Role of the spleen on the pathogenesis of schistosomal periportal (pipestem) fibrosis of the liver: an experimental approach.

Splenectomized and sham-operated mice with chronic Schistosoma mansoni infection were compared for the development of periportal fibrosis (pipestem fibrosis). Although this lesion appeared less frequently in splenectomized mice, it also developed in the absence of the spleen. The time of splenectomy, spleen weight, presence of anti-idiotypic antibodies, the number of eggs in the liver, and the type and size of periovular granulomas in the liver as evaluated by computerized morphometry did not show statistically significant differences between the two groups. It is concluded that the role of the spleen in the development of pipestem fibrosis seems ancillary and that multifactorial influences, including worm burden, hepatic vascular adjustment, and factors associated with the biology of extracellular matrix of the liver, probably play a more significant role.

The indeterminate phase of Chagas' disease: ultrastructural characterization of cardiac changes in the canine model.

The indeterminate phase of Chagas' disease is defined as the prolonged period of clinically silent infection that follows the phase of acute primary infection with Trypanosoma cruzi. The dog is the only experimental animal model in which the indeterminate phase progresses to the late phase of severe, chronic myocarditis. This report describes the cardiac histologic and ultrastructural findings in dogs that survived the acute phase of infection with T. cruzi, becoming clinically and electrocardiographically normal for up to 3.5 years, while maintaining positive serologic test results during this period of time. Most of the myocardium appeared morphologically normal; however, small foci of mild, chronic myocarditis were present, with interstitial edema, mild fibrosis, and infiltration by lymphocytes, macrophages, and plasma cells. No microvascular lesions and no areas of close contact between immune effector cells and endothelial cells or cardiac myocytes were present. These findings were in sharp contrast to those observed in the canine model during the acute infection with T. cruzi. In this model, acute myocyte damage and lesions in the microcirculation, including fibrin microthrombi, were associated with close contacts between immune effector cells and myocytes or endothelial cells. Focally inflamed interstitial tissue showed increased deposition of amorphous and collagenous extracellular matrix as well as evidence of breakdown of collagen. The features of the inflammatory cells in the indeterminate phase of Chagas' disease were interpreted as indicating a self-limited cycle of focal inflammatory changes, with modulation and suppression of cell-mediated immune responses. Thus, we consider the indeterminate phase of Chagas' disease to be a stage of host-parasite equilibrium rather than a process of progressive damage.

A novel receptor mediated ATP transport system regulated by tyrosine and serine/threonine phosphokinases in Trypanosoma cruzi trypomastigotes.

Trypanosoma cruzi has a plasma membrane ATP transport system that may consist of an exterior receptor domain (ATP-R) and an interior domain regulated by tyrosine and serine/threonine kinases. The addition of exogenous ATP to freely swimming trypomastigotes resulted in a receptor-mediated inward movement of the nucleotide, and the system obeyed mass action law (Km, 9.42 microM and Vmax, 77.7 nmol x min(-1) x 4 x 10(6) trypomastigotes(-1)). Preloaded [3H]ADPi was not exchanged for ATP following the addition of increasing concentrations of exogenous ATPo to swimming trypomastigotes. Trypomastigote [ATP]o <--> ATP-R --> [ATP]i transport was [ATP]o-dependent and saturable at 100 microM. [ATP]o <--> ATP-R --> [ATP]i transport was abrogated by the tyrosine kinase inhibitors, genistein and lavendustin A. [ATP]o <--> ATP-R --> [ATP]i transport was also inhibited by the serine/threonine kinase inhibitor, staurosporin. Suramin, the antagonist of P2x and P2y purinergic receptors and the inhibitor of tyrosine kinase growth factor receptors, was also a very effective competitive inhibitor of the trypomastigote ATP transport system. The action of exogenous [gamma32P]ATPo resulted in the initial and simultaneous phosphorylation of a 63-kDa polypeptide (p63) and of a 92.4-kDa polypeptide (p92.4), which was followed by the abrupt phosphorylation of many other substrate proteins. The trypomastigote p63/p92.4 polypeptides may represent substrate proteins of a putative ATP-R-related tyrosine phosphokinase, and ATP receptors may transmit their signals by phosphorylation of specific substrate proteins.

Myocardial changes in acute Trypanosoma cruzi infection. Ultrastructural evidence of immune damage and the role of microangiopathy.

