Search PubMed⌕ Search

Biomedical subjects

J M Stark

Publications and source records attributed to J M Stark.

At least 19 recordsLinked to original sources
PubMed2006

Risk of leukaemia among children living near the Solway coast of Dumfries and Galloway Health Board area, Scotland, 1975-2002.

OBJECTIVE: To investigate allegations of an excess risk of leukaemia among children living near the Solway Firth coast of Dumfries and Galloway Health Board area in Scotland, UK. METHODS: Incident cases of childhood leukaemia (International Classification of Diseases, 10th revision, C91-C95, patients aged 0-14 years) for two almost equal calendar periods of diagnosis (1975-89 and 1990-2002) were selected from the Scottish Cancer Registry database and allocated to predetermined study areas, on the basis of proximity of residence to the Solway coast. Expected numbers of childhood leukaemia cases for the study areas were calculated by applying Scotland's age-specific, sex-specific and calendar period-specific rates to estimates of the person-years at risk in each study area. The ratios of observed to expected cases or standardised incidence ratios (SIRs) were calculated overall and for each sex and calendar period category. Exact 95% confidence intervals (CIs) for the SIRs were calculated assuming a Poisson distribution for the observed number of cases of childhood leukaemia. RESULTS: No statistically significantly increased SIRs were found in boys, girls or both combined for any of the areas or periods of diagnosis studied. For the total period of observation (1975-2002), and the more immediate coastal area studied, the SIR for both sexes combined was 1.22 (95% CI 0.53 to 2.40). CONCLUSION: No statistically significant evidence was found of an excess risk of childhood leukaemia in the vicinity of the Solway Firth coast of Dumfries and Galloway Health Board area in Scotland.

Adolescent↗
PubMed2001

Decreased pulmonary clearance of S. pneumoniae following influenza A infection in mice.

In children, the incidence of complicated pneumonias (including empyemas and lung abscesses) associated with Streptococcus pneumoniae infection has increased in recent years. In many cases, these complicated pneumonias followed flu-like illnesses. To determine mechanisms behind this association, a murine model of sequential pulmonary infection has been developed. BALB/cJ mice infected with influenza A had mild pulmonary inflammation that resolved within 5-7 days. Seven days following their initial 'treatment' (mock infection or influenza exposure), mice were challenged with 10(6) cfu of S. pneumoniae, and their lungs were harvested at intervals for analysis. Lungs of influenza-exposed mice demonstrated greater colony counts 24 and 48 h following S. pneumoniae exposure compared to control mice. In addition, neutrophil numbers were significantly increased in the influenza/S. pneumoniae sequentially-infected animals compared to S. pneumoniae infection alone (1.4+/-0.6 x 10(6) vs. 0.06+/-0.07 x 10(6) cells, P < 0.05, 24 h). Influenza-exposed animals had greater levels of IL-1beta and TNF-alpha in lung homogenates following S. pneumoniae inoculation. These data demonstrate that mice exposed to influenza have enhanced inflammatory responses and increased bacterial burden following S. pneumoniae exposure than do control mice. This model will be useful in defining mechanisms behind the enhanced susceptibility to S. pneumoniae that occurs after influenza exposure.

Animals↗
PubMed2001

Embryonic neuronal death due to neurotrophin and neurotransmitter deprivation occurs independent of Apaf-1.

Apoptotic protease-activating factor-1 (Apaf-1), dATP, and procaspase-9 form a multimeric complex that triggers programmed cell death through the activation of caspases upon release of cytochrome c from the mitochondria into the cytosol. Although cell death pathways exist that can bypass the requirement for cytochrome c release and caspase activation, several gene knockout studies have shown that the cytochrome c-mediated apoptotic pathway is critical for neural development. Specifically, the number of neuronal progenitor cells is abnormally increased in Apaf-1-, caspase-9-, caspase-3-deficient mice. However, the role of the cytochrome c cell death pathway for apoptosis of postmitotic, differentiated neurons in the developing brain has not been investigated in vivo. In this study we investigated embryonic neuronal cell death caused by trophic factor deprivation or lack of neurotransmitter release by analyzing Apaf-1/tyrosine kinase receptor A (TrkA) and Apaf-1/Munc-18 double mutant mice. Histological analysis of the double mutants' brains (including cell counting and terminal (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining) reveals that neuronal cell death caused by these stimuli can proceed independent of Apaf-1. We propose that a switch between apoptotic programs (and their respective proteins) characterizes the transition of a neuronal precursor cell from the progenitor pool to the postmitotic population of differentiated neurons.

Animals↗
PubMed2001

Double-strand breaks and tumorigenesis.

The establishment of connections between biochemical defects and clinical disease is a major goal of modern molecular genetics. In this review, we examine the current literature that relates defects in the two major DNA double-strand-break repair pathways--homologous recombination and nonhomologous end-joining--with the development of human tumors. Although definitive proof has yet to be obtained, the current literature is highly suggestive of such a link.

Animals↗
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

Comparison of the fine structure of cortical and collicular terminals in the rat medial geniculate body.

Neurons throughout the rat medial geniculate body, including the dorsal and ventral divisions, display a variety of responses to auditory stimuli. To investigate possible structural determinants of this variability, measurements of axon terminal profile area and postsynaptic dendrite diameter were made on inferior colliculus and corticothalamic terminal profiles in the medial geniculate body identified by anterograde tracer labeling following injections into the inferior colliculus or cortex. Over 90% of the synapses observed were axodendritic, with few axosomatic synapses. Small (<0.5 microm(2)) and large (>1.0 microm(2)) collicular profiles were found throughout the medial geniculate, but were smaller on average in the dorsal division (0.49+/-0.49 microm(2)) than in the ventral division (0.70+/-0.64 microm(2)). Almost all corticothalamic profiles were small and ended on small-caliber dendrites (0.57+/-0.25 microm diameter) throughout the medial geniculate. A few very large (>2.0 microm(2)) corticothalamic profiles were found in the dorsal division and in the marginal zone of the medial geniculate. GABA immunostaining demonstrated the presence of GABAergic profiles arising from cells in the inferior colliculus. These profiles were compared with GABAergic profiles not labeled with anterograde tracer, which were presumed to be unlabeled inferior colliculus profiles or thalamic reticular nucleus profiles. The distributions of dendritic diameters postsynaptic to collicular, cortical and unlabeled GABAergic profiles were compared with dendritic diameters of intracellularly labeled medial geniculate neurons from rat brain slices. Our results demonstrate a corticothalamic projection to medial geniculate body that is similar to other sensory corticothalamic projections. However, the heterogeneous distributions of excitatory inferior collicular terminal sizes and postsynaptic dendritic diameters, along with the presence of a GABAergic inferior collicular projection to dendrites in the medial geniculate body, suggest a colliculogeniculate projection that is more complex than the ascending projections to other sensory thalamic nuclei. These findings may be useful in understanding some of the differences in the response characteristics of medial geniculate neurons in vivo.

Animals↗
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↗