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

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

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

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