Increased Trypanosoma cruzi invasion and heart fibrosis associated with high transforming growth factor beta levels in mice deficient in alpha(2)-macroglobulin.

Trypanosoma cruzi proteinases are involved in host cell invasion in human patients and in mouse models. In mice, murine alpha(2)-macroglobulin (MAM) and murinoglobulin are circulating plasma proteinase inhibitors that also have important roles in inflammation and immune modulation. To define their role in experimental Chagas disease, we investigated the susceptibility to T. cruzi infection of mice that are deficient only in alpha2-macroglobulins (AM-KO) or in both MAM and monomeric murinoglobulin-1 (MM-KO), relative to the wild type (WT). Despite the high parasite load, parasitemia was lower in AM-KO and MM-KO mice than in WT mice. Nevertheless, we observed a significantly higher parasite load in the hearts of AM-KO and MM-KO mice, i.e., more amastigote nests and inflammatory infiltrates than in WT mice. This result demonstrates a protective role for MAM in the acute phase of murine T. cruzi infection. We further demonstrated in vitro that human alpha2-macroglobulins altered the trypomastigote morphology and motility in a dose-dependent way, and that also impaired T. cruzi invasion in cardiomyocytes. Finally, we demonstrated that the levels of transforming growth factor beta in AM-KO mice increased significantly in the third week postinfection, concomitant with high amastigote burden and important fibrosis. Combined, these in vivo and in vitro findings demonstrate that the MAM contribute to the resistance of mice to acute myocarditis induced by experimental T. cruzi infection.

Differential expression of mRNA coding for the alpha-2-macroglobulin family and the LRP receptor system in C57BL/6J and C3H/HeJ male mice.

Expression of mouse A2M (MAM), murinoglobulin (MUG), the A2M receptor or LDL-Receptor related protein (A2MR/LRP) and the Receptor Associated Protein (RAP) were measured by northern blotting of mRNA isolated from liver, heart and peritoneal macrophages from C3H/HeJ and C57BL/6J (B6) mice. Marked differences between males of the two mouse strains were observed for MAM and MUG mRNA levels in liver, which were reflected in plasma levels of both proteinase inhibitors, as confirmed by immune-electrophoresis. C3H/HeJ mice had higher levels of the MAM and MUG mRNA and their corresponding plasma proteins than B6 mice. B6 mice expressed higher levels of LRP mRNA relative to C3H/HeJ mice but had lower levels of RAP mRNA. LRP receptor activity, assayed by fluoresceinated-A2M binding, was higher in B6 cells. The present data contribute to the knowledge of genetic background characteristics among male mouse of these two strains, which can take part in many biological events such as lipid metabolism, inflammation and immune response to different infectious agents.

Trypanosoma cruzi: acute infection affects expression of alpha-2-macroglobulin and A2MR/LRP receptor differently in C3H and C57BL/6 mice.

Although a complete cellular and humoral immune response is elicited in Chagas' disease, recent data suggest that other natural elements of innate immunity may also contribute to the initial host primary defense. alpha-Macroglobulins are a family of plasma proteinase inhibitors that are acute-phase reactants in Trypanosoma cruzi-infected mice and humans. Mice contain a tetrameric alpha-2-macroglobulin (MAM) and a monomeric murinoglobulin (MUG). Heterogeneity in their reactions was observed in murine T. cruzi-infected plasma A2M levels despite an overall increase. In addition, up-regulation of the A2M receptor (A2MR/LRP) was observed in peritoneal macrophages during T. cruzi infection. Here, we show that during T. cruzi infection (Y strain), the MAM and MUG hepatic mRNA levels and the corresponding plasma protein levels were up-regulated in C3H and C57BL/6 (B6) mice, but with different kinetics. On the contrary, A2MR/LRP mRNA levels increased in acutely infected C3H mice, but decreased in B6 mice, in both liver and heart. Immunocytochemistry of infected B6 heart cryosections confirmed a less intense endothelium labeling by the fluoresceinated ligand for A2MR/LRP. On the other hand, infected B6 spleen cells displayed higher F-A2M-FITC binding and MAC1 expression, confirming higher A2MR/LRP expression in macrophages. In uninfected mice, as well as after T. cruzi infection, higher A2M plasma levels were measured in C3H mice than in B6 mice. The lower tissue T. cruzi parasitism found in C3H-infected mice could reflect an inhibitory effect of A2M on parasite invasion. Our present data further contribute to clarifying aspects of the role of A2MR/LRP in a model of acute Chagas' disease in different mouse strains.

