Tofacitinib inhibits inflammatory cytokines from ulcerative colitis and healthy mucosal explants and is associated with pSTAT1/3 reduction in T-cells.

Poor translatability of animal disease models has hampered the development of new inflammatory bowel disorder (IBD) therapeutics. We describe a preclinical, ex vivo system using freshly obtained and well-characterized human colorectal tissue from patients with ulcerative colitis (UC) and healthy control (HC) participants to test potential therapeutics for efficacy and target engagement, using the JAK/STAT inhibitor tofacitinib (TOFA) as a model therapeutic. Colorectal biopsies from HC participants and patients with UC were cultured and stimulated with multiple mitogens ± TOFA. Soluble biomarkers were detected using a 29-analyte multiplex ELISA. Target engagement in CD3+CD4+ and CD3+CD8+ T-cells was determined by flow cytometry in peripheral blood mononuclear cells (PBMCs) and isolated mucosal mononuclear cells (MMCs) following the activation of STAT1/3 phosphorylation. Data were analyzed using linear mixed-effects modeling, t test, and analysis of variance. Biomarker selection was performed using penalized and Bayesian logistic regression modeling, with results visualized using uniform manifold approximation and projection. Under baseline conditions, 27 of 29 biomarkers from patients with UC were increased versus HC participants. Explant stimulation increased biomarker release magnitude, expanding the dynamic range for efficacy and target engagement studies. Logistic regression analyses identified the most representative UC baseline and stimulated biomarkers. TOFA inhibited biomarkers dependent on JAK/STAT signaling. STAT1/3 phosphorylation in T-cells revealed compartmental differences between PBMCs and MMCs. Immunogen stimulation increases biomarker release in similar patterns for HC participants and patients with UC, while enhancing the dynamic range for pharmacological effects. This work demonstrates the power of ex vivo human colorectal tissue as preclinical tools for evaluating target engagement and downstream effects of new IBD therapeutic agents.NEW & NOTEWORTHY Using colorectal biopsy material from healthy volunteers and patients with clinically defined IBD supports translational research by informing the evaluation of therapeutic efficacy and target engagement for the development of new therapeutic entities. Combining experimental readouts from intact and dissociated tissue enhances our understanding of the tissue-resident immune system that contribute to disease pathology. Bayesian logistic regression modeling is an effective tool for predicting ex vivo explant biomarker release patterns.

The CSF-1-receptor inhibitor, JNJ-40346527 (PRV-6527), reduced inflammatory macrophage recruitment to the intestinal mucosa and suppressed murine T cell mediated colitis.

BACKGROUND & AIMS: Originally believed to be primarily a disorder of T-cell signaling, evidence shows that macrophage-lineage cells also contribute to the pathogenesis of Crohn's disease (CD). Colony stimulating factor-1 (CSF-1) is a key regulator of the macrophage lineage, but its role in CD has not been well established. We examined transcriptional data from CD mucosa for evidence of CSF-1 pathway activation and tested JNJ-40346527 (PRV-6527), a small molecule inhibitor of CSF-1 receptor kinase (CSF-1R), for its ability to inhibit disease indices in murine colitis. METHODS: A CSF-1 pathway gene set was created from microarray data of human whole blood cultured ex vivo with CSF-1 and compared to a TNFα-induced gene set generated from epithelial-lineage cells. Gene set variation analysis was performed using existing Crohn's mucosa microarray data comparing patients who either responded or failed to respond to anti-TNFα therapy. Commencing day 14 or day 21, mice with T-cell transfer colitis were treated with vehicle or JNJ-40346527 until study termination (day 42). Endpoints included colon weight/length ratios and histopathology scores, and macrophage and T cells were assessed by immunohistochemistry. Mucosal gene expression was investigated using RNAseq. RESULTS: Both the CSF-1 and the TNFα gene sets were enriched in the colonic mucosal transcriptomes of Crohn's disease and in mouse colitis, and expression of both gene sets was highest in patients who did not respond to anti-TNFα therapy. In these patients neither set was reduced by therapy. In the mouse model, JNJ-40346527 inhibited the increase in colon weight/length ratio by ∼50%, reduced histological disease scores by ∼60%, and reduced F4/80+ mononuclear cell and CD3+ lymphocyte numbers. RNAseq analysis confirmed the CSF-1 gene set was sharply reduced in treated mice, as were gene sets enriched in "M1" inflammatory and "M0" resident macrophages and in activated T cells. CONCLUSIONS: CSF-1 biology is activated in Crohn's disease and in murine T cell transfer colitis. Inhibition of CSF-1R by JNJ-40346527 was associated with attenuated clinical disease scores and reduced inflammatory gene expression in mice. These data provide rationale for testing JNJ-40346527 (PRV-6527) in human inflammatory bowel disease.

