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Biomedical subjects

S D Sharma

Publications and source records attributed to S D Sharma.

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

Specific tolerance to an acetylcholine receptor epitope induced in vitro in myasthenia gravis CD4+ lymphocytes by soluble major histocompatibility complex class II-peptide complexes.

In autoimmune disorders, inactivation of pathogenic antigen-specific T cells, rather than global immunosuppression, would be highly desirable. One way to achieve this would be to deliver the first antigen-specific signal to the T cell in the absence of the second costimulatory signal. Myasthenia gravis (MG) is a well-characterized autoimmune disease in which T cell-dependent autoantibodies are directed against the acetylcholine receptor (A ChR) at the neuromuscular junction. AChR-specific T cells have been cloned from MG patients, and in this study, we have induced long-lasting tolerance in vitro in one particular clone (PM-A1) with a known peptide epitope (alpha 144-163) and MHC class II restriction (DR4 Dw14.2 or 4.2) by using soluble MHC-class II peptide complexes. Preincubation of PM-A1 T cells with such complexes induced death by apoptosis in < or = 40-50% of the AChR-specific cells. Surviving cells remained refractory to stimulation with AChR-derived synthetic peptides or recombinant polypeptides for < or = 38 d after complex treatment. These effects were highly specific, dose-dependent and required > 2 h preincubation. The T cells could be protected from the tolerizing effects of complex by coincubation with DR-matched or -mismatched antigen-presenting cells. This work shows that antigen-specific T cells can be selectively killed or anergized using soluble MHC class II: peptide complexes. Such an antigen-specific therapy offers a rational approach to the immunotherapy of autoimmune or allergic disease in vivo.

Adolescent↗
PubMed1993

Cloned T cells internalize peptide from bound complexes of peptide and purified class II major histocompatibility complex antigen.

Antigen presentation to helper T cells involves the formation of a trimolecular complex consisting of a class II major histocompatibility complex (MHC) antigen combined with an antigenic peptide on the surface of an antigen-presenting cell and a T cell receptor (TCR) on the T cell. The fate of the MHC class II, peptide, or TCR moieties of the ternary complex following antigen presentation is unknown. Using radiolabeled complexes of affinity-purified murine MHC class II molecules and peptides corresponding to T cell epitopes of myelin basic protein (MBP), this report presents evidence that the binding of preformed relevant MHC class II-peptide complexes to cloned T cells in vitro results in internalization of the peptide moiety. Neither the restricting MHC class II molecule nor the TCR moiety of the trimolecular complex was internalized by T cells. The specificity of peptide internalization was demonstrated using complexes of syngeneic MHC class II with an irrelevant MBP peptide analog and by cloned T cells restricted for a different epitope of the same MBP antigen. Furthermore, the peptide translocation mediated by MHC class II and TCR was demonstrated by antibody-blocking experiments using anti-class and anti-TCR monoclonal antibodies. The peptide internalization by T cells was markedly reduced when binding was performed at 4 degrees C as compared with 37 degrees C. In addition, a significant inhibition of peptide translocation was observed in the presence of a metabolic inhibitor (sodium azide) but not in the presence of cytochalasin B. These results together demonstrate that the in vitro interaction of soluble MHC II-peptide complexes with cloned T cells is an active process associated with uptake of the antigenic peptide.

Amino Acid Sequence↗
PubMed1993

Stimulation of T cells by antigenic peptide complexed with isolated chains of major histocompatibility complex class II molecules.

