Search

Nonalcoholic fatty liver disease (NAFLD) is a multifaceted disorder that ranges from simple fatty liver to nonalcoholic steatohepatitis (NASH) with or without fibrosis, which may evolve toward cirrhosis and hepatocellular carcinoma. It is currently considered a "global" and "epidemic" disease, whose prevalence is progressively increasing even in pediatric age. The incidence of NAFLD is very high in overweight/obese children, and a greater risk of disease progression is associated with severe obesity, highlighting the role of nutrition. To date, for NAFLD, there are few guidelines for diagnostic and follow-up methods, and scarce validated protocols for treatment. The initial indications consist of gradual weight loss and regular exercise, but in children, the difficulty of adhering to long-term behavioral changes makes this approach limited. The purpose of this narrative review is to examine the mechanism underlying the pathogenic mechanisms that lead to NAFLD in children, with a major focus on the role of nutrition. Because this is particularly relevant in light of the absence of pharmacological treatments suitable for children, we also overview clinical studies on the potential effects of nutritional supplementations, including vitamins, docosahexaenoic acid, and probiotics.in children. To this aim, updated search was conducted on PubMed and clinicaltrials.gov databases. Future research should consider additional clinical studies in pediatric NAFLD patients to validate the benefits of dietary supplements and to define the appropriate dosage and duration for intervention. Furthermore, experimental studies with -omics approaches could be helpful to deepen the related mechanisms and to search for a possible optimal supplement combination against NAFLD in children.

Nonalcoholic fatty liver disease (NAFLD) ranges from simple steatosis to steatohepatitis, which may progress to fibrosis, and cirrhosis, leading eventually to hepatocarcinoma development. Recently, cases of hepatocarcinoma have been diagnosed in steatotic patients without nonalcoholic steatohepatitis (NASH) and cirrhosis. The p53 protein, besides its function as tumor suppressor, is emerging as an important regulator of cellular metabolism, but its role in steatosis remains unclear. We induced steatosis in HepG2 (wt-p53) and Huh7.5.1 (Y220C-mutant p53) cells using free fatty acids. We observed a different modulation of p53, different intracellular lipid content, and similar down-regulation of the de novo lipid synthesis genes but opposite modulation of the fatty acid β-oxidation pathway between HepG2 and Huh7.5.1. Accordingly, we found a diverse amount of apoptosis and reactive oxygen species between the two cell lines. Transfection of the wt-p53 in Huh7.5.1 cells reverted the different lipid metabolism behavior observed in these cells. In conclusion, unlike the wt-p53, the Y220C mutant provides a specific protection against steatosis and potentially against its progression. Our findings highlight for the first time an unknown role of a p53 mutant in the setting of steatosis. Being this mutation very frequent in human cancers, this study could be a breakthrough in explaining the occurrence of hepatocarcinoma in steatotic patients without NASH and cirrhosis.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excess lipids in hepatocytes, due to excessive fatty acid influx from adipose tissue, de novo hepatic lipogenesis, in addition to excessive dietary fat and carbohydrate intake. Chronic hepatic lipid overload induces mitochondrial oxidative stress and cellular damage leading the development of NAFLD into a more severe liver disease condition, non-alcoholic steato-hepatitis (NASH). In turn, this can progress to cirrhosis and hepatocellular carcinoma (HCC). Among others, copper is one of the main bio-metals required for the preponderance of the enzymes involved in physiological redox reactions, which primarily occurs during mitochondrial respiration. Thus, copper homeostasis could be considered a target point for counteracting the progression of NAFLD. Accordingly, many diseases are correlated to unbalanced copper levels and, actually, some clinical trials are examining the use of copper chelating agents. Currently, no pharmacological interventions are approved for NAFLD, but nutritional and lifestyle modifications are always recommended. Fittingly, antioxidant food agents recognized to improve NAFLD and its complications have been described in the literature to bind copper. Therefore, this review describes the role of nutrition in the development and progression of NAFLD with a particular focus on copper and copper-binding antioxidant compounds against NAFLD.

