Anatomical Variation in Mesenteric Macrophage Phenotypes in Crohn's Disease.

INTRODUCTION: Clinical trials are currently investigating whether an extended mesenteric resection for ileocecal resections could reduce postoperative recurrence in Crohn's disease. Resection of the mesorectum, which contains proinflammatory macrophages, during proct(ocol)ectomy, is associated with reduced recurrent inflammation and improved wound healing. We aimed to characterize the macrophages in the ileocecal mesentery, which were compared with those in the mesorectum, to provide a biological rationale for the ongoing trials. METHODS: In 13 patients with Crohn's disease and 4 control patients undergoing a proctectomy, tissue specimens were sampled at 3 locations from the mesorectum: distal (rectum), middle, and proximal (sigmoid). In 38 patients with Crohn's disease and 7 control patients undergoing ileocecal resections, tissue specimens also obtained from 3 locations: adjacent to the inflamed terminal ileum, adjacent to the noninflamed ileal resection margin, and centrally along the ileocolic artery. Immune cells from these tissue specimens were analyzed by flow cytometry for expression of CD206 to determine their inflammatory status. RESULTS: In the mesorectum, a gradient from proinflammatory to regulatory macrophages from distal to proximal was observed, corresponding to the adjacent inflammation of the intestine. By contrast, the ileocecal mesentery did not contain high amounts of proinflammatory macrophages adjacent to the inflamed tissue, and a gradient toward a more proinflammatory phenotype was seen in the central mesenteric area. DISCUSSION: Although the mesentery is a continuous structure, the mesorectum and the ileocecal mesentery show different immunological characteristics. Therefore, currently, there is no basis to perform an extended ileocecal resection in patients with Crohn's disease.

Molecular Signaling and Dysfunction of the Human Reactive Enteric Glial Cell Phenotype: Implications for GI Infection, IBD, POI, Neurological, Motility, and GI Disorders.

BACKGROUND: Clinical observations or animal studies implicate enteric glial cells in motility disorders, irritable bowel syndrome, inflammatory bowel disease, gastrointestinal (GI) infections, postoperative ileus, and slow transit constipation. Mechanisms underlying glial responses to inflammation in human GI tract are not understood. Our goal was to identify the "reactive human enteric glial cell (rhEGC) phenotype" induced by inflammation, and probe its functional relevance. METHODS: Human enteric glial cells in culture from 15 GI-surgical specimens were used to study gene expression, Ca, and purinergic signaling by Ca/fluo-4 imaging and mechanosensitivity. A nanostring panel of 107 genes was designed as a read out of inflammation, transcription, purinergic signaling, vesicular transport protein, channel, antioxidant, and other pathways. A 24-hour treatment with lipopolysaccharide (200 µg/mL) and interferon-γ (10 µg/mL) was used to induce inflammation and study molecular signaling, flow-dependent Ca responses from 3 mL/min to 10 mL/min, adenosine triphosphate (ATP) release, and ATP responses. RESULTS: Treatment induced a "rhEGC phenotype" and caused up-regulation in messenger RNA transcripts of 58% of 107 genes analyzed. Regulated genes included inflammatory genes (54%/IP10; IFN-γ; CxCl2; CCL3; CCL2; C3; s100B; IL-1ß; IL-2R; TNF-α; IL-4; IL-6; IL-8; IL-10; IL-12A; IL-17A; IL-22; and IL-33), purine-genes (52%/AdoR2A; AdoR2B; P2RY1; P2RY2; P2RY6; P2RX3; P2RX7; AMPD3; ENTPD2; ENTPD3; and NADSYN1), channels (40%/Panx1; CHRNA7; TRPV1; and TRPA1), vesicular transporters (SYT1, SYT2, SNAP25, and SYP), transcription factors (relA/relB, SOCS3, STAT3, GATA_3, and FOXP3), growth factors (IGFBP5 and GMCSF), antioxidant genes (SOD2 and HMOX1), and enzymes (NOS2; TPH2; and CASP3) (P < 0.0001). Treatment disrupted Ca signaling, ATP, and mechanical/flow-dependent Ca responses in human enteric glial cells. ATP release increased 5-fold and s100B decreased 33%. CONCLUSIONS: The "rhEGC phenotype" is identified by a complex cascade of pro-inflammatory pathways leading to alterations of important molecular and functional signaling pathways (Ca, purinergic, and mechanosensory) that could disrupt GI motility. Inflammation induced a "purinergic switch" from ATP to adenosine diphosphate/adenosine/uridine triphosphate signaling. Findings have implications for GI infection, inflammatory bowel disease, postoperative ileus, motility, and GI disorders.

