Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells.

The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).

Erythropoietin non-viral gene therapy does not affect motility, viability, morphology or concentration of rabbit sperm.

Erythropoietin (EPO) gene therapy can be used for several purposes; however, its effects on reproductive performance are unknown. The aim of this study was to evaluate the toxicological effects of non-viral (EPO) gene transfer on sperm motility, viability, morphology and concentration. Rabbit EPO cDNA was cloned into a pTarget mammalian expression vector. Rabbits were administered with: (1) pTarget/EPO vector, (2) recombinant human EPO (rHuEpo) and (3) saline (control). Both pTarget/EPO and rHuEpo significantly increased (P < 0.05) hematocrit levels 1 week after injection and they remained significantly higher than the control for up to 5 weeks (P < 0.05), showing that both EPO treatments were effective in stimulating the production of red blood cells in rabbits. The EPO gene transfer or rHuEPO administration had no significant effect (P > 0.05) on sperm motility, vigor, viability, concentration or morphology in the testis.

Naïve donor responses to Schistosoma mansoni soluble egg antigens.

Schistosome infection induces profound Th-biasing and immune suppression. Although much has been examined in mice, few studies have examined responses of naïve humans to schistosome antigens. In this study, we examined the response of naïve human peripheral blood mononuclear cells (nPBMC) to stimulation with Schistosoma mansoni soluble egg antigen (SEA) using a priming in vitro (PIV) assay. We found that SEA induced a pronounced CD4+ T-helper cell response based on cytokine secretion and phenotyping markers. SEA-stimulated nPBMC (SEA cells) at day 7 post-priming and after the first recall consisted predominantly of Th0-like CD4+ T cells. Following the second recall, the majority of donor (10/12) responses were Th2-like. The cell population consisted of approximately 64% CD4+, 17% CD8(+high), 12% CD19+, and 7% CD23+ cells. The CD4+ population also expressed HLA-DR+, CD54+, CD45RO+ and CD25+ whereas the CD19+ cells expressed CD80 and CD86. Following priming, we detected high levels of IL-6, IFN-gamma, IL-12p40, IL-10 and IL-5. Upon restimulation, SEA cells secreted IL-5 and high levels of IL-10, typical of a Th2-like response. The data presented herein shows that the majority of naïve donor dendritic cells, following stimulation with SEA, prime and clonally expand SEA-specific T cells towards a Th2-type response. However, two donors responded with an atypical response, producing IFN-gamma coincident with low levels of IL-10. Whether this differential response was due to HLA or other genes was not determined but is currently under investigation.

Expression of the B-cell and T-cell epitopes of the rabies virus nucleoprotein in Mycobacterium bovis BCG and induction of an humoral response in mice.

Expression vectors containing rabies virus nucleoprotein B-cell and T-cell epitopes in Mycobacterium bovis BCG were constructed. The epitopes were subcloned into the M. leprae 18-kDa gene to ensure correct presentation to the host immune system. Expression of the 18-kDa::B+T epitope fusion protein was driven by either the hsp60 promoter, which is constitutively activated at a high level in M. bovis BCG, or the 18-kDa promoter, which is strongly induced in vivo. Mice were immunised intra-peritoneally with the recombinant BCG cultures and compared to a control group vaccinated with the commercial rabies vaccine Rai-SAD. Both of the expression vectors elicited a higher antibody titre than that of the rabies vaccine, with the highest response shown by M. bovis BCG (pUP203), expression controlled by the 18-kDa promoter. Immunisation with M. bovis BCG (pUP202), expression controlled by the hsp60 promoter, resulted in a continuously increasing antibody titre up to 60 days post immunisation. The mice antibodies were also capable of recognising the whole rabies virus and not only the synthetic peptide epitopes.