Biomedical subjects

G Hecht

Publications and source records attributed to G Hecht.

At least 37 records · Page 2Linked to original sources

PubMed1999

Human colonic epithelial cells express galanin-1 receptors, which when activated cause Cl- secretion.

Galanin is a peptide hormone widely expressed in the central nervous system and gastrointestinal (GI) tract. Within the GI tract galanin is present in enteric nerve terminals where it is known to modulate intestinal motility by altering smooth muscle contraction. Recent studies also show that galanin can alter intestinal short-circuit current (Isc) but with differing results observed in rats, rabbits, guinea pigs, and pigs. In contrast, nothing is known about the ability of galanin to alter ion transport in human intestinal epithelial tissues. By RT-PCR, we determined that these tissues express only the galanin-1 receptor (Gal1-R) subtype. To evaluate Gal1-R pharmacology and physiology, we studied T84 cells. Gal1-R expressed by these cells bound galanin rapidly (half time 1-2 min) and with high affinity (inhibitor constant 0.7 +/- 0.2 nM). T84 cells were then studied in a modified Ussing chamber and alterations in Isc, a measure of all ion movement across the tissue, were determined. Maximal increases in Isc were observed in a concentration-dependent manner around 2 min after stimulation with peptide, with 1 microM galanin causing Isc to rise more than eightfold and return to baseline occurring within 10 min. The increase in galanin-induced Isc was shown by 125I efflux studies to be due to Cl- secretion, which occurred independently of alterations in cAMP and phospholipase C. Rather, Cl- secretion is mediated via a Ca2+-dependent, pertussis toxin-sensitive mechanism. These data suggest that galanin released by enteric nerves may act as a secretagogue in the human colon by activating Gal1-R.

Calcium↗

PubMed1999

Enteropathogenic E. coli attenuates secretagogue-induced net intestinal ion transport but not Cl- secretion.

Enteric bacterial pathogens often increase intestinal Cl- secretion. Enteropathogenic Escherichia coli (EPEC) does not stimulate active ion secretion. In fact, EPEC infection decreases net ion transport in response to classic secretagogues. This has been presumed to reflect diminished Cl- secretion. The aim of this study was to investigate the influence of EPEC infection on specific intestinal epithelial ion transport processes. T84 cell monolayers infected with EPEC were used for these studies. EPEC infection significantly decreased short-circuit current (Isc) in response to carbachol and forskolin, yet 125I efflux studies revealed no difference in Cl- channel activity. There was also no alteration in basolateral K+ channel or Na+-K+-2Cl- cotransport activity. Furthermore, net 36Cl- flux was not decreased by EPEC. No alterations in either K+ or Na+ transport could be demonstrated. Instead, removal of basolateral bicarbonate from uninfected monolayers yielded an Isc response approximating that observed with EPEC infection, whereas bicarbonate removal from EPEC-infected monolayers further diminished Isc. These studies suggest that the reduction in stimulated Isc is not secondary to diminished Cl- secretion. Alternatively, bicarbonate-dependent transport processes appear to be perturbed.

Absorption↗

PubMed1999

Pathogenic Escherichia coli increase Cl- secretion from intestinal epithelia by upregulating galanin-1 receptor expression.

Galanin is widely distributed in enteric nerve terminals lining the human gastrointestinal (GI) tract. We have shown previously that galanin-1 receptors (Gal1-R) are expressed by epithelial cells lining the human GI tract, and upon activation cause Cl- secretion. Because expression of this receptor is transcriptionally regulated by nuclear factor-kappa B (NF-kappa B), which is activated by enteric pathogens as a part of the host epithelial response to infection, we investigated whether such bacterial pathogens could directly increase Gal1-R expression in the T84-cell model system. Pathogenic Escherichia coli, but not nonpathogenic E. coli, activate a p50/p65 NF-kappa B complex that binds to oligonucleotides corresponding to a recognition site located within the 5' flanking region of the human GAL1R gene. Pathogenic E. coli, but not normal commensal organisms, increase Gal1-R mRNA synthesis and [(125)I]galanin binding sites. Whereas galanin increases short-circuit current (Isc) approximately 5-fold in uninfected T84 cells, exposure to pathogenic, but not nonpathogenic, E. coli results in galanin increasing Isc approximately 20-fold. To confirm the validity of these in vitro observations, we also studied C57BL/6J mice infected with enterohemorrhagic E. coli (EHEC) by gavage. Infection caused a progressive increase in both NF-kappa B activation and Gal1-R expression, with maximal levels of both observed 3 days after gavage. Ussing chamber studies revealed that colons infected with EHEC, but not those exposed to normal colonic flora, markedly increased Isc in response to galanin. These data indicate that pathogen-induced increases in Gal1-R expression by epithelial cells lining the colon may represent a novel unifying pathway responsible for at least a portion of the excessive fluid secretion observed during infectious diarrhea.

