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.

Canine parvoviruses in New Zealand form a monophyletic group distinct from the viruses circulating in other parts of the world.

Canine parvovirus 2 (CPV-2) is a well-recognized cause of acute haemorrhagic enteritis in dogs worldwide. The aim of the current study was to identify which CPV-2 subtypes circulate among dogs in New Zealand, and to investigate the evolutionary patterns of contemporary CPV-2 viruses. Faecal samples were collected from 79 dogs with suspected CPV-2 infection over the period of 13 months, and tested for the presence of CPV-2 DNA by PCR. Of 70 positive samples, 69 were subtyped as CPV-2a and one as CPV-2. A majority of CPV-2 positive samples were collected from unvaccinated or not-fully vaccinated puppies ≤6 months of age. The haplotype network produced from New Zealand CPV-2 sequences showed no structure when assessed based on location, vaccination status or age of the animals sampled. International haplotype network indicated that, unlike CPV-2 from other countries, the population of CPV-2 in New Zealand appeared to be monophyletic.

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.

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.

Multinomial logit analysis of injury severity in U.S. underground bituminous coal mines, 1975-1982.

Relationships among mine and injured miner characteristics and degrees of injury are examined for 91 404 injuries in underground bituminous coal mines in the United States from 1975 through 1982. Injury severity varies by the mining system, geographical region, circumstances surrounding the injury, the injured miner's age, whether the miner was using powered equipment, the year in which the injury occurred and the location in the mine where the injury happened. Injury severity is not related to the injured miner's total mining experience, job experience and experience in the mine in which the injury occurred.

Relationship between preferences for part-time work and characteristics of unemployed youths.

Using data from the March 1979 Current Population Survey, the relationship was examined between preferences for part-time work and the personal characteristics of 2,087 unemployed 16- through 24-year olds in the civilian, noninstitutional population of the United States. Age and school enrollment were found to be the strongest correlates of desire for part-time work. Gender, race, veteran status, marital status, and educational attainment were related only slightly to part-time job preferences. These data support the notion that most unemployed youths searching for part-time work are 16 to 19 years old and enrolled in school; on the other hand, knowing merely this does not indicate that youth unemployment has little economic significance, as some analysts have suggested. Additional research on the consequences of teenage labor market problems on subsequent adult well-being is needed to determine the significance of youth unemployment.

Relationship between accreditation status and hourly wages of medical record technicians.

Using data from a 1975 hospital wage survey conducted by the Industry Wage Division of the U.S. Bureau of Labor Statistics, the relationship between accreditation status and hourly wages of 590 female full-time medical record technicians (MRTs) in four metropolitan areas was examined. Through multiple regression analysis, the hourly wages of Accredited Record Technicians were found to be $0.62 greater than those not accredited. Job location and hospital funding source also were related strongly to MRT hourly wages. Only 35 percent of the MRT's studied were accredited. These data reveal the willingness of employers to hire MRT's not accredited, while being willing to pay a premium for accreditation.

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.

In vitro maximum binding of antigenic peptides to murine MHC class II molecules does not always take place at the acidic pH of the in vivo endosomal compartment.

Presentation of Ag to the T cell requires binding of specific peptide fragments of the Ag to MHC II molecules. The ability of a peptide to bind to MHC class II appears to be pH dependent. Recent reports indicate that the binding of peptide to MHC class II molecules takes place primarily within an endosomal compartment of the cell at around pH 5. In this study, we have explored the in vitro pH dependence of peptide binding to different haplotypes of murine MHC class II molecules. The binding of peptides to MHC II was analyzed and quantitated by silica gel TLC, using radiolabeled peptides. The MBP peptide fragments, MBP(1-14)A4 and MBP(88-101)Y88, bound maximally at pH 8 to IAk and IAs, respectively. The binding of PLP peptide fragment, PLP(138-151)Y138, to IAs was maximal at around neutral pH. The maximum binding of an OVA peptide fragment, OVA(323-340)Y340, to IAd, was found to occur at pH 6. Results presented in this report thus suggest that the in vitro maximum binding of peptide is pH dependent and does not always occur at pH 5. The optimum pH range for maximum binding may depend on the nature and net charge of the peptide and its interaction with MHC class II molecules.

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.

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.

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.