Lower polyunsaturated fatty acid levels and FADS2 expression in adult compared to neonatal keratinocytes are associated with FADS2 promotor hypermethylation.

Keratinocytes produce lipids that are critical for the skin barrier, however, little is known about the impact of age on fatty acid (FA) biosynthesis in these cells. We have examined the relationship between keratinocyte FA composition, lipid biosynthetic gene expression, gene promoter methylation and age. Expression of elongase (ELOVL6 and 7) and desaturase (FADS1 and 2) genes was lower in adult versus neonatal keratinocytes, and was associated with lower concentrations of n-7, n-9 and n-10 polyunsaturated FA in adult cells. Consistent with these findings, transient FADS2 knockdown in neonatal keratinocytes mimicked the adult keratinocyte FA profile in neonatal cells. Interrogation of methylation levels across the FADS2 locus (53 genomic sites) revealed differential methylation of 15 sites in neonatal versus adult keratinocytes, of which three hypermethylated sites in adult keratinocytes overlapped with a SMARCA4 protein binding site in the FADS2 promoter.

Wrinkle reduction in post-menopausal women consuming a novel oral supplement: a double-blind placebo-controlled randomized study.

OBJECTIVE: The maintenance of youthful skin appearance is strongly desired by a large proportion of the world's population. The aim of the present study was therefore to evaluate the effect on skin wrinkling, of a combination of ingredients reported to influence key factors involved in skin ageing, namely inflammation, collagen synthesis and oxidative/UV stress. A supplemented drink was developed containing soy isoflavones, lycopene, vitamin C and vitamin E and given to post-menopausal women with a capsule containing fish oil. METHOD: We have performed a double-blind randomized controlled human clinical study to assess whether this cocktail of dietary ingredients can significantly improve the appearance of facial wrinkles. RESULTS: We have shown that this unique combination of micronutrients can significantly reduce the depth of facial wrinkles and that this improvement is associated with increased deposition of new collagen fibres in the dermis. CONCLUSION: This study demonstrates that consumption of a mixture of soy isoflavones, lycopene, vitamin C, vitamin E and fish oil is able to induce a clinically measureable improvement in the depth of facial wrinkles following long-term use. We have also shown, for the first time with an oral product, that the improvement is associated with increased deposition of new collagen fibres in the dermis.

Climbazole increases expression of cornified envelope proteins in primary keratinocytes.

OBJECTIVE: Dandruff is a troubling consumer problem characterized by flaking and pruritus of the scalp and is considered a multifactorial condition with sebum, individual susceptibility and the fungus Malassezia all thought to play a part. The condition is commonly treated with shampoo products containing antifungal ingredients such as zinc pyrithione and climbazole. It is hypothesized that these ingredients may be delivering additional scalp skin benefits besides their antifungal activity helping to relieve dandruff effectively. The objective of this study was to evaluate the anti-dandruff ingredient climbazole for potential skin benefits using genomics and in vitro assays. METHODS: Microarray analysis was performed to profile gene expression changes in climbazole-treated primary human keratinocyte cells. Results were independently validated using qPCR and analysis of protein expression using ELISA and immunocytochemistry. RESULTS: Microarray analysis of climbazole-treated keratinocytes showed statistically significant expression changes in genes associated with the gene ontology groups encompassing epidermal differentiation, keratinization, cholesterol biosynthesis and immune response. Upregulated genes included a number encoding cornified envelope proteins such as group 3 late-cornified envelope proteins, LCE3 and group 2 small-proline-rich proteins, SPRR2. Protein analysis studies of climbazole-treated primary keratinocytes using ELISA and immunocytochemistry were able to demonstrate that the increase in gene transcripts translated into increased protein expression of these cornified envelope markers. CONCLUSION: Climbazole treatment of primary keratinocytes results in an upregulation in expression of a number of genes including those encoding proteins involved in cornified envelope formation with further studies demonstrating this did translate into increased protein expression. A climbazole-driven increase in cornified envelope proteins may improve the scalp skin barrier, which is known to be weaker in dandruff. These studies suggest climbazole, besides its antifungal activity, is delivering positive skin benefits helping to relive dandruff symptoms effectively.

