Perfil clínico e epidemiológico de pacientes idosos com doença de Chagas atendidos entre 2005-2013 por um serviço de atenção farmacêutica no estado do Ceará, nordeste do Brasil

Controlando-se a transmissão da doença de Chagas, surge o desafio de prestar assistência a milhões de pacientes infectados que chegam à velhice. Neste estudo, foram avaliados os registros socioeconômicos, demográficos e de comorbidades de todos os pacientes chagásicos idosos acompanhados no Serviço de Atenção Farmacêutica do Laboratório de Pesquisa em Doença de Chagas. As informações relacionadas à forma clínica da doença foram obtidas a partir de registros médicos disponibilizados pelo Hospital Universitário Walter Cantídio. O perfil da população estudada foi de: mulheres (50,5%); idade média de 67 anos; aposentados (54,6%); casados (51,6%); alta taxa de analfabetismo (40,2%); e renda familiar de um salário mínimo (51,5%). As formas clínicas predominantes da doença de Chagas foram a cardíaca (65,3%) e a indeterminada (14,7%). As principais alterações eletrocardiográficas foram o bloqueio de ramo direito (41,0%), associado ou não ao bloqueio ântero superior esquerdo (27,4%). O número médio de comorbidades por paciente foi de 2,23 ± 1,54, sendo a hipertensão arterial sistêmica a principal encontrada (67,0%). Verificou-se que os idosos constituem grupo vulnerável de pacientes que associam o envelhecimento com as alterações cardíacas e/ou digestivas resultantes da evolução da doença de Chagas e outras comorbidades, o que exige atenção especial dos serviços de saúde para um atendimento médico e social mais adequado.By controlling the transmission of Chagas disease, the challenge of providing assistance to millions of infected patients that reach old age arises. In this study, the socioeconomic, demographic and comorbidity records of all elderly chagasic patients followed at the Pharmaceutical Care Service of the Chagas Disease Research Laboratory were assessed. The information related to the clinical form of the disease was obtained from medical records provided by the Walter Cantídio University Hospital. The profile of the studied population was: women (50.5%); mean age of 67 years; retired (54.6%); married (51.6 %); high illiteracy rate (40.2%); and family income equal to the minimum wage (51.5%). The predominant clinical forms of Chagas disease were cardiac (65.3%) and indeterminate (14.7%). The main electrocardiographic changes were the right bundle branch block (41.0%), associated or not with the anterosuperior left bundle branch block (27.4%). The average number of comorbidities per patient was 2.23 ± 1.54, with systemic arterial hypertension being the main one found (67.0%). It was found that the elderly comprise a vulnerable group of patients that associate aging with cardiac and/or digestive disorders resulting from the evolution of Chagas disease and other comorbidities, which requires special attention from health services to ensure more appropriate medical and social care

Humoral and Cellular Immune Responses to Trypanosoma cruzi-Derived Paraflagellar Rod Proteins in Patients with Chagas' Disease

Sera and peripheral blood mononuclear cells (PBMC) from patients displaying different clinical symptoms as well as from normal uninfected individuals (NI) were used to evaluate the humoral and cellular responses of Chagas' disease patients to Trypanosoma cruzi-derived paraflagellar rod proteins (PFR). Our results show that sera from both asymptomatic Chagas' disease patients (ACP) and cardiac Chagas' disease patients (CCP) have higher levels of antibodies to PFR than sera from NI. Immunoglobulin G1 (IgG1) and IgG3 were the main Ig isotypes that recognized PFR. We also tested three recombinant forms of PFR, named rPAR-1, rPAR-2, and rPAR-3, by Western blot analysis. Sera from seven out of eight patients with Chagas' disease recognized one of the three rPAR forms. Sera from 75, 50, and 37.5% of Chagas' disease patients tested recognized rPAR-3, rPAR-2, and rPAR-1, respectively. PFR induced proliferation of 100 and 70% of PBMC from ACP and CCP, respectively. Further, stimulation of cells from Chagas' disease patients with PFR enhanced the frequencies of both small and large CD4(+) CD25(+) and CD4(+) CD69(+) lymphocytes, as well as that of small CD8(+) CD25(+) lymphocytes. Finally, we evaluated the ability of PFR to elicit the production of gamma interferon (IFN-γ) by PBMC from patients with Chagas' disease. Fifty percent of the PBMC from ACP as well as CCP produced IFN-γ upon stimulation with PFR. PFR enhanced the percentages of IFN-γ-producing cells in both CD3(+) and CD3(−) populations. Within the T-cell population, large CD4(+) T lymphocytes were the main source of IFN-γ

