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. METHODS: C57BL/6J mice with a targeted disruption of IRF-1 and wild-type C57BL/6J controls received five 7-day cycles of 2% dextran sulfate sodium alternating with five 7-day cycles of water. Colonic tissue was formalin fixed for histological analysis and total RNA extracted for gene chip and SYBR green real-time polymerase chain reaction (PCR) analysis. RESULTS: Histological analysis revealed increased distortion of crypt architecture in the dextran sulfate sodium-treated, IRF-1 -/- animals as compared to dextran sulfate sodium-treated wild-type animals. Five of 15 dextran sulfate sodium-treated IRF-1 -/- mice, but only one of 14 dextran sulfate sodium-treated wild-type mice, developed colonic dysplasia. Microarray analysis comparing colonic gene expression in IRF-1 -/- and wild-type animals revealed decreased expression of caspases, genes involved in antigen presentation, and tumor suppressor genes in the IRF-1 -/- animals. Increased expression of genes involved in carcinogenesis and immunoglobulin and complement genes was also noted in the knock-out animals. CONCLUSIONS: Absence of IRF-1 is not protective in dextran sulfate sodium colitis.

Gene expression in mononuclear cells from patients with inflammatory bowel disease.

OBJECTIVES: Discovery of Nod2 as the inflammatory bowel disease 1 (IBD1) susceptibility gene has brought to light the significance of mononuclear cells in inflammatory bowel disease pathogenesis. The purpose of this study was to examine changes in gene expression in peripheral blood mononuclear cells in patients with untreated Crohn's disease (CD) and ulcerative colitis (UC) as compared to patients with other inflammatory gastrointestinal disorders and to healthy controls. METHODS: We used a 2400 gene cDNA glass slide array (MICROMAX) to examine gene expression in peripheral blood mononuclear cells from seven patients with Crohn's disease, five patients with ulcerative colitis, 10 patients with other inflammatory gastrointestinal disorders, and 22 age- and sex-matched controls. Results. Novel categories of genes differentially expressed in Crohn's disease and ulcerative colitis patients included genes regulating hematopoietic cell differentiation and leukemogenesis, lipid raft-associated signaling, the actin cytoskeleton, and vesicular trafficking. CONCLUSIONS: Altered gene expression in mononuclear cells may contribute to inflammatory bowel disease pathogenesis.

The effect of drug dose and drug exposure time on the binding, internalization, and cytotoxicity of radiolabeled somatostatin analogs.

BACKGROUND: Creation of protease-resistant somatostatin analogs has allowed development of these peptides as clinically useful drugs. Widespread diagnostic use of radiolabeled somatostatin analogs has enhanced interest in the binding and intracellular distribution of these peptides. The degree of drug internalization and length of drug retention may be critical for drug-induced cytotoxicity. We hypothesized that the ability of a radiolabeled peptide to bind to a cell, be internalized, and induce cytotoxicity is proportional to both the radioligand concentration and the exposure time. MATERIALS AND METHODS: To test this hypothesis, somatostatin receptor-expressing cells (IMR-32) were incubated with (111)In-pentetreotide, a sst 2 preferring somatostatin analogue. Radioligand exposure time and/or concentration were varied. RESULTS: Prolonged exposure to a fixed concentration of radioligand resulted in progressive increases in whole cell binding and internalization over time. Cells exposed to a relatively fixed number of microCi-Hr yielded constant whole cell binding and internalization. Increasing the microCi-Hr resulted in a proportionate increase in binding. Cytotoxicity was also proportional to the dose of radiation regardless of whether the exposure was internalized radiation (microCi-Hr from (111)In-pentetreotide) or from external beam radiation (cGy). CONCLUSION: Both drug exposure time and drug concentration contribute to cell binding and cytotoxicity in this model and their relative contributions are inversely related.

Short-term stretch translocates the alpha-1-subunit of the Na pump to plasma membrane.

Short-term (2 30 min) cyclic stretch activates the Na pump in cultured aortic smooth muscle cells (ASMCs). This effect of stretch involves the phosphotidylinositol 3-kinase (PI 3-kinase) participation. Presently, we investigated whether this stimulation is the result of translocation of Na(+),K(+)-ATPase from endosomes to the plasma membrane. ASMCs were stretched 20% for 5 min using the Flexercell Strain Unit. The plasma membrane and endosome fractions were isolated and Western blotted to localize the Na(+),K(+)-ATPase alpha-1-subunit protein. Membrane marker enzyme, 5' nucleotidase activity, and the early and recycling endosome markers Rab4 and Rab11 were used to verify the enrichment of these fractions. Stretch increased Na(+),K(+)-ATPase alpha-1 expression in plasma membrane fractions and decreased it in endosomes. PI 3-kinase inhibitors LY294002 and wortmannin blocked the stretch-induced translocation of the Na(+),K(+)-ATPase alpha-1-subunit. Rab4 and Rab11 were enriched in the endosomal fraction, whereas 5' nucleotidase activity was enriched in the plasma membrane fraction. We conclude that stimulation of the Na pump activity by shortterm cyclic stretch is the result, at least in part, of transport of the alpha-subunit of the enzyme from endosomes to the plasma membrane.

Effect of human Angiostatin protein on human angiogenesis in vitro.

