Ileocecal Anastomosis Type Significantly Influences Long-Term Functional Status, Quality of Life, and Healthcare Utilization in Postoperative Crohn's Disease Patients Independent of Inflammation Recurrence.

OBJECTIVES: Anastomotic reconstruction following intestinal resection in Crohn's disease (CD) may employ side-to-side anastomosis (STSA; anti-peristaltic orientation) or end-to-end anastomosis (ETEA). Our aim was to determine the impact of these two anastomotic techniques on long-term clinical status in postoperative CD patients. METHODS: We performed a comparative effectiveness study of prospectively collected observational data from consented CD patients undergoing their first or second ileocolonic bowel resection and re-anastomosis between 2008 and 2012, in order to assess the association between anastomosis type and 2-year postoperative quality of life (QoL), healthcare utilization, disease clinical or endoscopic recurrence, use of medications, and need for repeat resection. RESULTS: One hundred and twenty eight postoperative CD patients (60 STSA and 68 ETEA) were evaluated. At 2 years postoperatively, STSA patients had higher rates of emergency department visits (33.3% vs. 14.7%; P=0.01), hospitalizations (30% vs. 11.8%; P=0.01), and abdominal computed tomography scans (50% vs. 13.2%; P<0.001) with lower QoL (mean short inflammatory bowel disease questionnaire 47.9 vs. 53.4; P=0.007). There was no difference among the two groups in the 30 day surgical complications and 2-year patterns of disease activity, CD medication requirement, endoscopic recurrence, and need for new surgical management (all P > 0.05). CONCLUSIONS: At 2 years postoperatively, CD patients with ETEA demonstrated better QoL and less healthcare utilization compared with STSA, despite having similar patterns of disease recurrence and CD treatment. These findings suggest that surgical reconstruction of the bowel as an intact tube (ETEA) contribute to improved functional and clinical status in patients with CD.

Myocyte TLR4 enhances enteric and systemic inflammation driving late murine endotoxic ileus.

Myocytes are nonhemopoietic in origin and functionally essential in generating gastrointestinal motility. In endotoxemia, a rapid-onset nonhemopoietic mechanism potently triggers early ileus in a Toll-like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (MyD88)-dependent manner. Moreover, synergistically with hemopoietic cells, nonhemopoietic cells escalate late ileus via an IL-6 receptor-dependent inflammation-driven pathway. We therefore specifically investigated the role of myocytes in TLR4-triggered inflammation and ileus. TLR4(+/+), TLR4(-/-), bmTLR4(+/+)/TLR4(-/-) chimera, SM22-Cre(-/-)TLR4(flox/flox), and selective myocyte TLR4-deficient (SM22-Cre(+/-)TLR4(flox/flox)) mice were injected intraperitoneally with purified lipopolysaccharide. SM22-driven Cre recombinase activity was selectively detected in cardiac, gastrointestinal, skeletal, and vascular myocytes, of small-sized vessels in a two-color fluorescent Cre reporter mouse. In contrast to nonhemopoietic TLR4 deficiency, deletion of myocyte TLR4 signaling prevented neither endotoxin-induced suppression of spontaneous jejunal contractility in vitro nor early ileus in vivo at 6 h. Circulating plasma colony-stimulating factor 3 was greatly elevated during endotoxemia, independent of myocyte TLR4 signaling or time. TLR4 activation of myocytes contributed significantly to an early enteric IL-6 mRNA induction and systemic IL-6 release, as well as to a late increase in circulating chemokine (C-X-C motif) ligand 1 (CXCL1) and IL-17. Consequently, inhibition of myocyte TLR4 signaling allowed functional recovery of motility by preventing inflammation-driven late ileus at 24 h. Direct TLR4 activation of myocytes is not responsible for nonhemopoietic-mediated early ileus. However, myocytes are proinflammatory cells that potently drive enteric and systemic inflammation, subsequently fueling late mediator-triggered ileus. Specifically, the myocyte TLR4-dependent inflammatory signature of elevated plasma IL-6, CXCL1, and IL-17 is strongly associated with late rodent ileus.

