Intraportal Infusion Research Articles

CMV, EBV AND PARVOVIRUS STATUS IN PATIENTS WITH TYPE 1 DIABETES MELLITUS AWAITING ISLET CELL TRANSPLANTATION

P453 Background: CMV, EBV and Parvovirus (B19) status of transplant donors and recipients has important implications on post-transplantation antiviral therapy, morbidity, mortality, donor selection criteria and hospital stay. While relative risk for CMV serological positivity in patients with type 1 DM has been clearly established, no studies on CMV, EBV and B19 serological positivity have been reported. One patient who received a single islet cell infusion using a steroid free regimen at our institution developed B19 infection that led to severe anemia and graft failure. Immune Globulin (Gamimune) therapy was complicated by aseptic meningitis and conversion of immunosuppression failed to facilitate the clearance of the virus. This study compares the prevalence of positive CMV, EBV and B19 serology in type 1 diabetic patients listed for islet cell transplantation and tries to identify risk/protection factors for seropositivity in this group. Method: Serological status of CMV, EBV, and B19 was compared among type 1 diabetic patients screened for solitary islet cell transplantation at our institution. Age at onset of type 1 DM and time of screening, gender, HLA phenotype, ABO group, and auto-antibodies were investigated as risk factors. Results: CMV positivity was 39% in patients with type 1 diabetes (N=370), as compared to 98% for EBV (N=327; p<0.0001) and 64% for B19 (N=56; p<0.001). The mean age of patients was 40 +/-9.5 years, time of diabetes onset 14 +/-9.0 and duration 27 +/-10.4. Serostatus of CMV was not found to be significantly associated with serostatus of either EBV or B19 among these patients. CMV-negative patients were younger than CMV-positive patients at the time of screening (41.3 +/-9 vs. 45.3 +/-9 years; p<0.0001) and at the time of diabetes onset (13.6 +/- 8.5 vs. 15.7 +/- 10.4; p=0.03). The percentage of CMV positivity was higher in female than in male patients (69% vs. 62%; p=0.001). All other parameters studied had no significant impact on CMV, EBV and B19 positivity. Interestingly we have not observed CMV disease or infection despite positive donor to negative recipient combination in 22 of 29 subjects receiving intraportal islet infusions utilizing a steroid free regimen with daclizumab, sirolimus and sacrolimus in addition to gancyclovir or valgancyclovir prophylaxis for 90 days. Conclusions: These findings may suggest patients with type 1 diabetes either have factors that protect them from CMV primary infection, or are immunologically incompetent in their response to CMV infection and fail to produce detectable levels of anti-CMV antibodies. This peculiar characteristic is not observed within the same group of patients tested for EBV and B19 serology, demonstrating similar prevalence comparable to the general population. The discrepancy between CMV, EBV and B19 positivity in this group of potential transplant recipients and the donor population has important implications on tissue allocation as well as prophylactic/preemptive antiviral therapy in islet cell transplant recipients.

NEW STRATEGY FOR THE ABO-INCOMPATIBLE LIVING DONOR LIVER TRANSPLANTATION WITH ANTI-CD20 ANTIBODY (RITUXIMAB) AND PLASMA EXCHANGE

