Transforming growth factor (TGF)-ß1 and interferon (IFN)-γ differentially regulate ICAM-1 expression and adhesion of Toxoplasma gondii to human trophoblast (BeWo) and uterine cervical (HeLa) cells.

Toxoplasma gondii is a parasite able to infect various cell types, including trophoblast cells. Studies have demonstrated that interleukin (IL)-10, transforming growth factor (TGF)-ß1 and interferon (IFN)-γ are involved in the susceptibility of BeWo trophoblast cells to T. gondii infection. Furthermore, T. gondii is able to adhere to the plasma membrane of host cells through intercellular adhesion molecule (ICAM)-1. Thus, the present study aimed to assess the role of IL-10, TGF-ß1 and IFN-γ in the expression of ICAM-1 in BeWo and HeLa cells and to analyze the role of ICAM-1 in the adhesion and invasion of T. gondii to these cells under the influence of these cytokines. For this purpose, BeWo and HeLa cells were treated or not, before and after T. gondii infection, with rIL-10, rTGF-ß1 or rIFN-γ. For the BeWo cells, rIL-10 and rTGF-ß1 favored susceptibility to infection, but only rTGF-ß1 and rIFN-γ increased ICAM-1 expression, and TNF-α release. On the other hand, rIFN-γ downregulated the expression of ICAM-1 triggered by T. gondii in HeLa cells, leading to control of the infection. Moreover, we observed that upregulation of ICAM-1, mediated by cytokine's stimulation, in BeWo and HeLa cells resulted in a high number rate of both parasite adhesion and invasion to these cells, which were strongly reduced after ICAM-1 neutralization. Likewise, the blockage of ICAM-1 molecule also impaired T. gondii infection in human villous explants. Taken together, these findings demonstrate that TGF-ß1 and IFN-γ differentially regulate ICAM-1 expression, which may interfere in the adhesion/invasion of T. gondii to BeWo and HeLa cells for modulating susceptibility to infection.

Si-Accumulation In Artemisia annua Glandular Trichomes Increases Artemisinin Concentration, but Does Not Interfere In the Impairment of Toxoplasma gondii Growth.

Artemisia annua is used as a source of artemisinin, a potent therapeutic agent used for the treatment of infectious diseases, chiefly malaria. However, the low concentration (from 0.01 to 1.4% of dried leaf matter) of artemisinin in the plant obtained with the traditional cropping system makes it a relatively expensive drug, especially in developing countries. Considering that artemisinin and silicon (Si) are both stored in A. annua glandular trichomes, and that Si accumulation has never been investigated, this study aimed to look into Si effects on A. annua trichome artemisinin concentration, and whether leaf infusion from Si-treated A. annua plants is able to control Toxoplasma gondii growth. T. gondii is the etiologic agent of toxoplasmosis, a zoonotic parasitic disease whose traditional treatment shows significant side effects. The experimental design consisted of A. annua seedlings randomly planted in soil treated with different doses of calcium/magnesium silicate (0, 200, 400, 800, and 1600 kg ha-1). Analysis of foliar macronutrients showed significant increases of nitrogen content only at the highest dose of silicate. Foliar micronutrients, Si concentrations, and plant height were not affected by any of the silicate doses. However, the dose of 400 kg ha-1 of silicate increased the trichome size, which in turn raised artemisinin concentration in leaves and the infusion. In contrast, the 800 and 1600 kg ha-1 doses dramatically decreased artemisinin concentration. HeLa cell treatment with the infusion of A. annua grown in soil treated with 400 kg ha-1 of silicate decreased parasite proliferation in a dose-dependent manner when the treatment was carried out after or along with T. gondii infection. However, this effect was similar to A. annua grown in soil without silicate treatment. Thus, it can be concluded that, even though Si applied to the soil at 400 kg ha-1 has a positive effect on the A. annua glandular trichome size and the artemisinin concentration, this outcome cannot be directly associated with the efficiency of A. annua infusion on T. gondii growth, suggesting that other components from A. annua leaves could be acting in synergy with artemisinin.

Azithromycin is able to control Toxoplasma gondii infection in human villous explants.

