Search

• We cloned and produced recombinant Phosphotyrosyl phosphatase activator (PTPA) protein from Haemonchus contortus (rHCPTPA). • The pNPP phosphatase activity of bovine PP2A 1 was increased, due to the existence of rHCPTPA, dose-dependently. • rHCPTPA could be bound by goat PBMCs and exerts its immunomodulatory effects on multiple aspects. Suppression and modulation of the host immune response to parasitic nematodes have been extensively studied. In the present study, we cloned and produced recombinant phosphotyrosyl phosphatase activator protein from Haemonchus contortus (rHCPTPA), a parasitic nematode of small ruminants, and studied the effect of this protein on modulating the immune response of goat peripheral blood mononuclear cells. Enzymatic assays revealed that rHCPTPA enhanced the p-nitrophenylphosphate phosphatase activity of bovine PP2A 1. Immunohistochemical tests verified that the HCPTPA protein was localised mainly in the bowel wall and on the body surface of worms. It was also shown that serum produced by goats artificially infected with H. contortus successfully recognised rHCPTPA, which conjugated with goat peripheral blood mononuclear cells. The rHCPTPA was then co-incubated with goat peripheral blood mononuclear cells to assess its immunomodulatory effects on proliferation, apoptosis, cytokine secretion, migration and nitric oxide production. Our results showed that rHCPTPA suppressed the proliferation of goat peripheral blood mononuclear cells stimulated by concanavalin A and induced apoptosis in goat peripheral blood mononuclear cells. After rHCPTPA exposure, IFN-γ and IL-2 expression was markedly reduced, whereas secretion of IL-10 and IL-4 was significantly elevated, in goat peripheral blood mononuclear cells. Moreover, rHCPTPA down-regulated nitric oxide production and migration of goat peripheral blood mononuclear cells in a dose-dependent manner. These results illuminate the interaction between parasites and hosts at the molecular level, suggest a possible immunomodulatory target and contribute to the search for innovative proteins that might be candidate targets for drugs and vaccines. [ABSTRACT FROM AUTHOR]

• Fusion protein of C. perfringens Cpa247-370, Cpb108-305 and EtxH118P toxin was generated. • B. subtilis expressing C. perfringens recombinant toxin protein rCpa-b-x was constructed. • Immunization with recombinants can elicit cellular and humoral immune responses. • Immunization with recombinants protected mice from C. perfringens toxins. The anaerobic pathogen Clostridium perfringens is the most potent cause of intestinal diseases, such as enterotoxemia, hemorrhagic enteritis, and lamb dysentery, in sheep. Three toxinotypes (B, C, and D) are usually the cause of these diseases and are mainly mediated via three important exotoxins: alpha toxin (CPA), beta toxin (CPB), and epsilon toxin (ETX). We have designed a chimeric protein, rCpa-b-x, that contains the C-terminal binding region of CPA, partial sequence of CPB, and ETX (Cpa247-370, Cpb108-305, and EtxH118P, respectively) according to the principle of structural vaccinology. The rCpa-b-x protein was then expressed by pHT43 plasmid in vivo using Bacillus subtilis as a delivery vector (Bs-pHT43-Cpa-b-x). The immunological activity of the rCpa-b-x protein was verified by western blot and its immunological efficacy was evaluated in a murine model. Oral administration with a recombinant agent caused local mucosal and systemic immune responses, and serum lgG and intestinal mucosal secretory IgA (sIgA) antibody titers were significantly increased. Levels of IL-2, IL-4, and IFN-γ were significantly higher in lymphocytes isolated from the Bs-pHT43-Cpa-b-x group compared with levels from the control groups. The percentages of CD4+ and CD8+ T lymphocytes in the Bs-pHT43-Cpa-b-x and inactivated vaccine (IV) groups were in the normal range. Mice of vaccine groups and control groups were challenged with 1x LD 100 unit filtrate containing alpha, beta, and epsilon toxins. Mice in the Bs-pHT43-Cpa-b-x group were found to have lower rates of morbidity. The active immunization of mice with Bs-pHT43-Cpa-b-x still maintained 85% to 90% survival at the end of the 10-day observation period, whereas mice of control groups died within two to five days. The results of this study demonstrate the effectiveness of Bs-pHT43-Cpa-b-x in preventing C. perfringens infection in mice, and that Bs-pHT43-Cpa-b-x could be considered a potential vaccine against C. perfringens. [ABSTRACT FROM AUTHOR]

