Segments Of Human Colon Research Articles

Mice expressing fluorescent PAR2 reveal that endocytosis mediates colonic inflammation and pain

G protein-coupled receptors (GPCRs) regulate many pathophysiological processes and are major therapeutic targets. The impact of disease on the subcellular distribution and function of GPCRs is poorly understood. We investigated trafficking and signaling of protease-activated receptor 2 (PAR2) in colitis. To localize PAR2 and assess redistribution during disease, we generated knockin mice expressing PAR2 fused to monomeric ultrastable green fluorescent protein (muGFP). PAR2-muGFP signaled and trafficked normally. PAR2 messenger RNA was detected at similar levels in Par2-mugfp and wild-type mice. Immunostaining with a GFP antibody and RNAScope in situ hybridization using F2rl1 (PAR2) and Gfp probes revealed that PAR2-muGFP was expressed in epithelial cells of the small and large intestine and in subsets of enteric and dorsal root ganglia neurons. In healthy mice, PAR2-muGFP was prominently localized to the basolateral membrane of colonocytes. In mice with colitis, PAR2-muGFP was depleted from the plasma membrane of colonocytes and redistributed to early endosomes, consistent with generation of proinflammatory proteases that activate PAR2 PAR2 agonists stimulated endocytosis of PAR2 and recruitment of Gαq, Gαi, and β-arrestin to early endosomes of T84 colon carcinoma cells. PAR2 agonists increased paracellular permeability of colonic epithelial cells, induced colonic inflammation and hyperalgesia in mice, and stimulated proinflammatory cytokine release from segments of human colon. Knockdown of dynamin-2 (Dnm2), the major colonocyte isoform, and Dnm inhibition attenuated PAR2 endocytosis, signaling complex assembly and colonic inflammation and hyperalgesia. Thus, PAR2 endocytosis sustains protease-evoked inflammation and nociception and PAR2 in endosomes is a potential therapeutic target for colitis.

Hydrodynamic IL10 Gene Transfer in Human Colon: Results from an "EX VIVO" Study with Potential Clinical Application in Crohn's Disease.

The aim of this work is to evaluate the efficacy of hydrodynamic venous IL10 gene delivery to "ex vivo" human colon segments and to determine its potential interest in Crohn's disease treatment. Twenty human colon segments were obtained from surgical resections. Hydrodynamic transfection through the main vein of the pedicle with 50 mL of hIL10 plasmid (20 μg/mL) solution was performed on 13 of them. Tissue sections were cultured and DNA, RNA, and protein copies were determined after 1, 2, and 4 days. Data obtained were compared with 6 nontransfected specimens. Finally, 1 specimen was injected with gold nanoparticles, and their distribution was examined under electron microscope. IL10 DNA levels were higher in treated tissues than in controls (P < 0.001), decreasing along time. The amount of hIL10 RNA was significantly increased in treated tissues when compared with controls (P = 0.001). The indexes of protein IL10 translation in treated groups were much higher (P < 0.001) than the basal production. The protein expression was higher in transfected tissue (10-50-fold, with respect to control tissue); this difference being established during the first hours and maintained during, at least, 4 days. With electron microscopy, we hardly observed large (15 nm) gold nanoparticles within the tissue, always in the submucosa. However, multiple small (4 nm) nanoparticles were observed within the cytoplasm of enterocytes in mucosa. Hydrodynamic procedure efficiently delivers the IL10 gene to the human colon, achieving levels of tissue protein expression high enough to mediate pharmacological effects with interest in controlling immune response in patients with Crohn's disease.

Expression and localization of pannexin‐1 hemichannels in human colon in health and disease

