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PURPOSE: Regulatory T cells (Tregs) play a role in the immunosuppressive state in pancreatic cancer patients. We aimed to evaluate the changes of immune cells population including Tregs caused by gemcitabine (GEM)-based chemotherapy. METHODS: Fifty-three patients with pancreatic cancer were enrolled in this study, of which 32 received GEM- based chemotherapy. Blood samples were collected before and at least 2 weeks after the last dose of chemotherapy. The peripheral blood mononuclear cells (PBMCs) were subjected to flow cytometry analysis after labeling with anti-CD4, anti-CD25, and anti-Foxp3 antibodies. Other lymphocytes and NK cell markers were also measured. The proliferative capacity of PBMCs stimulated with anti-CD3 was analyzed using H(3) thymidine. RESULTS: The percentage and number of Tregs were significantly decreased after chemotherapy (p = 0.032, p = 0.003, respectively). The other immune cells and the proliferative capacity did not change. CONCLUSION: This study showed that GEM-based chemotherapy produced an immunomodulatory effect via the depletion of Tregs.

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This study evaluated the performance of the EIE-leishmaniose-visceral-canina-Bio-Manguinhos (EIE-LVC) kit and to compare it with that of the IFI-leishmaniose-visceral-canina-Bio-Manguinhos (IFI-LVC) kit. Four groups of dogs were studied: group 1 (G1), dogs with clinical signs indicative of CVL and testing positive for the parasite (n = 25); group 2 (G2), dogs with only a presumed diagnosis of CVL (n = 62); group 3 (G3), dogs that had never lived in an area where CVL is endemic and never received a blood transfusion (n = 16); group 4 (G4), dogs carrying other parasites: such as babesiosis (n = 4), ehrlichiosis (n = 6) and demodicosis (n = 1). G1 and G3 were used for the calculation of sensitivity and specificity, respectively. The EIE-LVC showed a sensitivity of 72% (IC 95%: 50.4-87.1%) and a specificity of 87.5% (IC 95%: 60.4-97.8%). The value of the kappa index was 0.975 (CI 95%: 0.926-1.024), which represents an excellent fit. For IFI-LVC, the sensitivity was 68.0% (CI 95%: 46.4-84.3%) and the specificity 87.5% (CI 95%: 60.4-97.8%). When the tests were conducted in parallel, sensitivity was 92.0% (CI 95%: 72.5-98.6%) and specificity 75.0% (CI 95%: 47.4-91.7%). However, when conducted consecutively, the tests showed a sensitivity of 48.0% (CI 95%: 28.3-68.2%) and a specificity of 100.0% (CI 95%: 75.9-99.4%). The analysis of clinically suspected dogs using IFI-LVC and EIE-LVC kits in parallel, revealed that 26/62 animals were positive. Cross-reaction was observed in a dog with demodicosis. These results lead to the following conclusions: (1) the performance of the EIE-LVC kit is not statistically different from the IFI-LVC and (2) the kits must be used in parallel if higher sensitivity is required, reducing the number of false-negative results.

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BACKGROUND AND OBJECTIVES: Serological screening for Chagas' disease in the blood banks of South America is carried out by using two different assays that generally show a high number of inconclusive results. To establish a combination of two tests that can minimize the number of inconclusive results, we compared a recombinant enzyme-linked immunosorbent assay (ELISA) with two conventional tests. MATERIALS AND METHODS: Serum samples from chagasic patients (n = 112), from non-chagasic individuals (n = 143) and from patients with other diseases (n = 32) were tested using three assays: recombinant ELISA (Rec-ELISA); conventional ELISA (Con-ELISA); and the indirect haemagglutination (IHA) test. RESULTS: When we evaluated the data by matching the Rec-ELISA and the IHA test, 52 inconclusive results were obtained. When Rec-ELISA and Con-ELISA were matched, only four inconclusive results were observed. CONCLUSIONS: Our investigation indicates that the use of two ELISAs with different antigen preparations provides an effective test combination for blood bank screening of Chagas' disease.

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The reactivities of sera from chronic chagasic patients against the trypomastigote excreted-secreted antigens (TESA) of Trypanosoma cruzi strains with different biodemes were analyzed by TESA-blot and TESA-enzyme-linked immunosorbent assay (ELISA). Although both tests presented high sensitivity and specificity, TESA-ELISA is more appropriate for screening a larger number of samples.

