Heikki Huhtinen

Mucosal Adhesion Properties of the Probiotic Lactobacillus rhamnosus GG SpaCBA and SpaFED Pilin Subunits

ABSTRACT Lactobacillus rhamnosus GG is a well-established Gram-positive probiotic strain, whose health-benefiting properties are dependent in part on prolonged residence in the gastrointestinal tract and are likely dictated by adherence to the intestinal mucosa. Previously, we identified two pilus gene clusters (spaCBA and spaFED) in the genome of this probiotic bacterium, each of which contained the predicted genes for three pilin subunits and a single sortase. We also confirmed the presence of SpaCBA pili on the cell surface and attributed an intestinal mucus-binding capacity to one of the pilin subunits (SpaC). Here, we report cloning of the remaining pilin genes (spaA, spaB, spaD, spaE, and spaF) in Escherichia coli, production and purification of the recombinant proteins, and assessment of the adherence of these proteins to human intestinal mucus. Our findings indicate that the SpaB and SpaF pilin subunits also exhibit substantial binding to mucus, which can be inhibited competitively in a dose-related manner. Moreover, the binding between the SpaB pilin subunit and the mucosal substrate appears to operate through electrostatic contacts and is not related to a recognized mucus-binding domain. We conclude from these results that it is conceivable that two pilin subunits (SpaB and SpaC) in the SpaCBA pilus fiber play a role in binding to intestinal mucus, but for the uncharacterized and putative SpaFED pilus fiber only a single pilin subunit (SpaF) is potentially responsible for adhesion to mucus.

Akkermansia muciniphila Adheres to Enterocytes and Strengthens the Integrity of the Epithelial Cell Layer

ABSTRACT Akkermansia muciniphila is a Gram-negative mucin-degrading bacterium that resides in the gastrointestinal tracts of humans and animals. A. muciniphila has been linked with intestinal health and improved metabolic status in obese and type 2 diabetic subjects. Specifically, A. muciniphila has been shown to reduce high-fat-diet-induced endotoxemia, which develops as a result of an impaired gut barrier. Despite the accumulating evidence of the health-promoting effects of A. muciniphila, the mechanisms of interaction of the bacterium with the host have received little attention. In this study, we used several in vitro models to investigate the adhesion of A. muciniphila to the intestinal epithelium and its interaction with the host mucosa. We found that A. muciniphila adheres strongly to the Caco-2 and HT-29 human colonic cell lines but not to human colonic mucus. In addition, A. muciniphila showed binding to the extracellular matrix protein laminin but not to collagen I or IV, fibronectin, or fetuin. Importantly, A. muciniphila improved enterocyte monolayer integrity, as shown by a significant increase in the transepithelial electrical resistance (TER) of cocultures of Caco-2 cells with the bacterium. Further, A. muciniphila induced interleukin 8 (IL-8) production by enterocytes at cell concentrations 100-fold higher than those for Escherichia coli, suggesting a very low level of proinflammatory activity in the epithelium. In conclusion, our results demonstrate that A. muciniphila adheres to the intestinal epithelium and strengthens enterocyte monolayer integrity in vitro, suggesting an ability to fortify an impaired gut barrier. These results support earlier associative in vivo studies and provide insights into the interaction of A. muciniphila with the host.

Type and location of tumor‐infiltrating macrophages and lymphatic vessels predict survival of colorectal cancer patients

The type of tumor‐infiltrating macrophages may be decisive in tumor immunity, lymphangiogenesis and in the clinical outcome of cancer. Here, we elucidated the prognostic significance of lymphatic vessels, different types of macrophages and the balance between different macrophage types in colorectal cancer. We analyzed the impact of density, type and location of macrophages on the clinical behavior of 159 primary colorectal carcinomas using CD68 as a pan‐macrophage marker and CLEVER‐1/Stabilin‐1 as a marker for regulatory/suppressive macrophages. Podoplanin was used as a pan‐lymphatic vessel marker. A high number of CLEVER‐1/Stabilin‐1+ peritumoral macrophages positively correlated with survival (p = 0.04). However, in more advanced disease (Stage IV), the patients with a high number of peritumoral or intratumoral CLEVER‐1/Stabilin‐1+ macrophages had a shorter disease‐specific survival (p = 0.05, and p = 0.008, respectively). Moreover, a low number of suppressive intratumoral CLEVER‐1/Stabilin‐1+ macrophages among high numbers of CD68+ macrophages correlated with a low number of distant recurrences (p = 0.01) and to fewer disease relapses exclusively in the liver as well (p = 0.006). A high number of intratumoral lymphatics correlated with poor survival (p = 0.03). The results of this work suggest that the type of macrophages, number of lymphatic vessels and their location contribute to the clinical behavior of colorectal cancer in a disease stage‐specific manner.