Combined Blockade of TNF-α and IL-17A Alleviates Progression of Collagen-Induced Arthritis without Causing Serious Infections in Mice.

The cytokines TNF-α and IL-17A are elevated in a variety of autoimmune diseases, including rheumatoid arthritis. Both cytokines are targets of several biologic drugs used in the clinic, but unfortunately many patients are refractory to these therapies. IL-17A and TNF-α are known to mediate signaling synergistically to drive expression of inflammatory genes. Hence, combined blockade of TNF-α and IL-17A represents an attractive treatment strategy in autoimmune settings where monotherapy is not fully effective. However, a major concern with this approach is the potential predisposition to opportunistic infections that might outweigh any clinical benefits. Accordingly, we examined the impact of individual versus combined neutralization of TNF-α and IL-17A in a mouse model of rheumatoid arthritis (collagen-induced arthritis) and the concomitant susceptibility to infections that are likely to manifest as side effects of blocking these cytokines (oral candidiasis or tuberculosis). Our findings indicate that combined neutralization of TNF-α and IL-17A was considerably more effective than monotherapy in improving collagen-induced arthritis disease even when administered at a minimally efficacious dose. Encouragingly, however, dual cytokine blockade did not cooperatively impair antimicrobial host defenses, as mice given combined IL-17A and TNF-α neutralization displayed infectious profiles and humoral responses comparable to mice given high doses of individual anti-TNF-α or anti-IL-17A mAbs. These data support the idea that combined neutralization of TNF-α and IL-17A for refractory autoimmunity is likely to be associated with acceptable and manageable risks of opportunistic infections associated with these cytokines.

Remodeling of Tight Junctions and Enhancement of Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer by Micronutrients.

The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed.

Matrix metalloproteinase-9 inhibition reduces inflammation and improves motility in murine models of postoperative ileus.

BACKGROUND & AIMS: Matrix metalloproteinase (MMP)-9, a member of the gelatinase family of MMPs, mediates leukocyte migration during inflammation. Inflammation contributes to development of postoperative ileus (POI), which is caused by physical disturbances to the bowel during abdominal surgery. We evaluated the role of MMP-9 in POI and investigated whether disruption of MMP-9 or administration of an inhibitor of MMP-9 activity reduced cellular inflammation and bowel dysmotility in rat and mouse models of POI. METHODS: Mice and rats underwent laparotomy and bowel manipulation; bowel tissues were collected 3 to 24 hours later and analyzed by real-time reverse-transcriptase polymerase chain reaction, immunoblot, in situ zymography, and functional analyses. RESULTS: Bowel manipulation resulted in a time-dependent increase in MMP-9 expression within the intestinal muscularis; increases in MMP-9 messenger RNA were inducible nitric oxide synthase dependent. Immunoblot analyses confirmed the presence of the proenzyme and the catalytically active form of MMP-9. Administration of MMP-2/MMP-9 II, a dual active-site inhibitor, reduced the number of myeloperoxidase-positive immune cells that infiltrated the muscularis and prevented the surgically induced reduction in bowel smooth muscle contractility. Zymography analysis, performed in muscularis whole mounts in situ, indicated that MMP-9 and not MMP-2 mediated the gelatinase activity observed in infiltrating cells. MMP-9 knockout mice were protected from the inflammation and dysmotility associated with POI. CONCLUSIONS: MMP-9 mediates cellular inflammatory responses within the intestinal muscularis in mouse and rat models of POI. Inhibition of MMP-9 activity reduced recruitment of immune cells to the intestinal muscularis, preventing loss of smooth muscle contractility. Induction of MMP-9 expression requires inducible nitric oxide synthase.

