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G Hashim

Publications and source records attributed to G Hashim.

At least 19 recordsLinked to original sources
PubMed2001

TCR peptide therapy in human autoimmune diseases.

Inflammatory Th1 cells reacting to tissue/myelin derived antigens likely contribute to the pathogenesis of diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), and psoriasis. One regulatory mechanism that may be useful for treating autoimmune diseases involves an innate second set of Th2 cells specific for portions of the T cell receptor of clonally expanded pathogenic Th1 cells. These Th2 cells are programmed to respond to internally modified V region peptides from the T cell receptor (TCR) that are expressed on the Th1 cell surface in association with major histocompatibility molecules. Once the regulatory Th2 cells are specifically activated, they may inhibit inflammatory Th1 cells through a non-specific bystander mechanism. A variety of strategies have been used by us to identify candidate disease-associated TCR V genes present on pathogenic Th1 cells, including BV5S2, BV6S5, and BV13SI in MS, BV3, BV14, and BV17 in RA, and BV3 and BV13S1 in psoriasis. TCR peptides corresponding to the mid region of these BV genes were found to be consistently immunogenic in vivo when administered either i.d. in saline or i.m. in incomplete Freund's adjuvant (IFA). In MS patients, repeated injection of low doses of peptides (100-300 microg) significantly boosted the number of TCR-reactive Th2 cells. These activated cells secreted cytokines, including IL-10, that are known to inhibit inflammatory Th1 cells. Cytokine release could also be induced in TCR-reactive Th2 cells by direct cell-cell contact with Th1 cells expressing the target V gene. These findings indicate the potential of regulatory Th2 cells to inhibit not only the target Th1 cells, but also bystander Th1 cells expressing different V genes specific for other autoantigens. TCR peptide vaccines have been used in our studies to treat a total of 171 MS patients (6 trials), 484 RA patients (7 trials), and 177 psoriasis patients (2 trials). Based on this experience in 824 patients with autoimmune diseases, TCR peptide vaccination is safe and well tolerated, and can produce significant clinical improvement in a subset of patients that respond to immunization. TCR peptide vaccination represents a promising approach that is well-suited for treating complex autoimmune diseases.

Arthritis, Rheumatoid↗
PubMed1994

Immunity to TCR peptides in multiple sclerosis. II. T cell recognition of V beta 5.2 and V beta 6.1 CDR2 peptides.

The biased expression of V beta 5.2 and V beta 6.1 by T cells specific for myelin basic protein (BP) has led to our use of TCR peptides from these V gene sequences to induce anti-TCR immunity in patients with multiple sclerosis (MS). Injection of V beta 5.2-39-59 or V beta 6.1-39-59 peptides significantly increased the peptide specific T cell frequency in 7 of 11 MS patients, often with an accompanying delayed hypersensitivity reaction at the injection site. Here, we validate these cellular immune responses by characterizing TCR peptide specific T cells from an MS patient with biased V beta 5.2 expression in BP reactive T cells before treatment with TCR peptides, and from two MS patients in whom the frequencies of anti-TCR peptide specific T cells were significantly boosted after injection with low doses of TCR peptides. In both cases, T cell lines were established with relative ease, especially after boosting with the peptides. A V beta 5.2-39-59 reactive line responded selectively to the boosting peptide and was restricted by both MHC class I (HLA-B7) and MHC class II (HLA-DR2) molecules. Characterization of 22 clonal isolates revealed that the responding T cells were predominantly activated CD4+CD8lo, circulating memory cells restricted by either HLA-B7 or HLA-DR2, that utilized mainly V beta 4, V beta 6, V beta 12, and V beta 14, but not V beta 5.2 in their TCR. T cell isolates specific for V beta 6.1-39-59 possessed similar characteristics but contained specificities cross-reactive with an N-terminal sequence on V beta 5.2-39-59. Upon stimulation with peptide or Con A, the TCR peptide specific T cell lines had increased message production for IFN-gamma, GM-CSF, IL-4, IL-5, and to a lesser degree, IL-2. This lymphokine mRNA profile differed from a BP-specific T cell line that produced message for IFN-gamma and GM-CSF but low or absent levels of IL-4 and IL-5. The extensive parallels between human T cells specific for V beta 5.2 and V beta 6.1 CDR2 peptides and rat T cells specific for V beta 8.2 CDR2 peptide that are highly protective against experimental encephalomyelitis strengthen the rationale for the therapeutic use of TCR peptides in human autoimmunity.

