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Renal transplant recipients experience premature cardiovascular disease and death. The association of inflammation, all-cause mortality, and cardiovascular events in renal transplant recipients has not been examined in a large prospective controlled trial.ALERT was a randomized, double-blind, placebo-controlled study of the effect of fluvastatin on cardiovascular and renal outcomes in 2102 renal transplant recipients. Patients initially randomized to fluvastatin or placebo in the 5- to 6-yr trial were offered open-label fluvastatin in a 2-yr extension to the original study. The association between inflammation markers, high-sensitivity C-reactive protein (hsCRP), and IL-6 on cardiovascular events and all-cause mortality was investigated.The baseline IL-6 value was 2.9 +/- 1.9 pg/ml (n = 1751) and that of hsCRP was 3.8 +/- 6.7 mg/L (n = 1910). After adjustment for baseline values for established risk factors, the hazard ratios for a major cardiac event and all-cause mortality for IL-6 were 1.08 [95% confidence interval (CI), 1.01 to 1.15, P = 0.018] and 1.11 (95% CI, 1.05 to 1.18, P0.001), respectively. The adjusted hazard ratio for hsCRP for a cardiovascular event was 1.10 (95% CI, 1.01 to 1.20, P = 0.027) and for all-cause mortality was 1.15 (95% CI, 1.06 to 1.1.25, P = 0.049).The inflammation markers IL-6 and hsCRP are independently associated with major cardiovascular events and all-cause mortality in renal transplant recipients.

The pharmacokinetics, safety, and preliminary efficacy of FTY720, a novel immunomodulator, were examined in de novo renal transplant patients. Both noncompartmental and population methods were used to estimate pharmacokinetic estimates in the patients. The steady-state plasma concentrations of FTY720 increased in accordance with maintenance dose level, indicating linearity in clearance and volume of distribution over the 0.25- to 2.5-mg dose range. The pharmacokinetics of FTY720 in de novo renal transplant patients were characterized by the long terminal phase half-life of approximately 200 hours across doses, high volume of distribution (>3000 L), and low clearance (10.8 L/h). The intersubject variation of clearance was 55%, and the intrasubject variation of FTY720 concentrations was 28%. The population analysis revealed significant positive relationships between baseline alkaline phosphatase and clearance, as well as between baseline body weight on apparent volume of distribution. There was no relationship between FTY720 concentrations within a given FTY720 dose cohort and the rate of allograft rejection.

Background. Therapeutic drug monitoring for cyclosporine microemulsion (CsA-ME) is often performed using either trough levels (C0) or levels at 2 h post-dose (C2). This analysis assessed changes in C0 and C2 and their relationship to CsA-ME dose over time posttransplant in renal transplant patients. Methods. Data were obtained from MO2ART, a prospective multicentre trial in which CsA-ME dose was adjusted based on C2 level. All 98 patients in whom C0 and C2 were available at day 5, month 3 and month 12 were included, out of 234 who completed the 12 month study. Normalized dose (ND) of CsA-ME, defined as dose per kilogram body weight, was calculated, together with C0/ND, C2/ND and C2/C0. Results. C0/ND and C2/ND both increased between day 5 and month 3: C0/ND from 33±15 to 53± 24 (ng/ml)/(mg/kg) and C2/ND from 161±64 to 248± 80 (ng/ml)/(mg/kg). Between month 3 and month 12, C2/ND remained stable but C0/ND decreased to 42±20 (ng/ml)/(mg/kg) while the C2/C0 ratio increased from 5.2±1.9 to 6.5±2.3, indicating an acceleration of drug elimination. The inter-individual coefficient of variation was higher for C0/ND than for C2/ND at 3 months (45 vs 32%, P

Renal transplantation is associated with an increased risk for premature cardiovascular disease. We analyzed the data in the placebo arm of Assessment of Lescol in Renal Transplantation (ALERT) to improve our understanding of the relationship between cardiovascular risk factors and outcomes in this unique population.We performed Cox survival analysis for myocardial infarction, cardiac death, and noncardiac death in 1,052 patients recruited to the placebo arm of ALERT. These subjects were aged 30 to 75 years, had stable graft function at least 6 months after transplantation, had a serum total cholesterol level between 155 and 348 mg/dL (4 and 9 mmol/L), and were receiving cyclosporine-based immunosuppression.The results confirm previous studies. In multivariate analysis, preexisting coronary heart disease (hazard ratio [HR], 3.69; P0.001), total cholesterol level (HR, 1.55 per 50 mg/dL; P = 0.0045), and prior acute rejection (HR, 2.36; P = 0.0023) were independent risk factors. Conversely, independent risk factors for cardiac death were age (HR, 1.58 per decade; P = 0.0033), diabetes (HR, 3.35; P = 0.0002), ST-T changes on the ECG (HR, 3.17; P = 0.0004), and serum creatinine level (HR, 2.65 per milligram per deciliter; P0.0001).This analysis confirms that renal transplant recipients share risk factors for myocardial infarction and cardiac death with the general population. However, the pattern of risk factors and their relationship with outcomes is atypical, highlighting the unique nature of cardiovascular risk in transplant recipients.

Renal-transplant recipients have shortened life expectancy primarily because of premature cardiovascular disease. Traditional and nontraditional risk factors for cardiovascular disease are prevalent in renal patients. In renal-transplant recipients, immunosuppressive therapy can be nephrotoxic and aggravate cardiovascular disease risk factors. Renal dysfunction has been established as a risk factor for cardiovascular disease and mortality in different populations. We evaluated the effects of baseline renal-transplant function on mortality and cardiovascular and renal endpoints in 1,052 placebo-treated patients of the Assessment of Lescol in Renal Transplantation trial.All renal-transplant recipients were on cyclosporine-based immunosuppressive therapy. Follow-up was 5 to 6 years, and endpoints included cardiac death, noncardiovascular death, all-cause mortality, major adverse cardiac event (MACE), stroke, nonfatal myocardial infarction, and graft loss.Baseline serum creatinine was strongly and independently associated with increased cardiac, noncardiovascular, and all-cause mortality, as well as MACE and graft loss. Serum creatinine was not a risk factor for stroke or nonfatal myocardial infarction.Elevated baseline serum creatinine in renal-transplant recipients is a strong and independent risk factor for all-cause, noncardiovascular and cardiac mortality, MACE, and graft loss.

