Evaluation of antifibrotic drugs in bleomycin-induced pulmonary fibrosis in hamsters

Endotracheal administration of bleomycin to hamsters causes severe pulmonary fibrosis. We have examined the utility of this response as a model for the screening of pulmonary antifibrotic agents. The time course of collagen synthesis after bleomycin administration was examined in neutral salt soluble and insoluble fractions by in vitro incubation of minced lung with [14C] proline. Collagen synthesis increased to approximately 250% above control in both neutral salt soluble and insoluble collagen fractions by day 6 after bleomycin. Noncollagenous protein synthesis was also increased but to a lesser amount. The early rise in collagen synthesis leads to accumulation of collagen that can be biochemically quantitated within 1 week. This time course is advantageous for short-term testing of antifibrotic agents. In the present study, beta-aminopropionitrile, D-penicillamine, and p-aminobenzoic acid were examined. All three agents were found to reduce significantly the accumulation of neutral salt insoluble collagen in bleomycin-treated animals.     

Fatty acid composition of erythrocyte phospholipids is related to insulin levels, secretion and resistance in obese type 2 diabetics on Metformin

BACKGROUND: Relationships between fatty acids in erythrocyte phospholipids and insulin parameters have been described in healthy and overweight individuals, but not in obese diabetics. We assessed whether erythrocyte phospholipid fatty acids are related to insulin parameters in obese type 2 diabetics on Metformin. METHODS: In 23 diabetics, the fractions of the different fatty acids in erythrocyte phospholipids were correlated with insulin levels, secretion, sensitivity, resistance and insulinemic response following a standardised breakfast. RESULTS: Fasting insulin levels and insulin resistance correlated positively with the fraction of alpha-linolenic and dihomo-gamma-linolenic acid and with the ratios of stearic to palmitic and dihomo-gamma-linolenic to linoleic acid and negatively with the fraction of palmitic acid in erythrocyte phospholipids. Insulin secretion correlated negatively with the fraction of palmitic acid. For this parameter, a positive correlation was also found with the sum of uneven fatty acids. Insulinemic response following a meal was negatively associated with the fraction of oleic acid in erythrocyte phospholipids. Insulin sensitivity did not correlate with erythrocyte fatty acids. CONCLUSIONS: The relationships found differ from those described in healthy and overweight individuals and may be characteristic for type 2 diabetics. They concur with the recommendations that saturated fat intake should be reduced and monounsaturated increased.     

In vivo persistence of retrovirally transduced murine long-term repopulating cells is not limited by expression of foreign gene products in the fully or minimally myeloablated setting

Many nonmalignant hematologic disorders could potentially be treated by genetic correction of as few as 5-10% of target lineage cells. However, immune system clearance of cells expressing gene products perceived as foreign could be limiting. There is evidence that tolerance to foreign proteins can result when myeloablative conditioning is used, but this limits the overall applicability of such techniques. Therefore, we sought to evaluate the engraftment of hematopoietic stem cells carrying a foreign transgene after low-dose irradiation by comparing in vivo survival of murine long-term repopulating cells (LTRC) transduced with either a retroviral vector expressing the bacterial neomycin phosphotransferase gene (neo) or a vector containing neo gene sequences but modified to prevent protein expression (nonexpression). First, marrow cells from congenic donors were transduced with either vector and transplanted into recipients treated with standard dose irradiation of 800 rads. High-level engraftment and gene marking resulted, without differences in the marking levels or pattern of persistence of the cells between cells transduced with either vector. Low-dose irradiation at 100 rads was tested using higher cell doses. Marking levels as high as 10% overall were obtained, again with no differences between mice receiving cells transduced with the neo versus the nonexpression vectors. To investigate a potentially more immunogenic protein, marrow cells were transduced with a vector containing the green fluorescent protein (GFP) gene, and their persistence was studied in recipient mice receiving 100 rads. Stable GFP expression in 5-10% of circulating cells was observed long term. We conclude that even with very low dose conditioning, engraftment by genetically modified LTRC cells at clinically significant levels can be achieved without evidence for clearance of cells known to be expressing immunogenic proteins.     

Multiple comparisons model-based clustering and ternary pattern tree numerical display of gene response to treatment: procedure and application to the preclinical evaluation of chemopreventive agents

Microarray technology has greatly aided the identification of genes that are expressed differentially. Statistical analysis of such data by multiple comparisons procedures has been slow to develop, in part, because methods to cluster the results of such comparisons in biologically meaningful ways have not been available. We isolated and analyzed, by Northern blot and GeneChip, replicate liver RNA samples (n = 4/group) from rats fed with control diet or diet containing one of three chemopreventive compounds, selected because their pharmacological activities, including RNA expression response, are relatively well understood. We report on a classification tree, based on the results of nonparametric multiple comparisons, which results in the bipolar hierarchical clustering of genes in relation to their response to treatment. In addition to identifying treatment-responsive genes, application of this procedure to our test study identified the known pharmacological relationships among the treatment groups without supervision. Also, small treatment-specific subsets of genes were identified that may be indicative of additional pharmacophores present in the test compounds.     

The Val432Leu polymorphism of the CYP1B1 gene is associated with differences in estrogen metabolism and bone density

Polymorphisms of the CYP450 genes that encode for the enzymes that metabolize estrogen are linked to hormone-related cancers. We investigated the impact of two polymorphisms of the CYP1B1 gene previously reported to be associated with hormone-related disorders on estrogen metabolism and bone mineral density (BMD), another hormone-dependent condition, in women from different ethnic backgrounds. Four hundred sixty-eight postmenopausal Caucasian women, 220 from St. Louis, MO, USA (mean age=63.5+/-0.53 years) and 248 from Palermo, Italy (mean age=72.9+/-0.44 years) participated in the study. Measurements of urinary estrogen metabolites by enzyme-linked immunoassay, serum estradiol by ultrasensitive radioimmnunoassay, and serum sex hormone-binding globulin by immunoradiometric assay were performed only in the American women, while BMD by dual energy X-ray absorptiometry and genotyping by pyrosequencing were performed in both American and Italian women. Differences in the levels of metabolites, free estradiol index and BMD were analyzed by analysis of covariance. Analysis among the American participants for the Valine432Leucine polymorphism showed that, compared to women with the Val/Val genotype, women with the Leu allele (Val/Leu and Leu/Leu) had significantly higher log-transformed values of total urinary estrogen metabolite (ng/mg-creatinine) levels (1.23+/-0.04, 1.35+/-0.02, and 1.34+/-0.03; p=0.03), and significantly lower BMD (gm/cm(2)) in the lumbar spine (1.009+/-0.02, 0.955+/-0.01 and 0.931+/-0.02; p=0.03) and the femoral neck (0.748+/-0.02, 0.717+/-0.01 and 0.693+/-001, p=0.03) for the Val/Val, Val/Leu and Leu/Leu genotypes respectively. There were no significant differences in the urinary metabolites and BMD in the different genotypes for the Alanine119Serine polymorphism among the American women. Meanwhile, a separate analysis among the Italian women revealed no significant differences in BMD among the different genotypes for the two polymorphisms investigated. In conclusion, women with the Leu allele for the CYP1B1 Val432polymorphism have increased estrogen catabolism, as indicated by higher urinary estrogen metabolites, compared to those with Val/Val genotype. This may lead to relative hypoestrogenism and lower BMD in the lumbar spine and femoral neck in these women. Our data suggest that through its effect on the rate of estrogen catabolism, the Val432Leu polymorphism of the CYP1B1 gene may represent as a possible genetic risk factor for osteoporosis in American women.     

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.