Dietary antioxidants, peroxidation and cardiovascular risks.

Most of the many epidemiological studies in the field strongly suggest that an equilibrated diet such as the so-called "mediterranean diet", is associated with protective effects against major diseases, and particularly, against cardiovascular risks. Since many reports also consider reactive oxygen species or free radical oxidations to be responsible for the accompanying disorders of most pathologies as well as for ageing, it is conceivable that natural plant metabolites such as polyphenols, are likely to play an important role in insuring this protection. Indeed, not only their presence, in particularly high amounts and varieties in foods of such a diet, but also, inter alia, their very potent antioxidant or radical scavenging properties, make polyphenols best accounting for the parodoxical part of the french paradox . Therefore, many efforts have been made to assess the mechanisms for such a cardiovascular disease protection. Whatever convincing were the polyphenols properties demonstrated by many in vitro experiments to support those theories, quite a great number of the results appeared somewhat contradictory when transposed to humans, in the in vivo situation. Some people totally refute this explanation, thinking that health benefits, as far as alcoholic beverages are concerned, originate from ethanol but also, with no doubt, some polyphenols even revealing to be pro-oxidants .

Polyphenols newly extracted in red wine from southwestern France by centrifugal partition chromatography.

Polyphenols from the ethyl acetate extracts of red wine were successfully fractionated using a four-step process (solvent extraction, ion-exchange column chromatography, centrifugal partition chromatography, and semipreparative HPLC), which resulted in the isolation of 22 compounds belonging to different classes of polyphenols (stilbenes, cinnamic acids, flavonoids). Five of them are red wine constituents reported for the first time. The newly isolated compounds include resveratrol dimers, dihydroflavonols, and a cinnamic derivative.

Growth inhibition of herpes simplex virus-type 1 in calphostin C-treated astrocytes.

The aim was to evaluate the effects of calphostin C (CC), a protein kinase C inhibitor, on lytic herpes simplex virus-type 1 infection of cultured rat astrocytes. At 24 h postinjection, the cell culture receiving CC treatment at 50 nM concentration showed decreased cell detachment and retraction versus untreated infected controls; likewise, the infective virus yield was significantly lower in a dose-dependent manner. In contrast, image analysis failed to disclose differences in viral antigen immunolabeling at low drug concentrations thus suggesting that CC-induced inhibition of cytopathic effects and infectivity taken place through mechanisms not involving viral protein synthesis. Given the low dose required and the apparent lack of cytotoxic effects, present findings encourage additional studies on CC antiviral potential in the whole organism.

Histometric demonstration of periastrocytic fibronectin loss during herpes simplex virus infection.

Since Herpes simplex virus-type 1 (HSV-1) is liable to induce modifications and/or loss of immunoreactive fibronectin (FN) in cultured cells, our present goal was to determine whether such loss is attributable to FN binding impairment rather than cell detachment secondary to viral cytopathic effect. For this purpose, we resorted herein to an histometric approach for statistical evaluation of FN pattern in the course of HSV-1 infection of astroglial cell monolayers. The length of FN positive fibers was calculated by means of their tracing on a digitizer tablet; in the same field, cell nucleus count was performed. Recorded data allowed the calculation of an index as the ratio of the length of FN positive fibers over cell nucleus number. On comparing the indices between infected and control cultures, matricial FN loss was found in the former at 48 h post-inoculation, accompanied by cell fusion and retraction, though without cell detachment. A significant loss of FN was thus demonstrated as an event prior to severe cytopathic effect induced by viral infection.

Histometric demonstration of periastrocytic fibronectin loss during herpes simplex virus infection.

Since Herpes simplex virus-type 1 (HSV-1) is liable to induce modifications and/or loss of immunoreactive fibronectin (FN) in cultured cells, our present goal was to determine whether such loss is attributable to FN binding impairment rather than cell detachment secondary to viral cytopathic effect. For this purpose, we resorted herein to an histometric approach for statistical evaluation of FN pattern in the course of HSV-1 infection of astroglial cell monolayers. The length of FN positive fibers was calculated by means of their tracing on a digitizer tablet; in the same field, cell nucleus count was performed. Recorded data allowed the calculation of an index as the ratio of the length of FN positive fibers over cell nucleus number. On comparing the indices between infected and control cultures, matricial FN loss was found in the former at 48 h post-inoculation, accompanied by cell fusion and retraction, though without cell detachment. A significant loss of FN was thus demonstrated as an event prior to severe cytopathic effect induced by viral infection.

