Immune regulation of protease-activated receptor-1 expression in murine small intestine during Nippostrongylus brasiliensis infection

Infection with gastrointestinal nematodes exerts profound effects on both immune and physiological responses of the host. Helminth infection induces a hypercontractility of intestinal smooth muscle that is dependent on the Th2 cytokines, IL-4 and IL-13, and may contribute to worm expulsion. Protease-activated receptors (PARs) are expressed throughout the gut, and activation of PAR-1 was observed in asthma, a Th2-driven pathology. In the current study we investigated the physiologic and immunologic regulation of PAR-1 in the murine small intestine, specifically 1) the effect of PAR-1 agonists on small intestinal smooth muscle contractility, 2) the effects of Nippostrongylus brasiliensis infection on PAR-1 responses, 3) the roles of IL-13 and IL-4 in N. brasiliensis infection-induced alterations in PAR-1 responses, and 4) the STAT6 dependence of these responses. We demonstrate that PAR-1 activation induces contraction of murine intestinal smooth muscle that is enhanced during helminth infection. This hypercontractility is associated with an elevated expression of PAR-1 mRNA and protein. N. brasiliensis-induced changes in PAR-1 function and expression were seen in IL-4-deficient mice, but not in IL-13- or STAT6-deficient mice, indicating the dependence of IL-13 on the STAT6 signaling pathway independent of IL-4.     

Formation and antiproliferative effect of prostaglandin E(3) from eicosapentaenoic acid in human lung cancer cells

We investigated the formation and pharmacology of prostaglandin E(3) (PGE(3)) derived from fish oil eicosapentaenoic acid (EPA) in human lung cancer A549 cells. Exposure of A549 cells to EPA resulted in the rapid formation and export of PGE(3.) The extracellular ratio of PGE(3) to PGE(2) increased from 0.08 in control cells to 0.8 in cells exposed to EPA within 48 h. Incubation of EPA with cloned ovine or human recombinant cyclooxygenase 2 (COX-2) resulted in 13- and 18-fold greater formation of PGE(3), respectively, than that produced by COX-1. Exposure of A549 cells to 1 microM PGE(3) inhibited cell proliferation by 37.1% (P < 0.05). Exposure of normal human bronchial epithelial (NHBE) cells to PGE(3), however, had no effect. When A549 cells were exposed to EPA (25 microM) or a combination of EPA and celecoxib (a selective COX-2 inhibitor), the inhibitory effect of EPA on the growth of A549 cells was reversed by the presence of celecoxib (at both 5 and 10 microM). This effect appears to be associated with a 50% reduction of PGE(3) formation in cells treated with a combination of EPA and celecoxib compared with cells exposed to EPA alone. These data indicate that exposure of lung cancer cells to EPA results in a decrease in the COX-2-mediated formation of PGE(2), an increase in the level of PGE(3), and PGE(3)-mediated inhibition of tumor cell proliferation.     

Diesel exhaust enhances virus- and poly(I:C)-induced Toll-like receptor 3 expression and signaling in respiratory epithelial cells

Prior exposure of respiratory epithelial cells to an aqueous-trapped solution of diesel exhaust (DE(as)) enhances the susceptibility to influenza infections. Here, we examined the effect of DE(as) on the Toll-like receptor 3 (TLR3) pathway, which is responsible for the recognition of and response to viruses and double-stranded RNA. Flow cytometric and confocal microscopy analyses showed that TLR3 is predominantly expressed in the cytoplasm of respiratory epithelial cells. To examine the effect of DE on TLR3 expression and function, differentiated human bronchial or nasal epithelial cells as well as A549 cells were exposed to DE(as) and then infected with influenza A or treated with polyriboinosinic acid-polyribocytidylic acid [poly(I:C)], a synthetic form of double-stranded RNA. Exposure to DE(as) before infection with influenza or stimulation with poly(I:C) significantly upregulated the expression of TLR3. Additionally, preexposure to DE(as) significantly increased the poly(I:C)-induced expression of IL-6. Overexpression of a dominant-negative mutant form of TNF receptor-associated factor 6 reversed the effects of DE(as) on poly(I:C)-induced IL-6 expression, suggesting that the response was TLR3 dependent. Similarly, preexposure to DE(as) significantly increased nuclear levels of interferon regulatory factor 3 and the expression of IFN-beta in response to poly(I:C). Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, was able to abate the effect of DE(as) on poly(I:C)-induced IFN-beta expression. Together, these results indicate that exposure of respiratory epithelial cells to DE(as) could potentially alter the response to viral infections by increasing the expression and function of TLR3.     

Functional importance of regional differences in localized gene expression of receptors for IL-13 in murine gut

IL-13 induces a STAT6-dependent hypercontractility of intestinal smooth muscle that is mediated by binding to the IL-13Ralpha1 component of the type 2 IL-4R that is linked to STAT6. IL-13 also binds to the IL-13Ralpha2 that is not linked to STAT6 and functions to limit the effects of IL-13 in vivo. In this study we assessed the contributions of regional and cellular differences in the distribution of the IL-13R components to the physiological regulation of smooth muscle function in wild-type mice and mice deficient in STAT6 or IL-13Ralpha2. The expression of IL-13 and IL-13Ralpha2 was higher in colon than in small intestine. Laser capture microdissection of specific cell types revealed that the expression of IL-13Ralpha2 was higher in the smooth muscle layer compared with levels in the epithelial cells of the mucosa. In contrast, there was a uniform distribution of IL-13alpha1 in smooth muscle, epithelia, and myenteric neurons. The significant hypercontractility of smooth muscle in mice deficient in IL-13Ralpha2, but not in STAT6, shows the physiological importance of IL-13 binding to IL-13Ralpha2. The pronounced differences in the expression of IL-13Ralpha2 suggest that the gut has developed sophisticated mechanisms for controlling the physiological and pathophysiological activities of IL-13.     

