The dynamic cellular and molecular features during the development of radiation proctitis revealed by transcriptomic profiling in mice.

BACKGROUND: Radiation proctitis (RP) is the most common complication of radiotherapy for pelvic tumor. Currently there is a lack of effective clinical treatment and its underlying mechanism is poorly understood. In this study, we aimed to dynamically reveal the mechanism of RP progression from the perspective of RNomics using a mouse model, so as to help develop reasonable therapeutic strategies for RP. RESULTS: Mice were delivered a single dose of 25 Gy rectal irradiation, and the rectal tissues were removed at 4 h, 1 day, 3 days, 2 weeks and 8 weeks post-irradiation (PI) for both histopathological assessment and RNA-seq analysis. According to the histopathological characteristics, we divided the development process of our RP animal model into three stages: acute (4 h, 1 day and 3 days PI), subacute (2 weeks PI) and chronic (8 weeks PI), which could recapitulate the features of different stages of human RP. Bioinformatics analysis of the RNA-seq data showed that in the acute injury period after radiation, the altered genes were mainly enriched in DNA damage response, p53 signaling pathway and metabolic changes; while in the subacute and chronic stages of tissue reconstruction, genes involved in the biological processes of vessel development, extracellular matrix organization, inflammatory and immune responses were dysregulated. We further identified the hub genes in the most significant biological process at each time point using protein-protein interaction analysis and verified the differential expression of these genes by quantitative real-time-PCR analysis. CONCLUSIONS: Our study reveals the molecular events sequentially occurred during the course of RP development and might provide molecular basis for designing drugs targeting different stages of RP development.

Endothelial Hey2 deletion reduces endothelial-to-mesenchymal transition and mitigates radiation proctitis in mice.

The current study evaluated the role of Hey2 transcription factor in radiation-induced endothelial-to-mesenchymal transition (EndoMT) and its impact on radiation-induced tissue damage in mice. Phenotypic modifications of irradiated, Hey2 siRNA- and Hey2 vector plasmid-transfected human umbilical vein endothelial cells (HUVECs) resembling EndoMT were monitored by qPCR, immunocytochemistry and western blots. Subsequently, in mice, a Cre-LoxP strategy for inactivation of Hey2 specifically in the endothelium was used to study the biological consequences. Total body irradiation and radiation proctitis were monitored to investigate the impact of conditional Hey2 deletion on intestinal stem cells and microvascular compartment radiosensitivity, EndoMT and rectal damage severity. We found that EndoMT occurs in irradiated HUVECs with concomitant Hey2 mRNA and protein increase. While Hey2 silencing has no effect on radiation-induced EndoMT in vitro, Hey2 overexpression is sufficient to induce phenotypic conversion of endothelial cells. In mice, the conditional deletion of Hey2 reduces EndoMT frequency and the severity of rectal tissue damage. Our data indicate that the reduction in mucosal damage occurs through decline in stem/clonogenic epithelial cell loss mediated by microvascular protection. EndoMT is involved in radiation proctitis and this study demonstrates that a strategy based on the reduction of EndoMT mitigates intestinal tissue damage.

Silk-elastinlike protein polymers enhance the efficacy of a therapeutic glycosaminoglycan for prophylactic treatment of radiation-induced proctitis.

Radiation-induced proctitis (RIP) is the most common clinical adverse effect for patients receiving radiotherapy as part of the standard course of treatment for ovarian, prostate, colon, and bladder cancers. RIP limits radiation dosage, interrupts treatment, and lowers patients' quality of life. A prophylactic treatment that protects the gastrointestinal tract from deleterious effects of radiotherapy will significantly improve patient quality of life and may allow for higher and more regular doses of radiation therapy. Semi-synthetic glycosaminoglycan (GAG), generated from the sulfation of hyaluronic acid, are anti-inflammatory but have difficulty achieving therapeutic levels in many tissues. To enhance the delivery of GAG, we created an in situ gelling rectal delivery system using silk-elastinlike protein polymers (SELPs). Using solutions of SELP 815K (which contains 6 repeats of blocks comprised of 8 silk-like units, 15 elastin-like units, and 1 lysine-substituted elastin-like unit) with GAG GM-0111, we created an injectable delivery platform that transitioned in <5min from a liquid at room temperature to a hydrogel at body temperature. The hydrogels released 50% of their payload within 30min and enhanced the accumulation of GAG in the rectum compared to traditional enema-based delivery. Using a murine model of radiation-induced proctitis, the prophylactic delivery of a single dose of GAG from a SELP matrix administered prior to irradiation significantly reduced radiation-induced pain after 3, 7, and 21days by 53±4%, 47±10%, and 12±6%, respectively. Matrix-mediated delivery of GAG by SELP represents an innovative method for more effective treatment of RIP and promises to improve quality of life of cancer patients by allowing higher radiotherapy doses with improved safety.

