Dental Caries and Brushing Techniques Attitude in School Students: An Original Research.

Introduction: Oral health plays an important role in the overall wellness of an individual. Hence in our study, we aim to evaluate the awareness and knowledge about dental caries and pattern of brushing among secondary school students. Materials and Methods: We conducted a questionnaire-based survey among 200 secondary school students to estimate their awareness and knowledge about dental caries and brushing pattern. Only those students with at least one filled, missing, or decayed tooth were considered. The data was presented as percentages. Results: We observed that knowledge regarding dental caries among students was 72.5%. 75.5% students had good knowledge of brushing teeth; nonetheless 30% brushed their teeth twice. Only 21.5% students visited the dental clinic. Conclusion: Though good knowledge about dental caries and brushing was appreciated among the students, very few students practiced good oral hygiene habits. Promotion of oral hygiene habits should be motivated at the school level.

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.

Anandamide exerts a suppressive effect on sperm binding to oviduct explants through CB1 receptors in the water buffalo (Bubalus bubalis).

An endocannabinoid system comprising of Anandamide (AEA) and its receptor has been shown to play a role in sperm acquisition of fertilizing potential and sperm-oviduct interaction. In the present study, we assessed the effect of sperm pre-treatment with AEA or co-incubation of sperm-oviduct explants with AEA in the presence or absence of CB1 receptor antagonist (SR141716A) on sperm-oviduct binding in the water buffalo. Cryopreserved spermatozoa from 3 Murrah buffalo bulls (3 ejaculates from each bull) were utilized for the study. Oviduct explants were prepared by overnight culture of epithelial cells in TCM- 199 and washed spermatozoa were added to the oviduct explants and incubated for 1h. Then, sperm-oviduct explants were stained with a fluorescent stain (JC-1) and sperm binding index (BI - No. of bound spermatozoa/unit area of oviduct explants) was assessed. The results indicate that BI decreased significantly (P<0.05) when spermatozoa were either pre-treated with AEA (14.16±0.87) or sperm-oviduct explants were co-incubated with AEA (16.27±0.86) at 1nM concentration compared to the control group (29.12±2.17), however such effect was not observed when AEA was used at 1µM concentration. Incorporation of SR141716A in the incubation medium inhibited the suppressive effect of AEA on BI. It was concluded that AEA, at 1nM concentration, decreased the number of spermatozoa bound to the oviduct explants and the suppressive effect of AEA on sperm-oviduct binding was inhibited by CB1 receptor antagonist suggesting that the effect of AEA was mediated through CB1 receptor in the water buffalo.

pH dependent binding of high and low affinity myelin basic protein peptides to purified HLA-DR2.

Major histocompatibility complex (MHC) class II molecules are cell surface glycoproteins and are known to display processed antigens on the surface of antigen presenting cells (APC). Within the APC, the loading of processed antigenic peptides to MHC class II molecules is known to take place in the endosomal compartment at acidic pH environment. The present study describes the in vitro effect of pH on binding of four biotinylated myelin basic protein (MBP) peptides to affinity purified HLA-DR2 containing a mixture of DRB1*1501 and DRB5*0101 beta chain. The binding affinity of the selected peptides are in the order of MBP(83-102)Y83 > MBP(124-143) > MBP(143-168) > MBP(1-14). Most of these peptides in association with HLA-DR2 are considered as immunodominant epitopes for human multiple sclerosis autoimmune disorder. One epitope, MBP(1-14), had almost no affinity to purified HLA-DR2 and was used as a control peptide in all binding assays. The quantitation of the bound peptide at various pH was carried out by antibody capture of complexes followed by avidin-alkaline phosphatase detection system. Among four peptides tested, only the highest affinity MBP(83-102)Y83 peptide showed maximum binding to purified HLA-DR2 at acidic pH. Two other epitopes, MBP(124-143) and MBP(143-168), showed maximum binding at basic and neutral pH values, respectively. The binding of only high affinity peptides, MBP(83-102)Y83 and MBP(124-143), was significantly affected by changing the pH of the binding buffer. Such alteration in pH of the binding buffer resulted in 100% occupancy of DR2 with both high affinity MBP peptides. In contrast, no significant increase in binding of the low affinity MBP(143-168) peptide was observed at altered pH values. The specificity of the increased binding of high affinity peptides to HLA-DR2 at optimum pH was demonstrated by competitive binding assays using non-biotinylated peptides. Finally, the stability of various MBP peptide bound complexes was tested at 4 degrees, 25 degrees and 37 degrees C which correlates well with their affinity to HLA-DR2. These results suggest that pH plays an important role in in vitro binding of antigenic peptides and such manipulation of binding conditions can be utilized in generating 100% loaded MHC class II with high affinity antigenic peptides. Since high affinity peptides are generally considered as major immunodominant epitopes, the in vitro pH dependent binding can be utilized in screening immunodominant epitopes of various autoantigens and generating complexes of defined composition.

