Martha N. Calderon

Inhibiting rotavirus infection by membrane-impermeant thiol/disulfide exchange blockers and antibodies against protein disulfide isomerase.

Objectives: Determining the effect of membrane-impermeant thiol/disulfide exchange inhibitors on rhesus rotavirus infectivity in MA104 cells and investigating protein disulfide isomerase (PDI) as a potential target for these inhibitors. Methods: Cells were treated with DTNB [5,5-dithio-bis-(2-nitrobenzoic acid)], bacitracin or anti-PDI antibodies and then infected with virus. Triple-layered particles (TLPs) were also pretreated with inhibitors before inoculation. The effects of these inhibitors on α-sarcin co-entry, virus binding to cells and PDI-TLP interaction were also examined. FACS analysis, cell-surface protein biotin-labeling, lipid-raft isolation and ELISA were performed to determine cell-surface PDI expression. Results: Infectivity became reduced by 50% when cells or TLPs were treated with 1 or 6 mM DTNB, respectively; infectivity became reduced by 50% by 20 mM bacitracin treatment of cells whereas TLPs were insensitive to bacitracin treatment; anti-PDI antibodies decreased viral infectivity by about 45%. The presence of DTNB (2.5 mM) or bacitracin (20 mM) was unable to prevent virus binding to cells and rotavirus-induced α-sarcin co-entry. Conclusions: It was concluded that thiol/disulfide exchange was involved in rotavirus entry process and that cell-surface PDI was at least a potential target for DTNB and bacitracin-induced infectivity inhibition.

Mycobacterium tuberculosis ESAT-6 antigen immunogenicity in Owl Monkeys

Several ESAT-6-based vaccines have been widely studied in different animal models, presenting potent ability to induce both cellular and humoral responses. As ESAT-6 is a well-characterized mycobacterial antigen, its capacity to induce immune responses in a nonhuman primate model has been evaluated. The immunization of recombinant ESAT-6 (rESAT-6) in Aotus nancymaae monkeys has elicited a strong cellular response, not just to rESAT-6, but also to the native protein present in Mycobacterium tuberculosis culture filtrate proteins measured as [3H]thymidine incorporation in lymphocyte proliferation assays. High humoral response was also observed, having antibody titers of 1:12,800 directed towards rESAT-6. The protein.s multi-epitope nature was further demonstrated since several peptide sequences were specifically recognized at both cellular and humoral level. The high immunogenicity observed, as well as the relatively high characterization of the Aotus. immune system at molecular level, are two advantage to propose Aotus nancymaae as animal model for studying M. tuberculosis infection; however, our results reveal an animal-to-animal variation in response to vaccination, this could be a disadvantage.

Interaction of rotavirus with protein disulfide isomerase in vitro and cell system

INTRODUCTION Rotavirus entry process involves a multi-step mechanism, the first of which is when the outermost viral proteins interact with four different integrins and Hsc70. Recently, rotavirus infection reportedly has been decreased after blocking cell surface protein disulfide isomerase (PDI). This suggested that this protein interacts with rotavirus during the entry process. OBJECTIVES The aim was to establish the rotavirus-PDI interaction in an in vitro system using PDI isolated from bovine liver, and in a cell system consisting of MA104 cells and mouse small intestinal villi. MATERIALS AND METHODS Protein disulfide isomerase was isolated from a bovine liver homogenate using anti-PDI antibodies coupled to agarose through hydrazone bonds. Purity of purified protein was assessed by SDS-PAGE and Western blot. The purified PDI was used to study its in vitro interaction with the rotavirus particles. This interaction was compared with that taking place in MA104 cells and small intestinal villi isolated from sucking mice ICR. RESULTS The purified PDI showed an electrophoretic homogeneity and was able to bind rotavirus particles in vitro. Rotavirus-PDI interaction was detected by capture ELISA using purified protein and rotavirus strains RRV and wild-type ECwt. Interaction between rotavirus particles and cellular PDI was detected by ELISA using cell lysates after virus inoculation. CONCLUSIONS Rotavirus-PDI interaction was demonstrated in vitro as well as inMA104 cells and intestinal villi from suckling mice.