Quality of life after surgery for rectal cancer with special reference to pelvic floor dysfunction.

Aim  Conventional outcomes such as survival, tumour recurrence and complication rates after surgery for rectal cancer have been rigorously assessed, but the importance of maintaining quality of life (QOL) after surgery for rectal cancer has received less attention. The aim of the current study was to analyse QOL and the occurrence of pelvic dysfunction after the surgical treatment of rectal cancer.

Antibacterial effects of human group IIA and group XIIA phospholipase A2 against Helicobacter pylori in vitro

Group IIA phospholipase A2 (PLA2‐IIA) is an enzyme which has important roles in inflammation and infection. Recently, a novel human secretory PLA2 called group XIIA PLA2 (PLA2‐XIIA) has been identified. Both PLA2‐IIA and PLA2‐XIIA are bactericidal against Gram‐positive bacteria like many other secretory PLA2s. However, PLA2‐XIIA is the only known PLA2 displaying significant bactericidal activity against the Gram‐negative bacterium Escherichia coli. We examined the antibacterial properties of recombinant human PLA2‐IIA and PLA2‐XIIA against Helicobacter pylori, a Gram‐negative bacterium, in vitro. PLA2‐IIA was not bactericidal against H. pylori, whereas PLA2‐XIIA effectively killed H. pylori at a concentration of 50 μg/ml but was not bactericidal at concentrations of 0.5 μg/ml and 5 μg/ml.

Elective laparoscopic cholecystectomy without routine intraoperative cholangiography: a retrospective analysis of 1101 consecutive cases.

Background: Laparoscopic cholecystectomy (LC) is today the operation of choice for symptomatic gallstone disease. Before the laparoscopic era intraoperative cholangiography (IOC) was generally considered as a fundamental step in cholecystectomy while nowadays the role of IOC is controversial: is there a need for IOC to specify anatomy of biliary tree in order to avoid bile duct injuries (BDI) and to detect possible common bile duct (CBD) stones or not? Patients and Methods: We studied retrospectively all the elective LCs done in Turku City Hospital for Surgery during the ten years (1992–2001). Cholecystectomy was performed to 1101 patients, 874 (79%) female and 227 (21%) male patients, mean age 53y (range 15–89). LC was possible in 1022 (93%) cases while 79 (7%) had to be converted to open procedure. The number and severity of bile duct injuries were recorded. The cases with endoscopic retrograde cholangiopancreatography (ERCP) and/or magnetic resonance cholangiopancreatography (MRCP) during the follow-up and the findings in ERCP and MRCP were recorded from patient records and radiological database. Results: IOC was performed in 32 operations (20 in LC and 12 after conversion) and CBD stones were found in seven patients. There were four primary BDIs: two CBD injuries and two minor bile leaks. During a mean follow-up of 72 months (range 36–144) ERCP was performed in 16 and MRCP in three patients. Three patients underwent both MRCP and ERCP. CBD stones were detected in ten patients and a postoperative late CBD stricture was found in one case. Conclusions: According to our data, both the incidence of BDIs (0.5%) and symptomatic postoperative CBD stones (0.9%) remain low without the routine use of IOC.

Adoption of self-expanding metallic stents in the palliative treatment of obstructive colorectal cancer-look out for perforations!

Self-expanding metallic stents (SEMSs) are increasingly used for the palliative treatment of inoperable colorectal cancer. The aim of the current study was to analyze the safety and efficacy of SEMS in the palliative treatment of obstructive colorectal cancer. Between 2003 and 2006, SEMS placement was attempted in 26 patients suffering from inoperable obstructive colorectal cancer. The recovery of the patients and the outcome of this treatment modality were analyzed prospectively. SEMS was successfully inserted in 19 (73%) of 26 patients. In 16 (84%) of these 19 cases, the placement of SEMS was the definitive treatment of colorectal obstruction and no additional surgical palliation was needed. There were 3 (16%) colonic perforations related to stent application. SEMS insertion seems to be an effective alternative in the palliative treatment of patients with malignant colorectal obstruction. However, perforation is a dangerous complication of the procedure.

