Análise do padrão de resposta Th17 e de células T regulatórias em pacientes com a forma digestiva da doença de Chagas crônica

A Doença de Chagas (DC), cujo agente etiológico é o protozoário Trypanosoma cruzi (T. cruzi), tornou-se nos últimos anos um grave problema de saúde pública, mesmo em países não-endêmicos. Um importante desafio para os pesquisadores da doença de Chagas é estabelecer os eventos que levam alguns pacientes a desenvolver a doença crônica assintomática, ou forma indeterminada, enquanto outros evoluem para uma doença severa, com lesões em tecidos cardíacos ou gastrointestinais. Alguns estudos têm sugerido que a ausência de sintomas clínicos em indivíduos com a forma crônica indeterminada está associada a um controle eficaz da resposta imune efetora contra o parasita, que evita uma resposta inflamatória excessiva, o que resultaria em lesão tecidual. Não existem estudos com o objetivo de confirmar este mecanismo em pacientes com a forma digestiva da DC, especialmente em relação ao controle modulador de mecanismos efetores envolvendo células Th17, por células Treg. No presente estudo, nós avaliamos em pacientes com a forma digestiva da DC e em pacientes com a forma indeterminada ou assintomática a taxa de ativação Th17/Treg, através da avaliação da freqüência de células Th17 e Treg por citometria de fluxo; da expressão do mRNA para Rorγt e Foxp3, fatores de transcrição envolvidos na diferenciação dessas células, respectivamente; bem como os níveis de IL-17a e IL-10 em sobrenadantes de cultura de células mononucleares de sangue periférico (PBMC’s). Foi demonstrado no presente estudo, que em pacientes com a forma digestiva da DC há um desequilíbrio na taxa de ativação de células Th17/Treg, favorecendo o perfil Th17, enquanto que em pacientes com a forma indeterminada, este desequilíbrio favorece o perfil Treg. Os nossos resultados apoiam a hipótese de que o desequilíbrio na taxa de ativação Th17/Treg observado no grupo de pacientes digestivos, favorecendo Th17 em detrimento de Treg, geraria um ambiente não regulador, que resulta em uma resposta inflamatória exacerbada, responsável pelas lesões dos tecidos gastrointestinais apresentadas por estes pacientes.Chagas disease (CD) whose etiological agent is the protozoan Trypanosoma cruzi (T. cruzi) in recent years became a serious problem of public health even in nonendemic countries. One important challenge for the researchers of Chagas disease is to establish the events that lead some patients to develop asymptomatic or indeterminate chronic disease, while others undergo severe disease with lesions in cardiac or gastrointestinal tissues. Some studies have suggested that absence of clinical symptoms in individuals with the indeterminate chronic form is associated to a fine control of effector immune responses against the parasite that avoid a perpetuated inflammatory process which results in tissue injury. There are no studies aiming to confirm this mechanism in patients with digestive form of the disease, particularly in relation to the modulatory control by Treg cells of effector mechanisms involving Th17. Here, we studied in patients with the digestive form of CD and in those with the indeterminate or asymptomatic form of the disease, the ratio Th17/Treg activation by evaluating the frequency of Th17 and Tregs cells by flow cytometry, mRNA expression for Rorγt and Foxp3, the transcription factors involved in the differentiation of these cells respectively as well as the levels of IL-17a and IL-10 in peripheral blood mononuclear cells culture supernatants. We showed that in patients with digestive form of the disease there is an imbalance in the ratio Th17/Treg cells activation in favor of Th17, while in patients with the indeterminate form, this imbalance favour Treg activation. Our findings support the hypothesis that in contrast with indeterminate patients a non regulatory environment observed in patients with digestive CD, which results in an exacerbated inflammatory response exerted by Th17, due to lower Treg activation, may be responsible for tissue lesions in these patients.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Indeterminate form of chagas disease and metabolic syndrome: a dangerous combination

Chagas’ disease (CD) has been a major concern in public health in Latin America countries and in Brazil there are about 3 million people suffering from this disease. With the social and economic changes which have been occurring in the last 6 decades in the country, there have been a lot of changes in the population life style with severe metabolic consequences, especially for those with Chagas' disease. The objective of this study was to evaluate the prevalence of metabolic syndrome in individuals with the indeterminate form of CD. A total of 74 individuals, mean age of 55.6 years, participated in the study. Anthropometric and biochemical evaluations were performed. Overweight/obesity was found in 86.5 % of individuals, increased waist circumference in 72.5%, and 67% had more than 30% of fat mass. Hyperglycemia and dyslipidemia were observed in 24.3% and 75.7% of patients, respectively. Metabolic syndrome was diagnosed in 48.2% of patients. The family history revealed high prevalence of cardiovascular diseases (80.3%), systemic arterial hypertension (57.1%) and diabetes mellitus (42.8%). A total of 90% of patients were overweight/obese, and it is well known that increased adipose tissue, specially visceral adipose tissue is highly associated with dyslipidemia and cardiovascular diseases, as well as imbalance in production of proinflammatory and antiinflammatory cytokines produced by that tissue. Adipocytes are also known as a reservoir for Trypanosoma cruzi, favoring an increase in parasite load and a possible reacutization of the disease. Therefore, the study individuals are at high risk of developing cardiovascular diseases as well as further symptomatic form of the Chagas' disease, mainlychagastic cardiopathy