Histological and ultrastructural studies of the hearts of dogs sacrificed 18 to 26 days after intraperitoneal inoculation with 4 x 10(5) blood forms of the 12 SF strain of Trypanosoma cruzi/kg of body weight disclosed myocarditis characterized by parasitic invasion of some myocytes, damage and necrosis of nonparasitized myocytes, and interstitial infiltration by mononuclear cells. Nonparasitized myocytes showed alterations ranging from mild edema to severe myocytolysis. These changes often were accompanied by contacts of myocytes with lymphocytes (both granular and agranular) and macrophages. These contacts were characterized by focal loss of the myocyte basement membrane and close approximation of the plasma membranes of the two cells. Contacts between lymphocytes and capillary endothelial cells were also frequent. Platelet aggregates and fibrin microthrombi were observed in some capillaries. Our findings suggest that immune effector cells play a major role in the pathogenesis of the myocyte damage and the microangiopathy in acute Chagas' disease.

Cytomorphology and immunocytochemistry in the diagnosis of non-Hodgkin lymphomas.

1. Lymph node aspirates from 17 patients with an initial cytologic diagnosis of lymphoma (11 cases) or with suspected lymphoma (6 cases) were studied by immunocytochemistry, which led to a final diagnosis. Immunocytochemical staining demonstrated a B-cell phenotype in 10 cases, one case of anaplastic large cell Ki-1+ lymphoma, one lymphoblastic lymphoma negative for B and T cell markers, one large-cell unclassified lymphoma, and one inconclusive case. Three of the cases with suspected lymphoma were diagnosed as reactive lymphadenitis. 2. Combined cytomorphology and cytochemistry permitted a conclusive diagnosis in 13 out of 14 cases of lymphoma. Histology and immunohistochemistry confirmed the cytologic diagnosis. The inconclusive case was diagnosed as a T pleomorphic, small-cell lymphoma by histology. 3. The accuracy of cytomorphology associated with immunocytochemistry is high. However, the diagnosis of low-grade lymphomas, especially of a T phenotype may be difficult.

Cytomorphology and immunocytochemistry in the diagnosis of non-Hodgkin lymphomas

1. Lymph node aspirates from 17 patients with an initial cytologic diagnosis of lymphoma (11 cases) or with suspected lymphoma (6 cases) were studied by immunocytochemistry, which led to a final diagnosis. Immunocytochemical staining demonstrated a B-cell phenotype in 10 cases, one case of anaplastic large cell Ki-1+ lymphoma, one lymphoblastic lymphoma negative for B and T cell markers, one large-cell unclassified lymphoma, and one inconclusive case. Three of the cases with suspected lymphoma were diagnosed as reactive lymphadenitis. 2. Combined cytomorphology and cytochemistry permitted a conclusive diagnosis in 13 out of 14 cases of lymphoma. Histology and immunohistochemistry confirmed the cytologic diagnosis. The inconclusive case was diagnosed as a T pleomorphic, small-cell lymphoma by histology. 3. The accuracy of cytomorphology associated with immunocytochemistry is high. However, the diagnosis of low-grade lymphomas, especially of a T phenotype may be difficult.

Parasite and egg burden, hepatic collagen and histologic pattern of liver granulomas in selection III high and low antibody responder mice infected with Schistosoma mansoni

Selection III mice have particular immunological characteristics: they are high (H III) or low (L III) antibody producer animals, yet both lines display similar T cell responses and macrophage activities. We submittedthese mice to infection with Schistosoma mansoni to assess in vivo parasite and egg burden, hepatic collagen and cellular composition of granulomas in both lines. Titration of anti-Schistosoma IgG by ELISA showed remarkably higher values inH III line, at both studied periods (8th and 12th weeks post-infection). Nevertheless, the number of adult worms recovered from the portal system was similar inboth lines, being not associated with anti-Schistosoma antibody levels. There isan increase in hepatic collagen from the 8th to the 12th weeks post-infection, which is paralleled by an increase in the number of eggs in the liver. This association apparently occurs at the same radio in H III and L III animals. The most important difference found between the two lines was the outstanding contrast interms of volume and eosinophil counts in the granulomas, with lesions from H IIImice clearly being larger and containing more of these cells than LIII lesions

Parasite and egg burden, hepatic collagen and histologic pattern of liver granulomas in selection III high and low antibody responder mice infected with Schistosoma mansoni.

Selection III mice have particular immunological characteristics: they are high (H III) or low (L III) antibody producer animals, yet both lines display similar T cell responses and macrophage activities. We submitted these mice to infection with Schistosoma mansoni to assess in vivo parasite and egg burden, hepatic collagen and cellular composition of granulomas in both lines. Titration of anti-Schistosoma IgG by ELISA showed remarkably higher values in H III line, at both studied periods (8th and 12th weeks post-infection). Nevertheless, the number of adult worms recovered from the portal system was similar in both lines, being not associated with anti-Schistosoma antibody levels. There is an increase in hepatic collagen from the 8th to the 12th weeks post-infection, which is paralleled by an increase in the number of eggs in the liver. This association apparently occurs at the same ratio in H III and L III animals. The most important difference found between the two lines was the outstanding contrast in terms of volume and eosinophil counts in the granulomas, with lesions from H III mice clearly being larger and containing more of these cells than LIII lesions.

