This GomesdosSantos_Watanabe_2020.txt file was generated on 2021-01-26 by ELIESER HITOSHI WATANABE GENERAL INFORMATION 1. Title of Dataset: GomesdosSantos_Watanabe_2020_FrontImmunol 2. Author Information A. Principal Investigator Contact Information Name: ELIESER HITOSHI WATANABE Institution: HOSPITAL DAS CLÍNICAS DA FACULDADE DE MEDICINA DA UNIVERSIDADE DE SÃO PAULO Address: AV DR ARNALDO, 455, CERQUEIRA CÉSAR, SÃO PAULO, SP, BRASIL Email: elieserhw@yahoo.com B. Associate or Co-investigator Contact Information Name: MARIA APARECIDA YASUDA Institution: HOSPITAL DAS CLÍNICAS DA FACULDADE DE MEDICINA DA UNIVERSIDADE DE SÃO PAULO Address: AV DR ARNALDO, 455, CERQUEIRA CÉSAR, SÃO PAULO, SP, BRASIL Email: masyasuda@yahoo.com.br C. Alternate Contact Information Name: LUIZ FERNANDO ONUCHIC Institution: HOSPITAL DAS CLÍNICAS DA FACULDADE DE MEDICINA DA UNIVERSIDADE DE SÃO PAULO Address: AV DR ARNALDO, 455, CERQUEIRA CÉSAR, SÃO PAULO, SP, BRASIL Email: lonuchic@usp.br 3. Date of data collection: 2015-2019 4. Geographic location of data collection: Brazil 5. Information about funding sources that supported the collection of the data: This work was supported by São Paulo Research Foundation (FAPESP) [#12/50273-0]; and by Conselho Nacional de Desenvolvimento Científico e Tecnológico - Programa Institucional de Bolsas de Iniciação Científica (CNPq - PIBIC) [AGS #100793/2016-9 from January to July 2016], and JOR # 157966/2015-1 from September to December 2015]; and by FAPESP (Technical Training Scholarship [CSO # 2014/07100-3 from May of 2014 to March 2015] and Secretary of Administration of São Paulo State Secretary [DTF # from March of 2014 to February of 2016], and for IL18 analyses by São Paulo Research Foundation (FAPESP) [#19/06363-4]. SHARING/ACCESS INFORMATION 1. Licenses/restrictions placed on the data: 2. Links to publications that cite or use the data: https://doi.org/10.3389/fimmu.2020.521409 3. Links to other publicly accessible locations of the data: 4. Links/relationships to ancillary data sets: 5. Was data derived from another source? no A. If yes, list source(s): 6. Recommended citation for this dataset: AUTHOR=Gomes dos Santos Alexandra, Watanabe Elieser Hitoshi, Ferreira Daiane Tomomi, Oliveira Jamille, Nakanishi Érika Shimoda, Oliveira Claudia Silva, Bocchi Edimar, Novaes Cristina Terra Gallafrio, Cruz Fatima, Carvalho Noemia Barbosa, Sato Paula Keiko, Yamashiro-Kanashiro Edite Hatsumi, Pontillo Alessandra, de Freitas Vera Lucia Teixeira, Onuchic Luiz Fernando, Shikanai-Yasuda Maria Aparecida TITLE=A Specific IL6 Polymorphic Genotype Modulates the Risk of Trypanosoma cruzi Parasitemia While IL18, IL17A, and IL1B Variant Profiles and HIV Infection Protect Against Cardiomyopathy in Chagas Disease JOURNAL=Frontiers in Immunology VOLUME=11 YEAR=2020 PAGES=2682 URL=https://www.frontiersin.org/article/10.3389/fimmu.2020.521409 DOI=10.3389/fimmu.2020.521409 ISSN=1664-3224 ABSTRACT=BackgroundChagas disease caused by Trypanosoma cruzi (T. cruzi) affects approximately six million individuals worldwide. Clinical manifestations are expected to occur due to the parasite persistence and host immune response. Herein we investigated potential associations between IL1B, IL6, IL17A, or IL18 polymorphism profiles and cardiomyopathy or T. cruzi parasitemia, as well as the impact of HIV infection on cardiopathy.MethodsTwo hundred twenty-six patients and 90 control individuals were analyzed. IL1B rs1143627 T>C, IL6 rs1800795 C>G, IL17A rs2275913 G>A, IL18 rs187238 C>G, and IL18 rs1946518 C>A SNVs were analyzed by real-time PCR and T. cruzi parasitemia by PCR.ResultsOur data revealed association between a cytokine gene polymorphism and parasitemia never previously reported. The IL6 rs1800795 CG genotype lowered the risk of positive parasitemia (OR = 0.45, 95% CI 0.24–0.86, P = 0.015). Original findings included associations between IL17A rs2275913 AA and IL18 s1946518 AA genotypes with decreased risk of developing cardiomyopathy (OR = 0.27, 95% CI 0.07–0.97, P = 0.044; and OR = 0.35, 95% CI 0.14–0.87, P = 0.023, respectively). IL18 rs1946518 AA and IL1B rs1143627 TC were associated with reduced risk for cardiomyopathy severity, including NYHA (New York Heart Association) class ≥ 