P520 Aims: The donor shortage problem encouraged us to perform living donor liver transplantation (LDLT) across the ABO blood barrier. The survival rate in ABO-incompatible transplantations was much poorer than in ABO-compatible recipients for the early experiences, but the introduction of novel immunosuppressive regimens and PE has yielded excellent results in ABO-incompatible transplantations. We have successfully performed 3 cases of ABO-incompatible LDLT by using rituximab. Methods: In our institution we experienced 10 ABO-incompatible recipients out of 79 LDLT and 3 out of 10 recipients were administered rituximab for rescue or prophylactic therapy. The age range of the 10 recipients was 7 months to 54 years, with median age of 12.8 years. IgM and IgG hemagglutinin titers were measured before and after Tx using serial dilution in saline. Pre-Tx PE was performed as necessary to maintain hemaggulutinin titers below 1:16 and post-Tx PE was performed when it was diagnosed as hyperacute rejection with high titers. Induction immunosuppression consisted of FK506, steroid and MMF. Results: Nine of the 10 recipients and all the 3 recipients who were administered rituximab were alive. Case1 : A 22 months old girl whose primary disease was BA was performed LDLT from her father (A to O). She was also performed PE for twice before Tx in order to reduce titers 16 or below. From POD 3 the titers and LFTs were elevated despite the PE. On POD 7 needle graft biopsy revealed the hyperacute rejection and she was treated by rituximab, DSG and steroid pulse therapy. Her titers and LFTs were improved drastically and she was discharged on POD 74. CD19 positive lymphocytes in her peripheral blood were less than 1% until 3 month after Tx. Currently, 22 months after Tx, she is in stable condition. Case2 : A 42 year old woman whose primary disease was PBC was performed LDLT from her brother in law (B to O). She was performed PE for 7 times before Tx because of the high titer (1:512) and was administered rituximab prophylactic just after Tx. She also had splenectomy and intrahepatic artery infusion of PGE1 and steroid for 2 weeks. One week after Tx the titers were elevated with slight deterioration of LFTs, steroid pulse with PE improved the LFTs. She was discharged on POD 102 due to biliary leakage. CD19 positive lymphocytes in her peripheral blood were less than 1% until 8 month after Tx. Currently, 12 months after Tx, she is in stable condition. Case3 : A 54 year old woman whose primary disease was LC(B) with HCC was performed LDLT from her son (A to O). She was performed PE for 3 times before Tx and was administered rituximab prophylactic just after Tx. Because her hepatic artery was dissected during the Tx, she had intraportal infusion of PGE1 and steroid for 2 weeks and she also had splenectomy. Until 1 month after Tx her LFTs was stable and no sings of rejection was occurred. CD19 positive lymphocytes in her peripheral blood were 6 % after 1 month. Conclusions: Rituximab has the possibility to become the new strategy for the ABO-incompatible liver transplantation.

REPEATED INTRAPORTAL WHOLE BONE MARROW CELL TRANSPLANTATION IN ANALBUMINEMIC RATS.

P683 Aims: Bone marrow cell (BMC) has been intensively investigated whether these cells could be used for the treatment of liver dysfunctions. However the effectivities of BMC transplantations is still not clear because survival period of transplanted cells were not long enough to support recipient liver functions. We have developed a novel technique which allows repeated infusion of BMC into the portal system via an indwelling catheter. In this study we have developed an effective intraportal transplantation protocol to extend survival time of transplanted cells. Methods: 6weeks old male Nagase Analbuminemic rats (NAR) were injected subcutaneously every 3.5 days with CCl4 dissolved in equal volume of olive oil at a dose of 2.0 ml/kg body weight for 8weeks. Repeated intraportal infusion of 5 x 10(6) rat BMC isolated from 6weeks old female enhanced green fluorescent protein transgenic (EGFP-tg)rat twice a week for the last 4weeks via an indwelling catheter connected to a subcutaneous port. Another NAR underwent repeated intraportal infusion of Dulbecco’s Modified Eagle’s Medium (DMEM). For assessment of liver function, Body and liver weight was measured and blood was obtained for total bilirubin, albumin, PT, transaminase and ammonia. We also evaluated the improvement of fibrosis by histology and repopulating cells by immunohistochemistry. Results: At 4 weeks after the start of BMC transplantation (BMT), we found no significant difference in their total body weight (BMT model: 170g, Control model: 166g). But their liver weight of BMT groups remarkably increased (BMT model: 14.7g, Control model: 11.2g). We also observed macroscopically different between them. Control group liver was white and atrophic, but the liver from BMT model was red and expanded. Microscopic studies revealed that the liver of rat receiving continuous BMT showed decreases in fibrosis, necrosis and inflammatory infiltration. And in the BMT rat, GFP positive cells were found around the portal vein. Prothrombin time(PT), total bilirubin, serum ammonia and transaminase were also significantly better in BMT model compared with control model. Conclusions: Repeated bone marrow cell transplantation via a portal catheter can decrease liver fibrosis and improve liver function of rats with liver cirrhosis and may be useful in the treatment of cirrhosis in humans.