Bradykinin regulates human colonic ion transport in vitro.

BACKGROUND AND PURPOSE: Kinins are acknowledged as important regulators of intestinal function during inflammation; however, their effects on human intestinal ion transport have not been reported. Here, we used muscle-stripped human colonic tissue and cultured T(84)-cell monolayers to study bradykinin (BK) actions on human intestinal ion transport. EXPERIMENTAL APPROACH: Ion transport was measured as changes in short-circuit current (I(sc)) across colonic epithelia mounted in Ussing chambers. KEY RESULTS: In intact tissue, there was a distinct polarity to BK-elicited I(sc) responses. Whereas basolateral BK stimulated sustained responses (EC(50)=0.5+/-0.1 microM), those to apical BK were more rapid and transient (EC(50)=4.1+/-1.2 nM). In T(84) cells, responses to both apical and basolateral BK were similar to those seen upon apical addition to intact tissues. Cross-desensitization between apical and basolateral domains was not observed. BK-induced responses were largely due to Cl(-) secretion as shown by their sensitivity to bumetanide and removal of Cl(-) from the bathing solution. Studies using selective agonists and antagonists indicate responses to BK are mediated by B(2) receptors. Finally, responses to basolateral BK in intact tissues were inhibited by tetrodotoxin (1 microM), atropine (1 microM), capsaicin (100 microM) and piroxicam (10 microM). BK-stimulated prostaglandin (PG)E(2) release from colonic tissue. CONCLUSIONS: BK stimulates human colonic Cl(-) secretion by activation of apical and basolateral B(2) receptors. Responses to apical BK reflect a direct action on epithelial cells, whereas those to basolateral BK are amplified by stimulation of enteric nerves and PG synthesis.

Cross-talk between calcium and cAMP-dependent intracellular signaling pathways. Implications for synergistic secretion in T84 colonic epithelial cells and rat pancreatic acinar cells.

Treatment of various cells with combinations of agents that increase either cAMP or cytosolic calcium can lead to synergistic responses. This study examined interactions, or cross-talk, between these two intracellular messengers and its implication for signaling in two secretory cell types, T84 human colonic epithelial cells and rat pancreatic acinar cells. T84 cell chloride secretion was measured in Ussing chambers. Acinar cell activation was monitored as amylase secretion. Cytosolic calcium was assessed via fura-2 microfluorimetry. A cell-permeant analogue of cAMP synergistically enhanced secretory responses to calcium-mobilizing hormones in both cell types, but paradoxically reduced overall calcium mobilization. The reduction in calcium mobilization could be attributed to an inhibition of calcium influx in T84 cells, although a different mechanism likely operates in acinar cells. The effects of the cAMP analogue were reproduced by other agents that increase cAMP. Furthermore, econazole, an inhibitor of calcium influx, potentiated secretory responses to calcium-dependent stimulation in T84 cells without itself inducing secretion. We conclude that there is cross-talk between calcium and cAMP-dependent signaling pathways at the level of second messenger generation in two secretory cell types. This cross-talk appears to regulate the extent of secretory responses.

Mast cells and histamine contribute to bile acid-stimulated secretion in the mouse colon.

Certain dihydroxy bile acids cause secretory diarrhea when present in the colonic lumen at inappropriately high concentrations. However, the mechanism underlying the secretagogue activity has not been fully elucidated. Experiments were performed to test whether mast cells and one of their major mediators, histamine, might contribute to the secretory effect. Chenodeoxycholic acid, a secretory bile acid, and ursodeoxycholic acid, a nonsecretory, hydrophilic bile acid, were compared for their ability to induce chloride secretion across segments of mouse colon mounted in Ussing chambers. Chenodeoxycholic acid, but not ursodeoxycholic acid, induced dose-dependent, biphasic chloride secretion that was greater after serosal than mucosal addition and was greater in distal versus proximal colonic segments. The secretory effect of chenodeoxycholic acid was inhibited by H1 histamine receptor antagonists and modified by the cyclooxygenase inhibitor indomethacin. However, it was unaffected by an H2 histamine receptor antagonist or by atropine. Secretory effects of chenodeoxycholic acid were diminished in magnitude and delayed in colonic tissues from mice with a genetic deficiency of tissue mast cells. Concentrations of chenodeoxycholic acid inducing secretion also released histamine from tissue segments. These data indicate that mast cells and histamine-mediated processes contribute significantly to the secretory effects of dihydroxy bile acids in the murine colon.

