Pharmacokinetic modeling of enterohepatic circulation of mycophenolic acid in renal transplant recipients.

Several factors contribute to mycophenolic acid (MPA) between-patient variability. Here we characterize the metabolic pathways of MPA and quantify the effect of combining genetic polymorphism of multidrug-resistant-associated protein-2, demographics, biochemical covariates, co-medication (cyclosporine (CsA) vs. macrolides), and renal function on MPA, 7-O-MPA-glucuronide (MPAG), and acyl-glucuronide (AcMPAG) disposition, in renal transplant recipients, after mycophenolate mofetil. Complete pharmacokinetic profiles from 56 patients (five occasions) were analyzed. Enterohepatic circulation was modeled by transport of MPAG to the absorption site. This transport significantly decreased with increasing CsA trough concentrations (CtroughCsA). MPAG and AcMPAG plasma clearances significantly decreased with renal function. No significant influence of multidrug-resistant-associated protein-2 C24T single-nucleotide polymorphism was found. The model adequately predicted the increase in MPAG/AcMPAG exposures in CsA and macrolide patients with decreased renal function. This resulted in higher MPA exposures in macrolide patients versus CsA patients, and increased MPA exposures with renal function from 25 to 10 ml/min, in macrolide patients, owing to enhanced MPAG enterohepatic circulation. Lower-percentage enterohepatic circulation occurred with higher CtroughCsA and renal function values. The lack of MPA protein-binding modeling did not permit evaluation of the impact of renal function and CtroughCsA on MPA exposures in CsA patients. Thus, dose tailoring of covariates is recommended for target MPA exposure.

Immunosuppressive properties of a series of novel inhibitors of the monocarboxylate transporter MCT-1.

We have recently described the immunosuppressive properties of AR-C117977 and AR-C122982, representatives of a group of compounds identified as inhibitors of lactate transporters (monocarboxylate transporters; MCTs). These compounds demonstrate the potential therapeutic usefulness of inhibiting MCT-1, but their physical and metabolic properties made them unsuitable for further development. We have therefore tried to find analogues with similar immunosuppressive efficacy and a more suitable profile for oral administration. Five analogues of AR-C117977 were synthesised and screened for binding to the transporter, for inhibition of proliferation of both human and rat lymphocytes, for in vivo activity in a model of graft-versus-host (GvH) response in the rat, and in high- and low-responder cardiac transplant models in the rat. There was a good correlation between levels of binding of the five analogues to MCT and their inhibition of lymphocyte proliferation in human and rat cells. Furthermore, activity in both the GvH response and the cardiac transplant models correlated well with the determined concentrations of test compound in plasma. These findings on new analogues of MCT-1 inhibitors have taken us further towards defining the pharmacokinetic properties that may help to identify future drug candidates among inhibitors of MCT-1.

Do drug transporter (ABCB1) SNPs and P-glycoprotein function influence cyclosporine and macrolides exposure in renal transplant patients? Results of the pharmacogenomic substudy within the symphony study.

The function of the efflux pump P-glycoprotein (Pgp) and ABCB1 single nucleotide polymorphisms (SNPs) should be considered as important tools to deepen knowledge of drug nephrotoxicity and disposition mechanisms. The aim of this study is to investigate the association of C3435T, G2677T, C1236T, and T129C ABCB1 SNPs with Pgp activity and exposure to different immunosuppressive drugs in renal transplant patients. Patients included in the Symphony Pharmacogenomic substudy were genotyped for ABCB1 SNPs. According to the design, patients were randomized into four immunosuppressive regimens: low and standard dose of cyclosporine (n = 30), tacrolimus (n = 13), and sirolimus (n = 23) concomitantly with mycophenolate and steroids. Pgp activity was evaluated in PBMC using the Rhodamine 123 efflux assay. TT carrier patients on C3435T, G2677T, and C1236T SNPs (Pgp-low pumpers) showed lower Pgp activity than noncarriers. Pgp-high pumpers treated with cyclosporine showed lower values of Pgp function than macrolides. There was a negative correlation between cyclosporine AUC and Pgp activity at 3 months. Results did not show any correlation between tacrolimus and sirolimus AUC and Pgp activity at 3 months. We found an important role of the ABCB1 SNPs Pgp function in CD3(+) peripheral blood lymphocytes from renal transplant recipients. Pgp activity was influenced by cyclosporine but not macrolides exposure.

