Circadian expression of clock- and tumor suppressor genes in human oral mucosa.

PURPOSE: Circadian rhythms are daily oscillations of multiple biological processes driven by endogenous clocks. Imbalance of these rhythms has been associated with cancerogenesis in humans. To further elucidate the role circadian clocks have in cellular growth control, tumor suppression and cancer treatment, it is revealing to know how clock genes and clock-controlled genes are regulated in healthy humans. MATERIALS AND METHODS: Therefore comparative microarray analyses were conducted investigating the relative mRNA expression of clock genes throughout a 24-hour period in cell samples obtained from oral mucosa of eight healthy diurnally active male study participants. Differentially expressed selected genes of interest were additionally evaluated using qRT-PCR. RESULTS: Microarray analysis revealed 33 significant differentially regulated clock genes and clock- controlled genes, throughout a one day period (6.00h, 12.00h, 18.00h, 24.00h). Hereof were 16 clock genes and 17 clock- controlled genes including tumor suppressor- and oncogenes. qRT-PCR of selected genes of interest, such as hPER2, hCRY1, hBMAL1, hCCRN4L and hSMAD5 revealed significant circadian regulations. CONCLUSION: Our study revealed a proper circadian regulation profile of several clock- and tumor suppressor genes at defined points in time in the participants studied. These findings could provide important information regarding genes displaying the same expression profile in the gastrointestinal tract amounting to a physiological expression profile of healthy humans. In the future asynchronous regulations of those genes might be an additional assistant method to detect derivations distinguishing normal from malignant tissue or assessing risk factors for cancer.

Extrinsic afferent nerve sensitivity and enteric neurotransmission in murine jejunum in vitro.

Enteric and extrinsic sensory neurons respond to similar stimuli. Thus they may be activated in series or in parallel. Because signal transmission via synapses or mediator release would depend on calcium, we investigated its role for extrinsic afferent sensitivity to chemical and mechanical stimulation. Extracellular multiunit afferent recordings were made in vitro from paravascular nerve bundles supplying the mouse jejunum. Intraluminal pressure and afferent nerve responses were recorded under control conditions and under four conditions designed to interfere with enteric neurotransmission. We found that phasic intestinal contractions ceased after switching perfusion to Ca(2+)-free buffer with or without a purinergic P2 receptor antagonist, pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) or cadmium (blocking all Ca(2+)-channels) but not following omega-conotoxin GVIA (N-type Ca(2+)-channel blocker). Luminal HCl (pH 2) and 5-HT (500 microM) evoked peak firing of 17 +/- 4 impulses per second (imp/s) (n = 10) and 21 +/- 4 imp/s (n = 13) under control conditions. These responses were reduced to 4 +/- 2 imp/s and 5 +/- 2 imp/s by cadmium (n = 7, P < 0.05), to 7 +/- 2 imp/s and 6 +/- 1 imp/s by Ca(2+)-free perfusion (n = 6, P < 0.05), and to 3 +/- 1 imp/s and 4 +/- 1 imp/s by Ca(2+)-free perfusion with PPADS (n = 6, P < 0.05). Responses were unchanged by omega-conotoxin GVIA. Mechanical ramp distension of the intestinal segment to 60 cmH(2)O was not altered by any of the experimental conditions. We concluded that HCl and 5-HT activate extrinsic afferents via a calcium-dependent mechanism, which is unlikely to involve enteric neurons carrying N-type calcium channels. Extrinsic mechanosensitivity is independent of enteric neurotransmission. It appears that cross talk from the enteric to the extrinsic nervous system does not mediate extrinsic afferent sensitivity.

Cholecystokinin-58 is more potent in inhibiting food intake than cholecystokinin-8 in rats.

INTRODUCTION: Several cholecystokinin (CCK) forms have been detected in plasma, but most studies on food intake investigated the effects of CCK-8 only. Recently, it has been demonstrated that CCK-58 is the only endocrine-active form of CCK in rats. METHODS: CCK-58 was synthesized with a peptide synthesizer using FMOC chemistry and CCK-58 effects on food intake were compared to CCK-8 in rats. RESULTS: Both CCK-58 and CCK-8 inhibited food intake in a dose-dependent manner and were equally potent at 30 min. CCK-58 showed a prolonged inhibition of food intake compared to CCK8 at the higher dose tested (7 nmol/kg), inhibiting food intake also at 60 min, and cumulative food intake was inhibited for up to 210 min by CCK-58. CONCLUSIONS: CCK-58 has the same potency in inhibiting food intake as CCK-8 in rats, but inhibits food intake longer. This might be due to its tertiary structure resulting in a delayed plasma degradation or a prolonged binding at the CCK receptor. As CCK-58 is the major CCK form in the gut wall and possibly in the circulating blood in humans, the effects of CCK on food intake might have been underestimated in the past.

