Biomedical subjects

A Abouhamze

Publications and source records attributed to A Abouhamze.

9 recordsLinked to original sources

PubMed1998

Enhanced collagen accumulation following direct transfection of the inducible nitric oxide synthase gene in cutaneous wounds.

Inducible nitric oxide synthase (iNOS) is expressed during cutaneous wound repair. Mounting evidence suggests that wound nitric oxide (NO) augments collagen accumulation. We hypothesized that in vivo transfection of wound cells with the iNOS gene would increase physiological wound NO levels and thus augment collagen accumulation. Polyvinyl alcohol sponges were instilled with a mammalian expression plasmid (pMP6) containing either the chloramphenicol acetyl transferase (CAT) reporter or murine iNOS gene driven by a CMV immediate-early promoter. Plasmid DNA was injected alone or in complex with cationic liposomes, and the sponges were placed subcutaneously in male Sprague-Dawley rats which had received a longitudinal dorsal midline incision. Animals were sacrificed at different time points post-wounding and the sponges assayed for CAT activity, transfected iNOS mRNA, total nitrate and nitrite concentration (NOx) (as an index of wound NO synthesis), and hydroxyproline content (as an index of sponge collagen accumulation). The results demonstrate that wound cells were more efficiently transfected by naked DNA than by liposome mediated transfection and that maximal expression of both iNOS and CAT occurred at 48 hrs with a rapid decline after this time point. After 7 days, iNOS transfected sponges had accumulated significantly more collagen than those transfected with CAT. We conclude that cutaneous wounds can be successfully transfected by direct injection of naked DNA and that increased iNOS expression precedes an increase in collagen synthesis.

Animals↗

PubMed1998

Leptin produces anorexia and weight loss without inducing an acute phase response or protein wasting.

The ob gene product leptin is known to produce anorexia and loss of body fat when chronically administered to both lean and genetically obese mice. The current study was undertaken to examine whether administration of recombinant leptin in quantities sufficient to produce decreases in food intake and body weight and alterations in body composition would elicit either an hepatic acute phase protein response or preferential loss of carcass lean tissue. Mice were administered increasing quantities of recombinant human leptin or human tumor necrosis factor-alpha as a positive control. Although leptin (at 10 mg/kg body wt) produced significant anorexia and weight loss (both P < 0.05), human leptin administration did not appear to induce an hepatic acute phase protein response in either lean or genetically obese mice, as determined by protein synthetic rates in the liver or changes in the plasma concentration of the murine acute phase protein reactants, amyloid A, amyloid P, or seromucoid (alpha1-acid glycoprotein). In addition, human leptin administration did not induce a loss of fat-free dry mass (protein) in lean or obese animals. The findings suggest that at doses adequate to alter food intake and body weight leptin is not a significant inducer of the hepatic acute phase response nor does leptin promote the preferential loss of somatic protein characteristic of a chronic inflammatory process.

Acute-Phase Reaction↗

PubMed1998

Pharmacokinetics, immunogenicity, and efficacy of dimeric TNFR binding proteins in healthy and bacteremic baboon.

Immunogenicity, pharmacokinetics, and therapeutic efficacy of three novel dimeric soluble tumor necrosis factor (TNF)-receptor I constructs [TNF-binding protein (bp)] were evaluated in 28 baboons, 12 of which were healthy and 16 were challenged with a lethal Escherichia coli bacteremia. The three constructs differed only in the number of extracellular domains of the TNF receptor I and were dimerized with polyethylene glycol. Although all three constructs had generally similar pharmacokinetics when administered to a naive animal, they differed quantitatively in their immunogenicity. Antibodies were detected more frequently, and titers were significantly higher (P < 0.05) in both healthy and septic baboons that received the 4.0-domain TNF-bp construct, compared with animals receiving the 2.6-domain construct. When the TNF-bp constructs were administered a second time (21 days later), the half-lives of the three constructs were significantly shorter in animals that had an antibody response after the first injection. In contrast, all three TNF-bp constructs were equally effective at improving outcome, blocking a systemic TNF-alpha response, and attenuating the cytokine responses when administered at a dose of 1.0 mg/kg body wt 1 h before a lethal E. coli infusion. The findings suggest that immunogenicity of TNF-bp constructs can be altered by changing the number of functional domains, without affecting their capacity to neutralize TNF-alpha and to abrogate TNF-mediated pathology.

Animals↗

PubMed1997

Involvement of 26-kDa cell-associated TNF-alpha in experimental hepatitis and exacerbation of liver injury with a matrix metalloproteinase inhibitor.

