Mast cells and sphingosine-1-phosphate underlie prelesional remodeling in a mouse model of eczema.

BACKGROUND: Atopic dermatitis (AD) is a chronic skin inflammation that affects children and adults worldwide, but its pathogenesis remains ill-understood. METHODS: We show that a single application of OVA to mouse skin initiates remodeling and cellular infiltration of the hypodermis measured by a newly developed computer-aided method. RESULTS: Importantly, we demonstrate that skin mast cell (MC) activation and local sphingosine-1-phosphate (S1P) are significantly augmented after OVA treatment in mice. Deficiency in sphingosine kinase (SphK)1, the S1P-producing enzyme, or in MC, remarkably mitigates all signs of OVA-mediated remodeling and MC activation. Furthermore, skin S1P levels remain unchanged in MC-deficient mice exposed to OVA. LPS-free OVA does not recapitulate any of the precursor signs of AD, supporting a triggering contribution of LPS in AD that, per se, suffice to activate local MC and elevate skin S1P. CONCLUSION: We describe MC and S1P as novel pathogenic effectors that initiate remodeling in AD prior to any skin lesions and reveal the significance of LPS in OVA used in most studies, thus mimicking natural antigen (Ag) exposure.

Mammary glands exhibit molecular laterality and undergo left-right asymmetric ductal epithelial growth in MMTV-cNeu mice.

Significant left-right (L-R) differences in tumor incidence and disease outcome occur for cancers of paired organs, including the breasts; however, the basis for this laterality is unknown. Here, we show that despite their morphologic symmetry, left versus right mammary glands in wild-type mice have baseline differences in gene expression that are L-R independently regulated during pubertal development, including genes that regulate luminal progenitor cell renewal, luminal cell differentiation, mammary tumorigenesis, tamoxifen sensitivity and chemotherapeutic resistance. In MMTV-cNeu(Tg/Tg) mice, which model HER2/Neu-amplified breast cancer, baseline L-R differences in mammary gene expression are amplified, sustained or inverted in a gene-specific manner and the mammary ductal epithelium undergoes L-R asymmetric growth and patterning. Comparative genomic analysis of mouse L-R mammary gene expression profiles with gene expression profiles of human breast tumors revealed significant linkage between right-sided gene expression and decreased breast cancer patient survival. Collectively, these findings are the first to demonstrate that mammary glands are lateralized organs, and, moreover, that mammary glands have L-R differential susceptibility to HER2/Neu oncogene-mediated effects on ductal epithelial growth and differentiation. We propose that intrinsic molecular laterality may have a role in L-R asymmetric breast tumor incidence and, furthermore, that interplay between the L-R molecular landscape and oncogene activity may contribute to the differential disease progression and patient outcome that are associated with tumor situs.

Assessing vascular permeability during experimental cerebral malaria by a radiolabeled monoclonal antibody technique.

Vascular endothelial integrity, assessed by Evans blue dye extrusion and radiolabeled monoclonal antibody leakage, was markedly compromised in the brain, lung, kidney, and heart during Plasmodium berghei infection, a well-recognized model for human cerebral malaria. The results for vascular permeability from both methods were significantly (P < 0.001) related.

Programmed cell death in rat intestine: effect of feeding and fasting.

BACKGROUND: The regulation of intestinal cell death by luminal factors is poorly understood. The objectives of this study were to determine whether a diurnal rhythm of intestinal apoptosis exists, and to determine the role that feeding and fasting play in this process. METHODS: Mucosal apoptotic death was measured in fed and 24-h fasted rats and at various times after feeding by DNA fragmentation and in situ immunohistochemical staining (TUNEL). RESULTS: In 24-h fasted rats, 32% of total mucosal DNA was fragmented as compared to 9% in fed animals. In both jejunal and ileal segments, the fragmented DNA exhibited characteristic apoptotic DNA ladders on agarose gels. Immunohistochemical staining revealed significant location of apoptotic cells at the upper third of the intestinal villus. In the duodenum, DNA fragmentation at 6-12 h post feeding was 20% and decreased to 4% at 24 h. In comparison, DNA fragmentation in the jejunum and ileum was low from 0 to 6 h post feeding (2%-9%) and significantly increased at 12 h (18% versus 12%) and 24 h (30% versus 32%), respectively. These results are consistent with a temporal relationship between percent fragmented DNA and time after feeding with greater cell death at longer fasting period. A postprandial rhythm of DNA fragmentation was evident in the jejunum and ileum, in which fragmentation was at a peak between 0900 h and 1200 h. CONCLUSION: Collectively, the data show that initiation of apoptosis in apical enterocytes is coincident with cessation of feeding and commencement of fasting, and is consistent with a rhythm of programmed cell death in these cells that parallels the cyclical pattern of feeding and fasting.

