PubMed Health⌕ Search

Biomedical subjects

J M Stark

Publications and source records attributed to J M Stark.

At least 19 recordsLinked to original sources
PubMed2000

Molecular analysis of CCR-3 events in eosinophilic cells.

CCR-3 is a major receptor involved in regulating eosinophil trafficking. Initial analysis of chemokine receptors has demonstrated unique receptor events in different cell types, indicating the importance of investigating CCR-3 events in eosinophilic cell lines. We now report that the eosinophilic cell line, acute myelogenous leukemia (AML) 14.3D10, expresses eosinophil granule proteins and eotaxin, but has no detectable expression of eosinophil chemokine receptors. Treatment of the cell line with butyric acid and IL-5 results in a dose-dependent synergistic induction of CCR-3 and, to a lesser extent, CCR-1 and CCR-5. Interestingly, using a luciferase reporter construct under the control of the hCCR-3 promoter, the uninduced and induced cells display high, but comparable, levels of promoter activity. Differentiated AML cells developed enhanced functional activation, as indicated by adhesion to respiratory epithelial cells and chemokine-induced transepithelial migration. Chemokine signaling did not inhibit adenylate cyclase activity even though calcium transients were blocked by pertussis toxin. Additionally, chemokine-induced calcium transients were inhibited by pretreatment with PMA, but not forskolin. Eotaxin treatment of differentiated AML cells resulted in marked down-modulation of CCR-3 expression for at least 18 h. Receptor internalization was not dependent upon chronic ligand exposure and was not accompanied by receptor degradation. Thus, CCR-3 is a late differentiation marker on AML cells and uses a signal transduction pathway involving rapid and prolonged receptor internalization, calcium transients inhibitable by protein kinase C but not protein kinase A, and the paradoxical lack of inhibition of adenylate cyclase activity.

Adenylate Cyclase Toxin↗
PubMed2000

Current concepts on pulmonary host defense mechanisms in children.

The respiratory tract is exposed continuously to noxious agents, microbial organisms, particles, and allergens. It has therefore evolved both innate and specific defense mechanisms. The innate host defense mechanisms include components such as collectins, beta-defensins, lactoferrin, and complement, all of which have an important role in modulating the immune response. Immune protection of the lungs by specific antibody is reviewed. The airways are protected by alveolar macrophages, neutrophils, and lymphocytes, and their origins, regulation, functions, and antimicrobial activity are summarized. Antimicrobial peptides and immune-modulating peptides are likely to have a significant therapeutic role for infection and inflammation in the respiratory tract.

Animals↗
PubMed1999

The relative strengths of SR protein-mediated associations of alternative and constitutive exons can influence alternative splicing.

We have characterized the functional role of SR protein-mediated exon/exon associations in the alternative splicing of exon 5 of chicken cardiac troponin T (cTnT). We have previously shown that SR proteins can promote the association of the alternative exon 5 with the flanking constitutive exon 6 of this pre-mRNA. In this study, we have shown that when exons 2, 3, and 4 of the cTnT pre-mRNA are spliced together, the composite exon 2/3/4 contains an additional SR protein binding site. Furthermore, we have found that SR proteins can also promote interactions between the pairs of exons 2/3/4-5 and 2/3/4-6. We then asked whether the SR protein binding sites in these exons play a role in cTnT alternative splicing in vivo. We found that the SR protein binding sites in exons 2/3/4 and 6 promote exon 5 skipping, and it has previously been shown that the SR protein binding site in exon 5 promotes exon 5 inclusion. Consistent with these results, we find that the SR protein-mediated association of exon 2/3/4 with 6 is preferred over associations involving exon 5, in that exons 2/3/4 and 6 are more efficient than exon 5 in competing an SR protein-mediated exon/exon association. We suggest that the relative strengths of SR protein-mediated associations of alternative and constitutive exons play a role in determining alternative splicing patterns.