Flagellin preconditioning enhances the efficacy of mesenchymal stem cells in an irradiation-induced proctitis model.

The success of mesenchymal stem cell transplantation for proctitis depends not only on cell donors but also on host microenvironmental factors, which play a major role in conditioning mesenchymal stem cell immunosuppressive action and repair. This study sought to determine if flagellin, a TLR5 ligand, can enhance the mesenchymal stem cell treatment efficacy in radiation-induced proctitis. With the use of a colorectal model of 27 Gy irradiation in rats, we investigated and compared the effects on immune capacity and remodeling at 28 d after irradiation of the following: 1) systemic mesenchymal stem cell (5 × 10(6)) administration at d 7 after irradiation, 2) administration of flagellin at d 3 and systemic mesenchymal stem cell administration at d 7, and 3) in vitro preconditioning of mesenchymal stem cells with flagellin, 24 h before their administration on d 7. The mucosal CD8(+) T cell population was normalized after treatment with flagellin-preconditioned mesenchymal stem cells or flagellin plus mesenchymal stem cells, whereas mesenchymal stem cells alone did not alter the radiation-induced elevation of CD8(+) T cell frequency. Mesenchymal stem cell treatment returned the irradiation-elevated frequency of CD25(+) cells in the mucosa-to-control levels, whereas both flagellin-preconditioned mesenchymal stem cell and flagellin-plus-mesenchymal stem cell treatment each significantly increased not only CD25(+) cell frequency but also forkhead box p3 and IL-2Rα expression. Specifically, IL-10 was overexpressed after flagellin-preconditioned mesenchymal stem cell treatment. Analysis of collagen expression showed that the collagen type 1/collagen type 3 ratio, an indicator of wound-healing maturation, was low in the irradiated and mesenchymal stem cell-treated groups and returned to the normal level only after the flagellin-preconditioned mesenchymal stem cell treatment. This was associated with a reduction in myofibroblast accumulation. In a proctitis model, flagellin-preconditioned mesenchymal stem cells improved colonic immune capacity and enhanced tissue remodeling.

Identification of Endothelial-to-Mesenchymal Transition as a Potential Participant in Radiation Proctitis.

The endothelial-to-mesenchymal transition (EndoMT) is a crucial cellular process during heart development necessary to the formation of cardiac valves. This embryonic process reappears in several pathological situations, such as vascular injury or organ fibrosis of various etiologies, as a mediator of extracellular matrix-producing cells. Because radiation induces both vascular damage and fibrosis, we investigated whether radiation exposure induces EndoMT in primary human intestinal microvascular endothelial cells (HIMECs) and whether EndoMT contributes to radiation-induced rectal damage in humans and in a preclinical model of radiation proctitis in mice. Irradiated HIMECs show phenotypic hallmarks of radiation-induced endothelial cell activation in vitro. Moreover, HIMECs undergo changes in molecular expression pattern compatible with EndoMT, with up-regulation of mesenchymal markers and down-regulation of endothelial markers via transforming growth factor/Smad pathway activation. In vivo, EndoMT readily occurs in the human rectum after radiation therapy for rectal adenocarcinoma. Finally, EndoMT was observed in rectal mucosal and submucosal microvessels in a preclinical model of radiation proctitis in Tie2-green fluorescent protein reporter-expressing mice all along radiation proctitis development, also associated with transforming growth factor/Smad pathway activation. In conclusion, radiation-induced cell activation and tissue inflammation constitute a setting that fosters the phenotypic conversion of endothelial cells into mesenchymal cells. Therefore, EndoMT is identified as a potential participant in radiation-induced gut damage and may represent an interesting therapeutic target in cases of radiation-induced pelvic disease.

