Eduardo F. Lascano

Histological study of the progression of herpes simplex virus in mice

SummaryThe progress of an experimental infection with Herpesvirus hominis type 1 was studied in newborn mice inoculated into the foot pad of the hind leg. To trace the viral antigen, the unlabeled antibody enzyme PAP (peroxidase/antiperoxidase) method was employed. The virus antigen appeared first in the epidermal and connective tissue cells of the inoculation site, and then progressed along the sciatic nerve. This nerve was studied by electron microscopy and showed active multiplication within the Schwann cells, with the production of virions, some of which were found in the intercellular spaces. No intra-axonal particles were observed. The infection then spread to the spinal ganglia and to the spinal cord. In this progression, the pia mater appeared to play an important role. From the spinal cord, the infection spread to the encephalon. The present study supports a mixed route for the neural transport of herpes simplex virus: a) by cell-to-cell transmission (Schwann and connective tissue cells in the sciatic nerve; meningeal cells, neurons and glial cells in the CNS); b) by a passive motion of the virions along the intercellular spaces. The inoculated virus also gave rise to viremia with viral multiplication in several viscera.

Melatonin Inhibits β-Adrenoceptor-Stimulated Cyclic AMP Accumulation in Rat Astroglial Cell Cultures

We investigated whether astroglial cells are a site of action for the effect of melatonin on brain cyclic AMP content. Rat astroglial cell subcultures, identified according to morphological and immunochemical criteria, were used. Addition of melatonin to the cultures did not result in changes of cyclic AMP content. However, melatonin at 0.1-1 microM concentrations was able to impair the cyclic AMP increase elicited by 1 microM norepinephrine or isoproterenol in astroglial cultures. This melatonin effect was also shared by its biologically active analogues 5-methoxytryptophol and 6-chloromelatonin. Serotonin was only effective at a 100-fold greater concentration, while the biologically inactive melatonin metabolite 6-hydroxymelatonin was devoid of activity at any concentration used. These results suggest that methoxyindoles modulate negatively beta-adrenoceptor-induced cyclic AMP accumulation in cultured rat astroglial cells.

Astrocytic reaction predominance in chronic encephalitis of junin virus-infected rats

Junin virus antigen distribution and astrocytic reaction to prolonged infection were characterized in rat brain by the PAP technique. During the acute stage of neurologic disease following intracerebral inoculation, Junin antigen was detected in 100% of animals, strongly in most neurons but also to a much lesser degree in scattered astrocytes, dropping to 20% of rats at 540 days postinfection. Initially labeled in all brain areas, viral antigen gradually disappeared from hippocampus but persisted irregularly in cerebral cortex, basal ganglia, Purkinje cells, pons, and medulla oblongata. Such a pattern suggests that specific neuronal subpopulations, in spite of apparently unaltered cell morphology, may persistently harbor the virus, leading on occasion to a delayed neurologic syndrome. During both the acute and chronic stages of disease, a mild inflammatory exudate was observed, characterized by the presence of T and B lymphocytes, as well as macrophages and unidentified round cells. GFAP immunostaining showed increased astrocytic reaction as infection lapsed into chronicity. Corpus callosum, hippocampus, and cerebellum exhibited the sharpest reactive astrocytosis, followed by basal ganglia, pons, and medulla oblongata, whereas in cerebral cortex it was considerably less. Astrocyte activation, which failed to correlate with viral antigen presence in neurons, seems to result from a generalized condition, possibly including diffusible brain factors triggered by viral infection. Such widespread astroglial reaction may thus contribute to the outcome of the late neurologic syndrome.

Effect of staggered cyclophosphamide-immunosuppression on resistance to experimental Junin virus infection

SummaryOtherwise resistant adult mice were rendered susceptible to intracerebral Junin virus (JV) infection only when a staggered cyclophosphamide (CY) schedule was used.Forty-five-day old Balb/c mice, intracerebrally JV-infected and immunosuppressed with four 50 mg/kg body weight CY doses at days −1, +1, +4, +6 (day 0: viral infection) developed a lethal disease (86.6 per cent mortality) with high CNS viral titers and brain lesions. Neutralizing antibodies were absent throughout, while immunofluorescent antibody levels were considerably diminished.The transfer of hyperimmune serum conferred partial though significant protection on CY-treated animals but no correlation was found between CNS viral titers and mortality since in both infected CY-treated and untreated mice similar brain viral content was found.This was also confirmed by immune spleen cell transfer at day 0 where the clearance achieved was unable to modify the time course of the disease.Feasible mechanisms explaining recovery from JV infection by means of the protective effect of antibodies and the cell-mediated clearance are discussed.

Brain inflammatory exudate in Junin virus-infected rats: Its characterization by the immunoperoxidase (PAP) technique

Morphologic changes in cyclophosphamide (CY)-suppressed vs. control non-suppressed new-born rats infected i.c. with XJC13 strain of Junin virus were compared and the cells involved in CNS lesions were identified by the PAP technique. Fifty per cent of the control rats exhibited widespread cerebral necrosis vs. only 15% of the immunosuppressed animals. The first cells to reach Junin virus-infected CNS in controls were T lymphocytes, which destroyed viral antigen-laden target neurons and astrocytes. B lymphocytes and macrophages, presumably attracted by viral antigen and/or by lymphokines, made their appearance a day or two later. Activated macrophages phagocytosed necrotic cells and perhaps exerted a cytotoxic effect upon target neural cells, whereas the actual role of B lymphocytes requires further explanation. In CY-treated rats, cerebral lesions were smaller and the cellular exudate, though similar, proved much scantier than in controls. A similar extent of cerebellar necrosis was observed in both groups.

