Elizabeth E. Mannick

PEROXYNITRITE-INDUCED APOPTOSIS IN T84 AND RAW 264.7 CELLS : ATTENUATION BY L-ASCORBIC ACID

The free radicals nitric oxide and superoxide react to form peroxynitrite (ONOO-), a potent cytotoxic oxidant. This study was designed to evaluate whether addition of L-Ascorbic acid (AsC) into the culture medium decreases peroxynitrite-induced apoptosis in human intestinal epithelial (T84) and murine macrophage (RAW 264.7) cell lines. In Experiment 1, T84 and RAW 264.7 cells were divided in two protocols: (1) treated with 100-300 microM ONOO- and incubated for 4 h, and (2) treated with 10-100 microM ONOO- and incubated overnight (14 h). In Experiment 2, T84 and RAW 264.7 cells were treated with 300 microM ONOO- and 500 microM AsC and incubated for 4 h. In Experiment 3, T84 and RAW 264.7 cells were preincubated for 2 h with 500 microM AsC then exposed to 300 microM ONOO- for 4 h. Cell viability (necrosis) was assessed by trypan blue dye exclusion. Apoptosis was quantified with a cell death detection ELISA assay. In the 4 h protocol, ONOO- induced apoptosis in T84 and RAW 264.7 cells, at levels of 100-300 microM. Concentrations of ONOO- greater than 300 microM caused necrosis. In contrast, extension of the protocol to 14 h indicated that ONOO- induced apoptosis at lower concentrations (50;-75 microM), with concentrations > 75 microM resulting in necrosis. AsC administered to the media or with preincubation plus washout, decreased peroxynitrite-induced apoptosis in T84 and RAW 264.7 cells. These results indicate that ONOO- may contribute to the pathophysiology of gut inflammation by promoting cell death and ascorbic acid may protect against peroxynitrie-induced damage.

Use of Infliximab in Pediatric Patients with Inflammatory Bowel Disease

BACKGROUND: The concentration of tumor necrosis factor, a proinflammatory cytokine, is increased in the gastrointestinal mucosa of patients with active Crohns disease (CD) and ulcerative colitis (UC). Neutralization of tumor necrosis factor decreases the mucosal inflammatory response of adults with CD. Little information is available on the use of monoclonal antibody to tumor necrosis factor (infliximab) in children and adolescents with CD or UC. OBJECTIVE: To evaluate the clinical response and side effects of patients to infliximab. METHODS: A retrospective review of data regarding 18 pediatric and adolescent patients with active CD (n = 15) and UC (n = 3) poorly controlled with conventional therapy. All patients received one to six intravenous infusions of infliximab 5 mg/kg, while receiving their usual medications. RESULTS: All patients experienced clinical improvement, including decrease in the frequency of stooling and resolution of extraintestinal symptoms such as arthropathy, malaise, and skin manifestations after treatment with infliximab. All but one patient had a documented decrease in the erythrocyte sedimentation rate. Prednisone dosage was tapered in all but two patients, and discontinued in seven patients. Intravenous infusion of infliximab was well tolerated. One patient developed a rash several days after the infusion. A patient who received six infliximab infusions developed recurrent Staphylococcus aureus infections, as well as septic arthritis and chronic osteomyelitis during the follow-up period, raising the issue of the long-term safety of infliximab. CONCLUSIONS: Treatment of our patients with refractory CD and UC with infliximab was associated with remarkable clinical improvement. Although the drug may have an important role in their management, further assessment of long-term safety and efficacy is needed.

Genistein and gut inflammation: role of nitric oxide.

Abstract Genistein, a principal soy isoflavone, has been identified as a protein kinase inhibitor that possesses immunosuppressive and anti-inflammatory properties. The aim of the study was to determine if genistein modified chronic ileitis in guinea pigs induced by the hapten trinitrobenzene sulfonic acid (TNBS), and the activity index of cultured macrophages (RAW 264.7 cells) stimulated with lipopolysaccharide (LPS). Genistein at low doses (0.1 mg/kg, s.c.) had mild anti-inflammatory effects in TNBS ileitis. Therapeutic benefit included a reduction in nitric oxide production, granulocyte infiltration and improved mucosal architecture. Genistein, at low doses, also appeared to attenuate immunohistochemical staining for inducible nitric oxide synthase (iNOS) and nitrotyrosine. The beneficial effects of genistein were not apparent at doses above 0.1 mg/kg. We found that genistein also inhibited LPS-induced nitrite production by cultured macrophages and protected against LPS-induced necrosis despite its ability to cause apoptosis. These results indicate that genistein displayed mild antiinflammatory properties which may, in part, involve an attenuation of nitric oxide release via inducible nitric oxide synthase, and the formation of peroxynitrite.

Gastroesophageal reflux and Nissen fundoplication following percutaneous endoscopic gastrostomy in children.

