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J M McCormack

Publications and source records attributed to J M McCormack.

17 recordsLinked to original sources
PubMed2000

Inducible expression of claudin-1-myc but not occludin-VSV-G results in aberrant tight junction strand formation in MDCK cells.

Occludin and 18 distinct members of the claudin family are tetra-span transmembrane proteins that are localized in cell-specific tight junctions (TJs). A previous study showed that expression of chick occludin in Madin-Darby canine kidney (MDCK) cells raised transepithelial electrical resistance (TER) and, paradoxically, increased mannitol flux. In the present study, we employed epitope tagged canine occludin expression, under the control of the tetracycline repressible transactivator, to determine the extent to which the unexpected parallel increase in TER and mannitol flux was related to a structural mismatch between avian and canine occludins, which are only 50% identical. To determine whether the paradoxical changes in permeability was specific to occludin, we assessed the effect of over-expressing epitope tagged murine claudin-1. Our data revealed that over-expression of either of the epitope tagged mammalian tight junction proteins increased TER, mannitol and FITC-dextran flux. We observed a 2- and up to 5.6-fold over-expression of occludin-VSV-G and claudin-1-myc, respectively, with no change in ZO-1, endogenous occludin or claudin-1 expression. Confocal microscopy revealed that occludin-VSV-G, claudin-1-myc and ZO-1 co-localized at the TJ. In addition, claudin-1-myc formed aberrant strands along the lateral cell surface without an underlying ZO-1 scaffold. In fracture labeled replicas these strands consisted of claudin-1-myc with little accompanying occludin. These observations suggest that in epithelial cells claudin-1 can assemble into TJ strands without the participation of either ZO-1 or occludin. The proximity of the myc tag to the COOH-terminal YV sequence of claudin-1 appeared to interfere with its interaction with ZO-1, since over-expression of non-tagged claudin-1 increased TER but had a minimal effect on solute flux and no aberrant strands formed. From our data we conclude that differences in structure between avian and mammalian occludin do not account for the observed paradoxical increase in mannitol flux. Levels of ZO-1 remained unchanged despite substantial increases in induced TJ integral protein expression, suggesting that an imbalance between levels of ZO-1 and occludin or claudin-1 leads to altered regulation of pores through which non-charged solute flux occurs. We suggest that ion and solute flux are differentially regulated at the TJ.

Animals↗
PubMed1997

Quality control for DNA contamination in laboratories using PCR-based class II HLA typing methods.

Quality control (QC) in laboratories performing molecular histocompatibility class II typing often includes a polymerase chain reaction (PCR) approach for monitoring DNA contamination. An oligonucleotide primer set was designed, (RBQBf/RBQBr), which is specific for nonpolymorphic regions of the DR-B, DQ-B, and DP-B consensus sequences with an expected PCR product size of 81 bp. RBQBf/RBQBr detected genomic DNA from reference cell lines LWAGS and BM21 (50 to 100 picograms) as well as DR-B, DP-B, and DQ-B amplicon (1 copy). Additionally, RBQBf/RBQBr detected SSP products from routine DR-B and DQ-B typings. Validation studies employing controlled DNA contamination of laboratory surfaces revealed that increasing amounts of wipe test samples (5% to 20% v/v) were inhibitory to the wipe test PCR, whereas lower amounts (1% to 2%), or alternatively, a diluted wipe test sample, increased the sensitivity of the test and optimized the results. Collectively, this study describes a primer set, RBQBf/RBQBr, which detects both genomic DNA and DR-B, DQ-B, or DP-B amplicon and furthermore illustrates the necessity of routine testing for potential inhibitory factors that may be introduced into the wipe test PCR.

Base Sequence↗
PubMed1995

Basolateral but not apical application of protease results in a rapid rise of transepithelial electrical resistance and formation of aberrant tight junction strands in MDCK cells.