Ambulatory blood pressure monitoring (ABPM) is a helpful tool to comprehensively identify and diagnose arterial hypertension. Moreover, it allows to better identify alterations in the circadian BP profile, as the nocturnal "nondipping" status, characterized by a lack of the physiological 10% night BP reduction and associated with a greater risk of target organ damage. However, ABPM has some limitations such as restricted availability, discomfort, particularly at night, cost implications, and reproducibility.Aim of the study was evaluate if the "nondipping" phenomenon may be related to low degree of tolerance to ABPM. Additionally, to determine whether self-reported events of sleep disorders and nighttime urinations may affect the "nondipping" status.From January 2013 to December 2015, we consecutively evaluated 1046 patients with arterial hypertension, performing ABPM, considering a tolerance index calculated on the basis of the patients' responses to a questionnaire.Thirty-eight out of 1046 patients showed complete lack of tolerance to the instrument during the day, whilst 126 during the night. There were no statistically significant differences in daytime and nighttime values of tolerance to the instrument between "dippers" and "nondippers," between "extreme-dippers" and the remaining patients or between "reverse-dippers" and the remaining patients. There were no statistically significant differences in the number of nocturnal awakenings between the groups. However, we found that the number of awakenings followed by urination was higher in "nondipping" patients and in "reverse-dipping" patients compared to the other groups.We found that the poor tolerance to the instrument does not seem to influence the BP "dipping" phenomenon among hypertensive individuals. Moreover, we think that in the evaluation of the ABPM data, factors, such as nocturnal urination and sleep disorders, need to be carefully taken into account, since may lead to a higher incidence of "nondipping" pattern.

This study investigates manufacturing procedures that affect islet isolation outcomes from donor pancreata standardized by the North American Islet Donor Score (NAIDS). Islet isolations performed at the University of Illinois, Chicago, from pancreata with NAIDS ≥65 were investigated. The research cohort was categorized into two groups based on a postpurification yield either greater than (group A) or less than (group B) 400,000 IEQ. Associations between manufacturing procedures and islet isolation outcomes were analyzed using multivariate logistic or linear regressions. A total of 119 cases were retrieved from 630 islet isolations performed since 2003. Group A is composed of 40 cases with an average postpurified yield of 570,098 IEQ, whereas group B comprised 79 cases with an average yield of 235,987 IEQ. One third of 119 cases were considered successful islet isolations that yielded >400,000 IEQ. The prepurified and postpurified islet product outcome parameters were detailed for future reference. The NAIDS (>80 vs. 65–80) [odds ratio (OR): 2.91, 95% confidence interval (CI): 1.27–6.70], cold ischemic time (≤10 vs. >10 h) (OR: 3.68, 95% CI: 1.61–8.39), and enzyme perfusion method (mechanical vs. manual) (OR: 2.38, 95% CI: 1.01–5.56) were independent determinants for postpurified islet yield ≥400,000 IEQ. The NAIDS (>80, p < 0.001), cold ischemic time (≤10 h, p < 0.05), increased unit of collagenase ( p < 0.01), and pancreatic duct cannulation time (

This prospective phase 1/2 trial investigated the safety and reproducibility of allogeneic islet transplantation (Tx) in type I diabetic (T1DM) patients and tested a strategy to achieve insulin-independence with lower islet mass. Ten C-peptide negative T1DM subjects with hypoglycemic unawareness received 1-3 intraportal allogeneic islet Tx and were followed for 15 months. Four subjects (Group 1) received the Edmonton immunosuppression regimen (daclizumab, sirolimus, tacrolimus). Six subjects (Group 2) received the University of Illinois protocol (etanercept, exenatide and the Edmonton regimen). All subjects became insulin- independent. Group 1 received a mean total number of islets (EIN) of 1460 080 +/- 418 330 in 2 (n = 2) or 3 (n = 2) Tx, whereas Group 2 became insulin- independent after 1 Tx (537 495 +/- 190 968 EIN, p = 0.028). All Group 1 subjects remained insulin free through the follow-up. Two Group 2 subjects resumed insulin: one after immunosuppression reduction during an infectious complication, the other with exenatide intolerance. HbA1c reached normal range in both groups (6.5 +/- 0.6 at baseline to 5.6 +/- 0.5 after 2-3 Tx in Group 1 vs. 7.8 +/- 1.1 to 5.8 +/- 0.3 after 1 Tx in Group 2). HYPO scores markedly decreased in both groups. Combined treatment of etanercept and exenatide improves islet graft function and facilitates achievement of insulin-independence with less islets.

Providing sufficient islet mass is important for successful islet transplantation. Apoptosis plays a major role in post-isolation islet cell death, and prevention of apoptosis could improve transplant outcomes. The purpose of this study was to determine whether increased concentration of human albumin (HA) in pre-transplantation culture of human islets would reduce apoptosis. Human islets were cultured in CMRL with 1.5 or 5% of HA for 24 h and apoptosis was evaluated indirectly by measuring caspase 3 activity and tetramethylrhodamine-ethyl-ester (TMRE) in dissociated islets. Islet function and viability were evaluated. Islets cultured in higher albumin concentration presented with lower caspase 3 activity (43.9 +/- 3.9 vs. 67.4 +/- 11.1, p = 0.011), and had increased insulin secretory capacity (Stimulation index 3.76 +/- 0.91 vs 1.23 +/- 0.21, p = 0.023). We conclude that an increase in albumin concentration can prevent apoptosis in isolated human islets. These findings may have implications for islet transplant outcomes.

