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.

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.

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.

Oesophageal web--a case report.

A case of posterior oesophageal web in an 18-year-old girl is being presented, in view of its rarity. The diagnosis could be established only after thoracotomy and exploration of the oesophagus. The clinical profile along with possible theories of aetiology are discussed and a brief review of literature is made.

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.

Peptide binding inhibits aggregation of soluble MHC class II in solution.

Affinity-purified major histocompatability complex (MHC) class II molecules are known to bind antigenic peptide in vitro. This peptide-bound MHC class II is known to undergo a change in structure upon stable binding of antigenic peptide. Previous results from our, and other laboratories, have suggested a relationship between MHC class II structure and peptide association that enables class II to enter into a stable conformation upon peptide binding. In this report we describe that stable binding of high-affinity antigenic peptide to MHC class II molecule results in transition of aggregated purified MHC class II proteins to a stable heterodimeric state. Such transition was demonstrated by using purified human HLA-DR2 class II molecule and high-affinity myelin basic protein (MBP) 83-102)Y83 peptide. Highly aggregated purified DR2 (high molecular weight; HMW) was first separated from heterodimer (low molecular weight: LMW) in the presence of 50-fold molar excess of MBP(83-102)Y83 peptide. We then show that the aggregated HMW preparation can be successfully converted into a stable dimer by further incubation with MBP(83-102)Y83 and changing various binding parameters such as pH, temperature, reducing agent, and peptide concentrations. Under optimized conditions, the highly aggregated inactive DR2 molecules can be completely loaded with the antigenic peptide. The transformed heterodimers with bound peptide prepared by this method are biologically active, as shown by their ability to induce the production of gamma-interferon by SS8T-transformed human T cells. These results suggest that in solution, MHC class II molecules may be aggregated in the absence of bound peptide. Such aggregated MHC class II molecules can be converted to stable and biologically active heterodimers in the presence of high-affinity antigenic peptide.

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.

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.

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.

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.

Blood conservation in small adults undergoing valve surgery.

OBJECTIVES: A substantial reduction in transfusion requirements for cardiac surgical procedures has been reported. Many of these reports have been described in patients undergoing coronary artery bypass grafting. Patients suffering from rheumatic heart disease in India are usually small and also anemic. This study was conducted to assess blood conservation methods for cardiac valve surgery in this subset of patients. DESIGN: This was a prospective, randomized study. SETTING: The study was performed in a New Delhi tertiary care hospital, and the patients were referred from the northern states of India. PARTICIPANTS: One hundred fifty consecutive patients undergoing elective valve surgery using cardiopulmonary bypass were included. The mean age was 27.7 years and mean weight was 45.2 kg. INTERVENTIONS: The patients were divided into three groups of 50 each. Group 1 received autologous fresh blood donated before bypass, and both a cell saver and membrane oxygenator were used. The oxygenator contents at the end of perfusion were processed by cell saver. Group 2 patients were reinfused with autologous blood only, and group 3 was a control group. In groups 2 and 3, the blood that remained in the oxygenator at the conclusion of cardiopulmonary bypass was reinfused. A hematocrit of less than 25% was considered an indication for transfusion in the postoperative period. MEASUREMENTS AND MAIN RESULTS: The mean preoperative hematocrit was 35.5%. A mean of 361.1 mL of autologous blood was collected from group 1 and 303.3 mL from group 2. Group 1 required 15 units of bank blood, group 2, 90 units (p < 0.001), and group 3, 102 units (p < 0.001). Seventy-eight percent of group 1 patients did not receive any donor blood. There was no significant difference in chest tube drainage among the three groups. CONCLUSIONS: In this unique group of patients whose mean body weight was only 45 kg, autologous blood alone did not decrease blood bank requirements but when combined with a cell saver and membrane oxygenator greatly reduced the need for donor blood.

Soluble MHC II-peptide complexes induce antigen-specific apoptosis in T cells.

