Phylogeny, processing and expression of the rat cathelicidin rCRAMP: a model for innate antimicrobial peptides.

A database search identified a rat cDNA clone which phylogenetic analysis revealed to encode a cathelicidin most similar to mouse cathelicidin CRAMP. The analysis also showed that the evolutionary pattern of the cathelicidin family is lineage specific. The rat cathelicidin is called rCRAMP. Its peptide was isolated from granulocytes, and determined to be 43 amino acids long by mass spectrometry and N-terminal sequencing. Synthetic rCRAMP had antimicrobial activity. The expression of rCRAMP was investigated by reverse-transcriptase polymerase chain reaction followed by Southern hybridization and by Western blot analysis. rCRAMP was identified in granulocytes, thymus, testis, lung, mouth mucosa, tongue, oesophagus, colon, caecum and small intestine, a distribution similar to cathelicidins of mouse and human. The rat is a small laboratory animal with additional disease models available compared to the mouse. Our results open up the possibility to use the rat as a model system to study responses connected to cathelicidin expression in health and disease.

Outcome of primary and secondary ileal pouch-anal anastomosis and ileorectal anastomosis in patients with familial adenomatous polyposis.

PURPOSE: The aim of this study was to present Swedish experiences of the ileal pouch-anal anastomosis in patients with familial adenomatous polyposis from the introduction in 1984. The study also compared the surgical and functional outcome of different anal continence preserving procedures: ileal pouch-anal anastomosis as primary surgery, ileal pouch-anal anastomosis as secondary surgery after colectomy and ileorectal anastomosis, and ileorectal anastomosis alone. METHODS: The material comprises all 120 patients with familial adenomatous polyposis reported to the Swedish Polyposis Registry who had undergone prophylactic colorectal surgery, including those operated on because of colorectal cancer from 1984 until the end of 1996. Anal continence preserving surgery was performed on 102 patients: 20 had ileal pouch-anal anastomosis as primary surgery at a median age of 24.5 years, 39 had ileal pouch-anal anastomosis as secondary surgery at a median age of 34 years, and 43 had ileorectal anastomosis alone, at a median age of 26 years, because 6 of the initially ileorectal anastomosis-operated patients were converted to ileal pouch-anal anastomosis as secondary surgery. Surgical outcome was assessed on the basis of hospital records. A questionnaire was used to evaluate the functional outcome. Fisher's exact probability test was used for statistical analysis. RESULTS: Complications occurred in 51 percent of the patients after ileal pouch-anal anastomosis: 40 percent after ileal pouch-anal anastomosis as primary surgery and 56 percent after ileal pouch-anal anastomosis as secondary surgery. When the previous ileorectal anastomosis was taken into account 67 percent of the patients suffered complications which was significantly more compared with ileal pouch-anal anastomosis as primary surgery. After ileorectal anastomosis, 26 percent had complications which was significantly less compared with all other procedures but ileal pouch-anal anastomosis as primary surgery. No cancer occurred after ileal pouch-anal anastomosis, either in the ileal pouch or in retained rectal mucosa, but two of the patients who had an ileorectal anastomosis developed rectal cancer. One pouch excision was performed compared with ten rectal excisions. Functional outcome did not differ between ileal pouch-anal anastomosis as primary surgery and ileal pouch-anal anastomosis as secondary surgery. However, ileorectal anastomosis-operated patients had significantly better bowel function with regard to nighttime stool frequency, continence and perianal soreness. CONCLUSION: These findings indicate that major advantages of ileal pouch-anal anastomosis are the low excision rate and, so far, no cancer in the ileal pouch. Moreover, the surgical outcome of ileal pouch-anal anastomosis as primary surgery is not significantly different from that of ileorectal anastomosis. However, the good surgical and functional outcome of ileorectal anastomosis, despite the long-range prognosis including rectal cancer and excision risks, has to be taken into consideration when selecting patients with familial adenomatous polyposis for primary surgery.