The therapeutic potential of targeting the glucagon-like peptide-2 receptor in gastrointestinal disease.

INTRODUCTION: Glucagon-like peptide-2 (GLP-2) is a pleiotropic intestinotrophic hormone that enhances digestive and absorptive capacity by acting through a limited population of intestinal GLP-2 receptors. The development of protease-resistant analogs or GLP-2/IgG fusion proteins confers a longer circulating half life than the native peptide. GLP-2 has garnered interest as a therapeutic most notably by reducing reliance on total parenteral nutrition in patients with short bowel syndrome. AREAS COVERED: The clinical evidence for benefit in conditions requiring longer term treatment with GLP-2 receptor agonists, for example short bowel syndrome and inflammatory bowel disease. Benefits of short-term GLP-2 treatment are emerging in pre-clinical models, such as post-operative ileus, GI mucositis and conditions of altered intestinal permeability. The therapeutic utility of GLP-2 receptor agonists is limited by concern that it predisposes patients to gastrointestinal cancers, or their re-occurrence in cancer patients. This affects the types of diseases treated and, possibly, the duration of dosing. EXPERT OPINION: GLP-2 is therapeutically attractive in diseases to enhance absorptive capacity, restore mucosal health and reduce inflammation. Long-term surveillance studies with a marketed therapeutic agent are needed to weigh the benefits of GLP-2 treatment against the potential effects on co-morbidities and increased risk of intestinal carcinogenesis.

GLP-2 receptor agonism ameliorates inflammation and gastrointestinal stasis in murine postoperative ileus.

Glucagon-like peptide 2 (GLP-2) is a pleiotropic intestinotrophic hormone that we hypothesized could lessen gastrointestinal inflammation associated with postoperative ileus (POI). To test this idea, the prophylactic timing and dose of a long-acting variant of human GLP-2 linked to the Fc portion of murine immunoglobulin G (IgG) (GLP-2/IgG) was optimized in a murine model of POI. Surgically treated mice received a single dose of GLP-2/IgG, IgG isotype control, or phosphate-buffered saline 1 to 48 h before small bowel surgical manipulation. The distribution of orally fed fluorescein isothiocyanate-dextran and histological analyses of myeloperoxidase-positive immune cells were determined 24 and 48 h postoperatively. TaqMan quantitative polymerase chain reaction was used to determine early changes in mRNA expression in the muscularis or mucosa. In normal mice, prolonged exposure to GLP-2 increased upper gastrointestinal (GI) transit and mucosal weight. When administered 1 or 3 h before surgery, GLP-2/IgG reduced the leukocyte infiltrate 24 and 48 h postoperatively and improved GI transit 48 h postoperatively. Surgical manipulation rapidly increased gene expression of proinflammatory cytokines and enzymes for kinetically active mediators in the mucosa and muscularis. GLP-2/IgG2a affected the expression of genes associated with mucosal inflammation and barrier function. We conclude that prophylactic treatment with a long-acting GLP-2 agonist ameliorates inflammation and improves intestinal dysmotility associated with surgical manipulation of the bowel. The action of GLP-2 is consistent with a lessening of inflammation, leading to a more rapid recovery.

Proinflammatory role of leukocyte-derived Egr-1 in the development of murine postoperative ileus.

