Soluble cell adhesion molecules in human Chagas' disease: association with disease severity and stage of infection.

Formation of inflammatory lesions, one of the pathologic consequences of infection with Trypanosoma cruzi, involves intricate cell-cell interactions in which cell adhesion molecules (CAMs) are involved. Sera from 56 Chagas' disease patients grouped according to disease severity were studied for the presence of soluble intercellular adhesion molecule-1 (s-ICAM-1), soluble endothelial selectin (s-E-selectin), soluble vascular cell adhesion molecule-1 (s-VCAM-1), soluble platelet selectin (s-P-selectin), and s-CD44 were studied to determine if they could be used alone or in different combinations as markers for specific diagnostic procedures. Comparisons were made between congenitally, acutely, and chronically infected patients and aged-matched, noninfected individuals, as well as between patients with chronic Chagas' disease grouped according to the severity of their heart-related pathology. No differences in levels of s-CAMs were detected between sera from children with congenital T. cruzi infection and sera from noninfected infants born from chagasic mothers. In contrast, titers of s-ICAM-1, s-VCAM-1, s-selectin, and s-CD44 but not s-P-selectin were significantly increased in sera from patients during the acute phase of infection with T. cruzi. Titers of s-VCAM-1 and s-P-selectin were increased in chronically infected patients. A positive association with disease severity in sera from patients with chronic disease was observed for the levels of s-P-selectin. In contrast, we found no association between clinical symptoms and levels of s-VCAM-1. Patients with chronic disease with severe cardiopathy also showed diminished levels of s-CD44 in comparison with healthy controls or patients with mild disease. The results are discussed in the context of pathology of Chagas' disease.

Epitopes common to Trypanosoma cruzi and mammalian tissues are recognized by sera from Chagas' disease patients: prognosis value in Chagas disease.

Monoclonal antibodies (MoAbs) raised against Trypanosoma cruzi microsomal fraction (Mc) and cross-reactive with mammalian tissues were used to evaluate the ability of cross-reactive T. cruzi antigens to induce an immune response in Chagas' disease. Thus, we studied the ability of sera from Chagas' disease patients (CDP) with different degrees of cardiac dysfunction to block the immune recognition of these MoAb to the target antigen determining for each serum an inhibition index (II). By means of this approach we inferred that blocking of monoclonal antibody binding to T. cruzi microsomes by subjects' serum represents antibodies with the same reactivity. After serological and medical examinations, individuals were separated into the following groups: Chagas' disease patients without manifest cardiac involvement (CDP-0), CDP with suspected or borderline cardiac disease (CDP-1), CDP with moderate myocardial dysfunction (CDP-2), CDP with overt cardiac dysfunction (CDP-3) and controls including healthy subjects (HS) and patients with idiopathic myocarditis (IMP). The reactivity between MoAb 5F2 and its target antigen was significantly (p < 0.05) inhibited by sera from CDP irrespective of the clinical stage [CDP: n = 46, 50 +/- 20, mean II +/- SD: control: n = 16, 18 +/- 8]. Moreover, 5F2 was able to distinguish (p < 0.05) sera from CDP with mild disease (CDP clinical grade 0/1: n = 26, 34 +/- 18) from that of CDP with severe disease (CDP clinical grade 2/3: n = 20, 67 +/- 7). Moreover, the inhibitory capacity of sera from asymptomatic CDP (CDP-0) correlated with patients age (r = 0.66, p < 0.05). CDP-0 below or equal 40 years of age had results (n = 15, 25 +/- 13) comparable (p > 0.05) to that of controls while mean inhibition of CDP-0 over 40 years of age (n = 5, 60 +/- 5) was indistinguishable (p > 0.05) from that of patients with severe disease. Competitive assay with MoAb 5A9B11 also showed significant differences (p < 0.05) between sera from CDP (n = 46, 46 +/- 24) and controls (n = 13, 5 +/- 5). On the contrary, the differences observed between CDP with different cardiac involvement was not significant (mild: n = 26, 31 +/- 22; severe: n = 20, 66 +/- 11). However a thorough study of data from asymptomatic sera revealed the existence of two levels of reactivity, with low and high capacity to inhibit the reaction of 5A9B11 against Mc. On the contrary, CDP sera showed a blocking activity for 1A10C11 comparable to that of controls (CDP: n = 25, 19 +/- 9; control: n = 12, 14 +/- 6). Some cross-reactive MoAbs recognized epitopes partially composed of carbohydrates. Interestingly, 5F2 and 5A9B11 epitopes did not appear to have carbohydrates moieties. In summary, immunoinhibition assays revealed differences in the immune response of chronic chagasic patients against parasite epitopes. These results have opened the possibility to identify a prognosis marker of the disease suggesting the clinical utility of monitoring levels of these anti-Mc antibodies in patients with chronic Chagas' disease.