Local short-chain fatty acids supplementation without beneficial effect on inflammation in excluded rectum.

BACKGROUND: Rectal instillation of short-chain fatty acids (SCFA), important nutrients for the colorectal mucosa, has been suggested to be of therapeutic value in distal intestinal inflammation. METHODS: In this study nine patients with Hartmann-closed rectum after colectomy for acute colitis were investigated. In a double-blind crossover trial an enema containing SCFA or a placebo solution was administered twice daily for 3 weeks. Before entry into the protocol, after each treatment period, and 6 weeks after the study period the patients' symptoms were evaluated, rectal endoscopy was performed, histologic samples were scored, and microbiologic analyses were carried out. RESULTS: No significant differences in symptoms, in mucosal inflammation, in histologic scoring, or in microbiologic studies were found between SCFA and placebo periods. Unexpectedly, all but one patient entirely lacked coliform bacteria in the rectum. CONCLUSIONS: In this study SCFA enemas had no beneficial effect on inflammation in excluded rectum in patients earlier submitted to colectomy for colitis. However, a different rectal flora was detected in these patients.

Pelvic reservoir with S-pouch: can surgeons evaluate the results of their own operations?

PURPOSE: This study was designed to evaluate whether operating surgeons could follow up the functional outcome of their own operations, without bias, by using standardized methods at follow-up. METHODS: Fifty-five patients who received a pelvic reservoir with an S-pouch were evaluated regarding functional outcome after at least one year postoperative follow-up. The functional surgical outcome was evaluated by an internist especially trained in gastroenterology and the operating surgeon by using a standardized scale comprising eight functional variables. RESULTS: None of the variables analyzed reached statistically significant difference between the two observers, and a high degree of agreement could be shown by using kappa and weighted kappa analysis. CONCLUSIONS: Our results indicate that it is possible for an operating surgeon to assess the postoperative surgical outcome using standardized methods at follow-up.

Lack of neurohumoral response to pneumoperitoneum for laparoscopic cholecystectomy.

BACKGROUND: Pneumoperitoneum (PP) for laparoscopic surgery induces prompt changes in circulatory parameters. The rapid onset of these changes suggests a reflex origin, and the present study was undertaken to evaluate whether release of vasopressor substances could be responsible for these alterations. The influence of two different anesthesia techniques was also evaluated. METHODS: American Society of Anesthesiologists (ASA) class I patients, scheduled for laparoscopic cholecystectomy, were investigated. The first group (n = 10) was anesthetized intravenously. The second group (n = 6) had inhalation anesthesia. Plasma vasopressin, catecholamines, and plasma renin activity were investigated as neurohumoral vasopressor markers of circulatory stress. The general stress response to surgery was assessed by analysis of plasma cortisol. RESULTS: Induction of pneumoperitoneum caused no apparent activation of vasopressor substances, although several hemodynamic parameters responded promptly. CONCLUSION: The hemodynamic alterations, seen at the establishment of PP during stable anesthesia, cannot be explained by elevation of vasopressor substances in circulating blood.

Bulking fibre prevents translocation to mesenteric lymph nodes of an efficiently translocating Escherichia coli strain in rats.

