Increase of reactive oxygen species by desferrioxamine during experimental Chagas' disease.

Oxidative stress is common in inflammatory processes associated with many diseases including Chagas' disease. The aim of the present study was to evaluate, in a murine model, biomarkers of oxidative stress together with components of the antioxidant system in order to provide an overview of the mechanism of action of the iron chelator desferrioxamine (DFO). The study population comprised 48 male Swiss mice, half of which were treated daily by intraperitoneal injection of DFO over a 35-day period, while half were administered sterile water in a similar manner. On the 14th day of the experiment, 12 DFO-treated mice and an equal number of untreated mice were experimentally infected with Trypanosoma cruzi. Serum concentrations of nitric oxide and superoxide dismutase and hepatic levels of total glutathione, thiobarbituric acid reactive species and protein carbonyl, were determined on days 0, 7, 14 and 21 post-infection. The results obtained revealed that DFO enhances antioxidant activity in the host but also increases oxidative stress, indicating that the mode of action of the drug involves a positive contribution to the host together with an effect that is not beneficial to the parasite

Trypanosoma cruzi: desferrioxamine decreases mortality and parasitemia in infected mice through a trypanostatic effect.

Desferrioxamine (DFO) is a potent iron chelator that is also known to modulate inflammation and act as an efficient antioxidant under normal conditions and under oxidative stress. Many in vitro and in vivo studies have shown the efficacy of DFO in the treatment of viral, bacterial and protozoan infections. DFO is known to reduce the intensity of Trypanosoma cruzi infections in mice even during a course of therapy that is not effective in maintaining anaemia or low iron levels. To further clarify these findings, we investigated the action of DFO on mouse T. cruzi infection outcomes and the direct impact of DFO on parasites. Infected animals treated with DFO (5 mg/animal/day) for 35 days, beginning 14 days prior to infection, presented lower parasitemia and lower cumulative mortality rate. No significant effect was observed on iron metabolism markers, erythrograms, leukograms or lymphocyte subsets. In the rapid method for testing in vivo T. cruzi susceptibility, DFO also induced lower parasitemia. In regard to its direct impact on parasites, DFO slightly inhibited the growth of amastigotes and trypomastigotes in fibroblast culture. Trypan blue staining showed no effects of DFO on parasite viability, and only minor apoptosis in trypomastigotes was observed. Nevertheless, a clear decrease in parasite mobility was detected. In conclusion, the beneficial actions of DFO on mice T. cruzi infection seem to be independent of host iron metabolism and free of significant haematological side effects. Through direct action on the parasite, DFO has more effective trypanostatic than trypanocidal properties

Coinfection with Different Trypanosoma cruzi Strains Interferes with the Host Immune Response to Infection

A century after the discovery of Trypanosoma cruzi in a child living in Lassance, Minas Gerais, Brazil in 1909, many uncertainties remain with respect to factors determining the pathogenesis of Chagas disease (CD). Herein, we simultaneously investigate the contribution of both host and parasite factors during acute phase of infection in BALB/c mice infected with the JG and/or CL Brener T. cruzi strains. JG single infected mice presented reduced parasitemia and heart parasitism, no mortality, levels of pro-inflammatory mediators (TNF-α, CCL2, IL-6 and IFN-γ) similar to those found among naïve animals and no clinical manifestations of disease. On the other hand, CL Brener single infected mice presented higher parasitemia and heart parasitism, as well as an increased systemic release of pro-inflammatory mediators and higher mortality probably due to a toxic shock-like systemic inflammatory response. Interestingly, coinfection with JG and CL Brener strains resulted in intermediate parasitemia, heart parasitism and mortality. This was accompanied by an increase in the systemic release of IL-10 with a parallel increase in the number of MAC-3+ and CD4+ T spleen cells expressing IL-10. Therefore, the endogenous production of IL-10 elicited by coinfection seems to be crucial to counterregulate the potentially lethal effects triggered by systemic release of pro-inflammatory mediators induced by CL Brener single infection. In conclusion, our results suggest that the composition of the infecting parasite population plays a role in the host response to T. cruzi in determining the severity of the disease in experimentally infected BALB/c mice. The combination of JG and CL Brener was able to trigger both protective inflammatory immunity and regulatory immune mechanisms that attenuate damage caused by inflammation and disease severity in BALB/c mice

Influência da utilização da desferrioxamina, quelante de ferro, sobre o curso da infecção pelo Trypanosoma cruzi em camundongos.

