Human intestinal epithelial cells promote the differentiation of tolerogenic dendritic cells.

OBJECTIVE: In mice, a subpopulation of gut dendritic cells (DCs) expressing CD103 drives the development of regulatory T (T(reg)) cells. Further, it was recently described that the cross-talk between human intestinal epithelial cells (IECs) and DCs helps in maintaining gut immune homeostasis via the induction of non-inflammatory DCs. In this study, an analysis was carried out to determine whether IECs could promote the differentiation of CD103+ tolerogenic DCs, and the function of primary CD103+ DCs isolated from human mesenteric lymph nodes (MLNs) was evaluated. METHODS: Monocyte-derived DCs (MoDCs) and circulating CD1c+ DCs were conditioned or not with supernatants from Caco-2 cells or IECs isolated from healthy donors or donors with Crohn's disease and analysed for their ability to induce T(reg) cell differentiation. In some cases, transforming growth factor beta (TGFbeta), retinoic acid (RA) or thymic stromal lymphopoietin (TSLP) were neutralised before conditioning. CD103+ and CD103- DCs were sorted by fluorescence-activated cell sorting (FACS) from MLNs and used in T(reg) cell differentiation experiments. RESULTS: It was found that human IECs promoted the differentiation of tolerogenic DCs able to drive the development of adaptive Foxp3+ T(reg) cells. This control was lost in patients with Crohn's disease and paralleled a reduced expression of tolerogenic factors by primary IECs. MoDCs differentiated with RA or IEC supernatant upregulated the expression of CD103. Consistently, human primary CD103+ DCs isolated from MLNs were endowed with the ability to drive T(reg) cell differentiation. This subset of DCs expressed CCR7 and probably represents a lamina propria-derived migratory population. CONCLUSIONS: A population of tolerogenic CD103+ DCs was identified in the human gut that probably differentiate in response to IEC-derived factors and drive T(reg) cell development.

Risk Factors for Small Bowel Obstruction After Laparoscopic Ileal Pouch-Anal Anastomosis for Inflammatory Bowel Disease: A Multivariate Analysis in Four Expert Centres in Europe.

BACKGROUND AND AIMS: Although laparoscopy is associated with a reduction in adhesions, no data are available about the risk factors for small bowel obstruction [SBO] after laparoscopic ileal pouch-anal anastomosis [IPAA]. Our aims here were to identify the risk factors for SBO after laparoscopic IPAA for inflammatory bowel disease [IBD]. METHODS: All consecutive patients undergoing laparoscopic IPAA for IBD in four European expert centres were included and divided into Groups A [SBO during follow-up] and B [no SBO]. RESULTS: From 2005 to 2015, SBO occurred in 41/521 patients [Group A; 8%]. Two-stage IPAA was more frequently complicated by SBO than 3- and modified 2-stage IPAA [12% vs 7% and 4%, p = 0.04]. After multivariate analysis, postoperative morbidity (odds ratio [OR] = 3, 95% confidence interval [CI] = 1.5-7, p = 0.002), stoma-related complications [OR = 3, 95% CI = 1-6, p = 0.03] and long-term incisional hernia [OR = 6, 95% CI = 2-18, p = 0.003] were predictive factors for SBO, while subtotal colectomy as first surgery was an independent protective factor [OR = 0.4, 95% CI = 0.2-0.8, p = 0.002]. In the subgroup of patients receiving restorative proctocolectomy as first operation, stoma-related or other surgical complications and long-term incisional hernia were predictive of SBO. In the patient subgroup of subtotal colectomy as first operation, postoperative morbidity and long-term incisional hernia were predictive of SBO, whereas ulcerative colitis and a laparoscopic approach during the second surgical stage were protective factors. CONCLUSIONS: We found that SBO occurred in less than 10% of patients after laparoscopic IPAA. The study also suggested that modified 2-stage IPAA could potentially be safer than procedures with temporary ileostomy [2- and 3-stage IPAA] in terms of SBO occurrence.

Different effects of vertical banded gastroplasty and Roux-en-Y gastric bypass on meal inhibition of ghrelin secretion in morbidly obese patients.

