Leonardo Baiocchi

A pilot study on the safety and effectiveness of immunosuppression without prednisone after liver transplantation

BACKGROUND Corticosteroids are commonly used in the immunosuppression therapy after liver transplantation, yet are associated with considerable side effects. Retrospective studies have shown that corticosteroids can be safely withdrawn from months to years after transplant. We prospectively investigated the effects of early immunosuppression without the use of corticosteroids on graft outcome and transplant complications. METHODS Forty-five patients undergoing liver transplantation were randomized to receive immunosuppression composed of cyclosporine microemulsion and azathioprine with (n=22) or without prednisone (n=23), in conventional doses. In those patients who received prednisone, this was withdrawn within 3 months after transplant. The median follow-up of survivors was 14 months (range: 6-24). The study end points were to determine graft survival and function, infectious complications, including hepatitis C virus (HCV)-RNA levels in HCV-infected recipients, acute rejection, kidney function, and metabolic complications. RESULTS Eleven deaths occurred, 6 of which were in the prednisone group. Two-year survival did not differ between patients treated with or without prednisone (70.2% vs. 78.3%, P=0.83), nor did the causes of death. No differences were observed with regard to graft function, renal function, and infectious complications. In the subset of patients who received transplants for HCV-related cirrhosis, the dynamics of virus replication HCV-RNA was faster among those treated with prednisone. The incidence and severity of acute rejection was similar in the two groups. More than 80% of acute rejections in both groups were classified as mild or moderate and underwent spontaneous resolution. Only two patients in each group had severe acute rejection requiring additional treatment with high-dose steroids. Patients receiving prednisone tended to have greater biochemical signs of cholestasis, higher serum cholesterol and glucose levels, and more frequent insulin requirement than those treated without corticosteroids. CONCLUSIONS Liver transplantation can be performed safely without using corticosteroids in the early postoperative course, and there is no need for routine aggressive steroid treatment of established acute rejections.

Gastrin inhibits cholangiocarcinoma growth through increased apoptosis by activation of Ca2+-dependent protein kinase C-α

BACKGROUND/AIMS We determined the role of gastrin in the regulation of cholangiocarcinoma growth. METHODS We evaluated for the functional presence of cholecystokinin (CCK)-B/gastrin receptors in the cholangiocarcinoma cell lines, Mz-ChA-1, HuH-28 and TFK-1. We determined the effect of gastrin on the growth of Mz-ChA-1, HuH-28 and TFK-1 cells. We evaluated the effect of gastrin on growth and apoptosis of Mz-ChA-1 in the absence or presence of inhibitors for CCK-A (L-364, 718) and CCK-B/gastrin (L-365, 260) receptors, the intracellular Ca2+ chelator (BAPTA/AM), and the protein kinase C (PKC)-alpha inhibitor, H7. We evaluated if gastrin effects on Mz-ChA-1 growth and apoptosis are associated with membrane translocation of PKC-alpha. RESULTS Gastrin inhibited DNA synthesis of Mz-ChA-1, HuH-28 and TFK-1 cells in a dose- and time-dependent fashion. The antiproliferative effect of gastrin on Mz-ChA-1 cells was inhibited by L-365, 260, H7 and BAPTA/AM but not L-364, 718. Gastrin induced membrane translocation of PKC-alpha. The inhibition of growth of Mz-ChA-1 cells by gastrin was associated with increased apoptosis through a PKC-dependent mechanism. CONCLUSIONS Gastrin inhibits the growth of Mz-ChA-1, HuH-28 and TFK-1 cells. Gastrin inhibits growth and induces apoptosis in Mz-ChA-1 cells through the Ca2+-dependent PKC-alpha. The data suggest a therapeutic role for gastrin in the modulation of cholangiocarcinoma growth.

