Permeability alterations of microvascular endothelia may be a factor in the plasma leakage produced by dengue virus infection. Confluent monolayers of the human dermal microvascular endothelial cell line HMEC-1 were utilized as an experimental model to study the cellular responses induced by the virus. Infected monolayers showed increased permeability for [ 3 H]mannitol, but no changes were observed for 4-70 kDa dextrans at 48 h post-infection (p.i.), a time at which viral titres reached maximal values and 40 % of the cells expressed viral proteins. A further increase in permeability occurred at 72 h, still without evident cytopathic effects on the monolayer. Coinciding with this, actin was reorganized in the infected cells and the tight junction protein occludin was displaced to the cytoplasm. Increments in the thickness of stress fibres and focal adhesions were observed in uninfected cells neighbouring infected cells. Culture medium from infected monolayers induced permeability changes and thickening of actin-containing structures in control cultures that resembled those observed 48 h p.i. Interleukin (IL) 8 was found in culture medium at concentrations ranging from 20 to 100 pg ml "1 . Neutralizing antibodies against IL8 partially inhibited the changes produced by the culture medium as well as those induced by addition of IL8. Genistein inhibited the effect of the culture medium and the phosphorylation of proteins associated with focal adhesions and indicated the participation of tyrosine kinases. These findings suggest that IL8 production by infected monolayers contributes to the virus-induced effect on the cytoskeleton and tight junctions and thereby modifies transendothelial permeability.

Biliary epithelia express high levels of CD44 in hepatobiliary diseases. The role of CD44-hyaluronic acid interaction in biliary pathology, however, is unclear. A rat model of hepatic cholestasis induced by bile duct ligation was employed for characterization of hepatic CD44 expression and extracellular hyaluronan distribution. Cell culture experiments were employed to determine whether hyaluronan can regulate cholangiocyte growth through interacting with adhesion molecule CD44. Biliary epithelial cells were found to express the highest level of CD44 mRNA among four major types of nonparenchymal liver cells, including Kupffer, hepatic stellate, and liver sinusoidal endothelial cells isolated from cholestatic livers. CD44-positive biliary epithelia lining the intrahepatic bile ducts were geographically associated with extracellular hyaluronan accumulated in the portal tracts of the livers, suggesting a role for CD44 and hyaluronan in the development of biliary proliferation. Cellular proliferation assays demonstrated that cholangiocyte propagation was accelerated by hyaluronan treatment and antagonized by small interfering RNA CD44 or anti-CD44 antibody. The study provides compelling evidence to suggest that proliferative biliary epithelia lining the intrahepatic bile ducts are a prime source of hepatic CD44. CD44-hyaluronan interaction, by enhancing biliary proliferation, may play a pathogenic role in the development of cholestatic liver diseases.

No abstract

Harvesting MDCK cells with trypsin-EDTA reduces potassium currents (IK) to a mere 10%, presumably by hydrolysis of K+ channels, but replating at confluence restores them in 12-18 hr, through a process that requires transcription, translation and exocytic fusion of intracellular membrane vesicles to the plasma membrane (Ponce & Cereijido, 1991; Ponce et al., 1991a). In the present work we find that this restoration of IK also requires cell-cell contacts and the presence of 1.8 mM Ca2+. The role of extracellular Ca2+ may be substituted by 2.0 microM TRH, 10 nM PMA or 200 micrograms/ml DiC8. drugs that stimulate the system of phospholipase C (PLC) and protein kinase C (PKC). Conversely, the recovery of IK triggered by Ca-dependent contacts can be blocked by 110 microM neomycin, 2.0 microM H7, and 250 nM staurosporine, inhibitors of PLC and PKC. These results suggest that the expression of new K+ channels depends on Ca(2+)-activated contacts with neighboring cells and that the information is conveyed through PLC and PKC, a process in keeping with changes in its enzymatic activity and cellular distribution of PKC. Plasma membrane is also reduced and restored upon harvesting and replating, and depends on Ca(2+)-activated contracts. However, the effects of the chemicals tested on IK differ from the ones they elicit on the recovery of plasma membrane, suggesting that cells can independently regulate their population of K+ channels and the surface of their membrane.

The effect of luteinizing (LH) and human chorionic gonadotropin (hCG) hormones on gap junctions (Gjs) and intercellular communication (ic) was evaluated in Leydig (interstitial) cells from mouse testes. Cell cultures enriched in Leydig cells were studied under control conditions and when maintained in the presence of 100 ng/mL LH, 10 ng/mL hCG, or 1 mM dibutiryl-cAMP (db-cAMP), for 8, 24, and 36 h. To monitor the extent of ic, Lucifer yellow (LY) was injected through a patch pipet into one cell of-small cell aggregates (6-10), and its transfer was evaluated using fluorescent microscopy. The expression of GJs was monitored using immunofluorescent (IF) labeling of connexin 43 (Cx43) with a specific antibody. Testosterone secretion was determined by radioimmunoassay. At all culture times, testosterone levels in the medium were higher in treated than in control cell cultures. In cell cultures of 8 h, LY transferred to most of the neighboring cells (93%) and cell membrane appositions showed abundant Cx43; no difference was found between control and treated cells. In contrast, in control cell cultures of 24 and 36 h, LY transferred to a reduced fraction of neighboring cells (46 and 21%, respectively) and Cx43 labeling was markedly decreased. Addition of LH, hCG, or db-cAMP, to cell cultures for 24 and 36 h completely prevented the decrease in ic and Cx43 expression. Immunoblot studies, from total protein homogenates of cell cultures of 36 h, showed that relative levels of 40- and 43-kDa bands, characteristic of Cx43, were higher in treated than in control cells. These results demonstrate that the expression of Cx43 and ic in Leydig cells is modulated by LH and hCG, and suggest that their effect is mediated by the second messenger of these hormones, cAMP.