Antigenic peptide binding to MHC class II molecules at increased peptide concentrations.

Affinity-purified major histocompatibility complex (MHC) class II molecules are known to bind antigenic peptides in vitro. The percentage of MHC class II molecules occupied with such peptides is usually very low and varies significantly depending upon the sequence and size of a given antigenic peptide. The present study describes a method by which complete saturation of affinity-purified MHC class II with antigenic peptide can be achieved by simply incubating purified MHC class II molecules at neutral pH in the presence of several 100-fold molar excess of antigenic peptide. Complexes of human HLA-DR2 and a peptide analog from human myelin basic protein MBP (83-102)Y83 were selected for this study. The on-rate kinetic results showed saturation of MHC class II occupancy at 300-500-fold molar excess peptide concentrations. The specificity of the MBP (83-102)Y83 peptide binding to HLA-DR2 at higher peptide concentration was demonstrated by incubating an equivalent amount of another epitope from myelin basic protein [MBP (1-14) peptide] as well as by competitive binding assays. The quantitation of bound peptide was carried out using biotinylated-MBP (83-102)Y83 peptide which showed 100-125% occupancy of HLA-DR2 with a recovery of 100%. The presence of a single peptide entity in purified complexes was confirmed by reverse-phase narrowbore HPLC analysis of the acid extracted supernatant and by mass spectrometry analysis. Two-dimensional gel electrophoresis (IEF/SDS) of purified HLA-DR2 and DR2.MBP (83-102)Y83 complexes showed the absence of various endogenous polypeptides in 100% loaded complexes. These results demonstrate that higher peptide concentrations can be useful in generating MHC class II-peptide complexes of defined composition. Such complexes of MHC class II occupied with a single peptide may have significant clinical relevance for antigen-specific therapy of various autoimmune diseases and may provide better understanding of MHC-peptide-TCR interactions.

Drug induced gingival overgrowth: a rare case report.

Gingival overgrowth is well documented side effect associated with three major classes of drugs viz, anticonvulsants, calcium channel blockers, and immunosuppressants. Despite our greater understanding of pathogenesis of Drug induced Gingival Overgrowth (DIGO), its treatment still remains a challenge for the periodontists and treatment is still largely limited to maintenance of improved level of oral hygiene and surgical removal of overgrown tissue. Dental Surgeons need to discuss this issue with their medical colleagues and to practice care while prescribing the drugs associated with gingival overgrowth. The aim of present article is to report a rare case where even after extraction of all teeth; the enlargement did not subsided for one month.

Preparative-scale purification and characterization of MHC class II monomers.

The MHC class II molecule is a heterodimeric glycoprotein consisting of one alpha and one beta polypeptide chain of almost identical molecular size. Recently it has been shown by others, and confirmed in our laboratory, that isolated monomers of murine MHC II molecules are capable of binding antigenic peptides like the alpha/beta intact heterodimer. In addition, preliminary results from our laboratory indicate that isolated single chain-peptide complexes of murine MHC class II molecules are capable of stimulating cloned T cells in an antigen specific manner. These results prompted us to isolate relatively large quantities of individual alpha and beta subunits of MHC II molecules for further in vitro and in vivo studies. Isolation of alpha and beta monomers proved to be difficult using conventional chromatographic methods. In this report we describe micro-preparative and preparative continuous flow electrophoresis methods by which milligram quantities of MHC II subunits can be purified. An optimal condition for the dissociation of heterodimeric MHC II into alpha and beta monomers was identified, and separation of human HLA DR2 and murine IAs monomers was accomplished. Both methods offer the resolving power of gel electrophoresis with the convenience of continuous sample elution. Purified MHC II subunits obtained by these methods were tested for their ability to bind antigenic peptides. Results presented in this study indicate that monomeric subunits of both human HLA-DR2 and murine IAs are equally active in specific binding of antigenic peptides like the native heterodimer.

