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The neglected tropical infirmity Chagas disease (CD) presents high mortality. Its etiological agent T. cruzi is transmitted by infected hematophagous insects. Symptoms of the acute phase of the infection include fever, fatigue, body aches, and headache, making diagnosis difficult as they are present in other illnesses as well. Thus, in endemic areas, individuals with undetermined pain may be considered for CD. Although pain is a characteristic symptom of CD, its cellular and molecular mechanisms are unknown except for demonstration of a role for peripheral TNF-α in CD pain. In this study, we evaluate the role of spinal cord glial cells in experimental T. cruzi infection in the context of pain using C57BL/6 mice. Pain, parasitemia, survival, and glial and neuronal function as well as NFκB activation and cytokine/chemokine production were assessed. T. cruzi infection induced chronic mechanical and thermal hyperalgesia. Systemic TNF-α and IL-1β peaked 14 days postinfection (p.i.). Infected mice presented increased spinal gliosis and NFκB activation compared to uninfected mice at 7 days p.i. Glial and NFκB inhibitors limited T. cruzi–induced pain. Nuclear phosphorylated NFκB was detected surrounded by glia markers, and glial inhibitors reduced its detection. T. cruzi–induced spinal cord production of cytokines/chemokines was also diminished by glial inhibitors. Dorsal root ganglia (DRG) neurons presented increased activity in infected mice, and the production of inflammatory mediators was counteracted by glial/NFκB inhibitors. The present study unveils the contribution of DRG and spinal cord cellular and molecular events leading to pain in T. cruzi infection, contributing to a better understanding of CD pathology.

We evaluated the influence of metabolic syndrome (MS) on acute Trypanosoma cruzi infection. Obese Swiss mice, 70 days of age, were subjected to intraperitoneal infection with 5 × 102 trypomastigotes of the Y strain. Cardiovascular, oxidative, inflammatory, and metabolic parameters were evaluated in infected and non-infected mice. We observed higher parasitaemia in the infected obese group (IOG) than in the infected control group (ICG) 13 and 15 days post-infection. All IOG animals died by 19 days post-infection (dpi), whereas 87.5% of the ICG survived to 30 days. Increased plasma nitrite levels in adipose tissue and the aorta were observed in the IOG. Higher INF-γ and MCP-1 concentrations and lower IL-10 concentrations were observed in the IOG compared to those in the ICG. Decreased insulin sensitivity was observed in obese animals, which was accentuated after infection. Higher parasitic loads were found in adipose and hepatic tissue, and increases in oxidative stress in cardiac, hepatic, and adipose tissues were characteristics of the IOG group. Thus, MS exacerbates experimental Chagas disease, resulting in greater damage and decreased survival in infected animals, and might be a warning sign that MS can influence other pathologies.

Long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) are known to modulate a variety of immune cell functions. On occasion, this has led to diminished host resistance to certain viral and bacterial infections. Little is known about the impact of n-3 PUFA on host resistance to parasitic infection, however, based on results from a small study conducted more than two decades ago, we hypothesized that providing mice LC n-3 PUFA will diminish host resistance to Trypanosoma cruzi, the parasitic pathogen responsible for Chagas disease. To investigate this, C57BL/6 mice were supplemented by gavage (0.6% v/w) with phosphate-buffered saline, corn oil (CO), or menhaden fish oil (FO, a fat source rich in LC n-3 PUFA) for 15 days prior to T cruzi (Y strain) challenge and throughout the acute phase of infection. FO supplementation was associated with a transient 2-fold greater peak of blood parasitemia at 7 days postinfection (dpi), whereas subsequent cardiac parasitemia was ~60% lower at 12 dpi. FO treatment also ameliorated the leukopenia and thrombocytopenia observed in the early stages of a T cruzi infection. FO supplementation reduced circulating and cardiac nitric oxide at 7 and 12 dpi, respectively. FO supplementation altered ex vivo prostaglandin E2 and cytokine and chemokine production by splenocytes isolated from uninfected and infected mice. Overall, our results suggest that oral administration of LC n-3 PUFA from FO can have beneficial effects on the host in the early course of a T cruzi infection.