In the presence of Ca2+, application of trypsin to the basolateral surface of confluent MDCK cell monolayers with formed tight junctions (TJ), induces the formation of basolaterally oriented aberrant TJ strands. Induction of aberrant TJ strands is accompanied by an increase in transepithelial electrical resistance (TER), up to 90%, which upon addition of trypsin inhibitor is maintained for up to 1 h. Thereafter TER returns slowly to baseline values. Under similar conditions, application of trypsin to the apical surface has little or no effect on either TER or the number of aberrant TJ strands. Confocal microscopy of monolayers, immunostained for ZO-1, revealed that this TJ associated cytoplasmic protein, extended below the TJ along the basolateral surface following brief exposure to trypsin. Removing Ca2+ after treatment of the monolayer with basolaterally applied trypsin resulted, after 20 min, in the increased partitioning of TJ particles onto the E fracture face, of both normal and aberrant TJ strands. Like the TJ strands themselves, therefore, aberrant strands may be linked to cytoskeletal elements. Aberrant TJ strands do not form when monolayers, maintained in low Ca2+ medium, are exposed to trypsin, suggesting that under these conditions TJ precursors, and/or trypsin-sensitive proteins regulating TJ strand assembly, are sequestered in a vesicular compartment that is inaccessible to exogenous trypsin. Prolonged exposure of the apical surface of an established, polarized epithelium with intact TJ to trypsin, had little effect on TJ integrity and did not induce aberrant strands.

Animals↗
PubMed1993

Macrophage progenitors from mouse bone marrow and spleen differ in their expression of the Ly-6C differentiation antigen.

Using mAb and magnetic and fluorescence-activated cell sorting, together with culturing in semisolid culture medium, we show that essentially all splenic macrophage progenitors are Ly-6ChiMac-1-. By contrast, bone marrow macrophage progenitors were more heterogeneous in Ly-6C expression, the majority being restricted to the Ly-6CdimMac-1- and the Ly-6C-Mac-1- subsets. These findings provide immunophenotype evidence that macrophages in the spleen and bone marrow arise from different progenitors.

Animals↗
PubMed1993

Alloantigen presentation by individual clones of mouse splenic macrophages. Selective expression of IL-1 alpha in response to CD8+ T cell-derived IFN-gamma defines the alloantigen-presenting phenotype.

Approximately one-third of mouse splenic macrophage (M theta) progenitors yield progeny that constitutively present MHC class I alloantigen to naive T cells, a response that is restricted to CD8+ T cells and is elicited in a CD4+ Th cell-independent manner. In addition, both the alloantigen-presenting (alloAP+) and nonpresenting (alloAP-) M theta subsets constitutively express similar levels of MHC class I molecules, and their alloAP phenotypes are unaffected by IFN-gamma, which enhances the expression of both class I and II MHC molecules. We therefore postulated the restricted expression of costimulator molecules to account for the alloAP+ phenotype. Using cytokine-specific antibodies, recombinant mouse cytokines, and polymerase chain reaction analyses (to detect specific cytokine mRNA transcripts), we identified the putative costimulators as IL-1 alpha, IL-6, and TNF-alpha. TNF-alpha transcripts were present in both the alloAP+ and alloAP- M theta subsets, but IL-1 alpha and IL-6 were not constitutively expressed by the alloAP+ subset of M theta; rather, they were induced by IFN-gamma, which was released from naive CD8+ T cells only during coculture with alloAP+ M theta. Although IFN-gamma induced IL-6 gene transcription in both alloAP+ and alloAP- M theta subsets, it induced IL-1 alpha transcripts only in the alloAP+ subset. Finally, CD8+ T cells exposed to alloAP- M theta were unresponsive when subsequently cultured with alloAP+ M theta. We conclude that the ability of some M theta to elicit IFN-gamma from CD8+ T cells and to respond to this cytokine by producing IL-1 alpha defines the alloAP phenotype of the cell population, and that alloAP- M theta induce a state of alloantigen-specific tolerance in naive CD8+ T cells.

Animals↗
PubMed1993

Poststreptococcal anti-myosin antibody idiotype associated with systemic lupus erythematosus and Sjögren's syndrome.