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.

Differential molecular and cellular immune mechanisms of postoperative and LPS-induced ileus in mice and rats.

Ileus is caused by the initiation of a complex cascade of molecular and cellular inflammatory responses within the intestinal muscularis, which might be species specific. Our objective was to investigate a possible immunological divergence in the mechanisms of postoperative- and endotoxin-induced ileus in C57BL/6 mice and Sprague-Dawley rats. Gastrointestinal transit (GIT) was measured at 24 h after the injurious stimulus. MPO-staining and F4/80 immunohistochemistry were used to quantify polymorphonuclear and monocyte infiltration of jejunal muscularis whole-mounts, and intestinal muscularis MCP-1, ICAM-1 and iNOS gene expression was assessed by RT-PCR. Intestinal muscularis subjected to in vivo surgical manipulation (SM) or LPS treatment was cultured for 24 h, and the liberation of nitric oxide and chemokines/cytokines into the culture medium was analyzed by Griess reaction and Luminex multiplex assay. Intestinal SM and lipopolysaccharide (LPS) (15 mg/kg) caused a significant delay in gastrointestinal transit, which was more severe in mice compared to rats in both injury models. Both SM- and LPS-triggered neutrophil and monocytic extravasation into the rat jejunal muscularis exceeded the cellular infiltration seen in mice. These results correlated with significantly greater increases in rat muscularis MCP-1 (syn. CCL2), ICAM-1 and iNOS message with more subsequent NO production after SM or LPS compared to mouse. The cultured muscularis obtained from SM mice released significantly more inflammatory proteins such as TNF-α, IL-1-α, IL-4 and GM-CSF compared to the manipulated rat muscularis. In contrast, LPS initiated the secretion of significantly more IL-1ß by the inflamed rat muscularis compared to the mouse, but GM-CSF (syn. CSF2) liberation from mouse muscularis was markedly higher compared to LPS-treated rat muscularis. The data indicate that mechanistically the development of ileus in rat is mediated predominately through a leukocytic pathway consisting of chemotaxis, cellular extravasation and NO liberation. Whereas, the more intense mouse ileus evolves via a potent but injury-specific local cytokine response.

Iatrogenic extracellular matrix disruption as a local trigger for postoperative ileus.

BACKGROUND: Active matrix metallopeptidase 9 (MMP-9) disruption of the extracellular matrix (ECM) plays an important role in inflammatory disorders. In this study, we investigated the inflammatory role of MMP-9 and the ECM breakdown product hyaluronan as a trigger for the postoperative intestinal inflammatory response of postoperative ileus. METHODS: We performed a standardized intestinal surgical manipulation on rats to produce ileus assessed by the oral non-digestible fluorescein isothiocyanate-dextran transit assay. We studied isolated intestinal muscularis extracts for mRNA expressions of interleukin 6 (IL-6), MMP-9 and CD44. We quantified peritoneal MMP-9 activity using zymography, and quantified peritoneal fluid and serum for hyaluronan and tissue inhibitor of metalloproteinase 1 levels by enzyme-linked immunosorbent assay (ELISA). We cultured peritoneal macrophages and exposed them to peritoneal fluid or synthetic hyaluronan for ELISA analysis of IL-6 and macrophage inflammatory protein-1α. RESULTS: Transit was significantly delayed after surgical manipulation, and extracts of the isolated jejunal and colonic muscularis demonstrated a significant induction of IL-6, MMP-9, and CD44 mRNAs compared with controls. Zymography confirmed significant MMP-9 activity in peritoneal fluid compared with controls. Enzyme-linked immunosorbent assay measurements showed a significant up-regulation in hyaluronan and tissue inhibitor of metalloproteinase 1 in the peritoneal fluid and serum. In addition, ELISA and reverse transcriptase-polymerase chain reaction measurements of peritoneal macrophages stimulated with postsurgical peritoneal fluid and synthetic hyaluronan resulted in higher expressions of IL-6 and macrophage inflammatory protein-1α in the macrophage supernatant. CONCLUSIONS: Our results confirm that MMP-9 disruption in the ECM with hyaluronan release and muscularis CD44 receptor induction has the potential to trigger muscularis proinflammatory cascades that cause postoperative ileus. Matrix metallopeptidase 9 inhibition may be a novel therapeutic approach to limit postoperative ileus.