Utilizaram-se camundongos Swiss machos com trinta dias de idade, divididos em quatro grupos experimentais: (1) controle não-tratado - CNT; (2) controle tratado - CT; (3) infectado com a cepa Y do T. cruzi e não-tratado - INT e (4) infectado com a cepa Y do T. cruzi e tratado - IT. Os animais tratados receberam 5mg/animal/dia de Desferrioxamina (DFA) durante os 14 dias que precederam a infecção (500 formas sanguíneas da cepa Y do T. cruzi via intraperitoneal). Após a infecção, o grupo IT recebeu DFA por mais 21 dias. Nos grupos infectados, tratados ou não, avaliaram-se a curva de parasitemia diária, período pré-patente, patente, pico de parasitemia, dia do pico de parasitemia, taxa de mortalidade, e nos animais que sobreviveram à fase aguda da infecção, hemocultura, PCR e ELISA. Nos quatro grupos experimentais foram realizadas a dosagem de ferro no fígado, ferro sérico e hemoglobina. Avaliou -se também o peso dos animais, o peso relativo do coração, fígado, baço e linfonodo e as alterações histopatológicas, bem como o parasitismo tecidual nestes órgãos. Os animais foram necropsiados no 14 o e 21 o dia após a infecção (DAI). No grupo IT, a média dos níveis de parasitemia, a taxa de mortalidade apresentaram-se menores em relação ao grupo INT. Não foram observadas diferenças significativas no período pré patente. O pico de parasitemia no grupo INT foi no 11 o dia e no grupo IT no 10 o . O período patente no grupo INT (11 dias) foi significativamente menor que no grupo IT (21 dias). Cinco animais do grupo IT e 1 animal do grupo INT sobreviveram à infecção. Todos os animais tratados apresentaram hemocultura negativa na fase aguda e crônica da infecção enquanto o animal não-tratado apresentou hemocultura positiva nas duas fases. A PCR apresentou-se positiva em todos os animais tratados avaliados aos 60 DAI e 3 destes apresentaram PCR negativa aos 240 DAI. O animal infectado e nãotratado apresentou PCR positiva aos 60 e 240. Todos os animais apresentaram ELISA positiva aos 60 e 240 DAI. Os animais infectados apresentaram menores níveis de ferro no fígado que os animais não-infectados no 14 o e 21 o DAI. Os animais do grupo INT apresentaram maiores concentrações de ferro sérico quando comparados aos animais dos grupos CNT e IT no 21 o DAI. No 14 o DAI, os animais do grupo IT apresentaram níveis mais baixos de hemoglobina quando comparados ao grupo CT. O tratamento com a DFA reduziu o peso corporal dos animais infectados ou não. Entre o 14 o e 21 o DAI, observou-se aumento de peso relativo do coração, baço, fígado e linfonodo apenas no grupo INT. Os animais infectados, tratados ou não apresentaram alterações histológicas semelhantes no coração. O fígado dos animais do grupo IT, apresentou menor intensidade de alterações e a avaliação dos órgãos linfóides demonstrou resposta mais precoce no grupo IT . Não foram observadas diferenças em relação ao parasitismo tecidual nos órgãos analizados dos animais infectados, tratados ou não com DFA. Estes resultados demonstram que a diminuição dos níveis de ferro do hospedeiro contribui para a melhora do quadro clínico da infecção.Male Swiss mice with thirty days of age, were divided in four experimental groups: (1) no-treated control - NTC; (2) treated control - TC; (3) no-treated and infected with Y strain of T. cruzi - NTI; (4) treated and infected with Y strain of T. cruzi - TI. Animals treated received daily doses (5mg/animal/day) of desferrioxamine (DF) during 14 days before the infection (500 blood forms of Y strain of T. cruzi via intraperitoneal). After infection, treated groups received DF for more 21 days. In TI and NTI groups parasitaemia, prepatent period, patent period, day of maximum parasitemia mortality were evaluated daily. Hemoculture, PCR and ELISA were performed in animals that had survived to the acute phase. In all groups iron evaluations were performed by means of the dosage of iron levels in the liver, hemoglobin and serum iron. One also evaluated the weight of the animals, the relative weight and the histopathological alterations, as well as tissue parasitism in the heart, liver, spleen and lymph nodes. These animals had been sacrificed in 14 o and 21 o days after the infection. Treatment produced a less severe disease. All treated animals that had survived to the acute phase of the infection presented negative hemoculture in the acute and chronic phase of the infection while no treated present positive hemoculture in both phases. All treated animals presented positive PCR assay in the acute phase and 3 of these presented negative PCR in the chronic phase. No treated animal presented positive PCR in both, acute and chronic phase. Treated and no treated animals presented positive ELISA in the acute and chronic phase. Infected groups presented minors levels of iron in the liver when compared to no infected groups, treated or no treated. NTI group presented greater levels of serum iron when compared to NTC and TI in 21 o days after infection. In 14 o day after infection, the TI presented lower levels of hemoglobin than NTI group. DF treatment diminished the corporal weight of the infected animals or not. Between 14 o and 21 o day after infection, were observed increase of relative weight of the heart, spleen, liver and lymph nodes only in the NTI group. The infected, treated or no treated animals presented similar histological alterations in the heart. The liver of the animals of IT group, presented minor intensity of alterations and the evaluation of the lymphatic organs demonstrated precocious reply in IT group. Differences in relation to tissue parasitism in the analyzed organs of the infected treated or no treated animals had not been observed. These results demonstrate that the reduction of the host iron levels contributes for the improvement of the clinical picture of the infection