Aerosol transfer of bladder urothelial and smooth muscle cells onto demucosalized colonic segments for bladder augmentation: in vivo, long term, and functional pilot study.

BACKGROUND: Bladder augmentation technique has changed over the years and the current practice has significant adverse health effects and long-term sequelae. Previously, we reported a novel cell transfer technology for covering demucosalized colonic segments with bladder urothelium and smooth muscle cells through an aerosol spraying of these cells and a fibrin glue mixture. OBJECTIVE: To determine the long-term durability and functional characteristics of demucosalized segments of colon repopulated with urothelial cells in the bladder of swine for use in augmentation cystoplasty. STUDY DESIGN: Nine swine were divided into three groups. The first group (control) underwent standard colocystoplasty; the second group underwent colocystoplasty with colonic demucosalization and aerosol application of fibrin glue and urothelial cell mixture; in the third group detrusor cells were added to the mixture described in group two. The animals were kept for 6 months. Absorptive and secretory function was assessed. Bladders were harvested for histological and immunohistochemical evaluation. RESULTS: All animals but one in the experimental groups showed confluent urothelial coverage of the colonic segment in the bladder without any evidence of fibrosis, inflammation, or regrowth of colonic epithelial cells. Ten percent of the instilled water in the bladder was absorbed within an hour in the control group, but none in experimental groups(p = 0.02). The total urine sediment and protein contents were higher in the control group compared with experimental groups (p < 0.05). DISCUSSION: Both study groups developed a uniform urothelial lining. Histologically, the group with smooth muscle had an added layer of submucosal smooth muscle. Six months after bladder augmentation the new lining was durable. We were also able to demonstrate that the reconstituted augmented segments secrete and absorb significantly less than the control colocystoplasty group. We used a non-validated simple method to evaluate permeability of the new urothelial lining to water. To determine if the aerosol transfer of bladder cells would have behaved differently in the neurogenic bladder population, this experiment should have been performed in animals with neuropathic bladders. CONCLUSION: Aerosol spraying of single cell suspension of urothelial and muscular cells with fibrin glue resulted in coverage of the demucosalized intestinal segment with a uniform urothelial layer. This new lining segment was durable without regrowth of colonic mucosa after 6 months. The new reconstituted segment absorbs and secretes significantly less than control colocystoplasty.

Proteomic study of the mucin granulae in an intestinal goblet cell model.

Goblet cells specialize in producing and secreting mucus with its main component, mucins. An inducible goblet-like cell line was used for the purification of the mucus vesicles stored in these cells by density gradient ultracentrifugation, and their proteome was analyzed by nanoLC-MS and MS/MS. Although the density of these vesicles coincides with others, it was possible to reveal a number of proteins that after immunolocalization on colon tissue and functional analyses were likely to be linked to the MUC2 vesicles. Most of the proteins were associated with the vesicle membrane or their outer surface. The ATP6AP2, previously suggested to be associated with vesicular proton pumps, was colocalized with MUC2 without other V-ATPase proteins and, thus, probably has roles in mucin vesicle function yet to be discovered. FAM62B, known to be a calcium-sensitive protein involved in vesicle fusion, also colocalized with the MUC2 vesicles and is probably involved in unknown ways in the later events of the MUC2 vesicles and their secretion.

An original endoluminal magnetic anastomotic device allowing pure NOTES transgastric and transrectal sigmoidectomy in a porcine model: proof of concept.