Major histocompatibility complex (MHC) class II molecules are heterodimeric glycoproteins with one alpha and one beta polypeptide chain of similar molecular size. In this report, we describe the binding of an acetylated N-terminal peptide of myelin basic protein, [Ala4]MBP-(1-14), to purified individual alpha and beta chains of murine I-Ak molecules. Purified complexes of isolated single chains and antigenic peptide bind to cloned T cells restricted by I-Ak and [Ala4]MBP-(1-14) tetradecapeptide. The binding is blocked by alpha/beta anti-T-cell receptor (TCR) monoclonal antibody. Cell triggering as measured by an increase in extracellular acidification rate is observed when cloned T cells are exposed to purified complexes of isolated chains and antigenic peptide. This increase in the extracellular acidification rate is antigen specific and MHC-restricted, as chains alone or irrelevant chain-peptide complexes do not trigger an increase in the metabolic acidification rate. These results together demonstrate that in vitro cloned T cells are triggered by complexes of specific antigenic peptides and isolated individual chains of their cognate MHC proteins.

Amino Acid Sequence↗
PubMed1993

Purified beta-chain of MHC class II binds to CD4 molecules on transfected HeLa cells.

The initial event triggering the activation of Th cells occurs when the TCR interacts with antigenic peptide in the context of the MHC II on APC. Various T cell accessory molecules including CD4, CD28, and LFA-1 participate and facilitate the activation event. Although some evidence for the interaction of MHC II and CD4 is available, the site of MHC class II (alpha-chain, beta-chain, or both chains) for CD4 interaction has not yet been clearly defined. Results from different laboratories had indicated the involvement of alpha 1, beta 1, and beta 2 domains of MHC class II molecules in CD4 interaction. Recently, a conserved site of DR beta 2 domain has been identified that involves CD4 interaction that is analogous to MHC class I binding site for CD8 molecule. In this report, direct binding of affinity-purified HLA-DR2 dimer and its isolated alpha- and beta-chains to CD4 was studied using a CD4-transfected HeLa cell line. Preferential binding of the beta-chain and intact MHC II dimer to the CD4-transfected cells was observed and found to be specifically inhibited by anti-CD4 mAb. In contrast, the isolated alpha-chain of HLA DR2 did not show significant binding to CD4-transfected cells. Complexes of radiolabeled DR2 dimer or beta-chain alone with an immunodominant epitope from myelin basic protein (83-102) did not show any further increase in binding of these molecules. Binding of the beta-chain to CD4+ cells was markedly inhibited by a DR beta 1 peptide (35-46) and was partially inhibited by a DR beta 2 peptide (134-148) of MHC class II molecule. These results suggest the involvement of at least two conserved regions of the beta polypeptide chain of MHC class II in CD4 interaction. Because in our experiments transfected cells lack TCR molecules and the binding of DR2 to the CD4-transfected cells was unaffected by added antigenic peptide, it is possible that the interaction of MHC class II to CD4 is independent of TCR occupancy.

Amino Acid Sequence↗
PubMed1993

Vaccination of mice with the protective F3G3 antigen of Toxoplasma gondii activates CD4+ but not CD8+ T cells and induces Toxoplasma specific IgG antibody.

A major cytoplasmic Toxoplasma gondii (T. gondii) antigen recognized by monoclonal antibody F3G3 (F3G3-Ag), as well as two surface antigens recognized by monoclonal antibodies 2G11 and 1E11 respectively (2G11-Ag; 1E11-Ag), were isolated from crude Toxoplasma sonicates using affinity chromatography. Purified F3G3-Ag induced long term protection against Toxoplasma infection in mice and induced Toxoplasma specific IgG antibody. CD4+ but not CD8+ T cells from immune animals proliferated and produced IL-2 upon restimulation with either Toxoplasma sonicate or F3G3-Ag in vitro. Furthermore, CD4+ T cells from mice immunized with F3G3-Ag responded to purified 2G11- and 1E11-Ag. In contrast, CD4+ T cells from mice immunized with 2G11-Ag responded to Toxoplasma sonicate and 2G11-Ag, but not to F3G3- or 1E11-Ag. The results may indicate that the protective F3G3-Ag shares immunogenic epitopes present also on 2G11- and 1E11-Ag, since the F3G3-Ag used for the vaccination did not contain detectable amounts of 2G11- or 1E11-Ag, and none of the antigens displayed any mitogenicity. Taken together the results show that the cytoplasmic F3G3-Ag of T. gondii induces CD4+ T helper cells, Toxoplasma specific IgG antibodies and long term protection against Toxoplasma infection, but does not induce detectable sensitization of the CD8+ T cell compartment.