BACKGROUND & AIMS: Early growth response gene-1 (Egr-1) is an important inflammatory transcription factor. We hypothesize that leukocyte-derived Egr-1 plays a key inflammatory role in causing postoperative ileus. METHODS: Wild-type, Egr-1 knockout, and chimera mice (constructed by irradiation followed by injection with Egr-1(+/+) or Egr-1(-/-) bone marrow) were subjected to surgical manipulation of the gastrointestinal tract to induce ileus. Reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry quantified and localized Egr-1. Lumenal transit of nonabsorbable fluorescein isothiocyanate-labeled dextran and in vitro organ bath techniques measured functional gastrointestinal motility. Inflammatory mediator expressions were measured by Griess reaction, enzyme-linked immunosorbent assay, and multiplex Luminex assay. RESULTS: Intestinal manipulation rapidly and significantly induced Egr-1 messenger RNA and protein within the inflamed muscularis externa. Egr-1 was colocalized early to smooth muscle and enteric neurons and later in extravasated monocytes after surgery when postoperative ileus was functionally prominent. The functional severity of postoperative ileus was significantly ameliorated in mice deficient in Egr-1(-/-) and chimera wild-type mice transplanted with Egr-1(-/-) bone marrow, whereas knockout mice with Egr-1(+/+) bone marrow again displayed significant ileus. Motility was mechanistically associated in Egr-1(-/-) gene deficiency with a down-regulation in the release of nitric oxide, prostanoids, monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, interleukin-6, interleukin-1, and granulocyte colony-stimulating factor, as well as a decrease in the recruitment of leukocytes into the manipulated muscle wall of the intestine compared with wild-type mice. CONCLUSIONS: Leukocyte-derived Egr-1 plays an early critical inflammatory role in the initiation of the postoperative inflammatory response, which leads to a prolonged decreased in gastrointestinal motility after intestinal surgery.

Endogenous endotoxin participates in causing a panenteric inflammatory ileus after colonic surgery.

OBJECTIVE: To investigate muscularis inflammation and endogenous endotoxin as causes of postoperative ileus. BACKGROUND: Postoperative inflammatory ileus of the colon is associated with a significant delay in gastrointestinal transit. We investigated whether these changes are caused by the downstream obstructive barrier of the surgically altered colon or by small intestinal muscularis inflammation itself. Furthermore, we evaluated the mechanistic role of gut derived endotoxin in the development of postoperative intestinal dysfunction. METHODS: Rats underwent surgical manipulation of the colon. Isolated gastrointestinal transit was analyzed in animals with ileostomy. The perioperative emigration of intracolonic particles was investigated by colonic luminal injection of fluorescently labeled LPS and microspheres. Mediator mRNA induction was quantified by real-time RT-PCR. Muscularis leukocytic infiltrates were characterized. In vitro circular muscle contractility was assessed in a standard organ bath. RESULTS: Ileostomy rats presented with a significant delay in small intestinal transit after colonic manipulation. This was associated with leukocyte recruitment and inflammatory mediator mRNA induction within the small intestinal muscularis. Colonic manipulation caused the transference of intracolonic LPS and microspheres into the intestinal muscularis. Postoperative in vitro small intestinal circular muscle contractility was impaired by 42% compared with controls. Gut decontamination and TLR-4 deletion significantly alleviated the small intestinal muscularis inflammation and prevented intestinal muscle dysfunction. CONCLUSIONS: Selective colonic manipulation initiates a distant inflammatory response in the small intestinal muscularis that contributes to postoperative ileus. The data provide evidence that gut-derived bacterial products are mechanistically involved in the initiation of this remote inflammatory cascade.

Altered inflammatory gene expression underlies increased susceptibility to murine postoperative ileus with advancing age.