INTRODUCTION: While experimental natural orifice transluminal endoscopic surgery (NOTES) sigmoid colectomies have been reported, pure NOTES anastomoses are restricted by the limited reach of commercially available circular staplers. MAGNAMOSIS is a set of self-orienting magnetic rings that can be delivered endoluminally throughout the colon to generate a compression anastomosis. Aim. To assess the feasibility of a pure NOTES transrectal (TR) and transgastric (TG) approach to perform any segmental colectomy. MATERIALS AND METHODS: One pig (50 kg) underwent the experimental procedure as follows: (a) creation of the TG access to the peritoneal cavity, (b) precise transluminal placement of the proximal MAGNAMOSIS ring, (c) creation of the TR access with the TEO and transrectal dissection of the sigmoid mesentery, (d) resection of the surgical specimen, (e) transrectal extraction of the specimen, (f) delivery and mating of the distal MAGNAMOSIS ring, and (g) closure of the TG and TR viscerotomies. The animal survived for 14 days at which time burst pressure and histology were performed. RESULTS: A pure NOTES TR and TG segmental colectomy was performed in 139 minutes. The postoperative course was uneventful. The animal had a formed bowel movement including the magnetic rings on postoperative day 5. Endoscopic examination at postoperative day 14 revealed a patent anastomosis. Necropsy revealed no abscess or signs of peritonitis. Burst pressure was >198 mm Hg. The histology showed a sealed anastomosis with mild inflammation. CONCLUSIONS: MAGNAMOSIS enabled a totally NOTES partial colectomy with combined TG and TR access. The flexible delivery options and low cost of manufacturing could make MAGNAMOSIS an attractive alternative to circular staplers.

Neo-regeneration of urinary bladder: a desired metaplasia of autologous membrane from rectosigmoid colon containing stem cells of intestinal crypts.

The current management of diseases of urinary bladder requiring resection is by augmentation cystoplasty or transplantation of ureters. Transplantation of ureters is associated with morbidity and mortality. Ideal management will be by regenerating urinary bladder in vivo. Neo-regeneration of tissues and organs like abdominal wall, aponeurosis etc., has been attempted and patented. After neo-regeneration of mesoderm tissues and organs, regeneration of urinary bladder (developed from endoderm) was. In vivo surgical techniques were developed in dogs. It is known that the embryonic morphogenesis of urinary bladder is from uro-genital sinus of hind gut. A membrane, containing endoderm stem cells in crypts of recto-sigmoid colon, was surgically isolated and colonized with remnant of urinary bladder wall after extensive resection. Experimental study was performed in dogs, for 60 days to one and a half year. Regeneration of all the layers of tissues of the wall of urinary bladder was observed. The neo-regeneration phenomenon has been recognized as "desired metaplasia". The regenerated neo tissue/organ on histological examination and cystometry studies was found compatible with normal urinary bladder. The hypothesis, neo-regeneration and desired metaplasia, is discussed.

Immune reconstitution in the sigmoid colon after long-term HIV therapy.

Early and profound CD4+ T-cell depletion in gut-associated lymphoid tissue (GALT) may drive Human Immunodeficiency Virus (HIV) immunopathogenesis, and GALT immune reconstitution on highly active antiretroviral therapy (HAART) may be suboptimal. Blood and sigmoid colon biopsies were collected from HAART-treated individuals with undetectable blood HIV RNA for > or =4 years and from uninfected controls. HIV proviral levels and T-cell phenotype/function were examined in both compartments. CD4+ T-cell reconstitution in the sigmoid, including CD4+ T cells expressing CCR5, exceeded that in blood and did not differ from uninfected controls. Sigmoid HIV proviral load was not correlated with CD4+ reconsitution, but was correlated with the degree of mucosal CD8+ T-cell immune activation. Colonic Gag-specific T-cell responses were common, but were not associated with proviral load or immune activation. In this select study population, long-term HAART was associated with complete CD4+ T-cell reconstitution in sigmoid colon. However, colonic immune activation may drive ongoing HIV replication.

Insulin-like growth factor I improves aspects of mycophenolate mofetil-impaired anastomotic healing in an experimental model.

