**5.4 Ultrastructural features**

136 Myocarditis

Fig. 6. A. Detail of the left bundle of His is shown. Immunostaining for T lymphocyte. Positive cells express CD45R0 antigen (brown); specialized myocardial cells have almost disappeared. Extensive mononuclear infiltrate, the majority of them being T lymphocytes. X20. B. Double immunostaining for the simultaneous demonstration of T lymphocytes (CD45RO) and macrophages (CD68). T lymphocytes (brown) in close contact with a macrophage (pinky cytoplasm). X1000. C. Immunostaining to show endothelial cells. Capillaries and small vessels are clearly showed by the expression of CD31. Vessels are midly or moderately disorted because of the surrounding fibrosis. X100. From Milei, 1996a. In our studies in endomyocardial biopsies, infiltrates were approximately 50% lymphocytes and 50% macrophages. Almost 80% of lymphocytic population were T lymphocytes, being only 20% B lymphocytes. Eosinophils were scarce in infiltrates reaching 5%, and were associated with areas of necrotic myocardium. Mast cells also were scarce or absent in

specialized and in contracting myocardium. (Milei, et al., 1996a, Milei, et al., 1992b)

Histological study of aneurisms showed a thinned wall 2-4 mm, with sclerotic plaques of thickened endocardium of up to 92% of total tissue and extensive mononuclear chronic inflammatory infiltrates and widespread fibrosis in myocardium. Myocytes were organized in thin bands or atrophic units surrounded by fibrotic tissue (Figure 5B) (Milei,

Autonomic ganglia showed above described Terplan's nodules, with satellite cell proliferation replacing degenerated autonomic neurons. As stated, these lesions, once considered patognomonic, can be found in other cardiomyopathies (Rossi L., et al., 1994).

Immunophenotyping of infiltrates allowed a better characterization of the cells participating in the inflammatory infiltrates, mainly macrophages (CD68+) and lymphocytes (CD45R+). In our works 26.5% percent of them were T lymphocytes (CD45R+, CD45R0+) and 10.5% were B lymphocytes (CD20+, light chains kappa and/or lambda+) (Figure 6A). Thirty percent of the infiltrate was composed of macrophages (CD68+). The remaining infiltrate was composed of mononuclear cells resembling macrophages and CLA-negative mononuclear cells. Contacts between CD68 positive cells and T lymphocytes were frequently found

et al., 1991a).

**5.3 Immunohistochemical findings** 

Myocardial fibers showed nuclear enlargement, nuclear membrane invaginations, lipofuscin deposits, myofibrils derangements and loss, swelling, mitochondrial atrophy, dilatation of sarcotubular system, and interstitial fibrosis (Carrasco, et al., 1982, Palacios-Prü, et al., 1982). These findings have been confirmed by our group in endomyocardial biopsies (Ferrans, et al., 1988, Milei, et al., 1992b). Platelet thrombi can be demonstrated within capillaries (Figure 7B).

Other striking alteration in these specimens was the thickening of the basement membranes of cardiac myocytes (Figures 7A, 7C), endothelium Figure 7C) and vascular smooth muscle up to 20 times their normal thickness of 500 Å (Ferrans, et al., 1988). The thickened basement membranes appeared structurally homogeneous, without being multilayered or subdivided into a lamina rara and a lamina densa. They were of relatively low electron density, had a finely fibrillar appearance at high magnification and measured up to 1 m in thickness. Using gold-conjugated antibodies, we could demonstrate the presence of laminin in the thickened basal membranes of myocytes and endothelium (Sanchez, et al., 1993).

Regarding the ultrastructure of aneurysms resected from chagasic patients we observed, hypertrophy of myocytes, with swelling, partial or complete loss of myofibrils, swelling of mitochondria, disruption of mitochondrial cristae, lipofuscin granules, and intact sarcolemmas . Basement membranes were thickened, as previously described (Milei, et al., 1991a)

Fig. 7. A. Myocardial fibre with thickened basement membrane. B. Platelet thrombus within a capillary. C. Thickened basement membranes in a myocardial fibre and a capillary. From Milei, et al., 2008.

Pathogenesis and Pathology of Chagas' Chronic Myocarditis 139

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