Selection of an optimal donor pancreas is the first key task for successful islet isolation. We conducted a retrospective multicenter study in 11 centers in North America to develop an islet donor scoring system using donor variables. The data set consisting of 1,056 deceased donors was used for development of a scoring system to predict islet isolation success (defined as postpurification islet yield >400,000 islet equivalents). With the aid of univariate logistic regression analyses, we developed the North American Islet Donor Score (NAIDS) ranging from 0 to 100 points. The c index in the development cohort was 0.73 (95% confidence interval 0.70–0.76). The success rate increased proportionally as the NAIDS increased, from 6.8% success in the NAIDS < 50 points to 53.7% success in the NAIDS ≥ 80 points. We further validated the NAIDS using a separate set of data consisting of 179 islet isolations. A comparable outcome of the NAIDS was observed in the validation cohort. The NAIDS may be a useful tool for donor pancreas selection in clinical practice. Apart from its utility in clinical decision making, the NAIDS may also be used in a research setting as a standardized measurement of pancreas quality.

The hepatic artery, through the peribiliary plexus, nourishes the intrahepatic biliary tree. During obstructive cholestasis, the nutritional demands of intrahepatic bile ducts are increased as a consequence of enhanced proliferation; in fact, the peribiliary plexus (PBP) displays adaptive expansion. The effects of hepatic artery ligation (HAL) on cholangiocyte functions during cholestasis are unknown, although ischemic lesions of the biliary tree complicate the course of transplanted livers and are encountered in cholangiopathies. We evaluated the effects of HAL on cholangiocyte functions in experimental cholestasis induced by bile duct ligation (BDL). By using BDL and BDL + HAL rats or BDL + HAL rats treated with recombinant-vascular endothelial growth factor-A (r-VEGF-A) for 1 wk, we evaluated liver morphology, the degree of portal inflammation and periductular fibrosis, microcirculation, cholangiocyte apoptosis, proliferation, and secretion. Microcirculation was evaluated using a scanning electron microscopy vascular corrosion cast technique. HAL induced in BDL rats 1) the disappearance of the PBP, 2) increased apoptosis and impaired cholangiocyte proliferation and secretin-stimulated ductal secretion, and 3) decreased cholangiocyte VEGF secretion. The effects of HAL on the PBP and cholangiocyte functions were prevented by r-VEGF-A, which, by maintaining the integrity of the PBP and cholangiocyte proliferation, prevents damage of bile ducts following ischemic injury.

Background/Aims We evaluated the role and mechanisms by which the GH/IGF1 axis modulates cholangiocyte proliferation. Methods GH-receptors (GH-R), IGF1, IGFBP3 (binding protein 3), IGF1-R and receptor substrates (IRS) were evaluated in cholangiocytes of normal or bile duct-ligated (BDL) rat livers. The effects of GH and IGF1 on proliferation of normal quiescent cholangiocytes and the transduction pathways involved were investigated. Results IGF1, GH-R, IGF1-R, IRS-1/2 were expressed in normal cholangiocytes and overexpressed in cholangiocytes proliferating after BDL which also secrete IGF1 in a higher amount than normal cells. IGFBP3, which may counter-regulate IGF1 effects, was decreased in BDL cholangiocytes. IGF1 promoted cholangiocyte proliferation in association with overexpression of p-IGF1R, IRS1, IRS-2, p-ERK1/2 and p-AKT. GH induced IGF1 expression and release in isolated cholangiocytes, and reproduced the effects of IGF1 but GH effects were abolished by IGF1-R blocking antibody, suggesting IGF1 as a mediator of GH. Finally, IGF1 and 17β-estradiol reciprocally potentiated their proliferative effects on cholangiocytes, and by interacting at both receptor and post-receptor levels. Conclusions Cholangiocytes respond to GH with production and release of IGF1 that modulates cell proliferation by transduction pathways involving IGF1-R, IRS1/2 and both ERK and PI3-kinase pathways. The biliary epithelium is a target of GH/IGF1 liver axis.