Detection of alpha-macroglobulin in the heart of mice infected with Trypanosoma cruzi.

The aim of this work was to investigate the presence of alpha-macroglobulin (AM) in the heart of mice during acute experimental Chagas' disease and to study its localization as related to the presence of the parasite and/or to their antigens. Frozen heart tissue sections obtained from Swiss albino male mice at different days postinfection with Trypanosoma cruzi were examined for triple immunofluorescence in response to parasite antigen (green), AM (red), and nuclei (blue) from both cells. AM was found in the heart of all the infected animals studied. Parasites were seen arranged in nests inside heart muscle cells. Usually, AM staining corresponded in position to parasite nests and to their antigens spread in large areas of the myocardium. The most intense staining of AM was observed at days 9 and 11 postinfection, when the highest tissue-infection level occurs. These observations relate the presence of AM to that of T. cruzi antigen in the same areas of the inflamed myocardium of the chagasic animals.

Alpha-2-macroglobulin receptor is differently expressed in peritoneal macrophages from C3H and C57/B16 mice and up-regulated during Trypanosoma cruzi infection.

Chagas' disease is caused by the protozoan Trypanosoma cruzi. The acute phase of T. cruzi infection, which can be conveniently studied in mouse models, is thought to be a determinant of survival and of the pathological features of the chronic phase. With regard to the occurrence of early death and parasitaemia levels C3H and C57/B16 mice are classically classified as 'susceptible' and 'resistant' to T. cruzi infection, respectively. Alpha-2-macroglobulin (A2M) is a physiological proteinase inhibitor found in tissues and in the plasma of mammals. Previous studies showed that A2M plasma levels increase in C3H mice acutely infected by T. cruzi but do not change in C57/B16 mice. This difference might involve two possible phenomena, concerning A2M synthesis and/or clearance by its receptor (A2M-R). In this study, we examined by flow cytometry the binding of A2M-trypsin conjugated with FITC to macrophages from normal and T. cruzi-infected C3H and C57/B16 mice. Our present results show for the first time that A2M-R is expressed more (by approximately 33%) in the surface of cells from normal C57/B16 as compared to C3H mice. We also show that A2M-R expression is up-regulated in both strains during acute T. cruzi infection, but at higher levels and earlier in C57/B16 mice. At the same time, peritoneal cells become activated as judged by: (1) increase of their size and granularity; (2) gradual increase of Fc gamma RII/III expression assayed by 2.4G2 binding; (3) down-modulation of F4/80 binding, a mAb that recognizes an antigen typically expressed in resident macrophages. Finally, our results indicate that as macrophages become activated in vivo a higher expression of A2M-R is observed.

Alpha-2-macroglobulin binds to the surface of Trypanosoma cruzi.

Trypanosoma cruzi, the causative agent of Chagas disease, infects vertebrate cells after an initial step of parasite/host-cell recognition. Alpha-2-macroglobulin (A2M), an important type of physiological proteinase inhibitor found in tissues and in the plasma of mammals, inhibits cell invasion by T. cruzi and accumulates in sites of the inflamed myocardium associated with parasite antigens. To study whether A2M would bind to T. cruzi, an indirect immunofluorescence reaction was performed using two different anti-mouse A2M sera. Intense labeling was observed in the membrane lining the cell body and the flagellum of bloodstream trypomastigotes obtained from experimentally infected mice in the peak of parasitemia, suggesting that the antisera recognize plasma A2M associated with the parasite surface. Metacyclic trypomastigotes obtained in a serum-free defined medium reacted with anti-A2M only after previous incubation with purified human A2M. Enzyme-linked immunosorbent assay (ELISA) studies were applied to characterize better the binding of native (N-A2M) and of proteinase-complexed (P-A2M) forms of A2M. The "in vitro" binding of N-A2M to trypomastigotes was better at pH 5.0, followed by pH 10.0 and pH 7.4. Cysteinly and serine proteinase inhibitors, E-64 and STI, respectively, inhibited the reaction. P-A2M also bound to T. cruzi in a dose-dependent way. Flow-cytometry studies showed that about 80% of the parasites stained with fluorescein isothiocyanate (FITC)-labeled P-A2M (50 micrograms/ml) with high affinity at pH 7.4 (but also at pH 10.0) in a process that was reverted by the addition of unlabeled P-A2M or the calcium-chelator agent EDTA and also by incubation at an acid pH (4.0). These results suggest that (a) native-A2M binds to T. cruzi proteinase(s) and (b) T. cruzi expresses a receptor(s) that binds proteinase-complexed A2M.

