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Biomedical subjects

J M Stark

Publications and source records attributed to J M Stark.

At least 37 records · Page 2Linked to original sources
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

Evidence that elevated CO2 levels can indirectly increase rhizosphere denitrifier activity.

We examined the influence of elevated CO2 concentration on denitrifier enzyme activity in wheat rhizoplanes by using controlled environments and solution culture techniques. Potential denitrification activity was from 3 to 24 times higher on roots that were grown under an elevated CO2 concentration of 1,000 micromoles of CO2 mol-1 than on roots grown under ambient levels of CO2. Nitrogen loss, as determined by a nitrogen mass balance, increased with elevated CO2 levels in the shoot environment and with a high NO3- concentration in the rooting zone. These results indicated that aerial CO2 concentration can play a role in rhizosphere denitrifier activity.

Ammonia↗
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↗
PubMed1996

Inhibition of viral replication reverses respiratory syncytial virus-induced NF-kappaB activation and interleukin-8 gene expression in A549 cells.

Previous studies have demonstrated that respiratory syncytial virus (RSV) infection of airway epithelial cells results in the expression of a number of cytokines, such as interleukin-8 (IL-8), that are transcriptionally regulated by nuclear factor kappaB (NF-kappaB). In the studies reported here, we demonstrate that treatment of RSV-infected A549 cells with 100 microg of ribavirin (a viral replication inhibitor) per ml results in reversal of RSV-induced NF-kappaB activation, IL-8 mRNA expression, and IL-8 protein production in A549 cells. These data confirm that viral replication is a key step in RSV-induced NF-kappaB activation and IL-8 production.

Antiviral Agents↗
PubMed1996

Mechanism of RSV-induced IL-8 gene expression in A549 cells before viral replication.

Previous studies demonstrated that respiratory syncytial virus (RSV) infection of A549 cells induced interleukin (IL)-8 gene expression and protein release from the cells as early as 2 h after treatment [M. A. Fiedler, K. Wernke-Dollries, and J. M. Stark. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L865-L872, 1995; J. G. Mastronarde, M. M. Monick, and G. W. Hunninghake. Am. J. Respir. Cell Mol. Biol. 13: 237-244, 1995]. Furthermore, the effects of RSV at the 2-h time point were not dependent on viral replication. The studies reported here were designed to test the hypothesis that active and inactive RSV induce IL-8 gene expression in A549 cells at the 2-h time point by a mechanism dependent on the activation of the nuclear transcription factor NF-kappa B Northern blot analysis indicated that IL-8 gene expression occurred independent of protein synthesis 2 h after A549 cells were treated with RSV. Analysis of nuclear extracts from RSV-treated A549 cells by electrophoretic mobility shift assays demonstrated that NF-kappa B was activated as early as 15 min after RSV was added to the cells and remained activated for at least 90 min. In contrast, baseline levels of NF-IL-6 and activator protein-1 (AP-1) did not change over this period of time. Deoxyribonuclease footprint analysis of a portion of the 5'-flanking region of the IL-8 gene demonstrated two potential regions for transcription factor binding, which corresponded to the potential AP-1 binding site, and potential NF-IL-6 and NF-kappa B binding sites. Mutational analysis of the 200-bp 5'-untranslated region of the IL-8 gene demonstrated that activation of NF-kappa B and NF-IL-6 were required for RSV-induced transcriptional activation of the IL-8 gene.

DNA Mutational Analysis↗
PubMed1995

The ability of membrane potential dyes and calcafluor white to distinguish between viable and non-viable bacteria.

Various dyes were assessed for their ability to discriminate between viable and non-viable bacteria. Two methods of killing were employed: by heat treatment or by gramicidin treatment. Staining was carried out in two ways; by staining directly in the medium or by washing cells prior to staining in buffer. Carbocyanine and rhodamine 123 dyes only exhibited small changes in fluorescence between viable and non-viable populations of bacteria. Both oxonol dye (bis 1,3-dibutylbarbituric acid trimethine oxonol) and calcafluor white proved much more useful.

Bacterial Physiological Phenomena↗
PubMed1995

Mechanisms for soil moisture effects on activity of nitrifying bacteria.

