Dimitrios Pantelis

Liver sinusoidal endothelial cells contribute to CD8 T cell tolerance toward circulating carcinoembryonic antigen in mice.

Immunity against cancer is impeded by local mechanisms promoting development of tumor‐specific T cell tolerance, such as regulatory T cells, myeloid‐derived suppressor cells, or immunosuppressive factors in the tumor microenvironment. The release of soluble antigens, such as carcinoembryonic antigen (CEA) from colorectal carcinoma (CRC) cells, has been investigated for diagnostic purposes, but not for its immunological consequences. Here, we address the question of whether soluble CEA influences tumor‐specific immunity. Mice were injected with soluble CEA protein, and CEA‐specific CD8 T cells were analyzed for their phenotype and functionality by means of restimulation ex vivo or antitumor efficacy in vivo. We furthermore characterized the CD8 T cell population in peripheral blood mononuclear cell (PBMCs) from healthy donors and colorectal carcinoma patients. In mice, circulating CEA was preferentially taken up in a mannose receptor–dependent manner and cross‐presented by liver sinusoidal endothelial cells, but not dendritic cells, to CD8 T cells. Such systemically circulating CEA promoted tolerization of CEA‐specific CD8 T cells in the endogenous T cell repertoire through the coinhibitory molecule B7H1. These CD8 T cells were not deleted but were rendered nonresponsive to antigen‐specific stimulation and failed to control growth of CEA‐expressing tumor cells. These nonresponsive CD8 T cells were phenotypically similar to central memory T cells being CD44highCD62LhighCD25neg. We found T cells with a similar phenotype in PBMCs of healthy donors and at increased frequency also in patients with colorectal carcinoma. Conclusion: Our results provide evidence for the existence of an unrecognized tumor immune escape involving cross‐presentation of systemically circulating tumor antigens that may influence immunotherapy of cancer. (HEPATOLOGY 2012;56:1924–1933)

Acute rejection and the muscularis propria after intestinal transplantation: the alloresponse, inflammation, and smooth muscle function.

Background. It has been shown that in transplantation the intestinal muscularis may act as an immunologically active layer via the activation of resident macrophages and the recruitment of leukocytes. Thus we hypothesized that inflammation within the intestinal muscularis is involved in the promotion of acute rejection in intestinal allografts and that this causes smooth muscle dysfunction. Methods. Orthotopic allogenic and small bowel transplantation (Brown-Norway rats–Lewis rats) was performed without immunosuppression. Animals were sacrificed 1, 4, and 7 days after small bowel transplantation. Isogenic transplanted grafts (Brown-Norway rats–Brown-Norway rats) as well as nontransplanted bowel served as controls. Mediator mRNA expression was determined by real-time reverse-transcriptase polymerase chain reaction. Leukocyte infiltration was evaluated in muscularis whole mounts by immunohistochemistry. Apoptosis was evaluated by TdT-mediated dUTP-X nick end labeling assay. Contractility was assessed in a standard organ bath under bethanechol stimulation. Statistical analysis was performed using a Student’s t test and one-way analysis of variance. Results. Transplanted animals showed a significant early inflammatory response within the graft muscularis because of reperfusion injury. Only allogenic transplanted animals exhibited a significant second molecular inflammatory peak in the muscularis during rejection (mRNA induction for interleukin (IL)-6, intercellular adhesion molecule-1, monocyte chemoattractant protein (MCP)-1, interferon-&ggr;, IL-2, tumor necrosis factor-&agr;, IL-10, inducible nitric oxide synthase). These findings were associated with significant leukocyte infiltration within the muscularis, increasing apoptotic cells and massive impairment of smooth muscle contractile activity by 78%. Conclusions. The data shows that transplantation results in an early and temporary inflammatory response within the intestinal graft muscularis, that is reactivated and intensified during acute allograft rejection. The immunoreaction within the intestinal muscularis leads to intestinal allograft smooth muscle dysfunction.

