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J H Robinson

Publications and source records attributed to J H Robinson.

At least 73 records · Page 4Linked to original sources
PubMed1994

Identification of two binding sites in staphylococcal enterotoxin B that confer specificity for TCR V beta gene products.

The enterotoxins produced by Staphylococcus aureus are potent mitogens. They stimulate T cells in an oligoclonal fashion that is dependent on the expression of particular variable region gene elements in the beta-chain of the TCR (V beta). The fourth hypervariable loop of the TCR beta-chain is generally regarded as the site of contact for both viral and microbial superantigens. Recently, residues 60 and 61 of staphylococcal enterotoxin B (SEB) have been highlighted as central to the interaction of this toxin with the TCR. We have, therefore, analysed a series of toxins with mutations at these positions to investigate how amino acid substitutions affect the ability of mutant toxins to stimulate both human and mouse T cells. Each of the variant toxins induced proliferation in a murine V beta 8.3 T cell clone, whereas a V beta 8.1 T cell clone only responded to native toxin. A panel of nine human T cell clones expressing six different V beta elements, all of which responded to native SEB, was tested for reactivity to the variant toxins. Only one V beta 19.1+ T cell clone was found to be sensitive to substitution at positions 60 and 61 in a manner analogous to the murine V beta 8.1 T cell clone. Semi-quantitative analysis of the TCR V beta expression of human T cell lines expanded with native and mutant SEB revealed that none of the variant toxins could stimulate T cells that expressed V beta 19.1. Taken together, these results suggest that the interaction of mouse V beta 8.1 and human V beta 19.1 TCRs with SEB differs from other TCRs. Sequence comparisons of the different TCR V beta chains indicated that residues in the second complementarity determining region (CDR2) interact with the 60-61 loop of SEB. Therefore, a minimum of two distinct binding modules confer specificity to the interaction of the TCR with SEB.

Amino Acid Sequence↗
PubMed1994

Identification of T cell autoepitopes that cross-react with the C-terminal segment of the M protein of group A streptococci.

Rheumatic fever (RF) follows a throat infection with different M-serotypes of beta-hemolytic group A streptococci (GAS) and can affect different tissues, predominantly the heart. It is thought to be an autoimmune illness. Although histological examination of affected heart shows an infiltrate consisting mainly of T cells, antigens or epitopes that could be putative targets of autoimmune T cells have not been identified. We have examined the T cell response to the conserved C-terminal region of the M protein--a streptococcal surface coiled-coil protein which is the target of opsonic antibodies and antibodies which cross-react with human heart tissue. Australian Aborigine, Caucasian and Thai patients, controls and mice were studied to define regions of the protein immunogenic for T cells, and T cell lines and clones were tested for cross-reactivity to myosin as well as an extract of RF-diseased mitral heart valve. Murine (B10, B10.D2, B10.BR) M peptide-specific T cells were often cross-reactive for other M peptides but did not cross-react with human heart antigens. Patients with RF or other heart diseases, or control subjects exposed more commonly to GAS were more likely to have T cell responses to the M protein, with many regions of the C-terminus being recognized. T cell lines and a clone specific for different M peptides were generated from five donors. Cross-reactivity could be shown between different M peptides, but unlike murine M peptide-specific T cells three of the human T cell lines reacted strongly to peptides representing homologous regions of cardiac and skeletal muscle myosins, and two of these lines also responded to porcine myosin and an extract of human rheumatic mitral valve. However, these last two lines were derived from a normal donor without history of RF or other heart disease. Our data demonstrate that regions of the M protein, including regions that are being considered as subunit vaccines, have the potential to stimulate pre-existing heart cross-reactive T cells, but that the ability of such T cells to cross-react (as measured in vitro) is not in itself sufficient to lead to disease.

Adolescent↗
PubMed1993

The use of active centre acylation to control the pharmacokinetic profile of a recombinant chimaeric plasminogen activator.

