABL mutants  and PKC4These findings also emphasize the critical need for re-biopsy of patients with cancer treated with molecularly targeted therapies at time of relapse. Tissue acquisition is more challenging in solid tumors than for hematopoietic malignancies, and may entail risk. Nonetheless, it is clear that data derived from such analyses will be essential to inform approaches to improving therapy for NSCLC and other solid tumors.


Thousands of genes in a genomewide data set are tested against some null hypothesis, for detecting differentially expressed genes in microarray experiments. The expected proportion of false positive genes in a set of genes, called the False Discovery Rate (FDR), has been proposed to measure the statistical significance of this set. Various procedures exist for controlling the FDR. However the threshold  is arbitrary and a specific measure associated with each gene would be worthwhile.p-values is available at .Using process intensity estimation methods, we define and give estimates of the local FDR, which may be considered as the probability for a gene to be a false positive. After a global assessment rule controlling the false positive error, the local FDR is a valuable guideline for deciding wether a gene is differentially expressed. The interest of the method is illustrated on three well known data sets. A R routine for computing local FDR estimates from The local FDR associated with each gene measures the probability that it is a false positive. It gives the opportunity to compute the FDR of any given group of clones (of the same gene) or genes pertaining to the same regulation network or the same chromosomic region. Microarrays are part of a new class of biotechnologies that allow the monitoring of the expression level of thousands of genes simultaneously. Among the applications of microarrays, an important task is the identification of differentially expressed genes, i.e genes whose expressions are associated with the status of the patient (treatment/control for example).The biological question of the identification of differentially expressed genes can be restated as a one (for paired data) or two-sample (for unpaired data) hypothesis testing procedure: is the gene differentially expressed between the two situations? However, when thousands of genes in a microarray data set are evaluated simultaneously by fold changes or significance tests approach, multiple testing problems immediately arise and lead to many false positive genes. In this 'one-by-one gene' approach the probability of detecting false positives rises sharply.p-values that guarantees that the FDR is less than a target level α. The same paper demonstrated that the BH procedure is more powerful than the Bonferroni method that controls the familywise error rate.The False Discovery Rate (FDR), is defined as the expected fraction of false rejections among those hypotheses rejected. In their seminal paper Benjamini & Hochberg  providedThe FDR gives an idea of the expected number of false positive hypotheses that a practitioner can expect if the experiment is done an infinite number of time. As usual with expectation, it gives very little information about the number of false discovery hypotheses in a given experiment.t, is arbitrary. Storey and Tibshirani [q-value,The value of 1, 5 or 10% for the FDR, which determines the threshold bshirani  stressedPi is the p-value of the ordered gene i, Ri is the total number of rejected genes whose p-values are less than the threshold t = Pi and  is an estimate of the total number of non differentially expressed genes, m0.where q-value is appealing because it gives a measure of significance that can be attached to each gene, but it must be stressed that it is not an estimate of the probability for the gene to be a false positive. The q-value is generally lower than the latter because it is computed using all the genes that are more significant than gene i. Obviously a gene whose p-value is near to the threshold t does not have the same probability to be differentially expressed than a gene whose p-value is close to zero. Therefore the q-value gives a too optimistic view of the probability for the gene to be a false positive.The p-values under H1.Therefore it is interesting to obtain an estimate of the FDR attached to each gene, called local FDR, from an inferential point of view and without any assumption about the distribution of the LetH0(i) = {gene i is not differentially expressed}.FDR(i) is the probability that a gene, whose p-value is Pi, is not differentially expressed, taking into account the whole set of tests. A raw local FDR estimate is defined in a first step. In a second step the local FDR estimate is defined as a smoothed value based on the raw values.Let the local FDR be the probability that a given gene is not differentially expressed. More specifically, P1 < … <Pm denote the ordered p-values for testing H0(i). The raw local FDR estimate for gene i is:Let whereW(λ) = #{i, Pi > λ}, see Storey [where λ is a tuning parameter and e Storey .p-values for the non-differentially expressed genes are independent. The raw local FDR estimate has the following properties:Assume that the H0(i) and H0(i - 1) and if E = m0, (i, λ) is unbiased with mean 1.• Under (i, m0) = m0(Pi - Pi-1). Under H0(i) and H0(i - 1) and if m0 is known, V((i, m0)) = /[(m0 + 1)2(m0 + 2)] ≈ 1, for m0 large enough. This value is a lower bound for V((i, λ)) when m0 is unknown.• Let H1 is generally much smaller than under H0.• The variance of the raw local FDR under  where qj is the q-value of gene j. The q-value may thus be viewed as the mean of the local FDR of the genes with p-values lower than Pj.• (i, λ) is generally a very variable estimator. Moreover the local FDR should increase with the p-value. This is not the case for the raw local FDR. Therefore it is necessary to use a smoothed estimate.i) isThe smoothed local FDR(fi is a smoothing function of the (j, λ) for j = 1, m, computed at position Pi.where (i, λ) gives a very valuable guideline for the choice of a threshold. One may consider the curve of the local FDR versus the index of the gene ordered by their p-values: a good candidate for the threshold should be a point with a high second order derivative, which corresponds to an abrupt change in the slope of the curve (see the examples of the following section). The second order derivative of the smoothed local FDR can be computed numerically using finite differences.p-values. The following sections demonstrate how the local FDR can be useful using the data of well known experiments.As an interesting application of the local FDR, it is possible to compute the FDR associated with a class of genes or clones by summing up the local FDR estimate of each clone or gene: one may consider for example clones corresponding to the same gene, genes known involved in a given regulatory network, or gene from the same chromosomic region, and associate a FDR with the whole class. These genes do not need to have consecutive Golub  were intp-values based on random permutations of the ALL/AML labels on Welch t-statistics for each gene, Dudoit [m0 is estimated with bootstrap method as suggested by Storey and Tibshirani and implemented in the library GeneTS of software R.Data are available in the R multtest package. We used the preprocessing proposed by the authors and the , Dudoit , on the (i) for ordered genes and Figure t = 0.15 for the p-value. This may be an indication about the threshold. The Figure p-values. We can see in Figure q-value equal to 0.0078 while the 438th gene has a local FDR equal to 0.027. It must be noticed that there is a big difference between the two measures of FDR because the numerous regulated genes with very small p-values have a great influence on the q-value, which is not the case of the local FDR  may be equal to 0 because the difference between two successive