BACKGROUND: Although Toxoplasma gondii infection is normally asymptomatic, severe cases of toxoplasmosis may occur in immunosuppressed patients or congenitally infected newborns. When a fetal infection is established, the recommended treatment is a combination of pyrimethamine, sulfadiazine and folinic acid (PSA). The aim of the present study was to evaluate the efficacy of azithromycin to control T. gondii infection in human villous explants. METHODS: Cultures of third trimester human villous explants were infected with T. gondii and simultaneously treated with either PSA or azithromycin. Proliferation of T. gondii, as well as production of cytokines and hormones by chorionic villous explants, was analyzed. RESULTS: Treatment with either azithromycin or PSA was able to control T. gondii infection in villous explants. After azithromycin or PSA treatment, TNF-α, IL-17A or TGF-ß1 levels secreted by infected villous explants did not present significant differences. However, PSA-treated villous explants had decreased levels of IL-10 and increased IL-12 levels, while treatment with azithromycin increased production of IL-6. Additionally, T. gondii-infected villous explants increased secretion of estradiol, progesterone and HCG+ß, while treatments with azithromycin or PSA reduced secretion of these hormones concurrently with decrease of parasite load. CONCLUSIONS: In conclusion, these results suggest that azithromycin may be defined as an effective alternative drug to control T. gondii infection at the fetal-maternal interface.

Modulation of mucosal/systemic antibody response after sublingual immunotherapy in mite-allergic children.

BACKGROUND: There have been no data on sublingual immunotherapy (SLIT) in Brazilian patients sensitized to house dust mites. This study aimed to evaluate the mucosal/systemic antibody response changes and clinical efficacy after SLIT using Dermatophagoides pteronyssinus (Dpt) allergens with or without bacterial extracts in mite-allergic Brazilian children. METHODS: Patients with allergic rhinitis and asthma were selected for a double-blind, placebo-controlled trial randomized to three groups: DPT (Dpt extract, n = 34), DPT+MRB (Dpt plus mixed respiratory bacterial extracts, n = 36), and Placebo (n = 32). Total symptom and medication scores for rhinitis/asthma, skin prick test (SPT) to Dpt, and measurements of Dpt-, Der p 1-, Der p 2-specific serum IgE, IgG4, IgG1, and specific salivary IgA were evaluated at baseline and after 12 and 18 months of treatment. RESULTS: A significant long-term decline in total symptom/medication scores was observed only in active groups (DTP and DPT+MRB). There was no significant change in SPT results in all groups. SLIT using Dpt allergen alone induced increased levels of serum IgG4 to Dpt, Der p 1, and Der p 2, serum IgG1 and salivary IgA to Dpt and Der p 1. SLIT with Dpt plus bacterial extracts was able to decrease IgE levels, particularly to Der p 2, to increase salivary IgA levels to Der p 1, but had no changes on specific IgG4 and IgG1 levels. CONCLUSIONS: All children undergoing SLIT showed clinical improvement, but a long-term reduction in symptom/medication scores with modulation of mucosal/systemic antibody responses were seen only in active groups (DPT and DPT+MRB).

Heme oxygenase-1 activity is involved in the control of Toxoplasma gondii infection in the lung of BALB/c and C57BL/6 and in the small intestine of C57BL/6 mice.

Heme oxygenase-1 (HO-1) is an enzyme that catabolizes free heme, which induces an intense inflammatory response. The expression of HO-1 is induced by different stimuli, triggering an anti-inflammatory response during biological stress. It was previously verified that HO-1 is able to induce indoleamine 2,3-dioxygenase (IDO), an enzyme that is induced by IFN-γ in Toxoplasma gondii infection. To verify the role of HO-1 during in vivo T. gondii infection, BALB/c and C57BL/6 mice were infected with the ME49 strain and treated with zinc protoporphyrin IX (ZnPPIX) or hemin, which inhibit or induce HO-1 activity, respectively. The results show that T. gondii infection induced high levels of HO-1 expression in the lung of BALB/c and C57BL6 mice. The animals treated with ZnPPIX presented higher parasitism in the lungs of both lineages of mice, whereas hemin treatment decreased the parasite replication in this organ and in the small intestine of infected C57BL/6 mice. Furthermore, C57BL/6 mice infected with T. gondii and treated with hemin showed higher levels of IDO expression in the lungs and small intestine than uninfected mice. In conclusion, our data suggest that HO-1 activity is involved in the control of T. gondii in the lungs of both mouse lineages, whereas the hemin, a HO-1 inducer, seems to be involved in the control of parasitism in the small intestine of C57BL/6 mice.