Humoral and cellular immune responses to Trypanosoma cruzi-derived paraflagellar rod proteins in patients with Chagas' disease.

Sera and peripheral blood mononuclear cells (PBMC) from patients displaying different clinical symptoms as well as from normal uninfected individuals (NI) were used to evaluate the humoral and cellular responses of Chagas' disease patients to Trypanosoma cruzi-derived paraflagellar rod proteins (PFR). Our results show that sera from both asymptomatic Chagas' disease patients (ACP) and cardiac Chagas' disease patients (CCP) have higher levels of antibodies to PFR than sera from NI. Immunoglobulin G1 (IgG1) and IgG3 were the main Ig isotypes that recognized PFR. We also tested three recombinant forms of PFR, named rPAR-1, rPAR-2, and rPAR-3, by Western blot analysis. Sera from seven out of eight patients with Chagas' disease recognized one of the three rPAR forms. Sera from 75, 50, and 37.5% of Chagas' disease patients tested recognized rPAR-3, rPAR-2, and rPAR-1, respectively. PFR induced proliferation of 100 and 70% of PBMC from ACP and CCP, respectively. Further, stimulation of cells from Chagas' disease patients with PFR enhanced the frequencies of both small and large CD4(+) CD25(+) and CD4(+) CD69(+) lymphocytes, as well as that of small CD8(+) CD25(+) lymphocytes. Finally, we evaluated the ability of PFR to elicit the production of gamma interferon (IFN-gamma) by PBMC from patients with Chagas' disease. Fifty percent of the PBMC from ACP as well as CCP produced IFN-gamma upon stimulation with PFR. PFR enhanced the percentages of IFN-gamma-producing cells in both CD3(+) and CD3(-) populations. Within the T-cell population, large CD4(+) T lymphocytes were the main source of IFN-gamma

Deletion of one <i>LMIT1</i> allele impairs temperature/pH-induced differentiation of promastigotes into axenic amastigotes.

<p>Late-log phase promastigotes were washed, resuspended in pH 4.5 at 2x10⁵ parasites/ml, and cultured at 32°C. (A) Numbers of wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1)</i> parasites following shift to amastigote medium. The data represent the mean +/- SD of triplicate determinations and are representative of four independent experiments. (B–D) wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) parasites incubated for 48 h in amastigote medium at 32°C were subjected to: (B) Quantification of viable rounded forms with a short flagellum. At least 400 FDA-stained parasites were counted microscopically in each sample. The data represent the mean ± SD of quadruplicate determinations. (C) SEM analysis of the morphology of parasites. Bars = 2 μm. (D) Determination of cell viability and membrane integrity by staining with FDA (green) and PI (red). Bars = 27 μm. (E) The iron content of whole cells and mitochondrial fractions was determined in parasites collected 24 h after induction of axenic differentiation (pH 4.5/ 32°C). Data represents the mean ± SD of three independent experiments (Student’s <i>t</i> test *p = 0.037, **<i>p</i> = 0.004). (F) Aconitase activity was determined in mitochondrial fractions from parasites collected 24 h after induction of axenic differentiation (pH 4.5/ 32°C). The data represent the mean ± SD of three independent experiments (**p≤ 0.008). (G) Determination of mitochondrial membrane potential (ΔΨ_m) with JC-1 with and without prior treatment with the mitochondrial uncoupler CCCP. The data represents the mean ± SD of three independent experiments (Student’s <i>t</i> test * <i>p</i> = 0.01; **<i>p</i> = 0.002). ((H) Viable amastigotes obtained by temperature/pH- induced axenic differentiation in wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) cultures were tested for their ability to infect BMMs. BMMs were infected and either fixed immediately or after further incubation for 24, 48 or 72 h and the number of intracellular parasites was determined microscopically. The data represent the mean ± SD of triplicate determinations and are representative of more than three independent experiments.</p

<i>LMIT1</i> single knockout parasites induced to differentiate axenically into amastigotes show reduced FeSOD activity and mitochondrial abnormalities.

