Biomedical subjects

J McCluskey

Publications and source records attributed to J McCluskey.

At least 91 records · Page 5Linked to original sources

PubMed1994

Comparison of outer-membrane proteins of Pasteurella haemolytica expressed in vitro and in vivo in cattle.

Outer-membrane protein (OMP) profiles of two serotype A1 isolates of Pasteurella haemolytica were compared by SDS-PAGE and Western blotting with bovine convalescent serum after growth (a) in vitro under iron-sufficient and -deficient conditions, (b) in vivo in the lungs of experimentally infected calves and (c) in vivo in diffusion chambers implanted into the peritoneal cavities of calves. Lung-grown bacteria differed from iron-sufficient in vitro-grown bacteria in having enhanced expression of the previously recognized 71, 77 and 100 kDa iron-regulated proteins, reduced expression of 18, 31, 39.5 and 50 kDa proteins, and expression of a 19 kDa protein. Differences were also apparent in the Western blot profiles of OMPs of in vitro- and lung-grown bacteria. These included the apparent lack of recognition of the 100 kDa protein in the lung-grown bacteria, but not in the in vitro-grown bacteria, and more intense staining of a 47 kDa protein in in vitro-grown bacteria, but not in lung-grown bacteria. The OMP profiles of the chamber-grown bacteria resembled those of the lung-grown bacteria in that expression of the 18, 19, 31 and 39.5 kDa proteins was similar. These similarities demonstrated that the chamber-grown bacteria had adapted to the in vivo environment, and that growth conditions within the chambers resembled, but not perfectly, those within the lungs. For example, expression of the three iron-regulated OMPs was very low in the chamber-grown bacteria compared to the lung-grown bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

How does autoimmunity to La and Ro initiate and spread?

Autoimmunity to La(SS-B) and Ro(SS-A) is a paradigm for understanding the normal mechanisms of B cell and T cell tolerance and development of autoimmunity to clinically important sequestered autoantigens. Epitope mapping experiments have indicated that the autoantibody response is largely self antigen-driven but have failed to elucidate why these particular autoantigens are selected. Abnormal trafficking of La or Ro peptides in polarised epithelial cells and their presentation to autoreactive T cells, or selective release of ribonucleoproteins by injured epithelial cells, could explain the targeting of salivary and lacrimal epithelium in Sjogren's syndrome. There appears to be little, if any, immune tolerance to La in the T helper and B cell compartments. Both intra-and inter-molecular spreading of the autoimmune response have been observed for La. We present a model of recruited autoimmunity whereby capture and internalisation of La/Ro ribonucleoproteins by B cells and subsequent presentation of La or Ro determinants to autoreactive T cells could lead to inter-molecular spreading of the response.

Autoantigens↗

PubMed1993

Class II-restricted presentation of a hen egg lysozyme determinant derived from endogenous antigen sequestered in the cytoplasm or endoplasmic reticulum of the antigen presenting cells.

A cDNA encoding a form of hen egg lysozyme (HEL) lacking a leader sequence and predicted to be localized in the cytoplasm, was transfected into MHC class II-positive B lymphoma cells. Cytoplasmically expressed HEL (cytHEL) had a half-life of less than 5 min and did not react with HEL specific mAb suggesting non-native conformation. Cells expressing cytoplasmic HEL, as well as cells previously reported to express a low level of HEL retained in the endoplasmic reticulum (ERHEL), constitutively presented the HEL determinant encoded by residues 46-61 to a sensitive class II-restricted T hybridoma (3A9). Constitutive presentation of HEL determinants was not detectable in cytHEL or ERHEL transfectants using T hybridomas with lower sensitivity to exogenous Ag. Constitutive presentation of HEL46-61 derived from cytoplasmic HEL was demonstrable in multiple transfected clones and was most obvious when a CMV rather than SV40 promoter was used to express the cytHEL gene. The presentation of HEL46-61 by cytHEL transfectants was not due to HEL reuptake by bystander cells because there was no biochemical evidence of cytHEL shedding and cytHEL supernatants added to indicator APC did not result in HEL46-61 presentation. Constitutive presentation of endogenous HEL46-61 by the cytHEL and ERHEL transfectants was inhibited by chloroquine, and recovery of presentation of endogenous HEL was slower in cytHEL compared with ERHEL transfectants. The findings indicate that class II-restricted presentation of Ag retained in the cytoplasm or endoplasmic reticulum does take place but probably requires abundant levels of intracellular Ag and is easily disrupted by lysosomotropic agents. These pathways of presentation may be important when high levels of foreign endoplasmic reticulum-retained or cytoplasmic Ag are present (e.g., viral infection), and during the acquisition of self-tolerance by highly sensitive developing T cells.

