Pierpaolo Sileri

Aryl hydrocarbon receptor-induced signals up-regulate IL-22 production and inhibit inflammation in the gastrointestinal tract.

BACKGROUND & AIMS The pathogenesis of inflammatory bowel disease (IBD) is believed to involve an altered balance between effector and regulatory T cells. Aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor that mediates the toxicity of dioxins, controls T-cell responses. We investigated the role of AhR in inflammation and pathogenesis of IBD in humans and mouse models. METHODS AhR expression was evaluated in intestinal tissue samples from patients with IBD and controls by real-time polymerase chain reaction (PCR) and flow cytometry. Intestinal lamina propria mononuclear cells (LPMCs) were activated in the presence or absence of the AhR agonist 6-formylindolo(3, 2-b)carbazole (Ficz). Colitis was induced in mice using trinitrobenzene sulfonic acid (TNBS), dextran sulfate sodium (DSS), or T-cell transfer. Mice were given injections of Ficz or the AhR antagonist 2-metyl-2H-pyrazole-3-carboxylic acid; some mice first received injections of a blocking antibody against interleukin (IL)-22. Cytokines were quantified by real-time PCR and flow cytometry. RESULTS Intestine tissue from patients with IBD expressed significantly less AhR than controls. In LPMCs from patients with IBD, incubation with Ficz reduced levels of interferon gamma (IFN)-γ and up-regulated IL-22. Mice injected with Ficz were protected against TNBS-, DSS-, and T-cell transfer-induced colitis; they had marked down-regulation of inflammatory cytokines and induction of IL-22. Mice given AhR antagonist produced more inflammatory cytokines and less IL-22 and developed a severe colitis. Neutralization of endogenous IL-22 disrupted the protective effect of Ficz on TNBS-induced colitis. CONCLUSIONS AhR is down-regulated in intestinal tissue of patients with IBD; AhR signaling, via IL-22, inhibits inflammation and colitis in the gastrointestinal tract of mice. AhR-related compounds might be developed to treat patients with IBDs.

Robotic-assisted laparoscopic donor nephrectomy for kidney transplantation

Background. Minimally invasive laparoscopic nephrectomy is a well-established alternative to open surgery in living donors for kidney transplantation. Donor mortality and morbidity rates as well as recipient outcome are comparable to the open approach. Furthermore, the procedure is associated with reduced donor discomfort, faster recovery, and improved cosmetic results. Recently, an advanced robotic system for laparoscopic surgery was approved for use in the United States. This system allows a greater freedom of movement and recreates the hand–eye coordination and three-dimensional vision that is lost in standard laparoscopic procedures. Methods. We report the first 12 successful cases of robotic-assisted laparoscopic living donor nephrectomy performed using the da Vinci Surgical System (Intuitive Surgical, Mountain View, CA). Results. Our initial experience has shown that the system allows the performance of donor nephrectomy in a safe and accurate fashion. Conclusions. As technology continues to evolve, robotic-assisted surgery has the potential to become a widely used attractive alternative to standard laparoscopic donor nephrectomy.

Bacterial translocation in clinical intestinal transplantation

Background Bacterial translocation (BT) has been suggested to be responsible for the high incidence of infections occurring after small bowel transplantation (SBTx). Bacterial overgrowth, alteration of the mucosal barrier function as a consequence of preservation injury or acute rejection (AR), and potent immunosuppression are all associated with BT. The aim of this study was to evaluate and quantify the correlation of BT with these events. Methods. Fifty pediatric SBTx recipients on tacrolimus and prednisone immunosuppression were analyzed. Blood, stool, and liver biopsies and peritoneal fluid were cultured (circa 4000 total specimens) when infection was clinically suspected or as part of follow-up. BT episodes were considered when microorganisms were found simultaneously in blood or liver biopsy and stool. Results. BT (average of 2.0 episodes/patient) was evident in 44% of patients and was most frequently caused by Enterococcus, Staphylococcus, Enterobacter, and Klebsiella. The presence of a colon allograft was associated with a higher rate of BT (75% vs. 33.3%). Furthermore, the transplantation procedure (colon vs. no colon) affected the rate of BT: SBTx=40% vs. 25%, combined liver and SBTx=100% vs. 30%, multivisceral transplantation=25% vs. 50%. AR was associated with 39% of BT episodes. BT followed AR in 9.6% of the cases. In 5.2% of the cases, positive blood cultures without stool confirmation of the bacteria were seen. Prolonged cold ischemia time (CIT) affected BT rate significantly (CIT>9 hr 76% vs. CIT<9 hr 20.8%). Conclusions. This study shows that 1) a substantial percentage of, but not all, BT is associated with AR, 2) the presence of a colon allograft increases the risk for BT, and 3) a long CIT is associated with a high incidence of BT after SBTx.