p-values is 0. The discrete structure of the p-values implies a departure from the theoretical continuous uniform distribution. This explains why the moving average smoothing creates discrete jumps which appear in Figure The H0 is correct, the p-values are distributed as a uniform distribution over [0, 1]. The empirical distribution of the high observed p-values (say above 0.5) is far from the uniform distribution. There are several non-exclusive possibilities to explain this: more than 50% of the genes are differentially expressed, the gene results for non-differentially expressed are correlated or there is a technical problem in the random permutations of the Welch t-statistics.If the distribution of the statistics under p-values, we have computed local FDR estimates. The three genes which have been analysed in detail by Storey and Tibshirani [Storey and Tibshirani , have anbshirani  are presq-value and gives a better idea of the probability that a gene is a false positive. For example, at the level of 5%, CTGF will be considered as differentially expressed on the basis of the q-value while it will be considered as non differentially expressed using the local FDR.One can see that the smooth local FDR estimate is generally greater than the (i) for ordered genes and Figure Setting λ = 0.5, Storey and Tibshirani  estimateq-value. We can see on the Figure q-values.The goal of the study is to identify genes with altered expression in the livers of two lines of mice with very low HDL cholesterol levels compared to inbred control mice. The mouse model is the apolipoprotein AI (ApoAI) knock-out mice. ApoAI is a gene known to play a pivotal role in HDL metabolism. The statistical analysis is described in Dudoit . Height (i) for ordered clones and Figure p-value for which it is crucial to obtain good estimates of the probability of being false positives. The lowess smoothing does not work well for the 50 first clones. In this particular case the default smoothing parameter f = 0.2 is not well suited and should be lower. However if it is chosen too low, the smoothing will not fit well the rest of the curve.Figure . This example shows that it is possible to estimate the local FDR of any group of clones. This opportunity provided by the local FDR is certainly one of its major advantage with many potential applications.There are two clones of the gene Apo-AI. If we want to estimate the FDR of these two clones taken in a whole, we compute the mean of the smoothed local FDR of the two clones (the first and the height top clones) and obtain a local FDR for the gene Apo-AI, which is equal to t maximizing the second derivative. Alternatively one can use a cost function and choose the threshold that minimizes the mean cost for a given cost function: using cost of the experiment, cost of false positive gene validation and the profit of discovering a differentially expressed gene, it is direct to compute the optimal strategy for choosing the threshold.The curve of the smoothed local FDR is an efficient tool to summarize the information about the number and the statistical significance of differentially expressed genes, and may also be used to give an indication about the validity of the statistical assumptions. Moreover it is a valuable tool to choose the threshold for separating the differentially expressed genes from the non-differentially expressed one: one can choose a value of FDR < 0.05. The local FDR is higher than the q-value. Therefore the first set is strictly included in the second one. The local FDR rule is therefore more conservative than the usual FDR one.Note that a decision rule based on the local FDR would lead to a different set of selected genes than the usual one obtained by controlling the FDR. Consider the set of tests for which the local FDR is below 0.05, say. This set is not identical to the set identified by the standard criterion that p-value gives the probability that a non differentially expressed gene would be as or more extreme than the gene under concern. The q-value indicates the estimated proportion of genes as or more extreme than the gene under concern that are a false positive. The local FDR gives the estimated proportion of genes around the gene under concern which are false positive. The latter may be used as the probability that the gene under concern is a false positive, taking into account the multiplicity of the test. One of the major interest of the local FDR is that it gives the opportunity to compute the FDR of any given group of clones (of the same gene) or genes pertaining to the same regulatory network or the same chromosome.The Basically, the various procedures proposed in the literature aim to test the null hypothesisH0(i) = {gene i is not differentially expressed}.p-values Pi. In the following we will use the classical property: the p-values corresponding to non differentially expressed genes are uniformly distributed over [0, 1]. Furthermore, we will assume, as often, that these p-values are independent. However, the independence of the p-values of differentially expressed genes is not required. Consider a multiple testing situation in which m tests are being performed. Let m0 be the number of non differentially expressed genes. Let I(t) be the set of the genes having a p-value lower than t: I(t) = {i : Pi ≤ t} and R(t) = #I(t), its cardinal. LetLet consider a particular experiment. We observed the differential expression of the genes and compute the associated ordered V(t) = #[I(t) ∩ (i ∈ H0)]andS(t) = #[I(t) ∩ (i ∈ H1)].t, the m genes can be classified according to the following 2 × 2 table Using a threshold The Family Wise Error Rate (FWER) is defined to beP [V(t) ≥ 1].FWER = A classical way to control FWER is given by the Bonferroni inequality. This quantity corresponds to the most direct extension from a test hypothesis procedure but can be very restrictive in a multiple testing procedure.Pi is an unobserved value. It is the same framework as point process (see for example [R(t) = V(t) + S(t) the sum of two counting processes. The first one V(t) is a counting process associated with non differentially expressed gene. Since the p-values under H0 are uniformly distributed, V(t) has a binomial distribution with parameter m0 and t. The intensity of V(t) is constant and proportional to m0. S(t) is the counting process associated with gene under H1 and very few can be said about its distribution. One may expect the intensity of S(t) to be decreasing with t. The false discovery rate is defined as:The status of the gene associated with the  example ). In facIt corresponds to the expected proportion of rejections that are incorrect.P1 < … <Pm denote the ordered p-values. Calculate k = maxi{Pi ≤ αi/m}. The procedure rejects all null hypotheses for which Pi ≤ Pk. If the tests are independent, this procedure ensures thatThe BH procedure works as follows. Let t) be the FDR when rejecting all null hypotheses with Pi ≤ t. Because the p-values of non-differentially expressed genes are uniformly distributed over [0, 1], a natural estimate of FDR(t) isLet FDR and R(t): FDR(t) = E[V(t)/max(R(t), 1)]. The expectation of V(t) is m0t and R(t) is observed. Therefore, Storey [FDR is defined as the expectation of the ratio of two counting processes , Storey  propose E((t, λ)) ≥ FDR(t) using a convexity argument.The ratio of the expectations differs from the expectation ratio but Storey  proved tV(t) and R(t) are counting (i.e. cumulative) processes. It would be very interesting to estimate the ratio of the local intensities of the two processes at point t. The intensity of process V(t) is equal to m0 and thus is known, provided that we know m0. The intensity of process R(t) is unknown, but R(t) is observed. Therefore, using point process methods it is possible to estimate its intensity at each point t.As stated before, t1 to t2:We first define the cumulative processes from t1 <t2, I(t1, t2) = {i : t1 <Pi ≤ t2},Let 0 ≤ R(t1, t2) = #I(t1, t2),V(t1, t2) = #[I(t1, t2) ∩ (i ∈ H0)]andS(t1, t2) = #[I(t1, t2) ∩ (i ∈ H1)].t1, t2) is defined as the expected ratio of V (t1, t2) and R(t1, t2):FDR (t1 = 0 and t2 = t then FDR(t1, t2) = FDR(t). So, the natural estimate of FDR(t1, t2) is:It is a generalization of the usual FDR: if t1 does not change the proof, so using the same convexity argument as Storey [The substitution of 0 by s Storey , we obtaE((t1, t2, λ)) ≥ FDR (t1, t2).t1, t2) for small intervals [t1, t2]. If we want to estimate the local FDR around the p-value of the gene i, the question can be restated as how to estimate the ratio of the intensities of two processes around a given point Pi.The local FDR is the FDR(R(t) has to be estimated at each value of t. It is possible to consider small windows of size h, or alternatively, to consider windows of different sizes corresponding to a fixed count for R(t). We have chosen the latter solution, for windows of variable size seem more appealing in the particular context.The intensity of process i) be the local FDR around Pi. To estimate FDR(i) we need to define a neighborhood around Pi. Let Vi = V(Pi-1, Pi). Remarking that R(Pi-1, Pi) = 1, we have FDR(i) = E(Vi). FurthermoreLet FDR(E(Vi) = P(Vi = 1)Vi is a binary variable. Thus FDR(i) provides an unbiased estimation of P(Vi = 1), the probability for gene i to be a false positive.since i is:The raw local FDR estimate for gene H0(i) and H0(i - 1) are true and E = m0. Therefore this estimate is unbiased with mean 1.Assume that Using definition (1), it is direct to obtain:q-value of gene j. The q-value may thus be viewed as the mean of the raw local FDR of the genes with p-values lower than Pj.which equals the H0, it is known that the differences between successive ordered values of independent realizations of the uniform([0,1]) distribution have a Beta distribution with parameters 1 and m0 2 (m0 + 2)] ≈ 1, for m0 large enough.Under the hypothesis  Johnson  Chap. 26H1 is generally much smaller than under H0  is a very variable estimator.The variance of estimates (1) under Pi-1, Pi[ is not symmetric. If we consider the neighborhood interval around Pi defined by t1 = (Pi-1 + Pi)/2, t2 = (Pi+1 + Pi)/2 then we obtain another estimate of the local FDR:This fact is well known in point process literature, . MoreoveNote that (2) is a moving average of order 2 of (1). It is well known that estimates provided by moving average (or kernel estimators) are more stable, see .i) byThis smoothing is generally not enough to obtain usable results and we can consider any kind of smoothing. We propose to estimate FDR(fi is a smoothing function of the (j, λ) for j = 1, m, computed at position Pi.where The smoothing method must be suited to the properties of the raw FDR:p-values corresponding to highly differentially expressed genes• its variance is low for low p-values corresponding to non differentially expressed genes• its variance is very high for p-values and large for p-values corresponding high p-values. The lowess smoothing method has a fixed number of neighbor points. Therefore its window size depends of the density of points around the p-value under concern. The density of points is higher for low p-values which in turn implies a shorter window size, which is a good property. However the adaptation of the window size is not sufficient in some cases such as in the Apo-AI example. Moreover the smoothed FDR should be an increasing function of the p-values, a property which is not satisfied by the lowess smoothing. Therefore we prefer to use an ad hoc moving average smoothing using the following algorithm for computing (i, λ): let 0 <t1 <t2 <t3 be three pre-definite thresholds and m1 <m2 <m3 <m4 four pre-definite integers.Therefore the window of smoothing should be short for low j≤i (j, λ) <t1 use a moving average of order min(2i - 1, m1)• if maxt1 < maxj≤i (j, λ) <t2 use a moving average of order min(2i - 1, m2)• if t2 < maxj≤i (j, λ) <t3 use a moving average of order min(2i - 1, m3).• if j≤i (j, λ) >t3 use a moving average of order min(2i - 1, m4).• if maxt1 = 0.01, t2 = 0.05, t3 = 0.2, m1 = 3, m2 = 5, m3 = 15 and  with the constraint that (i, λ) is not decreasing. This adaptative moving average method is quite empirical. This topic deserve some more work to build a well assessed smoothing method. This is one of our ongoing research project.We have obtained good empirical results on many data sets with Avner Bar-Hen, Jean-Jacques Daudin and Stephane Robin equally contributed to the statistical work and the redaction task. Julie Aubert coded the R-program and analyzed the three data sets.


Gastrointestinal stromal tumors (GIST) constitute a large majority of mesenchymal tumors of the gastrointestinal (GI) tract, which express the c-kit proto-oncogene protein, a cell membrane receptor with tyrosine kinase activity. GI stromal tumors of the stomach are usually associated with bleeding, abdominal pain or a palpable mass.A 75-year-old male presented with upper abdominal pain and palpable mass. Computed tomographic (CT) scan of the abdomen showed a large mass arising in the posterior aspect of fundus, body, and greater curvature of the stomach. Second day after the admission, there was significant reduction in the size of the tumor, clinically as well as radiologically. Endoscopic biopsy showed large bulge in fundus and corpus of the stomach posteriorly with an opening in the posterior part of the corpus, and biopsy from the edge of the opening reveled GIST. Patient underwent curative resection.Spontaneous ruptured of giant gastric stromal tumor is very rare presentation of stomach GIST. Thorough clinical examination and timely investigation can diagnose rare complication. Gastrointestinal stromal tumors (GIST) are the most common form of mesenchymal tumors arising from the gastrointestinal (GI) wall, mesentery, omentum or retroperitoneum that express the c-kit proto-oncogene protein . This exA 75-year-old diabetic male presented with dull upper abdominal pain of one-week duration. He noticed swelling in left upper abdomen. There was no history of vomiting, fever or gastrointestinal bleeding. He had no significant medical or family history and was non-smoker and non-alcoholic. Physical examination showed a 14 × 10 cm mass palpable in epigastrium and left hypochondrium with minimal intrinsic mobility.Routine biochemical investigations were normal. Ultrasonogram and CT-scan of the abdomen showed large heterogeneous mass of 13 × 10 cm extending from the tail of pancreas to anterior pararenal space, lesser sac to gastrosplenic ligament enveloping the posterior aspect of fundus, body and greater curvature Figure . Fluid aGI stromal tumors express c-kit protein also known as CD 117, and is considered as highly specific marker that differentiates GIST from other mesenchymal tumors such as leiomyomas -10. The Assessment of malignant potential of a primary GIST lesion is difficult in many cases; even as small as less than 2 cm size GIST has certain malignant potentials . Currentet al [Imatinib mesylate, competitive inhibitors of certain tyrosine kinases including the intracellular kinases ABL and BCR-ABL fusion proteins present in some leukemia's, and platelet-derived growth factor receptors , is the et al  had repoet al . The optet al . Role ofSpontaneous ruptured of giant gastric stromal tumor is very rare presentation of stomach GIST. Clinical examination and timely investigations can diagnose this rare complication.The author(s) declare that they have no competing interests.RM, AJ: Preparation of manuscriptNR: performed upper endoscopy and biopsy and helped in preparation of draft manuscript.SOV, SS, PD: surgical management, manuscript revision for scientific contentPD, BV: Revision of manuscript and preparation of final manuscript.All authors read and approved the final version of the manuscript.


