Immunoperoxidase technique in experimental chronic chagasic myocarditis

Chagas'disease has been described as the commonest form of chronic myocarditis. An immunologic pathogenesis has been discribed for this form of the disease. So far, no immunoperoxidase technique has been used for the detection of immunological deposits in chronic experimental Chagas'myocardiopathy. Forty-one Swiss mice, three months old were inoculated intraperitoneally with doses between 10 and 10(5) Tulahuen trypomastigotes. Mice were reinoculated one month after with doses between 10² and 10(5) and sacrificed at 6 (n=21) and 9 months (n=9) after the first inoculation. ECGs were recorded before sacrifice. Immunoperoxidase technique (peroxidase-antiperoxidase method), immunofluorescence (direct and indirect) as well as histological studies were performed in myocardiums and skeletal muscles of the surviving animals. The most sensitive methods for detecting chronic chagasic infection were the routine histologic studies (73%) and the ECGs 83% and 89% on 6 and 9 mo. post-infected mice, respectively. Myocardial involvement varied from interstitial mild focal lymphocyte infiltrates up to replacement of myocytes by loose connective tissue. Atrial myocardiums (21/23, 91%) were more affected than ventricles (9/23, 39%). Typical chagasic nests were rarely found. Skeletal muscle involvement (11/18 and 7/9) varied from mild to extensive lymphocyte and plasmacell infiltrates, and necrotic fibers. The involved antigen were shown in skeletal muscles by the immunoperoxidase technique as diffusely arranged granular intracytoplasmatic deposit for both IgC and total immunoglobulins. The coincidence between this technique and histologic muscle lesions was 11/18 (61(%) in 6 mo. and 6/8 (75%) at 9 mo. post-infection. In heart, delicate granular deposits of total immunoglobulins were seen diffusely arranged within the ventricular myocytes; coincidence between immunoperoxidase technique anl histologic involvement increased from 36 to 66% in animals sacrifeced 6 and 9 mo. post-infection. This strongly stressed the increase of immunologic phenomena with the chronification of infection. Concerning sensitivity, immunoperoxidase and direct immunofluorescence were highly sensitive in skeletal muscle (100%, p < 0.01). Conversely, direct immunofluorescence technique showed poor results in heart while immunoperoxidase increased its sensitivity from 21.4% (at 6 mo.) to 66.6% (at 9 mo.) post-infection (p < 0.001). Considering the necessity of obtaining an adequate vaccine in order to prevent this disease an experimental model like this, rendering immunological reactions as revealed by the immunoperoxidase technique, would be useful.

Cardiac sympathetic-parasympathetic balance in rats with experimentally-induced acute chagasic myocarditis

To clarify the mechanism responsible for the transient sinus tachycardia in rats with acute chagasic myocarditis, we have examined the cardiac sympathetic-parasympathetic balance of 29 rats inoculated with 200,000 parasites (Trypanosoma cruzi). Sixteen infected animals and 8 controls were studied between days 18 and 21 after inoculation (acute stage). The remaining 13 infected animals and 9 controls were studied between days 60 and 70 after inoculation (sub-acute stage). Under anesthesia (urethane 1.25 g/kg), all animals received intravenous atenolol (5 mg/kg) and atropine (10 mg/kg). Acute stage: The baseline heart rate of the infected animals was significantly higher than that of the controls (P < 0.0001). The magnitude of the negative chronotropic response to atenolol was 4 times that of the controls (P < 0.00001). This response correlated with the baseline heart rate (r= - 0.72, P < 0.001). The heart rate responses to the beta-blocker and to atropine, of the infected animals studied during the sub-acute stage, were not different from controls. These findings suggest that cardiac sympathetic activity is transiently enhanced and cardiac parasympathetic activity is not impaired, in rats with acute chagasic myocarditis. The transient predominance of cardiac sympathetic activity could explain, in part, the sinus tachycardia observed in the acute stage of experimentally-induced chagasic myocarditis.