LOGISTICS AND TRANSPLANT COORDINATION ACTIVITY IN THE GRAGIL SWISS-FRENCH MULTICENTER NETWORK OF ISLET TRANSPLANTATION

O454* Aims: Since the report of the Edmonton trial in 2000, increasing numbers of transplant centers are interested in implementing islet of Langerhans transplantation programs. Some of these centers have elected to associate with groups experienced in islet isolation, in multicenter networks such as the Swiss-French GRAGIL consortium. Methods: All pancreata offered to the University of Geneva Cell Isolation and Transplantation Center from hospitals within the GRAGIL network from 1.01.2002 to 31.12.2003 were reviewed. Organs accepted for processing were shipped by ambulance to Geneva. Islets were isolated using the automated Ricordi method. Islet preparations with a sufficient number of IEQ and meeting release criteria were attributed to the most suitable recipient in the centrally managed waiting list. Islet preparations were shipped by ambulance to the recipient hospital for percutaneous intraportal infusion. Pancreas offers and islet distribution were analyzed with respect to regions of origin and destination (Switzerland, and interregions II, III and IX in France). Results: Over the period of study, a total of 259 pancreata were offered for islet processing, from a total of 1304 cadaveric donors in the 4 regions (19.9%). Fifty-two patients were on the waiting list at any time during this 2-year period, 20 were transplanted (of which 15 became insulin independent) and 9 were removed from the list without transplantation. Percentage of organs offered varied in the range of 0.5%- 41% depending on region of origin, with a correlation with number of patients on the waiting list in each region. Of these, 103 (39.8%) were accepted for processing, 105 (40.5%) were rejected according to medical criteria, 41 (15.8%) for logistic reasons, and 10 (3.9%) for lack of potential recipient. Ninety-one pancreata were actually processed, resulting in 41 islet preparations being transplanted. Number of IEQ of transplanted preps was 380000 ± 106000 versus 175000 ± 116000 (p<0.0001) for non transplanted preps. Total cold ischemia time was 6 hours ± 2.1 for transplanted preps versus 6.75 hours ± 2.75 for non transplanted preps (ns). Conclusions: This study demonstrates that a high rate of pancreas offer, successful isolation and islet transplantation can be achieved in a multicenter network such as GRAGIL. Both the donor pool and the recipient population can be expanded with such an approach.

Hepatic failure at obstructive parasitogenic cholestasis

In Tomsk zonal hepatologic center 358 patients with obstructive parasitogenic jaundice have been operated (alveococcosis-based — 43, echinococcosis-based — 17, opisthorchosis-based — 298). Clinical presentations of hepatic failure before the surgery have been observed at 30,7% of patients, more often — under long and intensive cholestasis. Postsurgical hepatic failure has developed at 23,1%. Intraportal medicine infusions play an important role in prophylaxis and treatment of hepatic failure along with timely and optimal surgery. Infusions permit to decrease the hepatic failure progress probability from 60,6 to 30,5%, probability of suppurative complications from 10,3 to 3,1% and postsurgical lethality from 38,2 to 9,5%.

Vagal cooling and concomitant portal norepinephrine infusion do not reduce net hepatic glucose uptake in conscious dogs

We examined the role of efferent neural signaling in regulation of net hepatic glucose uptake (NHGU) in two groups of conscious dogs with hollow perfusable coils around their vagus nerves, using tracer and arteriovenous difference techniques. Somatostatin, intraportal insulin and glucagon at fourfold basal and basal rates, and intraportal glucose at 3.8 mg.kg(-1).min(-1) were infused continuously. From 0 to 90 min [period 1 (P1)], the coils were perfused with a 37 degrees C solution. During period 2 [P2; 90-150 min in group 1 (n = 3); 90-180 min in group 2 (n = 6)], the coils were perfused with -15 degrees C solution to eliminate vagal signaling, and the coils were subsequently perfused with 37 degrees C solution during period 3 (P3). In addition, group 2 received an intraportal infusion of norepinephrine at 16 ng.kg(-1).min(-1) during P2. The effectiveness of vagal suppression was demonstrated by the increase in heart rate during P2 (111 +/- 17, 167 +/- 16, and 105 +/- 13 beats/min in group 1 and 71 +/- 6, 200 +/- 11, and 76 +/- 6 beats/min in group 2 during P1-P3, respectively) and by prolapse of the third eyelid during P2. Arterial plasma glucose, insulin, and glucagon concentrations; hepatic blood flow; and hepatic glucose load did not change significantly during P1-P3. NHGU during P1-P3 was 2.7 +/- 0.4, 4.1 +/- 0.6, and 4.0 +/- 1.2 mg.kg(-1).min(-1) in group 1 and 5.0 +/- 0.9, 5.6 +/- 0.7, and 6.1 +/- 0.9 mg.kg(-1).min(-1) in group 2 (not significant among periods). Interruption of vagal signaling with or without intraportal infusion of norepinephrine to augment sympathetic tone did not suppress NHGU during portal glucose delivery, suggesting the portal signal stimulates NHGU independently of vagal efferent flow.