SAG2A protein from Toxoplasma gondii interacts with both innate and adaptive immune compartments of infected hosts.

BACKGROUND: Toxoplasma gondii is an intracellular parasite that causes relevant clinical disease in humans and animals. Several studies have been performed in order to understand the interactions between proteins of the parasite and host cells. SAG2A is a 22 kDa protein that is mainly found in the surface of tachyzoites. In the present work, our aim was to correlate the predicted three-dimensional structure of this protein with the immune system of infected hosts. METHODS: To accomplish our goals, we performed in silico analysis of the amino acid sequence of SAG2A, correlating the predictions with in vitro stimulation of antigen presenting cells and serological assays. RESULTS: Structure modeling predicts that SAG2A protein possesses an unfolded C-terminal end, which varies its conformation within distinct strain types of T. gondii. This structure within the protein shelters a known B-cell immunodominant epitope, which presents low identity with its closest phyllogenetically related protein, an orthologue predicted in Neospora caninum. In agreement with the in silico observations, sera of known T. gondii infected mice and goats recognized recombinant SAG2A, whereas no serological cross-reactivity was observed with samples from N. caninum animals. Additionally, the C-terminal end of the protein was able to down-modulate pro-inflammatory responses of activated macrophages and dendritic cells. CONCLUSIONS: Altogether, we demonstrate herein that recombinant SAG2A protein from T. gondii is immunologically relevant in the host-parasite interface and may be targeted in therapeutic and diagnostic procedures designed against the infection.

Adjuvant and immunostimulatory effects of a D-galactose-binding lectin from Synadenium carinatum latex (ScLL) in the mouse model of vaccination against neosporosis.

Vaccination is an important control measure for neosporosis that is caused by a coccidian parasite, Neospora caninum, leading to abortion and reproductive disorders in cattle and serious economic impacts worldwide. A D-galactose-binding lectin from Synadenium carinatum latex (ScLL) was recently described by our group with potential immunostimulatory and adjuvant effects in the leishmaniasis model. In this study, we evaluated the adjuvant effect of ScLL in immunization of mice against neosporosis. First, we investigated in vitro cytokine production by dendritic cells stimulated with Neospora lysate antigen (NLA), ScLL or both. Each treatment induced TNF-α, IL-6, IL-10 and IL-12 production in a dose-dependent manner, with synergistic effect of NLA plus ScLL. Next, four groups of C57BL/6 mice were immunized with NLA + ScLL, NLA, ScLL or PBS. The kinetics of antibody response showed a predominance of IgG and IgG1 for NLA + ScLL group, whereas IgG2a response was similar between NLA + ScLL and NLA groups. Ex vivo cytokine production by mouse spleen cells showed the highest IFN-γ/IL-10 ratio in the presence of NLA stimulation for mice immunized with NLA + ScLL and the lowest for those immunized with ScLL alone. After parasite challenge, mice immunized with NLA + ScLL or ScLL alone presented higher survival rates (70-80%) and lower brain parasite burden as compared to PBS group, but with no significant changes in morbidity and inflammation scores. In conclusion, ScLL combined with NLA was able to change the cytokine profile induced by the antigen or lectin alone for a Th1-biased immune response, resulting in high protection of mice challenged with the parasite, but with low degree of inflammation. Both features may be important to prevent congenital neosporosis, since protection and low inflammatory response are necessary events to guide towards a successful pregnancy.

Production, characterization and applications for Toxoplasma gondii-specific polyclonal chicken egg yolk immunoglobulins.