Reciprocal regulation of endothelial substrate adhesion and barrier function.

OBJECTIVE: To examine how cell-substrate adhesion is regulated during barrier changes produced by exposure to inflammatory mediators. METHODS: Lung microvascular endothelial monolayers were treated with test agents +/- blockers, and barrier was measured by transendothelial resistance; cell-substrate adhesion was assessed by surface area conservation after trypsin treatment of monolayers. Protein phosphorylation and distribution were assayed by immunoblotting and fluorescent microscopy, respectively. RESULTS: H2O2, histamine, bradykinin, and thrombin, decreased endothelial barrier function, and enhanced adhesion to the substratum. H2O2 enhanced cell adhesion to the substrate in a concentration (0-1 mM)- and time (0-60 minutes)-dependent fashion. This effect of H2O2 reversed within 120 minutes of removal of H2O2 and was blocked by the mean arterial pressure (MAP) kinase inhibitor, PD98059 and by chelating cytoplasmic Ca2+ but not PKC or PKG inhibition. H2O2 also stimulated tyrosine phosphorylation of several proteins and increased the association of the focal adhesive proteins paxillin, talin, and vinculin with the cytoskeleton and may promote localization of these proteins to junctions. CONCLUSIONS: Our data indicate that inflammatory mediators reduce cell-cell contact, contributing to reduced solute barrier and simultaneously enhanced substrate binding, which may be reciprocal events in barrier regulation in vitro and in vivo.

E-Selectin expression in a murine model of chronic colitis.

The objective of this study was to quantify E-selectin surface expression in the colon as well as other tissues in a CD4(+) T-cell model of chronic colitis in mice using the newly developed dual radiolabel monoclonal antibody technique. Male SCID mice were reconstituted with either 5 x 10(5) CD4(+) CD45RB(low) or CD45RB(high) T-cells isolated from normal CB-17 donor mouse spleens and subsequently monitored for clinical signs of colitis. We found that animals injected with CD45RB(high) but not CD45RB(low) T-cells nor PBS developed colitis at 6-8 weeks following reconstitution as assessed by loss of body weight, development of loose stools and/or diarrhea, and histopathology. Concurrent with the onset of distal bowel inflammation was enhanced expression of E-selectin compared to SCID mice injected with PBS or reconstituted with CD45RB(low) T-cells, both of which did not develop colitis. We also observed significant increases in E-selectin expression in cecum, small intestine, mesentery, and liver of colitic mice. Our data confirm that reconstitution of SCID mice with CD45RB(high) but not CD45RB(low) T-cells induces chronic colitis and demonstrate that this chronic colitis is associated with enhanced expression of an endothelial cell-specific adhesion molecule. Furthermore, our studies demonstrate that reconstitution of SCID mice with CD45RB(high) T-cells enhances E-selectin expression in a variety of tissues distant from the site of active inflammation.

FK506 attenuates developing and established joint inflammation and suppresses interleukin 6 and nitric oxide expression in bacterial cell wall induced polyarthritis.