BACKGROUND: starvation for 24 h prior to experimental haemorrhage increases bacterial translocation in rats. Forty-eight hours starvation alone causes pronounced microbiological changes in caecal contents and a marked increase in bacterial adherence to caecal epithelium. The aim of the present study was to examine whether bulking fibre prevents these microbiological changes induced by starvation, i.e. mucosal adherence and/or bacterial translocation with and without haemorrhage in rats. METHODS: 32 rats were inoculated with the translocating Escherichia coli strain Kl-C1. Groups of these rats were then starved for 48 h with or without access to bulking fibre. An additional group of rats was given bulking fibre and subjected to haemorrhage. A control group was untreated and given regular food. Samples were taken from caecal contents, caecal epithelium, mesenteric lymph nodes and blood. A biochemical fingerprinting method was used to characterize and compare E. coli strains in all samples. RESULTS: ingestion of bulking fibre alone for 48 h significantly reduced the frequency of Kl-C1 both in caecal contents and on caecal epithelium and completely prevented translocation of the strain, compared to starvation without bulking fibre for 48 h. Enforced stress (haemorrhage) increased bacterial translocation to the same level as starvation for 48 h. E. coli phenotypes found in mesenteric lymph nodes were also found adhering to the caecal epithelium. CONCLUSIONS: The presence of bulking fibre in gut lumen, by unknown mechanisms, reduces the frequency of an inoculated translocating strain of E. coli in caecal contents and on caecal epithelium and prevents its translocation to mesenteric lymph nodes.

Carbachol induces oscillations in membrane potential and intracellular calcium in a colonic tumor cell line, HT-29.

The patch-clamp technique was used to study the effects of carbachol (CCh) on HT-29 cells. During CCh exposure, the cells (n = 23) depolarized close to the equilibrium potential for Cl- (E(Cl-); -48 mV) and the membrane potential then started to oscillate (16/23 cells). In voltage-clamp experiments, similar oscillations in whole cell currents could be demonstrated. The whole cell conductance increased from 225 +/- 25 pS in control solution to 6,728 +/- 1,165 pS (means +/- SE, n = 17). In substitution experiments (22 mM Cl- in bath solution, E(Cl-) = 0 mV), the reversal potential changed from -41.6 +/- 2.2 mV (means +/- SE, n = 9) to -3.2 +/- 2.0 mV (means +/- SE, n = 7). When the cells were loaded with the calcium-sensitive fluorescent dye, fluo 3, and simultaneously patch clamped, CCh caused a synchronous oscillating pattern of fluorescence and membrane potential. In cell-attached patches, the CCh-activated currents reversed at a relative membrane potential of 1.9 +/- 3.7 mV (means +/- SE, n = 11) with control solution in the pipette and at 46.2 +/- 5.3 mV (means +/- SE, n = 10) with a 15 mM Cl- solution in the pipette. High K+ (144 mM) did not change the reversal potential significantly (P < or = 0.05, n = 8). In inside-out patches, calcium-dependent Cl- channels could be demonstrated with a conductance of 19 pS (n = 7). It is concluded that CCh causes oscillations in membrane potential that involve calcium-dependent Cl- channels and a K+ permeability.

Selective translocation of coliform bacteria adhering to caecal epithelium of rats during catabolic stress.

Adult conventional rats were starved for 48 h with or without haemorrhage at 24 h, and translocation of caecal coliforms to mesenteric lymph nodes (MLNs) was measured. Translocation was detected in three of 11 rats without haemorrhage, in 6 of 11 starved and sham-operated rats and in 12 of 22 rats after haemorrhage. In contrast, only one of 13 non-instrumented and fed control rats showed translocation. Translocation was associated with more coliforms adhering to caecal epithelium in rats. Coliform isolates from caecum, caecal epithelium and MLNs were characterised and grouped into different biochemical phenotypes (BPTs) by a biochemical fingerprinting method. Of 291 BPTs detected in the caecum of all rats, 108 were also found on caecal epithelium; 36 BPTs were detected in MLNs, of which 17 were not detected either in the caecum or on the caecal epithelium of the corresponding rats. One isolate from each of these 36 BPTs was selected and compared to the others. Four common (C) BPTs (i.e., C1-C4) were identified among them. Strains of C1 formed the majority of isolates from the caecum (79%), caecal epithelium (71%) and MLNs (91%). In contrast, C2-C4 had a significantly lower incidence both in the caecum and on the caecal epithelium, but not in the MLNs. These findings indicate that not all caecal coliforms adhere to the epithelium during catabolic stress and that for translocation to occur, other bacterial properties besides adhesion are needed. It is also concluded that coliforms with a low incidence in the caecum can translocate with the same efficiency as those with a high incidence.