Differential sensitization of afferent neuronal pathways during postoperative ileus in the mouse jejunum.

BACKGROUND: Postoperative ileus induces reflex inhibition of gastrointestinal motility and an intestinal inflammatory response. We aimed to determine whether afferent sensitivity is increased during postoperative ileus and whether alterations are cyclooxygenase-2 (COX-2)-dependent. METHODS: C57BL/6 mice underwent laparotomy followed by standardized small bowel manipulation to induce ileus or sham treatment. After 24 hours, extracellular multiunit mesenteric afferent nerve discharge was recorded in vitro from 2-cm segments of jejunum. Fos immunoreactivity was determined for neuronal activation in the vagal nucleus of the solitary tract (nTS) of the brain stem and leukocyte infiltration in the intestinal muscularis by myeloperoxidase stains. RESULTS: Serosal bradykinin (1 microM) was followed by an increase in afferent discharge to 65 +/- 5 imp x s(-1) in ileus segments compared with 37 +/- 6 imp x s(-1) in sham controls (P < 0.05). The response was attenuated to 31 +/- 7 imp x s(-1) after the selective COX-2 inhibitor 5,5-dimethyl-3-(flurorophenyl)-4-(4-methylsulfonyl) phenyl-2(5H)-furanone (DFU) in ileus segments. Afferent firing during ileus was augmented at luminal distension at 20 mm Hg but not at pressures up to 60 mm Hg. The number of Fos-positive neurons in the nTS was 110 +/- 45 during ileus compared with 7 +/- 4 in sham controls (-7.32 mm from bregma, P < 0.05) and did not differ after DFU. The intestinal muscularis contained more leukocytes during ileus compared with ileus segments after DFU and controls (both P < 0.05). CONCLUSION: This study provides direct evidence from afferent nerve recordings that sensitivity to bradykinin, which stimulates predominantly spinal afferents, is augmented during postoperative ileus involving a COX-2 pathway. Vagal afferents were also sensitized because low-threshold mechanosensitivity and neuronal activation in the nTS were increased.

Postoperative complications have little influence on long-term quality of life in Crohn's patients.

PURPOSE: The purpose of the study was to determine the influence of postoperative complications on long-term quality of life in patients after abdominal operations for Crohn's disease. MATERIALS AND METHODS: From 1996 to 2002, 305 Crohn's patients underwent abdominal surgery, and 66 patients developed postoperative complications. Quality of life was studied using a standardized questionnaire and four quality of life instruments. Sixty-six Crohn's patients with uneventful postoperative course matched for age, and follow-up time served as controls. RESULTS: Forty-eight patients (81%) in the complication group (32 major and 16 minor) and 43 patients (75%) in the control group answered the questionnaire. Postoperative follow-up time was 42 (10-94) and 41 months (13-94; median (range)). Quality of life was comparable between groups, except on the subscale "physical functioning" of the Short-form 36 on which patients with minor and major complications showed impaired quality of life compared to controls (67+/-6, 69+/-4, and 84+/-2%; mean+/-standard error of the mean; both p<0.05 vs controls). The incidence of Crohn's disease-related symptoms at follow-up was unaffected by complications (minor 63%, major 56% vs controls 70%; both not significant). CONCLUSION: Postoperative complications after abdominal operations for Crohn's disease do not impair long-term quality of life in general but may affect specific dimensions of quality of life like patients' physical function.

Long-term quality of life in patients with Crohn's disease and perianal fistulas: influence of fecal diversion.

