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Joyce Popoola,1,3 Helen Greene,2 Margaret Kyegombe,2 Iain A MacPhee1,3 1Department of Nephrology and Transplantation, 2Department of Pharmacy, St George’s Healthcare NHS Trust, 3St George’s University of London, London, UK Abstract: Transplantation has made a considerable difference to the lives of many patients. However, feedback from patients indicates that although having a transplant is a hugely positive experience, having to take medications indefinitely is one of the biggest challenges. An ideal scenario would be no medications following a transplant. A compromise would be a minimal number of medications, with minimal restrictions and as simple a regimen as possible. Although there is considerable research going into fine-tuning the management of the immune response to a transplant, to date there is no universal regimen that enables patients to remain free of immunosuppressant medications, making adherence paramount to maintain long-term allograft survival. This paper reviews the available immunosuppressant regimens and factors influencing choice from both the clinician’s and the patient’s perspective. Factors influencing the decision-making process, such as quality of life for patients, their satisfaction, acceptability, and adherence uptake are reviewed. We conclude with a further assessment of patient choice as a factor in regimen selection, its impact on adherence, and its implications. Keywords: adherence, transplant, allograft, immunosuppressants, patient involvement, satisfaction, decision-making

Hamid Abboudi, Iain AM MacPheeDivision of Clinical Sciences, Renal Medicine, St George's, University of London, London, UKAbstract: The immunosuppressive drugs used to prevent the rejection of transplanted organs have a narrow therapeutic index. Under treatment results in episodes of rejection leading to either damage or loss of the organ. Over immunosuppression increases the risk of infection and malignancy as well as drug specific complications including diabetes mellitus and nephrotoxicity. There is wide variation in the drug dose required to achieve target blood concentrations and there is often dissociation between pharmacokinetics and pharmacodynamics. Currently, immunosuppressive drug treatment is individualized based on a clinical assessment of the risk of rejection or toxicity. Therapeutic drug monitoring is routinely employed for several immunosuppressive drugs. Pharmacogenetics has the potential to complement therapeutic drug monitoring but clinical benefit has yet to be demonstrated. Novel biomarker-based approaches to risk stratification and pharmacodynamic monitoring are under development and are ready for clinical trials.Keywords: CYP3A5, immunosuppression, pharmacogenetics, transplantation

CYP3A5 (cytochrome P450, family 3, subfamily A, polypeptide 5) expression stimulates the sodium retentive actions of the mineralocorticoid receptor causative of hypertension, probably by means of its ability to substantially increase the level of 6β-hydroxylase activity. Most Black individuals are functional CYP3A5 expressers, and this is a candidate gene for the high incidence of hypertension in Black populations. The study investigates whether CYP3A5 expression results in higher blood pressure in a Ghanaian population. Real-time PCR was used to genotype 898 DNA samples for the CYP3A5*3 and CYP3A5*6 single-nucleotide polymorphisms with technically adequate genotyping for 881 samples. Of these, 803 were genetic CYP3A5 expressers, 44 nonexpressers and 34 uncertain (CYP3A5*3/*6). Although there was a trend in the proportion of hypertensive individuals as CYP3A5 expression decreased, using a two-sided t-test, no statistically significant relationship was established between systolic or diastolic pressure and CYP3A5*3 or CYP3A5*6 genotypes, or their haplotypes (Systolic confidence interval: -8.44 to -7.70, P=0.93, Diastolic confidence interval: -4.89 to 4.85, P=0.99). We conclude, therefore, that there is either no association between CYP3A5 expression and blood pressure or, if there is a relationship, the strength of the association is very small.