<p>(A) Log phase (~2x10⁷/ml) wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) parasites grown in regular promastigote medium (pH 7.4 / 26°C) were transferred to amastigote media (pH 4.5) and incubated at an elevated temperature (32°C) for 72 h, followed by determination of SOD activity in whole cell extracts. The data represents the mean ± SD of triplicate determinations and are representative of three independent experiments. (Student’s <i>t</i> test compared to <i>WT</i>: 24 h **<i>p</i> = 0.013, 48 h ***<i>p</i> = 0.0097 and *<i>p</i> = 0.093, 72 h ***<i>p</i> = .0.008 and **<i>p</i> = 0.026). (B) TEM micrographs of wild type (WT) and single knockout (<i>LMIT1/Δlmit1</i>) parasites incubated in amastigote media at 32°C for 48 h. (a-c) WT; (d-h) <i>LMIT1/Δlmit1</i>. m, mitochondria; k, kinetoplast; white arrows, normal mitochondrial cristae; black arrows, aggregates inside mitochondria; black arrowheads, enlarged mitochondria. Bars = 1 μm.</p

<i>LMIT1</i> single knockout metacyclic forms are strongly impaired in virulence for mice.

<p>(A) C57BL/6 female mice were inoculated with 1x10⁶ wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) or complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) purified metacyclic promastigotes in the left hind footpad and lesion development was measured weekly. The data represent the mean ± SD of 5 mice. (B) The parasite load in footpad tissues was determined 11 weeks after infection (n = 5). **, <i>p</i> = 0.009; *, <i>p</i> = 0.167 (Student’s t test). Relative levels of episomally expressed LMIT1-HA protein was determined in western blots using 10 μg of whole cell extracts prepared from <i>LMIT1/Δlmit1+LMIT1</i> parasites prior to footpad injection (injected) or recovered from lesions post-sacrifice (recovered) and detected with anti-HA antibody. (C) Balb/c female mice were inoculated with single knockout (<i>LMIT1/Δlmit1</i>) or complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) purified metacyclic promastigotes in the left hind footpad and lesion development was measured weekly. The data represent the mean ± SD of 5 mice. (D) Parasite load in footpad tissues was determined 12 weeks after infection (n = 5). **, <i>p</i> = 0.007 (Student’s t test).</p

Deletion of one <i>LMIT1</i> allele impairs <i>L</i>. <i>amazonensis</i> promastigote growth, enhances sensitivity to ROS and inhibits amastigote generation triggered by iron deprivation.

<p>(A) Growth curves of wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1)</i> promastigotes in regular growth medium. (B) The iron content in whole cells and mitochondrial fractions was determined in 4 day-old cultures of wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) promastigotes. The data represent the mean ± SD of three independent experiments. (C) Effect of menadione, an inducer of superoxide generation, on the survival of wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1</i>) promastigotes. The parasites were cultured for 48 h in the presence increasing concentrations of mitochondrial superoxide generator menadione and the number of viable cells was determined after staining with FDA. The data expressed as percentage of the number of viable cells in cultures without menadione, represent the mean ± SD of triplicate determinations and are representative of three independent experiments. (D) Growth curves of wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1)</i> promastigotes in iron-depleted medium. (E) Fraction of wild type (WT), single knockout (<i>LMIT1/Δlmit1</i>) and complemented single knockout (<i>LMIT1/Δlmit1+LMIT1)</i> parasites with a rounded morphology and short flagellum after 48 h of culture in iron-depleted medium.</p