Macrophage and dendritic cell subsets in IBD: ALDH+ cells are reduced in colon tissue of patients with ulcerative colitis regardless of inflammation.

Disruption of the homeostatic balance of intestinal dendritic cells (DCs) and macrophages (MQs) may contribute to inflammatory bowel disease. We characterized DC and MQ populations, including their ability to produce retinoic acid, in clinical material encompassing Crohn's ileitis, Crohn's colitis and ulcerative colitis (UC) as well as mesenteric lymph nodes (MLNs) draining these sites. Increased CD14(+)DR(int) MQs characterized inflamed intestinal mucosa while total CD141(+) or CD1c(+) DCs numbers were unchanged. However, CD103(+) DCs, including CD141(+)CD103(+) and CD1c(+)CD103(+) DCs, were reduced in inflamed intestine. In MLNs, two CD14(-) DC populations were identified: CD11c(int)HLADR(hi) and CD11c(hi)HLADR(int) cells. A marked increase of CD11c(hi)HLADR(int) DC, particularly DR(int)CD1c(+) DCs, characterized MLNs draining inflamed intestine. The fraction of DC and MQ populations expressing aldehyde dehydrogenase (ALDH) activity, reflecting retinoic acid synthesis, in UC colon, both in active disease and remission, were reduced compared to controls and inflamed Crohn's colon. In contrast, no difference in the frequency of ALDH(+) cells among blood precursors was detected between UC patients and non-inflamed controls. This suggests that ALDH activity in myeloid cells in the colon of UC patients, regardless of whether the disease is active or in remission, is influenced by the intestinal environment.

Infliximab inhibits activation and effector functions of peripheral blood T cells in vitro from patients with clinically active ulcerative colitis.

Many patients with inflammatory bowel disease (IBD) are undergoing therapy with infliximab, an antibody specific for TNF. However, the exact mechanisms of action of infliximab are not completely understood. The aim of this study was to determine the in vitro effects of infliximab on blood T cells derived from anti-TNF therapy-naïve ulcerative colitis (UC) patients with clinically active disease. Peripheral blood mononuclear cells were stimulated polyclonally or by antigen in the presence or absence of infliximab. The T cell phenotype was investigated by flow cytometry, cytokine secretion was determined by ELISA, and cell proliferation was determined by thymidine assay or CFSE dye. Presence of infliximab resulted in reduced expression of CD25 in CD4(+) and CD8(+) T cell populations and inhibited secretion of IFN-γ, IL-13, IL-17A, TNF as well as granzyme A. Infliximab also suppressed CD4(+) and CD8(+) T cell proliferation. These effects of infliximab were recorded both in T cells activated by polyclonal and antigen-specific stimulation. The effects of infliximab on T cell apoptosis and induction of FOXP3(+) CD4(+) T regulatory cells were ambiguous and depended on the originating cellular source and/or the stimulation mode and strength. In conclusion, infliximab is able to reduce T cell activation as measured by CD25, proliferation and cytokine secretion in vitro from UC patients with clinically active disease. These data suggest that suppression of T cell activity may be important for infliximab-mediated disease remission in patients with UC.

Ulcerative colitis patients in remission have an altered secretory capacity in the proximal colon despite macroscopically normal mucosa.

BACKGROUND: One of the hallmarks of acute colitis is loss of epithelial transport. For unknown reasons, many patients still suffer from GI symptoms during remission, indicating a sustained imbalance between absorption and secretion. We hypothesize that the colonic epithelium becomes more reactive to secretagogues to compensate for a failing barrier. METHODS: Biopsies from ascending colon and sigmoid colon of UC patients in remission and controls were mounted in Ussing chambers. Membrane current (Im) and epithelial capacitance (Cp) were used as markers for anion secretion and mucus exocytosis. Carbachol (1 mmol L(-1) ) and forskolin (10 µmol L(-1) ) were used to study Ca(2+) and cAMP-mediated secretion. KEY RESULTS: Baseline values showed segmental patterns with higher Im in ascending colon and higher Cp in sigmoid colon of both UC patients and controls, but the patterns did not differ between the groups. The Im response to forskolin was increased (+35%) in the ascending colon of UC patients and the Im response to carbachol was decreased (-40%) in the same segment. No group differences were seen in the distal colon for either the forskolin or carbachol-induced Im responses. The Cp response to carbachol was instead up-regulated in the distal colon of UC patients, but remained unaffected in the proximal colon. CONCLUSIONS & INFERENCES: The proximal colonic mucosa of UC patients in remission seems to shift its reactivity to secretagogues, becoming more sensitive to cAMP-dependent secretion and less sensitive to Ca(2+) -dependent secretion. This phenomenon may contribute to residual diarrhea in this patient group, despite resolution of inflammation.

