American cutaneous leishmaniasis: In situ immune response of patients with recent and late lesions.

TNF-α, IFN-γ, IL-10, IL-17, CD68 and CD57 were evaluated in biopsies of patients with American cutaneous leishmaniasis living in Sorocaba, Brazil. The analyses were performed considering the time of lesions from 23 patients with recent lesions (Group I) and 19 patients with late lesions (Group II). All patients were infected with Leishmania (Viannia) braziliensis. Immunostaining cells for CD68, CD57, TNF- α, IFN-γ, IL-10 and IL-17 were performed by immunohistochemistry. Except for CD68 and IL-17, the distribution of in situ for CD57, IL-10, TNF-α and IFN-γ showed that patients with recent lesions expressed higher levels than those with late lesions. The comparison of cytokine expression/group showed that IL-10 was significantly higher than IL-17 and IFN-γ (similar data were shown in IL-17 compared with TNF-α), suggesting an immunological balance between inflammatory-anti-inflammatory agents. This balance was similar for two groups of patients. In conclusion, these data suggested that (i) patients from Group I had recent lesions (in the beginning of chronic phase) compared to those from Group II and (ii) the modulation of inflammatory response in patients with recent American cutaneous leishmaniasis was correlated with IL-10 expression in skin lesions preventing the development of mucosal forms. The parasite treatment also prevented the evolution of severe forms.

Evaluation of gene expression levels for cytokines in ocular toxoplasmosis.

This study evaluated levels for mRNA expression of 7 cytokines in ocular toxoplasmosis. Peripheral blood mononuclear cells (PBMC) of patients with ocular toxoplasmosis (OT Group, n = 23) and chronic toxoplasmosis individuals (CHR Group, n = 9) were isolated and stimulated in vitro with T. gondii antigen. Negative controls (NC) were constituted of 7 PBMC samples from individuals seronegative for toxoplasmosis. mRNA expression for cytokines was determined by qPCR. Results showed a significant increase in mRNA levels from antigen stimulated PBMCs derived from OT Group for expressing IL-6 (at P < .005 and P < .0005 for CHR and NC groups, respectively), IL-10 (at P < .0005 and P < .005 for CHR and NC groups, respectively) and TGF-ß (at P < .005) for NC group. mRNA levels for TNF-α and IL-12 were also upregulated in patients with OT compared to CHR and NC individuals, although without statistical significance. Additionally, mRNA levels for IL-27 and IFN-γ in PBMC of patients with OT were upregulated in comparison with NC individuals. Differences between OT and NC groups were statistically significant at P < .05 and P < .0005, respectively.

Risk factors for ocular toxoplasmosis in Brazil.

The aim of this study was to investigate risk factors for ocular toxoplasmosis (OT) in patients who received medical attention at a public health service. Three hundred and forty-nine consecutive patients, treated in the Outpatient Eye Clinic of Hospital de Base, São José do Rio Preto, São Paulo state, Brazil, were enrolled in this study. After an eye examination, enzyme-linked immunosorbent assay (ELISA) was used to determine anti-Toxoplasma gondii antibodies. The results showed that 25.5% of the patients were seronegative and 74.5% were seropositive for IgG anti-T. gondii antibodies; of these 27.3% had OT and 72.7% had other ocular diseases (OOD). The presence of cats or dogs [odds ratio (OR) 2.22, 95% confidence interval (CI) 1.24-3.98, P = 0.009] and consumption of raw or undercooked meat (OR 1.77, 95% CI 1.05-2.98, P = 0.03) were associated with infection but not with the development of OT. Age (OT 48.2 ± 21.2 years vs. OOD: 69.5 ± 14.7 years, P < 0.0001) and the low level of schooling/literacy (OT vs. OOD: OR 0.414, 95% CI 0.2231-0.7692, P = 0.007) were associated with OT. The presence of dogs and cats as well as eating raw/undercooked meat increases the risk of infection, but is not associated with the development of OT.

