Human T cell responses against the major cysteine proteinase (cruzipain) of Trypanosoma cruzi: role of the multifunctional alpha 2-macroglobulin receptor in antigen presentation by monocytes.

Chagas' disease patients (CDP) develop both humoral and cellular immune responses against the major cysteine proteinase (cruzipain) from Trypanosoma cruzi. Here we demonstrate that complexes formed by cruzipain and alpha 2-macroglobulin (alpha 2M) are efficiently internalized by human monocytes, and that this process results in enhanced presentation of cruzipain peptides to CD4+ T cells from CDP. Purified or serum alpha 2M binds to polymorphic cruzipains, but only a fraction of the proteinases become covalently linked. Once bound to alpha 2M, fluorescein-labeled cruzipain (FITC-cruzipain) or [125I]cruzipain were more efficiently internalized by normal peripheral blood mononuclear cells (PBMC) or monocytes; this effect was abolished by (I) pre-treating the cells with receptor-associated protein (rRAP), a known antagonist the of alpha 2M receptor (alpha 2MR/LRP), and (II) inactivating [125I]cruzipain's active site prior to the reaction with alpha 2M, indicating that the exposure of receptor binding sites on alpha 2M complexes required bait region cleavage. We then sought to determine if the alpha 2MR/LRP-dependent uptake of alpha 2M:cruzipain by monocytes resulted in increased CD4+ T cell responses of PBMC-CDP (n = 13). These effects were only revealed after depletion of CD19+ B lymphocytes from PBMC-CDP; the threshold of T cell stimulation was far lower in cultures stimulated with alpha 2M:cruzipain, as compared to antigen alone. Myocardial specimens from CDP with chronic myocardiopathy (three necropsies) were analyzed by immunohistochemistry with mAb anti-cruzipain or anti-alpha 2MR/LRP (CD81+). Extracellular depots of cruzipain were localized amidst inflammatory mononuclear infiltrates, part of which contained CD91+ macrophage-like cells. Ongoing studies should clarify if T. cruzi cysteinyl proteinases play a role in the pathogenesis of Chagas' heart disease.

Immunohistochemical expression of atrial natriuretic peptide (ANP) in the conducting system and internodal atrial myocardium of human hearts.

Expression and distribution of atrial natriuretic peptide (ANP) were studied immunohistochemically in the conducting system and internodal atrial myocardium of 5 adult human hearts. Myocytes from the sinus node and compact atrioventricular node were usually ANP-negative; only a very few cells exhibited ANP immunoreactivity. These ANP-positive myocytes were small and did not appear to be trapped working atrial myocytes which are larger than nodal cells. The transitional cell zones of the sinus node and the atrioventricular node were composed of bundles of ANP-positive myocytes, intermingled with non-reactive myocytes. The internodal atrial myocardium exhibited a comparable intensity of myocyte staining in each case examined. Thus, morphologically distinct connecting pathways between the sinus node and the atrioventricular node with regard to myocyte ANP immunoreactivity could not be demonstrated, reinforcing the notion that they actually do not exist. The penetrating bundle, branching bundle and bundle branches were usually composed of ANP-negative myocytes although some ANP-positive myocytes were observed in the branching bundle and bundle branches in 4 cases. Myocytes from the ventricular conducting tissue presenting ANP immunoreactivity have been designated Purkinje fibers and have been found in several mammalian species.

An in situ quantitative immunohistochemical study of cytokines and IL-2R+ in chronic human chagasic myocarditis: correlation with the presence of myocardial Trypanosoma cruzi antigens.

Inflammatory cells positive for the cytokines IL-2, IL-4, IL-6, TNF-alpha, and IFN-gamma and for IL-2R, as well as CD8+ and CD4+ T cells and B cells were quantified using an immunoperoxidase technique in 25 fresh myocardial fragments from patients presenting with chronic chagasic cardiomyopathy. The presence of Trypanosoma cruzi antigens (Ags) in the myocardium was also investigated. The cases were grouped into group A (no Ag), group B (scarce extramyocardial fiber Ags), and group C (intramyocardial pseudocysts and extramyocardial fiber Ags). IL-2 was detected in very few cells (0.30 +/- 0.40 positive cells/hpf), suggesting immunological imbalance in chronic chagasic patients. IFN-gamma+ was the cytokine most frequently demonstrated (7.52 +/- 5.87 positive cells/hpf) and there was a good correlation between the number of IFN-gamma+ cells and CD8+ T cells in group A. IL-4+ cells were present in higher numbers in group C (2.78 +/- 1.49 positive cells/hpf). TNF-alpha+ (1.59 +/- 1.68 positive cells/hpf) and IL-6+ (2.76 +/- 2.32 positive cells/hpf) cells were present in moderate numbers. Fewer B cells were present, not related with the intensity of T. cruzi Ags. These results suggest that cytokines, as they occur in other infectious diseases, play a fundamental role in the control of T. cruzi in chronic human chagasic disease. A fatal outcome seems to be associated with the increased production of cytokines derived from the Th2 subpopulation of the CD4+ T cells.