TNF-alpha is a pleiotropic cytokine that exists both as a 26-kDa cell-associated and a 17-kDa soluble form. Recently, a class of matrix metalloproteinase inhibitors has been identified that can prevent the processing by TNF convertase of 26-kDa TNF-alpha to its 17-kDa form and can reduce mortality from normally lethal doses of D-galactosamine plus LPS (D-GalN/LPS). Here we report that a matrix metalloproteinase inhibitor, GM-6001, improves survival but does not protect against liver injury from D-GalN/LPS-induced shock in the mouse. In Con A-induced hepatitis, GM-6001 actually exacerbates hepatocellular necrosis and apoptosis despite greater than 90% reduction in plasma TNF-alpha concentrations. Treatment with GM-6001 also has minimal effect on the concentration of membrane-associated TNF-alpha in the livers of animals with Con A induced hepatitis. In contrast, a TNF binding protein (TNF-bp), which neutralizes both membrane-associated and soluble TNF-alpha, prevents D-GalN/LPS- and Con A-induced hepatitis. Our studies suggest that cell-associated TNF-alpha plays a role in the hepatocellular necrosis and apoptosis that accompany D-GalN/LPS- or Con A-induced hepatitis, and that matrix metalloproteinase inhibitors are ineffective in preventing this hepatic injury.

Animals↗

PubMed1997

Evidence that brief stress may induce the acute phase response in rats.

Exposing rats to a single session of inescapable tail shock (IS) reduces corticosteroid binding globulin (CBG) 24 h later (Fleshner et al., Endocrinology 136: 5336-5342, 1995). The present experiments examined whether reductions in CBG are differentially affected by controllable vs. identical uncontrollable tail shock, are mediated by IS-induced glucocorticoid elevation, or reflect IS-induced activation of the acute phase response and whether IS produces fever. The results demonstrate that 1) equivalent reductions in CBG are observed in response to escapable tail shock or yoked IS, 2) IS-induced CBG reduction is not blocked by adrenalectomy in rats that receive basal corticosteroid replacement or by pretreatment with RU-38486, and 3) IS appears to activate the acute phase response, since IS reduces serum levels of an acute-phase negative reactant (CBG), increases serum levels of acute-phase positive reactants (haptoglobin and alpha 1-acid glycoprotein), and increases core body temperature 20-24 h later.

Adrenalectomy↗

PubMed1996

A role for tumor necrosis factor-alpha in the increased mortality associated with Vibrio vulnificus infection in the presence of hepatic dysfunction.

OBJECTIVE: The present study was designed to evaluate whether pre-existing hepatic dysfunction (cirrhosis) leads to increased morbidity and mortality, in part through an inappropriate in vivo tumor necrosis factor-alpha response. SUMMARY BACKGROUND DATA: Vibrio vulnificus is the most commonly isolated member of the noncholera Vibrio sp., responsible for fulminant and frequently fatal septicemia. A strong clinical association exists between hepatic dysfunction and increased morbidity and mortality from Vibrio sp. infection. However, the underlying mechanism behind this association has not been fully delineated. METHODS: Cirrhosis was induced in C57BL/6 (15 to 20 g) mice using thrice-weekly injections of carbon tetrachloride (CCl4) for 7 weeks. Either a 7.0 to 9.5 X 10(7) (low dose) or a 0.8 to 1.2 X 10(9) colony-forming unit (high dose) of V. vulnificus was administered through a mini-laparotomy incision via transgastric puncture into both cirrhotic and control animals. RESULTS: Mortality in cirrhotic mice to low- and high-dose Vibrio infection was 88% (7/8) and 100% (8/8), respectively, whereas mortality in control animals was 0% (0/8) and 12% (1/8), respectively (p<0.01). Tumor necrosis factor-alpha mRNA could be detected by reverse transcriptase polymerase chain reaction in livers and lungs from infected animals 2 and 4 hours after Vibrio administration in both control and cirrhotic animals. Lung and liver tumor necrosis factor-alpha bioactivity, however, was significantly lower in cirrhotic animals infected with Vibrio when compared with controls. Serum tumor necrosis factor-alpha was only sporadically detected in both groups of Vibrio-infected animals. When cirrhotic mice challenged with a low dose of Vibrio sp. were pretreated with 1.0 mg/kg body weight of a novel tumor necrosis factor-alpha receptor immunoadhesin, the increased mortality was completely prevented. CONCLUSIONS: Cirrhotic mice show increased mortality to Vibrio infection, and this increased mortality is dependent on an in vivo tumor necrosis factor-alpha response.

Animals↗

PubMed1994

Glucocorticoids regulate intestinal glutamine synthetase gene expression in endotoxemia.