Increased expression and secretion of ICAM-1 during experimental infection with Trypanosoma cruzi.

In the present study we demonstrate that spleens and hearts from BALB/c mice infected with the virulent Tulahuén or the low virulent CA-I strains of Trypanosoma cruzi, contain substantially higher ICAM-1 transcripts than uninfected controls. ICAM-1 expression in heart cells was also increased in the protein level, as measured by flow cytometry, ELISA and immunohistochemistry. The adhesive receptor was observed not only on inflammatory cells but also on sarcolemma of cardiac myocytes from T. cruzi infected mice. ICAM-1 expression was higher during the acute phase than in the chronic phase of infection, and paralleled the density of inflammatory leukocytes. Elevated titres of soluble ICAM-1 (s-ICAM-1) were detected in sera from mice during the acute phase of infection with CA-I or Tulahuén parasites. Cytokines, including IFN-gamma, IL-1 alpha, IL-6 and TNF-alpha have been shown to modulate expression of ICAM-1. Spleens and hearts from mice infected with CA-I or Tulahuen strains showed increased accumulation of mRNAs specific for these cytokines, which peaked during the acute phase of infection. However, IFN-gamma activity was not necessary for ICAM-1 induction, as its levels were also increased during infection in IFN-gamma receptor knock-out (IFN-gamma R- ) mice. Upregulation of ICAM-1 expression might be a direct consequence of parasite infection, since its density on cell lines of different lineages was enhanced after 24 or 48 h of infection with T. cruzi.

[Role of cytokines in resistance and pathology in Trypanosoma cruzi infection]. / Papel de las citoquinas en la resistencia y patología durante la infección con Trypanosoma cruzi.

Chagas disease is associated with several immunological alterations. Although resistance against infection with Trypanosoma cruzi has been shown to be influenced by the immune system, its participation in the development of the disease remains unclear. In this regard, cytokines play a fundamental role since they are involved in the regulation of hemopoiesis, lymphopoiesis and affect the function of all cell types involved in an immune response. Interferon gamma (IFN-gamma) has been extensively involved as a protective lymphokine against T. cruzi. Macrophages activated by IFN-gamma result in the release of reactive oxygen metabolites (ROS) and nitric oxide (NO). On the other hand, interleukin 4 (IL-4), interleukin 10 (IL-10) and transforming growth factor beta (TGF-beta) are able to down-regulate the intracellular control of T. cruzi infection by IFN-gamma-activated macrophages, to inhibit NO release and to down-regulate the activity of the TH1 subset of cells (IFN-gamma producers). While TNF-alpha has been implicated in the resistance as well as in the generation of tissue damage, interleukin 6 (IL-6) and interleukin 1 (IL-1) are associated with a variety of alterations in endothelial cell function which may be responsible for the microvascular spasm seen in chagasic myocardiopathy. Several cytokines, including IFN-gamma, IL-1 alpha, IL-6 and TNF-alpha have been shown to modulate the expression of adhesion molecules which participate in inflammatory process by recruitment of lymphocytes into inflammatory sites, contributing to the progression of the local inflammatory reaction in chagasic cardiomyopathy. Thus, it has been shown that acute infection with different strains of T. cruzi induced enhanced expression of ICAM-1 not only on infiltrating leukocytes but also on sarcolemma of cardiocytes and paralleled the production of proinflammatory cytokines. Experimental infection with T. cruzi induces cytokine production which in time modulates the resistance against the parasite and probably the development of chronic Chagas disease. Therefore, it can be postulated that an alteration in quantity and/or quality of cytokine production may be the cause of chronic Chagas disease.

