Anti-B2M monoclonal antibody, clone F21-21

In previous studies, we have demonstrated that very virulent plus Marek's disease viruses (vv+MDV) are highly immunosuppressive in commercial meat-type chickens. The specific objectives of this work were to evaluate if vv+MDV immunosuppression (MDV-IS) is induced by reduction of lymphocyte responsiveness and/or viability. Three experiments were conducted to (i) compare vv+MDV 686 with a partially attenuated 686-BAC; (ii) compare vv+MDV strains (648A and 686) with vMDV (GA) and vvMDV (Md5); and (iii) compare chickens vaccinated with Md5-BAC Delta MEQ and with CVI988 + HVT. In each experiment, spleens were collected at 28-30 days post infection and lymphocytes were isolated and investigated in three ways: their proliferative response to Concanavalin A (ConA) was analysed by MTT proliferation assay; cell death, and expression of CD45 and MHC-I was studied by flow cytometry; and MHC-IA and beta-2 microglobulin (B2M) expression was evaluated by real time RT-PCR. Splenocytes of chickens inoculated with vv+MDV were severely impaired to proliferate when exposed to ConA. Furthermore, vv+MDV induced severe splenocyte death that did not occur after infection with v or vvMDV strains. Vaccination with CVI988 + HVT, and at less level with Md5-BAC Delta MEQ reduced these negative effects. This is in contrast to our previous results in which Md5-BAC Delta MEQ but not CVI988 + HVT protected against MDV-IS suggesting that although cell death and decrease lymphocyte function seem to be related to MDV virulence and certainly will be associated with immunosuppression, they might not fully explain the previously reported MDV-IS.

Background. Bone marrow micro-environment changes during hematopoietic stem cell transplantation (HSCT) with subsequent alteration of genes expression. Quantitative polymerase chain reaction (q-PCR) is a reliable and reproducible technique for the analysis of gene expression. To obtain more accurate results, it is essential to find a reference during HSCT. However, which gene is suitable during HSCT remains unclear. This study aimed to identify suitable reference genes for mRNA studies in bone marrow after HSCT. Methods. C57BL/6 mice were treated with either total body irradiation (group T) or busulfan/cyclophosphamide (BU/CY) (group B) followed by infusion of bone marrow cells. Normal mice without treatments were served as a control. All samples (group T + group B + control) were defined as group G. On days 7, 14, and 21 after transplantation, transcription levels of 7 candidate genes, ACTB, B2M, GAPDH, HMBS, HPRT, SDHA, and YWHAZ, in bone marrow cells were measured by use of real-time quantitative PCR. The expression stability of these 7 candidate reference genes were analyzed by 2 statistical software programs, GeNorm and NormFinder. Results. Our results showed that ACTB displayed the highest expression in group G, with lowest expression of PSDHA in group T and HPRT in groups B and G. Analysis of expression stability by use of GeNorm or NormFinder demonstrated that expression of B2M in bone marrow were much more stable during HSCT, compared with other candidate genes including commonly used reference genes GAPDH and ACTB. Conclusions. ACTB could be used as a suitable reference gene for mRNA studies in bone marrow after HSCT.