OBJECTIVE: To determine the efficacy of therapeutic administration of FK506 (Tacrolimus) in suppressing developing and established joint inflammation, proinflammatory cytokine expression, and nitric oxide (NO) production in peptidoglycan-polysaccharide (PG/PS) induced experimental polyarthritis in rats. METHODS: Chronic joint inflammation was induced by intraperitoneal injection of PG/PS, and joint inflammation was quantified using arthritis index and paw volume. Serum and joint levels of interleukin 6 (IL-6) were measured by bioassay and Western blot analysis respectively, and serum levels of NO production were determined by the Griess procedure and the expression of the inducible isoform of nitric oxide synthase (i-NOS) in the joints was determined by Western blot analysis. RESULTS: Arthritis induced by PG/PS is biphasic, progressing through an initial acute phase and a remission phase, which is followed by a persistent chronic phase. Daily administration of FK506 initiated during the remission phase significantly attenuated the onset and development of chronic joint inflammation. We observed a significant reduction in joint inflammation and swelling, an apparent suppression of pannus development, and minimal erosive damage to the articular cartilage and subchondral bone. Fully established chronic joint inflammation was also ameliorated by daily administration of FK506. Joint swelling and inflammation was significantly reduced by 5 days post-treatment with FK506 and the erosive activity associated with the pannus appeared diminished. The elevated expression of IL-6 and NO characteristic of chronic joint inflammation in the serum and in joint tissue was significantly reduced by FK506 treatment. CONCLUSION: Therapeutic administration of FK506 has a profound antiinflammatory effect on the development of the chronic, erosive arthritis induced by PG/PS. This attenuation in joint inflammation was associated with suppression of IL-6 and NO production systemically and locally in the joints. Our data suggest that FK506 may be effective in the treatment of chronic joint inflammation associated with rheumatoid arthritis.

Indomethacin induced gastropathy in CD18, intercellular adhesion molecule 1, or P-selectin deficient mice.

BACKGROUND: Neutrophil-endothelial cell interactions are thought to play a critical role in the pathophysiology of non-steroidal anti-inflammatory drug (NSAID) induced gastropathy. AIMS: To optimise a mouse model of NSAID induced gastropathy and to evaluate the importance of adhesion molecules using adhesion molecule deficient mice. METHODS: Gastropathy was induced in C57BL/6 mice or their adhesion molecule deficient counterparts via oral administration of indomethacin (20 mg/kg). Lesion scores, mucosal permeability, and histopathology were used to assess gastric mucosal injury. RESULTS: Intragastric administration of indomethacin induced linear haemorrhagic mucosal lesions, primarily in the corpus of the stomach that were first observed at six hours. These lesions continued to develop over the next six hours with maximal lesion scores and mucosal permeabilities at 12 hours. When indomethacin was administered to mice deficient in CD18, intercellular adhesion molecule 1 (ICAM-1), or P-selectin, there were significant decreases in lesion scores compared with their C57BL/6 controls. In addition, mucosal permeabilities were found to be significantly lower in CD18 or ICAM-1 deficient mice observed at 12 hours. CONCLUSION: Certain leucocyte and endothelial cell adhesion molecules are important determinants for full expression of indomethacin induced gastropathy. It is proposed that this modification of the mouse model may be useful for the investigation of other pathophysiological mechanisms of NSAID induced gastropathy.

Selective proteasome inhibitors as anti-inflammatory agents.

Nuclear factor kappaB (NF-kappaB) is an ubiquitous transcription factor and pleiotropic regulator of numerous inflammatory and immune responses. Once activated, NF-kappaB translocates from the cytosol to the nucleus of the cell, where it binds to its consensus sequence on the promoter-enhancer region of different genes. By so doing, this activates the transcription of a variety of different pro-inflammatory cytokines, adhesion molecules and specific enzymes, such as the inducible forms of nitric oxide synthase and cyclooxygenase. A number of different cytokines, bacterial products and oxidants activate NF-kappaB via selective phosphorlyation, polyubiquitination and degradation of the inhibitor protein, IkappaB. Since the 26S proteasome complex degrades the post-translationally modified IkappaB, thereby liberating the transcriptionally active p50/p65 heterodimeric NF-kappaB, this proteolytic complex represents a critical step in the activation of NF-kappaB. This review discusses the basic biology of the ubiquitin-proteasome pathway as it relates to the inflammatory response, and highlights those studies demonstrating that selective proteasome inhibitors are effective anti-inflammatory agents in vivo.