BACKGROUND: Toxoplasma gondii may cause abortions, ocular and neurological disorders in warm-blood hosts. Immunized mammals are a wide source of hyperimmune sera used in different approaches, including diagnosis and the study of host-parasite interactions. Unfortunately, mammalian antibodies present limitations for its production, such as the necessity for animal bleeding, low yield, interference with rheumatoid factor, complement activation and affinity to Fc mammalian receptors. IgY antibodies avoid those limitations; therefore they could be an alternative to be applied in T. gondii model. METHODOLOGY/PRINCIPAL FINDINGS: In this study we immunized hens with soluble tachyzoite antigens of T. gondii (STAg) and purified egg yolk antibodies (IgY) by an inexpensive and simple method, with high yield and purity degree. IgY anti-STAg antibodies presented high avidity and were able to recognize a broad range of parasite antigens, although some marked differences were observed in reactivity profile between antibodies produced in immunized hens and mice. Interestingly, IgY antibodies against Neospora caninum and Eimeria spp. did not react to STAg. We also show that IgY antibodies were suitable to detect T. gondii forms in paraffin-embedded sections and culture cell monolayers. CONCLUSIONS/SIGNIFICANCE: Due to its cost-effectiveness, high production yield and varied range of possible applications, polyclonal IgY antibodies are useful tools for studies involving T. gondii.

Immunoproteomics of Brucella abortus reveals differential antibody profiles between S19-vaccinated and naturally infected cattle.

Brucella abortus is a Gram-negative intracellular bacterium that causes infectious abortion in food-producing animals and chronic infection in humans. This study aimed to characterize a B. abortus S19 antigen preparation obtained by Triton X-114 (TX-114) extraction through immunoproteomics to differentiate infected from vaccinated cattle. Three groups of bovine sera were studied: GI, 30 naturally infected cows; GII, 30 S19-vaccinated heifers; and GIII, 30 nonvaccinated seronegative cows. One-dimensional (1D) and two-dimensional electrophoretic profiles of TX-114 hydrophilic phase antigen revealed a broad spectrum of polypeptides (10-79 kDa). 1D immunoblot showed widespread seroreactivity profile in GI compared with restricted profile in GII. Three antigenic components (10, 12, 17 kDa) were recognized exclusively by GI sera, representing potential markers of infection and excluding vaccinal response. The proteomic characterization revealed 56 protein spots, 27 of which were antigenic spots showing differential seroreactivity profile between GI and GII, especially polypeptides <20 kDa that were recognized exclusively by GI. MS/MS analysis identified five B. abortus S19 proteins (Invasion protein B, Sod, Dps, Ndk, and Bfr), which were related with antigenicity in naturally infected cattle. In conclusion, immunoproteomics of this new antigen preparation enabled the characterization of proteins that could be used as tools to develop sensitive and specific immunoassays for serodiagnosis of bovine brucellosis, with emphasis on differentiation between S19 vaccinated and infected cattle.

Analysis of IgG subclasses (IgG1 and IgG3) to recombinant SAG2A protein from Toxoplasma gondii in sequential serum samples from patients with toxoplasmosis.

The kinetics of the humoral immune response was evaluated using the recombinant SAG2A protein comparatively to soluble Toxoplasma antigen (STAg) by ELISA in sequential serum samples of patients with toxoplasmosis up to 12 months of illness onset. The follow up of IgM and IgA levels to STAg showed a gradual decrease, with the majority of patients (88%) seropositive for IgM up to 12 months of infection, whereas IgA seropositivity was relatively low (78%) compared to IgM (100%) in the first 3 months of infection. The follow up of IgG and IgG1 antibodies showed a similar increasing profile for both SAG2A and STAg, with slightly higher seropositivity for STAg. The kinetics of IgG3 to STAg was similar to that of IgG1, contrasting with the kinetics of IgG3 to SAG2A that showed high levels up to 6 months of infection, with continuous decreasing over the time. Higher IgG3 seropositivity to SAG2A than STAg was also observed in the initial phases of infection. A higher IgG3/IgG1 ratio for SAG2A than STAg was detected in the first 3 months of infection, with decreasing profile over the time. The associations of IgG3/IgG1 ratio>1.0 with positive IgM or IgA antibodies were predominantly found in the first 3 months of infection, whereas associations of IgG3/IgG1 ratio<1.0 with positive IgM or negative IgA antibodies were mostly observed from 3 to 12 months of infection. In conclusion, our results demonstrate a differential kinetics of IgG3 antibodies to SAG2A and STAg in patients with toxoplasmosis up to 12 months of infection. Also, the IgG3/IgG1 ratio to SAG2A in association with classical serological markers of acute phase could be potential tools to distinguish early acute from convalescent phases of Toxoplasma gondii infection.