CD206-positive M2 macrophages that express heme oxygenase-1 protect against diabetic gastroparesis in mice.

BACKGROUND & AIMS: Gastroparesis is a well-recognized complication of diabetes. In diabetics, up-regulation of heme oxygenase-1 (HO1) in gastric macrophages protects against oxidative stress-induced damage. Loss of up-regulation of HO1, the subsequent increase in oxidative stress, and loss of Kit delays gastric emptying; this effect is reversed by induction of HO1. Macrophages have pro- and anti-inflammatory activities, depending on their phenotype. We investigated the number and phenotype of gastric macrophages in NOD/ShiLtJ (nonobese diabetic [NOD]) mice after onset of diabetes, when delayed gastric emptying develops, and after induction of HO1 to reverse delay. METHODS: Four groups of NOD and db/db mice were studied: nondiabetic, diabetic with normal emptying, diabetic with delayed gastric emptying, and diabetic with delayed gastric emptying reversed by the HO1 inducer hemin. Whole mount samples from stomach were labeled in triplicate with antisera against F4/80, HO1, and CD206, and macrophages were quantified in stacked confocal images. Markers for macrophage subtypes were measured by quantitative polymerase chain reaction. RESULTS: Development of diabetes was associated with an increased number of macrophages and up-regulation of HO1 in CD206(+) M2 macrophages. Onset of delayed gastric emptying did not alter the total number of macrophages, but there was a selective loss of CD206(+)/HO1(+) M2 macrophages. Normalization of gastric emptying was associated with repopulation of CD206(+)/HO1(+) M2 macrophages. CONCLUSIONS: CD206(+) M2 macrophages that express HO1 appear to be required for prevention of diabetes-induced delayed gastric emptying. Induction of HO1 in macrophages might be a therapeutic option for patients with diabetic gastroparesis.

Nonhemopoietic cell TLR4 signaling is critical in causing early lipopolysaccharide-induced ileus.

Endotoxin-mediated ileus is poorly understood. Our objective was to mechanistically investigate the role of cell-specific TLR4 expression/signaling in causing gastrointestinal dysmotility. TLR4 chimeras and CSF-1-dependent macrophage-deficient mice were subjected to i.p. ultrapure (UP)-LPS (5 mg/kg). At 6 h, gastric emptying and gastrointestinal transit assessed in vivo motility, and jejunal circular muscle contractility was measured in vitro. Muscularis infiltration of neutrophils and monocytes were counted, and intestinal muscularis inflammatory mediators were quantified by quantitative PCR. Demonstrating TLR4 dependency, UP-LPS-induced gastric stasis and ileus of TLR4(WT) mice were absent in mutant TLR4(LPS-d) mice. Unexpectedly, engraftment of TLR4-mutant bone marrow into TLR4-competent mice (bmTLR4(LPS-d)/TLR4(WT)) exhibited a significant transit delay to UP-LPS similar to bmTLR4(WT)/TLR4(WT) mice. CSF-1(-/-) mice were not protected from ileus. Contrary, UP-LPS-treated bmTLR4(WT)/TLR4(LPS-d) and bmTLR4(LPS-d)/TLR4(LPS-d) mice had normal transit. No leukocytic infiltration was detected at 6 h. Spontaneous jejunal contractions were markedly suppressed in UP-LPS-treated TLR4-competent mice, but bethanechol-stimulated contractions were not altered by UP-LPS in any group. UP-LPS-induced inflammatory mRNAs in a TLR4-dependent manner, but TLR4 mRNA itself was not significantly altered. In chimera mice, UP-LPS induction of IL-1beta and IL-10 were hemopoietic dependent, and GM-CSF was nonhemopoietic dependent, whereas IL-6 and inducible NO synthase were derived from both cell types. Hemopoietic and nonhemopoietic cells contribute to TLR4-sensitive muscularis inflammatory signaling, but nonhemopoietic TLR4 signaling plays an exclusive primary role in causing functional UP-LPS-induced gastric stasis and ileus. Direct LPS suppression of spontaneous contractility participates in mediating early TLR4-transduced dysmotility.

