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Biomedical subjects

G Harcourt

Publications and source records attributed to G Harcourt.

16 recordsLinked to original sources
PubMed2006

Diminished frequency of hepatitis C virus specific interferon gamma secreting CD4+ T cells in human immunodeficiency virus/hepatitis C virus coinfected patients.

BACKGROUND: Human immunodeficiency virus/hepatitis C virus (HIV/HCV) coinfection is a common and complex clinical problem in which loss of immunological control of HCV occurs, with increased HCV viral load and more aggressive liver disease. Cellular immune responses, particularly secretion of interferon gamma (IFN-gamma) appear to be important in the control of HCV, and a detectable HCV specific CD4 response is associated with clearance of the virus. HCV specific CD8+ T cell responses, weak in chronic HCV infection, have been shown to be further impaired in HIV coinfection and this CD8+ T cell deficiency is related to the decline in CD4 T cell count. AIMS: To compare the CD4 T cell response to HCV in HIV/HCV coinfected and HCV monoinfected individuals and to determine the relationship of responses with declining CD4 count. PATIENTS: The study subjects were a cohort of 68 HCV monoinfected and 67 HCV/HIV coinfected haemophiliac children and adolescents (the Hemophilia Growth and Development Study) who were followed for a seven year period. METHODS: We analysed IFN-gamma secreting CD4+ responses to HCV proteins and peptides and HIV p24 antigen using an ELISpot assay. RESULTS: We found a significant decrease in HCV specific responses among those who were HIV coinfected (10/67 v 36/68; p<0.0001) both in numbers of responders and frequency of specific cells. This did not appear to be closely related to CD4 count. CONCLUSIONS: The reduction in HCV specific CD4 T cells in coinfection provide a cellular mechanism for the loss of control of HCV in coinfected individuals, even in those with relatively preserved CD4+ T cell counts and CD4+ T cell responses to HIV.

Adolescent↗
PubMed2001

Effect of HLA class II genotype on T helper lymphocyte responses and viral control in hepatitis C virus infection.

Hepatitis C virus (HCV) infection is very common worldwide, but has a broad range of outcomes. A minority of patients are able to clear infection spontaneously, and this is thought to be due to the emergence and maintenance of effective cell-mediated immunity, particularly CD4+ T lymphocyte responses. Furthermore, genetic studies have indicated that HLA class II genotype strongly influences the outcome of infection. We have therefore investigated the influence of the protective HLA class II haplotype (DQB1*0301, which is in tight linkage disequilibrium with DRB1*1101) on the CD4+ T lymphocyte responses to HCV. We observe a strong association between this genotype and maintenance of a multispecific CD4+ T helper response. The effect on T helper responses was also maintained after combination interferon-alpha/ribavirin therapy, although the latter influenced the pattern of viral antigens to which patients responded. This is the first disease in which an association of HLA genotype with clinical outcome has been linked to an alteration of the immunological phenotype. The selection of protective peptides in those with the favourable HLA class II genotype may point in the direction of suitable vaccine candidates.

Adult↗
PubMed1995

Antigen presentation by thymoma epithelial cells from myasthenia gravis patients to potentially pathogenic T cells.

Thymomas associate strongly with myasthenia gravis (MG). We now show that cultured thymoma epithelial cells can present synthetic acetylcholine receptor (AChR) peptides to HLA-sharing responder T cell lines/clones nearly as efficiently as blood mononuclear cells. Responses depended strictly on the specific antigen added. Processing of longer recombinant AChR polypeptides was clearly less efficient than by blood mononuclear cells, and was selectively abolished by preculture with chloroquine. The T cell responses depended on the presence of LFA-3 on the thymoma cells. This study demonstrates that thymoma epithelial cells have the capacity to stimulate T cells and perhaps, therefore, to autosensitize against AChR in vivo.

Antigen-Presenting Cells↗
PubMed1993

Detection of alpha-subunit isoforms in human muscle acetylcholine receptor by specific T cells from a myasthenia gravis patient.

The nicotinic acetylcholine receptor (AChR) is both the best-characterized transmitter receptor-ion channel and the target for the pathogenic antibodies in the human autoimmune disease myasthenia gravis (MG). In cloning and sequencing its components in man, we found that the alpha-subunit was transcribed in two isoforms, with (P3A+) or without (P3A-) a 75 base pair exon that had not been described in other species. While studying the human T lymphocyte response to recombinant AChR, we found that part of this P3A insert was recognized by one T cell line (from an MG patient), whereas another line only recognized the uninterrupted insertion site. To establish whether this exon is also translated in normal human muscle, we initially raised anti-peptide antibodies to the relevant amino acid sequences, but these failed to bind native AChR (affinity-purified from muscle on alpha-neurotoxin columns). We therefore exploited the great sensitivity and specificity of these T cells to detect the two isoforms after unfolding by antigen-presenting cells, and have been able to show that both are expressed in affinity-purified human muscle AChR.

Adult↗
PubMed1993

Approaches for studying the pathogenic T cells in autoimmune patients.