Trans-sialidase delivered as a naked DNA vaccine elicits an immunological response similar to a Trypanosoma cruzi infection.

Trypanosoma cruzi. the protozoan parasite that causes Chagas' disease, does not synthesize sialic acid, but expresses a trans-sialidase (TS) that catalyzes the transfer of sialic acid from host glycoconjugates to the parasite surface. Here, we review studies that characterize the immune response to the catalytic domain of the enzyme in humans during Chagas' disease or in mice following immunization with the TS gene. In both cases, there are antibodies that strongly inhibit the enzymatic activity and generation of interferon-gamma-producing T cells.

American cutaneous leishmaniasis: In situ immune response of patients with recent and late lesions

TNF­α, IFN­Î³, IL­10, IL­17, CD68 and CD57 were evaluated in biopsies of patients with American cutaneous leishmaniasis living in Sorocaba, Brazil. The analyses were performed considering the time of lesions from 23 patients with recent lesions (Group I) and 19 patients with late lesions (Group II). All patients were infected with Leishmania (Viannia) braziliensis. Immunostaining cells for CD68, CD57, TNF­ α, IFN­Î³, IL­10 and IL­17 were performed by immunohistochemistry. Except for CD68 and IL­17, the distribution of in situ for CD57, IL­10, TNF­α and IFN­Î³ showed that patients with recent lesions expressed higher levels than those with late lesions. The comparison of cytokine expression/group showed that IL­10 was significantly higher than IL­17 and IFN­Î³ (similar data were shown in IL­17 compared with TNF­α), suggesting an immunological balance between inflammatory­anti­inflammatory agents. This balance was similar for two groups of patients. In conclusion, these data suggested that (i) patients from Group I had recent lesions (in the beginning of chronic phase) compared to those from Group II and (ii) the modulation of inflammatory response in patients with recent American cutaneous leishmaniasis was correlated with IL­10 expression in skin lesions preventing the development of mucosal forms. The parasite treatment also prevented the evolution of severe forms.

TNF-α, IFN-γ, IL-10, IL-17, CD68 e CD57 foram avaliados em biópsias de pacientes com leishmaniose tegumentar americana residentes em Sorocaba, Brasil. As análises foram realizadas considerando o tempo de lesão de 23 pacientes com lesões recentes (Grupo I) e 19 pacientes com lesões tardias (Grupo II). Todos os pacientes foram infectados com Leishmania (Viannia) braziliensis. As células de imunocoloração para CD68, CD57, TNF-α, IFN-γ, IL-10 e IL-17 foram realizadas por imuno-histoquímica. Exceto para CD68 e IL-17, a distribuição in situ de CD57, IL-10, TNF-α e IFN-γ mostrou que pacientes com lesões recentes expressavam níveis mais altos do que aqueles com lesões tardias. A comparação da expressão / grupo de citocinas mostrou que a IL-10 foi significativamente maior que a IL-17 e IFN-γ (dados semelhantes foram mostrados na IL-17 em comparação com o TNF-α), sugerindo um equilíbrio imunológico entre agentes inflamatórios e anti-inflamatórios . Esse equilíbrio foi semelhante para dois grupos de pacientes. Em conclusão, esses dados sugeriram que (i) pacientes do grupo I apresentavam lesões recentes (no início da fase crônica) em comparação com os do grupo II e (ii) a modulação da resposta inflamatória em pacientes com leishmaniose tegumentar americana recente estava correlacionada com Expressão da IL - 10 em lesões cutâneas impedindo o desenvolvimento de formas mucosas. O tratamento do parasita também impediu a evolução de formas graves.