Heart rate responses to a muscarinic agonist in rats with experimentally induced acute and subacute chagasic myocarditis

We administered arecoline to rats, with experimentally induced chagasic myocarditis, in order to study the sinus node sensitivity to a muscarinic agonist. Sixteen month old rats were inoculated with 200,000 T. cruzi parasites ("Y" strain). Between days 18 and 21 (acute stage), 8 infected rats and 8 age-matched controls received intravenous arecoline as a bolus injection at the following doses: 5.0, 10.0, 20.0, 40.0, and 80.0 mug/kg. Heart rate was recorded before, during and after each dose of arecoline. The remaining 8 infected animals and 8 controls were subjected to the same experimental procedure during the subacute stage, i.e., days 60 to 70 after inoculation. The baseline heart rate, of the animals studied during the acute stage (349 ± 68 bpm, mean ± SD), was higher than that of the controls (250 ± 50 bpm, p < 0.005). The heart rate changes were expressed as percentage changes over baseline values. A dose-response curve was constructed for each group of animals. Log scales were used to plot the systematically doubled doses of arecoline and the induced-heart rate changes. The slope of the regression line for the acutely infected animals (r = - 0.99, b =1.78) was not different from that for the control animals (r = - 0.97, b = 1.61). The infected animals studied during the subacute stage (r = - 0.99, b = 1.81) were also not different from the age-matched controls (r = - 0.99, b = 1.26, NS). Consequently, our results show no pharmacological evidence of postjunctional hypersensitivity to the muscarinic agonist arecoline. Therefore, these results indirectly suggest that the postganglionic parasympathetic innervation, of the sinus node of rats with autopsy proved chagasic myocarditis, is not irreversibly damaged by Trypanosoma cruzi.

Human chronic chagasic myocarditis: quantitative study of CD4 + and CD8 + lymphocytes in the inflammatory infiltrate

Myocardialexsudate CD4+ andCD8+ lymphocytes were counted in transmural left ventricular free wall frozen sections taken from 10 necropsied chronic cardiac chagasic patients. The cells were labeled with monoclonal antibodies using a streptavidin-biotin technique. We counted: 1) lymphocytes in the total exsudate (LTE) and, separately, 2) the lymphocytes touching orvery near to my oc ells (LTVNM). Lymphocytes were considered very near whenever their own nuclear shortest nuclear diameter was larger than their distance from myocells. CD8+ lymphocytes were more numerous than CD4+ lymphocytes, especially among the LTVNM. The LTE CD4/CD8 ratio was 0,37 ± 0,20, but the LTVNM CD4/CD8 ratio was smaller (0,23 ± 0,11). Among theLTE, 34 ± 11% ofCD8+ (against24 + 12% of CD4+) were LTVNM. All these differences were statistically significant. Both subtypes ofT-lymphocytes were found to have an intimate relationship with both ruptured and unruptured myocells, and parasites were not seen. These findings are in accordance with the idea that the myocardial cell lesions in the cardiac form of human Chagas' disease are mediated mainly by T- cytotoxic lymphocytes.

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

T cells, adhesion molecules and modulation of apoptosis in visceral leishmaniasis glomerulonephritis

Background: Immune complex deposition is the accepted mechanism of pathogenesis of VL glomerulopathy however other immune elements may participate. Further in the present study, no difference was seen between immunoglobulin and C3b deposit intensity in glomeruli between infected and non-infected dogs thus T cells, adhesion molecules and parameters of proliferation and apoptosis were analysed in dogs with naturally acquired VL from an endemic area. The dog is the most important domestic reservoir of the protozoa Leishmania (L.) chagasi that causes visceral leishmaniasis (VL). The similarity of VL manifestation in humans and dogs renders the study of canine VL nephropathy of interest with regard to human pathology. Methods: From 55 dogs with VL and 8 control non-infected dogs from an endemic area, kidney samples were analyzed by immunohistochemistry for immunoglobulin and C3b deposits, staining for CD4+ and CD8+ T cells, ICAM-1, P-selectin and quantified using morphometry. Besides proliferation marker Ki-67, apoptosis markers M30 and TUNEL staining, and related cytokines TNF-alpha, IL-1 alpha were searched and quantified. Results: We observed similar IgG, IgM and IgA and C3b deposit intensity in dogs with VL and non-infected control dogs. However we detected the Leishmania antigen in cells in glomeruli in 54, CD4+ T cells in the glomeruli of 44, and CD8+ T cells in 17 of a total of 55 dogs with VL. Leishmania antigen was absent and T cells were absent/scarse in eight non-infected control dogs. CD 4+ T cells predominate in proliferative patterns of glomerulonephritis, however the presence of CD4+ and CD8+ T cells were not different in intensity in different patterns of glomerulonephritis. The expression of ICAM-1 and P-selectin was significantly greater in the glomeruli of infected dogs than in control dogs. In all patterns of glomerulonephritis the expression of ICAM-1 ranged from minimum to moderately severe and P-selectin from absent to severe. In the control animals the expression of these molecules ranged from absent to medium intensity. It was not observed any correlation between severity of the disease and these markers. There was a correlation between the number of Leishmania antigen positive cells and CD4+ T cells, and between the number of CD4+ T cells and CD8+ T cells. In dogs presenting different histopathological patterns of glomerulonephritis, parameters of proliferation and apoptosis were studied. Ki-67, a proliferative marker, was not detected locally, but fewer apoptotic cells and lower TNF-alpha expression were seen in infected animals than in non-infected controls. Conclusion: Immunopathogenic mechanisms of VL glomerulonephritis are complex and data in the present study suggest no clear participation of immunoglobulin and C3b deposits in these dogs but the possible migration of CD4+ T cells into the glomeruli, participation of adhesion molecules, and diminished apoptosis of cells contributing to determine the proliferative pattern of glomerulonephritis in VL.