PURPOSE: Symptomatic perianal fistulas impair quality of life in patients with Crohn's disease. Fecal diversion improves symptoms but may impair quality of life. This study was designed to compare long-term quality of life in patients with Crohn's disease with symptomatic perianal fistulas who were treated with or without fecal diversion. METHODS: From 1996 to 2002, perianal fistulas were treated in 116 patients with Crohn's disease. A questionnaire, including four quality of life instruments, was mailed to each patient (Short-Form General Health Survey, Gastrointestinal Quality of Life Index, Cleveland Global Quality of Life Score, Short Inflammatory Bowel Disease Questionnaire). RESULTS: Questionnaires were returned by 77 of 116 patients (66 percent). Thirty-four of these patients had undergone fecal diversion, whereas 43 had not. Median follow-up was 49 (range, 18-97) months in diverted and 44 (range, 14-98) months in undiverted patients (not significant). In the diverted group, 44 percent complained of Crohn's disease-related symptoms, which was less compared with 79 percent in undiverted patients (P < 0.05). Diverted patients achieved 68 +/- 1 percent of the maximum possible score on the Gastrointestinal Quality of Life Index compared with 60 +/- 2 percent in undiverted patients (mean +/- standard error of the mean; P < 0.001); diverted patients scored better on the subscale "gastrointestinal symptoms" of the Gastrointestinal Quality of Life Index (81 +/- 1 percent vs. 67 +/- 2 percent; P < 0.001). There was no difference in the Short Inflammatory Bowel Disease Questionnaire between diverted and undiverted patients except for the subscale "bowel function" (91 +/- 2 percent vs. 76 +/- 2 percent; P < 0.0001). No difference in quality of life was detected by the Short-Form General Health Survey and Cleveland Global Quality of Life Score. CONCLUSIONS: In the investigated population of patients with Crohn's disease, quality of life seems to be similar or potentially superior in diverted patients suffering from perianal fistulas compared with undiverted patients. A diverting stoma, therefore, may improve quality of life in patients with severe perianal Crohn's disease.

Enteral immunonutrition during sepsis prevents pulmonary dysfunction in a rat model.

BACKGROUND: Sepsis often results in severe pulmonary dysfunction. Via the thoracic duct, the lung is the first organ exposed to gut-derived inflammatory mediators released into mesenteric lymph during sepsis. AIM: To investigate whether an enteral immunonutrition during sepsis improves pulmonary function. METHODS: Mesenteric lymph was obtained from lymph fistula donor rats after intra peritoneal (i.p.) saline (control lymph) or lipopolysaccharide (sepsis lymph) injection. Sepsis lymph was also collected during enteral immunonutrition with omega-3 enriched, long-chain fatty acids (SMOF lipid). Control, sepsis, or sepsis-SMOF lymph was reinfused into the jugular vein of separate recipient rats. The lungs were then harvested, stained with hematoxylin-eosin, and analyzed for: (1) perpendicular parenchyma thickness of the alveolar wall; (2) myeloperoxidase-positive cells; and (3) terminal deoxynucleotidyl transferase Biotin-dUTP nick end labeling (TUNEL)-positive cells. RESULTS: Enteral immunonutrition during sepsis reduced the release of TNFalpha into mesenteric lymph by about 4.5-fold within the first 2 h. Infusion of sepsis lymph into recipient rats induced thickening of alveolar walls, inflammatory reaction, and apoptosis. Infusion of sepsis lymph obtained during enteral immunonutrition did not cause anatomical changes, induced only a mild inflammatory reaction, and prevented apoptosis in the lungs of recipient rats. CONCLUSIONS: Mediators in sepsis lymph induce pulmonary dysfunction such as an increased distance for oxygen transport, inflammatory reaction, and apoptosis. The lung may be protected by an enteral immunonutrition containing long-chain fatty acids.

Olive oil is more potent than fish oil to reduce septic pulmonary dysfunctions in rats.

BACKGROUND: Abdominal sepsis is frequently the cause of severe pulmonary dysfunction. Via the thoracic duct, the lung is the first organ exposed to gut-derived mediators released into the mesenteric lymph. AIM: The aim of this study is to investigate whether an enteral immunonutrition with long chain triglycerides prevents septic pulmonary dysfunctions. MATERIALS AND METHODS: Mesenteric lymph was obtained from lymph fistula donor rats during sepsis (lipopolysaccharides [LPS], 5 mg/kg i.p.) with or without enteral immunonutrition (1% of olive oil or 1% of fish oil). Sepsis lymph was then reinfused into the jugular vein of separate recipient rats. Thereafter, the lung tissue was analyzed for the distance of oxygen diffusion, inflammatory response, and cell apoptosis. RESULTS: Sepsis significantly increased TNFalpha release into the mesenteric lymph, whereas an enteral immunonutrition with olive oil significantly reduced the TNFalpha release into the mesenteric lymph by more than five-fold. Sepsis lymph induced a significant increase in alveolar wall thickness, inflammatory reaction, and apoptosis; whereas sepsis lymph collected during olive oil resorption prevented the thickening of the alveolar walls and induced only a mild inflammation, being more potent than fish oil to reduce septic pulmonary dysfunction. CONCLUSIONS: Mediators in the sepsis lymph induce pulmonary dysfunction. The lung may be protected by an enteral immunonutrition containing long chain triglycerides such as olive oil.

