Imaging techniques used for the detection of 8-oxoguanine adducts and DNA repair proteins in cells and tissues.

The presence of 8-oxoguanine (8-oxoG) in DNA is considered a marker of oxidative stress and DNA damage. Numerous biochemical techniques have been described for its detection in cells or tissues. Although these approaches are quantitative, they do not provide insights into whether the lesion occurs in mitochrondrial versus genomic DNA. In addition, biochemical techniques are not amenable to the evaluation of individual cells or archival tissues. Antibodies have been raised against 8-oxoG, which may circumvent some of these issues. In this review, we described the use of in situ imaging techniques to detect oxidative DNA damage including the comet assay. We will review our previous work that describes the utility of an antibody fragment (Fab) engineered to recognize 8-oxoG in DNA. Furthermore, we will discuss the analysis of DNA repair enzymes in the assessment of oxidative DNA damage. Finally, advantages and potential concerns associated with immunodetection of 8-oxoG are discussed.

Nitrogen dioxide induces death in lung epithelial cells in a density-dependent manner.

Nitrogen dioxide (*NO2) is commonly known as an indoor and outdoor air pollutant. Inhalation of *NO2 is associated with epithelial cell injury, inflammation, and the aggravation of asthma. *NO2 can also be formed during inflammation, by the metabolism of nitric oxide. We describe a gas-phase exposure system for in vitro exposure of lung epithelial cells to *NO2. Immunofluorescence revealed 3-nitrotyrosine immunoreactivity of rat alveolar type II epithelial cells exposed to 5 parts per million of *NO2 for 4 h. Comparative analysis of log-phase and confluent cultures demonstrated that cell death occurred extensively in log-phase cells, whereas minimal death was observed in confluent cultures. Peroxynitrite (ONOO-) or the ONOO- generator 3-morpholinosydnonimine (SIN-1) caused similar amounts of death. Further, exposure of wounded cell cultures to *NO(2) or SIN-1 revealed that death was restricted to cells repopulating a wounded area. Cycloheximide or actinomycin D, inhibitors or protein and messenger RNA synthesis, respectively, significantly reduced terminal transferase reactivity, suggesting that a new protein(s) may be required for cell death. These results suggest that during restitution after pulmonary injury, epithelium may be sensitive to cell death by reactive nitrogen species.

Effects of glutamine supplementation on circulating lymphocytes after bone marrow transplantation: a pilot study.

Glutamine (Gln) is an important nutrient substrate for lymphocytes and macrophages in vitro. This pilot study evaluated effects of Gln supplementation on circulating lymphocytes and lymphocyte subsets after allogeneic bone marrow transplantation (BMT). Adult patients received either parenteral nutrition supplemented by L-Gln (Gln-PN) or standard Gln-free PN after BMT. Leukocyte and total lymphocyte counts were determined during hospitalization, and flow cytometry studies of peripheral mononuclear cells were performed 1 to 2 weeks after hospital discharge. The Gln-PN group demonstrated a higher percentage of blood lymphocytes during hospitalization. At flow cytometry, patients who received Gln-PN had an increased total lymphocyte count (332 +/- 50 versus 590 +/- 71 cells/microL, P = 0.010); greater numbers of total T lymphocytes (54 +/- 19 versus 229 +/- 70 cells/microL, P = 0.030); and higher CD4+ and CD8+ T-lymphocyte counts in peripheral blood compared with controls. Gln-PN may support lymphocyte recovery after BMT.

A new treatment for patients with short-bowel syndrome. Growth hormone, glutamine, and a modified diet.

OBJECTIVE: The purpose of this study was to initially determine if growth hormone or nutrients, given alone or together, could enhance absorption from the remnant small bowel after massive intestinal resection. If clinical improvement were observed, this therapy would then be used to treat patients with the short-bowel syndrome over the long term. SUMMARY BACKGROUND DATA: Patients who undergo extensive resection of the gastrointestinal tract frequently develop malabsorption and require long-term parenteral nutrition. The authors hypothesized that the administration of growth factors and/or nutrients could enhance further compensation of the remnant intestine and thereby improve absorption. Specifically, animal studies have shown that there is enhanced cellularity with the administration of growth hormone (GH) or glutamine (GLN), or a fiber-containing diet. METHODS: Initially, 17 studies were performed in 15 total parenteral nutrition (TPN)-dependent short-bowel patients over 3 to 4 weeks in the clinical research center; the first week served as a control period, and during the next 1 to 3 weeks, the specific treatment was administered and evaluated. Throughout the study, food of known composition was provided and all stool was collected and analyzed to determine absorption across the remaining bowel. The effect of a high-carbohydrate, low-fat diet (DIET), the amino acid glutamine (GLN) and growth hormone (GH) administered alone or in combination with the other therapies (GH + GLN + DIET) was evaluated. The treatment was expanded to 47 adults (25 men, 22 women) with the short-bowel syndrome, dependent on TPN for 6 +/- 1 years. The average age was 46 +/- 2 years, and the average jejunal-ileal length was 50 +/- 7 cm (median 35 cm) in those with all or a portion of colon and 102 +/- 24 cm (median 102 cm) in those with no colon. After 28 days of therapy, the patients were discharged on only GLN + DIET. RESULTS: The initial balance studies indicated improvement in absorption of protein by 39% accompanied by a 33% decrease in stool output with the GH + GLN + DIET. In the long-term study, 40% of the group remain off TPN and an additional 40% have reduced their TPN requirements, with follow-up averaging a year and the longest being over 5 years. CONCLUSION: GH + GLN + DIET offers a potential method for providing cost-effective rehabilitation of surgical patients who have the short-bowel syndrome or other complex problems of the gastrointestinal tract. This therapeutic combination also may be useful to enhance bowel function in patients with other gastrointestinal diseases and those requiring extensive intestinal operations, including transplantation.