Combined liver and islet transplantation using steroid-free immunossupression

Introduction
HCV cirrhosis currently represents the most common indication for both living donor and cadaver liver transplantation (LT) worldwide, accounting for more than 50% of cases (1). Some studies demonstrated that patients with liver cirrhosis and diabetes mellitus had a higher rate of mortality and morbidity after LT than non-diabetic cirrhotic patients (2, 3). Combined liver and islet transplantation is a complex procedure with only 14 cases reported in the literature (4-7). One third of the cases were represented by islet transplantation after upper abdominal exenteration and liver replacement (8).
We present the case of combined liver and pancreatic islets transplantation performed in a patient with HCV liver cirrhosis associated with insulin-dependent diabetes. This is also the first case of islet allotransplantation in Romania.
Case presentation
A 40-year-old male (85 kg, body mass index 24.8 kg/m2) had been diagnosed with chronic hepatitis C, genotype 1 in 1995. He underwent combined antiviral therapy with standard interferon and ribavirin for 12 months, but he relapsed after cessation of therapy. During the regular biochemical evaluations, starting from July 1999, blood glucose (BG) values increased progressively to 105 mg/dl in August 1999, 152 mg/dl in November 1999, 176 mg/dl in February 2000 and 293 mg/dl in May 2000. In May 2000, glycated hemoglobin (HbA1c) was 8.2%. Due to the poor metabolic control and because of the presence of the hepatic disease, subcutaneous insulin 26 U/day (Humalog Mix 25) was started in June 2000: 16 units in the morning and 10 units in the evening. In 2002, the patient underwent an emergency laparoscopic cholecystectomy for acute calculous cholecystitis. Postoperatively he developed mild-to-moderate ascites, jaundice, impaired synthetic function and was included on the waiting list for liver transplantation as minimal eligibility criteria were met (Child Pugh score B, MELD score 17). Biochemical and virological findings revealed an active liver disease with marked hepatocytolysis (transaminases x 3 normal value, gamma-glutamyl transpeptidase x 2 normal value) and high HCV RNA (1,840,000 IU/mL). Because he had only mild thrombocytopenia and normal levels of hemoglobin and leucocytes, a second antiviral treatment was initiated using a low dose pegylated interferon-a2a (Pegasys®) 90 µg/week and ribavirin (Copegus®) 1000 mg/day. After one month of therapy with good hematological tolerability, the dose of Peginterferon-a2a was increased to 180µg/week. After 12 weeks he had no early virological response, but the viremia dropped by more than 1 log10 (89,200 IU/mL) and the aminotransferases decreased to 1.5 x normal value; after 24 weeks he had normal levels of aminotransferases but still a very low positive viral load (3,700 IU/mL). It has been therefore decided to initiate a long-term maintenance regimen with 90 µg/week Pegasys.
Between 2002 and 2004, blood glucose values increased (while on two Humalog Mix insulin injections) progressively and parallel with HbA1c from 8.6% to 9.1%. In the same time, due to the high blood glucose values, insulin requirement increased from 26 U to 40 U per day (2003 - 2004).
In November 2005, a sudden increase of aminotransferases levels (2-2.5 x normal value) and viral load (1,220,000 IU/mL) was noted while still on therapy. At that moment, the risk of disease decompensation triggered by the high viral load urged the procedure of liver transplantation (LT) for HCV induced liver cirrhosis (MELD score of 14). A sudden increase in insulin need (64 U/ day) was also noted.
Due to the association of severe hepatic disease and insulin-treated diabetes, a liver and islet cell transplantation was decided for this patient. Metabolic variables and liver tests before and after transplantation are summed up in Table 1.
On 5 December 2005, liver and pancreas were harvested from a cadaveric multi-organ donor. The cold ischemia time was 8 hours and for preservation, a Custodiol® solution at 4ºC was used.