Acute Chagas' disease: plasma levels of alpha-2-macroglobulin and C-reactive protein in children under 13 years in a high endemic area of Bolivia.

We measured the levels of two human acute phase proteins (APP), alpha-2-macroglobulin (A2M) and C-reactive protein (CRP), in sera of 56 healthy and 84 acute chagasic children aged from 1 to 13 years old, from a highly endemic area in Bolivia. In such areas, children are continuously exposed to vectors and the frequency of acute cases increases with age. Quantitation of A2M and CRP were performed using sandwich ELISAs, that were shown to be sensitive, reproducible and suited for studying many samples rapidly. A2M levels observed were higher in healthy younger children, decreasing with age until children reached their teens, and their distribution suggested a relationship between A2M concentration and age that could be consistently expressed by a power function. The same does not occur with CRP levels. Concentrations of A2M were age-adjusted to allow comparison using sera collected from children with different ages. Both A2M and CRP were significantly increased in acute chagasic children. Since parasites are commonly present in blood and tissues during the acute phase of Chagas' disease, it is possible that the high levels of A2M may act as inhibitors of a high load of proteinases, derived either from the parasites, from host cell damage or from both.

Acute-phase proteins and serologic profiles of chagasic children from an endemic area in Bolivia.

Chagas' disease represents a major public health problem in Latin America. In endemic areas, it is important to detect acute and even asymptomatic infections in children so that specific therapy can be started immediately. We studied 203 sera from children from the region of Cochabamba, Bolivia. A high percentage of seropositive individuals was found in the three villages studies. Levels of alpha-2 macroglobulin (A2M) and C-reactive protein (CRP) increased in a significant number of children with acute Chagas' disease. The combined analysis of serologic and biochemical parameters can define the different stages of acute infection by Trypanosoma cruzi: 1) an early stage, with an increase only in specific immunoglobulin M (IgM) levels; 2) intermediate stages, with high specific IgM and IgG levels and/or high anti-galactose (anti-Gal) levels and increased A2M and/or CRP levels; and 3) a late acute stage, with low IgM levels but high A2M, CRP, anti-Gal, and specific IgG levels. The detection of high IgG levels alone is indicative of the chronic/indeterminate stage of Chagas' disease. We also show serologic differences between seropositive asymptomatic villagers and symptomatic patients undergoing medical care; asymptomatic cases presented higher levels of A2M and lower levels of specific antibodies.

Cloning, sequencing, and phenotypic analysis of laf1, encoding the flagellin of the lateral flagella of Azospirillum brasilense Sp7.

Azospirillum brasilense can display a single polar flagellum and several lateral flagella. The A. brasilense Sp7 gene laf1, encoding the flagellin of the lateral flagella, was isolated and sequenced. The derived protein sequence is extensively similar to those of the flagellins of Rhizobium meliloti, Agrobacterium tumefaciens, Bartonella bacilliformis, and Caulobacter crescentus. An amino acid alignment shows that the flagellins of these bacteria are clustered and are clearly different from other known flagellins. A laf1 mutant, FAJ0201, was constructed by replacing an internal part of the laf1 gene by a kanamycin resistance-encoding gene cassette. The mutant is devoid of lateral flagella but still forms the polar flagellum. This phenotype is further characterized by the abolishment of the capacities to swarm on a semisolid surface and to spread from a stab inoculation in a semisolid medium. FAJ0201 shows a normal wheat root colonization pattern in the initial stage of plant root interaction.