There is a dearth of data on end-stage renal disease (ESRD) in Africa. Several national renal registries have been established but have not been sustainable because of resource limitations. The African Association of Nephrology (AFRAN) and the African Paediatric Nephrology Association (AFPNA) recognize the importance of good registry data and plan to establish an African Renal Registry. This article reviews the elements needed for a successful renal registry and gives an overview of renal registries in developed and developing countries, with the emphasis on Africa. It then discusses the proposed African Renal Registry and the first steps towards its implementation. A registry requires a clear purpose, and agreement on inclusion and exclusion criteria, the dataset and the data dictionary. Ethical issues, data ownership and access, the dissemination of findings and funding must all be considered. Well-documented processes should guide data collection and ensure data quality. The ERA-EDTA Registry is the world's oldest renal registry. In Africa, registry data have been published mainly by North African countries, starting with Egypt and Tunisia in 1975. However, in recent years no African country has regularly reported national registry data. A shared renal registry would provide participating countries with a reliable technology platform and a common data dictionary to facilitate joint analyses and comparisons. In March 2015, AFRAN organized a registry workshop for African nephrologists and then took the decision to establish, for the first time, an African Renal Registry. In conclusion, African nephrologists have decided to establish a continental renal registry. This initiative could make a substantial impact on the practice of nephrology and the provision of services for adults and children with ESRD in many African countries.

Tacrolimus is the mainstay immunosuppressant drug used after solid organ and hematopoietic stem cell transplantation. Individuals who express CYP3A5 (extensive and intermediate metabolizers) generally have decreased dose-adjusted trough concentrations of tacrolimus as compared with those who are CYP3A5 nonexpressers (poor metabolizers), possibly delaying achievement of target blood concentrations. We summarize evidence from the published literature supporting this association and provide dosing recommendations for tacrolimus based on CYP3A5 genotype when known (updates at www.pharmgkb.org)., (© 2015 American Society for Clinical Pharmacology and Therapeutics.)

Introduction: There is currently no accurate method of measuring glomerular filtration rate (GFR) during acute kidney injury (AKI). Knowledge of how much GFR varies in stable subjects is necessary before changes in GFR can be attributed to AKI. We have designed a method of continuous measurement of GFR intended as a research tool to time effects of AKI. The aims of this crossover trial were to establish accuracy and precision of a continuous infusion of low dose Iohexol (CILDI) and variation in GFR in stable volunteers over a range of estimated GFR (23-138 mL/min/1.73 m(2))., Methods: We randomised 17 volunteers to GFR measurement by plasma clearance (PC) and renal clearance (RC) of either a single bolus of Iohexol (SBI; routine method), or of a continuous infusion of low dose Iohexol (CILDI; experimental method) at 0.5 mL/h for 12 h. GFR was measured by the alternative method after a washout period (4-28 days). Iohexol concentration was measured by high performance liquid chromatography/electrospray tandem mass spectrometry and time to steady state concentration (Css) determined., Results: Mean PC was 76.7 ± 28.5 mL/min/1.73 m(2) (SBI), and 78.9 ± 28.6 mL/min/1.73 m(2) (CILDI), p = 0.82. No crossover effects occurred (p = 0.85). Correlation (r) between the methods was 0.98 (p < 0.0001). Bias was 2.2 mL/min/1.73 m(2) (limits of agreement -8.2 to 12.6 mL/min/1.73 m(2)) for CILDI. PC overestimated RC by 7.1 ± 7.3 mL/min/1.73 m(2). Mean intra-individual variation in GFR (CILDI) was 10.3% (p < 0.003). Mean ± SD Css was 172 ± 185 min., Conclusion: We hypothesise that changes in GFR >10.3% depict evolving AKI. If this were applicable to AKI, this is less than the 50% change in serum creatinine currently required to define AKI. CILDI is now ready for testing in patients with AKI., Trial Registration: This trial was registered with the European Union Clinical Trials Register ( https://www.clinicaltrialsregister.eu/ ), registration number: 2010-019933-89 .

