Transcriptome changes in renal allograft protocol biopsies at 3 months precede the onset of interstitial fibrosis/tubular atrophy (IF/TA) at 6 months.

BACKGROUND: Interstitial fibrosis and tubular atrophy (IF/TA) in renal transplants are the major morphological correlates of progressive graft deterioration. Early diagnosis of IF/TA is a pre-requisite for a timely therapeutic intervention in patients at risk. To evaluate events occurring before the overt onset of IF/TA, gene expression profiling of 3-month protocol biopsies from patients with IF/TA was performed in a patient group (n = 8) who developed mild IF/TA [chronic allograft nephropathy (CAN) grade I, by the Banff scoring system] in the subsequent 6-month protocol biopsy ('progressors'), and in 12 patients without IF/TA at 6 months ('non-progressors'). METHODS: RNA was extracted, labelled and hybridized to human specific genome wide DNA microarrays. Normalized data were subjected to gene-centric and pathway-centric statistical methods. RESULTS: Compared to the non-progressors, the 3-month biopsies of the progressor group showed overexpression of several genes that are important in the T- and B-cell activation and immune response. Genes involved in pro-fibrotic processes were identified in the biopsies of the progressors that preceded the observed IF/TA at 6 months. Furthermore, several genes with transporter and metabolic functions were underrepresented in the progressors in the 3-month biopsies. CONCLUSION: Gene expression profiling of early protocol biopsies identified changes in the transcriptome of grafts, which may be important for the development of IF/TA. Such early detection of transcriptome changes can facilitate the identification of patients at risk shifting the intervention time point well before the histological diagnosis of irreversible IF/TA.

Tabebuia avellanedae extracts inhibit IL-2-independent T-lymphocyte activation and proliferation.

In order to identify new, immune modulating compounds, aqueous extracts of plants pre-selected on ethno-pharmacological knowledge were screened for inhibitory effects in an anti-CD3 driven lymphocyte proliferation assay (MTT-assay). We found for the extract of the inner bark of Tabebuia avellanedae (Tabebuia) dose dependent and reproducible inhibitory effects on lymphocyte proliferation. We further analyzed Tabebuia in flow cytometry based whole blood T-cell function assays. We found that Tabebuia inhibited dose dependent ConA stimulated T-cell proliferation. Decreased T-lymphocyte proliferation was associated with dose dependent reduction of CD25 and CD71 expression on T-lymphocytes. In contrast Tabebuia exerted no effects on cytokine expression (Il-2 and TNF-alpha) by PMA/Ionomycin stimulated T-lymphocytes. Concentrations of Tabebuia used were not toxic for lymphocytes as verified by trypan blue exclusion assay. Further experiments showed that the immune inhibitory effects by Tabebuia were not mediated by its pharmacological lead compound beta-lapachone and only observed in aqueous but not in ethanol plant extracts.

Transplantation of an eight-organ multivisceral graft in a patient with frozen abdomen after complicated Crohn's disease.

To report an extended multivisceral transplantation (MVTx) including right kidney and ascending colon in a patient with complicated Crohn's disease (CD). A 36-year old female suffering from short bowel syndrome and frozen abdomen due to fistulizing CD after multiple abdominal operations underwent MVTx of eight organs including stomach, pancreatoduodenal complex, liver, intestine, ascending colon, right kidney, right adrenal gland, and greater omentum in November 2003. Immunosuppression consisted of alemtuzumab, tacrolimus and steroids. The patient was off parenteral nutrition by postoperative wk 3. She experienced one episode of pneumonia. The patient recovered completely and discharged 2.5 mo and was doing well 30 mo after MVTx. This is one of the very rare cases in which a complete mulitivisceral graft of eight abdominal organs was transplanted orthotopically.

Sirolimus (rapamycin) monotherapy prevents graft vascular disease in nonhuman primate recipients of orthotopic aortic allografts.