Role of the proteasome and NF-kappaB in streptococcal cell wall-induced polyarthritis.

The transcription factor NF-kappaB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-kappaB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IkappaB is degraded by the ubiquitin-proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-kappaB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin-proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-kappaB and the subsequent transcription of genes that are regulated by NF-kappaB. Significantly, PS-341 displayed similar effects in vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IkappaBalpha degradation and NF-kappaB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin-proteasome pathway and NF-kappaB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect.

Tumor necrosis factor-alpha in ischemia and reperfusion injury in rat lungs.

The effects of both recombinant rat tumor necrosis factor-alpha (TNF-alpha) and an anti-TNF-alpha antibody were studied in isolated buffer-perfused rat lungs subjected to either 45 min of nonventilated [ischemia-reperfusion (I/R)] or air-ventilated (V/R) ischemia followed by 90 min of reperfusion and ventilation. In the I/R group, the vascular permeability, as measured by the filtration coefficient (Kfc), increased three- and fivefold above baseline after 30 and 90 min of reperfusion, respectively (P < 0.001). Over the same time intervals, the Kfc for the V/R group increased five- and tenfold above baseline values, respectively (P < 0.001). TNF-alpha measured in the perfusates of both ischemic models significantly increased after 30 min of reperfusion. Recombinant rat TNF-alpha (50,000 U), placed into perfusate after baseline measurements, produced no measurable change in microvascular permeability in control lungs perfused over the same time period (135 min), but I/R injury was significantly enhanced in the presence of TNF-alpha. An anti-TNF-alpha antibody (10 mg/rat) injected intraperitoneally into rats 2 h before the lung was isolated prevented the microvascular damage in lungs exposed to both I/R and V/R (P < 0.001). These results indicate that TNF-alpha is an essential component at the cascade of events that cause lung endothelial injury in short-term I/R and V/R models of lung ischemia.

ICAM-1 and P-selectin expression in a model of NSAID-induced gastropathy.

A growing body of experimental evidence suggests that neutrophilic polymorphonuclear leukocyte (PMN)-endothelial cell interactions play a critical role in the pathophysiology of nonsteroidal anti-inflammatory drug (NSAID)-induced gastropathy. The objective of this study was to directly determine whether the expression of endothelial cell adhesion molecules is enhanced in a model of NSAID-induced gastropathy. Gastropathy was induced in male Sprague-Dawley rats via oral administration of indomethacin (Indo, 20 mg/kg). Lesion scores, blood-to-lumen clearance of 51Cr-EDTA (mucosal permeability), and histological analysis (epithelial necrosis) were used as indexes of gastric mucosal injury. Gastric mucosal vascular expression of intercellular adhesion molecule 1 (ICAM-1) or P-selectin were determined at 1 and 3 h after Indo administration using the dual radiolabeled monoclonal antibody (MAb) technique. For some experiments, a blocking MAb directed at either ICAM-1 (1A29) or P-selectin (RMP-1) or their isotype-matched controls was injected intravenously 10 min before Indo administration. We found that P-selectin expression was significantly increased at 1 h but not 3 h after Indo administration, whereas ICAM-1 expression was significantly increased at both 1 and 3 h after Indo treatment. The blocking ICAM-1 and P-selectin MAbs both inhibited Indo-induced increases in lesion score, mucosal permeability, and epithelial cell necrosis. However, the Indo-induced gastropathy was not associated with significant PMN infiltration into the gastric mucosal interstitium, nor did Indo reduce gastric mucosal blood flow. We propose that NSAID-induced gastric mucosal injury may be related to the expression of P-selectin and ICAM-1; however, this mucosal injury does not appear to be dependent on the extravasation of inflammatory cells or mucosal ischemia.

Effects of nitric oxide synthase inhibition or sulfasalazine on the spontaneous colitis observed in HLA-B27 transgenic rats.

