NOTCH1 and DLL4 are involved in the human tuberculosis progression and immune response activation.

Tuberculosis (TB) is the leading cause of mortality among infectious diseases worldwide. The study of molecular targets for therapy and diagnosis suggested that Notch signaling is an important pathway for the maintenance of the immune response during Mycobacterium tuberculosis (Mtb) infection. We evaluated the participation of the Notch pathway in the modulation of immune response during Mtb infection, and observed that patients with active TB had increased DLL4 expression in intermediate and non-classic monocytes. Further, patients with moderate and advanced lung injury have higher Notch1 expression in CD4+ T cells when compared to patients with a minimal lung injury. When we considered the severity of disease in active TB patients, the expression of the DLL4 in intermediate monocytes and the expression of Notch1 in CD4+ T cells are positively correlated with the degree of lung injury. In vitro, PBMCs treated with the Notch pharmacological inhibitor reduced the production of IL-17A and IL-2, whereas anti-hDLL4 treatment promoted a significant increase in TNF-α and phagocytosis. We suggest that Notch1 and DLL4 are associated with immune response activation in human tuberculosis, and can be a novel target to be exploited in the future in the searching of biomarkers.

Quantificação de subpopulações linfocitárias no sangue periférico de cães sadios adultos / Lymphocyte subsets in peripheral blood of adult healthy dogs

A citometria de fluxo vem se firmando como uma ferramenta útil na prática médico-veterinária, particularmente na clínica de pequenos animais. Tal conhecimento tem ensejado o estabelecimento de valores fisiológicos para diferentes subpopulações linfocitárias, indispensáveis à compreensão da dinâmica da celularidade linfóide, em diversas situações patológicas. Assim sendo, o presente ensaio teve como objetivo imunomarcar, por intermédio da técnica citométrica, as subpopulações linfocitárias CD5+CD4+, CD5+CD8+ e CD21+, em quatro diferentes raças de cães domésticos e sadios, de tal forma a agregar informações sobre o perfil imunológico das diferentes raças. Foram utilizados 40 cães adultos (2-7 anos), machos e fêmeas, das raças Beagle (G1, n=10), Golden Retriever (G2, n=10), Bulldog Inglês (G3, n=10) e sem raça definida (SRD) (G4, n=10). As colheitas de sangue foram realizadas por venipunção jugular, utilizando-se sistema de frascos a vácuo (K2-EDTA). O hemograma e o processamento das amostras para citometria de fluxo foram realizados num prazo máximo de 24 horas após a colheita do sangue. As amostras foram analisadas no citofluorômetro FACSCANTO (Becton Dickinson, San Jose, CA, USA). Utilizou-se o programa FACSDiva (Becton Dickinson, San Jose, CA, USA), para identificar e quantificar as células CD5+CD4+, CD5+CD8+ e CD21+, que forneceu o histograma e respectiva tabela com a quantidade de células detectadas pela imunofenotipagem. Os dados obtidos para contagem de linfócitos T auxiliares, T citotóxicos/supressores e linfócitos B foram tabulados e submetidos a Análise de Variância pelo teste F. O teste de Tukey a 5% de probabilidade foi utilizado para comparação das médias entre as diferentes raças de cães. Os valores médios de contagens de células CD5+CD8+ no sangue periférico do G1, G2, G3 e G4 foram de 155, 206, 544 e 503 células/µL, respectivamente. Os valores médios de contagens de células CD5+CD4+ no sangue periférico do G1, G2, G3 e G4 foram de 746, 642, 1101 e 855 células/µL, respectivamente. Os valores médios de contagens de células CD21+ no sangue periférico do G1, G2, G3 e G4 foram de 171, 299, 494 e 403 células/µL, respectivamente. Assim sendo, o número médio de células obtido para a subpopulação de linfócitos T citotóxicos foi significativamente maior (p<0,05) nos Bulldogs Ingleses e cães SRD, comparativamente aqueles encontrados nos grupos de Beagles e Golden Retrievers. Ademais, observou-se que o número médio de células obtido para a subpopulação de linfócitos B foi significavamente maior (p<0,05) nos Bulldogs Ingleses, quando comparado àquele dos Beagles.(AU)