Acetylcholine potentiates secretin-stimulated ductal secretion by Ca(2+)-calcineurin-mediated modulation of adenylyl cyclase. D2 dopaminergic receptor agonists inhibit secretin-stimulated ductal secretion via activation of protein kinase C (PKC)-gamma. No information exists regarding the effect of adrenergic receptor agonists on ductal secretion in a model of cholestasis induced by bile duct ligation (BDL). We evaluated the expression of alpha-1A/1C, -1beta and beta-1 adrenergic receptors in liver sections and cholangiocytes from normal and BDL rats. We evaluated the effects of the alpha-1 and beta-1 adrenergic receptor agonists (phenylephrine and dobutamine, respectively) on bile and bicarbonate secretion and cholangiocyte IP(3) and Ca(2+) levels in normal and BDL rats. We measured the effect of phenylephrine on lumen expansion in intrahepatic bile duct units (IBDUs) and cyclic adenosine monophosphate (cAMP) levels in cholangiocytes from BDL rats in the absence or presence of BAPTA/AM and Gö6976 (a PKC-alpha inhibitor). We evaluated if the effects of phenylephrine on ductal secretion were associated with translocation of PKC isoforms leading to increased protein kinase A activity. Alpha-1 and beta-1 adrenergic receptors were present mostly in the basolateral domain of cholangiocytes and, following BDL, their expression increased. Phenylephrine, but not dobutamine, increased secretin-stimulated choleresis in BDL rats. Phenylephrine did not alter basal but increased secretin-stimulated IBDU lumen expansion and cAMP levels, which were blocked by BAPTA/AM and Go6976. Phenylephrine increased IP(3) and Ca(2+) levels and activated PKC-alpha and PKC-beta-II. In conclusion, coordinated regulation of ductal secretion by secretin (through cAMP) and adrenergic receptor agonist activation (through Ca(2+)/PKC) induces maximal ductal bicarbonate secretion in liver diseases. (Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Background & Aims: We tested the hypothesis that during bile duct obstruction, increased biliary bile acids trigger cholangiocyte proliferation and secretion by a phosphatidylinositol 3-kinase (PI3-K)–dependent pathway. Methods: In bile duct–incannulated (BDI) rats, bile duct obstruction present for 7 days was relieved for 24 hours by external bile drainage. During the 24-hour drainage period, animals received either Krebs Ringer Henseleit (the bile-depleted group), or sodium taurocholate (the bile-depleted, taurocholate-infused group). We evaluated cholangiocyte proliferation and secretin-stimulated ductal secretion. Apical bile acid transporter (ABAT) expression and bile acid transport activity was determined. In pure preparations of cholangiocytes, we examined the effect of taurocholate (in the absence or presence of wortmannin or PI 3,4-bisphosphate the lipid product of PI3-K) on cholangiocyte proliferation and secretin-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) levels. Results: Bile depletion reduced cholangiocyte proliferation and secretin-stimulated ductal secretion and ABAT expression and bile acid transport activity compared with 1-week BDI control rats. In bile-depleted, taurocholate-infused rats, cholangiocyte proliferation and secretion and ABAT expression and bile acid transport activity were maintained at levels similar to those seen in BDI control rats. In vitro, taurocholate stimulation of DNA replication and secretin-stimulated cAMP levels was blocked by wortmannin. The inhibitory effect of wortmannin on taurocholate stimulation of cholangiocyte proliferation and secretion was prevented by PI 3,4-bisphosphate. Conclusions: Bile acid uptake by ABAT and the PI3-K pathway are important for bile acids to signal cholangiocyte proliferation. In bile duct obstruction, increased biliary bile acid concentration and ABAT expression initiate increased cholangiocyte proliferation and secretion. GASTROENTEROLOGY 2002;123:1226-1237

Eight manufacturing facilities participating in the National Institutes of Health-sponsored Clinical Islet Transplantation (CIT) Consortium jointly developed and implemented a harmonized process for the manufacture of allogeneic purified human pancreatic islet (PHPI) product evaluated in a phase 3 trial in subjects with type 1 diabetes. Manufacturing was controlled by a common master production batch record, standard operating procedures that included acceptance criteria for deceased donor organ pancreata and critical raw materials, PHPI product specifications, certificate of analysis, and test methods. The process was compliant with Current Good Manufacturing Practices and Current Good Tissue Practices. This report describes the manufacturing process for 75 PHPI clinical lots and summarizes the results, including lot release. The results demonstrate the feasibility of implementing a harmonized process at multiple facilities for the manufacture of a complex cellular product. The quality systems and regulatory and operational strategies developed by the CIT Consortium yielded product lots that met the prespecified characteristics of safety, purity, potency, and identity and were successfully transplanted into 48 subjects. No adverse events attributable to the product and no cases of primary nonfunction were observed.

This report summarizes a 5-year phase 1/2 allogeneic islet transplantation clinical trial conducted at the University of Illinois at Chicago (UIC). Ten patients were enrolled in this single center, open label, and prospective trial in which patients received 1–3 transplants. The first four subjects underwent islet transplantation with the Edmonton immunosuppressive regimen and the remaining six subjects received the UIC immunosuppressive protocol (Edmonton plus etanercept and exenatide). All 10 patients achieved insulin independence after 1–3 transplants. At five years of follow-up, six of the initial 10 patients were free of exogenous insulin. During the follow-up period, 7 of the 10 patients maintained positive C-peptide levels and a composite hypoglycemic (HYPO) score of 0. Most patients maintained HbA1c levels < 6.0% (42.1 mmol/mol) and a significantly improved β-score. In conclusion, this study demonstrated long-term islet graft function without using T-cell depleting induction, with an encouraging outcome that includes 60% of patients remaining insulin independent after five years of initial transplantation.