Meaningful or redundant complexity - mechanisms behind cyclic changes in gastroduodenal pH in the fasting state.

The antroduodenal region is probably the site of the most common chronic infection of mankind, helicobacter-induced antral gastritis. After meals, the remaining gastric contents are evacuated by an interdigestive motor programme, the so-called migrating motor complex (MMC). The most characteristic feature of the MMC is phase III, a series of contractions at slow wave frequency (3 min⁻¹ in the stomach, approx. 12 min⁻¹ in the duodenum). Phase III is associated with complex changes in antroduodenal pH, the most prominent feature being a rapid alkalinization of the antral lumen immediately after the end of antral phase III. Before and during antral phase III (late phase II), gastric acid secretion increases and reflux of bile-containing fluid from the duodenum frequently occurs. At the start of duodenal phase III, the pacemaker driving the motor waves is located proximally in the contracting segment, and the motor waves are uniformly antegrade. After passing the papilla, the pacemaker which is now in the middle of the contracting segment stops its migration and waves passing the papilla hence become retrograde. Bile is diverted into the gall bladder. Duodenal phase III activates electrogenic chloride and bicarbonate secretion and release of secretory IgA. During the second half of phase III, there is accordingly reflux of bile-free fluid, bicarbonate and secretory IgA containing fluid from the duodenum into the stomach. Possible physiological and pathophysiological implications of this complex system, in particular the role of the gastric mucus layer in antral Helicobacter infection, will be discussed.

Indirect evidence for increased mechanosensitivity of jejunal secretomotor neurones in patients with idiopathic bile acid malabsorption.

AIM: The interdigestive motor rhythm, the migrating motor complex (MMC), is accompanied by active secretion of chloride during periods of distally propagating maximal motor activity (MMC phase III). We studied the behaviour of this system in bile acid malabsorption (BAM), a relative common cause of chronic diarrhoea. We measured motor activity and transmucosal potential difference (PD, reflecting active chloride secretion), in the proximal jejunum in healthy controls (n = 18) and in a group of patients with BAM (n = 11). The phase III-generated voltage was related to the degree of BAM quantified by the (75)SeHCAT test. METHODS: We used a multi-channel intestinal infusion system to simultaneously measure jejunal pressure and PD. Saline passing calomel half-cells was infused into the jejunum and subcutaneously. Pressure and PD were recorded in the fasting state and after a test meal. RESULTS: In the absence of motor activity, jejunal PD was not significantly different from zero in either group. During MMC phase III, PD reached significantly higher mean and peak levels in BAM patients. The product of MMC phase III length multiplied by voltage, over 3 h, was also significantly higher in BAM patients (controls: median 307 mV x cm, range 70-398; BAM: median 511, range 274-2271, P < 0.01). This value was also significantly correlated with the degree of BAM as reflected by the (75)SeHCAT test (P < 0.05). CONCLUSION: Phase III induced jejunal secretion may be upregulated in BAM patients, resulting in overload of colonic reabsorption capacity.

B-cell activation in patients with irritable bowel syndrome (IBS).