Flow cytometry has established itself as a useful tool in veterinary practice, particularly for small animals practice. Such knowledge has made necessary the establishment of physiological values for different lymphocyte subpopulations, indispensable to understanding the dynamics of lymphoid cellularity in various pathological conditions. In this sense, the objective of this study was to determine, through cytometric technique, lymphocyte subsets of CD5+CD4+, CD5+CD8+ and CD21+ in four different breeds of domestic dogs, so to add information about the immunological profile of different breeds. A total of 40 adult dogs were used (2-7 years), being males and females of Beagles (G1, n=10), Golden Retrievers (G2, n=10), English Bulldogs (G3, n=10) and crossbreed dogs (G4, n=10). Blood samples were collected by jugular venipuncture, using vacuum flasks system (K2-EDTA). Hemogram and processing of samples for flow cytometry were performed within a maximum of 24 hours after blood collection. Samples were analyzed using FACSCanto device (Becton Dickinson, San Jose, CA, USA). FACSDiva software (Becton Dickinson, San Jose, CA, USA) was used to identify and quantify the CD5+ CD4+, CD5+CD8+, and CD21+, which provided the histogram and the respective table with the number of cells identified by immunophenotyping. The data obtained for T helper lymphocyte count, T cytotoxic/suppressor lymphocyte count and B lymphocytes were tabulated and submitted to analysis of variance by F test. Tukey test at 5% probability was used to compare means between the different breeds of dogs. The average values for CD5+CD8+ cell count in peripheral blood in G1, G2, G3 and G4 were of 155, 206, 544 and 503 cells/uL, respectively. The average values for CD5+CD4+ cell count in peripheral blood in G1, G2, G3 and G4 were of 746, 642, 855 and 1101 cells/uL, respectively. The average values for CD21 + cell count in peripheral blood for G1, G2, G3 and G4 were of 171, 299, 494 and 403 cells/uL, respectively. Therefore, The average number of cells obtained for the subsets of cytotoxic T lymphocytes was significantly higher (p<0,05) in the British Bulldogs and crossbreed dogs compared to those found in Beagles and Golden Retrievers. Furthermore, it was observed that the average number of cells obtained for the subsets of B lymphocytes was significantly higher (p<0,05) in English Bulldogs compared to that of Beagles.(AU)

Immunomodulatory activity of Tityus serrulatus scorpion venom on human T lymphocytes.

BACKGROUND: Tityus serrulatus scorpion venom (TsV) contains toxins that act on K(+) and Na(+) channels and account for the venom's toxic effects. TsV can activate murine peritoneal macrophages, but its effects on human lymphocytes have been poorly investigated. Considering that lymphocytes may play an important role in envenomation, we assessed whether TsV affects the expression of phenotypic (CD3, CD4, and CD8) and activation (CD69, CD25, and HLA-DR) markers, cell proliferation, and cytokine production in peripheral blood mononuclear cells. METHODS: Cytotoxicity of TsV was evaluated via the MTT assay. Cell proliferation, expression of phenotypic and activation markers, and release of cytokines were assessed using flow cytometry, after treatment with non-cytotoxic concentrations of TsV. The combined use of carboxyfluorescein diacetate succinimidyl ester and monoclonal antibodies against phenotypic and activation markers enabled us to simultaneously assess cell proliferation extent and cell activation status, and to discriminate among cell subpopulations. RESULTS: TsV at concentrations of 25 to 100 µg/mL were not cytotoxic towards peripheral blood mononuclear cells. TsV did not induce significant changes in lymphocyte subpopulations or in the expression of activation markers on CD4(+) and CD8(+) T cells. TsV inhibited the phytohemagglutinin-stimulated lymphocyte proliferation, particularly in the CD8(+) CD25(+) T lymphocyte subset. TsV alone, at 50 and 100 µg/mL, did not induce peripheral blood mononuclear cell proliferation, but elicited the production and release of IL-6, a proinflammatory cytokine that plays an important role in innate and adaptive immune responses. CONCLUSIONS: TsV is a potential source of molecules with immunomodulatory action on human T lymphocytes.

