Immunostimulating activity of Uncaria tomentosa in RAW 264.7 macrophages.

Uncaria tomentosa is a plant native to the Amazon that has immunomodulatory and antitumor properties due to the alkaloids found in the plant, being able to modify the immune response by potentiating or suspending the action of cytokines secreted by macrophages that induce the immune response, either by the classical route (M1) or through the alternative route (M2). Macrophages activated by M1 convert L-arginine into L-citrulline and nitric oxide (NO), whereas macrophages activated by the M2 pathway use the enzymatic activity of arginase to convert the same substrate into L-ornithine and urea. The aim of this work was to evaluate the immunostimulating activity of the crude hydroalcoholic extract from the bark of the U. tomentosa stem in RAW 264.7 macrophages. Concentrations of 0.2, 0.1 and 0.05 mg/mL of U. tomentosa extract associated with LPS, INF-γ and IL-4 inducers were tested by determining NO production and arginase enzyme activity. Nitric oxide production was enhanced by the extract when associated with LPS and LPS + INF-γ inducers. In the activity of the arginase enzyme, the extract decreased the stimulation of IL-4 on the enzyme, mainly at 0.2 mg/mL concentration. Therefore, it is concluded that the crude hydroalcoholic extract of the stem bark of U. tomentosa in RAW 264.7 cells, at a concentration of 0.2 mg/mL, showed considerable pro-inflammatory activity.

Protein from Hevea brasiliensis "Hev b 13" latex attenuates systemic inflammatory response and lung lesions in rats with sepsis / Proteína do latex de Hevea brasiliensis "Hev b 13" atenua resposta inflamatória sistêmica e lesão pulmonar de ratos com sepse

Abstract Sepsis induces a severe systemic inflammatory response that may result in multiple organ dysfunction and death. Studies using a protein derived from natural Hevea brasiliensis (rubber tree) latex, denominated Hev b 13, have demonstrated important anti-inflammatory effects, but no data have been published regarding its effects on sepsis. The aim of this study was to investigate the effects of Hev b 13 on the inflammatory response and lung lesions of septal rats. Male Wistar rats were submitted to cecal ligation and puncture (CLP), randomized into groups and treated with subcutaneously administered doses of 0.5/2.0/3.0 mg/Kg of Hev b 13. Next, animals were subdivided into three different points in time (1, 6 and 24 hours after treatments) for collection of blood samples and euthanasia accompanied by organ removal. Total and differential leukocyte counts, cytokine dosage and histological assessment were analyzed. Treatment with Hev b 13 resulted in a significant decline in total and differential leukocytes as well as suppression of TNF-α and IL-6 production, associated with the increase in IL-10 and IL-4 in plasma and lung tissue. Moreover, it reduced morphological and pathological changes found in the lungs, including neutrophil infiltration, edema and alveolar thickening. The present study concluded that Hev b 13 exerts anti-inflammatory effects and attenuates lung lesions in septal rats, showing potential for clinical application.

Resumo Sepse induz uma resposta inflamatória sistêmica grave podendo resultar em disfunção de múltiplos órgãos e morte. Pesquisas utilizando uma proteína derivada do látex natural de Hevea brasiliensis (seringueira), denominada Hev b 13 tem demonstrado importantes efeitos anti-inflamatórios, mas nenhum dado foi publicado dos seus efeitos na sepse. O objetivo deste estudo foi investigar os efeitos da Hev b 13 na resposta inflamatória e na lesão pulmonar de ratos com sepse. Ratos machos da linhagem Wistar foram submetidos a ligação e perfuração do ceco (LPC), randomizados em grupos e tratados com as doses 0,5/2,0/3,0 mg/Kg de Hev b 13 subcutâneo. Após subdividiu-se os animais em três pontos diferentes de tempo (1, 6 e 24 horas após os tratamentos) para coleta de amostras sanguíneas e eutanásia com remoção dos órgãos. Contagem total e diferencial de leucócitos, dosagem de citocinas e avaliação histológica foram analisadas. O tratamento com a Hev b 13 resultou em diminuição significativa de leucócitos totais e diferenciais bem como suprimiu a produção de TNF-α e IL-6, associado ao aumento de IL-10 e IL-4 no plasma e tecido pulmonar. Além disso, reduziu as alterações morfológicas e patológicas encontradas nos pulmões, incluindo infiltrado de neutrófilos, edema e espessamento alveolar. Este estudo concluiu que a Hev b 13 tem efeitos anti-inflamatórios e atenua lesões pulmonares em ratos com sepse, apresentando potencialidades para aplicabilidade clínica.