Made available in DSpace on 2018-12-11T17:08:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação Araucária American Tegumentar Leishmaniasis (ATL) is an infectious disease caused by Leishmania parasites with ineffective treatment. The properties of propolis have been studied in different experimental studies, however, few works have investigated the effects of propolis on human-derived peripheral blood mononuclear cells (PBMC) in leishmaniasis models. Thus, we investigate the immunomodulatory effects of propolis treatment on PBMC from ATL patients and on PBMC from healthy donors infected with Leishmania braziliensis. Our data demonstrate that propolis pretreatment shows immunomodulatory effects on both healthy donors and ATL patients adherent cells, increasing IL-4 and IL-17 and decreasing IL-10, in either the presence or absence of the L. braziliensis infection, demonstrating that propolis contributes with the decrease of the inflammation and could also contribute with parasite control. Departamento de Ciências Patológicas Centro de Ciências Biológicas Universidade Estadual de Londrina Laboratório de Mediadores Inflamatórios Universidade do Oeste do Paraná UNIOESTE Ambulatório de Especialidades do Hospital Universitário de Londrina Laboratório de Virologia Molecular Instituto Carlos Chagas/Fundação Oswaldo Cruz Departamento de Microbiologia e Imunologia Instituto de Biociência Universidade Estadual Paulista UNESP Departamento de Microbiologia e Imunologia Instituto de Biociência Universidade Estadual Paulista UNESP

Infection with the protozoanTrypanosoma cruzicauses Chagas disease and consequently leads to severe inflammatory heart condition; however, the mechanisms driving this inflammatory response have not been completely elucidated. Nitric oxide (NO) is a key mediator of parasite killing inT. cruzi-infected mice, and previous studies have suggested that leukotrienes (LTs) essentially regulate the NO activity in the heart. We used infected 5-lipoxygenase-deficient mice (5-LO−/−) to explore the participation of nitric oxide synthase isoforms, inducible (iNOS) and constitutive (cNOS), in heart injury, cytokine profile, and oxidative stress during the early stage ofT. cruziinfection. Our evidence suggests that the cNOS of the host is involved in the resistance of 5-LO−/−mice duringT. cruziinfection. iNOS inhibition generated a remarkable increase inT. cruziinfection in the blood and heart of mice, whereas cNOS inhibition reduced cardiac parasitism (amastigote nests). Furthermore, this inhibition associates with a higher IFN-γproduction and lower lipid peroxidation status. These data provide a better understanding about the influence of NO-interfering therapies for the inflammatory response towardT. cruziinfection.

O objetivo deste trabalho foi determinar a presença de parasitas intestinais em amostras de alfaces e almeirões comercializadas em feira livre e supermercado da cidade de Londrina, Paraná. Foram coletadas e analisadas oito amostras de alface (Lactuca sativa) e oito amostras de almeirão (Cichirium intybus). A análise foi realizada no Laboratório de Extensão e Pesquisas em Enteroparasitoses – L.E.P.En. Foram encontrados cistos de Endolimax nana, Entamoeba coli, Entamoeba histolytica, Balantidium coli, larvas de Strongyloides stercoralis, larvas e ovos de Ancilostomídeos e ovos de Ascaris spp. Esses resultados demonstram a necessidade de medidas higiênico-sanitárias, visando à educação da comunidade, possibilitando a melhoria na condição de vida da população.

The intracellular protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a serious disorder that affects millions of people in Latin America. Cell invasion by T. cruzi and its intracellular replication are essential to the parasite's life cycle and for the development of Chagas disease. Here, we present evidence suggesting the involvement of the host's cyclooxygenase (COX) enzymes during T. cruzi invasion. Pharmacological antagonists for COX-1 (aspirin) and COX-2 (celecoxib) caused marked inhibition of T. cruzi infection when rat cardiac cells were pretreated with these nonsteroidal anti-inflammatory drugs (NSAIDs) for 60 min at 37°C before inoculation. This inhibition was associated with an increase in the production of NO and interleukin-1β and decreased production of transforming growth factor β (TGF-β) by cells. Taken together, these results indicate that COX-1 more than COX-2 is involved in the regulation of anti- T. cruzi activity in cardiac cells, and they provide a better understanding of the influence of TGF-β-interfering therapies on the innate inflammatory response to T. cruzi infection and may represent a very pertinent target for new therapeutic treatments of Chagas disease.

The intracellular protozoan parasiteTrypanosoma cruzicauses Chagas disease, a serious disorder that affects millions of people in Latin America. Cell invasion byT. cruziand its intracellular replication are essential to the parasite’s life cycle and for the development of Chagas disease. Here, we present evidence suggesting the involvement of the host’s cyclooxygenase (COX) enzyme duringT. cruziinvasion. Pharmacological antagonist for COX-1, aspirin (ASA), caused marked inhibition ofT. cruziinfection when peritoneal macrophages were pretreated with ASA for 30 min at 37°C before inoculation. This inhibition was associated with increased production of IL-1βand nitric oxide (NO∙) by macrophages. The treatment of macrophages with either NOS inhibitors or prostaglandin E2(PGE2) restored the invasive action ofT. cruziin macrophages previously treated with ASA. Lipoxin ALX-receptor antagonist Boc2 reversed the inhibitory effect of ASA on trypomastigote invasion. Our results indicate that PGE2,NO∙, and lipoxins are involved in the regulation of anti-T. cruziactivity by macrophages, providing a better understanding of the role of prostaglandins in innate inflammatory response toT. cruziinfection as well as adding a new perspective to specific immune interventions.