BACKGROUND: Patients taking immunosuppressants after transplantation may require intestinal surgery. Mycophenolate mofetil (MMF) has been found to impair the healing of colonic anastomoses in rats. This study examined whether insulin-like growth factor (IGF) I prevents MMF impairment of anastomotic healing. METHODS: Sixty-three rats were divided into three groups (MMF, MMF/IGF and control). Animals underwent a sigmoid colon anastomosis with a 6/0 suture, and were killed on days 2, 4 and 6 after surgery. Investigations included bursting pressure measurement, morphometric analysis, and assessment of mucosal proliferation by 5-bromo-2'-deoxyuridine and Ki67 immunohistochemistry of the anastomoses. RESULTS: The leak rate was three of 21, one of 20 and two of 20 in the MMF, MMF/IGF-I and control groups respectively. Anastomotic bursting pressures were significantly lower in the MMF group than in the control group on days 2 and 4, but there was no significant difference by day 6. Values in the MMF/IGF-I and control groups were similar. Colonic crypt depth was significantly reduced in MMF-treated animals on days 2 and 4, but this impairment was attenuated by IGF-I on day 4. Similarly, IGF-I reduced the negative impact of MMF on mucosal proliferation on days 2 and 6. CONCLUSION: Exogenous IGF-I improves some aspects of MMF-impaired anastomotic healing.

Dihydroxy bile acids increase mucosal permeability and bacterial uptake in human colon biopsies.

OBJECTIVE: Bile acids in mM concentrations are known to increase chloride secretion and alter mucosal permeability in animal colon. Increased mucosal permeability is believed to play an important role in the development of intestinal inflammation. The aim of this study was to investigate the influence of microM concentrations of dihydroxy bile acids on permeability and bacterial uptake in the normal human colon. MATERIAL AND METHODS: Endoscopic biopsies from the sigmoid colon of 18 subjects with normal colonic histology were mounted in modified Ussing chambers. Chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) were added to the mucosal compartment. Short-circuit current (Isc) and transepithelial resistance (TER) were studied for 120 min. Cr-EDTA and horseradish peroxidase (HRP) were used to assess paracellular and transcellular permeability, respectively. The transmucosal passage of chemically killed Escherichia coli was quantified and investigated using confocal microscopy. RESULTS: A significant decrease in TER was seen after 60 min of exposure to 1000 micromol/l CDCA and DCA. The combination of E. coli and 100 micromol/l CDCA gave a decrease in TER compared to controls (p = 0.06). DCA showed a dose-related increase in Cr-EDTA permeability, which was most pronounced at 1000 micromol/l (p = 0.02). Increased E. coli uptake was induced by 500 micromol/l (p = 0.01) and 1000 micromol/l CDCA (p = 0.04). Bacterial uptake was increased at 100 micromol/l by exposure to DCA (p = 0.03). Confocal microscopy revealed the presence of E. coli bacteria in the lamina propria after 15 min of exposure to 1000 micromol/l CDCA and DCA. CONCLUSIONS: Our study suggests that dihydroxy bile acids in microM concentrations alter barrier function in normal human colon biopsies, causing increased antigen and bacterial uptake; thereby bile acids may contribute to the development of intestinal inflammation.

A novel role of VIP in colonic motility function: induction of excitation-transcription coupling in smooth muscle cells.

BACKGROUND & AIMS: Vasoactive intestinal polypeptide (VIP) relaxes smooth muscle by generation of cAMP and activation of protein kinase A (PKA). However, PKA activation also phosphorylates the transcription factor CREB. The aim of this study was to investigate whether the phosphorylation of CREB induces gene expression of the pore-forming alpha(1C) subunit of Ca(v)1.2 channels (L-type calcium channels), whose promoter has 2 binding sites for CREB. METHODS: The experiments were performed on primary cultures of human colonic circular smooth muscle cells and freshly obtained human and rat colonic circular muscle strips. RESULTS: The incubation of human colonic circular smooth muscle cells or muscle strips with VIP for 24 hours enhanced the expression of alpha(1C) protein and mRNA as well as the contractile response to acetylcholine and KCl. On the contrary, incubation of the muscle strips with VIP antagonist for 24 hours suppressed cell contractility. The incubation of the cells with VIP caused sustained generation of cAMP for 24 hours, but PKA activation and CREB phosphorylation were transient. The inhibition of PKA by H-89 or silencing of CREB gene with targeted RNAi blocked the transcription of alpha(1C). Progressive 5' deletions of halpha(1C)1b promoter and site-directed mutations of the 2 CREB binding cis-elements indicated that most of alpha(1C) transcription was mediated by the 5' cAMP response element. CONCLUSIONS: The excitation-transcription coupling stimulated by VIP induces expression of the Ca(v)1.2 channels. The influx of calcium through these channels is a critical step in excitation-contraction coupling in smooth muscle cells.