Angiostatin, a 38-kD fragment of plasminogen, inhibits angiogenesis in both animal tumor models and in vitro endothelial cell models. However, human Angiostatin has not been tested in vitro against an intact human tissue target to determine its ability to inhibit the initiation or subsequent promotion of the human angiogenic response. We hypothesized that high doses of human Angiostatin would inhibit the development of an angiogenic response in an intact human vessel target, and would suppress the subsequent growth of blood vessels following the initiation of an angiogenic response. To test these hypotheses, full-thickness human placental vein disks were cultured for 15 days in an in vitro fibrin-thrombin clot assay. This assay system had been used to evaluate the efficacy of a wide variety of compounds. Vessels were obtained from three placentas. Treatments included a control medium plus fetal bovine serum (FBS), heparin-steroid (300 micro g/ml heparin and 350 micro g/ml hydrocortisone; a treatment known to inhibit angiogenesis) and Angiostatin at doses from 1 x 10(-4) to 1 x 10(-9) M. In the control groups, 81% of vessels initiated an angiogenic response compared to 53% of the vessels treated with heparin-steroid. Angiostatin (10(-4)-10(-9) M) decreased the initiation of an angiogenic response, but this was not statistically significant. Of the disks that initiated an angiogenic response, the mean ( +/- standard error of the mean (SEM)) semi-quantitative visual angiogenic index (AI) of the control vessels was 9 +/- 1.7 on day 15. In comparison, the mean AI of heparin-steroid treated vessels was 3.7 +/- 0.4. Angiostatin at doses of 10(-4)-10(-9) M also failed to inhibit blood vessel growth after initiation of the angiogenic response. Based on these observations, we cannot demonstrate significant activity of human Angiostatin (10(-4)-10(-9) M) against the initiation or promotion of a human angiogenic response in this in vitro model of angiogenesis using an intact human vessel target.

Inhibition of angiogenic initiation and disruption of newly established human vascular networks by juice from Morinda citrifolia (noni).

noni, the juice of the fruit from the Morinda citrifolia plant, has been used for centuries as a medicinal agent. We tested the effects of noni juice in a three-dimensional fibrin clot matrix model using human placental vein and human breast tumor explants as sources for angiogenic vessel development. Noni in concentrations of 5% (vol/vol) or greater was highly effective in inhibiting the initiation of new vessel sprouts from placental vein explants, compared with initiation in control explants in media supplemented with an equivalent amount of saline. These concentrations of noni were also effective in reducing the growth rate and proliferation of newly developing capillary sprouts. When used at a concentration of 10% in growth media, noni was able to induce vessel degeneration and apoptosis in wells with established capillary networks within a few days of its application. We also found that 10% noni juice in media was an effective inhibitor of capillary initiation in explants from human breast tumors. In tumor explants which did show capillary sprouting, the vessels rapidly degenerated (2-3 days) in those exposed to media supplemented with 10% noni.

Effect of human recombinant Endostatin protein on human angiogenesis.

Tumor growth and metastasis are dependent on the development of new blood vessels. Inhibitors of new vessel growth have been widely investigated as anti-tumor agents. Endostatin, a 20 kDa C-terminal fragment of collagen XVIII inhibits endothelial cell proliferation, induces endothelial cell apoptosis, and can both inhibit and reverse tumor growth in mice. However, human recombinant endostatin has had limited testing against human tissue targets. To investigate the effect of human endostatin on a human vessel target over a broad range of concentrations (10(-l2)-10(-4) M), human placental vein disks were grown for a period of 2 weeks in a 0.3% fibrin clot overlayed with growth medium. Disks from five individual placentas were tested. For each placenta utilized, a control (medium and 20% fetal bovine serum [FBS]) group and a group treated with heparin (300 microg/ml) and hydrocortisone 21-phosphate (350 microg/ml) (heparin-steroid) at a dose known to inhibit angiogenesis were included. Endostatin was tested at concentrations of 10(-12)-10(-4) M in medium containing 20% FBS. The rate of initiation and the angiogenic growth index (on a visually graded semi-quantitative scale of 0-16) were determined for all experimental conditions. Endostatin inhibited angiogenesis in our model only in high concentrations. At 10(-5) M, endostatin did not alter the percent of wells that initiated an angiogenic response, but significantly inhibited subsequent vessel growth. At 10(-4) M, endostatin was able to inhibit both initiation and subsequent new vessel growth. Human endostatin can inhibit the initiation of a human angiogenic response and inhibit the subsequent proliferation of human neovessels when used at high doses in a continuous exposure model.

Population change in adult obesity and blood lipids in American Samoa from 1976-1978 to 1990.

Obesity in American Samoan adults in 1990 was compared to that in 1976-1978 to evaluate population changes concomitant with modernization. Body weight, stature, the body mass index (BMI), and two skinfolds were measured in 1990 in 830 males and females 25-74 years old, and were compared to corresponding data from 1976 and 1978 for 1,621 adults. Mean BMI and skinfold thicknesses increased markedly from 1976-1978 to 1990 in males at all ages. Mean BMI for 45-54 year old males was approximately 3.6 kg/m2 higher (P < 0.0001) in 1990 than in 1976-1978, but was only 0.6 kg/m2 higher in females of the same age. The prevalence of overweight increased significantly from 66% in 1976-1978 to 85% in 1990 (P < 0.001) in 35-44 year old males, but remained about the same, 91%, in females of that age. Similar sex differences in temporal change were found in skinfolds. Fasting serum total and high density lipoprotein (HDL) cholesterol and triglycerides were obtained for a random subsample of 67 males 40-49 years old and were compared to lipid levels in a 1978 sample of American Samoan males of similar age and residence. Both total and HDL cholesterol were significantly different between 1978 and 1990, 178 vs. 205 mg/dl (P < 0.02), and 43 vs. 37 mg/dl (P < 0.01), respectively. Triglycerides were higher in 1990 than in 1978, 169 vs. 128 mg/dl. The results suggest that obesity and adiposity increased more over 12-14 years among adult males than among females, who in 1976-1978 were already massively overweight. © 1993 Wiley-Liss, Inc.