Biomedical subjects
Publications and source records attributed to P M Cossio.
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Calomys musculinus, a wild cricetid rodent, is one of the main reservoirs of Junin virus. Six of these animals were infected by being placed in close contact with animals that had been experimentally infected with the virus. They were sacrificed at 10, 15 and 20 months after contact, and their salivary glands were studied by ultrastructural, immunohistochemical and virological methods. Two animals developed chronic viremia and low titers of complement-fixing antibodies. These animals were the only ones that had high viral titers in salivary glands and blood and viral antigen and particles in salivary glands. Although some of the other animals had viremia at the beginning of the experiment, it was absent 5 months later. Complement-fixing antibodies developed in all animals. On the basis of these findings, we assumed that the salivary gland is an important site of viral synthesis and excretion. This type of chronic infection, with persistent viremia and virus shedding, is possibly important for virus perpetuation in nature and transmission to man.
Immunofluorescent anti-Junin virus antibodies were detected in 15 nonfatal cases of Argentine hemorrhagic fever between the 2nd and 3rd week after onset of symptoms. In most cases, antibodies appeared by the day of clinical improvement, or between 2 and 4 days later. It was interesting to note that in 5 of 11 cases studied, the first positive serum sample presented anti-Junin virus immunofluorescent antibodies in IgA. In 2 of these 5 cases, IgA was the only immunoglobulin with antibody activity in the early positive serum sample.
An antibody reacting with Schwann sheaths of myelinated somatic and unmyelinated autonomic peripheral nerve was found in sixty-one out of seventy-one chronic, and nine out of ten acute, Chagas' disease sera. Indirect immunofluorescence (IFL) was carried out on rat, mouse and human somatic nerves and rat sympathetic nerves with initial serum dilutions of 1 : 10, and the staining reached a final titre of 1 : 320 in some cases. The antibodies fixed complement and were absorbed out by lyophilized epimastigotes of T. cruzi. Lipid extraction of the tissue sections enhanced the staining of myelinated nerve, whereas unfixed unmyelinated sympathetic nerve was strongly reactive. Central nervous tissue did not display any positive staining on neurons, glial cells or periaxonal sheaths. Furthermore, by using a double-labelled IFL technique, it was possible to show that a rabbit antiserum raised against guinea-pig spinal cord and the chagasic anti-nerve antibodies reacted with different structures in the rat sciatic nerve. These findings suggest that the reactive antigen(s) could be located on Schwann cells. The majority, but not all, of the chagasic individuals with anti-nerve antibodies also showed the sarcolemmal and endothelial staining (EVI) previously described in Chagas' disease. The possible recognition of Schwann cell antigens by circulating antibodies in Chagas' disease could be relevant, since an autonomic denervation has been postulated as a pathogenic mechanism of cardiomyopathy and megaviscera in this condition.
We investigated immunologic mechanisms and the role of complement in the pathogenesis of Argentine hemorrhagic fever, a disease caused by the Junin virus, a member of the arenavirus group. Total serum complement activity was reduced to 68 per cent of control values in patients with severe or moderate disease (P less than 0.001). C2, C3 and C5 values were also low (12 to 60 per cent) during the early acute period of the disease. However, serum C4 content was increased to 160 per cent of the control values in the same patients. Total complement activity returned to normal with clinical and laboratory recovery, at the time of detection of antibodies against Junin virus. C1q reactive material was found in four of 19 cases and no relation to the evolution of the disease could be established. These results suggest that immune complexes are not important in the pathogenesis of Argentine hemorrhagic fever, but that activation of the complement system has a role.
One hundred fifty-six of 1,250 sera from patients with presumed connective tissue and related diseases showed vascular staining on mouse liver cryostat sections when they were routinely checked for antinuclear factor by the indirect immunofluorescence test. In a third of the cases, the vascular immunofluorescent pattern was given by the EVI antibody reacting with the plasma membrane of striated muscle fibers and endothelial cells, as has been recently described to occur in Chagas' disease. This led to the detection of previously unsuspected Trypanosoma cruzi infection in 67.8% of the serum samples in which the EVI antibody was detected after observation of a positive vascular pattern with mouse liver cryostat sections. On the other hand, no significant relationship between Chagas infection and sera with other anti-striated-muscle immunofluorescent patterns that also showed a vascular staining on mouse liver cryostat sections was established. Consideration of the vascular pattern observed with the EVI antibody on mouse liver cryostat sections can be helpful in detection of previously ignored T. cruzi infection in patients who have connective-tissue diseases and related conditions. This is of interest in view of the fact that anergic immunodepressive therapy, often used in these patients, significantly alters the host-parasite relationship and may lead to severe dissemination of the parasite.
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Antibodies reacting against endothelial cells, vascular structures, and heart and skeletal muscle cells (EVI antibodies) were studied in 10 patients (one to 14 years of age) treated with Nifurtimox. The patients were observed for several months to two years after the onset of symptoms of acute infection with Trypanosoma cruzi. Although these 10 patients were selected because after treatment their sera became negative for antibodies to T. cruzi as detected by the immunofluorescence test, sera from six patients remained positive for EVI antibodies. It is suggested that EVI antibodies may be self-perpetuated in the absence of infection. Further studies are needed to determine the pathogenic significance of EVI antibodies.