• rHCETFα suppressed the proliferation of goat PBMCs stimulated by concanavalin A. • rHCETFα significantly induced apoptosis in goat PBMCs. • rHCETFα modulated the secretion of cytokines in goat PBMCs. • rHCETFα up-regulated NO production in goat PBMCs. • rHCETFα inhibited phagocytosis of goat monocytes. Suppression and modulation of the host immune response to parasitic nematodes have been extensively studied. In the present study, we cloned and produced recombinant electron transfer flavoprotein α subunit (ETFα) protein from Haemonchus contortus (rHCETFα), a parasitic nematode of small ruminants, and studied the effect of this protein on modulating the immune response of goat peripheral blood mononuclear cells (PBMCs) and monocytes. Immunohistochemical tests verified that the HCETFα protein was localized mainly in the intestinal wall and on the body surface of worms. Immunoblot analysis revealed that rHCETFα was recognized by the serum of goats artificially infected with H. contortus. Immunofluorescence analysis indicated that rHCETFα bound to the surface of PBMCs. rHCETFα was co-incubated with goat PBMCs to observe the immunomodulatory effects exerted by HCETFα on proliferation, apoptosis, cytokine secretion and nitric oxide (NO) production. The results showed that rHCETFα suppressed the proliferation of goat PBMCs stimulated by concanavalin A and induced apoptosis in goat PBMCs. After rHCETFα exposure, IL-2, IL-4, IL-17A and TNF-α expression was markedly reduced, whereas secretion of TGF-β1 was significantly elevated, in goat PBMCs. Moreover, rHCETFα up-regulated NO production in a dose-dependent manner. FITC-dextran internalization assays showed that rHCETFα inhibited phagocytosis of goat monocytes. These results elucidate the interaction between parasites and hosts at the molecular level, suggest a possible immunomodulatory target and contribute to the search for innovative proteins that may be candidate targets for drugs and vaccines. [ABSTRACT FROM AUTHOR]

Background: Modulation of the host immune response by nematode parasites has been widely reported. Rhodaneses (thiosulfate: cyanide sulfurtransferases) are present in a wide range of organisms, such as archaea, bacteria, fungi, plants and animals. Previously, it was reported that a rhodanese homologue could be bound by goat peripheral blood mononuclear cells (PBMCs) in vivo. Methods: In the present study, we cloned and produced a recombinant rhodanese protein originating from Haemonchus contortus (rHCRD), a parasitic nematode of small ruminants. rHCRD was co-incubated with goat PBMCs to assess its immunomodulatory effects on proliferation, apoptosis and cytokine secretion. Results: We verified that the natural HCRD protein localized predominantly to the bowel wall and body surface of the parasite. We further demonstrated that serum produced by goats artificially infected with H. contortus successfully recognized rHCRD, which bound to goat PBMCs. rHCRD suppressed proliferation of goat PBMCs stimulated by concanavalin A but did not induce apoptosis in goat PBMCs. The production of TNF-α and IFN-γ decreased significantly, whereas secretion of IL-10 and TGF-β1 increased, in goat PBMCs after exposure to rHCRD. rHCRD also inhibited phagocytosis by goat monocytes. Moreover, rHCRD downregulated the expression of major histocompatibility complex (MHC)-II on goat monocytes in a dose-dependent manner, but did not alter MHC-I expression. Conclusions: These results propose a possible immunomodulatory target that may help illuminate the interactions between parasites and their hosts at the molecular level and reveal innovative protein species as candidate drug and vaccine targets. [ABSTRACT FROM AUTHOR]