Recombinant hybrid plasminogen activators consisting of the "A" chain of plasminogen linked to the "B" chain of t-PA that are inhibited rapidly by plasma protease inhibitors have recently been described (Robinson et al. Circulation 1992; 86: 548-552). We have now shown that following bolus administration of native hybrid to guinea pigs, fibrinolytic activity was cleared rapidly from the circulation. Active centre acylation appeared to protect the hybrid from inhibition and allowed material to circulate as potentially active species for prolonged periods. Clearance rates of a range of acyl derivatives of the hybrid were 7-35-fold slower than for native hybrid and 20-100-fold slower than for t-PA. Clearance rates were influenced markedly by deacylation rate, such that clearance half-life correlated well with deacylation half-life. We have thus shown that it is feasible to control the pharmacokinetic profile of a recombinant hybrid plasminogen activator over a wide range by selection of an appropriate acyl group for attachment to the active site. Such control is not possible with plasminogen activators that are cleared predominantly by mechanisms other than inhibition.

Acylation↗
PubMed1993

Deletion of residues K296-G302 from the slowly-cleared tissue-type plasminogen activator t-PA del (G) leads to partial loss of plasminogen activating activity.

Plasmids encoding tissue-type plasminogen activators lacking residues K296-G302 were constructed and were expressed in a Hela cell transient expression system. Conditioned media from the cultures were tested in a number of systems designed to detect function or antigen. Functional assays comprised the chromogenic substrate S2288 and three plasminogen activation assays. All t-PA variants were active in all assays but to varying degrees. The results are consistent with the conclusion that deletion of residues K296-G302 from the slowly-cleared t-PA mutant t-PA del(G) adversely affects the plasminogen activating ability of the molecule without altering the integrity of the active site. This is in contrast to the situation in native t-PA where the same mutation has little overall effect on activity.

Binding Sites↗
PubMed1993

Characterization of a conserved helper-T-cell epitope from group A Streptococcal M proteins.

We have previously defined major histocompatibility complex (MHC) class II-restricted T-cell epitopes from the carboxy-terminal region of group A streptococcal type 5 M protein. In this report, T-cell responses to one of these epitopes have been characterized in detail. T-cell clones from recombinant M5-immunized mice and popliteal lymph node cells from peptide-immunized mice were used to show that sM5[300-319] is recognized in the context of I-A alleles of four of nine independent MHC class II haplotypes: I-Ad, I-Af, I-Ak, and I-As. This epitope was also recognized by both helper (Th2) and inflammatory (Th1) subsets of murine T cells. The I-Ad-restricted epitope recognized by BALB/c mice was mapped to the 12-amino-acid peptide sM5[308-319] and was shown to provide helper function for an immunoglobulin G anti-peptide antibody response in BALB/c mice. Anti-peptide antibody was shown to be specific for M5[304-315] but failed to recognize intact rM5, suggesting that the conformation of the epitope differed between peptide and protein. However, the results demonstrate that overlapping epitopes can be the focus for both immunoglobulin G antibodies and the T cells which augment their production. Comparison of the available sequences for M proteins indicated that the T-cell epitope within M5[300-319] was highly conserved between M types and hence may elicit helper function for protective antibody responses to a wide range of M types. T-cell epitopes from conserved regions of M proteins which are recognized in the context of multiple MHC haplotypes have potential for the design of multivalent streptococcal vaccines.

Amino Acid Sequence↗
PubMed1992

The role of nicotine in tobacco use.

The 1988 US Surgeon General's Report titled "Nicotine Addiction", is cited frequently in the literature as having established the "fact" that nicotine derived from cigarette smoke is addictive in the same sense as "classic" addicting drugs such as heroin and cocaine. This manuscripts critically evaluates key research findings used in support of this claim and identifies shortcomings in the data that seriously question the logic of labeling nicotine as "addictive". In addition, the manuscript argues that the role of nicotine in tobacco use is not like the role of cocaine in coca leaf use as argued by the 1988 Surgeon General's Report, but is, in fact, more like the role of caffeine in coffee drinking as concluded in the 1964 US Surgeon General's Report.