Role of intestinal epithelial cells in the host secretory response to infection by invasive bacteria. Bacterial entry induces epithelial prostaglandin h synthase-2 expression and prostaglandin E2 and F2alpha production.

Increased intestinal fluid secretion is a protective host response after enteric infection with invasive bacteria that is initiated within hours after infection, and is mediated by prostaglandin H synthase (PGHS) products in animal models of infection. Intestinal epithelial cells are the first host cells to become infected with invasive bacteria, which enter and pass through these cells to initiate mucosal, and ultimately systemic, infection. The present studies characterized the role of intestinal epithelial cells in the host secretory response after infection with invasive bacteria. Infection of cultured human intestinal epithelial cell lines with invasive bacteria, but not noninvasive bacteria, is shown to induce the expression of one of the rate-limiting enzymes for prostaglandin formation, PGHS-2, and the production of PGE2 and PGF2alpha. Furthermore, increased PGHS-2 expression was observed in intestinal epithelial cells in vivo after infection with invasive bacteria, using a human intestinal xenograft model in SCID mice. In support of the physiologic importance of epithelial PGHS-2 expression, supernatants from bacteria-infected intestinal epithelial cells were shown to increase chloride secretion in an in vitro model using polarized epithelial cells, and this activity was accounted for by PGE2. These studies define a novel autocrine/paracrine function of mediators produced by intestinal epithelial cells in the rapid induction of increased fluid secretion in response to intestinal infection with invasive bacteria.

Effect of the diglyceride lipase inhibitor, RG80267, on epithelial chloride secretion induced by various agents.

We previously reported that a diglyceride lipase inhibitor, RG80267, inhibits chloride secretion stimulated by adenosine agonists, stimuli whose effects appear unrelated to cAMP, cGMP or cytosolic calcium. Here, the effect of RG80267 on Cl- secretory responses to agents which do utilize these messengers was examined. RG80267 inhibited responses to vasoactive intestinal polypeptide, forskolin (cAMP-dependent) and E. coli heat stable enterotoxin (cGMP-dependent), but not to prostaglandin E1 or cholera toxin (cAMP-dependent). RG80267 enhanced responses to histamine (calcium-dependent). The inhibitory effect of RG80267 was not due to inhibition of cAMP accumulation. Arachidonic acid release may participate in chloride secretion. Vasoactive intestinal polypeptide, but not prostaglandin E1, released radiolabel from cells preloaded with [3H]arachidonic acid. There may thus be differences between mechanisms of various cyclic nucleotide-dependent chloride secretory responses. Arachidonic acid release may modulate the extent of secretion elicited by some secretagogues.

Possible mechanisms of diarrheal side effects associated with the use of a novel chemotherapeutic agent, flavopiridol.

The novel cyclin-dependent kinase inhibitor flavopiridol has recently completed Phase I trials for the treatment of refractory neoplasms. The dose-limiting toxicity observed with this agent was severe diarrhea. Because the compound otherwise showed promise, the present study sought to determine possible mechanisms underlying the diarrheal side effects. Flavopiridol was tested for its ability to modify chloride secretory responses of the human colonic epithelial cell line, T84. Studies were conducted in vitro in modified Ussing chambers. High concentrations of flavopiridol (10(-4) M), above those likely to be clinically relevant, had a direct stimulatory effect on chloride secretion, probably ascribable to an increase in cyclic AMP. Lower, clinically relevant concentrations of flavopiridol (10(-6) M) had no effect on chloride secretion by themselves but potentiated responses to the calcium-dependent secretagogue, carbachol. The drug also potentiated responses to thapsigargin and taurodeoxycholate and reversed the inhibitory effects of carbachol and epidermal growth factor on calcium-dependent chloride secretion. Pretreatment with the cyclic AMP-dependent secretagogue, forskolin, potentiated responses to flavopiridol, but not vice versa. Thus, diarrheal side effects induced by flavopiridol are likely multifactorial in origin and may involve interactions with endogenous secretagogues such as acetylcholine and bile acids. A better understanding of the diarrhea induced by flavopiridol should allow optimization of therapy with this otherwise promising drug and/or the development of related agents with improved toxicity profiles.