Morbidity associated with intraportal islet transplantation

Introduction Complications associated with intraportal islet infusion have been reported. In this study, we analyzed the relationship between occurrence of complications and islet preparation characteristics/infusion technique. Methods We reviewed all intraportal islet infusions from 1992 to 2003. Results Sixteen islet autotransplantations were performed without infusion-related complications. The tissue volume injected was 13 ± 11 mL with basal and peak portal pressures of 13 ± 6 and 21 ± 6 mm Hg. Seventy-seven intraportal islet allotransplantations were performed in 51 patients. Fifteen islet infusions were done by laparotomy during simultaneous islet/kidney transplantation without complication. Among 62 percutaneous transhepatic injections, nine complications (two portal branch thrombosis and seven intra-abdominal hemorrhages) were recorded. Rise in portal pressure was related to tissue volume injected ( P < .05). Basal and peak portal pressures were 14 ± 5 and 18 ± 6 mm Hg in uncomplicated infusions, 14 ± 9 and 18 ± 9 mm Hg in the thrombosis group, and 13 ± 7 and 18 ± 5 mm Hg in the hemorrhage group ( P > .05). Complications occurred only after percutaneous islet infusion ( P < .03). Conclusions Procedure-related morbidity of intraportal islet infusion is low. Changes in portal pressure are related to volume of tissue injected but do not seem to be associated with the occurrence of complications. Percutaneous infusion is a minimally invasive procedure, but this advantage must be balanced by the higher rate of complications.

Intraportal nicotine infusion in rats decreases hepatic blood flow through endothelin-1 and both endothelin A and endothelin B receptors

Smoking has been demonstrated to aggravate liver injury. Nicotine, a major pharmacological component of tobacco smoke, affects a multitude of functions. Smoking and nicotine induce synthesis of endothelin (ET)-1. The effect of intraportal infusion of nicotine on hepatic circulation and an involvement of ET-1 and ET receptor in the action of nicotine were investigated in rats. Nicotine (0–100 μg/kg/h) was infused into the portal vein of urethane-anesthetized rats, and changes of hepatic blood flow were evaluated. Intraportal infusion of nicotine dose-dependently decreased hepatic blood flow and increased portal pressure without any alteration of heart rate or arterial blood pressure. This action of intraportal nicotine was completely abolished by pretreatment of ET-1 antibody. Either BQ485 (ET A receptor antagonist) or BQ788 (ET B receptor antagonist) partially reversed the effect of nicotine, and combination of BQ788 and BQ485 completely abolished it. These findings suggest that nicotine inhibits hepatic circulation through ET-1, and ET A and ET B receptor.

Insulin action during late pregnancy in the conscious dog.

Our aim was to assess the magnitude of peripheral insulin resistance and whether changes in hepatic insulin action were evident in a canine model of late (3rd trimester) pregnancy. A 3-h hyperinsulinemic (5 mU.kg(-1).min(-1)) euglycemic clamp was conducted using conscious, 18-h-fasted pregnant (P; n = 6) and nonpregnant (NP; n = 6) female dogs in which catheters for intraportal insulin infusion and assessment of hepatic substrate balances were implanted approximately 17 days before experimentation. Arterial plasma insulin rose from 11 +/- 2 to 192 +/- 24 and 4 +/- 2 to 178 +/- 5 microU/ml in the 3rd h in NP and P, respectively. Glucagon fell equivalently in both groups. Basal net hepatic glucose output was lower in NP (1.9 +/- 0.1 vs. 2.4 +/- 0.2 mg.kg(-1).min(-1), P < 0.05). Hyperinsulinemia completely suppressed hepatic glucose release in both groups (-0.4 +/- 0.2 and -0.1 +/- 0.2 mg.kg(-1).min(-1) in NP and P, respectively). More exogenous glucose was required to maintain euglycemia in NP (15.2 +/- 1.3 vs. 11.5 +/- 1.1 mg.kg(-1).min(-1), P < 0.05). Nonesterified fatty acids fell similarly in both groups. Net hepatic gluconeogenic amino acid uptake with high insulin did not differ in NP and P. Peripheral insulin action is markedly impaired in this canine model of pregnancy, whereas hepatic glucose production is completely suppressed by high circulating insulin levels.

Intestinal absorption and hepatic extraction of propranolol and metoprolol in rats with bilateral ureteral ligation.