Chronic chagasic cardiopathy: the product of a turbulent host-parasite relationship.

The pathogenesis of chronic chagasic cardiopathy is still a debated matter. In this review, the main theories raised about it since the first description of the disease in 1909 by Carlos Chagas, are considered. The scarcity of T.cruzi parasites into the myocardium and the apparent lack of correlation between their presence and the occurrence of myocardial inflammatory infiltrate, have originated many theories indicating that chronic Chagas' cardiopathy is an autoimmune disease. Recently however, papers using immunohistochemical technique or PCR have demonstrated a strong association between moderate or severe myocarditis and presence of T.cruzi Ags, indicating a direct participation of the parasite in the genesis of chronic chagasic myocarditis. Different patterns of cytokine production seem to have important role in the outcome of the disease. Participation of the microcirculatory alterations and fibrosis as well as the relationship with the parasite are also emphasized. Finally, the author suggests that the indeterminate form of the disease occurs when the host immunological response against the parasite is more efficient while the chronic cardiopathy occurs in patients with hyperergic and inefficient immune response.

Adventitial layer enlargement correlates with the percentage of medial thickness in peripheral pulmonary arteries from patients with congenital heart defects.

Arterial walls undergo modifications during the course of pulmonary hypertension, particularly in the medial and intimal layers, leading to progressive occlusion of the lumen. Adventitial layer enlargement has been described as being present in the experimental hypoxic model and in the persistent pulmonary hypertension of the newborn. It was suggested that this enlargement may be related to stimulating factors derived from the medial smooth muscle cells. This study was designed to verify if different degrees of medial hypertrophy are correlated to the volume density of the adventitial layer in pulmonary hypertension secondary to congenital heart defects. Reviewing 21 lung biopsies from patients with congenital heart defects, we concluded that there is a statistically significant positive linear correlation between the mean percentage of medial arterial thickness and the volume density of the adventitial layer in the biopsies showing isolated medial hypertrophy. On the other hand, in biopsies showing frequent intimal proliferative lesions and irregular medial layer hypertrophy the correlation coefficient was lower. These findings suggest that the adventitial layer participates in the arterial remodeling process in secondary pulmonary hypertension, and that its enlargement depends on the qualitative degree of pulmonary vaso-occlusive disease.

Restricted heterogeneity of T cell receptor variable alpha chain transcripts in hearts of Chagas' disease cardiomyopathy patients.

The role of autoimmunity in the pathogenesis and progression of heart lesions in the chronic phase of Chagas' disease is controversial. In the absence of parasites in situ, the T cell infiltrate seen in heart lesions may be the primary determinant of tissue damage ultimately leading to heart failure and death. We used the polymerase chain reaction to amplify each known T cell receptor (TCR) V alpha and V beta subfamily-specific sequence in transcripts derived from heart samples obtained from Chagas' cardiomyopathy patients. The average number of TCR V alpha subfamilies (7.1 per tissue sample) was significantly lower than that for TCR V beta subfamilies (15.1 per sample). The average percentage of tissue samples positive per TCR V alpha and V beta subfamily was respectively 39.6% vs. 73.5%. These data suggest that, in Chagas' heart lesions, the detectable TCR V alpha repertoire is significantly narrower than TCR V beta repertoire. On the other hand, in normal heart tissue, diversity of V alpha and V beta TCR is similar among the scarce circulating T cell population. Such evidence of restricted TCR V region repertoire has been described in experimental and human autoimmune diseases. Our results are consistent with the possibility that T cells responsible for heart damage in chronic Chagas' cardiomyopathy may be recognizing a few heart-specific antigenic targets.