Bases moleculares e inmunológicas para el desarrollo de una vacuna contra la enfermedad de Chagas / Molecular and immunological bases for the development of a vaccine against Chagas'disease

Diferentes fracciones subcelulares de epimastigotes de T.cruzi fueron ensayadas en su capacidad de inducir protección o agresión en animales experimentales. La fracción flagelar (F) tuvo las mejores propiedades protectoras, sin efectos agresivos sobre los tejidos. Se prepararon varios anticuerpos monoclonales contra esta fracción. Dos de ellos, FCH-F8-1 y 4, mostraron capacidad de neutralizar la infectividad de tripomastigotes sanguíneos, de producir la lisis mediada por complemento de tripomastigotes de cultivo y de reconocer antígenos de la superfície de ambas formas epi y tripomastigotes. El anticuerpo FCH-F8-1, reconoce por inmunoprecipitación, una proteína de 85 kDa en tripomastigotes, mientras que en "blotting" reaccionó con una molécula de 43 kDa, en ambas formas del parásito. El otro anticuerpo, FCH-F8-4 reaccionó por esta última técnica, con varias proteínas de peso molecular entre 50 y 150 kDa, en epimastigotes y sólo con dos (15 y 48 kDa) en tripomastigotes. Ratones inmunizados con antígenos purificados por cromatografia de afinidad usando FCH-F8-4, fueron protegidos contra el desafio de formas infectantes. En una biblioteca de ADNc de epimastigotes de T. cruzi construída en el vector I gt11 se detectaron varios clones, tres con FCH-F8-4 y dos con FCH-F8-1. Dos clones, uno de cada grupo fueron estudiados, y (FCH-F8-1) 1 y (FCH-F8-4) 1. El tamaño de los insertos para ambos fue de 150 pares de bases y utilizados como sondas detectaron ARNm de epimastigotes de 3,5 y 5,0...

Bases moleculares e inmunológicas para el desarrollo de una vacuna contra la enfermedad de Chagas / Molecular and immunological bases for the development of a vaccine against Chagasdisease

Diferentes fracciones subcelulares de epimastigotes de T.cruzi fueron ensayadas en su capacidad de inducir protección o agresión en animales experimentales. La fracción flagelar (F) tuvo las mejores propiedades protectoras, sin efectos agresivos sobre los tejidos. Se prepararon varios anticuerpos monoclonales contra esta fracción. Dos de ellos, FCH-F8-1 y 4, mostraron capacidad de neutralizar la infectividad de tripomastigotes sanguíneos, de producir la lisis mediada por complemento de tripomastigotes de cultivo y de reconocer antígenos de la superfície de ambas formas epi y tripomastigotes. El anticuerpo FCH-F8-1, reconoce por inmunoprecipitación, una proteína de 85 kDa en tripomastigotes, mientras que en "blotting" reaccionó con una molécula de 43 kDa, en ambas formas del parásito. El otro anticuerpo, FCH-F8-4 reaccionó por esta última técnica, con varias proteínas de peso molecular entre 50 y 150 kDa, en epimastigotes y sólo con dos (15 y 48 kDa) en tripomastigotes. Ratones inmunizados con antígenos purificados por cromatografia de afinidad usando FCH-F8-4, fueron protegidos contra el desafio de formas infectantes. En una biblioteca de ADNc de epimastigotes de T. cruzi construída en el vector I gt11 se detectaron varios clones, tres con FCH-F8-4 y dos con FCH-F8-1. Dos clones, uno de cada grupo fueron estudiados, y (FCH-F8-1) 1 y (FCH-F8-4) 1. El tamaño de los insertos para ambos fue de 150 pares de bases y utilizados como sondas detectaron ARNm de epimastigotes de 3,5 y 5,0... (AU)

[Molecular and immunologic bases for the development of a vaccine against Chagas disease]. / Bases moleculares e inmunológicas para el desarrollo de una vacuna contra la enfermedad de Chagas.