To investigate the mechanism responsible for the increased bioavailability of propranolol in bilateral ureter-ligated (BUL) rats, the intestinal absorption and hepatic extraction of propranolol and metoprolol were evaluated. The initial absorption rate of these drugs after intra-intestinal administration was only slightly increased in the BUL rats, whereas the blood drug concentration in these rats was higher than that in control rats. The blood propranolol and metoprolol concentrations during intra-portal infusion in the BUL rat were significantly higher than that in the control rat. In the presence of NADPH, the intrinsic metabolic activity of metoprolol in hepatic microsomes was not altered by BUL. On the other hand, the NADPH generation rate in the hepatic cytosol in the BUL group was lower than that in the control group. These results indicate that the absorption rate-dependent decrease in hepatic first-pass clearance of propranolol and metoprolol due to saturation kinetics is marginal, and that the hepatic metabolic activity and extraction of the drugs is significantly decreased in BUL rats probably due to the reduced NADPH generation rate in the liver.

Living donor liver transplantation with special reference to ABO-incompatible grafts and small-for-size grafts.

Living donor liver transplantation (LDLT) has developed on the basis of increased safety of conventional liver surgery and the need for expanding donor sources, especially in children. Indications for LDLT were soon extended to adult patients in Japan, where cadaveric donation was limited. The right liver is now routinely transplanted to adults to avoid small-for-size graft syndrome, even though the right liver graft has the disadvantages of less remaining donor liver and the question of donor safety. Assessing the suitable size or quality of the graft, as well as of the remnant donor liver, is one of the most important problems in adult LDLT. Although several tactics have been proposed to manage the small-for-size syndrome, their efficacy remains a question. We suggest that small-for-size syndrome is preventable by engaging in careful donor selection or using effective agents for hepatic microcirculatory disturbance control. Sometimes for LDLT only ABO-incompatible grafts are available from relatives, but they must be transplanted despite the expected poor outcome in adults and older children. To overcome the problems in this situation, we developed a novel protocol including intraportal infusion therapy with methylprednisolone, prostaglandin E1, and gabexate mesylate. Two adult patients undergoing ABO-incompatible LDLT have now survived 53 and 35 months after transplantation with good liver function. However, the other two patients suffered thrombotic microangiopathy postoperatively and died owing to cerebral hemorrhage or multiple organ failure, respectively. Further investigation is needed to improve the outcome of liver transplantation across the ABO blood group barrier.

Alterations in hepatic microcirculation, mitochondrial redox state and tissue oxygenation after trauma and sepsis

Introduction: Hepatic microcirculatory dysfunction and mismatch of blood supply to metabolic demand play an important role in liver failure after stress. Here, we assessed hepatic microcirculation in response to the vasoregulator Endothelin-1 (ET-1) after femur fracture (FFx) and cecal ligation and puncture (CLP). We hypothesized that sequential stress causes hepatic microcirculatory dysfunction leading to altered mitochondrial redox state and tissue PO2 (tPO2). Methods: Male Sprague-Dawley rats (200–300 g, n = 4/groups) underwent sham surgery, FFx, CLP and FFx+CLP. Using intravital microscopy, hepatic microcirculation was assessed by sinusoidal volumetric flow (pL/sec) and perfusion index (PI, pL/sec/200μm). Changes in hepatic mitochondrial redox state and liver tPO2 were determined by NADH autofluorescence (AU, arbitrary unit) and ruthenium (Ru, AU) fluorescence, respectively. Western blot analysis was used to determine ETA/ETB receptor protein expression (AU). Differences among groups were compared using one-way ANOVA followed by Student-Newman-Keuls post hoc test. Results: After 10 minutes of intraportal ET-1 infusion, liver microcirculation was altered in CLP and FFx+CLP groups (Table, values are mean ± SE). TABLE—ABSTRACT 24GroupFlowPINADHRuETAETBSham21 ± 3160 ± 19109 ± 7177 ± 50 ± 09 ± 3FFx19 ± 1155 ± 15101 ± 10148 ± 80 ± 038 ± 8CLP9 ± 2∗69 ± 14∗153 ± 10∗190 ± 6∗0 ± 036 ± 16FFx+CLP5 ± 0.8∗28 ± 4∗160 ± 12∗202 ± 7∗52 ± 8§58 ± 12§∗p < 0.05 vs. sham and FFx,§p < 0.05 vs. sham, FFx, and CLP. Sequential stress also led to increased NADH and Ruthenium fluorescence intensity, indicating a reduction in O2 availability. Vasoactive receptor proteins ETA and ETB were upregulated after injury. Conclusion: Our data suggest that sepsis and sequential stress result in liver microcirculatory disturbances leading to altered global hepatic perfusion, and contribute toward a reduction in O2 supply. These changes in the liver microcirculation, mediated in part by Endothelin-1, may play an important role in the development of liver failure after inflammatory stress.

Embolization by sinusoidal lining cells obstructs the microcirculation in rat sinusoidal obstruction syndrome.