Animals↗
PubMed1993

Is combined therapy of sympathomimetics and theophylline indicated?

Bronchodilator effect and toxicity of theophylline 300 mg twice a day (R1), salbutamol 4 mg tid (R2), their combination in higher (200/4mg, R3), and lower doses (100/2mg R4), and placebo (calcium lactate 300 mg) tid (R5) were compared in 25 patients with bronchial asthma in a randomized crossover trial. Statistically significant improvement in forced expiratory volume in one second (FEV1) was observed in all the active treatment groups (R1 to R4) compared with placebo (R5). The mean improvement in FEV1 was 29.0%, 22.0%, 28.0%, 30.0%, and 0.73% in regimen R1, R2, R3, R4, and R5, respectively day 1, and corresponding improvement was 30.0%, 24.0%, 29.0%, 34.0%, and 4.4% on completion of one week therapy. On intergroup statistical comparison, mean improvement in pulmonary function test values were statistically significant or highly significant in regimens R1 to R4, as compared with placebo. However, improvement between any two regimens was not statistically significant in any of the regimens (R1-R4). Almost all the regimens were tolerated well and no patient showed major adverse reactions or cardiotoxicity necessitating withdrawal of the drug. On the other hand, minor adverse reactions were common and the high dose combination (R3) was found to have more adverse reactions than the low dose combination and either drug used alone.

Adolescent↗
PubMed1992

N-linked oligosaccharides of murine major histocompatibility complex class II molecule. Role in antigenic peptide binding, T cell recognition, and clonal nonresponsiveness.

To evaluate the functional role of the N-linked oligosaccharides of major histocompatibility complex (MHC) class II molecules, affinity-purified murine IAs class II molecules were deglycosylated in the presence of asparagine amidase enzyme. The deglycosylated IAs molecules were characterized by 12% SDS-polyacrylamide gel analysis under reduced and native conditions and the complete enzymatic removal of all three N-linked sugar components from the alpha/beta heterodimer was confirmed by lectin-link Western blot analysis. Like the native IAs molecules, the deglycosylated IAs molecules were fully capable of binding an antigenic peptide from myelin basic protein MBP(89-101). The kinetics of dissociation of preformed complexes of IAs.MBP(89-101) and deglycosylated IAs.MBP(89-101) were compared at 4 and at 37 degrees C. Both complexes were equally stable at 4 degrees C; however, at 37 degrees C the deglycosylated IAs.MBP(89-101) complexes showed an increased rate of dissociation as compared with the native IAs.MBP(89-101) complexes. When tested for their ability to recognize the T cell receptor on T cells, both complexes bound to cloned HS-1 T cells that recognize and respond to IAs.MBP(89-101). Finally, the complexes of deglycosylated IAs.MBP(89-101) were tested for the induction of in vitro nonresponsiveness and compared with native IAs.MBP(89-101) complexes. Both complexes were capable of inducing 95-100% nonresponsiveness in a proliferation assay. These results suggest that the N-linked oligosaccharide of MHC class II molecules may not be essential for either antigenic peptide binding or T cell recognition. In addition results obtained here provide evidence that the carbohydrate moities of MHC class II molecules may not be involved in induction of T cell clonal anergy.

Amidohydrolases↗
PubMed1992

Antigen-specific stimulation of T cell extracellular acidification by MHC class II-peptide complexes.

