Detection of annexin A8 antibodies in serum of patients with antiphospholipid syndrome

Introduction: Antibodies specific for annexin A8 (AnxA8) have not been investigated in patients suffering from antiphospholipid syndrome (APS) yet. The aim of this study was to compare the presence of AnxA8 antibodies in serum of APS patients with that of age-matched healthy controls and to investigate whether AnxA8 antibodies are potential biomarkers for APS. Materials and methods: We enrolled 22 APS patients and 22 healthy controls in this case-control study. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblot to investigate the presence of AnxA8 antibodies, and we applied enzyme-linked immunosorbent assay to investigate the presence of cardiolipin (CL) and beta-2-glycoprotein I (ß2GPI) antibodies. Results: The serum of 9/22 APS patients showed AnxA8 IgG isotype antibody reactivity compared to serum of 2/22 healthy controls (P = 0.034). When we also included weak immunoblot signals, 12/22 APS patients exhibited AnxA8 IgG isotype antibody reactivity compared to 3/22 healthy controls (P = 0.005). We also investigated the presence of AnxA8 IgM isotype antibodies in the serum of APS patients but found no statistically significant difference between the APS patient group and healthy control group (P = 0.500). We further investigated the presence of ß2GPI and CL IgG and IgM isotype antibodies. AnxA8 IgG isotype antibodies were present in APS patients in a similar frequency as the APS “criteria” antibody against CL (P = 0.764). Conclusion: We demonstrated that AnxA8 IgG isotype antibodies are potential biomarkers for the diagnosis of APS

Up-Regulation of the Cardiac Lipid Metabolism at the Onset of Heart Failure

Chronic pressure overload and atherosclerosis are primary etiologic factors for cardiac hypertrophy and failure. However, mechanisms underlying the transition from hypertrophy to heart failure are incompletely understood. We analyzed the development of heart failure in mice with chronic pressure overload induced by aortic constriction and compared the results with aged apolipoprotein E-deficient mice suffering from advanced atherosclerosis. We combined cardiac function analysis by echocardiography and invasive hemodynamics with a comprehensive microarray gene expression study (GSE25765-8). The microarray data showed that the onset of heart failure induced by pressure overload or advanced atherosclerosis was accompanied by a strong up-regulation of key lipid metabolizing enzymes involved in fat synthesis, storage and oxidation. Cardiac lipid overload may be involved in the progression of heart failure by enhancing cardiomyocyte death. Up-regulation of the cardiac lipid metabolism was related to oxygen and ATP depletion of failing hearts because anti-ischemic treatment with ranolazine normalized the cardiac lipid metabolism and improved cardiac function. Vice versa, inhibition of cellular respiration and ATP generation by mild thiol-blocking with cystamine triggered the cardiac lipid metabolism and caused signs of heart failure. Cardiac tissue specimens of patients with heart failure also showed high protein levels of key fat metabolizing enzymes as well as lipid accumulation. Taken together, our data strongly indicate that up-regulation of the cardiac lipid metabolism and myocardial lipid overload are underlying the development of heart failure

Gene expression analysis of nuclear factor I-A deficient mice indicates delayed brain maturation

BACKGROUND: Nuclear factor I-A (NFI-A), a phylogenetically conserved transcription/replication protein, plays a crucial role in mouse brain development. Previous studies have shown that disruption of the Nfia gene in mice leads to perinatal lethality, corpus callosum agenesis, and hydrocephalus. RESULTS: To identify potential NFI-A target genes involved in the observed tissue malformations, we analyzed gene expression in brains from Nfia(-/- )and Nfia(+/+ )littermate mice at the mRNA level using oligonucleotide microarrays. In young postnatal animals (postnatal day 16), 356 genes were identified as being differentially regulated, whereas at the late embryonic stage (embryonic day 18) only five dysregulated genes were found. An in silico analysis identified phylogenetically conserved NFI binding sites in at least 70 of the differentially regulated genes. Moreover, assignment of gene function showed that marker genes for immature neural cells and neural precursors were expressed at elevated levels in young postnatal Nfia(-/- )mice. In contrast, marker genes for differentiated neural cells were downregulated at this stage. In particular, genes relevant for oligodendrocyte differentiation were affected. CONCLUSION: Our findings suggest that brain development, especially oligodendrocyte maturation, is delayed in Nfia(-/- )mice during the early postnatal period, which at least partly accounts for their phenotype. The identification of potential NFI-A target genes in our study should help to elucidate NFI-A dependent transcriptional pathways and contribute to enhanced understanding of this period of brain formation, especially with regard to the function of NFI-A

