Inducible Nitric Oxide Synthase in Heart Tissue and Nitric Oxide in Serum of Trypanosoma cruzi-Infected Rhesus Monkeys: Association with Heart Injury

Chagas disease, a neglected tropical disease caused by the protozoan Trypanosoma cruzi, afflicts from 8 to 15 million people in the Latin America. Chronic chagasic cardiomyopathy (CCC) is the most frequent manifestation of Chagas disease. Currently, patient management only mitigates CCC symptoms. The pathogenic factors leading to CCC remain unknown; therefore their comprehension may contribute to develop more efficient therapies. In patients, high nitric oxide (NO) levels have been associated with CCC severity. In T. cruzi-infected mice, NO, mainly produced via inducible nitric oxide synthase (iNOS/NOS2), is proposed to work in parasite control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, infected rhesus monkeys and iNOS/NOS2-deficient mice were used to explore the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection. Chronically infected monkeys presented electrical abnormalities, myocarditis and fibrosis, resembling the spectrum of human CCC. Moreover, cardiomyocyte lesion correlated with iNOS/NOS2+ cells infiltrating the cardiac tissue. Our findings support that parasite-driven iNOS/NOS2+ cells accumulation in the cardiac tissue and NO overproduction contribute to cardiomyopathy severity, mainly disturbing the pathway involved in electrical synchrony in T. cruzi infection

Motivos de pais e responsáveis para a não adesão à vacinação contra o Papilomavírus Humano: Revisão de Escopo

Considerando os impactos causados pelo papilomavírus humano (HPV), a vacina quadrivalente foi incorporada ao Programa Nacional de Imunização, PNI do Brasil. Esta atua protegendo contra lesões genitais pré-cancerosas além de verrugas genitais, em mulheres e homens. Apesar do Ministério da Saúde não poupar esforços para que mais pessoas se beneficiassem da imunoprevenção, a baixa cobertura vacinal é um problema perceptível em todo território nacional. Nesse sentido, este estudo tem o objetivo de identificar e sintetizar as evidências científicas sobre os motivos que levam os responsáveis a não vacinarem as crianças/adolescentes contra o HPV. Trata-se de uma revisão de escopo, com estratégia de busca aplicada em bancos de dados, apresentando extração e análise dos dados realizados em pares e os dados analisados e sintetizados em forma narrativa

iNOS/NOS2 and NO status influence heart parasitism and cardiomyocyte integrity in <i>T. cruzi</i>-infected mice.

<p>The mice were infected with 100 blood trypomastigotes of the Colombian <i>T. cruzi</i> strain and analyzed at 40 dpi. The presence of iNOS/NOS2⁺ cells, parasite nests, inflammatory cells and Cx43 in the myocardium was immunohistochemically detected, NO concentration was evaluated by a Griess-based method and CK-MB activity levels in the serum was biochemically determined. (<b>A</b>) Increased NO levels in serum of <i>T. cruzi</i>-infected C57BL/6 mice in comparison with noninfected controls (NI). (<b>B</b>) Photomicrograph of iNOS/NOS2⁺ cells in the cardiac tissue of infected C57BL/6 mice and quantification of iNOS/NOS2⁺ cells in the cardiac tissue of infected C57BL/6 mice in comparison with noninfected controls. (<b>C</b>) Photomicrographs and quantification of parasite nests showing increased heart parasitism in <i>Nos2</i>^−/− compared with <i>T. cruzi</i>-infected C57BL/6 mice. (<b>D</b>) Similar number of inflammatory cells in the heart tissue of C57BL/6 and <i>Nos2</i>^−/−<i>T. cruzi</i>-infected mice. (<b>E</b>) CK-MB activity levels in the serum of noninfected and <i>T. cruzi</i>-infected mice revealing increased CK-MB activity in <i>T. cruzi</i>-infected mice when compared with noninfected controls. Decreased CK-MB activity in the serum of <i>Nos2</i>^−/− compared with C57BL/6 <i>T. cruzi</i>-infected mice. (<b>F</b>) Preserved expression of Cx43 in the heart tissue of <i>Nos2</i>^−/− compared with C57BL/6 infected mice. Analysis at 40 dpi of 3–5 noninfected and 5–8 infected mice/group. * <i>p</i><0.05 and ** <i>p</i><0.01. Bar = 100 µm (<b>C</b>). Bar = 50 µm (<b>F</b>).</p

Cardiomyocyte injury in <i>T. cruzi</i>-infected rhesus monkeys.