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A kit based on an enzyme immunoassay, EIE-Recombinant-Chagas-Biomanguinhos, developed by the Oswaldo Cruz Foundation, was evaluated for the serodiagnosis of chronic Chagas disease. Evaluation was performed with 368 serum samples collected from individuals living in an endemic area for Chagas disease: 131 patients in the chronic phase with confirmed clinical, epidemiological, and serological diagnosis (indirect immunofluorescence, indirect hemagglutination or enzyme-linked immunosorbent assay) and 237 nonchagasic seronegative individuals were considered negative control. The EIE-Recombinant-Chagas-Biomanguinhos kit showed high sensitivity, 100% (CI 95%: 96.4-100%) and high specificity, 100% (CI 95%: 98-100%). The data obtained were in full agreement with clinical and conventional serology data. In addition, no cross-reaction was observed with sera from patients with cutaneous (n=14) and visceral (n=3) leishmaniasis. However, when these sera were tested by conventional serological assays for Chagas disease, cross-reactions were detected in 14.3% and 33.3% of the patients with cutaneous and visceral leishmaniasis, respectively. No cross-reactions were observed when sera from nonchagasic seronegative patients bearing other infectious disease (syphilis, n=8; HTLV, n=8; HCV, n=7 and HBV, n=12) were tested. In addition, sera of patients with inconclusive results for Chagas disease by conventional serology showed results in agreement with clinical evaluation, when tested by the kit. These results are relevant and indicate that the referred kit provides a safe immunodiagnosis of Chagas disease and could be used in blood bank screening.

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The polypeptides of 46 and 58 kDa were recognized in different T. cruzi strains (Y, WSL and Colombiana) by serum of all chagasic patients studied. These polypeptides were isolated from T. cruzi Y strain and used in ELISA. The sensitivity and specificity were 97.6% [CI 95%: 86-100%] and 100% [CI 95%: 89.3-100%], respectively when Tc 46 was used. When Tc 58 was used the sensitivity and specificity were 100% [CI 95%: 89.6-100%] and 90.2% [CI 95%: 75.9-96.8%], respectively.

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A 72 kDa Trypanosoma cruzi glycoprotein recognized by the 164C11 monoclonal antibody (IgM isotype) was purified by preparative electrophoresis. The antigenic preparation obtained, named TcY 72, was used to immunize C57Bl/10 mice. The following results were observed after immunization: (1) induction of higher titres of IgG than IgM antibodies, as evaluated by indirect immunofluorescence; (2) significant DTH after injection of epimastigotes in mice footpads; (3) peak parasitemia in immunized mice was significantly reduced and animals were negative by 13 days post-infection, although the mice still succumb to infection; (4) the phenotypic analysis of spleen cell populations showed a decrease in the CD4/CD8 ratio in immunized mice. Taken as a whole, these findings indicate that TcY 72 is immunogenic and potentially important for protective immunity.

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Various parasitic nematodes secrete acetylcholinesterase (AChE). In this study, the localization of AChE in the nematode Nippostrongylus brasiliensis and the secretory forms of AChE in culture fluid were examined. A thiocholine method revealed that AChE activity was localized in the subventral glands, which have a secretory and excretory function via a duct connected to the excretory pore. By electron microscopy, AChE activity was found mainly in the matrix of secretory granules, and sometimes in the Golgi apparatus in the subventral gland cells. These results show that nematode AChE is produced and stored in the subventral glands. Monoclonal antibodies against AChE of human erythrocytes or electric rays also bound to the nematode subventral gland, suggesting immuno-cross-reactivity of AChE among these species. When AChE activity in the nematode excretory-secretory product was examined by SDS polyacrylamide gel electrophoresis combined with the thiocholine method, intense activity was demonstrated as a single band at 74 kDa. Immunoblot analysis showed specific recognition of this molecule by IgE and IgG1 antibodies, but not by IgG2a antibody, in nematode-infected rat sera. These results indicate that the nematode AChE molecule produced in and secreted from the subventral glands is antigenic for the production of IgE/IgG1 in host animals.

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IgE, IgG and mast cell responses were studied in rats infected weekly with 10 larvae of Nippostrongylus brasiliensis (NB). Worm recovery at 8 weeks of repeated infections was six-fold greater than that of a single infection with 10 larvae, suggesting the accumulation of worms during the repeated infections. Total serum IgE was increased after 2 weeks of infection, and further increased after repeated infections: at 6 weeks of infection the level was four to six times higher than that after a single infection. Anti-NB IgG1 levels were also significantly higher after repeated infections than after a single infection. On the other hand, there was no significant difference in the level of anti-NB IgE between single and repeated infections, as determined by ELISA, as well as by passive cutaneous anaphylaxis (PCA) reaction. Mastocytosis was induced in the small intestine after both single and repeated infections, but the levels did not differ between the two. These results indicate that total IgE and specific IgG1 production are augmented by repeated helminth infections, but specific IgE and mast cell responses are not. This pattern of response may minimize the development of IgE-dependent hypersensitivity reactions with repeated helminth infections.