A specific T cell response to a preformed complex of detergent-solubilized MHC class II molecule and cognate antigenic peptide was observed by monitoring the extracellular acidification. An increase in this rate was observed when the resting 4R3.9 T cell clone specific for the peptide fragment MBP(1-14) of myelin basic protein was exposed to preformed detergent-solubilized IAk-MBP(1-14)A4 complexes. MBP peptide alone, IAk alone, or complexes of IAs-proteolipid protein(139-151) and IAd-OVA(323-339), did not cause significant increases in the acidification rates of the MBP(1-14)-restricted 4R3.9 T cell clone. In addition, BW 5147 T lymphoma cells, which lack TCR, did not show any increase in rate when exposed to IAk-MBP(1-14)A4 complexes. Similar increases in acidification rate were observed in the presence of IL-2, anti-CD3 and anti-TCR antibodies. The enhanced acidification responses were blocked by genistein, a tyrosine kinase inhibitor.

Acids↗
PubMed1992

Race, ethnicity and birth-weight: Hawaii 1983 to 1986.

A study of racial/ethnic-specific mean infant birth-weights reported on 1968 to 1972 live birth-weight certificates for Oahu, Hawaii is reviewed here. The 1983 to 1986 data confirm those earlier results: (a) The Hawaiian group is significantly heavier in mean birth-weights than other cohorts during the preterm period (33 to 36 weeks gestational age); (b) statistically significant differences also were present in mean birth-weights for the term and post-term periods (37 to 45 weeks gestational age); and (c) the ranking of cohorts from heaviest to lightest in mean birth-weights is Caucasian, Hawaiian, Japanese and Filipino respectively. The data raises 2 questions: (1) Which birth-weight standards are most applicable for Asian and Pacific Islanders; and (2) what are the implications of the race/ethnic-specific mean birth-weight differences relative to mortality, morbidity and developmental outcome?

Birth Weight↗
PubMed1991

Antigen-specific therapy of experimental allergic encephalomyelitis by soluble class II major histocompatibility complex-peptide complexes.

Experimental allergic encephalomyelitis is a T-cell-mediated, major histocompatibility complex (MHC) class II gene-linked autoimmune demyelinating disease of the central nervous system. To develop therapies that will specifically inactivate only the autoantigen-reactive T cells, mice were treated with soluble MHC class II molecules that had been complexed with encephalitogenic peptides. Intravenous injections of 300 micrograms of complexes consisting of encephalitogenic peptide 91-103 of myelin basic protein plus I-As protein on day 0, 4, and 7 were effective in preventing experimental allergic encephalomyelitis. Similarly, administration of 45 micrograms of I-As protein complexed to peptide 139-151 from proteolipoprotein on day 1, 4, and 7 prevented mortality and significantly reduced paralysis induced by immunization with the encephalitogenic proteolipoprotein peptide. Histological examination of sections of animal brains revealed that treatment with I-As protein plus myelin basic protein 91-103 peptide prevents the development of inflammatory lesions characteristic of experimental allergic encephalomyelitis. Thus, treatment with MHC-self-peptide complexes could serve as a highly specific therapeutic modality in treating autoimmune disease when the putative autoantigen and the MHC restricting elements are known.

Animals↗
PubMed1991

An in vitro model system to evaluate pulmonary macrophage, endothelial cell, and neutrophil interactions.

Pasteurella haemolytica, the cause of fibrinous pleuropneumonia in cattle, produces extensive microvascular endothelial cell (EC) damage. We have developed an in vitro model system to study the inflammatory process of this disease involving the interaction of pulmonary alveolar macrophages (AM), neutrophils (PMN), and EC. Bovine EC are grown to confluency in 24 well tissue culture plates. To mimic the vascular component, 10(6) PMN are later added to the EC monolayers. Bovine AM are plated onto millicell inserts and placed into the wells containing EC and PMN. The millicell insert serves as a semi-permeable barrier between AM and EC, allowing the exchange of only diffusible material. Preliminary work demonstrates that P. haemolytica stimulated AM resulted in EC damage presumably due to both soluble lipopolysaccharide and AM secreted products.

Animals↗
PubMed1991

Designing peptide and protein ligands for biological receptors.