Patients with irritable bowel syndrome (IBS) may have a low grade immune activation. However, little is known about the properties of B cells of IBS patients. We therefore investigated activation level and antigen presenting phenotype of blood B cells of IBS patients. We also examined B-cell responses to lipopolysaccharide (LPS) and probiotic bacteria. Blood samples were obtained from 74 IBS patients and 30 healthy subjects. Peripheral blood mononuclear cells were isolated and stimulated with LPS or an UV-light inactivated bacterial cocktail consisting of the probiotic Gram-positive strains; Lactobacillus paracasei ssp. paracasei 19, Lactobacillus acidophilus La5, Bifidobacterium lactis B612. The phenotype of CD19(+) B cells was investigated by flow cytometry before and after 72 h cell culture. Furthermore, IBS symptom severity was assessed. B cells isolated from blood of IBS patients displayed an amplified activation level as demonstrated by increased cell surface expression of IgG, and also the costimulatory molecules CD80 and CD86. Expression of antigen presenting HLA-DR and costimulatory molecule CD40 on B cells was, however comparable in IBS patients and controls. B cells of IBS patients displayed an impaired ability to increase expression of CD80, but not CD86, in response to both LPS as well as probiotic bacteria stimulations. To conclude, blood B cells of IBS patients have an increased activation level. Bacterial component induced expression of the costimulatory molecule CD80, regarded as important for tolerance induction, is impaired. These data suggest that B-cell antigen presentation in IBS patients is associated with altered capacity of providing costimulation to T cells.

Elevated motility-related transmucosal potential difference in the upper small intestine in the irritable bowel syndrome.

The pathophysiology of irritable bowel syndrome (IBS) is complex and incompletely known. Very little has been studied regarding the role of submucous neuronal activity. We therefore measured small intestinal transmural potential difference (PD, reflecting mainly electrogenic chloride secretion), and its linkage with fasting motor activity [migrating motor complex (MMC)] in controls (n = 16) and patients with IBS [n = 23, 14 diarrhoea predominant (d-IBS) and nine constipation predominant (c-IBS)]. Transmural-PD and its relation to MMC phase III was measured by modified multilumen manometry for 3 h in the fasting state using one jejunal and one duodenal infusion line as flowing electrodes. The amplitude and duration of motor phase III was similar in controls and IBS patients, but the propagation speed of phase III was higher in IBS patients. In IBS patients, maximal PD during MMC phase III was significantly elevated in both the duodenum and jejunum (P < 0.05) and the PD decline after phase III was significantly prolonged in the jejunum (P < 0.01). The PD elevation was seen in both duodenum and jejunum in d-IBS patients, but only in the jejunum in the c-IBS patients. On the basis of previous modelling studies, we propose that the enhanced secretion may reflect disturbed enteric network behaviour in some patients with IBS.

CD4+CD25+ regulatory T cells in irritable bowel syndrome patients.

The aetiology of the irritable bowel syndrome (IBS) is incompletely understood. A low-grade colonic inflammation is frequently seen, but it is unclear to what extent this phenomenon contributes to the pathophysiology of IBS. CD4(+)CD25(+) regulatory T cells (Treg) are implicated to play an important role in suppressing intestinal inflammation. We, therefore, examined whether the intestinal inflammatory process in IBS patients is the result of an altered function and/or frequency of CD25(+) Treg cells. Patients with IBS (n = 34), fulfilling the Rome II criteria, were compared with controls (n = 26). The suppressive activity of blood CD25(+) Treg cells was determined and the frequency of colonic and blood CD25(+) Treg cells was analysed by flow cytometry. The expression of the Treg marker, FOXP3 mRNA, in colonic biopsies was determined by reverse transcription-polymerase chain reaction. Blood CD25(+) Treg cells from IBS patients suppressed the proliferation of blood CD4(+)CD25(low/-) T cells. Similar frequencies of CD25(+) Treg cells were recorded in mucosa and blood of IBS patients and controls. FOXP3 mRNA was equally expressed in the colonic mucosa of patients with IBS and controls. In conclusion, the low-grade intestinal inflammation recorded in patients with IBS is not associated with an altered function or frequency of CD25(+) Treg cells.

Serotonin and vasoactive intestinal peptide antagonists attenuate rotavirus diarrhoea.