Chagas' disease is accompanied by severe anemia and oxidative stress, which may contribute to mortality. In this study, we investigated the role of 5-lipoxygenase (5-LO) in the control of parasitism and anemia associated with oxidative damage of erythrocytes in experimental Trypanosoma cruzi infection. Wild-type C57BL/6, 129Sv mice treated or not with nordihydroguaiaretic acid (NDGA, 5-LO inhibitor), mice lacking the 5-LO enzyme gene (5-LO(-/-)) and inducible nitric oxide synthase gene (iNOS(-/-)) were infected with the Y strain of T. cruzi. Impairment of 5-LO resulted in increased numbers of trypomastigote forms in the blood and amastigote forms in the heart of infected mice. We assessed oxidative stress in erythrocytes by measuring oxygen uptake, induction time and chemiluminescence following treatment with tert-butyl hydroperoxide (TBH). Our results show that 5-LO metabolites increased lipid peroxidation levels in erythrocytes during the early phase of murine T. cruzi infection. NDGA treatment reduced oxidative damage of erythrocytes in C57BL/6 T. cruzi-infected mice but not in C57BL/6 iNOS(-/-) infected mice, showing that the action of NDGA is dependent on endogenous nitric oxide (NO). In addition, our results show that 5-LO metabolites do not participate directly in the development of anemia in infected mice. We conclude that 5-LO products may not only play a major role in controlling heart tissue parasitism, i.e., host resistance to acute infection with T. cruziin vivo, but in the event of an infection also play an important part in erythrocyte oxidative stress, an NO-dependent effect.

HPV is one of the most frequent causes for the development of cervical cancer. It is known that chemokines are important determinants of early inflammatory responses. The CC chemokine receptor 5 (CCR5) gene is involved in the chemotaxis of leukocytes toward inflammation sites. In the present study, polymerase chain reactions (PCR) in genomic DNA samples, using specific CCR5 oligonucleotide primers surrounding the breakpoint deletion, detected a 225 bp product from the normal CCR5 allele and a 193 bp product from the 32 bp deletion allele. The wild type genotype was prevalent in both group, but it was not statistically significant, with χ2 = 1.519 (2 degrees of freedom; p > 0.05). As there are a small number of 32 allele carriers, further studies are needed to clarify the role of CCR5 in the cervical cancer.HPV is the most responsible of cervical cancer. It is known that chemokines are important determinants of the early inflammatory response. The CC chemokine receptor 5 (CCR5) gene is involved in the chemotaxis of leukocytes toward inflammation sites. In the present study, polymerase chain reactions (PCR) in genomic DNA samples, using specific CCR5 oligonucleotide primers surrounding the breakpoint deletion, detected a 225bp product from the normal CCR5 allele and a 193bp product from the 32bp deletion allele. The wild type genotype was prevalent in both group, but it wasn’t statistically significant with χ² =1,519 (2 degrees of freedom; p>0.05). Once there is a small number of 32 allele carriers, further studies are needed to clarify the role of CCR5 in the cervical cancer.