Osteopontin knockout does not influence the severity of rectal damage in a preclinical model of radiation proctitis in mice.

BACKGROUND: Radiation damage to the normal gut is a dose-limiting factor in the application of radiation therapy to treat abdominal and pelvic cancers. All tissue cell types react in concert to orchestrate an acute inflammatory reaction followed by a delayed chronic scarring process. Osteopontin (OPN) is a matricellular protein known to be involved in various physiological but also pathological processes such as tissue inflammation and fibrosis. AIMS: The aim of our study was to determine whether OPN knockout influences the severity of radiation proctitis and to investigate the role of OPN in the development of radiation-induced gut damage. RESULTS: Here we show that human radiation proctitis is associated with increased immunostaining of the intracellular and extracellular/matrix-linked isoforms of OPN. Moreover, endothelial cells in vitro and rectal tissue in a preclinical model of radiation proctitis in mice both respond to radiation exposure by a sustained increase in OPN mRNA and protein levels. Genetic deficiency of OPN did not influence radiation-induced rectal damage and was associated with significantly decreased animal survival. The acute and late radiation injury scores were similar in OPN-null mice compared with their control littermates. CONCLUSION: This study shows that in our model and given the pleiotropic actions of OPN in tissue inflammation and fibrosis, further studies are necessary to understand the precise roles of OPN in radiation-induced proctitis and to determine whether OPN is a useful therapeutic tool in prevention of radiation-induced intestinal tissue injury.

Randomized clinical trial of the effect of gum chewing on postoperative ileus and inflammation in colorectal surgery.

BACKGROUND: Postoperative ileus (POI) is a common complication following colorectal surgery that delays recovery and increases length of hospital stay. Gum chewing may reduce POI and therefore enhance recovery after surgery. The aim of the study was to evaluate the effect of gum chewing on POI, length of hospital stay and inflammatory parameters. METHODS: Patients undergoing elective colorectal surgery in one of two centres were randomized to either chewing gum or a dermal patch (control). Chewing gum was started before surgery and stopped when oral intake was resumed. Primary endpoints were POI and length of stay. Secondary endpoints were systemic and local inflammation, and surgical complications. Gastric emptying was measured by ultrasonography. Soluble tumour necrosis factor receptor 1 (TNFRSF1A) and interleukin (IL) 8 levels were measured by enzyme-linked immunosorbent assay. RESULTS: Between May 2009 and September 2012, 120 patients were randomized to chewing gum (58) or dermal patch (control group; 62). Mean(s.d.) length of hospital stay was shorter in the chewing gum group than in controls, but this difference was not significant: 9·5(4·9) versus 14·0(14·5) days respectively. Some 14 (27 per cent) of 52 analysed patients allocated to chewing gum developed POI compared with 29 (48 per cent) of 60 patients in the control group (P = 0·020). More patients in the chewing gum group first defaecated within 4 days of surgery (85 versus 57 per cent; P = 0·006) and passed first flatus within 48 h (65 versus 50 per cent; P = 0·044). The decrease in antral area measured by ultrasonography following a standard meal was significantly greater among patients who chewed gum: median 25 (range -36 to 54) per cent compared with 10 (range -152 to 54) per cent in controls (P = 0·004). Levels of IL-8 (133 versus 288 pg/ml; P = 0·045) and TNFRSF1A (0·74 versus 0·92 ng/ml; P = 0·043) were lower among patients in the chewing gum group. Fewer patients in this group developed a grade IIIb complication (2 of 58 versus 10 of 62; P = 0·031). CONCLUSION: Gum chewing is a safe and simple treatment to reduce POI, and is associated with a reduction in systemic inflammatory markers and complications. REGISTRATION NUMBER: NTR2867 (http://www.trialregister.nl).