Starvation increases the number of coliform bacteria in the caecum and induces bacterial adherence to caecal epithelium in rats.

OBJECTIVE: To investigate the impact of starvation for 24 and 48 h on the number of coliform bacteria in the caecal contents, on the mucosal adherence of coliform bacteria, and on bacterial translocation in rats. DESIGN: Open prospective study. SETTING: University departments of surgery and microbiology, Sweden. MATERIAL: 46 adult male Sprague-Dawley rats. INTERVENTIONS: 19 rats served as controls, and were fed until samples were taken. Six animals were starved for 24 h and another 15 for 48 h, with free access to water, and then anaesthetised before blood, mesenteric lymph nodes (MLN), caecum, and caecal contents were sampled. To verify bacterial translocation in this strain of rats, another six rats underwent controlled haemorrhage for 60 min to reduce the blood pressure to 55 mm Hg mean arterial pressure (MAP). These rats had free access to food and water before haemorrhage but were allowed only water until samples were taken 24 h after haemorrhage. MAIN OUTCOME MEASURES: Presence and number of coliform bacteria in samples taken from caecal contents, caecal epithelium, MLN, and blood. RESULTS: Starvation for 24 h increased the number of coliform bacteria (colony forming units (CFU)/g) in the caecal contents 25-fold (p < 0.05). Starvation for 48 h further increased the number by a factor of 100. The number of coliform bacteria that adhered to the caecal epithelium increased 3,000 times in rats that had been starved for 48 h (p < 0.001). There was no significant difference in translocation (as indicated by cultures from MLN) between rats that had been fed and those that had been starved for 48 h. In 4 of the 6 rats that were bled and then starved for 24 h there were signs of bacterial translocation, which was significantly more than the 1/19 in fed rats (p < 0.05). CONCLUSION: Starvation increases the number of bacteria in the caecal contents and increases bacterial adherence to the caecal epithelium. These changes may contribute to the previously reported increase in bacterial translocation in starved compared wit fed rats that were subjected to stress. The same changes in the gut were observed in animals subjected to haemorrhagic stress in addition to starvation, and in which bacterial translocation was evident.

Enhanced prevalence of T cell receptor V beta 7 gene family expression in human intestine-associated T lymphocytes.

Relative levels of expression of T cell receptor variable (V) beta and joining (J) beta gene segments were determined in T cells derived from intestinal biopsies of healthy mucosal areas, mesenteric lymph nodes and peripheral blood of the same individuals. Samples taken from patients suffering from inflammatory (n = 8) and non-inflammatory (n = 8) bowel diseases were analyzed by semi-quantitative polymerase chain reaction-based methods. In the intestine, fewer (median = 3.5) V beta gene segments constituted more than 50% of the T cell receptor V beta repertoire compared to that of peripheral blood T cells (median = 7, P < 0.001). Interestingly, in all sixteen individuals studied, intestinal T lymphocytes (IL-T) expressed the V beta 7 gene family to a higher degree than did T cells in the paired peripheral blood and mesenteric lymph nodes (P < 0.001). T cell receptor J beta gene segment analyses of V beta 7+ T cells revealed no significant difference in oligoclonality rates between peripheral blood (4/16) and intestine (7/16) (P = 0.46). Hence, overexpression of intestinal TCR V beta 7 message does not seem to be due to oligoclonal expansions in the majority of the samples.

Perineal excision of the rectum.

An intersphincteric and perimuscular approach was employed for perineal excision of the rectum in 33 patients who had previously undergone total colectomy with preservation of the rectum. Laparotomy was avoided in 28 of the 29 patients who had had closure of the rectal stump with ileostomy. Of the four patients (two with ileorectal anastomoses and two with sigmoid mucous fistulas) for whom laparotomy was planned, this was considered to be much less extensive than would otherwise have been required. It is concluded that in patients who have previously undergone total colectomy for inflammatory bowel disease, subsequent perineal excision of the rectal stump without laparotomy is frequently possible.