Soluble major histocompatibility (MHC) class II molecules in association with antigenic peptide recognize T cell receptors (TCRs) on CD4+ T cells. Such recognition of MHC II-peptide complexes by T cells in the absence of costimulatory signals is known to induce T cell nonresponsiveness. The present study describes that recognition of TCRs by MHC class II-peptide complexes induces antigen-specific apoptosis in a T cell clone independently of nonresponsiveness. Apoptosis was demonstrated in a murine T cell clone (4R3.9) restricted for IAk in association with a peptide analog of myelin basic protein [MBP(1-14)A4]. A dose- and time-dependent T cell death was observed upon incubation of 4R3.9 T cells with purified IAk-MBP(1-14)A4 complexes. The specificity of T cell apoptosis was shown by incubating 4R3.9 T cells with irrelevant IAs-MBP(90-101) complexes. The DNA fragmentation as a result of apoptosis was demonstrated by agarose gel electrophoresis and by pulsing T cells with BrdU followed by the detection of BrdU-labeled DNA fragments using an antibody enzyme-linked immunosorbent assay. The expression level of two regulatory intracellular proteins, bcl-2 and bax, involved in apoptosis showed a decrease in bcl-2 and an increase in bax with time. Finally, the nuclear shrinkage and chromatin condensation, typical hallmark of apoptosis, have been demonstrated by transmission electron microscopy of complex-treated T cells. Since the T cell clone (4R3.9) used in this study failed to show nonresponsiveness by IAk-MBP(1-14)A4 complexes, our results suggest that apoptosis induced by purified MHC class II-peptide complexes may involve distinct pathways rather than T cell nonresponsiveness. Such antigen-specific apoptosis may have significant clinical relevance in deleting autoreactive T cells in various autoimmune diseases.

Functionally active recombinant alpha and beta chain-peptide complexes of human major histocompatibility class II molecules.

Major histocompatibility (MHC) class II molecules are cell surface heterodimeric (alphabeta) glycoproteins that display processed antigens to T cell receptors (TCRs) of CD4-positive T cells. The present study describes that individual recombinant alpha and beta chains of human MHC class II molecules lacking the transmembrane region (alpha-Tm and beta-Tm) are capable of binding antigenic peptide and that these complexes of chain-peptide are recognized by TCRs to induce antigen-specific apoptosis in restricted T cells. The alpha-Tm and the beta-Tm of human HLA-DR2 (DRB5*0101) were cloned, expressed in Escherichia coli, and purified in large scale by conventional chromatographic methods. The in vitro binding of an immunodominant epitope from the myelin basic protein (MBP-(83-102)Y83) to purified DR2 alpha-Tm and DR2 beta-Tm was demonstrated with biotinylated and fluoresceinated MBP-(83-102)Y83 peptide. The specificity of the MBP-(83-102)Y83 peptide binding to both DR2 alpha-Tm and DR2 beta-Tm was demonstrated in a competitive peptide binding assay. When exposed to a transformed T cell clone (SS8T) restricted to DR2(DRB5*0101) and MBP-(84-102) peptide, complexes of DR2 alpha-Tm and DR2 beta-Tm with MBP-(83-102)Y83 peptide were able to specifically recognize TCRs as measured by the increase in gamma-interferon (gamma-IFN) cytokine. Such recognition of TCRs by soluble alpha-MBP-(83-102)Y83 and beta-MBP-(83-102)Y83 complexes led to the induction of antigen-specific apoptosis in SS8T cells as measured by double fluorescence flow cytometry and electron microscopy. These results provide the first evidence that soluble complexes of antigenic peptide and individual chains of human MHC class II molecules lacking the transmembrane region can recognize TCRs and induce antigen-specific apoptosis in T cells. Since activated CD4-positive T cells are involved in pathogenesis of various autoimmune diseases, the apoptosis triggered by individual soluble chain-peptide complexes has significant potential for eliminating autoreactive T cells.

Antigen-specific apoptosis in immortalized T cells by soluble MHC class II-peptide complexes.

