Cristiana Rastellini

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.

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.

Pyruvate inhibits hepatic ischemia-reperfusion injury in rats

BACKGROUND Ischemia/reperfusion (I/R) injury is a limiting factor in liver transplantation. We have recently shown that pyruvate (PY) inhibits intestinal and renal I/R injury. This study aims to evaluate the protective effect of PY on hepatic I/R injury. METHODS ACI rats were treated with PY, whereas control animals received placebo. Rats were killed after 60 min of partial hepatic ischemia and after 2, 6, 24, and 48 hr of reperfusion. For each time point, serum aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were measured, and liver biopsy specimens were obtained to evaluate morphology, DNA fragmentation, and apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling). RESULTS The survival rate 48 hr after I/R was 83% in the control group, and 100% in the PY-treated group (P>0.05). Increased enzymatic levels and histologic findings showed increased liver damage in the untreated group compared with PY. In control rats, apoptosis was enhanced after 1 hr of ischemia and peaked after 2 hr of reperfusion, to decrease gradually 48 hr after reperfusion; in the PY group apoptosis was delayed and reduced. After 1 hr of ischemia, the number of apoptotic nuclei was significantly increased in control livers compared with normal preischemic livers, whereas the number was significantly reduced by PY. After 2 hr of reperfusion, the maximum number of apoptotic cells was observed, whereas PY significantly reduced the amount of apoptotic cells (P<0.05). Apoptosis was delayed in PY-treated livers to 6 hr after reperfusion, peaking at a significantly lower count compared with placebo-treated controls (P<0.05). CONCLUSION These data indicate that PY has a protective effect on I/R injury of the liver.

Gene silencing in the endocrine pancreas mediated by short-interfering RNA

Objectives: RNA interference as mediated by short-interfering RNA (siRNA) offers a nonviral means to silence genes in tissue; however, few data exist about gene therapy using siRNA in pancreas tissue. To determine if siRNA treatment could silence an endogenous gene in pancreatic islets, we developed a murine model using the endocrine pancreas. Methods: The insulin 2 (Ins2) gene was targeted with siRNA, and quantitative RT-PCR, fluorescent microscopy, and FACS were used to measure transcript levels and siRNA cellular uptake and transfection efficiency. Isolated pancreatic islets were transfected with siRNA in vitro using a liposomal delivery method in a dose titration (50-400 nM) or pooled from BALB/c mice having received siRNA (100 μg) via hydrodynamic tail vein injection. Results: The Ins2 transcript level was significantly reduced by 55% in vitro with FACS data showing a transfection efficiency over 45% with the 400 nM concentration. In vivo delivery of siRNA to pancreatic islets revealed a 33% reduction in Ins2 mRNA levels, although siRNA was able to be detected in 19% of isolated islet cells. Conclusion: We have successfully used RNA interference to silence an endogenous tissue-specific gene (Ins2) in pancreatic islets when transfected in vitro or administered in vivo.

Revascularization and remodelling of pancreatic islets grafted under the kidney capsule

The revascularization and the structural changes resulting from interactions between the graft and the host were investigated in transplanted pancreatic islets under the kidney capsule. Islets were isolated from mice pancreata and transplanted in syngeneic diabetic animals. Graft‐bearing kidneys were collected on different days post‐transplant and processed for light microscopy, immunohistochemistry and transmission electron microscopy. A numerical analysis was performed in order to compare the percentage number of the different types of cells in native islets and at different time points after the transplant. Recipient animals reversed diabetes within 4 days. An intraperitoneal glucose tolerance test was performed to determine islet functionality under stressful conditions. During the initial few days post‐transplant, the islets showed peculiar shapes and the graft tended to aggregate along the vessels. Starting at days 4–7 post‐transplant, islets were revascularized from vessels connected to both the cortical and the capsular vascular network of the kidney. From day 7–14 post‐transplant, the vessels progressively appeared more similar in features and size to those of in situ pancreatic islets. Both the percentage number of the different cell types and the distribution of Alpha, Beta and Delta cells inside the graft were significantly different as compared with intact islets, demonstrating quantitative and structural changes after the engraftment. No concomitant proliferation of Beta cells was detected using a bromodeoxyuridin staining method. Despite the fact that quick revascularization preserved a large mass of tissue, the remodelling process of the graft and the newly formed vascularization led to a different organization of the endocrine tissue as compared with intact in situ islets. This constitutes the morphological basis for alterations of the normal intercellular interactions and may explain the altered secretory cell function often observed in transplant.