Repeated autologous bone marrow-derived mesenchymal stem cell injections improve radiation-induced proctitis in pigs.

The management of proctitis in patients who have undergone very-high-dose conformal radiotherapy is extremely challenging. The fibrosis-necrosis, fistulae, and hemorrhage induced by pelvic overirradiation have an impact on morbidity. Augmenting tissue repair by the use of mesenchymal stem cells (MSCs) may be an important advance in treating radiation-induced toxicity. Using a preclinical pig model, we investigated the effect of autologous bone marrow-derived MSCs on high-dose radiation-induced proctitis. Irradiated pigs received repeated intravenous administrations of autologous bone marrow-derived MSCs. Immunostaining and real-time polymerase chain reaction analysis were used to assess the MSCs' effect on inflammation, extracellular matrix remodeling, and angiogenesis, in radiation-induced anorectal and colon damages. In humans, as in pigs, rectal overexposure induces mucosal damage (crypt depletion, macrophage infiltration, and fibrosis). In a pig model, repeated administrations of MSCs controlled systemic inflammation, reduced in situ both expression of inflammatory cytokines and macrophage recruitment, and augmented interleukin-10 expression in rectal mucosa. MSC injections limited radiation-induced fibrosis by reducing collagen deposition and expression of col1a2/col3a1 and transforming growth factor-ß/connective tissue growth factor, and by modifying the matrix metalloproteinase/TIMP balance. In a pig model of proctitis, repeated injections of MSCs effectively reduced inflammation and fibrosis. This treatment represents a promising therapy for radiation-induced severe rectal damage.

Increased expression of VEGF and CD31 in postradiation rectal tissue: implications for radiation proctitis.

Background. Inflammation mediators related to radiation proctitis are partially elucidated, and neovascularization is thought to play a key role. Objectives. To investigate the expression of vascular endothelial growth factor (VEGF) and CD31 as angiogenetic markers in postradiation rectal tissue. Methods. Rectal mucosa biopsies from 11 patients who underwent irradiation for prostate cancer were examined immunohistochemically for the expression of VEGF and CD31 at three time settings-before, at the completion of, and 6 months after radiotherapy. VEGF expressing vascular endothelial cells and CD31 expressing microvessels were counted separately in 10 high-power fields (HPFs). VEGF vascular index (VEGF-VI) and microvascular density (MVD) were calculated as the mean number of VEGF positive cells per vessel or the mean number of vessels per HPF, respectively. Histological features were also evaluated. Results. VEGF-VI was significantly higher at the completion of radiotherapy (0.17 ± 0.15 versus 0.41 ± 0.24, P = 0.001) declining 6 months after. MVD increased significantly only 6 months after radiotherapy (7.3 ± 3.2 versus 10.5 ± 3.1, P < 0.005). The histopathological examination revealed inflammatory changes at the completion of radiotherapy regressing in the majority of cases 6 months after. Conclusions. Our results showed that in postradiation rectal biopsy specimens neoangiogenesis seems to be inflammation-related and constitutes a significant postradiation component of the tissue injury.

Claudin-2 regulates colorectal inflammation via myosin light chain kinase-dependent signaling.

BACKGROUND: Claudins have been demonstrated to be associated with inflammatory bowel disease (IBD), but the specific role of claudin-2 in colorectal inflammation remains undefined. AIMS: We aimed to determine the role of claudin-2 in TNFα-induced colorectal inflammation. METHODS: We used claudin-2 (-/-) mice to assess the role of claudin-2 in colon. The mice were intraperitoneally injected with 3 µg of recombinant murine TNFα, and the NF-κB signaling and mRNA expression levels of proinflammatory cytokines and myosin light chain kinase (MLCK) were evaluated. Moreover, in claudin-2 (-/-) mice, colitis was induced by the administration of dextran sodium sulfate (DSS). The involvement of claudin-2 in colorectal inflammation was also investigated using the Caco-2 human colon adenocarcinoma cell line, and the expression of claudin-2 was downregulated using claudin-2 siRNA. RESULTS: TNFα-induced colorectal inflammation via NF-κB signaling activation was enhanced in claudin-2 (-/-) mice compared with that in claudin-2 (+/+) mice. MLCK expression level in the colon tissue of claudin-2 (-/-) mice treated with TNFα was enhanced in comparison to that of the claudin-2 (+/+) mice. DSS-induced colitis was more severe in the claudin-2 (-/-) mice than in the claudin-2 (+/-) mice. In in vitro experiments, the decreased expression of claudin-2 enhanced the expressions of IL-6, IL-1ß and MLCK. CONCLUSIONS: Our findings concerning the role of claudin-2 in epithelial inflammatory responses enrich our collective understanding of mucosal homeostasis and intestinal diseases such as IBD. Furthermore, the results of this study indicate that claudin-2 and MLCK are potential therapeutic targets for treatments against intestinal disease.