Dominant role of the MyD88-dependent signaling pathway in mediating early endotoxin-induced murine ileus.

TLR4 ligation by pathogen-associated molecular patterns, such as Gram-negative bacteria-derived LPS, triggers a nonhematopoietic cell-mediated ileus during early endotoxemia. Our objective was to investigate the quantitative contributions of the two downstream signaling pathways of TLR4, namely the adapter proteins myeloid differentiation primary response gene 88 (MyD88) and Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing IFN-beta (TRIF). Six hours after intraperitoneal injection of highly purified LPS (UP-LPS, 5 mg/kg), in vivo gastrointestinal transit and intestinal muscularis gene transcripts of inflammatory mediators chemokine (C-X-C motif) ligand 10, synonymous IP-10 (CXCL10), granulomonocyte colony stimulating factor (GM-CSF, synonymous CSF-2), IL-1beta, IL-6, IL-10, and inducible NO synthase (iNOS) were assessed in mice with transgenic loss-of-function for MyD88 or TRIF. LPS-induced MyD88 and TRIF mRNA upregulation was quantified within the intestinal muscularis of TLR4-competent and TLR4-mutant mice, and MyD88 mRNA levels were additionally measured in TLR4 bone marrow chimeras. MyD88 deficiency completely protected mice from early endotoxin-induced ileus, while TRIF deficiency partially ameliorated ileus severity. LPS induction of the primary downstream signaling element MyD88 was TLR4 dependent and was derived in equal amounts from both the hematopoietic and the nonhematopoietic cells. Conversely, no induction of TRIF mRNA was detectable. Significant gene induction of all inflammatory mediators was dependent on intracellular signal transduction by MyD88, while the TRIF MyD88-independent pathway predominantly regulated the molecular levels of CXCL10. In summary, MyD88 and TRIF are nonredundant signaling pathways in early endotoxin-induced rodent ileus, but MyD88 is the essential adaptor molecule for transduction of early TLR4-induced ileus and inflammatory signaling. The dependency of ileus on individual adaptor protein pathways is also reflected in the manifestation of specific molecular inflammatory events within the intestinal muscularis externa.

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.

IL-10 deficiency augments acute lung but not liver injury in hemorrhagic shock.

In hemorrhagic shock and trauma, patients are prone to develop systemic inflammation with remote organ dysfunction, which is thought to be caused by pro-inflammatory mediators. This study investigates the role of the immuno-modulatory cytokine IL-10 in the development of organ dysfunction following hemorrhagic shock. Male C57/BL6 and IL-10 KO mice were subjected to volume controlled hemorrhagic shock for 3h followed by resuscitation. Animals were either sacrificed 3 or 24h after resuscitation. To assess systemic inflammation, serum IL-6, IL-10, KC, and MCP-1 concentrations were measured with the Luminex multiplexing platform; acute lung injury (ALI) was assessed by pulmonary myeloperoxidase (MPO) activity and lung histology and acute liver injury was assessed by hepatic MPO activity, hepatic IL-6 levels, and serum ALT levels. There was a trend towards increased IL-6 and KC serum levels 3h after resuscitation in IL-10 KO as compared to C57/BL6 mice; however this did not reach statistical significance. Serum MCP-1 levels were significantly increased in IL-10 KO mice 3 and 24 h following resuscitation as compared to C57/BL6 mice. In IL-10 KO mice, pulmonary MPO activity was significantly increased 3 h following resuscitation and after 24 h histological signs of acute lung injury were more apparent than in C57/BL6 mice. In contrast, no significant differences in any liver parameters were detected between IL-10 KO and C57/BL6 mice. Our data indicate that an endogenous IL-10 deficiency augments acute lung but not liver injury following hemorrhagic shock.