Protein from Hevea brasiliensis "Hev b 13" latex attenuates systemic inflammatory response and lung lesions in rats with sepsis.

Sepsis induces a severe systemic inflammatory response that may result in multiple organ dysfunction and death. Studies using a protein derived from natural Hevea brasiliensis (rubber tree) latex, denominated Hev b 13, have demonstrated important anti-inflammatory effects, but no data have been published regarding its effects on sepsis. The aim of this study was to investigate the effects of Hev b 13 on the inflammatory response and lung lesions of septal rats. Male Wistar rats were submitted to cecal ligation and puncture (CLP), randomized into groups and treated with subcutaneously administered doses of 0.5/2.0/3.0 mg/Kg of Hev b 13. Next, animals were subdivided into three different points in time (1, 6 and 24 hours after treatments) for collection of blood samples and euthanasia accompanied by organ removal. Total and differential leukocyte counts, cytokine dosage and histological assessment were analyzed. Treatment with Hev b 13 resulted in a significant decline in total and differential leukocytes as well as suppression of TNF-α and IL-6 production, associated with the increase in IL-10 and IL-4 in plasma and lung tissue. Moreover, it reduced morphological and pathological changes found in the lungs, including neutrophil infiltration, edema and alveolar thickening. The present study concluded that Hev b 13 exerts anti-inflammatory effects and attenuates lung lesions in septal rats, showing potential for clinical application.

Evaluation of an rK39-based immunochromatographic test for the diagnosis of visceral leishmaniasis in human saliva.

Visceral leishmaniasis (VL) is a tropical neglected disease endemic in 98 countries and affects more than 58 000 individuals per year. Several serological tests are available for VL diagnosis, including an immunochromatographic (IC) test with the rK39 antigen and finger prick-collected blood, a rapid and low-invasive test. Here, we investigate the possibility to use saliva as a non-invasive source of biological material for the rK39 IC test. Blood samples from 84 patients with suspected VL were screened by the rK39 IC test, and 29 were confirmed as being infected by a positive rK39 IC test and the presence of amastigotes on smears slides or parasite DNA (detected using PCR-RFLP) from bone marrow aspirate. The rK39 IC test using saliva samples was positive for 17 of the 29 confirmed VL cases (58.6%). The amount of Leishmania-specific IgG or total IgG, as evaluated by an immunoenzymatic assay, was higher in the saliva of patients who had rK39 IC test positivity using saliva, whereas the amount of Leishmania-specific IgA or total IgA was similar to the healthy donors. These results suggest that saliva is not an appropriated material for diagnosing VL with this test.

Evaluation of an rK39-based immunochromatographic test for the diagnosis of visceral leishmaniasis in human saliva

Visceral leishmaniasis (VL) is a tropical neglected disease endemic in 98 countries and affects more than 58 000 individuals per year. Several serological tests are available for VL diagnosis, including an immunochromatographic (IC) test with the rK39 antigen and finger prick-collected blood, a rapid and low-invasive test. Here, we investigate the possibility to use saliva as a non-invasive source of biological material for the rK39 IC test. Blood samples from 84 patients with suspected VL were screened by the rK39 IC test, and 29 were confirmed as being infected by a positive rK39 IC test and the presence of amastigotes on smears slides or parasite DNA (detected using PCR-RFLP) from bone marrow aspirate. The rK39 IC test using saliva samples was positive for 17 of the 29 confirmed VL cases (58.6%). The amount of Leishmania-specific IgG or total IgG, as evaluated by an immunoenzymatic assay, was higher in the saliva of patients who had rK39 IC test positivity using saliva, whereas the amount of Leishmania-specific IgA or total IgA was similar to the healthy donors. These results suggest that saliva is not an appropriated material for diagnosing VL with this test.

Leishmania braziliensis amastigotes stimulate production of IL-1ß, IL-6, IL-10 and TGF-ß by peripheral blood mononuclear cells from nonendemic area healthy residents.