Identification of core structure and critical T cell receptor contact residues in an antigenic peptide by measuring acidification rates.

A silicon-based biosensor microphysiometer measures real time cell response by monitoring an increase in extracellular acidification rate in response to ligands for specific membrane receptors. We used the microphysiometer to identify the minimal structure and critical residues of an antigenic peptide for its interaction with T cell receptor (TCR) using a synthetic peptide analog of human myelin basic protein (MBP) corresponding to residues 84-102 [MBP(83-102)Y83]. MBP(83-102)Y83 peptide analogs were allowed to interact with TCRs on a DRB5*0101-restricted Herpes virus saimiri (HVS) transformed human T cell clone (SS8T) which also contains major histocompatibility complexes (MHC) class II (DR2) molecules. Cultured SS8T cells were exposed to 11 N-terminus and 11 C-terminus truncated peptides separately in the microphysiometer chambers to determine the minimal amino acid residues required for the T cell response. In parallel, 13 analogs of the MBP(83-102)Y83 peptide with single alanine substitutions were tested in this assay to identify critical amino acid residues involved in TCR interactions. A minimal core length of MBP(91-100) peptide and residues F-91, K-93, N-94, I-95 and V-96 were essential for TCR interaction. Acidification rate measurements correlated well with enhanced levels of gamma-IFN (interferon gamma) and TNF-beta cytokine production and suggested that the increase in the extracellular acidification rate is a direct result of early T cell signaling events.

Novel methods to rapidly and sensitively analyze antigenic peptide binding to MHC class II molecules.

One of the important steps for antigen presentation by MHC class II molecules involves binding of a peptide fragment of the antigen to the class II molecule followed by recognition of the resulting complex by T cells. The most commonly used methods for studying binding of peptide to MHC II are: equilibrium dialysis, gel filtration chromatography, HPLC and polyacrylamide gel electrophoresis. Each of these methods has some limitations and is time consuming. In addition, each requires a considerable amount of native MHC class II, which is always difficult to obtain. In this report, we describe three different sensitive methods using radiolabeled peptide to study peptide binding to murine MHC class II molecules. These are: nitrocellulose filter binding, thin-layer chromatography (TLC) using plate-supported silica gel or PEI cellulose, and paper electrophoresis using Sepraphor cellulose polyacetate paper. All three methods are rapid, highly sensitive and require only ng quantities of affinity pure MHC class II molecules and peptides. These methods can be used to calculate the peptide occupancy of MHC class II molecules.

Separation of complexes of major histocompatibility class II molecules and known antigenic peptide by metal chelate affinity chromatography.

A small fraction of affinity-purified MHC class II molecules are known to bind antigenic peptides in vitro. No simple method with acceptable recovery exists for separation of complexes of a known antigenic epitope and MHC class II from empty MHC class II and complexes of MHC class II and endogenously bound peptide. Here we describe an one step metal chelate affinity chromatography method to purify complexes of MHC class II and antigenic peptide of known composition. Complexes of human HLA-DR2 (DRB1*1501/DRB5*0101) and a peptide analog from human myelin basic protein MBP(84-102) containing a 6 histidine tag (6 x His) and a tyrosine residue at the N-terminus end [6 x His-MBP(83-102)Y83] were prepared and purified. The absence of residual free 6 x His-MBP peptide in the complex preparations were confirmed by gel filtration and TLC analyses. The purified complexes were applied onto Ni2+.nitrilotriacetic acid (Ni2+.NTA)-agarose affinity support and 6 x His-tagged peptide class II complexes were selectively eluted with imidazole-containing buffer. The quantitation of bound peptide in the eluted complexes showed 100% occupancy of HLA-DR2 (DRB1*1501/DRB5*0101) with [6 x His-MBP(83-102)Y83] peptide with a recovery of 50-75%. The presence of a single peptide entity in the eluted complexes was confirmed by reverse-phase narrowbore HPLC analysis of the acid-extracted supernatant and by amino acid sequencing analyses. As expected, no endogenous polypeptide was detected in the Ni2+.NTA eluted complexes when analyzed by two-dimensional IEF gel electrophoresis. Finally, we demonstrate that both MBP(84-102) and [6 x His-MBP(83-102)Y83] peptides were equally capable of stimulating restricted T cell line in the presence of autologous antigen presenting cells (APCs). These results demonstrate that metal chelate affinity chromatography can be used to prepare MHC class II-peptide complexes containing single peptide. Such complexes of class II molecules containing known peptide have significant clinical relevance for antigen-specific therapy of various autoimmune diseases and may provide better understanding of the trimolecular interaction between MHC class II, antigenic peptide and T cell receptor (TCR).

