Immunization with extracellular vesicles excreted by Toxoplasma gondii confers protection in murine infection, activating cellular and humoral responses.

The study aim was to analyze whether microvesicles and exosomes, named extracellular vesicles (EVs), purified from Toxoplasma gondii are able to stimulate the protective immunity of experimental mice when administered, as challenge, a highly virulent strain. EVs excreted from T. gondii tachyzoites (RH strain) were purified by chromatography and used for immunization assays in inbred mouse groups (EV-IM). Chronic infected (CHR) and naive (NI) mice were used as control groups, since the immune response is well known. After immunizations, experimental groups were challenged with 100 tachyzoites. Next, parasitemias were determined by real-time PCR (qPCR), and survival levels were evaluated daily. The humoral response was analyzed by detection of IgM, IgG, IgG1 and IgG2a, and opsonization experiments. The cellular response was evaluated in situ by immunohistochemistry on IFN-γ, IL-10, TNF-α and IL-17 expression in cells of five organs (brain, heart, liver, spleen and skeletal muscles). EV immunization reduced parasitemia and increased the survival index in two mouse lineages (A/Sn and BALB/c) infected with a lethal T. gondii strain. EV-IM mice had higher IgG1 levels than IgM or IgG2a. IgGs purified from sera of EV-IM mice were able to opsonize tachyzoites (RH strain), and mice that received these parasites had lower parasitemias, and mortality was delayed 48 h, compared with the same results from those receiving parasites opsonized with IgG purified from NI mice. Brain and spleen cells from EV-IM mice more highly expressed IFN-γ, IL-10 and TNF-α. In conclusion, EV-immunization was capable of inducing immune protection, eliciting high production of IgG1, IFN-γ, IL-10 and TNF-α.

Gene expression profile of cytokines produced in biopsies from patients with American cutaneous leishmaniasis

American cutaneous leishmaniasis (ACL) causes a local inflammatory process, inducing expression of several cytokine genes. Particularly, IFN-γ can predict to disease susceptibility. Based in these data, this study was aimed to investigate the gene expression profile of IFN-γ, IL-10, IL-27, TNF-γ, TGF-ß and IL-6 produced in biopsies from ACL patients; and whether the gene expression profile of IFN-γ could determine the disease evolution. Gene expression of 6 cytokines was investigated in 40 formalin-fixed paraffin embedded (FFPE) biopsies from patients with cutaneous leishmaniosis (CL); and 10 FFPE biopsies from patients with mucosal leishmaniasis (ML) (control). All 50 patients were infected with Leishmania (Viannia) braziliensis. Gene expression was determined by qPCR; and a normal control group was used for calculations (5 normal biopsies). Values were expressed as Relative Quantification (RQ). The 40 CL patients were classified into 2 groups. CLlowIFN-γ, 35 patients with RQ for IFN-γ below 100; and CLhighIFN-γ, 5 (12.5%) patients with RQ above 100. Significant increase of mRNA levels of IFN-γ, IL-10 and IL-27 was shown in CLhighIFN-γ group when compared with CLlowIFN-γ and ML groups. TNF-α levels in CLlowIFN-γ group were higher than CLhighIFN-γ and ML groups. TGF-ß and IL-6 were similar in 3 groups. Comparison of cytokine expression/group showed that CLlowIFN-γ group had an equilibrium between the cytokines analyzed. In ML group, IFN-γ was over-expressed; but in CLhighIFN-γ group, besides IFN-γ, IL-27 was also over-expressed. The immune response to Leishmania induces to identification of some markers, which can be determined by analysis by gene expression of cytokines produced in biopsies

Gene expression profile of cytokines produced in biopsies from patients with American cutaneous leishmaniasis.

American cutaneous leishmaniasis (ACL) causes a local inflammatory process, inducing expression of several cytokine genes. Particularly, IFN-γ can predict to disease susceptibility. Based in these data, this study was aimed to investigate the gene expression profile of IFN-γ, IL-10, IL-27, TNF-γ, TGF-ß and IL-6 produced in biopsies from ACL patients; and whether the gene expression profile of IFN-γ could determine the disease evolution. Gene expression of 6 cytokines was investigated in 40 formalin-fixed paraffin embedded (FFPE) biopsies from patients with cutaneous leishmaniosis (CL); and 10 FFPE biopsies from patients with mucosal leishmaniasis (ML) (control). All 50 patients were infected with Leishmania (Viannia) braziliensis. Gene expression was determined by qPCR; and a normal control group was used for calculations (5 normal biopsies). Values were expressed as Relative Quantification (RQ). The 40 CL patients were classified into 2 groups. CLlowIFN-γ, 35 patients with RQ for IFN-γ below 100; and CLhighIFN-γ, 5 (12.5%) patients with RQ above 100. Significant increase of mRNA levels of IFN-γ, IL-10 and IL-27 was shown in CLhighIFN-γ group when compared with CLlowIFN-γ and ML groups. TNF-α levels in CLlowIFN-γ group were higher than CLhighIFN-γ and ML groups. TGF-ß and IL-6 were similar in 3 groups. Comparison of cytokine expression/group showed that CLlowIFN-γ group had an equilibrium between the cytokines analyzed. In ML group, IFN-γ was over-expressed; but in CLhighIFN-γ group, besides IFN-γ, IL-27 was also over-expressed. The immune response to Leishmania induces to identification of some markers, which can be determined by analysis by gene expression of cytokines produced in biopsies.