Cerebral and ocular toxoplasmosis related with IFN-y, TNF-a, and IL-10

This study analyzed the synthesis of Interferon gamma (IFN-γ), Tumor Necrosis Factor alpha (TNF-α), and Interleukin 10 (IL-10) in chronically infected patients which developed the symptomatic disease as cerebral or ocular toxoplasmosis. Blood from 61 individuals were divided into four groups: Cerebral toxoplasmosis/AIDS patients (CT/AIDS group) (n = 15), ocular toxoplasmosis patients (OT group) (n = 23), chronic toxoplasmosis individuals (CHR group) (n = 13) and healthy individuals (HI group) (n = 10). OT, CHR, and HI groups were human immunodeficiency virus (HIV) seronegative. The diagnosis was made by laboratorial (PCR and ELISA) and clinical subjects. For cytokine determination, peripheral blood mononuclear cells (PBMC) of each patient were isolated and stimulated in vitro with T. gondii antigen. IFN-γ, TNF-α, and IL-10 activities were determined by ELISA. Patients from CT/AIDS and OT groups had low levels of IFN-γ when were compared with those from CHR group. These data suggest the low resistance to develop ocular lesions by the low ability to produce IFN-γ against the parasite. The same patients, which developed ocular or cerebral toxoplasmosis had higher TNF-α levels than CHR individuals. High TNF-α synthesis contribute to the inflammatory response and damage of the choroid and retina in OT patients and in AIDS patients caused a high inflammatory response as the TNF-α synthesis is not affected since monocytes are the major source this cytokine in response to soluble T. gondii antigens. IL-10 levels were almost similar in CT/AIDS and OT patients but low when compared with CHR individuals. The deviation to Th2 immune response including the production of anti-inflammatory cytokines, such as IL-10 may promote the parasite's survival causing the tissue immune destruction. IL-10 production in T. gondii-infected brains may support the persistence of parasites as down-regulating the intracerebral immune response. All these indicate that OT and CT/AIDS patients produced low levels of IL-10 (Th2 response) and IFN-γ (Th1 response). They produced high TNF-α suggesting a high inflammatory response triggered by the parasite.

Sera from chronic Chagasic patients and rodents infected with Trypanosoma cruzi inhibit trans-sialidase by recognizing its amino-terminal and catalytic domain.

We investigated whether sera from chronic Chagasic patients and animals infected with Trypanosoma cruzi inhibit the removal of sialic acid from human erythrocytes and the transfer of sialic acid from sialyllactose to [14C]lactose in the reactions catalyzed by the parasite trans-sialidase. Sera from Swiss mice and Calomys callosus animals infected with three different T. cruzi strains inhibit both reactions. Inhibition increases during the infection, reaching maximal levels when the parasitemia decreases. Among 44 sera of untreated chronic Chagasic patients, 40 inhibit both reactions. Inhibition is observed with total, defatted sera or with purified immunoglobulins. Whereas most of the inhibitory antibodies from Chagasic patients react with the papain fragment of trans-sialidase in immunoblots, a few patients have noninhibitory antibodies that react only with the entire trans-sialidase. These findings may be relevant for the pathology of Chagas' disease.

A sialidase activity in the midgut of the insect Triatoma infestans is responsible for the low levels of sialic acid in Trypanosoma cruzi growing in the insect vector.

Trypanosoma cruzi expresses a unique trans-sialidase that is responsible for the transfer of sialic acid from host glycoproteins and glycolipids to mucin-like glycoprotein acceptors on the parasite surface. The enzyme and the sialic acid acceptors are present in the mammalian forms of the parasite and in the parasite forms that grow in axenic cultures, which correspond to the developmental stages found in the insect vectors. Here we show that parasite forms growing in the vector Triatoma infestans express trans-sialidase in the hindgut portions of the insect. However, the sialic acid acceptors are poorly sialylated due to the low concentration of sialic acid donors in the gut lumen of T.infestans, which feeds exclusively on blood that is rich in sialic acid donors. These low levels of sialic acid donors are due to a novel sialidase activity present mainly in the anterior midgut with high specificity for alpha-2,3-sialyllactose, but not for alpha-2,6-sialyllactose. The activity is present in starved insects or insects fed with culture medium, indicating that it did not originate from the blood meal. Enzyme activity does not decrease in insects fed with antibiotics, is present in the salivary glands, and the few bacteria isolated from the gut and faeces of T.infestans did not display sialidase activity, indicating that the enzyme is not derived from a commensal organism. This novel activity could have a nutritional role in the gut of haematophagous insects and indicates that acquisition of sialic acid is not required for parasite development in the gut of T.infestans.