Effect of different immunosuppressive regimens on the evolution of distinct metabolic parameters: evidence from the Symphony study.

BACKGROUND: The metabolic syndrome (MS) is an important risk factor for graft dysfunction and patient death after renal transplantation. The aim of this sub-analysis of the Symphony study was to assess the progression of the laboratory parameters associated with MS in the first year after transplantation. METHODS: Data collected from the Symphony study were used; 1645 patients were randomized to receive standard-dose cyclosporine (Stand-CsA), low-dose cyclosporine (Low-CsA), tacrolimus (Low-Tac) or sirolimus (Low-SRL), in addition to mycophenolate mofetil (MMF) and corticosteroids. Data were collected for levels and progression over the first year post-transplantation of systolic and diastolic blood pressure, uric acid, triglycerides, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and fasting glucose levels by treatment arm. RESULTS: The low-SRL group had significantly higher levels of triglycerides and LDL. The two CsA arms were associated with the highest uric acid levels at each time point. There were no significant differences in overall levels or changes in glucose or HDL. Patients in the standard-CsA arm had significantly higher diastolic blood pressure than those in the Low-SRL and Low-Tac arms. Systolic blood pressure was higher in the Low-CsA arm than in the Low-Tac arm. The use of antihypertensive and antidiabetic agents was similar between the treatment arms. In the Low-SRL arm, more patients were treated with lipid-lowering therapy. Mean daily steroid doses were the highest in the Low-SRL arm. CONCLUSIONS: This sub-analysis demonstrates that there is a difference in metabolic parameters between immunosuppressive groups. CsA therapy was associated with the highest values of uric acid and systolic and diastolic blood pressure. Patients on SRL therapy had the worst lipaemic control. A possible effect of Tac on new-onset diabetes could not be excluded.

Current status of transplantation and organ donation in the Balkans--could it be improved through the South-eastern Europe Health Network (SEEHN) initiative?

Organ donation and transplantation activity in the majority of Balkan countries (Albania, Bosnia and Herzegovina, Croatia, Macedonia, Moldova, Montenegro, Serbia, Romania and Bulgaria) are lagging far behind international averages. Inadequate financial resources, unclear regional data and lack of government infrastructure are some of the issues which should be recognized to draw attention and lead to problem-solving decisions. The Regional Health Development Centre (RHDC) Croatia, a technical body of the South-eastern Europe Health Network (SEEHN), was created in 2011 after Croatia's great success in the field over the last 10 years. The aim of the RHDC is to network the region and provide individualized country support to increase donation and transplantation activity in collaboration with professional societies (European Society of Organ Transplantation, European Transplant Coordinators Organization, The Transplantation Society and International Society of Organ Donation and Procurement). Such an improvement would in turn likely prevent transplant tourism. The regional data from 2010 show large discrepancies in donation and transplantation activities within geographically neighbouring countries. Thus, proposed actions to improve regional donation and transplantation rates include advancing living and deceased donation through regular public education, creating current and accurate waiting lists and increasing the number of educated transplant nephrologists and hospital coordinators. In addition to the effort from the professionals, government support with allocated funds per deceased donation, updated legislation and an established national coordinating body is ultimately recognized as essential for the successful donation and transplantation programmes. By continuous RHDC communication and support asked from the health authorities and motivated professionals from the SEEHN initiative, an increased number of deceased as well as living donor kidney transplantations in the future should be more realistic.

Challenges and considerations in diagnosing the kidney disease in deteriorating graft function.

Despite significant reductions in acute-rejection rates with the introduction of calcineurin inhibitor (CNI)-based immunosuppressive therapy, improvements in long-term graft survival in renal transplantation have been mixed. Improving long-term graft survival continues to present a major challenge in the management of kidney-transplant patients. CNIs are a key component of immunosuppressive therapy, and chronic CNI toxicity has been widely thought to be a major factor in late graft failure. However, recent studies examining the causes of late graft failure in detail have challenged this view, highlighting the importance of antibody-mediated rejection and other factors. In addition, the diagnosis of CNI nephrotoxicity represents a challenge to clinicians, with the potential for over-diagnosis and an inappropriate reduction in immunosuppressive therapy. When graft function is deteriorating, accurately determining the cause of the kidney disease is essential for effective long-term management of the patient. Diagnosis requires a thorough clinical investigation, and in the majority of cases a specific cause can be identified.

