Sequence context analysis of 8.2 million single nucleotide polymorphisms in the human genome

We analyzed n-mers (n=3-8) in the local environment of 8,249,446 human SNPs and compared their distribution with that in the genome reference sequences. The results revealed that the short sequences, which contained at least one CpG dinucleotide, occurred

Ex Vivo Expansion of Human Outgrowth Endothelial Cells Leads to IL-8-Mediated Replicative Senescence and Impaired Vasoreparative Function

Harnessing outgrowth endothelial cells (OECs) for vasoreparative therapy and tissue-engineering requires efficient ex-vivo expansion. How such expansion impacts on OEC function is largely unknown. In this study, we show that OECs become permanently cell-cycle arrested after ex-vivo expansion, which is associated with enlarged cell size, ß-galactosidase activity, DNA damage, tumour suppressor pathway activation and significant transcriptome changes. These senescence hallmarks were coupled with low telomerase activity and telomere shortening, indicating replicative senescence. OEC senescence limited their regenerative potential by impairing vasoreparative properties in-vitro and in-vivo. Integrated transcriptome-proteome analysis identified inflammatory signalling pathways as major mechanistic components of the OEC senescence programme. In particular, IL8 was an important facilitator of this senescence; depletion of IL8 in OECs significantly extended ex-vivo lifespan, delayed replicative senescence and enhanced function. While the ability to expand OEC numbers prior to autologous or allogeneic therapy remains a useful property, their replicative senescence and associated impairment of vasorepair needs to be considered. The current study also suggests that modulation of the senescence-associated secretory phenotype (SASP) could be used to optimise OEC therapy.

Expression of toll-like receptors by human muscle cells in vitro and in vivo:TLR3 is highly expressed in inflammatory and HIV myopathies, mediates IL-8 release and up-regulation of NKG2D-ligands

The particular microenvironment of the skeletal muscle can be the site of complex immune reactions. Toll-like receptors (TLRs) mediate inflammatory stimuli from pathogens and endogenous danger signals and link the innate and adaptive immune system. We investigated innate immune responses in human muscle. Analyzing TLR1-9 mRNA in cultured myoblasts and rhabdomyosarcoma cells, we found constitutive expression of TLR3. The TLR3 ligand Poly (I:C), a synthetic analog of dsRNA, and IFN-gamma increased TLR3 levels. TLR3 was mainly localized intracellularly and regulated at the protein level. Poly (I:C) challenge 1) activated nuclear factor-kappaB (NF-kappaB), 2) increased IL-8 release, and 3) up-regulated NKG2D ligands and NK-cell-mediated lysis of muscle cells. We examined muscle biopsy specimens of 6 HIV patients with inclusion body myositis/polymyositis (IBM/PM), 7 cases of sporadic IBM and 9 nonmyopathic controls for TLR3 expression. TLR3 mRNA levels were elevated in biopsy specimens from patients with IBM and HIV-myopathies. Muscle fibers in inflammatory myopathies expressed TLR3 in close proximity of infiltrating mononuclear cells. Taken together, our study suggests an important role of TLR3 in the immunobiology of muscle, and has substantial implications for the understanding of the pathogenesis of inflammatory myopathies or therapeutic interventions like vaccinations or gene transfer.

Distribution of the human intracellular serpin protease inhibitor 8 in human tissues.

Ovalbumin-like serine protease inhibitors are mainly localized intracellularly and their in vivo functions are largely unknown. To elucidate their physiological role(s), we studied the expression of one of these inhibitors, protease inhibitor 8 (PI-8), in normal human tissues by immunohistochemistry using a PI-8-specific monoclonal antibody. PI-8 was strongly expressed in the nuclei of squamous epithelium of mouth, pharynx, esophagus, and epidermis, and by the epithelial layer of skin appendages, particularly by more differentiated epithelial cells. PI-8 was also expressed by monocytes and by neuroendocrine cells in the pituitary gland, pancreas, and digestive tract. Monocytes showed nuclear and cytoplasmic localization of PI-8, whereas neuroendocrine cells showed only cytoplasmic staining. In vitro nuclear localization of PI-8 was confirmed by confocal analysis using serpin-transfected HeLa cells. Furthermore, mutation of the P(1) residue did not affect the subcellular distribution pattern of PI-8, indicating that its nuclear localization is independent of the interaction with its target protease. We conclude that PI-8 has a unique distribution pattern in human tissues compared to the distribution patterns of other intracellular serpins. Additional studies must be performed to elucidate its physiological role.