Mechanisms leading to the obstruction of the microcirculation in sinusoidal obstruction syndrome (SOS) have been unclear. Because this occurs at the onset of disease, this is a potential key target for therapeutic intervention. Rats were treated with monocrotaline with or without continuous intraportal infusion of glutathione and were studied at 0.5, 1, 2, 4, 6, and 10 days after monocrotaline treatment with the use of in vivo microscopy and transmission electron microscopy. Sinusoidal perfusion decreased from days 1 through 10 with a nadir on day 4. At 12 h, numerous swollen sinusoidal endothelial cells (SECs) were observed. Subsequently, red blood cells penetrated into the space of Disse through gaps between and through swollen SEC and dissected the sinusoidal lining away from the parenchymal cells. Sinusoidal blood flow was obstructed by an embolism of aggregates of sinusoidal lining cells, red blood cells, and adherent monocytes. All changes were prevented by glutathione infusion, notably the initial swelling of SEC. SOS is initiated by changes in SEC. Microcirculatory obstruction is due to dissection of the sinusoidal lining, followed by embolization of the sinusoid by sinusoidal lining cells, compounded by aggregates of monocytes adherent in the sinusoids. Glutathione prevents SOS by preserving an intact sinusoidal barrier.

Effect of intraportal glucagon-like peptide-1 on glucose metabolism in conscious dogs

Arteriovenous difference and tracer ([3-(3)H]glucose) techniques were used in 42-h-fasted conscious dogs to identify any insulin-like effects of intraportally administered glucagon-like peptide 1-(7-36)amide (GLP-1). Each study consisted of an equilibration, a basal, and three 90-min test periods (P1, P2, and P3) during which somatostatin, intraportal insulin (3-fold basal) and glucagon (basal), and peripheral glucose were infused. Saline was infused intraportally in P1. During P2 and P3, GLP-1 was infused intraportally at 0.9 and 5.1 pmol. kg(-1). min(-1) in eight dogs, at 10 and 20 pmol. kg(-1). min(-1) in seven dogs, and at 0 pmol. kg(-1). min(-1) in eight dogs (control group). Net hepatic glucose uptake was significantly enhanced during GLP-1 infusion at 20 pmol. kg(-1). min(-1) [21.8 vs. 13.4 micromol. kg(-1). min(-1) (control), P < 0.05]. Glucose utilization was significantly increased during infusion at 10 and 20 pmol. kg(-1). min(-1) [87.3 +/- 8.3 and 105.3 +/- 12.8, respectively, vs. 62.2 +/- 5.3 and 74.7 +/- 7.4 micromol. kg(-1). min(-1) (control), P < 0.05]. The glucose infusion rate required to maintain hyperglycemia was increased (P < 0.05) during infusion of GLP-1 at 5.1, 10, and 20 pmol. kg(-1). min(-1) (22, 36, and 32%, respectively, greater than control). Nonhepatic glucose uptake increased significantly during delivery of GLP-1 at 5.1 and 10 pmol. kg(-1). min(-1) (25 and 46% greater than control) and tended (P = 0.1) to increase during GLP-1 infusion at 20 pmol. kg(-1). min(-1) (24% greater than control). Intraportal infusion of GLP-1 at high physiological and pharmacological rates increased glucose disposal primarily in nonhepatic tissues.

Intraportal infusion of 99mtechnetium-macro-aggregrated albumin particles and hepatocytes in rabbits: assessment of shunting and portal hemodynamic changes

Partial correction of metabolic liver disease by hepatocyte transplantation requires infusion of a large number of cells into the portal vein. Uncontrolled infusion of cells leads to extrahepatic shunting. Obstruction of the sinusoidal space may result in hemodynamic changes and impairment of liver function. Catheters connected to a port were placed into the caudal mesenteric vein of rabbits. After injection of 99mtechnetium-macroaggregated albumin (99mTc-MAA) surrogates or 99mTc-MAA/hepatocyte (Hc) mixtures (1:125), shunting into the lung was scintigraphically monitored. Volume flow (mL/min) and maximum velocity of the portal vein were recorded by color-coded Doppler ultrasound during intraportal application of 2.5 x 10(7) MAA particles, 2.5 x 10(7) isolated hepatocytes, and saline solution without particles or cells. 99mTc-MAA particles (2.5 x 10(7)) or equivalent MAA/Hc mixtures were completely retained in the liver. With additional application of 2.5 x 10(7) particles, shunting into the lung was observed in two animals of the MAA group. All animals in the hepatocyte group have received 5 x 10(7) MAA/Hc mixtures, and three of these received 10(8) mixtures without shunting. Maximum velocity and volume flow increased with saline infusion. Hepatocyte suspended in the same volume blunted the increase observed in the control group, but parameters remained normal. Liver enzymes increased after hepatocyte application but returned to normal values within 5 days. Sinusoidal uptake capacity for hepatocyte or MAA particles varies at a wide range in normal rabbits. Scintigraphic monitoring of transplanted cells allows efficient monitoring of cell translocation into the lungs. No significant impairments of portal hemodynamics and liver function were detected.