To dress or not to dress surgical wounds? Patients' attitudes to wound care after major abdominal operations.

OBJECTIVE: To find out what patients' attitudes were to wound care after abdominal operations to ensure that patients' own views were not violated by introducing a new method of wound care. DESIGN: Randomised study. SETTING: University hospital, Sweden. PATIENTS: 68 consecutive patients operated for benign gastrointestinal disease. INTERVENTIONS: Patients were randomised to have their abdominal wounds dressed or exposed. On their last day in hospital their attitudes were assessed by visual analogue score and questionnaire. MAIN OUTCOME MEASURES: Attitudes to wound care. RESULTS: There were no differences between the two groups in any of the variables studied. The visual analogue scores for postoperative pain were higher than had been anticipated. CONCLUSION: Most patients do not mind whether their wounds are covered with a dressing or not after abdominal operations.

Hyperosmotic glucose infusion during hemorrhage does not reduce bacterial translocation in 24 hour-starved rats.

Food deprivation 24 h before stress increases bacterial translocation in hemorrhage. Presently it tested whether hyperosmolality, induced by exogenous glucose infusion to improve plasma refill, prevents or reduces bacterial translocation after experimental hemorrhage in 24 h food-deprived rats. Rats were given an i.v. infusion of either 2 mL of 30% glucose (G) or the same volume of .9% NaCl (C) while simultaneously being submitted to a standardized 60 min hemorrhage period, of moderate or more severe hemorrhage. Blood was not reinfused. Despite development of marked hyperglycemia (p < .001, G vs. C) resulting in significantly greater reductions in packed cell volume (p < .001, G vs. C), bacterial translocation was detected similarly in both groups regardless of whether moderate (10/12-G, 9/12-C) or severe (15/19-G, 15/18-C) hemorrhage was inflicted. It was concluded that hyperglycemic hyperosmolality did not prevent bacterial translocation in these models of hemorrhagic stress in 24 h-starved rats.

Role of bile in regulation of gut motility.

Bile empties into the duodenum not only after a meal but also in the interdigestive state. In man, interdigestive biliary emptying is related to fasting motor activity, the migrating motor complex (MMC), in the stomach and small bowel and generally occurs during phase 2 preceding a gastroduodenal phase 3 activity (activity front). It seems that the main regulatory peptide to initiate phase 3 is motilin. During a period with 13 phase 3 activities of MMC, 18 episodes of gall-bladder emptying and 19 motilin peaks were observed. Such a peak of plasma motilin usually took place 25 +/- 11 min after onset of biliary emptying. In conclusion, data indicate that motilin is released to the circulation by the biliary output and induces phase 3 of MMC. The induced phase 3 propels bile acids along the gut to promote their absorption in the distal intestine. The choleretic action of recycling of bile acids may cause subsequent episodes of biliary emptying with motilin release by the action of the enterohepatic circulation of bile acids. In such a manner the MMC may be withheld as a recycling motility pattern.

Inhibition of the migrating motor complex by duodenal drainage in man.

