Neospora caninum: Early immune response of rat mixed glial cultures after tachyzoites infection

Neospora caninum causes neurologic disease in dogs and abortion in cattle. Little is known about the immune response of the CNS against this protozoan. The aim of this study was to evaluate production of IL-6, IL-10, TNF-α, IFN-γ, and NO in rat mixed glial cell cultures infected by N. caninum. IFN-γ was not observed. The mean cytokine released after 24 and 72 h of infection were 3.8 ± 0.6 and 3.7 ± 0.6 pg TNF-α/mg protein and 2.7 ± 0.69 and 4.1 ± 0.64 pg IL-10/mg protein, respectively, and more than 8.0 pg IL-6/mg protein for both time points. NO levels increased 24 h post-infection (2.3 ± 0.8 pg/mg protein) until 72 h (4.2 ± 1.1 pg/mg protein) and the number of tachyzoites reduced with the time. Our results show high levels of regulatory cytokines that may suppress the harmful effects of IFN-γ; high levels of TNF-α and NO may represent an effective response by infected glial cells against N. caninum.     

Functional activation of T cells by dendritic cells and macrophages exposed to the intracellular parasite Neospora caninum

Neospora caninum is an intracellular protozoan pathogen that causes abortion in cattle. We studied how the interaction between murine conventional dendritic cells or macrophages and N. caninum influences the generation of cell-mediated immunity against the parasite. We first explored the invasion and survival ability of N. caninum in dendritic cells and macrophages. We observed that protozoa rapidly invaded and proliferated into these two cell populations. We then investigated how Neospora-exposed macrophages or dendritic cells distinguish between viable and non-viable (heat-killed tachyzoites and antigenic extract) parasites. Viable tachyzoites and antigenic extract, but not killed parasites, altered the phenotype of immature dendritic cells. Dendritic cells infected with viable parasites down-regulated the expression of MHC-II, CD40, CD80 and CD86 whereas dendritic cells exposed to N. caninum antigenic extract up-regulated the expression of MHC-II and CD40 and down-regulated CD80 and CD86 expression. Moreover, only viable tachyzoites and antigenic extract induced IL-12 synthesis by dendritic cells. MHC-II expression was up-regulated and CD86 expression was down-regulated at the surface of macrophages, regardless of the parasitic form was encountered. However, IL-12 secretion by macrophages was only observed under conditions using viable and heat-killed parasite. We then analysed how macrophages and dendritic cells were involved in inducing T-cell responses. T lymphocyte IFN-γ-secretion in correlation with IL-12 production occurred after interactions between T cells and dendritic cells exposed to viable tachyzoites or antigenic extract. By contrast, for macrophages IFN-γ production was IL-12-independent and only occurred after interactions between T cells and macrophages exposed to antigenic extract. Thus, N. caninum-induced activation of murine dendritic cells, but not that of macrophages, was associated with T cell IFN-γ production after IL-12 secretion.     

Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host-parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.     

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.     

Toxoplasma gondii: serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris

The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti-Toxoplasma human serum, pooled anti-Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n = 20), acute (IgM positive, IgG positive; n = 20) and chronic (IgM negative, IgG positive; n = 20) toxoplasmosis patients, and toxoplasmosis negative control patients (n = 20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients’ serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P < 0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the P. pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti-Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge.     

Differential susceptibility of human trophoblastic (BeWo) and uterine cervical (HeLa) cells to Neospora caninum infection

Neospora caninum is an apicomplexan parasite, closely related to Toxoplasma gondii, and causes abortion and congenital neosporosis in cattle worldwide. Trophoblast cells act in mechanisms of innate immune defense at the fetal-maternal interface and no data are available about the interaction of Neospora with human trophoblasts. Thus, this study aimed to verify the susceptibility of human trophoblastic (BeWo) compared with uterine cervical (HeLa) cell lines to N. caninum. BeWo and HeLa cells were infected with different parasite:cell ratios of N. caninum tachyzoites and analyzed at different times after infection for cell viability using thiazolyl blue tetrazole and lactate dehydrogenase assays. Both cell lines were also evaluated for cytokine production and parasite infection/replication assays when pre-treated or not with Neospora lysate antigen (NLA) or human recombinant IFN-γ. Cell viability was increased up to 48 h of infection in both types of cells, suggesting that infection could inhibit early cell death and/or induce cell proliferation. Neospora infection induced up-regulation of the macrophage migration inhibitory factor (MIF), mainly in HeLa cells, which was enhanced by cell pre-treatment by NLA or IFN-γ. Conversely, parasite infection induced down-regulation of the transforming growth factor (TGF-β), mostly in BeWo cells, which was decreased with NLA or IFN-γ pre-treatment. HeLa cells were more susceptible to Neospora infection than BeWo cells and IFN-γ pre-treatment resulted in reduced infection indices in both cell lines. Control of parasite growth was mediated by IFN-γ through an indoleamine-2,3-dioxygenase-dependent mechanism in HeLa cells alone. Based on these results, we concluded that BeWo and HeLa cells are readily infected by N. caninum, although presenting differences in susceptibility to infection, cytokine production and cell viability. Thus, these host cells can be considered in comparative approaches to understand strategies used by N. caninum to survive at the maternal-fetal interface.     