The role of N-linked oligosaccharides of MHC class II antigens in T cell stimulation.

A specific increase in T cell extracellular acidification rate has been demonstrated recently when complexes of purified MHC class II molecules and antigenic peptides interact with T cell receptors (TCRs) on cloned T cells. The present study shows that such measurements of an increase in extracellular acidification rate can be used to evaluate the functional role of various N-linked oligosaccharides of MHC class II antigens. Affinity-purified murine IAk and IAs were deglycosylated in the presence of aspargine-amidase enzyme and were characterized by SDS-polyacrylamide gel electrophoresis. The complete removal of all three N-linked oligosaccharides from the alpha/beta heterodimer was confirmed by four different lectin-linked Western blot analyses. Similar to the native heterodimer, both deglycosylated IAk and deglycosylated IAs were fully capable of binding synthetic antigenic peptides derived from myelin basic protein (MBP). When equivalent amount of glycosylated and deglycosylated class II-peptide complexes were exposed to restricted cloned T cells, identical increases in T cell extracellular acidification rates were observed. The specificity of such increases in extracellular acidification rate was demonstrated by exposing cloned T cells to irrelevant complexes of glycosylated and deglycosylated class II and antigenic peptides. These results show how measurement of extracellular acidification rate can be used to study structure-function correlations of ligand-receptor interactions, and support an earlier observation that N-linked oligosaccharides of murine MHC class II molecules are not involved in either antigenic peptide binding or T cell recognition.

Vaccination with peptides from MHC class II beta chain hypervariable region causes allele-specific suppression of EAE.

In our earlier studies we showed that successful immunotherapy of EAE in SJL/J mice can be achieved either by the use of antibodies to MHC class II antigens or by vaccination with synthetic peptide analogs of the beta chain of MHC class II molecules. We proposed that inhibition of EAE following vaccination with synthetic peptides derived from the beta chain of mouse I-A, was in part due to the generation of auto-anti-MHC class II antibodies that interfered with T cell sensitization. In our present study we show that suppression of EAE following vaccination results in poor sensitization of MBP reactive T cells, and that the lack of immune response is allele-specific. In F1(SJL(I-AS) x Balb/cI-Ad) mice, in which susceptibility to EAE is linked closely to the I-AS allele, vaccination with peptides from beta chain of I-AS results in inhibition of proliferative response to MBP and prevents the development of EAE. Vaccination with peptide from the beta chain of I-Ad did not affect either the development of immune response to MBP or the induction of EAE, indicating allele-specific suppression. Since global immunosuppression is not induced by vaccination with I-A peptides, we propose that this strategy can be extended to human autoimmune diseases wherein a clear association between certain MHC class II alleles and autoimmune disease is evident.

Localization of soluble major histocompatibility class II-peptide complexes on T cell surface.

Affinity purified major histocompatibility (MHC)-peptide complexes are heterodimeric cell surface glycoproteins and are known to recognize antigen-specific CD4(+) T cell receptors (TCRs). In general, the affinity of MHC-peptide complexes to TCRs are considered very low with a K(D) of 5 x 10(-5) M and, therefore, stabilization of these complexes on T cell surface was not reported earlier. This could be due to (1) incomplete occupancy of MHC molecules with antigenic peptides, (2) variability of the binding constant of peptides to MHC molecules, (3) presence of endogenously bound peptides in MHC preparations, or (4) a combination of these. Using well-characterized HLA-DR2 complex loaded with a high affinity immunodominant epitope analog from human myelin basic protein (MBP), which shows release of gamma-IFN by specific stimulation of transformed human T cell clone (SS8T). The present report demonstrates a method for the localization of bound MHC class II-peptide complexes on T cell surface by backscatter electron imaging using in-lens Field Emission Scanning Electron Microscopy (FESEM). The localization is specific to the complex recognized by the TCR on MHC class II (DR2) and MBP peptide restricted human T cells.