Morphological changes of isolated rat pancreatic islets: a structural, ultrastructural and morphometric study

Improved techniques for pancreatic islet extraction can yield a reasonable number of transplantable cells. However, the isolation and purification process may damage the islets and impair their physiological functions. The aim of this study was to determine the effect of the isolation procedure on the structure of isolated islets and to correlate this with their functionality. Islets were isolated from rat pancreata and purified by Eurocollins‐Ficoll discontinuous density gradient processing, and then processed for light microscopy, and scanning and transmission electron microscopy. Morphometric analysis was also performed. Islet functionality was determined by reversal of streptozotocin‐induced diabetes and the intraperitoneal glucose tolerance test in a syngeneic rat model of pancreatic islet transplantation. Fragments of variable size and shape comprised a relatively large proportion (26%) of the isolated endocrine tissue. Isolated islets showed slight alterations of cell ultrastructure. Major damage (including breakage of the plasma membrane) and loss of cells were observed in the peripheral cells of the isolated islets. An equal mass of islet equivalent (IEq, islets with an average diameter of 150 µm), but with a different islet equivalent/islet number ratio, was transplanted in diabetic animals. When larger and more complete islets were transplanted (higher ratio), better function of the graft was observed by reversal of hyperglycaemia and response to the glucose tolerance test as compared with the functionality and response of smaller (fragmented) islets transplanted (lower ratio). Digestion, trauma and hypoxia during isolation are responsible for qualitative and quantitative changes of isolated islets. Alterations in normal secretory function after the transplant were related to lower islet equivalent/islet number ratio. The incomplete integrity of the islets may explain the failure of the fine glycaemic metabolic regulation.

Oridonin inhibits hepatic stellate cell proliferation and fibrogenesis

BACKGROUND Liver fibrosis is a common response to liver injury and, in severe cases, leads to cirrhosis. The hepatic stellate cells (HSCs) become activated after liver injury and play a significant role in fibrogenesis. The activated HSC is characterized by increased proliferation, overexpression of α smooth muscle actin, and excessive production of extracellular matrix (ECM) proteins. Oridonin, a naturally occurring diterpenoid, has been shown to induce apoptosis in liver and gastric cancer cells. However, its effects on the HSC are unknown. METHODS We tested the effects of oridonin on the activated human and rat HSC lines LX-2 and HSC-T6, and the human hepatocyte cell line C3A. Transforming growth factor β1 (TGF-β1) was used to stimulate LX-2 cells. RESULTS Oridonin significantly inhibited LX-2 and HSC-T6 proliferation. In contrast, oridonin had no antiproliferative effect on C3A cells at our tested range. Oridonin induced apoptosis and S-phase arrest in LX-2 cells. These findings were associated with an increase in p53, p21, p16, and cleaved Poly (ADP-ribose) Polymerase (PARP), and with a decrease in Cyclin-dependent kinase 4 (Cdk4). Oridonin markedly decreased expression of α smooth muscle actin and ECM protein type I collagen and fibronectin, blocked TGF-β1-induced Smad2/3 phosphorylation and type I collagen expression. CONCLUSIONS Oridonin induces apoptosis and cell cycle arrest involving the p53-p21 pathway in HSC and appears to be nontoxic to hepatocytes. In addition, oridonin suppressed endogenous and TGF-β1-induced ECM proteins. Thus, oridonin may act as a novel agent to prevent hepatic fibrosis.