Trypanosoma cruzi : treatment with the iron chelator desferrioxamine reduces parasitemia and mortality in experimentally infected mice.

The effects of prolonged treatment with iron chelator (desferrioxamine) on the development of infection in mice inoculated with Y Trypanosoma cruzi were determined. Infected/treated mice presented lower levels of parasitemia and reduced mortality rate compared with infected/non-treated animals. The five out of twenty infected/treated mice that survived the acute phase of infection showed negative hemoculture and positive ELISA in the acute and chronic phases and positive PCR in the acute phase: in the chronic phase, three of the animals presented negative PCR. The single surviving infected/non-treated animal exhibited positive hemoculture, PCR and ELISA in both phases of infection. Infected groups presented lower levels of iron in the liver compared with treated/non-infected or non-treated/ non-infected animals. The serum iron levels of the infected/non-treated group were higher on the 21st day post-infection in comparison with control and infected/treated groups. These results suggest that decrease of iron in the host leads to T. cruzi infection attenuation

Trypanosoma cruzi : effect of benznidazole therapy combined with the iron chelator desferrioxamine in infected mice.

Iron chelators have been employed in various studies aimed at evaluating the relationship between the iron status of the host and the development of infection. In the present study, the effects of benznidazole (BZ) therapy in combination with the iron chelator desferrioxamine (DFO) on the development of infection in mice inoculated with Trypanosoma cruzi Y strain have been investigated. Infected mice treated with DFO presented lower levels of parasitemia compared with infected untreated animals. Therapy with BZ for 21 days, with or without DFO, led to decreased parasitemia and reduced mortality, but BZ in combination with DFO treatment for 35 days (BZ/DFO-35) gave 0% mortality. All infected groups presented lower levels of iron in the liver, but serum iron concentrations were greater in DFO-35 and BZ/DFO-35, whereas hemoglobin levels were higher in BZ/DFO-35 and lower in DFO-35 compared with other treated groups. The percentage cure, determined from negative hemoculture and PCR results in animals that had survived for 60 days post-infection, was 18% for BZ and BZ/DFO-35, 42% for BZ combined with DFO for 21 days, and 67% for DFO-35. The results demonstrate that modification in iron stores increases BZ efficacy

Trypanosoma cruzi : desferrioxamine decreases mortality and parasitemia in infected mice through a trypanostatic effect.