Immunoregulatory effects of a glucogalactan from the root of Panax quinquefolium L.

In this research we purified one homogeneous glucogalactan from the roots of Panax quinquefolium, with a molecular weight of 54 kDa estimated by high-performance gel permeation chromatography (HPGPC). The monosaccharide composition of PPQ was composed of Glc (glucose) and Gal (galactose) in a molar ratio of 2.1:1, as determined by gas chromatography (GC). In order to evaluate the anti-lung carcinoma and immunoregulatory effects of this glucogalactan in mice, we established a Lewis lung carcinoma model in C57BL/6 mouse. The results showed that PPQ not only could inhibit the growth of lung tumor, but also enhance the thymus and spleen indices, as well as the level of IFN-γ, IL-10 and IL-2, indicating PPQ could have a possible cancer therapeutic potential.     

Counter-regulatory anti-parasite cytokine responses during concurrent Plasmodium yoelii and intestinal helminth infections in mice

Malaria and helminth infections are two of the most prevalent parasitic diseases globally. While concomitant infection is common, mechanisms contributing to altered disease outcomes during co-infection remain poorly defined. We have previously reported exacerbation of normally non-lethal Plasmodium yoelii malaria in BALB/c mice chronically infected with the intestinal trematode Echinostoma caproni. The goal of the present studies was to determine the effect of helminth infection on IFN-γ and other key cytokines during malaria co-infection in the P. yoelii-E. caproni and P. yoelii-Heligmosomoides polygyrus model systems. Polyclonally stimulated spleen cells from both E. caproni- and H. polygyrus-infected mice produced significantly lower amounts of IFN-γ during P. yoelii co-infection than malaria-only infected mice. Furthermore, the magnitude of IFN-γ suppression was correlated with the relative amounts of IL-4 induced by these helminths (E. caproni = low; H. polygyrus = high), but not IL-10. Concurrent malaria infection also suppressed helminth-associated IL-4 responses, indicating that immunologic counter-regulation occurs during co-infection with malaria and intestinal helminths.     

Anti-inflammatory effects of IL-17A on Helicobacter pylori-induced gastritis

Helicobacter pylori-induced immune responses are skewed toward a T helper (Th) 1 phenotype. IL-17-producing Th17 cells have recently been discovered, and we examined the role of IL-17A in H. pylori-induced gastritis. Six months after inoculation with H. pylori, the mice received an intraperitoneal injection of recombinant IL-17A, anti-IL-17A antibody or irrelevant IgG_2a for 3 days. H. pylori infection markedly increased mRNA for IL-17A. Double immunofluorescence studies showed that IL-17A proteins were expressed on CD4⁺ T cells, macrophages, and dendritic cells. H. pylori infection elevated mRNAs for IL-12, IFN-γ, and TNF-α with increase in myeloperoxidase activity, whereas it did not affect mRNAs for IL-4 and IL-5. Neutralization of IL-17A elevated mRNAs for IFN-γ and TNF-α, and myeloperoxidase activity, whereas recombinant IL-17A had a tendency to reduce these parameters. In conclusion, IL-17A exerts anti-inflammatory effects on H. pylori-induced gastritis through suppression of Th1 differentiation.     

Australian dingoes are definitive hosts of Neospora caninum

To provide objective data on the potential role of dingoes (Canis lupus dingo) in the life cycle of Neospora caninum in Australia, the production of N. caninum oocysts by experimentally infected canids was investigated. Three dingo pups raised in captivity and three domestic dogs were fed tissue from calves infected with an Australian isolate of N. caninum, Nc-Nowra. Oocysts of N. caninum, confirmed by species-specific PCR, were shed in low numbers by one dingo pup at 12-14 days p.i. The remaining animals did not shed oocysts. Furthermore, the blood from two out of three dingoes tested positive for DNA of N. caninum using PCR tests at 14 and 28 days p.i. Oocyst shedding from the intestinal tract of a dingo demonstrates that dingoes are definitive hosts of N. caninum and horizontal transmission of N. caninum from dingoes to farm animals and wildlife may occur in Australia.     