BACKGROUND: Delayed treatment with sirolimus (SRL) halts progression of graft vascular disease (GVD) in nonhuman primate (NHP) aortic allograft recipients. In this study, we investigated whether SRL monotherapy prevents the development of GVD. METHODS AND RESULTS: Pairs of 3-cm infrarenal aortic segments were exchanged between mixed lymphocyte reaction-mismatched, blood group-compatible NHPs (n=12). Six NHPs were untreated controls, and 6 were treated orally with SRL starting on the day of transplantation. Follow-up was 105 days. SRL doses were adjusted individually by assessing SRL blood concentrations, immune function, and clinical status. The severity of GVD was determined every 3 weeks by intravascular ultrasound, which quantified intimal area (IA) and intimal volume (IV) for the middle 1-cm graft segments. The mean+/-SEM SRL plasma levels were 14.5+/-9 ng/mL. In grafts from treated NHPs, IA and IV values on days 63, 84, and 105 were significantly lower than for controls (P<0.05 to P<0.001). On day 105, in the grafts from SRL-treated NHPs compared with grafts from controls, values (mean+/-SEM) were IA, 2.9+/-0.9 versus 5.5+/-0.7 mm2, P<0.001 and IV, 29.6+/-4.6 versus 55.2+/-2.8 mm3, P<0.001; IA and IV values for grafts from SRL-treated NHPs did not increase significantly between days 21 and 105. CONCLUSIONS: We show that SRL monotherapy prevented GVD in NHP aortic allograft recipients, suggesting the value of SRL for controlling GVD in clinical transplantation.

Indications of mycophenolate mofetil in liver transplantation.

Mycophenolate mofetil (MMF) is approved for prophylaxis of acute rejection after kidney, heart, and liver transplantation as well as for pediatric patients after kidney transplantation. MMF, a noncompetitive inhibitor of inosine monophosphate dehydrogenase (IMPDH), blocks de novo purine synthesis which leads to an effective inhibition of proliferation selectively in T and B lymphocytes, smooth muscle cells, and fibroblasts. MMF shows additional effects with inhibition of the expression of activating and adhesion molecules on the surface of lymphocytes. The beneficial safety profile with distinct side effects compared to calcineurin inhibitors (CNI) enable efficacious combination with ciclosporin or tacrolimus as de novo therapy after liver transplantation. Furthermore, recent studies show the possibility to reduce CNI induced toxicities by adding MMF to primary immunosuppression. MMF is also used to enable early steroid withdrawal after liver transplantation. MMF can increase efficacy of immunosuppressive therapy and thereby support the treatment of steroid resistant acute rejections, chronic rejections and chronic graft dysfunction.

Extended indications in living-donor liver transplantation: bile duct cancer.

The advantages of living donor liver transplantation are an individually available graft and a tremendously reduced waiting time until transplantation. One consequence is that many centers have extended the pretransplant selection criteria, especially for potential recipients suffering from hepatocellular carcinoma. In contrast, reports on living donor liver transplantation for cholangiocarcinoma are restricted to few case reports. We have analyzed our experience with seven patients suffering from cholangiocarcinoma (Klatskin tumors, n=5; intrahepatic cholangiocarcinoma, n=2). During a median follow-up of 20 months (range 2-46 months), all patients are alive except for one posttransplant death. Four patients suffering from Klatskin tumors are alive without recurrence; both patients suffering from intrahepatic cholangiocarcinoma are alive with bone and peritoneal metastases. Living donor liver transplantation may be beneficial in selected patients suffering from Klatskin tumors, whereas caution should prevail when considering intrahepatic cholangiocarcinoma.

Endocrine tumours of the gastrointestinal tract. Transplantation in the management of metastatic endocrine tumours.

Patients with neuroendocrine tumours often present with synchronous liver metastases or develop hepatic metastases in the course of their disease. A complete removal of liver metastases with an intention to cure may be accomplished by liver resection or, if hepatic disease is disseminated or hormonal symptoms and pain cannot be controlled medically, by total hepatectomy and transplantation. The indications for orthotopic liver transplantation for metastatic neuroendocrine tumour disease should be anchored in a multimodal and multidisciplinary therapeutic approach. Approximately, 120-130 cases of orthotopic liver transplantation for neuroendocrine tumours have been published so far, but follow-up after transplantation has been limited, and most reports comprise a small number of patients. After considering published studies and data, some recommendations may be given, although these are based on a low level of evidence. After excluding extrahepatic tumour manifestations by imaging procedures and diagnostic laparoscopy, the indication should be chosen restrictively. Few prognostic markers, for example age below 50 years and absence of concurrent extensive surgery, were identified by multivariate analysis in a large retrospective analysis. The prognostic impact of primary tumour localisation is still controversial. However, further indicators of favourable long-term prognosis are needed. Tumour biology characterised by Ki67 and E-cadherin expression may help to identify patients with a favourable outcome so that patient selection can be improved, but this needs further evaluation in larger patient cohorts. Orthotopic liver transplantation for patients with remission of disease or stable disease under medical treatment, and orthotopic liver transplantation for palliative reasons, should be restricted to selected individual cases.

