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INTRODUCTION The genetic causes of limb-girdle muscular dystrophy (LGMD) have been studied in numerous countries, but such investigations have been limited in Egypt. METHODS A cohort of 30 families with suspected LGMD from Assiut, Egypt, was studied using immunohistochemistry, homozygosity mapping, Sanger sequencing, and whole exome sequencing. RESULTS Six families were confirmed to have pathogenic mutations, 4 in SGCA and 2 in DMD. Of these, 3 families harbored a single nonsense mutation in SGCA, suggesting that this may be a common mutation in Assiut, Egypt, originating from a founder effect. CONCLUSIONS The Assiut region in Egypt appears to share at least several of the common LGMD genes found in other parts of the world. It is notable that 4 of the 6 mutations were ascertained by means of whole exome sequencing, even though it was the last approach adopted. This illustrates the power of this technique for identifying causative mutations for muscular dystrophies. Muscle Nerve 54: 690-695, 2016.

Background: We investigated the reversibility of liver fibrosis induced with a CCl4 injection and the role of stem cells in reversing the hepatic injury. Furthermore, the most effective cell fraction among bone marrow cells (BMCs) in the repair process was analysed. Methods: C57BL/6 mice were divided into four groups after 5 weeks of injection of CCl4: control, sacrificed after 5 weeks, sacrificed at 10 weeks and sacrificed 5 weeks later after GFP-donor BM transplantation. Liver function tests and real-time polymerase chain reaction (PCR) of markers indicating liver fibrosis were compared between the groups. To identify the most effective BMC fraction that repairs liver injury, the mice were divided into three groups after the injection of CCl4 for 2 days: granulocyte colony stimulating factor (G-CSF) only, mononuclear cell (MNC) transplantation and Lin-Sca-1+c-kit+haematopoietic stem cell (HSC) transplantation. Eight days after transplantation, the mice were harvested and morphometric, immunohistochemical analyses were performed to compare the expression of extracellular matrix and liver fibrosis-related factors. Results: The liver fibrosis induced by CCl4 was not spontaneously recovered but was persistent until 10 weeks, but the group injected with BMCs had less fibrosis and better liver function. Mobilization with G-CSF increased the recovery of the injured liver and the best results were seen in those mice administered the MNC fraction and Lin-Sca-1+c-kit+HSC fraction, with no difference between the two groups. Conclusion: BMC transplantation and stem cell mobilization with G-CSF effectively treats liver injury in mice. These are promising techniques for autologous transplantation in humans with liver fibrosis.

Restricted numbers and poor regenerative properties limit the use of adult stem cells. We tested the effect of hypoxic treatment as a method by which to increase cell migration. Bone marrow cells (BMCs) were cultured under oxygen saturations of 0.1, 3, and 20% for 24h. After hypoxic treatment, BMCs of apoptotic fraction were decreased. The expression of CXCR4 was noticeably increased in the hypoxia-treated BMCs and their migration in response to SDF-1alpha was enhanced compared with cells cultured under normoxic condition. Hypoxic BMCs had a higher degree of engraftment to the CCl(4)-injured liver than the normoxic cells. Hypoxic treatment of BMCs may have merits in decreasing apoptosis of those cells as well as in enhancing cellular migration to SDF-1alpha, the chemokine which binds to BMCs expressed CXCR4 and to the injured tissue, such as CCl(4) damaged liver.

Keratinocytes constitute the first-line barrier against exogenous antigens and contain Toll-like receptors (TLRs), which function as pattern-recognition molecules to activate antimicrobial innate immune responses. In an effort to ascertain whether or not filaggrin (filament-aggregating protein) expression affected the TLR-mediated responses of keratinocytes, we transfected filaggrin siRNA into HaCaT human keratinocyte cells and determined that thymic stromal lymphopoietin (TSLP) and IL-6 secretion were increased by poly(I:C) stimulus. Additionally, TSLP expression is increased in filaggrin knockdown as well as TLR3 stimulation in reconstituted human epidermal layers. Therefore, the findings of this study show that reduced filaggrin levels may influence innate immune responses via TLR stimuli and may contribute to the pathogenesis of inflammatory skin disease via TSLP expression.

Endothelial cells are major targets of Orientia tsutsugamushi. To examine the consequences of the infection of endothelial cells with O. tsutsugamushi, we used human endothelial cell line ECV304. Persistent infection was established and infected cultures could be maintained for over seven months without the addition of normal cells. The heavily infected cells became round and floated in the culture medium, harboring large numbers of organisms inside them. Some of the infected ECV304 cells showed features of apoptotic cells, as determined by the terminal deoxytransferase-mediated dUTP nick end-labeling reaction and DNA fragmentation. We also found that O. tsutsugamushi increased transcription of the mRNAs of proinflammatory cytokines such as IL-6 and IL-8. These results show the first evidence of in vitro-persistent infection by O. tsutsugamushi, which may be related to in vivo persistence reported previously.