New cavity perturbation technique for microwave measurement of dielectric constant.

A new cavity perturbation technique is presented for microwave measurement of dielectric constant, which uses a modified cylindrical reentrant cavity. Though suitable for only low dielectric constants, the method has the advantages, (a) sample area does not appear in the calculations, (b) only the ratio of frequency shifts due to two samples of same area and different thickness is involved, and (c) calibration of the measuring system with known dielectric is not necessary.

Identification of the elongation factor Tu binding site on 70S E. coli ribosomes by chemical crosslinking.

Elongation factor Tu (EF-Tu), in the presence of Phe-tRNA, GMPPCP, and Poly (U), binds to 70S ribosomes at the recognition (R) site. In order to identify the ribosomal proteins adjacent to the EF-Tu occupying the R site, EF-Tu:Phe-tRNA:GMPPCP:ribosome complexes were crosslinked by modification with 2-iminothiolane and mild oxidation to form disulfide bridges between neighbouring proteins whose endogenous or introduced SH groups were appropriately located. The binding of Phe-tRNA to the ribosome was shown to be largely dependent on the presence of Poly(U). The total protein from the complexes was extracted and separated by two-dimensional gel electrophoresis by non-equilibrium pH gradient electrophoresis (NEpHGE) in the first dimension, followed by gradient SDS gel electrophoresis in the second dimension. Comparison of control samples crosslinked without Poly(U) to those crosslinked with Poly(U) present showed a single crosslinked complex in the region of the gel near EF-Tu. No cross-links in the vicinity of EF-Tu were visible in the absence of Poly(U). The crosslinked proteins in this region were recovered by electroelution, radiolabeled and their identity was confirmed by 2D gel electrophoresis and immunoblot analyses. Two major 50S ribosomal proteins, L7/L12 and L10 were found to be covalently linked to EF-Tu. The isolated crosslinked complex did not contain any protein from the 30S subunit. These results demonstrate that L7/L12 and L10 are the major, if not only, ribosomal protein cross-links to EF-Tu in the R site. In contrast to previous crosslinking results obtained by others, our results define a unique location for the EF-Tu binding site, one compatible with functional data and near that of the EF-G binding site on the ribosome.

T-cell receptor-mediated signal transduction in transformed human T-cells.

Recently it has been shown that purified complexes of major histocompatibility (MHC) class II and antigenic peptide can recognize T-cell receptors (TCRs) on virally transformed CD4+ T-cells in vitro. It is not clearly understood whether peptide bound to purified MHC II molecules (MHC-P), or to MHC II molecules on the surface of antigen-presenting cells (APC-peptide), initiate similar or different signals in transformed human T-cells. To address this question, the expression of protein tyrosine kinases (PTKs), their phosphorylation, and the effect of various kinase inhibitors were investigated using transformed T- and B-cells. HLA-DR2- and MBP(84-102)-restricted cloned T-cells (SS8T) immortalized with herpes saimiri virus (HSV) and DR2-expressing lymphoblastoid B cells transformed with Epstein-Barr virus (EBV) were utilized in this study. The expression and phosphorylation of three major PTKs (1ck-56, fyn-59, zap-70) involved in signaling through the TCR were analyzed by enhanced chemiluminescence blots. T-cells exposed to soluble MHC-P complex did not show altered expression of 1ck-56 protein. In contrast, a decrease in 1ck expression was observed in SS8T cells when TCRs were engaged with APC-peptide. Upon interaction with the TCR, both MHC-P complex and APC-peptide showed increased fyn-59 protein expression and phosphorylation. In our experiments using immortalized T- and B-cells, the expression of zap-70 protein remained unchanged. When T-cells were exposed to herbimycin and H-7, inhibitors of PTKs and protein kinase C (PKC) pathways, respectively, a dose-dependent decrease in gamma-IFN levels was observed with both systems. However, in the presence of genestein, another PTK inhibitor, such decrease in gamma-IFN was observed only in the case in which T-cells were exposed to MHC-P complexes. These results together suggest that the occupancy of TCRs in transformed T-cells by soluble MHC-P complex and APC-peptide differs with respect to the 1ck expression, although both can induce signals that lead to increased fyn activity and its phosphorylation. In addition, genestein showed differential inhibitory effect on gamma-IFN production by T-cells exposed to APC-peptide and MHC-P complexes, suggesting that the TCR occupancy by MHC-P complex and APC-peptide have subtle differences in PTK pathways.

