Antiphospholipid and antioangiogenic activity in females with recurrent miscarriage and antiphospholipid syndrome.

Background Antiphospholipid syndrome is an autoimmune disease characterized by thrombosis, fetal losses and thrombocytopenia associated to antiphospholipid antibodies. They are directed to phospholipids, such as cardiolipins (anticardiolipin) and lupus anticoagulant or to complexes formed by phospholipids and protein cofactors, such as ß2 glycoprotein 1 (a-ß2GP1) and annexin V (a-annexin V). These auto-antibodies may be considered as a family of antibodies involved in thrombotic events and antiphospholipid activity. On the other hand, some proangiogenic factors are involved in the normal development of placental vasculature, such as the vascular endothelial growth factor. Overexpression of vascular endothelial growth factor receptor in its soluble form (sVEGFR-1) has been associated to a higher antiangiogenic activity. Our aim was to analyse the association between anticardiolipin, lupus anticoagulant, a-ß2GP1, a-annexin V and sVEGFR-1 with recurrent miscarriage before week 10 of gestation in females with antiphospholipid syndrome. Methods We studied 24 females (primary or secondary antiphospholipid syndrome), who were divided into two groups: females with recurrent miscarriage before week 10 of gestation (M; n = 12) and females with no history of fetal loss (NM; n = 12). Anticardiolipin, a-ß2GP1, a-annexin V and sVEGF-R1 concentrations were assessed by ELISA, while lupus anticoagulant was assessed by screening and confirmatory tests. Results A significant association was observed between the number of positive biomarkers and the belonging group ( P < 0.05). Besides, a positive result for lupus anticoagulant and a-ß2GP1 was found to be significantly associated to the M group ( P < 0.05). Conclusions Lupus anticoagulant and a-ß2GP1 may be implicated in pregnancies complicated by recurrent miscarriage in females with antiphospholipid syndrome.

Reference genes for real-time PCR quantification of microRNAs and messenger RNAs in rat models of hepatotoxicity.

Hepatotoxicity is associated with major changes in liver gene expression induced by xenobiotic exposure. Understanding the underlying mechanisms is critical for its clinical diagnosis and treatment. MicroRNAs are key regulators of gene expression that control mRNA stability and translation, during normal development and pathology. The canonical technique to measure gene transcript levels is Real-Time qPCR, which has been successfully modified to determine the levels of microRNAs as well. However, in order to obtain accurate data in a multi-step method like RT-qPCR, the normalization with endogenous, stably expressed reference genes is mandatory. Since the expression stability of candidate reference genes varies greatly depending on experimental factors, the aim of our study was to identify a combination of genes for optimal normalization of microRNA and mRNA qPCR expression data in experimental models of acute hepatotoxicity. Rats were treated with four traditional hepatotoxins: acetaminophen, carbon tetrachloride, D-galactosamine and thioacetamide, and the liver expression levels of two groups of candidate reference genes, one for microRNA and the other for mRNA normalization, were determined by RT-qPCR in compliance with the MIQE guidelines. In the present study, we report that traditional reference genes such as U6 spliceosomal RNA, Beta Actin and Glyceraldehyde-3P-dehydrogenase altered their expression in response to classic hepatotoxins and therefore cannot be used as reference genes in hepatotoxicity studies. Stability rankings of candidate reference genes, considering only those that did not alter their expression, were determined using geNorm, NormFinder and BestKeeper software packages. The potential candidates whose measurements were stable were further tested in different combinations to find the optimal set of reference genes that accurately determine mRNA and miRNA levels. Finally, the combination of MicroRNA-16/5S Ribosomal RNA and Beta 2 Microglobulin/18S Ribosomal RNA were validated as optimal reference genes for microRNA and mRNA quantification, respectively, in rat models of acute hepatotoxicity.

The eSS rat, a nonobese model of disordered glucose and lipid metabolism and fatty liver.

BACKGROUND: eSS is a rat model of type 2 diabetes characterized by fasting hyperglycemia, glucose intolerance, hyperinsulinemia and early hypertriglyceridemia. Diabetic symptoms worsen during the second year of life as insulin release decreases. In 12-month-old males a diffuse hepatic steatosis was detected. We report the disturbances of lipid metabolism of the model with regard to the diabetic syndrome. METHODS: The study was conducted in eight 12-month-old eSS male rats and seven age/weight matched eumetabolic Wistar rats fed with a complete commercial diet al libitum. Fasting plasmatic glucose, insulin, triglycerides, total cholesterol, low-density and high-density lipoprotein, and nonesterified fatty acids levels were measured. Very low density and intermediate-density lipoproteins were analyzed and hepatic lipase activity was determined. RESULTS: eSS rats developed hyperglycemia and hyperinsulinemia, indicating insulin resistance. Compared with controls, diabetic rats exhibited high plasmatic levels of NEFA, triglycerides (TG), total cholesterol (Chol) and LDL-Chol while high-density lipoprotein (HDL) cholesterol values were reduced. eSS rats also displayed TG-rich VLDL and IDL particles without changes in hepatic lipase activity. CONCLUSION: The nonobese eSS rats develop a syndrome characterized by glucose and lipid disorders and hepatic steatosis that may provide new opportunities for studying the pathogenesis of human type 2 diabetes.

Novel experimental effects on bone material properties and the pre- and postyield behavior of bones may be independent of bone mineralization.

In this article, we summarize the results of six different tomographic/biomechanical rat studies involving hypophysectomy (Hx), ovariectomy, treatment with rhGH, olpadronate, alendronate, and toxic doses of aluminum and the development of a genetic diabetes in the eSS strain. All these conditions induced some interesting and rarely reported effects on postyield bone strength. These effects were generally related neither to the degree of mineralization or the elastic modulus of the bone tissue nor to the preyield behavior of the bones. In two particular cases (Hx, eSS), the elastic modulus of bone tissue varied independently of its degree of mineralization. These results suggest the involvement of some microstructural factor(s) of bone tissue resistance to crack progression (a postyield feature of bone behavior), rather than to crack initiation (the yield-determining factor) in the corresponding mechanism. Changes in collagen or crystal structure may play that role. These changes are relevant to the mechanism of fracture production during plastic deformation, a feature of bone strength that might be independent from mineralization. Therefore, these changes might help to explain some effects of novel treatments on bone strength unrelated to bone mineralization. This questions the belief that the remaining bone mass in metabolic osteopenias is biologically and mechanically normal.