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Biomedical subjects

L C Rogers

Publications and source records attributed to L C Rogers.

13 recordsLinked to original sources
PubMed1995

Correction of the cystic fibrosis defect by gene complementation in human intrahepatic biliary epithelial cell lines.

BACKGROUND/AIMS: Hepatobiliary disease is the second most common cause of mortality in patients with cystic fibrosis (CF). In the liver, only the intrahepatic biliary epithelial (IBE) cells express cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The aim of this study was to determine whether human CF-derived IBE cells can be infected with adenovirus and the CF phenotype complemented. METHODS: IBE cells were isolated from 2 patients with CF and immortalized using retrovirus transduction of SV40 large T antigen. Immortalized cells were infected with the adenovirus vector Ad2/CFTR2 and assayed 2-31 days postinfection for cyclic adenosine monophosphate (cAMP)-induced halide efflux. Halide efflux was measured in single cells using fluorescence microscopy and the fluorescent probe 6-methoxy-N-(3-sulfopropyl)-quinolinium. RESULTS: CF-derived IBE cell lines express biliary specific markers and express no cAMP-inducible halide efflux. Following infection with the adenovirus vector Ad2/CFTR2, a cAMP-induced halide efflux was observed for 31 days, although the number of responsive cells decreased with time. CONCLUSIONS: Human CF-IBE cells can be infected by adenovirus and the defective CFTR complemented. The loss of responsive cells with time could be due to loss of construct and/or a reduced growth of cells that are overexpressing CFTR. These CF-IBE cell lines offer an opportunity to determine the mechanisms responsible for hepatobiliary disease in the patients with CF.

3T3 Cells↗
PubMed1995

Purinoceptor P2U identification and function in human intrahepatic biliary epithelial cell lines.

The mechanisms that regulate ion and fluid transport by the human intrahepatic bile duct have not been well defined. Human intrahepatic biliary cell lines that we have developed were used to identify and characterize purinoceptors based on increases in intracellular calcium in response to ATP and other nucleotides. Intracellular free calcium was measured in cell suspensions using the fluorescent probe Fura-2 and a fluorescence spectrophotometer. Halide efflux was measured in single cells using fluorescence microscopy and the fluorescent probe SPQ. Intracellular calcium increases equivalently in response to ATP and UTP, peaking, then diminishing to a new, elevated baseline. The peak elevation of calcium is the result of both the release of intracellular stores of calcium and the influx of extracellular calcium. The purinoceptor P2U-subtype was identified based on the potency rank order of ATP-analogues. Halide efflux increases with P2U-purinoceptor stimulation which is consistent with the opening of a Ca(2+)-sensitive Cl- channel. The physiological significance of P2U-purinoceptor activation and its effect on the ionic content and flow rate of bile remains to be determined.

Adenosine Triphosphate↗
PubMed1995

Continuous epithelial cell lines from ADPKD liver cysts exhibit characteristics of intrahepatic biliary epithelium.

We have produced continuous cell lines using retroviral transduction of SV40 large T antigen into epithelial cells removed from the lumen of liver cysts from four female patients with autosomal dominant polycystic kidney disease (ADPKD). Liver cyst-derived epithelial (LCDE) cell lines are grown in a hormonally supplemented medium in the presence of lethally irradiated NIH/3T3 fibroblast coculture. LCDE cells maintain their epithelial appearance and are positive for the biliary-specific markers cytokeratin 7 and 19 and gamma-glutamyl transpeptidase while being negative for hepatocyte markers. SV40 large T antigen is localized to the cell nucleus. LCDE cells have been grown continuously for periods exceeding 12 mo and 25 passages (170 population doublings). LCDE cells exhibit intracellular pH regulatory pathways that, with one exception, are similar to those found in normal intrahepatic biliary epithelium. These LCDE cell lines exhibit impaired alkalinization in response to Cl- substitution. This finding is suggestive of decreased function or abundance of a Cl-/HCO3- anion exchanger and could account for the failure of ADPKD hepatic cysts to secrete HCO3- in response to secretin.

3T3 Cells↗
PubMed1994

Anthrax edema toxin differentially regulates lipopolysaccharide-induced monocyte production of tumor necrosis factor alpha and interleukin-6 by increasing intracellular cyclic AMP.

Bacillus anthracis exotoxins mediate most of the symptomatology of severe anthrax. In addition to a clinical syndrome reminiscent of septic shock, which may be mediated by cytokines produced by macrophages stimulated with lethal toxin, infected patients show profound edema at sites of infection. Edema is mediated by edema toxin (ET), which comprises of a binding molecule, protective antigen, and an active moiety, edema factor, which possesses intrinsic adenylyl cyclase activity. Intracellular cyclic AMP (cAMP) regulates the production of several cytokines that modulate edema formation and play important roles in host defense against invading bacteria. To determine whether ET enhanced the accumulation of cAMP in monocytes and thereby influenced cytokine production, we cultured human monocytes with endotoxin (lipopolysaccharide [LPS]) and dilutions of ET and determined the levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) in culture supernatant fluids. We further estimated cytokine-specific mRNA accumulation in monocytes by reverse transcription PCR and examined intracellular cAMP concentrations following treatment with ET. ET and LPS each induced monocytes to secrete comparable amounts of IL-6. ET did not inhibit and in most experiments modestly enhanced LPS-induced IL-6 production. In contrast to this stimulatory effect on IL-6 production, ET induced little or no TNF-alpha production. Moreover, ET profoundly inhibited LPS-induced TNF-alpha synthesis. These regulatory phenomena were also observed at the mRNA level in association with dose-related enhancement of intracellular cAMP in ET-treated monocytes. Monocytes treated with dibutyryl cAMP, an active analog of cAMP, produced cytokines in a pattern identical to that of cells treated with ET. The disruption of cytokine networks as a consequence of unregulated, ET-induced cAMP accumulation in human monocytes may impair cellular antimicrobial responses and contribute to clinical signs and symptoms.