Arginine, ornithine, and proline interconversion is dependent on small intestinal metabolism in neonatal pigs.

We have previously shown that arginine deficiency is exacerbated by the removal of dietary proline in orally, but not parenterally, fed piglets. Therefore, we hypothesized that the net interconversions of proline, ornithine, and arginine primarily occur in the small intestine of neonatal piglets. Ten intragastrically fed piglets received either intraportal (IP) or intragastric (IG) primed, constant infusions of [guanido-(14)C]arginine and [U-(14)C]ornithine + [2,3-(3)H]proline. By infusing amino acid isotopes via the stomach compared with the portal vein, we isolated small intestinal first-pass metabolism in vivo. During IP infusion, fractional net conversions (%) from proline to ornithine (0), ornithine to arginine (11 +/- 6), and ornithine to proline (5 +/- 1) were lower (P < 0.05) than during IG infusion (39 +/- 8, 18 +/- 6, and 42 +/- 12, respectively); we speculate that these data are due to the localization of ornithine aminotransferase to the gut. The balance of these conversions indicated a large synthesis of arginine (70.0 micromol. kg(-1). h(-1)) by the gut, with a corresponding degradation of ornithine (70.8 micromol. kg(-1). h(-1)) and no change in proline balance. Gut synthesis of arginine from proline (48.1 micromol. kg(-1). h(-1)) was 50% of its requirement, whereas proline synthesis from arginine (33.0 micromol. kg(-1). h(-1)) amounted to 10% of its requirement. Overall, arginine synthesis is more dependent on the gut than proline synthesis. In situations in which gut metabolism is compromised, such as during parenteral nutrition or gastrointestinal disease, arginine and proline are individually indispensable because their biosyntheses are negligible.

The effects of different growth factors on human bone marrow stromal cells differentiating into hepatocyte-like cells.

Stem cells are self-renewable and are thought to be the pluripotent cells that can differentiate into a variety of cells or tissue types. For many years, adult stem cells harvested from bone marrow have been used therapeutically. In 1999, Pittenger et al. published the methods to induce differentiation of human mesenchymal stem cells into different cell types of mesodermal lineages. Theise et al. have demonstrated that hepatocytes, from endodermal lineage, can be derived from bone marrow in human and mice. Hepatocyte engraftment from bone marrow cells have been confirmed by double FISH (fluorescence in situ hybridization) after bone marrow transplantation. Lagasse et al. further demonstrated that purified hematopoietic stem cells from adult mouse bone marrow rescued fumarylacetoacetate hydrolase (FAH) deficient mice and restored the biochemical function of their liver. Alison et al.5showed that the human adult hematopoietic stem cell population is capable of yielding an epithelial lineage in chronically damaged liver. ß2m-/Thy-1+cells from rat and human bone marrow were integrated with hepatic cell plates and were differentiated into mature hepatocytes after intraportal infusion into rat livers studied by Avital et al.6ßzm7Thy-1+cells also differentiated into mature hepatocytes in a culture system simulating liver regeneration and containing cholestatic serum. Oh et al.’ and Schwartz et al. have succeeded in differentiating rat bone marrow cells and multipotent adult progenitor cells from bone marrow into hepatocyte-like cells by addition of growth factors in the culture medium. Hamazaki et al. induced hepatic maturation in mouse embryonic stem cellsin vitro.Other studies suggest that the microenvironment of embryonic liver, including growth factors and cell-cell interactions, is critical for regulating liver development. Acidic FGF and HGF were known to play important roles in the early-and mid-developmental stages of liver development. So far, none have succeeded to induce human bone marrow stromal cells (BMSC) differentiation into hepatocytes in culture.KeywordsHepatocyte Growth FactorHuman Mesenchymal Stem CellHuman Bone Marrow Stromal CellMature HepatocyteHepatic DifferentiationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Impact of chronic fructose infusion on hepatic metabolism during TPN administration.