Anti-myosin antibodies are found in acute rheumatic fever (ARF), a sequela of group A streptococcal infection. An antiidiotypic serum was produced that was specific for idiotopes expressed by anti-myosin antibodies in ARF (anti-My1). Studies indicated that idiotypic determinants detected with this serum were present in anti-myosin antibodies and absent from normal human immunoglobulins that lacked specificity for myosin. Anti-My1 was tested against sera from patients with other types of autoimmune diseases as well as uncomplicated streptococcal infections. Sera from systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), and poststreptococcal acute glomerulonephritis patients demonstrated idiotypic reactivity with anti-My1. Affinity-purified anti-myosin antibodies from SLE, SS, and ARF sera also reacted strongly with anti-My1, indicating that immunoglobulins produced in these diseases share idiotypic determinants. The data demonstrated an association of the My1 idiotype with poststreptococcal sequelae and the two autoimmune diseases SLE and SS.

Autoantibodies↗
PubMed1992

Mouse splenic macrophage cell lines with different antigen-presenting activities for CD4+ helper T cell subsets and allogeneic CD8+ T cells.

A panel of seven mouse splenic macrophage cell lines, derived from cloned progenitors, was compared for their ability to present antigen to Th1 or Th2 helper T cell lines and hybridomas, as well as to naive T cells, and to provide accessory cell function for the synthesis of antibody from primed B cells. One of the cell lines expressed MHC class II molecules and was the only line with constitutive antigen-presenting activity for Th1 cells. It may represent a subset of splenic macrophages responsible for the activation of naive Th1 helper cells in situ. The remaining six cell lines responded to INF-gamma by up-regulating their class II expression and acquiring Th1 antigen-presenting activity. They may represent cells which, in situ, lack constitutive antigen-presenting activity but are promoted to presenting status by Th1-derived INF-gamma. Five of the cell lines provided accessory cell function to Th2 cells, as indicated by antibody synthesis in suspensions of spleen cells from primed mice depleted of their antigen-presenting cells. One of the cell lines lacking accessory cell activity had constitutive antigen-presenting activity for Th1 cells. This reciprocal expression of antigen-presenting activity supports the idea that Th1 and Th2 helper cells are activated by different antigen-presenting cells. Finally, the cell lines differed in their ability to constitutively induce an allogeneic response; a response that was limited to CD8+ T cells occurred in a CD4+ helper cell-independent manner and was unaffected by the addition of INF-gamma. The alloantigen-presenting macrophage cell lines also possessed the most efficient accessory cell activity for antibody synthesis. These cell lines, which represent a spectrum of antigen-presenting activities in the spleen afford models for defining the roles of macrophages in the induction of immune responses and for resolving issues concerning their development.

Animals↗
PubMed1992

Phenotypes and alloantigen-presenting activity of individual clones of microglia derived from the mouse brain.

To clarify the origin and function of the microglia residing in the central nervous system, we cloned brain cells from newborn and adult mice in soft agar containing the macrophage-specific growth factor, colony-stimulating factor-1 and expanded the cells from individual colonies in liquid culture medium. The results of molecular, immunophenotypic and functional analyses showed that the clones consisted of microglia derived from the macrophage family of cells. For instance, the microglia contain mRNA transcripts for the receptor for colony-stimulating factor-1 and truncated CD4 transcripts similar to those found in mouse macrophages but not T helper cells. About a third of the microglial progenitors gave rise to progeny that constitutively induced the selective proliferation of naive allogeneic CD8+ T cells in a CD4+ T cell-independent manner, a response that was inhibited by monoclonal antibodies to major histocompatibility complex (MHC) class I molecules on the microglia. Since all microglia expressed similar levels of MHC class I molecules, the basis for the alloantigen presentation likely resides in the ability of some clones of microglia to synthesize co-stimulator molecules that are required for CD8+ T cell proliferation. Thus, at least some microglia in mouse brain arise from endogenous progenitors and appear capable of specialized functions.

