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Biomedical subjects

J McCluskey

Publications and source records attributed to J McCluskey.

At least 73 records · Page 4Linked to original sources
PubMed1995

Antigen processing and presentation by a murine myoblast cell line.

The ability of non-professional antigen-presenting cells (APC) to process and present antigen to the immune system has been the subject of debate in autoimmunity and tumour immunology. The role of muscle cells in the processing and presentation of antigen to T cells via class I and class II MHC pathways is of increasing interest. Muscle cells are the targets of autoimmune attack in the inflammatory muscle diseases, and direct intramuscular injection of antigen-expressing DNA constructs is under scrutiny as a means of vaccination. Furthermore, the immunological properties of muscle cells are of relevance in attempts to transfer myoblasts as replacement cells in dystrophic diseases or as depot cells for the secretion of certain molecules in deficiency states. Using class I and class II MHC transfectant clones of the C2C12 myoblast cell line, myoblasts have been shown to be capable of presenting antigen to, and stimulating secretion of IL-2 by, T cell hybridomas via both of these pathways. The epitopes which are dominantly presented by professional APC after processing of native antigens were also presented by the myoblast cell line after processing of either ovalbumin (class I) or hen egg lysozyme (class II). Further, antigen processing and presentation via the class II pathway were enhanced by pretreatment of the myoblasts with interferon-gamma (IFN-gamma). Up-regulation of invariant chain expression by this treatment may have contributed to this enhanced presentation, but an effect of IFN-gamma on the expression of other molecules such as H-2 DM may have also played a role. The demonstration of the antigen-presenting properties of these myoblasts is of relevance to all three areas mentioned above. In each situation myoblasts comprise a significant population within muscle. In the case of inflammatory muscle diseases the process of muscle degeneration and regeneration is on-going, while in the vaccination procedure some muscle damage occurs, and vaccination is more effective when muscle damage has preceded inoculation.

Animals↗
PubMed1995

HLA class I variation in Australian aborigines: characterization of allele B*1521.

Traditional methods of serological typing have largely used antisera of Caucasoid origin, which can overlook HLA heterogeneity in non-Caucasoid populations. Therefore, we have used molecular techniques to evaluate potential polymorphism in HLA class I molecules of Aborigines from the central desert and northern coast of Australia. The DNA sequence of common Aboriginal HLA-A and B antigens were compared with serological reaction patterns which suggested new polymorphisms. Although serological data indicated that long and short variants of A34 may exist, regardless of the serological pattern, all individuals carried the A*3401 allele. Therefore, the variation in A34 reaction pattern observed serologically was not attributable to primary sequence variation in the HLA A*3401 allele. Similarly, there was no detectable polymorphism in the sequences of selected HLA-B alleles, even though some of these alleles showed unusual serological reaction patterns. However, a new allele of B15 (B*1521) was detected in two individuals carrying this serotype. The cells from both of these individuals showed ambiguous reaction patterns with monospecific B62 and B75 sera. cDNA sequencing of the HLA B15 gene from these cells revealed a B15 allele that differed from B*1502 by a single nucleotide change. This change occurred at position 272, resulting in a C to G substitution at residue 67 in the consensus B15 cDNA sequence. Hence, the Australian Aborigines as an ethnic group show very little primary sequence polymorphism within the class I loci, consistent with the results obtained from previous serological studies.

Amino Acid Sequence↗
PubMed1995

Matching for MHC haplotypes results in improved survival following unrelated bone marrow transplantation.

Unrelated bone marrow donor-recipient pairs were assessed retrospectively for matching of the HLA-B, -C region (beta-block) and HLA-DR, DQ region (delta block) of the major histocompatibility complex (MHC) using a new DNA-based method referred to as MHC-block typing. The method utilises non-HLA DNA polymorphisms in the MHC as markers of blocks of ancestral haplotypes. Kaplan-Meier analysis of recipients who were matched at both the beta- and delta-blocks revealed a 6 months survival of 54%. Survival was better than for patients who were matched only by conventional criteria, including SSO-typing for class II.

Adult↗
PubMed1995

Identification and synthesis of altered peptides modulating T cell recognition of a synthetic peptide antigen.

In studies of T cell responses to synthetic peptides we have observed agonist and antagonist activities associated with contaminants identified within the parent synthesis. The synthesis of two candidate analogues implied by a peptide contaminant formed during the synthesis of La 51-58 (IMIKFNRL) has been carried out. The peptide contaminant was 17-18 Da smaller than the parent peptide consistent with a modified asparagine residue at position 6 and so we synthesised both an aspartimide and a nitrile analogue, representing cyclisation or dehydration of the asparagine residue. The candidate aspartimide and nitrile analogues both bound empty MHC class I molecules to form allo determinants recognised by monoclonal antibodies. These results demonstrate that altered synthetic peptides can bind class I MHC molecules and prompt caution in the use of synthetic peptides as a source of immunising antigen.