Osteoidosis leads to altered differentiation and function of osteoclasts

In patients with osteomalacia, a defect in bone mineralization leads to changed characteristics of the bone surface. Considering that the properties of the surrounding matrix influence function and differentiation of cells, we aimed to investigate the effect of osteoidosis on differentiation and function of osteoclasts. Based on osteomalacic bone biopsies, a model for osteoidosis in vitro (OIV) was established. Peripheral blood mononuclear cells were differentiated to osteoclasts on mineralized surfaces (MS) as internal control and on OIV. We observed a significantly reduced number of osteoclasts and surface resorption on OIV. Atomic force microscopy revealed a significant effect of the altered degree of mineralization on surface mechanics and an unmasking of collagen fibres on the surface. Indeed, coating of MS with RGD peptides mimicked the resorption phenotype observed in OIV, suggesting that the altered differentiation of osteoclasts on OIV might be associated with an interaction of the cells with amino acid sequences of unmasked extracellular matrix proteins containing RGD sequences. Transcriptome analysis uncovered a strong significant up-regulation of transmembrane glycoprotein TROP2 in osteoclastic cultures on OIV. TROP2 expression on OIV was also confirmed on the protein level and found on the bone surface of patients with osteomalacia. Taken together, our results show a direct influence of the mineralization state of the extracellular matrix surface on differentiation and function of osteoclasts on this surface which may be important for the pathophysiology of osteomalacia and other bone disorders with changed ratio of osteoid to bone

Detection of multiple annexin autoantibodies in a patient with recurrent miscarriages, fulminant stroke and seronegative antiphospholipid syndrome.

Anti-phospholipid syndrome (APS) is one of the main causes for recurrent miscarriages. The diagnosis of APS is based on the occurrence of clinical symptoms such as thrombotic events or obstetric complications as well as the detection of antiphospholipid antibodies directed against ß2-glycoprotein I and cardiolipin, or a positive lupus anticoagulant assay. However, there is a subpopulation of patients with clinical symptoms of APS, but the lack of serological markers (seronegative APS). In addition, a large proportion of patients with unexplained recurrent miscarriages exist. These cases may be attributed, at least in part, to a seronegative APS. The presence of autoantibodies against annexins is potentially associated with APS. Here we used immunoassays and immunoblots to detect autoantibodies directed against annexin A1-5, and A8, respectively, in a patient with a seronegative APS and a history of six recurrent pregnancy losses and fulminant stroke. We found strong IgM isotype antibody reactivity directed against annexin A2 and annexin A8, and moderate to weak IgM isotype antibody reactivity directed against annexin A1, A3, and A5. Further studies will evaluate the diagnostic value of IgM isotype antibodies against annexin A1-A5, and A8 for seronegative APS and recurrent miscarriages

Negative Regulation of Bone Formation by the Transmembrane Wnt Antagonist Kremen-2

Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders

ARSB IS REQUIRED FOR BONE REMODELING

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Identification of differentially expressed microRNAs in human male breast cancer

<p>Abstract</p> <p>Background</p> <p>The discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases.</p> <p>Methods</p> <p>The expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods.</p> <p>Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer.</p> <p>Results</p> <p>Unsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer.</p> <p>Conclusions</p> <p>Male breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.</p

Genome-Wide Gene Expression Analysis in Cancer Cells Reveals 3D Growth to Affect ECM and Processes Associated with Cell Adhesion but Not DNA Repair

Cell morphology determines cell behavior, signal transduction, protein-protein interaction, and responsiveness to external stimuli. In cancer, these functions profoundly contribute to resistance mechanisms to radio- and chemotherapy. With regard to this aspect, this study compared the genome wide gene expression in exponentially growing cell lines from different tumor entities, lung carcinoma and squamous cell carcinoma, under more physiological three-dimensional (3D) versus monolayer cell culture conditions. Whole genome cDNA microarray analysis was accomplished using the Affymetrix HG U133 Plus 2.0 gene chip. Significance analysis of microarray (SAM) and t-test analysis revealed significant changes in gene expression profiles of 3D relative to 2D cell culture conditions. These changes affected the extracellular matrix and were mainly associated with biological processes like tissue development, cell adhesion, immune system and defense response in contrast to terms related to DNA repair, which lacked significant alterations. Selected genes were verified by semi-quantitative RT-PCR and Western blotting. Additionally, we show that 3D growth mediates a significant increase in tumor cell radio- and chemoresistance relative to 2D. Our findings show significant gene expression differences between 3D and 2D cell culture systems and indicate that cellular responsiveness to external stress such as ionizing radiation and chemotherapeutics is essentially influenced by differential expression of genes involved in the regulation of integrin signaling, cell shape and cell-cell contact