Subcellular fractions of T. cruzi epimastigotes (Epi) were studied for their capability to induce protective or aggressive effects in animals. The flagellar fraction (F) showed the best immunoprotective properties without tissular aggression. Monoclonal antibodies were raised against F. Two of them, FCH-F8-1 and 4, were able to neutralize the infectivity of bloodstream forms, to mediate lysis by complement of cell culture derived[trypomastigotes (Tripo) and to recognize the surface of Tripo and Epi. FCH-F8-1 reacted with a 85 kDa protein from Tripo (assayed by immunoprecipitation) and with peptides of 43 kDa on Epi and Tripo (tested by immunoblotting). FCH-F8-4 recognized several proteins ranging from 50 to 150 kDa on Epi and two molecules of 15 and 48 kDa on Tripo. Mice immunized with antigens purified by affinity chromatography by using FCH-F8-4 were protected against the infection. Several recombinant clones were detected on a cDNA lambda gt11 expression library constructed from T. cruzi Epi (Tulahuén strain): three with FCH-F8-4 and two with FCH-F8-1. One clone recognized by each monoclonal antibody was studied gamma (FCH-F8-1) 1 and gamma (FCH-F8-4) 1. Both inserts were of 150 base pairs each; they detected a 3.5 and 5.0 kilobases Epi mRNA, respectively. Both inserts were sequenced, and the amino acid sequences were inferred. gamma (FCH-F8-4) 1 codified for a 19 aa peptide, PAFLGCSSRFSGSFSGVEP, and gamma (FCH-F8-1) 1 for a 29 aa peptide EFLERGRISCORHSYTSYTSCSDEHNVTPFC. The whole 19 aa peptide was synthesized. This peptide (SP4) inhibited the ELISA reactivity against the parasite of chronically infected and F immunized mouse sera.(ABSTRACT TRUNCATED AT 250 WORDS)

Bases moleculares e inmunológicas para el desarrollo de una vacuna contra la enfermedad de Chagas. / [Molecular and immunologic bases for the development of a vaccine against Chagas disease]

Subcellular fractions of T. cruzi epimastigotes (Epi) were studied for their capability to induce protective or aggressive effects in animals. The flagellar fraction (F) showed the best immunoprotective properties without tissular aggression. Monoclonal antibodies were raised against F. Two of them, FCH-F8-1 and 4, were able to neutralize the infectivity of bloodstream forms, to mediate lysis by complement of cell culture derived[trypomastigotes (Tripo) and to recognize the surface of Tripo and Epi. FCH-F8-1 reacted with a 85 kDa protein from Tripo (assayed by immunoprecipitation) and with peptides of 43 kDa on Epi and Tripo (tested by immunoblotting). FCH-F8-4 recognized several proteins ranging from 50 to 150 kDa on Epi and two molecules of 15 and 48 kDa on Tripo. Mice immunized with antigens purified by affinity chromatography by using FCH-F8-4 were protected against the infection. Several recombinant clones were detected on a cDNA lambda gt11 expression library constructed from T. cruzi Epi (Tulahuén strain): three with FCH-F8-4 and two with FCH-F8-1. One clone recognized by each monoclonal antibody was studied gamma (FCH-F8-1) 1 and gamma (FCH-F8-4) 1. Both inserts were of 150 base pairs each; they detected a 3.5 and 5.0 kilobases Epi mRNA, respectively. Both inserts were sequenced, and the amino acid sequences were inferred. gamma (FCH-F8-4) 1 codified for a 19 aa peptide, PAFLGCSSRFSGSFSGVEP, and gamma (FCH-F8-1) 1 for a 29 aa peptide EFLERGRISCORHSYTSYTSCSDEHNVTPFC. The whole 19 aa peptide was synthesized. This peptide (SP4) inhibited the ELISA reactivity against the parasite of chronically infected and F immunized mouse sera.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme immunoassay in cell monolayers for evaluation of in vitro activity of chemotherapeutic agents against Trypanosoma cruzi.

A standardized enzyme-linked immunosorbent assay (ELISA), performed directly on monolayers of mouse peritoneal macrophages or L 929 fibroblasts, was used to evaluate the activity of chemotherapeutic agents against four different stocks of Trypanosoma cruzi. Absorbance readings, performed in an automatic ELISA reader, were directly related to the number of intracellular parasites as determined by microscopic examination of tissue culture slides run in parallel. Results were highly reproducible in replicate wells and in repeated experiments. Results with nifurtimox, ketoconazole, and formycin B, compounds known to have in vivo activity against T. cruzi, revealed that the ELISA technique was capable of detecting small dose-response variations in the rate of phagocytosis of different life cycle stages of T. cruzi by normal and activated mouse macrophages.