Influence of MRP2 on MPA pharmacokinetics in renal transplant recipients-results of the Pharmacogenomic Substudy within the Symphony Study.

BACKGROUND: The aim of this study was to determine the relationship between single-nucleotide polymorphisms (SNPs) in MRP2 genes and mycophenolic acid (MPA) pharmacokinetics in renal transplant recipients of the Symphony Pharmacogenomic substudy. METHODS: Sixty-six renal transplant recipients of eight Spanish centres were randomized into four branches of immunosuppressive regimen: low dose of cyclosporine, standard dose of cyclosporine, tacrolimus and sirolimus, all in addition to mycophenolate mofetil and steroids. Fifty-five patients were genotyped for SNPs in MRP2, C24T and C3972T. Pharmacokinetic sampling was done before MPA administration and up to 12 h post-dose at Day 7, 1 month and 3 months post-transplant. Relationships of area under the curve (AUC) of MPA and MPAG plasma sampling with the presence of MRP2 SNPs and with the immunosuppressive regimens were studied. RESULTS: At steady-state conditions, MPA-reduced exposure was observed in C24T variant allele in MRP2 (CC: 68.73 ± 6.78; *T: 48.12 ± 4.90, P = 0.023); no significant differences linked to C3972T SNP were observed. Taking into account groups of treatment, lower MPA AUC in variant allele of C24T was only found under macrolides treatment with statistically significant differences at Month 3 (Tac and SRL, CC: 86.52 ± 10.98 versus *T: 41.99 ± 4.82, P = 0.001; CsA, CC: 52.31 ± 5.30 versus *T: 54.24 ± 8.30, P = 0.772); for C3972T, the same tendency was found but differences at steady state did not reach statistical significance. CONCLUSIONS: Renal transplant recipients T carriers of C24T MRP2 with macrolides treatment were associated with reduced MPA AUC in steady-state conditions. Patients treated with cyclosporine lost the effect of this polymorphism.

KDIGO clinical practice guideline for the care of kidney transplant recipients: a summary.

The 2009 Kidney Disease: Improving Global Outcomes (KDIGO) clinical practice guideline on the monitoring, management, and treatment of kidney transplant recipients is intended to assist the practitioner caring for adults and children after kidney transplantation. The guideline development process followed an evidence-based approach, and management recommendations are based on systematic reviews of relevant treatment trials. Critical appraisal of the quality of the evidence and the strength of recommendations followed the Grades of Recommendation Assessment, Development, and Evaluation (GRADE) approach. The guideline makes recommendations for immunosuppression and graft monitoring, as well as prevention and treatment of infection, cardiovascular disease, malignancy, and other complications that are common in kidney transplant recipients, including hematological and bone disorders. Limitations of the evidence, especially the lack of definitive clinical outcome trials, are discussed and suggestions are provided for future research. This summary includes a brief description of methodology and the complete guideline recommendations but does not include the rationale and references for each recommendation, which are published elsewhere.

Reduced exposure to calcineurin inhibitors in renal transplantation.