Pannexin-1 (Panx1) proteins can function as channels for adenosine triphosphate (ATP) release, but there have been limited studies investigating their potential role in the human intestine. The aim of this study was to characterize Panx1 expression and distribution in the human colon and its potential involvement in inflammatory bowel diseases (IBD). Human colon segments were dissected into mucosa and muscularis layers, and evaluated for Panx1 expression by real-time PCR and Western blotting. Immunohistochemistry was conducted to localize the cellular distribution of Panx1 in intact tissues. In the colonic muscularis of ulcerative colitis (UC), Panx1 mRNA expression showed a 3.5-fold reduction compared with control (P=0.0015), but no change was seen in UC mucosa. In contrast, down-regulation of Panx1 mRNA was observed in both muscularis and mucosa of Crohn's disease (CD), showing a 2.7- and 1.8-fold reduction, respectively (P<0.05). There was reduced Panx1 protein expression in CD muscularis, but no change in CD mucosa, UC muscularis, or UC mucosa. Pannexin-1 immunoreactivity was mainly localized to enteric ganglia, blood vessel endothelium, erythrocytes, epithelial cells, and goblet cells. Inflammatory bowel disease samples showed a similar overall pattern of Panx1 staining, but in UC myenteric ganglia, there was a significant reduction in Panx1 immunoreactivity. Significant Panx1 positive leukocyte infiltrations were seen at the sites of inflammation. The presence of Panx1 in the colon and changes to its distribution in disease suggests that Panx1 channels may play an important role in mediating gut function and in IBD pathophysiology.

Distinct differences in tachykinin gene expression in ulcerative colitis, Crohn’s disease and diverticular disease: a role for hemokinin‐1?

In the intestine, the tachykinins substance P (SP) and neurokinin A (NKA) are found in neurons and have key roles in motility, secretion, and immune functions. A new tachykinin, hemokinin (HK-1), has been identified in non-neuronal cells in recent years and its role in intestinal inflammation is unclear. We aimed to examine the expression of genes encoding tachykinin peptides and receptors in colon from patients with ulcerative colitis (UC), Crohn's disease (CD), and acute diverticular disease (DD). Human colon segments were dissected into mucosa and muscle, and evaluated for tachykinin and tachykinin receptor gene expression by real-time PCR. In UC mucosa, the TAC4 gene (encoding HK-1) was 10-fold more abundant than in control mucosa (P < 0.01). Similarly, TAC1 (encoding SP and NKA) and TACR1 (encoding NK1 receptor) displayed 6-fold and 12-fold upregulation, respectively, in UC mucosa, but no change occurred in UC muscle. In contrast to UC, no difference was observed for any tachykinin genes in CD mucosa. In CD muscle, expression of TAC1 (P < 0.01), TAC4 and TACR1 (both P < 0.05) were moderately upregulated. In DD, there was a decrease in TACR1 (P < 0.05), and TACR2 (encoding NK2 receptor, P < 0.0001) in muscle compared with control. Histological staining showed increased collagen fibers between muscle bundles in DD smooth muscle. We provide evidence for the first time that HK-1, like SP, may be involved in the pathophysiology of inflammatory bowel disease. Distinctly different expression patterns of tachykinin-related genes occur in UC, CD and DD.

Differential protein abundance and function of UT-B urea transporters in human colon

Facilitative UT-B urea transporters enable the passage of urea across cell membranes. Gastrointestinal urea transporters are thought to play a significant role in the urea nitrogen salvaging process that occurs between mammalian hosts and their gut bacteria. This study investigated the expression of UT-B urea transporters in different segments of human colon. Immunoblot analysis showed that human colon expressed a 35-kDa glycosylated UT-B protein in the colonic mucosa. The 35-kDa UT-B transporter was predominantly located in plasma membrane-enriched samples (P < 0.001; n = 6), and its expression was greater in the ascending colon compared with the descending colon (P < 0.01; n = 3). At the cellular level, UT-B transporters were located throughout colonocytes situated in the upper portion of the colonic crypts. Bidirectional trans-epithelial urea transport was significantly greater in the ascending colon than the descending colon (P < 0.05; n = 6). In addition, the facilitative urea transporter inhibitor 1,3,dimethylurea significantly reduced urea transport in the ascending colon (P < 0.05; n = 6) but had no effect in the descending colon (NS; n = 6). These results illustrate differential protein abundance of functional UT-B protein in different sections of the human colon, strongly correlating to regions that contain the largest populations of intestinal bacteria. This study suggests an important role for UT-B urea transporters in maintaining the symbiotic relationship between humans and their gut bacteria.