Susceptibility to postoperative ileus following abdominal surgery increases with advancing age. The mechanisms underlying this phenomenon are unknown. This study compares functional and molecular endpoints between young-adult (2 mo old), middle-aged (15 mo old), and elderly mice (26-30 mo old) to identify potential mechanisms. Susceptibility to ileus was assessed by measuring gastrointestinal transit (geometric center) 24 h after anesthesia, laparotomy, and light manipulation (LM) of the small bowel. Proinflammatory (IL-6, COX-2, inducible nitric oxide synthase) and anti-inflammatory (IL-10, heme oxygenase-1) gene and protein expressions were determined by real time RT-PCR, Western blot, and ELISA. LM did not alter gastrointestinal transit in young animals (geometric center = 8.8 +/- 0.9), but transit was increasingly delayed in middle-aged (6.9 +/- 0.8, P = 0.03) and elderly animals (4.7 +/- 0.6, P = 0.013). Despite the lack of LM effect on transit in young mice, IL-6 and COX-2 mRNA expressions were significantly increased postoperatively (165 +/- 24-fold and 2.9 +/- 0.3-fold, respectively). Expressions were increased further in middle-aged mice (1,103 +/- 187-fold; 4.4 +/- 0.7-fold) and further still in elderly mice (1,218 +/- 168-fold; 6.9 +/- 0.3-fold). IL-10 and heme oxygenase-1 gene expressions were also elevated postoperatively in young mice (4.8 +/- 0.5-fold and 13.0 +/- 1.3-fold, respectively) and were further increased in middle-aged mice (7.5 +/- 0.6-fold; 21.8 +/- 3.2-fold). However, inductions in elderly mice were significantly blunted (5.8 +/- 0.9-fold; 16.9 +/- 0.8-fold). There is both an age-dependent increase in the proinflammatory mediator expression and an age-dependent decrease in anti-inflammatory mediator expressions following minor insult to the bowel. Such imbalances between pro- and anti-inflammatory mechanisms may form the basis for increased susceptibility to ileus and for the increased severity and duration of ileus observed in the elderly.

Leukocyte-derived inducible nitric oxide synthase mediates murine postoperative ileus.

OBJECTIVE: To provide evidence that iNOS expression solely in leukocytes plays a role in postoperative ileus. SUMMARY BACKGROUND DATA: Intestinal handling initiates a molecular and cellular muscularis inflammation that has been associated with iNOS expression and ileus. The specific cellular source of iNOS is a matter of speculation. METHODS: Chimeric mice were constructed that selectively express the iNOS gene only in their leukocytes or only in their parenchymal cells by lethal radiation and reconstitution with reciprocal bone marrow. Mild intestinal manipulation was used to induce postoperative ileus. RESULTS: Intestinal manipulation caused a significant leukocyte extravasation into the muscularis of all groups. Postoperative iNOS mRNA expression was evident in iNOS and transplanted iNOS mice with iNOS bone marrow but not in iNOS animals. The loss of the iNOS gene in leukocytes of iNOS mice reduced iNOS mRNA expression by 59%. iNOS-deficient mice and iNOS animals with iNOS leukocytes presented with a significant improvement in postoperative intestinal transit and in vitro smooth muscle contractility, whereas the replacement with iNOS bone marrow in iNOS mice completely reversed this improvement. CONCLUSION: These results clearly show that iNOS expressed in leukocytes within the intestinal muscularis plays a major role in mediating smooth muscle dysfunction and subsequently postoperative ileus.

Biliverdin protects against polymicrobial sepsis by modulating inflammatory mediators.

Highly inducible heme oxygenase (HO)-1 is protective against acute and chronic inflammation. HO-1 generates carbon monoxide (CO), ferrous iron, and biliverdin. The aim of this study was to investigate the protective effects of biliverdin against sepsis-induced inflammation and intestinal dysmotility. Cecal ligation and puncture (CLP) was performed on Sprague-Dawley rats under isoflurane anesthesia with and without intraperitoneal biliverdin injections, which were done before, at the time of CLP, and after CLP. In vivo gastrointestinal transit was carried out with fluorescein-labeled dextran. Jejunal circular muscle contractility was quantified in vitro using organ bath-generated bethanechol dose-response curves. Neutrophilic infiltration into the muscularis externa was quantified. The jejunal muscularis was studied for cytokine mRNA expressions [interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, inducible nitric oxide synthase, cyclooxygenase-2, biliverdin, IL-10, and HO-1] using real-time RT-PCR. Biliverdin treatment prevented the sepsis-induced suppression of gastrointestinal muscle contractility in vivo and in vitro and significantly decreased neutrophilic infiltration into the jejunal muscularis. Inflammatory mRNA expressions for small bowel IL-6 and MCP-1 were significantly reduced after biliverdin treatment in CLP-induced septic animals compared with untreated septic animals. The anti-inflammatory mediator expression of small bowel IL-10 was significantly augmented after CLP at 3 h compared with untreated septic animals. These findings demonstrate that biliverdin attenuates sepsis-induced morbidity to the intestine by selectively modulating the inflammatory cascade and its subsequent sequelae on intestinal muscularis function.