Glycosylation is involved in the correct folding, targeting, bioactivity and clearance of therapeutic glycoproteins. With the development of transgenic animals as expression systems it is important to understand the impact of different genetic backgrounds and lactations on glycosylation. We have evaluated the glycosylation of recombinant antithrombin produced in several transgenic goat lines, from cloned animals and from different types of lactation including induced lactations. Our results show glycosylation patterns from the protein expressed in animals, derived from the same founder goat, are mostly comparable. Furthermore, the protein expressed in two cloned goats had highly consistent oligosaccharide profiles and similar carbohydrate composition. However, there were significantly different oligosaccharide profiles from the proteins derived from different founder goats. Artificial induction of lactation did not have significant effects on overall carbohydrate structures when compared to natural lactation. The only major difference was that recombinant antithrombin from induced lactations contained a slightly higher ratio of N-acetylneuraminic acid to N-glycolylneuraminic acid and less amount of oligosaccharides containing N-glycolylneuraminic acid. The oligosaccharides from all animals were a mixture of high mannose-, hybrid- and complex-type oligosaccharides. Sialic acid was present as alpha-2,6-linkage and no alpha-1,3-linked galactose was observed. These results indicate that transgenic animals with closely related genetic backgrounds express recombinant protein with comparable glycosylation.

Effect of fluvastatin on renal end points in the Assessment of Lescol in Renal Transplant (ALERT) trial.BackgroundHyperlipidemia is a risk factor for long-term renal transplant dysfunction, but no prospective clinical trials have investigated the effects of statin treatment on graft function in renal transplant recipients. The aim of the present study was to evaluate the effect of fluvastatin on long-term renal transplant function and development of chronic allograft nephropathy in the ALERT (Assessment of Lescol in Renal Transplantation) study.MethodsALERT was a randomized, double-blind, placebo-controlled study of the effect of fluvastatin, 40 mg and 80 mg daily, in renal transplant recipients. Patients were randomized to receive either fluvastatin (N = 1050) or placebo (N = 1052) and followed for five to six years. Renal end points included graft loss or doubling of serum creatinine or death; glomerular filtration rate (GFR) was also measured during follow-up in a subset of patients (N = 439).ResultsThere were 283 patients (13.5%) with graft loss, mainly due to chronic rejection (82%), yielding an annual rate of 2.4%. Fluvastatin treatment significantly lowered mean low-density lipoprotein (LDL)-cholesterol levels by 32% (95% CI -33 to -30) compared with placebo, but had no significant effect on the incidence of renal graft loss or doubling of serum creatinine, or decline in GFR throughout follow-up in the whole study population. Neither was any treatment effect by fluvastatin found in any of the subgroups analyzed.ConclusionFluvastatin treatment significantly improves lipid values in renal transplant recipients but has no effect on graft loss or doubling of serum creatinine.

Background. MO2ART (monitoring of 2-hr absorption in renal transplantation) is the first prospective, multicenter trial of cyclosporine (CsA) blood level 2 hr postdose (C 2 ) monitoring in de novo kidney recipients receiving CsA microemulsion (ME) (Neoral; Novartis, Basel, Switzerland). Efficacy and safety results from the first 3 months are presented here. Methods. MO2ART is a 12-month, open-label, randomized study involving 296 patients. In all patients, the dose of CsA-ME was adjusted to achieve protocol-defined C 2 targets of 1.6 to 2.0 μg/mL for the first month, with subsequent tapering. Randomization into two target groups occurred at 3 months. All patients received steroids and mycophenolate mofetil (89%) or azathioprine. For patients with delayed graft function, the protocol permitted reduced C 2 targets and prophylactic administration of antibodies. Results. At 3 months, overall incidence of biopsy-proven acute rejection was 11.5%. Median serum creatinine was 132 μmol/L. Patient and graft survival were 96.6% and 91.2%, respectively. C 2 levels greater than 1.6 μg/mL were achieved within 5 days by 60.6% of patients with immediate graft function and 19.5% of patients with delayed graft function. Prophylactic antibodies were used in 15% of the total population. Twenty-four patients (8.1%) experienced serious adverse events with a suspected relation to CsA, and 26 patients (8.8%) discontinued the study because of adverse events (n=15) or after a switch in immunosuppression after rejection episodes (n=11). Conclusions. Patient management by C 2 monitoring resulted in a low incidence of biopsy-proven acute rejection in standard risk de novo kidney recipients, 85% of whom did not receive prophylactic antibodies. CsA-ME with C 2 monitoring provides excellent short-term efficacy and safety among de novo renal transplant patients.

BACKGROUND Cyclosporine monitoring using the 2-hr postdose sample, C2, has been shown to have advantages in monitoring de novo renal transplant recipients. The purpose of this study was to assess cyclosporine exposure, using C2, in stable renal transplant patients previously monitored by C0 to determine the effect of dose reduction on patients with C2 more than 10% above target and the course of those with C2 at and more than 10% below target, whose dose was not modified. METHODS One hundred and seventy-five patients, three or more months after transplantation, had C2 assessed. The relationship of C2 to C0 and of both to renal function was analyzed by linear regression. Blood pressure, serum creatinine level, and lipids were followed for a mean of 15+/-2.6 months. RESULTS Eighty-five patients had values more than 10% above target, 42 were within 10% of target, and 48 were more than 10% below target. Cyclosporine dose was reduced in all patients above target. In this group, serum creatinine level was stable overall, but fell significantly in 46 (54%) of 85 from 153+/-55 to 132+/-49 microM. Blood pressure also fell in that group from 135/82 to 131/77. Serum creatinine level was stable in the remaining two groups of patients. CONCLUSIONS These data suggest that dose reduction in many overexposed patients leads to improvements in renal function and blood pressure. Further study is required to confirm the long-term benefits of this strategy.