Moisture may limit microbial activity in a wide range of environments including salt water, food, wood, biofilms, and soils. Low water availability can inhibit microbial activity by lowering intracellular water potential and thus reducing hydration and activity of enzymes. In solid matrices, low water content may also reduce microbial activity by restricting substrate supply. As pores within solid matrices drain and water films coating surfaces become thinner, diffusion path lengths become more tortuous, and the rate of substrate diffusion to microbial cells declines. We used two independent techniques to evaluate the relative importance of cytoplasmic dehydration versus diffusional limitations in controlling rates of nitrification in soil. Nitrification rates in shaken soil slurries, in which NH(inf4)(sup+) was maintained at high concentrations and osmotic potential was controlled by the addition of K(inf2)SO(inf4), were compared with rates in moist soil incubations, in which substrate supply was controlled by the addition of NH(inf3) gas. Comparison of results from these techniques demonstrated that diffusional limitation of substrate supply and adverse physiologic effects associated with cell dehydration can explain all of the decline in activity of nitrifying bacteria at low soil water content. However, the relative importance of substrate limitation and dehydration changes at different water potentials. For the soil-microbial system we worked with, substrate limitation was the major inhibiting factor when soil water potentials were greater than -0.6 MPa, whereas adverse physiological effects associated with cell dehydration were more inhibiting at water potentials of less than -0.6 MPa.

Journal Article↗
PubMed1995

Respiratory syncytial virus increases IL-8 gene expression and protein release in A549 cells.

The mechanism of respiratory syncytial virus (RSV)-induced inflammation in the airways of infants and children is not fully understood. We hypothesized that RSV directly induces interleukin (IL)-8 gene expression in airway epithelial cells, independent of IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) production. Exposure of A549 cells (an airway epithelial cell line) to RSV resulted in increased IL-8 mRNA expression and IL-8 protein release from the cells as early as 2 h after treatment. Neither IL-1 beta nor TNF-alpha (mRNA or protein) were detected. Viral replication was not necessary for the effects of RSV on IL-8 mRNA expression and protein release early in the infectious process. However, sustained levels of increased IL-8 production required RSV replication. A dose-response relationship was observed between the multiplicity of infection and IL-8 production with both active and nonreplicative RSV at the 2-h time point. Both active RSV and nonreplicative RSV increased the transcriptional activity of the 1.6-kb 5' flanking region of the IL-8 gene. Neither active RSV nor nonreplicative RSV increased the stability of the IL-8 mRNA in A549 cells. We conclude that RSV increases IL-8 gene expression in A549 cells in a biphasic pattern independent of viral replication early (2 h) but dependent on viral replication late (24 h).

Animals↗
PubMed1995

Leukotriene B4 formation: human neutrophil-airway epithelial cell interactions.

Leukotriene B4 (LTB4) is a potent inflammatory mediator involved in the pathogenesis of many pulmonary diseases. Although the neutrophil is the predominant source of LTB4, other cells can also interact with neutrophils and increase LTB4 formation. In this study, we investigated whether human neutrophil-airway epithelial cell interactions can increase LTB4 formation. Neutrophils were cocultured with transformed airway epithelial cells (9HTEo- cells), and LTB4 and leukotriene A4 (LTA4) degradation product release was measured by high-performance liquid chromatography and ultraviolet spectrometry. When stimulated with the calcium ionophore A-23187, neutrophil-9HTEo- cell cocultures released more LTB4 and less LTA4 degradation products in a time- and dose-related manner than did neutrophils alone. This increase in LTB4 release involved the metabolism of neutrophil-derived LTA4 to LTB4 by 9HTEo- cells and was affected by the neutrophil-to-epithelial cell ratio. Enhanced LTB4 release required proximity between neutrophils and 9HTEo- cells but not specific cell-cell adhesion. Our data demonstrate that human neutrophil-airway epithelial cell interactions can increase LTB4 formation through transcellular arachidonic acid metabolism.

Cell Adhesion↗
PubMed1995

Adhesion of activated eosinophils to respiratory epithelial cells is enhanced by tumor necrosis factor-alpha and interleukin-1 beta.

