The rat mast cell line RBL-2H3.1 contains an 85-kDa cytosolic phospholipase A2 (cPLA2) that is very likely involved in liberating arachidonate from membrane phospholipid for the synthesis of eicosanoids following stimulation with either calcium ionophore or IgE/antigen. In this study, the intracellular location of cPLA2 was determined using immunofluorescence microscopy and immuno-gold electron microscopy. In nonstimulated cells, cPLA2 is distributed throughout the cytosol and is excluded from the nucleoplasm. Following cell activation with calcium ionophore, most of the cPLA2 translocates to the nuclear envelope, and the enzyme remains there during the entire period that ionophore is present. With IgE/antigen stimulation for 5 min, approximately 20-30% of the cPLA2 translocates to the nuclear envelope, and after 30 min of stimulation, most of the enzyme returns to the cytosol. Measurement of intracellular calcium using the dye Fura-2/AM shows that the level of calcium rises immediately after antigen is added, remains high for about 30 s, and then declines back to resting levels. Activation with calcium ionophore produces a 10-fold larger release of arachidonate than does stimulation with IgE/antigen. Thus, the results suggest that the extent of membrane binding of cPLA2 correlates with the release of arachidonate and that the site of arachidonate liberation is the nuclear envelope where many of the enzymes that oxygenate this fatty acid are located.

Although infection by group A streptococc is a model of extracellular mucosal pathogenesis, these organisms can be associated with highly invasive infections resulting in sepsis and shock. Over the last 6 yr this species has renewed its reputation as a si nt cause of sepsis and has piqued interest in the m nim by which some strains are better able to breach mucosal barriers to gain access to the bloodstream than are others. An ite tion assay was developed on the basis of resistance of intacellular streptococci to penicillin and gentamicin. Experiments showed that stationary-phase, as opposed to logarithmic-phase, bacteria are efficiently internalized and can persist in cultured human cells. Electron microscopy confirmed that streptococci were contained within intraceflular vacuoles. Various strains of streptococci revealed signicant differences in their capacity to be intenalied. Two type Ml streptococci Isolated from blood infections were internalized at frequencies equal to those reported for Sabnonella and Listeria monocytogenes and greater than the frequency of a cional variant from a case of pharyngitis.Numerous mucosal pathogens that readily invade the bloodstream have the capacity to be internalized and to persist within human epithelial cells (1). Although the importance of intracellular invasion in pathogenesis is debated by some, there is general agreement that this process can lead to invasion of deeper tissue and blood. The capacity ofListeria (2), Shigella (3), Salmonella (4), and several Yersinia species (5) to invade epithelial cells is entirely consistent with the pathophysiology associated with their infections. Listeria monocytogenes has become the paradigm for Gram-positive intracellular infections (6). L. monocytogenes possesses the adhesin internalin that mediates internalization and shares structural similarities to streptococcal surface proteins (6). In addition, both group A streptococci and L. monocytogenes express highly related thiol-activated hemolysins (7). Until recently, streptococcal species were traditionally considered to be extracellular pathogens of the oral or vaginal mucosa, even though some species have long been recognized to cause deadly blood or meningeal infections. Group A streptococci are commonly associated with pharyngitis and impetigo, but the recent resurgence of blood infections caused by this species has reminded the public health community of their potential to invade deeper tissues and the bloodstream and to cause significant morbidity (8,9 RPMI-1640 (1 ml) containing gentamicin (100 Ag/ml) and penicillin (5 pg/ml) was then added to wells to eliminate extracellular bacteria. Neither antibiotic penetrates eukaryotic cells (16). Infected monolayers were incubated at 370C for various time periods depending on the experiment before they were washed and dispersed by addition of 100 A4 of 0.25% trypsin/l mM EDTA. Streptococci were released from disrupted monolayers by the addition of 400 pA of 0.025% Triton X-100 (10). In later experiments, monolayers were...

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Our data suggest that topical application of linoleate-enriched oil such as sunflower seed oil might enhance skin barrier function and improve outcome in neonates with compromised barrier function. Mustard oil, used routinely in newborn care throughout South Asia, has toxic effects on the epidermal barrier that warrant further investigation.

Topical therapy to enhance skin barrier function may be a simple, low‐cost, effective strategy to improve outcome of preterm infants with a developmentally compromised epidermal barrier, as lipid constituents of topical products may act as a mechanical barrier and augment synthesis of barrier lipids. Natural oils are applied topically as part of a traditional oil massage to neonates in many developing countries. We sought to identify inexpensive, safe, vegetable oils available in developing countries that improved epidermal barrier function. The impact of oils on mouse epidermal barrier function (rate of transepidermal water loss over time following acute barrier disruption by tape‐stripping) and ultrastructure was determined. A single application of sunflower seed oil significantly accelerated skin barrier recovery within 1 h; the effect was sustained 5 h after application. In contrast, the other vegetable oils tested (mustard, olive and soybean oils) all significantly delayed recovery of barrier function compared with control‐ or Aquaphor‐treated skin. Twice‐daily applications of mustard oil for 7 d resulted in sustained delay of barrier recovery. Moreover, adverse ultrastructural changes were seen under transmission electron microscopy in keratin intermediate filament, mitochondrial, nuclear, and nuclear envelope structure following a single application of mustard oil. Conclusion: Our data suggest that topical application of linoleate‐enriched oil such as sunflower seed oil might enhance skin barrier function and improve outcome in neonates with compromised barrier function. Mustard oil, used routinely in newborn care throughout South Asia, has toxic effects on the epidermal barrier that warrant further investigation.