Animals↗

PubMed1998

Galanin causes Cl- secretion in the human colon. Potential significance of inflammation-associated NF-kappa B activation on galanin-1 receptor expression and function.

Galanin is widely distributed in enteric nerves and nerve terminals throughout the gastrointestinal (GI) tract. Within the GI tract galanin is best known for its ability to alter smooth muscle contractility and regulate intestinal motility. However, recent studies also indicate that galanin can modulate epithelial ion transport. We previously showed that epithelial cells lining the human GI tract, including those of colonic origin, express Gal1 galanin receptors (Gal1-R). We herein demonstrate that epithelial cells lining the human colon only express Gal1-R receptors and do not express other galanin receptor subtypes. We previously showed that Gal1-R expression was transcriptionally regulated by the transcription factor NF-kappa B. Consistent with this transcription factor being activated in a number of inflammatory conditions, we show increased colonic Gal1-R expression in patients with colitis due to a variety of causes. To further evaluate the physiology of Gal1-R activation, we studied this receptor expressed by the human colon epithelial cell line T84. Gal1-R activation resulted in a dose-dependent increase in Cl- secretion; whereas infection of T84 cells with pathogens known to activate NF-kappa B augmented Gal1-R expression and Cl- secretion. Thus, galanin acts as a secretagogue in epithelial cells lining the human colon, with alterations in Gal1-R expression possibly playing an important role in the diarrhea associated with various inflammatory processes affecting the GI tract.

Base Sequence↗

PubMed1998

Exposure to chloramines in a green salad processing plant.

Acute eye and upper respiratory irritation was reported at an industrial facility that processes green salads in water containing hypochlorite. The irritant agents were chloramines resulting from the reaction of hypochlorite and nitrogen compounds coming from the sap proteins released when the vegetables were cut. Specific sampling and analytical methods were developed in order to assess the workers' exposure. Monochloramine, dichloramine and nitrogen trichloride were found in the atmosphere, which can explain the irritation phenomena of which the workers complained. In the washing room (where salads are processed), the total chloramine concentration ranged from 0.4 to 16 mg.m-3, depending on the day and the location of the area sampling points. The exposure of workers determined by personal sampling ranged from 0.2 to 5 mg.m-3. In a previous study, with swimming pool instructors exposed to the same pollutants, irritation phenomena generally appeared at chloramine values of around 0.5 mg.m-3.

Chloramines↗

PubMed1998

Electrophysiological characterization of human distal colon epithelium isolated using a novel technique.

Electrophysiological studies of human colonic epithelia traditionally have been hampered by the lack of tissue availability and by poor tissue quality. Human colonic epithelium is usually obtained surgically from individuals with underlying disease, while surgery itself can injure or alter the resected tissue. As a result, a wide range in electrophysiological parameters is reported in previous studies of human colonic epithelium. Such factors may also account for differences in measurements between humans and the few other species studied. We therefore devised a novel and rapid endoscopic technique, endoscopic mucosal resection (EMR), that allows for the removal and study of intestinal mucosal epithelium from normal volunteers. Using EMR we rapidly (7.2+/-2.4 min) isolated surgical-sized epithelial sheets from the distal colon (1.4+/-0.4 by 1.3+/-0.4 cm) that were readily mounted in a 0.64-cm2 Ussing chamber. We observed stable resistance (289+/-30 omega cm2), potential difference (1.6+/-0.6 mV), and I(SC)(24+/-9 microA/cm2) for at least 90 min, after which all experiments were terminated. Exposure to carbachol increased I(SC)2.2+/-0.5-fold, while forskolin increased I(SC) 4.4+/-0.5-fold. These data show that the electrophysiological characteristics of the human distal colon removed by EMR more closely approximate values reported for other mammals than when removed using other techniques. Thus EMR represents a significant advance over traditional techniques for isolating human tissues and will increase the availability of this tissue for future studies.