Identification of nuclear matrix protein alterations associated with human colon cancer.

The early diagnosis of colorectal cancer and the early detection of recurrence are central to effective treatment, as prognosis is directly related to the stage of the disease. When colorectal cancer is diagnosed at an early, localized stage, 5-year survival is 90%. With regional lymph node metastases, survival drops to 45-60%, and with distant metastases, 5-year survival is <5%. Development of tumor markers that can detect colon cancer at an early stage should have a major impact in mortality from this disease. The nuclear matrix is the structural scaffolding of the nucleus, and specific nuclear matrix proteins (NMPs) have been identified as an oncological "fingerprint" for bladder, renal, and prostate cancers. We have successfully used this approach to develop an immunoassay that detected bladder cancer early in a clinical trial with a sensitivity of 96.4% and a specificity of 100%. The objective of the present study was to identify the existence of a specific NMP fingerprint for human colon cancer, using high-resolution, two-dimensional gel electrophoresis, and thereby identify unique human colon cancer NMPs. Ten matched colon cancer and adjacent normal samples and 4 normal donor samples were analyzed. Analysis of multiple gels for each sample identified four proteins present in all tumor samples that were not present in the matched normal adjacent and normal colon tissue and six proteins present only in normal adjacent and normal colon tissue. Additionally, two proteins were found in all cancer and normal tissues, but not in the normal adjacent tissue. Data provided here demonstrate that examination of the nuclear matrix composition allows differentiation of colon cancer tissue from normal adjacent and normal colon tissue. Development of an assay to detect these specific NMPs by examining tissue, serum, and stool specimens is a promising modality for early detection of colorectal cancer. In addition, the functional characterization of these proteins and their early detection through the generation of NMP antibodies could significantly impact on the understanding of cancer progression and its diagnosis.

Interleukin 10 Restores Gastric Emptying, Electrical Activity, and Interstitial Cells of Cajal Networks in Diabetic Mice.

BACKGROUND & AIMS: Gastroparesis is a complication of diabetes characterized by delayed emptying of stomach contents and accompanied by early satiety, nausea, vomiting, and pain. No safe and reliable treatments are available. Interleukin 10 (IL10) activates the M2 cytoprotective phenotype of macrophages and induces expression of heme oxygenase 1 (HO1) protein. We investigated whether IL10 administration could improve gastric emptying and reverse the associated cellular and electrical abnormalities in diabetic mice. METHODS: Nonobese diabetic mice with delayed gastric emptying were given either IL10 (0.1-1 µg, twice/day) or vehicle (controls). Stomach tissues were isolated, and sharp microelectrode recordings were made of the electrical activity in the gastric muscle layers. Changes to interstitial cells of Cajal (ICC), reduced nicotinamide adenine dinucleotide phosphate diaphorase, and levels and distribution of HO1 protein were determined by histochemical and imaging analyses of the same tissues. RESULTS: Gastric emptying remained delayed in vehicle-treated diabetic mice but returned to normal in mice given IL10 (n = 10 mice; P < .05). In mice given IL10, normalization of gastric emptying was associated with a membrane potential difference between the proximal and distal stomach, and lower irregularity and higher frequency of slow-wave activity, particularly in the distal stomach. Levels of HO1 protein were higher in stomach tissues from mice given IL10, and ICC networks were more organized, better connected, and more evenly distributed compared with controls. CONCLUSIONS: IL10 increases gastric emptying in diabetic mice and has therapeutic potential for patients with diabetic gastroparesis. This response is associated with up-regulation of HO1 and repair of connectivity of ICC networks.