Background: The use of donation after circulatory death (DCD) kidneys for transplantation is increasing. Subsequent delayed graft function is related to ischaemia/reperfusion injury (I/R), warm ischaemia (WI) being one of the main contributing factors. This proteomics study aimed to identify candidate biomarkers of WI., Methods: Termination biopsies were obtained over 180min in 6 pigs. Proteins were subjected to differential in-gel electrophoresis (DIGE) and identified using LC MS/MS., Results: Thirty nine protein spots showed significant changes in expression (ANOVA, p<0.05). Peroxiredoxin-3 and -6 (PRX3 and PRX6) were expressed with a fold change (FD) of +1.8 (p=0.03 and 0.02 respectively). A significant upregulation of Alpha-2-HS-glycoprotein (A2HSG, FD+1.9, p=0.047) and heat-shock protein 70-1b (HSP70-1b, FD+2.1 p=0.002) was recorded., Conclusions: The expression of PRX3, PRX6 and HSP70-1b during the first 30min of WI may be critical in measuring cellular responses. This is the first large animal model to describe the novel candidate biomarker, structural protein A2HSG. A2HSG upregulation during WI alone in this study is encouraging and further assessment in a DCD auto-transplant model is warranted., Biological Significance: Warm ischaemia (WI) during donation after circulatory death (DCD) organ retrieval is associated with higher rates of post transplant organ dysfunction. The cellular and molecular mechanism of this paradigm is poorly reported. The work carried out in this large animal study has been performed to enable better understanding of protein expression during DCD WI at the time of retrieval. We have identified differential increased expression of PRX3, PRX6 and HSP70 during the first 30min of WI. Observation of this behaviour has not been reported before. Application of these results in a reperfusion model or autograft animal study would further help study of the named proteins as clinical biomarkers of WI. Alpha 2-HS Glycoprotein (A2HSG) species were also differentially expressed during the WI period. This remains a novel finding. Assessment of A2HSG is also recommended for further study in a reperfusion context. Previous reports of A2HSG have suggested an association in chronic kidney disease and diabetes, but no association with WI has previously been noted in either small or large animals., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aims: An algorithm based on the CYP3A5 genotype to predict tacrolimus clearance to inform the optimal initial dose was derived using data from the DeKAF study (Passey et al. Br J Clin Pharmacol 2011; 72: 948-57) but was not tested in an independent cohort of patients. Our aim was to test whether the DeKAF dosing algorithm could predict estimated tacrolimus clearance in renal transplant recipients at our centre., Methods: Predicted tacrolimus clearance based on the DeKAF algorithm was compared with dose-normalized trough whole-blood concentrations (estimated clearance) on day 7 after transplantation in a single-centre cohort of 255 renal transplant recipients., Results: There was a weak correlation (r = 0.431) between clearance based on dose-normalized trough whole-blood concentrations and DeKAF algorithm-predicted clearance. The means of the tacrolimus clearance predicted by the DeKAF algorithm and the estimated tacrolimus clearance based on the dose-normalized trough blood concentrations were plotted against the differences in the clearance as a Bland-Altman plot. Logarithmic transformation was performed owing to the increased difference in tacrolimus clearance as the mean clearance increased. There was a highly significant systematic error (P < 0.0005) characterized by a sloped regression line [gradient, 0.88 (95% confidence interval, 0.75-1.01)] on the Bland-Altman plot., Conclusions: The DeKAF algorithm was unable to predict the estimated tacrolimus clearance accurately based on real tacrolimus doses and blood concentrations in our cohort of patients. Other genes are known to influence the clearance of tacrolimus, and a polygenic algorithm may be more predictive than those based on a single genotype., (© 2013 The Authors. British Journal of Clinical Pharmacology © 2013 The British Pharmacological Society.)

We present a rare complication of transplant renal artery stenting in a patient who developed subcapsular hematoma and presented as acute page kidney. We discuss possible mechanisms and successful novel management using radiological-assisted decompression. We propose considering this alternative option before proceeding with surgical exploration and renal capsulotomy.

The concept of hepatorenal syndrome is well recognized, although incompletely understood. The converse clinical problem of hepatic dysfunction in patients with acute kidney injury (AKI) is less well recognized yet may be a contributor to the high patient morbidity and mortality seen in this group. This review draws together the available evidence for AKI's effect on the liver from animal models, pharmacological studies and recent clinical data. It examines liver function beyond clinically used blood tests, to determine the effect of AKI on hepatic synthetic function, acute phase response and drug metabolism. Parallels are drawn with other organ crosstalk in AKI and with liver-kidney interactions in chronic kidney disease. Definition of the pathophysiology of renohepatic crosstalk may lead to improved management strategies for this vulnerable patient group.