Amino Acid Sequence↗

PubMed1993

Characterization of the mouse autoantigen La (SS-B). Identification of conserved RNA-binding motifs, a putative ATP binding site and reactivity of recombinant protein with poly(U) and human autoantibodies.

To facilitate the study of autoimmunity to the nuclear Ag La (SS-B) we have isolated and characterized cDNA encoding the mouse La (SS-B) protein. Mouse La (SS-B) protein has 76.7% identity to both human and bovine La (SS-B) proteins and the previously recognized RNP-binding motifs were noted to be highly conserved across the three species. Examination of the primary sequence of human and bovine La (SS-B) newly identifies the site of a potential ATP-binding motif G/AXXXXGK which is preserved in mouse La (SS-B) despite a unique 16 amino acid insertion present in this region of the mouse protein. Analysis of mouse genomic DNA suggests a single gene encodes mouse La (SS-B) and no restriction fragment polymorphisms were identified in the three mouse strains investigated. Two alternate 5' untranslated regions were identified in mouse La (SS-B) cDNA and La (SS-B) mRNA was identified in a wide range of murine tissues consistent with its ubiquitous expression. Recombinant mouse La (SS-B) protein purified from Escherichia coli was shown to bind to poly(U) agarose and to react with sera containing anti-La (SS-B) antibodies obtained from patients with Sjögren's syndrome. The ability to produce recombinant mouse La (SS-B) should provide a valuable reagent for the study of the cellular autoimmune response as well as immunologic tolerance to La (SS-B) Ag in mice.

Adenosine Triphosphate↗

PubMed1993

Electroporation and commercial liposomes efficiently deliver soluble protein into the MHC class I presentation pathway. Priming in vitro and in vivo for class I-restricted recognition of soluble antigen.

Class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte responses to ovalbumin (OVA) were evaluated following delivery of soluble antigen mixed with commercial liposomes or by electroporation of soluble protein into target cells. Splenic antigen presenting cells (APC) and transfected L cell lines were sensitised for recognition by OVA-specific, class I-restricted T hybridomas when antigen was introduced by either method into live cells. Delivery of soluble OVA by both electroporation and commercial liposomes proved more efficient than osmotic loading in sensitising for class I presentation. OVA-specific cytotoxic T lymphocytes (CTL) were effectively primed in naive mice following reinjection of spleen cells pulsed with soluble OVA encapsulated by liposomes or electroporated in vitro. These CTL recognised the well defined OVA257-264 determinant in association with H-2Kb and were derived under conditions where CTL activity obtained from cross priming by soluble OVA alone was undetectable. In addition, using electroporation and commercial liposomes, the loading of APC for OVA recognition required intact MHC-linked antigen presentation genes deleted in the T2-Kb cell line. Antigen delivery to APC by electroporation and commercial liposomes provides a simple and efficient way of studying class I-restricted T cell recognition of soluble protein antigens.

Animals↗

PubMed1993

Changes at peptide residues buried in the major histocompatibility complex (MHC) class I binding cleft influence T cell recognition: a possible role for indirect conformational alterations in the MHC class I or bound peptide in determining T cell recognition.

Recent crystallographic studies on two peptide complexes with the mouse Kb molecule have shown that peptide binding appears to alter the conformation of the class I alpha-helical regions that flank the antigen binding cleft. Given that this study also showed that much of the foreign peptide is buried within the class I binding cleft with only a small portion accessible for direct interaction with the components of the T cell receptor, this finding suggests that at least some component of T cell specificity may arise as a consequence of peptide-induced conformational changes in the class I structure. To assess this possibility, we have made systematic substitutions at residues within the Kb-restricted determinant from ovalbumin (OVA257-264) that are thought to be buried on binding to the class I molecule. We have found that changes in this determinant at the completely buried second residue (P2) can influence T cell recognition without affecting binding to Kb, suggesting that the substitutions may indirectly determine T cell recognition by altering the conformation of the class I molecule or the bound peptide.

Amino Acids↗

PubMed1993

Expression and functional conservation of the human La (SS-B) nuclear autoantigen in murine cell lines.