BACKGROUND: Immunosuppressive regimens with the fewest possible toxic effects are desirable for transplant recipients. This study evaluated the efficacy and relative toxic effects of four immunosuppressive regimens. METHODS: We randomly assigned 1645 renal-transplant recipients to receive standard-dose cyclosporine, mycophenolate mofetil, and corticosteroids, or daclizumab induction, mycophenolate mofetil, and corticosteroids in combination with low-dose cyclosporine, low-dose tacrolimus, or low-dose sirolimus. The primary end point was the estimated glomerular filtration rate (GFR), as calculated by the Cockcroft-Gault formula, 12 months after transplantation. Secondary end points included acute rejection and allograft survival. RESULTS: The mean calculated GFR was higher in patients receiving low-dose tacrolimus (65.4 ml per minute) than in the other three groups (range, 56.7 to 59.4 ml per minute). The rate of biopsy-proven acute rejection was lower in patients receiving low-dose tacrolimus (12.3%) than in those receiving standard-dose cyclosporine (25.8%), low-dose cyclosporine (24.0%), or low-dose sirolimus (37.2%). Allograft survival differed significantly among the four groups (P=0.02) and was highest in the low-dose tacrolimus group (94.2%), followed by the low-dose cyclosporine group (93.1%), the standard-dose cyclosporine group (89.3%), and the low-dose sirolimus group (89.3%). Serious adverse events were more common in the low-dose sirolimus group than in the other groups (53.2% vs. a range of 43.4 to 44.3%), although a similar proportion of patients in each group had at least one adverse event during treatment (86.3 to 90.5%). CONCLUSIONS: A regimen of daclizumab, mycophenolate mofetil, and corticosteroids in combination with low-dose tacrolimus may be advantageous for renal function, allograft survival, and acute rejection rates, as compared with regimens containing daclizumab induction plus either low-dose cyclosporine or low-dose sirolimus or with standard-dose cyclosporine without induction. (ClinicalTrials.gov number, NCT00231764 [ClinicalTrials.gov].).

Cyclosporine, tacrolimus and sirolimus retain their distinct toxicity profiles despite low doses in the Symphony study.

BACKGROUND: Reducing side effects of immunosuppressive regimens has become a priority in transplantation medicine because of the large number of patients and grafts that succumb to infection in the short term and cardiovascular disease in the long term. The Symphony study was a 12-month prospective, randomized, open-label, multi-centre, four parallel arm study that aimed to evaluate the safety and efficacy of low-dose immunosuppressive regimens compared with a standard-dose regimen in renal transplant recipients. This sub-analysis focuses on specific toxicities observed with the low-dose regimens. METHODS: Adult patients (n = 1645) scheduled to undergo renal transplantation received low-dose cyclosporine (CsA), tacrolimus (Tac) or sirolimus (SRL) in addition to daclizumab induction or standard-dose cyclosporine without induction. All patients received mycophenolate mofetil and corticosteroids. We evaluated the incidence of adverse events (AEs), tested specific group differences and assessed the relationship of selected AEs with drug levels. RESULTS: The four arms had similar incidences of AEs, but serious AEs were more common with low-dose SRL and led to more discontinuations. Infections were the most common AEs, with the highest incidence in the standard-dose CsA group, in particular, cytomegalovirus (CMV) infections. Low-dose Tac had the most reports of new-onset diabetes, leucopenia and diarrhoea. Low-dose SRL negatively influenced triglycerides, wound healing, lymphocele and anaemia. We found only weak relationships between specific AEs and drug levels. CONCLUSIONS: Despite the low doses, CsA, Tac and SRL retained distinct and different toxicity profiles. These findings may be of relevance for tailoring specific immunosuppressive regimens to patients with particular needs.

Combination of antibodies inhibits accelerated rejection mediated by memory T cells in xenoantigen-primed mice.

BACKGROUND: Donor-reactive memory T cells are known to accelerate allograft rejection; in our previous study, we reported that combined monoclonal antibodies (mAbs) could prolong islet allograft survival in alloantigen-primed mice. In this study, we examine the effects of donor-reactive memory T cells on the xenograft survival and methods to prolong the islet graft survival. METHODS: To collect donor-reactive T cells, we performed full-thickness rat skin xenografting on BALB/c mice and isolated the T cells from the mice after 6-8 weeks. These cells were then adoptively transferred to syngenic mice 1 day before rat-to-mouse islet transplantation. Three experimental groups were established in the adoptive transfer model: recipient mice treated with isotype mAbs (isotype group); mice treated with anti-CD40L mAb (anti-CD40L group); and mice treated with anti-CD40L, anti-OX40L, and anti-CD122 mAbs (3-combined group). RESULTS: Lewis rat islet xenografts transplanted in naïve mice showed a mean survival time (MST) of 12.8 days, while the graft rejection was accelerated if the recipient mice were treated with adoptively transferred donor-reactive T cells (MST, 8.67 days). Treatment with anti-CD40L mb could not reverse the accelerated rejection (MST, 9.3 days). However, when anti-CD40L mb was combined with anti-OX40L and anti-CD122 mAbs, there was a considerable increase in the MST, which was 72.2 days. Compared to the isotype group, the 3-combined group had significantly lesser proportion of memory T cells and greater proportion of regulatory T cells (Tregs) in the spleen. Meanwhile, in the 3-combined group, the production of anti-rat antibodies was markedly inhibited. CONCLUSION: Treatment with a combination of antibodies could significantly reverse the accelerated rejection mediated by donor-reactive memory T cells by inhibiting cellular and humoral immune responses.

