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Articles by Barbara Barbaro in JoVE
Other articles by Barbara Barbaro on PubMed
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Dopaminergic Inhibition of Secretin-stimulated Choleresis by Increased PKC-gamma Expression and Decrease of PKA Activity
American Journal of Physiology. Gastrointestinal and Liver Physiology.
Apr, 2003 |
Pubmed ID: 12505882 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 Ca(2+)-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 Ca(2+)-dependent PKC-gamma but not other PKC isoforms. The dopaminergic nervous system counterregulates secretin-stimulated ductal secretion in experimental cholestasis.
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Alpha-1 Adrenergic Receptor Agonists Modulate Ductal Secretion of BDL Rats Via Ca(2+)- and PKC-dependent Stimulation of CAMP
Hepatology (Baltimore, Md.).
Nov, 2004 |
Pubmed ID: 15486932 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).
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Administration of R-VEGF-A Prevents Hepatic Artery Ligation-induced Bile Duct Damage in Bile Duct Ligated Rats
American Journal of Physiology. Gastrointestinal and Liver Physiology.
Aug, 2006 |
Pubmed ID: 16574985 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.
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Estrogens and Insulin-like Growth Factor 1 Modulate Neoplastic Cell Growth in Human Cholangiocarcinoma
The American Journal of Pathology.
Sep, 2006 |
Pubmed ID: 16936263 We investigated the expression of estrogen receptors (ERs), insulin-like growth factor 1 (IGF-1), and IGF-1R (receptor) in human cholangiocarcinoma and cholangiocarcinoma cell lines (HuH-28, TFK-1, Mz-ChA-1), evaluating the role of estrogens and IGF-1 in the modulation of neoplastic cell growth. ER-alpha, ER-beta, IGF-1, and IGF-1R were expressed (immunohistochemistry) in all biopsies (18 of 18) of intrahepatic cholangiocarcinoma. ER-alpha was expressed (Western blot) only by the HuH-28 cell line (intrahepatic cholangiocarcinoma), whereas ER-beta, IGF-1, and IGF-1R were expressed in the three cell lines examined. In serum-deprived HuH-28 cells, serum readmission induced stimulation of cell proliferation that was inhibited by ER and IGF-1R antagonists. 17beta-Estradiol and IGF-1 stimulated proliferation of HuH-28 cells to a similar extent to that of MCF7 (breast cancer) but greater than that of TFK-1 and Mz-ChA-1, inhibiting apoptosis and exerting additive effects. These effects of 17beta-estradiol and IGF-1 were associated with enhanced protein expression of ER-alpha, phosphorylated (p)-ERK1/2 and pAKT but with decreased expression of ER-beta. Finally, transfection of IGF-1R anti-sense oligonucleotides in HuH-28 cells markedly decreased cell proliferation. In conclusion, human intrahepatic cholangiocarcinomas express receptors for estrogens and IGF-1, which cooperate in the modulation of cell growth and apoptosis. Modulation of ER and IGF-1R could represent a strategy for the management of cholangiocarcinoma.
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Human Islet Isolation Outcomes from Pancreata Preserved with Histidine-Tryptophan Ketoglutarate Versus University of Wisconsin Solution
Transplantation.
Oct, 2006 |
Pubmed ID: 17038916 This study was designed to compare Histadine-Tryptophan-Ketogluterate (HTK) with University of Wisconsin (UW) solution. Pancreata from extended criteria donors were flushed and transported with HTK (n=41) or UW (n=45). Isolation outcomes were determined by islet yields, viability and in vitro and in vivo function. Final yields were similar between two groups (HTK: 383,085 vs. UW: 328,514 EIN, P=0.14). In the HTK group, 63.4% (26/41) of isolations resulted in a yield of over 300,000, and in the UW group this was achieved in 46.7% (21/45; P=0.12). Viability results were similar (HTK: 82.9 vs. UW: 82.7%, P=0.93). Stimulation index in the HTK and UW groups were comparable (5.28 vs. 4.91, P=0.62). Ten out of 41 islet preparations in HTK and 4 of 45 in UW group were suitable for clinical transplantation (P=0.05). Our study shows HTK is equivalent to UW solution in the preservation of pancreata for islet isolation.
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Improved Human Pancreatic Islet Purification with the Refined UIC-UB Density Gradient
Transplantation.
Nov, 2007 |
Pubmed ID: 17998877 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.
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Encapsulation of Human Islets in Novel Inhomogeneous Alginate-ca2+/ba2+ Microbeads: in Vitro and in Vivo Function
Artificial Cells, Blood Substitutes, and Immobilization Biotechnology.
2008 |
Pubmed ID: 18925451 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.
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Highly Purified Versus Filtered Crude Collagenase: Comparable Human Islet Isolation Outcomes
Cell Transplantation.
Mar, 2011 |
Pubmed ID: 21396158 This study was designed to retrospectively compare the impact of crude Sigma V collagenase (Sigma V, n=52) with high-purified Serva NB1 collagenase (Serva NB1, n=42) on human islet isolation outcomes. A three-step filtration was applied to the crude Sigma V to remove endotoxin contamination and impurities; in addition, this process was used as a lot prescreening tool. Isolation outcomes were determined by digestion efficacy, islet yields, purity, viability, glucose-stimulated insulin release, and endotoxin content. The digestion efficacy between Sigma V and Serva NB1 was statistically significant (Sigma V: 64.71% vs. Serva NB1: 69.71%, p=0.0014). However, the islet yields were similar (Sigma V: 23422.58 vs. Serva NB1: 271097 IEq, p=0.23) between groups. There was no significant purity difference observed in fractions with purities greater than 75%. Viability (Sigma V: 93.3% vs. Serva NB1: 94.8%, p=0.061) and stimulation indexes (Sigma V: 3.41 vs. Serva NB1: 2.74, p=0.187) were also similar between the two groups. The impact of cold ischemia and age on the isolation outcome in the Sigma V group was comparable to the Serva NB1 group. The endotoxin content of the final products in the filtered Sigma V group was significantly less than that in the high-purified Serva NB1 group (0.022 EU/ml vs. 0.052 EU/ml, p=0.003). Additionally, in the Sigma V group there was minimal lot to lot variation and no significant loss of enzymatic activity after filtration. These findings indicate that the use of Sigma V or other crude enzyme blends for research pancreata is warranted to reduce isolation costs and increase the amount of islets available for critical islet research. These findings also validate the need for a systematic enzyme analysis to resolve these inconsistencies in overall enzyme quality once and for all.
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