Histometric demonstration of periastrocytic fibronectin loss during herpes simplex virus infection

Since Herpes simplex virus-type 1 (HSV-1) is liable to induce modifications and/or loss of immunoreactive fibronectin (FN) in cultured cells, our present goal was to determine whether such loss is attributable to FN binding impairment rather than cell detachment secondary to viral cytopathic effect. For this purpose, we resorted herein to an histometric approach for statistical evaluation of FN pattern in the course of HSV-1 infection of astroglial cell monolayers. The length of FN positive fibers was calculated by means of their tracing on a digitizer tablet; in the same field, cell nucleus count was performed. Recorded data allowed the calculation of an index as the ratio of the length of FN positive fibers over cell nucleus number. On comparing the indices between infected and control cultures, matricial FN loss was found in the former at 48 h post-inoculation, accompanied by cell fusion and retraction, though without cell detachment. A significant loss of FN was thus demonstrated as an event prior to severe cytopathic effect induced by viral infection.

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.

Extracellular matrix remodelling after coxsackievirus B3-induced murine myocarditis.

Weanling inbred Balb/c mice were intraperitoneally inoculated with a myocarditic variant of coxsackievirus B3. At days 1, 2, 4, 6, 8, 10, 14, 24 and 30 post-infection (p.i.), myocardial tissue was harvested for viral infectivity titrations and histological studies, including routine techniques (haematoxylin-eosin, Masson trichrome and von Kossa) and specialized procedures (silver impregnation for reticulin, picrosirius red stain for collagen and immunoperoxidase labelling for laminin). Virus was isolated as from day 2, reached maximal infectivity at days 6-8 and decreased gradually to become undetectable by day 14. Early histological findings during the 1st week consisted mainly of scattered foci of necrotic myocytes showing calcium deposits; slight mononuclear cell infiltration and fragmentation of both reticulin fibres and pericellular laminin were also present. From the 2nd up to 4th week p.i., inflammatory reaction abated concomitantly with the gradual development of fibrosis, as evidenced by reticulin fibre thickening, irregular laminin distribution and collagen fibre increase. Our results suggest that viral-induced necrosis is able to trigger marked extracellular matrix remodelling even in the case of minimal inflammation.

Fibronectin involvement following herpes simplex infection of cultured rat astrocytes.

To evaluate the potential impact of a strongly neurotropic virus on the matricial fibronectin (FN) of a given neural cell type, first subculture of rat astrocytes were inoculated with either a low (0.1-0.5) or a high MOI (10-100) of Herpes simplex virus-type 1 (HSV-1). Infected samples, as well as matched controls, were harvested from 12 to 72 h post-inoculation and processed for extracellular FN labeling by the peroxidase-antiperoxidase (PAP) method. Following low MOI, multinucleated cell formation and syncytial development drastically modified peripheral FN, since the typical loose network was largely replaced by a patch-like appearance. At high MOI, when viral action caused further cytoplasmic retraction leading to rounded or stellate cells, the FN pattern was remarkably altered, even with total FN loss around highly retracted cells. It may be concluded that a virus such as HSV-1, capable of modifying astrocyte morphology, induces a redistribution or loss of pericellular FN.

Influence of cell fixatives and incubation schedule on PAP labeling of fibronectin in cultivated fibroblasts.

A simple combined method for differential PAP labeling of fibronectin (FN) in mouse embryo fibroblast cultures was developed. Methanol-5% glacial acetic acid in dry ice-fixed cell monolayers showed mainly intracellular FN staining. Fixation with neutral paraformaldehyde before labeling, developed membrane- and extracellular matrix-associated FN. A combination of both procedures, which required incubation with primary antibody, fixation with paraformaldehyde followed by chilled acid methanol, and re-incubation with primary antibody, yielded sharp intracellular and extracellular FN labeling. The outlined methods can be readily employed in association with other staining techniques.