Adult↗
PubMed1994

Healthy monozygous twins do not recognize identical T cell epitopes on the myelin basic protein autoantigen.

The T cell response against myelin basic protein (MBP) has been extensively studied in humans because of its putative role in the pathophysiology of multiple sclerosis (MS). Higher concordance rates in monozygous twins as well as an increased risk in relatives suggest the role of genetic factors in MS susceptibility. Very little is known about the shaping of T cell repertoire towards self antigens in humans and their contribution to disease susceptibility in autoimmune disorders. Here we report the comparative T cell epitope recognition patterns towards the MBP auto-antigen in healthy identical twins. We have established MBP-specific T cell lines from eight sets of twins and characterized their fine epitope specificity. Intra-pair comparison showed the co-existence of shared as well as distinct epitopes in six of eight pairs and a complete absence of concordant epitope recognition within two other pairs. These findings indicate that important differences in T cell repertoires against a self antigen may be observed between genetically identical healthy individuals, rendering difficult the interpretation of the differences which may be observed between identical twins discordant for an autoimmune disease.

Adult↗
PubMed1994

Human myelin basic protein (MBP) epitopes recognized by mouse MBP-selected T cell lines from multiple sclerosis patients.

We investigated whether myelin basic protein (MBP)-reactive T cells from multiple sclerosis (MS) patients can recognize mouse MBP since this is an expected requirement for the transfer of experimental autoimmune encephalomyelitis (EAE) into severe combined immunodeficiency (SCID) mouse-human chimeras. Peripheral blood mononuclear cells from 11 MS patients were analyzed for in vitro proliferation to mouse MBP. Six patients (55%) responded to mouse MBP at the first or second stimulation. Five T cell lines, selected with mouse MBP from five MS patients, were analyzed for their proliferation to mouse and human MBP and to a panel of synthetic peptides of human MBP. Four of the five lines recognized mouse MBP. In vitro proliferation was restricted by MHC class II in one line tested for MHC restriction. One of the five lines recognized whole human MBP and all five of the lines responded to at least one of the five synthetic peptides corresponding to human MBP residues 8-28, 67-90, 84-102, 87-99 or 130-149. These results show that MS patient T cells recognize mouse MBP and suggest that distinct human MBP epitopes are immunologically cross-reactive with epitopes of mouse MBP.

Amino Acid Sequence↗
PubMed1993

Where, when, and how to detect biased expression of disease-relevant V beta genes in rats with experimental autoimmune encephalomyelitis.

The biased use of V beta 8.2 and V beta 6 in rats by encephalitogenic T cells specific for the S72-89 and S87-99 epitopes of guinea pig basic protein (Gp-BP) has allowed the use of anti-V beta antibodies and synthetic TCR peptides for treatment of experimental autoimmune encephalomyelitis (EAE). Striking V gene biases also occur in human autoimmune diseases, raising the question of to what degree these biases reflect potentially pathogenic T cells. To address this question, we evaluated the expression of the EAE-associated marker V beta 8.2 and V beta 6 molecules in the periphery, spinal cord (SC), and cerebrospinal fluid (CSF) during the course of EAE, in unselected, IL-2-expanded, and Gp-BP-restimulated populations. In CSF cells, there was a strong bias for the marker V beta before the onset of EAE, but this bias was not enhanced by IL-2, which skewed the CSF population to > 80% CD8+ T cells. In SC, the marker V beta were expressed optimally during the onset of EAE, even in unselected cells, and this bias could be enhanced sequentially by IL-2 expansion and Gp-BP restimulation. During the recovery phase, however, the marker V beta 8.2 bias was obfuscated by the appearance of a heterogeneous V beta T cell population. Biased expression of the marker V genes was not detected in unselected or IL-2-expanded peripheral cells at any time during EAE. These data suggest that peripheral T cells bearing the disease-relevant V genes first appeared in CSF before disease onset and then migrated to SC beginning on the first day of clinical signs. During the recovery phase of the disease, these cells were diluted by an influx of T cells bearing other V beta genes, requiring restimulation with Gp-BP to observe the V beta 8.2 bias. These data have important implications for the interpretation of V beta gene biases that have been reported in human autoimmune diseases.