Serum neuraminidase activity and hematological alterations in acute human Chagas' disease.

Neuraminidase activity was detected in serum 12 days after an accidental laboratory infection with Trypanosoma cruzi, peaked at the time when clinical symptoms appeared, and dropped to undetectable values by the day antibodies specific for T. cruzi were first demonstrated. A seric factor blocking neuraminidase activity was demonstrated when antibodies were first detected and persisted with high titers for at least 12 weeks thereafter. Erythrocyte and white blood cell counts as well as hemoglobin and hematocrit were below the lower limit of normality when seric neuraminidase activity was at its peak.

Circulating Trypanosoma cruzi from the same cloned population show differences in the ability to infect cells and to cause lethal infection in mice.

Two subpopulations of circulating parasites displaying different abilities to infect mammalian cells and to cause lethal infection when inoculated into normal mice were demonstrated in the blood of mice acutely infected with T. cruzi. Parasites of one subpopulation rapidly penetrated mouse fibroblasts and were readily phagocytized by normal mouse peritoneal macrophages whereas parasites of the other subpopulation showed little ability to invade non-phagocytic cells and resisted phagocytosis. Inoculation of organisms of this latter population into mice resulted in infections with lower parasitemias and longer time to death as compared to controls inoculated with organisms from a population containing both types of parasites. When a population of parasites containing both types of trypanosomes was cultured in acellular medium at 28 degrees C a decrease in the number of parasites was noted to occur in the initial days of culture. This decrease was not noted when parasites of the subpopulation of trypanosomes resistant to phagocytosis were cultured similarly.

Chagas' disease: humoral response to subcellular fraction of Trypanosoma cruzi in symptomatic and asymptomatic patients.

The capacity of antibodies in serum from individuals with chronic Chagas' disease to react with antigens in different subcellular fractions of Trypanosoma cruzi varied according to the clinical status of the patients. Antibodies in serum of asymptomatic patients were directed mostly against antigens in the citosol of the parasite, whereas in overtly cardiopathic patients antibodies were directed mostly against antigens in the microsomal fractions.

Mechanism of cell invasion by Trypanosoma cruzi: importance of sialidase activity.

The sialidase activity of trypomastigotes of Trypanosoma cruzi and its relationship to the ability of different stocks of the organism to infect cultured cells was examined. Sialidase activity in lysates of trypomastigotes was confirmed and shown to be present in organisms of four different stocks of T. cruzi. In addition, sialidase activity was detected in sera of mice acutely infected with organisms of each of the stocks of T. cruzi examined. Erythrocytes from these mice were agglutinated by peanut lectin, suggesting sialidase activity in vivo. Treatment of normal mouse peritoneal macrophages with sera from acutely infected mice resulted in an increased capacity of the cells to internalize blood trypomastigotes. IgM or IgG antibodies specific to T. cruzi were not detected in the sera displaying sialidase activity. Treatment of parasites and/or normal mouse macrophages with Vibrio cholerae neuraminidase, however, had little effect in the rate of internalization of parasites. Treatment of L 929 mouse fibroblasts with neuraminidase reduced significantly the rate of infection of the cells with blood trypomastigotes. Anti-sialidase activity developed and was detected in sera of infected mice and humans, suggesting that the neuraminidase activity of the parasite may play a significant role in the invasion of host cells only during the initial phase of the infection.

Activity of recombinant tumor necrosis factor on Toxoplasma gondii and Trypanosoma cruzi.

Activated macrophages produce tumor necrosis factor (TNF), a cytokine with anti-tumor and anti-plasmodia activities. This study revealed that recombinant TNF (rTNF) inhibits intracellular multiplication of blood trypomastigotes of Trypanosoma cruzi in murine peritoneal macrophages. rTNF did not have any apparent direct effect on the survival of extracellular T. cruzi or on its ability to infect mammalian cells. The degree of inhibition of the intracellular multiplication of T. cruzi was found to be a function of the time of exposure of the infected cells to rTNF. rTNF induced a comparable effect when different strains of the parasite were used. In contrast to its activity on T. cruzi, rTNF did not affect intracellular multiplication of Toxoplasma gondii tachyzoites or bradyzoites in normal murine peritoneal macrophages or in human fibroblasts. Killing of Toxoplasma tachyzoites by activated macrophages was not enhanced by rTNF.