A infecção por Trypanosoma cruzi produz uma intensa resposta inflamatória em diversos tecidos, inclusive no coração. A reação inflamatória é crítica para o controle da proliferação dos parasitos e evolução da Doença de Chagas (DC). Antiinflamatórios não esteróides liberadores de NO (AINES-NO) compõem uma nova classe de fármacos disponível para o tratamento de doenças inflamatórias. Pesquisas realizadas em nosso laboratório mostraram que antiinflamatórios não esteroides (AINES) convencionais como indometacina, aspirina e celecoxibe promovem aumento da carga parasitária no sangue e no tecido cardíaco de camundongos BALB/c e C57BL/6 infectados por T. cruzi. A capacidade do óxido nítrico (NO) em mediar a atividade anti-T. cruzi e o estresse oxidativo em eritrócitos durante a fase aguda da infecção, nos levou a investigar qual o impacto da administração de doadores de NO sobre a anemia, carga parasitária e estresse oxidativo em eritrócitos no inicio da infecção por T. cruzi. Camundongos C57BL/6 foram infectados por T. cruzi (Cepa Y, 5 x 103 formas tripomastigotas sanguíneos) via intraperitoneal. Após 30 minutos de infecção e nos subsequentes 12 dias, os animais foram tratados com indometacina-NO (indo-NO, 50 ppm/animal) ou sal de Angeli 15 minutos após infecção e diariamente por 12 dias (6 ?g, 60 ?g ou 600 ?g) ip. O grupo controle recebeu 100 ?L de PBS pela mesma via. O tratamento com indo-NO mostrou diminuição da sobrevida, aumento da anemia e do estresse oxidativo, diminuição da produção de NO ?in vivo? e ?in vitro?, além do aumento de internalização e saída de formas tripomastigotas de T. cruzi em macrófagos. O tratamento com sal de Angeli na concentração de 60?g/Kg diminuiu a parasitemia, o número de ninhos no tecido cardíaco, a produção de NO plasmático, e o estresse oxidativo. Por outro lado, melhorou a leucopenia e a trombocitopenia. O tratamento ?in vitro? na concentração de 60 ?M foi capaz de reduzir a produção de NO em cultura de macrófagos, diminuir a internalização e saída de formas tripomastigotas de macrófagos e diminuir a produção de IL-10. Trypanosoma cruzi produces an intense inflammatory response in several tissues, including the heart. The inflammatory response is critical for controlling parasites proliferation and evolution of Chagas disease (CD). Non-steroidal anti-inflammatory releasing NO (NO-NSAIDs) constitute a new class of drugs available for the treatment of inflammatory diseases. Research conducted in our laboratory have shown that anti-inflammatory drugs (NSAIDs) such as conventional indomethacin, aspirin and celecoxib do not promote increased parasite burden in blood and heart tissue of BALB/c and C57BL/6 mice infected with T. cruzi. The ability of nitric oxide (NO) in mediating the activity of anti-T. cruzi and oxidative stress in erythrocytes during the acute phase of infection, led us to investigate the impact of administration of NO donors on anemia, parasite burden and oxidative stress in erythrocytes at the beginning of infection by T. cruzi. C57BL/6 mice were infected with T. cruzi (Y strain, 5 x 103 trypomastigotes) intraperitoneally. After 30 minutes of infection and the subsequent 12 days, the animals were treated with indomethacin-NO (Indo-NO, 50 ppm/animal) or Angeli's salt 15 minutes after infection and daily for 12 days (6 ?g, 60 ?g, or 600 ?g/animal) ip. The control group received 100 uL of PBS by the same route. Treatment with Indo-NO showed decreased survival, increased anemia and oxidative stress, decreased NO production in vivo and in vitro; apart from increased internalization and output trypomastigotes of T. cruzi in macrophages. The results obtained with treatment with Angeli's salt at a concentration of 60 ?g/kg decreased parasitemia and the number of nests in the cardiac tissue, decreased production of plasma NO, decreased oxidative stress and improved leukopenia and thrombocytopenia observed in acute infection. The in vitro at a concentration of 60 ?M treatment was able to reduce NO production in macrophage culture, internalization, release of trypomastigotes and decrease the production of IL-10.

Trypanosoma cruzi is the causative agent of Chagas disease. Approximately 8 million people are thought to be affected with this disease worldwide. T. cruzi infection causes an intense inflammatory response, which is critical for the control of parasite proliferation and disease development. Nitric oxide-donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs) are an emergent class of pharmaceutical derivatives with promising utility as chemopreventive agents. In this study, we investigated the effect of NO-indomethacin on parasite burden, cell invasion, and oxidative stress in erythrocytes during the acute phase of infection. NO-indomethacin was dissolved in dimethyl formamide followed by i.p. administration of 50 ppm into mice 30 min after infection with 5×10(3) blood trypomastigote forms (Y strain). The drug was administered every day until the animals died. Control animals received 100 μL of drug vehicle via the same route. Within the NO-indomethacin-treatment group, parasitemia and mortality (100%) were higher and oxidative stress in erythrocytes, anemia, and entry of parasites into macrophages were significantly greater than that seen in controls. Increase in the entry and survival of intracellular T. cruzi was associated with inhibition of nitric oxide production by macrophages treated with NO-indomethacin (2.5 μM). The results of this study provide strong evidence that NO-NSAIDs potently inhibit nitric oxide production, suggesting that NO-NSAID-based therapies against infections would be difficult to design and would require caution.

Trypanosoma cruzi, the causative agent of Chagas’ disease (CD), is a substantial public health concern in Latin America. Laboratory mice inoculated with T. cruzi have served as important animal models of acute CD. Host hypoferremic responses occur during T. cruzi infection; therefore, it has been hypothesized that T. cruzi requires iron for optimal growth in host cells and, unlike extracellular pathogens, may benefit from host hypoferremic responses. Recent technological improvements of X-ray fluorescence are useful for diagnostics or monitoring in biomedical applications. The goal of our study was to determine whether the iron availabilities in Swiss and C57BL/6 mice differ during the acute phase of T. cruzi infection and whether the availability correlates with oxidative stress in the susceptible and resistant phenotypes identified in these mice. Our results showed that the decrease in iron levels in the skin of resistant infected mice correlated with the increase in oxidative stress associated with anemia and the reduction in parasite burden.

