MULTIPRED: A computational system for prediction of promiscuous HLA binding peptides

MULTIPRED is a web-based computational system for the prediction of peptide binding to multiple molecules ( proteins) belonging to human leukocyte antigens (HLA) class I A2, A3 and class II DR supertypes. It uses hidden Markov models and artificial neural network methods as predictive engines. A novel data representation method enables MULTIPRED to predict peptides that promiscuously bind multiple HLA alleles within one HLA supertype. Extensive testing was performed for validation of the prediction models. Testing results show that MULTIPRED is both sensitive and specific and it has good predictive ability ( area under the receiver operating characteristic curve A(ROC) > 0.80). MULTIPRED can be used for the mapping of promiscuous T-cell epitopes as well as the regions of high concentration of these targets termed T-cell epitope hotspots. MULTIPRED is available at http:// antigen.i2r.a-star.edu.sg/ multipred/.

CandiVF - Candida albicans virulence factor database

Candida albicans is a pathogen commonly infecting patients who receive immunosuppressive drug therapy, long-term catheterization, or those who suffer from acquired immune deficiency syndrome (AIDS). The major factor accountable for pathogenicity of C. albicans is host immune status. Various virulence molecules, or factors, of are also responsible for the disease progression. Virulence proteins are published in public databases but they normally lack detailed functional annotations. We have developed CandiVF, a specialized database of C. albicans virulence factors (http://antigen.i2r.a-star.edu.sg/Templar/DB/CandiVF/) to facilitate efficient extraction and analysis of data aimed to assist research on immune responses, pathogenesis, prevention, and control of candidiasis. CandiVF contains a large number of annotated virulence proteins, including secretory, cell wall-associated, membrane, cytoplasmic, and nuclear proteins. This database has in-built bioinformatics tools including keyword and BLAST search, visualization of 3D-structures, HLA-DR epitope prediction, virulence descriptors, and virulence factors ontology.

A side order of stem cells: The SP phenotype

A defining property of murine hematopoietic stein cells (HSCs) is low fluorescence after staining with Hoechst 33342 and Rhodamine 123. These dyes have proven to be remarkably powerful tools in the purification and characterization of HSCs when used alone or in combination with antibodies directed against stem cell epitopes. Hoechst low cells are described as side population (SP) cells by virtue of their typical profiles in Hoechst red versus Hoechst blue bivariate fluorescent-activated cell sorting dot plots. Recently, excitement has been generated by the findings that putative stem cells from solid tissues may also possess this SP phenotype. SP cells have now been isolated from a wide variety of mammalian tissues based on this same dye efflux phenomenon, and in many cases this cell population has been shown to contain apparently multipotent stem cells. What is yet to be clearly addressed is whether cell fusion accounts for this perceived SP multipotency. Indeed, if low fluorescence after Hoechst staining is a phenotype shared by hematopoietic and organ-specific stem cells, do all resident tissue SP cells have bone marrow origins or might the SP phenotype be a property common to all stem cells? Subject to further analysis, the SP phenotype may prove invaluable for the initial isolation of resident tissue stem cells in the absence of definitive cell-surface markers and may have broad-ranging applications in stem cell biology, from the purification of novel stem cell populations to the development of autologous stem cell therapies.

Papillomavirus virus-like particles activate the PI3-kinase pathway via alpha-6 beta-4 integrin upon binding

We have previously shown that human papillomavirus virus-like particles (VLPs) are able to activate the Ras/MAP kinase pathway. Ras can also elicit an anti-apoptotic signal via PI3-kinase so we investigated this further. Here we show that binding of VLPs from HPV types 6b, 18, 3 1, 35 and BPV1 results in activation of PI3-kinase. Activation was achieved by either L1 or L1/L2 VLPs and was dependent on both VLP-cell interaction and correct conformation of the virus particle. VLP-induced PI3-kinase activity resulted in efficient downstream signaling to Akt and consequent phosphorylation of FKHR and GSK3 beta. We also present evidence that PV signaling is activated via the alpha 6 beta 4 integrin. These data suggest that papillomaviruses use a common receptor that is able to signal through to Ras. Combined activation of the Ras/MAP kinase and PI3-kinase pathways may be beneficial for the virus by increasing cell numbers and producing an environment more conducive to infection. (c) 2006 Elsevier Inc. All rights reserved