Central nervous system involvement in Chagas' disease: an updating

A review was made of the available literature on central nervous system (CNS) involvement in Chagas' disease. Thirty-one works concerning the acute nervous form and 17 others dealing with the chronic nervous form, all presenting neuropathologic studies, were critically analysed. Based on this analysis, an attempt was made to establish the possible natural history of CNS involvement in Chagas' disease. Among others, the following facts stand out: 1) the initial, acute phase of Trypanosoma cruzi infection is usually asymptomatic and subclinical; 2) only a small percentage of cases develop encephalitis in the acute phase of Chagas' disease; 3) the symptomatic acute forms accompanied by chagasic encephalitis are grave, with death ensuing in virtually all cases as a result of the brain lesions per se or of acute chagasic myocarditis, this being usually intense and always present; 4) individuals with the asymptomatic acute form and with the mild symptomatic acute form probably have no CNS infection or, in some cases, they may have discrete encephalitis in sparse foci. In the latter case, regression of the lesions may be total, or residual inflammatory nodules of relative insignificance may persist. Thus, no anatomical basis exists that might characterize the existence of a chronic nervous form of Chagas' disease; 5) reactivation of the CNS infection in the chronic form of Chagas' disease is uncommon and occurs only in immunosuppressed patients.

Central nervous system involvement in experimental trypanosomiasis cruzi

A review of the available literature on central nervous system involvement in experimental trypanosomiasis cruzi is undertaken. From a critical analysis of 26 works on experimental infections with Trypanosoma cruzi (23 on the acute phase, 2 on the chronic phase, and one describing sequentially both phases), all supported by neuropathologic studies, it can be concluded that: 1) central nervous system involvement during the acute phase, in the form of encephalitis in multiple foci, with variable intensity of the parasitism and inflamatory changes, is frequent and well documented; 2) in animals with more severe central nervous system involvement death occurs as a result of the brain lesions or acute chagasic myocarditis, the latter being always present; 3) in animals with more discrete brain involviment death during the acute phase is due to complications not related to the nervous system, among which congestive heart failure second to acute chagasic myocarditis, a condition that is always present, regardless of whether or not the central nervous system is infected; 4) it is possible that in surviving animals that had mild encephalitis the inflammatory changes from the acute phase usually regress as the infection progress to the chronic phase.

Chronic experimental infection by Trypanosoma cruzi in Cebus apella monkeys

Twenty young male Cebus apella monkeys were infected with CAl Trypanosoma cruzi strain and reinfected with CA l or Tulahuen T.cruzi strains, with different doses and parasite source. Subpatent parasitemia was usually demonstrated in acute and chronic phases. Patent parasitemia was evident in one monkey in the acute phase and in four of them in the chronic phase after re-inoculations with high doses of CAl strain. Serological conversion was observed in all monkeys; titers were low, regardless of the methods used to investigate anti-T. cruzi specific antibodies. Higher titers were induced only when re-inoculations were perfomed with the virulent Tulahuén strain or high doses of CAl strain. Clinical electrocardiographic and ajmaline test evaluations did not reveal changes between infected and control monkeys. Histopathologically, cardiac lesions were always characterized by focal or multifocal mononuclear infiltrates and/or isolated fibrosis, as seen during the acute and chronic phases; neither amastigote nests nor active inflammation and fibrogenic processes characteristic of human acute and chronic myocarditis respectively, were observed. These morphological aspects more closely resemble those found in the "indeterminate phase" and contrast with the more diffuse and progressive pattern of the human chagasic myocarditis. All monkeys survived and no mortality was observed.