The subclinical form of experimental visceral leishmaniasis in dogs

Pathological aspects of a subclinical form of experimental canine leishmaniasis is reported here for the first time. Fifteen mongrel dogs were used in the present study. Eight dogs were infected and seven were used as control. Four of the control dogs were inoculated with spleen cells from non-infected hamsters. The eight mongrel dogs inoculated intravenously with amastigotes forms of Leishmania chagasi envolved for periods as long as 25 months without any clinical characteristic sign of classical Visceral Leishmaniasis (VL). Most of the laboratory test results were compatible to those of the seven control animals but culture of bone marrow aspirated material and serologic testing (IIF) demonstrated or provided evidence that the animals were infected. The most important and predominant histopathological lesion in infected animals were epitheloid granulomas presented in the liver, spleen, adrenal gland and lung of some animals. Channels containing erythrocytes in some granulomas of the liver suggeste that these granulomas are formed inside sinusoidal capillaries. Despite the animals were proved to be infected and presented characteristic histologic lesions, they did not present external signs of disease. The granulomatous aspect of the lesions indicates a good immunologic reactivity and suggest that a host-parasite equilibrium does exist in the dog experimental model

The subclinical form of experimental visceral leishmaniasis in dogs.

Pathological aspects of a subclinical form of experimental canine leishmaniasis is reported here for the first time. Fifteen mongrel dogs were used in the present study. Eight dogs were infected and seven were used as control. Four of the control dogs were inoculated with spleen cells from non-infected hamsters. The eight mongrel dogs inoculated intravenously with amastigotes forms of Leishmania chagasi evolved for periods as long as 25 months without any clinical characteristic sign of classical Visceral Leishmaniasis (VL). Most of the laboratory test results were compatible to those of the seven control animals but culture of bone marrow aspirated material and serologic testing (IIF) demonstrated or provided evidence that the animals were infected. The most important and predominant histopathological lesion in infected animals were epithelioid granulomas presented in the liver, spleen, adrenal gland and lung of some animals. Channels containing erythrocytes in some granulomas of the liver suggest that these granulomas are formed inside sinusoidal capillaries. Despite the animals were proved to be infected and presented characteristic histologic lesions, they did not present external signs of disease. The granulomatous aspect of the lesions indicates a good immunologic reactivity and suggest that a host-parasite equilibrium does exist in the dog experimental model.

Evaluation of spleen cell population and effect of splenectomy on granuloma modulation in BALB/c mice infected with Schistosoma mansoni

A kinetic study of the cells present in the spleen of BALB/c mice infected with Schistosoma mansoni was carried out. The lymphocytes were evaluated phenotypically with monoclonal antibodies and the effect of splenectomy on the modulation of periovular granuloma was also investigated. The infected mice had proportional increases in the numbers of neutophils, plasma cells, macrophages and eosinophils in the spleen. The largest number of neutrophil, plasma cells and macrophage were found between the 8th and the 12th week of infection, while the amount of eosinophils were higher later on, around the 20th week. The lymphocytes phenotipically characterized as Thy 1.2, Lyt 1.2 (CD4) increased mildly in proportional numbers. However, the percentage of lymphocytes with the Lyt 2.2 (CD8) phenotype, which is characteristic of supressor and cytotoxic T cells, increased significantly with the progress of the disease. The numbers of B lymphocytes, which comprise 50% of the mononuclear cells present in the spleen, increased significantly till the 16th week they began to decrease. The mean diameters of periovular granulomas were comparatively similar in both experimental groups (splenectomized and non-splenectomized mice). However, the evolutive types of granuloma (exudative, intermediate and fibrous) in splenectomized mice were proprtionally different from those of non splenectomized mice in the 16th and 24th week of infection. It is inferred that lymphonodes or other secondary lymphoide organs, in the abscence of the spleen, assume a modulating action on periovular granulomas, although the evolution of the granulomas is somehow delayed in splenectomized mice

Evaluation of spleen cell population and effect of splenectomy on granuloma modulation in BALB/c mice infected with Schistosoma mansoni.