Our provisional conclusions from this work are as follows. (1) For screening responses of established lines, native human AChR is not prohibitively scarce, especially if it is concentrated onto beads, and class II-transfected TE671 cells may be useful too; both may give vital evidence of AChR-specificity, but it is still crucial to confirm that with synthetic peptides. (2) For mapping epitopes, panels of full-length and shorter recombinant human polypeptides, and of synthetic peptides, are invaluable complementary material: longer peptides tend to stimulate particularly strongly. (3) Initial selection with pooled synthetic peptides can easily generate interesting lines from both patients and controls, but they may depend on the artificial processing sites that are an inevitable consequence of arbitrarily chosen start and stop points. Of course, these might conceivably be employed in unusual antigen-presenting cells (such as thymic myoid cells), so we cannot totally dismiss such "cryptic" epitopes. This system can sometimes select T cells responding to "natural" epitopes too, as now reported for tetanus toxin. Nevertheless, for these and other reasons, at present, we strongly favor using the longest human recombinant material possible, because it is apparently processed more naturally. This must be combined with rigorous screening for reactivity to E. coli-derived contaminants plus concomitant mapping of epitopes as above. Use of intact AChR for initiating lines may yet become feasible. (4) The T cells thus isolated and characterized so far are proving to be heterogeneous in the epitopes and presenting class II molecules they recognize, and in their T-cell receptor gene usage. It is premature to claim key myasthenogenic epitopes or clonotypes, but HLA-DR3 and the linked -DQw2 do not appear to monopolize presentation. (5) Assessing the disease-relevance of these T cells is a separate problem, highlighted by their apparent similarity in healthy controls. In the meantime, to test their potential pathogenicity, we are assaying their cytokine profiles and ability to help specific antibody production in vitro. In the hope that they do prove to be relevant, we are also using some of them to test possible therapeutic strategies that might prove applicable in the patients.

Amino Acid Sequence↗
PubMed1992

Stimulation of human T cells by sparse antigens captured on immunomagnetic particles.

The acetylcholine receptor (AChR) of muscle is the target of the pathogenic antibodies in the human autoimmune disease myasthenia gravis (MG). For studies on the autoreactive T cells presumed to be responsible, use of intact human autoantigen would be optimal, but it was thought to be prohibitively scarce. However, adsorption to the surface of immunomagnetic particles (Dynabeads) of intact AChR from whole muscle extracts or from affinity-purified preparations, using mouse anti-human AChR Mabs, largely overcomes this problem. Together with antigen presenting cells (APC), this bead-bound AChR has consistently and maximally stimulated an established MG T cell line (previously selected with recombinant human AChR alpha subunit) that recognises the 144-156 region of the human alpha sequence (Ong et al., 1991). For equivalent T cell stimulation, bead-bound AChR was at least 10(3) times more potent than soluble AChR or recombinant alpha subunit, and 10(6) times more potent than peptide 144-156, implying that antigen in this form is targetted very efficiently to APC and thus to T cells. Finally, we have obtained similar results with T cells specific for other antigens suggesting that this method may have wider applications.

Amino Acid Sequence↗
PubMed1992

T cell responses to human recombinant acetylcholine receptor-alpha subunit in myasthenia gravis and controls.

Antibodies against the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction are detectable in most patients with myasthenia gravis (MG) and assumed to participate in the destruction of the AChR, thereby, causing the characteristics signs and symptoms of the disease. The extent and importance of T cell responses to AChR and its subunits in MG are still unsettled. We have now examined T cell reactivities using human recombinant AChR-alpha subunit as antigen. Upon recognition of appropriate antigen in an MHC-class II-restricted fashion, memory T cells secrete interferon-gamma (IFN-gamma). Adopting this principle in an immunospot assay we found that 73% of MG patients had recombinant human AChR-alpha subunit-reactive T cells at a median value of 1 per 56,000 blood mononuclear cells, while only 27% of the MG patients responded to the alpha subunit in a conventional lymphocyte proliferation assay. This compares with even lower numbers of AChR-reactive T cells and 14% positivity in the proliferation assay among control subjects. The T cell responses to the control antigens purified protein derivative and myelin basic protein did not differ between MG and controls, underlining the specificity of an augmented T cell reactivity to AChR-alpha subunit in MG. Alpha Subunit-specific T cell lines and clones propagated from patients with MG and healthy controls yielded a high proportion of alpha subunit-reactive T cells in the IFN-gamma immunospot assay. Their appearance was inhibited by the addition of monoclonal anti-MHC class II antibodies, demonstrating that an MHC-restricted T cell response was measured. Our data underline that the AChR-alpha subunit is a major T cell autoantigen in MG.

Adolescent↗
PubMed1989

T-cell reactivity in myasthenia gravis.

In a proliferation assay, peripheral blood lymphocytes (PBL) from a relatively small proportion of myasthenia gravis (MG) patients and from controls responded to Torpedo acetylcholine receptor (T-AChR), which shows approximately 75% homology with the human AChR. Over 50% of MG patients responded to recombinant human AChR alpha-subunit (r37-437) however, compared with 9% of controls. A proportion of MG PBL respond to synthetic peptides of the extracellular portion of the human alpha-subunit, but only MG patients (18%) responded to the juxta-membrane sequence p257-269. MG T-cell lines raised against native T-AChR failed to respond to the synthetic peptides. These results underline the need to use human AChR sequences to test relevant T-cell reactivity in MG. T-cell lines raised from three MG patients to human alpha-subunit r37-437 have shown Stimulation Index (SI) values of 3.5-22. Three clones derived from one of these had SI values of 100-500. Preliminary testing of responsiveness in one of these clones showed reactivity to several recombinant polypeptides including r37-437 and r37-181, as in the parent line. The epitope(s) within this latter sequence have not yet been identified, but the experimental approach used here should make it possible to define critical T-cell epitopes in MG, and to determine their functional relevance by investigating the ability of AChR-reactive T-cell clones to provide specific help in anti-AChR antibody production.

Animals↗