Elaine V. Lourenço

Leukotrienes play a role in the control of parasite burden in murine strongyloidiasis

It is clear that leukotrienes mediate inflammatory response; new aspects of leukotriene function have recently been described. In this study, we demonstrate that leukotrienes are key chemical mediators in the control of parasite burdens in mice infected with Strongyloides venezuelensis. High leukotriene levels were detected in the lungs and small intestines of Swiss mice. In infected Swiss mice treated with MK886, a leukotriene synthesis inhibitor, numbers of adult worms, and eggs/g/feces were greater than in infected-only animals. The MK886 treatment inhibited leukotriene B4 production in the lungs and small intestines, albeit on different postinfection days. Similarly, parasite burdens and eggs/g/feces were greater in 5-lipoxygenase−/− mice than in wild-type animals. These observation were confirmed by histopathological study of the duodena. We subsequently observed significant lower numbers of eosinophils and mononuclear cells in the blood, peritoneal cavity fluid, and bronchoalveolar lavage fluid of Swiss mice treated with MK886. In the lung parenchyma of infected animals, MK886 significantly inhibited synthesis of IL-5 at the beginning of infection, whereas levels of IL-12 increased progressively throughout the postinfection period. However, levels of leukotriene C4, PGE2, TNF-α, IL-3, IL-4, IFN-γ, and IL-10 were comparable between the treated and untreated groups. Nevertheless, IgE and IgG1 (but not IgG2a) synthesis was also significantly inhibited by MK886 administration. Therefore, in S. venezuelensis-infected mice, adult worm and egg burdens are leukotriene dependent. These findings indicate potential immunostimulatory strategies involving leukotriene administration, and may serve as an alert to physicians treating Strongyloides stercoralis-infected patients presenting asthma-like symptoms because use of 5-lipoxygenase inhibitors may worsen the infection.

Myosin Va phosphorylated on Ser1650 is found in nuclear speckles and redistributes to nucleoli upon inhibition of transcription

Nuclear actin and nuclear myosins have been implicated in the regulation of gene expression in vertebrate cells. Myosin V is a class of actin-based motor proteins involved in cytoplasmic vesicle transport and anchorage, spindle-pole alignment and mRNA translocation. In this study, myosin-Va, phosphorylated on a conserved serine in the tail domain (phospho-ser(1650) MVa), was localized to subnuclear compartments. A monoclonal antibody, 9E6, raised against a peptide corresponding to phosphoserine(1650) and flanking regions of the murine myosin Va sequence, was immunoreactive to myosin Va heavy chain in cellular and nuclear extracts of HeLa cells, PC12 cells and B16-F10 melanocytes. Immunofluorescence microscopy with this antibody revealed discrete irregular spots within the nucleoplasm that colocalized with SC35, a splicing factor that earmarks nuclear speckles. Phospho-ser(1650) MVa was not detected in other nuclear compartments, such as condensed chromatin, Cajal bodies, gems and perinucleolar caps. Although nucleoli also were not labeled by 9E6 under normal conditions, inhibition of transcription in HeLa cells by actinomycin D caused the redistribution of phospho-ser(1650) MVa to nucleoli, as well as separating a fraction of phospho-ser(1650) MVa from SC35 into near-neighboring particles. These observations indicate a novel role for myosin Va in nuclear compartmentalization and offer a new lead towards the understanding of actomyosin-based gene regulation.

Strongyloides venezuelensis: The antigenic identity of eight strains for the immunodiagnosis of human strongyloidiasis

The antigens of eight strains of Strongyloides venezuelensis were identified by means of the indirect immunofluorescence antibody (IFAT), enzyme-linked immunosorbent assay (ELISA) and immunoblot (IB) tests. Infective larvae (L3) from these strains were obtained from Rattus norvegicus feces cultures. For IFAT, sections of L3 were used while the ELISA and IB, tests were conducted with alkaline extract. Ninety serum samples were tested: 30 from patients with S. stercoralis, 30 from patients with other parasitic diseases, and 30 from healthy subjects (free of parasites). Average sensitivity and specificity among all eight strains, both for IFAT and ELISA, were, respectively, 93% and 100%. In the IB, anti-S. stercoralis IgG recognized a single antigenic fraction with 45 kDa. Serum samples from patients with S. stercoralis revealed antigens from different strains of S. venezuelensis, indicating antigenic identity for possible use in the synthesis of recombinant antigen that could be useful in immunodiagnosis and vaccine against this parasite.