A decrease in ghrelin plasma levels in morbidly obese patients subjected to bariatric surgery has been considered to help increase body weight loss. Contradictory results have been described after Roux-en-Y gastric bypass (RYGBP), and no study to date has compared RYGBP and vertical banded gastroplasty (VBG), the two main operations performed in the United States. We investigated the effects of RYGBP (10 patients) and VBG (12 patients) on basal and postmeal ghrelin plasma levels in 22 morbidly obese patients (20 F and 2 M), mean age 42.1 +/- 3.7 years, mean weight 115 +/- 3.9 kg, mean body mass index (BMI) 43.5 +/- 1.7. Before surgery and after a 20% reduction in BMI, ghrelin concentrations (pg/mL; radioimmunoassay [RIA], DRG Diagnostics, Germany) were measured in all patients 45 min before and for 3 h after a standard liquid meal (Osmolite RTH solution, 500 mL, 504 kcal). The results were expressed as mean +/- SD. Differences between times and groups were evaluated by Student's t-test and one-way analysis of variance (ANOVA). We found that basal ghrelin plasma levels were reduced after RYGBP (to 73.1 +/- 6 pg/mL, p < .05) but increased after VBG (to 172 +/- 26 pg/mL, p < .0009). After a standard liquid meal, ghrelin plasma levels decreased significantly over 1 h in VBG patients, whereas they remained unchanged in RYGBP patients. Since these results were obtained under the same metabolic and anthropometric conditions, we conclude that RYGBP acts through permanent inhibition of ghrelin secretion, whereas VBG merely restores the mechanisms of ghrelin regulation by nutrients.

Diagnosis and Treatment of Biliary Fistulas in the Laparoscopic Era.

Biliary fistulas are rare complications of gallstone. They can affect either the biliary or the gastrointestinal tract and are usually classified as primary or secondary. The primary fistulas are related to the biliary lithiasis, while the secondary ones are related to surgical complications. Laparoscopic surgery is a therapeutic option for the treatment of primary biliary fistulas. However, it could be the first responsible for the development of secondary biliary fistulas. An accurate preoperative diagnosis together with an experienced surgeon on the hepatobiliary surgery is necessary to deal with biliary fistulas. Cholecystectomy with a choledocoplasty is the most frequent treatment of primary fistulas, whereas the bile duct drainage or the endoscopic stenting is the best choice in case of minor iatrogenic bile duct injuries. Roux-en-Y hepaticojejunostomy is the extreme therapeutic option for both conditions. The sepsis, the level of the bile duct damage, and the involvement of the gastrointestinal tract increase the complexity of the operation and affect early and late results.

Vertical banded gastroplasty modifies plasma ghrelin secretion in obese patients.

BACKGROUND: Restrictive bariatric surgery causes weight loss through substantial decline of appetite with satiety after meals. Reduction of plasma ghrelin levels after Roux-en-Y gastric bypass and laparoscopic adjustable gastric banding could contribute to these effects, although contradictory results have been reported. The only restrictive operation still not yet investigated is vertical banded gastroplasty (VBG). We studied the effects of VBG on basal plasma ghrelin levels and meal-mediated inhibition. METHODS: 12 morbidly obese patients, 11 female and 1 male, were studied before and after VBG, when the BMI fell by 20%. The control group consisted of 6 lean volunteers. Active ghrelin was determined by RIA after overnight fasting and after the administration of a liquid meal. RESULTS: Obese patients preoperatively had significantly lower basal plasma ghrelin levels than lean volunteers, and the meal did not inhibit ghrelin secretion. After VBG and 20% BMI loss, basal plasma ghrelin levels increased and the reduction caused by a meal recovered. CONCLUSIONS: Weight loss caused by VBG is associated with higher plasma ghrelin levels in obese patients. The operation restores the normal adaptation of the A- cells of the stomach to a meal.

Comparison of the gastric cytoprotective properties of atropine, ranitidine and PGE2 in rats.

In view of the controversy as to whether antisecretory agents such as H2 antagonists and antimuscarinics might be cytoprotective like the PGs, the oral activity of atropine, ranitidine and PGE2 against absolute ethanol-induced lesions was evaluated in rats. The results showed that atropine and PGE2, but not ranitidine, were effective in preventing absolute ethanol-induced gastric damage. The effects were related to the doses of the ulcerogenic agent and of the cytoprotective compound. The anti-ulcer activity of atropine is considered to be an expression of cytoprotection, since the pathogenesis of ethanol-induced gastric damage was independent of gastric pH and atropine, like PGE2, does not affect basal acid secretion at a fully cytoprotective dose. Some studies were undertaken to elucidate the mechanism of gastric cytoprotection by atropine. The possibility that the anti-muscarinic agent might work as a mild irritant was ruled out since, like PGE2, the agent was still effective in PG-deficient rats. The evidence that neostigmine markedly aggravated gastric damage caused by low doses of absolute ethanol and that atropine completely prevented this damage postulates mechanisms involving specific muscarinic receptor interactions.