Recently, tissue engineering has merged with stem cell technology with interest to develop new sources of transplantable material for injury or disease treatment. Eminently interesting, are bone and joint injuries/disorders because of the low self-regenerating capacity of the matrix secreting cells, particularly chondrocytes. ES cells have the unlimited capacity to self-renew and maintain their pluripotency in culture. Upon induction of various signals they will then differentiate into distinctive cell types such as neurons, cardiomyocytes and osteoblasts.1, insulin and ascorbic acid were identified as signals that together with BMP-2 induce a chondrocytic phenotype that is characterized by increased expression of cartilage marker genes in a timely co-ordinated fashion. Expression of collagen type IIB and aggrecan, indicative of a fully mature state, continuously ascend until reaching a peak at day 32 of culture to approximately 80-fold over control values. Sox9 and scleraxis, cartilage specific transcription factors, are highly expressed at very early stages and show decreased expression over the time course of EB differentiation. Some smaller proteoglycans, such as decorin and biglycan, are expressed at earlier stages. Overall, proteoglycan biosynthesis is up-regulated 7-fold in response to the supplements added. BMP-2 induced chondrocytes undergo hypertrophy and begin to alter their expression profile towards osteoblasts. Supplying mineralization factors such as β-glycerophosphate and vitamin D3 with the culture medium can facilitate this process. Moreover, gene expression studies show that adipocytes can also differentiate from BMP-2 treated ES cells.We present here that BMP-2 can drive ES cells to the cartilage, osteoblast or adipogenic fate depending on supplementary co-factors. TGFβUltimately, we have found that ES cells can be successfully triggered to differentiate into chondrocyte-like cells, which can further alter their fate to become hypertrophic, and adipocytes. Compared with previous reports using a brief BMP-2 supplementation early in differentiation, prolonged exposure increased chondrogenic output, while supplementation with insulin and ascorbic acid prevented dedifferentiation. These results provide a foundation for the use of ES cells as a potential therapy in joint injury and disease. Articular cartilage is composed of extracellular matrix (ECM), the matrix-secreting chondrocyte and water, which all account for the tissue's characteristic rigidity as well as its flexibility. These features are necessary in order to warrant life-long survival of the cartilage tissue, especially in the joint where it has to endure pressure forces caused by movement. Chondrocytes arise from a mesenchymal progenitor during development, the same progenitor that gives rise to other mesenchymal cell types including osteoblasts, adipocytes and myocytes. Bone formation can either be endochondral, when chondrocytes mature and calcify to provide a matrix for the invading osteoprogenitors, or intramembraneous involving ossification directly from a mesenchymal ancestor. All these diverse cell types may arise from the same precursor, but are distinguished by specific morphological features and with that, a certain set of characteristic proteins including transcription factors that control their differentiation.Two chondrocyte-specific transcription factors have been identified, Sox9, a member of the SOX-family of transcription factors, and Scleraxis, a member of the basic helix-loop-helix transcription factors ,2. Howev1 and BMPs [For decades, the treatment of degenerative cartilage and bone diseases has been a challenge for orthopaedic surgeons due to the apparent inability of cartilage and bone to repair itself. Arthritis, a degenerative joint condition, is one of the most prevalent chronic health conditions in North America. Arthritis can devastate people, but to date there is no effective therapy available and patients can only be helped by surgical joint replacement. An inherent major concern is the limited availability of autografts, which significantly reduces the choice of treatable defects. However, new approaches to cell grafting are being developed in this field: increased yields of cells are achieved by the usage of bioreactors and growth factor administration, such as TGFβand BMPs ,9. Addit3 forces ES cells to undergo osteogenesis [Embryonic stem cells represent a valuable source for cell transplantation since their characteristic features include an unlimited self-renewing capacity and a multilineage differentiation potential ,11. In fogenesis . Kramer ogenesis . Those Eogenesis .1, insulin and ascorbic acid leads to improved chondrogenesis in vitro. Compared to the brief supplementation described by Kramer et al. [3 induced ES-derived osteoblasts [3 starting at day 20 rescued the osteoblast phenotype. In many differentiations, we also observed an accumulation of lipid droplets and an up-regulation of adipocyte-specific genes. This direction towards adipocyte differentiation varied with the use of specific co-factors, suggesting that in the future, such spurious differentiation may be controlled, once the pathways involved in adipogenesis are better understood.We show here, that prolonged treatment of differentiating ES cell cultures with BMP-2 in synergy with TGFβr et al. , the expr et al. , ES-derieoblasts . Yet, su1, insulin and ascorbic acid show typical morphological changes compared to the untreated cultures. Starting with the fourth week of culture, aggregates consisting of small round cells formed in the supplemented cultures, which stained positive with alcian blue  evoked a 2-fold increase in collagen II expression for both splice forms. The synergistic effect of both growth factors, TGFβ1 and BMP-2 (combination C) began to show an increased expressions of aggrecan, link protein and COL IIA compared to cultures that were treated with one supplement alone. Additional supplementation with insulin and ascorbic acid between culture days 3 and 5 (combination D) barely increased aggrecan, link protein and COL II expression, but when given from day 5 onwards enhanced both the BMP-2 and TGFβ1 effects (supplement combinations E and H). Addition of insulin and ascorbic acid to BMP-2 induced cultures increased collagen type IIA and aggrecan expression minimally to 1.2-fold, link protein and collagen type IIB were induced 2.7- to 2.8-fold compared to BMP-2 alone. The TGFβ1 response was increased 3- to 4-fold. When cultures were induced with BMP-2 and TGFβ1 in suspension (d3–5) and supplemented with insulin and ascorbic acid starting on day 5 onwards (combination F), aggrecan, link protein and COL II were up-regulated 10-fold compared to controls.Since BMP-2 is believed to play a role in late chondrogenesis , we studng agent , did not1, insulin and ascorbic acid.Surprisingly, when BMP-2 supplementation was maintained throughout the culture period (combination G), a dramatic increase of marker gene expression was seen. Aggrecan and COL IIB were up-regulated over 80-fold above controls and link protein and COL IIA expression was increased to 35- and 16-fold, respectively. Here, scleraxis and Sox9 expression behaved conversely. Supplement combination G showed significant decreases of scleraxis and Sox9 expression to 50 and 87% of the control, respectively (P = 0.001). Deferral of the expression of these transcription factors in favour of chondrogenic differentiation characterizes BMP-2 induced differentiation, whereupon chondrogenic processes are furthermore enhanced by TGFβ1 alone (combination B) did not alter proteoglycan levels compared to controls, and in combination with BMP-2 decreased the proteoglycan content of EBs compared to BMP-2 alone (combination C). Insulin