Background: The risk of long-term chronic allograft nephropathy and graft loss after kidney transplantation is increased in patients with a high intrapatient variability in the clearance of tacrolimus. CYP3A5 genotype has a significant influence on the oral bioavailability of tacrolimus and is a potential influence on variability of exposure., Methods: The study population consisted of 118 renal transplant recipients with stable renal function 12 months after transplant. The intrapatient variability of tacrolimus concentration was calculated. The patients were divided into low- and high-intraindividual variability groups using the median variability of tacrolimus clearance as the cutoff value., Results: No differences in baseline characteristics were observed between the expressers (n = 37) and nonexpressers (n = 81) except for ethnicity, which is in line with previous observations. Tacrolimus dose requirement was significantly higher in patients expressing CYP3A5, confirming earlier observations (P < 0.0001). However, intraindividual variability of tacrolimus clearance was not related to CYP3A5 genotype (P = 0.3331)., Conclusions: The intrapatient variability of tacrolimus clearance was not associated with CYP3A5 genotype in stable renal transplant recipients.

Introduction. RIFLE and AKIN provide a standardised classification of acute kidney injury (AKI), but their categorical rather than continuous nature restricts their use to a research tool. A more accurate real-time description of renal function in AKI is needed, and some published data suggest that equations based on serum creatinine that estimate glomerular filtration rate (eGFR) can provide this. In addition, incorporating serum cystatin C concentration into estimates of GFR may improve their accuracy, but no eGFR equations are validated in critically ill patients with AKI. Aim. This study tests whether creatinine or cystatin-C-based eGFR equations, used in patients with CKD, offer an accurate representation of 4-hour creatinine clearance (4CrCl) in critically ill patients with AKI. Methods. Fifty-one critically ill patients with AKI were recruited. Thirty-seven met inclusion criteria, and the performance of eGFR equations was compared to 4CrCl. Results. eGFR equations were better than creatinine alone at predicting 4CrCl. Adding cystatin C to estimates did not improve the bias or add accuracy. The MDRD 7 eGFR had the best combination of correlation, bias, percentage error and accuracy. None were near acceptable standards quoted in patients with chronic kidney disease (CKD). Conclusions. eGFR equations are not sufficiently accurate for use in critically ill patients with AKI. Incorporating serum cystatin C does not improve estimates. eGFR should not be used to describe renal function in patients with AKI. Standards of accuracy for validating eGFR need to be set.

The immunosuppressive drugs used for solid organ transplantation all have a narrow therapeutic index with wide variation between individuals in the blood concentration achieved by a given dose. Therapeutic drug monitoring is employed routinely but may not allow optimisation of drug exposure during the critical period two to three days following transplantation. A key factor in the inter-individual variability for tacrolimus, and probably sirolimus, is whether an individual is genetically predicted to express the drug metabolising enzyme cytochrome P450 3A5 (CYP3A5). Individuals predicted to express CYP3A5 by possession of at least one wild-type CYP3A5*1 allele require 1.5-2 times higher doses of tacrolimus to achieve target blood concentrations than individuals homozygous for the CYP3A5*3 allele who are functional non-expressers of CYP3A5. Planning the initial tacrolimus dose based on the CYP3A5 genotype has been shown to allow more rapid achievement of target blood concentrations after transplantation than a standard dose given to all patients. However, it remains to be demonstrated that use of this approach as an adjunct to therapeutic drug monitoring can reduce either efficacy failure (transplant rejection) or toxicity. Use of a pharmacogenetic approach to dosing sirolimus awaits testing and it is unlikely to be useful for ciclosporin or everolimus., (Copyright © 2011. Published by Elsevier B.V.)

