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S Hawke

Publications and source records attributed to S Hawke.

26 records · Page 2Linked to original sources
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↗
PubMed1991

The pseudotumor syndrome. Disorders of cerebrospinal fluid circulation causing intracranial hypertension without ventriculomegaly.

We report a series of eight cases that show a close resemblance to, but are not identical with, pseudotumor cerebri (PTC) as normally defined. The majority of these cases are characterized by raised intracranial pressure without ventriculomegaly. They include two cases of cranial venous outflow obstruction in which clinical or radiologic abnormalities precluded the diagnosis of PTC proper (cases 1 and 2); one case of chronic meningitis in which an abnormal cerebrospinal fluid (CSF) composition precluded the diagnosis of PTC (case 3); two cases without either papilledema or a measured increase of CSF pressure, which in other respects, particularly in response to treatment, resembled PTC (cases 4 and 5); and three cases of what is thought to represent an infantile form of PTC (cases 6 through 8). The purpose of the analysis of these cases is twofold. First, it is argued that these cases throw light on the mechanism of PTC itself, supporting a concept of a disturbance of CSF circulation in this condition, and that they are themselves illuminated by considerations of typical PTC. Second, the cases are used to frame a proposed classification of the pseudotumor syndrome aimed at broadening the diagnostic criteria applied currently to PTC. It is suggested that the pseudotumor syndrome has a single underlying mechanism (disturbed CSF circulation) and that recognition of this mechanism not only clarifies the pathophysiologic processes of PTC but also has important diagnostic and therapeutic implications.

Adolescent↗