and ascorbic acid did not enhance the proteoglycan synthesis in combination with TGFβ1, but caused a 4.2-fold increase in combination with BMP-2 (combination E, P = 0.01). In agreement with aggrecan gene expression, combinations F and G generated a massive 7-fold induction of proteoglycans in EBs (P = 0.001).The determination of proteoglycan content was used to confirm the results obtained by quantitative RT-PCR for all supplement combinations fig. . TGFβ1 a1 10 ng/ml, BMP-2 10 ng/ml; d3–32: BMP-2 10 ng/ml, ascorbic acid 50 μg/ml and insulin 1 μg/ml]. Green fluorescing chondrocytes in both cultures appeared either organized in clusters or scattered as seen in figure Genetically manipulated ES cells that express GFP under the control of chondrocyte-specific aggrecan promotor were then used to quantify chondrocyte yield using fluorescence-activated cell sorting. ES cells were differentiated along the chondrocytic lineage using BMP-2 at 2 ng/ml or supplement combination G [d3–5: TGFβ1, insulin and ascorbic acid supplementation was then examined over the 35-day culture period using quantitative RT-PCR of various cartilage matrix genes . Previously, we have described the induction of mineralization in osteoblasts derived from ES cells with vitamin D3, which reach maturity at day 32 of culture [3 induced ES-derived osteoblasts could be rescued in the chondrocytes by adding VD3 on day 20 of differentiation, a time when chondrocytes could be morphologically identified in the cultures (11.18 mg/dl, P < 0.001). As shown by quantitative RT-PCR, VD3 treated ES cell derived chondrocytes could alter their expression profile to that of d32 ES cell derived osteoblasts  however, alkaline phosphatase and Cbfa1 were also significantly up-regulated above controls (5.4- and 4-2-fold, respectively, P < 0.001). As we noted earlier, BMP-2 induced osteogenesis with or without VD3 supplementation did not meet the levels that were attained by VD3 alone, but the late addition of VD3 on day 20 rescued bone-specific gene expression arguing for an involvement for BMP-2 in endochondral bone formation.It has been shown by others that the BMP-2 induced alteration in cell fate is both concentration- and time-dependent . Lower cDuring treatment of EBs with various chondrocyte differentiation inducing factors, we noticed accumulation of lipid droplets, which could not be found in untreated controls. Those droplets could indeed be characterized as lipid-containing by means of Oil-Red-O staining fig. . Quantit1. In the early phase of differentiation, BMP-2 operates by directing differentiation towards the cartilage lineage and acting on chondroprogenitors. During later differentiation adding mineralizing agents can trigger hypertrophy and mineralization of the ES-derived chondrocytes. In the embryo, maturation of chondrocytes in the process of endochondral ossification follows a timely regulated developmental program, whereby cellular stages can be delimitated molecularly. During ES cell differentiation into chondrocytes, developmental processes follow the same pattern, as judged by the gene expression patterns observed. Chondrocytes, osteoblasts and adipocytes are thought to arise from the same mesenchymal progenitor. Based on our previous observations around VD3-induced osteogenesis, we have already hypothesized that during the first 5 days of differentiation mesodermal progenitors develop, which then are susceptible to the VD3 treatment [1 at days 3–5 may augment the number of mesenchymal progenitors, as TGFβ1 is thought to inhibit the proliferation of most cells, but to stimulate some mesenchymal cells such as osteoblasts and chondrocytes [1 promoted mesodermal lineage.In this study, we demonstrate an improved method for driving ES cells to a cartilaginous fate when stimulated with BMP-2 and TGFβreatment . Treatmedrocytes . It was in vivo adipogenesis were met in the EBs treated with BMP-2, TGFβ1, insulin and ascorbic acid. Indeed, a future challenge for improving adipogenic cultures will be the discovery of regulatory pathways in adipogenesis. Once identified, such pathways may be antagonized in order to enhance ES differentiation into chondrocytes.The gain of adipose characteristics in culture is hallmarked by: a) the appearance of cytoplasmic lipid droplets, b) the acquisition of insulin sensitivity with regard to glucose uptake (GLUT4) and c) the expression and secretion of numerous bioactive molecules . All of 1, BMP-2, IGF-1 and FGF-2 had no affect on the immediate regulation of these genes. In our differentiation system, Sox9 is elevated very early during differentiation at day 5 (data not shown) underlining its role as an early controller of chondrogenesis. We have shown here that BMP-2 regulates later processes in cartilage development. Marker gene expression levels reached upon overexpression of the Sox trio do not meet the levels we have observed in this study, suggesting that Sox9 may be necessary but not sufficient do direct all progenitors to the chondrocytic lineage.During the revision of this paper, a study was published describing that the overexpression of the sox triad, namely Sox9, Sox5 and Sox6, markedly increased chondrocyte marker gene expression (collagen type 2, aggrecan) in ES cells within 3 days . Here, TEarlier this year, another study portrayed chondrogenesis in ES cells using encapsulation in alginate . The usaArticular cartilage has been refractory to repair following degeneration. Despite its limited capacity to self-repair, cartilage is replete with cells capable of undergoing mitotic division . CurrentPluripotent ES cells are now being contemplated as a new cell source for tissue engineering since they are the most multifaceted cells amongst all stem cells. While their pluripotency offers a huge potential for cell therapies directed against a wide spectrum of degenerative diseases that are ineffectively treated by traditional approaches, it also poses challenge for controlling developmental fate in the recipient. Understanding the developmental pathways regulating ES cell differentiation, will enable the creation of a cell source that can be manipulated to correct for a particular defect.in vitro development of functional cartilage as this represents a first critical step in applying ES cells clinically. Additionally, the presented in vitro model of chondrogenesis may be useful for in vitro embryotoxicity tests [in vitro models of chondrogenesis, this model incorporates all steps of development beginning with a pluripotent, uncommitted cell and thus might further our understanding of normally unamendable stages of development.This study was designed to improve upon the number of differentiated cells needed for the ty tests  as also 1, insulin and ascorbic acid. By examining gene expression responses and cell sorting for GFP-expressing chondrocytes, we demonstrate that using a higher concentration of BMP-2 in combination with appropriate co-factors, we can significantly enhance ES cell derived chondrogenesis compared to known protocols. In addition, we document that ES-derived chondrocytes behave naturally as they undergo hypertrophy.This study was particularly designed to improve chondrocyte yields from ES cells by investigating the effect of higher BMP-2 concentrations and the complementary effects of TGFβin vitro. This observation is consistent with knowledge that adipocytes, chondrocytes and osteoblasts arise from the same mesenchymal ancestor. Accordingly, in the future, it will be necessary to understand how to discriminate these populations for cytotherapeutic applications. At this time, the presented in vitro model allows the study of mechanisms involved in BMP-2 induced chondrogenesis, osteogenesis and adipogenesis.We show that EBs supplemented with BMP-2 