Decreased interstitial cells of Cajal in the neorectum after anterior resection of the rectum.

BACKGROUND/AIMS: Patients who have undergone anterior resection for rectal carcinoma often complain of anorectal and defecatory dysfunction postoperatively. The aim of this study was to examine the expression of interstitial cells of Cajal (ICCs) in the sigmoid colon used for constructing the neorectum after anterior resection of the rectum. METHODOLOGY: As the neorectum group, we assessed 12 patients with local and anastomotic recurrence or new neoplasm in the neorectum after anterior resection of the rectum. The control group consisted of 16 patients who underwent sigmoid colon resection for sigmoid colon carcinoma. All resected specimens were investigated with immunohistochemical staining, using c-kit antibody for ICCs. The correlation between the number of ICCs and defecatory symptoms was assessed for the neorectum. RESULTS: The total number of ICCs significantly decreased in the neorectum group as compared to the control group. In particular, a significant difference was noted between the two groups as to the number of ICCs found between the layers of the myenteric plexus in histological studies, as well as in the circular and longitudinal muscles. There was no correlation between the number of ICCs and the time interval from the initial anterior resection to the resection of the neorectum, nor was there any relationship between the number of ICCs and defecatory symptoms. CONCLUSIONS: The expression of ICCs in the neorectum was reduced in the early stages after anterior resection of the rectum. Expression of ICCs in the neorectum did not recover to preoperative levels over time.

In vivo recording of colonic motility in conscious rats with deficiency of interstitial cells of Cajal, with special reference to the effects of nitric oxide on colonic motility.

BACKGROUND: We recorded in vivo colonic motility in rats with a deficiency of interstitial cells of Cajal (ICC) (Ws/Ws rats) and in wild-type rats (+/+ rats), with special reference to the effects of nitric oxide (NO) on colonic motility in both types of rats, in order to ascertain the role of ICC in colonic motility, and the relationship between NO and ICC in regard to colonic motility. METHODS: Miniature strain-gauge force transducers were sutured on the surface of the ascending and sigmoid colon of Ws/Ws rats and +/+ rats as controls. After 1 week and a fasting period of 24 h, colonic motility in +/+ and Ws/Ws rats was recorded. We also studied the effect of NO on colonic motility in both types of rats, by means of the administration of N-nitro-L-arginine methyl ester (L-NAME) or L-arginine. RESULTS: In +/+ rats, there were contractions with high amplitude and long duration in both the ascending and sigmoid colon. The number, amplitude, and duration of contractions in the ascending colon were 9.9/20 min, 6.1 g, and 22.7 s, respectively. These findings in the sigmoid colon were 5.2/20 min, 5.2 g, and 23.0 s, respectively. The number of contractions in the ascending and sigmoid colon in Ws/Ws rats (2.3 and 1.0/20 min) was significantly lower than that in +/+ rats (P < 0.05). The number of contractions in the ascending and sigmoid colon in +/+ rats (9.7 and 5.1/20 min before treatment) was significantly increased by L-NAME administration (28.7 and 13.9/40-60 min after treatment; P < 0.05), but that in Ws/Ws rats was not influenced. The number of contractions in the ascending and sigmoid colon in +/+ rats (10.2 and 5.2/20 min before treatment) was significantly decreased by L-arginine administration (3.6 and 2.1/40-60 min after treatment; P < 0.05), but that in Ws/Ws rats was not influenced. CONCLUSIONS: ICC must be related to the occurrence of a normal number of colonic contractions. NO may be involved in the inhibitory regulation of colonic motility, and the effect of NO on the occurrence of contractions appears to be mediated by ICC.

Aerosol transfer of bladder urothelial and smooth muscle cells onto demucosalized colonic segments for porcine bladder augmentation in vivo: a 6-week experimental study.