Counterregulation of Th2 immunity by interleukin 12 reduces host defenses against Strongyloides venezuelensis infection.

The aim of this study was to investigate the role of interleukin 12 (IL-12) during Strongyloides venezuelensis infection. IL-12(-/-) and wild-type C57BL/6 mice were subcutaneously infected with 1500 larvae of S. venezuelensis. On days 7, 14, and 21 post-infection, we determined eosinophil and mononuclear cell numbers in the blood and broncoalveolar lavage fluid (BALF), Th2 cytokine secretion in the lung parenchyma, and serum antibody levels. The numbers of eggs in the feces and worm parasites in the duodena were also quantified. The eosinophil and mononuclear cell counts and the concentrations of IL-3, IL-5, IL-10, IL-13, and IgG1 and IgE antibodies increased significantly in infected IL-12(-/-) and wild-type mice as compared with uninfected controls. However, the number of eosinophils and mononuclear cells in the blood and BALF and the Th2 cytokine levels in the lungs of infected IL-12(-/-) mice were greater than in infected wild-type C57BL/6 mice. In addition, serum IgE and IgG1 levels were also significantly enhanced in the infected mice lacking IL-12. Meanwhile, parasite burden and fecal egg counts were significantly decreased in infected IL-12(-/-) mice. Together, our results showed that the absence of IL-12 upregulates the Th2 immune response, which is important for control of S. venezuelensis infection.

Comparison of immune responses in mice infected with different strains of Strongyloides venezuelensis.

In human hosts and in murine models, the immune response to Strongyloides spp. is Th2 type. In addition, the profile of the host immune response follows various symptoms induced by Strongyloides spp. In the present study, we demonstrated that the L2 and L49 strains of Strongyloides venezuelensis obtained from Bolomys lasiurus and Nectomys squamipes induced significant and similar increases in eosinophil/mononuclear cell counts in the blood, peritoneal cavity fluid and bronchoalveolar lavage fluid when compared with uninfected mice. However, in the first 3 days of infection, IL‐4, IL‐5 and IFN‐γ levels were higher in the lungs of mice infected with the L2 strain, which also presented greater production of IgG and IgG1 than did mice infected with the L49 strain. The higher antibody and cytokine levels induced by the L2 strain correlated with a decrease in the number of female parasites recovered in the faeces of mice on post‐infection day 7. The results demonstrate that the L2 strain was a more potent stimulant of the humoral immune response, which can result in more efficient antibody‐dependent cell‐mediated cytotoxicity, a mechanism involved in eosinophil activation and parasite elimination. Further studies are needed in order to elucidate the molecular differences among parasites.

Cyclooxygenase-derived mediators regulate the immunological control of Strongyloides venezuelensis infection.

The aim of this study was to define the immunoregulatory role of prostaglandins in a mouse model of Strongyloides venezuelensis infection. Strongyloides venezuelensis induced an increase of eosinophils and mononuclear cells in the blood, peritoneal cavity fluid, and bronchoalveolar lavage fluid. Treatment with the dual cyclooxygenase (COX-1/-2) inhibitors indomethacin and ibuprofen, and the COX-2-selective inhibitor celecoxib partially blocked these cellular responses and was associated with enhanced numbers of infective larvae in the lung and adult worms in the duodenum. However, the drugs did not interfere with worm fertility. Cyclooxygenase inhibitors also inhibited the production of the T-helper type 2 (Th2) mediators IL-5, IgG1, and IgE, while indomethacin alone also inhibited IL-4, IL-10, and IgG2a. Cyclooxygenase inhibitors tended to enhance the Th1 mediators IL-12 and IFN-gamma. This shift away from Th2 immunity in cyclooxygenase inhibitor-treated mice correlated with reduced prostaglandin E(2) (PGE(2)) production in infected duodenal tissue. As PGE(2) is a well-characterized driver of Th2 immunity, we speculate that reduced production of this lipid might be involved in the shift toward a Th1 phenotype, favoring parasitism by S. venezuelensis. These findings provide new evidence that cyclooxygenase-derived lipids play a role in regulating host defenses against Strongyloides, and support the exploration of eicosanoid signaling for identifying novel preventive and therapeutic modalities against these infections.