Ethyl pyruvate ameliorates ileus induced by bowel manipulation in mice.

BACKGROUND: Ethyl pyruvate (EP) improves survival, decreases proinflammatory cytokine expression, and ameliorates organ dysfunction in mice who have lethal sepsis or were subjected to hemorrhagic shock. Herein, we tested the hypothesis that treatment with EP can prevent the development of ileus after bowel manipulation, a phenomenon that is mediated by an inflammatory response in the bowel wall. METHODS: C57Bl/6 mice underwent operative manipulation of the small intestine or were subjected to a sham procedure. Some of the mice subjected to gut manipulation were pre- and post-treated with 4 doses of EP (40 or 80 mg/kg per dose), whereas others received similar volumes of the vehicle for EP. Gastrointestinal transit of a nonabsorbable marker was assessed by gavaging the mice with the tracer 24 hours after operation and assessing its concentration 90 minutes later in bowel contents from the stomach, 10 equally long segments of small intestine, the cecum, and 2 equally long segments of colon. The contractile responses of ileal circular smooth muscle to graded concentrations of bethanechol were assessed by using standard organ bath methodology. Expression of interleukin-6 and inducible nitric oxide synthase transcripts in ileal muscularis propria was assessed by using the semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: In sham-operated controls, the mean (+/- SE) geometric center for the transit marker was 10.0 +/- 0.5, whereas for vehicle-treated mice subject to bowel manipulation, the value for this parameter was 3.5 +/- 0.1 (P < .05). When mice subjected to bowel manipulation were treated with several 40 mg/kg doses of EP, the geometric center was 7.3 +/- 1.0 (P < .05 vs sham-operated group). Gut manipulation impaired intestinal smooth muscle contractility in vitro and increased steady-state levels of interleukin-6 and inducible nitric oxide synthase messenger RNA. Treatment with EP ameliorated these effects of gut manipulation. CONCLUSIONS: EP warrants further evaluation as a therapeutic agent to ameliorate postoperative ileus.

Brief inhalation of low-dose carbon monoxide protects rodents and swine from postoperative ileus.

OBJECTIVE: Carbon monoxide (CO), an endogenous byproduct of heme metabolism, is produced at high levels in injured tissue via induction of heme-oxygenase-1 activity, where it contributes to the modulation of proinflammatory processes. Alone, CO has potent anti-inflammatory effects in models of acute and chronic inflammation. In rodents, inhalation of low concentrations of CO (250 ppm) for 24 hrs protects against postoperative gastrointestinal ileus. The current study determined whether shorter exposures and lower concentrations were equally protective and whether CO treatment would be effective in a large animal species (swine) managed under conditions approximating the clinical setting. DESIGN: Dosing studies were first performed in rats by exposing them to CO (30-250 ppm) or air by inhalation for 1 or 3 hrs before anesthesia. An effective dosing regimen was then selected for testing in swine. Postoperative ileus in both species was induced by laparotomy and mild compression (running) of the small intestine. MEASUREMENTS AND MAIN RESULTS: In rats, inhalation of 75 ppm CO for 3 hrs before anesthesia and surgery ameliorated the surgically induced delay in gastrointestinal transit to levels achieved using 250 ppm for 24 hrs. Swine treated with 250 ppm CO for the same time period exhibited significantly improved postoperative intestinal circular muscle contractility in vitro and gastrointestinal transit in vivo. Carboxyhemoglobin concentrations measured after termination of CO exposure averaged 5.8% (baseline, 1.5%). No deleterious effects on heart rate, oxygen saturation, blood chemistries, and serum electrolytes were observed. CONCLUSIONS: These findings demonstrate that inhalation of a low concentration of CO before surgery attenuates postoperative ileus in rodents and, more importantly, in a large animal species without risk to well-being during surgery or perioperatively. Exposures need not be prolonged, with significant benefit occurring with a 3-hr pretreatment.