Immunomodulatory activity of Tityus serrulatus scorpion venom on human T lymphocytes

Background Tityus serrulatus scorpion venom (TsV) contains toxins that act on K + and Na + channels and account for the venom's toxic effects. TsV can activate murine peritoneal macrophages, but its effects on human lymphocytes have been poorly investigated. Considering that lymphocytes may play an important role in envenomation, we assessed whether TsV affects the expression of phenotypic (CD3, CD4, and CD8) and activation (CD69, CD25, and HLA-DR) markers, cell proliferation, and cytokine production in peripheral blood mononuclear cells. Methods Cytotoxicity of TsV was evaluated via the MTT assay. Cell proliferation, expression of phenotypic and activation markers, and release of cytokines were assessed using flow cytometry, after treatment with non-cytotoxic concentrations of TsV. The combined use of carboxyfluorescein diacetate succinimidyl ester and monoclonal antibodies against phenotypic and activation markers enabled us to simultaneously assess cell proliferation extent and cell activation status, and to discriminate among cell subpopulations. Results TsV at concentrations of 25 to 100 μg/mL were not cytotoxic towards peripheral blood mononuclear cells. TsV did not induce significant changes in lymphocyte subpopulations or in the expression of activation markers on CD4 + and CD8 + T cells. TsV inhibited the phytohemagglutinin-stimulated lymphocyte proliferation, particularly in the CD8 + CD25 + T lymphocyte subset. TsV alone, at 50 and 100 μg/mL, did not induce peripheral blood mononuclear cell proliferation, but elicited the production and release of IL-6, a proinflammatory cytokine that plays an important role in innate and adaptive immune responses. Conclusions TsV is a potential source of molecules with immunomodulatory action on human T lymphocytes.(AU)

Immunomodulatory activity of Tityus serrulatus scorpion venom on human T lymphocytes

Background Tityus serrulatus scorpion venom (TsV) contains toxins that act on K + and Na + channels and account for the venoms toxic effects. TsV can activate murine peritoneal macrophages, but its effects on human lymphocytes have been poorly investigated. Considering that lymphocytes may play an important role in envenomation, we assessed whether TsV affects the expression of phenotypic (CD3, CD4, and CD8) and activation (CD69, CD25, and HLA-DR) markers, cell proliferation, and cytokine production in peripheral blood mononuclear cells. Methods Cytotoxicity of TsV was evaluated via the MTT assay. Cell proliferation, expression of phenotypic and activation markers, and release of cytokines were assessed using flow cytometry, after treatment with non-cytotoxic concentrations of TsV. The combined use of carboxyfluorescein diacetate succinimidyl ester and monoclonal antibodies against phenotypic and activation markers enabled us to simultaneously assess cell proliferation extent and cell activation status, and to discriminate among cell subpopulations. Results TsV at concentrations of 25 to 100 g/mL were not cytotoxic towards peripheral blood mononuclear cells. TsV did not induce significant changes in lymphocyte subpopulations or in the expression of activation markers on CD4 + and CD8 + T cells. TsV inhibited the phytohemagglutinin-stimulated lymphocyte proliferation, particularly in the CD8 + CD25 + T lymphocyte subset. TsV alone, at 50 and 100 g/mL, did not induce peripheral blood mononuclear cell proliferation, but elicited the production and release of IL-6, a proinflammatory cytokine that plays an important role in innate and adaptive immune responses. Conclusions TsV is a potential source of molecules with immunomodulatory action on human T lymphocytes.