A murine model for the study of molecular pathogenesis of radiation proctitis.

PURPOSE: To establish a novel mouse brachytherapy model with which to study the role of inflammation in the pathogenesis of radiation proctitis. METHODS AND MATERIALS: The distal rectums of BALB/c and C57BL/6 mice were irradiated with three to five fractions of 5.5 to 8 Gy. Tissues were harvested and evaluated for histopathology, using the radiation injury score (RIS). Cytokine mRNA expression was assessed using real-time PCR. RESULTS: Fifty percent of the mice treated with 22 Gy delivered in four fractions of 5.5 Gy died as a result of anorectal stenosis and distal bowel obstruction prior to the time of scheduled sacrifice, with a latency period of 4 to 10 weeks for the BALB/c and 3 to 4 weeks for the C57BL/6 mice. The RISs were 7, 12, and 8 at 2, 6, and 11 weeks, respectively, in the BALB/c mice and was 8.7 in the C57BL/6 mice on week 6. A 100- to 300-fold increase in interleukin-1beta (IL-1beta) (p = 0.04) and IL-6 mRNA (p = 0.07) and a 5- to 6-fold increase in transforming growth factor (TGF) and tumor necrosis factor-alpha mRNA expression levels (p < 0.001 and p = 0.01) were observed at 2 to 6 weeks after radiation. Cytokine mRNA tissue expression correlated positively with radiation dose (p < 0.0001). The RIS correlated well with IL-1beta and IL-6 mRNA levels in the BALB/c mice and with IL-1beta, IL-6, and TGF mRNA levels in C57BL/6 mice. Analysis of receiver operating characteristic curve showed that IL-1beta and IL-6 have the largest area under the curve and therefore are good markers of radiation proctitis (p < 0.001). CONCLUSIONS: Radiation-induced proctitis was associated with a dose-dependent, characteristic proinflammatory cytokine response pattern in a novel mouse model suitable for interventional studies.

Up-regulation of endothelin type a receptor in human and rat radiation proctitis: preclinical therapeutic approach with endothelin receptor blockade.

PURPOSE: Rectum radiation damage and fibrosis are often associated with radiation therapy of pelvic tumors. The endothelin (ET) system has been implicated in several fibrotic diseases but never studied in the context of gastrointestinal radiation damage. This study assessed modifications in ET type 1 (ET-1), ET type A receptor (ET(A)), and ET type B receptor (ET(B)) localization and/or expression in irradiated human rectal tissue and in a rat model of delayed colorectal injury. We also evaluated the therapeutic potential of long-term ET receptor blockade. METHODS AND MATERIALS: Routine histological studies of sections of healthy and radiation-injured human rectum tissue were done; the sections were also immunostained for ET(A) and ET(B) receptors. The rat model involved the delivery of 27 Gy in a single dose to the colons and rectums of the animals. The ET-1/ET(A)/ET(B) expression and ET(A)/ET(B) localization were studied at 10 weeks postexposure. The abilities of bosentan and atrasentan to protect against delayed rectal injury were also investigated. RESULTS: The immunolocalization of ET(A) and ET(B) in healthy human rectums was similar to that in rat rectums. However, strong ET(A) immunostaining was seen in the presence of human radiation proctitis, and increased ET(A) mRNA levels were seen in the rat following colorectal irradiation. Immunostaining for ET(A) was also strongly positive in rats in areas of radiation-induced mucosal ulceration, atypia, and fibroproliferation. However, neither bosentan nor atrasentan prevented radiation damage to the rectum when given long term. The only effect seen for atrasentan was an increased number of sclerotic vessel sections in injured tissues. CONCLUSIONS: As the result of the overexpression of ET(A), radiation exposure deregulates the endothelin system through an "ET(A) profile" in the human and rodent rectum. However, therapeutic interventions involving mixed or specific ET(A) receptor blockade do not prevent radiation damage. Further studies are necessary to identify the precise roles of ET in the gastrointestinal response to radiation exposure.