Recent clinical trials of primary and secondary prevention of cardiovascular disease have demonstrated that lowering plasma cholesterol with 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors ('statins') reduces morbidity and mortality from coronary heart disease in diverse patient populations.The aim of the present ALERT (Assessment of Lescol in Renal Transplantation) study is to determine whether renal transplant recipients would also benefit from statin therapy. ALERT is a multicentre, randomized, double-blind, placebo-controlled trial to assess the effect of fluvastatin in renal transplant recipients with mild-to-moderate hypercholesterolaemia. The primary objective is to investigate the effects of fluvastatin on major adverse cardiac events (MACE). In addition, the effects on cardiovascular and all-cause mortality, as well as renal function, will be addressed.The study population contains patients with functioning renal allografts of more than 6 months' duration, recruited from 75 centres in Northern Europe and Canada. Patients of both sexes, aged 30-75 years, with a total cholesterol level of 4.0-9.0 mmol/l (155-348 mg/dl) were included, except for those with a history of myocardial infarction, where the upper limit for inclusion was 7.0 mmol/l (270 mg/dl).A total of 2100 patients were recruited by the end of October 1997 and will be followed for up to 6 years. This report presents the design features of the study (recruitment, follow-up, sample size, data analysis and study organization), along with baseline results. ALERT is the first large-scale prospective, randomized, double-blind study to address the prevention of cardiovascular mortality in renal transplant patients receiving an HMGCoA reductase inhibitor.

Despite the need to expand the donor pool, it is unclear what parameters should be used. The value of donor renal pathology and calculated creatinine clearance (CrCl) in determining recipient outcome was assessed in 57 kidney transplants from 34 donors in whom pretransplant renal biopsies were performed because of ageor =60, hypertension, and/or vascular disease. We retrospectively compared clinical outcomes in these recipients and 57 control recipients selected to have the same baseline demographics but receiving transplants from low risk donors who were significantly younger (32+/-13.9 vs. 61+/-7.3 years) and lighter weight (71+/-18.1 vs. 84+/-20.2 kg) than the high-risk donors (P.001 for both).Recipients of high-risk kidneys had a higher incidence of delayed graft function, defined by a10% fall in serum creatinine (Cr) in the first 24 hr, (56% vs. 30%, P.01), a higher incidence of rejection (60% vs. 37%, P = .02) and a higher Cr level (197+/-64 vs. 144+/-54 micromol/L at 18 months, P.005). Graft and patient survival were similar; 12% and 5% vs. 91% and 9% in high-risk vs. control groups, respectively (P = NS). Donor renal pathology was scored 0-3 (none to severe disease) in four areas: glomerulosclerosis, interstitial fibrosis, tubular atrophy, and vascular disease. A donor vessel score of 3/3 was associated with a 100% incidence of delayed graft function and a mean 1-year Cr level of 275+106 micromol/L (compared with 43% and 192+54 micromol/L in those with lower vessel scores, P.05). Calculated donor CrCl100 ml/min was associated with higher recipient Cr levels at 1 year, 240+/-95 micromol/L vs. 180+/-54 micromol/L in recipients of kidneys from donors with CrCl levels100 ml/min (P.05). The mean 1-year Cr level was 320+/-102 micromol/L in recipients with both a vascular score of 3/3 and a donor CrCl100 ml/min and 184+/-63 micromol/L in those with neither factor (P = .001).Calculated donor CrCl and donor vascular pathology predict recipient graft function and may be helpful in selecting high-risk donors for single kidney transplantation.

Commercially available human plasma-derived preparations of the serine protease inhibitor antithrombin (AT) were shown to contain low levels of oxidation, and we sought to determine whether oxidation might be a means of regulating the protein's inhibitory activity. A recombinant form of AT, with similarly low levels of oxidation as purified, was treated with hydrogen peroxide in order to study the effect of oxidation, specifically methionine oxidation, on the biochemical properties of this protein. AT contains two adjacent methionine residues near the reactive site loop cleaved by thrombin (Met314 and Met315) and two exposed methionines that border on the heparin binding region of AT (Met17 and Met20). In forced oxidations with hydrogen peroxide, the methionines at 314 and 315 were found to be the most susceptible to oxidation, but their oxidation did not affect either thrombin-inhibitory activity or heparin binding. Methionines at positions 17 and 20 were significantly oxidized only at higher concentrations of peroxide, at which point heparin affinity was decreased. However at saturating heparin concentrations, activity was only marginally decreased for these highly oxidized samples of AT. Structural studies indicate that highly oxidized AT is less able to undergo the complete conformational change induced by heparin, most probably due to oxidation of Met17. Since this does not occur in less oxidized, and presumably more physiologically relevant, forms of AT such as those found in plasma preparations, oxidation does not appear to be a means of controlling AT activity.

Background. The safety, tolerability, and pharmacokinetics of conventional cyclosporine (ConCsA) and cyclosporine microemulsion (MeCsA) were compared under conditions of normal clinical practice in a prospective, randomized, concentration-controlled, pharmacoepidemiologic study. Methods. Between September 1994 and March 1995, 1097 stable renal transplant recipients in 14 Canadian centers were randomized 2:1 to treatment with MeCsA or ConCsA Patients were commenced on each study drug at a dose equal to their previous therapy with ConCsA, and the dose was adjusted to maintain predose whole blood cyclosporine concentrations within the therapeutic range established for each center. Prednisone and azathioprine were continued unless dose adjustment was required for clinical reasons. Results. The mean cyclosporine concentration was comparable in both treatment groups at all time points throughout the 6 months of follow-up. The mean dose of cyclosporine was 3.6 mg/kg/day in both treatment groups at entry to the study, and declined by 0.3% and by 2.8% in patients receiving ConCsA and MeCsA, respectively. The nature and severity of adverse events were similar in both treatment groups, but there was a transient increase in neurological and gastrointestinal complications in the group receiving MeCsA within the first month after conversion (P