Animals↗
PubMed1991

A subset of mouse splenic macrophages can constitutively present alloantigen directly to CD8+ T cells.

About a third of mouse splenic macrophage (M phi) progenitors give rise to cloned progeny that constitutively induce the selective proliferation of naive allogeneic CD8+ T cells in a CD4+ helper cell-independent manner--a response that is inhibited by mAb to the MHC class I molecules present on the M phi. Colony-mixing experiments indicated that the failure of most M phi clones to present allo-Ag was not due to their suppression of the ability of CD8+ cells to respond, nor did the nonpresenting clones interfere with the activity of the allo-Ag presenting M phi. The allo-Ag presenting phenotypes were found to be a stable characteristic in a panel of cell lines derived from individual clones of M phi. Analysis of the cell lines revealed that the differential expression of allo-APC activity could not be attributed to the levels of MHC class I molecules; rather, the cell lines and the primary M phi clones differ in their expression of a cell-associated costimulator molecule that likely functions to induce the expression of the IL-2R on and the secretion of IL-2 from the T cells.

Animals↗
PubMed1991

Immortalization of growth factor-dependent mouse splenic macrophages derived from cloned progenitors.

We describe a nonviral transformation strategy for the establishment of permanent cell lines derived from the progeny of individual mouse splenic macrophage (M phi) progenitors. These colony stimulating factor-1 (CSF-1)-dependent cell lines possess many features of mature M phi s, including antibody-dependent phagocytic and cellular cytotoxic activities, ability to secrete lysozyme, and expression of the Mac-1 antigen and mRNA for the CSF-1 receptor. It was also possible to immortalize selected clones of splenic M phi s differing in their constitutive antigen-presenting activities with the retention of the antigen-presenting phenotype in the resultant cell lines. The approach described in this report should be useful in obtaining additional cell lines of M phi s expressing other phenotypes of interest.

Animals↗
PubMed1989

Human and murine antibodies cross-reactive with streptococcal M protein and myosin recognize the sequence GLN-LYS-SER-LYS-GLN in M protein.

Molecular mimicry or epitope similarity between group A streptococcal M proteins and myosin may contribute to the presence of heart reactive antibodies in acute rheumatic fever. In our study overlapping synthetic peptides copying the entire sequence of PepM5 protein were used to map the myosin cross-reactive epitopes of streptococcal M protein recognized by mouse and human mAb and affinity purified myosin-specific antibodies from acute rheumatic fever and rheumatic heart disease sera. Overlapping M protein peptides SM5(164-197)C and SM5(184-197)C inhibited the murine mAb reactions with PepM5 protein. The human mAb and affinity purified myosin-specific antibodies reacted exclusively with SM5(184-197)C. However, one of the five different purified myosin-specific antibodies not only reacted with SM5(184-197)C but also reacted with SM5(84-116)C. The synthetic subpeptides SM5(175-184)C and SM5(188-197C) did not react with any of the antibodies to PepM5 and myosin demonstrating a requirement of the 184-188 amino acid sequence for antibody recognition. A heptapeptide containing the sequence SM5(183-189) was also found to inhibit selected human myosin-specific antibodies and a human antimyosin mAb. Therefore, the majority of mouse and human myosin crossreactive antibodies recognized an epitope within the 14 residue carboxy terminus of PepM5 which appeared to involve the GLN-LYS-SER-LYS-GLN sequence.

Amino Acid Sequence↗
PubMed1988

Human monoclonal antibodies reactive with antigens of the group A Streptococcus and human heart.