Acute rejection in low-toxicity regimens: clinical impact and risk factors in the Symphony study.

The Symphony study assessed whether mycophenolate mofetil (MMF)-based regimens containing reduced doses of adjunct immunosuppressants could reduce toxicity while maintaining efficacy. Here, we examined the impact of acute rejection and associated risk factors. The incidence of biopsy-proven acute rejection in the low-dose tacrolimus group was approximately half that of the standard-dose cyclosporine and low-dose cyclosporine groups, and a third of that in the low-dose sirolimus group. The low-dose cyclosporine group had more severe rejection episodes (≥grade II) compared with other groups. Acute rejection was associated with a 10 mL/min glomerular filtration rate (GFR) reduction and a 5.3% absolute increase in graft loss at 12 months. Overall, the highest GFR was found in both rejecters and non-rejecters receiving low-dose tacrolimus, both in an intent-to-treat analysis and in patients successfully treated according to the protocol. In Cox regression models, human leukocyte antigen (HLA) mismatches and expanded criteria donors increased the acute rejection risk, while recipient age, living related donor, and MMF dose were associated with a reduced risk. Acute rejection was associated with worse outcome but did not entirely explain the differences among the treatment groups. The 2 g MMF plus low-dose tacrolimus combination appears to be the most efficient of all regimens examined regardless of acute rejection.

Costimulation blockade induces tolerance to HESC transplanted to the testis and induces regulatory T-cells to HESC transplanted into the heart.

In order to study the ability of costimulation blockade to induce tolerance to human embryonic stem cells (HESC), severe combined immunodeficient (SCID), and immunocompetent C57BL/6 mice treated with costimulation blockade received intratesticular and intramyocardial HESC transplants. All SCID mice with intratesticular HESC transplants developed teratoma. When SCID mice were transplanted intramyocardially, only two of five mice developed teratoma-like tumors. C57BL/6 mice transplanted intratesticularly and treated with costimulation blockade all developed teratoma and were surrounded by CD4(+)CD25(+)Foxp3(+) T-cells, while isotype control treated recipients rejected their grafts. Most C57BL/6 mice transplanted intramyocardially and treated with costimulation blockade demonstrated lymphocytic infiltrates 1 month after transplantation, whereas one maintained its graft. Isolation of regulatory T-cells from intramyocardial transplanted recipients treated with costimulation blockade demonstrated specificity toward undifferentiated HESC and down-regulated naive T-cell activation toward HESC. These results demonstrate that costimulation blockade is sufficiently robust to induce tolerance to HESC in the immune-privileged environment of the testis. HESC specific regulatory T-cells developed to HESC transplanted to the heart and the success of transplantation was similar to that seen in SCID mice.

The pharmacokinetics of mycophenolate mofetil in renal transplant recipients receiving standard-dose or low-dose cyclosporine, low-dose tacrolimus or low-dose sirolimus: the Symphony pharmacokinetic substudy.