Metastatic melanoma of the gallbladder.

A 38-year-old man arrived at our clinic with symptoms and investigation results (U.S. scan and MR) suggestive of acute calculous cholecystitis. He gave a past history of excision of a stage I melanoma of the shoulder. Metastatic disease was suspected following measurement of CA 19.9 levels and the CT scan. The patient underwent laparotomy and cholecystectomy; pathological examination confirmed the presence of a malignant melanoma metastatic lesion of the gallbladder.

Discontinuous oral absorption of cimetropium bromide, a new antispasmodic drug.

The pharmacokinetic profiles of cimetropium bromide, after either intravenous injection of 10 mg or oral ingestion of 200 mg, were determined in eight healthy volunteers. After intravenous administration, the plasma levels and urinary excretion indicated that the drug is distributed and eliminated at a rapid rate (terminal half-life, 50 +/- 8 min) and that urinary excretion is not the exclusive route of elimination (46 +/- 2%) of the administered dose). After oral administration, a low percentage of the drug is absorbed (1-4% of the administered dose), however, the amount is sufficient for therapeutic effect. The absorption is discontinuous, with two distinct phases, and ends abruptly during the second phase.

The mechanisms of blood vessel closure in humans by the application of ultrasonic energy.

BACKGROUND: The use of the ultrasonically activated scalpel (UAS) for vessel closure has attained widespread acceptance in many surgical fields. The aim of our study was to investigate the electron microscopic changes to the blood vessels after the application of UAS. METHODS: We collected 10 arterial and 10 venous segments from vessels that had previously been closed by UAS during abdominal operations. The samples were then prepared for ultramicroscopic analysis. Pathological changes in the lumen and the three wall layers of the blood vessel were examined under scanning and transmission electron microscopy. RESULTS: All of the vessel segments showed similar changes: the presence of a blood clot, endothelial cell condensation, coagulative necrosis of the wall, and charring of the vessel at its tip. The edge of the cut vessel were closed by the coagulation bond, which was tied up by collagen fibrils escaped from denaturation. CONCLUSION: When ultrasonic energy is applied to tissues, it changes their structure so as to make a new extracellular matrix.

New cyclosporine microemulsion randomized, cross-over bioequivalence steady-state study in renal transplanted patients.

A new microemulsion formulation of cyclosporine was compared with the marketed formulation in 18 stable renal transplanted patients. Aim of the study was not only to determine the bioequivalence between the two pharmaceutical preparations, but also to ascertain whether tested drug could maintain stable blood concentrations of cyclosporine. Renal transplanted patients under cyclosporine treatment from at least 12 months at a well individualized dosage (resulting in 90-200 ng/mL of blood level drug) have been selected. Patients received the same preceding dose of cyclosporine through both the two preparations according to a cross-over, randomized schedule during 4 weeks in two equally divided daily administrations. Serial blood samples were obtained over a 24-hour period at steady-state of each formulation. Cyclosporine concentrations were determined by a specific immunoassay method (FPIA) n whole blood taken in the last day of each cycle of treatment. Statistical comparisons of cyclosporine levels (using pharmacokinetic parameters) were cross-performed between formulations and days of blood test. Tested drug resulted bioequivalent with the reference marketed formulation. Furthermore, the study showed that tested drug maintained satisfactory stable blood concentrations of cyclosporine.

Effects of warfarin on thrombosis of the jugular veins of rabbits induced by prolonged stasis.

We have studied the effects of warfarin pretreatment on blood coagulation tests, thrombosis and endothelial damage in the rabbit after 2 h of subsequent venous stasis. We found that 3 mg/kg warfarin significantly modified the results of prothrombin time, activated partial thromboplastin time and ProComplex tests. The incidence of thrombosis was not reduced by warfarin, but the weight of the thrombi were inversely related to the effects of the drug on blood coagulation. The endothelial cells showed some degree of damage. In conclusion, warfarin neither prevents endothelial damage nor reduces the incidence of thrombosis after venous stasis, but retards the growth of thrombi.

Paradoxical effects of intra-arterial administration of basic fibroblast growth factor on inflammatory angiogenesis in rats.