2 (OR = 0.21, 95% CI 0.06–0.68, P = 0.009; and OR = 0.48, 95% CI 0.24–0.95, P = 0.036, respectively) and LVEF (left ventricular ejection fraction) <45% for IL18 rs1946518 AA (OR = 0.22, 95% CI 0.05–0.89, P = 0.034). A novel, unexpected protective effect of HIV infection against development/progression of cardiomyopathy was identified, based on a lower risk of developing cardiopathy (OR = 0.48, 95% CI 0.23–0.96, P = 0.039), NYHA class ≥ 2 (OR = 0.15, 95% CI 0.06–0.39, P < 0.001), and LVEF < 45% (OR = 0.03, 95% CI 0.00–0.25, P = 0.001). Digestive involvement was negatively associated with NYHA ≥ 2 and LVEF < 45% (OR = 0.20, 95% CI 0.09–0.47, P < 0.001; and OR = 0.24, 95% CI 0.09–0.62, P = 0.004, respectively).ConclusionsOur data support a protective role of IL17A AA, IL18 AA, and IL1B TC genotypes against development/progression of cardiomyopathy and a modulatory effect of the IL6 CG genotype on the risk of parasitemia in Chagas disease. Notably, HIV infection was shown to protect against development/progression of cardiopathy, potentially associated with a synergistic effect of HIV and highly active antiretroviral therapy (HAART), attenuating a Th1-mediated response in the myocardium. This proposed hypothesis requires confirmation, however, in larger and more comprehensive future studies. DATA & FILE OVERVIEW 1. File List: GomesdosSantos_Watanabe_2020_FrontImmunol 2. Relationship between files, if important: 3. Additional related data collected that was not included in the current data package: 4. Are there multiple versions of the dataset? yes A. If yes, name of file(s) that was updated:REPOSITORY_Chagas_Sep2020 i. Why was the file updated? include a README file with your data set that defines the variables and allowable values ii. When was the file updated? 2021-01-27 METHODOLOGICAL INFORMATION 1. Description of methods used for collection/generation of data: Study Subjects Our cohort included 206 patients with Chagas disease: 78 from the Heart Institute of Hospital das Clínicas da Faculdade de Medicina, University of São Paulo (HCFMUSP), and 129 from the Infectious Diseases Division of HCFMUSP, 49 of whom with HIV co-infection. These 49 cases were followed at Serviço de Extensão e Atendimento aos Paciente com Infecção por HIV/Aids of the same Division. All participants were adults. The diagnosis of trypanosomiasis was based on the 2nd Brazilian Guidelines for Chagas Diseases (1). Two positive tests of the following ones, therefore, were used to establish the diagnosis: ELISA—enzyme linked immunosorbent assay, indirect immunofluorescence, and hemagglutination (47). All patients from the Infectious Diseases Division with positive epidemiology were sent to the HIV/Aids Clinic, where HIV infection was confirmed by ELISA and immunoblot (31). Patients from the Heart Institute were tested for HIV as previously described (31). When included in the current protocol, 26 of the 49 HIV patients were not on regular HAART therapy or had therapeutic failure due to antiretroviral resistance (three patients). In 23 of them, the median viral load was 14,000 (2,618–100,000, 25th–75th percentiles) DNA viral copies/µl while the median CD4 count was 340 (93–510) cells/mm3. Data from 23 patients under HAART for 1 to >5 years showed a median viral load of 0.0 (0.0–0.0) DNA viral copies/µl. In these 23 patients, the median CD4 count was 631 (439–715) cells/mm3. The patients underwent electrocardiography, echocardiography, and thorax, esophagus, and colon radiological examinations, being then classified within one of the clinical forms of Chagas disease (1). Individuals without signs and symptoms and with no alteration in the mentioned tests were classified as having the indeterminate form (n = 51), patients with abnormalities suggestive of Chagas disease on electrocardiography or on dynamic electrocardiography and without digestive involvement as with the cardiac form (n = 96), the cases with abnormal findings on esophagus and/or colon without Chagas cardiac manifestation as presenting the digestive form (n = 32). Twenty-seven patients presented both digestive and cardiac alterations, constituting the cardio-digestive group. Cardiac involvement was detected in 123 patients (cardiac and