We have studied the cellular expression and functional conservation of the human La autoantigen across species by introducing genes encoding human La antigen into cultured murine cell lines. In transfected murine fibroblasts and lymphoid cell lines human La was expressed as a predominantly nuclear antigen, with a typical pattern of nuclear speckling. Radiolabeled human La was of the predicted molecular mass (48 kDa) when expressed in murine cells and was associated intracellularly with murine 60 kDa Ro antigen. Under certain conditions of cell lysis the association between human La and murine Ro was disrupted, whereas human La and human Ro complexes remained intact, suggesting the possibility of species-specific protein interaction between La/Ro antigens. Expression of human La from the genomic gene construct was associated in some transfectants with the presence of additional high molecular weight bands reactive with anti-La monoclonal antibodies. Cell surface La determinants were not detected on any of the transfected cells. Human La protein associated with many of the same radiolabeled RNAs as the murine La antigen in transfected murine cells, indicating intact RNA-binding function of La protein across species. The findings indicate that despite 23% non-identity within the primary structure of human and murine La, the human molecule is functionally highly conserved across species.

Animals↗

PubMed1993

A study of wound healing in the E11.5 mouse embryo by light and electron microscopy.

In this paper we report our light and electron microscopic studies of the healing of a simple excisional lesion to the E11.5 mouse embryo hindlimb. The wounded living embryo is cultured in a roller bottle and under such conditions the lesion is completely re-covered with epithelium by 24 hr. We discuss how our studies of such a simple wound healing model may offer insight into the mechanisms of tissue repair generally.

Animals↗

PubMed1993

Threshold detection of self-antigen/MHC class II complexes formed in vivo: constitutive presentation of an immunodominant epitope of hen egg lysozyme (HEL) in HEL-transgenic mice.

Hen egg lysozyme (HEL)-specific T hybridomas were used to study the presentation of HEL as a self component in tolerant, HEL-transgenic mice expressing different serum levels of HEL (AL3, 3-7 nM: ML5, 1-3 nM). Constitutive presentation of the HEL46-61 determinant was detectable in antigen presenting cell (APC)-enriched preparations of spleen, thymus, and lymph node from AL3 transgenic mice. The most likely cell responsible for constitutive presentation of HEL antigen was a non-B cell. In addition, constitutive presentation of HEL46-61 was clearly evident in thymic rosettes (complexes of macrophages or dendritic cells and lymphoid cells) derived from AL3 HEL-transgenic mice, but not from ML5 HEL-transgenic mice. In contrast to the HEL46-61 determinant, constitutive presentation of the HEL25-43 and HEL112-129 determinants was not detectable on APC-enriched preparations from either of the HEL-transgenic lines tested. Thus, although T cell tolerance was evident in transgenic mice expressing a range of serum HEL concentrations, constitutive presentation of processed HEL antigen was not detectable on APCs from mice expressing less than 3-7 nM. This threshold of detectable antigen presentation is consistent with the notion that the concentration of nominal antigen needed for tolerizing T cells is less than that required for activation of mature T cells.

Animals↗

PubMed1993

Murine cytomegalovirus interacts with major histocompatibility complex class I molecules to establish cellular infection.

The expression of stable, correctly folded major histocompatibility complex class I molecules conferred susceptibility to murine cytomegalovirus (MCMV) in cells which were previously resistant to infection, demonstrating that these molecules interact critically with MCMV to initiate infection. All class I molecules could potentiate MCMV infection but H-2Dd and Kb molecules were most efficient. Monoclonal antibodies specific for the alpha 1 and/or alpha 2 domains of Dd and Kb inhibited infection. Infection of L cells transfected with hybrid major histocompatibility complex class I molecules demonstrated that allelic control of susceptibility to MCMV mapped to the alpha 1 domain of Dd when in correct configuration with the alpha 2 and alpha 3 domains. In MCMV-resistant RMA-S cells, an improvement in the conformation of class I molecules introduced susceptibility to infection.

Animals↗

PubMed1993

Identification of a human-specific epitope in a conserved region of the La/SS-B autoantigen.