Segmental living related small bowel transplantation in adults

The advent of small bowel transplantation has provided selected patients with chronic intestinal irreversible failure with a physiologic alternative to total parenteral nutrition. Recently a standardized technique for living related small bowel transplantation (LR-SBTx) has been developed. Three patients with short bowel syndrome underwent LR-SBTx at our institution. All donors were ABO compatible with a good human leukocyte antigen match. A segment of 180 to 200 cm of ileum was harvested and transplanted with its vascular pedicle constituted by the ileocolic artery and vein. The grafts were transplanted with a short cold and warm ischemia time. The immunosuppression regimen consisted of oral FK-506, prednisone, and intravenous induction with atgam. Serial biopsies of the intestinal grafts were performed to evaluate rejection or viral infections. The postoperative course was uneventful for all donors. All of the recipients are currently alive and well. Two of three patients are off total parenteral nutrition and tolerating an oral diet with no limitations on daily activity. In the third patient, the graft was removed 6 weeks after transplantation. At the time of enterectomy, no technical or immunologic complications were documented. Absorption tests for D-xylose and fecal fat studies were performed showing functional adaptation of the segmental graft. All biopsies were negative for acute rejection. A well-matched segmentai ileal graft from a living donor can provide complete rehabilitation for patients with short bowel syndrome. Our initial experience suggests that the risk of acute rejection and infection is greatly reduced compared to cadaveric bowel transplantation. Further clinical application of this procedure is warranted.

Total Pancreatectomy With Islet Autotransplantation: Summary of an NIDDK Workshop

A workshop sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases focused on research gaps and opportunities in total pancreatectomy with islet autotransplantation (TPIAT) for the management of chronic pancreatitis. The session was held on July 23, 2014 and structured into 5 sessions: (1) patient selection, indications, and timing; (2) technical aspects of TPIAT; (3) improving success of islet autotransplantation; (4) improving outcomes after total pancreatectomy; and (5) registry considerations for TPIAT. The current state of knowledge was reviewed; knowledge gaps and research needs were specifically highlighted. Common themes included the need to identify which patients best benefit from and when to intervene with TPIAT, current limitations of the surgical procedure, diabetes remission and the potential for improvement, opportunities to better address pain remission, GI complications in this population, and unique features of children with chronic pancreatitis considered for TPIAT. The need for a multicenter patient registry that specifically addresses the complexities of chronic pancreatitis and total pancreatectomy outcomes and postsurgical diabetes outcomes was repeatedly emphasized.

Risk factors for failure to meet listing requirements in liver transplant candidates with alcoholic cirrhosis.

Background. The majority of liver transplant centers require a 6-month abstinence period before listing candidates for liver transplantation with alcoholic cirrhosis and a persistent sobriety thereafter. We attempted to identify risk factors for failure to comply with these requirements. Methods. Ninety-nine consecutive patients with alcoholic cirrhosis were referred for liver transplant evaluation between September 1996 and May 1998. The mean age was 49 years, 74% were male, and 54% were hepatitis C virus positive. To be listed, patients had to meet the following requirements. All patients received extensive psychosocial evaluations and were frequently monitored with random urine and blood alcohol tests; patients found positive were excluded or removed from the liver transplant waiting list. Detailed patient information was entered into a computerized database, and 36 discreet variables were analyzed in relation to success (patient listed and remained on the list) or failure (not listed or removed from the list based on noncompliance). Results. Forty-nine patients were successfully listed. Nineteen received a transplant, with a 95% 1-year patient and graft survival rate and 21% alcohol relapse rate after transplantation. Twenty-two patients had either medical contraindication and/or died before transplant listing. Twenty-four patients were never listed and four were removed from the list due to recurrent alcoholism, for a total of 28 failures. Our statistical analysis identified five significant risk factors for failure: (I) living arrangement (alone/family versus community/friend), P =0.006; (II) history of suicide ideation, P =0.03; (III) history of previous alcohol-related hospitalization, P =0.01; (IV) lack of previous alcoholic rehabilitation before transplant evaluation, P =0.001; and (V) failure to accept further alcoholic rehabilitation before orthotopic liver transplantation, P =0.01. Conclusions. Our experience confirms that transplantation can be extremely successful in properly selected patients with alcoholic cirrhosis. We identified several predictive psychosocial factors of early alcoholic recidivism in transplant candidates.