The effects of oxygen free radicals on the carbohydrate moiety of IgG

The CH2-linked glycoform of rheumatoid IgG is abnormal in having a reduced galactose content. This has been postulated to be a synthetic defect due to a decrease in the level of rheumatoid B cell galactosyltransferase. However, more recent work has indicated that agalactosylation may be common to chronic inflammatory diseases. In this work we have investigated the effect of oxygen free radicals (OFRs), which are generated by activated phagocytic cells at inflammatory sites, on the carbohydrate moiety of IgG. Radiolytically generated peroxy (ROO.) and hydroxyl radicals (OH.) but not superoxide anion radicals (O2.-) were found to destroy galactose on IgG. After OH. attack, this was associated with an increase in the availability of N-acetylglucosamine, possibly due to its presence as a terminal residue. These results suggest that the agalactosylation associated with chronic inflammation may not only be synthetic in nature, but may also be a consequence of post-synthetic degradation by OFRs.

MHC class II binding prediction:a little help from a friend

Vaccines are the greatest single instrument of prophylaxis against infectious diseases, with immeasurable benefits to human wellbeing. The accurate and reliable prediction of peptide-MHC binding is fundamental to the robust identification of T-cell epitopes and thus the successful design of peptide- and protein-based vaccines. The prediction of MHC class II peptide binding has hitherto proved recalcitrant and refractory. Here we illustrate the utility of existing computational tools for in silico prediction of peptides binding to class II MHCs. Most of the methods, tested in the present study, detect more than the half of the true binders in the top 5% of all possible nonamers generated from one protein. This number increases in the top 10% and 15% and then does not change significantly. For the top 15% the identified binders approach 86%. In terms of lab work this means 85% less expenditure on materials, labour and time. We show that while existing caveats are well founded, nonetheless use of computational models of class II binding can still offer viable help to the work of the immunologist and vaccinologist.

Unexpected role of surface transglutaminase type II in celiac disease

Background & Aims: In celiac disease (CD), transglutaminase type II (TG2) has 2 fundamental roles: (1) as the autoantigen recognized by highly specific autoantibodies and (2) the modifier of pathogenic gliadin T-cell epitopes. It follows that inhibition of TG2 might represent an attractive strategy to curb the toxic action of gliadin. Here we studied the validity of this strategy using the organ culture approach. Methods: Duodenal biopsy specimens from 30 treated patients with CD, 33 untreated patients with CD, and 24 controls were cultured with or without gliadin peptides p31-43, pα-9, and deamidated pα-9 for 20 minutes, 3 hours, and 24 hours. In 31 patients with CD and 16 controls, TG2 inhibitor R283 or anti-TG CUB 7402 or anti-surface TG2 (6B9) mAbs were used in cultures. T84 cells were also cultured with or without peptides with or without TG inhibitors. Mucosal modifications after culture were assessed by immunofluorescence, in situ detection of TG activity, confocal microscopy, and fluorescence-activated cell sorter analysis. Results: The enzymatic inhibition of TG2 only controlled gliadin-specific T-cell activation. The binding of surface TG2 contained gliadin-specific T-cell activation and p31-43-induced actin rearrangement, epithelial phosphorylation, and apoptosis, both in organ cultures and T84 cells. Conclusions: These data indicate a novel and unexpected biological role for surface TG2 in the pathogenesis of CD suggesting a third role for TG2 in CD. These results have a specific impact for celiac disease, with wider implications indicating a novel biologic function of TG2 with possible repercussions in other diseases. © 2005 by the American Gastroenterological Association.