Biologics in the treatment of transplant rejection and ischemia/reperfusion injury: new applications for TNFalpha inhibitors?

Tumor necrosis factor (TNF)-alpha inhibitors have proven efficacy in various autoimmune diseases such as Crohn disease, rheumatoid arthritis, psoriasis, and ankylosing spondylitis. Indeed, some TNFalpha inhibitors have already been approved for the management of the inflammatory manifestations associated with Crohn disease and rheumatoid arthritis. These agents are increasingly used for treatment of corticosteroid-resistant graft-versus-host disease after bone marrow transplantation, and case reports have documented their efficacy in treating corticosteroid- and muromonab-resistant rejection after intestinal transplantation. Thus, the potential role of TNFalpha inhibitors in transplantation of other vascularized solid organs is worthy of investigation. Experimental evidence indicates that TNFalpha plays a key role in mediating ischemia/reperfusion (IR) injury after liver, kidney, intestine, heart, lung, and pancreas transplantation. TNFalpha was also identified as a marker cytokine during organ rejection. Single-center studies evaluating the role of TNFalpha inhibitors in kidney transplantation have been initiated but the results are not yet available. TNFalpha is known to be a contributing factor in kidney allograft rejection, and may have value in predicting the onset of steroid-resistant acute rejection after liver transplantation. Experimental and preliminary clinical data have shown that circulating levels of TNFalpha are increased during cardiac graft rejection, and indicate that TNFalpha plays a role in the pathogenesis of acute cardiac allograft rejection. Anti-TNFalpha therapy was shown to prolong cardiac allograft survival when used alone or in combination with other drugs. TNFalpha genotype has been strongly associated with mortality in humans due to acute cell-mediated heart transplant rejection. In addition, there is evidence for a genetic predisposition toward acute rejection after kidney and simultaneous kidney-pancreas transplantation. TNFalpha inhibition has been used successfully as part of an induction therapy for pancreatic islet cell transplantation. Apart from IR injury and acute rejection after lung transplantation, TNFalpha was also found to be involved in the pathoimmunology of obliterative bronchiolitis. In conclusion, a substantial body of experimental evidence and preliminary clinical data suggest that TNFalpha inhibitors may play an important role in solid-organ transplantation, both in the amelioration of IR injury and in the treatment and prevention of acute rejection. Pharmacodynamic monitoring and pharmacogenetic screening may help to identify patients most likely to benefit from TNFalpha blockade. Randomized controlled trials in patients undergoing solid-organ transplantation are needed to further elucidate the clinical value of TNFalpha inhibition.

Motivation for living-donor liver transplantation from the donor's perspective: an in-depth qualitative research study.

BACKGROUND: There has been a lack of systematic in-depth research on the motives of living liver donors before transplantation that could contribute to an advanced understanding of their situation and to a more precise psychosocial evaluation, to protect the autonomy for decision, and to prevent psychosocial complications. METHODS: Twenty-eight living liver donors were assessed preoperatively through a semistructured clinical interview. The taped and transcribed interviews were analyzed using a combination of grounded theory and empirically grounded type construction. RESULTS: Various factors contribute to the donor's motivation for donation: the relationship to the recipient, the personal attitude of the donor, his or her personal history, family dynamics, the donor's personal profit, and the exceptional situation of the recipient's life-threatening disease combined with the life-rescuing possibility of living-donor liver transplantation (LDLT). In reference to this, five "ideal types" of living donors emerged from the authors' data. CONCLUSIONS: A complete absence of coercion on the decision to donate seems unrealistic because of the dynamics initiated by the life-threatening condition of the recipient. It is important that donors feel they are gaining something by donation to be sufficiently motivated and that their profit is of an emotional or moral nature (i.e., the donation being set in an emotionally meaningful context). A mature relationship with the recipient usually provides such a context. The role of the clinician as a part of LDLT dynamics has a decisive influence.

Impact of human leukocyte antigen matching in liver transplantation.