Switch to 1.5 grams MMF monotherapy for CNI-related toxicity in liver transplantation is safe and improves renal function, dyslipidemia, and hypertension

Although mycophenolate mofetil (MMF) monotherapy has been successfully used in liver transplant recipients suffering from calcineurin‐inhibitor (CNI)‐related chronic toxicity, still no consensus has been reached on its safety, efficacy and tolerability. We attempted the complete weaning off CNI in 42 individuals presenting chronic renal dysfunction and/or dyslipidemia and/or arterial hypertension and simultaneously introduced 1.5 gm/day MMF. CNI could be completely withdrawn in 41 cases. A total of 32 (75%) patients are currently on ≤1.5 gm/day of MMF. Mean follow‐up from the introduction of MMF is 31.5 months and mean length of follow‐up from the beginning of MMF monotherapy is 27.3 months. Renal function improved in 31/36 (89%) cases. Blood levels of cholesterol and triglycerides decreased in 13 of 17 (76%) and 15 of 17 (89%) patients, respectively. Arterial hypertension improved in 4 of 5 (80%) cases. A total of 8 patients showed a single episode of fluctuation of liver function tests during tapering off CNI. This feature was interpreted as an acute rejection (AR), based on the resolution of the clinical setting after escalation of MMF daily dose to 2 gm. A further patient developed a biopsy‐proven AR insensitive to MMF adjustment, requiring reinstitution of the CNI dose. No deaths or major toxicity requiring MMF discontinuation occurred. In conclusion, low dose MMF monotherapy is safe, effective, and well tolerated. Liver Transpl, 2007.

The Tor Vergata weaning off immunosuppression protocol in stable HCV liver transplant patients: The updated follow up at 78 months

BACKGROUND We report the update of the Tor Vergata immunosuppression (IS) weaning protocol in stable hepatitis C virus (HCV) liver transplant (LT) recipients. METHODS The weaning off IS was attempted in 34 patients who had received a LT 63.5+/-20.1 month earlier, for HCV-related end stage liver disease. Patients were observed over a period of 6.5 years. During this time, yearly protocol liver biopsies were performed. Primary endpoints were determined as the feasibility of weaning off IS and its impact on the long term disease progression. Secondary endpoints were defined as the impact on patient morbidity and quality of life. RESULTS Of the 8 originally tolerant patients, 7 remain alive and in good condition, while 1 died of severe HCV recurrence 10 years post-LT and 6 years after complete removal of IS. Four out of 26 intolerant individuals died of HCV recurrence (2x), lung carcinoma (1x) and acute myocardial infarction (1x), after a mean follow up period from LT of 115 (range 100-124). The 10-year survival from LT was comparable (89% vs. 87.5%). Liver graft pathology showed no significant differences between the two groups in terms of staging, fibrosis progression rate, and grading. Quantitative HCV RNA assay showed a significant non-logarithmic difference between the two groups (p = 0.03). The two groups were comparable in terms of liver function tests and lipid profile, whereas they differed with regards to glycaemia. While all tolerant individuals were euglicemic, 11 intolerant individuals developed new onset diabetes that required specific treatment (p = 0.03). Finally, significantly more intolerant patients are suffering from either cardiovascular (14/22 vs. 0/7, p = 0.01) or infectious diseases (13/22 vs. 0/7, p = 0.01). CONCLUSIONS After a 6.5-year follow up, the complete withdrawal of IS in HCV LT recipient remains safe and beneficial to patients, because it reduces the IS-related morbidity and increases the quality of life. The impact on HCV disease recurrence is less marked than after 3.5 years.

Regulation of cholangiocyte bile secretion

he intrahepatic biliary system is comprised of aset of anastomosing and progressively largertubes resembling a tree lined by cholangiocytes (1,2).The major function of the biliary system is modifi-cation of canalicular bile by secretory and reabsorptiveprocesses in cholangiocytes as bile passes through bileducts before reaching the duodenum (1,3–8). In con-trast to hepatocytes, where secretion is primarily un-regulated and constant (9), bile duct secretion is modi-fied by hormones, peptides, nerves and biliary con-stituents (1,3–8,10–24). The bile duct system attractsattention from clinicians, pathologists, physiologistsand cell biologists because it is the target for a varietyof liver diseases including primary biliary cirrhosis(PBC), primary sclerosing cholangitis (PSC), graft