Acidity induces c-Fos expression in a subpopulation of human colonic submucosal neurons

Enteric neurons responding to chemical challenge of the mucosa have been characterized in animal models mainly in the myenteric plexus. However, in humans, the existence of enteric neurons responding to chemical stimulation of the mucosa remains currently unknown. Therefore, the aim of our study was to identify and characterize human submucosal neurons activated by mucosal challenge with butyrate or hydrochloric acid. Segments of human colon were placed in a modified Ussing chamber and incubated on the mucosal side with butyric acid (20 mM, pH 6.5), sodium butyrate (20 mM, pH 7.5), hydrochloric acid (10 mM, pH 6.5) or culture medium (pH 7.5). After 90 min of culture, tissues were fixed and microdissected to obtain whole mount preparation of submucosa containing the Meissner's plexus. Neuron specific enolase (NSE), c-Fos, vasoactive intestinal peptide (VIP) and substance P (SP) were detected using immunohistochemical methods. Tetrodotoxin (TTX, 1 μM) was used to inhibit neuronal activity. After 90 min of culture, butyric acid induced a significant 5.6-fold increase in the proportion of c-Fos-immunoreactive neurons compared to control (19 ± 4% versus 4 ± 1%, respectively, p < 0.001). 41 ± 5% of c-Fos-immunoreactive neurons were VIP-immunoreactive and 3 ± 2% were SP-immunoreactive. Butyric acid did not modify the proportion of VIP-immunoreactive neurons. The increase in c-Fos-immunoreactive neurons induced by butyric acid was reproduced with hydrochloric acid at the same pH but not with sodium butyrate. Finally, preincubation of the tissue with TTX prevented the effect of butyric acid. In conclusion, our results demonstrate that acidic mucosal challenge induced the activation of a population of human submucosal neurons with a specific neurochemical coding.

Preservative solution for freeze-storage of surgically excised human colon to enable study of smooth muscle function in vitro

We have compared the reactivity to carbachol and high potassium of circular smooth muscle isolated from segments of human colon which was freeze-stored in different preservative solutions for more than one month following surgical resection. Concentration-dependent contractions in response to carbachol were reduced in terms of both their sensitivity (pEC50) and reactivity (Emax), depending on the preservative solutions used. Similar reduction of reactivity to 100 mM KCl was also observed. The best responsiveness was shown when the tissue was freeze-stored in SFM101. It is concluded that the freeze-storage of surgically excised human colon in SFM101 or phosphate buffer solution for more than one month provided the best preservation of smooth muscle function for in vitro pharmacological examination.

Toxin B of Clostridium difficile activates human VIP submucosal neurons, in part via an IL-1beta-dependent pathway.

This study investigated whether toxin B of Clostridium difficile can activate human submucosal neurons and the involved pathways. Isolated segments of human colon were placed in organ culture for 3 h in the presence of toxin B or IL-1beta. Whole mounts of internal submucosal plexus were stained with antibodies against c-Fos, neuron-specific enolase (NSE), vasoactive intestinal polypeptide (VIP), and substance P (SP). The membrane potential (Vm) response of submucosal neurons to local application of toxin B and IL-1beta was determined by a multisite optical recording technique. Toxin B (0.1 to 10 ng/ml) increased the proportion of c-Fos-positive neurons dose dependently compared with the control. In the presence of toxin B (10 ng/ml), most c-Fos-positive neurons were immunoreactive for VIP (79.8 +/- 22.5%) but only 19.4 +/- 14.0% for SP. Toxin B induced a rapid rise in IL-1beta mRNA level and a sixfold increase in IL-1beta protein in supernatant after 3 h of incubation. c-Fos expression induced by toxin B was reduced dose dependently by IL-1 receptor antagonist (0.1-10 ng/ml). IL-1beta significantly increased c-Fos expression in submucosal neurons compared with the control (34.2 +/- 10.1 vs. 5.1 +/- 1.3% of NSE neurons). Microejection of toxin B had no effect on the Vm of enteric neurons. Evidence of a direct excitatory effect of IL-1beta on Vm was detected in a minority of enteric neurons. Therefore, toxin B of C. difficile activates VIP-positive submucosal neurons, at least in part, via an indirect IL-1beta-dependent pathway.

Doxorubicin activity is enhanced by hyperthermia in a model of ex vivo vascular perfusion of human colon carcinoma.