Immunoperoxidase tracing of Junin virus neural route after footpad inoculation

SummaryTo determine the pathway adopted by peripherally inoculated Junin virus (JV) to reach the CNS, rat tissues were serially harvested to trace the sequence of viral progression from right hind footpad to brain. Immunoperoxidase (PAP) labeling of viral antigen, concomitantly with infectivity assays and histological examination of each selected sample, were carried out. As from the 2nd week post-infection (pi), neurological disease inducing 100% mortality at 1 month was evident. At day 5 pi, viral antigen was first detected at footpad level in epidermic and dermic cells, as well as in neighbouring myocytes; labeled macrophages infiltrating small nerve branches were also disclosed. As from 10–15 days pi, viral antigen became apparent along ipsilateral sciatic nerve structures and within lumbar spinal ganglion neurons, followed by a fast viral spread throughout CNS neurons that involved spinal cord and brain.Concurrent histopathology featured minimal inflammatory reaction together with generalized astrocytic activation. Hematogenous viral transport was negligible, since JV was isolated much earlier and in higher infectivity titers in neural tissues than in blood. It may be concluded that after viral replication in footpad, JV neural route was demonstrated by its PAP labeling from peripheral nerves to cerebral cortex.

A Controlled Silver Impregnation Method to Characterize Cultured Cardiomyocytes

A morphological characterization of cultured cardiomyocytes was attempted using a modification of a silver impregnation technique originally described for connective tissue. Cardiac cells, obtained from newborn rats and grown as dissociated cultures on plastic surfaces, were fixed in methanol plus 5% glacial acetic acid, treated with potassium permanganate, decolorized in oxalic acid, sensitized with potassium bichromate, impregnated with a silver-ammonium complex, reduced in gelatin-formalin preparation, toned with gold chloride and fixed in sodium thiosulfate. The cultured cardiac cells tended to form a monolayer, although many myocytes remained isolated. Spherical nuclei, sharply stained with silver, were centrally located and surrounded by relatively plentiful cytoplasm packed with well delineated myofibrils. Contaminating fibroblasts were readily distinguished by their spindle-shaped nuclei and the presence of overstained collagen fibers, as well as the absence of myofibrils. In the absence of specific antibody for immunocytochemical identification of cardiomyocytes, morphological characterization of cell type and degree of differentiation by the controlled silver impregnation procedure described here provides a viable alternative, both in short- and long-term studies.

Diagnosis of Junin virus in cell cultures by immunoperoxidase staining

SummaryVero cells grown in Leighton tubes were infected with blood taken from Argentine Hemorrhagic Fever patients. In 11 out of 12 cases, and between the 2nd–8th days p.i. of the monolayers, Junin viral antigen was detected by the PAP method.

Experimental myocarditis by adenoviruses. I. Electron microscopy and infectivity of Aura virus in the mouse heart.

The Aura virus myocarditis of the new-born mouse was studied by virus assay and electron microscopy. High titers of virus were found in heart and blood from intracerebrally and subcutaneously infected mice, at times when virus particles and lesions were more frequently detected by electron microscopy (on the third postinoculation day). The cardiac myocyte of the Aura virus-infected mouse showed virus precursors within the cytoplasm and budding particles from the plasma membranes and from walls of sarcotubules. Mature virus particles were found in the intercellular space, within the lumina of sarcotubules and in membrane-bound intracellular spaces. Cell-free viral aggregates were observed within the lumina of myocardial capillaries. Incipient pathologic changes of the myocyte (swelling of mitochondria, sarcotubules and cytoplasmic matrix, and margination of chromatin in the nucleus) were relatively frequent findings. On the other hand, advanced lesions (loss of periodic bands of myofibrils, disorderly disposition of myofilaments, necrotic myocytes) were found in scarce, small foci.The Aura virus myocarditis of the new-born mouse was studied by virus assay and electron microscopy. High titers of virus were found in heart and blood from intracerebrally and subcutaneously infected mice, at times when virus particles and lesions were more frequently detected by electron microscopy (on the third postinoculation day). The cardiac myocyte of the Aura virus-infected mouse showed virus precursors within the cytoplasm and budding particles from the plasma membranes and from walls of sarcotubules. Mature virus particles were found in the intercellular space, within the lumina of sarcotubules and in membrane-bound intracellular spaces. Cell-free viral aggregates were observed within the lumina of myocardial capillaries. Incipient pathologic changes of the myocyte (swelling of mitochondria, sarcotubules and cytoplasmic matrix, and margination of chromatin in the nucleus) were relatively frequent findings. On the other hand, advanced lesions (loss of periodic bands of myofibrils, disorderly disposition of myofilaments, necrotic myocytes) were found in scarce, small foci.

Modification of Junin Virus Neurotropism in Mice by Selective Brain or Spinal Cord Passaging

The percentage of suckling mice that developed paralysis after intracerebral Junin virus (XJ-JV pathogenic strain) inoculation (13.8%) consistently increased after 5 serial passages of virus-infected brain or spinal cord obtained from paralytic animals, reaching 37.9 and 45.7%, respectively. As expected, all paralytic mice exhibited an identical spinal cord histologic picture, with widespread JV antigen in spinal cord astrocytes and neurons, particularly the large motor neurons of the anterior horn. These findings strongly support the existence of a motor neurotropic viral particle subpopulation in parental XJ-JV stock.