Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is u...Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti-diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of BCG which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into BCG cells by electroporation. Mice immunized with recombinant BCG expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rBCGs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rBCGpUS977dtbPW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay.展开更多
Almost 110 years after the first studies by Dr. Carlos Chagas describing an infectious disease that was named for him, Chagas disease remains a neglected illness and a death sentence for infected people in poor countr...Almost 110 years after the first studies by Dr. Carlos Chagas describing an infectious disease that was named for him, Chagas disease remains a neglected illness and a death sentence for infected people in poor countries. This short review highlights the enormous need for new studies aimed at the development of novel and more specific drugs to treat chagasic patients. The primary tool for facing this challenge is deep knowledge about the similarities and differences between the parasite and its human host.展开更多
AIM To investigate the modulatory effect of B-1 cells on murine peritoneal macrophages infected with Leishmania major(L. major) in vitro.METHODS Peritoneal macrophages obtained from BALB/c andBALB/c XID mice were infe...AIM To investigate the modulatory effect of B-1 cells on murine peritoneal macrophages infected with Leishmania major(L. major) in vitro.METHODS Peritoneal macrophages obtained from BALB/c andBALB/c XID mice were infected with L. major and cultured in the presence or absence of B-1 cells obtained from wild-type BALB/c mice. Intracellular amastigotes were counted, and interleukin-10(IL-10) production was quantified in the cellular supernatants using an enzymelinked immunosorbent assay. The levels of the lipid mediator prostaglandin E2(PGE2) were determined using a PGE2 enzyme immunoassay kit(Cayman Chemical, Ann Arbor, MI), and the number of lipid bodies was quantified in the cytoplasm of infected macrophages in the presence and absence of B-1 cells. Culturing the cells with selective PGE2-neutralizing drugs inhibited PGE2 production and confirmed the role of this lipid mediator in IL-10 production. In contrast, we demonstrated that B-1 cells derived from IL-10 KO mice did not favor the intracellular growth of L. major.RESULTS We report that B-1 cells promote the growth of L. major amastigotes inside peritoneal murine macrophages. We demonstrated that the modulatory effect was independent of physical contact between the cells, suggesting that soluble factor(s) were released into the cultures. We demonstrated in our co-culture system that B-1 cells trigger IL-10 production by L. major-infected macrophages. Furthermore, the increased secretion of IL-10 was attributed to the presence of the lipid mediator PGE2 in supernatants of L. major-infected macrophages. The presence of B-1 cells also favors the production of lipid bodies by infected macrophages. In contrast, we failed to obtain the same effect on parasite replication inside L. major-infected macrophages when the B-1 cells were isolated from IL-10 knockout mice. CONCLUSION Our results show that elevated levels of PGE2 and IL-10 produced by B-1 cells increase L. major growth, as indicated by the number of parasites in cell cultures.展开更多
基金The authors gratefully acknowledge supported by Bio-Manguinhos/FIOCRUZ,PAPESII/FIOCRUZ,FAPERJ,CNPq,CAPES,Programa de Nucleo de Excelencia(PRONEX/MCT/CNPq)We thank Fundacao Ataulfo de Paiva for the strain of BCG.
文摘Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti-diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of BCG which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into BCG cells by electroporation. Mice immunized with recombinant BCG expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rBCGs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rBCGpUS977dtbPW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay.
文摘Almost 110 years after the first studies by Dr. Carlos Chagas describing an infectious disease that was named for him, Chagas disease remains a neglected illness and a death sentence for infected people in poor countries. This short review highlights the enormous need for new studies aimed at the development of novel and more specific drugs to treat chagasic patients. The primary tool for facing this challenge is deep knowledge about the similarities and differences between the parasite and its human host.
文摘AIM To investigate the modulatory effect of B-1 cells on murine peritoneal macrophages infected with Leishmania major(L. major) in vitro.METHODS Peritoneal macrophages obtained from BALB/c andBALB/c XID mice were infected with L. major and cultured in the presence or absence of B-1 cells obtained from wild-type BALB/c mice. Intracellular amastigotes were counted, and interleukin-10(IL-10) production was quantified in the cellular supernatants using an enzymelinked immunosorbent assay. The levels of the lipid mediator prostaglandin E2(PGE2) were determined using a PGE2 enzyme immunoassay kit(Cayman Chemical, Ann Arbor, MI), and the number of lipid bodies was quantified in the cytoplasm of infected macrophages in the presence and absence of B-1 cells. Culturing the cells with selective PGE2-neutralizing drugs inhibited PGE2 production and confirmed the role of this lipid mediator in IL-10 production. In contrast, we demonstrated that B-1 cells derived from IL-10 KO mice did not favor the intracellular growth of L. major.RESULTS We report that B-1 cells promote the growth of L. major amastigotes inside peritoneal murine macrophages. We demonstrated that the modulatory effect was independent of physical contact between the cells, suggesting that soluble factor(s) were released into the cultures. We demonstrated in our co-culture system that B-1 cells trigger IL-10 production by L. major-infected macrophages. Furthermore, the increased secretion of IL-10 was attributed to the presence of the lipid mediator PGE2 in supernatants of L. major-infected macrophages. The presence of B-1 cells also favors the production of lipid bodies by infected macrophages. In contrast, we failed to obtain the same effect on parasite replication inside L. major-infected macrophages when the B-1 cells were isolated from IL-10 knockout mice. CONCLUSION Our results show that elevated levels of PGE2 and IL-10 produced by B-1 cells increase L. major growth, as indicated by the number of parasites in cell cultures.