Human mAb were produced from tonsillar or PBL of normal individuals or patients infected with group A streptococci. Lymphocytes were purified on Ficoll-Hypaque gradients and stimulated in vitro with purified group A streptococcal membranes or M protein extracts. The mAb were selected for study based on their reaction with group A streptococci, pep M5 protein, and/or M6 Escherichia coli protein. Further analysis by Western immunoblot or competitive inhibition ELISA revealed that there were two types of antibodies: one type that reacted with myosin and DNA and the other type that reacted with myosin, keratin, and/or actin. The specificities of these human mAb are similar to specificities observed in our previous studies of murine mAb reactive with group A streptococci and heart Ag. For comparison, anti-myosin antibodies were affinity purified from the sera of infected or acute rheumatic fever patients and were shown to react with myosin and DNA as well as with group A streptococci and M protein. To affinity purify these antibodies from normal sera, five times the amount of sera was required to obtain detectable quantities. These data suggest that the human mAb reactive with group A streptococci and myosin reflect the antibodies seen in sera from infected patients or acute rheumatics and that the B lymphocyte clones capable of producing these cross-reactive antibodies are also present in normal individuals.

Antibodies, Bacterial↗
PubMed1988

Recombinant human interleukin 2 (rIL-2) enhancement of antibody production by human-human hybridomas.

We studied the effect of highly purified recombinant interleukin 2 from Escherichia coli (rIL-2) on antibody production by our human hybridomas. Increasing concentrations of rIL-2 were directly related to increased production of immunoglobulin, which reached peak levels of 13-25 micrograms/ml, a 10- to 20-fold increase over untreated hybridomas. We were unable to demonstrate large quantities of the IL-2 receptor on the hybridomas as measured by an anti-TAC monoclonal antibody. The data suggest that the antibody enhancement phenomenon is mediated by IL-2 and that is a new, effective way to achieve higher levels of immunoglobulin from human hybridomas.

Antibodies, Bacterial↗
PubMed1987

Induction of a macrophage-suppressive lymphokine by soluble cryptococcal antigens and its association with models of immunologic tolerance.

Soluble extracts of Cryptococcus neoformans were examined for their ability to induce a macrophage-regulatory T-suppressor cell known to appear in the spleens of mice infected with cryptococci. Suppressor cells were induced by injection of extracts of encapsulated or thinly encapsulated strains of cryptococci. Dose-response analysis showed that as little as 25 micrograms of soluble capsular polysaccharide antigen could induce significant suppressor cell activity, with maximum suppression occurring at a dose of 100 micrograms. The suppressor cells appeared within 1 week of injection of antigen and persisted for at least 2 months. Suppressor cells were induced in animals given tolerogenic doses of levan, human gamma globulin, and soluble capsular polysaccharide antigen. When these same antigens were administered in immunogenic form, no suppressor cell activity was detected. Therefore, the suppressive mechanism was common to models of immunologic tolerance and was not unique to cryptococcal disease or cryptococcal capsular polysaccharide antigen. The phagocytosis-inhibiting lymphokine produced by the suppressor cell population completely inhibited the phagocytic activity of only a portion of peritoneal exudate cells. Other macrophages in the population were not totally inhibited but exhibited a reduction in the number of yeast cells engulfed.

Animals↗
PubMed1984

Intercellular junctions in upper airway submucosal glands of the rat: a tracer and freeze fracture study.

The structure of intercellular tight junctions of rat airway submucosal glands was examined by freeze fracture techniques and their permeability assessed by the use of colloidal lanthanum. The submucosal glands were organized into three distinct regions: a) serous tubules and b) mucous tubules lined, respectively, by serous and mucous cells, and c) ducts lined by cuboidal epithelial cells, containing few secretory granules, and some ciliated cells. The mean number of parallel fibrils constituting the tight junctions between serous cells was 3.6 +/- 0.4, which was significantly smaller than those between any of the other cell types. Colloidal lanthanum permeated the tight junctions between serous cells up to the level of the acinar lumen. There was a progressive increase in the mean number of parallel fibrils of tight junctions between mucous (5.1 +/- 0.6), ductal (5.4 +/- 0.5), and ciliated cells (8.5 +/- 0.7); none of these junctions was permeated by colloidal lanthanum. These results imply that tight junctions between serous cells are more permeable to small water-soluble solutes than those present in the more proximal portions of the gland. Gap junctions were observed between serous cells and between mucous cells, suggesting that these secretory cells may be electotronically and metabolically coupled.

Animals↗