THROMBOPROPHYLAXIS FOLLOWING SURGERY FOR COLORECTAL CANCER -IS IT WORTHWHILE AFTER HOSPITAL DISCHARGE?

Background: The efficacy of low-molecular-weight heparin (LMWH) in preventing venous thromboembolism (VTE) after surgery for colorectal cancer is well documented, but the optimal duration of postoperative thromboprophylaxis is not known. The aim of this retrospective study was to assess the occurrence of symptomatic VTE after surgery for colorectal cancer in patients in whom LMWH was continued only until hospital discharge. Methods: During 2003–2006 a total of 494 patients underwent abdominal surgery for colorectal cancer at our institution. Enoxaparin (Klexane® 40mg s.c.) prophylaxis was started 12 hours before surgery and continued once a day until hospital discharge. The median duration of thromboprophylaxis was 11 days. The follow-up data were collected retrospectively from electronic archives and analyzed up to three months after the operation. Results: Only three (0.6%) symptomatic VTEs occurred during the follow-up period. One patient presented with pulmonary embolism, while the remaining two had proximal deep-vein thrombosis. The 30-day-mortality was 1.6%. None of the deaths were obviously associated with VTE. Conclusion: LMWH given for a median of 11 days until hospital discharge seems to provide sufficient thromboprophylaxis after surgery for colorectal cancer combined with the use of graded compression stockings and early mobilization.

Phospholipases A2 in Gastric Juice of Helicobacter pylori – Positive and Negative Individuals

Abstract Gastric juice is known to have phospholipase A2 catalytic activity. Helicobacter pylori (H. pylori) has been reported to produce phospholipase A2, which is believed to hydrolyse the protective layer of gastric mucosal phospholipids and to promote mucosal damage. The current study aimed at identifying secretory phospholipase A2 subtypes (pancreatic group I phospholipase A2 and synovial-type group II phospholipase A2) in gastric juice and their relation to the presence of H. pyloriin gastric mucosal biopsies in the same individuals. Gastric juice was collected from 29 individuals during gastroscopy. Biopsies were taken from the antrum and body of the stomach to determine the H. pylori status. We found catalytically active phospholipase A2 and both group I and group II phospholipases A2 in the gastric juice samples. The catalytic activity and the mass concentrations of group I and group II phospholipases A2 correlated significantly with the pH value in gastric juice. The gastric juice of H. pylori positive individuals did not contain higher amounts of phospholipases A2 than the juice of H. pylori negative individuals. Rather, the mass concentration of group II phospholipase A2 in gastric juice seemed to be somewhat lower in individuals with H. pylori infection than in uninfected individuals. The results of the current study show that both group I and group II phospholipases A2 are present in gastric juice. The main sources of phospholipases A2 in gastric juice are probably other than H. pylori.

Late complications related to palliative stenting in patients with obstructing colorectal cancer.

Abstract Introduction: Self-expanding metal stents (SEMSs) are increasingly used for the palliative treatment of incurable obstructing colorectal cancer. The aim of the current study was to evaluate clinical outcome, including technical and clinical success of stenting, and to identify factors associated with late complications of SEMS in palliation of incurable obstructing colorectal cancer. Material and methods: Between 2003 and 2010 details of 56 patients who underwent an attempt of SEMS insertion for obstructive incurable colorectal cancer at Turku University Hospital were recorded to our database prospectively and analyzed retrospectively. Results: Technical success was achieved in 42 patients (75%) and clinical success in 39 patients (70%). Late complications related to SEMS occurred in 13 patients (31%). Ten patients (24%) needed re-intervention because of a complication: Eight ostomies, one Hartmann´s procedure due to late perforation and one re-stenting because of stent migration. Three patients with stent-related complications were treated conservatively. Chemotherapy and prolonged survival were risk factors for SEMS-related late complications and re-intervention. Conclusion: SEMS insertion is a feasible procedure for the palliative treatment of obstructing colorectal cancer in patients with severe comorbidities and short life expectancy. However, for patients who are candidates for chemotherapy and have a longer life expectancy, other treatment options such as palliative tumor resection should also be considered.