Alternative Splicing↗
PubMed1999

Induction of CD95 (Fas) and apoptosis in respiratory epithelial cell cultures following respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) infection is associated with epithelial cell death and vigorous inflammation. In mouse models, and in immunosuppressed patients, CD8(+) T cells are necessary for RSV clearance. In vitro, RSV has been shown to induce expression of several proteins on the respiratory epithelial cell, including RSV proteins, ICAM-1, and MHC class I, that can potentially interact with CD8(+) T cells in initiating apoptosis of the target cell. One mechanism of T-cell-directed cell death is the interaction of FasL on the CD8(+) T lymphocytes and Fas expressed on the target cell. In order to determine the ability of RSV to induce Fas on the respiratory epithelium, we studied the RSV infection of a human respiratory epithelial cell line (A549) in vitro. Fas mRNA and protein levels are increased two-to-fourfold following RSV infection, and transcriptional upregulation of Fas was demonstrated using promoter/reporter gene constructs. RSV infection directly resulted in cellular apoptosis, and the frequency of apoptotic cells was further increased by cross-linking with antibodies to Fas. These data demonstrate that RSV infection induces cellular apoptosis and suggest that interactions of surface Fas with T cells may further augment this process in vivo.

Animals↗
PubMed1999

Interleukin-1 receptor antagonist inhibits interleukin-8 expression in A549 respiratory epithelial cells infected in vitro with a replication-deficient recombinant adenovirus vector.

In an earlier study, we showed that a recombinant adenovirus vector with deletions in the E1 and E3 regions of the viral genome (AV1LacZ4) induces expression of interleukin (IL)-8 in A549 cells (a human respiratory cell line). IL-8 can be induced through several pathways, including activation by IL-1. We tested the hypothesis that the induction of IL-8 by the AV1LacZ4 adenovirus is accomplished by means of the IL-1/IL-8 activation pathway, which could be blocked by IL-1 receptor antagonist (IRAP). Viral infections of A549 cells were performed at a multiplicity of infection (MOI) of 50 in the presence and absence of IRAP (50 ng/ml). A549 cells were also stimulated with tumor necrosis factor (TNF)-alpha (100 ng/ml), a known stimulant of IL-8, in the presence and absence of IRAP. IL-8 expression was evaluated by Northern blot analysis and enzyme-linked immunosorbent assay. Levels of IL-8 protein and messenger RNA (mRNA) were greater in the infected cells than in the uninfected ones at 24, 48, and 96 h (P < 0.01). Virus-infected cells treated with IRAP expressed 75% less IL-8 mRNA and protein (P < 0.01) than did untreated cells, whereas IRAP pretreatment of TNF-alpha-stimulated cells did not affect IL-8 production. IL-1 production by the virus-infected cells was detectable by concentration of the supernatants and reverse transcription-polymerase chain reaction. We conclude that IL-8 is produced by virus vector-infected cells, partly through IL-1 activation that can be downregulated by IRAP.

Adenoviridae↗
PubMed1998

The heat shock response inhibits RANTES gene expression in cultured human lung epithelium.

The chemokine RANTES is thought to be involved in the pathophysiology of inflammation-associated acute lung injury. Although much is known regarding signals that induce RANTES gene expression, relatively few data exist regarding signals that inhibit RANTES gene expression. The heat shock response, a highly conserved cellular defense mechanism, has been demonstrated to inhibit a variety of lung proinflammatory responses. We tested the hypothesis that induction of the heat shock response inhibits RANTES gene expression. Treatment of A549 cells with TNF-alpha induced RANTES gene expression in a concentration-dependent manner. Induction of the heat shock response inhibited subsequent TNF-alpha-mediated RANTES mRNA expression and secretion of immunoreactive RANTES. Transient transfection assays involving a RANTES promoter-luciferase reporter plasmid demonstrated that the heat shock response inhibited TNF-alpha-mediated activation of the RANTES promoter. Inhibition of NF-kappaB nuclear translocation with isohelenin inhibited TNF-alpha-mediated RANTES mRNA expression, indicating that RANTES gene expression is NF-kappaB dependent in A549 cells. Induction of the heat shock response inhibited degradation of the NF-kappaB inhibitory protein, I-kappaBalpha but did not significantly inhibit phosphorylation of I-kappaBalpha. We conclude that the heat shock response inhibits RANTES gene expression by a mechanism involving inhibition of NF-kappaB nuclear translocation and subsequent inhibition of RANTES promoter activation. The mechanism by which the heat shock response inhibits NF-kappaB nuclear translocation involves stabilization of I-kappaBalpha, without significantly affecting phosphorylation of I-kappaBalpha.