The impaired pregnancy outcome in murine congenital toxoplasmosis is associated with a pro-inflammatory immune response, but not correlated with decidual inducible nitric oxide synthase expression.

Congenital toxoplasmosis is associated with adverse pregnancy outcome. Despite the type 1 immune response, C57BL/6 mice are more susceptible than BALB/c mice to Toxoplasma gondii infection. Additionally, successful pregnancy appears to be correlated with type 2 T helper maternal immunity and regulatory T cells. In order to investigate the mechanisms of susceptibility/resistance to congenital toxoplasmosis in mice with different genetic backgrounds and the influence of inducible nitric oxide synthase in pregnancy outcome, groups of C57BL/6, BALB/c and C57BL/6 iNOS(-/-) females were orally infected with T. gondii ME-49 strain on day 1 of pregnancy and were sacrificed on day 8 p.i. and day 19 p.i. The uterus and placenta were evaluated for the foetal resorption rate, parasite load, immunological and histological changes. C57BL/6 mice presented inflammatory foci in the decidua (endometrium) of the uterus at a higher frequency than BALB/c mice on day 8 p.i., and a large number of pregnant C57BL/6 mice presented necrotic implantation sites. The parasite was seldom found in the uterus or placenta of either lineage of mice. Interestingly, there was no observed difference in inducible nitric oxide synthase expression in the uterus and placenta of infected mice. In addition, higher levels of TNF-α were detected in serum samples from C57BL/6 mice compared with BALB/c mice. Accordingly, C57BL/6 mice presented with levels of 90% abortion compared with 50% in BALB/c mice on day 19 p.i. C57BL/6 iNOS(-/-) mice showed low placental parasite counts and high absorption rates, similar to wild type mice. The data suggest that the impaired pregnancy outcome due to T. gondii infection in C57BL/6 mice could be associated with a higher inflammatory response leading to cell apoptosis and necrosis of implantation sites compared with BALB/c mice, and this phenomenon was not due to inducible nitric oxide synthase expression in the decidua.

Spirometric parameters and levels of interferon gamma and IL-5 in induced sputum from patients with allergic rhinitis or asthma.

BACKGROUND: This study was designed to analyze spirometric parameters and cytokine (interferon [IFN] gamma and IL-5) levels in induced sputum from patients with asthma or allergic rhinitis (AR) and nonatopic subjects. METHODS: Thirty-three subjects aged 18-60 years were enrolled in the present study. Eight patients had asthma without AR symptoms, 16 had AR without asthma symptoms or history, and both groups had positive skin-prick test (SPT) to aeroallergens. The nine remaining subjects were healthy nonatopic subjects with negative SPT to aeroallergens. Spirometry was performed by evaluating the forced vital capacity (FVC), forced expiratory volume in the 1st second (FEV(1)), and forced expiratory flow between 25 and 75% (FEF(25-75)) of FVC before and after bronchodilator use. Induced sputum samples were also collected for measuring cytokine (IFN-gamma and IL-5) levels by enzyme-linked immunosorbent assay. RESULTS: Significant pre- and postbronchodilator change was observed only for FEV(1) with higher variation values in asthmatic subjects compared with patients with AR (p < 0.05) and nonatopic subjects (p < 0.01). There was no significant difference in pre- and postbronchodilator spirometric parameters among the three groups, although asthmatic patients showed lower prebronchodilator FEF(25-75) values (p = 0.065). IL-5 levels were higher in induced sputum from patients with asthma and AR compared with nonatopic subjects (p = 0.020 and p = 0.032, respectively), but IFN-gamma levels showed no significant difference between the groups. CONCLUSION: Spirometric measurements were not able to show any occurrence of bronchial constriction in patients with AR, but predominant IL-5 levels in induced sputum from these patients reinforce the role of Th2-type immune response in lower respiratory airways that could contribute to the concept of "one airway, one disease."