Macrophages and lymphocyte responiveness to mitogen

Purified B-cells fail to proliferate in response to the strong thymus-independent (TI) antigen Lipopolysaccharide (LPS) in the absence of macrophages (Corbel and Melchers, 1983). The fact that macrophages, or factors derived from them are required is supported by the inability of marginal zone B-cells in infants to respond to highly virulent strains of bacteria such as Neisseria meningitidis and Streptococcus pneumoniae (Timens, 1989). This may be due to the lack of CD21 expression on B-cells in infants which could associate with its co-receptor (C3d) on adjacent macrophages. It is not clear whether cell surface contacts and/or soluble products are involved in lymphocyte-macrophage interactions in response to certain antigens. This thesis describes the importance of the macrophage in lymphocyte responses to T-dependent (TD) and TI antigens. The major findings of this thesis were as follows: (1). Macrophages were essential for a full proliferative response to a range of T - and B-cell mitogens and TI-1 and TI-2 antigens, including Concanavalin A, LPS, Pokeweed mitogen (PWM), Dextran sulphate, Phytohaemagglutinin-P (PHA-P) and Poly[I][C]. (2). A ratio of 1 macrophage to 1000 lymphocytes was sufficient for the mitogens to exert their effects. (3). The optimal conditions were established for the activation of an oxidative burst in cells of the monocyte/macrophage lineage as measured by luminometry. The order of ability was OpZ >PMA/lonomycin >f-MLP >Con A >DS >PHA >Poly[I][C] >LPS >PWM. Responses were only substantial and protracted with OpZ and PMA. Peritoneal macrophages were the most responsive cells, whereas splenic and alveolar macrophages were significantly less active and no response could be elicited with Kupffer cells, thus demonstrating heterogeneity between macrophages. (4). Activated macrophages that were then fixed with paraformaldehyde were unable to restore mitogenic responsiveness, even with a ratio of 1 macrophage to 5 lymphocytes. (5). Although highly purified T- and B-cells could respond to mitogen provided live macrophages were present, maximum activation was only observed when all 3 cell types were present. (6). Supernatants from purified macrophage cultures treated with a range of activators were able to partially restore lymphocyte responses to mitogen in macrophage-depleted splenocyte cultures, and purified T - and B-cell cultures. In fact supernatants from macrophages treated with LPS for only 30 minutes could restore responsiveness. Supernatants from OpZ treated macrophages were without effect. (7). Macrophage supernatants could not induce proliferation in the absence of mitogen. They therefore provide a co-mitogenic signal required by lymphocytes in order to respond to mitogen. (8). Macrophage product profiles revealed that LPS and Con A-treated macrophage supernatants showed elevated levels of IL-1β, TNF -α L TB4 and TXB2. These products were therefore good candidates as the co-mitogenic factor. The possible inhibitory factors secreted by OpZ-treated macrophages were PGE2, IL-10 and NO. (9). The removal of cytokines, eicosanoids and TNF-α from LPS-treated macrophage supernatants using Cycloheximide, Dexamethasone and an MMPI respectively, resulted in the inability of these supernatants to restore macrophage-depleted lymphocyte responses to mitogen. (10). rIL-1β and rTNF-α are co-mitogenic factors, as macrophage-depleted lymphocytes incubated with rIL-1β and rTNF-α can respond to mitogen.

Antenatal corticosteroids impact the inflammatory rather than the antiangiogenic profile of women with preeclampsia

Circulating antiangiogenic factors and proinflammatory cytokines are implicated in the pathogenesis of preeclampsia. This study was performed to test the hypothesis that steroids modify the balance of inflammatory and proangiogenic and antiangiogenic factors that potentially contribute to the patient’s evolving clinical state. Seventy singleton women, admitted for antenatal corticosteroid treatment, were enrolled prospectively. The study group consisted of 45 hypertensive women: chronic hypertension (n=6), severe preeclampsia (n=32), and superimposed preeclampsia (n=7). Normotensive women with shortened cervix (<2.5 cm) served as controls (n=25). Maternal blood samples of preeclampsia cases were obtained before steroids and then serially up until delivery. A clinical severity score was designed to clinically monitor disease progression. Serum levels of angiogenic factors (soluble fms-like tyrosine kinase-1 [sFlt-1], placental growth factor [PlGF], soluble endoglin [sEng]), endothelin-1 (ET-1), and proinflammatory markers (IL-6, C-reactive protein [CRP]) were assessed before and after steroids. Soluble IL-2 receptor (sIL-2R) and total immunoglobulins (IgG) were measured as markers of T- and B-cell activation, respectively. Steroid treatment coincided with a transient improvement in clinical manifestations of preeclampsia. A significant decrease in IL-6 and CRP was observed although levels of sIL-2R and IgG remained unchanged. Antenatal corticosteroids did not influence the levels of angiogenic factors but ET-1 levels registered a short-lived increase poststeroids. Although a reduction in specific inflammatory mediators in response to antenatal steroids may account for the transient improvement in clinical signs of preeclampsia, inflammation is unlikely to be the major contributor to severe preeclampsia or useful for therapeutic targeting.