Aged↗

PubMed1997

Enteropathogenic Escherichia coli-induced myosin light chain phosphorylation alters intestinal epithelial permeability.

BACKGROUND & AIMS: Infection of epithelial cells with enteropathogenic Escherichia coli (EPEC) induces phosphorylation of the 20-kilodalton myosin light chain (MLC20). The physiological consequence of this biochemical observation, however, has not been discerned. The aim of this study was to determine if EPEC-induced phosphorylation of MLC20 was involved in the associated perturbation of intestinal epithelial barrier function. METHODS: Cultured intestinal epithelial cells, T84, were infected with EPEC. The effects of protein kinase inhibitors on EPEC-induced perturbation of barrier function were assessed using electrophysiological techniques. Alterations in MLC20 phosphorylation were correlated with functional responses. RESULTS: Inhibition of myosin light chain kinase, but not protein kinase C or tyrosine kinase, prevented the decrease in resistance caused by EPEC infection and significantly diminished EPEC-induced MLC20 phosphorylation. Epithelial cell monolayers genetically manipulated to constitutively increase MLC20 phosphorylation were relatively resistant to the effects of EPEC on barrier function. CONCLUSIONS: For the first time, these data show that a physiological consequence of the long-recognized increase in MLC20 phosphorylation by EPEC is perturbation of intestinal epithelial barrier function, which probably contributes to the diarrhea associated with this infection.

Bacterial Adhesion↗

PubMed1997

The toxin of diarrheic shellfish poisoning, okadaic acid, increases intestinal epithelial paracellular permeability.

BACKGROUND & AIMS: Diarrhea associated with shellfish poisoning is poorly understood. The responsible toxin, dinophysistoxin 1, has been identified as okadaic acid, a potent phosphatase inhibitor, but its effects on intestinal epithelia have not been examined. The aim of this study was to investigate the effect of okadaic acid on intestinal epithelial function, both Cl- secretion and barrier function. METHODS: Cultured human intestinal epithelial T84 cell monolayers were used. The effect of okadaic acid on these monolayers was assessed by measuring electrophysiological parameters, lactate dehydrogenase release, and 22Na+ and [3H]mannitol flux rates. Protein phosphorylation studies were performed to identify potentially involved proteins. RESULTS: Okadaic acid does not directly stimulate Cl- secretion from intestinal epithelial cells. On the contrary, the response to well-characterized secretagogues is attenuated by okadaic acid. However, it does decrease transepithelial electrical resistance in a polarized fashion without inducing cytotoxicity. Sodium-mannitol flux studies suggest that the observed decrease in resistance is attributable to an increase in paracellular permeability. CONCLUSIONS: Okadaic acid, the toxin responsible for diarrheic shellfish poisoning, does not stimulate Cl- secretion but increases the paracellular permeability of intestinal epithelia. This alteration in intestinal epithelial physiology may contribute to the diarrhea of shellfish poisoning.

Cell Membrane Permeability↗

PubMed1997

Review article: Effector role of epithelia in inflammation--interaction with bacteria.

The common response to infection is infiltration of the affected tissue by inflammatory cells. It is now recognized that the epithelium plays a crucial role in this immunological process by producing an array of proinflammatory cytokines including interleukin-8, tumour necrosis factor-alpha, monocyte chemotactic protein-1, granulocyte-macrophage colony-stimulating factor, extractable nuclear antigen-78 and others. The response of the intestinal epithelium to bacterial pathogens is particularly intriguing because it is literally bathed by normal bacterial flora and bacterial components/products yet remains immunologically quiescent despite this potentially hostile environment. In contrast, when challenged by bacterial pathogens, intestinal epithelial cells exhibit a vigorous immunological response. Our laboratory has, therefore, focused on the immune response of intestinal epithelial cells when confronted by a specific bacterial pathogen, enteropathogenic Escherichia coli.