Donor's pancreas and liver were selected based on ABO blood type identity, negative cross-match test between donor and recipient, and HLA antigen matching. Orthotopic LT was performed, with side-to-side cavo-caval anastomosis, end-to-end porto-portal anastomosis and end-to-end choledocho-choledochostomy; the arterial reconstruction of the donor's celiac trunk was performed using the superior mesenteric artery. At the end of the procedure, a 4 French catheter was implanted to enable islet transplantation into the main portal vein via the inferior mesenteric vein, and two epiploic pockets were created also for islet implantation.
The immunosuppressive protocol was initiated shortly before LT consisting of Daclizumab (Zenapax®) as induction therapy (1mg/kg twice weekly) and Sirolimus (Rapamycin®) and low-dose Tacrolimus (Prograf®) as maintenance treatment. Sirolimus plasma level was monitored to obtain a maintenance target level of 12-15 ng/mL during the first 3 months and 8-10 ng/mL afterwards, and the tacrolimus level was maintained between 3 and 6 ng/mL (9).
The islets were isolated by a two-step method as previously described (10). Isolated islets were maintained in CMRL 1066 media (Sigma-Aldrich), supplemented with insulin transferrin and selenium (ITS), linoleic acid, human serum albumin, nicotinamide, vitamin E (Sigma Aldrich) held overnight in an atmosphere of 5% carbon dioxide (CO2) and 95% air at 37ºC, the temperature being further lowered to 24ºC within 72 hours. The results of the micro-biological sterility test were negative. The cultured islets were then resuspended in an infusion medium and shipped to Fundeni Clinical Institute in Bucharest within 3 hours. The total islet yield was 284 000 islet equivalents (IEQ), with 80% pretransplantation viability. Seventy-two hours after liver transplantation, in the intensive care unit, the islets were slowly (over 20 minutes) infused intraportally and into the epiploic pockets through a catheter via syringe. The portal venous pressure was monitored before and every 5 minutes during the infusion.
The Ethics Board of the Fundeni Clinical Institute approved all protocols. The patient signed the informed consent for all procedures.
On postoperative day 4, the patient underwent surgery to remove a large subhepatic hematoma and to check for potential sources of bleeding; on this occasion the catheters for islet infusion were removed.
A transient increase of the liver enzyme levels (aspartat aminotransferase, alanine aminotransferase) with onset in the 4th post-islet transplantation day (considered to be related to islet infusion) was noted. A percutaneous liver biopsy has been performed on post-LT day 13 and acute cellular rejection was ruled out; the histological findings (liver parenchyma specimen with preserved architecture of two portal spaces with minimal intraportal plasma cell inflammatory infiltrate) were considered a reaction to the infused islets. After 2 weeks, the levels of liver enzymes decreased progressively towards normalization, noted 20 days later.
In the following 2 months after transplantation, blood glucose levels decreased progressively with a parallel decrease of HbA1c, from 7.5% in June 2006 to 6.1% in September 2006. During this period, the insulin requirements decreased from 64 U/day (pre-transplantation, in December 2005) to 40 U/day in February 2006 and 20 U/day in September 2006. An attempt to decrease insulin dose to 16 U per day was associated with a slight deterioration of the metabolic control (blood glucose level occasionally above 150 mg/dl and HbA1c of 7.5%). Increase of the insulin dose back to 20 U/day was followed by a stabilization of the metabolic control (blood glucose <130 mg/dl and a decrease of HbA1c to 6.9%). Metabolic evaluation was performed by measuring plasmatic glucose levels (fasting blood glucose levels, 2-hours postprandial levels), basal C-peptide levels and HbA1c concentrations). A 72 hours continuous plasma glucose monitoring (Medtronic MiniMed Continuous Glucose Monitoring System - CGMS) was performed in February 2006.
The GMS recording (Fig. 1) indicates a medium range glycemia of about 115.8 + 33.2 mg/dL for all the three days. Records of blood glucose (BG) values showed that only one measurement yielded a level over 200 mg/dL, but multiple values were <70 mg/dL. Fasting blood glucose concentrations were <126 mg/dL. These findings proved an excellent metabolic control in accordance with the HbA1c value.