Serum and salivary IgE, IgA, and IgG4 antibodies to Dermatophagoides pteronyssinus and its major allergens, Der p1 and Der p2, in allergic and nonallergic children.

Allergic rhinitis (AR) is a public health problem with high prevalence worldwide. We evaluated levels of specific IgE, IgA, and IgG4 antibodies to the Dermatophagoides pteronyssinus (Dpt) house dust mite and to its major allergens (Der p1 and Der p2) in serum and saliva samples from allergic and nonallergic children. A total of 86 children were analyzed, from which 72 had AR and 14 were nonallergic healthy children. Serum IgE and serum/salivary IgG4 levels to Dpt, Der p1, and Der p2 were higher in allergic children whereas serum/salivary IgA levels to all allergens were higher in nonallergic children. IgE levels positively correlated with IgG4 and IgA to all allergens in allergic children, while IgA levels negatively correlated with IgG4 to Dpt and Der p1 in nonallergic children. In conclusion, mite-specific IgA antibodies predominate in the serum and saliva of nonallergic children whereas mite-specific IgE and IgG4 are prevalent in allergic children. The presence of specific IgA appears to have a key role for the healthy immune response to mucosal allergens. Also, specific IgA measurements in serum and/or saliva may be useful for monitoring activation of tolerance-inducing mechanisms during allergen specific immunotherapeutic procedures, especially sublingual immunotherapy.

ArtinM, a D-mannose-binding lectin from Artocarpus integrifolia, plays a potent adjuvant and immunostimulatory role in immunization against Neospora caninum.

ArtinM and Jacalin (JAC) are lectins from the jackfruit (Artocarpus integrifolia) that have important role in modulation of immune responses to pathogens. Neospora caninum is an Apicomplexa parasite that causes neuromuscular disease in dogs and reproductive disorders in cattle, with economic impact on the livestock industry. Hence, we evaluated the adjuvant effect of ArtinM and JAC in immunization of mice against neosporosis. Six C57BL/6 mouse groups were subcutaneously immunized three times at 2-week intervals with Neospora lysate antigen (NLA) associated with lectins (NLA+ArtinM and NLA+JAC), NLA, ArtinM and JAC alone, and PBS (infection control). Animals were challenged with lethal dose of Nc-1 isolate and evaluated for morbidity, mortality, specific antibody response, cytokine production by spleen cells, brain parasite burden and inflammation. Our results demonstrated that ArtinM was able to increase NLA immunogenicity, inducing the highest levels of specific total IgG and IgG2a/IgG1 ratio, ex vivo Th1 cytokine production, increased survival, the lowest brain parasite burden, along with the highest inflammation scores. In contrast, NLA+JAC immunized group showed intermediate survival, the highest brain parasite burden and the lowest inflammation scores. In conclusion, ArtinM presents stronger immunostimulatory and adjuvant effect than Jacalin in immunization of mice against neosporosis, by inducing a protective Th1-biased pro-inflammatory immune response and higher protection after parasite challenge.

IgG avidity in differential serodiagnosis of human strongyloidiasis active infection.