Expression of cytochrome P450 and MDR1 in patients with proctitis.

BACKGROUND: The aim of this study was to investigate the effect of inflammation on the gene expression of three cytochrome P450's (CYP) and P-glycoprotein (P-gp) in the rectal and colonic mucosa in patients with proctitis. METHODS: Biopsies were obtained from inflamed and normal mucosa in association with routine sigmoidoscopy in patients with proctitis. The biopsies were snap-frozen in liquid nitrogen. Real time PCR (polymerase chain reaction) was used for quantitative analyses of mRNA specific for the CYP2E1, CYP3A4 and CYP3A5 gene and the MDR1 genes. Values were normalised based on gene expression of beta-actin to enable comparisons between samples. RESULTS: The gene expression of CYP2E1 and CYP3A4 was lower in mucosa with severe inflammation vs normal mucosa (p<0.05). For CYP3A5 and P-gp there was no significant difference when comparing normal and inflammatory changed mucosa. CONCLUSION: Our study suggests that at least for some of the CYP enzymes the expression decreases in response to the inflammatory process in the gastrointestinal tract.

Local release of human neutrophil lipocalin (HNL), IL-8, and TNF-alpha is decreased as response to topical prednisolone treatment in distal ulcerative colitis and proctitis.

The local release of human neutrophil lipocalin, considered to be highly specific for neutrophil granulocyte activation, and interleukin-8 and tumor necrosis factor-alpha were studied in 11 patients with distal ulcerative colitis and proctitis before and during treatment with steroid enemas. A rectal perfusion technique for sampling and specific immunoassays for analysis were used. In responders (N = 8) the concentrations of all proteins decreased during the study. There was a close correlation between human neutrophil lipocalin concentrations and treatment response. Tumor necrosis factor-alpha showed an initial decline in concentrations irrespective of treatment outcome and preceded the decline of human neutrophil lipocalin and interleukin-8. We conclude that decreased neutrophil degranulation is correlated with treatment outcome. Furthermore, an important role of tumor necrosis factor-alpha in the process of stimulating neutrophil activation and degranulation in ulcerative colitis is suggested.

Transient P-cadherin expression in radiation proctitis; a model of mucosal injury and repair.

Morphology at both cellular and glandular levels in the colon is dependent to an extent on cell-cell adhesion mediated by cadherin-catenin complexes. Alterations in the expression of E-cadherin, the cadherin normally present in colon, have been shown to be implicated in tissue remodelling within the gastrointestinal tract. Furthermore, it has previously been shown that P-cadherin, normally present only in stratified epithelia and placenta, is expressed in colitis and during neoplastic change in the colon. The morphological features of mucosal injury induced by pre-operative radiotherapy in the non-neoplastic rectal mucosa were studied in patients with rectal adenocarcinoma. Three characteristic phases of radiation proctitis were defined on histological grounds (acute injury, and early and late regenerative phases) essentially correlating with the time interval between radiotherapy and surgery; such features were mirrored by alterations in cadherin-catenin expression and localization in rectal crypts. On immunohistochemistry and western blotting, P-cadherin was highly expressed in the acute injury and early regenerative phases, with a decreased level of expression during late regeneration. E-cadherin and associated catenins were translocated from membrane to cytoplasm in degenerating crypts, with return of normal membranous expression in regenerating crypts. In conclusion, radiation-induced proctitis represents an in vivo model of mucosal injury and regeneration and provides a valid model in which to study events during epithelial injury and repair. Altered cadherin expression, in particular transient aberrant P-cadherin expression, is intimately associated with these processes.