Adenocarcinoma↗
PubMed1998

SR proteins are sufficient for exon bridging across an intron.

We have developed a defined system to characterize the role of SR proteins and exonic enhancers in directly promoting splice-site interactions across an intron. Using RNA affinity chromatography, we find that SR proteins alone are sufficient to promote the specific association of the enhancer-containing exon 5 with the adjoining exon 6 from avian cardiac troponin-T. Direct visualization of this exon/exon association by electron spectroscopic imaging shows it to be highly specific. Furthermore, using in vivo characterized mutants of exon 5, we also show that this exon/exon association depends on the splicing enhancer within exon 5. These results suggest a model by which SR proteins may function through exonic enhancers to directly promote exon bridging.

Alternative Splicing↗
PubMed1998

Immunological adjuvance of metabolic origin: oxidative stress, postulated impaired function of thiol proteases and immunogenicity.

Autoimmune disease may sometimes arise because of dysfunction of thiol proteases, which are vulnerable to oxidation of their sulphydryl groups. This may be initially signalled by hyperinsulinaemia, regarded here as a telltale phenomenon of oxidative stress and indicating difficulty in protein catabolism. Initial immunogenic sensitization may take place when antigen processing is altered by a metabolic process, which has locally overwhelmed the antioxidant systems and led to diminished thiol-protease digestion and to the repeated survival in critical cells of immunogenic peptide fragments. From this it follows that an immunological host may be either prejudiced towards or against tolerance by agents, respectively, which stabilize or destabilize antioxidant homeostasis.

Autoimmune Diseases↗
PubMed1998

Essential roles of NF-kappaB and C/EBP in the regulation of intercellular adhesion molecule-1 after respiratory syncytial virus infection of human respiratory epithelial cell cultures.

To determine the molecular mechanism(s) of respiratory syncytial virus (RSV)-induced intercellular adhesion molecule-1 (ICAM-1) upregulation in respiratory epithelial cells (REC; A549 cell cultures), we investigated the roles of the transcription factors NF-kappaB and C/EBP. Increases in ICAM-1 message required de novo mRNA synthesis. ICAM-1 promoter constructs (luciferase reporter gene) transfected into A549 monolayers demonstrated promoter activation following RSV infection. Activation was abolished by site-specific mutation of the NF-kappaB (-228) or C/EBP (-239) sites. These data support the critical role of the activation of NF-kappaB and C/EBP in RSV-induced ICAM-1 expression by REC.

CCAAT-Enhancer-Binding Proteins↗
PubMed1998

Generation of a transgenic mouse with lung-specific overexpression of the human interleukin-1 receptor antagonist protein.

The purpose of the studies described here was to test the hypothesis that overexpression of the human interleukin-1 receptor antagonist (IL-1ra) in the distal airway epithelia of mice would result in amelioration of the inflammatory effects of IL-1alpha. The coding region of the human IL-1ra gene was placed under transcriptional control of the 5' flanking region of the human SP-C gene. Transgenic mice were generated by pronuclear injection of the transgene and identified by Southern blot analysis of genomic DNA. RNA expression of the transgene was confirmed by Northern blot analysis. In order to determine whether expression of the transgene conferred protection against inflammatory stimuli, control and transgenic mice were treated with IL-1alpha by intratracheal instillation. Six hours after treatment, bronchoalveolar lavage was performed, which revealed a statistically significant decrease in the degree of neutrophilia in the transgenic mice as compared with control mice. Furthermore, there was a significant reduction in the whole-lung myeloperoxidase concentration. Reverse transcription-polymerase chain reaction analysis of whole-lung RNA revealed a significant reduction in the messenger RNA/beta-actin ratio of macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-2 in the transgenic animals as compared with controls. The results of these studies indicate that distal airway epithelial cell expression of human IL-1ra results in partial protection from IL-1alpha-induced airway inflammation and injury.

Animals↗
PubMed1998

Inhibition of TNF-alpha-induced NF-kappaB activation and IL-8 release in A549 cells with the proteasome inhibitor MG-132.