IgG avidity assays have been developed for several parasitic diseases although there are no researches focused in strongyloidiasis diagnosis. Definitive diagnosis of strongyloidiasis is based on the presence of Strongyloides larvae in stool, but majority of cases involve low and irregular larval output. While limitations of serological assays for strongyloidiasis are well known, characteristics of persons who are misdiagnosed based on negative coproparasitological tests have been little explored. The aim of the present study was to evaluate the use of IgG avidity to detect patients with active strongyloidiasis and to characterize sources of disagreement between serology and coproparasitology. A total of 80 serum samples was analyzed, 40 from patients with Strongyloides larvae in stool (G1) and 40 from individuals with negative coproparasitology, but positive serology (G2). Serum samples were analyzed in an indirect IgG avidity ELISA using urea 6M in serial double dilutions from 1:80 to 1:2560. Avidity index (AI) was calculated to each serum dilution and analyzed as screening AI (serum dilution of 1:160) or mean AI of different serum dilutions that had a positive result. Statistical analyzes were performed by Mann-Whitney's (U) and Fisher's exact tests. At screening dilution, median of AI was 68% in G1 and 88% in G2 (P<0.0001), whereas median of mean AI in G1 was 72% and in G2 94% (P<0.0001), but there was no significant differences between both AI in each patient group. A cut off value established at AI of 75% demonstrated a significant difference between groups, with G1 sera showing AI<75% and G2 sera with AI>75% (P<0.0001). In conclusion, IgG avidity assays may distinguish active infection with Strongyloides stercoralis from suspect or serologically false positive cases.

Bothrops pirajai snake venom L-amino acid oxidase: in vitro effects on infection of Toxoplasma gondii in human foreskin fibroblasts

The effect of an L-amino acid oxidase isolated from Bothrops pirajai snake venom (BpirLAAO-I) was investigated on infection of Toxoplasma gondii in human foreskin fibroblasts (HFF). The cytotoxic activity of BpirLAAO-I on HFF cells showed a dose-dependent toxicity with median cytotoxic dose (TD50) of 11.8 µg/mL. BpirLAAO-I induced considerable dose-dependent decrease in the T. gondii infection index under two different conditions, treatment of tachyzoites before infection or treatment of HFF cells after infection. A maximal inhibition of infection (56 percent) was found for treatment before infection, with a median inhibitory dose (ID50) at 1.83 µg/mL and selectivity index (SI) at 6.45. For treatment after infection, it was observed a maximal inhibition of infection at 65 percent, ID50 of 1.20 µg/mL and SI of 9.83. The treatment before infection was also effective to reduce intracellular parasitism up to 62 percent, although presenting higher values of ID50 (3.14 µg/mL) and lower values of SI (3.76). However, treatment after infection was not effective, suggesting that the enzyme seems to have no effect on the parasite intracellular replication for this condition. In conclusion, BpirLAAO-I was more effective to inhibit the infection of neighboring cells and consequently parasite dissemination than primary infection and parasite replication. Thus, the effect of BpirLAAO-I described herein could be taken into account for the development of new synthetic anti-parasite therapeutic agents.

Spirometric parameters and levels of interferon gamma and IL-5 in induced sputum from patients with allergic rhinitis or asthma.

BACKGROUND: This study was designed to analyze spirometric parameters and cytokine (interferon [IFN] gamma and IL-5) levels in induced sputum from patients with asthma or allergic rhinitis (AR) and nonatopic subjects. METHODS: Thirty-three subjects aged 18-60 years were enrolled in the present study. Eight patients had asthma without AR symptoms, 16 had AR without asthma symptoms or history, and both groups had positive skin-prick test (SPT) to aeroallergens. The nine remaining subjects were healthy nonatopic subjects with negative SPT to aeroallergens. Spirometry was performed by evaluating the forced vital capacity (FVC), forced expiratory volume in the 1st second (FEV1), and forced expiratory flow between 25 and 75% (FEF25-75) of FVC before and after bronchodilator use. Induced sputum samples were also collected for measuring cytokine (IFN-gamma and IL-5) levels by enzyme-linked immunosorbent assay. RESULTS: Significant pre- and postbronchodilator change was observed only for FEV1 with higher variation values in asthmatic subjects compared with patients with AR (p < 0.05) and nonatopic subjects (p < 0.01). There was no significant difference in pre- and postbronchodilator spirometric parameters among the three groups, although asthmatic patients showed lower prebronchodilator FEF25-75 values (p = 0.065). IL-5 levels were higher in induced sputum from patients with asthma and AR compared with nonatopic subjects (p = 0.020 and p = 0.032, respectively), but IFN-gamma levels showed no significant difference between the groups. CONCLUSION: Spirometric measurements were not able to show any occurrence of bronchial constriction in patients with AR, but predominant IL-5 levels in induced sputum from these patients reinforce the role of Th2-type immune response in lower respiratory airways that could contribute to the concept of "one airway, one disease."