Introduction: Acute kidney injury (AKI) is a common and serious complication increasing morbidity and mortality from all causes of hospital admission. We have previously shown that AKI decreases midazolam metabolism, a substrate of the cytochrome P450 3A (CYP3A) enzymes and our primary aim was to determine if this effect is dependent on the severity of AKI. We also present preliminary data on the functional impact of different genotypes of CYP3A., Methods: Critically ill patients at risk of AKI and admitted to a general intensive care unit were categorised after initial resuscitation according to the RIFLE criteria for AKI. Midazolam (1mg) was administered and the serum concentration of midazolam measured at 4 h. Samples were taken for CYP3A genotyping., Results: Seventy-three patients were assigned to categories R, I and F of the RIFLE criteria or C (controls). Midazolam concentrations (ng mL(-1)) increased significantly (p = 0.002) as the severity of AKI worsened [control 3.1 (1.4-5.9), risk 4.7 (1.3-10.3), injury 3.9 (2.0-11.1) and failure 6.8 (2.2-113.6)] and were predicted by the duration of AKI (p = 0.000) and γ-glutamyl transferase (p = 0.005) concentrations. Increasing BMI negatively predicted the midazolam concentration (p = 0.001). Preliminary data suggest this effect is diminished if the patient expresses functional CYP3A5., Conclusion: Increasing severity and duration of AKI are associated with decreased midazolam elimination. We propose that this is caused by impaired CYP3A activity secondary to AKI. The exact mechanism remains to be elucidated. This may have important implications for our drug treatment of critically ill patients.

Background: The association of CYP3A5, CYP3A4, and ABCB1 single nucleotide polymorphisms (SNPs) with cyclosporine (CsA) pharmacokinetics is controversial. The authors studied the influence of these SNPs on CsA pharmacokinetics as well as on the incidence of biopsy-proven acute rejection (BPAR) and renal function after kidney transplantation., Method: One hundred seventy-one patients participating in an international, randomized controlled trial were genotyped for CYP3A5*3, CYP3A4*1B and the ABCB1 1236 C>T, 2677 G>T/A, and 3435 C>T SNPs. The patients were treated with CsA, mycophenolate mofetil, and glucocorticoids. CsA was dosed to reach predose concentrations (C0) or two hours postdose concentrations (C2). Pharmacokinetic parameters were measured on Days 3 and 10 and Months 1, 3, 6, and 12 after transplantation. Renal function was assessed by measuring serum creatinine and calculating the creatinine clearance. The incidence of BPAR and delayed-graft function was recorded., Results: CYP3A5, CYP3A4, and ABCB1 genotype were not associated with dose-adjusted CsA C0 or C2. The incidence of BPAR in this cohort was 16% and was comparable between the different ABCB1 genotype groups. No significant difference in the incidence of BPAR was found between CYP3A5 expressers (10%) and nonexpressers (18%) (P = 0.24) nor was there a difference in the incidence of BPAR between CYP3A4*1 homozygotes (5%) versus CYP3A4*1B carriers (18%) (P = 0.13). There were no differences with regard to creatinine clearance between the different CYP3A and ABCB1 genotype groups., Conclusion: According to the results, determination of CYP3A and ABCB1 SNPs pretransplantation is not helpful in determining the CsA starting dose and does not aid in predicting the risk of BPAR or worse renal function in an individual patient.

Pharmacogenetic strategies offer promise as an adjunct to therapeutic drug monitoring in achieving target blood concentrations of the immunosuppressive drugs as early as possible after transplantation. To date, the only strategy to have been tested in a clinical trial is the use of the cytochrome P450 3A5 (CYP3A5) genotype to predict tacrolimus dose. Other potential candidates are CYP3A5 and sirolimus and UGT1A9 for mycophenolate. There are also genetic predictors of pharmacodynamics, including IMPDH1 for mycophenolate and ABCB1 for cyclosporine that may identify individuals at particular risk of efficacy failure or toxicity with a given drug. As pharmacogenetic testing moves into routine clinical practice, standards for service delivery and reporting of results need to be established.

The immunosuppressive drugs used in organ transplantation typically have a narrow therapeutic index, with wide variation in the blood concentration achieved from a given dose observed between individuals. This issue has been addressed through the use of therapeutic drug monitoring (TDM), but it may take 5 to 7 days to reach target blood concentrations using this approach. This timeline is not conducive to achieving sufficiently high concentrations in all patients to prevent graft rejection without exposing the patient to excessive toxicity over the critical 2- to 3-day period following transplantation. SNPs in drug-metabolizing enzymes and transporter proteins have been associated with the pharmacokinetic and pharmacodynamic characteristics of immunosuppressive drugs. Data suggest that genetic prediction of the optimal initial drug dose leads to earlier attainment of target blood concentrations compared with using the standard initial dose. The pharmacogenetic strategy that is closest to translation into clinical practice is the use of the cytochrome P450 (CYP)3A5 genotype to predict the optimal initial dose for tacrolimus. Genetic prediction of the optimal dose may be particularly useful for drugs with a long half-life, such as sirolimus, which require several days to achieve a steady state following the implementation of a change in drug dosing, resulting in a long response-time for TDM. The influence of genetic factors on intracellular drug concentrations and the consequences for efficacy and toxicity are an emerging area of research. The SNPs described in this process could be added to existing molecular tissue typing methodology at minimal extra financial expense.