also result in a small amount of adipogenesis 1 [2 ng/ml], insulin [1 μg/ml] and ascorbic acid [50 μg/ml] in various combinations on the differentiation of chondrocytes were examined. Medium was changed every second day.Cells of the mouse ES cell line D3 (American Type Culture Collection, Rockville, Maryland, USA) were kept in permanent culture as described ,18 with 2 containing 0.05% Alcian blue for 48 h. Wash steps in 3% acetic acid, 3% acetic acid/25% ethanol and 3% acetic acid/50% ethanol reduced unspecific binding of the dye.Proteoglycans secreted by ES-cell derived chondrocytes were stained with Alcian blue. Cultures were fixed in 2.5% glutaraldehyde, 25 mM sodium acetate, 0.4 M MgClProteoglycan content of differentiated cultures was determined with the DMMB-assay . Proteog1, insulin and ascorbic acid and fixed on day 32 with ice-cold methanol:aceton (7:3) at -20°C for 10 min. For staining with anti-collagen type II (Chemicon, MAB 8887) cultures were digested with pepsin for 15 min at 37°C. Staining with anti-adult proteoglycan (Chemicon MAB 2015) was performed after a 1 h digest with chondroitinase ABC at room temperature. Cells were overlaid with the appropriate dilution of the first antibodies in PBS, 10% FCS at 4°C over night. The corresponding secondary antibody, an AlexaFluor 488 goat anti-mouse IgG (H+L), F(ab')2 fragment (A-11006, Molecular Probes, Leiden, The Netherlands) was incubated with the cells for 2 h at room temperature. Cultures were observed in a Leica Fluovert FU fluorescent microscope (Leitz, Wetzlar) with an excitation wavelength of 495 and an emission wavelength of 519 nm.Embryoid bodies were differentiated to the chondrocyte lineage with BMP-2, TGFβ® 7700 Sequence Detector. The accumulation of reaction products during PCR was monitored by measuring the increase in fluorescence caused by the binding of SYBR® Green (Applied Biosystems, Perkin Elmer, Weiterstadt, Germany) to double-stranded DNA. Expression analysis of collagen type II A and B was done in the same PCR run with two differently labelled probes specific for either the A isoform (5'-CGAGATCCCCTTCGGAGAGTGCTGT-3'/VIC) or the B isoform (5'-CCAGGATGCCCGAAAATTAGGGCCAA-3'/FAM) [T-values were standardized against GAPDH expression and induction of expression in treated EBs was normalized to control EBs. Primer sequences for murine GAPDH were 5'-GCACAGTCAAGGCCGAGAAT-3' and 5'-GCCTTCTCCATGGTGGTGAA-3' (Ta = 60°C).Total RNA was isolated from 20 EBs per probe using the RNeasy Midi Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions with on-column DNase I digestion. The amount of RNA was determined using the RiboGreen™ RNA quantitation reagent and kit (Molecular Probes). 275 ng total EB RNA was used as a template for cDNA synthesis with Superscript II (Invitrogen, Paisley, Scotland) as described  in a totThe GFP expressing plasmid pEGFP-1 (Clontech) under the control of the Aggrecan promotor (kind gift of John R. Matyas) was stably transfected into D3 ES cells using Invitrogen's Effectene system followed by neomycin selection. Genomic integration of the reporter was analyzed by RT-PCR with specific GFP primers (data not shown). ES cells were differentiated along the chondrocyte lineage, trypsinized into a single cell suspension and subjected to fluorescence-activated cell sorting on day 32 using a FACS Calibur instrument and the CellQuest software from Becton Dickinson (Germany). Ten thousand events were registered per sample and analysis of whole cells was performed using appropriate scatter gates to avoid cellular debris and aggregates.Cells were washed with PBS and without any fixation directly overlaid with Oil-red-O working solution (0.18% Oil-Red-O dye/60% propanol) After a staining period of 15 minutes, ES cell cultures were rinsed in distilled water until desired colour was achieved.Data analysis was performed with ONE-WAY ANOVA on n = 3 experiments using SigmaStat version 2.03 (SPSS Inc., San Rafael, CA, USA).ADD1 Adipocyte determination- and differentiation-dependent factor 1aP2 Fatty acid-binding proteinBMP Bone Morphogenetic ProteinCbfa1 Core binding factor alpha 1C/EBP CCAAT/enhancer-binding proteinCol CollagenDMMB DimethylmethylenblueECM extracellular matrixES embryonic stemEB embryoid bodyFCS Fetal calf serumFGF Fibroblast growth factorGAPDH Glyceraldehyde-3-phosphate dehydrogenaseGLUT4 Glucose transporter 4IGF Insulin-like growth factorPPAR Peroxisome proliferator-activated receptorSCD Steroyl CoA desaturaseSox Sry-related high mobility group boxTGFβ Transforming Growth Factor beta3 Vitamin D3VDNZN carried out cell culture, biochemical and molecular studies and drafted the manuscript. GK, DER and HJA participated in its design and coordination. All authors read and approved the final manuscript.


A paper published over 20 years ago by Susan Iversen and Mortimer Mishkin on reversal learning continues to inform cognitive neuroscience today One of the hallmarks of human nature is our remarkably flexible behaviour, especially in the social domain, which is perhaps also a major reason for our relative evolutionary success. Our social skills are already being honed in childhood and early adolescence, when we quickly become very adept at forming and breaking alliances within and between groups and spend much of our time engaged in complex social interactions. At best, these interactions enrich our society; at worst, they become ‘Machiavellian’ and exploitative. While science might appear removed from such politics, many scientists would probably agree that science is in fact a social enterprise, sharing many characteristics with other human pursuits, and that any claim to greater scientific truth can only be accorded over decades, even centuries.I have always been fascinated by social intelligence, particularly of the ‘Machiavellian’ kind, and found myself wondering at the start of my doctoral research how one might use neuroimaging to study social intelligence in the human brain. I was also interested by the fact that some of this flexible behaviour is shared with other primates such as chimps, bonobos, and even monkeys, who also spend inordinate amounts of time in social interactions, working out social hierarchies. However, it was not immediately obvious how one might go about designing experiments that would address these somewhat intangible issues of social behaviour.Trawling the scientific literature, I came across the concept of reversal learning. While it is obviously important that we can learn arbitrary associations between stimuli and actions, it is also extremely important that we can relatively easily break these associations and learn others. If we learn that choosing a certain object leads to a reward, it would be rather maladaptive to keep choosing this object when it was no longer associated with a reward but, say, a punishment instead. In order to accommodate complex behaviour, we need to be able to adapt or reverse the learning patterns when things change.For a long time, it was thought that complex behaviour depended crucially on the prefrontal cortex of the brain, but it was not clear which parts were important for reversal learning. This was investigated in a classic paper by the eminent neuroscientists Susan This was not the case for monkeys who had had the medial parts of the orbitofrontal cortex lesioned. These monkeys were not completely unaffected by the lesion, but showed moderate impairment on all but the first of the object discrimination reversals. Furthermore, they had moderate difficulty withholding response between trials on an auditory differentiation task. These results strongly suggested a differential role for the lateral and medial parts of the orbitofrontal cortex.Although the paper was not published in a high-profile journal, this elegant