Leukotrienes are important mediators of inflammatory responses. In this study, we investigated the effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on levels of cytokines, nitric oxide (NO) and iNOS expression in cardiac tissue of mice infected with Trypanosoma cruzi, the agent of Chagas’ disease. NO is a key mediator of parasite killing in mice experimentally infected with T. cruzi, and previous studies have suggested that leukotrienes, such as LTB4, induces NO synthesis in T. cruzi-infected macrophages and plays a relevant role in the killing of parasite in a NO-dependent manner. We therefore investigated whether leukotrienes would have a similar role in vivo in controlling the parasite burden by regulating NO activity. We have made the striking observation that absence of 5-LO-derived leukotrienes results in increased NO and IL-6 production in the plasma with a concomitant decrease in the expression of iNOS in the cardiac tissue on day 12 after T. cruzi infection. These findings indicate that endogenous leukotrienes are important regulators of NO activity in the heart and therefore influence the cardiac parasite burden without exerting a direct action on IL-6 production in the acute phase of infection with T. cruzi.

HPV is the most responsible of cervical cancer. It is known that chemokines are important determinants of the early inflammatory response. The CC chemokine receptor 5 (CCR5) gene is involved in the chemotaxis of leukocytes toward inflammation sites. In the present study, polymerase chain reactions (PCR) in genomic DNA samples, using specific CCR5 oligonucleotide primers surrounding the breakpoint deletion, detected a 225bp product from the normal CCR5 allele and a 193bp product from the 32bp deletion allele. The wild type genotype was prevalent in both group, but it wasn’t statistically significant with χ² =1,519 (2 degrees of freedom; p>0.05). Once there is a small number of 32 allele carriers, further studies are needed to clarify the role of CCR5 in the cervical cancer.

Prostaglandins are known to be produced by macrophages when challenged with Trypanosoma cruzi, the etiological agent of Chagas' disease. It is not known whether these lipid mediators play a role in oxidative stress in host defenses against this important protozoan parasite. In this study, we demonstrated that inducible cyclooxygenase-mediated prostaglandin production is a key chemical mediator in the control of parasite burden and erythrocyte oxidative stress during T. cruzi infection in C57BL/6 and BALB/c mice, prototype hosts for the study of resistance and susceptibility in murine Chagas' disease. The results suggested the existence of at least two mechanisms of oxidative stress, dependent or independent with regard to the nitric oxide and cyclooxygenase pathway, where one or the other is more evident depending on the mouse strain.