There is little information on the pharmacokinetics and pharmacodynamics of doxorubicin (DXR) administered during locoregional treatments of colon carcinoma under hyperthermic conditions. The aim of this study was to evaluate distribution and activity of DXR in healthy tissue and tumor tissues under hyperthermic conditions by using an experimental model of ex vivo isolated vascular perfusion of human colon segments bearing primary carcinoma. The influence of topoisomerase-II alpha (TPI2 alpha) expression on the anti-cancer activity of DXR combined with heat was evaluated as well. Twenty seven colon segments surgically resected for primary carcinoma were perfused ex vivo under physiological conditions (group A, n = 7), with DXR (group B, n = 6), under hyperthermic conditions (group C, n = 6), and with the combination DXR-hyperthermia (group D, n = 8). Samples of perfusate and tissues (normal and pathologic) were collected during 90 minutes of perfusion. Doxorubicin concentration in perfusate and tissues was assessed with high-performance liquid chromatography. Protein expression of TPI2 alpha, the main molecular target of DXR, was measured by image analysis in normal mucosa and tumor samples. Drug uptake was increased by heat equally in healthy and diseased tissue. Under hyperthermic conditions, DXR activity was significantly higher in pathologic mucosa than in normal mucosa. Furthermore, the activity of DXR combined with heat correlated with baseline TPI2 alpha levels in tumor tissue. From these findings, it appears that heat sensitizes tumor cells-but not normal mucosa-to DXR activity. Furthermore, protein levels of TPI2 alpha in pretreatment samples could predict tumor sensitivity to DXR.

Distribution of neuropeptide Y-like immunoreactivity in the normoganglionic and aganglionic segments of human colon

The localisation and distribution of neuropeptide Y (NPY)-like immunoreactivity were studied by use of immunohistochemical methods in gut tissues from 19 patients with Hirschsprung's disease, including 4 cases of long segment aganglionosis. In the normoganglionic segment, immunoreactive cell bodies and nonvaricose processes were seen within both myenteric and submucous plexuses. A scarce supply of varicose fibres was found in the lamina propria mucosae, muscularis mucosae and longitudinal muscle layer. NPY fibres were more frequently encountered in the circular muscle layer, although with a weakly immunostaining intensity. In addition, blood vessels in the submucosal connective tissue were surrounded by a typical plexus of varicose, NPY-positive fibres. Immunoreactive endocrine cells could be detected in the colonic epithelium. In the aganglionic segment, numerous nerve fasciculi comprising a small to moderate number of NPY fibres with varicosities were observed throughout the entire layer of the colonic wall. A few varicose, NPY-positive fibres were also contained in the relatively large, hypertrophic nerve fasciculi located in the intermuscular zone and submucosal connective tissue. NPY-immunoreactive fasciculi were more densely distributed in the distal aganglionic segment than in the proximal aganglionic one. On the other hand, the distribution of NPY-positive fibres in long segment aganglionosis was quite different from that in short segment type; in cases of long segment type, no immunoreactive nerve fibres were detected within the circular muscle layer of the proximal aganglionic segment near the oligoganglionic segment and only a few fibres were observed within the hypertrophic nerve bundle of the intermuscular zone.(ABSTRACT TRUNCATED AT 250 WORDS)

Segmental variability of membrane conductances in rat and human colonic epithelia

The membrane conductances in proximal and distal segments of rat and human colon were studied with microelectrodes, nystatin, ion channel blockers and Cl replacement. The results reveal that (1) in rat colon, total conductance (Gt) is greater in the proximal segment than in the distal segment, reflecting greater values of apical (Ga) and paracellular shunt (Gs) conductances in the proximal segment; in contrast, in human colon, Gt and its individual membrane components are similar in the proximal and distal segments, and lower than the corresponding values in rat colon; (2) amiloride sensitive apical Na conductances are absent in rat proximal colon, rat distal colon, and human proximal colon, but in human distal colon amiloride produces changes consistent with blockade of an apical Na conductance and inhibition of electrogenic Na transport; (3) a TEA-sensitive apical K conductance may be present in rat proximal colon (a K secretory epithelium), but not in rat distal colon (a K absorptive epithelium) or in either segment of human colon; and (4) in rat colon, replacement of mucosal and serosal Cl produces changes consistent with a substantial paracellular shunt permeability to Cl which is more marked in the proximal segment, whereas in human colon Cl replacement results in changes which suggest a relatively small paracellular shunt permeability to Cl which is similar in both segments. These data indicate marked segmental differences in Na, K and Cl transport in rat and human colon, and emphasise the hazards of applying models of colonic electrolyte transport in one species to another.