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A method for detecting Junin virus viremia in guinea pigs is described. The method consists of infecting BHK-21 cells with blood samples from infected guinea pigs; 48 h later, Junin virus antigens are detected in the cells by indirect immunofluorescence. Application of this technique to patients with Argentine hemorrhagic fever may lead to the quickest method for the virologic diagnosis of this disease.
Immunopathologic and morphologic studies at the light and transmission electron microscope levels were carried out in myocardial biopsies of 4 chagasic individuals with circulating antibodies reacting with plasma membrane of striated muscle and endothelial cells (EVI antibody). Two cases did not present clinical evidences of heart involvement, and 2 cases showed chronic heart disease. In viv deposits of immunoglobulins were found at the plasma membrane of working myocardial cells and endothelial cells. The cytologic location of the in vivo bound gamma-globulin was coincident with the specificity of the EVI antibody. Ultrastructural studies showed intracellular alterations compatible with hypoxia of the fibers; these lesions, although they were more severe in the 2 cases with heart disease, were also present in the asymptomatic individuals. These results are congruent with a possible pathogenic effect of the EVI antibody. In 2 patients with Chagas' heart disease, foci of mononuclear infiltrates were examined by transmission electron microscopy. At that level, a close relationship between lymphoctes and muscle cells was observed, with imbrication of the plasma membranes and disappearance of the basal laminae. In the neighborhood of the lymphocytes, definite muscle cell abnormalities were found. These observations are also congruent with the recently suggested possibility that a lymphocyte-mediated immune response against heart tissue may participate in some of the pathogenetic mechanisms of chronic chagasic cardiopathy.
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Immunopathological and ultrastructural studies were carried out on kidney biopsies of eight children with a persistent nephropathy (PN) following the haemolytic-uremic syndrome (HUS). Significant amounts of in vivo-bound immunoglobulins and C3 were demonstrated by immunofluorescence methods in the glomeruli of all the cases, with a nodular pattern along the capillary walls. In four cases studied, C1q and C4 were also demonstrated, with an identical distribution. Transmission electron-microscope studies revealed a marked thickening of the glomerular basement membrane, and the existence of an electron-dense material with an intramembranous and subepithelial localization. With the exception of one case, serum complement studies did not present major modifications. Coagulation studies reveal that alterations observed in acute HUS were not present in the PN. Results of the present study suggest that an immune mechanism of glomerular damage operates in the pathogenesis of the PN observed after HUS, leading to a normocomplementemic, chronic glomerulonephritis. Although a hypothetical antigen (or perhaps several antigens) remains to be demonstrated, immunofluorescence and electron-microscope studies suggest the deposition of immune complexes in the renal lesions.
An antibody reacting with the plasma membrane of working myocardial cells, skeletal muscle fibres, and endothelial cells (EVI antibody) has been described in the sera of patients with Chagas' disease. In the present study of rat isolated atrial preparations beating in ddifferent media, direct immunofluorescence and ultrastructural immunohistochemical procedures indicate that the antibody can interact with the living tissue, becoming fixed to the plasma membranes. Transmission electronmicroscopy studies also showed the presence of sarcolemmal alterations. These observations suggest a possible pathogenic effect of the EVI antibody. The presence of EVI-positive sera in the beating medium leads to a significant increase in the frequency of contractions; no significant effects of EVI-positive sera in contractile force were seen. The increase in frequency could be prevented by previous treatment with a b-adrenergic blocking agent (MJ-1999), but not by an x-blocker (phentolamine) or by an anti-histamine compound (cyproheptadine). The changes described were observed only in those atrial preparations which were beating in media containing EVI-positive sera. In those atria beating in control media (KR,KR plus normal human serum, KR plus EVI-negative chagasic serum), neither immunological nor morphological or functional changes wersence of EVI-positive chagasic serum diminished atrial stimulation after added norepinephrine. These results suggest the possibility that the EVI antibody may act as a b-adrenergic agonist at the cell plasma membrane level. Such an effect might account for some of the clinical features of chronic Chagas' heart disease.
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Ultrastructural and immunohistochemical studies on tissues from five patients with Argentine hemorrhagic fever revealed previously undetected lesions caused by the viral infection. Two types of particle were seen in the cells of all organs examined. The particles had some characteristics similar to those described for arenaviruses. However, the virus-like particles were intracellular, had a single membrane, and apparently originated by a process of budding into the endoplasmic reticulum cisternae. Intranuclear bodies and three types of cytopolasmic change were observed in conjunction with the virus-like particles; Antigenic determinants of Junin virus were demonstrated in cells of all organs examined. Immunohistochemical experiments also indicated alterations in the cellular mechanisms of protein synthesis. Until now the pathogenesis of human diseases produced by arenaviruses has not been established. The results of this study suggest that in Argentine hemorrhagic fever the virus is responsible for a direct pathogenic action.