Antigens, Bacterial↗
PubMed1994

Regulation of intracellular pH by immortalized human intrahepatic biliary epithelial cell lines.

We have produced continuous cell lines using retroviral transduction of SV40 large T antigen into human intrahepatic biliary epithelial (IBE) cells from three different normal individuals. These IBE cell lines grow in a hormone-supplemented medium in the presence of NIH/3T3 fibroblast coculture. These cells maintain their epithelial appearance and are positive for the biliary-specific markers cytokeratins 7 and 19 and gamma-glutamyl transpeptidase while being negative for the hepatocyte markers albumin and asialoglycoprotein receptor. To evaluate ion transport pathways in IBE cell lines, we utilized intracellular pH (pHi) measurements obtained using the intracellular fluorescent indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. In the absence of HCO3(-)-CO2, an amiloride-sensitive Na(+)-H+ exchanger participated in the regulation of basal pHi. In the presence of HCO3(-)-CO2, a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive, Na-, Cl-, and HCO3(-)-dependent acid extrusion mechanism accounted for approximately 60% of pHi recovery from acidic pHi; this mechanism is most consistent with the presence of a Na-dependent Cl-HCO3- exchanger (Na+HCO3(-)-Cl-H+). Under basal conditions, Cl- depletion revealed a DIDS-sensitive alkalinization consistent with a Na-independent Cl(-)-HCO3- exchanger. These model systems will allow the opportunity to study the normal mechanisms of IBE function and to study the pathobiology of IBE processes in disease states.

3T3 Cells↗
PubMed1993

Pasteurella multocida pneumonia in a man with AIDS and nontraumatic feline exposure.

A case of acute pneumonia due to Pasteurella multocida ssp multocida occurred in a young man with AIDS and chronic sinusitis. The pneumonia was diagnosed by bronchoscopy and responded to treatment with aztreonam. Epidemiologic investigation revealed the case was temporally related to nontraumatic exposure to cat secretions that the patient presumably had acquired via an aerosol. The cat's oral cavity was cultured and an isolate of P multocida ssp multocida with identical biochemical reactions, DNA restriction patterns, and nearly identical fatty acid profile to that of the patient's isolate was obtained suggesting they were identical strains and therefore epidemiologically linked. A control strain with identical biochemical reactions and antibiotic sensitivities exhibited different patterns. To our knowledge, this is the first such reported infection in a patient infected with human immunodeficiency virus.

AIDS-Related Opportunistic Infections↗
PubMed1986

The Stratus immunofluorometric assay system evaluated for quantifying human choriogonadotropin in serum.

We evaluated the Stratus (American Dade, Miami, FL), an automated immunofluorometric assay system, for the quantification of human choriogonadotropin (hCG) in serum or plasma. The assay is based on the "sandwich" (two-site) immunoassay methodology: use of two monoclonal antibodies, one specific for the alpha subunit and the other for the beta subunit, results in an assay that is specific for the intact hCG molecule. Results for the first sample are obtained in 7 min; subsequent additional values are produced at 1-min intervals. Inter-run precision (CV), estimated from replicate determinations of sera, was 4.5% at an hCG concentration of 38 int. units/L, 4.9% at 114, and 6.1% at 194. Intrarun CV was less than 2% at all three concentrations. Correlations of results for 127 specimens analyzed in duplicate with the Stratus (y) and by a radioimmunoassay (x) for beta hCG (Gamma Dab M [cf931125I] beta-hCG, Travenol-Genentech Diagnostics, Cambridge, MA) yielded the following regression equation: y = 0.969x - 6.0 (r = 0.995). The Stratus immunofluorometric system provides a rapid and convenient assay of hCG in serum or plasma.

Antibodies, Monoclonal↗
PubMed1985

Atrial natriuretic factor inhibits dehydration- and angiotensin II-induced water intake in the conscious, unrestrained rat.

Peptides isolated from atrial extracts possess potent natriuretic and diuretic activities. In general, these peptides, called atrial natriuretic factors (ANFs), oppose the actions of the water-conservatory peptides angiotensin II and vasopressin and are released from the heart in response to atrial stretch as a consequence of increased venous return. The recent description of ANF-like immunoreactivity in brain regions associated with the control of water intake suggested a role for these peptides in the neurogenic mechanisms of thirst. Intracerebroventricular (third ventricle) infusion of 1.0 or 2.0 nmol of ANF in conscious, overnight-dehydrated rats significantly inhibited subsequent water intake over a 2-hr test period. Intravenous infusion of 2.0 nmol, but not 1.0 nmol, of ANF resulted in a similar inhibitory action, suggesting that ANF released from the heart might act centrally to inhibit water intake by an action at one or more of the circumventricular organs. Water intake induced by central infusion of angiotensin II (9.6 and 25 pmol) in normally hydrated rats was significantly inhibited by prior infusion of 2.0 nmol of ANF. Water intake induced by higher doses of angiotensin II was not altered significantly by prior infusion of ANF. These results indicate a possible physiologic role for ANF in the hypothalamic control of water intake and reveal that the cardiac peptides can act centrally, as well as peripherally, to assist in the normalization of extracellular fluid volume.

Angiotensin II↗