The authors studied the effects of basic fibroblast growth factor (b-FGF) on inflammatory angiogenesis in rats. In the corneal cauterization model b-FGF was given intra-arterially (i.a.) (carotid) and in the mesenteric window angiogenesis model, topically (i.e., intraperitoneally (i.p.)). The corneal cauterization was done under anaesthesia by topical application of silver nitrate. Mesenteric window angiogenesis was induced by injection of saline or b-FGF for four days. There were the same two groups of treatment in both models b-FGF 2.5 micrograms/kg/day or saline 1.2-5 ml/kg/day. The area of neovessels and the number of polymorphonuclear cells/field were considered for the corneal angiogenesis, the total length of neovessels was measured for the mesenteric window angiogenesis. The results were expressed as mean values (s.d.). When given i.a., b-FGF significantly reduced the number of polymorphonuclear cells three days after corneal cauterization (from 107 +/- 27 to 41.8 +/- 26, p < 0.01) and inhibited the area covered by neovessels (30 +/- 7.7% vs 51 +/- 20%, p < 0.01) after five days. In contrast, given through the extracellular space, it significantly stimulated the length of mesenteric window microvessels (169 +/- 60 mm vs 90 +/- 31 mm, p < 0.05). These results suggest that b-FGF stimulates inflammatory angiogenesis through interaction with extracellular matrix components, but inhibits it directly when given intra-arterially.

The effects of L-sulpiride on reflux oesophagitis.

Prokinetic drugs are commonly used for treatment of reflux oesophagitis. Although much data has been collected in clinical trials, their therapeutic effects are still uncertain. In this study the effects of L-sulpiride, if any, were examined when used to treat reflux oesophagitis in thirty patients. The patients were divided into two groups: a control group and a group given 25 mg t.i.v./day, p.o. of L-sulpiride for 30 days. They were treated as outpatients and had endoscopic, histological and ultrastructural examinations on the 30th and 60th days of treatment. It was found that the symptoms of patients with reflux oesophagitis were alleviated and the endoscopic and ultrastructural lesions of patients with minor oesophagitis were also decreased. In other patients, symptoms improved without resolution of the lesions. The authors conclude, therefore, that L-sulpiride would be appropriate treatment for Grade I cases.

Pharmacokinetics of cimetidine in patients with unresponsive duodenal ulcer.

The pharmacokinetics of cimetidine after an oral dose of 400 mg were measured in 18 patients with duodenal ulcer, 9 refractory and 9 responders. The peak plasma concentration of cimetidine (2.13 +/- 0.17 micrograms/ml vs 1.43 +/- 0.04 micrograms/ml), the area under the plasma concentration curve (A.U.C.) between 0 to 8 hours after cimetidine (8.49 +/- 0.29 micrograms/ml/h vs 5.83 +/- 0.25 micrograms/ml/h), and the time span in which cimetidine was above 0.5 micrograms/ml (I.C.50) (401 +/- 8.86 min vs 296 +/- 20 min) were all found to be greater in responding patients than in non-responders to the therapy. No differences were detectable between the two groups in urinary excretion, T 1/2 of cimetidine or percentage inhibition (1%) of maximal pentagastrin-stimulated acid output (MAO). The results indicate that clinical healing of duodenal ulcer after cimetidine is related principally to the drug's pharmacokinetics, i.e. to its absorption from the small bowel, and that some other therapeutic approaches might be tried before surgery in cases of duodenal ulcer refractory to cimetidine.

Pouch ileitis in excluded reservoir: an unusual complication of restorative proctocolectomy for ulcerative colitis.

One case of pouch ileitis after restorative proctocolectomy for ulcerative colitis is described. Diagnosis was made by endoscopy, histology and electron microscopy. The most prominent feature was intense inflammation of the mucosa and submucosa, with atrophy of the villi, and colonic metaplasia, occurring before closure of the loop ileostomy. The patient improved after a course of metronidazole therapy, but ileostomy closure was postponed. It appears that the ileum mucosa of patients with ulcerative colitis is highly prone to the development of inflammation and careful, regular follow-up is recommended.

The role of mast cells in wound healing.

Mast cells are known to participate in three phases of wound healing: the inflammatory reaction, angiogenesis and extracellular-matrix reabsorption. The inflammatory reaction is mediated by released histamine and arachidonic acid metabolites. Compound 48/80 and disodium-cromoglycate are both able to increase skin breaking strength shortly after wounding. Under light and electron microscopy we found that small, granule-poor, irregular mast cells (MLMC) accumulate in the wound. This suggests that the small MLMC (mucosal-like mast cells) migrate into the skin during wound healing, and that both CTMC (connective-tissue mast cells) and MLMC are involved in tissue repair. Moreover, there is some evidence that mast cells participate in angiogenesis, since heparin is able to stimulate endothelial-cell migration and proliferation in vitro, and protamine to inhibit these processes and also angiogenesis in vivo. When the effect of protamine on wound breaking strength was examined, we encountered a decrease which was not prevented by heparin administration. Further studies are needed to demonstrate that protamine is specifically involved in inhibiting heparin-mediated angiogenesis in wounded tissue. Finally, mast cells may play a role in the extracellular matrix remodelling, on the basis of in-vitro experiments (but there are still no in-vivo data).