Application of murine T-T hybridoma cells to in vitro potency assay of human synthetic peptide vaccines.

Recently, it has been shown that the immunization of mice with an 18 amino acid synthetic peptide corresponding to the third hypervariable region of MHC class II beta chain can induce a specific antibody response against MHC class II molecules, and can be utilized in the prevention and treatment of experimental allergic encephalomyelitis (EAE) [Proc. Natl. Acad. Sci. 1994, 91, 8005-8009]. Based on this finding, a chemically-modified synthetic peptide with the amino acid sequence corresponding to residues of beta 57-76 from human HLA-DR4Dw4 (DR4/1 peptide) is being clinically investigated for the treatment of rheumatoid arthritis in human. The present study describes the development of a novel in vitro potency assay for human HLA-DR4/1 peptide using cloned murine T-T hybridoma cells. Several mouse strains were immunized with the DR4/1 peptide and their lymph node T cell proliferation was measured in the presence of syngeneic APCs and the DR4/1 peptide. T cells isolated from the peptide primed-B10. PL mouse strain, which showed the highest recall response in this assay, were fused with BW5147 lymphoma cells to generate DR4/1 peptide-specific T-T hybridoma clones. Cloned hybridoma cells were characterized for peptide specificity and MHC class II restriction, and used to monitor the biological activity of various DR4/1 peptide preparations. The potency of peptide batches were assessed by measuring the IL-2 secretion of cloned T-T hybridoma cells upon TCR engagement in an antigen-specific manner. The quantitative detection of IL-2 was performed by measuring [3H]thymidine incorporation of HT-2 cells or directly by ELISA. These results demonstrate that peptide-specific murine T-T hybridoma clones can be successfully utilized to monitor biological activity of synthetic peptides by measuring T cell-mediated immunological responses. Development of such in vitro potency assay for synthetic peptides may have broad applications for vaccines related to immunological disorders.

Differential expression of protein tyrosine kinases and their phosphorylation in murine Th1 cells anergized with class II MHC-peptide complexes.

In resting T cell clones, antigen presentation with immobilized anti-CD3 or anti-T cell receptor (TCR) is known to result in a state of anergy as characterized by unresponsiveness to normal antigenic restimulation. Similarly, T cell unresponsiveness could be induced by immobilized (plate-coated) complexes of purified class II MHC and antigenic peptide. It is not clearly defined whether the engagement of TCR by immobilized anti-TCR or immobilized class II MHC-peptide complexes generates similar or differential signals during the induction of T cell unresponsiveness. In order to address the initial signalling events induced by TCR occupancy with anti-TCR and class II MHC-peptide molecules, the expression of three critical protein tyrosine kinases (PTK) and their phosphorylation were investigated in the present study using a murine T cell clone (HS17) restricted for IAS and myelin basic protein (MBP (91-103)) peptide. The anergic T cells induced by immobilized IAS-MBP (91-103) complex or anti-TCR (H57) showed differential expression of lck (56 kDa) and Zap-70 (70 kDa) proteins. In both systems, however, the induction of T cell unresponsiveness was accompanied by increased level of fyn (59 kDa) expression. When analysed for the total tyrosine phosphorylation of PTK, anergic HS17 T cells induced by both molecules showed increased phosphorylation associated with only the fyn protein. These results suggest that the signal transduction events induced by immobilized class II MHC-peptide complexes and anti-TCR are distinct, although both can initiate signals that lead to increased fyn expression and phosphorylation. In addition, the present study supports the evidence for the important functional association of fyn protein with direct TCR engagement in T cell signalling.