and very significant result has had a huge influence on subsequent research. The paper, like many other great papers, was ahead of its time, and it took almost a decade before the citations started to pick up (at last online count, on February 1, 2004, of the ISI database, the paper had generated 229 citations since 1981).There was also some evidence that humans with lesions to the orbitofrontal cortex have problems with reversal learning, but the lesions, caused by neurological insult, were not very clean or focal . In addiThese interesting but nonconclusive results in humans spurred us on to use neuroimaging on a modified version of a probabilistic reversal learning task designed by Julia Hornak and John O'Doherty , whose pWe used functional magnetic resonance imaging to show that dissociable activity in the medial orbitofrontal cortex was correlated with the magnitude of the monetary gains received, while activity in the lateral orbitofrontal cortex was correlated with the monetary losses incurred . This diOne way to solve these problems was to use facial expressions rather than money as the reinforcing stimuli. This made sense, given that the key to social intelligence is the ability to detect subtle changes in communication and act upon these changes rapidly as they occur. Such changes in social behaviour are often based on facial expression and come so naturally to humans (and are in place so early in child development) that some might argue that this functionality is essentially innate. However, our human social behaviour is sufficiently flexible that we can easily learn to adapt our behaviour to most facial expressions. For example, other people's neutral expressions do not normally indicate that our behaviour should change, but it is easy to think of social contexts in which a neutral expression does indeed imply that our current behaviour is inappropriate and should change.I designed a reversal task in which the subject's overall goal was to keep track of the mood of two people presented in a pair and, as much as possible, to select the ‘happy’ person, who would then smile. Over time, the person with the ‘happy’ mood (who would smile when selected), changed his/her mood to ‘angry’. This person thus no longer smiled when selected, but instead changed to a facial expression that signalled that he/she should no longer be selected. In the main reversal task, the facial expression used to cue reversal was an angry expression , while in the second, control, version of the reversal task, a neutral expression was used. By using two different reversal tasks in which different facial expressions signalled that behaviour must change, we were able to determine which brain areas were specific to general reversal learning, rather than just to reversal following a particular expression, such as anger.We used functional magnetic resonance imaging to show that the ability to change behaviour based on facial expression is not reflected in the activity of the fusiform face area , but that general reversal learning is specifically correlated with activity in the lateral orbitofrontal and anterior cingulate/paracingulate cortices  .This result confirmed and extended the results from Iversen and Mishkin's original paper. Further confirmation came from the neuropsychological testing, carried out by Julia Hornak on human patients with surgical lesions to the orbitofrontal cortex, which showed that bilateral  lesions to the lateral orbitofrontal cortex produce significant impairments in reversal learning . Yet, asThe Prince, Niccolò Machiavelli offers a rather pessimistic view on human nature, in which ‘love is held by a chain of obligation which, since men are bad, is broken at every opportunity for personal gain’. It may be that our capacity for rapid reversal learning is sometimes used for less than noble pursuits, both in science and in interpersonal relations in general, but we would be in real trouble if we couldn't learn to change.In his masterpiece,


In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholders It is a great pity when vested interest and dogma dominate what should be a well-informed and rational debate. The essay by In the United Kingdom, we have had an almost continuous debate since the mid 1980s on topics relating to research on early human embryos. I myself have been involved in some of this debate, especially over the last few years, relating to human embryonic stem cells and nuclear transfer. I will not dwell on the political outcomes of this debate, which are widely known, but I want to stress that it has been one that has been very well informed, with contributions from all sides, including many highly respected moral philosophers and bioethicists. These include notable individuals such as Dame Mary Warnock and bodies such as the Nuffield Bioethics Council, who have been especially valuable because of their independence.So why are the conclusions reached by bioethicists in the UK, who are generally supportive of research involving human embryos, different from those of the President's Council on Bioethics? The same scientific information is available on both sides of the Atlantic. The rules of logic are the same. So it has to be the way the information is interpreted or filtered. This implies bias or vested interest or the input of dogma that is based on belief rather than rational thought. Some examples of this are discussed in the Blackburn and Rowley essay, and they are very worrying. The scare mongering about preimplantation genetic diagnosis is ridiculous—simple mathematics shows that it is implausible to use this technique to screen the usual number of embryos obtained in one round of in vitro fertilisation for more than two or three genetic traits, while we know that intelligence must rely on many more. I am a great fan of science fiction, but I can recognise it as such. I worry that some members of the President's Council seem unable to do this. Many of these daft ideas were already promoted in a book by Francis It is certainly very unfortunate if the input of real science in the Council is to be reduced. The scientific issues are complex. For example, we certainly do not know nearly enough about either adult or embryonic stem cells to say which will be the best for therapies, and of course it is possible that both will turn out to be useful for different problems. Both also offer exciting new ways to explore human disease and the influence of genetics and environment without having to rely on human experimentation. But any committee looking into what is ethically acceptable has to be provided with a balanced view of what will be possible in the near future. There is no point in being too speculative, in part because it is also difficult to predict what will be ethically acceptable in the future. If cures come from the use of human embryonic stem cells, then I suspect that there will be widespread acceptance, as happened with heart transplants and with in vitro fertilisation, both of which were initially greeted with horror by many.It is impossible to have an informed debate without accurate and appropriate information, and there seems little point in having a debate that is not informed. Because of various sensitivities, it seemed to me before the creation of the President's Council on Bioethics that for far too long the issues relating to embryo research had not been considered properly within the United States. The President's Council was therefore an opportunity to redress this situation. But from the evidence I fear it will not succeed. Moreover, it does the general public a disservice to pretend to have a serious committee exploring issues of bioethics when that committee fails to live up to the ideals of impartiality and rationality.