The HCV RNA serum level was 4,080,000 IU/mL at 7 weeks, 5,420,000 IU/mL at 4 months and 6,300,000 IU/mL at 6 months after LT. Although during this time interval the transaminases remained within normal range, a liver biopsy was performed 6 months post-LT showing mild portal lympho-cytic infiltrate indicating recurrent hepatitis C. During the 10th post-LT month there was a sudden increase in the transaminase values (4 x normal value) concomitantly with a high increase of the viral load (HCV RNA = 36,700,000 IU/mL). The liver biopsy showed histological lesions of marked portal, periportal and lobular inflammation, focal perisinusoidal collagenisation, frequent areas of confluent necrosis, mild macrovesicular steatosis. Antiviral therapy with pegylated interferon a2a (Pegasys®, Roche, Basel, Switzerland) 180 µg/week and ribavirin (Copegus®, Roche, Basel, Switzerland) 1000 mg/day was started with good biochemical and viral response within one month. The transaminases normalized and the HCV RNA decreased to 1,500,000 IU/mL. At this time, the decision to introduce mycophenolate mofetil (MMF, Roche®) instead of sirolimus was taken due to proved antifibrotic and anti-inflammatory effects of MMF in HCV recipients; tacrolimus was maintained at a target level of 2 to 4 ng/mL. As side effects, due to sirolimus the patient had increased levels of cholesterol and triglycerides with increased cardiovascular risk, also taking into account the presence of concomitant diabetes mellitus and arterial hypertension. The pegylated interferon was continued up to 3 months, but the viral load increased to 2,850,000 IU/mL (in the second month of antiviral therapy) and 2,129,000 IU/mL (in the third month of antiviral therapy). Because of the concomitantly increase of transaminases a liver biopsy was performed showing a moderate inflammatory necrosis activity and progression of fibrosis (periportal fibrosis), perisinusoidal collagenisation and hepatic steatosis grade 1. The antiviral therapy was stopped due to lack of efficacy.
On posttransplant month 14th (February 2007), the patient developed right pleural effusion and low-grade fever, and based on pleural biopsy he was diagnoses with tuberculosis. Antituberculosis treatment was started using a triple-drug regimen and the patient developed jaundice and ascites. The liver biopsy reconfirmed HCV infection, and the serological tests revealed an acute CMV infection [IgM (+) and PP65 CMV (+)] with HIV (-). The patient was relisted for retransplantation, but no liver was available while his liver failure progressed and he subsequently died in May 2007.
The necroptic examination of the liver reported a persistent chronic hepatitis with modified tissular architecture by diffuse and localized fibrosis, with massive chronic inflammatory infiltration of the portal and periportal spaces and focal intralobular inflammatory infiltration, biliary duct proliferation, multiple binucleate hepatocytes, moderate intrasinusoidal biliary stases and interface hepatitis over 50%.

Discussions
In this case the diabetes chronologically occurred after the HCV chronic hepatitis and the diabetes phenotype was probably a form of non-autoimmune type 1 diabetes with a significant residual beta cell function. Arguments for this are the young age at onset (35 years), absence of overweight (BMI 24.8 Kg/m2), the relatively slow but constant progressive increase of hyperglycemia, the absence of the HLA genes characteristic for autoimmune type 1 diabetes as well as the absence of markers for beta cell autoimmunity. He was AB blood group and HLA typing showed an A*02/A*68; B*15/B*18; DRB1*13/DRB1*13; DQB1*03/DQB1*06 genotype. The absence of anti beta cell autoimmunity could explain also the good tolerance for the transplanted islets, proved by the rapid and durable decrease of the insulin need, from 64 U/day to 20 U/day at one-month post-transplantation, dose that was maintained up to the end. This low dose allowed an excellent metabolic control, proved by the CGMS recording of BG values (Fig. 1) that showed the absence of hyperglycemic peaks but also of severe hypoglycemic reactions. The fact that insulin-independence was not obtained after islet transplantation can be explained by the relatively small number of viable islets (~ 284.000 IEQ), obtained from a single cadaveric donor, a figure that is well below half the number of islets that allow insulin-independence according to Edmonton protocol (~ 800.000 IEQ) (11). It should be noted that in Edmonton series, sometimes two or three supplementary transplantations were needed in order to maintain the insulin-independence. In what diabetes therapy is concerned, was important that only low-dose insulin therapy was needed (~0.2 U/kg), dose which represents less than 50% of the usual insulin need for a normal subject (~4.5 U/day), suggesting that the difference of insulin was supplied by the function of transplanted islets. The last unexpectedly high C-peptide level (1.04 nmol/l) after a substantial decrease in the immediate post-transplantation period (0.17 and 0.11 nmol/l) may suggest that the new metabolic conditions created by the transplanted liver led to a stimulation of the regenerative power of the residual pancreatic beta cells. It is appreciated that metabolic stability after transplantation is a marker associated with a good long-term prognostic (11). A non-invasive method for beta cell mass imaging using positron emission tomography was only recently reported (11) but this was not available for our patient.