Impaired intestinal wound healing in Fhl2-deficient mice is due to disturbed collagen metabolism

Four and one half LIM domain protein FHL2 participates in many cellular processes involved in tissue repair such as regulation of gene expression, cytoarchitecture, cell adhesion, migration and signal transduction. The repair process after wounding is initiated by the release of peptides and bioactive lipids. These molecules induce synthesis and deposition of a provisional extracellular matrix. We showed previously that sphingosine-1-phosphate (S1P) triggers a signal transduction cascade mediating nuclear translocation of FHL2 in response to activation of the RhoA GTPase. Our present study shows that FHL2 is an important signal transducer influencing the outcome of intestinal anastomotic healing. Early wound healing is accompanied by reconstitution and remodelling of the extracellular matrix and collagen is primarily responsible for wound strength. Our results show that impaired intestinal wound healing in Fhl2-deficient mice is due to disturbed collagen III metabolism. Impaired collagen III synthesis reduced the mechanical stability of the anastomoses and led to lower bursting pressure in Fhl2-deficient mice after surgery. Our data confirm that FHL2 is an important factor regulating collagen expression in the early phase of wound healing, and thereby is critically involved in the physiologic process of anastomosis healing after bowel surgery and thus may represent a new therapeutic target.

Oral CPSI-2364 Treatment Prevents Postoperative Ileus in Swine without Impairment of Anastomotic Healing

Background: Postoperative ileus (POI) is an iatrogenic complication of abdominal surgery, mediated by a severe inflammation of the muscularis externa (ME). We demonstrated that orally applicated CPSI-2364 prevents POI in rodents by blockade of p38 MAPK pathway and abrogation of NO production in macrophages. In the present experimental swine study we compared the effect of orally and intravenously administered CPSI-2364 on POI and examined CPSI-2364 effect on anastomotic healing. Methods: CPSI-2364 was administered preoperatively via oral or intravenous route. POI was induced by intestinal manipulation of the small bowel. ME specimens were examined by quantitative PCR for CCL2 chemokine gene expression and myeloperoxidase activity. Functional analyzes included measurement of ileal smooth-muscle ex vivo contractility, in vivo intestinal and colonic transit. Furthermore, anastomotic healing of a rectorectostomy after CPSI-2364 treatment was assessed by perianastomotic hydroxyproline concentration, a histochemically evaluated healing score and anastomotic bursting pressure (ABP). Results: CPSI-2364 abolished inflammation of the ME and improved postoperative smooth muscle contractility and intestinal transit independently of its application route. Hydroxyproline concentration and ABP measurement revealed no wound healing disturbances after oral or intravenous CPSI-2364 treatment whereas histological scoring demonstrated delayed anastomotic healing after intravenous treatment. Conclusion: CPSI-2364 effectively prevents POI in swine independently of its application route. Impairment of anastomotic healing could be observed after intravenous but not oral preoperative CPSI-2364 treatment. Subsumed, an oral preoperative administration of CPSI-2364 appears to be a safe and efficient strategy for prophylaxis of POI.

Prophylactic total gastrectomy in the management of hereditary tumor syndromes

PurposeGermline mutations in several genes confer a relevant lifetime risk of gastric cancer. In this context, an increasing involvement of a surgeon can be seen, mainly with the question of performing a prophylactic operation.MethodsPatients with hereditary tumor syndromes predisposing for gastric cancer who received care leading to prophylactic total gastrectomy in our Center for Hereditary Tumor Syndromes were analyzed. For each patient, the multidisciplinary decision-making process, the perioperative course, and the histopathologic findings were assessed. Short-term morbidity was evaluated based on the medical reports.ResultsThe analysis includes nine patients (six female, three male) with a median age of 41.6 (range 23–60)xa0years. Indication for prophylactic total gastrectomy was based on family history and genetic analysis (eight patients with a germline mutation of the CDH1 gene and one patient with a SMAD4 mutation). Removal of the entire gastric mucosa was documented intraoperatively by fresh frozen section examination. Extended (DII) lymphadenectomy was performed in four patients. Histopathologic examination of gastrectomy specimens revealed six patients (6/9, 67xa0%) with multifocal signet ring cell carcinomas. In our series, prophylactic total gastrectomy was a safe procedure without mortality and low morbidity.ConclusionsPatients with hereditary syndromes predisposing for gastric cancer should be evaluated for this curative procedure in a specialized center. Further research is necessary, and the implementation of nationwide registers including patients with prophylactic gastrointestinal operations due to hereditary tumor syndrome is advisable.