Human neutrophil lipocalin is a unique marker of neutrophil inflammation in ulcerative colitis and proctitis.

BACKGROUND AND AIM: Accumulation and infiltration by neutrophil granulocytes is a prominent feature in the local inflammatory process in ulcerative colitis (UC). The present study was performed to evaluate human neutrophil lipocalin (HNL) as a specific neutrophil marker in the inflamed lesions of the colon and rectum in patients with colitis and proctitis. METHODS: The activity of intestinal neutrophils with respect to release of granule proteins was studied in 18 patients with UC (10 with colitis and eight with isolated proctitis) and in 18 healthy controls using perfusion fluid and biopsies from the sigmoid colon and rectum. The released amounts of the neutrophil granule proteins HNL and myeloperoxidase (MPO) were determined by radioimmunoassays, and the location of HNL and MPO in biopsies from colonic mucosa was examined by immunohistochemistry. RESULTS: Mucosal release of HNL and MPO was increased 10-55-fold in patients with colitis and proctitis compared with controls. Their bowel biopsies demonstrated that only neutrophils were stained with anti-HNL. We also found correlations between HNL and levels of granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin 8 (IL-8) in perfusion fluids from the sigmoidal segments of patients with proctitis, between HNL and GM-CSF in rectal segments in patients with proctitis, and in sigmoidal segments in patients with colitis. CONCLUSION: We conclude that the increased release of HNL and MPO in colorectal perfusion fluids indicates neutrophil involvement in the local inflammatory process, and suggest that HNL may serve as a specific marker of intestinal neutrophil activation in UC. GM-CSF, and to some extent IL-8, may play a role in neutrophil accumulation and priming in this disease.

Increased intraluminal release of eosinophil granule proteins EPO, ECP, EPX, and cytokines in ulcerative colitis and proctitis in segmental perfusion.

OBJECTIVE: The role of the eosinophil granulocyte in bowel mucosa in inflammatory bowel disease still remains obscure. The present study was performed in order to elucidate the local eosinophil activity and activating cytokines in the inflamed lesions of colon and rectum in patients with ulcerative colitis and proctitis. METHODS: The activity of intestinal eosinophils with respect to the release of granule proteins was studied in 18 patients (10 with colitis and 8 with isolated proctitis) and 18 healthy controls, using intraluminal segmental perfusion of the sigmoid colon and rectum. The released amounts of eosinophil granule proteins: eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil protein X (EPX) to perfusion fluid were determined by radioimmunoassays. The intraluminal release of possible eosinophil priming cytokines granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin 8 (IL-8), were analyzed by immunoassays. RESULTS: The mucosal release of ECP, EPO, and EPX was increased 10- to 20-fold in patients with colitis and proctitis compared with controls. The intraluminal release of GM-CSF and IL-8, was several-fold enhanced in patients with colitis and proctitis. We also found a correlation between all three eosinophil granule proteins and the levels of IL-8/GM-CSF in the sigmoidal segments of patients with colitis. CONCLUSIONS: We conclude that the increased release of ECP, EPO, and EPX to colorectal perfusion fluid indicate eosinophil involvement in the local disease in patients with colitis and proctitis. IL-8 and GM-CSF may play a role in eosinophil accumulation and priming in colitis.

Antioxidant effects of aminosalicylates and potential new drugs for inflammatory bowel disease: assessment in cell-free systems and inflamed human colorectal biopsies.