Hepatitis C virus (HCV) infection is associated with hepatic and extrahepatic manifestations, including immunological disorders. Chronic Hepatitis C (CHC) is often characterized by cholesterol and lipid metabolism alterations, leading to hepatic steatosis. Cholesterol metabolism, in fact, is crucial for the viral life cycle. Recent works described that a higher dietary cholesterol intake is associated with the progression of HCV-related liver disease. CHC patients have increased levels of T helper 17 (Th17)-cells, a lymphocytic population involved in the pathogenesis of liver inflammation and autoimmune hepatitis. The balance between Th17 and regulatory T (Treg) cells is crucial for chronic inflammation and autoimmunity. Th17-cell differentiation is deeply influenced by the activation LXRs, nuclear receptors modulating cholesterol homeostasis. Moreover, HCV may affect these nuclear receptors, and cholesterol metabolism, through both direct and indirect mechanisms. On these bases, we hypothesized that modulation of cholesterol levels through Normocaloric Low Cholesterol Diet (NLCD) may represent an innovative strategy to reduce the progression of HCV infection, through the modulation of peripheral Th17/Treg balance. To this end, we performed a pilot study to investigate whether a Normocaloric Low Cholesterol Diet may be able to modulate Th17/Treg balance in patients affected by chronic HCV infection. After 30 days of NLCD CHC patients showed a significant reduction in Th17 cells frequency, which correlated with strong reduction of IL-17 and IL-22 serum levels. At the same time, we appreciated an increase in the percentage of Treg cells, thus improving Treg/Th17balance. Moreover, we observed an increased expression of LXRs and their target genes: SREBP-1c and ABCA-1. In conclusion, NLCD finely regulates Th17/Treg balance, improving immune system response in CHC patients. This study could pave the way for new treatments of CHC patients, suggesting that change in lifestyle could support the management of these patients, promoting well-being and possibly hindering disease progression. Trial Registration ClinicalTrials.gov NCT02038387

The liver is crucial for human life, and the health of this organ often mirrors the health of the individual. The liver can be the target of several diseases, the most prevalent of which, as a consequence of development and changes in human lifestyles, is the nonalcoholic fatty liver disease (NAFLD). NAFLD is a multifactorial disease that embraces many histo-pathologic conditions and is highly linked to metabolic derangements. Technological progress and industrialization have also had the consequence of releasing pollutants in the environment, for instance pesticides or solvents, as well as by-products of discharge, such as the particulate matter. In the last decade, a growing body of evidence has emerged, shedding light on the potential impact of environmental pollutants on liver health and, in particular, on NAFLD occurrence. These contaminants have a great steatogenic potential and need to be considered as tangible NAFLD risk factors. There is an urgent need for a deeper comprehension of their molecular mechanisms of action, as well as for new lines of intervention to reduce their worldwide diffusion. This review wishes to sensitize the community to the effects of several environmental pollutants on liver health.

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37°C overnight culture before islets were transplanted or used for research.

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37 degrees C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4 degrees C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Microencapsulation may allow for immunosuppression-free islet transplantation. Herein we investigated whether human islets can be shipped safely to a remote encapsulation core facility and maintain in vitro and in vivo functionality. In non-encapsulated islets before and encapsulated islets after shipment, viability was 88.3+/-2.5 and 87.5+/-2.7% (n=6, p=0.30). Stimulation index after static glucose incubation was 5.4+/-0.5 and 6.3+/-0.4 (n=6, p=0.18), respectively. After intraperitoneal transplantation, long-term normoglycemia was consistently achieved with 3,000, 5,000, and 10,000 IEQ encapsulated human islets. When transplanting 1,000 IEQ, mice returned to hyperglycemia after 30-55 (n=4/7) and 160 days (n=3/7). Transplanted mice showed human oral glucose tolerance with lower glucose levels than non-diabetic control mice. Capsules retrieved after transplantation were intact, with only minimal overgrowth. This study shows that human islets maintained the viability and in vitro function after encapsulation and the inhomogeneous alginate-Ca(2+)/Ba(2+) microbeads allow for long-term in vivo human islet graft function, despite long-distance shipment.

