Emad A. Ahmed

Type I interferon (IFN-α/β) rescues B-lymphocytes from apoptosis via PI3Kδ/Akt, Rho-A, NFκB and Bcl-2/BclXL

Although IFN-alpha was reported to promote the survival of peripheral B-lymphocytes via the PI3-kinase-Akt pathway, the triggered signalling pathways involved in the protection of B cell from apoptosis need to be clarified. Using flow cytometry and western blot analysis, we have found that type 1 IFNs (IFN-alpha/beta) protect human B cells in culture from spontaneous apoptosis and from apoptosis mediated by anti-CD95 agonist, in a dose- and time-dependant manner. IFN-alpha/beta-mediated anti-apoptotic effect on human B cells was totally abrogated by blockade of IFNR1 chain. Our data indicate that PI3Kdelta, Rho-A, NFkappaB and Bcl-2/Bcl(XL) are active downstream of IFN receptors and are the major effectors of IFN-alpha/beta-rescued B cells from apoptosis. Furthermore, immunohistochemical results show marked reduction in numbers of CD20 positive B cell in both spleen and Peyers patches from mice treated with anti-IFNR1 blocking antibody compared with control group. Moreover, ultrastructural observations of these organs show an obvious increase in apoptotic cells from mice treated with anti-IFNR1 blocking antibody. Our results provide more details about the triggered signalling pathways and the phosphorylation cascade which are involved in the protection of B cell from apoptosis after treatment with IFN-alpha/beta.

T lymphocytes from malnourished infants are short-lived and dysfunctional cells.

To investigate T-cell functional molecules and inflammatory cytokines and to assess T-cell apoptosis in malnourished infants, 64 infants from undernourished women and 28 healthy control infants were recruited to the study. Malnourished infants showed a significant decrease in the levels of circulating IL-2 and IL-7 and increases in the levels of IL-1β, IL-6, IL-10 and TNF-α, as measured by flow cytometry. There was a significant reduction in the number of CD3(+) T cells and an increase in apoptotic T cells, which was associated with an up-regulation of CD95 and PD-1 expression on CD3(+) T cells in malnourished compared to control infants. Significant reductions were also observed in the phosphorylation of AKT and STAT5 and in the expression of CCR7 and CXCR4 receptors in malnourished children, and these reductions were associated with a significant reduction in T-cell migratory capacity to their ligands CCL21 and CXCL12, respectively, as measured using an in vitro chemotaxis assay. Taken together, these data suggest that lymphocytes from malnourished infants are short-lived and dysfunctional.

DNA Double Strand Break Response and Limited Repair Capacity in Mouse Elongated Spermatids.

Spermatids are extremely sensitive to genotoxic exposures since during spermiogenesis only error-prone non homologous end joining (NHEJ) repair pathways are available. Hence, genomic damage may accumulate in sperm and be transmitted to the zygote. Indirect, delayed DNA fragmentation and lesions associated with apoptotic-like processes have been observed during spermatid elongation, 27 days after irradiation. The proliferating spermatogonia and early meiotic prophase cells have been suggested to retain a memory of a radiation insult leading later to this delayed fragmentation. Here, we used meiotic spread preparations to localize phosphorylate histone H2 variant (γ-H2AX) foci marking DNA double strand breaks (DSBs) in elongated spermatids. This technique enabled us to determine the background level of DSB foci in elongated spermatids of RAD54/RAD54B double knockout (dko) mice, severe combined immunodeficiency SCID mice, and poly adenosine diphosphate (ADP)-ribose polymerase 1 (PARP1) inhibitor (DPQ)-treated mice to compare them with the appropriate wild type controls. The repair kinetics data and the protein expression patterns observed indicate that the conventional NHEJ repair pathway is not available for elongated spermatids to repair the programmed and the IR-induced DSBs, reflecting the limited repair capacity of these cells. However, although elongated spermatids express the proteins of the alternative NHEJ, PARP1-inhibition had no effect on the repair kinetics after IR, suggesting that DNA damage may be passed onto sperm. Finally, our genetic mutant analysis suggests that an incomplete or defective meiotic recombinational repair of Spo11-induced DSBs may lead to a carry-over of the DSB damage or induce a delayed nuclear fragmentation during the sensitive programmed chromatin remodeling occurring in elongated spermatids.