BACKGROUND AND AIMS: The mechanisms underlying intestinal secretion in rotavirus diarrhoea remain to be established. We previously reported that rotavirus evokes intestinal fluid and electrolyte secretion by activation of the enteric nervous system. We now report that antagonists for the 5-hydroxytryptamine 3 receptor (5-HT(3)) and vasoactive intestinal peptide (VIP) receptor, but not antagonists for 5-hydroxytryptamine 4 receptor or the muscarinic receptor, attenuate rotavirus induced diarrhoea. METHODS: Neurotransmitter antagonists were administered to wild-type or neurokinin 1 receptor knockout mice infected with homologous (EDIM) or heterologous (RRV) rotavirus. RESULTS: While RRV infected mice had diarrhoea for 3.3 (0.2) days (95% confidence interval (CI) 3.04-3.56), the 5-HT(3) receptor antagonist (granisetron) and the VIP receptor antagonist (4Cl-D-Phe(6),Leu(17))-VIP both reduced the total number of days of RRV induced diarrhoea to 2.1 (0.3) (95% CI 1.31-2.9) (p<0.01). EDIM infected mice treated with granisetron had a significantly shorter duration of diarrhoea (5.6 (0.4) days) compared with untreated mice (8.0 (0.4) days; p<0.01). Experiments with neurokinin 1 receptor antagonists suggest that this receptor may possibly be involved in the secretory response to rotavirus. On the other hand, rotavirus diarrhoea was not attenuated in the neurokinin 1 receptor knockout mice. CONCLUSIONS: Our results suggest that the neurotransmitters serotonin and VIP are involved in rotavirus diarrhoea; observations that could imply new principles for treatment of this disease with significant global impact.

Segmentation induced by intraluminal fatty acid in isolated guinea-pig duodenum and jejunum.

Small intestinal movements depend on the composition of the chyme with mixing predominating at high nutrient levels and propulsion being prevalent at low nutrient levels. The mechanisms coupling nutrients to motility are unknown. We used computer analysis of video recordings of isolated guinea-pig duodenum, jejunum and ileum to examine movements induced by a fatty acid, decanoic acid. Increasing intraluminal pressure past a threshold using control saline consistently evoked propulsive reflexes: lumen-occluding constrictions appeared at the oral end propagating at 20.4 +/- 2.4 mm s(-1) (mean +/-s.d., jejunum) to the anal end before being repeated until the intraluminal pressure was returned to control. Subthreshold pressure increases sometimes evoked a transient series of constrictions appearing at the oral end and propagating anally at 18.4 +/- 4.7 mm s(-1) (jejunum). At basal pressures, decanoic acid dose-dependently induced motor activity consisting of 40-60 s episodes of constrictions separated by 40-200 s periods of quiescence and lasting up to 2 h. Five contraction patterns were identified within episodes including localized stationary constrictions; constrictions that propagated slowly (5-8 mm s(-1)) for short distances orally or anally; and constrictions that propagated orally or anally for the length of the preparation at 14-20 mm s(-1). Decanoic acid induced motor activity was reversibly abolished by tetrodotoxin (3 microm), hyoscine (1 microm) and hexamethonium (100 microm), but was insensitive to blockade of P2 purinoceptors by PPADS (60 microm). Thus, decanoic acid induces motor activity equivalent to segmentation in guinea-pig small intestine in vitro and this depends on intrinsic neural pathways.

Enteric neurones modulate the colonic permeability response to luminal bile acids in rat colon in vivo.

BACKGROUND: The mechanisms behind microscopic colitis and exacerbations of ulcerative colitis are incompletely understood. It seems highly likely that both luminal antigens and bile are involved. The aim of this study was to test the hypothesis that bile acids increase colonic mucosal permeability by activating enteric neurones. METHOD: The effect of 4 mM deoxycholic acid (DCA) on the appearance rate of intravenously administered (3)H-mannitol and (14)C-urea into the lumen of the proximal and distal rat colon was measured in vivo and expressed as clearance. The nerve blocking agents atropine and hexamethonium were given intravenously, and lidocaine was applied onto the serosal surface of the colon, before and after DCA exposure RESULTS: DCA markedly increased clearance of the permeability probes into the lumen in both colonic segments and also the ratio of mannitol/urea clearance, particularly in the distal colon. Pretreatment with atropine, hexamethonium, and lidocaine significantly inhibited the increase in clearance by approximately 65-80% but did not affect the clearance ratio. In the distal colon, the inhibitory effect of lidocaine was not statistically significant. Also, administration of atropine and hexamethonium after DCA exposure significantly inhibited the DCA effect on clearance of the probes. CONCLUSION: The results suggest that in vivo, the permeability increase induced by a moderate concentration of bile acid is to a large extent mediated by a neural mechanism involving muscarinic and nicotinic receptors. This mechanism may be a link between the central nervous system and colonic mucosal barrier function, and may be a new target for treatment.

Inhibition of nitric oxide synthesis potentiates the colonic permeability increase triggered by luminal bile acids.