BACKGROUND: Human leukocyte antigen (HLA) compatibilities are beneficial in the setting of kidney transplantation but have demonstrated inconclusive results after liver transplantation. On the basis of recent controversial reports, the authors analyzed the impact of HLA matching in their patients after liver transplantation under modern immunosuppressive drug regimens and new HLA typing techniques with respect to outcome and adverse immunologic events. METHODS: Data from 924 transplants with complete donor-recipient HLA typing were retrospectively analyzed. Immunosuppression was commenced as either cyclosporine A- or tacrolimus-based therapy in different protocols. The follow-up period ranged from 1 to 144.8 months (median, 66 months). RESULTS: The actuarial graft survival was 88% after 1 year and 78.7% after 5 years and was similar in tacrolimus- and cyclosporine A-treated patients. However, cyclosporine A-treated patients underwent significantly more rejection episodes. The number of HLA compatibilities had no influence on graft survival, whereas the number of acute rejections was significantly less in transplants with more HLA compatibilities (P<0.05). Graft survival tended to be improved in patients with chronic hepatitis B and more HLA compatibilities (P=0.05). In contrast, graft survival in transplants for primary sclerosing cholangitis was significantly impaired in the presence of one or two HLA-DR compatibilities (P<0.05). In addition, in autoimmune hepatitis, survival tended to be lower in the presence of more HLA compatibilities (P=0.1). Overall graft survival or frequency of adverse immunologic events was not influenced by any specific donor-recipient HLA allele. CONCLUSION: This study demonstrated fewer acute rejections in transplants with more HLA compatibilities. However, in liver transplantation, a more specific investigation of HLA typing may be necessary, because in some indications HLA antigens play a role in the nature of the disease. Therefore, recurrence of autoimmune disease may be more severe in patients sharing HLA antigens.

Treatment with humanized monoclonal antibodies against CD80 and CD86 combined with sirolimus prolongs renal allograft survival in cynomolgus monkeys.

BACKGROUND: Co-stimulatory blockade has been shown to prolong allograft survival in different transplant models. We investigated the effect of combining humanized anti-CD80 and anti-CD86 monoclonal antibodies (mAb) with sirolimus in cynomolgus monkey renal transplant recipients. METHODS: After renal transplantation, groups of four animals were treated daily with sirolimus, sirolimus and nine weekly doses of mAb, two weekly doses of mAb, or sirolimus and two weekly doses of mAb. RESULTS: Survival was significantly better in monkeys treated with the combination of sirolimus and mAb when compared with treatment with either agent alone (P=0.0067 by log-rank analysis). When combined with sirolimus, nine weekly doses of mAb did not result in an additional survival benefit compared with only two mAb doses (P=0.74). None of the treatment regimens used in this study resulted in development of transplantation tolerance. CONCLUSIONS: Sirolimus can be successfully combined with humanized mAb against CD80 and CD86. Induction with a short course of mAb is effective in prolonging allograft survival in combination with sirolimus.

Mycophenolatemofetil for immunosuppression after liver transplantation: a follow-up study of 191 patients.

BACKGROUND: Mycophenolatemofetil (MMF) combined with calcineurin inhibitors (CNIs) as immunosuppression after orthotopic liver transplantation (OLT) is still under discussion. We retrospectively investigated the immunosuppressive potency of MMF for treatment of steroid-resistant acute rejection (AR) or chronic rejection (CR), chronic graft dysfunction, and CNI-induced toxicity in patients after OLT. METHODS: Between 1988 and 2001 we performed 1386 OLTs in 1258 patients. Since 1995, 191 patients have received MMF after OLT for steroid-resistant AR or CR, chronic graft dysfunction (115 patients), and CNI-induced toxicity (76 patients). The mean follow-up time was 56 months. RESULTS: Of 47 patients with steroid-resistant AR, 12 had been treated with OKT3, without resolving the rejection. Overall, bilirubin and transaminases decreased significantly within 2 weeks after the addition of MMF, and liver function normalized in 38 patients. Five of eight patients with CR demonstrated stable liver function after a follow-up of 55+/-8 months; 52 of 60 patients with chronic graft dysfunction improved within 3 months; and 46 of 59 patients with CNI-induced nephrotoxicity improved after MMF treatment and a reduction of CNIs (with a significant decrease in serum creatinine within 2 weeks and an increase of creatinine clearance within 3 months). Clinical symptoms improved in 10 of 12 patients with neurotoxicity and four of five patients with hepatotoxicity. Side effects of MMF, such as gastrointestinal disorders or bone marrow toxicity, occurred in 60 patients (31.4%). The incidence of infections did not increase. Patient survival was 93%, and graft survival was 88.2%. CONCLUSIONS: MMF is a potent and safe immunosuppressive agent in OLT recipients for rescue therapy in AR, CR, or chronic graft dysfunction and helps to reduce the serious toxic side effects of CNIs.

