Induction of enlarged intestinal lymphoid aggregates during acute glutathione depletion in a murine model.

Glutathione is a nonenzymatic antioxidant synthesized by most animal cells and is depleted in inflammatory bowel disease. The effects of glutathione depletion on intestinal histology and inhibitory neurochemicals was examined in a mouse model. Glutathione depletion in A/J mice involved inhibition of gamma-glutamylcysteine synthetase using L-buthionine-(S,R)-sulfoximine (BSO) for 10 days. Ileum and colon were obtained from saline-control mice, BSO-treated mice, and BSO-treated mice receiving ascorbate or glutathione monoethylester. Glutathione, lipid peroxides, and nicotineamide adenine dinucleotide phosphate diaphorase activity were measured by colorimetric assays. Vasoactive intestinal peptide was measured by radioimmunoassay. Glutathione depletion induced enlargement of mucosal-submucosal lymphoid aggregates without germinal centers in ileum and colon. These aggregates were prevented by supplementation with glutathione monoethylester but not ascorbate. Tissue levels of inhibitory neurochemicals were unchanged. Depletion of glutathione appears to induce enlarged lymphoid aggregates by recruitment of lymphocytes from the peripheral circulation. A component of the inflammation that develops in inflammatory bowel disease could be related to depletion of tissue levels of glutathione.

Nitric oxide synthase is increased following small intestinal transplantation in the rat.

Transplantation of the small intestine is a neural model that could include extrinsic denervation, loss of intrinsic enteric neurons, or loss of intrinsic neural pathways. Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity was measured in normal rat ileum, ileum 3 months after resection of the jejunum, and ileum 3 months after isotransplantation of the ileum. The distribution of NADPH diaphorase activity and immunoreactive neuronal nitric oxide synthase were examined. Nicotinamide adenine dinucleotide phosphate diaphorase activity was increased in transplanted ileum (16.5+/-3.5 mU/mg protein) compared to normal controls (6.6+/-0.7) and resection controls (6.8+/-0.6) (P < 0.05, ANOVA). Histologically, NADPH diaphorase activity and immunoreactive nitric oxide synthase appeared increased within nerve cell bodies following transplantation. These findings may represent an adaptive response of the enteric nervous system to extrinsic denervation. Loss of intrinsic neural pathways was not supported as a mechanism.

Chronic glutathione depletion alters expression of enteric inhibitory neurochemicals in the mouse.

Antioxidants may delay or prevent neural diseases. Depletion of the non-enzymatic antioxidant, glutathione, in a mouse model was produced by inhibiting its rate-limiting enzyme, gamma-glutamylcysteine synthetase, for 7 weeks. Ileum and colon were obtained from treated and control (saline) mice. Glutathione levels and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity were determined by spectrophotometric assays; vasoactive intestinal peptide (VIP) levels were measured by radioimmunoassay. Glutathione levels were higher in ileum than colon. Colonic glutathione was decreased in treated mice compared to controls; there were no differences in ileal glutathione levels. VIP was decreased in ileum compared to controls, while NADPH diaphorase activity was decreased in colon compared to controls. In this chronic mouse model, glutathione appeared to regulate expression of enteric inhibitory nerve cell products.

Expression of mRNA for vasoactive intestinal peptide in normal human colon and during inflammation.

The availability of colon provides a ready source of human neurons. Among the products of nerve cell bodies, vasoactive intestinal peptide is a neuropeptide that serves as a marker of non-adrenergic, non-cholinergic inhibitory nerves in colon. These nerves have been proposed to be involved in regulation of immune function, secretion, and smooth muscle function. In previous work, we identified decreased tissue levels of vasoactive intestinal peptide in a disorder of chronic colonic mucosal inflammation, ulcerative colitis. We hypothesized that diminished gene expression of vasoactive intestinal peptide could result in decreased tissue levels of this neuropeptide. Sigmoid colon was obtained at surgery from controls (n = 6) and patients with ulcerative colitis (n = 6). Vasoactive intestinal peptide mRNA was quantified by Northern blot hybridization and tissue levels of vasoactive intestinal peptide were determined by radioimmunoassay. Tissue vasoactive intestinal peptide was decreased only in the mucosal-submucosal layer of ulcerative colitis (p = .02). There was a single 1.7 kbase vasoactive intestinal peptide transcript identified in both control colon and ulcerative colitis. Normalized vasoactive intestinal peptide mRNA levels were increased by 260% in ulcerative colitis compared to controls (p < .01). These observations suggest that decreased vasoactive intestinal peptide gene expression or abnormal post-transcriptional processing are not primary defects in this disorder of chronic inflammation. The findings support the alternative hypothesis that axonal degeneration in ulcerative colitis could result in increased expression of neuronal vasoactive intestinal peptide mRNA.