AIM: Experiments were performed in anaesthetized rats to clarify the role of nitric oxide (NO) in the control of colonic permeability. METHODS: Colonic luminal pressure, the transmucosal potential difference (PD) and the clearance of [3H] mannitol and [14C] urea from blood to lumen were measured. NO synthesis was blocked with Nomega-nitro-L-arginine (L-NNA) i.v. and mucosal permeability was increased by deoxycholic acid (DCA, 4 mm). The involvement of histamine in the response was studied by giving the histamine H1 receptor blocker pyrilamine. RESULTS: In proximal colon, L-NNA per se increased luminal pressure and PD but had no significant effect on clearance. DCA per se increased luminal pressure, had no significant effect on PD, but increased mannitol and urea clearance and the clearance ratio. L-NNA and pyrilamine both blocked the luminal pressure effect of DCA but L-NNA had no significant effect on the clearance response to DCA. In distal colon, L-NNA per se had no significant effect on pressure and clearance, but increased PD like in proximal colon. DCA had no significant effect on luminal pressure, but markedly reduced PD and increased both clearance and clearance ratio. In this segment, L-NNA significantly potentiated the clearance response to DCA, and further increased clearance ratio to a value not significantly different from unity (1.00 +/- 0.05). CONCLUSION: The data suggest that in vivo, moderate concentrations of bile acids increase colonic permeability in rats via a mechanism that is inhibited by NO in distal but not in proximal colon. In distal colon, NO may contribute to the maintenance of epithelial barrier function.

Computational model of the migrating motor complex of the small intestine.

The migrating motor complex (MMC) is a cyclic motor pattern with several phases enacted over the entire length of the small intestine. This motor pattern is initiated and coordinated by the enteric nervous system and modulated by extrinsic factors. Because in vitro preparations of the MMC do not exist, it has not been possible to determine the intrinsic nerve circuits that manage this motor pattern. We have used computer simulation to explore the possibility that the controlling circuit is the network of AH/Dogiel type II (AH) neurons. The basis of the model is that recurrent connections between AH neurons cause local circuits to enter a high-firing-rate state that provides the maximal motor drive observed in phase III of the MMC. This also drives adjacent segments of the network causing slow migration. Delayed negative feedback within the circuit, provided by activity-dependent synaptic depression, forces the network to return to rest after passage of phase III. The anal direction of propagation is a result of slight anal bias observed in projections of AH neurons. The model relates properties of neurons to properties of the MMC cycle: phase III migration speed is governed by neuron excitability, MMC cycle length is governed by the rate of recovery of synaptic efficacy, and phase III duration is governed by duration of slow excitatory postsynaptic potentials in AH neurons. In addition, the model makes experimental predictions that can be tested using standard techniques.

Quantitative assessment of motility-associated changes in gastric and duodenal luminal pH in humans.

BACKGROUND: Interdigestive pain relieved by food is a common feature of ulcer disease. We tested the hypothesis that the duodenal bulb is intermittently acidified in association with phase III of the interdigestive motility cycle, and tried to quantify the balance between acid and duodenal bicarbonate secretion during this particular period. METHODS: The experiments were performed in Helicobacter-negative healthy volunteers. Gastric and duodenal luminal pH was measured with a triple antimon electrode before, during, and after phase III of the migrating motor complex. Gastric acid secretion rate was measured in real time with a perfusion system and duodenal bicarbonate secretion was estimated from a continuous recording of the transmucosal potential difference (PD) in the duodenal bulb. RESULTS: No significant changes in bulb pH occurred before, during, or after phase III. During the studied time window, the stomach produced 2.24 +/- 0.55 mmol of acid at a peak pH of 1.74 +/- 0.10. Basal HCO3- secretion calculated from bulb PD was 0.82 +/- 0.12 mmol x 30 min(-1) to which was added 0.47 +/- 0.07 mmol of HCO3- during duodenal phase III. The contribution of retroperistalsis-driven HCO3- reflux was small (0.08 +/- 0.02 mmol). CONCLUSIONS: Both the pH recording and the quantitative assessment of secretion rates show that in healthy subjects, fasting gastric acid production and duodenal bicarbonate secretion are of similar magnitude and dynamically coordinated. The mechanism behind the linkage may be reflex activation by motor activity, or a luminal PCO2 rise during phase III activity.