Recombinant human PRL was produced in a murine C127 cell expression system and purified to greater than 97% homogeneity using anion and cation exchange chromatography. This material was biologically equivalent to pituitary-derived PRL in both an enzyme-linked immunosorbent assay and the Nb2 lymphoma cell proliferation assay. The predominant PRL forms were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting as being 23 and 25 kilodaltons (kDa). These mass values were confirmed by electrospray mass spectroscopy. Glycosidase digestions indicated that the 25-kDa PRL is N-glycosylated and sialylated, whereas 23-kDa PRL is nonglycosylated. Glycosylated and nonglycosylated forms of the hormone were individually purified to greater than 95% homogeneity using novel cation exchange chromatography. Isoelectric focusing demonstrated that both forms consist of multiple charge isomers, with the charge heterogeneity of the glycosylated form primarily due to differences in sialylation. Monosaccharide analysis of the glycosylated form suggested a minimal complex oligosaccharide chain that may be fucosylated and partially sialylated. Oligosaccharide mol wt were determined by electrospray ionization mass spectroscopy. Analysis of the oligosaccharides by fluorophore-assisted carbohydrate electrophoresis indicated that bi- and triantennary oligosaccharide forms are predominant and have multiple combinations of terminal sialylation. Both forms of PRL were active in the Nb2 lymphoma cell proliferation assay; however, the 23-kDa nonglycosylated form was 3-4 times more active in this assay than the 25-kDa glycosylated form.

We have genetically engineered a cell line, and developed a reproducible process, for the expression and purification of biologically active recombinant human thyroid stimulating hormone (rhTSH).rhTSH was expressed by co-transfecting a human alpha-subunit cDNA with a human beta-subunit partial genomic clone into Chinese Hamster Ovary (CHO) cells. Stable transfectants which expressed high levels of rhTSH were selected, and subsequently cultured on microcarrier beads. The rhTSH-containing media, produced under serum-free conditions, was clarified and purified by a combination of ion exchange, dye and gel filtration chromatographies. Individual step recoveries were greater than 90% with the exception of a very conservative pooling of the final gel filtration step (78% recovery) that resulted in a cumulative yield of 54% for the purification process. Purity of the final bulk material was judged to be > 99% by SDS polyacrylamide gel electrophoresis (SDS-PAGE), reverse phase HPLC, and size exclusion chromatography. Initial characterization of the oligosaccharide composition indicated the presence of partially sialylated bi- and triantenary complex oligosaccharides. Purified rhTSH was active in a thyroid membrane bioactivity assay with a specific activity of 8.2 IU/mg. The in vivo activity of rhTSH in cynomolgus monkeys appeared to be equal to or greater than that reported for bovine TSH (bTSH) in human subjects. The rapid clearance phase half-life of rhTSH was approximately 35 minutes while the post-distribution phase half life was approximately 9.8 hours. Furthermore, the monkeys showed cumulative increases in minimum plasma rhTSH levels when given three daily intramuscular (IM) rhTSH injections; a phenomenon not observed when bTSH had been administered to humans. The rhTSH showed no evidence of toxic or adverse effects when administered at doses up to 7.2 IU/kg and 0.52 IU/kg in rat and monkey, respectively. These are 50X and 4X multiples of the bTSH doses of 0.143 IU/kg (10 IU/70kg) previously administered to humans.

The incidence of stroke and risk factors for different subtypes of cerebrovascular (CBV) events in renal transplant recipients have not been examined in any large prospective controlled trial.The Assessment of Lescol in Renal Transplantation was a randomized, double-blind, placebo-controlled study of the effect of fluvastatin (40-80 mg) daily on cardiovascular, and renal outcomes in renal transplant recipients. Patients initially randomized to fluvastatin or placebo in the 5 to 6 year trial was offered open-label fluvastatin in a 2-year extension to the original study. We investigated the incidence of stroke and risk factors for ischemic and hemorrhagic CBV events in 2102 renal graft recipients participating in the Assessment of Lescol in Renal Transplantation core and extension trial with a mean follow-up of 6.7 years.The incidence and type of CBV events did not differ between the lipid lowering arm and the placebo arm. A total of 184 (8.8%, 95% confidence interval 4.6-12.9) of 2102 patients experienced a CBV event during follow-up, corresponding to an incidence of 1.3% CBV event per year. The mortality for patients experiencing a hemorrhagic stroke was 48% (13 of 27), whereas the mortality for ischemic strokes was 6.0% (8 of 133). Diabetes mellitus, previous CBV event, age, and serum creatinine were independent risk factors for cerebral ischemic events. The risk of a hemorrhagic cerebral event was increased by diabetes mellitus, polycystic kidney disease, left ventricular hypertrophy, and systolic blood pressure.Risk factors for CBV events in renal transplant recipients differ according to subtype.

Recombinant baboon and monkey prolactins were expressed in murine C127 cells. The hormones were purified from the conditioned media of these cells using a combination of cation, anion, and gel filtration chromatographies. This purification scheme provided approximately a 20-fold purification of the proteins with a 40% cumulative yield. Sodium dodecyl sulfate gel electrophoresis of the purified hormones in conjunction with Coomassie blue staining and immunoblotting procedures revealed three major prolactin-related bands with molecular weights corresponding to Mr 16,000, 23,000, and 27,000. Based on these analyses the samples were judged to be greater than 90% pure. Amino terminal sequence analysis of the purified baboon and monkey hormones provided three distinct prolactin-related sequences for each preparation. The predominant sequence corresponded to the predicted amino terminal sequences of the hormones which began with leucine at position 1. Two minor sequences, individually representing approximately 10-20% of the total population, were also identified; one starting at position 11 and the other at position 133. Carbohydrate compositional analysis of the proteins suggested that greater than 50% of the population were glycosylated with a fucosylated complex oligosaccharide. Analysis of the specific bioactivity of the recombinant hormones in the Nb2 cell proliferation assay showed them to be comparable to the NIH and WHO human pituitary-derived standards.