The acute phase of Trypanosoma cruzi infection is attenuated in 5-lipoxygenase-deficient mice.

In the present work we examine the contribution of 5-lipoxygenase- (5-LO-) derived lipid mediators to immune responses during the acute phase of Trypanosoma cruzi infection in 5-LO gene knockout (5-LO(-/-)) mice and wild-type (WT) mice. Compared with WT mice, the 5-LO(-/-) mice developed less parasitemia/tissue parasitism, less inflammatory cell infiltrates, and a lower mortality. This resistance of 5-LO(-/-) mice correlated with several differences in the immune response to infection, including reduced PGE2 synthesis; sustained capacity of splenocytes to produce high levels of interleukin (IL)-12 early in the infection; enhanced splenocyte production of IL-1ß, IL-6, and IFN-γ; rapid T-cell polarization to secrete high quantities of IFN-γ and low quantities of IL-10; and greater numbers of CD8(+)CD44(high)CD62L(low) memory effector T cells at the end of the acute phase of infection. The high mortality in WT mice was associated with increased production of LTB4/LTC4, T cell bias to produce IFN-γ, high levels of serum nitrite, and marked protein extravasation into the peritoneal cavity, although survival was improved by treatment with a cys-LT receptor 1 antagonist. These data also provide evidence that 5-LO-derived mediators negatively affect host survival during the acute phase of T. cruzi infection.

L-amino acid oxidase isolated from Bothrops pirajai induces apoptosis in BCR-ABL-positive cells and potentiates imatinib mesylate effect.

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder characterized by the presence of Philadelphia chromosome and by BCR-ABL1, which encodes the BCR-ABL oncoprotein. Although imatinib mesylate (IM) is effective for CML treatment, patients in accelerated and blastic phases of the disease are often refractory to this therapy, and there are also cases of IM resistance in patients in the chronic phase. Therefore, potential new drugs are being investigated to improve the efficiency of the therapy of CML such as snake venoms and their compounds. In this investigation, Bothrops pirajai L-amino acid oxidase (BpirLAAO-I) effect on normal peripheral blood mononuclear cells (PBMC) and on BCR-ABL(+) cell line was assessed to explore its potential against leukaemic cells. MTT viability assay, lymphocyte subsets quantification and cell activation markers expression were performed to evaluate BpirLAAO-I effect on normal PBMC. The effect of BpirLAAO-I on HL-60 and HL-60.BCR-ABL cell lines was assessed by apoptosis detection. BpirLAAO-I was able to induce apoptosis in HL-60 and HL-60.BCR-ABL cell lines in a dose-dependent manner, promoted caspases 3, 8 and 9 activation and enhanced IM effect while not affecting the viability of normal cells. In addition, BpirLAAO-I promoted immune cells activation and lymphocytes subsets changes on normal PBMC. The results indicate that BpirLAAO-I induces apoptosis and potentiates IM effect on BCR-ABL(+) cells.

A DNA vaccine candidate encoding the structural prM/E proteins elicits a strong immune response and protects mice against dengue-4 virus infection.

A DNA vaccine expressing dengue-4 virus premembrane (prM) and envelope (E) genes was produced by inserting these genes into a mammalian expression plasmid (pCI). Following a thorough screening, including confirmation of protein expression in vitro, a recombinant clone expressing these genes was selected and used to immunize BALB/c mice. After 3 immunizations all the animals produced detectable levels of neutralizing antibodies against dengue-4 virus. The cytokines levels and T cell proliferation, detected ex vivo from the spleen of the immunized mice, showed that our construction induced substantial immune stimulation after three doses. Even though the antibody levels, induced by our DNA vaccine, were lower than those obtained in mice immunized with dengue-4 virus the levels of protection were high with this vaccine. This observation is further supported by the fact that 80% of the vaccine immunized group was protected against lethal challenge. In conclusion, we developed a DNA vaccine employing the genes of the prM and E proteins from dengue-4 virus that protects mice against this virus.