Construction of a Festuca pratensis BAC library for map-based cloning in Festulolium substitution lines.

Introgression in Festulolium is a potentially powerful tool to isolate genes for a large number of traits which differ between Festuca pratensis Huds. and Lolium perenne L. Not only are hybrids between the two species fertile, but the two genomes can be distinguished by genomic in situ hybridisation and a high frequency of recombination occurs between homoeologous chromosomes and chromosome segments. By a programme of introgression and a series of backcrosses, L. perenne lines have been produced which contain small F. pratensis substitutions. This material is a rich source of polymorphic markers targeted towards any trait carried on the F. pratensis substitution not observed in the L. perenne background. We describe here the construction of an F. pratensis BAC library, which establishes the basis of a map-based cloning strategy in L. perenne. The library contains 49,152 clones, with an average insert size of 112 kbp, providing coverage of 2.5 haploid genome equivalents. We have screened the library for eight amplified fragment length polymorphism (AFLP) derived markers known to be linked to an F. pratensis gene introgressed into L. perenne and conferring a staygreen phenotype as a consequence of a mutation in primary chlorophyll catabolism. While for four of the markers it was possible to identify bacterial artificial chromosome (BAC) clones, the other four AFLPs were too repetitive to enable reliable identification of locus-specific BACs. Moreover, when the four BACs were partially sequenced, no obvious coding regions could be identified. This contrasted to BACs identified using cDNA sequences, when multiple genes were identified on the same BAC.

Tyrphostin AG 126 inhibits development of postoperative ileus induced by surgical manipulation of murine colon.

Manipulation of the bowel during abdominal surgery leads to a period of ileus, which is most severely manifested after procedures that directly involve the colon. Ileus is associated with the increased expression of proinflammatory cytokines and chemokines, a leukocytic infiltration into the muscularis, and the release of mediators from resident and infiltrating leukocytes that directly inhibit intestinal smooth muscle contractility. Phosphorylation of tyrosine residues on regulatory proteins by protein tyrosine kinases (PTKs) occurs at multiple steps in the signaling cascades that regulate the expression of proinflammatory genes. The purpose of this study was to determine whether inhibition of PTK activity will attenuate the inflammatory response associated with colonic ileus and lead to improved function. Using a rodent model of colonic postoperative ileus, we demonstrate that a single bolus injection of the PTK inhibitor tyrphostin AG 126 (15 mg/kg sc) before surgery significantly attenuates the surgically induced impairment of colonic contractility both in vivo and in vitro. Improvement in function was associated with a reduction in magnitude of inflammatory cell infiltrate and with a decrease in transcription of genes encoding proinflammatory mediators IL-1beta and monocyte chemoattractant protein (MCP)-1, inducible nitric oxide synthase, and cyclooxygenase-2. Furthermore, tyrphostin AG 126 pretreatment significantly inhibited activation of multifactorial transcription factor NF-kappaB, which could form the basis for reduction in proinflammatory mediator expression. These data demonstrate for the first time that inhibition of PTK activity may represent a novel approach for management of ileus in the clinical setting.

Potassium currents regulating secretion from Brunner's glands in guinea pig duodenum.