BACKGROUND: Exposure to mycophenolic acid (MPA), the primary active metabolite of mycophenolate mofetil (MMF), is correlated with therapeutic efficacy of MMF but varies depending on the concomitantly administered immunosuppressive drugs. METHODS: A 3-month pharmacokinetic substudy of the prospective, randomized, multicentre, open-label Symphony study was performed. Eighty-three adult renal transplant patients received standard-dose cyclosporine, MMF 2 g/day and corticosteroids, or daclizumab induction, MMF 2 g/day and corticosteroids plus low-dose cyclosporine, low-dose tacrolimus or low-dose sirolimus. The area under the concentration-time curve (AUC(0-12)) of MPA and its metabolites between treatment groups was compared. Pharmacokinetic sampling was performed before MMF administration and at 20, 40, 75 min; 2, 3, 6, 8, 10 and 12 h post-dose on Day 7 and Months 1 and 3. RESULTS: Compared with standard-dose cyclosporine, patients receiving low-dose tacrolimus or low-dose sirolimus had significantly higher AUC(0-12) values for MPA at Day 7 and Month 1 and for free MPA at Day 7, and significantly lower AUC(0-12) values for 7-O-MPA-glucuronide (MPAG) at Month 1 and for acyl-glucuronide at Months 1 and 3 (P < 0.05). AUC(0-12) of MPA and free MPA was significantly greater with low-dose tacrolimus and low-dose sirolimus than with low-dose cyclosporine in the first month (P < 0.05). The ratio of MPA to MPAG exposure was significantly higher in the three low-dose groups than in the standard-dose cyclosporine group (P < 0.05). CONCLUSIONS: Standard- and low-dose cyclosporine reduces the exposure of MPA and free MPA compared to low-dose tacrolimus or low-dose sirolimus in patients given the same dose of MMF.

Combined costimulation blockade inhibits accelerated rejection mediated by alloantigen-primed memory T cells in mice.

Donor-reactive memory T cells threaten the survival of transplanted organs via multiple pathways. This study was undertaken to induce tolerance of cardiac allografts in mice, in which alloreactive memory T cells were adoptively transferred, by combined costimulatory blockade of both effector and memory T cells. We found that the median survival time (MST) of the grafts was 5.17 days in the untreated group, 10.33 days in the CTLA4Ig- and anti-CD40L-treated (2-combined) group, and more than 100 days in the CTLA4Ig-, anti-CD40L-, anti-LFA-1-, and anti-OX40L-treated (4-combined) group. Histological analysis revealed that the mean rejection level was Grade 4 in the untreated group, Grade 3 in the 2-combined treatment group, and Grade 0 in the 4-combined treatment group. CD44(high) T cells were detected only in the untreated group. The in vitro proliferation of lymphocytes of both untreated and 2-combined group was higher than that of the 4-combined treatment group (p < 0.01). Compared with the untreated group, the expression levels of IL-2, IFN-gamma, and Foxp3 were lower in the 2-combined treatment group; the expression levels of these genes were the lowest in the 4-combined treatment group. IL-10 expression was significantly higher in the 4-combined treatment group than in the other groups. These results demonstrate the inhibition efficacy of combined costimulation blockade in accelerated-rejection models and the possible mechanisms underlying the suppression of cellular immunity in mice receiving grafts as well as in inducing the activation of IL-10-producing Tr1 cells in grafts.

Low toxicity regimens in renal transplantation: a country subset analysis of the Symphony study.

Regional transplant practices may affect clinical outcomes within multinational studies. This study evaluated whether the overall results from the Symphony study can be generalized to the participating countries. De novo adult renal transplant recipients (n = 1645) were randomized to receive standard-dose cyclosporine, or daclizumab induction plus low-dose cyclosporine, low-dose tacrolimus,or low-dose sirolimus, all in addition to mycophenolate mofetil and steroids. Data for the highest patient-recruiting countries, Spain (n = 275),Germany (n = 316) and Turkey (n = 258), were compared. Patient transplant characteristics were different among the country subsets; only deceased donors in Spain, more expanded criteria donors in Germany, and mainly living donors in Turkey. Efficacy results for the three countries were consistent with that of the overall study - renal function and biopsy-proven acute rejection (BPAR)rates were superior with low-dose tacrolimus. Turkey had higher mean calculated glomerular filtration rate across all treatment groups (60.6-72.2 ml/min)compared with that of Spain (51.1-57.5 ml/min) and Germany (51.3-62.9 ml/min). Spain and Turkey had lower BPAR rates across the four treatment groups compared with the overall study; Germany had much higher rates(21.0-54.2%). These findings confirm the general applicability of the Symphony study results and highlight the importance of inclusion of patients from different geographic origins in randomized clinical trials.

Variations in graft and patient survival after kidney transplantation in Sweden: caveats in interpretation of center effects when benchmarking.