Eosinophilic infiltration and damage to airway epithelium are characteristic features of asthma. To assess possible interactions between eosinophils and airway epithelium, Percoll-purified human peripheral blood eosinophils were evaluated for their ability to adhere to respiratory epithelial cell (REC) cultures. REC (an immortalized cell line, A549, and primary bronchial epithelial cells) were grown in 96-well tissue culture plates, treated with proinflammatory cytokines (TNF-alpha or IL-1 beta), and eosinophil adhesion to these tissues was determined. Cytokine treatment of the REC cultures significantly increased expression of intercellular adhesion molecule-1 (ICAM-1) (P < 0.01). Eosinophils demonstrated a variable baseline adhesion to untreated REC which was then significantly increased following activation with phorbol myristate acetate (PMA) (P < 0.01). Furthermore, treatment of REC monolayers with TNF-alpha or IL-1 beta significantly increased adhesion of PMA-stimulated eosinophils (P < 0.01). To delineate the adhesion proteins involved in the cell-cell interactions, assays were performed in the presence of specific blocking monoclonal antibodies to eosinophil CD18, CD11a, or CD11b, and REC ICAM-1 molecules. Blocking antibodies to ICAM-1 had no significant effect on levels of eosinophil adhesion. In contrast, antibodies to CD18, CD11a, and CD11b significantly decreased (P < 0.01) eosinophil adhesion, thus demonstrating pivotal roles for the CD11/CD18 (beta 2) integrins, but not necessarily for ICAM-1, in interactions between the REC and eosinophils. These data demonstrate that TNF-alpha and IL-1 beta increase eosinophil adhesion to human respiratory epithelial cell cultures by induction of ligands recognized by eosinophil beta 2 integrins.

Asthma↗
PubMed1994

Rapid estimation of bacterial antibiotic susceptibility with flow cytometry.

Bacterial antibiotic susceptibility was rapidly estimated for Escherichia coli and Staphylococcus spp. by flow cytometry. This was achieved by measuring the uptake of a negatively charged membrane potential sensitive dye bis-(1,3-dibutyl-barbituric acid) trimethine oxonol and observing changes in low-angle light scatter (excitation light scattered by up to 15 degrees). Estimations of ampicillin, gentamicin and ciprofloxacin susceptibilities were possible within 2-5 h from a plate culture, depending on the species and antibiotic used. This includes the time necessary to establish steady-state growth in liquid culture.

Anti-Bacterial Agents↗
PubMed1993

Presence and possible significance of immunocytochemically demonstrable metallothionein over-expression in primary invasive ductal carcinoma of the breast.

Metallothioneins (MTs) are ubiquitous low-molecular-weight proteins with a high affinity for heavy metal ions such as zinc, copper and cadmium. MT over-expression has been associated with resistance against anticancer drugs. In the present study we investigated 86 cases (45 cases of tumour category pT1 and 41 of category pT2) of routinely fixed and paraffin-embedded primary breast carcinomas immunohistochemically with a monoclonal antibody to an epitope of MT shared by its I and II isoforms. Immunohistochemically demonstrated MT over-expression was found in the invasive components of 7 of 32 pT1 and 17 of 28 pT2 invasive ductal carcinomas, whereas all 26 invasive lobular carcinomas gave weak or negative results. Fourteen of 17 pT2 and 2 of 7 pT1 invasive ductal carcinomas with MT over-expression developed metastases during follow-up with poor prognostic outcome. In contrast only 3 of 11 pT2 and none of the 25 pT1 cases without MT over-expression had a poor clinical course (P < 0.001). It is concluded that MT over-expression is associated with significantly poor prognosis particularly in pT2 invasive ductal breast carcinomas.

Adult↗
PubMed1993

Lung infections in children.

Airway infections in children is a considerably broad topic. This discussion focuses on several common nonbacterial causes of lower respiratory tract infection in children, including respiratory syncytial virus, Mycoplasma pneumoniae, and Chlamydia pneumoniae. In addition, the occurrence of two important bacterial causes of lower respiratory illness (Bordetella pertussis and Mycobacterium tuberculosis) is increasing. This review focuses on current information on the prophylaxis, treatment, and diagnosis of these agents. Finally, consideration is given to infections in immunocompromised children: the effects of respiratory syncytial virus infections in immunosuppressed transplant patients, and prevention and diagnosis of opportunistic infections (including Pneumocystis carinii) in children with human immunodeficiency virus.

Bronchiolitis↗