Immunoenzymatic Quantitative Analysis of Antigens Expressed on the Cell Surface (Cell-ELISA)

Cell-enzyme-linked immunosorbent assay (cell-ELISA) is an useful technique for the quantitative analysis of cell surface antigen expression that was developed on the basis of enzyme immunohistochemistry (EIH) and ELISA. Since its development, which was made possible by the establishment of monoclonal antibody technology, a wide range of cell types and surface molecules were analyzed by cell-ELISA. Here we show four variants of this method and provide a brief comparison of cell-ELISA with flow cytometry (FACS) and radioimmunobinding assay (RIA), which are other methods for the quantitative detection of cell-surface molecules. We describe step-by-step procedures for both direct and indirect cell-ELISA using either adherent or nonadherent live cells.

Toxoplasma gondii 70 kDa Heat Shock Protein: Systemic Detection Is Associated with the Death of the Parasites by the Immune Response and Its Increased Expression in the Brain Is Associated with Parasite Replication

The heat shock protein of Toxoplasma gondii (TgHSP70) is a parasite virulence factor that is expressed during T. gondii stage conversion. To verify the effect of dexamethasone (DXM)-induced infection reactivation in the TgHSP70-specific humoral immune response and the presence of the protein in the mouse brain, we produced recombinant TgHSP70 and anti-TgHSP70 IgY antibodies to detect the protein, the specific antibody and levels of immune complexes (ICs) systemically, as well as the protein in the brain of resistant (BALB/c) and susceptible (C57BL/6) mice. It was observed higher TgHSP70-specific antibody titers in serum samples of BALB/c compared with C57BL/6 mice. However, the susceptible mice presented the highest levels of TgHSP70 systemically and no detection of specific ICs. The DXM treatment induced increased parasitism and lower inflammatory changes in the brain of C57BL/6, but did not interfere with the cerebral parasitism in BALB/c mice. Additionally, DXM treatment decreased the serological TgHSP70 concentration in both mouse lineages. C57BL/6 mice presented high expression of TgHSP70 in the brain with the progression of infection and under DXM treatment. Taken together, these data indicate that the TgHSP70 release into the bloodstream depends on the death of the parasites mediated by the host immune response, whereas the increased TgHSP70 expression in the brain depends on the multiplication rate of the parasite.

Infectivity of Strongyloides venezuelensis is influenced by variations in temperature and time of culture

The present research investigated the influence of temperature and time of larvae culture on the infectivity of Strongyloides venezuelensis. Mice were infected s.c. with 1500 larvae of S. venezuelensis maintained at 28 °C for three days of culture (dc), 28 °C for seven dc or 18 °C for seven dc. On days 1, 3, 5, 7, 14 and 21 post-infection the animals were sacrificed and cell numbers in the blood, peritoneal cavity fluid (PCF), broncoalveolar fluid (BALF), cytokines, immunoglobulins, number of parasites and eggs/g of feces were quantified. Results demonstrated an increase in eosinophils and mononuclear cells in the blood, PCF and BALF of infected mice. Larvae at 28 °C/3dc induced earlier eosinophils in the PCF and BALF as opposed to larvae at 28 °C/7dc and 18 °C/7dc. Larvae at 28 °C/7dc induced higher synthesis of IL-4, IL-5 and IL-10 on days 5 and 7 post-infection. Larvae at 28 °C/3dc in culture induced higher synthesis of IL-12 than larvae of seven dc, but time in culture induced better synthesis of IFN-γ after larval migration had ceased and only adult worms were present. Larvae at 28 °C/3dc in culture induced higher synthesis of IgG and IgG1 and expelled less female parasites than larvae cultivated for seven days. In conclusion, it was observed that the infectivity of S. venezuelensis is influenced by variations in temperature and time of culture.

O ensino de História encontra seu passado: memórias da atuação docente durante a ditadura civil-militar

A ditadura civil-militar que se instalou no Brasil a partir de 1964 influenciou no cotidiano escolar e na atuacao dos professores do periodo. As transformacoes se deram desde a nova legislacao, implantada com a Lei 5692/71, ate as mudancas que ocorreram em funcao da vigilância que os professores sentiam ao exercer sua profissao. As memorias aqui analisadas, de docentes da escola publica paulista no periodo, mostram diferentes percepcoes em torno dessa vigilância, o que implica diversas formas de aceitacao ou de resistencia. O que se pretende e mostrar que, a despeito de nao haver uma repressao mais direta as escolas, como havia as universidades, os docentes da escola basica tambem se sentiam vigiados, o que se pode perceber nas suas praticas escolares e nas suas concepcoes de ensino.