Th17-type cytokines, IL-6 and TNF-α synergistically activate STAT3 and NF-kB to promote colorectal cancer cell growth

Colorectal cancers (CRCs) often show a dense infiltrate of cytokine-producing immune/inflammatory cells. The exact contribution of each immune cell subset and cytokine in the activation of the intracellular pathways sustaining CRC cell growth is not understood. Herein, we isolate tumor-infiltrating leukocytes (TILs) and lamina propria mononuclear cells (LPMCs) from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of patients who underwent resection for sporadic CRC and show that the culture supernatants of TILs, but not of LPMCs, potently enhance the growth of human CRC cell lines through the activation of the oncogenic transcription factors signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-kB). Characterization of immune cell complexity of TILs and LPMCs reveals no differences in the percentages of T cells, natural killer T cells, natural killer (NK) cells, macrophages and B cells. However, T cells from TILs show a functional switch compared with those from LPMCs to produce large amounts of T helper type 17 (Th17)-related cytokines (that is, interleukin-17A (IL-17A), IL-17F, IL-21 and IL-22), tumor necrosis factor-α (TNF-α) and IL-6. Individual neutralization of IL-17A, IL-17F, IL-21, IL-22, TNF-α or IL-6 does not change TIL-derived supernatant-driven STAT3 and NF-kB activation, as well as their proproliferative effect in CRC cells. In contrast, simultaneous neutralization of both IL-17A and TNF-α, which abrogates NF-kB signaling, and IL-22 and IL-6, which abrogates STAT3 signaling, reduces the mitogenic effect of supernatants in CRC cells. IL-17A, IL-21, IL-22, TNF-α and IL-6 are also produced in excess in the early colonic lesions in a mouse model of sporadic CRC, associated with enhanced STAT3/NF-kB activation. Mice therapeutically given BP-1-102, an orally bioavailable compound targeting STAT3/NF-kB activation and cross-talk, exhibit reduced colon tumorigenesis and diminished expression of STAT3/NF-kB-activating cytokines in the neoplastic areas. These data suggest that strategies aimed at the cotargeting of STAT3/NF-kB activation and interaction between them might represent an attractive and novel approach to combat CRC.

Involvement of interleukin-21 in the regulation of colitis-associated colon cancer

IL-21 expression is increased in the gut of patients with colitis-associated colon cancer, and genetic ablation or antibody neutralization of IL-21 reduces tumor size and inflammation in mice treated with dextran sulfate sodium and azoxymethane.

Acute pancreatitis and bacterial translocation.

Infectious complications are the most frequent and severe complications of acute narcotizing pancreatitis (AP) with a mortality rate up to 80%. Although experimental and clinical studies suggest that the microbiologic source of pancreatic infection could be enteric, information in this regard is scant. This study evaluated bacterial translocation (BT) using mild and severe models of AP. Mild AP was induced by 6-hr continuous intravenous infusion of cerulein, while severe AP was induced by additional infusion of glycodeoxycholic acid into the biliopancreatic duct. BT was evaluated with organ cultures performed when animals were killed (24 hr). To confirm the gastrointestinal origin of the translocating microorganisms, fluorescent microspheres were also given to the animals in drinking water 24 hr before induction of AP. At the time of death beads were counted with a (fluorescence-activated cell sorter) (FACS) in peritoneal lavages and with fluorescent microscopy in frozen sections of the pancreata. Morphology of the distal small bowel showed significant changes in the animals with AP compared to controls, such as reduction of villus high and altered microvasculature. Mild AP induced BT to the pancreas in 100% of the animals, compared to pancreata from control groups. Severe AP induced increased BT to the pancreas. BT to liver and spleen was also significantly increased with AP. The presence of fluorescent microspheres confirmed their enteric derivation. This study provides evidence for the enteric origin of microorganisms responsible for pancreatic infectious complications during AP. The evidence of BT after laparotomy suggests an increased risk of infections with the association of these conditions. This could provide an explanation for the high mortality associated with laparotomy in course of AP.

Epigenetically silenced miR-34b/c as a novel faecal-based screening marker for colorectal cancer

Background:MicroRNAs are tiny non-coding small endogenous RNAs that regulate gene expression by translational repression, mRNA cleavage and mRNA inhibition. The aim of this study was to investigate the hypermethylation of miR-34b/c and miR-148a in colorectal cancer, and correlate this data to clinicopathological features. We also aimed to evaluate the hypermethylation of miR-34b/c in faeces specimens as a novel non-invasive faecal-DNA-based screening marker.Methods:The 5-aza-2′-deoxycytidine treatment and methylation-specific PCR were carried out to detect the hypermethylation of miR-34b/c and miR-148a.Results:The miR-34b/c hypermethylation was found in 97.5% (79 out of 82) of primary colorectal tumours, P=0.0110. In 75% (21 out of 28) of faecal specimens we found a hypermethylation of miR-34b/c while only in 16% (2 out of 12) of high-grade dysplasia. In addition, miR-148a was found to be hypermethylated in 65% (51 out of 78) of colorectal tumour tissues with no significant correlation to clinicopathological features. However, a trend with female gender and advanced age was found, P=0.083. We also observed a trend to lower survival rate in patients with miR-148a hypermethylation with 10-year survival probability: 48 vs 65%, P=0.561.Conclusions:These findings show that aberrant hypermethylation of miR-34b/c could be an ideal class of early screening marker, whereas miR-148a could serve as a disease progression follow-up marker.