PURPOSE: Although glutamine is required to maintain gut mucosal metabolism and function, intestinal glutamine uptake from the gut lumen and from the bloodstream is decreased during sepsis. We hypothesized that endogenous mucosal glutamine biosynthesis is increased during endotoxemia, and we attempted to define the "stress" mediators that regulate the activity of small intestinal glutamine synthetase (GS), the principal enzyme of de novo glutamine biosynthesis in the gut. METHODS: Adult rats received Escherichia coli lipopolysaccharide (LPS) (7.5 mg/kg intraperitoneally), RU 38486 (a glucocorticoid antagonist; 10 mg/kg by gavage) 2 hours prior to LPS administration, antibody to tumor necrosis factor (TNF) (4 mg/kg intraperitoneally) prior to LPS administration, or ketorolac tromethamine (a prostaglandin synthesis inhibitor; 1 mg/kg intraperitoneally) followed by LPS administration. Mucosal GS activity was assayed 12 hours after LPS administration. In a separate set of studies, cultured intestinal mucosal cells (Caco-2) were exposed to LPS, interleukin 1 (IL-1), IL-6, TNF-alpha, interferon-gamma, prostaglandin E2, or dexamethasone. Twelve hours later, GS activity was assayed and messenger RNA was extracted. The GS transcripts were labeled with a GS complementary DNA probe radiolabeled with phosphorus 32, were quantitated by phosphoimaging, and were normalized to beta-actin. RESULTS: In vivo LPS treatment increased mucosal GS activity by 250%. Pretreatment with antibody to TNF or ketorolac did not inhibit the LPS-induced increase in mucosal GS, whereas pretreatment with RU 38486 attenuated the increase in gut GS activity by 60%. Lipopolysaccharide, IL-1, IL-6, TNF-alpha, gamma-interferon, and prostaglandin E2 did not increase GS activity in Caco-2 cells, whereas dexamethasone increased GS activity and messenger RNA 2.5-fold and threefold, respectively. These data indicate that cytokines and prostaglandins (prostaglandin E2) do not regulate mucosal GS expression during endotoxemia. Glucocorticoids, however, stimulate GS gene expression directly. CONCLUSIONS: This hormonally mediated response may support de novo mucosal GS during septic states when uptake of glutamine from the lumen and blood is decreased.

Animals↗

PubMed1994

Cytokines decrease glutaminase expression in human fibroblasts.

BACKGROUND: Glutamine metabolism in fibroblasts is essential for energy production, nucleotide biosynthesis, and growth during wound healing. Because cytokines can impair fibroblast proliferation, we tested the hypothesis that cytokines impair glutamine metabolism. We studied the influence of several cytokines on the expression of glutaminase, the major enzyme of intracellular glutamine metabolism in fibroblasts. METHODS: Human foreskin fibroblasts were incubated for 6 and 12 hours with varying doses (10, 100, or 1000 units/ml) of interleukin (IL)-1, IL-6, tumor necrosis factor-alpha, or gamma-interferon. Cell lysates were assayed for glutaminase-specific activity, and glutaminase protein content was measured by Western blotting with a polyclonal antibody. Total cellular RNA was extracted, and relative glutaminase messenger RNA levels were determined by Northern blotting with a 32P-labeled glutaminase complement DNA-derived probe. These mRNA levels were normalized by blotting with a beta-actin cDNA-derived probe as control. Cell nuclei were isolated, and nuclear run-ons were used to determine relative glutaminase mRNA transcription rates. RESULTS: IL-1, IL-6, tumor necrosis factor-alpha, and gamma-interferon decreased glutaminase activity and protein concentration after a 12-hour incubation in a dose-independent fashion. No difference was noted at 6 hours. Western blot analysis showed a 30% to 60% reduction in glutaminase protein in treated cells. These cytokines also decreased glutaminase mRNA levels, consistent with transcriptional regulation. This was confirmed by nuclear run-on assays that showed a decrease in the number of glutaminase transcripts. CONCLUSIONS: A variety of different pro-inflammatory cytokines decrease glutaminase expression in cultured human fibroblasts. This cytokine-mediated inhibition of glutamine metabolism may limit the availability of key glutamine-derived intermediates and impair fibroblast proliferation in certain patients.

Cells, Cultured↗

PubMed1992

Glucocorticoids regulate intestinal glutaminase expression.

BACKGROUND: The metabolism of glutamine by the small intestinal mucosal cells is highly dependent on the glutaminase enzyme. Because mucosal glutamine utilization is increased after operation, we hypothesized that the elevated glucocorticoid hormones that occur after surgical stress regulate expression of mucosal glutaminase at the molecular level. METHODS: Adult rats received saline solution or dexamethasone (0.5 mg/kg, one dose) and were sacrificed at various times after treatment. Jejunal mucosal total RNA was extracted for Northern hybridization with an alpha-32P-labeled rat glutaminase cDNA. The mRNA of the constitutively expressed gene beta-actin was the control for RNA loading. Quantitation of glutaminase transcripts was determined by densitometric scanning and normalized to actin. Glutaminase activity (micromoles per milligram of protein per hour) and its kinetic parameters, maximal transport velocity (micromoles per milligram of protein per hour) and Michaelis-Menten constant (micromoles per liter), were also determined. RESULTS: Dexamethasone increased glutaminase mRNA (twofold at 4 hours, sixfold at 12 hours; p less than 0.01) and glutaminase-specific activity. The increase in message preceded the increase in activity by 4 hours, consistent with de novo RNA synthesis followed by protein synthesis. The increase in glutaminase activity was the result of a 21% increase in the maximal enzyme capacity (maximal transport velocity = 8.6 +/- 0.5 mumol/mg protein/hr in control rats vs 10.4 +/- 0.3 mumol/mg protein/hr in rats treated with dexamethasone; p less than 0.01) rather than a change in enzyme affinity (Michaelis-Menten constant). CONCLUSION: Glucocorticoids may accelerate intestinal glutamine utilization by increasing glutaminase expression, an adaptive response that could provide more energy for mucosal cells in stress states.

Animals↗