<p>Cardiomyocyte damage was assessed by immunohistochemical detection of Cx43 in the myocardium of the left ventricle and CK-MB activity levels in the serum of noninfected and chronically <i>T. cruzi</i>-infected rhesus monkeys. (<b>A</b>) Photomicrograph of myocardium section of the left ventricle of the noninfected monkey #94 showing normal pattern of Cx43 expression in intercalated discs. (<b>B</b>) Photomicrograph of myocardium section of the left ventricle of the <i>T. cruzi</i>-infected monkey #64 (23 ypi) showing normal aspect. (<b>C</b>) Photomicrograph of left ventricle section of the <i>T. cruzi</i>-infected monkey #99 (20 ypi) showing normal Cx43 pattern. (<b>D</b>) Photomicrograph of section of left ventricle of the <i>T. cruzi</i>-infected monkey #90 (20 ypi) revealing Cx43 loss in myocardial area lacking inflammation. (<b>E–F</b>). Photomicrographs of left ventricle section of the cardiopatic <i>T. cruzi</i>-infected monkey #95 (20 ypi) showing Cx43 loss in area with (<b>E</b>) intense diffuse inflammation and (<b>F</b>) the substitution of cardiomyocytes by mesenchymal cells. (<b>G</b>) Frequency of stained Cx43 area in heart sections of noninfected and chronically <i>T. cruzi</i>-infected monkeys (20–23 ypi). (<b>H</b>) Detection of CK-MB activity in the serum of noninfected and chronically <i>T. cruzi</i>-infected monkeys (20–23 ypi). (<b>I</b>) Correlation between the number of iNOS/NOS2⁺ cells in heart tissue and CK-MB activity levels in serum of rhesus monkeys. Bar = 100 µm.</p

Increased collagen deposits in the myocardium of <i>T. cruzi</i>-infected rhesus monkeys.

<p>Collagen deposition was used to assess fibrosis in the myocardium of the left ventricle of noninfected and <i>T. cruzi</i>-infected monkeys. (<b>A</b>) Noninfected monkey #94, normal slight collagen deposits. (<b>B</b>) Monkey #67 (41 dpi), increased collagen deposition. (<b>C</b>) Monkey #45 (3 ypi), normal collagen deposits. (<b>D</b>) Monkey #64 (23 ypi), increased collagen deposition. (<b>E</b>) Monkey #99 (20 ypi) normal slight collagen deposits. (<b>F</b>) Monkey #95 (20 ypi), increased interstitial matrix deposits. <b>G.</b> Percentage of cardiac section area occupied by collagen deposits in the myocardium of the left ventricle of noninfected and <i>T. cruzi</i>-infected monkeys. (<b>H–K)</b> Serial heart sections of monkey #95 (20 ypi) showing: (<b>H</b>) intense infiltrates of mononuclear inflammatory cells (<b>I</b>) paralleling fibrosis, and (<b>J</b>) substitution of cardiomyocytes by mesenchymal cells in (<b>K</b>) an area of intense fibrosis. <b>A–F</b>, <b>I</b> and <b>K</b>, Picro-Sirius red stain. <b>H</b> and <b>J</b>, H&E. Bar = 100 µm.</p

iNOS/NOS2⁺ cells in the myocardium and NO in the serum of <i>T. cruzi</i>-infected rhesus monkeys.

<p>The presence of iNOS/NOS2⁺ cells in the myocardium of the left ventricles was immunohistochemically detected and NO concentration was evaluated by a Griess-based method. Photomicrograph of myocardium section of the left ventricle (<b>A</b>) and (<b>B</b>) of the noninfected monkey #81, which showed a few iNOS/NOS2⁺ cells. Photomicrograph of myocardioum section of the left ventricle (<b>C</b>) and (<b>D</b>) of the <i>T. cruzi</i>-infected monkey #95 (20 ypi). (<b>E</b>) Number of iNOS/NOS2⁺ cells in 100 microscopic fields in heart sections. (<b>F</b>) Concentration of NO in the serum of noninfected controls and chronically (20–23 ypi) <i>T. cruzi</i>-infected monkeys. (<b>G</b>) Concentration of NO in the serum of monkey #95 prior to infection (day 0), during the acute phase when parasitemia was positive (56 dpi) and negative (163 dpi) and during the chronic phase (16 ypi and 20 ypi). Bar = 100 µm in (<b>A</b>) and (<b>C</b>); Bar = 25 µm in (<b>B</b>) and (<b>D</b>).</p

Electrocardiographic patterns detected in <i>Trypanosoma cruzi</i>-infected rhesus monkeys during acute and chronic infection.