Successful infliximab treatment of steroid and OKT3 refractory acute cellular rejection in two patients after intestinal transplantation.

Acute rejection resistant to established immunosuppressive rescue protocols remains the most prominent risk factor after intestinal transplantation. In two patients presenting with steroid-resistant severe acute cellular rejection 9 months and 2 years after intestinal transplantation, complete resolution was not achieved despite 5 and 10 days of OKT3 treatment, respectively, and high-dose triple baseline immunosuppression with tacrolimus, rapamycin, and steroids. There was a dissociated course of rejection with persistent moderate to severe rejection in the terminal portion of the graft despite complete recovery from rejection in the proximal parts. Both patients were treated with four subsequent infusions of infliximab (3 mg/kg body weight), a chimeric anti-tumor necrosis factor-alpha antibody. There was an immediate response regarding macroscopic appearance, graft histology, and clinical symptoms. Both patients recovered. In conclusion, infliximab has proven to be an effective rescue therapy in a selected group of patients with steroid and OKT3 refractory severe acute rejection after intestinal transplantation.

Sustained suppression of peripheral blood immune functions by treatment with mycophenolate mofetil correlates with reduced severity of cardiac allograft rejection.

BACKGROUND: We tested the hypothesis that sustained suppression of immune functions by mycophenolate mofetil (MMF) throughout the dosing interval reduces the severity of rejection. METHODS: Four groups of rat heart allograft recipients were treated orally daily through Day 5 with either: "low-dose" MMF, 10 mg/kg once daily (QD) or 5 mg/kg twice daily (BID); or "high-dose" MMF, 20 mg/kg QD or 10 mg/kg BID. The following were determined for all animals on Day 6: pharmacokinetics (PK, using high-performance liquid chromatography) of mycophenolic acid (MPA); pharmacodynamics (PD, by flow cytometry quantitation of whole blood mitogen-stimulated lymphocyte proliferation and expression of diverse T-cell surface activation molecules); and histologic graft rejection scores (RS). RESULTS: RS correlated with PD for suppression of both lymphocyte proliferation and transferrin receptor expression (r2 = 0.85 and 0.81, respectively) more highly than with MPA plasma levels (r2 = 0.45), which shows the validity of PD as surrogate markers of MMF efficacy. MMF 5 mg/kg BID produced greater (p < 0.001) suppression of lymphocyte proliferation (area under the PD effect-time curve, AUE = 2,010% inhibition. hour) and sustained trough (E0) PD effect (86% suppression) than MMF 10 mg/kg QD (AUE = 1,436% inhibition. hour, E0 = 55%). RS did not differ between the 20 mg/kg QD and 10 mg/kg BID "high-dose" groups, because PD was maximally suppressed. CONCLUSIONS: PD were surrogate markers for MMF immunosuppressive efficacy. MMF 5 mg/kg BID produced more sustained suppression of both PD and rejection than MMF 10 mg/kg QD.

How pharmacokinetic and pharmacodynamic drug monitoring can improve outcome in solid organ transplant recipients.

Within the field of solid organ transplantation there is an unprecedented interest in therapeutic drug monitoring of immunosuppressive drugs. Ideally therapeutic drug monitoring should cost-effectively lead to improved efficacy of the drug and to a reduction in side effects. Therapeutic drug monitoring will be most effective if there is a large interpatient variability and a small intrapatient variability. Therapeutic drug monitoring in transplantation is largely based on correlations between drug concentrations and toxicity or between drug concentrations and efficacy. Pharmacodynamic monitoring of immunosuppressive drugs has not reached the stage of widespread clinical application. In part this is caused by the fact that most of the pharmacodynamic assays are time-consuming, costly and in some cases only give a result after several days of incubation. Another reason for the limited interest in pharmacodynamic monitoring is the lack of data showing improved outcome if dose adjustment is based on pharmacodynamics rather than pharmacokinetics. On the other hand, such data are also lacking for pharmacokinetic monitoring. Prospective investigations on the contribution of therapeutic drug monitoring may result in further improvement of the safety and efficacy of our immunosuppressive regimens and more refined methods for therapeutic drug monitoring. There is no contest between pharmacokinetic and pharmacodynamic monitoring. Most likely the results of both ways of monitoring will be complementary.