Desferrioxamine (DFO) is a potent iron chelator that is also known to modulate inflammation and act as an efficient antioxidant under normal conditions and under oxidative stress. Many in vitro and in vivo studies have shown the efficacy of DFO in the treatment of viral, bacterial and protozoan infections. DFO is known to reduce the intensity of Trypanosoma cruzi infections in mice even during a course of therapy that is not effective in maintaining anaemia or low iron levels. To further clarify these findings, we investigated the action of DFO on mouse T. cruzi infection outcomes and the direct impact of DFO on parasites. Infected animals treated with DFO (5 mg/animal/day) for 35 days, beginning 14 days prior to infection, presented lower parasitemia and lower cumulative mortality rate. No significant effect was observed on iron metabolism markers, erythrograms, leukograms or lymphocyte subsets. In the rapid method for testing in vivo T. cruzi susceptibility, DFO also induced lower parasitemia. In regard to its direct impact on parasites, DFO slightly inhibited the growth of amastigotes and trypomastigotes in fibroblast culture. Trypan blue staining showed no effects of DFO on parasite viability, and only minor apoptosis in trypomastigotes was observed. Nevertheless, a clear decrease in parasite mobility was detected. In conclusion, the beneficial actions of DFO on mice T. cruzi infection seem to be independent of host iron metabolism and free of significant haematological side effects. Through direct action on the parasite, DFO has more effective trypanostatic than trypanocidal properties

Flow cytometry analysis of splenic MAC-3⁺ and NK cells from BALB/c mice infected with JG and/or CL Brener.

<p>Groups of mice were infected with 100 trypomastigotes of JG or CL Brener (single infection) or coinfected with 100 trypomastigotes of both <i>T. cruzi</i> populations in a 1∶1 ratio via the intraperitoneal route, and flow cytometry analysis of splenic MAC-3⁺ and NK cells was performed at 7, 14 and 21 days p.i. Symbols as follows: (A) TNF-α⁺/MAC-3⁺; (B) TNF-α⁺/NK; (C) IL-10⁺/MAC-3⁺; white bar: Naïve mice; light gray bar: JG infected mice; black bar: CL Brener infected mice; and dark gray bar: JG and CL Brener infected mice. Values are expressed as the mean ± SEM of three mice per group (representative of two independent experiments). *, † and ‡ represent <i>P</i><0.05 when compared with naïve, JG and CL Brener mouse groups, respectively.</p

Representative flow cytometry charts illustrating the cytokine synthesis by spleen MAC-3⁺ and CD4⁺ T cells from BALB/c mice infected with JG and/or CL Brener.

<p>Results are presented in density plot format. The analyses were performed by quadrant statistics expressed as the percentage of cytokine⁺ cells within gated MAC-3⁺ at 14 days p.i., and CD4⁺ cells at 14 and 21 days p.i. in splenocytes cultures from naïve, JG single infected, CL Brener single infected and JG and CL Brener coinfected mice. Cytokine flow cytometry charts demonstrate the enhanced percentage of cytokine⁺ cells in all infected mice. Outstanding levels of TNF-α-producing CD4⁺ T cells were contra balanced by high frequency of IL-10-producing MAC-3⁺ and CD4⁺ T cells in coinfected mice.</p

Flow cytometry analysis of splenic CD4⁺ T and CD8⁺ T cells from BALB/c mice infected with JG and/or CL Brener.

<p>Groups of mice were infected with 100 trypomastigotes of JG or CL Brener (single infection) or coinfected with 100 trypomastigotes of both <i>T. cruzi</i> populations in a 1∶1 ratio via the intraperitoneal route, and flow cytometry analysis of splenic CD4⁺ T and CD8⁺ T cells were performed at 7, 14 and 21 days p.i. Symbols as follow: (A) TNF-α⁺/CD4⁺; (B) TNF-α⁺/CD8⁺; (C) IL-10⁺/CD4⁺; (D) IL-10⁺/CD8⁺; white bar: Naïve mice; light gray bar: JG infected mice; black bar: CL Brener infected mice; and dark gray bar: JG and CL Brener infected mice. Values are expressed as the mean ± SEM of three mice per group (representative of two independent experiments). *, † and ‡ represent <i>P</i><0.05 when compared with naïve, JG and CL Brener mice groups, respectively.</p