Benchmarking and comparisons between transplantation centers are becoming more common. A crude comparison indicated a 50% difference in patient survival between centers in Sweden. A 'task group' was formed to refute or confirm and learn from this observation. Patient survival and graft survival of 5 933 patients transplanted at three different transplantation centers in Sweden (Stockholm, Göteborg, and Malmö) were followed up until February 2007. Patient survival and graft survival were compared between the centers with and without consideration being given to important covariates such as time period, type of donation (living or deceased donor), gender, and age. A refined cohort of 2,956 adult patients that had been transplanted for the first time between 1991 and 2007 was assessed in more detail using Cox regression analysis. The difference in patient and transplant outcome observed in the crude comparison diminished considerably after adjustment for differences in case mix and time period of transplantation, and was neither evident nor significant after 1999. Patient survival and graft survival have improved considerably during the time period since 1991. The adjusted hazards ratio for mortality was 0.39 (95% CI 0.29-0.53) for patients who were transplanted after 1999 when compared with those transplanted between 1991 and 1994. Crude comparisons between results from transplantation centers may be severely confounded not only by case mix but also by differences in the proportion of patients transplanted during different time periods. Patient outcome and graft outcome have improved considerably since 1991, and after 1999 center effects were no longer apparent in Sweden.

Downregulation of tissue factor (TF) by RNA interference induces apoptosis and impairs cell survival of primary endothelium and tumor cells.

Tissue factor (TF) has been implicated in the thrombotic complications seen during vascular rejection of allografts and may contribute to intimal hyperplasia in chronic allograft vasculopathy. Downregulation of endothelial TF expression post-transplantation could therefore be of therapeutic value. Lentivirus-mediated RNA interference was used in primary endothelial cells (EC) to investigate its effects on TF protein expression and functional activity. Lentivirus-mediated expression of a TF-specific short-interfering (si) RNA with green fluorescent protein as a reporter gene (siRNATF-GFP) resulted in a 42 +/- 3.9% reduction in EC surface-expressed TF as compared with cells expressing a scrambled siRNATF sequence (P = 0.025). The TF content in EC lysates was reduced from 6.85 +/- 1.99 ng to 3.05 +/- 0.82 ng (P = 0.006). Factor X (FX) activation was not impaired on the apical EC surface. The subendothelial matrix of ECs with low TF expression showed significantly reduced TF activity compared with non-transduced cells or with cells harboring the empty vector. ECs expressing siRNATF-GFP exhibited reduced reporter gene (GFP) expression and cell density and an altered morphology. Transfection of control cells with high (J82 cells) or low (MiaPaCa-2 cells) TF expression with siRNATF oligonucleotides caused apoptosis of the J82 but not of the MiaPaCa-2 cells. Thus, lentivirus-mediated RNA interference reduces the TF expression of activated ECs but does not affect FX activation by TF/FVIIa expressed on the apical surface. The downregulation has nevertheless substantial negative effects on the viability of ECs and TF-expressing control cells. These findings imply that certain levels of TF are required for the maintained viability and growth of endothelium and TF-expressing tumor cells.

Costimulation blockade in transplantation of nerve allografts: long-term effects.

Costimulation blockade can prevent rejection of nerve allografts in short-term studies. We tested if costimulation blockade also prevented rejection of nerve allografts in long-term experiments, thereby improving functional recovery. A 7-mm sciatic nerve defect in C57/BL6 mice was bridged either by nerve allografts from Balb/C mice or by isogenic nerve grafts (isografts) from C57/BL6 mice. Costimulation blockade in the form of a triple treatment with anti-LFA-1, anti-CD40L, and CTLA4Ig was given at post-operative days 0, 2, 4, and 6 (intraperitoneal). Control mice (placebo; allografts) with nerve grafts were treated with isotype antibodies during the same time period. After 49 days, tetanic muscle force, wet weight of gastrocnemius muscle, histology, and morphometry in the tibial nerve were evaluated. Costimulation blockade diminished rejection of the nerve allografts. Axons bridged the graft. Treatment increased wet weight of the gastrocnemius muscle and resulted in a higher mean myelin area/nerve fiber in the tibial nerve distal to the nerve grafts. Tetanic muscle force and number of axons in tibial nerve showed no differences between groups. We conclude that rejection is suppressed by costimulation blockade. Treatment improves recovery of target muscle and myelination after nerve allografting.