The relationship between HCV and diabetes involves complex mechanisms that include, on one hand, the diabetogenic influence of HCV and on the other hand, the diabetogenic effect of antiviral therapy (especially interferon). The specific mechanisms by which HCV affects the ß cell function are not fully understood, but an increase of insulin resistance, associated with both steatosis and over-production of proinflammatory cytokines (12, 13, 14) could play an important role. An attractive theoretic link between HCV and type 1 diabetes has been sustained by Honeyman et al. (15), exploiting the existence of a shared regional amino acid homology between HCV and GAD antibody. However, in our patient GAD antibodies were negative, making less probable an autoimmune mechanism in this case. Recently, Simo et al. (13) have shown that eradication of HCV infection using interferon alpha -2ß alone or with ribavirin significantly reduces the incidence of glucose abnormalities in hepatitis C patients (14.6% vs 34.1% - in patients without such a treatment), suggesting that HCV infection is a more important diabetogenic factor than antiviral treatment.
Unfortunately, without viral eradication before transplantation, HCV recurrence is universal and is associated with poor graft and patient survival (16).
Histologic evidence of recurrence is apparent in approximately 50% of hepatitis C virus infected recipients in the first postoperative year (17). Accelerated decompensation has also been noted when compared with cirrhosis in non-transplant patients (18). Approximately 10% of HCV-infected recipients will die or lose their allograft due to hepatitis C-associated allograft failure, especially with genotype 1b (17). A recent study (19) on 354 patients with LT for HCV cirrhosis with over 1 year survival period showed that 56% of deaths were caused by severe HCV recurrence. Berenguer M et al (20) showed a dramatic worsening in the outcome of HCV patients transplanted after 1998 with only a 60% survival at 1 year, fact explained by the combined detrimental effect of the increasing age of the donors and the use of more potent induction immunosuppression.
Different treatment strategies are available for recurrent HCV infection post-LT, the antiviral therapy being the mainstay of managing recurrent HCV disease in transplant recipients, but the response rates are modest, with poor tolerability and the risk of rejection as well as graft loss. Anti-HCV immunoglobulin therapy to prevent graft infection with HCV has no established role at present but studies are ongoing. Prophylactic interferon-based antiviral therapy in the early postoperative period to prevent graft infection was shown to have low response rates and high rates of adverse effects (18). Prophylactic therapy in the form of polyclonal HCV antibodies has not been effective at prevention of HCV re-infection, but higher-dose therapy may modify the severity of early disease recurrence (21). Treatment of established recurrent HCV infection with combination peginterferon (pegylated interferon) and ribavirin is associated with only 10-59% sustained virological response (18) and should be considered for treatment of recipients with histologically apparent recurrence of hepatitis C before total bilirubin exceeds 3 mg/dL (17). Histologic and biochemical improvements generally are more frequent than virologic responses. Overall, the treatment of HCV disease in transplant recipients leaves much to be desired and there is an urgent need of new HCV therapies in this patient population (21).

Conclusion
Islet transplantation can be associated to liver transplantation in order to improve the associated diabetes in cirrhotic patients.

Acknowledgments
This study was supported by grant CEEX 211/2006 from the National Institutes of Research.

References
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