Diagnostic Potential of Anti-rNcp-43 Polyclonal Antibodies for the Detection of Neospora caninum

Neosporosis is a disease caused by the apicomplexan parasite Neospora caninum, which is closely related to Toxoplasma gondii. N. caninum infection represents an important cause of reproductive failure in sheep, goats, horses, and cattle worldwide. The diagnosis of neosporosis is based on the detection of pathogen-specific antibodies in animal sera or the presence of tissue cysts. However, morphological similarities and serological cross-reactivity between N. caninum and T. gondii can result in the misdiagnosis. In this study, the N. caninum tachyzoite surface protein Ncp-43 was expressed in a recombinant form to elicit polyclonal antibodies (pAb) response. The pAb was purified and conjugated to horseradish peroxidase (HRP) or fluorescein isothiocyanate (FITC) to detect the recombinant and native Ncp-43 proteins, respectively. The pAb and pAb/HRP were able to recognize rNcp-43 by dot blot and ELISA, and pAb/FITC immunolabeled the apical complex of tachyzoites. A blocking enzyme-linked immunosorbent assay (b-ELISA) was performed to evaluate pAb/HRP as a diagnostic tool. The mean percent inhibition for the positive and negative serum samples from cattle with neosporosis was significantly different (P < 0.0001). These results suggest that the pAb may bind to the same epitopes of Ncp-43 as anti-N. caninum antibodies in the positive samples tested. The b-ELISA using the pAb/HRP can facilitate diagnostic testing for neosporosis, since fewer steps are involved, and cross-reactivity with secondary antibodies is avoided. In summary, this report describes the production of antibodies against N. caninum, and evaluates the potential of these tools for the development of new diagnostic tests for neosporosis.     

Stimulus (polyphenol, IFN-gamma, LPS)-dependent nitric oxide production and antileishmanial effects in RAW 264.7 macrophages

The effects of interferon (IFN-γ), lipopolysaccharide (LPS), and some polyphenols as individual stimuli, as well as in various combinations on NO production in non-infected and infected macrophage-like RAW 264.7 cells were investigated, with emphasis on the NO/parasite kill relationship. In non-infected and in Leishmania parasitized cells, gallic acid significantly inhibited the IFN-γ and LPS-induced NO detected in the supernatant. This effect was less prominent in IFN-γ- than in LPS-stimulated cells. Interestingly, and in contrast to non-infected cells, gallic acid inhibited NO production only when added within 3 h after IFN-γ + LPS. Addition of gallic acid following prolonged incubation with IFN-γ + LPS periods (24 h) no longer inhibited, sometimes even enhanced NO release. Notably, an excellent NO/parasite kill relationship was evident from all the experiments. This study was extended to a series of polyphenols (3-O-shikimic acid, its 3,5-digalloylated analogue, catechin, EGCG, and a procyanidin hexamer) with proven immunostimulatory activities. Although these compounds themselves were found to be weak NO-inducers, the viability of intracellular Leishmania parasites was considerably reduced. Furthermore, their dose-dependent effects on macrophage NO release was determined in the presence of IFN-γ and/or LPS. Again, non-infected and infected cells differed significantly in the NO response, while inhibition of IFN-γ and/or LPS-induced NO production by the tested polyphenols strongly depended on the given time of exposure and the sequence of immunological stimuli. A strong inverse correlation between NO levels and intracellular survival rates of Leishmania parasites supported the assumption that the observed inhibition of NO was not simply due to interference with the Griess assay used for detection.     

Partial protective responses induced by a recombinant cysteine proteinase from Leishmania (Leishmania) amazonensis in a murine model of cutaneous leishmaniasis

A 500 bp fragment encoding an isoform of cysteine proteinase from Leishmania (Leishmania) amazonensis was subcloned and expressed in the pHis vector, resulting in a recombinant protein of 24 kDa, rLacys24. In Western blots of L. (L.) amazonensis extracts, antibodies directed to rLacys24 recognized a cysteine proteinase isoform of 30 kDa. Analysis by fluorescence-activated cell sorter showed a significantly higher expression of CD8⁺ lymphocytes in animals immunized with rLacys24 plus CFA, whereas a low expression of CD4⁺ lymphocytes was observed in these animals. The cytotoxicity of lymphocytes isolated from mice immunized with rLacys24 plus CFA on L. (L.) amazonensis-infected macrophages was significantly higher than that observed in the presence of lymphocytes from control animals. Immunization of BALB/c mice with rLacys24 plus CFA resulted in a low but significant decrease of foot lesions after challenge with L. (L.) amazonensis compared to those exhibited by control mice.     

Natural killer cell IFN-γ-activity is associated with Plasmodium falciparum infection during pregnancy

The ability of natural killer (NK) cells to produce gamma interferon (IFN-γ) after ex vivo stimulation with crude schizont lysate of Plasmodium falciparum was studied in uninfected and P. falciparum-infected pregnant Gabonese women segregated according to the gravidity at the time of delivery. This activity was measured in purified NK cells as well as in whole blood from the periphery and cord. Crude schizont lysate-stimulated NK cells from primiparous women produced significantly more IFN-γ than those from multiparous women (P < 0.001). Women with malaria infection produced more IFN-γ than negative women in peripheral blood (P < 0.001) indicating that immunological determinants regulating the susceptibility to malaria in pregnant women are parasite-specific. These findings reveal that NK cells are major source of IFN-γ when exposed to P. falciparum antigens in vitro in absence of any other co-stimulant.     