Secretin activation of the apical Na+-dependent bile acid transporter is associated with cholehepatic shunting in rats

The role of the cholangiocyte apical Na+‐dependent bile acid transporter (ASBT) in bile formation is unknown. Bile acid absorption by bile ducts results in cholehepatic shunting, a pathway that amplifies the canalicular osmotic effects of bile acids. We tested in isolated cholangiocytes if secretin enhances ASBT translocation to the apical membrane from latent preexisting intracellular stores. In vivo, in bile duct–ligated rats, we tested if increased ASBT activity (induced by secretin pretreatment) results in cholehepatic shunting of bile acids. We determined the increment in taurocholate‐dependent bile flow and biliary lipid secretion and taurocholate (TC) biliary transit time during high ASBT activity. Secretin stimulated colchicine‐sensitive ASBT translocation to the cholangiocyte plasma membrane and 3H‐TC uptake in purified cholangiocytes. Consistent with increased ASBT promoting cholehepatic shunting, with secretin pretreatment, we found TC induced greater‐than‐expected biliary lipid secretion and bile flow and there was a prolongation of the TC biliary transit time. Colchicine ablated secretin pretreatment‐dependent bile acid–induced choleresis, increased biliary lipid secretion, and the prolongation of the TC biliary transit. In conclusion, secretin stimulates cholehepatic shunting of conjugated bile acids and is associated with increased cholangiocyte apical membrane ASBT. Bile acid transport by cholangiocyte ASBT can contribute to hepatobiliary secretion in vivo. (HEPATOLOGY 2005.)

Protective role of tauroursodeoxycholate during harvesting and cold storage of human liver: a pilot study in transplant recipients.

Background. Ischemia-reperfusion injury is a major cause of early graft dysfunction after liver transplantation. Tauroursodeoxycholic acid (TUDCA), a natural amidated hydrophilic bile salt, protects from cholestasis and hepatocellular damage in a variety of experimental models, as well as from ischemia-reperfusion injury. We investigated in the human liver transplantation setting the effect of the addition of TUDCA at time of liver harvesting and cold storage on the intra- and postoperative enzyme release and liver histopathology at the end of cold storage, at reperfusion, and 7 days after transplantation. Methods. Eighteen patients undergoing elective liver transplantation were studied, including 6 serving as controls. In six patients, TUDCA was added to the University of Wisconsin solution used during harvesting and cold storage, to reach final concentrations of 2 mM. In three of these patients, TUDCA (3 g) was infused in the portal vein of the donor before organ explantation; in the other three cases, TUDCA was given through both routes. Results. The use of TUDCA did not cause adverse events. The release of aspartate aminotransferase in the inferior vena cava blood during liver flushing was significantly lower (P =0.05) in TUDCA-treated than in control grafts, as were cytolytic enzyme levels in peripheral blood during the first postoperative week (P <0.02). At electron microscopy, an overt endothelial damage (cytoplasmic vacuolization, cell leakage, and destruction with exposure of hepatocytes to the sinusoidal lumen) was invariably found in control grafts, both at reperfusion and at day 7 after transplant. These features were significantly ameliorated by TUDCA (P <0.001). Several ultrastructural cytoplasmic abnormalities of hepatocytes were seen. Among these, damage to mitochondria matrix and crystae was significantly reduced in TUDCA-treated versus control grafts (P <0.01). Mild to severe damage of bile canaliculi was a constant feature in control biopsies, with dilatation of canalicular lumen and loss of microvilli. Both these abnormalities were markedly ameliorated (P <0.001 by TUDCA). The best preservation was observed when TUDCA was given through both routes. Conclusions. The use of TUDCA during harvesting and cold storage of human liver is associated with significant protection from ischemia-reperfusion injury. The clinical significance of this findings must be studied.