Leishmania (Viannia) braziliensis causes cutaneous and mucosal leishmaniasis in several countries in Latin America. In mammals, the parasites live as amastigotes, interacting with host immune cells and stimulating cytokine production that will drive the type of the specific immune responses. Generation of Th17 lymphocytes is associated with tissue destruction and depends on IL-1ß, IL-6, TGF-ß and IL-23 production, whereas IL-10 and TGF-ß are associated with tissue protection. Here, we evaluate whether amastigotes stimulate peripheral blood mononuclear cells (PBMCs) from healthy donors to produce the major cytokines responsible for the generation of Th17. Seven L. (V.) braziliensis isolates from patients with different clinical forms of leishmaniasis were expanded in interferon-γ knockout mice to obtain amastigotes and in culture to get promastigotes. The parasites were used to stimulate PBMCs from healthy donors, and cytokine production was evaluated by ELISA or qPCR. Amastigotes and promastigotes induced IL-10 production in PBMCs; however, only amastigotes induced IL-1ß, IL-6 and TGF-ß. These data demonstrate for the first time that L. (V.) braziliensis amastigotes directly stimulate production of a unique pattern of cytokines that could contribute to the generation of Th17.

Neutrophils have a protective role during early stages of Leishmania amazonensis infection in BALB/c mice.

Neutrophils are involved in the early stages of immune responses to pathogens. Here, we investigated the role of neutrophils during the establishment of Leishmania amazonensis infection in BALB/c and C57BL/6 mice. First, we showed an accumulation of neutrophils between 6 and 24 h post-infection, followed by a reduction in neutrophil numbers after 72 h. Next, we depleted neutrophils prior to infection using RB6-8C5 or 1A8 mAb. Neutrophil depletion led to faster lesion development, increased parasite numbers and higher arginase activity during the first week of infection in BALB/c mice, but not in C57BL/6 mice. Increased susceptibility was accompanied by augmented levels of anti-L. amazonensis IgG and increased production of IL-10 and IL-17. Because IL-10 is a mediator of susceptibility to Leishmania infection, we blocked IL-10 signalling in neutrophil-depleted mice using anti-IL-10R. Interestingly, inhibition of IL-10 signalling abrogated the increase in parasite loads observed in neutrophil-depleted mice, suggesting that parasite proliferation is at least partially mediated by IL-10. Additionally, we tested the effect of IL-17 in inflammatory macrophages and observed that IL-17 increased arginase activity and favoured parasite growth. Taken together, our data indicate that neutrophils control parasite numbers and limit lesion development during the first week of infection in BALB/c mice.

Kinetics of the inflammatory response in subcutaneous cysticercosis induced in mice by Taenia crassiceps.

The larval stage of Taenia crassiceps has been used to study human cysticercosis as these larvae have antigenic similarity to the cysticerci of Taenia solium. The aim of this study was to evaluate the histopathological and immunological changes that followed the inoculation of T. crassiceps cysticerci into the subcutaneous tissue of C57BL/6 mice. Microscopically, granulomas formed of neutrophils and macrophages developed at the sites of inoculation. The serum concentration of the cytokine interferon (IFN)-γ increased throughout the course of infection, while the serum concentration of interleukin-4 increased during the period of transition from the initial phase (7-30 days postinoculation [dpi]) to the late phase (60-90 dpi) of infection. Destruction of the parasite therefore appears to be associated with an increase in IFN-γ, suggesting that a type 1 immune response is important in the control of the parasite.

Germ-free mice produce high levels of interferon-gamma in response to infection with Leishmania major but fail to heal lesions.

In order to investigate the importance of the host microbiota on differentiation of T cell subsets in response to infection, Swiss/NIH germ-free mice and conventional (microbiota-bearing) mice were infected with Leishmania major, and lesion development, parasite loads, and cytokine production were assessed. Germ-free mice failed to heal lesions and presented a higher number of parasites at the site of infection than their conventional counterparts. In addition, histopathological analysis indicated a higher density of parasitized macrophages in lesions from germ-free mice than in conventional mice. The initial production of interleukin (IL)-12 and interferon-gamma (IFN-gamma) in germ-free mice was comparable to the conventional controls. Also, germ-free mice produced elevated levels of IFN-gamma and lower levels of IL-4 throughout the course of infection, suggesting the development of a Th1 response. Macrophages from germ-free mice exposed to IFN-gamma and infected with amastigotes in vitro were not as efficient at killing parasites as macrophages from conventional animals. These observations indicate that the microbiota is not essential for the development of Th1 immune responses, but seems to be important for macrophage activation.