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-ß), 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.

Antibody and cytokine responses to house dust mite allergens and Toxoplasma gondii antigens in atopic and non-atopic Brazilian subjects.

According to hygiene hypothesis, a lower exposure to infection is associated with increased prevalence of allergic diseases. This study aimed to investigate the association between atopy and Toxoplasma gondii (Tg) infection by analyzing the antibody and cytokine responses to house dust mite allergens and T. gondii antigens in Brazilian subjects. A total of 275 individuals were assessed and divided into atopics (n=129) and non-atopics (n=146) based on markers of allergy (positive skin prick test and ELISA-IgE to mite allergens) or Tg-seropositive (n=116) and Tg-seronegative (n=159) groups according to infection markers (positive ELISA-IgG to T. gondii). Tg-seropositive individuals presented lower allergenic sensitization (37%) to mite allergens than Tg-seronegative subjects (54%). A significant association was found between atopy and negative serology to T. gondii (OR: 2.0; 95% CI: 1.23-3.26; P<0.05). Proliferative responses and cytokine production after antigenic stimulation showed predominant synthesis of Th1-cytokines as IFN-gamma in Tg-seropositive patients, whether atopics or non-atopics. Conversely, Th2-cytokines as IL-5 prevailed in atopics compared to non-atopics, regardless the seropositivity to T. gondii. Levels of IL-10, IL-13, IL-17, and TGF-beta were not able to discriminate the groups. Hence, a negative association between atopy and infection by T. gondii was demonstrated for the first time in Brazilian subjects, focusing on the antibody and cytokine responses and indicating that the immunomodulation induced by the parasite may play a protective role in the development of allergic diseases.

Evaluation of indirect enzyme-linked immunosorbent assays and IgG avidity assays using a protein A-peroxidase conjugate for serological distinction between Brucella abortus S19-vaccinated and -infected cows.

This study aimed to evaluate the use of protein A-peroxidase (horseradish peroxidase [HRPO]) in indirect enzyme-linked immunosorbent assays (iELISAs) and IgG avidity assays for serological distinction between Brucella abortus S19-vaccinated and -infected cows. Four groups were analyzed: GI, 41 nonvaccinated seropositive cows; GII, 79 S19-vaccinated heifers analyzed at 3 months postvaccination; GIII, 105 S19-vaccinated cows analyzed after 24 months of age; and GIV, 278 nonvaccinated seronegative cows. IgG levels and avidity to B. abortus smooth lipopolysaccharide (S-LPS) were determined using anti-bovine IgG-HRPO or protein A-HRPO conjugates. Similar levels of IgG anti-S-LPS were found with GI using both conjugates. Lower IgG levels were detected with GII, GIII, and GIV using protein A-HRPO. Both conjugates showed high performance in discriminating GI from GIII, with high sensitivity (Se; 97.6%) and specificity (Sp; 97.1%). Protein A-HRPO was better in distinguishing GI from GIV (Se, 97.6%; Sp, 94.6%) and GI from GII (Se, 80.5%; Sp, 94.9%). Protein A-HRPO excluded a higher number of positive samples with GII and GIV. IgG avidity showed that protein A-HRPO, but not anti-IgG-HRPO, was able to distinguish nonvaccinated from vaccinated cattle, showing a higher avidity index (AI) with GI than with GII, with 78% of serum samples in GII showing an AI of <50%. Therefore, the iELISA using B. abortus S-LPS antigen and protein A-HRPO conjugate for preferential detection of the IgG2 subclass was shown to be suitable for serological distinction between S19-vaccinated and -infected cows. Also, antibodies generated after vaccination showed lower avidity, suggesting a role for the IgG2 subclass as an antibody of higher-affinity maturation after infection, constituting an additional tool for differentiating vaccinated from infected cattle.