Effects of the malononitrilamide FK778 on immune functions in vitro in whole blood from non-human primates and healthy human volunteers.

BACKGROUND: FK778, a malononitrilamide analog of leflunomide, is currently being investigated for use in clinical transplantation. METHODS: Whole blood from cynomolgus monkeys (n=4) and healthy human volunteers (n=4) was incubated with different concentrations of FK778 and stimulated with mitogens in culture medium. Lymphocyte proliferation was assessed by tritium-labeled thymidine incorporation and by flow-cytometric analysis of expression of proliferating cell nuclear antigen on cells in S/G(2)M phase. Flow cytometry was also used to assess expression of T and B lymphocyte activation surface antigens and production of intracellular cytokines by T cells. RESULTS: Not only lymphocyte proliferation, but also expression of various T cell surface antigens (CD25, CD11a, CD95, CD154) was suppressed by FK778. Fifty percent effective concentration values for the different immune functions were lower in human blood than in blood from cynomolgus monkeys. CONCLUSIONS: FK778 inhibits multiple immune functions. Their flow cytometric evaluation can be used to assess the effects of the drug in vivo.

Parathyroid hormone venous sampling before reoperative surgery in renal hyperparathyroidism: comparison with noninvasive localization procedures and review of the literature.

OBJECTIVES: To analyze the predictive values of selective venous sampling (SVS) in our own experience and in a systematic meta-analysis of the international literature and to compare them with the results of noninvasive localization studies before reoperative parathyroid surgery. DATA SOURCES: Twenty-one consecutive patients with persistent or recurrent renal hyperparathyroidism underwent preoperative SVS and noninvasive imaging. These data were added to a systematic review of the literature on localization studies before reoperative surgery. The literature search included localization studies, recurrent hyperparathyroidism, and reoperation. STUDY SELECTION Prospective and retrospective studies that provided at least the true-positive rate of 1 procedure were included. Data from initial surgery, hyperfunctioning autografts, and case reports were excluded. DATA EXTRACTION: Thirty-one publications reported on SVS (n = 22), technetium Tc 99m sestamibi scintigraphy (n = 17), thallium-technetium scintigraphy (n = 11), ultrasonography (n = 18), magnetic resonance imaging (n = 12), and computed tomography (n = 13). The overall analysis was performed by dividing the overall number of true- and false-positive results by the total number of patients. DATA SYNTHESIS: Localization by SVS was correct in 20 of 21 patients. In 1 patient with 2 localizations, only 1 was predicted correctly. Therefore, the sensitivity of SVS was at least 90%, with no false-positive results. Overall true- and false-positive rates, respectively, in 31 studies were 71% and 9% for SVS, 69% and 7% for technetium Tc 99m sestamibi scintigraphy, 54% and 16% for magnetic resonance imaging, 55% and 15% for thallium-technetium scintigraphy, 50% and 18% for ultrasonography, and 45% and 14% for computed tomography. CONCLUSIONS: With its high sensitivity, SVS is the gold standard in patients with persistent or recurrent renal hyperparathyroidism and negative results of noninvasive localization procedures. The noninvasive procedure of choice is now technetium Tc 99m sestamibi scintigraphy, with high sensitivity and a low rate of false-positive results.

Validation of immunological biomarkers for the pharmacodynamic monitoring of immunosuppressive drugs in humans.

Pharmacodynamic monitoring (PD) can evaluate the efficacy of immunosuppressive drug therapies. In this study, the expressions of PD biomarkers [lymphocyte proliferation, CD25 and CD71 expression, interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-alpha) synthesis] were determined in whole-blood assays and were validated for their application in PD of immune modulators in future clinical trials. Initially, the assay conditions were re-evaluated. The measurement of T-lymphocyte proliferation and activation marker expression in whole-blood cultures resulted in optimized stimulation for 72 hours with 7.5 microg/mL concavalin A. Intracellular cytokine expression of CD3+ T-cells received optimized stimulation for 4 hours with 15 ng/mL phorbol 12-myristate 13-acetate and 0.75 microg/mL ionomycin. Statistical assay parameters (intra-assay, intra-individual, and interindividual variabilities) were determined. It was found that blood storage for up to 24 hours is possible without any change in biomarker expression. Dosage effects of immunosuppressive drugs (tacrolimus, cyclosporin A, sirolimus, mycophenolic acid, and methylprednisolone) were evaluated in vitro and the assay was applied successfully to dialysis, renal transplant, and liver transplant patients. We conclude that these biomarkers used in whole-blood assays are suitable for PD of immune modulators in clinical trials.