A infecção com o T. cruzi provocou aumento na porcentagem de reticulócitos no sangue periférico dos camundongos C57BL/6 em relação aos camundongos normais (12,5 ± 1,4 vs 7,6 ± 0,6). Os camundongos C57BL/6 infectados e tratados com celecoxibe aprsentaram no dia 12 pós infecção um aumento no número de reticulócitos (22 ± 2,2 vs 12,5 ± 1,4) em relação aos controles (infectados não tratados), o que não foi verificado com os outros inibidores de COX utilizados. A infecção também provocou aumento na porcentagem de reticulócitos no sangue periférico dos camundongos BALB/c em comparação aos animais normais (14,0 ± 1,5 vs 6,8 ± 2,9). O tratamento com indometacina aumentou cerca de 1,5 vezes a porcentagem de reticulócitos no sangue daqueles animais. Estes dados sugerem que produtos do metabolismo gerados via COX-2 estariam suprimindo a produção de reticulócitos durante a fase aguda da infecção experimental com o T. cruzi. Em adição, nossos dados mostram claramente que macrófagos obtidos da cavidade peritoneal de camundongos BALB/c infectados produzem espontaneamente muito menos óxido nítrico (NO) em relação aos macrófagos oriundos de camundongos C57BL/6 também infectados. O aumento da carga parasitária e morte precoce dos camundongos C57BL/6 e a tendência de queda da parasitemia e prolongamento da sobrevivência dos camundongos BALB/c, quando tratados com indometacina ou Asa podem ser mais bem compreendidos quando consideramos às diferenças na produção de NO mencionadas anteriormente e os efeitos dos diferentes inibidores utilizados sobre a produção de NO. O tratamento com indometacina não modificou a produção espontânea de NO por macrófagos obtidos de camundongos C57BL/6, mas provocou o aumento da produção de NO em macrófagos obtidos de BALB/c (que apresentaram tendência de diminuição da parasitemia no sangue e no aumento da sobrevida). Já o tratamento com Asa provocou diminuição da produção de NO por macrófagos de camundongos C57BL/6 (esses apresentaram 50% de morte no período do experimento). O tratamento de camundongos BALB/c infectados com Asa provocou aumento de NO o que poderia explicar a tendência observada no controle da parasitemia por parte destes animais Em adição, há evidências sugerindo que as miocardites chagásicas são devidas aos danos induzidos pelo estresse oxidativo, podendo contribuir para a evolução da doença de Chagas em humanos e animais de laboratório. Em doenças infecciosas, a formação de espécies reativas do oxigênio (ROS) é, principalmente, derivada de danos celulares mediados pela invasão e replicação do patógeno e por reações citotóxicas mediadas pelo sistema imunológico. No entanto, como as ROS são formadas e sua função no estresse oxidativo na cardiomiopatia chagásica (CCM) não estão completamente elucidadas. As evidências atuais dão ênfase para as anormalidades mitocondriais como a disfunção da cadeia de transporte de elétrons para explicar o aumento do estresse oxidativo na doença de Chagas. A análise do estresse oxidativo por quimiluminescência iniciada por tert-butil hidroperóxido, mostrou claramente que o estresse oxidativo em eritrócitos que se desenvolve no camundongo C57BL/6 infectado com T. cruzi é atenuado com o bloqueio farmacológico tanto de COX-1 como de COX-2. Em relação ao camundongo BALB/c a quimiluminescência mostrou que o tratamento com as NSAIds utilizadas não foi capaz de reduzir o estresse oxidativo em eritrócitos de camundongos infectados. Estes dados sugerem que o mecanismo gerador do estresse oxidativo que ocorre na fase aguda da infecção com T. cruzi depende da capacidade do hospedeiro em responder á infecção por meio da alta produção de NO e ROS. CONCLUSÕES: Nossos dados não deixam dúvidas de que na fase inicial da infecção com T. cruzi ocorre um aumento da lipoperoxidação de eritrócitos. Mostramos que o bloqueio de COX atenua a lipoperoxidação de eritrócitos no início da infecção com T cruzi (12º dia pós-infecção). The T. cruzi infection caused an increase in the percentage of reticulocytes in C57BL/6 mice’s peripheral blood in relation to normal mice (12,5 ± 1,4 vs 7,6 ± 0,6). C57BL/6 mice that were infected and treated with celecoxib presented an increase in reticulocytes (22 ± 2,2 vs 12,5 ± 1,4) on day 12 post-infection, in relation to the controls (infected but not treated), which was not verified with others COX inhibitors used. The infection also caused an increase in the percentage of reticulocytes in BALB/c mice’s peripheral blood in comparison to normal animals (14,0 ± 1,5 vs 6,8 ± 2,9). Treatment with indomethacin increased the percentage of reticulocytes in those animals by 1,5 times. This data suggests that metabolism products generated via COX-2 were suppressing the reticulocyte production during the acute phase of the T. cruzi’s experimental infection. In addition, our data clearly show that macrophages obtained in the peritoneal cavity of infected BALB/c mice spontaneously produce much less nitric oxide (NO) in relation to macrophages originated in infected C57BL/6 mice. The increase in parasite load and premature death of C57BL/6 mice, the decrease in parasitemia pattern and the survival prolongation of BALB/c mice; when treated with indomethacin or Asa can better understood when we consider the differences in NO production previously mentioned and the effects of different inhibitors used over NO production. The indomethacin treatment did not change the spontaneous production of NO by macrophages obtained in C57BL/6 mice, but it caused an increase in NO production in macrophages obtained in BALB/c mice (which presented decrease in blood parasitemia and survival increase tendencies). The treatment with Asa caused a decrease in NO production by C57BL/6 mice macrophages (these presented 50% death rate during the experiment). The treatment of infected C57BL/6 mice with Asa caused an increase in NO, which could explain the pattern observed in parasitemia control in these animals. In addition, there is evidence suggesting that chagasic myocarditis occur due to damage induced by oxidative stress, allowing the evolution of Chagas disease in humans and lab animals. In infectious diseases, the formation of reactive oxygen species (ROS) is mainly derived from cell damage mediated by pathogen invasion and replication, and by cytotoxic reactions mediated by the immune system. Therefore, since ROS are formed and its role in oxidative stress in chagasic cardiomyopathy (CCM) is not completely clear. Actual evidence gives emphasis to mitochondrial abnormalities such as electron transport chain dysfunction in order to explain the increase in oxidative stress in Chagas disease. The analysis of oxidative stress via tert-butyl hydroperoxide-induced chemiluminescence clearly showed that the oxidative stress in erythrocytes that develops in T. cruzi infected C57BL/6 mice is weakened with either COX-1 or COX-2 pharmacological blockage. In regard to the BALB/c mice, the chemiluminescence showed that treatment with the NSAIds used were not able to reduce the oxidative stress in infected mice’s erythrocytes. This data suggests that oxidative stress producing mechanism that occurs in T. cruzi’s infection acute phase depends on the host’s capacity to respond to the infection through high production of NO and ROS. CONCLUSIONS: Our data shows that in T. cruzi infections’ initial phase there is an increase in lipid peroxidation in erythrocytes. We show that blocking COX weakens lipid peroxidation in erythrocytes at the beginning of T. cruzi infections (12th day post-infection).