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The differences were examined between IgE, IgG1 and IgG2a antibody responses against two kinds of nematode antigens in rats infected with Nippostrongylus brasiliensis. With ELISA studies, remarkable IgE and IgG1 antibody responses were observed against antigens in excretory/secretory products (ES) of N. brasiliensis, whereas the IgG2a antibody response against ES was negligible. On the other hand, antibody response to antigens in an extract of homogenized adult worm (AW) was observed mainly in IgG2a, with little response in IgE or IgG1. Immunohistochemical studies showed that IgE- and IgG1-binding antigens were localized almost exclusively in the subventral glands, a secretory apparatus in N. brasiliensis, while IgG2a-binding antigens were found mainly in the nematode wall along the body cavity. Immunoblot analysis revealed that the major IgE- and IgG1-binding molecules in ES were identical. On the other hand, some, but not all, of the major IgG2a-binding molecules in AW were different from the IgE/IgG1-binding molecules in ES. The findings suggest that the IgE/IgG1 and IgG2a antibody responses in N. brasiliensis-infected rats are induced by different groups of nematode antigens. Thus, it is presumed that the production of each class of antibody might be dependent, at least in part, on the nature of the antigen or antigen-linked molecules.

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A simple rapid method of enzyme labeled anti-isotype assay (ELIA) for detection of monoclonal isotype on hybridoma cells is proposed. This alternative method was first carried out on hybridoma cell lines 147C11 and 257C11 produced against Trypanosoma cruzi and male accessory secretion of Panstrongylus megistus, respectively. The monoclonal antibodies produced by these hybridoma were characterized by this method as IgM (147C11) and IgG1 (257C23) isotypes, allowing evaluation of isotype without having to wait until the concentration of antibody present in the supernatant itself rises. Results were confirmed by Ouchterlony immunodiffusion. The proposed method offers the advantages of a permanent rapid procedure for light microscopy.

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Specific and non-specific IgE antibody responses were studied in SD rats infected with between 5 and 2500 Nippostrongylus brasiliensis (NB) larvae. In rats with 2500 NB larvae, specific IgE antibody, measured by enzyme-linked immunosorbent assay (ELISA) using NB excretory/secretory substance as antigen, reached a peak at 4 weeks of infection and gradually declined. On the other hand, in rats infected with 10 or 100 NB larvae, specific IgE was induced at 4 weeks of infection and the level continued to rise until at least 8 weeks after infection. The level at 8 weeks was significantly higher in rats infected with 10 or 100 larvae than in rats infected with 2500 larvae. The results indicate that the low-level infection induced a much more sustained specific IgE response than that induced after heavy infection. However, the level of specific IgG was correlated with the dose of infection, and reached a plateau 6 weeks after infection. Total serum IgE increased significantly even in rats infected with five larvae, a dose which did not induce detectable specific IgE. The kinetics of the production of total IgE was different in rats with light and heavy infections. In rats infected with five or 10 larvae, total IgE increased slowly and reached a plateau 4 weeks after infection. On the other hand, rats infected with more than 500 larvae showed a remarkable rise in total IgE at 2 weeks of infection; this rise gradually declined thereafter. Six weeks after infection, total IgE levels were almost the same (2-3 micrograms/ml) in rats infected with 10-2500 NB larvae. These results show that low-level NB infection induces a significant and sustained specific and non-specific IgE response in rats.

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Infection with the nematode Nippostrongylus brasiliensis (NB) induces the intense production of specific and non-specific immunoglobulin E (IgE) in rats. In the present study, we analysed NB-derived allergenic substances and the variability of IgE antibody production in response to these allergens in outbred Sprague-Dawley rats. Two kinds of crude allergens were used: the excretory-secretory products (ES) of adult NB, and an extract of homogenized adult worms (AW). ELISA showed that IgE antibody titres to ES were more than five times higher than the titres to AW. In the homologous passive cutaneous anaphylaxis (PCA) reaction using serum from infected rats, as little as 50 micrograms of ES had a maximal PCA activity, while even 1 mg of AW still gave a slightly lower PCA titre. Thus, it appeared that ES contained more allergen than AW at the same amount of total proteins. By immunoblot analysis, at least six components were recognized by IgE antibodies from infected animals, and these components were exactly the same in both ES and AW. The results indicated that the allergenic components in ES and AW were the same molecules, and that only those molecules which could be excreted or secreted from living worms were allergenic. Among the array of allergens, 130,000 and 70,000 molecular weight (mw) molecules were commonly recognized by IgE from all serum samples examined, while other components of the allergens were recognized variably by IgE antibodies from individual animals. These findings suggested that individual animals varied considerably in their IgE antibody production to the different constituents of the nematode allergens.

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