During chronic total parenteral nutrition (TPN), net hepatic glucose uptake (NHGU) is markedly elevated. However, NHGU is reduced by the presence of an infection. We recently demonstrated that a small, acute (3-h) intraportal fructose infusion can correct the infection-induced impairment in NHGU. The aim of this study was to determine whether the addition of fructose to the TPN persistently enhances NHGU in the presence of an infection. TPN was infused continuously into the inferior vena cava of chronically catheterized dogs for 5 days. On day 3, a bacterial clot was implanted in the peritoneal cavity, and either saline (CON, n = 5) or fructose (+FRUC, 1.0 mg. kg(-1). min(-1), n = 6) infusion was included with the TPN. Forty-two hours after the infection was induced, hepatic glucose metabolism was assessed in conscious dogs with arteriovenous and tracer methods. Arterial plasma glucose concentration was lower with chronic fructose infusion (120 +/- 4 vs. 131 +/- 3 mg/dl, +FRUC vs. CON, P < 0.05); however, NHGU was not enhanced (2.2 +/- 0.5 vs. 2.8 +/- 0.4 mg. kg(-1). min(-1)). Acute removal of the fructose infusion dramatically decreased NHGU (2.2 +/- 0.5 to -0.2 +/- 0.5 mg. kg(-1). min(-1)), and net hepatic lactate release also fell (1.6 +/- 0.3 to 0.5 +/- 0.3 mg. kg(-1). min(-1)). This led to an increase in the arterial plasma glucose (Delta13 +/- 3 mg/dl, P < 0.05) and insulin (Delta5 +/- 2 micro U/ml) concentrations and to a decrease in glucagon (Delta-11 +/- 3 pg/ml) concentration. In conclusion, the addition of chronic fructose infusion to TPN during infection does not lead to a persistent augmentation of NHGU.

Contribution of presystemic hepatic extraction to the low oral bioavailability of green tea catechins in rats.

Green tea and green tea catechins have been shown to possess potent cancer-preventive activities in rodent cancer models. At present, epidemiological evidence of the protective effect of green tea consumption against the development of human cancers is not conclusive. Oral bioavailability of green tea catechins has been shown to be low in animals and possibly in humans. This study is designed to determine the contribution of first-pass hepatic elimination to the low oral bioavailability of green tea catechins. Green tea catechin mixture was dosed to rats by intravenous or intraportal infusion. Blood samples were collected after dosing and analyzed using high-performance liquid chromatography with the coulometric electrode array detection system. The systemic clearance of epigallocatechin gallate (EGCG), epigallocatechin (EGC), and epicatechin (EC) was 8.9, 6.3, and 9.4 ml/min, respectively. The steady state volume of distribution (V(ss)) of EGCG, EGC, and EC was 432, 220, and 187 ml, respectively. We found that high percentage of green tea catechins escaped first-pass hepatic elimination, with 87.0, 108.3, and 94.9% of EGCG, EGC, and EC, respectively, available in the systemic blood following intraportal infusion. Our results suggest that factors within the gastrointestinal tract such as limited membrane permeability, transporter mediated intestinal secretion, or gut wall metabolism may contribute more significantly to the low oral bioavailability of green tea catechins.

Intrahepatic bile ducts transport water in response to absorbed glucose

The physiological relevance of the absorption of glucose from bile by cholangiocytes remains unclear. The aim of this study was to test the hypothesis that absorbed glucose drives aquaporin (AQP)-mediated water transport by biliary epithelia and is thus involved in ductal bile formation. Glucose absorption and water transport by biliary epithelia were studied in vitro by microperfusing intrahepatic bile duct units (IBDUs) isolated from rat liver. In a separate set of in vivo experiments, bile flow and absorption of biliary glucose were measured after intraportal infusion of D-glucose or phlorizin. IBDUs absorbed D-glucose in a dose- and phlorizin-dependent manner with an absorption maximum of 92.8 +/- 6.2 pmol. min(-1). mm(-1). Absorption of D-glucose by microperfused IBDUs resulted in an increase of water absorption (J(v) = 3-10 nl. min(-1). mm(-1), P(f) = 40 x 10(-3) cm/sec). Glucose-driven water absorption by IBDUs was inhibited by HgCl(2), suggesting that water passively follows absorbed D-glucose mainly transcellularly via mercury-sensitive AQPs. In vivo studies showed that as the amount of absorbed biliary glucose increased after intraportal infusion of D-glucose, bile flow decreased. In contrast, as the absorption of biliary glucose decreased after phlorizin, bile flow increased. Results support the hypothesis that the physiological significance of the absorption of biliary glucose by cholangiocytes is likely related to regulation of ductal bile formation.