congresos y reuniones científicas
Differential response to VIP in non pregnant and pregnant prediabetic nod mice
Congreso; Annual European Congress of Rheumatology; 2006
Background Non obese diabetic (NOD) mice offer a suitable model to study the modulatory role of cytokines in the uterus signalling and function since in the prediabetic stage they develop a Th1 response against exocrine glands similar to Sjögren?s syndrome. Among the factors that contribute to the success of embryonic implantation and pregnancy are progesterone levels, a Th2 cytokine profile and a suitable balance between nitric oxide (NO) and prostaglandins (PG) in the uterus (8, 6). VIP is a neuroimmunopeptide that promotes secretion in glandular epithelium, contributes to vasodilatation in the uterus through NO production (1; 4) and promotes Th2 cytokine and anti-inflammatory responses (2). Objectives: The aim of the present study was to investigate the effect of VIP on NO and PGE 2 production in the uterus and NO and cytokines in macrophages of NOD mice with an emerging Th1 cytokine response. Methods: NOD and BALB/c female mice were bred and maintained in the Central Animal Care facility at the School of Exact and Natural Sciences, University of Buenos Aires. Normally cycling non-pregnant and 8 days pregnant mice of 15-16 weeks were fasted overnight with water ad libitum before sacrificed and tissues and blood were obtained and processed immediately after. All studies were conducted according to standard protocols of the Animal Care and Use Committee of the School of Exact and Natural Sciences, University of Buenos Aires. Nitric oxide synthase (NOS) activity was measured in uteri using L- [U-14C]-arginine as substrate as described earlier (7). NOS activity was calculated as total activity minus that measured in the presence of 500 µM L-NMMA. When used, VIP was added to one horn in the last 15 min. PGE2 was quantified by specific radioimmunoassay using rabbit antiserum as previously described (3). Results were expressed as ng of PG synthesized during 1 h/mg protein. When used, VIP was added from the beginning of the incubation. Purified peritoneal macrophages (0.5 x 106 cells/well) were cultured with RPMI 1640 medium, LPS (10 µg/ml), INF g (100 U/ml) in the presence or absence of VIP (10-7 M) for 24h at 37 °C. NO produced by macrophages was measured as nitrite (NO2-) in the culture supernatant using the Griess method. The concentration of NO2- was deduced from a standard nitrate curve. Results were expressed as µmol/106 cells/24h. Cytokines from serum, tissues and macrophage supernatant were determined with a capture ELISA assay as previously described (5). Statistical significance of differences was determined by the two-tailed t test for independent populations. When multiple comparisons were necessary, the Student-Newman-Keuls test was used after analysis of variance. Differences between groups were considered significant at P