Human islet isolation outcomes were compared between two purification methods; 32 pancreases were processed by conventional Biocoll purification method (SM, standard method) and 132 pancreases by a refined University of Illinois at Chicago UW/Biocoll method (UIC-UB). There was no difference in donor characteristics between the two study groups. The prepurification equivalent islet number was similar between the groups (359,425+/-40,794 equivalent islet number in SM vs. 370,682+/-17,579 in UIC-UB). SM purified islets were mostly collected in only 2 of 12 fractions (68.9% and 36.3% purity). With the UIC-UB, highly purified islets were collected in 6 of 12 separate fractions (fractions 3-8 with purity of 84.8%, 82.5%, 72.0%, 59.3%, 46.8%, and 36.2%). UIC-UB yielded significantly greater islet yield compared with SM (368,419+/-18,245 vs. 260,908+/-37,835, P=0.017). Islet recovery rate was superior in UIC-UB (84.9% vs. 64.5%, P=0.04). Our study demonstrates a superior recovery of highly pure human pancreatic islets after purification using the refined method of UIC-UB gradient.

During isolation, islets are exposed to warm ischemia. In this study, intraductal administration of oxygenated polymerized, stroma-free hemoglobin-pyridoxalated (Poly SFH-P) was performed to improve O2 delivery. Rat pancreata subjected to 30-min warm ischemia were perfused intraductally with collagenase in oxygenated Poly SFH-P/RPMI or RPMI (control). PO2 was increased by Poly SFH-P (381.7 +/- 35.3 mmHg vs. 202.3 +/- 28.2, p = 0.01) and pH maintained within physiological range (7.4-7.2 vs. 7.1-6.6, p = 0.009). Islet viability (77% +/- 4.6 vs. 63% +/- 4.7, p = 0.04) was improved and apoptosis lower with Poly SFH-P (caspase-3: 34,714 +/- 2167 vs. 45,985 +/- 1382, respectively, p = 0.01). Poly SFH-P improved islet responsiveness to glucose as determined by increased intracellular Ca2+ levels and improved insulin secretion (SI 5.4 +/- 0.1 vs. 3.1 +/- 0.2, p = 0.03). Mitochondrial integrity was improved in Poly SFH-P-treated islets, which showed higher percentage change in membrane potential after glucose stimulation (14.7% +/- 1.8 vs. 9.8 +/- 1.4, respectively, p0.05). O2 delivery by Poly SFH-P did not increase oxidative stress (GSH 7.1 +/- 2.9 nm/mg protein for Poly SFH-P vs. 6.8 +/- 2.4 control, p = 0.9) or oxidative injury (MDA 1.8 +/- 0.9 nmol/mg protein vs. 6.2 +/- 2.4, p = 0.19). Time to reach normoglycemia in transplanted diabetic nude mice was shorter (1.8 +/- 0.4 vs. 7 +/- 2.5 days, p = 0.02), and glucose tolerance improved in the Poly SFH-P group (AUC 8106 +/- 590 vs. 10,863 +/- 946, p = 0.03). Oxygenated Poly SFH-P improves islet isolation and transplantation outcomes by preserving mitochondrial integrity.

Background & Aims: Vascular endothelial growth factor (VEGF) is secreted by several epithelia and modulates cellular functions by autocrine and paracrine mechanisms. The role of VEGF in cholangiocyte pathophysiology is unknown. We evaluated the role of VEGF in the regulation of cholangiocyte proliferation in rats that underwent bile duct ligation. Methods: The expression of VEGF-A and VEGF-C and their receptors in cholangiocytes from normal and BDL rats was evaluated. Normal or BDL rats were treated with recombinant-VEGF-A or recombinant-VEGF-C or anti-VEGF antibodies, and proliferation of cholangiocytes was evaluated in situ by morphometry and in vitro by proliferating cell nuclear antigen immunoblots and MTS assay. In vitro, normal rat cholangiocyte cultures were stimulated with r-VEGF-A or r-VEGF-C and proliferation and signal transduction were evaluated. Results: We found that (1) cholangiocytes express messenger RNA and protein for VEGF-A, VEGF-C, VEGF receptor 2 (VEGFR-2), and VEGF receptor 3 (VEGFR-3) and secrete VEGF; (2) secretion of VEGF and expression of VEGFR-2 and VEGFR-3 increases in BDL cholangiocytes; (3) blocking VEGF in vivo by anti–VEGF-A or anti–VEGF-C antibodies decreases cholangiocyte proliferation; (4) the in vivo administration of r-VEGF-A or r-VEGF-C induces cholangiocyte proliferation in normal rats; and (5) in vitro, VEGF-A increases normal rat cholangiocyte culture proliferation by activation of inositol 1,4,5-triphosphate/Ca 2+ /protein kinase C α and phosphorylation of Src/ERK1/2. Conclusions: Cholangiocytes secrete VEGF and express VEGFR-2 and VEGFR-3, all of which are amplified in BDL cholangiocytes. VEGF induces cholangiocyte proliferation by activation of inositol 1,4,5-triphosphate/[Ca 2+ ] i /protein kinase C α and phosphorylation of Src/ERK1/2. VEGF mediates the adaptive proliferative response of cholangiocytes to cholestasis.