Protoporphyrin and long-wave ultraviolet light modulate metabolic events in rat peritoneal mast cells.

We have previously shown that protoporphyrin (PP) plus long-wave ultraviolet light (UVA) has an inhibitory effect on the release of histamine from rat peritoneal mast cells in response to various stimuli, without compromising cell viability. In the present study, we observed that protoporphyrin at a noncytolytic dose (3 ng/ml) plus UVA irradiation (0.038 J/cm2) is also able to suppress prostaglandin D2 generation by rat peritoneal mast cells in response to calcium ionophore A23187, compound 48/80, or anti-IgE antibody by 64%, 92%, and 100%, respectively. Because of the participation of protein kinase C in stimulus-secretion coupling in mast cells, we also investigated the effect of PP plus UVA on the release of histamine induced by the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA). PP plus UVA inhibited histamine release induced by PMA. The release of histamine induced by the synergistic combination of PMA (50 nM) and a low dose of calcium ionophore A23187 (0.1 microM) was also inhibited. PP plus UVA inhibited the release of histamine induced by the non-fluorescent calcium ionophore, 4-Br-A23187, by 47.8%, but had essentially no effect on changes in intracellular calcium induced by this stimulus. In contrast, both the release of histamine and changes in intracellular calcium stimulated by compound 48/80 were inhibited. We conclude from these results that PP plus UVA may affect both early and late biochemical events involved in mast cell mediator release.

Studies on histamine secretion from enzymically dispersed cutaneous mast cells of the rat.

A method has been developed for the enzymic dissociation of rat skin into its component cells. The resulting suspensions contained 3-5% mast cells. The latter were intact as judged by light microscopy and exhibited a low spontaneous release of histamine. Cells obtained from actively sensitized animals released histamine on challenge with specific antigen. The process was rapid, being essentially complete within 1 min, and was both calcium-and temperature-dependent. The cells also responded to antirat IgE and to calcium ionophores but showed a selective, time-dependent reactivity toward defined chemical histamine liberators. On the basis of these results the properties of the cutaneous mast cell are compared with those previously reported for mastocytes from other sources and discussed in terms of the general heterogeneity of this cell population.

Activation by calcium alone of chloride secretion in T84 epithelial cells.

1. The goal of this study was to determine if an increase in cytoplasmic calcium concentration ([Ca2+]i), in the absence of additional second messengers derived from membrane phospholipid turnover, is a sufficient signal to induce chloride secretion across monolayers of the human colonic epithelial line, T84. 2. Thapsigargin was used to increase [Ca2+]i by inhibiting the endomembrane Ca(2+)-ATPase. [Ca2+]i was monitored in monolayers by fura-2 fluorescence spectroscopy, chloride secretion by measuring changes in short circuit current (Isc) in modified Ussing chambers, and inositol phosphates were measured by radio-h.p.l.c. of extracts of cells prelabelled with [3H]-inositol. 3. Thapsigargin increased [Ca2+]i and Isc in parallel, without increasing any inositol phosphates. The effect of thapsigargin on Isc was abolished by the intracellular calcium chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N"-tetraacetic acid (BAPTA). 4. Increasing [Ca2+]i with thapsigargin did not prevent a subsequent calcium response to carbachol or histamine if extracellular calcium was available. In the absence of extracellular calcium, only one such release of calcium to hormonal stimulation occurred when cells were pretreated with thapsigargin, and a second response to either carbachol histamine was essentially abolished. 5. Addition of carbachol or histamine to thapsigargin-treated cells mounted in Ussing chambers caused a transient further increase in Isc followed by termination of the response, even though [Ca2+]i continued to rise. 6. We conclude that an elevation in [Ca2+]i is a sufficient signal to induce chloride secretion in T84 cells. Rather than being required to stimulate secretory responses, additional second messengers induced by hormonal secretagogues (such as inositol phosphates) may in fact serve to limit the secretory response.