Interferon-gamma-expressing cells are a major source of interleukin-21 in inflammatory bowel diseases.

Background: We previously demonstrated that in inflammatory bowel disease (IBD) there is enhanced production of interleukin (IL)‐21, a cytokine that activates multiple pathways that sustain mucosal inflammation. However, the phenotype of IL‐21‐producing cells in IBD, and the cytokine(s) they coproduce, is not known. We here characterized the cell source of IL‐21 and determined which factors regulate IL‐21 in the human gut. Methods: Cytokines were analyzed in CD4+ T intestinal lamina propria lymphocytes (T‐LPL) isolated from IBD patients and controls by flow cytometry. Moreover, IL‐21 was evaluated in mucosal T follicular cells (TFH). To assess the involvement of IL‐12 and IL‐23 in the production of IL‐21, T‐LPL were activated in the presence or absence of IL‐12 or IL‐23. Results: The proportion of IL‐21‐producing CD4+ T‐LPL was increased in IBD compared to controls. The majority of IL‐21‐producing T‐LPL coexpressed interferon (IFN)‐&ggr;, and to a lesser extent IL‐4 or IL‐17A. Activation of CD4+ T‐LPL with IL‐12 but not IL‐23 enhanced the fraction of cells coexpressing IL‐21 and IFN‐&ggr;. TFH cells in LPL were identified by CXCR5 expression and expressed IL‐21 both in IBD and controls; however, the fraction of IL‐21‐positive TFH cells was higher in Crohns disease than in ulcerative colitis and controls. Treatment of CD4+ T‐LPL with IL‐12 enhanced the frequency of CXCR5+ IL‐21‐producing TFH cells. Conclusions: These findings indicate that in IBD IL‐21 is mostly produced by CD4+ T‐LPL coexpressing IFN‐&ggr;, reinforcing the concept that distinct subsets of T cells can produce IL‐21. Inflamm Bowel Dis 2010

Antibiotic prophylaxis in elective laparoscopic cholecystectomy: lack of need or lack of evidence?

Background: The need to administer antibiotic prophylaxis (ABP) during laparoscopic cholecystectomy (LC) is still a matter of significant controversy. The purpose of this study was to resolve this issue by performing a meta-analysis of the available randomized controlled trials (RCT) on this topic. Methods: Papers identified via a systematic literature search were evaluated according to standard criteria. Data regarding the patient sample, study methods, and outcomes were abstracted and summarized across studies. The outcome measures were the rates of all perioperative infections, the rates of surgical site infections, and the rates of infections at other sites. Results were examined for 974 patients randomized to ABP or placebo prior to LC in six RCT published from 1997 to 2001. Results: The cumulative rates of all infections were 2.8% in the ABP group and 4.4% in the placebo group. The pooled odds ratio (OR) (95% confidence interval [CI]) was 0.69 (0.34–1.43; p = 0.32). The cumulative rates of surgical site infections were 2.1% in the ABP group and 2.9% in the placebo group. The pooled OR (95% CI) was 0.82 (0.36–1.86; p = 0.63). The cumulative rates of infections at other sites were 0.7% in the ABP group and 1.5% in the placebo group. Pooled OR (95% CI) was 0.82 (0.18–1.90; p = 0.37). No significant heterogeneity was found in any data pooling. Conclusions: Based on the available evidence, there appears to be no need to administer routine ABP to low-risk patients during LC. However, the number of patients enrolled to date into RCT is insufficient to avoid a type II error. A large and well-designed trial is urgently needed to find a conclusive answer to this question.

Bacterial translocation and intestinal morphological findings in jaundiced rats

The susceptibility to sepsis in obstructive jaundice may be related to bacterial translocation (BT) from the gastrointestinal tract. We evaluated BT to visceral organs and morphological changes of the intestinal mucosa in a rat model of obstructive jaundice. Animals were randomly divided into two groups: in group A the common bile duct was tied and divided, while group B had the bile duct mobilized but not tied. After seven days, peritoneal swabs and liver, spleen, pancreas, lung, mesenteric lymph nodes (MLN), cecum, and terminal ileum biopsies were obtained for cultures. Light and electron microscopy were performed on intestinal samples. The TUNEL assay was performed to detect apoptosis. Data were analyzed using Fisher exact test and Student ttest. Bile duct obliteration resulted in an increased incidence of BT. Seven days after duct obliteration, BT to the peritoneal cavity was evident in 37.5% of the animals in group A and 25% in group B. The respective BT rates for the two groups were: 42.8% vs 37.5% to MLN, 71.4% vs 25% to liver, 42.8% vs 12.5% to spleen, 28.6% vs 0% to pancreas and 14.3% vs 0% to lungs. Despite a trend, this was not statistically significant. Cecal counts did not differ statistically among the groups, while ileal counts were significantly higher in jaundiced rats (P < 0.05). Structural and ultrastructural abnormalities were evident only in the mucosa of the terminal ileum of jaundiced rats. Apoptosis was significantly increased in the terminal ileum of jaundiced rats (P < 0.002). This study suggests the possible association of biliary obstruction and BT. The nonspecific physical injury observed may contribute to breakdown of gastrointestinal barrier function thus promoting BT.