Leptin decreases apoptosis and alters BCL-2:bax ratio in clonal rodent pancreatic beta-cells

The adipocyte derived peptide hormone leptin is known to regulate apoptosis and cell viability in several cells and tissues, as well as having several pancreatic islet beta-cell specific effects such as inhibition of glucose-stimulated insulin secretion. This study investigated the effects of leptin upon apoptosis induced by serum depletion and on expression of the apoptotic regulators B-cell leukaemia 2 gene product (BCL-2) and BCL2-associated X protein (Bax) in the glucose-responsive BRIN-BD11 beta-cell line.

Significant alterations in peripheral blood lymphocyte subsets in patients with somatoform disorder

Objective: Previous studies have suggested that somatoform disorders (SFD) might be associated with changes in the function of the central and autonomic nervous systems. The aim of this study was to examine the possible immunological differences between SFD and healthy controls. Methods: Twenty-four patients with SFD and 13 healthy individuals completed the psychological questionnaires to assess symptom reporting [Symptom Checklist-90 Revised (SCL-90-R)] and to diagnose for SFD [Screening for Somatoform Symptoms scale (SOMS-scale)]. Participants also provided a blood sample taken in the morning, which was analysed with an automated cell counter to determine the number of leucocytes per μl and with flow cytometry to determine lymphocyte subsets. Results: With the exception of a higher T4/T8 ratio in the patient group, which was mainly because of lower CD8 counts, there were no significant differences in the absolute number of lymphocytes (subsets) between patients with SFD and healthy subjects. A positive correlation between B-lymphocyte subsets (CD19+CD22+, CD19+CD5+, CD19+CD3-) to all scales of the SCL-90-R, except somatisation, were found in SFD. Additionally, a positive correlation was found in SFD between CD14+CD16+ monocytes and somatisation (0.573) on the SCL-90-R scale. Conclusion: These data indicate that patients with SFD have an enhanced humoral immunity as shown by increased B-cell numbers and furthermore an elevated T4/T8 ratio because of lower CD8 suppressor cells. Further studies will be required to determine whether these alterations in lymphocyte subsets are directly involved in the pathophysiology of SFD. © 2007 Blackwell Munksgaard.

Predicting class II MHC-Peptide binding:a kernel based approach using similarity scores

Background - Modelling the interaction between potentially antigenic peptides and Major Histocompatibility Complex (MHC) molecules is a key step in identifying potential T-cell epitopes. For Class II MHC alleles, the binding groove is open at both ends, causing ambiguity in the positional alignment between the groove and peptide, as well as creating uncertainty as to what parts of the peptide interact with the MHC. Moreover, the antigenic peptides have variable lengths, making naive modelling methods difficult to apply. This paper introduces a kernel method that can handle variable length peptides effectively by quantifying similarities between peptide sequences and integrating these into the kernel. Results - The kernel approach presented here shows increased prediction accuracy with a significantly higher number of true positives and negatives on multiple MHC class II alleles, when testing data sets from MHCPEP [1], MCHBN [2], and MHCBench [3]. Evaluation by cross validation, when segregating binders and non-binders, produced an average of 0.824 AROC for the MHCBench data sets (up from 0.756), and an average of 0.96 AROC for multiple alleles of the MHCPEP database. Conclusion - The method improves performance over existing state-of-the-art methods of MHC class II peptide binding predictions by using a custom, knowledge-based representation of peptides. Similarity scores, in contrast to a fixed-length, pocket-specific representation of amino acids, provide a flexible and powerful way of modelling MHC binding, and can easily be applied to other dynamic sequence problems.