The objective of this study was to determine the effects that certain nitric oxide synthase inhibitors have on the spontaneous intestinal and colonic inflammation that develops in HLA-B27 transgenic rats and compare these data to those obtained using sulfasalazine (SZ). In an attempt to more closely mimic the clinical situation, drug treatment was begun after the onset of colitis. HLA-B27 male rats that developed clinical signs of colitis (diarrhea/loose stools) at 17 wk of age were randomized into fours groups consisting of one untreated colitic group and three treatment groups that received either aminoguanidine (AG; 52 micromol/kg/day), NG-nitro-L-arginine methyl ester (L-NAME; 45 micromol/kg/day) or SZ (130 mg/kg/day) in their drinking water for 14 days. Aged-matched Fisher 344 male rats were used as healthy controls. After 3 wk of treatment, ileal and colonic mucosal permeabilities, granulocyte infiltration and nitric oxide were quantified using blood-to-lumen clearance of 51Cr-EDTA, tissue myeloperoxidase activity, and plasma levels of nitrate and nitrite, respectively. We found that both AG and L-NAME but not SZ significantly attenuated the increases in plasma nitrate and nitrite levels. Interestingly, all three drugs were effective at significantly attenuating the increases in myeloperoxidase activity in the distal colon. Treatment with AG and SZ but not L-NAME were effective at significantly attenuating the increase in ileal and colonic permeabilities. Quantitative histological analysis revealed that AG and L-NAME but not SZ significantly attenuated the increase in the mucosal thickness and crypt depth in the distal colon compared to untreated colitis. Taken together, these data demonstrate that oral administration of certain nitric oxide synthase inhibitors or SZ to animals with active colitis attenuates the colonic inflammation by at least two different mechanisms. One mechanism appears to be dependent on inhibition of NO production whereas the other mechanism does not.

Proteasome inhibition attenuates nitric oxide synthase expression, VCAM-1 transcription and the development of chronic colitis.

The objectives of this study were to (1) assess the role of the 26S proteasome complex in regulating the expression of the inducible isoform of nitric oxide synthase (iNOS) and vascular cell adhesion molecule-1 (VCAM-1) in a model of chronic granulomatous colitis in vivo and (2) determine the role of the proteasome in regulating the inflammatory response observed in this model of chronic gut inflammation. The selective proteasome inhibitor MG-341 (0.3 mg/kg) was administered by gavage beginning immediately before the induction of colitis and continuing daily thereafter for the entire 14-day experimental period. We found that chronic proteasome inhibition using MG-341 significantly attenuated the peptidoglycan/polysaccharide (PG/PS)-induced up-regulation of iNOS in the colon and spleen and the consequent increase in plasma levels of nitrate and nitrite. Furthermore, we found that the proteasome inhibitor suppressed the up-regulation of the adhesion molecule VCAM-1 in the colon. We also found that MG-341 attenuated PG/PS-induced increases in macroscopic colonic inflammation, bowel wall thickness, colonic dry weight and colonic MPO activity. Treatment with MG-341 also significantly reduced PG/PS-induced increases in macroscopic spleen inflammation, spleen weight and spleen MPO activity. We conclude that the 26S proteasome complex plays an important role in regulating the PG/PS-induced up-regulation of iNOS and VCAM-1 in vivo and appears to be important in regulating colonic and splenic inflammation.

Cytokine and nitric oxide production in the acute phase of bacterial cell wall-induced arthritis.

We have investigated the temporal relationship among proinflammatory cytokine expression, nitric oxide (NO) production and joint inflammation in the acute phase of bacterial cell wall-derived peptidoglycan polysaccharide (PG/PS)-induced arthritis. Acute joint inflammation was induced in female LEW/N rats by a single intraperitoneal injection of PG/PS. Arthritis index and paw volume were quantified and joint histopathology was evaluated during acute joint inflammation (0-10 days). Tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) were determined by bioassay whereas nitric oxide (NO) was quantified by measuring serum nitrate/nitrite levels via the Griess procedure. We found that serum levels of TNF and serum IL-1 preceded the increase in IL-6 and NO production. Furthermore, the production of these proinflammatory cytokines and NO preceded bone erosion and osteoclast activity. Erosion of subchondral bone preceded pannus formation and cellular synovitis in the acute phase of PG/PS-induced arthritis. The temporal expression of TNF, IL-1, IL-6 and NO suggest a cascade of inflammatory mediators in which monocytes and macrophages respond to PG/PS with enhanced synthesis of TNF and IL-1, which may in turn promote the synthesis of IL-6 and NO. We postulate that one or more of these inflammatory events are responsible for initiating the subchondral bone erosion observed in acute joint inflammation.