Mice infected with Trypanosoma cruzi, the agent of Chagas disease, rapidly develop anemia and thrombocytopenia. These effects are partially promoted by the parasite trans-sialidase (TS), which is shed in the blood and depletes sialic acid from the platelets, inducing accelerated platelet clearance and causing thrombocytopenia during the acute phase of disease. Here, we demonstrate that oral immunization of C57BL/6 mice with Phytomonas serpens, a phytoflagellate parasite that shares common antigens with T. cruzi but has no TS activity, reduces parasite burden and prevents thrombocytopenia and leukopenia. Immunization also reduces platelet loss after intraperitoneal injection of TS. In addition, passive transfer of immune sera raised in mice against P. serpens prevented platelet clearance. Thus, oral exposure to P. serpens attenuates the progression of thrombocytopenia induced by TS from T. cruzi. These findings are not only important for the understanding of the pathogenesis of T. cruzi infection but also for developing novel approaches of intervention in Chagas disease.

A doença de Chagas, causada por Trypanosoma cruzi, é uma doença tropical negligenciada que ainda apresenta limitações quanto ao tratamento, como o aparecimento de efeitos colaterais graves e eficácia variável. Na busca por terapias alternativas, a utilização de produtos naturais e estratégias como a combinação de fármacos representam alternativas para introdução de novos medicamentos para essa doença. O trabalho teve como objetivo avaliar o óleo de copaíba e o ácido caurenóico quanto a sua atividade tripanocida e imunomoduladora em macrófagos infectados por T. cruzi, além de analisar o efeito combinatório destes produtos naturais com o benzonidazol. Ambos os compostos foram capazes de eliminar o parasito, promovendo diminuição significativa da taxa de infecção e proliferação das formas amastigotas de maneira dose dependente. A eliminação destas formas intracelulares ocorreu por mecanismos independentes da produção de espécies reativas de oxigênio e nitrogênio e das vias relacionadas ao metabolismo do ferro e defesa antioxidante. Apesar dos testes mostrarem que o óleo de copaíba e o ácido caurenóico modulam a resposta imune em macrófagos, o efeito tripanocida pode estar ocorrendo devido à ação direta destes compostos sobre o parasito, uma vez que os tratamentos exibiram um perfil anti-inflamatório. Quando os compostos foram associados ao benzonidazol observou-se efeito sinérgico contra todas as formas de T. cruzi para o óleo de copaíba e para o ácido caurenóico apenas para as formas epimastigotas e amastigotas. Em tripomastigotas o efeito da associação foi aditivo. As concentrações utilizadas nos testes mostraram efeito antagônico para células LLCMK2, não indicando, portanto, toxicidade. Além disso, os tratamentos com os compostos sozinhos ou em combinação com benzonidazol levaram a diversas alterações funcionais e estruturais nas formas epimastigotas, como aumento na produção de espécies reativas, formação de estruturas membranosas e vacuolização no interior das células, peroxidação lipídica entre outras características indicativas de processo autofágico. Portanto, nossos resultados mostram que óleo de copaíba e o ácido caurenóico são capazes de eliminar T. cruzi, modular a resposta imune e diminuir a concentração de benzonidazol necessária para causar a morte do parasito, o que poderia diminuir a toxicidade deste medicamento, sendo então, uma alternativa promissora no tratamento da doença de Chagas. Chagas disease, caused by Trypanosoma cruzi, is a neglected tropical disease that still has treatment limitations, such as the appearance of serious side effects and variable efficacy. In search for alternative therapies, the use of natural products and strategies such as drug combinations represent alternatives for introduction of new drugs for this disease. The study aimed to evaluate the copaiba oil and Kaurenoic acid as its trypanocidal and immunomodulatory activity in macrophages infected with T. cruzi, as well as analyzing the combinatorial effect of these natural products with benznidazole. Both compounds were able to eliminate the parasite, promoting a significant reduction in the rate of infection and proliferation of the amastigote forms in a dose dependent manner. The elimination these forms intracellular occurred by independent mechanisms of production of reactive oxygen species and nitrogen and pathways related to iron metabolism and antioxidant defense. Although tests showed that both copaiba oil and kaurenoic acid modulate the immune response in macrophages, the trypanocidal effect may be occurring due to the direct action of compounds on parasite, since treatments exhibited an anti-inflammatory profile. When compounds were associated with benznidazole a synergistic effect was observed against all forms of T. cruzi for copaiba oil and for kaurenoic acid, only for the epimastigote and amastigote forms. In trypomastigotes association effect was additive. The concentrations used in tests showed an antagonistic effect for LLCMK2 cells, thus not indicating toxicity for these cells. In addition, treatments with compounds alone or in combination with benzonidazole led to several functional and structural changes in epimastigote forms, such as increased production of reactive species, formation of membranous structures and vacuolization in the cells, lipid peroxidation among other indicative characteristics of autophagic process. Therefore, our results show that copaiba oil and kaurenoic acid are able to eliminate T. cruzi, modulate the immune response and decrease the concentration of benznidazole necessary to cause death of parasite, which could decrease the toxicity of this drug, and so, a promising alternative in the treatment of Chagas disease.

