Dynamics of hepatitis B virus quasispecies heterogeneity in association with nucleos(t)ide analogue treatment determined by MALDI-TOF MS.

Minor drug-resistant variants may preexist in every subject infected with hepatitis B virus (HBV). However, understanding the dynamic of genotypic evolution within the HBV population requires accurately following allele frequencies through time. We used MALDI-TOF MS (matrix-assisted laser desorption-ionization time-of-flight mass spectrometry) for localization and quantitative allele frequency detection to investigate preexisting HBV quasispecies and the genotypic evolution of drug-resistant variants during nucleos(t)ide analogue therapy. We found a significant difference between the genotypic evolution of drug-resistant variants depending on response to treatment.

Increased circulating endothelial progenitor cells in patients with haemorrhagic and ischaemic stroke: the role of endothelin-1.

Ischaemic stroke induces endothelial progenitor cell (EPC) mobilisation from bone marrow into peripheral blood. Circulating EPCs play an important role in post-injury regeneration of vasculature, whereas endothelial cells (ECs) have been shown to reflect endothelial damage and may be responsible for increased Endothelin-1 (ET-1) expression. We investigated herein the association between numbers of circulating ECs and EPCs, the levels of soluble factors regulating their migration and function, and the clinical outcome in patients with haemorrhagic (HS) or ischaemic stroke (IS). Sixteen patients with HS and eighteen with IS were assessed during the first 24h, day 3, and day 7 after stroke and compared them with twenty-three control subjects. We found elevated EPC and EC concentrations using flow cytometry and increase in VEGF, SDF-1, HGF, and ET-1 plasma levels by ELISA in the HS patients, while ET-1 mRNA expression in peripheral blood cells was elevated in the IS patients. Significant correlations were observed between EPCs or ECs and Big ET-1 protein or mRNA levels in HS but not in the IS patients. We suggest that ET-1 may play a role in pathophysiology of stroke and subsequent EPC mobilisation; however, further studies aimed at the precise elucidation of this issue are required.

Prostaglandin E2 (PGE2) and thromboxane A2 (TXA2) synthesis is regulated by conjugated linoleic acids (CLA) in human macrophages.

Conjugated linoleic acid (CLAs) are positional and geometric isomers of linoleic acid with have a potential anti-atherosclerotic and anti-inflammation properties. Metabolites of arachidonic acid--prostaglandins and thromboxans--are endogenous mediators of inflammation. Prostaglandin E(2) and thromboxan A(2) which are a products of two izoformes of cyclooxygenases (COX-1 and COX-2) in macrophages, play an important role in this process. COX-1 is a constitutive enzyme, whereas the COX-2 is inducible and its amount in the cell rapidly increases during inflammation (e.g. via NF kappaB pathway). The aim of the study was to test the effect of CLAs on cyclooxygenases (COX-1 and COX-2) activity, their mRNA expression and protein content in macrophages. Additionally the active form of the kappaB (NF kappaB) transcription factor was measured. For the experiments monocytes from monocytic cell line (THP-1) and from human venous blood were used. Monocytes were differentiated to macrophages and cultured with 30 muM CLAs or linoleic acid for 48 h. The COX-1 and COX-2 products - PGE(2) and TXB(2), were measured by ELISA method. The enzymes (COX-s) activity were estimated by spectroscopic method. mRNA expression and protein analysis were analysed by real-time PCR and Western blot technique. In macrophages cultured with CLAs reduction of TXB(2) and PGE(2) concentration was observed. Significant change in COX-2 expression in cells cultured with trans-10, cis-12 CLA (in macrophages obtained from peripheral blood) was observed. COX-1 inhibition was resulting from competition of CLA and linoleic acid with arachidonic acid.

The effect of beta carotene supplementation on the beta carotene and vitamin A levels of blood plasma and some fertility indices of dairy cows.

Seasonal variations in beta carotene intake and low levels of beta carotene and vitamin A in blood plasma were found in cows fed ration based mainly on maize silage. The supplementation of daily winter rations with 300 mg Rovimix-beta carotene per cow, beginning 14 days before parturition and 60 days after calving increased slighty the beta carotene and vitamin A concentrations of blood plasma and improved some fertility indices: the number of inseminations per cow was reduced and the percentage of conception rate was significantly higher. It may be assumed that beta carotene content in feeds and it's utilization rate generally reflected in beta carotene blood plasma level and in the improvement of fertility.

Immunogenicity of methylcholanthrene-induced tumors tested by draining lymph node assay in syngeneic rats.

The immunogenic properties of MC-induced and not metastasizing tumors were tested in inbred Wistar and August strains by the aid of lymphocyte stimulation in syngeneic draining lymph nodes in the comparison with the opposite ones. The lymphocyte stimulation was estimated in vitro by the spontaneous incorporation of 3HTdR. It was found that the peak of the first response of draining lymph nodes was noted on the 7th day after injection of syngeneic tumor cells. This antitumor response was specific because: 1. the second immunological response of draining lymph nodes to the same tumor cells developed earlier and was higher than the first response and 2. the rats previously immunized with MC-induced tumor, and then challenged with syngeneic sarcoma or syngeneic fibroblasts exhibited the lack of the second response and only some cross-reactivity was observed by the use of the other MC-induced sarcoma. MC-induced tumors appeared earlier and more frequently in August than in Wistar strain, the level of syngeneic draining lymph node response to immunogenic tumors was higher in August than in Wistar rats despite of the lack of difference between the percentages of immunogenic tumors arising in these two rat strains.