On the mechanism of killing of Trypanosoma cruzi by human polymorphonuclear leukocytes.

The temperature-dependence of some processes involved in the killing of sensitized T. cruzi epimastigotes by human polymorphonuclear leukocytes (PMN) was determined. The rate of the reactions was related to the temperature of incubation according to the Arrhenius equation and the apparent energies of activation (Ea) were calculated. The Ea values separated these complex reactions into two groups: one with Ea of about 10 kcal/mol for the phagocytosis of the parasites and the release of lysosomal enzymes by PMN, and the other with Ea of about 22 kcal/mol for the cytotoxicity against sensitized T. cruzi, the rate of oxygen consumption by PMN, and the lysis of the parasites with added hydrogen peroxide.

Cell-mediated reactivity against human and Trypanosoma cruzi antigens according to clinical status in Chagas' disease patients.

The presence of cellular reactivity against homologous tissues and subcellular fractions of Trypanosoma cruzi was investigated in Chagas' disease patients (CDP). CDP were grouped in asymptomatic (CDP-1) and with probable (CDP-2) and overt (CDP-3) cardiomyopathy. Healthy and non-Chagasic cardiomyopathic subjects were studied as controls. Lymphoproliferative reactions against heart tissue extracts were detected in 42% of 72 CDP studied, with similar prevalence of positive reactions in all groups, and correlated with reactivity to both liver and kidney homologous tissues (P less than 0.001). These results confirm the existence of cellular immune reactivity against tissues in CDP, and indicate the lack of organ specificity of this reaction as well as the absence of relation with the clinical state of patients. Cellular reactivity to subcellular fractions of T. cruzi showed a definite pattern according to the clinical status of CDP. Although prevalence of T. cruzi stimulation appeared similar in all groups (70% in CDP-1, 82% in CDP-2 and 75% in CDP-3), CDP-3 showed a significantly higher reactivity to flagellar (69%) and cytosol (63%) fractions than CDP-1 (38 and 27%, respectively). These findings suggest a variable modulation of immune response according to the clinical state of T. cruzi infected subjects.

Trypanosoma cruzi: parasite-induced release of lysosomal enzymes by human polymorphonuclear leukocytes.

The release of beta-glucuronidase and lysozyme from human polymorphonuclear leukocytes (PMN) engaged in phagocytosis and lysis of Trypanosoma cruzi epimastigotes was studied in the presence or absence of chagasic serum. Lysosomal enzyme release was enhanced when parasites were sensitized with serum from a chronic Chagas' patient, increased up to 3 hr of incubation at 28 C, and depended on the PMN:parasite ratio. The release of lysosomal enzymes was determined by the presence of 2 mM cyanide, 2 microM azide, 3 mM amobarbital, and 1 mM phenylbutazone. These drugs inhibited the killing of sensitized T. cruzi by interfering with the oxidative microbicidal mechanisms of PMN without affecting the uptake of the parasites. Lysosomal enzyme release occurred in the presence of cyanide and azide, indicating that in these cases the enzymatic release was unrelated to the killing of the parasites. Amobarbital and phenylbutazone, which stabilize PMN membranes, inhibited the release of beta-glucuronidase and lysozyme by PMN. The addition of 10 micrograms/ml of cytochalasin B inhibited the phagocytosis and killing of sensitized T. cruzi by PMN but increased the enzymatic release by effector cells. Since cytochalasin B did not affect the close contact between PMN and parasites, it appears that the enzymes released to the extracellular milieu were not toxic to noningested parasites. Furthermore, the lysosomal enzymes did not lyse bystander unsensitized parasites. Therefore, the release of lysosomal enzymes during the interaction of T. cruzi epimastigotes and PMN seems to be related to the triggering event of the phagocytic process and does not bear a cause-effect relationship with parasite death.