A kinetic study of the cells present in the spleen of BALB/c mice infected with Schistosoma mansoni was carried out. The lymphocytes were evaluated phenotypically with monoclonal antibodies and the effect of splenectomy on the modulation of periovular granuloma was also investigated. The infected mice had proportional increases in the numbers of neutrophils, plasma cells, macrophages and eosinophils in the spleen. The largest number of neutrophil, plasma cells and macrophage were found between the 8th and the 12th week of infection, while the amount of eosinophils were higher later on, around the 20th week. The lymphocytes phenotypically characterized as Thy 1.2, Lyt 1.2 (CD4) increased mildly in proportional numbers. However, the percentage of lymphocytes with the Lyt 2.2 (CD8) phenotype, which is characteristic of suppressor and cytotoxic T cells, increased significantly with the progress of the disease. The numbers of B lymphocytes, which comprise 50% of the mononuclear cells present in the spleen, increased significantly till the 16th week when they began to decrease. The mean diameters of periovular granulomas were comparatively similar in both experimental groups (splenectomized and non-splenectomized mice). However, the evolutive types of granuloma (exudative, intermediate and fibrous) in splenectomized mice were proportionally different from those of non splenectomized mice in the 16th and 24th week of infection. It is inferred that lymphnodes or other secondary lymphoid organs, in the absence of the spleen, assume a modulating action on periovular granulomas, although the evolution of the granulomas is somehow delayed in splenectomized mice.

Experimental chemotherapy of Trypanosoma cruzi infection: persistence of parasite antigens and positive serology in parasitologically cured mice.

Mice infected with Trypanosoma cruzi, but parasitologically cured after specific chemotherapy, continued to exhibit positive indirect immunofluorescence serological tests 3-6 months after the therapy. Treatment of trypanosome antigens with monospecific antisera produced in rabbits, and examination by immunoelectron-microscopy following peroxidase labelling disclosed the presence of membrane deposits in cell processes in the spleens of the mice. Similar deposits were observed in the external membranes of T. cruzi amastigotes in the spleens of acutely infected mice, but not in normal control mice. No reaction occurred in tissues not previously treated with the monospecific anti-T. cruzi serum. Positive cells in treated and cured mice, as well as in the not cured or untreated control mice, were located in germinal centres of the splenic white pulp and presented long and branching cytoplasmic processes, which are indicative of dendritic cells of the lymphoid follicles of the spleen.

Demostracion de la infeccion placentaria por T. Cruzi a traves de la tecnica de inmunoperoxidasa / Demostration of transplacental infections for by T. cruzi inmunoperoxidease technique

Los autores en el presente trabajo, muestran la utilidad y especificidad de la tecnica de la inmuno-peroxidasa aplicada para la identificacion del T. Cruzi y sus formas degeneradas (exoantigenos). Tecnicas realizadas en cortes de tejido de placenta, cordon y membrana incluidos en parafina y procedentes de madres con infeccion chagasica cronica

Association of elevated anti-sarcolemma, anti-idiotype antibody levels with the clinical and pathologic expression of chronic Chagas myocarditis.

Antibody F(ab')2 fragments derived from the sera of four patients with histology-proven chronic Chagas myocarditis [cF(ab')2]-complexed antibody F(ab')2 fragments of children with acute Trypanosoma cruzi infection [aF(ab')2] in significantly higher molar ratios than those measured with F(ab')2 antibody fragments of normal subjects [nF(ab')2] from nonendemic areas (p less than 0.05). Anti-idiotype [cF(ab')2 X aF(ab')2] immune-complex formation was significantly blunted by preabsorption of cF(ab')2 with porcine heart atria sarcolemma (PAMs) immobilized on sepharose beads (inhibition, mean, 78.1 +/- 2.4%, n = 4). cF(ab')2 X nF(ab')2] immune-complex formation was also inhibited by pretreatment of cF(ab')2 with PAMs (inhibition, mean, 48.7 +/- 7.5%, n = 4). The sera of three groups of subjects from a geographic zone endemic for T. cruzi infection in the northeast of Brazil were assayed for free and immune-complexed IgG anti-acute T. cruzi infection F(ab')2. The indexed levels of free IgG anti-idiotype antibody activity and levels of IgG anti-idiotype immune complexes (IC') were markedly elevated in hospitalized patients with severe, decompensated chronic Chagas myocarditis (n = 23), and their IC' indexes were significantly higher than those measured in asymptomatic seropositive subjects from a nearby endemic village of the northeast of Brazil (Moniz Ferreira, n = 92, p less than 0.001) and in healthy seronegative villagers (n = 84, p less than 0.001). There exists a strong correlation between elevated IgG anti-sarcolemma, anti-idiotype activity levels and the clinical and pathologic expression of chronic Chagas myocarditis.