Passive transfer of a monoclonal antibody specific for a sialic acid-dependent epitope on the surface of Trypanosoma cruzi trypomastigotes reduces infection in mice.

Trypanosoma cruzi, the parasite that causes Chagas' disease, proliferates in the cytosol of mammalian cells. When the trypomastigote forms exit the infected cell, they become extensively sialylated because the parasite contains an enzyme called trans-sialidase. This enzyme efficiently catalyzes the transfer of bound sialic acid residues from host glycoconjugates to acceptors containing terminal beta-galactosyl residues on the parasite surface. The sialic acid acceptors are developmentally regulated mucin-like glycoproteins that are extremely abundant on the trypomastigote surface. In the present study, we determined whether passive transfer of monoclonal antibodies specific for sialic acid acceptors could reduce the acute infection induced by T. cruzi in a highly susceptible mouse strain. We found that passive transfer to naive mice of an immunoglobulin G1 monoclonal antibody directed to a sialylated epitope of these mucin-like glycoproteins significantly decreased parasitemia and the number of tissue parasites as measured by a DNA probe specific for T. cruzi. Upon challenge with trypomastigotes, mice which received this antibody also had a significant increase in survival. A statistically significant reduction in parasitemia could be accomplished with relatively small doses of immunoglobulin, and Fab fragments alone could not mediate protective immunity. The precise mechanism of parasite elimination is unknown; however, this monoclonal antibody does not lyse trypomastigotes in vitro in the presence of human complement or mouse spleen cells.

Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase.

Trypanosoma cruzi, the agent of Chagas disease, expresses on its surface a trans-sialidase that catalyzes preferentially the transference of alpha-2,3-linked sialic acid to acceptors containing terminal beta-galactosyl residues, instead of the typical hydrolysis reaction, found in most sialidases. The trans-sialidase is responsible for the acquisition of the host sialic acid by this protozoan parasite, which does not synthesize sialic acids. Here, we have studied some kinetic properties of a recombinant trans-sialidase expressed in Escherichia coli. We found that it has sequential-type kinetics for the transferase reaction, as shown for the parasite-derived enzyme. The rates of sialic acid transfer to water (hydrolysis), and to beta-galactosyl residues have a unique behavior with respect to the reaction temperature. While the hydrolysis rate of sialyllactose increases continuously up to 35 degrees C, the temperature for the maximal rate of trans-glycosylation depends on the acceptor concentration. At low acceptor concentrations the rate of trans-glycosylation is maximal at 13 degrees C and independent of the amount of sialic acid donors. With increasing acceptor concentrations, maximal rates of trans-glycosylation are shifted to higher temperatures. This finding is explained by an 8-fold increase in the Km for the acceptor from 13 degrees C to 33 degrees C. Differences in hydrolysis and transfer rates were also obtained by using 4-methylumbelliferyl-N-acetyl-neuraminic acid. However, its hydrolysis rate is much higher than the rate of transference to lactose, suggesting that a long-lived enzyme-sialosyl intermediate is not formed. In addition, lactose does not increase the rate of methyl-umbelliferone release at any temperature, indicating that the rate limiting step is the aglycon release. Based on these results we propose that transglycosylation in T. cruzi sialidase is favored by the existence of a binding site for beta-galactosyl residues, which accepts the new glycosidic bond as sialic acid is released from the donor. With increasing temperature the affinity for the acceptor decreases, resulting in a concomitant increase in the rate of transfer to water, which, in turn, can be suppressed by increasing the acceptor concentration.

DNA sequences encoding CD4+ and CD8+ T-cell epitopes are important for efficient protective immunity induced by DNA vaccination with a Trypanosoma cruzi gene.