The effect of varying bile acid output on fasting small intestinal motility was investigated in healthy male volunteers. Biliary output was manipulated by jejunal infusion of isotonic mannitol, which resulted in increased output, and by prolonged drainage of duodenal contents, which resulted in decreased output. Intestinal motility was measured by manometric recordings performed at four levels in the proximal small intestine. A marker dilution technique was used to measure pancreatico-biliary output. There were three experimental groups: duodenal drainage, non-drainage and control. Both duodenal drainage and non-drainage groups underwent jejunal saline infusion, followed by mannitol infusion. The control group did not receive drainage or infusions. In the drainage group, 0.41 (0.13-0.68) activity fronts of the migrating motor complex (MMC) per hour were recorded during saline infusion, but only 0.06 (0-0.19) activity fronts per hour were observed during mannitol infusion. In the non-drainage group, 0.71 (0.61-0.81) activity fronts per hour were observed during saline infusion and 0.50 (0.18-0.82) activity fronts per hour were recorded during mannitol infusion. In the control group, 0.58 (0.33-0.84) activity fronts per hour were recorded during the first 4-h session and 0.58 (0.45-0.71) activity fronts per hour during the second session. There was no difference between the number of activity fronts per hour observed in the control group and those observed in the saline infusion of the drainage group.(ABSTRACT TRUNCATED AT 250 WORDS)

Food deprivation increases bacterial translocation after non-lethal haemorrhage in rats.

OBJECTIVE: To investigate whether brief fasting before the induction of hypotension by non-lethal haemorrhage may induce translocation of enteric bacteria to mesenteric lymph nodes or blood in rats. DESIGN: Laboratory experiment. SETTING: University departments of surgery and microbiology, Sweden. MATERIAL: 39 Male Sprague-Dawley rats. INTERVENTIONS: 20 animals were fasted for 24 hours, all 39 then underwent controlled haemorrhage for 60 minutes that reduced the blood pressure to 55 mm Hg. MAIN OUTCOME MEASURES: Differences in blood loss, blood glucose concentrations, and packed cell volume; and aerobic cultures of mesenteric lymph nodes and blood. RESULTS: Fasted rats (n = 20) lost 2.3% of blood volume compared with 2.8% in fed rats (p < 0.001). Packed cell volume dropped by 11.3% in fasted rats and 16.5% in fed rats (p < 0.001). Glucose concentrations rose by 7.0 mmol/l in fasted rats compared with 21.0 mmol/l in fed rats (p < 0.001). Mesenteric lymph nodes contained enteric bacteria in 14/20 fasted rats compared with 6/19 fed rats (p < 0.05). In 4 fasted rats blood cultures grew pathogenic bacteria compared with no fed rats (p = 0.11). The number of bacteria found in mesenteric lymph nodes was significantly greater in fasted than in fed rats (p = 0.01). CONCLUSIONS: Brief fasting before hypotension caused by non-lethal haemorrhage was associated with significantly increased bacterial translocation compared with fed animals. Increases in blood glucose concentrations and plasma refill may have had a protective effect in fed rats. These experiments may be of clinical relevance as elective operations are usually preceded by overnight fasting.

Glutamine supplementation does not prevent bacterial translocation after non-lethal haemorrhage in rats.

OBJECTIVE: To find out whether supplementation of an enteral diet with glutamine would reduce translocation of bacteria to mesenteric lymph nodes or blood after major haemorrhage in rats. DESIGN: Open randomised study. SETTING: University departments of surgery and microbiology, Sweden. MATERIAL: 49 Sprague-Dawley rats. INTERVENTIONS: Rats were fed enterally for 7 days on diets supplemented with either glutamine or an isonitrogenous amount of non-essential amino acids. After feeding, 8 experimental and 8 control rats underwent sham operation; 9 and 7, respectively, underwent moderate haemorrhage (to 65 mm Hg); and 9 and 8, respectively, underwent severe haemorrhage (50 mm Hg) without reinfusion. MAIN OUTCOME MEASURES: Microbiological analyses of samples of blood and mesenteric lymph nodes taken 24 hours after haemorrhage. RESULTS: The median (interquartile) number of colony forming units/mesenteric lymph nodes after moderate haemorrhage in animals who were given glutamine supplementation was 11 (0-34) and in control animals 20 (0-178). After severe haemorrhage the corresponding figures were 199 (10-310) and 22 (0-187). No pathogens were isolated from blood cultures. CONCLUSION: Glutamine supplementation before haemorrhage did not reduce bacterial translocation to mesenteric lymph nodes in this rat model.