Humans↗
PubMed1992

Enhancement of continuous performance task reaction time by smoking in non-deprived smokers.

In a test of the withdrawal-deficit hypothesis of the cognitive effects of cigarette smoking, non-deprived smokers participated in two sessions held on consecutive days. In both sessions, subjects performed two 20-min continuous performance tasks (CPTs). The CPT was a relatively "easy" version designed to require minimal practice (digit 0 target response; digits 1-9 nontarget response; 19% of stimuli targets). In one session, subjects smoked a cigarette prior to each CPT; in the other session they did not smoke (session order counterbalanced). Reaction time (RT) was significantly faster in the smoking session than in the non-smoking session with no difference in number of incorrect responses, a finding incompatible with the withdrawal-deficit hypothesis. Further, light inhalers (as assessed by pre-smoking to post-task increase in expired air carbon monoxide) appeared to process nontarget stimuli faster than deep inhalers, especially in the no smoking session. The results also indicated that, at least during the first CPT of each session, the performance of females in the no smoking session was poorer than in the smoking session and poorer than males regardless of session. In the latter part of the first CPT, the performance of males in the smoking session was better than their performance in the no smoking session. No clear pattern emerged for the second CPT.

Adolescent↗
PubMed1992

Psychopharmacological effects of smoking a cigarette with typical "tar" and carbon monoxide yields but minimal nicotine.

Five male smokers were tested, after 48-h abstention from tobacco-product use, smoking a leading "lights" category cigarette (Control-FTC nicotine yield 0.6 mg) and another cigarette yielding similar amounts of "tar" and carbon monoxide (CO), but only 0.06 mg nicotine (Test). Heart rate (HR) and the electroencephalogram (EEG) were monitored before, during and after the smoking of each cigarette. Other measures obtained included the subjects' puffing and breathing behaviors during smoking, plasma nicotine concentrations, blood carboxyhemoglobin concentrations and expired-air CO. The results indicated no significant differences in the way the subjects puffed and inhaled the two cigarettes and they were therefore assumed to have inhaled similar amounts of particulate matter and gas-phase components. Plasma nicotine concentrations were significantly higher following smoking of the Control cigarette. HR (percent relative change) increased following smoking of either cigarette; however, HR increase was significantly greater following smoking of the Control cigarette. Smoking the Test cigarette had no effect on the EEG. Smoking the Control cigarette produced a significant increase in beta 2 magnitude and a significant decrease in delta magnitude. This indicates that the effects of smoking on the EEG are a function of nicotine absorbed from cigarette smoke upon inhalation and not a function of inhaled particulate matter, CO, or other gas-phase components.

Analysis of Variance↗
PubMed1992

Group A streptococcal M proteins: virulence factors and protective antigens.

Rebecca Lancefield described group A streptococcal M proteins over 50 years ago, and they have remained at the forefront of investigations into streptococcal pathogenicity to the present day. As described in this review, they form cell surface fibrils with several functions, ranging from resisting phagocytosis and inducing host-crossreactive antibodies, to presenting the host immune system with an accessible protective antigen.

Animals↗
PubMed1992

A recombinant, chimeric enzyme with a novel mechanism of action leading to greater potency and selectivity than tissue-type plasminogen activator.