Immunohistochemical characterization of infiltrating cells in human chronic chagasic myocarditis: comparison with myocardial rejection process.

Cellular subpopulations that infiltrate the heart in human chronic chagasic myocarditis were defined immunohistochemically in endomyocardial biopsy (EMB) specimens. T cells formed 96.3% of the inflammatory infiltrate, predominantly CD8+ (cytotoxic/suppressor) T cells. The mean numbers of CD8+ and CD4+ (helper) T cells in the myocarditis were compared to those present in the myocardial rejection process. Mean numbers of CD8+ T cells were similar in both groups of EMB specimens while CD4+ T cell counts, CD4+/CD8+ ratios and CD4+ antigen expression were significantly lower in the chagasic group compared to the myocardial rejection group (P < 0.002). The persistent lower number and diminished expression of CD4+ T cells suggest an immunological imbalance in patients with chronic chagasic myocarditis. A possible participation of Trypanosoma cruzi parasites in the development of such immunological abnormalities is also discussed.

Correlation between Trypanosoma cruzi parasitism and myocardial inflammatory infiltrate in human chronic chagasic myocarditis: Light microscopy and immunohistochemical findings.

Trypanosoma cruzi parasites are only rarely identified in conventional histological sections of hearts from chronic chagasic patients. This finding suggests that T. cruzi plays no important direct role in the chronic myocarditis that accordingly has been considered mainly an autoimmune process. We reinvestigated this issue using a polyclonal anti-T. cruzi antibody serum to map immunohistochemically the T. cruzi antigen(s) in 9 different regions of 8 necropsy hearts and 24 septal fragments from 24 hearts from chronic chagasic patients. T. cruzi antigen(s) were identified in 7 (87%) of the 8 mapped hearts and in 14 (58%) of the 24 septal fragments. There was a statistically significant correlation between the presence of T. cruzi antigen(s) and moderate or severe inflammatory infiltrate (p = 0.005). When staining revealed amastigotes within intact myocardial fibers, there was no surrounding inflammatory infiltrate. However, when T. cruzi antigen(s) were found in macrophages either as amastigotes, diffusely in the macrophages cytoplasm, or free in the interstitium as round structures similar to amastigotes, there was a heavy inflammatory infiltrate. In the case in which no parasite was detected, a mild inflammatory infiltrate was present in the myocardium. Foci of fibrosis did not stain for T. cruzi antigen. These findings do not exclude a role of autoimmunity in chronic chagasic cardiopathy. However, the striking correlation between the presence of T. cruzi antigen(s) with the severity of site of the inflammatory infiltrate supports a direct role for the parasite in the perpetuation of myocardial inflammation in Chagas' disease. The destruction of microvessels and occasional endothelial cells with parasitism among dense inflammatory infiltrate favors the concept that microcirculatory injury, induced by T. cruzi, also contributes to the lesions of chronic Chagas' disease.

[Hemorrhagic myocardial infarct. An analysis of the distribution of the hemorrhage and of the permeability of the artery related to the infarct]. / Infarto hemorrágico do miocárdio. Análise da distribuição da hemorragia e da perviabilidade da artéria relacionada ao infarto.

PURPOSE: To study the extent of hemorrhagic myocardial infarction (HMI) and patency of the infarct related artery. METHODS: Forty seven cases of HMI diagnosed by necropsy (patient age range 30-81 years, mean 59) were studied retrospectively. Hemorrhagic extent was evaluated by microscopic analysis of myocardial sections of the infarcted areas and coronary patency was studied by angiography and by serial coronary sections at necropsy. RESULTS: In 12 cases hemorrhage extended outside the infarcted area and in the remaining cases it was restricted to the necrotic zone. Coronary patency was spontaneous in 8 of 24 cases, secondary to thrombolytic therapy or angioplasty in 8 and post coronary artery bypass in 15. Recent occlusive thrombus was diagnosed in 26 of 44 cases. Grouping all cases according to angiographic or macro and microscopic evidences of coronary patency, it was found that 35 of 47 studies cases (74.4%) had the infarct related coronary artery free of occlusion. In most cases of HMI myocardial hemorrhage restricted to the infarcted necrotic zone but in almost 25% it could reach areas beyond the infarcted necrotic zone probably resulting in deleterious consequences. CONCLUSION: Reperfusion is frequent and it plays a role in the hemorrhagic event but it was not seen in 25% of these studied cases. These findings suggest that other mechanisms could participate of the pathogenesis of HMI.