cardiac/digestive forms) while heart disease was not identified in 83 patients, a group composed of individuals with the indeterminate and digestive forms. Patients with cardiomyopathy were evaluated for heart failure according to the New York Heart Association (NYHA) classification (48), which includes the following criteria: class 1. Patients with cardiac disease without limitation of physical activity; class 2. Patients with cardiac disease with mild limitation of physical activity and symptomatic in routine physical activity; class 3. Patients with cardiac disease with marked limitation of physical activity and symptomatic in less than ordinary physical activity; and class 4. Patients with cardiac disease and unable to carry any physical activity without discomfort, symptoms may be present even at rest. For the purposes of our study, however, Chagas patients were classified according to two different scenarios related to their cardiac status, being divided in two groups in each of the cases: A) based on the NYHA classification/absence of cardiopathy—group 1: patients without cardiopathy (no CA) and with mild cardiopathy (NYHA 1), and group 2: patients with more severe cardiopathy (NYHA ≥ 2); and B) Based on the left ventricular ejection fraction (LVEF) yielded by echocardiography—group 1: patients with LVEF ≥ 45%, and group 2: patients with LVEF < 45%. The control group for the SNV analyses consisted of 90 healthy individuals with negative serological tests for T. cruzi antigens. Controls and patients comprised a total population of 296 individuals, including 149 females and 147 males: 215 white and 73 non-white. This classification was not available for eight control individuals. Among the 206 patients included in this study, 135 were classified within these groups according to self-declaration; 71 of them, in turn, following a distinct institutional policy, were classified according to their government-issued identification document or by the hospital employee who processed their hospital registration. Ethics Statement The present study was approved by the Ethics Committee for Research Project Analysis of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP), São Paulo, Brazil (CAPPesq 0174/11). All subjects gave written informed consent in accordance with the Declaration of Helsinki. Evaluation of Parasitemia Patients’ peripheral blood samples were collected in EDTA (ethylenediaminetetraacetic acid) tubes. DNA extraction was performed using the QIAmp DNA Mini Kit (Qiagen). DNA concentration and purity were analyzed with a spectrophotometer. The parasitemia status was determined employing a qualitative polymerase chain reaction (PCR) performed with the S35/S36 primers designed to the kinetoplastic DNA of T. cruzi (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), as previously described (49). DNA Extraction and Genotyping In most patients genomic DNA was extracted from peripheral blood samples using the QIAampTM DNA Mini Kit (Qiagen, Hilden, Germany). For the cases the DNA concentration was inappropriately low, an additional extraction was carried out using the salt precipitation method (DTAB/CTAB; Sigma-Aldrich, Merck, St. Louis, MO, USA) (50). The DNA samples underwent spectrophotometric evaluation to assess yield and purity. SNVs were investigated by real-time PCR with the TaqMan® Genotyping Master Mix assay and the corresponding SNV-specific primers in the StepOnePlus platform (Applied Biosystems, Thermo Fisher Scientific, Foster City, CA, USA). The investigated SNPs included IL1B −31 rs1143627 T>C, IL6 −174 rs1800795 C>G, IL17A −152 rs2275913 G>A, IL18 −137 rs187238 C>G, and IL18 −607 rs1946518 C>A. Serum IL-18 Quantification IL-18 levels were measured using commercial ELISA Kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Sera from 43 patients, 15 HIV-infected and 28 non-infected, were stored at −70°C. Twenty-four of such patients had cardiac disease. At the time of blood collection, the median viral load in the 15 HIV-infected patients was 0.0 DNA copies/µl. In 14 of them the median of CD4 count was 873.5 (769.8–974.0) cells/mm3. Three of the 15 HIV-infected patients were not on regular antiretroviral therapy. Two had detectable viremia, with