The recognition of T cell receptors (TCR) by purified major histocompatibility complex (MHC) class II-peptide complexes in the absence of costimulatory signals leads to the induction of T cell non-responsiveness or anergy. In a recent study using human T cell clones, it was observed that prolonged incubation of resting T cells with soluble MHC II-peptide complexes appears to result in T cell apoptosis. The present study shows that the engagement of TCR by soluble MHC II-peptide complexes also results in antigen-specific apoptosis in immortalized T cells. Apoptosis was demonstrated in a herpes saimiri virus (HSV) transformed human T cell clone (SS8T) restricted for HLA-DR2 in association with an epitope from the myelin basic protein [MBP(84-102)]. A dose- and time-dependent T cell death was observed upon incubation of SS8T cloned T cells with purified complexes of native human HLA-DR2 and MBP(83-102)Y83 peptide. The specificity of T cell apoptosis was demonstrated by exposing SS8T cells with DR2 alone and DR2 bound to another high affinity epitope [MBP(124-143)] from the same MBP. Recently, we have shown that the complexes of HLA-DR2 and [MBP(83-102)Y83] can be reconstituted by refolding Escherichia coli expressed individual DR2 alpha and beta (B5*0101) polypeptide chains lacking the transmembrane region. When SS8T cloned T cells were exposed to purified reconstituted rDR2.MBP(83-102)Y83 complexes, similar apoptosis of T cells was observed. Agarose gel analysis of T cells incubated with complexes showed a degradation of celluar deoxyribonucleic acid (DNA) to oligonucleosomal bands, a characteristic of apoptosis. The quantitative detection of DNA strand breaks was performed by pulsing T cells with 5-bromo-2'-deoxyuridine (BrdU) followed by the detection of BrdU-labelled DNA fragments using an antibody sandwich enzyme-linked immuno assay (ELISA). The fragmentation of DNA was also measured by double fluorescence flow cytometry by 3' end labelling of fragmented DNA with biotinylated-deoxyuridine triphosphate (dUTP) in the presence of terminal deoxynucleotide transferase (TdT) enzyme. The expression of the bcl-2 protein in SS8T cells following TCR engagement by soluble MHC II-peptide complexes was monitored by chemiluminescence blot analysis using anti-bcl-2 monoclonal antibody. Finally, the nucleosomal condensation of T cells following complex treatment, characteristics of typical apoptosis, was demonstrated by transmission electron microscopy. These results suggest that the binding of soluble MHC class II-peptide complexes to TCR induces antigen-specific apoptosis in transformed CD4 positive T cells in vitro. Such induction of apoptosis by soluble MHC II-peptide complexes may provide a novel therapeutic strategy to delete autoreactive T cells in various autoimmune diseases.

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.

Induction of tolerance in experimental autoimmune myasthenia gravis with solubilized MHC class II:acetylcholine receptor peptide complexes.

Stimulation of T lymphocytes through the T cell receptor in the absence of costimulatory signal(s) induces a state of unresponsiveness to subsequent antigen presentation. We have employed solubilized complexes consisting of rat class II MHC molecules containing an immunodominant peptide of the acetylcholine receptor (AChR alpha 100-116) to induce unresponsiveness in the autoreactive T lymphocytes mediating an animal model of myasthenia gravis. In vitro incubation of rat T cell lines specific for peptide AChR alpha 100-116 with solubilized complexes of MHC II and AChR alpha 100-116 (MHC II:AChR alpha 100-116) rendered the T cells unresponsive to subsequent stimulation by antigen presenting cells and the peptide. T cell lines with a broader specificity to the entire AChR protein pentamer had an 81% reduction in proliferation to AChR following a preincubation with solubilized MHC II:AChR alpha 100-116. Treatment with the solubilized MHC II:AChR alpha 100-116 induced phosphatidylinositol 4,5-bisphosphate hydrolysis, an early signalling event associated with binding to the TCR. Rats primed with AChR and injected intravenously with MHC II:AChR alpha 100-116 had reduced in vitro T cell proliferation to the AChR alpha 100-116 peptide and to whole AChR. Solubilized MHC II:AChR alpha 100-116 injected i.v. into rats exhibiting serological clinical symptoms of experimental autoimmune myasthenia gravis (EAMG) prevented death in 67% of the treated animals, compared to a 0-20% survival rate in all other control groups. These results demonstrate that solubilized MHC II complexed with an immunodominant autoantigenic peptide is tolerogenic and improves the survival rate of rats with EAMG, suggesting the basis for an antigen-specific therapy in autoimmune diseases such as MG.