A Leishmaniose Tegumentar Americana (LTA) causada por protozoários do gênero Leishmania é caracterizada pelo surgimento de lesões que podem se apresentar nas formas clínicas cutânea, cutânea difusa e mucocutânea. Essas manifestações clínicas são dependentes da interação entre as espécies do parasito envolvido, bem como a resposta imune apresentada pelo hospedeiro. Em humanos, a resposta imune à infecção por Leishmania não é tão bem caracterizada como na resposta em camundongos, em virtude do envolvimento de citocinas, de moléculas co-estimulatórias, assim como da saliva do flebotomíneo, dificultando o entendimento de como ocorre a resposta imunológica por parte do hospedeiro. No entanto, os estudos sugerem que em todas as formas clínicas da LTA, a resposta imune é dependente de células T e, de maneira geral, se aceita que a diferença entre resistência e susceptibilidade à infecção por Leishmania esteja relacionada com o predomínio da resposta celular Th1 e Th2 respectivamente. Os medicamentos disponíveis para o tratamento da LTA apresentam toxicidade elevada, longo período de tratamento, aliado a relatos de pacientes que desenvolvem resistência a tais terapias, estes fatores tem promovido a busca por alternativas terapêuticas. Estudos têm demonstrado que a própolis apresenta grande potencial para o auxilio no tratamento da leishmaniose, devido à suas propriedades anti-inflamatória, imunomoduladora, antiparasitária e de reparo tecidual. Este trabalho teve como objetivo analisar, in vitro, a atividade biológica do extrato de própolis brasileira em células mononucleares do sangue periférico (PBMC) humano de pacientes com leishmaniose e doadores saudáveis. Células mononucleares do sangue periférico de pacientes e indivíduos saudáveis foram cultivadas na presença ou ausência desses compostos com ou sem infecção com L. braziliensis. O sobrenadante e o soro de pacientes e doadores saudáveis foram utilizados para dosagem de óxido nítrico e citocinas. As células aderentes tratadas com própolis foram utilizadas para marcação com imunocitoquímica. Foi demonstrado que a própolis estimula a produção de citocinas pró-inflamatórias em células de pacientes com leishmaniose sendo potencial regulador na imunossupressão induzida por Leishmania braziliensis. The American Cutaneous Leishmaniasis (ACL) caused by protozoa of the genus Leishmania is characterized by the appearance of lesions that can appear in the clinical forms: cutaneous, diffuse cutaneous and mucocutaneous. These clinical manifestations are dependent on the interaction between the species of parasite involved and the immune response provided by the host. In humans, the immune response to Leishmania infection is not as well characterized as the response in mice, because of the involvement of cytokines, co-stimulatory molecules, as well as sandfly saliva, hampering the understanding of how the immune response is by the host. However, studies suggest that in all clinical forms of ACL, the immune response is T cell-dependent and, in general, it is accepted that the difference between resistance and susceptibility to Leishmania infection is related to the cellular response predominance Th1 and Th2, respectively. Available drugs for the treatment of ACL show high toxicity, prolonged periods of treatment, along with reports of patients developing resistance to such therapies, these factors have promoted the search for alternative treatment. Studies have shown that the propolis resin produced by bees, has great potential for the development of new drugs due to their anti-inflammatory, immunomodulatory, anti-parasitic and tissue repair. This study aimed to analyze, in vitro, the biological activity of extract of Brazilian propolis in mononuclear cells from peripheral blood (PBMC) of patients with leishmaniasis and healthy donors. Peripheral blood mononuclear cells from patients and healthy individuals were cultured in the presence or absence of these compounds with or without infection with L. braziliensis. The supernatant and serum from patients and healthy donors were used for measurement of nitric oxide and cytokines. The adherent cells were treated with propolis used for labeling with immunocytochemistry. Propolis has been shown to stimulate the production of proinflammatory cytokines in cell leishmaniasis patients with potential regulator in Leishmania braziliensis immunosuppression.