[Myocardial infarction: comparison of anatomo-pathological findings from hearts with and without severe coronary atherosclerosis in 194 necropsy cases]. / Infarto do miocárdio: comparação dos achados anátomo-patológicos em corações com e sem aterosclerose coronária grave em 194 casos de necrópsia.

PURPOSE: To compare morphological features of myocardial infarction (MI) from patients with any epicardial coronary artery narrowed at some point more than 70% (severe coronary atherosclerosis--SCA) with those from patients with either no coronary atherosclerosis or only mild (less than 70%) atherosclerosis. METHODS: Necropsy findings from 194 patients who died due to MI, 174 patients with and 20 without SCA. Ages ranged from 21 to 82 (mean 60) years. RESULTS: Mean age was 60 years in the cases with SCA and 56 in the case without it; nevertheless, age distribution was different (p = 0.023), due to the existence of more patients under age 40 in the group without SCA. There was no significant difference concerning sex (31.0% of female patients in the group with SCA and 35.0% in the other group, p = 0.718), mean heart weight (500 g and 506 g), distribution of cases according to time of evolution of MI in recent only, old only or both (cases with SCA--36.2%, 28.2% and 35.6%; cases without SCA--45.0% and 20%; p = 0.666), left ventricular wall committed by the MI (p = 0.715), incidence of hemorrhagic infarction (with SCA--8.6%; without SCA--15.0%; p = 0.406), left ventricular rupture (with SCa--5.17%, without SCA--10.0%; p = 0.719) and left ventricular aneurysms (with SCA--12.1%, without SCA--15.0%; p = 0.316). An association was found between coronary atherosclerosis and recent (p = 0.046) and recanalized (p less than 0.001) thrombosis, but absent when only recent thrombosis and non-operated cases with recent MI were considered (p = 0.091). CONCLUSION: Necropsy of fatal cases of MI were not significantly different in the presence or absence of severe atherosclerotic narrowing (greater than 70%) of epicardial coronary arteries, suggesting that this factor does not modify the natural history of MI.

[Histologic response of the myocardium to various immunosuppressor schedules in patients with dilated cardiomyopathy and diagnosis of myocarditis at endomyocardial biopsy]. / Resposta histológica do miocárdio a diferentes esquemas imunossupressores em pacientes com cardiomiopatia dilatada e diagnóstico de miocardite biópsia endomiocárdica.

PURPOSE: To analyse the histological evolution of endomyocardial biopsies from children with active myocarditis, submitted or not to immunosuppressive therapy. PATIENTS AND METHODS: Four groups of patients were compared, clinically treated as follows: group I--anticongestive drugs (4 patients); group II--prednisone (5 patients); group III--prednisone plus azathioprine (9 patients); group IV--prednisone and cyclosporine (5 patients). RESULTS: No patient from group I presented any histological improvement during a mean period of 9 months, while evident histological improvement occurred in 25% of patients from group I, 67% from group III and 80% from group IV. The microscopical aspect of resolving myocarditis was only observed in patients from groups III and IV, after treatment. CONCLUSION: The immunosuppressive therapy with azathioprine or cyclosporine plus prednisone leads to decrease of active myocarditis intensity in a higher proportion of cases than the treatment with only prednisone or no immunosuppressive drugs.

[Myocardial lesions after snake bites by the Crotalus durissus terrificus species (rattlesnake). A case report]. / Lesão miocárdica em acidente ofídico pela espécie Crotalus durissus terrificus (cascavel). Relato de caso.

Extensive and severe myocardial lesions are reported in a patient who died due to snakebite (Crotalus Durissus Terrificus). These lesions are documented by clinical, electrocardiographic, enzymatic and histological evidences. The main pathological features are represented by sarcoplasmatic vacuoles, densely clumped myofibrils and amorphous acidophilic mass into the cardiac fibers. These lesions are identical to those which have already been described in skeletal muscle after snakebite. This seems to be the first case report that shows undoubt myocardial lesions due to snakebite with anatomopathological documentation.

Histopathological criteria of myocarditis--a study based on normal heart, chagasic heart and dilated cardiomyopathy.