Human anti-La/SS-B autoantibodies are known to react with highly conserved epitopes suggested to be functional or active sites on the La/SS-B polypeptide. This study was designed to determine whether the autoantibodies also react with poorly conserved regions of La/SS-B as predicted by an antigen-driven autoimmune response. Binding of human autoantibodies to purified human, mouse, and bovine recombinant fragments representing immunodominant regions of the La/SS-B polypeptide was compared using Western blotting and ELISA. A cross-reactive epitope was located in the highly conserved NH2-terminal region of La/SS-B. Significantly, human-specific epitopes were identified in both the conserved RNA-recognition motif and a poorly conserved COOH-terminal fragment, providing direct evidence for an autoantigen-driven response. The lack of autoantibody cross-reactivity with a conserved domain of mouse and bovine La/SS-B implies that a small number of residues in human autoepitopes may be critical for autoimmunogenicity.

Amino Acid Sequence↗

PubMed1992

Restricted epitope recognition by precipitin-negative anti-La/SS-B-positive sera.

OBJECTIVE: To determine whether anti-La/SS-B-positive sera that are precipitin negative show a distinct B cell epitope pattern. METHODS: Serum reactivity was tested with recombinant La/SS-B fusion proteins. RESULTS: Among the 18 precipitin-negative anti-La/SS-B-positive sera, reactivity was confined to the full-length recombinant protein (La33.3) in 8 (44%); 5 of 18 (28%) reacted only with La33.3 and with the first 107 N-terminal amino acids (LaA), and 4 (22%) reacted with La33.3, LaA, and the middle region of the La molecule (LaC; amino acids 111-242). One serum reacted with La33.3 and LaC. None of the 18 precipitin-negative sera was positive on a carboxy-terminal fragment (LaL2/3; amino acids 346-408). In contrast, all 26 precipitin-positive anti-La/SS-B-positive sera reacted with La33.3, LaA, and LaC, and 92% reacted with LaL2/3. Rheumatoid factor and serum IgG levels were significantly lower in the precipitin-negative group, providing further evidence of a distinct serologic subset. CONCLUSION: The restricted epitope recognition by these sera may explain the lack of precipitin formation and may represent an early autoantibody response to La/SS-B.

Autoantibodies↗

PubMed1992

Impaired expression of chimaeric major histocompatibility complex transgenes associated with plasmid sequences.

Plasmid vector sequences were retained (vector+), or removed (vector-) from hybrid major histocompatibility complex gene constructs prior to microinjection of fertilized ova for the production of transgenic mice. In transgenic mice containing integrated vector+ gene constructs, low levels of class II cell surface determinants were detected on splenocytes from only two out of six independent lines. Class II membrane determinants were not detectable on splenocytes from the remaining four vector+ transgenic lines. Expression of transgene products did not correlate with transgene copy number which ranged from 1-10 copies. Low levels of mRNA transcripts were detected in thymic mRNA from vector+ lines. In contrast, high levels of thymic and splenic mRNA transcripts were detected in offspring from all four vector- transgenic lines. Spleen cells from the vector- transgenic animals also expressed high levels of the hybrid major histocompatibility complex transgene products. These results implicate plasmid vector sequences in the inhibition of expression of the hybrid class II-class I major histocompatibility complex genes in transgenic mice. This putative inhibition of transgene expression presumably occurs at the level of gene transcription.

Animals↗

PubMed1992

Growth factors and cutaneous wound repair.

The healing of an adult skin lesion is a well studied but complex affair of some considerable clinical interest. Endogenous growth factors, including the EGF, FGF, PDGF and TGF beta families, are released at the wound site and presumed to be a necessary part of the natural wound healing machinery. Moreover, members of each of these families have been shown to enhance healing if added exogenously to a wound site. In this review we shall briefly discuss what is known about the mechanics and cell biology of adult wound healing, describe the normal cellular source of growth factors during the healing process and, with reference to their known capacities in tissue culture, speculate as to how particular growth factors might be able to enhance healing.

Animals↗

PubMed1992

Stable expression of the human immunodeficiency virus type 1 envelope glycoprotein in transfected L cells.

An SV40-based expression vector was used to generate CD4-negative murine L cell lines which stably expressed the human immunodeficiency virus envelope glycoprotein (env). Despite the presence of abundant intracellular envelope glycoprotein, the expression of env gp120/41 was not detected on the cell surface. Pulse-chase studies showed that the majority of the gp120 detected at the end of a 20-h chase was in the culture medium. Therefore gp120 was shed and/or secreted from these cells. Transfected L cells (H-2k) served as targets for specific lysis by CTL raised against vaccinia virus-encoded env gp160. The discrepancy in relative levels of intracellular versus surface expression of env was probably due to the highly inefficient processing of newly synthesized gp160, as well as the apparent instability of the gp120/41 complex in the transfected cell lines. Digestion of immunoprecipitated gp120 and gp160 with endoglycosidase H and peptide N-glycosidase F revealed that the envelope glycoprotein in transfected L cells possessed both high mannose and complex N-glycans, analogous to the posttranslational modification of the mature envelope glycoprotein in infected T cells. These studies indicate that the relatively inefficient processing of env gp160 occurs in the absence of CD4, and that the stable surface expression of envelope gp120/41 complex may require additional factors not present in transfected cells.