This study examined the role of outward K(+) currents in the acinar cells underlying secretion from Brunner's glands in guinea pig duodenum. Intracellular recordings were made from single acinar cells in intact acini in in vitro submucosal preparations, and videomicroscopy was employed in the same preparation to correlate these measures with secretion. Mean resting membrane potential was -74 mV and was depolarized by high external K(+) (20 mM) and the K(+) channel blockers 4-aminopyridine (4-AP), quinine, and clotrimazole. The cholinergic agonist carbachol (60-2,000 nM; EC(50) = 200 nM) caused a concentration-dependent initial hyperpolarization of the membrane and an associated decrease in input resistance. This hyperpolarization was significantly decreased by 20 mM external K(+) or membrane hyperpolarization and increased by 1 mM external K(+) or membrane depolarization. It was blocked by the K(+) channel blockers tetraethylammonium (TEA), 4-AP, quinine, and clotrimazole but not iberiotoxin. When videomicroscopy was employed to measure dilation of acinar lumen in the same preparation, carbachol-evoked dilations were altered in a parallel fashion when external K(+) was altered. The dilations were also blocked by the K(+) channel blockers TEA, 4-AP, quinine, and clotrimazole but not iberiotoxin. These findings suggest that activation of outward K(+) currents is fundamental to the initiation of secretion from these glands, consistent with the model of K(+) efflux from the basolateral membrane providing the driving force for secretion. The pharmacological profile suggests that these K(+) channels belong to the intermediate conductance group.

Long-term function and morphology of intestinal autografts and allografts in outbred dogs.

Although small bowel transplantation (SBTx) has become a clinical option, there have been few studies of long-term function and histopathology of intestinal grafts. Unrelated mongrel dogs received autologous (n = 4) or allogeneic (n = 11) orthotopic SBTx under oral cyclosporine. Intestinal graft function and routine/immunohistopathology of full-thickness intestine were studied. Six allograft and all isograft recipients had comparable body weight gain and are currently alive (> 420 days). Five allograft recipients were sacrificed because of significant body weight loss and malnutrition at a median of 119 days. Analyses of intestinal function in long-surviving recipients revealed marginal reduction of D-xylose/cyclosporine absorption, intestinal transit time, in vitro muscle contractility, and mucosal enzyme activity compared with normal dogs. However, these changes were insignificant and no statistical difference was seen between auto and long-surviving allografts. In histopathological analysis, long-surviving allografts had normal mucosa with submucosal, muscularis propria, and perineural (Auerbach's plexus) inflammation. Five allorecipients with malnutrition had mucosal atrophy/erosion and significantly reduced intestinal absorption and motility. Thus, denervated intestinal allografts are able to efficiently digest and absorb nutrients to support life. Results also indicate that these allografts experienced low-grade chronic rejection as evidenced in the submucosa and muscle layers, despite the lack of clinical symptoms.

Inhaled carbon monoxide suppresses the development of postoperative ileus in the murine small intestine.

BACKGROUND & AIMS: The induction of heme oxygenase (HO-1), the rate-limiting enzyme in heme metabolism, is protective against injury in acute and chronic inflammation. Inhalation of low levels of carbon monoxide (CO), a byproduct of heme metabolism, has anti-inflammatory effects equal to HO-1 induction. This study examined whether inhaled CO was protective against the development of postoperative ileus. METHODS: Ileus was induced by surgical anesthesia and gentle manipulation of the mouse small intestine. Animals were exposed to CO (250 ppm) in air 1 hour before and continuously for 24 hours after surgery. RESULTS: CO inhalation prevented the manipulation-induced suppression of circular muscle contractility in vitro, and significantly improved gastrointestinal transit in vivo. Proinflammatory messenger RNA (mRNA) expression (interleukin [IL]-6, IL-1beta, cyclooxygenase 2 [COX-2], inducible nitric oxide [iNOS]) and anti-inflammatory mediator expression (IL-10 and HO-1) were elevated 3 to 6 hours after surgery relative to controls. CO treatment reduced IL-1beta and iNOS peak expression by 75%, but not IL-6 or COX-2. In manipulated mice treated with CO, HO-1 expression peaked earlier (3 hours after surgery) and at levels 300% higher than in mice not exposed to CO. IL-10 expression at 3 hours also was 300% higher after CO treatment. CONCLUSIONS: These findings suggest that CO attenuates postoperative ileus by inhibiting selective elements within the inflammatory cascade and by enhanced induction of the anti-inflammatory cytokine IL-10. In addition, the early and enhanced induction of HO-1 potentially amplifies the anti-inflammatory effects of the HO-1 pathway by protection from free radical stress and by increasing the tissue availability of CO directly at the sites of inflammation.