There has been no significant increase in the number of deceased organ donors in Canada over the past decade. Canada's donation and transplant system will be restructured with the formation of a new national organization to oversee activity in provincially governed donation and transplantation services. We review the current status of deceased organ donation, highlight issues contributing to the current stagnation in donation and identify changes that will enable success in a new Canadian system. Determining Canada's organ donation performance is difficult because the data required to calculate meaningful metrics of donation performance are not available. Canadians wait longer for transplantation than Americans, and Canada is falling further behind the United States primarily because of fewer donations after cardiac death. The ongoing divide between intergovernmental jurisdictional domains limits national initiatives to improve Canada's donation system. The success of a new national system will be enabled by uniform provincial legislation to ensure that all patients are offered the option to donate, commitment of resources to support organ donation by provincial governments, transparent reporting of comparable metrics of donation performance, establishment of processes to introduce and implement new initiatives and alterations to reimbursement models for organ donation and recovery.

Background: Cardiovascular (CV) disease is the foremost cause of mortality and an important cause of morbidity in renal transplant recipients. The disease burden is likely to increase as older patients are accepted for transplantation. The outcome of these high-CV risk patients after renal transplantation, especially with known pre-transplant coronary artery disease (CAD), has not been studied. Hence, we looked at the CV outcome in patients with known pre-transplant CAD. Methods: All renal transplants performed between 1998 and 2002 at our center, followed up to 2005, were divided into high- and low-risk groups, based on the presence of one or more of the following: pre-transplant angina, myocardial infarction, and positive coronary angiogram. The two groups were compared for post-transplant cardiac events and patient and graft survival. The factors predictive of post-transplant cardiac events were also determined by Cox-regression multivariate analysis. Results: Forty-five patients (10.5%), out of 429, had post-transplant cardiac events; 31.3% in the high risk, and 6.5% in the low-risk group (p = 0.001). Five-yr patient survival was lower in the high-risk group (82.8% vs. 93.1%, p = 0.004), while five-yr overall graft survival and death censored graft survival were statistically not different (74.8% vs. 84.1%, p = 0.08 and 87.3% vs. 90%, p = 0.25). Forty-one percent of patients who were treated with angioplasty plus stenting or bypass graft prior to transplantation had post-transplant cardiac events, as compared with 28% of those without intervention in the high-risk group and 6.5% of patients in the low-risk group (p = 0.001). Age, pre-transplant cardiac disease, arrhythmias, and low-ejection fraction (≤40%) were significant independent predictors of post-transplant cardiac events. Conclusion: Post-transplant survival of high-CV risk patients (with known CAD) is lower than that of low-risk recipients but remains acceptable. Cardiac interventions may reduce perioperative risk but do not reduce the probability of post-transplant cardiac events to that of low-risk group.

The calcineurin inhibitors (CNI) cyclosporine and tacrolimus remain the backbone of immunosuppression for most kidney transplant recipients. Despite many years of experience, protocols that optimize efficacy with minimal toxicity remain a subject of debate. Nevertheless, studies of the pharmacokinetic properties of the CNI, particularly cyclosporine, have led to improved dosing strategies. The purpose of this article is to review the current understanding of CNI pharmacokinetics and its relevance to proper dosing and monitoring of these medications. This article also reviews the trials that have helped to define the optimal dosages and discusses the effect of adjunctive immunosuppressive agents on CNI pharmacokinetics and dosing.

Background. There are few data directly comparing the effects of two-hour postingestion monitored cyclosporine (C2-CsA) vs. trough-monitored tacrolimus (C0-Tac) on renal function and cardiovascular risk factors. Methods. We studied 378 (202 C2-CsA vs. 176 CO-Tac) incident kidney transplant recipients in Toronto, Canada, from August 1,2000 and December 31,2003. Outcomes included changes in estimated glomerular filtration rate (eGFR at 1 and 6 months by modification of diet in renal disease four-variable equation), mean arterial pressure (MAP), total cholesterol (TC), and new-onset diabetes mellitus (NODM) at six months posttransplant. The independent effect of treatment/monitoring strategies on continuous outcomes and time-to-NODM was modeled using linear and Cox regression, respectively. Results. Mean eGFR was 59.5 vs. 62.9 ml/min at one month and 50.6 vs. 61.2 ml/min at six months for C2-CsA vs. CO-Tac, respectively. Multiple linear regression revealed the slope of eGFR to be 0.93 ml/min/month lower in C2-CsA patients. This was equivalent to an adjusted average eGFR difference of 4.64 ml/min between months one and six posttransplant. There was no significant difference in average MAP and TC. In a stepwise multivariable Cox model and a propensity score analysis, there was no significant association between the type of treatment/monitoring strategy and time-to-NODM. Conclusions. There was a greater decline in eGFR for patients on C2-CsA (vs. CO-Tac) between one and six months posttransplant. However, MAP, TC, and the risk of NODM were comparable in both treatment/monitoring groups. The long-term impact of short-term reductions in eGFR as a function of the type of treatment/monitoring strategy requires further study.

Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. LAL defects cause Wolman disease (WD) and CE storage disease (CESD). An LAL null (lal−/−) mouse model closely mimics human WD/CESD, with hepatocellular, Kupffer cell and other macrophage, and adrenal cortical storage of CEs and TGs. The effect on the cellular targeting of high-mannose and complex oligosaccharide–type oligosaccharide chains was tested with human LAL expressed in Pichia pastoris (phLAL) and CHO cells (chLAL), respectively. Only chLAL was internalized by cultured fibroblasts, whereas both chLAL and phLAL were taken up by macrophage mannose receptor (MMR)–positive J774E cells. After intraperitoneal injection into lal−/− mice, phLAL and chLAL distributed to macrophages and macrophage-derived cells of various organs. chLAL was also detected in hepatocytes. Ten injections of either enzyme over 30 d into 2- and 2.5-mo-old lal−/− mice produced normalization of hepatic color, decreased liver weight (50%–58%), and diminished hepatic cholesterol and TG storage. Lipid accumulations in macrophages were diminished with either enzyme. Only chLAL cleared lipids in hepatocytes. Mice double homozygous for the LAL and MMR deficiences (lal−/−;MMR−/−) showed phLAL uptake into Kupffer cells and hepatocytes, reversal of macrophage histopathology and lipid storage in all tissues, and clearance of hepatocytes. These results implicate MMR-independent and mannose 6-phosphate receptor–independent pathways in phLAL uptake and delivery to lysosomes in vivo. In addition, these studies show specific cellular targeting and physiologic effects of differentially oligosaccharide-modified human LALs mediated by MMR and that lysosomal targeting of mannose-terminated glycoproteins occurs and storage can be eliminated effectively without MMR.