Immature macrophages derived from mouse bone marrow produce large amounts of IL-12p40 after LPS stimulation.

Production of IL-12 is an important indicator of the macrophage's ability to regulate immune responses. In this study, we investigated the IL-12 production by macrophages in different developmental stages. To this end, macrophages were generated in vitro from precursors stimulated with M-CSF, GM-CSF or IL-3. Density separation yielded populations enriched in different maturation stages. Invariably, only cells banding at the 40-50% Percoll interface produced large amounts of IL-12p40 when stimulated with LPS, whereas only low levels of IL-12p70 were produced. These cells represented immature macrophages, as indicated by the absence of precursor markers CD31/ER-MP12, Ly-6C/ER-MP20 and ER-MP58, and by the low level of expression of mature-cell markers like ER-HR3, scavenger receptor and CD11b/Mac-1. Upon further maturation, the macrophages' ability to produce IL-12p40 decreased, coinciding with increased nitric oxide production upon LPS stimulation. These results show that immature macrophages produce high levels of IL-12p40 and thus may either contribute to IL-12p70 production or regulate it.

Immunochemotherapy in American cutaneous leishmaniasis: immunological aspects before and after treatment

In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-gamma production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-gamma at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-gamma producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine

Monoassociation with Lactobacillus acidophilus UFV-H2b20 stimulates the immune defense mechanisms of germfree mice

Probiotics are formulations containing live microorganisms or microbial stimulants that have some beneficial influence on the maintenance of a balanced intestinal microbiota and on the resistance to infections. The search for probiotics to be used in prevention or treatment of enteric infections, as an alternative to antibiotic therapy, has gained significant impulse in the last few years. Several studies have demonstrated the beneficial effects of lactic acid bacteria in controlling infection by intestinal pathogens and in boosting the host's nonspecific immune response. Here, we studied the use of Lactobacillus acidophilus UFV-H2b20, a lactic acid bacterium isolated from a human newborn from Viçosa, Minas Gerais, Brazil, as a probiotic. A suspension containing 108 cells of Lactobacillus acidophilus UFV-H2b20 was inoculated into groups of at least five conventional and germfree Swiss mice to determine its capacity to stimulate the host mononuclear phagocytic activity. We demonstrate that this strain can survive the stressing conditions of the intestinal tract in vivo. Moreover, the monoassociation of germfree mice with this strain for seven days improved the host's macrophage phagocytic capacity, as demonstrated by the clearance of a Gram-negative bacterium inoculated intravenously. Monoassociated mice showed an undetectable number of circulating E. coli, while 0.1 percent of the original inoculum was still present in germfree animals. Mice treated with viable or heat-killed Lactobacillus acidophilus UFV-H2b20 presented similarly improved clearance capacity when compared with germfree controls. In addition, monoassociated mice had twice the amount of Kupffer cells, which are responsible for the clearance of circulating bacteria, compared to germfree controls. These results suggest that the L. acidophilus strain used here stimulates a nonspecific immune response and is a strong candidate to be used as a probiotic

Induction of cell-mediated immunity during early stages of infection with intracellular protozoa

Toxoplasma gandii and Trypanosoma cruzi are intracellular parasites which, as part of their life cycle, induce a potent cell-mediated immunity (CMI) maintained by Th1 lymphocytes and IFN-gamma. In both cases, induction of a strong CMI is thought to protect the host against rapid parasite multiplication and consequent pathology and lethality during the acute phase of infection. However, the parasitic infection is not eliminated by the immune system and the vertebrate host serves as a parasite reservoir. In contrast, Leishmania sp, which is a slow growing parasite, appears to evade induction of CMI during early stages of infection as a strategy for surviving in a hostile environment (i.e., inside the macrophages which are their obligatory niche in the vertebrate host). Recent reports show that the initiation of IL-12 synthesis by macrophages during these parasitic infections is a key event in regulating CMI and disease outcome. The studies reviewed here indicate that activation/inhibition of distinct signaling pathways and certain macrophage functions by intracellular protozoa are important events in inducing/modulating the immune response of their vertebrate hosts, allowing parasite and host survival and therefore maintaining parasite life cycles.