PURPOSE: In a pilot study we developed a cell transfer technology for populating demucosalized colonic segments with bladder urothelium. This process was achieved through aerosol transfer of a single cell suspension consisting of bladder urothelial cells, smooth muscle cells and fibrin glue onto demucosalized colonic segments. We further evaluate this new concept in a controlled study. MATERIALS AND METHODS: The study was performed on 20 piglets (20 kg). In all animals 50% of the bladder with excised, and a 10 cm segment of the sigmoid was isolated. Animals were then equally divided into 5 groups of 1) colocystoplasty only, 2) demucosalized colocystoplasty, 3) demucosalized colocystoplasty plus covering of the demucosalized sigmoid with fibrin glue only, 4) aerosol application of fibrin glue with single cell suspension of urothelial cells only to the demucosalized colon, and 5) aerosol application of fibrin glue with urothelial and smooth muscle cells to the demucosalized colon. The 4 corners of the augmented segments were marked with 5-zero polypropylene sutures. Animals were sacrificed 6 weeks later and the surface area of the augmented segment was measured. Segments were submitted to histological and immunohistochemical analysis. RESULTS: The surface area of the augmented segments showed an increase in group 1 animals, stabilization in groups 4 and 5, and marked reduction in groups 2 and 3. On hematoxylin and eosin, and Masson trichrome staining all group 1 animals showed normal colonic epithelium of the augment. All animals in groups 2 and 3 showed excessive scarring with urothelial coverage only at the augment periphery, while the central augment area showed no epithelium. Segments from groups 4 and 5 showed confluent epithelial covering with no fibrosis. There was no evidence of colonic epithelial re-growth in any animal in groups 2 to 5. Cytokeratin 7 and uroplakin III staining demonstrated complete coverage of the augmented segment with urothelium only in groups 4 and 5. CONCLUSIONS: The addition of aerosolized cells of urological origin is a viable augmentation approach that appears to achieve the much sought after inhibition of intrinsic fibrosis and contraction of colonic segments when incorporated into the urinary tract without this cellular component. Moreover, this technique appears to provide a histologically normal, confluent urothelium, which sets the stage for prevention of the well-documented biochemical aberrations inherent in augments containing gastrointestinal epithelium. While successful in this model regardless of the incorporation of urological smooth muscle cells, chronic studies are now warranted to validate the short-term results as well as determine whether the urological mesenchymal population (smooth muscle) will be required to sustain the uroepithelial phenotype in the long term.

Endoscopic biopsies in Ussing chambers evaluated for studies of macromolecular permeability in the human colon.

OBJECTIVE: Studies of mucosal permeability to protein antigens in humans are limited to in vitro techniques. The use of surgical specimens for such studies has major shortcomings. Endoscopic biopsies in Ussing chambers have been introduced as a means of studying secretion and transepithelial permeability, but have not been evaluated for studies of protein antigen uptake in human intestine. MATERIAL AND METHODS: Standard forceps biopsies from the sigmoid colon of 24 healthy volunteers were mounted in Ussing chambers with an exposed tissue area of 1.76 mm2. 51Cr-EDTA (paracellular probe) and horseradish peroxidase (HRP; 45 kDa protein antigen) were used as permeability markers. Mucosal permeability, electrophysiology, histology and energy contents of the biopsies were studied over time. To evaluate the ability of the technique to detect permeability changes, the mucosa was modulated with capric acid, a medium-chain fatty acid, known to affect tight junctions. RESULTS: In the Ussing chamber the mucosal biopsies were viable for 160 min with stable levels of ATP and lactate, and only minor changes in morphology. Steady-state permeability with low variability was seen for both markers during the 30-90 min period. Exposure to capric acid induced a rapid decrease in short-circuit current (Isc) and a slower reversible decrease in transepithelial resistance (TER), as well as an increased permeability to 51Cr-EDTA and HRP. CONCLUSIONS: Endoscopic biopsies of human colon are viable in Ussing chambers and are reliable tools for studies of mucosal permeability to protein antigens. The technique offers a broad potential for studies of mucosal function in the pathophysiology of human gastrointestinal diseases.

Polyethylene glycol versus sodium phosphate in bowel cleansing for colonoscopy: a randomized trial.