Antigens, Nuclear↗

PubMed1997

Activation of NF-kappaB in intestinal epithelial cells by enteropathogenic Escherichia coli.

The initial response to infection is recruitment of acute inflammatory cells to the involved site. Interleukin (IL)-8 is the prototypical effector molecule for this process. Transcription of the IL-8 gene is primarily governed by the nuclear transcription factor (NF)-kappaB. Intestinal epithelial cells produce IL-8 in response to infection by enteric pathogens yet remain quiescent in a milieu where they are literally bathed in normal bacterial flora. We therefore sought to investigate NF-kappaB activation in response to enteropathogenic Escherichia coli (EPEC), nonpathogenic E. coli, and bacterial lipopolysaccharide in an intestinal epithelial cell (T84) model and to determine whether EPEC-induced activation of NF-kappaB factor is causally linked to IL-8 production. We report herein that NF-kappaB is activated by EPEC, yet such a response is not extended to nonpathogenic organisms or purified E. coli lipopolysaccharide. Transcription factor decoys significantly diminished IL-8 production in response to EPEC, demonstrating a causal relationship. Furthermore, deletion of specific EPEC virulence genes abrogates the NF-kappaB-activating property of this pathogen, suggesting that specific bacterial factors are crucial for inducing this response. These studies show for the first time that infection of intestinal epithelial cells with EPEC activates NF-kappaB, which in turn initiates IL-8 transcription, and highlight the differential response of these cells to bacterial pathogens vs. nonpathogens.

Base Sequence↗

PubMed1996

Exposure of contractors to chemical pollutants during the maintenance shut-down of a chemical plant.

The exposure of employees of different contracting firms to chemical pollutants was assessed during the five-week biennial maintenance shut-down of an isocyanate (TDI) synthesis plant. This assessment was mainly based on personal sampling, but work-related constraints occasionally required that area samples be used instead. Many tasks were carried out during the shut-down procedure (dismantling of reactors, installation of insulation, refection of kilns, cleaning, painting, electricity, etc.), thereby causing the employees of the contracting firms to be exposed to different kinds of pollutants, including products used or manufactured in the process (solvents, TDI and its synthesis intermediates), and products specific to the contractors' activity (welding fumes, crystalline silica, mineral fibers of heat insulation). The highest level of exposure (mainly to TDI) was found for the mechanical engineering activities undertaken during the dismantling of reactors and for cleaning activities. It was demonstrated that there was much variability in day-to-day exposure levels, and occasionally in the exposure levels encountered during a given shift. Cases of concomitant exposure to TDI (mainly short-term exposures) and other pollutants due to simultaneous activities from different tasks in the same area (co-activity) were also observed. Both the need for, and limitations of personal protective equipment (mainly respiratory) are stressed. Special emphasis is put on the responsibility of the owner of the facilities in helping the contractors in the organization of a health and safety policy.

Air Pollutants, Occupational↗

PubMed1996

Attachment of a noninvasive enteric pathogen, enteropathogenic Escherichia coli, to cultured human intestinal epithelial monolayers induces transmigration of neutrophils.