Amino Acid Sequence↗
PubMed1994

Antigen presentation and assembly by mouse I-Ak class II molecules in human APC containing deleted or mutated HLA DM genes.

The behavior of mouse I-Ak molecules was studied in the human Ag presentation mutants T2 and 9.5.3, which contain deleted or mutated HLA DM genes. HLA class II molecules expressed by these APC are defective in presentation of native Ag and are mostly complexed with class II-associated invariant chain peptides (CLIP). In contrast to human class II molecules, a significant proportion of mouse I-Ak molecules expressed in T2 and 9.5.3 were associated with antigenic peptides, indicating that I-Ak/peptide assembly is possible in the absence of the Dm proteins. Thus, the presentation of determinants derived from hen egg lysozyme (HEL), keyhole limpet hemocyanin, and conalbumin was normal in 9.5.3Ak and a conalbumin determinant was presented normally by T2.Ak. However, the keyhole limpet hemocyanin determinant was not presented by T2.Ak, and HEL46-61 was only presented at a low level by these APC. SDS-stable, dimeric I-Ak molecules were expressed by both T2.Ak and 9.5.3Ak and formed late in their intracellular transport. Presentation of HEL46-61 was partially inhibited by disrupting vacuolar acidification in 9.5.3Ak, consistent with I-Ak/peptide assembly in a post-Golgi endosomal compartment. Accordingly, Dm is not an obligatory requirement for MHC class II/peptide assembly. We propose that Dm influences the displacement of CLIP from recently synthesized class II molecules, a process that is likely to be less critical for I-Ak because of its low affinity for CLIP.

Animals↗
PubMed1994

MHC class II molecules that lack cytoplasmic domains are associated with the cytoskeleton.

MHC class II molecules, composed of alpha- and beta-chain heterodimers, are required for Ag presentation. The carboxyl-terminal domains of class II molecules are believed to mediate the location of class II in the plasma membrane and are important for signal transduction and Ag presentation. These domains contain typical transmembrane sequences, and cytoplasmic sequences of 12 or 18 amino acids for the alpha- and beta-chains, respectively. We examined these domains to determine whether they linked class II molecules to the actin-based cytoskeleton. Our analyses of class II-cytoskeleton interactions, such as a colocalization with actin filaments during capping, association with the detergent-insoluble cytoskeleton, and direct binding of filamentous actin, revealed that both the cytoplasmic and transmembrane domains contributed to class II interactions with the cytoskeleton. Detergent-extracted and immunoprecipitated full-length class II molecules had quantitatively stronger interactions with the cytoskeleton than did molecules with deleted cytoplasmic domains. A secondary Ab, which was used to cross-link primary Ab bound to class II, up-regulated the class II-cytoskeletal associations. This association was efficiently inhibited by dihydrocytochalasin B, but only partially disrupted by chlorpromazine. The mechanism of interaction with actin filaments after ligation of class II occurred without a measurable increase in filamentous actin levels. This suggested that enhanced class II-cytoskeleton associations involved a rearrangement of existing actin filaments, possibly through the multiple kinases that are activated after class II transmembrane signaling.

Actins↗
PubMed1994

Determinant selection of major histocompatibility complex class I-restricted antigenic peptides is explained by class I-peptide affinity and is strongly influenced by nondominant anchor residues.

The contribution of major histocompatibility complex (MHC) class I-peptide affinity to immunodominance of particular peptide antigens (Ags) in the class I-restricted cytotoxic T lymphocyte (CTL) response is not clearly established. Therefore, we have compared the H-2Kb-restricted binding and presentation of the immunodominant ovalbumin (OVA)257-264 (SIINFEKL) determinant to that of a subdominant OVA determinant OVA55-62 (KVVRFDKL). Immunodominance of OVA257-264 was not attributable to the specific T cell repertoire but correlated instead with more efficient Ag presentation. This enhanced Ag presentation could be accounted for by the higher affinity of Kb/OVA257-264 compared with Kb/OVA55-62 despite the presence of a conserved Kb-binding motif in both peptides. Kinetic binding studies using purified soluble H-2Kb molecules (Kbs) and biosensor techniques indicated that the Kon for association of OVA257-264-C6 and Kbs at 25 degrees C was integral of 10-fold faster (5.9 x 10(3) M-1 s-1 versus 6.5 x 10(2) M-1 s-1), and the Koff approximately twofold slower (9.1 x 10(-6) s-1 versus 1.6 x 10(-5) s-1), than the rate constants for interaction of OVA55-62-C6 and Kbs. The association of these peptides with Kb was significantly influenced by multiple residues at presumed nonanchor sites within the peptide sequence. The contribution of each peptide residue to Kb-binding was dependent upon the sequence context and the summed contributions were not additive. Thus the affinity of MHC class I-peptide binding is a critical factor controlling presentation of peptide Ag and immunodominance in the class I-restricted CTL response.

