Prolactin (PRL) is a pleiotropic polypeptide hormone produced by the anterior pituitary gland and negatively controlled by dopamine. Some researchers have associated the immune regulatory functions of PRL with some infectious diseases like Toxoplasma gondii and T. cruzi. This work aimed to analyze the possible immuno-modulatory effects of this hormone through the subcutaneous administration of PRL during the experimental Chagas disease. On the 14th day post-infection (dpi), PRL triggered increased percentages of NK cells in treated infected animals as compared to the infected and untreated ones. For early and late apoptosis, our results showed that in chronically infected groups, PRL counteracted splenocyte apoptosis as revealed by the reduced percentages of both, early and late apoptosis. Reduced percentages of spleen CD4+ and CD8+ T cells were detected in infected PRL treated rats (60 days post-infection). Concerning to B cells, a significant increased percentage of these cells was found for all PRL treated infected animals (14th dpi), but no statistically significant alteration was observed on the 60th days post-infection. Furthermore, PRL treatment triggered a significant increase in the percentage of CD4+ T lymphocytes IFN-γ producers, while on the 60th dpi, a reduced percentage of IFN-γ in these cells was observed in prolactin-treated rats compared to infected and untreated ones. Enhanced serum IL-12 levels were detected in infected and PRL treated subjects (60th dpi). Only on 7th day post-infection, the flow cytometric analysis of CFSE-stained CD3+ T cells showed an enhanced proliferation of polyclonal stimulated T cells in PRL-treated and infected animals. In this study, we demonstrated that PRL can influence many aspects of the immune response during the experimental Chagas' disease, and this substance could be used as a supporting trial along with the conventional drug treatment.

RESUMO: 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.

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.

Tumor-associated macrophages (TAMs) have an ambiguous and complex role in the carcinogenic process, since these cells can be polarized into different phenotypes (proinflammatory, antitumor cells or anti-inflammatory, protumor cells) by the tumor microenvironment. Given that the interactions between tumor cells and TAMs involve several players, a better understanding of the function and regulation of TAMs is crucial to interfere with their differentiation in attempts to skew TAM polarization into cells with a proinflammatory antitumor phenotype. In this study, we investigated the modulation of macrophage tumoricidal activities by the lectin jacalin. Jacalin bound to macrophage surface and induced the expression and/or release of mainly proinflammatory cytokines via NF-κB signaling, as well as increased iNOS mRNA expression, suggesting that the lectin polarizes macrophages toward the antitumor phenotype. Therefore, tumoricidal activities of jacalin-stimulated macrophages were evaluated. High rates of tumor cell (human colon, HT-29, and breast, MCF-7, cells) apoptosis were observed upon incubation with supernatants from jacalin-stimulated macrophages. Taken together, these results indicate that jacalin, by exerting a proinflammatory activity, can direct macrophages to an antitumor phenotype. Deep knowledge of the regulation of TAM functions is essential for the development of innovative anticancer strategies.

In previous studies, we have demonstrated that inoculation with a Trypanosoma cruzi marinkellei (avirulent RM1 strain) was able to reduce parasitemia in mice challenged with T. cruzi, although it was not able to prevent histopathological lesions. Th1 response stimulation by immunization is necessary for T. cruzi infection control, but the resistance is also dependent on immunoregulatory mechanisms, which can be induced by adjuvants. Thus, we evaluated whether inoculation of T. cruzi marinkellei associated with administration of different adjuvants would be capable of inducing different patterns of immune response to maximize the immune response against T. cruzi (virulent Romildo strain) infection. Two hundred eighty nonisogenic mice were divided into 14 groups according to the immunization scheme and the subsequent challenge with virulent Romildo T. cruzi strain. Nonimmunized groups and animals inoculated without adjuvants were also included. Immune protection was not observed with Th2 adjuvants (incomplete Freund's adjuvant [IFA] and Alum) due to high parasitemia. Th1/Th2-polarizing adjuvants also did not induce immune protection because inulin was unable to maintain survival, and immune-stimulating complexes induced intense inflammatory processes. Animals sensitized with RM1 strain without adjuvants were able to reduce parasitemia, increase survival, and protect against severe histological lesions, followed by adequate cytokine stimulation. Finally, our results demonstrate that the early and balanced IFN-γ production becomes critical to promote protection and that Th1 adjuvant elicited a controversial infection control due to increased histopathological damage. Therefore, the host's immunomodulation remains one of the most important challenges in the research for effective protection against T. cruzi infection. Similarly, the identification of protective antigens in the RM1 strain of T. cruzi marinkellei may contribute to further studies on vaccine development against human Chagas disease.

The immunomodulatory effects of melatonin and zinc during chronic experimental Chagas' disease were studied. Early and late apoptosis by Annexin V-propidium iodide staining were evaluated. The expression of CD28, CD80, CD86, CD45RA and CD4(+)T and CD8(+)T cells were also evaluated by flow cytometry analysis. The combination of zinc and melatonin notably reduced the apoptotic ratios of splenic cells in the infected and treated animals when compared to untreated rats, during early and late stages of apoptosis. The percentages of CD8(+)T cells in Zn, Mel or Zn and Mel treated rats were reduced when compared to infected and untreated animals. Higher percentages of CD28 expression in CD4(+) and CD8(+) T cell populations were observed in control and infected Zn-treated group as compared to untreated ones. Zn, Mel or the combination of both did not induce any statistically significant differences for B cells when comparing to treated control and infected groups. Zinc or Mel-treated animals presented a lower expression of CD86 when compared to untreated counterparts. According to our data, this work strongly suggest that the modulation of the immune system operated by zinc and melatonin administration affected the balance among T cell immune response, apoptosis and expression of co-stimulatory molecules during chronic Trypanosoma cruzi infection, inducing important changes in the host's immune response against the parasite. Future experiments in this field should be focused in improving our understanding of the key mechanisms underlying the involvement of melatonin and zinc in the immune response during chronic Chagas' disease.