Sequence-based typing (SBT) is one of the most comprehensive methods utilized for HLA typing. However, one of the inherent problems with this typing method is the interpretation of ambiguous allele combinations which occur when two or more different allele combinations produce identical sequences. The purpose of this study is to investigate the probability of this occurrence. We performed HLA-A,-B SBT for Exons 2 and 3 on 676 donors. Samples were analyzed with a capillary sequencer. The racial distribution of the donors was as follows: 615-Caucasian, 13-Asian, 23-African American, 17-Hispanic and 8-Unknown. 672 donors were analyzed for HLA-A locus ambiguities and 666 donors were analyzed for HLA-B locus ambiguities. At the HLA-A locus a total of 548 total ambiguous allele combinations were identified (548/1344 = 41%). Most (278/548 = 51%) of these ambiguities were due to the fact that Exon 4 analysis was not performed. At the HLA-B locus 322 total ambiguous allele combinations were found (322/1332 = 24%). The HLA-B*07/08/15/27/35/44 antigens, common in Caucasians, produced a large portion of the ambiguities (279/322 = 87%). A large portion of HLA-A and B ambiguous allele combinations can be addressed by utilizing a group-specific primary amplification approach to produce an unambiguous homozygous sequence. Therefore, although the prevalence of ambiguous allele combinations is high, if the resolution of these ambiguities is clinically warranted, methods exist to compensate for this problem. The precise identification of HLA Class I and Class II alleles is critical for successful hematopoietic progenitor transplants, the development of peptide based viral and cancer vaccines, and investigating immune response ,2. DNA sThere exist two main types of ambiguous typing results obtained with SBT. The first is when a heterozygous sequence can be explained by more than one possible pair of alleles within the region analyzed. The second exists when alleles are defined by a polymorphism outside the region analyzed. In addition to these two situations, a third type of ambiguity arises when an allele has an incomplete sequence in the region analyzed.The prevalence of ambiguities in HLA typing relates to the nature of polymorphisms which exists in the sequence of the major histocompatibility complex (MHC) Class I and Class II genes. The majority of polymorphisms that distinguish one MHC allele from another are oftentimes due to gene conversion, recombination and exon shuffling events. Due to this, polymorphic motifs at given positions are generally shared among several alleles.Sequence-based typing involves PCR amplification and sequencing of specific HLA exons, which are known to be polymorphic, from genomic DNA. For each HLA locus both alleles are amplified and sequenced; therefore, it is not always possible to determine exactly which two alleles were responsible for sequence results. For example two or more different allele combinations can combine to produce identical sequences due to the heterozygous base pair combinations, the first type of ambiguity. More specifically, in the Class I region, HLA-B*070201, 3503 would have the same nucleotide sequence as HLA-B*0724, 3533 in positions 559 and 560 . However, for investigations of genetic inheritance or disease association, the definition of all polymorphisms may be significant.The purpose of this study was to summarize the incidence, nature, and cause of ambiguous HLA SBT results. This represents an important step toward developing strategies to reduce or eliminate this problem.® isolation kit (Gentra Systems, Minneapolis, MN, U.S.A). The DNA was resuspended in Tris HCl buffer (pH 8.5) and the concentration was measured using a Pharmacia Gene Quant II Spectrophotometer. The DNA was then stored at -70°C until testing.Genomic DNA was isolated from peripheral blood using the Gentra PUREGENE® 3700 DNA Analyzer with Dye Set file: Z and mobility file: DT3700POP6 {ET}.The primary PCR amplification reaction consists of a 1.5 kb reaction encompassing exon 1 through intron 3 of the HLA region. All reagents necessary for primary amplification and sequencing are supplied in the HLA-A or HLA-B AlleleSEQR Sequenced Based Typing Kits . The primary amplification PCR products were purified from excess primers, dNTPs, and genomic DNA using ExoSAP-IT (Amersham Life Science, Cleveland, OH, U.S.A.) Each template was sequenced in the forward and reverse sequence orientation for exon 2 and Exon 3 according to protocols supplied with the SBT kit. Excess dye terminators were removed from the sequencing products utilizing an ethanol precipitation method with absolute ethanol. The reaction products were reconstituted with 15 μl of Hi-Di™ Formamide (PE Applied Biosystems/Perkin-Elmer, Foster City, CA, U.S.A.) and analyzed on the ABI PrismSequence based typing analysis of HLA-A and B alleles was performed on a population of 676 normal donors. The racial distribution of the subjects studied was: 615 Caucasian, 13 Asian, 23 African American, 17 Hispanic and 8 Unknown. 672 of the 676 subjects were analyzed for the presence of HLA-A locus ambiguities and 666 were analyzed for HLA-B locus ambiguities. Each allele was counted separately in this analysis in order to determine the total percentage of ambiguous allele combinations. Four new potential alleles were found.At the HLA-A locus a total of 548 ambiguous allele combinations were found. This represented 41% of all HLA-A alleles 548 of 1344)  of the HLA-A ambiguities cannot be resolved with Exon 4 analysis Table .Review of the HLA-B locus results revealed 322 ambiguous allele combinations among the 1332 total HLA-B alleles 24%). Antigens HLA-B*07/08/15/27/35/44, common in Caucasians, produced the largest portion of the ambiguities (279 of 322 or 87%). Each of these ambiguities had an independent reason for occurring. Table %. Antige maintains an updated listing of all ambiguous possibilities [While SBT provides the best available typing of HLA-A and B antigens, it is limited by sequence results that don't allow the precise identification of alleles. We found that 41% of HLA-A alleles and 24% of HLA-B alleles were ambiguously typed. The ambiguities involve some of the most frequent HLA-A and HLA-B antigens: A*01, A*02, A*24, B*07, B*08, B*15, B*27, B*35, and B*44. However, ambiguous allele combinations occur in all loci tested in HLA. The IMGT/HLA Sequence Database The need to initiate additional testing to clarify ambiguous allele combinations must consider whether it is practical to obtain the information and if the information is useful and valuable. The clinical need for the highest resolution HLA typing possible is an important variable that must be considered. When typing is performed for cancer and viral vaccine development studies, high resolution allele data may be necessary to determine if a subject has an HLA type that is appropriate for a study. Utilization of high resolution data may also have implications for hematopoietic progenitor cell transplantation. Transplants involving partially mismatched or unrelated donor-recipient pairs require a higher resolution typing, but those involving HLA identical siblings may not.If it is necessary to resolve an ambiguous typing, a variety of different methods can be used. If the ambiguity is due to an allele that has not been completely sequenced or because the ambiguity is outside the region amplified by the SBT assay, the resolution is dependent on the nature and complexity of the ambiguity. Traditionally, for Class I sequencing purposes most laboratories have performed Exon 2 and Exon 3 analysis alone and for Class II sequencing only Exon 2 analysis. Many of the ambiguities can be resolved by sequencing Exon 4. In fact, in this study the largest portion of the typing ambiguities can be resolved by sequencing exon 4. However, many polymorphisms in exon 4 have no functional significance, so it may not be worthwhile resolving most ambiguities involving exon 4. The requirement for the analysis of Exon 4 to reduce the incidence of typing ambiguity has now been realized by commercial kit manufacturers. Both Celera Diagnostics (Alameda, CA) and Forensic Analytical/Atria Genetics (South San Francisco, CA) now include reagents for analysis of exon 4 in the HLA-A and -B kits. As the use of SBT increases, more data may become available from non-traditional exons in addition to those that have been traditionally sequenced and the number of ambiguities due to unknown sequences will decrease.If the ambiguity is due to an identical heterozygote sequence, as shown in figure Other methods which have been utilized to produce a homologous sequence include cloning, reference strand conformational analysis (RSCA), Pyrosequencing™ and denaturing high-performance liquid chromatography (DHPLC) -11. PyroIn conclusion, although the prevalence of ambiguous allele combinations is high, methods exist to compensate for this problem. As the HLA field continues the discovery of new alleles, alternative approaches to discerning ambiguous allele combinations will need to be investigated in order to reduce the ever-growing number of ambiguities.