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.

Stimulation of T cells by antigen-presenting cells is kinetically controlled by antigenic peptide binding to major histocompatibility complex class II molecules.

Activation of CD4+ T cells by antigenic peptide involves the interaction of major histocompatibility complex (MHC) class II-peptide complexes on the surface of antigen-presenting cells (APCs) with T-cell receptors. This report describes the kinetics of T-cell triggering by exogenous antigenic peptides in the presence of APCs. A rapid specific increase in extracellular acidification rate is observed within minutes upon exposure of A.E7 T cells (restricted for IEk and moth cytochrome c peptide containing residues 88-103) and 4R3.9 T cells (restricted for IAk and myelin basic protein peptide containing residues 1-14 [AcMBP-(1-14)]) to their cognate peptides in the presence of CH27 cells bearing both IAk and IEk MHC class II molecules. Pretreatment of cloned T cells, but not APCs, with herbimycin A resulted in complete inhibition of triggering events, indicating that the acidification response is mediated by T-cell second messenger pathways. This rapid assay for 4R3.9 T-cell stimulation showed increased T-cell triggering activity for AcMBP-(1-14)-A4 and MBP-(1-14)-M4 peptides compared to the native AcMBP-(1-14)-K4. By using the previously determined kinetic constants for MBP-(1-14)-A4 reactions with IAk, it is possible to show that at the lowest peptide concentrations the kinetics of T-cell triggering are limited by the kinetics of the peptide binding to MHC class II molecules.

Refolding and reconstitution of functionally active complexes of human leukocyte antigen DR2 and myelin basic protein peptide from recombinant alpha and beta polypeptide chains.

Major histocompatibility complex (MHC) class II molecules are cell surface heterodimeric glycoproteins consisting of one alpha and one beta polypeptide chain of similar size. These molecules play a critical role in immune recognition by displaying processed antigens to CD4-positive T helper cells. Several attempts to express the MHC class II molecules by recombinant methods in various systems resulted in either failure or poor recovery of the intact heterodimer. The present study describes our successful effort to refold and reconstitute HLA DR2 heterodimer from individually expressed alpha and beta polypeptide chains lacking the transmembrane hydrophobic regions in Escherichia coli, in the presence of an immunodominant epitope analog from human myelin basic protein (b-MBP(83-102)Y83). The reconstituted DR2 heterodimer complex was selectively purified from unfolded alpha and beta chains using heterodimer-specific monoclonal antibody (L243) coupled to a solid support. The detection of two polypeptide chains in the purified refolded DR2-peptide complex preparations was accomplished by Western blot analysis and enzyme-linked immunosorbent assay using heterodimer- and chain-specific polyclonal antibodies, and the presence of equimolar amounts of both alpha chain and beta chain in the reconstituted complex preparation was confirmed by a double label experiment. The quantitation of the bound peptide in complex preparation was measured by incubating two chains in the presence of 125I-labeled peptide. An increase in the yield of refolded and reconstituted DR2-peptide complexes was observed with increasing peptide concentration in the reaction mixture. Finally, the functional activity of the reconstituted DR2 complexes was measured by their ability to stimulate gamma-interferon production by SS8T cloned T cells in an antigen-specific and dose-dependent manner. These results demonstrate that biologically active complexes of human DR2.b-MBP (83-102)Y83 can be prepared by proper folding of human leukocyte antigen DR2 alpha and beta chains in the presence of antigenic peptide. The yield of such DR2 heterodimers with bound peptide is several thousand-fold higher over native DR2 purified from transformed B cells. Since purified MHC class II-peptide complexes have been shown to prevent autoimmune diseases in various animal models, reconstituted heterodimer complexes may have significant clinical relevance in antigen-specific treatment of various autoimmune diseases. In addition, such complexes with increased yield will provide better understanding of the trimolecular interactions between MHC-peptide and T cell receptor.