Responses of sawgrass and spikerush to variation in hydrologic drivers and salinity in Southern Everglades marshes

Aboveground net primary production (ANPP) by the dominant macrophyte and plant community composition are related to the changing hydrologic environment and to salinity in the southern Everglades, FL, USA. We present a new non-destructive ANPP technique that is applicable to any continuously growing herbaceous system. Data from 16 sites, collected from 1998 to 2004, were used to investigate how hydrology and salinity controlled sawgrass (Cladium jamaicense Crantz.) ANPP. Sawgrass live biomass showed little seasonal variation and annual means ranged from 89 to 639 gdw m⁻². Mortality rates were 20–35% of live biomass per 2 month sampling interval, for biomass turnover rates of 1.3–2.5 per year. Production by C. jamaicense was manifest primarily as biomass turnover, not as biomass accumulation. Rates typically ranged from 300 to 750 gdw m⁻² year⁻¹, but exceeded 1000 gdw m⁻² year⁻¹ at one site and were as high as 750 gdw m⁻² year⁻¹ at estuarine ecotone sites. Production was negatively related to mean annual water depth, hydroperiod, and to a variable combining the two (depth-days). As water depths and hydroperiods increased in our southern Everglades study area, sawgrass ANPP declined. Because a primary restoration goal is to increase water depths and hydroperiods for some regions of the Everglades, we investigated how the plant community responded to this decline in sawgrass ANPP. Spikerush (Eleocharis sp.) was the next most prominent component of this community at our sites, and 39% of the variability in sawgrass ANPP was explained by a negative relationship with mean annual water depth, hydroperiod, and Eleocharis sp. density the following year. Sawgrass ANPP at estuarine ecotone sites responded negatively to salinity, and rates of production were slow to recover after high salinity years. Our results suggest that ecologists, managers, and the public should not necessarily interpret a decline in sawgrass that may result from hydrologic restoration as a negative phenomenon.     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.     

A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells

Background  

Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis.     

Disabled-2 protein facilitates assembly polypeptide-2-independent recruitment of cystic fibrosis transmembrane conductance regulator to endocytic vesicles in polarized human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel expressed in the apical plasma membrane of fluid-transporting epithelia, where the plasma membrane abundance of CFTR is in part controlled by clathrin-mediated endocytosis. The protein networks that control CFTR endocytosis in epithelial cells have only been partially explored. The assembly polypeptide-2 complex (AP-2) is the prototypical endocytic adaptor critical for optimal clathrin coat formation. AP-2 is essential for recruitment of cargo proteins bearing the YXXΦ motif. Although AP-2 interacts directly with CFTR in vitro and facilitates CFTR endocytosis in some cell types, it remains unknown whether it is critical for CFTR uptake into clathrin-coated vesicles (CCVs). Disabled-2 (Dab2) is a clathrin-associated sorting protein (CLASP) that contributes to clathrin recruitment, vesicle formation, and cargo selection. In intestinal epithelial cells Dab2 was not found to play a direct role in CFTR endocytosis. By contrast, AP-2 and Dab2 were shown to facilitate CFTR endocytosis in human airway epithelial cells, although the specific mechanism remains unknown. Our data demonstrate that Dab2 mediates AP-2 independent recruitment of CFTR to CCVs in polarized human airway epithelial cells. As a result, it facilitates CFTR endocytosis and reduces CFTR abundance and stability in the plasma membrane. These effects are mediated by the DAB homology domain. Moreover, we show that in human airway epithelial cells AP-2 is not essential for CFTR recruitment to CCVs.     

Precursors and inhibitors of hydrogen sulfide synthesis affect acute hypoxic pulmonary vasoconstriction in the intact lung

The effects of hydrogen sulfide (H(2)S) and acute hypoxia are similar in isolated pulmonary arteries from various species. However, the involvement of H(2)S in hypoxic pulmonary vasoconstriction (HPV) has not been studied in the intact lung. The present study used an intact, isolated, perfused rat lung preparation to examine whether adding compounds essential to H(2)S synthesis or to its inhibition would result in a corresponding increase or decrease in the magnitude of HPV. Western blots performed in lung tissue identified the presence of the H(2)S-synthesizing enzymes, cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST), but not cystathionine β-synthase (CBS). Adding three H(2)S synthesis precursors, cysteine and oxidized or reduced glutathione, to the perfusate significantly increased peak arterial pressure during hypoxia compared with control (P < 0.05). Adding α-ketoglutarate to enhance the 3-MST enzyme pathway also resulted in an increase (P < 0.05). Both aspartate, which inhibits the 3-MST synthesis pathway, and propargylglycine (PPG), which inhibits the CSE pathway, significantly reduced the increases in arterial pressure during hypoxia. Diethylmaleate (DEM), which conjugates sulfhydryls, also reduced the peak hypoxic arterial pressure at concentrations >2 mM. Finally, H(2)S concentrations as measured with a specially designed polarographic electrode decreased markedly in lung tissue homogenate and in small pulmonary arteries when air was added to the hypoxic environment of the measurement chamber. The results of this study provide evidence that the rate of H(2)S synthesis plays a role in the magnitude of acute HPV in the isolated perfused rat lung.