Induction of IL-6 within the rodent intestinal muscularis after intestinal surgical stress.

BACKGROUND: Postoperative ileus is a poorly understood surgical problem characterized by leukocyte extravasation into the intestinal muscularis and suppression in muscle function. The study objective was to delineate a mechanistic inflammatory cascade initiated by intestinal manipulation. METHODS: ACI and Sprague-Dawley rats, and IL-6 +/+ and IL-6 -/- mice were subjected to intestinal manipulation. One group of rats received adhesion molecule-blocking antibodies (1A29 and WT.3) before intestinal manipulation. Interleukin-6 (IL-6) messenger RNA (mRNA) levels and electrophoretic mobility shift assay for signal transducers and activators of transcription (STAT) activation were measured in tissue extracts. IL-6 protein levels were assessed by immunohistochemistry and enzyme-linked immunosorbent assay. RESULTS: IL-6 mRNA from muscularis extracts demonstrated a significant induction after intestinal manipulation. No IL-6 induction was observed in mucosal extracts. Adhesion molecule blockade resulted in a marked decrease of cellular infiltration but did not change IL-6 mRNA expression in muscularis extracts. Resident macrophages in the muscularis stained for IL-6 by immunohistochemistry after intestinal manipulation. The isolated manipulated muscularis demonstrated a significant increase in IL-6 release. Electrophoretic mobility shift assay of manipulated muscularis showed a marked increase in IL-6-dependent Stat3 activation. CONCLUSIONS: This study demonstrates that manipulation of the small bowel during an abdominal operation initiates downstream induction, translation, release, and functional activity of IL-6 within the muscularis.

Nuclear matrix protein alterations associated with colon cancer metastasis to the liver.

PURPOSE: The development of colon cancer markers that can detect liver metastases early and predict which patients are at risk to develop liver metastases would have a major impact on this disease. We have previously identified G. Brunagel, et al., Cancer Research, 62:2437-2442, 2002, nuclear matrix proteins (NMPs), which are associated with colon cancer. The objective of this study is to identify the existence of a specific NMP "fingerprint" for human liver metastasis from colon cancer. EXPERIMENTAL DESIGN: Using high-resolution two-dimensional gel electrophoresis, we analyzed the NMP expression of 12 matched liver metastases and adjacent normal samples and three normal donor liver samples. These were compared with colon cancer NMP patterns, along with several primary cell systems and lines. RESULTS: Analysis of multiple gels for each sample revealed three proteins present in all liver metastases, which are not present in normal liver tissue and normal hepatocytes. These three proteins were also present in colon cancer samples. CONCLUSION: Data provided here demonstrate that the NMP composition is able to differentiate liver metastases from normal liver tissue and normal hepatocytes and that these proteins are also expressed in colon cancer. These results further show that the adjacent normal liver tissue changes its NMP pattern of expression. Development of an assay to detect these specific NMPs in tissue and/or serum specimens is a promising modality for early detection of liver metastases from colon cancer or potentially as a prognostic tool. In addition the functional characterization of these proteins will significantly enhance our understanding of the development of liver metastases of this disease.

Protective effects of ex vivo graft radiation and tacrolimus on syngeneic transplanted rat small bowel motility.