Heterogeneity in the synthesis of alpha-macroglobulins in outbred Swiss albino mice acutely infected with Trypanosoma cruzi.

Alpha-Macroglobulins (AM) are protease inhibitors with important roles in inflammation and in immunomodulation that behave as acute-phase proteins in many experimental models. In the present work the levels of AM in the plasma of outbred Swiss albino mice acutely infected with Trypanosoma cruzi were studied. The results showed that increased levels of AM were present in the majority of the infected mice and that AM levels increased independently of the rise in parasitaemia. There was a high degree of heterogeneity in the intensity of the modulation of AM levels as well as in the kinetics of AM synthesis. This heterogeneity was related neither with the intensity of infection nor with the sex of the host. No correlation between AM levels and survival to the acute phase could be observed in the outbred mice. The consequence of such a heterogeneity is unclear, although AM as immunoregulatory molecules could play a role in the development of the symptoms of the chronic phase of Chagas' disease.

An inhibition ELISA to determine alpha macroglobulin levels in mouse plasma.

A sensitive method for quantifying mouse plasma alpha-macroglobulins (AM) using an inhibition ELISA is described. AM are important plasma proteinase inhibitors that possibly act also as immunomodulatory molecules. The standard protocol developed in our experiments involves coating well with 10 micrograms/ml A2M in carbonate buffer, followed by incubation with a 1:1 (v/v) mixture of the plasma to be tested (diluted 1/1000) and goat anti-AM (diluted 1/1250). This is followed by further incubation, first with the enzyme-conjugated antibody and with the substrate prior to the reading of absorbance levels of the reaction products. Standard curve samples must be included in each plate, employing known amounts of the purified Murine Alpha-2-Macroglobulin (MuA2M) used for coating, with concentrations ranging from 0.001 to 10 micrograms/ml. Using test samples in triplicates and a 6-point standard curve in a single ELISA plate, 25 plasma samples can be tested accurately. The method offers an useful tool for establishing AM levels in small samples of mouse plasma.

Heterogeneity in the plasma levels of two acute-phase proteins in mice from inbred strains infected with Trypanosoma cruzi.

We analyzed the variations observed in the plasma levels of both alpha-macroglobulins (AM) and serum amyloid P (SAP) in mice from three different inbred strains (C3H, Balb/C and C57black/6) acutely infected with Trypanosoma cruzi. SAP levels increased in C57black/6 and Balb/C mice but not C3H mice. AM levels increased in all C3H mice but not in C57black/6 mice and rose slightly in only 43% of the Balb/C mice. AM and SAP levels are differently modulated in patterns that may be strain-determined.

Trypanosoma cruzi: enhanced alpha-macroglobulin levels correlate with the resistance of BALB/cj mice to acute infection.

Trypanosoma cruzi proteinases are very likely involved in host-cell invasion. Physiological plasma-proteinase inhibitors from the macroglobulin (MG) family, among them alpha-2-macroglobulin (A2M), are found in tissues and in the plasma of mammals. By complexing to all classes of proteinases, MGs inhibit their action on high-molecular-weight substrates. In vitro studies have shown that A2M impairs T. cruzi proteases and, consequently, the parasite's ability to invade host cells and enhances the phagocytic and microbicidal actions of resident macrophages against T. cruzi. To test the hypothesis of a putative "protective" effect for MG, we quantified it in BALB/cj mice during the course of an experimental T. cruzi infection, comparing a posteriori the levels in mice that died with those in animals that survived, which were considered as being susceptible and resistant to the infection, respectively. The results showed that surviving mice showed an increase in plasma concentrations of MG during the first few weeks after the infection, whereas the levels in mice that died during the acute phase did not differ significantly from those in non-infected mice. These findings and the previous in vitro data indicate a role for physiological proteinase inhibitors, particularly alpha-macroglobulins, in resistance to T. cruzi infection, whereby a balance between parasite proteases and host protease inhibitors may be crucial. MG may thus participate in the complex network of reactions involved in the early acute phase of the disease and contribute by conferring to the host an ability to survive the infection.