T cell antigen receptors in autoimmunity

Three mAb to variable region determinants of the alpha/beta-chain TCR were used to detect discrete populations of peripheral blood T cells. T cells sharing a TCR determinant defined by such an antibody presumably use the same or similar TCR V or J genes for their alpha- or beta-chains. Thus analysis with these mAb provides a tool to investigate TCR gene usage and expression. Since autoantigen specific T cells may play an important role in initiating autoimmune diseases, TCR were analyzed in different autoimmune diseases and control groups including rheumatoid arthritis, Graves disease, idiopathic thrombocytopenic purpura, psoriasis, SLE, insulin-dependent diabetes mellitus, and in nonautoimmune control diseases and normals. Purified T cells were stained by indirect immunofluorescence with three mAb to TCR variable regions: mAb S511 stains 1.8 +/- 0.9% (mean +/- 2 SD), mAb C37 stains 3.4 +/- 1.5% and mAb OT145 stains from 0 to 6% of T cells from normal donors. Several individuals were identified with expanded subsets of positive T cells. One patient with adult ITP followed during a 12-mo period consistently had elevated percentages of T cells staining with the mAb OT145 (15.9 to 24.5%). These cells were found to be exclusively CD8+. By Southern blotting DNA prepared from these OT145+, CD8+ cells, but not DNA from the patient's OT145- T cells, revealed a clonal rearrangement using a beta-chain C region probe. Thus this patient had a monoclonal expansion of CD8+, OT145+ cells. Hyperexpression of a TCR variable region, as defined by the available mAb, could not be associated with any of the diseases studied. Examination of T cells at the site of autoimmunity, such as T cells from rheumatoid arthritis synovial fluid, revealed normal percentages of cells staining with these mAb. Immunoperoxidase staining of psoriatic lesional skin showed no striking enrichment of T cells bearing one or the other TCR type.     

The oral neutrophil alteration test in drug allergy

Oral leukocyte alteration method was established for the diagnosis of drug allergy. This method is 68% positive in patients with drug allergy and 7% positive in healthy persons. The whole procedure is simple and takes only an hour and a half.     

Dendrimerized cellulose as a scaffold for artificial antigens with applications in drug allergy diagnosis

Cellulose-based dendrimerized material was prepared and its quality was assessed by determining the number of amine functional groups incorporated. Based on the results for a series of preparations, the material was obtained in a highly reproducible manner thanks to the particular chemical construction method used. The number of amine groups incorporated and the amount of dendrimer attached are directly related to the dendrimer generation. The combination of the properties of the cellulose polymer and those of the dendrimeric state provides biocompatible materials amenable to easy chemical characterization. The proposed method provides an effective tool for developing clinically testable materials with a view to studying adverse immunological responses to drugs in humans.     

Pattern of changes in clinico-immunological indicators of patients with drug allergy during treatment with autologous blood

An efficient method for the treatment of drug allergy was proposed and practically implemented. The method consists in autologous venous blood being lyzed with sterile bidistilled water at a ratio of 5:1 and injected subcutaneously and partially intradermally into the reflexogenic zones of the back 2-3 cm from the spinal column. During the first week of treatment, increasing doses were injected (3 to 10 ml), whereas during the second week the doses decreased from 10 to 3 ml. Following treatment the patients felt better and featured enhanced working ability as well as markedly declined susceptibility to common colds. The most informative immunological indicators were chosen to evaluate the efficiency of therapy.     

Adenosine receptors as drug targets

There are four adenosine receptors, A₁, A_2A, A_2B and A₃, together forming a defined subgroup of G protein coupled receptors. They are well conserved and widely expressed. The endogenous agonist, adenosine, has a minimal concentration in body fluids (20-200 nM) that is sufficient to slightly activate the receptors where they are very highly expressed—as in the basal ganglia, on fat cells and in the kidney. Here adenosine can play a physiological role and here antagonists such as caffeine can have effects in healthy individuals. Adenosine levels rise in stress and distress (up to 30 μM in ischemia) and tend to minimize the risk for adverse outcomes by increasing energy supply and decreasing cellular work, by stimulating angiogenesis, mediating preconditioning and having multiple effects on immune competent cells. These pathophysiological roles of adenosine also offer some potential drug targets, but the fact that adenosine receptors are involved in so many processes does not simplify drug development.     