Amino Acid Sequence↗
PubMed1993

Surface expression of alpha 4 integrin by CD4 T cells is required for their entry into brain parenchyma.

Cloned CD4 T cell lines that recognize the Ac1-16 peptide of myelin basic protein bound to I-Au were isolated and used to analyze the immunopathogenesis of experimental autoimmune encephalomyelitis (EAE). T helper type 1 (Th1) clones induced disease, while Th2 clones did not. Using variants of a single cloned Th1 line, the surface expression of alpha 4 integrins (very late antigen 4 [VLA-4]) was identified as a major pathogenic factor. Encephalitogenic clones and nonencephalitogenic variants differ by 10-fold in their level of surface expression of alpha 4 integrin and in their ability to bind to endothelial cells and recombinant vascular cell adhesion molecule 1 (VCAM-1). The alpha 4 integrin-high, disease-inducing cloned Th1 T cells enter brain parenchyma in abundance, while alpha 4 integrin-low, nonencephalitogenic Th1 cells do not. Moreover, antibodies to alpha 4 integrin, its ligand VCAM-1, and intercellular adhesion molecule 1 all influence the pathogenicity of this encephalitogenic clone in vivo. The importance of the expression of VLA-4 for encephalitogenicity is not unique to cloned T cell lines, as similar results were obtained using myelin basic protein-primed lymph node T cells. alpha 4 integrin levels did not affect antigen responsiveness or production of the Th1 cytokines interleukin 2, interferon gamma, and lymphotoxin/tumor necrosis factor beta; and antibodies against alpha 4 integrin did not block antigen recognition in vitro. Thus, we conclude that surface expression of alpha 4 integrin is important in CD4 T cell entry into brain parenchyma. A general conclusion of these studies is that alpha 4 integrins may be crucial in allowing activated effector T cells to leave blood and enter the brain and other tissues to clear infections.

Animals↗
PubMed1992

TCR peptide therapy decreases the frequency of encephalitogenic T cells in the periphery and the central nervous system.

The V beta 8 CDR2 consensus peptide, residues 44-54, is highly effective in the treatment of clinical experimental autoimmune encephalomyelitis (EAE) in Lewis rats. To monitor immunological changes during EAE resulting from TCR peptide therapy, the frequencies of encephalitogenic and regulatory T cells were quantitated in lymph nodes, blood, and spinal cord. The frequency of T cells specific for basic protein and its major encephalitogenic epitope, residues 72-89, increased during EAE to about 1 cell per 100,000 lymph node or blood cells at the peak of clinical disease, and then declined. In contrast, the frequency of these T cells in spinal cord was highest, 50 per 100,000, prior to onset of clinical signs, and then decreased rapidly prior to spontaneous recovery. Injection of 100 micrograms of TCR V beta 8-44-54 peptide caused a decrease within 1-5 days in the frequencies of guinea pig basic-protein (GP-BP) and 72-89-reactive T cells in blood and spinal cord, and in the total number of infiltrating cells in spinal cord. In lymph nodes, 72-89-reactive T cells decreased as T cells specific for a protective epitope, residues 55-69 of GP-BP increased, suggesting epitope switching at the site of GP-BP immunization. Conversely, the frequency of T cells specific for the V beta 8-44-54 peptide increased, especially in blood and spinal cord, whereas T cell frequencies to control antigens were unchanged. These data document the critical presence of encephalitogenic T cells within the spinal cord during clinical EAE, and demonstrate that rapid and profound changes in T cell frequencies in the periphery and spinal cord are triggered by TCR peptide therapy.