BACKGROUND AND STUDY AIMS: There have been conflicting results regarding the adverse effects of established bowel cleansing regimens. The aim of the present study was to compare the effects of three bowel cleansing regimens on subjective well-being, electrolyte balance, cardiac arrhythmia, and the microscopic post-cleansing appearance of the colonic mucosa. PATIENTS AND METHODS: A total of 231 consecutive outpatients were randomly assigned to receive bowel preparation for colonoscopy with either 4 l polyethylene glycol (PEG; group I, n = 76); 2 l PEG plus 10 mg Bisacodyl (group II, n = 71); or 90 ml sodium phosphate (group III, n = 84). After bowel preparation, the participants completed a questionnaire on symptoms. Endoscopists blinded to the regimen used gave scores for the quality of cleansing at endoscopy, ranging from poor (0) to very good (5). Blood samples were taken before and after bowel cleansing, electrocardiographic monitoring was used during colonoscopy, and mucosal biopsy samples were taken in the sigmoid colon. RESULTS: Bowel preparation in group II was poorer (mean score 3.26) than in groups I (3.88) and III (4.01); P < 0.001 (II vs. III), P < 0.001 (I vs. II). The frequency of arrhythmias and post-cleansing mucosal inflammation was similar in all three groups. Lower serum potassium and higher serum phosphate concentrations were found in group III in comparison with the other groups ( P < 0.001). CONCLUSIONS: No differences were detected regarding the effectiveness and safety of bowel preparation with PEG alone and sodium phosphate in individuals without cardiac, renal, or hepatic failure, despite a significantly stronger alteration of the electrolyte balance with sodium phosphate.

A role for chromogranin A (4-16), a vasostatin-derived peptide, on human colonic motility. An in vitro study.

The hypothesis that CgA-derived peptides may be involved in mechanisms modulating motility was tested. Human colonic smooth muscles were studied using an organ bath technique. Acetic acid (AA) effects were characterized on spontaneous mechanical activities (SMA) and on responses to transmural nerve stimulation (NS). AA induced a significant decrease in tone and abolished SMA; this effect was insensitive to either TTX or L-NAME/apamin. The AA-induced inhibitory effects were significantly reduced in the presence of CgA4-16. This effect was insensitive to TTX or L-NAME/apamin. Furthermore, AA-induced effects were blocked in the presence of BAYK8644 and CgA4-16 together. The inhibitory effect of nifedipine was delayed in the presence of CgA4-16. NS induced a triphasic response. Only the excitatory components were reduced in the presence of AA. This effect was dose-related and remained unchanged in the presence of CgA4-16 alone, but was blocked in the presence of simultaneous administration of CgA4-16 and L-NAME/apamin. AA application induced inhibition of human colon motility in vitro. This effect may be mediated through an action on L-type calcium channels. CgA4-16 may display a protective role, which prevents the inhibition of motility due to AA to occur, by acting on both smooth muscle and afferent terminals.

Decreased interstitial cells of Cajal in the sigmoid colon of patients with slow transit constipation.

BACKGROUND AND AIMS: Slow transit constipation (STC) is a colonic motor disorder that is characterized by measurably delayed movement of materials through the colon. Although abnormalities in the neuronal networks of the colon have been demonstrated in patients with STC, the etiology of STC remains unclear. Interstitial cells of Cajal (ICC) have been shown to be the pacemaker cells of the intestine and have been implied in the pathogenesis of a number of gastrointestinal motility dysfunctions, including idiopathic STC. This study aimed to determine the normal distribution of ICC within the colon of the Chinese and also to determine if ICC are decreased in Chinese STC patients. PATIENTS AND METHODS: Twelve patients with STC and eight age-matched normal controls were studied. Specimens of sigmoid colon were obtained immediately after resection. ICC were identified with a monoclonal antibody to c-kit by an indirect immunofluorescence method. Immunostained tissues were examined with a laser scanning confocal microscope and the area occupied by ICC was calculated with an image analysis system. RESULTS: ICC were located in the external muscle layers including myenteric plexus (MP) and submucosal border (SMB). Two types of Kit-positive ICC were observed: bipolar cells characterized by one or two long processes and multipolar cells characterized by long stellate processes extending in various directions. A higher percentage of ICC was present in the MP regions and circular muscle (CM) layers compared with the SMB and longitudinal muscle (LM) layers. Tissues from STC patients showed a considerable decrease in the number of ICC located in the four regions (ICC-LM, ICC-MP, ICC-CM, ICC-SMB), especially the ICC-SMB, in which ICC almost completely disappeared. CONCLUSIONS: Similar distribution of ICC was observed in the normal sigmoid colon of the Chinese. Decreased area of c-kit+ ICC may play an important role in the pathophysiology of STC. It remains to be determined whether the loss of ICC is primary or secondary to another lesion.