Effects of cyclosporine or FK506 in chronic colitis.

The objective of this study was to quantitatively characterize the effects FK506 on the pathophysiology observed in a model of chronic granulomatous colitis in rats and compare these effects to those obtained with cyclosporin A (CyA). Chronic granulomatous colitis was induced in female Lewis rats via intramural (subserosal) injections of peptidoglycan/polysaccharide (PG/PS) into the distal colon. Rats then received daily injections (i.m.) of either vehicle for CyA (0.5 ml/kg cremophor), CyA in vehicle (25 mg/kg), saline (0.5 ml/kg) or FK506 (1 mg/kg in saline), beginning 7 days after PG/PS injection and continuing for an additional 2 weeks. On day 21, we found that the intramural injection of PG/PS produced a chronic colitis that was associated with hepatic and splenic granulomatous inflammation. Daily treatment with CyA or FK506 beginning 7 days after the induction of colitis resulted in significant inhibition in colonic mucosal permeability, colonic myeloperoxidase activity and plasma nitrate/nitrite levels when compared with their vehicle or untreated controls. In some instances, we noticed a significant vehicle-dependent anti-inflammatory activity. The incidence of peritoneal adhesions as well as the presence of hepatic and splenic granulomas induced by PG/PS were also significantly reduced in both the CyA- and FK506-treated groups. Taken together, these data suggest that immunosuppressive therapy is effective at attenuating both the colitis as well as the extraintestinal inflammation induced by PG/PS. We conclude that FK506 may be useful in the treatment of certain types of inflammatory bowel disease.

Ethanol-induced depletion of lymphocytes from the mesenteric lymph nodes of C57B1/6 mice is associated with RNA but not DNA degradation.

The effects of ethanol (EtOH) consumption by adult female C57B1/6 mice on lymphocyte populations of the mesenteric lymph nodes (MLNs) were determined by feeding mice with the Lieber-DeCarli liquid diet by a pair-feeding paradigm. Histological analysis of the MLNs of EtOH-fed mice showed a progressive loss of lymphocytes from the medullary regions at 3, 5, and 7 days after initiation of the EtOH diet. The stromal cells in the medullary region also demonstrated a progressive alteration in stellate morphological features at times corresponding to those of loss of lymphocytes from this region. Microscopic evaluation of the follicle regions of MLNs obtained from mice fed an EtOH-containing diet showed no appreciable alterations in morphological characteristics. The number of tingible body macrophages in the germinal centers of the follicles, however, was increased after 3 days of EtOH diet feeding and declined progressively after this time. Flow cytometric analysis of isolated lymphocytes showed a depletion of both T and B cell populations from the MLNs. In contrast to B cells, however, T cells were depleted through 7 days of EtOH diet feeding. Total RNA isolated from the MLNs of mice consuming the EtOH-containing diet was progressively degraded. No degradation of DNA was observed. These study results establish that continuous consumption of dietary EtOH adversely affects the cellularity of MLN, resulting in a progressive loss of lymphocytes that is associated with degradation of total RNA.

Modulation of endothelial cell permeability by lung carcinoma cells: a potential mechanism of malignant pleural effusion formation.