TNF/TNFR1 signaling up-regulates CCR5 expression by CD8+ T lymphocytes and promotes heart tissue damage during Trypanosoma cruzi infection: beneficial effects of TNF-α blockade

In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-α) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-α levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-α, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-α+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-α treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-α-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-α treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.

Miocardite chagásica em caninos no Estado do Rio Grande do Sul

Neste trabalho, são relatados dois casos de morte súbita por doença de Chagas aguda em caninos da zona rural de Porto Alegre, Rio Grande do Sul, Brasil. Os cães, um macho Pit Bull com nove meses (canino 1) e uma fêmea Labrador Retriever com dois anos (canino 2), morreram em janeiro de 2005 e maio de 2008, respectivamente. As necropsias revelaram aumento cardíaco em ambos os casos. O coração do canino 2 apresentou formato globoso com múltiplas áreas pálidas na musculatura cardíaca, mais evidentes no ventrículo direito e câmaras cardíacas dilatadas, principalmente as da direita. Ao exame histológico, ambos os casos apresentaram alterações semelhantes caracterizadas por infiltrado inflamatório difuso não-purulento acentuado, predominantemente linfocitário intersticial. Nas fibras miocárdicas, havia grande número de pseudocistos, repletos de formas amastigotas do Trypanosoma cruzi. Ao teste sorológico TESA-blot, amostra do canino 2 foi positiva para anticorpos IgM e IgG anti-T.cruzi, achado característico da fase aguda da miocardite chagásica. Os resultados indicam que a doença de Chagas deve ser investigada em casos de morte súbita em cães na região Sul do Brasil e que a espécie pode servir como reservatório e sentinela da doença em humanos.

Gene expression changes associated with myocarditis and fibrosis in hearts of mice with chronic chagasic cardiomyopathy

Chronic chagasic cardiomyopathy is a leading cause of heart failure in Latin American countries. About 30% of Trypanosoma cruzi-infected individuals develop this severe symptomatic form of the disease, characterized by intense inflammatory response accompanied by fibrosis in the heart.We performed an extensive microarray analysis of hearts from a mouse model of this disease and identified significant alterations in expression of ~12% of the sampled genes. Extensive up-regulations were associated with immune-inflammatory responses (chemokines, adhesion molecules, cathepsins, and major histocompatibility complex molecules) and fibrosis (extracellular matrix components, lysyl oxidase, and tissue inhibitor of metalloproteinase 1). Our results indicate potentially relevant factors involved in the pathogenesis of the disease that may provide newtherapeutic targets in chronic Chagas disease. © 2010 by the Infectious Diseases Society of America.

IL-17 Produced during Trypanosoma cruzi Infection Plays a Central Role in Regulating Parasite-Induced Myocarditis

Background: Chagas disease is a neglected disease caused by the intracellular parasite Trypanosoma cruzi. Around 30% of the infected patients develop chronic cardiomyopathy or megasyndromes, which are high-cost morbid conditions. Immune response against myocardial self-antigens and exacerbated Th1 cytokine production has been associated with the pathogenesis of the disease. As IL-17 is involved in the pathogenesis of several autoimmune, inflammatory and infectious diseases, we investigated its role during the infection with T. cruzi. Methodology/Principal Findings: First, we detected significant amounts of CD4, CD8 and NK cells producing IL-17 after incubating live parasites with spleen cells from normal BALB/c mice. IL-17 is also produced in vivo by CD4(+), CD8(+) and NK cells from BALB/c mice on the early acute phase of infection. Treatment of infected mice with anti-mouse IL-17 mAb resulted in increased myocarditis, premature mortality, and decreased parasite load in the heart. IL-17 neutralization resulted in increased production of IL-12, IFN-gamma and TNF-alpha and enhanced specific type 1 chemokine and chemokine receptors expression. Moreover, the results showed that IL-17 regulates T-bet, ROR gamma t and STAT-3 expression in the heart, showing that IL-17 controls the differentiation of Th1 cells in infected mice. Conclusion/Significance: These results show that IL-17 controls the resistance to T. cruzi infection in mice regulating the Th1 cells differentiation, cytokine and chemokine production and control parasite-induced myocarditis, regulating the influx of inflammatory cells to the heart tissue. Correlations between the levels of IL-17, the extent of myocardial destruction, and the evolution of cardiac disease could identify a clinical marker of disease progression and may help in the design of alternative therapies for the control of chronic morbidity of chagasic patients.