An intense inflammatory cell infiltrate, consisting primarily of polymorphonuclear leukocytes (PMN), accompanies enteric infection by enteropathogenic Escherichia coli (EPEC). The mechanism(s) by which this pathogen elicits PMN recruitment has not been studied. To determine whether EPEC infection of intestinal epithelial cells induces PMN to transmigrate, an in vitro model consisting of cultured human intestinal epithelial monolayers (T84), a human EPEC strain (E2348/69), and isolated human PMN was used. Results of these studies showed that EPEC attachment to T84 monolayers stimulated the transepithelial migration of PMN in a dose-dependent fashion. This event was not attributable to the classic bacterial chemoattractants, n-formylated peptides, or other soluble bacterial factors. A nonadherent EPEC strain, JPN15, was unable to cause PMN to cross the epithelial monolayer. Epithelial protein synthesis was required for maximum EPEC-induced PMN transmigration to occur. Transfer assays demonstrated the presence of a chemokine in sterilized medium from infected monolayers. Neutralizing antibodies to interleukin 8 ablated approximately 50% of the chemotactic activity. Studies with EPEC mutant strains revealed that the eaeB gene, required for the activation of signal transduction pathways in host cells, was crucial for eliciting PMN transmigration. These data show for the first time that attachment of a noninvasive enteric pathogen to intestinal epithelial cells induces PMN transmigration. These findings strongly suggest that EPEC attachment to target host cells activates a signal transduction cascade which ultimately leads to the expression and release of an epithelium-derived chemotactic factor(s) for PMN.

Bacterial Adhesion↗

PubMed1996

Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability.

Contractile events resulting from phosphorylation of the 20-kDa myosin light chain (MLC20) have been implicated in the regulation of epithelial tight junction permeability. To address this question, Madin-Darby canine kidney cells were transfected with a murine leukemia retroviral vector containing DNA encoding either the catalytic domain of myosin light chain kinase (tMK) or the beta-galactosidase gene (beta-gal). Autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of myosin immunoprecipitated from 32Pi-labeled transfected cells demonstrated that MLC20 phosphorylation was increased 3.1 +/- 0.9-fold in cells expressing tMK compared with cells expressing beta-gal. Phosphopeptide mapping confirmed that myosin light chain kinase was responsible for the increased MLC20 phosphorylation. Transepithelial electrical resistance, a measurement of barrier function, of tMK cell monolayers was consistently < 10% (123 +/- 20 omega.cm2) of that of monolayers comprised of wild-type cells (1,456 +/- 178 omega.cm2) or cells expressing beta-gal (1,452 +/- 174 omega.cm2). Dual 22Na+ and [3H]mannitol flux studies indicated that the decrease in resistance in tMK cells was attributable to increased paracellular flow. These data support the idea that MLC20 phosphorylation by myosin light chain kinase is involved in regulating epithelial tight junction permeability.

Animals↗

PubMed1995

Enteropathogenic Escherichia coli adherence to intestinal epithelial monolayers diminishes barrier function.

The mechanism by which enteropathogenic Escherichia coli (EPEC) causes diarrhea remains elusive. Several alterations within the host cell have been demonstrated to occur following EPEC attachment including increases in intracellular Ca2+ concentration and rearrangement and phosphorylation of several cytoskeletal proteins. The consequences of these intracellular perturbations on host cell function, however, have not been determined. The aim of this study was to examine the effect of EPEC adherence on intestinal epithelial barrier function. T84 cell monolayers were infected with either wild-type EPEC or a nonadherent isogenic derivative. Transepithelial electrical resistance, a measure of barrier function, decreased 33.5 +/- 6.4% after a 6-h incubation with the wild-type strain. Electron microscopy revealed ultrastructurally normal cells, and lactate dehydrogenase release assays failed to demonstrate cytotoxicity. Dual 22Na+ and [3H]mannitol flux studies localized the permeability defect to tight junctions. In addition, cumulative flux of the paracellular marker mannitol was four- to fivefold greater across monolayers infected with wild-type EPEC. Sequestration of intracellular calcium stores by dantrolene completely abrogated the resistance drop associated with EPEC attachment. These data demonstrate that adherence of EPEC to intestinal epithelial cell monolayers disrupts tight junction barrier function via a calcium-requiring event.

Bacterial Adhesion↗

PubMed1994

The effect of dexamethasone administration on rat intestinal permeability: the role of bacterial adherence.