Long-term follow-up after recurrence of primary biliary cirrhosis after liver transplantation in 100 patients.

Orthotopic liver transplantation (OLT) is the only effective curative therapy for end-stage primary biliary cirrhosis (PBC). Survival after OLT is excellent, although recent data have shown a recurrence rate of PBC of up to 32% after transplantation. The aim of this study is to investigate the course after disease recurrence, particularly with regard to liver function and survival in a long-term follow-up. Between April 1989 and April 2003, 1,553 liver transplantations were performed in 1,415 patients at the Charité, Virchow Clinic. Protocol liver biopsies were taken after one, three, five, seven, 10 and 13 yr. One hundred (7%) patients suffered from histologically proven PBC. Primary immunosuppression consisted of cyclosporine (n = 54) or tacrolimus (Tac) (n = 46). Immediately after OLT, all patients received ursodeoxycholic acid. Corticosteroids were withdrawn three to six months after OLT. The median age of the 85 women and 15 men was 55 yr (range 25-66 yr). The median follow-up after liver transplantation was 118 months (range 16-187 months) and after recurrence 30 months (range 4-79 months). Actuarial patient survival after five, 10 and 15 yr was 87, 84 and 82% respectively. Ten patients (10%) died after a median survival time of 32 months. Two of these patients developed organ dysfunction owing to recurrence of PBC. Histological recurrence was found in 14 patients (14%) after a median time of 61 months (range 36-122 months). Patients with Tac immunosuppression developed PBC recurrence more often (p < 0.05) and also earlier (p < 0.05). Fifty-seven patients developed an acute rejection and two patients a chronic rejection episode. Liver function did not alter within the first five yr after histologically proven PBC recurrence. Multivariate analysis of the investigated patients showed that the recipient's age and Tac immunosuppression were significant risk factors for PBC recurrence. Long-term follow-up of up to 15 yr after liver transplantation, owing to PBC, in addition to maintenance of liver function, shows excellent organ and patient survival rates. Although protocol liver biopsies revealed histological recurrence in 14 (14%) patients, only two patients developed graft dysfunction. Tac-treated patients showed more frequently and also earlier histologically proven PBC recurrence; however, in our population we could not observe an impact on graft dysfunction and patient's survival.

Long-term results of mycophenolate mofetil as part of immunosuppressive induction therapy after liver transplantation.

BACKGROUND: The addition of mycophenolate mofetil (MMF) to the induction protocol resulted in a lower incidence of rejection episodes. However, the question whether MMF should be administered in combination with tacrolimus or cyclosporine has not been answered yet. In our study, we report on the long-term results of triple induction therapy after orthotopic liver transplantation (OLT), consisting of MMF and low-dose corticosteroids, in combination with either tacrolimus or cyclosporine. METHODS: Between March 1996 and April 1997, 120 consecutive patients, who underwent OLT at our institution, were enrolled in this study. Of these patients, 80 received triple induction therapy consisting of cyclosporine and MMF (40) or tacrolimus and MMF (40), in combination with low-dose corticosteroids, whereas the remaining 40 patients served as 'MMF-free' control group receiving dual induction therapy with tacrolimus and corticosteroids. Besides the eight-yr follow-up of patient and graft survival, clinical data were also reviewed for episodes of rejection and infection. Additionally, the early post-operative pharmacokinetics of mycophenolic acid (MPA, immunological active metabolite of MMF) were evaluated. RESULTS: Long-term results provided higher patient and graft survival after tacrolimus/MMF-based induction therapy than after cyclosporine/MMF-based induction therapy. However, the tacrolimus-based control protocol yielded similar results and, therefore, no significantly superior effect was observed when MMF was added. The same observation was made for incidence of rejection and infection episodes. AUC and C(max) of MPA increased in combination with tacrolimus compared with cyclosporine. CONCLUSIONS: Although pharmacological synergy between tacrolimus and MMF was observed, MMF showed no significant beneficial effects in the immunosuppressive induction protocol, neither in combination with tacrolimus nor with cyclosporine.