Effects of motility on epithelial transport in the human descending duodenum.

The aim of the study was to determine how motility affects the balance between absorptive and secretory ion transport in the proximal human small intestine. Thirty-two healthy subjects and 16 patients (eight with villus atrophy, eight with normal duodenal biopsies) were studied. The absorptive sodium flux was estimated by measurement of bicarbonate absorption with a double-lumen perfusion technique. The secretory chloride flux was calculated from the ratio between the continuously recorded transmural potential difference (PD) and the epithelial parallel resistance, which was measured in forceps biopsies by square-wave current analysis. Graded variations in contraction frequency were obtained by recording during defined time periods before, during and after phase III of the migrating motor complex (MMC). Bicarbonate was absorbed by a process that led to CO2 formation, and both bicarbonate absorption and luminal PCO2 increased with contraction frequency. The motility-related PCO2 rise was reduced in patients with villus atrophy and by removal of bicarbonate from the perfusate. A higher motor activity was also associated with a larger PD (more lumen negative). Both the absorptive and the secretory fluxes were thus enhanced by motility. The estimated absorptive flux was approximately twice as large as the secretory flux during periods of low motor activity, and four times as large during submaximal motor activity. We conclude that motor activity affects both absorptive and secretory mucosal function in a quantifiable manner. Information about the behaviour of the respective linkage functions may make it possible to model the intestinal absorption process in vivo.

Effects of neural blocking agents on motor activity and secretion in the proximal and distal rat colon: evidence of marked segmental differences in nicotinic receptor activity.

BACKGROUND: Neuromodulation may be a new therapeutic approach in inflammatory bowel disease, but very little is known about neural control of colonic secretion in vivo. We therefore determined the effects of neural blockade on colonic motor activity and mucosal secretion in anaesthetized rats. METHODS: A proximal and a distal colonic segment were isolated in four groups of chloralose-anaesthetized rats (n = 8 in each group), and we measured luminal pressure and transmural potential difference (PD) as a marker of electrogenic chloride secretion. Recordings were made from proximal and distal segments simultaneously, which made it possible to directly compare response patterns. RESULTS: Under control conditions luminal pressure waves were associated with phasic, lumen-negative increases in PD which had a significantly greater magnitude and longer duration in the distal colon. Atropine blocked both pressure waves and PD waves in the proximal colon, but some PD waves, although of lower magnitude, remained in the distal colon. Hexamethonium abolished pressure waves in both segments and induced a marked reduction in PD in the distal but not in the proximal colon. Lidocaine also reduced PD, more so in the distal colon, and dissociated the pressure-PD linkage. CONCLUSION: In the distal but not in the proximal colon, there is a strong nicotinic, neurogenic 'tone' that maintains a high basal secretory activity. The results encourage the search for neuromodulatory agents in the treatment of colonic secretory disease.

Pressure and frequency dependent linkage between motility and epithelial secretion in human proximal small intestine.

BACKGROUND: Motor disturbances are sometimes associated with diarrhoea by unknown mechanisms. AIM: To determine if there is a quantitative link between intestinal motility and epithelial secretion. SUBJECTS: Experiments were performed in 21 healthy volunteers and three patients with villus atrophy. METHODS: Duodenal and jejunal motor activities were registered in the fasted state by open tip manometry. Secretion was measured directly by marker perfusion and indirectly by recording transmural potential difference (PD). RESULTS: A significant correlation was found between "low pass filtered" pressure and PD, but no correlation was found between amplitudes of isolated contractions and PD changes. During repeated phasic contractions (phase III of migrating motor complex), PD increased at a rate that was higher in the duodenum than in the jejunum, and higher in patients with villus atrophy than in healthy controls. After reaching a peak, PD decreased despite continuing phasic motor activity, provided that there was no concomitant increase in mean pressure. Fluid secretion increased roughly in parallel with PD, except at the very end of the cycle. CONCLUSIONS: To explain these findings, one has to postulate participation of at least two types of receptor: a slowly adapting pressure sensitive receptor and another mechanoreceptor, possibly a mucosal touch receptor, to account for the run down phenomenon. This model predicts that short lasting trains of contractions, so called discrete clusters, will be a particularly potent stimulus for activation of mucosal secretion.