Aerosol transfer of bladder urothelial and smooth muscle cells onto demucosalized colonic segments: a pilot study.

PURPOSE: We developed a cell transfer technology for covering demucosalized colonic segments with bladder urothelium. This covering would be achieved through aerosol spraying of single cell suspension of bladder urothelial and smooth muscle cells with fibrin glue onto the demucosalized colonic segments. MATERIALS AND METHODS: In 6 piglets (20 kg.) a 4 cm.2 area of bladder was excised. Single cell suspension of bladder urothelial and smooth muscle cells was prepared. A segment of detubularized sigmoid colon was isolated on its vascular pedicle and demucosalized. The single cell suspensions were combined with an equal volume of fibrin glue and sprayed over the raw submucosal surface of the sigmoid segment. The sigmoid segment was retubularized and sutured to the posterior peritoneum. Animals were sacrificed 4 weeks later, and the segment was submitted to histological and immunohistochemical analysis. RESULTS: Sigmoid segments appeared grossly intact with no reduction in surface area. Hematoxylin and eosin architecture revealed an intact urothelial layer. Deep to this layer was a randomly aligned but distinctly segregated layer of smooth muscle cells. The urological new smooth muscle layer stained positive for calponin and the urothelial layer was cytokeratin-7 and uroplakin III positive. CONCLUSIONS: Separation, cell suspension and aerosol delivery of bladder urothelial and smooth muscle cells in fibrin glue can successfully transfer these urological cell populations to a new host tissue commonly used in urological reconstruction. In vivo co-culture of bladder smooth muscle and urothelial cells results in coverage of a large area of demucosalized gut providing new potential for transfer and reconstitution of urologically functionally appropriate tissue to the bladder itself.

Intestinal permeability in humans is increased after radiation therapy.

PURPOSE: Irradiation inflicts acute injuries to the intestinal mucosa with rapid apoptosis induction and subsequent reduction in epithelial surface area. It may therefore be assumed that the intestinal barrier function is affected. The aim of this study was to compare the mucosal permeability in irradiated rectum and nonirradiated sigmoid colon from patients subjected to radiation therapy before surgical treatment for rectal cancer. METHODS: Segments from sigmoid colon and rectum obtained from irradiated and nonirradiated patients were stripped from the serosa-muscle layer and mounted in Ussing diffusion chambers. The mucosa-to-serosa passage of the marker molecules 14C-mannitol, fluorescein isothiocyanate-dextran 4,400, and ovalbumin was followed for 120 minutes. RESULTS: The permeability to the markers was size-dependent and increased linearly across time in all specimens. The passage of all markers was increased in irradiated rectum compared with nonirradiated sigmoid colon, whereas in specimens from nonirradiated patients there were no differences between rectum and sigmoid colon. Histologic signs of crypt and mucosal atrophy were found in the irradiated rectal specimens. CONCLUSIONS: Early gastrointestinal complications after radiation therapy may be the result of mucosal atrophy in addition to mucosal damage, with a loss of barrier integrity.

Gammadelta T cell receptor repertoire in blood and colonic mucosa of rhesus macaques.

Although their precise roles are not well defined, gammadelta T lymphocytes are recognized as regular components of immune responses. These cells express a limited T cell receptor repertoire and they can be stimulated by soluble ligands without conventional processing and presentation by major histocompatibility antigens. Progress in this area has been limited by the substantial differences between murine and human gammadelta T cells and the lack of knowledge about these cells in nonhuman primates. We used molecular analysis of T cell receptor diversity to characterize gammadelta T cell populations from peripheral blood and colon of rhesus macaques (Macaca mulatta). The gammadelta T cell receptor diversity was limited and distinct for these tissue compartments, particularly in the TCRGV2 family. Furthermore, the TCRDV1 + subset of peripheral blood gammadelta T cells showed signs of progressive oligoclonalization as a function of age. Similar observations have been reported for human tissue samples and our results validate rhesus macaques as an appropriate animal model for studying primate gammadelta T cell populations.