Postoperative colonic motility after tropisetron and a standardized meal in patients undergoing conventional colorectal surgery.

BACKGROUND: Early postoperative enteral nutrition is advantageous for the recovery of colonic motility but may be limited by abdominal distension, nausea, and vomiting. We aimed to investigate the tolerance of a standardized meal after pretreatment with the 5-hydroxytryptamine-3-receptor antagonist tropisetron and to study the concomitant colonic motility. METHODS: Colonic motility and tone were recorded on postoperative day 1 to 3 with a combined manometry/barostat recording catheter in 12 patients who underwent open colorectal surgery with an anastomosis in the distal colon or rectum. The study protocol consisted of 30 min of baseline recordings followed by 5 mg of tropisetron intravenously. Then, motility was recorded for another 30 min before patients ingested a standardized meal to trigger the gastrocolonic response. Postprandial motility was recorded for the subsequent 60 min. RESULTS: The colonic motility index increased after administration of tropisetron on all three postoperative days (day 1: 34+/-11 vs 122+/-48, day 2: 55+/-19 vs 101+/-25, and day 3: 42+/-16 vs 93+/-33 mmHg/min; p<0.05). No further increase of the motility index was observed postprandially. Frequency and amplitude of contractions were virtually unaffected by tropisetron and the meal. Barostat bag volume decreased postprandially in the proximal bag on the third, and in the distal bag on the first and second postoperative day (p<0.05). Patients' condition was unaffected by the standardized meal after tropisetron administration. CONCLUSIONS: Tropisetron may enhance colonic motility in the early postoperative period; however, the gastrocolonic response was impaired thereafter. High caloric food intake is well tolerated early after surgery after tropisetron pretreatment.

Systemic capsaicin inhibits neuronal activation in the brainstem during postoperative ileus in the mouse.

INTRODUCTION: Neuronal inhibitory reflex mechanisms contribute to postoperative ileus after abdominal surgery. During this condition, sensory neurons in the brainstem are activated. We aimed to determine the contribution of capsaicin-sensitive afferents to central vagal sensitivity in mice during postoperative ileus. MATERIALS AND METHODS: Under enflurane anesthesia, C57BL/6 mice were laparotomized and the small bowel was manipulated to induce ileus or was left untouched as a sham-treatment group. A subgroup of ileus animals was pre-treated with Capsaicin (1 microm/kg, i.p.) 48 h before small bowel manipulation. The animals were killed 24 h later and the brainstem was removed for Fos immunohistochemistry, which was quantified in the nucleus of the solitary tract (nTS). Spontaneous jejunal motility was recorded in vitro. Leukocyte infiltration in the intestinal muscularis was studied by myeloperoxidase staining as an index of postoperative inflammation. RESULTS: There were 30+/-9 Fos-positive neurons counted in the nTS after ileus and 6+/-2 in sham controls (Bregma -7.70 mm, P=0.01). A reduction to 8+/-3 was observed after Capsaicin pre-treatment in ileus animals (P<0.05). Peak amplitudes of spontaneous jejunal motility were 2+/-0.3 cmH2O during postoperative ileus, 3+/-0.6 cmH2O after ileus with capsaicin pre-treatment, and 10+/-2 cmH2O in control animals (N=6, both P<0.05). The number of leukocytes infiltrating the muscularis was 39+/-9/mm2 during ileus and 1.8+/-1/mm2 in controls (mean+/-SEM, P<0.01, N=6). After capsaicin, this number increased to 72+/-28/mm2 in ileus animals (P<0.05 vs control animals, N=7). CONCLUSION: The inhibition of capsaicin-sensitive vagal afferent pathways appears to boost rather than to attenuate the inflammatory response during postoperative ileus, while intestinal motility remained unchanged. This suggests a protective role of the capsaicin-sensitive afferent innervation for the inflammatory phase of postoperative ileus.