The effects of oxygen free radicals on wound healing.

We have examined the effects of oxygen free radicals, generated by xenobiotics administration, ischaemia-reperfusion or sepsis, on the healing of skin or intestinal wounds in rats. We found that 5 days after operation there was a significant decrease in the wound breaking strength in rats treated with phenazine methosulfate, zymosan, ischaemia-reperfusion or retroperitoneal infection. These changes were specifically prevented by administration of superoxide-dismutase (SOD), aprotinin and (in some models) allopurinol. On the contrary, none of these measures was effective when a local trauma caused the decrease in breaking strength. Our results suggest that oxygen free radicals mediate the inhibition of wound healing following ischaemia-reperfusion and sepsis.

Prostaglandin cytoprotection in man: effectiveness of rosaprostol.

We studied the effect of a new prostaglandin analogue, 9-hydroxy-19,20-bis-norprostanoic acid (rosaprostol), on the duodenal mucosa of humans after administration of 40% ethanol. Eighteen healthy volunteers entered the study, which followed a blind cross-over design. At time 0 all the volunteers received rosaprostol or placebo; 5 minutes later, 40% ethanol (50 ml dose) was given. The mucosa was examined for lesions 3, 60 and 180 min later under endoscopy, light and scanning microscopy. It was found that: (i) 40% ethanol damaged the duodenum, with blood extravasation, inflammation and necrosis of the mucosa, (ii) rosaprostol significantly protected the mucosa 3 min after 40% ethanol, and (iii) the damage became worse after 3 h when placebo was given, whereas it was reduced after 1 h when PG was administered. The results suggest that PGs not only protect the mucosa against ethanol damage, but also stimulate its recovery.

Low molecular weight hyaluronic acid prevents oxygen free radical damage to granulation tissue during wound healing.

Hyaluronic acid protects granulation tissue from oxygen free radical damage and stimulates wound healing, but its molecular weight prevents it from permeating the epidermal barrier A low molecular weight hyaluronic acid preparation is able to permeate the skin, but it is unknown whether or not it retains the scavenging effects of oxygen free radicals in granulation tissue. Our experiments were conducted in rats with excisional or incisional wounds. Wound contraction over 11 days and breaking strength on the fifth day were measured. Oxygen free radical production was induced by intraperitoneal administration of two different xenobiotics: phenazine methosulfate and zymosan. The wounds were treated topically with low molecular weight hyaluronic acid (0.2%) cream or placebo. In the incisional wound group, the effects of superoxide dismutase were also determined. Absolute controls received wounds and placebo but no xenobiotics. Wound healing was significantly slower in the xenobiotic group than in the control groups. These effects were strongly reduced by topical administration of low molecular weight hyaluronic acid (0.2%) cream and in incisional wounds by topically injected superoxide dismutase. Low molecular weight hyaluronic acid is effective as the native compound against oxygen free radicals. Its pharmacological effects through transdermal administration should be tested in appropriate models.

Hyaluronic acid prevents oxygen free-radical damage to granulation tissue: a study in rats.

Oxygen free-radicals are known to impair wound healing after ischaemia-reperfusion or polymorphonuclear cell stimulation. Furthermore, they reduce the breaking strength of all recent wounds and might be a cause of wound leakage. This study was performed to evaluate whether or not hyaluronic acid can reduce the risk of wound impairment caused by free-radicals, in rats with abdominal sepsis, polymorphonuclear cell stimulation or cytochrome C function derangement produced by xenobiotics. Male Sprague-Dawley rats with open wounds received phenazine methosulfate or zimosan, or had abdominal sepsis to induce oxygen free-radical generation. There were three groups of treatment: hyaluronic acid cream, hyaluronic acid ethyl ester gel, and placebo. The reduction in wound size was measured from the 1st to the 11th postoperative day; biopsies were taken for histological evaluation. Every other day, a gentle debridement was performed in all the groups of animals. We found that hyaluronic acid and its ethyl ester derivative significantly improved the wound healing of rats subjected to an increased generation of oxygen free-radicals. It remains to be established whether or not hyaluronic acid acts as a scavenger of free-radicals.