Amino Acid Sequence↗
PubMed1994

Anergic B cells constitutively present self antigen: enhanced immunoglobulin receptor-mediated presentation of antigenic determinants by B cells is hierarchical.

Presentation of hen egg lysozyme (HEL) by HEL-specific B cells was studied in transgenic mice expressing anti-HEL immunoglobulin (Ig-transgenic). In T hybridoma assays, presentation of the HEL46-61 determinant by B cells from Ig-transgenic mice required 10(3)-10(4)-fold lower concentrations of HEL than were required for presentation by B cells from non-transgenic mice. In contrast, presentation of the HEL determinants 112-129 and 25-43 by HEL-specific B cells was either not significantly enhanced, or enhanced only 10-fold compared with B cells from non-transgenic mice. Enhanced presentation of HEL determinants by B cells from Ig-transgenic donors was specific for HEL, since keyhole limpet hemocyanin or synthetic HEL46-61 peptide were presented comparably by B cells from Ig-transgenic mice and non-transgenic littermates. A minimum of 1-4% Ig-transgenic B cells was required to detect enhanced presentation of HEL46-61 in vitro. Constitutive presentation of the HEL46-61 determinant, but not the HEL25-43 or HEL112-129 determinants, was detectable on anergic HEL-specific B cells from double (HEL/Ig)-transgenic mice. In the presence of exogenously added HEL, anergic B cells presented all three HEL determinants. Constitutively presented HEL46-61 was not due to endogenous synthesis of HEL antigen by anergic B cells from double-transgenic mice, as comparable levels of the HEL46-61 determinant were constitutively presented by B cells from Ig-Tg-->HEL-Tg irradiation bone marrow chimeric mice. Firstly, these results indicate that the enhanced antigen presentation mediated by Ig receptors on B cells is not equivalent for all antigenic determinants. Secondly, the data demonstrate that anergic, autoreactive B cells efficiently process and present nominal antigens in addition to constitutively presenting specific self antigen in vivo.

Animals↗
PubMed1994

Transfection and overexpression of the human 60-kDa Ro/SS-A autoantigen in HEp-2 cells.

A full-length cDNA encoding human 60-kDa Ro/SS-A protein was transfected into and overexpressed in the human cell line HEp-2, with the aim of developing an improved reagent for indirect immunofluorescence (IF) detection of anti-Ro autoantibodies. Stable transfectants were analyzed by IF using a panel of 20 precipitin-positive anti-Ro human sera. Transfectants showed bright finely speckled nuclear and nucleolar staining. No surface membrane expression was detected despite marked overexpression of 60-kDa Ro. All human anti-Ro sera reacted with the transfectants with titers ranging from 1:320 to 1:40,960. The same sera tested on untransfected cells showed titers from negative (3 sera) to 1:160. These transfectants dramatically increase the sensitivity of IF testing (mean increase in titer of 41-fold) and allow detection of specific anti-Ro antibodies in samples negative or equivocal on untransfected cells. The staining patterns of antisera of other important specificities remained unaltered. In a study of sera from 22 patients with systemic lupus erythematosus, anti-60-kDa Ro autoantibodies were detected in all sera when tested on 60-kDa Ro-transfected HEp-2 cells; however, in 12 of 22 sera autoantibodies were undetectable by recombinant 60-kDa Ro ELISA. The 60-kDa Ro transfectants are a simple and sensitive method for detection of anti-Ro antibodies in patients with systemic rheumatic diseases.

Autoantibodies↗
PubMed1994

Development of an intraperitoneal implant chamber for the study of in vivo-grown Pasteurella haemolytica in cattle.

An intraperitoneal implant chamber was developed for the study of the in vivo growth of Pasteurella haemolytica in calves. The chamber had a volume of approximately 100 ml and featured an external sampling port which allowed multiple and sequential sampling of the chamber contents. A single polycarbonate diffusion membrane with a pore size of 0.22 micron allowed host peritoneal fluid to diffuse into the chamber and maintained the bacterial population free of white blood cells. Chambers were implanted into the peritoneal cavities of four five-month-old dairy-cross calves, demonstrated to be sero-negative by indirect haemagglutination assay. Three days later, four different P. haemolytica isolates, of serotypes A1 or A2, were inoculated into the chambers. In all cases, there was a slow decline in the viable bacterial numbers within the chambers. Western blot analysis of the antibody content of the chamber fluids revealed IgG antibodies to P. haemolytica OMPs in the fluid prior to inoculation and both 9 and 15 days after inoculation. Furthermore, there was no significant change in the IgG antibody content of the chamber fluid, either quantitatively or qualitatively, during the course of the experiment. Analysis of the bactericidal activity of pre-inoculation chamber fluid against the corresponding bacterial isolate suggested that an antibody-dependent complement-mediated process was not responsible for the decline in bacterial numbers. Overall, the chamber design was demonstrated to be extremely effective for in vivo studies of P. haemolytica in calves, allowing easy and regular sampling of the chamber contents and maintaining bacteria free of white blood cells. Although there was a slow decline in bacterial numbers over time, sufficient numbers of cells could be obtained for analysis of cell-surface antigens.