one of them displaying a CD4 count below 350 cells/mm3. The third is an HIV-1-infected patient with an undetectable viral load and high CD4 level despite the absence of HAART therapy, belonging to the rare group named as “elite suppressor” (51). Statistical Analyses The calculations of Hardy-Weinberg equilibrium (HWE) were performed using chi square tests in MS Excel 365 (Microsoft Corporation, Redmond, WA). Multiple contingency analyses were performed to investigate potential associations among elected dependent variables and covariates. Fisher’s exact test was used for 2x2 contingency tables; otherwise the chi-square test was applied. When chi square test was used and was applicable, we performed a post-hoc test using standardized and adjusted residues with the Bonferroni correction. The P values obtained with single association tests (Fisher exact test or Pearson chi square test) were used as triage to elect candidate variables for multivariable analyses. We included in the logistic regression all variables that reached P < 0.2 in the single variable association test with the dependent variable. In the genetic models, when more than one model reached P < 0.2, we assumed the model with lower P value as the best-fit model and included it in the logistic regression analysis. To increase the modeling performance, continuous variables were categorized using a ROC (receiving operator characteristic) curve and clinical/biological criteria. Some variables presented missing values in 1–20% of cases and displayed a non-monotone missing pattern. In such cases, we performed multiple imputations including 100 imputed values for each missing one (52). This step was accomplished using MCMC (Markov chain-Monte Carlo simulation) for value imputation. The genotypes were clustered according to four possible models of genetic effects: additive (each genotype was analyzed separately), dominant (the presence of at least one SNV allele is associated with the effect), heterozygous (the heterozygous genotype is associated with the effect on phenotype), and recessive (the presence of SNV homozygosity is associated with the effect on phenotype). We adopted the best-fit model in the chi-square/Pearson exact test analyses, defined by the most significant P-value. Since rs1946518 and rs187238 are just 470 bp apart from each other in the IL18 promoter, we investigated linkage disequilibrium between these two SNVs using the Haploview software (53) and evaluated potential associations involving the haplotypes. Chagas cardiopathy, NYHA (New York Heart Association) score, LVEF, and parasitemia were selected upfront as dependent variables for the multiple logistic regressions. Since there was potential interference of the clinical form in the analyzed endpoints, we considered the digestive form as an independent variable in addition to traditional potential confounding factors. To address this point, such a variable was included in the triage as a potential predictor in the model applied to the logistic regression analysis. Continuous variables were tested for normality with the Shapiro-Wilk test. Continuous parametric variables were analyzed using t test to compare two groups, with the Welch correction if the Levine test pointed to unequal variance between the groups. Continuous non-parametric variables were analyzed using the Mann-Whitney U test when comparing two groups, or Kruskal-Wallis test when comparing more than two groups, followed by post-hoc analysis with the Bonferroni correction. Categorical variables were compared using the chi-square or Fisher exact test. Categorical variables are expressed as the number of cases and percentages while continuous variables as mean ± standard deviation when parametric, and median (25–75% range) when non-parametric. We accepted an α risk of 5% in this exploratory research. The statistical analyses were run in SPSS 24 2. Methods for processing the data: 3. Instrument- or software-specific information needed to interpret the data: SPSS 24 4. Standards and calibration information, if appropriate: 5. Environmental/experimental conditions: Evaluation of Parasitemia Patients’ peripheral blood samples were collected in EDTA (ethylenediaminetetraacetic