BACKGROUND/AIMS: Previous studies have shown that dexamethasone administration to rats results in diminution of biliary secretory immunoglobulin A concentration and alters intestinal barrier function to bacteria. The aims of this study were to examine and characterize the effect of dexamethasone on intestinal permeability and to determine the possible influence of bacterial adherence to the mucosa in this process. METHODS: Groups of adult Fisher rats were studied, and the effects of dexamethasone administration and bowel decontamination on bacterial adherence and intestinal permeability were determined in various bowel segments. RESULTS: Dexamethasone administration was associated with a significant increase in bacterial adherence to the mucosa, which was most pronounced in the cecum (4.33 +/- 0.2 colony forming units (cfu) (log10) per gram of mucosa for control vs. 6.23 +/- 0.2 for dexamethasone). This was accompanied by significant alterations in intestinal permeability as measured by a decrease in transepithelial electrical resistance (49.5 +/- 4.1 omega.cm2 for control vs. 25.4 +/- 2.5 omega.cm2 for dexamethasone). Dual sodium-mannitol flux analysis showed this defect to be predominantly paracellular. Antibiotic decontamination of the intestine completely abrogated the intestinal permeability defect observed in this model. CONCLUSIONS: Bacterial-mucosal cell interactions may be responsible for alterations in intestinal permeability after dexamethasone administration.

Animals↗

PubMed1994

Exposure to metallic catalyst dust: manufacturing and handling of catalysts in the chemical industry.

The exposure of workers to metallic catalyst dust was assessed in manufacturing and reprocessing industries, and during catalyst handling in the chemical industry. The level of exposure was found to be generally low during the manufacturing and reprocessing activities taking place in permanent installations, and extremely variable during catalyst handling performed by workers of sub-contracting specialized firms. The difficulties in treating the occupational hygiene problems of these workers are pointed out.

Catalysis↗

PubMed1994

Reversible disassembly of an intestinal epithelial monolayer by prolonged exposure to phorbol ester.

This article describes a model of reversible disassembly of a cultured human intestinal epithelial monolayer by prolonged exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Prolonged phorbol ester exposure reduces protein kinase C (PKC) levels in numerous cell types including T84, as shown here. Under PKC-downregulated conditions, T84 monolayers, which simulate the highly organized structure of native intestinal crypt cells, become disassembled into 2 or 3 layers of rounded cells. Proliferation does not account for these morphological changes as assessed by thymidine incorporation studies. The effects of structural disorganization on epithelial barrier function was also examined. The permeability of disassembled monolayers was significantly greater than that of controls. Flux studies localized the permeability defect to the tight junction. PKC-associated alterations in the perijunctional ring of actin and myosin, one of the putative regulators of flow across the tight junction, were found to correlate with the observed functional changes. Most interesting was the fact that monolayer reassembly to the original columnar epithelial phenotype and reestablishment of barrier function occurred upon normalization of PKC levels. This model of reversible monolayer disassembly will allow investigation into the relationship between epithelial structure and function and examination of factors that govern monolayer formation.

Actins↗

PubMed1994

Causes and consequences of bacterial adherence to mucosal epithelia during critical illness.

The interaction between host and pathogen during catabolic stress represents a dynamic battlefield, where the microbe's strategy to ensure its survival and multiplication come face to face with the repertoire of the host's immune system. From the microbe's point of view, the ICU offers the least optimal living condition, representing at times an outright holocaust for entire populations of fellow bacteria. This situation is especially true for intestinal bacteria, which can be exposed to all sorts of extreme conditions, such as starvation, turgor pressure, altered temperature, antibiotics, and osmolality changes. These conditions may act as environmental cues and elicit changes in bacterial gene expression that lead to effective coping responses. Starvation countermeasures can be especially strong signals for bacteria to follow chemical trails in search of nutrients. Harming the host is not the microbe's intent; its goal is to prevail. Injury to the host by a microbe struggling to survive is the inadvertent consequence of a threatening environment. This struggle between host and pathogen is particularly apparent at the mucosal surface, especially in the gastrointestinal tract, where microbes have enjoyed the leisurely life of a stable temperature and a constant food supply. The focus of this discussion is on one small aspect of the manner in which microbes ensure their survival during catabolic stress by attaching to host epithelial cells. The conditions and consequences of this process are discussed.

Anti-Bacterial Agents↗