*<p><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001644#pntd.0001644-BoneciniAlmeida1" target="_blank">[15]</a>.</p>†<p>ECG patterns were evaluated using the following standard criteria: AVB - atrioventricular block, AVR - Abnormal ventricular repolarization, LBBB - Left bundle branch block - second-degree His bundle, LvQRS - Low voltage QRS, RBBB - right bundle branch block - first-degree His bundle, RAD - right QRS axis deviation.</p

Persistence of <i>T. cruzi</i> in chronically infected rhesus monkeys.

<p>The persistence of <i>T. cruzi</i> parasite and antigens was evaluated by immunohistochemistry, PCR and antibody response. (<b>A</b>) Photomicrographs of section of myocardium of left ventricle of monkey #95 (20 ypi). Immunohistochemistry for <i>T. cruzi</i> antigens (black arrows and insert) associated (dotted square) or not associated (black arrow) with focal inflammation. Inflammatory infiltrates lacking parasite antigens (white arrow heads). <b>B–C</b>. PCR for <i>T. cruzi</i> kDNA (∼330 bp) in blood of noninfected controls (NI) and <i>T. cruzi</i>-infected rhesus monkeys at (<b>B</b>) 16–20 ypi and (<b>C</b>) 20–23 ypi. (<b>D</b>) PCR for <i>T. cruzi</i> kDNA (∼330 bp) in fragments of the left ventricle (LV) of the heart of noninfected controls and <i>T. cruzi</i>-infected rhesus monkeys at 20–23 ypi. Negative (−) and positive (+) controls were heart fragments of noninfected and <i>T. cruzi</i>-infected C57BL/6 mice, respectively. (<b>E</b>) Real time qPCR for the <i>T. cruzi</i> satellite DNA sequences Cruzi1/Cruzi2 in heart and spleen of noninfected controls and <i>T. cruzi</i>-infected rhesus monkeys at 20–23 ypi. (<b>F</b>) Standard curve of 10-fold serial dilution of DNA of epimastigote forms of the Colombian <i>T. cruzi</i> strain (10⁶ to 10 parasites/mL) used for the absolute quantification by real time qPCR. The linear regression curve, coefficient of determination (r² = 0.995) and qPCR efficiency (E = 80%) are indicated. The melting curve is also shown. (<b>G</b>) Serology for IgG anti-<i>T. cruzi</i> in rhesus monkeys prior to infection, during the acute phase (AP), when parasitemia was positive (+) and negative (−), and during the chronic phase (CP; at the end-point 20 ypi). Bar = 100 µm; Bar = 25 µm in insert in (<b>A</b>).</p

Electrocardiograph parameters of C57BL/6 and <i>Nos2</i>^−/− mice infected with the Colombian <i>T. cruzi</i> strain.

†<p>ECG parameters were evaluated at 40 dpi, using the following standard criteria: (i) heart rate (monitored by beats per minute (bpm), and (ii) the variation of the P wave and PR, QRS and QT intervals, all measured in milliseconds (ms); Brad, bradycardia; AVB1, first-degree atrioventricular block; AVB2, second-degree atrioventricular block.</p>‡<p>These data represent two independent experiments, with 5–7 mice/group.</p>§<p>, p<0.05;</p>§§<p>, p<0.01; comparison between the values for C57BL/6 and <i>Nos2</i>^−/− noninfected groups of mice;</p>*<p>, p<0.05;</p>**<p>, p<0.01;</p>***<p>, p<0.001; comparison between the values for noninfected and <i>T. cruzi</i>-infected groups of mice;</p>#<p>, p<0.05;</p>##<p>, p<0.01;</p>###<p>, p<0.001; comparison between the values for C57BL/6 and <i>Nos2</i>^−/−<i>T. cruzi</i>-infected mice.</p