Mucosal and systemic immunity in mice after intranasal immunization with recombinant Lactococcus lactis expressing ORF6 of PRRSV

The purpose of the study was to construct mucosal vaccine of a recombinant Lactococcus lactis expressing PRRSV ORF6 gene and evaluate mucosal and systemic immune response against PRRSV in mice after intranasal immunization. The result show that the vaccine can stimulate mice to produce specific IgG in serum and remarkable special s-IgA in lung lavage fluid, at the same time, the contents of cytokines IL-2 and IFN-γ of the experimental group were significant higher than those of the control group (P < 0.01), however, the contents of cytokines IL-4 was not different to the all groups. In summary, the constructed mucosal vaccine can significantly induce mucosal immune, humoral immunity and cellular immunity involved Th1 type cytokines, which will lay a theoretical foundation on immune mechanism and new efficient vaccines for PRRSV.     

Toxoplasma gondii-induced foetal resorption in mice involves interferon-gamma-induced apoptosis and spiral artery dilation at the maternofoetal interface

The severity of congenital toxoplasmosis depends on the stage of the pregnancy at which infection takes place. Infection during the first trimester generally leads to miscarriage, through an unknown mechanism. Toxoplasma gondii infection is normally controlled by a strong Th1-type response with IFN-γ production. To investigate the mechanisms of foetal resorption induced by T. gondii, pregnant Swiss-Webster mice were infected 1 day post coïtum with the avirulent Me49 strain. Mated recipients were examined at mid-gestation. Few parasites and no cytolytic effects were detected 10 days post coïtum in implantation sites undergoing resorption. Resorption was accompanied by haemorrhage, spiral artery dilation, hypocellularity of the decidua basalis, apoptosis of placental cells, a decline in uterine mature natural killer cell numbers, increased indoleamine 2,3-dioxygenase mRNA levels and reduced IL-15 mRNA levels. Given the role of IFN-γR^−/− in non-infectious abortive processes, IFN-γR^−/− mice were used to investigate its local role in T. gondii-induced foetal resorption. IFN-γR^−/− mice showed 50% less foetal resorption than their wild-type counterparts, and spiral artery dilation and placental cell apoptosis were both abolished. These results strongly suggest that, at least in mice, T. gondii-induced abortion in early gestation is not due to a direct action of the parasite at the maternofoetal interface but rather to massive IFN-γ release.     

Prevention of multiple low-dose streptozotocin (MLD-STZ) diabetes in mice by an extract from gum resin of Boswellia serrata (BE)

Type 1-diabetes is an autoimmune disease, where a chronic inflammatory process finally causes β-cell death and insulin deficiency. Extracts from gum resin of Boswellia serrata (BE) have been shown to posses anti-inflammatory properties especially by targeting factors/mediators related to autoimmune diseases. Multiple low dose-streptozotocin (MLD-STZ) treatment is a method to induce diabetes in animals similar to Type 1 diabetes in humans.It was aimed to study whether or not a BE could prevent hyperglycemia, inflammation of pancreatic islets and increase of proinflammatory cytokines in the blood in MLD-STZ treated mice.In BK+/+ wild type mice, 5 days of daily treatment with 40 mg/kg STZ i.p. produced permanent increase of blood glucose, infiltration of lymphocytes into pancreatic islets (CD3-stain), apoptosis of periinsular cells (staining for activated caspase 3) after 10 days as well as shrinking of islet tissue after 35 days (H&E staining). This was associated with an increase of granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF) and proinflammatory cytokines (IL-1A, IL-1B, IL-2, IL-6, IFN-γ, TNF-α) in the blood. Whereas BE alone did not affect blood glucose in non diabetic mice, in STZ treated mice simultaneous i.p. injection of 150 mg/kg of BE over 10 days prevented animals from increase of blood glucose levels. Histochemical studies showed, that i.p. injection of 150 mg/kg BE for 10 days starting with STZ treatment, avoided lymphocyte infiltration into islets, apoptosis of periinsular cells and shrinking of islet size 35 days after STZ. As far as the cytokines tested are concerned, there was a significant inhibition of the increase of G-CSF and GM-CSF. BE also significantly prevented the increase of IL-1A, IL-1B, IL-2, IL-6, IFN-γ and TNF-α. It is concluded that extracts from the gum resin of Boswellia serrata prevent islet destruction and consequent hyperglycemia in an animal model of type 1 diabetes probably by inhibition of the production/action of cytokines related to induction of islet inflammation in an autoimmune process.