Promoter-Proximal Chromatin Domain Insulator Protein BEAF Mediates Local and Long-Range Communication with a Transcription Factor and Directly Activates a Housekeeping Promoter in Drosophila.

BEAF (Boundary Element-Associated Factor) was originally identified as a Drosophila melanogaster chromatin domain insulator-binding protein, suggesting a role in gene regulation through chromatin organization and dynamics. Genome-wide mapping found that BEAF usually binds near transcription start sites, often of housekeeping genes, suggesting a role in promoter function. This would be a nontraditional role for an insulator-binding protein. To gain insight into molecular mechanisms of BEAF function, we identified interacting proteins using yeast two-hybrid assays. Here, we focus on the transcription factor Serendipity δ (Sry-δ). Interactions were confirmed in pull-down experiments using bacterially expressed proteins, by bimolecular fluorescence complementation, and in a genetic assay in transgenic flies. Sry-δ interacted with promoter-proximal BEAF both when bound to DNA adjacent to BEAF or > 2-kb upstream to activate a reporter gene in transient transfection experiments. The interaction between BEAF and Sry-δ was detected using both a minimal developmental promoter (y) and a housekeeping promoter (RpS12), while BEAF alone strongly activated the housekeeping promoter. These two functions for BEAF implicate it in playing a direct role in gene regulation at hundreds of BEAF-associated promoters.

Oral administration of recombinant Lactococcus lactis expressing HSP65 and tandemly repeated P277 reduces the incidence of type I diabetes in non-obese diabetic mice.

Diabetes mellitus type 1 (DM1) is an autoimmune disease that gradually destroys insulin-producing beta-cells. We have previously reported that mucosal administration of fusion protein of HSP65 with tandem repeats of P277 (HSP65-6P277) can reduce the onset of DM1 in non-obese diabetic (NOD) mice. To deliver large amounts of the fusion protein and to enhance long-term immune tolerance effects, in the present study, we investigated the efficacy of using orally administrated L. lactis expressing HSP65-6P277 to reduce the incidence of DM1 in NOD mice. L. lactis strain NZ9000 was engineered to express HSP65-6P277 either constitutively or by nisin induction. After immunization via gavage with the recombinant L. lactis strains to groups of 4-week old female NOD mice for 36 weeks, we observed that oral administration of recombinant L. Lactis resulted in the prevention of hyperglycemia, improved glucose tolerance and reduced insulitis. Immunologic analysis showed that treatment with recombinant L. lactis induced HSP65- and P277- specific T cell immuno-tolerance, as well as antigen-specific proliferation of splenocytes. The results revealed that the DM1-preventing function was in part caused by a reduction in the pro-inflammatory cytokine IFN-γ and an increase in the anti-inflammatory cytokine IL-10. Orally administered recombinant L. lactis delivering HSP65-6P277 may be an effective therapeutic approach in preventing DM1.

Enhanced antitumor efficacy by combination treatment with a human umbilical vein endothelial cell vaccine and a tumor cell lysate-based vaccine.

Angiogenesis inhibitors combined with other anticancer drugs have been shown to inhibit tumor growth in animal models and some of them were recently used in clinical trials. In the present study, a whole hepatocellular carcinoma cell lysate based vaccine with diphtheria toxin (DT) and two tandem repeats of microbial HSP70 peptide epitope 407-426 (2 mHSP70407-426, M2) as adjuvant, which was called HDM, was combined with a whole human umbilical vein endothelial cell (HUVEC) vaccine to develop a combination treatment regimen. This combination treatment regimen was named HUVEC-HDM, which was supposed to enhance its antitumor efficiency. HUVEC-HDM was administrated subcutaneously in both prophylactic and therapeutic procedures. Compared to either single vaccine, HUVEC-HDM induced a more significant inhibition on the growth and metastasis of H22 hepatocellular carcinoma in mice and prolonged the survival of tumor-bearing mice. Besides, HUVEC-HDM immunization elicited strong humoral and cellular immune responses targeting tumor cell as well as tumor angiogenesis, which could be responsible for the enhanced antitumor effect. Moreover, histochemistry analysis showed that HUVEC-HDM induced large areas of continuous necrosis within tumors, correlating well with the extent of tumor inhibition. These results not only highlight the superiority of the combined HUVEC-HDM treatment regimen, but also support the translation of such approaches into the clinic for the treatment of patients with hepatocellular carcinoma.

Vaccination of non-obese diabetic mice with a fragment of peptide P277 attenuates insulin-dependent diabetes mellitus.

P277 is a peptide derived from the HSP60 regions, have potent immunological effect on insulin-dependent diabetes mellitus (IDDM) and its phase III clinical trials are currently under investigation. However, we recently discovered a positive correlation between anti-P277 autoantibodies and the presence of endothelial cells damage in inducing vascular leak syndrome. Therefore, the aim of our study was to demonstrate the critical peptide epitope of P277 to IDDM and to highlight the effects of this peptide therapy on inflammation of the islets. Groups of 4-week old female non-obese diabetic (NOD) mice were immunized one time every three weeks for three times with a residue of P277, showing a significant effect of down-regulating immunity to P277 protein and preventing the development of IDDM. Immunologic results including the suppression of T-cell proliferation, the increase of interleukin-10 (IL-10) production and reduction of interferon-γ (IFN-γ) production caused immune tolerance to P277. Hence, a functional role of the key epitope in P277 peptide capable of preventing IDDM is suggested, which could be modified to develop a novel safe and effective peptide vaccine against IDDM by reconstructing P277 in the further studies.