Renal transplant recipients (RTR) have shortened life expectancy, primarily due to premature cardiovascular disease (CVD). Traditional CVD risk factors are highly prevalent. In addition, several non-traditional risk factors may contribute to the high risk. The aim of the study was to evaluate the effects of renal dysfunction on mortality and cardiovascular complications in 1052 placebo-treated patients of the Assessment of LEscol in Renal Transplantation (ALERT) trial. Follow-up was 5-6 years and endpoints included cardiac death, non-cardiovascular death, all-cause mortality, major adverse cardiac event (MACE), non-fatal myocardial infarction (MI) and stroke. The effects of serum creatinine at baseline on these endpoints were evaluated. Elevated serum creatinine in RTR was a strong and independent risk factor for MACE, cardiac, non-cardiovascular, and all-cause mortality, but not for stroke or non-fatal MI alone. Serum creatinine was associated with increased mortality and MACE, independent of established CVD risk factors. Graft loss resulted in increased incidences of non-cardiovascular death, all-cause mortality, MACE and non-fatal MI. In conclusion, elevated serum creatinine is a strong risk factor for all-cause, non-cardiovascular and cardiac mortality, and MACE, independent of traditional risk factors, but not for stroke or non-fatal MI alone.

Background. Renal transplant recipients have a significantly reduced life expectancy, largely due to premature cardiovascular disease. The aim of the current analysis was to investigate the importance of time of initiation of therapy after transplantation, on the benefits of statin therapy. Methods. 2102 renal transplant recipients with total cholesterol levels of 4.0-9.0 mmol/l were randomly assigned to treatment with fluvastatin (n = 1050) or placebo (n = 1052) and followed for a mean time of 5.1 years. The end-points were major cardiac events. The average median time from transplantation to randomization was 4.5 years (range: 0.5-29 years). Results. In patients starting treatment with fluvastatin 6 years, respectively. The risk reduction for patients initiating therapy with fluvastatin at years 0-2 (compared with >6 years) following transplantation was 59% (RR: 0.41; 95% CI: 0.18-0.92; P = 0.0328). This is also reflected in total time on renal replacement therapy: in patients in the first quartile (120 months) (P = 0.033). Conclusions. Our data support an early introduction of fluvastatin therapy in a population of transplant recipients at high risk of premature coronary heart disease

Background. The clinical benefits of C 2 monitoring of cyclosporine microemulsion have been demonstrated, but C 2 targets in renal transplant recipients during the first year require validation. Methods. MO2ART was a prospective, multicenter study of renal transplant recipients managed by C 2 monitoring of cyclosporine microemulsion with steroids and mycophenolate mofetil or azathioprine. Patients were randomized on day 3 to two groups, which were managed from month 3 with higher or lower C 2 target ranges (months 4-6, 1,0001,200 ng/mL vs. 800-1,000 ng/mL; months 7-12, 800-1,000 ng/mL vs. 600-800 ng/mL, respectively). The primary endpoint was the glomerular filtration rate (GFR) at month 12. Results. A total of 296 patients were recruited, of whom 250 remained in the study at 3 months (higher-C 2 , n=131; lower-C 2 , n=119). GFR at 12 months did not differ between the higher- and lower-C 2 groups (65±17 mL/min vs. 66±14 mL/min). When patients were regrouped according to C 2 achieved by months 8 to 12, those with the lowest C 2 (

The aim of the study was to identity risk factors for long-term renal transplant function and development of chronic allograft nephropathy (CAN) in renal transplant recipients included in the Assessment of Lescol in Renal Transplantation (ALERT) trial.The ALERT trial was a randomized, double-blind, placebo-controlled study of the effect of fluvastatin, 40 and 80 mg/day, in renal transplant recipients who were randomized to receive fluvastatin (Lescol) (n = 1,050) or placebo (n = 1,052) over 5 to 6 years of follow-up. Renal endpoints including graft loss or doubling of serum creatinine or death were analyzed by univariate and multivariate regression analysis in the placebo group.There were 137 graft losses (13.5%) in the placebo group, mainly caused by CAN (82%). Univariate risk factors for graft loss or doubling of serum creatinine were as follows: serum creatinine, proteinuria, hypertension, pulse pressure, time since transplantation, donor age, human leukocyte antigen-DR mismatches, treatment for rejection, low high-density lipoprotein cholesterol, and smoking. Multivariate analysis revealed independent risk factors for graft loss as follows: serum creatinine (relative risk [RR], 3.12 per 100-microM increase), proteinuria (RR, 1.64 per 1-g/24 hr increase), and pulse pressure (RR, 1.12 per 10 mm Hg), whereas age was a protective factor. With patient death in the composite endpoint, diabetes mellitus, smoking, age, and number of transplantations were also risk factors.Independent risk factors for graft loss or doubling of serum creatinine or patient death are mainly related to renal transplant function, proteinuria, and blood pressure, which emphasizes the importance of renoprotective treatment regimens in this population.