This study examined the hypothesis that tumor cells metastatic to the pleura secrete a soluble factor(s) that directly increases endothelial cell permeability. Nitrocellulose filters were endothelialized with bovine pulmonary artery endothelial cells and exposed to conditioned media from either human lung adenocarcinoma (Calu-3), human lung squamous cell carcinoma (SK-MES-1), or control media for 16 h. The diffusional permeability (Pd x 10(-5) cm/sec) to [14C]albumin was then determined for each monolayer with Ussing-type chambers. Both adenocarcinoma conditioned media (ACCM) and squamous cell carcinoma conditioned media (SCCM) caused a two- to threefold increase in endothelial monolayer permeability. The addition of indomethacin (10 micrograms/ml) blocked the observed permeability increase in ACCM but not in SCCM, suggesting that the increase in permeability by ACCM was secondary to the production of prostaglandins. To confirm this, a variety of prostanoids previously shown to be produced by the Calu-3 cell line were added directly to the endothelial monolayer. Prostaglandin F2 alpha (PGF2 alpha) in both low (10 ng/ml) and high (100 ng/ml) concentrations for 16 h resulted in a three- to fourfold increase in permeability. Prostaglandin E2 (PGE2) resulted in a small increase in [14C]albumin permeability but only at high concentrations (100 ng/ml). PGF2 alpha production by the two tumor cell lines was measured using radioimmunoassay. Baseline adenocarcinoma production of PGF2 alpha was 117.5 pmol/10(6) cells and fell to 24.2 pmol/10(6) cells hours following incubation with indomethacin. The decrease in PGF2 alpha occurred in parallel with the changes in permeability. Concomitant, reversible changes in cell shape and F-actin distribution were detected in endothelial cells exposed to ACCM. No significant production of PGF2 alpha by the squamous cell carcinoma cell line was detected. These results suggest that both adenocarcinoma and squamous cell carcinoma secrete a soluble factor(s) that directly increases endothelial cell permeability to albumin and that in the case of adenocarcinoma this soluble factor may be a prostanoid such as PGF2 alpha.

Increased susceptibility of experimental animals to infectious organisms as a consequence of ethanol consumption.

It has been shown by a number of studies that chronic alcohol abuse is associated with an increased incidence of infections. The mechanism of this increased susceptibility to infectious agents is multifactorial and certainly includes abuse of other drugs, smoking, malnutrition and other factors. Recently, it has become apparent that consumption of alcohol by human beings and experimental animals is associated with changes in the immune system. The increased incidence of tuberculosis and opportunistic bacterial and fungal infections described in alcoholics has led to the suggestion that alcohol affects the cell-mediated arm of the immune response. The present report will review the data that shows the effects of alcohol on the immune system. We will also review the data from our laboratory and other laboratories that show the effects of alcohol on host defense mechanisms to infectious agents. Where possible, mechanisms of increased susceptibility will be discussed or postulated.

Growth hormone-enhanced acid production and glutamate and glutamine utilization in LLC-PK-F+ cells.

Growth hormone (GH), recombinant human (rh) GH, effects on transepithelial proton gradient, acid production, and glutamine (Gln) and glutamate (Glu-) utilization were studied in LLC-PK-F+ cells. Monolayers were incubated in Dulbecco's modified Eagle's media containing either 2 mM Gln or Glu- and 5 mM glucose plus 10% fetal bovine serum for 4 h, after which samples were taken for metabolite and acid-base parameter measurements. Monolayers grown on porous supports in Glu- media responded to 32 nM rhGH by decreasing Glu- net uptake 29% (470 +/- 135 to 336 +/- 98 nmol/4 h, P < 0.05), reflecting elimination of basolateral uptake (189 +/- 80 to 2 +/- 35 nmol/4 h, P < 0.05) as ammonium production decreased. In 2 mM Gln, rhGH decreased Gln net uptake 84% (1,096 +/- 215 to 155 +/- 150 nmol/4 h, P < 0.05), with basolateral uptake reversing to release; Glu- formation increased as ammonium production decreased. Monolayers grown on porous supports required rhGH to generate a transepithelial proton gradient within 4 h (0.01 +/- 0.05 control vs. 0.27 +/- 0.11 U for rhGH, P < 0.05); in addition, rhGH increased acid production (16.7 +/- 2.0 to 21.5 +/- 0.6 mumol x 4 h-1 x mg protein-1, P < 0.05). Adding insulin-like growth factor I (IGF-I) to the basolateral media decreased pH and enhanced Glu- and Gln uptake from that compartment.(ABSTRACT TRUNCATED AT 250 WORDS)