The working hypothesis of the studies described herein was that inhibition of proteasome-mediated IkappaB degradation would inhibit TNF-alpha-induced nuclear factor-kappaB (NF-kappaB) activation, interleukin-8 (IL-8) gene transcription, and IL-8 protein release in A549 cells. Mutational analysis of the 5' flanking region of the IL-8 gene confirmed that an intact NF-kappaB site is necessary for TNF-alpha-induced IL-8 gene transcription. The addition of TNF-alpha to A549 cells resulted in rapid loss of IkappaB from the cytoplasm of cells, associated with a corresponding increase in NF-kappaB-binding activity in nuclear extracts from the cells. However, pretreatment of the cells with the proteasome inhibitor N-cbz-Leu-Leu-leucinal (MG-132, 10 microM) reversed the effects of TNF-alpha on IL-8 release from A549 cells (as determined with an enzyme-linked immunosorbent assay [ELISA]) and on IL-8 gene transcription (as determined with reporter-gene assays). MG-132 reversed the effects of TNF-alpha on IkappaB degradation as determined by Western blot analysis. IkappaB phosphorylation and ubiquination were not altered by MG-132, which implies that the effects of MG-132 were secondary to proteasome inhibition. MG-132 also reversed the increase in NF-kappaB binding in nuclear extracts from TNF-alpha-treated cells. These studies show that inhibition of proteasome-mediated IkappaB degradation results in inhibition of TNF-alpha induced IL-8 production in A549 cells by limiting NF-kappaB-mediated gene transcription.

Cell Line↗
PubMed1998

Surfactant protein-A-deficient mice are susceptible to Pseudomonas aeruginosa infection.

To determine the role of surfactant protein-A (SP-A) in host defense, the murine SP-A locus was targeted by homologous recombination to produce mice lacking SP-A. SP-A-/- and wild-type mice were infected with mucoid Pseudomonas aeruginosa by intratracheal instillation. Pulmonary bacterial loads were greater in SP-A-/- than in wild-type mice, with increased numbers of mucoid P. aeruginosa in lung homogenates at 6 and 24 h after infection. Pulmonary infiltration with polymorphonuclear leukocytes (PMN) was similar in both groups; however, an earlier influx of PMN into the lung occurred in the SP-A-/- mice. The number of bacteria phagocytosed by alveolar macrophages was decreased in the SP-A-/- mice at 1 h after infection. Superoxide-radical generation by PMN was similar for the SP-A-/- and wild-type mice, but nitrite levels were increased in SP-A-/- mice. Concentrations of tumor necrosis factor-alpha, interleukin-6, and macrophage inflammatory protein-2 (proinflammatory cytokines) were greater in bronchoalveolar lavage fluid at 2 h after infection in SP-A-/- mice. SP-A plays an important role in the pathogenesis of mucoid P. aeruginosa infection in the lung in vivo by enhancing macrophage phagocytosis and clearance of bacteria, and by modifying the inflammatory response.

Animals↗
PubMed1997

A flow cytometric study of antibiotic-induced damage and evaluation as a rapid antibiotic susceptibility test for methicillin-resistant Staphylococcus aureus.

Flow cytometry using the anionic membrane potential-sensitive fluorescent probe, bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)), enabled assessment of antibiotic-induced membrane perturbation in five clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and two antibiotic-sensitive reference strains, NCTC 6571 and 8325-4, after establishment of steady-state growth in liquid cultures inoculated from single colonies. Flow cytometric indications of the enhanced DiBAC4(3) uptake after treatment with vancomycin at 0.1, 1, 4 and 10 x MIC showed excellent comparison with viability losses quantified as cfu on solid agar in MRSA isolate QC. The antibiotic susceptibility patterns to benzylpenicillin, methicillin and vancomycin for all isolates used in this study could be determined in 2-4 h from an overnight plate culture. This technique thus provides a rapid and reproducible antibiotic sensitivity test which may be applicable in routine clinical practice.

Anti-Bacterial Agents↗
PubMed1997

Receptive field changes after strokelike cortical ablation: a role for activation dynamics.