Regulation of chloride secretion. Novel pathways and messengers.

The capacity for active chloride secretion, thereby driving the secretion of fluid, is an important property of the intestinal epithelium. Chloride secretion is stimulated by mechanisms involving increases in either cyclic nucleotide or cytoplasmic calcium concentrations. The calcium-dependent response is transient and limited in its magnitude, implying that negative signaling events may restrict the overall extent of this mode of chloride transport. We have uncovered a number of negative signaling mechanisms intrinsic to the epithelium that uncouple increases in calcium from the downstream response of chloride secretion. These involve various kinase cascades, the generation of messengers derived from membrane phospholipids, and interactions of G protein-coupled receptors with those for peptide growth factors such as epidermal growth factor. This chapter will review emerging information on the details of these negative signaling mechanisms, as well as points of convergence and divergence. The possible physiological and pathophysiological significance of such signaling will also be discussed.

Clinical usefulness of white blood cell count after cesarean delivery.

OBJECTIVE: To examine changes in white blood cell (WBC) count after cesarean and estimate risk of postoperative infection. METHODS: We measured complete blood cell counts at admission and on postoperative day 1 for 458 women who had cesareans. Information from charts was abstracted, and definitions of infectious outcomes and fever were applied by three physicians masked to laboratory results. We examined changes in absolute and relative WBC counts by labor status. Likelihood ratios for postoperative infection were calculated for statistically distinct categories of percentage changes. RESULTS: We excluded 60 women with chorioamnionitis. Of the remainder, 34 (8.5%) developed endometritis and three (0.8%) pneumonia. Women who labored before cesarean (n = 198) had higher antepartum (P <.001) and postoperative day 1 (P <.001) WBC counts than those who did not (n = 200). However, change in WBC count after cesarean relative to antepartum was similar for both groups (P =.41), averaging a 22% increase. We grouped percentage changes into the following three levels: up to 24%, 25-99%, and at least 100%. The lowest level (n = 246) corresponded to a category-specific likelihood ratio for diagnosis of serious postpartum infection of 0. 5 (95% confidence interval [CI] 0.3, 0.8), the midlevel (n = 141) to a category-specific likelihood ratio of 1.7 (95% CI 1.2, 2.3), and the highest level (n = 11) to a category-specific likelihood ratio of 5.8 (95% CI 1.8, 18.7). CONCLUSION: Labor influenced postcesarean WBC counts but did not obscure changes associated with infection. Information gained from changes in WBC counts can be used to assess risk of infection.

The immune response to nematode parasites: modulation of mast cell numbers and function during Strongyloides stercoralis infections in nonhuman primates.

Mucosal mast cell numbers are modulated in the intestines of rodents during parasitic infections. These mast cells can degranulate in response to worm antigens, and this event has been suggested to play a protective role for the host. To examine whether mast cells in higher animals play a role in protecting from disseminated parasitic disease, mast cell numbers and responsiveness to parasite antigens were evaluated in 5 Erythrocebus patas infected with the human intestinal nematode Strongyloides stercoralis. Initial infection and subsequent challenge infections were associated with increase in jejunal histamine and mast cell numbers, and these mast cells could release histamine in response to parasite antigens. Jejunal mast cell numbers returned to normal during a chronic phase of infection. The cells lost their ability to respond to antigenic stimulation following limited steroid treatment. Subsequent activation of chronic infections to fatal disseminated disease by more prolonged steroid treatment was associated with a marked decrease in jejunal mast cell numbers and histamine. In one animal which succumbed to severe disease without steroid treatment, jejunal mast cells were refractory to worm antigens.

Inhibition of IgE-mediated mast cell degranulation by sulphasalazine.