BACKGROUND: The therapeutic efficacy of 5-aminosalicylic acid in inflammatory bowel disease may be related to its antioxidant properties. AIM: To compare in vitro the antioxidant effects of conventional drugs (5-aminosalicylic acid, corticosteroids, metronidazole), with new aminosalicylates (4-aminosalicylic acid, balsalazide) and other potential therapies (ascorbate, N-acetylcysteine, glutathione, verapamil). METHODS: Compounds were assessed for efficacy in reducing the in vitro production of reactive oxygen species by cell-free systems (using xanthine/xanthine oxidase, with or without myeloperoxidase) and by colorectal biopsies from patients with ulcerative colitis using luminol-amplified chemiluminescence. RESULTS: 5-aminosalicylic acid and balsalazide were more potent antioxidants than 4-aminosalicylic acid or N-acetyl-5-aminosalicylic acid in cell-free systems. 5-aminosalicylic acid (20 mM) and balsalazide (20 mM) inhibited rectal biopsy chemiluminescence by 93% and 100%, respectively, compared with only 59% inhibition by 4-aminosalicylic acid (20 mM). Hydrocortisone, metronidazole and verapamil had no significant effect on chemiluminescence in any system. Ascorbate (20 mM) inhibited chemiluminescence by 100% in cell-free systems and by 60% in rectal biopsies. N-acetyl cysteine (10 mM), and both oxidized and reduced glutathione (10 mM), completely inhibited chemiluminescence in cell-free systems, but not with rectal biopsies. CONCLUSIONS: The antioxidant effects of compounds varies between cell-free systems and inflamed colorectal biopsies. The effect of drugs on the chemiluminescence produced by these two assay systems is useful for screening potentially new antioxidant treatments for inflammatory bowel disease. Ascorbate seems worth further study as a novel therapy.

Ropivacaine gel in active distal ulcerative colitis and proctitis -- a pharmacokinetic and exploratory clinical study.

BACKGROUND: Local anaesthetics have anti-inflammatory effects as indicated by preclinical and explorative clinical data. OBJECTIVE: To investigate the pharmacokinetics, tolerability and clinical efficacy of the new local anaesthetic ropivacaine in active distal ulcerative colitis. METHODS: Twelve patients were openly given 200 mg ropivacaine gel rectally twice daily for 2 weeks in this open study. RESULTS: Mean peak total plasma concentrations, Cmax, were 1.37, 1.26, 1.03 and 0.99 mg/L on treatment days 1, 3, 7 and 14. The mean unbound plasma concentrations at Cmax were 0.071, 0.058, 0.050 and 0.045 mg/L. The decrease in Cmax (P < 0.01) as well as in the area under the plasma concentration-time curve, AUC (P < 0.01), may be due to a decreased absorption but an increased metabolism cannot be excluded. The median time of Cmax was around 2 h and the mean terminal half-life was around 2.7 h. Mucosal inflammation assessed endoscopically at the most severely affected site decreased after 2 weeks of treatment (P < 0.01; blinded) and there was also a trend towards histological improvement (P = 0.06). Clinical symptoms, including total number of stools, blood in stools and diarrhoea increased (P < 0.05) during the study. The treatment was, in general, well tolerated with few gastrointestinal complaints and there were no unequivocal signs of systemic effects. CONCLUSIONS: Ropivacaine given rectally as a gel, 200 mg twice daily does not accumulate over a 2-week treatment period and carries a low risk for systemic adverse effects. The results suggest a therapeutic efficacy in active distal ulcerative colitis.

Cytokine production in pouchitis is similar to that in ulcerative colitis.

PURPOSE: Controversy exists as to whether pouchitis represents a reactivation of the immunologic mechanisms that lead to ulcerative colitis (UC). The aims of this study were to determine local levels of the cytokines: interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF alpha) in the mucosa of patients with "asymptomatic" ileoanal pouch (n = 25), pouchitis (n = 9), active UC (n = 20), normal ileum (n = 15), proctitis (n = 10), and normal colon (n = 15). METHODS: Lamina propria mononuclear cells were isolated from mucosal biopsies by enzymatic dispersion and cultured for 48 hours. Proinflammatory cytokine levels were measured in the supernatants by enzyme-linked immunosorbent assay. RESULTS: IL-1 beta, IL-6, IL-8, and TNF alpha secretions were significantly greater in pouchitis and active UC than in the noninflamed ileoanal pouch and normal controls (P < 0.001). There was significant correlation (r = 0.63, P < 0.05) between levels of cytokines expressed in pouchitis and active UC. CONCLUSIONS: Increased cytokine expression occurs in both active UC and pouchitis and to a lesser extent in the long-standing ileoanal pouch.