Renal transplant recipients have a greatly increased risk of premature cardiovascular disease. The ALERT study was a multicenter, randomized, double-blind, placebo-controlled trial of fluvastatin (40-80 mg/day) in 2102 renal transplant recipients followed for 5-6 years. The main study used a composite cardiac end-point including myocardial infarction, cardiac death and cardiac interventions. Although reduced by fluvastatin, this primary end-point failed to achieve statistical significance thus precluding analysis of predefined subgroups. Therefore, in the present survival analysis, we used an alternative primary end-point of cardiac death or definite nonfatal myocardial infarction (as used in other cardiac outcome trials) which was significantly reduced by Fluvastatin therapy and permits subgroup analysis. Fluvastatin reduced LDL-cholesterol by 1 mmol/L compared with placebo, and the incidence of cardiac death or definite myocardial infarction was reduced from 104 to 70 events (RR 0.65; 95% CI 0.48, 0.88; p = 0.005). Fluvastatin use was associated with reduction in cardiac death or nonfatal myocardial infarction, which achieved statistical significance in many subgroups. The subgroups included patients at lower cardiovascular risk, who were younger, nondiabetic, nonsmokers and without pre-existing CVD. These data support the early introduction of statins following renal transplantation.

The availability of Neoral in place of Sandimmun offered better pharmacokinetics and improved results but emphasized the poor accuracy of trough levels as a tool for monitoring drug exposure and dosage adjustment. Subsequent studies confirmed that Neoral exposure correlated with clinical events and it was found that C2 was the single point that correlated best with exposure as determined by AUC0–4h. Single and multicenter studies have now shown that C2 monitoring is practically feasible and that its use resulted in very low rates of acute rejection with excellent renal function. One retrospective comparison suggested that rates of acute rejection were lower with C2 than with C0 monitoring. This tool has now been shown to be effective in managing African-American renal transplants and those with delayed graft function. A very recent international randomized trial has shown equivalent rates of acute rejection in liver transplant recipients treated with both Neoral and Prograf. Studies now in progress will permit further refinement of target levels with the potential to improve long-term results.

Posttranslational modification can influence the biologic activity of recombinant proteins. The effects of beta-subunit C-terminal truncation, oligosaccharide heterogeneity, and chemical oxidation on the in vitro activity of recombinant human thyroid-stimulating hormone (rhTSH) were investigated. beta-Subunit C-terminal truncation up to residue 113 did not effect the in vitro activity of the hormone. The relationship between the heterogeneity of oligosaccharide structures on rhTSH and specific activity of the glycoprotein hormone was also examined. Oligosaccharide profiles were generated for preparations of rhTSH containing similar sialic acid levels. A weak correlation was observed between relative levels of monosialylated biantennary, bisialylated biantennary, and trisialylated triantennary oligosaccharide species and in vitro activity of the recombinant hormone (p < 0.05). To examine the effect of chemically induced methionine oxidation on the activity of rhTSH, the hormone was treated with tert-butyl hydroperoxide and then characterized. Using peptide mapping and mass spectrometry, the degree of oxidation of the five methionine residues within rhTSH was measured. Met-71 in the alpha-subunit was the most susceptible to oxidation whereas Met-9 in the beta-subunit was the most resistant. Also, after tert-butyl hydroperoxide treatment, levels of oxidation of Met-32 in the beta-subunit, and Met-29 and Met-47 in the alpha-subunit were less than half of that observed for Met-71. The in vitro activity of rhTSH initially declined with increasing oxidation; however, the loss in activity plateaued at approximately 50% of the control sample activity. In summary, despite the possible effects that posttranslational modifications may have on the bioactivity of a protein, a limited degree of variation in bioactivity was observed for the rhTSH preparations described in this study.

Anti-glomerular basement membrane (GBM) disease is a disorder characterized by antibodies against an epitope of type IV collagen found on the GBM. The major clinical sequela is rapidly progressive glomerulonephritis, which may be accompanied by pulmonary haemorrhage (Goodpasture’s syndrome). Glomerulonephritis secondary to anti-GBM disease frequently progresses to end-stage renal disease (ESRD) in the subset of patients who present with markedly impaired renal function. Renal transplantation is performed for ESRD due to anti-GBM disease, although most centres delay transplantation until patients are anti-GBM antibody negative for at least 12 months. Although early case series showed frequent recurrence in the allograft [1], modern therapeutic approaches have made recurrent disease very rare, and only four cases have been reported [2–5]. The effect of therapy for recurrent allograft disease is not well described. We report a case of recurrent anti-GBM disease in a renal allograft with successful salvage of the allograft.

Summary Background Renal transplant recipients are at increased risk of premature cardiovascular disease. Although statins reduce cardiovascular risk in the general population, their efficacy and safety in renal transplant recipients have not been established. We investigated the effects of fluvastatin on cardiac and renal endpoints in this population. Methods We did a multicentre, randomised, double-blind, placebo-controlled trial in 2102 renal transplant recipients with total cholesterol 4·0–9·0 mmol/L. We randomly assigned patients fluvastatin (n=1050) or placebo (n=1052) and follow up was for 5–6 years. The primary endpoint was the occurrence of a major adverse cardiac event, defined as cardiac death, non-fatal myocardial infarction (MI), or coronary intervention procedure. Secondary endpoints were individual cardiac events, combined cardiac death or non-fatal MI, cerebrovascular events, non-cardiovascular death, all-cause mortality, and graft loss or doubling of serum creatinine. Analysis was by intention to treat. Findings After a mean follow-up of 5·1 years, fluvastatin lowered LDL cholesterol concentrations by 32%. Risk reduction with fluvastatin for the primary endpoint (risk ratio 0·83 [95% Cl 0·64-1·06], p=0·139) was not significant, although there were fewer cardiac deaths or non-fatal Ml (70 vs 104, 0·65 [0·48–0·88] p=0·005) in the fluvastatin group than in the placebo group. Coronary intervention procedures and other secondary endpoints did not differ significantly between groups. Interpretation Although cardiac deaths and non-fatal Ml seemed to be reduced, fluvastatin did not generally reduce rates of coronary intervention procedures or mortality. Overall effects of fluvastatin were similar to those of statins in other populations. Published online June 3, 2003 http://image.thelancet.com/extras/03art4377web.pdf