A clearer understanding of structure-function relationships of protein hormone-receptor systems is emerging from the increased use of molecular biology approaches. On the other hand, the introduction of rationally designed conformation constraints into peptide hormones and neurotransmitters is leading to the development of highly receptor-selective ligands that allow further investigations into the topographic modulation of their bioactivities and rational design principles for peptide antagonists.

Amino Acid Sequence↗
PubMed1991

[D-Pen2,4'-125I-Phe4,D-Pen5]enkephalin: a selective high affinity radioligand for delta opioid receptors with exceptional specific activity.

[D-Pen2,4'-125I-Phe4,D-Pen5]enkephalin ([125I]DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. [125I]DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 +/- 67 pM and a receptor density (Bmax) value of 36.4 +/- 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM [125I] DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 +/- 0.88 X 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM [3H DPDPE and 0.8 nM [3H] [D-Pen2,4'-Cl-Phe4, D-Pen5]enkephalin, respectively. The dissociation rate of [125I]DPDPE (0.917 +/- 0.117 X 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of [125I]DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes [125I]DPDPE a valuable new radioligand for studies of delta opioid receptors.

Animals↗
PubMed1991

Distribution and analgesia of [3H][D-Pen2, D-Pen5]enkephalin and two halogenated analogs after intravenous administration.

To improve pharmacological characteristics of the delta-selective, cyclic peptide [D-Pen2, D-Pen5]enkephalin (DPDPE), modification by halogenation at the Phe4 residue was undertaken. The present study was to determine the extent [3H]DPDPE, [3H][p-Cl-Phe4]DPDPE and [p-125IPhe4]DPDPE crosses the blood-brain barrier, elicits analgesia and to characterize selective organ distribution and stability after i.v. administration. A significantly greater percentage of total [3H][p-Cl-Phe4]DPDPE reached the brain after 10, 20 and 40 min as compared to [3H]DPDPE and both peptides were significantly displaced by pretreatment with naloxone or naltrindole. The amount of [3H]DPDPE detected in the brain was greater than that of [p-125IPhe4]DPDPE. Distribution results revealed large amounts of the administered peptides were sequestered rapidly in the gall bladder and secreted into the small intestine. Hot-plate antinociception tests 5 min after i.v. administration (30 and 60 mg/kg) revealed [p-Cl-Phe4]DPDPE to elicit a much greater analgesic effect as compared to DPDPE or [p-125IPhe4]DPDPE. These results provide evidence that [p-Cl-Phe4]DPDPE has a greater apparent distribution to the brain and has a greater effect on the antinociception threshold as tested on the hot-plate than DPDPE or [p-125IPhe4]DPDPE. Stability of unlabeled and tritiated DPDPE and [p-Cl-Phe4]DPDPE was determined both in vitro and in vivo; both unlabeled and tritiated DPDPE and [p-Cl-Phe4]DPDPE remain intact.

Analgesics↗
PubMed1989

Lymphocyte proliferative responses of patients with ocular toxoplasmosis to parasite and retinal antigens.

In vitro lymphocyte proliferative responses to purified toxoplasma antigens and the retinal S-antigen were evaluated in 40 patients with ocular toxoplasmosis; 16 (40%) had a positive in vitro response to the retinal S-antigen. The proliferative responses to the 2G11 (p22) toxoplasma membrane antigen approached that of a crude antigen preparation of Toxoplasma gondii, whereas the response to the 1E11 immunodominant p30 membrane antigen was considerably less striking. HLA typing was also performed, and none correlated to S-antigen or toxoplasma reactivity. The evidence strongly suggests that patients with a clearly infectious ocular disorder may have part of their disease mediated by a putative autoimmune mechanism. An immune response to the 2G11 toxoplasma antigen may increase the risk of developing ocular disease or a substrain of toxoplasma with 2G11 as its immunodominant membrane antigen may have a greater affinity for the retina.

Adolescent↗