Oxidative stress during islet isolation induces a cascade of events injuring islets and hampering islet engraftment. This study evaluated islet isolation and transplantation outcomes after intra-ductal glutamine administration. Human pancreata deemed unsuitable for pancreas or islet transplantation were treated with either a 5 mM solution of l-glutamine (n = 6) or collagenase enzyme alone (n = 6) through the main pancreatic duct. Islet yield, viability, in vitro function; markers of oxidative stress [malondialdehyde (MDA) and Glutathione (GSH)] and apoptosis were assessed. Islet yields were significantly increased in the glutamine group compared to controls (318, 559 +/- 25, 800 vs. 165, 582 +/- 39, 944 mean +/- SEM, p < 0.01). The amount of apoptotic cells per islet was smaller in the glutamine group than the control. The percentage of nude mice rendered normoglycemic with glutamine-treated islets was higher than the controls (83% n = 10/12 vs. 26% n = 6/23; p < 0.01), and the time to reach normoglycemia was decreased in the glutamine group (1.83 +/- 0.4 vs. 7.3 +/- 3 days; p < 0.01). Glutamine administration increased GSH levels (7.6 +/- 1.7 nmol/mg protein vs. 4.03 +/- 0.5 in control, p < 0.05) and reduced lipid-peroxidation (MDA 2.45 +/- 0.7 nmol/mg of protein vs. 6.54 +/- 1.7 in control; p < 0.05). We conclude that intra-ductal administration of glutamine reduces oxidative injury and apoptosis and improves islet yield and islet graft function after transplantation.

Lipoxin A4(LXA4) is a potent eicosanoid that inhibits IL-1β-induced activation of human fibroblast-like synoviocytes (FLS) via the LXA4receptor (ALXR). Serum amyloid A (SAA) is an acute phase reactant with cytokine-like properties. SAA has been shown to bind the same seven transmembrane G protein-coupled receptor ligated by LXA4. Here we compared the inflammatory responses of lipid (LXA4) and peptide (SAA) ligands in human FLS via the shared ALX and characterized their downstream signaling. LXA4induced stimulation of tissue inhibitors of metalloproteinase-2, whereas SAA induced interleukin-8 and matrix metalloproteinase-3 production. SAA up-regulated NF-kB and AP-1 DNA binding activity, while LXA4markedly inhibited these responses after IL-1β stimulation. A human IL-8 promoter luciferase construct was transfected into CHO cells stably expressing ALXR in order to determine the role of NF-kB and/or AP-1 in the regulation of IL-8 gene expression. The NF-kB pathway proved to be the preeminent for the biological responses elicited by both ligands. These findings suggest that two endogenous molecules, targeting a common receptor, could participate in the pathogenesis of inflammatory arthritis by differentially regulating inflammatory responses in tissues expressing the ALXR.

Background/Aims : Taurohyodeoxycholate (THDCA) and tauroursodeoxycholate (TUDCA) induce more bile flow per molecule excreted compared to endogenous bile acids. The aim of this study is to determine if the hypercholeretic effect of tauroursodeoxycholate or taurohyodeoxycholate in normal and bile duct ligated (BDL) rats is due to increased ductal secretion. Methods : Normal or BDL rats were infused with tauroursodeoxycholate or taurohyodeoxycholate and bile flow, bicarbonate, bile salt, cholesterol, and phospholipid secretion were measured. Cholangiocytes were stimulated with taurohyodeoxycholate or tauroursodeoxycholate, and secretin-stimulated secretion was measured. Results : Taurohyodeoxycholate and tauroursodeoxycholate increased bile flow more in BDL than normal rats. Tauroursodeoxycholate increased bicarbonate secretion more in BDL compared to normal rats. Taurohyodeoxycholate when infused with taurocholate increased bile flow (but not phospholipid excretion) to a greater degree in BDL compared to normal rats. Taurohyodeoxycholate and tauroursodeoxycholate decreased secretin-stimulated cholangiocyte secretion. Conclusions : Consistent with a ductal origin for bile acid-induced hypercholeresis, taurohyodeoxycholate and tauroursodeoxycholate produced a greater hypercholeresis in BDL than normal rats. Tauroursodeoxycholate- (but not taurohyodeoxycholate-) stimulated hypercholeresis is associated with increased HCO 3 − secretion. Tauroursodeoxycholate increases biliary HCO 3 − secretion by a mechanism unrelated to secretin-stimulated cholangiocyte secretion. Taurohyodeoxycholate-induced hypercholeresis in BDL rats is unrelated to enhanced phospholipid excretion.