Hyperbaric oxygen therapy and squamous cell carcinoma cell line growth.

Hyperbaric oxygen (HBO) promotes tissue healing by increasing oxygenation. Therefore, HBO therapy is clinically useful for some patients who have undergone major cancer resection and/or radiotherapy to the head and neck. For individual patients, however, there might be undetected viable tumor present at the time of therapy. This study was performed to determine if increased tissue oxygen had a measurable effect on the growth of squamous carcinoma xenotransplants which had been derived from head and neck cancers. After the successful growth of two well-established human squamous cell carcinoma cell lines (183 and 1483), each tumor was transplanted into 20 mice. Every mouse received four transplants of 10(6) cells. Ten mice with 40 xenotransplants in each group were treated with HBO daily for 90 minutes at a pressure of 2 atm, whereas the other 10 formed the control group. The mice transplanted with cell line 1483 were treated for 21 days; mice transplanted with cell line 183 were treated for 28 days. The tumor weight, volume, and histology were evaluated. No significant difference was found between experimental groups. This study suggests that increased tissue oxygen neither significantly increases nor decreases the growth of squamous cell carcinoma.

Effects of fractionated doses of ionizing radiation on colonic motor activity.

The colonic motor effects of fractionated irradiation were studied in five conscious dogs. Seven colonic and two ileal strain gauge transducers were implanted. After control recordings, an abdominal dose of 250 cGy was administered three times a week on alternate days for three successive weeks (total dose 2,250 cGy). Recordings were then continued for 3 wk after the completion of radiation. Colonic giant migrating contractions (GMCs) occurred at a frequency of 0.15 +/- 0.05 contractions/h in the control state. Only one of these contractions (8.3%) originated in the small bowel and propagated into the colon. Abdominal field irradiation significantly increased the incidence of colonic GMCs to 0.51 +/- 0.11 contractions/h (P < 0.05). Fifty-four percent of GMCs originated in the small intestine. GMCs during the radiation schedule were associated with explosive diarrhea on seven occasions. Irradiation did not alter the frequency of colonic migrating motor complexes, but the mean duration of contractile states decreased in the middle and distal colon. Diarrhea occurred as early as the second dose of radiation. Pathological changes in the colon were correlated with motor activity. Both small intestinal and colonic GMCs reverted to control frequencies after cessation of radiation exposure. Abdominal irradiation significantly altered the contractile activity of the colon. These changes are associated with abdominal cramping and diarrhea.

Quantitative differential agglutination method using the Coulter Counter to measure survival of compatible but identifiable red blood cells.

The method described here using a centrifuge and Coulter Cell Counter for the quantitative differential agglutination of human red cells uses commercial anti-A and anti-B antisera for the ABO system, and for the Rh system a commercial anti-D serum, a low ionic strength solution and an anti-human IgG antiserum. We compared this Coulter Counter method with the Technicon AutoAnalyzer method which utilizes bromelin and polyvinyl pyrrolidone, and anti-A, anti-B and anti-CD antisera, and found this new method to be the simpler of the two. The nonagglutinable count with the Coulter Counter was 1.07% for A1 red cells, 2.26% for A2 red cells, 1.06% for B red cells, and 1.78% for Rh-positive red cells, results similar to those seen with the Technicon AutoAnalyzer. Results with the Coulter Counter method were consistently accurate whether the ACD red cells were studied on the day of collection, after 10 days of 4 degrees C storage, or after 4 degrees C storage for up to 6 days followed by cryopreservation with 40% (w/v) glycerol at-80 degrees C, thawing and washing. In this study, red cell samples obtained from recipients who had received compatible but identifiable donor red cells were frozen with 40% glycerol and stored at-80 degrees C for 10 months, thawed and washed. Survival measurements on these washed previously frozen red cells were similar to the values in liquid-stored red cells.