Antenatal corticosteroids impact the inflammatory rather than the antiangiogenic profile of women with preeclampsia

Circulating antiangiogenic factors and proinflammatory cytokines are implicated in the pathogenesis of preeclampsia. This study was performed to test the hypothesis that steroids modify the balance of inflammatory and proangiogenic and antiangiogenic factors that potentially contribute to the patient's evolving clinical state. Seventy singleton women, admitted for antenatal corticosteroid treatment, were enrolled prospectively. The study group consisted of 45 hypertensive women: chronic hypertension (n=6), severe preeclampsia (n=32), and superimposed preeclampsia (n=7). Normotensive women with shortened cervix (<2.5 cm) served as controls (n=25). Maternal blood samples of preeclampsia cases were obtained before steroids and then serially up until delivery. A clinical severity score was designed to clinically monitor disease progression. Serum levels of angiogenic factors (soluble fms-like tyrosine kinase-1 [sFlt-1], placental growth factor [PlGF], soluble endoglin [sEng]), endothelin-1 (ET-1), and proinflammatory markers (IL-6, C-reactive protein [CRP]) were assessed before and after steroids. Soluble IL-2 receptor (sIL-2R) and total immunoglobulins (IgG) were measured as markers of T- and B-cell activation, respectively. Steroid treatment coincided with a transient improvement in clinical manifestations of preeclampsia. A significant decrease in IL-6 and CRP was observed although levels of sIL-2R and IgG remained unchanged. Antenatal corticosteroids did not influence the levels of angiogenic factors but ET-1 levels registered a short-lived increase poststeroids. Although a reduction in specific inflammatory mediators in response to antenatal steroids may account for the transient improvement in clinical signs of preeclampsia, inflammation is unlikely to be the major contributor to severe preeclampsia or useful for therapeutic targeting. © 2014 American Heart Association, Inc.

Predicting class I major histocompatibility complex (MHC) binders using multivariate statistics:comparison of discriminant analysis and multiple linear regression

The accurate in silico identification of T-cell epitopes is a critical step in the development of peptide-based vaccines, reagents, and diagnostics. It has a direct impact on the success of subsequent experimental work. Epitopes arise as a consequence of complex proteolytic processing within the cell. Prior to being recognized by T cells, an epitope is presented on the cell surface as a complex with a major histocompatibility complex (MHC) protein. A prerequisite therefore for T-cell recognition is that an epitope is also a good MHC binder. Thus, T-cell epitope prediction overlaps strongly with the prediction of MHC binding. In the present study, we compare discriminant analysis and multiple linear regression as algorithmic engines for the definition of quantitative matrices for binding affinity prediction. We apply these methods to peptides which bind the well-studied human MHC allele HLA-A*0201. A matrix which results from combining results of the two methods proved powerfully predictive under cross-validation. The new matrix was also tested on an external set of 160 binders to HLA-A*0201; it was able to recognize 135 (84%) of them.

Toward prediction of class II mouse major histocompatibility complex peptide binding affinity:in silico bioinformatic evaluation using partial least squares, a robust multivariate statistical technique

The accurate identification of T-cell epitopes remains a principal goal of bioinformatics within immunology. As the immunogenicity of peptide epitopes is dependent on their binding to major histocompatibility complex (MHC) molecules, the prediction of binding affinity is a prerequisite to the reliable prediction of epitopes. The iterative self-consistent (ISC) partial-least-squares (PLS)-based additive method is a recently developed bioinformatic approach for predicting class II peptide−MHC binding affinity. The ISC−PLS method overcomes many of the conceptual difficulties inherent in the prediction of class II peptide−MHC affinity, such as the binding of a mixed population of peptide lengths due to the open-ended class II binding site. The method has applications in both the accurate prediction of class II epitopes and the manipulation of affinity for heteroclitic and competitor peptides. The method is applied here to six class II mouse alleles (I-Ab, I-Ad, I-Ak, I-As, I-Ed, and I-Ek) and included peptides up to 25 amino acids in length. A series of regression equations highlighting the quantitative contributions of individual amino acids at each peptide position was established. The initial model for each allele exhibited only moderate predictivity. Once the set of selected peptide subsequences had converged, the final models exhibited a satisfactory predictive power. Convergence was reached between the 4th and 17th iterations, and the leave-one-out cross-validation statistical terms - q2, SEP, and NC - ranged between 0.732 and 0.925, 0.418 and 0.816, and 1 and 6, respectively. The non-cross-validated statistical terms r2 and SEE ranged between 0.98 and 0.995 and 0.089 and 0.180, respectively. The peptides used in this study are available from the AntiJen database (http://www.jenner.ac.uk/AntiJen). The PLS method is available commercially in the SYBYL molecular modeling software package. The resulting models, which can be used for accurate T-cell epitope prediction, will be made freely available online (http://www.jenner.ac.uk/MHCPred).