The human immunodeficiency virus (HIV1) protease inhibitor sanquinavir activates autophagy and removes lipids deposited in lipid droplets.

Reduction in mortality and increased average life span of the human immunodeficiency virus (HIV)-infected patients treated with antiretroviral therapy (ART) are associated with the risk of unwanted effects, such as insulin resistance and dyslipidemia with cardiovascular complications. Antiretroviral therapy may also be associated with lipodystrophy characterized as peripheral lipoatrophy with central fat accumulation. Understanding the molecular mechanisms of lipodystrophy caused by ART is important for therapeutic strategy and the prediction of side-effects. Influence of protease inhibitor saquinavir (SQV) on preadipocyte differentiation was analyzed in in vitro human Chub-S7 cell line model. For measurement of the effects of SQV the drug was added to differentiated or non-differentiated cells. The influence of SQV on changes in the profile of gene expression was verified by microarray and changes in lipid species content were analyzed using GC-MS/MS. Results were confirmed by real-time PCR and analysis of autophagy. Addition of SQV to differentiated Chub-S7 cells lead to removal of lipids deposited in lipid droplets, down-regulation of expression of transcription factors and markers of adipocyte differentiation. Antiviral activity of SQV based on its non-selective inhibition of proteases resulted in proteasome inhibition, induction of endoplasmic reticulum stress and induction of macroautophagy. This activity was accompanied by an increase in PI, PEPL, PC lipid species especially with MUFA and PUFA. Additionally up-regulation of miR-100-3p, miR-222-5p, miR-483-5p were found, which correlated with obesity, insulin resistance, increasing insulin secretion and activation of lipolysis. Our results indicated that SQV, by inhibition of proteasome protein degradation, activated the unfolded protein response resulting in autophagic breakdown of lipids deposited in adipose tissue causing lipodystrophy.

Melatonin metabolite, N(1)-acetyl-N(1)-formyl-5-methoxykynuramine (AFMK), attenuates acute pancreatitis in the rat: in vivo and in vitro studies.

Melatonin protects the pancreas from inflammation and free radical damage but the effect of the melatonin metabolite: N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) on acute pancreatitis is unknown. This study assessed the effects of AFMK on acute pancreatitis (AP) in the rats in vivo and on pancreatic cell line AR42J in vitro. AFMK (5, 10 or 20 mg/kg) was given intraperitoneally to the rats 30 min prior to the induction of AP by subcutaneous caerulein infusion (25 µg/kg). Lipid peroxidation products (MDA + 4-HNE) and the activity of an antioxidant enzyme glutathione peroxidase (GPx) were measured in pancreatic tissue. Blood samples were taken for evaluation of amylase activity and TNF-α concentration. GPx, TNF-α, proapoptotic Bax protein, antiapoptotic Bcl-2 protein and the executor of apoptosis, caspase-3, were determined by Western blot in AR42J cells subjected to AFMK or to melatonin (both used at 10(-12), 10(-10), or 10(-8)M), without or with addition of caerulein (10(-8)M). AP was confirmed by histological examination and by serum increases of amylase and TNF-α (by 800% and 300%, respectively). In AP rats, pancreatic MDA + 4-HNE levels were increased by 300%, whereas GPx was reduced by 50%. AFMK significantly diminished histological manifestations of AP, decreased serum amylase activity and TNF-α concentrations, reduced MDA + 4-HNE levels and augmented GPx in the pancreas of AP rats. In AR42J cells, AFMK combined with caerulein markedly increased protein signals for GPx, Bax, caspase-3 and reduced these for TNF-α and Bcl-2. In conclusion, AFMK significantly attenuated acute pancreatitis in the rat. This may relate to the antioxidative and anti-inflammatory effects of this molecule and possibly to the stimulation of proapoptotic signal transduction pathway.

Human adipose tissue stromal vascular fraction cells differentiate depending on distinct types of media.

OBJECTIVES: Angiogenesis, the process of formation of blood vessels, is essential for many physiological as well as pathological processes. It has been shown that human adipose tissue contains a population of non-characterized cells, called stromal-vascular fraction (SVF) cells, which are able to differentiate into several lineages. The aim of this study was to determine conditions for promoting differentiation of human adipose tissue progenitors towards endothelial cells, as well as to show that SVF cells cooperate with differentiated endothelium in capillary network formation. MATERIALS AND METHODS: Stromal vascular fraction cells were isolated according to modified Hauner's method and after adaptation they were cultured in pro-angiogenic or pro-adipogenic medium. Cells were characterized by presence of surface antigens by flow cytometry, and by expression of genes characteristic for endothelial cells or for adipocytes, quantitative real-time polymerase chain reaction. A number of tests were performed to verify their differentiation. RESULTS: Differentiation of human SVF cells towards endothelium was stimulated by the presence of serum and absence of adipogenic factors, documented by the pattern of gene expression as well as different functional in vitro assays. SVF cells were found to work together with human umbilical vein endothelial cells to form capillary networks. CONCLUSIONS: Here, we show that differentiation of SVF cells to endothelial cells or adipocyte-like cells depended on the medium used. Our work provides a clear model for analysing the differentiation capacity of SVF cells.

The Wagner revision stem in alloarthroplasty of the hip.

Forty-one Wagner revision stems were implanted at the Orthopedic Department of the University of Tübingen between July 1990 and January 1993. We report the results of 37 patients at an average follow-up of 27 months (13-48 months) postoperatively. The main indication was stem loosening with considerable loss of bone. In addition, we used the implant 4 times in primary arthroplasty. At follow-up examination 33 patients (89%) were satisfied with the postoperative outcome. According to the Merle D'Aubigné score (12-point scale), 32 patients showed a poor functional result of less than 6 points preoperatively. Postoperatively, the results of 36 patients could be classified as very good to good. To categorise the radiological destruction of the implant bed, we used the femoral shaft defect classification of the DGOT (Deutsche Gesellschaft für Orthopädie und Traumatologie) in conjunction with the classification of Pak and Paproski [5, 11]. Twenty patients presented with trochanteric and calcar defects, and 11 patients with a combination of a calcar and shaft defect. We found a circular shaft defect in 2 patients. In 7 cases we assessed the bone remodelling postoperatively as very good, with strong newly formed bone structures, and in 25 cases as good, with remodelling of the old stem bed and bony structuring of the osteolyses. A secondary sinking in of the Wagner stem was seen in 7 cases. Only one stem had to be revised because of pain symptoms and loosening; in all other cases a secondary stabilisation of the revision-stem took place. With the Wagner revision stem, there is the possibility of achieving mechanical stability even in situations with massive bone loss. The evacuation of bone cement and granulation tissues is facilitated by the transfemoral approach, bony remodelling is accelerated, and bone grafting is often not necessary. As our short-term results show, the concept is a promising one. Nevertheless, we will be very careful in following these patients in the long term, as we have noticed stem sinkage in a small percentage of cases.