Amino Acid Sequence↗
PubMed1992

An antibody specific for component 8 of myelin basic protein from normal brain reacts strongly with component 8 from multiple sclerosis brain.

Myelin basic protein (MBP) consists of several components or charge isomers (C-1 through C-8) generated by one or a combination of posttranslational modifications. One of these, C-8, has been shown to contain citrulline (Cit) at defined sites formed by deimination of six arginyl residues. This unusual modification has allowed us to raise antibodies specific for this charge isomer only. To do this, a synthetic peptide, Gly-Cit-Cit-Cit-Cit, was coupled to keyhole limpet hemocyanin and injected into rabbits. The antibodies so generated reacted only with C-8 and not with any of the other charge isomers. A second antibody fraction was raised against the synthetic peptide ACitHGFLPCitHR naturally occurring between residues 24 and 33 of C-8 (all other charge isomers contain R instead of Cit at positions 25 and 31). These antibodies preferred C-8 but reacted with the other charge isomers, to the extent of approximately 25-30% of the reactivity shown with C-8. In studies with C-8 from multiple sclerosis (MS) MBP, much greater reactivity was obtained with these antibodies when compared with their reactivity with C-8 from normal MBP. Because the total number of Cit residues in C-8 from MS and normal MBP is the same, the difference in reactivity may be related to structural factors. The antibodies raised with the tetra-Cit peptide were reacted with three pairs of synthetic peptides: 24ARHGFLPRHR33 and ACitHGFLPCitHR; 120GQRPGFGYGGRAS132 and GQCitPGFGYGGCitAS; and 157GGRDSRSGSPMARR170 and GGCitDSRSGSPMACitR. They reacted only with the Cit-containing peptides in the order 157-170 greater than 120-130 greater than 24-33.(ABSTRACT TRUNCATED AT 250 WORDS)

Alzheimer Disease↗
PubMed1991

T cell lines specific for an immunodominant epitope of human basic protein define an encephalitogenic determinant for experimental autoimmune encephalomyelitis-resistant LOU/M rats.

The LOU/M rat (RT-1w) haplotype, although resistant to an encephalitogenic challenge of guinea pig myelin basic protein (Gp-BP)/CFA and unresponsive to Gp-BP, responded strongly to human (Hu)-BP. Both T cell and antibody responses focused on the 110-129 determinant of Hu-BP, and T cells specific for this epitope transferred clinical and histologic experimental autoimmune encephalomyelitis (EAE) to naive LOU/M rats. Moreover, EAE could be induced actively with Hu-BP and a synthetic Hu-S110-129 peptide in CFA, but only with co-immunomodulation by pertussis toxin or cyclophosphamide. Analysis of TCR V region genes revealed the predominant use of the V beta 8.5-J beta 2.3 gene combination, with extensive N region additions to both D beta 1 and D beta 2. These results define the Hu-BP 110-129 peptide sequence as the major encephalitogenic epitope for the LOU/M strain of rat previously considered resistant to EAE, and support the idea that the encephalitogenic property of BP and other CNS Ag for a given MHC is encompassed within immunodominant T cell epitopes. Furthermore, the TCR sequence data indicate the predominant use of a different V beta 8 subfamily member (V beta 8.5) than the V beta 8.2 gene used preferentially by several other rat strains and the PL/J mouse in the T cell response to BP.

Amino Acid Sequence↗
PubMed1991

T cell response to myelin basic protein epitopes in multiple sclerosis patients and healthy subjects.