This work is a detailed study of the relevance of three sets of criteria to define myocarditis: Dallas meeting criterion, Edwards criterion and Dallas meeting criterion modified by the authors. Two groups were evaluated: normal autopsied hearts and endomyocardial biopsy from chronic chagasic patients at high risk of having myocarditis. Furthermore, endomyocardial biopsies from patients with dilated cardiomyopathy (DCM) were also evaluated. Applying the Edwards criterion, incidences of myocarditis in normal and chagasic hearts were 0% and 67% while with Dallas meeting criterion they were 0% and 42% and using our criterion the incidences were 0% and 92% respectively. In endomyocardial biopsies from DCM patients, the incidence of myocarditis was 7% with Edwards criterion, 22% with Dallas meeting and 33% with the authors own criterion. The authors concluded that their criterion, which defines myocarditis as the presence of inflammatory mononuclear cells enclosing more than 2 lymphocytes/400X aggregated to the cardiac fiber sarcolemma, is the most appropriate criterion of the three. Myocarditis was found in 33% of the 27 endomyocardial biopsy specimens from patients with DCM.

[Differences between men and women in fatal cases of myocardial infarction: study of 200 necropsies]. / Diferenças entre homens e mulheres em casos fatais de infarto do miocárdio. Estudo de 200 necrópsias.

PURPOSE: To compare morphological characteristics from myocardial infarction (IM) in men and women in 200 consecutive necropsies. MATERIAL AND METHODS: Necropsy heart findings from 62 female cases compared with those from 138 male cases, from patients who died from transmural myocardial infarction. Age ranged from 21 to 82 (mean 60) years. RESULTS: Concerning the coronary arteries, the number of 3 major (right, left anterior descending and left circumflex) epicardial coronary arteries narrowed at some point greater than 70% in cross-sectional area by atherosclerotic plaque was not significantly different; on the other hand, there were more severe lesions in the left main coronary artery in men (10.33%) than in women (1.64%) (p = 0.050). 33.33% of men and 43.55% of women had only recent myocardial infarction (1 month or less); 33.33% of men and 14.52% of women had only old myocardial infarction (more than 1 month); 33.33% of men and 41.94% of women had both recent and old myocardial infarction. 12.90% of women and 2.17% of men showed rupture of the left ventricle (p = 0.0220). 15.22% of men and 6.45% of women had left ventricular aneurysms (p = 0.830). CONCLUSION: There are more deaths during the acute phase of MI, in the first infarction and from rupture of the left ventricle in female patients; in men, there are more deaths occurring in chronic phases of the disease and with previous myocardial infarction; and more severe narrowing from the left main coronary artery.

[Endomyocardial biopsy and myocardial imaging with 67-gallium in the diagnosis of active myocarditis in children with dilated myocardiopathy]. / Biópsia endomiocárdica e mapeamento miocárdico com gálio-67 no diagnóstico de miocardite ativa em crianças portadoras de miocardiopatia dilatada.

PURPOSE: This study was designed to compare 67Ga imaging and endomyocardial biopsy (EB) in children with severe dilated cardiomyopathy (DC), as well as to evaluate the results in a group of patients with active myocarditis submitted to immunosuppressive therapy. PATIENTS AND METHODS: Forty-four pediatric patients with severe DC were studied. Twenty males and 24 females from 10 months to 15 year old (median = 2.6 years). All patients were submitted to a protocol including 67Ga uptake and EB. In patients submitted to immunosuppressive therapy these procedures were repeated after six months. RESULTS: In 32 patients (72.7%) the EB revealed presence of inflammatory process; 21 (65.6%) of these had a positive 67Ga uptake and 11 (34.4%) negative. Twelve patients with no evidence of inflammatory process in the EB, nine (75%) presented negative 67Ga uptake. However, when the intensity of myocardial inflammatory was analysed (mild, moderate and severe) and correlated with 67Ga imaging, was observed that the majority of patients with negative 67Ga uptake (11 patients) had mild inflammatory infiltration (nine patients). In this way the 67Ga uptake demonstrated a good correlation in the diagnosis of moderate and severe inflammatory process in children with DC. This is important because the use of immunosuppressive drugs is indicated only in these group. CONCLUSION: The 67Ga imaging is a noninvasive diagnostic method with a good sensitivity to the diagnostic method with a good sensitivity to the diagnosis of AM in children with severe DC, demonstrating to be very useful in the therapeutic approach.