BACKGROUND: Intestinal transplantation is unduly complicated by the nontolerogenic properties of the gut-associated lymphoid tissue. Because simultaneous graft irradiation and bone marrow infusion significantly prolong the survival of the small bowel transplanted animal, our objective was to determine the functional motility effects of the immune modulating, graft irradiation procedure in the presence and absence of tacrolimus immunosuppression. METHODS: Four groups of syngeneic orthotopic small bowel transplanted animals were studied 48 hours after operations (untreated, tacrolimus, ex vivo graft irradiation, and tacrolimus + irradiation) and compared with controls. Histologic analysis was performed for mucosal apoptosis and neutrophilic infiltration into the muscularis externa. Gastrointestinal in vivo transit and in vitro circular muscle strip contractions were quantified in response to bethanechol (0.3-300 micromol/L). RESULTS: Graft irradiation ex vivo alone or in the presence of tacrolimus significantly increases (> 10-fold) the number of apoptotic mucosal cells after transplantation. Functional measurements showed that transplantation resulted in a significant delay in gastrointestinal transit and a decrease in muscle strip contractility. Tacrolimus and graft irradiation significantly ameliorated the transplant-induced dysfunction. CONCLUSIONS: Given the endowed propensity of mucosal regeneration, the immunologic and functional benefits of ex vivo graft irradiation appear to outweigh the detrimental effects to the mucosa.

Protective effect of carbon monoxide inhalation for cold-preserved small intestinal grafts.

BACKGROUND: Heme oxygenase (HO)-1 system has been shown to provide protection against oxidative stress through the degradation of heme to biliverdin, free iron, and carbon monoxide (CO). This study investigated cytoprotective efficacy of CO at a low concentration on cold ischemia/reperfusion (I/R) injury of transplanted intestine. METHODS: Lewis rat recipients of syngenic orthotopic small intestinal transplantation with 6 hours UW cold preservation were either kept in room air (air-treated control) or exposed to CO (250 ppm) for 1 hour before and 24 hours after surgery. RESULTS: In air-treated grafts, mRNA levels for interleukin-6, intracellular adhesion molecule-1, cyclooxygenase-2, and inducible nitric oxide synthase promptly increased. Sequential histopathologic analysis of untreated grafts revealed initial rapid epithelial loss, subsequent recruitment of inflammatory infiltrates, and local hemorrhage in the lamina propria, which extended downward to the epithelial crypt and muscle layer with time. CO effectively blocked proinflammatory cascade during I/R injury, inhibited upregulation of inflammatory molecules and ameliorated intestinal tissue injuries. Beneficial effects of CO were associated with improved graft blood flow without inhibiting endogenous HO-1 activity. Recipient animal survival was significantly improved with CO to 100% versus 58% in air-treated controls. CONCLUSIONS: These results indicate a significant role for CO in protecting the intestine from cold I/R injury associating with small intestinal transplantation.

Hydrogen-enriched preservation protects the isogeneic intestinal graft and amends recipient gastric function during transplantation.

BACKGROUND: Inhaled hydrogen gas exerts antioxidant and anti-inflammatory effects in rat intestinal transplantation. Here, we investigated whether ex vivo donor organ treatment with dissolved hydrogen would prevent intestinal graft injury. METHODS: Isogeneic intestinal transplantation was performed in Lewis rats with vascular flush, luminal preservation, and cold graft storage in nitrogen-bubbled (SITxN2) or hydrogen-bubbled (SITxH2) preservation solution. Lactated Ringer's solution and 3-hr cold ischemia time were used for mechanistic investigations, whereas survival experiments were performed with University of Wisconsin solution and 6-hr cold ischemia time. RESULTS: During the early phase of ischemia-reperfusion injury, hydrogen-enriched solution significantly preserved mucosal graft morphology, diminished graft malondialdehyde levels demonstrating substantial reduction potential and blunted proinflammatory molecular responses (early growth response gene [EGR-1], interleukin [IL]-6, IL-1ß, and inducible nitric oxide synthase) within the reperfused intestinal graft muscularis. During the late phase of ischemia-reperfusion injury, circulating IL-6 protein and lactate dehydrogenase levels were significantly ameliorated in SITxH2 animals, which were associated with a favorable functional outcome in in vivo liquid gastrointestinal transit and recipient solid gastric emptying of chrome steel balls, and marked prevention of the posttransplant associated suppression of in vitro muscarinic jejunal contractility. Reflecting improved graft preservation, hydrogen preloading of grafts increased recipient survival rates from 41% to 80%. Anti-inflammatory and antiapoptotic heme oxygenase-1 was significantly upregulated in the hydrogen-treated graft muscularis but not mucosa before reperfusion. CONCLUSIONS: Graft preloading with hydrogen demonstrated superior morphologic and functional graft protection in rodent intestinal transplantation, ultimately facilitating recipient survival. Antioxidant capacity and muscularis heme oxygenase-1 upregulation are possible protective mechanisms.