Biochemical and nutritional evaluation of patients with visceral leishmaniasis before and after treatment with leishmanicidal drugs

Introduction Visceral leishmaniasis (VL) is caused by the intracellular protozoan Leishmania donovani complex. VL may be asymptomatic or progressive and is characterized by fever, anemia, weight loss and the enlargement of the spleen and liver. The nutritional status of the patients with VL is a major determinant of the progression, severity and mortality of the disease, as it affects the clinical progression of the disease. Changes in lipoproteins and plasma proteins may have major impacts in the host during infection. Thus, our goal was evaluate the serum total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, glucose, albumin, globulin and total protein levels, as well as the body composition, of VL patients before and after treatment. Methods Nutritional evaluation was performed using the bioelectrical impedance analysis (BIA) to assess body composition. Biochemical data on the serum total cholesterol, HDL, LDL, triglycerides, glucose, albumin, globulin and total protein were collected from the medical charts of the patients. Results BIA indicated that both pre-treatment and post-treatment patients exhibited decreased phase angles compared to the controls, which is indicative of disease. Prior to treatment, the patients exhibited lower levels of total body water compared to the controls. Regarding the biochemical evaluation, patients with active VL exhibited lower levels of total cholesterol, HDL, LDL and albumin and higher triglyceride levels compared to patients after treatment and the controls. Treatment increased the levels of albumin and lipoproteins and decreased the triglyceride levels. Conclusions Our results suggest that patients with active VL present biochemical and nutritional changes that are reversed by treatment

The Involvement of TLR2 and TLR4 in Cytokine and Nitric Oxide Production in Visceral Leishmaniasis Patients before and after Treatment with Anti-Leishmanial Drugs - Table 1

<p>* <i>Glucantime (M-metyl-glucamine)scheme</i>: <i>20 mg/Kg/day for 20 days</i></p><p>** <i>Deoxycholate Amphotericin B scheme</i>: <i>1 mg/Kg/day for 20 days</i></p><p>*** <i>Liposomal Amphotericin B scheme</i>: <i>5 mg/Kg/day for 5 days</i></p><p>The Involvement of TLR2 and TLR4 in Cytokine and Nitric Oxide Production in Visceral Leishmaniasis Patients before and after Treatment with Anti-Leishmanial Drugs - Table 1 </p

NO production after stimulation with LPS and PGN agonists.

<p>Levels of NO in supernatants were analyzed after 24 hours of PBMCs cultured (1x10⁶ cells/ml), obtained from patients with VL pre-treatment, post-treatment and control subjects, and stimulated or not with LPS ultra-purified (1 μg/ml), PGN (5 μg/ml) and LPS+PGN. Lowercase letters represent significant differences among agonists in the same group: p<0.05 <b>a</b>—PBMC <i>vs</i>. LPS, PGN, LPS+PGN. Capital letters represent significant differences in the same agonists among groups: p<0.05 <b>A</b>—pre-treatment <i>vs</i>. post-treatment; <b>B</b>—pre-treatment <i>vs</i>. control individuals. Each dot represent a different patient and each bar represents the median. NO levels were measured by the levels of nitrite/nitrate and the data are representative of triplicates.</p

Cytokine production after stimulation with LPS and PGN agonists.

<p>Levels of TNF-α (A), IFN-γ (B), IL-17 (C), IL-10 (D) and TGF-β (E) in supernatants were analyzed after 24 hours of PBMCs cultured (1x10⁶ cells/ml), obtained from patients with VL pre-treatment, post-treatment and control subjects, and stimulated or not with LPS ultra-purified (1 μg/ml), PGN (5 μg/ml) and LPS+PGN. Lowercase letters represent significant differences among agonists in the same group: p<0.05 <b>a</b>—PBMC <i>vs</i>. LPS, PGN, LPS+PGN; <b>b</b>—PBMC, PGN <i>vs</i>. LPS, LPS+PGN; <b>c</b>—PBMC, LPS <i>vs</i>. PGN, LPS+PGN; <b>d</b>—LPS <i>vs</i>. LPS+PGN. Capital letters represent significant differences in the same agonists among groups: p<0.05 <b>A</b>—pre-treatment <i>vs</i>. post-treatment; <b>B</b>—pre-treatment <i>vs</i>. control individuals; <b>C</b>—post-treatment <i>vs</i>. control individuals. Cytokine levels were measured by CBA. Each dot represent a different patient and each bar represents the median.</p

Expression of surface TLR2 and TLR4.

<p>Frequency of CD3⁺ and CD14⁺ cells expressing TLR2, TLR4 and co-expressing TLR2 and TLR4 (A) and mean of fluorescence intensity (B) of TLR2 and TLR4 in CD3⁺ and CD14⁺ cells in whole blood from patients with visceral leishmaniasis pre-treatment, post-treatment and control subjects. Gating strategies to distinguish between cell populations analyzed by flow cytometry. FSC-SSC profile was used to distinguish total lymphocytes and monocytes and these subtype cells were gated according to light scatter profile and the expression of CD3 or CD14. Representative histograms plots of TLR2 and TLR4 expression in CD3⁺ (C,D) and CD14⁺ (E,F) cells. The results are expressed as the mean and standard deviation. *p<0.05 compared pre-treatment <i>vs</i>. post-treatment and control subjects; ▪ p<0.05 compared pre-treatment <i>vs</i>. control subjects and • p<0.05 compared post-treatment <i>vs</i>. control subjects.</p