Chromoendoscopy in combination with random biopsies does not improve detection of gastric cancer foci in CDH1 mutation positive patients

Background and study aims: Hereditary diffuse gastric cancer (HGGC), an autosomal dominant tumor-syndrome, accounts for 1u200a% to 3u200a% of gastric cancers worldwide. Presumably 30u200a% to 40u200a% of all patients fulfilling the clinical guidelines for HDGC are carriers of a pathogenic mutation in the CDH1 gene. Patients often show multiple foci of signet ring cell carcinoma at early age and are advised to undergo prophylactic total gastrectomy (PTG). Our aim was to improve the endoscopic detection of HDGC by using an enhanced endoscopic protocol. Patient and methods: Patients with a proven CDH1 germline mutation identified in our institute were prospectively included. Patients were advised to undergo PTG and offered a baseline endoscopic examination prior surgery. Examination was performed by using high-resolution white-light endoscopy and pan-gastric chromoendoscopy with indigo carmine as dye combined with targeted and multiple random biopsies assessed by an expert histopathologist. Postoperative histopathology was compared with results from endoscopic biopsies. Results: Between September 2012 and November 2014 8 patients with a proven CDH1 germline mutation were included. We conducted 44 targeted (6.3/patient) and 225 random (32.1/patient) biopsies in 7 patients. We detected 1 gastric cancer by random biopsy (14u200a%). All other examinations showed no signs of cancer. Histopathology of gastrectomy specimen revealed multiple foci of gastric carcinoma in 6 patients (86u200a%) with a total number of 27 cancer foci. Conclusions: Examination with targeted and random biopsies combined with chromoendoscopy is not able to detect small foci of gastric cancer in CDH1 mutation carriers. Therefore PTG is advocated in these patients.

Chirurgische Therapie und prognostische Faktoren des Papillenkarzinoms

BACKGROUNDnCarcinoma of ampulla of Vater are rare tumours of the GI-tract with an improved prognosis compared to other periampullary tumours. Analysis of survival and prognostic factors are limited due to the low incidence of the carcinoma. The intention of this study in patients with papillary carcinoma was to evaluate short- and long-term survival and to identify prognostic factors for pancreatectomy and reconstruction using pancreatogastrostomy as treatment of carcinoma of Vaters ampulla.nnnPATIENTS AND METHODSnBetween 1989 and 2008 76 patients with a carcinoma of the ampulla of Vater were treated by oncological resection followed by pancreatogastrostomy. Various factors such as demographics, perioperative factors, histopathological findings as well as short- and long-term survival were evaluated retrospectively. Data were analysed statistically using Kaplan-Meier estimates of survival with log-rank test and uni- and multivariate analysis with Cox regression.nnnRESULTSnThe overall 5-year survival was 46u200a%, the 10-year survival 26u200a% for resected patients. By univariate analysis we could demonstrate that lymph node metastasis is the only predictor for outcome. In the multivariate analysis, age, sex, grading and especially lymph node status were a significant predictor for the survival of patients.nnnCONCLUSIONnIn the current patient cohort lymph node status was the most important independent predictor of outcome after resection of carcinoma of Vaters papilla.

Immunmodulatorische Aspekte bei der Entstehung, Prophylaxe und Therapie des postoperativen Ileus

Postoperative ileus (POI) is defined as a transient episode of impaired gastrointestinal motility after abdominal surgery, which prevents effective transit of intestinal contents or tolerance of oral intake. This frequent postoperative complication is accompanied by a considerable increase in morbidity and hospitalisation costs. The aetiology of POI is multifactorial. Besides a suppression of peristalsis by inhibitory neuronal signalling and administration of opioids, particularly in the prolonged form, immunological processes play an important role. After surgical trauma, resident macrophages of the muscularis externa (ME) are activated leading to the liberation of proinflammatory mediators and a spreading of the inflammation along the entire gastrointestinal tract. To date, no prophylaxis or evidence-based single approach exists to treat POI. Since none of the current treatment approaches (i.e., prokinetic drug treatment) has provided a benefit in randomised trials, immunoregulatory interventions appear to be more promising in POI prevention or treatment. The present contribution gives an overview of immunological mechanisms leading to POI focusing on current and future therapeutic and prophylactic approaches.