Intraesophageal manganese superoxide dismutase-plasmid liposomes ameliorates novel total-body and thoracic radiation sensitivity of NOS1-/- mice.

The effect of deletion of the nitric oxide synthase 1 gene (NOS1(-/-)) on radiosensitivity was determined. In vitro, long-term cultures of bone marrow stromal cells derived from NOS1(-/-) were more radioresistant than cells from C57BL/6NHsd (wild-type), NOS2(-/-) or NOS3(-/-) mice. Mice from each strain received 20 Gy thoracic irradiation or 9.5 Gy total-body irradiation (TBI), and NOS1(-/-) mice were more sensitive to both. To determine the etiology of radiosensitivity, studies of histopathology, lower esophageal contractility, gastrointestinal transit, blood counts, electrolytes and inflammatory markers were performed; no significant differences between irradiated NOS1(-/-) and control mice were found. Video camera surveillance revealed the cause of death in NOS1(-/-) mice to be grand mal seizures; control mice died with fatigue and listlessness associated with low blood counts after TBI. NOS1(-/-) mice were not sensitive to brain-only irradiation. MnSOD-PL therapy delivered to the esophagus of wild-type and NOS1(-/-) mice resulted in equivalent biochemical levels in both; however, in NOS1(-/-) mice, MnSOD-PL significantly increased survival after both thoracic and total-body irradiation. The mechanism of radiosensitivity of NOS1(-/-) mice and its reversal by MnSOD-PL may be related to the developmental esophageal enteric neuronal innervation abnormalities described in these mice.

Interaction of hemorrhagic shock and subsequent polymicrobial sepsis on gastrointestinal motility.

Understanding "two-hit" experimental models is crucial for the rational development of therapies for hemorrhagic shock (HS). We modeled the clinical scenario of HS followed by polymicrobial sepsis (cecal ligation and puncture [CLP]) to investigate the molecular and functional alterations that occur within the gastrointestinal tract. Control, HS, CLP, simultaneous HS + CLP, and HS + delayed CLP by 24 h groups of Sprague-Dawley rats were studied for gastrointestinal transit and in vitro colonic circular muscle contractility to bethanechol. Reverse transcription-polymerase chain reaction quantified IL-6, IL-10, and heme oxygenase 1 messenger RNA expression in the isolated colonic muscularis 6 h after insult. Myeloperoxidase-positive neutrophils were quantified in colonic muscularis whole mounts. Mortality at 24 h was significantly increased in simultaneous mild HS + CLP (88%) over control, mild HS, CLP alone, or HS + delayed CLP. Cecal ligation and puncture significantly delayed transit compared with controls and HS alone. Hemorrhagic shock + delayed CLP animals had normal transit. Colonic contractions were suppressed by 50% after CLP compared with controls and HS. In contrast, HS + delayed CLP displayed control levels of contractile responses to bethanechol. Cecal ligation and puncture and simultaneous HS + CLP caused significant inflammatory messenger RNA induction of IL-6, iNOS, IL-10, and heme oxygenase 1 compared with control and HS, and these responses were significantly suppressed in HS + delayed CLP colonic muscularis extracts. Neutrophils were significantly recruited into the colonic muscularis following CLP after 24 h compared with control and HS. This recruitment was significantly less in the HS + delayed CLP animals. These data demonstrate the ability of mild HS to precondition the animal and protect it against a delayed, but not simultaneous, polymicrobial event.

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.