Immunization of BALB/c mice with a plasmid containing the gene for Trypanosoma cruzi trans-sialidase (TS) induced antibodies that inhibited TS enzymatic activity, CD4+ Th1 and CD8+ Tc1 cells, and protective immunity against infection. We used this model to obtain basic information on the requirement of CD4 or CD8 or B-cell epitopes for an effective DNA-induced immunity against T. cruzi infection. For that purpose, mice were immunized with plasmids containing DNA sequences encoding (i) the entire TS protein, (ii) the TS enzymatic domain, (iii) the TS CD4+ T-cell epitopes, (iv) the TS CD8+ T-cell epitope, or (v) TS CD4+ and CD8+ T-cell epitopes. Plasmids expressing the entire TS or its enzymatic domain elicited similar levels of TS-inhibitory antibodies, gamma interferon (IFN-gamma)-producing T cells, and protective immunity against infection. Although the plasmid expressing TS CD4 epitopes was immunogenic, its protective efficacy against experimental infection was limited. The plasmid expressing the CD8 epitope was poorly immunogenic and provided little protective immunity. The reason for the limited priming of CD8+ T cells was due to a requirement for CD4+ T cells. To circumvent this problem, a plasmid expressing both CD4+ and CD8+ T-cell epitopes was produced. This plasmid generated levels of IFN-gamma-producing T cells and protective immunity comparable to that of the plasmid expressing the entire catalytic domain of TS. Our observations suggest that plasmids expressing epitopes recognized by CD4+ and CD8+ T cells may have a better protective potential against infection with T. cruzi.

Chagasic patients develop a type 1 immune response to Trypanosoma cruzi trans-sialidase.

Infective forms of Trypanosoma cruzi, the parasite that causes Chagas' disease, express on their surface an enzyme denominated trans-sialidase (TS). The present study was designed to evaluate the naturally acquired immune responses to a bacterial recombinant protein representing the catalytic domain of TS in chronically infected chagasic individuals. The cellular immune response was measured by in-vitro T-cell proliferation and by interferon (INF)-gamma, interleukin (IL)-4 and IL-10 production in response to a whole-parasite homogenate and the recombinant protein. The peripheral blood mononuclear cells of 78.6% of 28 chagasic patients responded to the recombinant protein as estimated by T-cell proliferation. With respect to cytokine production, 88% of the cells of the chagasic individuals produced IFN-gamma on stimulation with the recombinant protein. In contrast, IL-4 or IL-10 were minimally produced in response to TS. The cellular immune response was specific because most healthy individuals never exposed to T. cruzi failed to react with this recombinant protein. The plasma of 71.4% or 100% of chagasic patients had IgG antibodies as determined by ELISA or by the presence of TS inhibitory antibodies, respectively. We conclude that the catalytic domain of TS is recognized by IFN-gamma producing type 1 cells and antibodies in a large proportion of patients infected with T. cruzi.

Comparison of antibody and protective immune responses against Trypanosoma cruzi infection elicited by immunization with a parasite antigen delivered as naked DNA or recombinant protein.

Immunization with naked DNA represents an attractive strategy for development of vaccines against a variety of infections including those caused by protozoan parasites. Recently, we have described that immunization with a plasmid containing the trans-sialidase (TS) gene induced protective immunity against Trypanosoma cruzi infection in BALB/c mice. The present study was aimed at examining and comparing the effectiveness of immunization using either plasmid or recombinant delivered antigens in a mouse strain highly susceptible to infection (A/Sn). Two plasmids were generated containing the coding region for the catalytic domain of TS. TS gene was inserted into pcDNA3 vector with or without the coding region for TS signal peptide. These two plasmids were found to be equally immunogenic at inducing antibodies to TS or inhibition of T. cruzi infection. A third plasmid, in which the TS gene was inserted into the vector VR1012, was as immunogenic as the two others. Immunization with a TS recombinant protein in alum generated a significantly higher antibody response as measured by ELISA or inhibition of TS enzymatic activity. Most relevant, this immunization reduced the mortality due to acute infection.

Trypanosoma cruzi defined antigens in the serological evaluation of an outbreak of acute Chagas disease in Brazil (Catolé do Rocha, Paraíba).