After one century of the discovery of Chagas' disease and the development of an efficient drug with amplitude of actions both in the acute and chronic phase is still a challenge. Alternative immune modulators have been exhaustively used. For that purpose, melatonin and zinc were administered during chronic Trypanosoma cruzi‐infected Wistar rats and several endpoints were assessed. Melatonin has a remarkable functional versatility, being associated with important antioxidant, anti‐inflammatory, and anti‐apoptotic effects. The cross‐talk between zinc and the immune system includes its ability to influence the production and signaling of numerous inflammatory cytokines in a variety of cell types. Our study showed that zinc triggered a decrease in the generation of IFN‐γ for TCD4+ cells. Reduced percentage of CD4+T cells producing TNF‐α was observed in control melatonin or zinc‐and‐melatonin‐treated animals as compared with untreated rats. On the other hand, a significant increase in the percentage of IL‐4 from CD4+ and CD8+ T lymphocytes producers was observed 60 days after infection, for all zinc‐treated animals, whether infected or not. Melatonin and zinc therapies increased the percentages of CD4+ and CD8+ T lymphocytes IL‐10 producers. CD4+CD25highFoxp3+ T cells were also elevated in zinc‐ and melatonin‐treated animals. The modulation of the immune system influenced by these molecules affected cytokine production and the inflammatory process during chronic T. cruzi infection. Elucidation of the interplay between cytokine balance and the pathogenesis of Chagas’ disease is extremely relevant not only for the comprehension of the immune mechanisms and clinical forms but, most importantly, also for the implementation of efficient and adequate therapies.

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.

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 PGE 2 synthesis; sustained capacity of splenocytes to produce high levels of interleukin (IL)-12 early in the infection; enhanced splenocyte production of IL-1 β , 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 + CD44highCD62Llowmemory effector T cells at the end of the acute phase of infection. The high mortality in WT mice was associated with increased production of LTB 4 /LTC 4 , 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.

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.

Este estudo visou a determinar os valores eritroleucométricos e quantificar as subpopulações linfocitárias no sangue do cordão umbilical (SCU) e no sangue jugular de eqüinos neonatos. Foi realizada a colheita de SCU e do sangue jugular de 20 potros ao nascimento. As amostras foram submetidas às determinações dos valores eritroleucométricos e à quantificação de subpopulações de linfócitos-T, pela técnica citofluorométrica. Não foram verificadas diferenças significativas (P<0,05) entre os valores médios de tais parâmetros, entre o sangue jugular de neonatos e o SCU eqüino. O valor total para neutrófilos segmentados, no SCU e na jugular dos neonatos, foi inferior ao reportado para eqüinos ao nascimento. As contagens de linfócitos CD5+ e CD4+ no SCU e jugular de neonatos eqüinos foram inferiores àquelas admitidas para o sangue periférico de eqüinos adultos, indicando um componente imunológico imaturo. No entanto, a contagem de linfócitos CD8+ foi semelhante à descrita em sangue periférico de eqüinos adultos. A proporção CD4:CD8 obtida nesse ensaio, tanto para o SCU (2,64±0,91), como no sangue jugular de eqüinos neonatos (2,41±0,81), demonstrou uma dominância das células T CD4+ sobre os linfócitos T CD8+.The objective of the present study was to determine erythometric and leukometic values and quantify subpopulations of lymphocytes in umbilical cord blood (UCB) and jugular blood of neonate foals. UCB and jugular blood were collected from 20 foals at birth and processed for determination of erythometric and leukometric values and quantification of subpopulations of T lymphocytes by cytofluorometry. No significant difference (P<0.05) was noted between average values in neonate jugular blood and equine UCB. Total values observed for segmented neutrophils in UCB and neonate jugular blood were lower than those reported for horses at birth. The quantity of CD5+ and CD4+ lymphocytes was lower in UCB and neonate jugular blood than in peripheral blood of adult horses, which indicates immunological immaturity. The frequency of CD8+ lymphocytes, however, was similar to that described for peripheral blood of adult horses. CD4:CD8 ratio observed in the present study both for UCB (2.64±0.91) and for neonate jugular blood (2.41±0.81) demonstrated the predominance of CD4+ T lymphocytes over CD8+ T cells.

Este estudo visou a determinar os valores eritroleucométricos e quantificar as subpopulações linfocitárias no sangue do cordão umbilical (SCU) e no sangue jugular de eqüinos neonatos. Foi realizada a colheita de SCU e do sangue jugular de 20 potros ao nascimento. As amostras foram submetidas às determinações dos valores eritroleucométricos e à quantificação de subpopulações de linfócitos-T, pela técnica citofluorométrica. Não foram verificadas diferenças significativas (P<0,05) entre os valores médios de tais parâmetros, entre o sangue jugular de neonatos e o SCU eqüino. O valor total para neutrófilos segmentados, no SCU e na jugular dos neonatos, foi inferior ao reportado para eqüinos ao nascimento. As contagens de linfócitos CD5+ e CD4+ no SCU e jugular de neonatos eqüinos foram inferiores àquelas admitidas para o sangue periférico de eqüinos adultos, indicando um componente imunológico imaturo. No entanto, a contagem de linfócitos CD8+ foi semelhante à descrita em sangue periférico de eqüinos adultos. A proporção CD4:CD8 obtida nesse ensaio, tanto para o SCU (2,64±0,91), como no sangue jugular de eqüinos neonatos (2,41±0,81), demonstrou uma dominância das células T CD4+ sobre os linfócitos T CD8+.