Possible Protective Effects of Quercetin and Sodium Gluconate Against Colon Cancer Induction by Dimethylhydrazine in Mice.

Micronutrients in food have been found to have chemopreventive effects, supporting the conclusions from epidemiologie studies that consumption of fresh fruits and vegetables reduces cancer risk. The present study was carried out to evaluate the role of querctin (Q) and sodium gluconate (GNA) supplementation separately or in combination in ameliorating promotion of colon tumor development by dimethyl-hydrazine (DMH) in mice. Histopathological observation of colons in mice treated with DMH showed goblet cell dysplasia with inflammatory cell infiltration. This pathological finding was associated with significant alteration in oxidative stress markers in colon tissues and carcinoembryonic antigen (CEA) levels in plasma. Mice co-treated with GNA and Q showed mild changes of absorptive and goblet cells and inflammatory cell infiltration in lamina properia, with improvement in oxidative stress markers. In conclusion, findings of the present study indicate significant roles for reactive oxygen species (ROS) in pathogenesis of DMH-induced colon toxicity and initiation of colon cancer. Also, they suggest that Q, GNA or the combination of both have a positive beneficial effect against DMH induced colonic cancer induction in mice.

Camel Whey Protein Protects B and T Cells from Apoptosis by Suppressing Activating Transcription Factor-3 (ATF-3)-Mediated Oxidative Stress and Enhancing Phosphorylation of AKT and IκB-α in Type I Diabetic Mice.

Background: Diabetes mellitus (DM) is associated with severe immune system complications. Camel whey protein (CWP) decreases free radicals (ROS) and modulates immune functions, but its effect on DM-impaired immune systems has not been studied. We investigated the impact of CWP on the immune system in a Type 1 diabetes mouse model. Methods: Three experimental groups were used: (1) non-diabetic control; (2) diabetic; and (3) CWP-treated diabetic mice. Results: Induction of diabetes by streptozotocin was associated with reduction of body weight and insulin level, increase in glucose level and pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), and reduction in IL-2 and IL-4 levels. Upregulated ATF-3 expression was followed by a marked elevation in ROS levels. Lymphocytes from diabetic mice exhibited increased apoptosis through decreased phosphorylation of AKT and IκB-α, increased infiltration of T cells in the spleen and thymus, and decreased B cell numbers in the spleen. Supplementation with CWP decreased the levels of proinflammatory cytokines, ROS, and ATF-3 expression, and increased the levels of IL-4. Treatment with CWP decreased apoptosis by enhancing the phosphorylation of AKT and IκB-α as well as T-cell and B-cell distribution in the spleen and thymus. Conclusions: Our findings suggest the beneficial effects of CWP supplementation during diabetes on decreasing and orchestrating the redox status and subsequently rescuing the immune cells from exhaustion.

Spermatocyte Spreading during Meiotic Cell Preparation is a Two StepProcess

Cytological techniques have been instrumental for the investigation of meiosis and gametogenesis. Especially high resolution chromatin spreads of male and female germline cells provide for detailed insights into the molecular mechanisms acting during germ cell differentiation. Most spreading techniques for germ cells are done with a hypotonic detergent solution followed by an extended drying period. Using video microscopy and we monitored the course of mouse spermatocyte spreading after exposure to graded concentrations of the ionic detergent mixture Lipsol that is known to efficiently spread meiocytes. Our analysis disclosed that spreading of meiotic cells is optimal at a final detergent concentration of about 0.7% and occurs in two phases. First, cells undergo slow swelling in the dilute detergent solution (lasting up to >1 h) followed by rapid dispersion of the nuclear chromatin over the glass surface just prior to the final evaporation of the water in the solution. These results provide a better understanding of an important technique in meiosis research and identify factors determining the spreading process.