María I. Berría

Antibodies against astrocyte M1 and M2 muscarinic cholinoceptor from schizophrenic patients' sera.

We demonstrated the presence of circulating antibodies from schizophrenic patients able to interact with cultured astrocytes activating muscarinic acetylcholine receptors (mAChRs). Sera and purified IgG from 15 paranoid schizophrenic and 15 age‐matched normal subjects were studied by indirect immunofluorescence (IFI), flow cytometry, dot blot, enzyme immunoassay (ELISA), and radioligand competition assays. Astrocyte membranes and/or a synthetic peptide, with identical amino acid sequence of human M1 and M2 mAChR, were used as antigens. By IFI and flow cytometry procedures, we proved that serum purified IgG fraction from schizophrenic patients, reacted to astrocyte cell surface. The same antibodies were able to inhibit the binding of the specific mAChR radioligand 3H‐QNB. Using synthetic peptide for dot blot and ELISA, we demonstrated that these antibodies reacted against the second extracellular loop of human cerebral M1 and M2 mAChR. Also, the corresponding affinity‐purified antipeptide antibody displayed an agonistic‐like activity associated to specific M1 and M2 mAChR activation, increasing inositol phosphates accumulation and decreasing cyclic AMP production, respectively. This article gives support to the participation of an autoimmune process in schizophrenia disease.

Endothelial cell function alteration after Junin virus infection

Hematologic involvement is the main feature of Argentine hemorrhagic fever (AHF), an endemo-epidemic disease caused by Junin virus (JV). Since endothelial dysfunction could play a role in AHF-altered hemostasis, we studied human umbilical vein endothelial cell (HUVEC) infection with a virulent (JVv) and a non-virulent (JVa) JV strain. Cells were infected by the two JV variants with no detectable apoptosis or cytopathic effect. Both viral variants up-regulated ICAM-1 and VCAM-1 levels, while von Willebrand factor (VWF) production was decreased. Prostacyclin (PGI2) release and decay accelerating factor (DAF) expression were greater in JVv- than in JVa-infected or control cells. Furthermore, nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) expression was only raised in JVv-infected supernatants. Significant NO and PGI2 values were also detected in AHF patient sera. These data demonstrate that endothelial cell responses are triggered subsequently by JV infection, suggesting that such alterations play a major role in the pathogenesis of AHF and perhaps in other viral-induced hemorrhagic diseases.

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.

A triple staining procedure to evaluate phagocytic role of differentiated astrocytes

To determine whether phagocytic activity is affected by a viral infection known to induce astrocyte differentiation, a triple procedure (PAP labeling for GFAP, PAS reaction for added bakers yeast cells and hematoxylin for nuclear staining of the whole monolayer) was applied to Junin virus-inoculated cultures, as well as matched controls. The three-step staining simplified yeast cell count for subsequent statistical analysis, which discerned preferential uptake by differentiated rather than immature astrocytes. Accordingly, greater cell maturation induced by Junin virus was concomitant with early enhancement of phagocytic activity.

Differential Behaviour in Pancreas and Heart of Two Coxsackievirus B3 Variants

In order to characterize viral kinetics and pathogenic properties of two intratypic variants of coxsackievirus B type 3, Balb/c mice were intraperitoneally inoculated and serial samples harvested from days 2 to 28. Although both CB3o (amyocarditic) and CB3m (myocarditic) variants induced similar early infectivity titres in pancreas, only the latter led to severe acinar necrosis, followed in turn by patent viraemia and subsequent focal myocarditis. Nevertheless, when both variants were inoculated in cultured cardiac cells, neither infectivity nor cell death rate differed noticeable. Therefore, our findings indicate that overt myocarditis is not attributable to contrasting cardiomyocyte susceptibility to the tested variants but rather to prior viral events in pancreatic tissues.

Increased in vitro glial fibrillary acidic protein expression, telomerase activity, and telomere length after productive human immunodeficiency virus-1 infection in murine astrocytes

Although HIV‐associated neurocognitive disorders (HAND) result from injury and loss of neurons, productive infection routinely takes place in cells of macrophage lineage. In such a complex context, astrocytosis induced by local chemokines/cytokines is one of the hallmarks of HIV neuropathology. Whether this sustained astrocyte activation is able to alter telomere‐aging process is unknown. We hypothesized that interaction of HIV with astrocytes may impact astrocyte telomerase activity (TA) and telomere length in a scenario of astrocytic activation measured by expression of glial fibrillary acidic protein (GFAP). To test this hypothesis, cultured murine astrocytes were challenged with pseudotyped HIV/vesicular stomatitis virus (HIV/VSV) to circumvent the absence of viral receptors; and GFAP, telomerase activity, and telomere length were quantified. As an early and transient event after HIV infection, both TA activity and telomere length were significantly augmented (P < 0.001). Later, a strong negative correlation (−0.8616, P < 0.0001) between virus production and telomerase activity was demonstrated. Once HIV production had reached a peak (7 dpi), the TA decreased, showing levels similar to those of noninfected cells. In contrast, the astrocyte became activated, exhibiting significantly increased levels of GFAP expression directly related to the level of HIV/VSV replication (P < 0.0001). Our results suggest that HIV‐infected astrocytes exhibit early disturbance in their cellular functions, such as telomerase activity and telomere length, that may attenuate cell proliferation and enhance the astrocyte dysregulation, contributing to HIV neuropathogenesis. Understanding the mechanisms involved in HIV‐mediated persistence by altering the telomere‐related aging processes could aid in the development of therapeutic modalities for neurological complications of HIV infection.

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.

Glial fibrillary acidic protein (GFAP) immunochemical profile after Junin virus infection of rat cultured astrocytes

Cultured astrocytes derived from newborn rat brain were inoculated with Junin virus (JV) to characterize their response to infection by means of their glial fibrillary acidic protein (GFAP) immunochemical profile. Samples from 1 to 11 days post-inoculation (pi), as well as matched controls, were serially harvested for GFAP labeling by peroxidase-antiperoxidase (PAP) method. It was only at day 3 that significantly greater values of GFAP staining (P < 0.05) were disclosed by three complementary approaches: image analysis, ELISA and immunoblot densitometry. Since such increase was abolished by Triton X-100 treatment, soluble GFAP fraction appeared responsible for the early though transient enhancement of GFAP immunoreactivity that followed viral inoculation.

Differential Astrocyte Response to Theiler’s Murine Encephalomyelitis Virus Infection

Objectives: We aimed to address if selective astrocyte apoptosis is involved in the lack of murine demyelinating disease following infection by the L*-1 variant of Theiler’s virus. In addition, we investigated whether L*-1-infected astrocytes were able to selectively express molecules whose effects would play a role as pathogenic factors. Methods: Murine cultured astrocytes were infected with two Theiler viruses, the DA strain and the mutated DA variant L*-1, which does not synthesize the out of frame L* protein. Results: Neither DA nor L*-1 provoked apoptosis, although they replicated in astrocytes inducing GFAP and iNOS expression, as well as subsequent nitric oxide production. In addition, both viruses caused an enhanced expression of ICAM-1, VCAM-1 and decay accelerating factor (DAF). In this connection, values of VCAM-1 and DAF induced by L*-1 were higher and lower, respectively, than those induced by DA. Conclusions: Since no apoptosis was found, such mechanism would not be involved in the lack of TMEV-induced demyelinating disease by L*-1. In contrast, selective expression of VCAM-1 and DAF molecules induced by L*-1 could have a role in virus clearance from the central nervous system.