BACKGROUND: Early intervention with thrombolytic agents has been shown unequivocally to reduce mortality after acute myocardial infarction. Presently used agents have disadvantages such as short half-life, immunogenicity, hypotension, and bleeding complications. Therefore, there is a need to develop improved thrombolytic drugs with novel mechanisms of action leading to improved properties. METHODS AND RESULTS: Hybrid plasminogen/tissue-type plasminogen activator (t-PA) complementary DNA was constructed and expressed in Chinese hamster ovary cells. The chimeric protein, comprising the fibrin-binding domains of plasminogen covalently linked to the catalytic domain of t-PA, was purified and evaluated in vitro and in vivo. The hybrid was inhibited rapidly in human and animal plasmas. The mediator of this rapid inhibition was shown to be alpha 2-antiplasmin. The active center of the hybrid could be protected by reversible active center acylation with a novel inverse acylating agent, 4'-amidinophenyl-4-chloroanthranilic acid (AP-CLAN). An acylated (CLAN-) hybrid was cleared from the bloodstream of guinea pigs at 0.35 +/- 0.02 ml/min.kg-1 compared with a clearance rate of 36 +/- 4 ml/min.kg-1 for t-PA. The CLAN-plasminogen/t-PA hybrid was evaluated in a quantitative, "humanized" guinea pig pulmonary embolism model and shown to be approximately threefold more potent when given by bolus than an infusion of t-PA. Furthermore, the acylated hybrid was more fibrin selective than t-PA as determined by the relation between clot lysis and fibrinogen degradation. CONCLUSIONS: An acylated, recombinant plasminogen/t-PA hybrid has sufficiently slow clearance to be administered by bolus and is more potent and fibrin selective than t-PA in vivo.

Acylation↗
PubMed1992

Palmitic acid conjugation of a protein antigen enhances major histocompatibility complex class II-restricted presentation to T cells.

The effect on antigenicity of covalent attachment of lipid groups to a protein antigen was investigated. Coupling of palmitic acid to ovalbumin (OVA) enhanced major histocompatibility complex (MHC) class II-restricted presentation to most OVA-specific murine T-cell clones in vitro. The enhanced antigenicity of palmitoylated antigen was localized to the level of presentation of the synthetic peptide epitope, OVA 323-339. T-cell responses to palmitoylated antigen were more difficult to block with anti-MHC class II antibodies than responses to native antigen. However, T-cell proliferation to palmitoyl (p)-OVA and native (n)-OVA were blocked equally by anti-CD4 antibodies. Taken together, the results suggest that lipid conjugation of a protein antigen leads to the formation of a lipopeptide T-cell epitope with increased affinity of binding to MHC class II and/or T-cell receptor (TcR). These results have implications for the design of synthetic peptide vaccines.

Adjuvants, Immunologic↗
PubMed1992

T-cell tolerance induced in nude mice grafted with thymic epithelium.

This study investigated the ability of foetal thymic epithelium prepared by 24 degrees culture (24 degrees-TE) or treatment with deoxyguanosine (dGuo-TE), to induce tolerance in nude mice. Thymic chimeras were constructed in which the thymic epithelium differed from the host at both major histocompatibility complex (MHC) antigens and multiple minor histocompatibility antigens (mHa), or at mHa only. Peripheral CTL from nude mice receiving dGuo-TE disparate for mHa, or both MHC antigens and mHa, were uniformly tolerant of thymic mHa. CTL from nude mice grafted with 24 degrees-TE or dGuo-TE were tolerant of host MHC antigens, but the two treatments differed in the efficiency with which they induced tolerance to thymic MHC antigens. CTL responses specific for thymic MHC antigens could be generated in vitro from dGuo-TE grafted mice but not from those receiving 24 degrees-TE. The addition of concanavalin A (Con A) supernatant had no effect on the CTL tolerance observed in 24 degrees-TE grafted mice, suggesting that the lack of CTL responses was not due to tolerance in MHC class II restricted 'helper' cells. However, CTL responses against the thymic MHC antigens of dGuo-TE grafted mice displayed high sensitivity to blocking by anti-CD8 antibodies, indicating that these CTL were of low affinity. These results suggest that 24 degrees-TE induces tolerance in most thymic MHC-specific CTL precursors, whereas dGuo-TE induces tolerance only in CTL with high affinity for thymic MHC antigens. Therefore, 24 degrees-TE and dGuo-TE are both capable of inducing CTL tolerance, consistent with the previously reported acceptance of thymic donor-type skin grafts by nude recipients of dGuo-TE treatment. We conclude that MHC class I molecules on thymic epithelium play a role in negative selection of the developing T-cell repertoire.

Animals↗