T cell lines and clones specific for human myelin basic protein (BP) were selected from three multiple sclerosis (MS) patients and two healthy subjects and tested for their proliferative responses to a battery of synthetic peptides, 9 to 21 amino acid residues long. The combined amino acid sequence of the peptides spanned the complete sequence of the human BP. The results suggest the development of T cells sensitized to at least four independent regions of the human BP, indicating some diversity of the human T cell repertoire to BP. However, an immunodominant T cell epitope was located in the C-terminal region, defined by residues 149-162. This epitope was recognized by T cells from three subjects out of five (one MS patient and both healthy controls) in the context of different DR specificities. Another epitope (located in the 57-75 region) which triggered one MS patient's T cell response was also recognized by a mycobacteria-specific T cell clone cross-reacting with BP.

Epitopes↗
PubMed1991

Specificity of human T cell clones reactive to immunodominant epitopes of myelin basic protein.

Several recently discovered lines of evidence support the involvement of myelin basic protein (BP)-specific T cells in multiple sclerosis (MS). To identify potentially relevant immunodominant T cell epitopes, human BP (Hu-BP)-reactive T cell lines were selected from MS and normal donors and tested for reactivity to cleavage fragments and synthetic peptides of Hu-BP. The MS T cell lines responded to more Hu-BP epitopes than did normal lines, showing biased recognition of the N terminal half of the molecule, and one region in the C terminal half, suggesting increased sensitization to BP. The MS lines also differed from normal lines in their decreased percentage of CD8+ T cells. One hundred nine T cell clones isolated from these lines confirmed the reactivity pattern of the lines but did not reflect the mixed phenotype, since all but three clones tested were CD4+. T cell clones from HLA-DR2 homozygous donors responded to a variety of epitopes, indicating that this molecule was permissive in its ability to restrict T cell responses. Other epitopes, including the immunodominant 149-170 sequence, were restricted by several different major histocompatibility complex (MHC) molecules from both MS and normal donors. T cell receptor (TCR) V gene products could be identified on six of 38 clones tested using monoclonal antibodies. From one HLA-DR2 homozygous donor, four of eight clones utilized V beta 5.2 in response to different BP epitopes, providing initial support for the preferential use of a limited set of V region genes in the human response to BP. Preferential TCR V gene use in MS patients would provide the rationale to regulate selectively BP-reactive T cells through immunity directed at the TCR and thus test for the first time the hypothesis that BP-reactive T cells play a critical role in the pathogenesis of MS.

Adult↗
PubMed1991

Characterization of a panel of neurofilament antibodies recognizing N-terminal epitopes.

Peptides corresponding to sequences from the amino-terminal "head" regions of the low, middle, and high molecular weight neurofilament proteins (NF-L, NF-M, and NF-H) were synthesized by a modification of the Merrifield solid-phase method, and a panel of polyclonal antibodies to these epitopes were prepared in rabbits by the injection of synthetic peptides conjugated to the carrier protein keyhole limpet hemocyanin (KLH). An additional, monoclonal antibody recognizing both glial fibrillary acidic protein (GFAP) and vimentin was also produced, by fusion of cells of the mouse myeloma line NS-1 with spleen cells from a mouse immunized with cytoskeletal extracts. Antibody specificities were confirmed by a combination of Western blotting against cytoskeletal extracts and immunofluorescence using both rat brain sections and fibroblasts transfected with fully encoding cDNAs for each neurofilament protein, driven by viral promoters.

Amino Acid Sequence↗
PubMed1990

Preferential peptide specificity and HLA restriction of myelin basic protein-specific T cell clones derived from MS patients.

A panel of 17 myelin basic protein (MBP)-specific T lymphocyte clones were generated from four multiple sclerosis (MS) patients. All T cell clones expressed CD4 phenotype and 14 clones exhibited substantial cytotoxic activity on MBP-coated target cells. T cell recognition sites of the clones on human MBP were identified by using MBP fragments and synthetic peptides. Despite the fact that at least three epitopes were defined, these T cell clones displayed a striking bias to the C-terminal peptide 149-171 independent of differences in HLA-DR and DQ expression. In addition, the T cell responses of the clones appeared to be restricted by HLA-DR molecules irrespective of peptide specificities. The present study suggests an immunodominant property of the C-terminal peptide for HLA-DR-restricted T cell responses to MBP. However, its association with encephalitogenicity in humans and its potential pathologic importance in MS await further clarification.