The reorganization of neural activity that takes place after stroke is of paramount importance in producing functional recovery. Experimental stroke models have suggested that this reorganization may have two phases, but physiology alone cannot fully resolve what causes each phase. Computer modeling suggests that these phases might involve an initial change in dynamics occurring immediately, followed by synaptic plasticity. We combined physiological recording from macaque middle temporal cortex (area MT) with a neural network computer model to examine this first phase of altered cortical function after a small, experimentally induced cortical lesion. Major receptive field (RF) changes seen in the first few days postlesion included both expansion and contraction of receptive fields. Although only expansion could be reproduced in an initial model, addition of inhibitory interneuron loss in a ring around the primary ablation, suggested by immunohistochemical examination, permitted contraction to be replicated as well. We therefore predict that this immunochemical observation reflects an immediate extension of the lesion rather than a late response. Additionally our model successfully predicted a correlation between increased firing rate and RF size. Our model suggests that activation dynamics alone, without anatomic remodeling, can cause the large receptive field changes that allow the rapid behavioral recovery seen after middle temporal lesions.

Animals↗
PubMed1996

Infection of A549 cells with a recombinant adenovirus vector induces ICAM-1 expression and increased CD-18-dependent adhesion of activated neutrophils.

A significant number of pulmonary exacerbations in patients with cystic fibrosis (CF) and asthma are associated with respiratory virus infections. The molecular mediators of this process are beginning to be understood. Viral infection of respiratory epithelial cultures in vitro leads to the production of intercellular adhesion molecule-1 (ICAM-1) (a ligand for inflammatory cell adhesion and activation) and a number of proinflammatory cytokines. Human gene therapy vectors derived from human adenoviruses (AV) are currently under evaluation for CF transmembrane regulator (CFTR) gene delivery to the airway epithelium of CF patients. However, studies in animal models using these AV vectors demonstrate pulmonary inflammation following AV exposure. Using an in vitro model, we examined the hypothesis that exposure of respiratory epithelial cells to AV vectors results in upregulation of ICAM-1 gene expression. Infections were performed using a replication-deficient, first-generation AV vector. A549 cells (a human pulmonary adenocarcinoma cell line) were exposed to AV at multiplicity of infection of 50-150 plaque-forming units/cell (resulting in > 90% of cells expressing the reporter gene by 48 hr following exposure). Measurements of ICAM-1 expression were made at time intervals following virus exposure using enzyme immunoassay, flow cytometry, and Northern blot analysis. Cell-bound ICAM-1 was significantly increased 96 hr following vector exposure, two to four times control, p < 0.001). The AV-exposed A549 cells also supported increased levels of adhesion of activated neutrophils 96 hr following AV exposure (four times control, p < 0.001) that was blocked by antibody to CD18. AV exposure of A549 monolayers increases expression of biologically active ICAM-1. Strategies to minimize host cellular proinflammatory responses to the replication-deficient AV vectors may improve their safety for gene therapy.

Adenocarcinoma↗
PubMed1996

Respiratory syncytial virus infection enhances neutrophil and eosinophil adhesion to cultured respiratory epithelial cells. Roles of CD18 and intercellular adhesion molecule-1.

Respiratory syncytial virus (RSV) infections in children precipitate acute episodes of respiratory obstruction that are associated with influx of inflammatory cells into the airway. Since RSV can induce the expression of adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1), by the respiratory epithelium, the hypothesis has been proposed that ICAM-1 expression contributes to airway inflammation by supporting adhesion and retention of infiltrating inflammatory leukocytes. To test this hypothesis, A549 cells (an immortalized human alveolar epithelial type II cell-like fine) were infected with RSV, and the ability of these infected monolayers to support adhesion by human neutrophils (NEUT) and eosinophils (EOS) was measured. RSV infection significantly increased ICAM-1 expression by A549 monolayers (p < 0.001). Although NEUT adhesion to A549 monolayers was significantly enhanced following RSV infection (p < 0.001), infection alone resulted in little change in EOS adherence. However, if EOS were first activated with phorbol ester (PMA), adhesion to both control and RSV-infected A549 cells was enhanced, with greater levels of adhesion supported by RSV-infected cultures (p < 0.001). The requirement for EOS activation (but not for NEUT activation) before adhesion remained when NEU and EOS were prepared and compared from the same donor. Despite this difference, NEUT and EOS adhesion was reduced by blocking Abs to epithelial ICAM-1 or granulocyte CD18 adhesion proteins (p < 0.01). However, only NEUT adhesion was blocked by Ab to CD11a. Our results show that RSV infections of respiratory epithelial monolayers can promote inflammatory cell adherence which could, in turn, potentially contribute to the airway injury and obstruction that accompanies bronchiolitis.

Adult↗