The effect of sulphasalazine on two mast cell populations and human peripheral leukocytes is reported. Sulphasalazine inhibited histamine release from mouse and rat mast cells, but it caused a potentiation of secretion in human peripheral leukocytes. The drug alone did not induce histamine release when administered without an anaphylactic stimulus. The results are discussed in terms of a possible mode of action of sulphasalazine in the treatment of inflammatory bowel disease.

Effects of quercetin on epithelial chloride secretion.

The effect of the flavonoid quercetin on epithelial chloride secretion has been studied in vitro. These studies were performed using monolayers of the human colonic epithelial cell line T84, mounted in modified Ussing chambers. Chloride secretion was assessed as changes in short circuit current (I(SC)) both in basal conditions as well as in response to different secretagogues: carbachol (100 microM), vasoactive intestinal polypeptide (VIP) (10 nM) and prostaglandin E2 (1 microM). Secretion was also induced via protein kinase C by adding phorbol myristate acetate (PMA, 100 nM) in the presence of the calmodulin inhibitor W13 (50 microM). Quercetin (100 microM) was able to promote secretion when the flavonoid was added to the mucosal side of the monolayer. On the contrary, serosal addition of quercetin was devoid of secretory activity and at concentration of 10 microM it was able to inhibit chloride secretion in response to carbachol, prostaglandin E2 and PMA/W13, but not that induced by VIP. We conclude that the effect of quercetin on epithelial chloride secretion is dual, secretory and antisecretory, depending on both the concentration and the side of the monoloayer where the addition of the flavonoid is made.

The effects of aspartame on mast cells and basophils.

The artificial sweetener aspartame was studied to determine whether it had any direct effects on mast cells and basophils. Aspartame was not shown to be a direct mast cell or basophil secretagogue in vitro, or in vivo as assessed by skin testing. During an acute incubation, aspartame did not affect IgE-mediated histamine release from mast cells. However, mast cells cultured in aspartame for periods of up to 9 days showed enhanced rates of proliferation and decreased responsiveness to releasing stimuli. The effect of aspartame on proliferation of cells in culture could be ascribed to a non-specific enhancing effect of its constituent amino acids.

Expression of acid-sensing ion channels in intestinal epithelial cells and their role in the regulation of duodenal mucosal bicarbonate secretion.

AIMS: As little is currently known about acid-sensing ion channels (ASICs) in intestinal epithelial cells, the aims of the present study were to investigate the expression and function of ASICs in intestinal epithelial cells, particularly their physiological role in the acid-stimulated duodenal mucosal bicarbonate secretion (DMBS). METHODS: RT-PCR and digital Ca²(+) imaging were used to determine the expression and function of ASICs in HT29 cells and SCBN cells, intestinal epithelial crypt cell lines. The acid-stimulated DMBS was measured in C57 black mice in vivo to study the role of ASICs in this physiological process. RESULTS: ASIC1a mRNA expression was detected in the duodenal mucosa stripped from mice and epithelial cell lines, in which cytoplasmic free Ca²(+) ([Ca²(+) ](cyt)) in response to extracellular acidosis was also increased. In Ca²(+) -containing solutions, acidosis (pH 6.0-5.0) raised [Ca²(+) ](cyt) in both HT29 cells and SCBN cells in a similar pH-dependent manner. Acidosis-induced increase in [Ca²(+) ](cyt) was markedly inhibited by amiloride (an ASICs blocker), SK&F96365 (a blocker for non-selective cation channels), or in Ca²(+) -free solutions; but was abolished by amiloride in Ca²(+) -free solutions. However, acidosis-induced increase in [Ca²(+) ](cyt) was slightly affected by U73122 (a PLC inhibitor), or nifedipine (a voltage-gated Ca²(+) channel blocker). After acidosis raised [Ca²(+) ](cyt) , stimulation of purinergic receptors with ATP further increased [Ca²(+) ](cyt) , but acidosis-induced increase in [Ca²(+) ](cyt) was not altered by suramin. Moreover, acid-stimulated murine DMBS was significantly attenuated by amiloride. CONCLUSION: Therefore, ASICs are functionally expressed in intestinal epithelial cells, and may play a role in acid-stimulated DMBS through a Ca²(+) signalling pathway.