Lymphocytes express a diverse array of specific receptors for sulfated polysaccharides

Lymphocyte receptors for sulfated polysaccharides were detected in two ways, namely, by the ability of lymphocytes to form rosettes with sheep red blood cells (SRBC) coupled with one of fourteen different sulfated polysaccharides, and by the ability of cholate extracts of lymphocytes to hemagglutinate the same sulfated polysaccharide-coupled SRBC. It was found that murine lymphocytes lacked receptors for a number of glycosaminoglycans, such as hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate, but reacted strongly with heparin, arteparon, and a number of sulfated polysaccharides of plant and bacterial origin. In each case receptor activity was demonstrated by rosetting and by the ability of lymphocyte lysates to strongly agglutinate sulfated polysaccharide-coupled SRBC. The receptors exhibited a high degree of diversity as evidenced by (a) only subpopulations of lymphocytes, particularly splenic B cells, expressing receptors for some of the sulfated polysaccharides and (b) hemagglutination-inhibition analyses revealing numerous subsets of receptors with different binding specificities. Receptor diversity was further highlighted by a 48% difference in the hemagglutination-inhibiton results between thymus and spleen. It is proposed that these receptors are involved in cell-cell communication and lymphocyte homing and recirculation. The likely target structures for the receptors in vivo are the heparan sulfates, a ubiquitous and structurally diverse family of sulfated glycosaminoglycans.     

Triamcinolone acetonide inhibits lymphocyte differentiation in B cells decorated with artificial antigen receptors

We examined the effects of triamcinolone acetonide (TA) on T cell independent antigen-induced differentiation of human B cells. Purified human B cells artificially decorated with palmitate-conjugated monoclonal IgA antibody specific for 2,4-dinitrophenyl differentiated polyclonally when challenged with optimum concentrations of dinitrophenyl-derivatized polymerized flagellin. This B cell response was reduced by the synthetic corticosteroid TA at a concentration of 10(-6) M. This suggests that TA can inhibit in vitro B lymphocyte differentiation independent of T cells.     

Rational design of T cell receptors with enhanced sensitivity for antigen

Enhancing the affinity of therapeutic T cell receptors (TCR) without altering their specificity is a significant challenge for adoptive immunotherapy. Current efforts have primarily relied on empirical approaches. Here, we used structural analyses to identify a glycine-serine variation in the TCR that modulates antigen sensitivity. A G at position 107 within the CDR3β stalk is encoded within a single mouse and human TCR, TRBV13-2 and TRBV12-5 respectively. Most TCR bear a S107. The S hydroxymethyl side chain intercalates into the core of the CDR3β loop, stabilizing it. G107 TRBV possess a gap in their CDR3β where this S hydroxymethyl moiety would fit. We predicted based on modeling and molecular dynamics simulations that a G107S substitution would increase CDR3β stability and thereby augment receptor sensitivity. Experimentally, a G107S replacement led to an ～10-1000 fold enhanced antigen sensitivity in 3 of 4 TRBV13-2(+) TCR tested. Analysis of fine specificity indicated a preserved binding orientation. These results support the feasibility of developing high affinity antigen specific TCR for therapeutic purposes through the identification and manipulation of critical framework residues. They further indicate that amino acid variations within TRBV not directly involved in ligand contact can program TCR sensitivity, and suggest a role for CDR3 stability in this programming.     

Modelling G-protein-coupled receptors for drug design

The G-protein coupled receptors form a large and diverse multi-gene superfamily with many important physiological functions. As such, they have become important targets in pharmaceutical research. Molecular modelling and site-directed mutagenesis have played an important role in our increasing understanding of the structural basis of drug action at these receptors. Aspects of this understanding, how these techniques can be used within a drug-design programme, and remaining challenges for the future are reviewed.