Thyroid stimulating hormone (TSH), a pituitary glycoprotein hormone, is a potent inducer of intracellular cAMP production. Two methods for measuring TSH bioactivity were evaluated and compared. One assay is based on using a radioimmunoassay (RIA) to measure the recombinant human TSH-induced increase in cAMP using a bovine thyroid membrane isolate. The other is based on a Chinese hamster ovary (CHO) cell line that has been transfected with the TSH receptor and a cAMP-responsive luciferase reporter. The within-assay coefficient of variation for the membrane-based assay was determined to be approximately 35% compared with approximately 25% for the cell-based assay. Twenty-one preparations of recombinant human TSH (rhTSH) were tested using both methods. No significant difference was detected between the data sets and no assay bias was present. Both assay systems provide a suitable means for measuring the activity of rhTSH. The advantage of the membrane-based assay is the relatively small quantity of TSH needed for analysis. However, the average time required to analyse a sample using the membrane-based method was more than twice as long as that needed to test a sample in the cell-based assay. Other advantages of the cell-based method include the use of a 96-well format, which facilitates the analysis of several concentrations of rhTSH within one assay plate, and the use of a non-radioactive endpoint.

Over 50 proteins have been expressed in the milk of transgenic animals. This chapter focuses on the status of expression of recombinant proteins in such milk. The chapter discusses the expression of therapeutically useful proteins in mammalian milk, the emphasis being on dairy animals as the production systems of choice. The chapter presents an overview of the expression constructs, milk-specific transgenes, transgene insertion, transgenic animal production, milk, and protein purification. The quality regulatory issues related to this are also discussed. The chapter also states that the mammary bioreactor is capable of most posttranslational modifications and protein folding, and can be used to produce complex proteins. The chapter presents the current status where transgenic dairy animals are providing a bulk protein production system. This system is capable of making proteins available that are either not available today or are only recoverable from other sources. Transgenic production may thus enable a move from “on-demand” patient treatment to prophylaxis and far wider indications and use of many proteins. In addition, the production of proteins in the milk of transgenic dairy animals is highly cost-effective, opening up real possibilities for nutraceutical product development.

There has been a steady increase in the utilization of aged donor kidneys for dual transplantation during the past several years. As the follow-up of these dual kidney recipients accrues, it is clear that the long-term graft survival rate approaches that seen in recipients of single kidneys transplanted from younger donors. Because the kidneys used for dual kidney transplants would have otherwise been discarded, it is imperative to recognize that kidneys from cadaver donors that fall outside the normal acceptance criteria are a valuable resource and can provide excellent long-term function when properly placed. Reducing cold storage time may be the single most important aspect to insuring long-term graft survival in recipients of aged dual kidney transplants.

The presence and specific structures of the oligosaccharides on TSH have been shown to be important for its production and bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the biological activity and metabolic clearance of a preparation of purified recombinant human (rh) TSH derived from a stable transfectant of Chinese hamster ovary cells. Carbohydrate compositional analysis of this rTSH showed it to be more highly sialylated than a nonrecombinant, cadaver-derived pituitary hTSH. In addition, no N-acetyl galactosamine was detectable in rhTSH, which implies the absence of terminal sulfate moieties, both of which are present in pituitary-derived TSH. The immunologic activity and porcine TSH receptor-binding activity of the preparation of rhTSH were 3- to 4-fold lower than those of a standard pituitary hTSH. The rhTSH showed a maximum stimulatory activity similar to that of pituitary hTSH in two different in vitro bioassays. However, rhTSH elicited about 3-fold and 5-fold less cAMP than pituitary TSH after stimulation of adenylyl cyclase in bovine thyroid membranes and the rat FRTL-5 cell line, respectively. Removal of sialic acid did not alter the immunologic activity of rhTSH. However, the potencies of rhTSH in receptor-binding, adenylyl cyclase, and FRTL-5 assays were increased 2.4-, 2.6- and 26.7-fold, respectively after sialic acid removal. These data suggest that the in vitro biological activity of rhTSH is influenced by its highly sialylated oligosaccharide chains. The rhTSH had a 2-fold lower metabolic clearance rate than pituitary TSH, resulting in a greater than 10-fold higher serum concentration of rhTSH at 3 h as compared to pituitary hTSH. After sialic acid removal, the rhTSH was cleared faster (7.5-fold) than pituitary hTSH, showing that its longer plasma half-life was due to its higher sialylation. Biologically active rhTSH should be of clinical value in the diagnosis and treatment of patients with thyroid cancer and as a pure hTSH reference preparation.

Transferrin-membrane protein complexes were solubilized either with 0.4% sodium dodecyl sulfate (SDS), 1% Triton X-100 or 0.5% sulfobetaine 3-14 from the plasma membranes of rabbit reticulocytes previously labeled with 125I and then incubated with 131-labeled transferrin. When the solubilized membranes were analyzed by gel filtration fractionation, marked variation in the preservation of transferrin-transferrin receptor interaction was noted between the three detergents. After SDS solubilization, more than 80% of the 131I-labeled transferrin remained associated with membrane proteins with apparent molecular weight of the transferrin-receptor complexes of 1400 000 and 240 000. In contrast, after Triton X-100 solubilization only 40% of the transferrin was still complexed to membrane proteins with an apparent molecular weight of the complex of 450 000. Dissociation of transferrin from its receptor was most marked following sulfobetaine solubilization, with less than 30% of the transferrin still complexed. Following gel filtration 131I-labeled transferrin-125I-labeled membrane protein complexes were immunoprecipitated with goat specific anti-rabbit transferrin antibodies. The immunoprecipitates were analyzed under stringent dissociating conditions by two SDS-polyacrylamide gel electrophoretic techniques. In a linear 5-25% polyacrylamide gradient the 125I-labeled receptor obtained after membrane solubilization with all three detergents had an apparent molecular weight of 80 000. In contrast, in a different system using 10% polyacrylamide gel two 125I-labeled receptor components were detected wih apparent molecular weights of 90 000 and 80 000. These results demonstrate that estimates of the molecular weight of the transferrin receptor depended on the conditions of electrophoresis and suggest that the transferrin receptor is partially modified, perhaps by glycosylation.