Animals↗

PubMed1992

CD8 expression alters the fine specificity of an alloreactive MHC class I-specific T hybridoma.

The influence of CD8 on the fine specificity of MHC class I-restricted T cell allorecognition was evaluated by comparing the reactivity of CD8- and CD8-transfected forms of an allospecific, H-2Kb-restricted T hybridoma. The CD8- T hybridoma responded to cells expressing H-2Kb, H-2Kbm6, and the individual H-2Kb----bm10 back mutations 165V----M, 173K----E, and 174N----L. Under the same conditions the CD8- T hybridoma responded poorly or not at all to cells expressing H-2Kbm10, H-2Kbm8, the individual H-2Kb----bm10 back mutants 163T----A and 167W----S, and the individual H-2Kb----bm8 back mutations 22Y----F and 24E----S. In contrast, T hybridoma cells expressing high levels of CD8 reacted strongly with antigen presenting cells (APC) expressing H-2Kb and H-2Kbm6 molecules, as well as APC expressing H-2Kbm10 (weakly), H-2Kbm8, and all five individual H-2Kb----bm10 and the two H-2Kb----bm8 back mutants 22Y----F and 24E----S. The mutations which distinguish the T cell recognition of both H-2Kbm10 and H-2Kbm8 from H-2Kb are predicted to control the interaction of these class I molecules with antigenic peptides in the binding site, implying an important role for peptide antigen in T cell allorecognition. Nonetheless, CD8 expression by the H-2Kb-restricted T cells conferred novel or enhanced alloreactivity with cells expressing H-2Kbm10, H-2Kbm8, and each of the individual H-2Kb----bm10 and H-2Kb----bm8 back mutants. These findings reflect an important role for CD8 in influencing the fine specificity of MHC class I recognition by T cells and may indicate a limited structural role for peptide antigen in defining the ligand recognized by these alloreactive T cells.

Animals↗

PubMed1991

Class II-restricted presentation of an endogenously derived immunodominant T-cell determinant of hen egg lysozyme.

An in vitro model was used to investigate the potential for different structural forms of endogenous antigen to be processed and presented by major histocompatibility complex class II molecules. For this purpose the class II-restricted presentation of an immunodominant epitope of hen egg lysozyme [HEL-(46-61)] was studied in class II-positive B-lymphoma cells (M12.C3) transfected with genes encoding HEL molecules either (i) secreted in high (hi) or low (lo) amounts as soluble antigen [sHEL(hi/lo)], (ii) localized within the endoplasmic reticulum (ER)/salvage compartment (ER-HEL), or (iii) anchored on the cell surface as an integral membrane protein (mHEL). The corresponding sHEL, ER-HEL, and mHEL gene products were expressed as predicted except that HEL determinants accumulated in the culture supernatant as well as on the cell membrane of mHEL-transfected cells. Class II-positive cells endogenously expressing all three forms of HEL antigen constitutively presented the immunodominant HEL-(46-61) determinant with differential efficiency (mHEL, sHEL greater than ERHEL) to a class II-restricted T hybridoma. A second T hybridoma recognized endogenous HEL-(46-61) determinants constitutively presented on sHEL(hi) and mHEL transfectants but not on sHEL(lo) or ERHEL transfectants. The formation of HEL-(46-61)/I-Ak complexes in the ERHEL and sHEL(lo) transfectants was therefore limiting. Mixing experiments with different antigen-presenting cells indicated that the HEL-(46-61) determinant was derived from endogenous antigen rather than by reuptake of shed or secreted HEL determinants. We conclude that MHC class II molecules can present some antigenic determinants derived from endogenous proteins that are sequestered in the ER/salvage compartment as well as distally transported in the form of secretory or membrane antigens.

Animals↗

PubMed1991

Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen: intracellular targeting to the amyloplast.

We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain.

Allergens↗