We investigated the expression of glucocorticoid receptors (GcRs) in the intrahepatic biliary epithelium and the role of corticosteroids in the regulation of cholangiocyte secretion.GcR was studied by immunohistochemistry, reverse-transcription polymerase chain reaction, and Western blots. The effects of dexamethasone and budesonide on biliary bicarbonate excretion and H+/HCO3- transport processes were investigated in bile fistula rats, isolated intrahepatic bile duct units (IBDUs), and purified cholangiocytes.GcRs were expressed by rat cholangiocytes. Although acute administration of corticosteroids showed no effect, treatment for 2 days with dexamethasone or budesonide increased (P0.05) biliary bicarbonate concentration and secretion, which were blocked by the specific GcR antagonist, RU-486. IBDUs isolated from rats treated with dexamethasone or budesonide showed an increased (P0.05) activity of the Na+/H+ exchanger (NHE1 isoform) and Cl-/HCO3- exchanger (AE2 member), which was blocked by RU-486. Protein expression of NHE1 and AE2 and messenger RNA for NH1 but not AE2 were increased (P0.05) in isolated cholangiocytes by dexamethasone treatment.The intrahepatic biliary epithelium expresses GcR and responds to corticosteroids by increasing bicarbonate excretion in bile. This is caused by corticosteroid-induced enhanced activities and protein expression of transport processes driving bicarbonate excretion in the biliary epithelium.

Serum amyloid A (SAA) is an acute-phase protein and inflammatory marker of rheumatoid arthritis, which has recently become recognized as a cytokine-like reactant. SAA has been shown to bind to the ALXR (FPRL1/LXA4R) receptor and to elicit proinflammatory activities. Indeed, we have determined that SAA elicits the proinflammatory activation of human fibroblast-like synoviocytes, with a profile of activity similar to that of IL-1β. Uteroglobin is a ubiquitously expressed secretory protein with anti-inflammatory properties that shares specific sequence motifs with annexin-1, a steroid-induced anti-inflammatory peptide that is also capable of interacting with ALXR. Data obtained by flow cytometry, ELISA and plasmon resonance, using ALXR stably transfected CHO cells, suggest that uteroglobin can downmodulate SAA proinflammatory effects by specifically interfering at the ALXR receptor level. In agreement with the ability to bind to the same receptor used by SAA, a functional antagonism between uteroglobin and SAA was observed, with uteroglobin causing inhibition of SAA-induced release of both IL-8 and IL-6, and blockage of SAA-induced PLA2 activation. Beside the receptor antagonism between proinflammatory ALXR ligands (SAA, urokinase proteolytic plasminogen peptides [uPA], MK1, etc.) and anti-inflammatory ALXR ligands (LXA4, annexin-1, uteroglobin) so far identified, results also suggest that each of these mediators can modify ALXR expression levels as well as other genes. In fact, present studies suggest that SAA acts in a positive autoregulatory feedback mechanism by increasing SAA 1 + 2 expression, while maintaining unchanged levels of its constitutively expressed isoform SAA4, as demonstrated by RT-PCR. Following SAA treatment, upregulation is extended to the expression of the ALXR mRNA, a response common to LXA4 and IL-1β. The antagonistic activities of SAA, uPA, uteroglobin, annexin-1 and LXA4 might constitute a novel complex signaling mechanism by which these mediators sharing a common receptor could achieve opposing roles in the regulation of the joint inflammatory processes.

To determine the role and mechanisms of action by which dopaminergic innervation modulates ductal secretion in bile duct-ligated rats, we determined the expression of D1, D2, and D3 dopaminergic receptors in cholangiocytes. We evaluated whether D1, D2 (quinelorane), or D3 dopaminergic receptor agonists influence basal and secretin-stimulated choleresis and lumen expansion in intrahepatic bile duct units (IBDU) and cAMP levels in cholangiocytes in the absence or presence of BAPTA-AM, chelerythrine, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H7), or rottlerin. We evaluated whether 1) quinelorane effects on ductal secretion were associated with increased expression of Ca2+-dependent PKC isoforms and 2) increased expression of PKC causes inhibition of PKA activity. Quinelorane inhibited secretin-stimulated choleresis in vivo and IBDU lumen space, cAMP levels, and PKA activity in cholangiocytes. The inhibitory effects of quinelorane on secretin-stimulated ductal secretion and PKA activity were blocked by BAPTA-AM, chelerythrine, and H7. Quinelorane effects on ductal secretion were associated with activation of the Ca2+-dependent PKC-γ but not other PKC isoforms. The dopaminergic nervous system counterregulates secretin-stimulated ductal secretion in experimental cholestasis.