Animals↗
PubMed1994

The structural influence of individual residues located within peptide antigen depends upon their sequence context.

Individual residues derived from antigenic peptides are believed to control certain mAb epitopes on MHC class I molecules. To further evaluate this influence we studied the antibody recognition of H-2Kb complexed with the antigenic peptides OVA257-264, OVA55-62 and their reciprocally substituted analogs. The mAb 20.8.4, Y-3, EH144 and AF6 reacted strongly with Kb complexed to both OVA peptides and their analogs. However, mAb 28.8.6 bound to Kb/OVA257-264 but not Kb/OVA55-62 complexes. Recognition of Kb/OVA55-62 by mAb 28.8.6 was restored by a single OVA55-62-->OVA257-264 substitution at position 1 (OVA55-62P1K-->S). Recognition by mAb 5F1 was also peptide-dependent in that Kb complexed to either the P4 substituted OVA257-264-->OVA55-62 peptide (OVA257-264P4N-->R) or the P1 substituted OVA55-62-->OVA257-264 peptide (OVA55-62P1K-->S) was not permissive for 5F1 recognition even though Kb complexed to the parental peptides OVA55-62 (P4R) and OVA257-264 (P1S) reacted with this mAb. These data demonstrate that the same peptide residues located within defined positions can exert a negative influence on mAb recognition in one sequence context but be permissive for antibody binding in another context. These findings might be explained by conformational effects on class I heavy chain structure, the extended structure of the peptide or altered orientation of specific peptide side chains located at the same position but within different sequence contexts. Therefore the sequence context of defined amino acids within peptide antigen can strongly influence the fine structural contributions of these residues to MHC-peptide conformation.

Amino Acid Sequence↗
PubMed1994

Repair of excisional wounds in the embryo.

Wound healing in the embryo, just as in the adult, comprises two tissue movements: re-epithelialisation and connective-tissue contraction. In this brief review we describe our recent studies of these two movements in both chick and rodent embryo model systems. In the chick we have evidence that the embryonic wound epidermis is drawn forwards by contraction of an actin pursestring extending around the circumference of the wound, rather than by lamellipodial crawling as in adult healing. Significant connective-tissue contraction also occurs. In the rat and mouse embryo we have examined expression of transcription factors and growth factors at the wound edge. We discuss our observations that the immediate-early gene c-fos and the growth factor transforming growth factor beta-1 are rapidly induced at the embryonic wound margin, and the possibility that these signals may trigger proliferation of wound edge cells and contraction of the exposed wound mesenchyme.

Animals↗
PubMed1994

Variation in outer-membrane protein and lipopolysaccharide profiles of Pasteurella haemolytica isolates of serotypes A1 and A2 obtained from pneumonic and healthy cattle.

The outer-membrane protein (OMP) and lipopolysaccharide (LPS) profiles of 29 isolates of Pasteurella haemolytica serotypes A1 (18 isolates) and A2 (11 isolates), obtained from pneumonic (13 isolates) or healthy (16 isolates) cattle, were compared by SDS-PAGE and Western blot analysis. Coomassie-blue-stained OMP profiles of serotype A1 and A2 isolates could be distinguished from each other by differences in both major and minor proteins. Whereas the OMP profiles of the serotype A1 isolates were extremely uniform in stained gels, there was variation in the mobilities of high-molecular-mass minor proteins and one of the major proteins of serotype A2 isolates. Differences in the OMP profiles of isolates within both the A1 and A2 serotypes were more clearly distinguished by Western blotting than by staining after SDS-PAGE. Thus, by Western blot analysis, four distinct OMP profiles were identified within the serotype A1 and A2 isolates, respectively. The profiles of the serotype A1 isolates were designated OMP types 1.1, 1.2, 1.3 and 1.4; those of the serotype A2 isolates were designated OMP types 2.1, 2.2, 2.3 and 2.4. Three distinct LPS profiles were recognized among the isolates which, by comparison with previously described LPS types, were identified as smooth LPS type 1 and rough LPS types 3 and 5. Isolates of serotype A1 consisted of LPS type 1 only. whereas isolates of serotype A2 consisted of LPS types 3 or 5. OMP and LPS analysis of P. haemolytica has applications in epidemiological and virulence studies.

Animals↗