Immunoglobulin G and M humoral response to recombinant protein B13 and glycoconjugate LPPG Trypanosoma cruzi defined antigens was evaluated by ELISA in 18 patients in the acute phase of Chagas disease, who were contaminated on the same occasion. LPPG showed 100% positivity detecting both IgM and IgG antibodies, while positivity of 55-65% was observed for B13. An epimastigote alkaline extract (EPI) also showed high sensitivity for acute IgM (100%) and IgG (90%) antibodies. However LPPG had better discriminatory reactivity since with EPI two patients showed negative IgG, and several other sera presented OD values for IgG and IgM antibodies very close to the cutoff. Thus, it is suggested that detection of IgM antibodies by LPPG may be used for diagnosis of the acute phase of Chagas disease. An intense decline of IgG and IgM antibodies to the three antigens was observed in response to anti-T. cruzi chemotherapy in all acute phase patients. After treatment, six (30%) individuals maintained IgG positivity to EPI, LPPG, and B13 with lower reactivity than that measured at the acute phase. For comparison, serology of a group of 22 patients in the chronic phase of Chagas disease and also submitted to chemotherapy was determined. Positive IgM antibodies to EPI, LPPG and B13 were detected in only 5-9% cases. In all chronic-phase patients IgG antibodies highly reactive to the three antigens were present and no significant decrease resulted after benznidazole administration. These observations reinforce previous reports that treatment in the acute phase may reduce or eliminate the parasite.

Immunization with a plasmid DNA containing the gene of trans-sialidase reduces Trypanosoma cruzi infection in mice.

Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, does not synthesize sialic acid, but expresses a trans-sialidase that catalyses the transfer of sialic acid from host glycoconjugates to the parasite surface. Several lines of evidence suggest that this enzyme is a virulence factor implicated in the establishment of infection. Here we studied whether immunization with a plasmid DNA containing a gene encoding for the catalytic domain of the enzyme could elicit protective immunity against T. cruzi infection in mice. We observed that immunization with this plasmid DNA generated antibody and T-cell mediated immune responses. Antibodies recognized the native enzyme and inhibited its activity in vitro. Upon challenge with bloodstream trypomastigotes, immunized animals displayed reduced parasitemia and mortality.

Mucin-like molecules form a negatively charged coat that protects Trypanosoma cruzi trypomastigotes from killing by human anti-alpha-galactosyl antibodies.

In the presence of sialic acid donors Trypanosoma cruzi acquires up to 10(7) sialic acid residues on its surface, in a reaction catalyzed by its unique trans-sialidase. Most of these sialic acid residues are incorporated into mucin-like glycoproteins. To further understand the biological role of parasite sialylation, we have measured the amount of mucin in this parasite. We found that both epimastigote and trypomastigote forms have the same number of mucin molecules per surface area, although trypomastigotes have less than 10% of the amount of glycoinositol phospholipids, the other major surface glycoconjugate of T. cruzi. Based on the estimated surface area of each mucin, we calculated that these molecules form a coat covering the entire trypomastigote cell. The presence of the surface coat is shown by transmission electron microscopy of Ruthenium Red-stained parasites. The coat was revealed by binding of antibodies isolated from Chagasic patients that react with high affinity to alpha-galactosyl epitopes present in the mucin molecule. When added to the trypomastigote, these antibodies cause an extensive structural perturbation of the parasite coat with formation of large blebs, ultimately leading to parasite lysis. Interestingly, lysis is decreased if the mucin coat is heavily sialylated. Furthermore, addition of MgCl2 reverses the protective effect of sialylation, suggesting that the sialic acid negative charges stabilize the surface coat. Inhibition of sialylation by anti-trans-sialidase antibodies, found in immunized animals, or human Chagasic sera, also increase killing by anti-alpha-galactosyl antibodies. Therefore, the large amounts of sialylated mucins, forming a surface coat on infective trypomastigote forms, have an important structural and protective role.