Correlation Between Liver Fibrosis and Inflammation in Patients Transplanted for HCV Liver Disease

Hepatitis C virus (HCV) re‐infection after liver transplantation (LT) is characterized by an accelerated disease progression in recent years with unclear mechanisms. We evaluate the relationship between progression of liver fibrosis and histological necro‐inflammation in HCV recipients, according to age of transplant. Fifty‐five patients transplanted (1993–2002) for HCV liver disease, were included in the study. Recipients were retrospectively stratified in three different age of transplant, of 40 months each: group 1) from January 1993 to May 1996; group 2) from June 1996 to august 1999; group 3) from September 1999 to December 2002. Grading (necro‐inflammation) and staging (fibrosis) scores were evaluated in liver biopsies at 1, 2 and 3 years from LT (Ishak classification). For all age of transplant the main factor associated with fibrosis progression, was grading score (p < 0.05). However mean staging score for each point of grading increased from 0.3 ± 0.2 in older LT to 0.7 ± 0.5 in newer ones (p = 0.01). In conclusion in HCV–LT patients (1) liver fibrosis is strictly associated to histological necro‐inflammation; (2) the proportion of this relationship has been changing in recent years since newer LT patients, show an increased amount of fibrosis in comparison with the older ones, for similar grading score.

TUDCA prevents cholestasis and canalicular damage induced by ischemia‐reperfusion injury in the rat, modulating PKCα–ezrin pathway

Cholestasis, induced by liver ischemia‐reperfusion injury (IRI), is characterized by dilatation of bile canaliculi and loss of microvilli. Tauroursodeoxycholic acid (TUDCA) is an anti‐cholestatic agent, modulating protein kinase C (PKC) α pathway. PKC reduces ischemic damage in several organs, its isoform α modulates ezrin, a key protein in the maintenance of cell lamellipoidal extensions. We evaluated the effects of TUDCA on cholestasis, canalicular changes and PKCα–ezrin expression in a rat model of liver IRI. Livers flushed and stored with Belzer solution or Belzer + 10 mm TUDCA (4 °C for 6 h) were reperfused (37 °C with O2) with Krebs–Ringer bicarbonate + 2.5 μmol/min of Taurocholate or TUDCA. Bile was harvested for bile flow assessment. Liver tissue was employed for Electron Microscopy (EM) and for PKCα and ezrin immunoblot and immunofluorescence. The same experiments were conducted with the PKCα inhibitor Go‐6976. TUDCA‐treated livers showed increased bile flow (0.25 ± 0.17 vs. 0.042 ± 0.02 μl/min/g liver, P < 0.05) and better preservation of microvilli and bile canalicular area at EM. These effects were associated with increased PKCα and ezrin expression (P = 0.03 and P = 0.04 vs. control respectively), as also confirmed by immunofluorescence data. PKCα inhibition abolished these TUDCA effects. TUDCA administration during IRI reduces cholestasis and canalicular damage in the liver modulating PKCα–ezrin pathway.

The Tor Vergata weaning of immunosuppression protocols in stable hepatitis C virus liver transplant patients: the 10-year follow-up.

We report herein the 10‐year outcome of the Tor Vergata weaning off immunosuppression protocol in hepatitis C virus (HCV) liver transplant patients. Thirty‐four patients who had received a liver graft for HCV‐related cirrhosis were enrolled in a prospective study in which they were progressively weaned off immunosuppression. The primary endpoints were feasibility and safety of the weaning; the second aim was to assess fibrosis progression. At the 10‐year follow‐up, of the eight original tolerant patients, six remained IS‐free. Of the 26 individuals who could not be weaned, 22 were alive. When the baseline biopsies were compared with the 10‐year biopsies, the tolerant group showed no differences in staging, whereas the nontolerant group showed a significant increase in staging. The fibrosis progression rates calculated for the tolerant and the nontolerant groups were −0.06 ± 0.12 and 0.1 ± 0.2, respectively (P = 0.04). Furthermore, with the last taken biopsies, nine nontolerant patients were showing frank cirrhosis versus no cirrhosis among the tolerant patients. After a 10‐year follow‐up of a Tor Vergata weaning protocol, 6/34 patients completed follow‐up without reinstitution of immunosuppression and this appeared beneficial regarding a reduction in fibrosis progression.