BACKGROUND Abnormal gastroesophageal reflux after percutaneous endoscopic gastrostomy is a serious problem in neurologically impaired children. Protective fundoplication has been advocated. Whether esophageal pH monitoring before percutaneous endoscopic gastrostomy will predict later problems with gastroesophageal reflux is unclear. METHODS Eighty-five mostly neurologically impaired pediatric patients who underwent percutaneous endoscopic gastrostomy were studied retrospectively regarding complications, success of nutritional rehabilitation, and the incidence of pathologic gastroesophageal reflux. Follow-up period was 1 to 4 years. Twenty-four-hour esophageal pH monitoring was performed in 46 patients before percutaneous endoscopic gastrostomy. RESULTS There were no deaths. Two major complications occurred that required surgical intervention, and 14 minor complications occurred related to the procedure. Z-scores for weight increased significantly after percutaneous endoscopic gastrostomy. pH probe results were normal in 22 patients (group 1). Five required medical treatment for gastroesophageal reflux after percutaneous endoscopic gastrostomy, but only 1 (5%) later required Nissen fundoplication. pH probe results were abnormal in 24 patients (group 2). Nineteen required medical therapy for gastroesophageal reflux, and 7 (29%) later needed fundoplication (p < 0.05, incidence of fundoplication group 1 vs. group 2). Improvement in Z-scores was similar in patients requiring and not requiring fundoplication. CONCLUSIONS Percutaneous endoscopic gastrostomy is a safe and effective technique for long-term nutritional support in children. Abnormal gastroesophageal reflux is common. Normal findings in an esophageal pH study before percutaneous endoscopic gastrostomy may be predictive of a favorable outcome with respect to gastroesophageal reflux. This is in contrast to patients with abnormal results in pH studies before percutaneous endoscopic gastrostomy of whom a relatively large percentage may later require fundoplication. Improved nutritional status after percutaneous endoscopic gastrostomy does not appear to have an impact on the severity of gastroesophageal reflux.

Altered phenotype of dextran sulfate sodium colitis in interferon regulatory factor‐1 knock‐out mice

Background and Aims: Interferon regulatory factor‐1 (IRF‐1) is a transcription factor with antiviral, proinflammatory and tumor suppressor properties. We examined the role of IRF‐1 in dextran sulfate sodium colitis, a murine model of inflammatory bowel disease, to determine if absence of the gene would protect against colitis.

Living related liver transplant in a patient with argininosuccinic aciduria and cirrhosis: metabolic follow-up.

Argininosuccinic aciduria (ASAuria) is an autosomal recessive urea cycle disorder (UCD) caused by deficiency of argininosuccinate lyase (ASL). Argininosuccinate lyase catalyzes the conversion of argininosuccinic acid (ASA) to arginine and fumarate and is expressed in the liver, kidney, and other tissues. Patients with ASAuria have elevations of plasma ASA, citrulline, and glutamine, as well as a decrease in arginine. During the neonatal period, patients often present with hyperammonemic coma and are at high risk for subsequent developmental problems. Long-term treatment includes protein restriction and arginine supplementation. Exogenous arginine stimulates incorporation of ammonia into the urea cycle allowing for excretion of waste nitrogen as ASA. Arginine supplementation also may be important for the body’s needs owing to impairment of its synthesis in the kidneys and other tissues (1). Liver disease, including cirrhosis, is a potential complication of ASAuria. The pathogenesis of liver disease remains unknown (2). Serum transaminases, alkaline phosphatase, and coagulation studies often are abnormal and provide a potential indication for liver transplantation. Orthotopic liver transplantation (OLT) has been demonstrated to correct hyperammonemia in patients with UCDs (3–6). Orthotopic liver transplantation would not be expected, however, to correct metabolic disturbances attributable to any urea-cycle enzyme deficiencies in extrahepatic tissues. We present the 4-year follow-up of a patient with ASAuria who developed cirrhosis and underwent suc-

Association of the T allele of an intronic single nucleotide polymorphism in the colony stimulating factor 1 receptor with Crohn's disease: a case-control study

BackgroundPolymorphisms in several genes (NOD2, MDR1, SLC22A4) have been associated with susceptibility to Crohns disease. Identification of the remaining Crohns susceptibility genes is essential for the development of disease-specific targets for immunotherapy. Using gene expression analysis, we identified a differentially expressed gene on 5q33, the colony stimulating factor 1 receptor (CSF1R) gene, and hypothesized that it is a Crohns susceptibility gene. The CSF1R gene is involved in monocyte to macrophage differentiation and in innate immunity.MethodsPatients provided informed consent prior to entry into the study as approved by the Institutional Review Board at LSU Health Sciences Center. We performed forward and reverse sequencing of genomic DNA from 111 unrelated patients with Crohns disease and 108 controls. We also stained paraffin-embedded, ileal and colonic tissue sections from patients with Crohns disease and controls with a polyclonal antibody raised against the human CSF1R protein.ResultsA single nucleotide polymorphism (A2033T) near a Runx1 binding site in the eleventh intron of the colony stimulating factor 1 receptor was identified. The T allele of this single nucleotide polymorphism occurred in 27% of patients with Crohns disease but in only 13% of controls (X2 = 6.74, p < 0.01, odds ratio (O.R.) = 2.49, 1.23 < O.R. < 5.01). Using immunohistochemistry, positive staining with a polyclonal antibody to CSF1R was observed in the superficial epithelium of ileal and colonic tissue sections.ConclusionsWe conclude that the colony stimulating factor receptor 1 gene may be a susceptibility gene for Crohns disease.

Oxidative Damage During the Gastric Precancerous Process

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) may play a role in human carcinogenesis. Epidemiologic studies have indicated that infection with Helicobacter pylori increases the risk of gastric cancer. This chapter explores the possibility that such an association is the result of long-standing inflammation and the release of ROS and RNS by white blood cells.

PEROXYNITRITE-INDUCED APOPTOSIS IN HUMAN EPITHELIAL CELLS (T84) IS ATTENUATED BY ASCORBIC ACID OR 5-AMINOSALICYLIC ACID. • 496

PEROXYNITRITE-INDUCED APOPTOSIS IN HUMAN EPITHELIAL CELLS (T84) IS ATTENUATED BY ASCORBIC ACID OR 5-AMINOSALICYLIC ACID. • 496