Ion transport in human colon in vitro

Ion transport in the human colon was studied in vitro under short-circuit conditions. The proximal, transverse, and distal colon all actively absorbed Na and Cl at similar rates. Tissue conductance was lower in proximal colon, but there were no other regional differences in basal electrophysiologic parameters. There was a gradient of amiloride-sensitive electrogenic Na transport. Whereas amiloride had only a minimal effect in proximal colon, it inhibited 70% of short-circuit current and 50% of net Na absorption in distal colon. Ion substitution experiments demonstrated an electroneutral, coupled Na-Cl cotransport system in proximal and distal colon. Neither amphotericin nor impermeant anions had a consistent stimulatory effect on short-circuit current in human colon. Theophylline (10−3M), increased short-circuit current by 4 μEq · cm−2 · h−1, stimulated net Cl secretion, but did not block net Na absorption. Epinephrine, via an α2-adrenergic mechanism, significantly decreased short-circuit current but did not alter Na or Cl transport. These results suggest that (a) all segments of human colon actively absorb Na and Cl, (b) Na absorption occurs by both electrogenic Na absorption and electroneutral Na-Cl cotransport, (c) there is an aboral gradient of increasing electrogenic Na transport, (d) theophylline stimulates secretion in a pattern most consistent with electrogenic Cl secretion, and (e) epinephrine does not increase Na-Cl cotransport in human distal colon. These studies demonstrate that human colon in vitro has distinct transport properties that must be considered both in clinical situations and in comparison to animal models.

Retinoic acid-binding protein in experimental and human colon tumors

Retinoic acid-binding protein is present in metastatic murine colon tumors as well as in Lewis lung tumors and in lungs and brains of mice bearing these tumors; however, this protein is below the limits of detection in weakly-metastatic carcinomas and in normal lung, colon, or brains. These observations are interesting since they concern the possibility of measuring the binding protein levels of colon tumors in clinical specimens as biochemical markers in human malignancy. A total of thirty-three human colon tumors and related materials were analyzed for the presence of the binding protein. The interfering serum albumin, which nonspecifically binds retinoic acid, was eliminated by affinity chromatography. Of the twenty colon, cecum, and rectum tumors analyzed, 80% contained the binding protein in detectable amounts, and 20% showed nondetectable or marginally detectable amounts. Twenty-two percent of the human colon segments isolated from patients suspected for colon tumors contained the binding protein in readily detectable amounts, whereas 78% revealed nondetectable to marginally detectable amounts. The retinoic acid-binding protein of human colon tumor shared the same ligand specificity, thiol functions in ligand-binding, and sedimentation coefficient as the binding protein isolated from chick embryo skin. However, the human protein exhibited altered isoelectric pH.

Synthesis of the first component of human complement in vitro.

Isolated segments of human colon and to a lesser extent ileum were capable of synthesizing hemolytically active C'1. This conclusion was based on the following evidence: After elimination of C'1 from tissue with EDTA, we found that segments of the intestinal tract in short-term organ culture showed a 50-1000-fold increase in C'1 activity. The rate of production of C'1 in human intestine was highly temperature dependent; C'1 production was reversibly inhibited by puromycin and actinomycin D. Furthermore, (14)C-labeled amino acids were incorporated into molecules which behaved like C'1. No significant C'1(hu) synthesis was observed in isolated segments of jejunum, stomach, liver, kidney, lung, spleen, lymph node, and thymus.

Abstracts of Papers Presented at the Third International Complement Workshop, Harvard Medical School, Boston, Massachusetts, June 3–5, 1968

Abstract We have presented evidence that the first component of guinea pig complement (C′1gp) is synthesized by columnar epithelial cells of the small intestine. We now report that isolated segments of human colon and to a lesser extent ileum are capable of synthesizing hemolytically active C′1hu. This conclusion is based on the following evidence: After elimination of C′1hu from tissue with EDTA, we found that segments of the intestinal tract in short-term organ culture showed a 100- to 1000-fold increase in C′1hu activity. The rate of production of C′1hu in human intestine was highly temperature dependent; C′1hu production was reversibly inhibited by puromycin and actinomycin D. Furthermore, 14C-labeled amino acids were incorporated into molecules which behaved like C′1. No significant C′1hu synthesis was observed in isolated segments of jejunum, stomach, liver, kidney, lung, spleen or lymph node.