Spätkomplikationen permanenter intestinaler Stomata

INTRODUCTIONnComplications following the creation of permanent intestinal ostomies are common and lead to serious problems in the stoma care of affected patients. The aim of this prospective, single-centre follow-up study was to record the rate of late complications in our own patient group and to identify potential risk factors.nnnMETHODSnAll patients who received a permanent intestinal ostomy in our clinic within the period 2006u200a-u200a2016 were included in the study. 50 patients gave their informed consent and participated in our follow-up (14 female [28%], 36 male [72%]). The analysis of stoma-associated complications was performed by review of medical records and a systematic follow-up (standardised questionnaire, clinical examination, and ultrasound of the abdominal wall).nnnRESULTSnIndications included malignancy (nu2009=u200927; 54%), anastomotic leakage (nu2009=u200910; 20%), acute diverticulitis (nu2009=u20097; 14%), IBD (nu2009=u20095; 10%) and rarer indications. The top 3 late complications were peristomal skin irritation (nu2009=u200925, 50%), parastomal hernia (nu2009=u200914; 28%) and prolapse (nu2009=u20099; 18%). Acute diverticulitis resulted in increased stomal retraction (pu2009=u20090.012). Double-barreled stomata were associated with increased herniation rates (pu2009=u20090.044) and prolapse (pu2009=u20090.047). Ileostomies were associated with peristomal skin irritation (pu2009=u20090.021). Age, sex or emergency stoma creation did not constitute independent risk factors for the development of late complications in our group of patients.nnnCONCLUSIONnProfessional pre- and postoperative stoma therapy and care includes preoperative marking of a stoma site and structured stoma-specific follow-up by stoma therapists, surgeons and general practitioners, as well as stringent and early treatment of structural complications. This can prevent and mitigate late complications of permanent intestinal stomata. Interestingly, stoma placement was not an independent risk factor for late complications in an emergency situation.

Significance of PITX2 Promoter Methylation in Colorectal Carcinoma Prognosis

Background New treatment modalities and a growing understanding of the complex genetic tumor landscape have improved the outcome of colorectal cancer (CRC) patients. Nonetheless, more individualized treatment regimens, taking individual tumor characteristics into account, have been recently postulated and prognostic biomarkers are needed. We therefore evaluated the prognostic potential of paired‐like homeodomain transcription factor 2 (PITX2) promoter methylation in CRC patients. Materials and Methods Data of 2 independent cohorts were investigated. Tissue specimens of cohort A (n = 179) were analyzed for their methylation in the PITX2 promoter region using quantitative methylation‐specific polymerase chain reaction and compared with publicly available data (PITX2 promoter methylation and PITX2 mRNA expression levels) from “The Cancer Genome Atlas Research Network” (cohort B, n = 443). Data were correlated with clinicopathological parameters and outcome. Results Tumor samples of both cohorts showed a decreased PITX2 promoter methylation level (both P < .001) compared with nonmalignant tissue. Additionally, PITX2 promoter hypomethylation was prognostic in univariate and multivariate analysis (hazard ratio [HR], 1.97 [95% confidence interval (CI), 1.12‐3.47], P = .018 and HR, 1.89 [95% CI, 1.09‐3.29], P = .023), and Kaplan–Meier analysis (median overall survival, 53.2 vs. 70.4 months, P = .004). Subanalysis of high‐risk vs. low‐risk stage II CRC patients also showed a PITX2 hypomethylation of the promoter region in the high‐risk group (P = .006). Conclusion Our results suggest a prognostic role of PITX2 promoter methylation in CRC as biomarker for risk stratification in stage II CRC patients although the results need to be independently validated. Micro‐Abstract Recent developments in colorectal cancer (CRC) patients have raised the need for prognostic biomarkers. Using 2 independent cohorts (cohort A, n = 179 and B, n = 443), we found that paired‐like homeodomain transcription factor 2 promotor methylation level might be used as prognostic marker in CRC.