acid) tubes. DNA extraction was performed using the QIAmp DNA Mini Kit (Qiagen). DNA concentration and purity were analyzed with a spectrophotometer. The parasitemia status was determined employing a qualitative polymerase chain reaction (PCR) performed with the S35/S36 primers designed to the kinetoplastic DNA of T. cruzi (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), as previously described (49). DNA Extraction and Genotyping In most patients genomic DNA was extracted from peripheral blood samples using the QIAampTM DNA Mini Kit (Qiagen, Hilden, Germany). For the cases the DNA concentration was inappropriately low, an additional extraction was carried out using the salt precipitation method (DTAB/CTAB; Sigma-Aldrich, Merck, St. Louis, MO, USA) (50). The DNA samples underwent spectrophotometric evaluation to assess yield and purity. SNVs were investigated by real-time PCR with the TaqMan® Genotyping Master Mix assay and the corresponding SNV-specific primers in the StepOnePlus platform (Applied Biosystems, Thermo Fisher Scientific, Foster City, CA, USA). The investigated SNPs included IL1B −31 rs1143627 T>C, IL6 −174 rs1800795 C>G, IL17A −152 rs2275913 G>A, IL18 −137 rs187238 C>G, and IL18 −607 rs1946518 C>A. Serum IL-18 Quantification IL-18 levels were measured using commercial ELISA Kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Sera from 43 patients, 15 HIV-infected and 28 non-infected, were stored at −70°C. Twenty-four of such patients had cardiac disease. At the time of blood collection, the median viral load in the 15 HIV-infected patients was 0.0 DNA copies/µl. In 14 of them the median of CD4 count was 873.5 (769.8–974.0) cells/mm3. Three of the 15 HIV-infected patients were not on regular antiretroviral therapy. Two had detectable viremia, with one of them displaying a CD4 count below 350 cells/mm3. The third is an HIV-1-infected patient with an undetectable viral load and high CD4 level despite the absence of HAART therapy, belonging to the rare group named as “elite suppressor” (51) 6. Describe any quality-assurance procedures performed on the data: 7. People involved with sample collection, processing, analysis and/or submission: Alexandra Gomes dos Santos, Elieser Hitoshi Watanabe, Daiane Tomomi Ferreira,Jamille Oliveira, Erika Shimoda Nakanishi, Claudia Silva Oliveira, Edimar Bocchi, Cristina Terra Gallafrio Novaes, Fatima Cruz, Noemia Barbosa Carvalho,Paula Keiko Sato, Edite Hatsumi Yamashiro-Kanashiro, Alessandra Pontillo,Vera Lucia Teixeira de Freitas, Luiz Fernando Onuchic and Maria Aparecida Shikanai-Yasuda DATA-SPECIFIC INFORMATION FOR: [GomesdosSantos_Watanabe_2020_FrontImmunol] 1. Number of variables: 33 2. Number of cases/rows: 206 3. Variable List: IDENTIFICATION "RACE White [1] Negro [2] Brown [3] Mulatto [4]" AGE RANGE <35 [1] 35-50 [2] >50 [3] CAUCASIAN [1] NOT CAUCASIAN [0] HIV + [1] HIV - [0] IL1B rs1143627 T>C TT [0] TC [1] CC [2] IL1B rs1143627 Dominant C_[1] TT [0] IL1B rs1143627 Heterozigous TC [1] TT/CC[0] IL1B rs1143627 Recessive CC [1] T_ [0] IL6 rs1800795 G>C CC [0] CG [1] GG [2] IL6 rs1800795 Dominant G_ [1] CC [0] IL6 rs1800795 Heterozigous CG [1] CC/GG [0] IL6 rs1800795_Recessive GG [1] C_ [0] IL17 rs2275913 G>A GG [0] GA [1] AA [2] IL17 rs2275913 DominantA_ [1] GG [0] IL17 rs2275913 Heterozigous GA [1] GG/AA [0] IL17rs2275913 Recessive AA [1] G_ [0] IL18 607 rs1946518 C>A TT [0] TG [1] GG [2] IL18 607 rs1946518_Dominant G_ [1] TT [0] IL18 607 rs1946518_HeterozygousTG [1] GG/TT [0] IL18 607 rs1946518_RecessiveTT [1] G_ [0] IL18 137 rs187238_C>G CC [0] CG [1] GG [2] IL18 137 rs187238_Dominant G_ [1] CC [0] IL18 137 rs187238_Heterozygous CG [1] CC/GG [0] I L18 137 rs187238_Recessive GG [1] C_ [0] NYHA 1 without CI [1] NYHA >2 [0] PARASITEMIA Positive [1] Negative [0] CHAGAS CARDIOPATHY Yes [1] No [0] CLINICAL FORM Cardiac Form [FC], Cardiodigestive Form [FCD] Indeterminate Form [FI] Digestive Form [FD] Left Ventricular ejection fraction (LVEF) <0,45 Yes [1] No [0] LVEF values SOURCE HIV [1] MI [2] INCOR [3] Control [4] IL-18 serum LEVELS (pg/mL) CHAGAS CARDIOPATHY IL18 levels analyses Yes [1] No [0] 4. Missing data codes: NA: not available NP: not pertinent 5. Specialized formats or other abbreviations used: