GSNO attenuates EAE disease by S-nitrosylation-mediated modulation of endothelial-monocyte interactions

S-Nitrosoglutathione (GSNO) is an endogenous nitric oxide carrier and recently, has been documented for its anti-inflammatory effects in rat model of cerebral ischemia (Khan et al. (2005) J Cereb Blood Flow Metab 25:177-192). Here, we explored the neuroprotective effects mediated by GSNO in Lewis rat model of EAE and its mechanism of action using in vitro model of monocyte-endothelial cell interaction. Oral administration of GSNO attenuated the clinical disease course in EAE animals by inhibiting the infiltration of vascular immune cells in the CNS that subsequently led to the reduction in the expression of proinflammatory cytokines and consequently limited demyelination. Based on the inhibition in infiltration of immune cells, we hypothesized that GSNO modulated endothelial cell activation that led to reduce cellular infiltration in the CNS. Using an in vitro model, we established that GSNO inhibited monocyte adhesion to the activated endothelial cell, which was mediated by down regulation of endothelial cell adhesion molecules (CAMs). The mechanism by which GSNO modulated CAMs expression appeared to be via S-nitrosylation of p65, which consequently inhibited nuclear factor kappa B (NF-kappaB) activation in endothelial cells. These observations suggest that GSNO exerts its protective effects in EAE by inhibition of cellular infiltration into the CNS by S-nitrosylation of p65, thereby modulating NF-kappaB-CAMs pathway in endothelial cells.     

Interspecies difference in liver-specific functions and biotransformation of testosterone of primary rat,porcine and human hepatocyte in an organotypical sandwich culture

Interspecies difference is an important issue in toxicology research. We compared the potential in vitro metabolism of human, porcine and rat hepatocytes over 2 weeks in culture in an organotypical culture model which reflects the in vivo situation. All three species show similar LDH-rates. Albumin measurements showed that rat cells are about twice as active as human and porcine hepatocytes. The ethoxyresorufin-O-deethylase (EROD) activity of the rat hepatocytes is with about 14 μU/10⁶ cells distinctly higher than those of porcine and human cells (1.8 and 0.5 μU/10⁶ cells respectively), furthermore, the activity of the rat EROD increases slightly during the prolonged time in culture, whereas those of porcine and human enzymes slightly decrease. Concerning ethoxycoumarin-O-deethylase (ECOD), the enzyme activities are found to be in three different ranges where rat cells show the highest activity with 66 μU/10⁶ cells, porcine hepatocytes exhibit an activity of about 23 μU/10⁶ cells, and human activity is lowest with 0.7 μU/10⁶ cells. All three species show a similar decreasing trend of ECOD during the period of study. Regarding the biotransformation of testosterone, human and porcine liver cells form three major metabolites whereas rat cells form a mixture of all measured metabolites. Hence, in vitro metabolism using porcine hepatocytes would be much more scientific sense than one using rat hepatocytes since the metabolic pathways are much closer to human metabolism.     

Assessing the safety of post-exposure rabies immunization in pregnancy

Fourteen pregnant women who received rabies post-exposure prophylaxis (PEP) at the anti-rabies clinic (ARC) of Kempegowda Institute of Medical Sciences (KIMS) were followed up for assessing the safety of modern rabies vaccines and equine rabies immunoglobulin (ERIG) in pregnancy. The women were in the age range of 18-28 years, mostly from urban area (64%) and exposed to suspect rabid dogs (86%). They had received purified vero cell rabies vaccine (Verorab = 8 and Abhayrab = 4), purified chick embryo cell vaccine (Rabipur = 2) by Essen regimen; and equine rabies immunoglobulin (Equirab = 7 and Pasteur anti-rabies serum = 1). None of the pregnant women reported any adverse events to either vaccine or equine rabies immunoglobulin. All had safe vaginal deliveries and in all cases both the mother and the child were found to be healthy and normal.     

The metabolomics of (+/-)-arecoline 1-oxide in the mouse and its formation by human flavin-containing monooxygenases

The alkaloid arecoline is a main constituent of areca nuts that are chewed by approximately 600 million persons worldwide. A principal metabolite of arecoline is arecoline 1-oxide whose metabolism has been poorly studied. To redress this, synthetic (+/-)-arecoline 1-oxide was administered to mice (20mg/kg p.o.) and a metabolomic study performed on 0-12h urine using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-TOFMS) with multivariate data analysis. A total of 16 mass/retention time pairs yielded 13 metabolites of (+/-)-arecoline 1-oxide, most of them novel. Identity of metabolites was confirmed by tandem mass spectrometry. The principal pathways of metabolism of (+/-)-arecoline 1-oxide were mercapturic acid formation, with catabolism to mercaptan and methylmercaptan metabolites, apparent CC double-bond reduction, carboxylic acid reduction to the aldehyde (a novel pathway in mammals), N-oxide reduction, and de-esterification. Relative percentages of metabolites were determined directly from the metabolomic data. Approximately, 50% of the urinary metabolites corresponded to unchanged (+/-)-arecoline 1-oxide, 25% to other N-oxide metabolites, while approximately, 30% corresponded to mercapturic acids or their metabolites. Many metabolites, principally mercapturic acids and their derivatives, were excreted as diastereomers that could be resolved by UPLC-TOFMS. Arecoline was converted to arecoline 1-oxide in vitro by human flavin-containing monooxygenases FMO1 (K(M): 13.6+/-4.9muM; V(MAX): 0.114+/-0.01nmolmin(-1)microg(-1) protein) and FMO3 (K(M): 44.5+/-8.0microM; V(MAX): 0.014+/-0.001nmolmin(-1)microg(-1) protein), but not by FMO5 or any of 11 human cytochromes P450. This report underscores the power of metabolomics in drug metabolite mining.     

Clinicopathologic Features and Long-Term Outcomes of 293 Phyllodes Tumors of the Breast

BACKGROUND: Phyllodes tumors (PT) are rare fibroepithelial neoplasms of the breast with unpredictable behavior. We reviewed our single institution experience with PT over 51 years to identify factors predictive of local recurrence (LR) and metastasis. METHODS: From 1954 to 2005, a total of 352 cases of PT were identified; 293 had follow-up. All available pathology slides (90%) were rereviewed for margins, borders, fibroproliferation in the surrounding breast tissue, stromal pattern, stromal cellularity, frequency of mitoses, and necrosis. RESULTS: All cases occurred in women, with a median age of 42, with 203 originally categorized as benign and 90 as malignant. Median follow-up was 7.9 years. A total of 35 patients developed LR at a median of 2 years. In univariate analyses, a higher actuarial LR rate was associated with positive margins (P = .04), fibroproliferation (P = .001), and necrosis (P = .006). PT classified as malignant did not have a higher risk of LR (P = .79). Five patients developed distant disease at a median of 1.2 years. These patients constituted 71% of the seven patients who had uniformly aggressive pathologic features, including large tumor size (>or=7.0 cm), infiltrative borders, marked stromal overgrowth, marked stromal cellularity, high mitotic count, and necrosis. CONCLUSIONS: Positive margins, fibroproliferation in the surrounding breast tissue, and necrosis are associated with a marked increase in LR rates. Efforts should be made to achieve negative surgical margins to reduce risk of LR. Death from PT is rare (2%), and only PT that demonstrate uniformly aggressive pathologic features seem to be associated with mortality.