Amino Acid Sequence↗
PubMed1990

Myelin basic protein, MHC restriction molecules and T cell repertoire.

Our laboratory has developed a broad base of information in rats and mice describing the properties of encephalitogenic T cells. Using the T cell line selection technique to identify dominant T cell specificities, we were the first to identify new encephalitogenic epitopes and MHC restriction molecules in the Lewis rat and the SJL mouse. Furthermore, using the soft agar technique, we have isolated encephalitogenic Lewis rat T cell clones specific for defined synthetic epitopes of BP. With our colleagues, we have identified and sequenced the V region genes, and deduced the amino acid sequence of proteins which these clones use preferentially in their T cell receptor. Furthermore, we have successfully induced protection against EAE by the lymphocyte vaccination approach, using attenuated encephalitogenic T cells as the vaccine. Using this information, we have made significant progress in characterizing and understanding human T lymphocyte responses to BP. Recent results indicate that MBP reactivity is more frequent in MS patients than in controls, suggesting a greater degree of sensitization, and a potential involvement of BP as a target antigen. Since human BP responses also appear to be directed at immunodominant epitopes (as we have observed in rodents), it is probable that human T cells will also use preferential combinations of T cell receptor V genes. The ability to induce specific regulation or removal of autoreactive T cells will allow for the first time an assessment of cause and effect in human diseases. Immunoregulation directed at the TCR polypeptides may allow selective suppression of encephalitogenic clones, and may have direct application in human diseases such as MS, or in other autoimmune situations where preferential TCR V gene usage can be identified.

Amino Acid Sequence↗
PubMed1989

Immunization with a synthetic T-cell receptor V-region peptide protects against experimental autoimmune encephalomyelitis.

T cells expressing the alpha beta T-cell receptor (TCR) for antigen can elicit anti-idiotypic antibodies specific for the TCR that regulate T-cell function. Defined sequences of the TCR, however, have not been used to elicit specific antibodies and the role of cellular immunity directed against TCR determinants has not been studied. We immunized Lewis rats with a synthetic peptide representing a hypervariable region of the TCR V beta 8 molecule. Subsequent induction of experimental autoimmune encephalomyelitis, a paralytic disease of the central nervous system mediated primarily by V beta 8+ T cells specific for myelin basic protein was prevented. T cells specific for the TCR V beta 8 peptide conferred passive protection against the disease to naive rats, apparently by shifting the predominant T-cell response away from the major encephalitogenic epitope of basic protein. This is the first report demonstrating the use of a synthetic TCR V-region peptide to induce specific regulatory immunity and has important implications for the regulation of human disease characterized by common TCR V-gene usage.

Animals↗
PubMed1989

Selection of encephalitogenic rat T-lymphocyte clones recognizing an immunodominant epitope on myelin basic protein.

Using the soft agar-cloning technique, we isolated 13 T-cell clones from guinea pig basic protein (GP-BP)-specific T-cell lines derived from Lewis rats. The clonal frequency was approximately 2.5 x 10(-5). Each of these clones had a similar but not identical pattern of response to a battery of synthetic peptides representing overlapping epitopes in the encephalitogenic region for Lewis rats (69-89 sequence). All clones responded to the minimal encephalitogenic sequence (residues 72-84) restricted by I-A but not I-E molecules, and all transferred clinical experimental autoimmune encephalomyelitis (EAE) and delayed-type hypersensitivity (DTH) reaction to naive rats. Phenotypically, the clones were W3/13+ (total T), W3/25+ (T helper), and OX-22+ (DTH associated). This report demonstrates for the first time the applicability of the soft agar-cloning technique for obtaining encephalitogenic T-cell clones. The exclusive recovery of 72-84-specific T-cell clones after only two rounds of stimulation with GP-BP indicates the immunodominance of this epitope and the power of the line selection technique for obtaining encephalitogenic T-cell specificities.

Agar↗