Background: African American transplant recipients have poorer long-term outcomes than Caucasian Americans. This difference was not found in French patients, suggesting socialized medicine overcame this disparity. It has also been hypothesized that the difference relates to the higher prevalence of Black individuals who express the metabolic enzyme cytochrome P4503A5 (CYP3A5), with consequent altered handling of immunosuppressive drugs., Methods: Records of 555 (50 Black; 505 non-Black) sequential renal transplant recipients from a single UK centre were analysed., Results: Outcomes were significantly worse for Black patients: death-censored graft survival (5-year 66% versus 87%, P = 0.001); halving of year one estimated glomerular filtration rate (mean 8.8 versus 10.8 years, P = 0.008); first-year graft loss (12% versus 3.8%, P = 0.02); and death-censored graft survival in patients surviving the first year with functioning grafts (5-year 77% versus 94%, P = 0.02). Death-censored 5-year graft survival was poorer in Black CYP3A5 expressers than in non-Black CYP3A5 expressers (62% versus 93%, P = 0.002). Following multivariate analysis, the Black group demonstrated poorer graft survival as compared to the non-Black group (hazard ratio 0.46, 95% CI 0.25-0.85, P = 0.002). In a subgroup of genotyped transplant recipients, ethnicity (hazard ratio 0.31, 95% CI 0.15-0.64, P = 0.002), and not CYP3A5 expresser status, persists as an independent risk factor for graft survival following multivariate analysis., Conclusion: In this cohort of patients with socialized medicine, Black recipients had poorer long-term outcomes than individuals from other ethnic groups. This was independent of CYP3A5 expresser status.

Objective: Critical illness and associated sequelae can cause severe metabolic disturbances. The effects these have on hepatic drug metabolism are poorly understood. In vivo, enzyme specific drug probes are used to measure changes in hepatic drug metabolism but they require multiple blood sampling and are time consuming. We suggest that a single measurement, 4 h after intravenous administration of midazolam is a reliable indicator of integral plasma midazolam exposure or area under the curve (AUC) in critically ill patients. We also explore the hypothesis that acute kidney injury (AKI) directly impairs hepatic metabolism of drugs in critically ill patients., Methods: A prospective study in 20 critically ill patients who were not taking specific enzyme inhibitors or inducers or benzodiazepines. Correlation between 4 h midazolam concentration and AUC was calculated. We also assessed the difference in metabolism between the patients with normal renal function and those with AKI., Results: Four hour midazolam concentration correlated with AUC r = 0.956 (p < 0.0001). In addition, the 4 h midazolam concentration was greater in critically ill patients with AKI than those with normal renal function p = 0.023., Conclusion: A single-time-point determination of plasma midazolam concentration is a reliable predictor of integral plasma midazolam exposure in critically ill patients. This tool can now be used to assess the effects of critical illness on hepatic drug metabolism. Using this method, we suggest that AKI reduces the hepatic metabolism of midazolam in critically ill patients.

The immunosuppressive drugs used in solid-organ transplantation are potent and toxic agents with narrow therapeutic ranges. Underdosing is associated with immunological rejection of the transplanted organ, whereas overdosing results in infections, malignancy and direct toxicity to a number of organs. Pharmacokinetic heterogeneity makes initial dose determination difficult, as there is a poor correlation between dose and blood concentration. Therapeutic drug monitoring is available but the pharmacokinetic-pharmacodynamic association is imperfect and it does not help in achieving target blood concentrations during the critical early 2-3 days after transplantation. Genetic polymorphisms in drug targets, drug-metabolizing enzymes and drug efflux pumps have been identified as potential targets for developing a pharmacogenetic strategy to individualize initial drug choice and dose. To date, use of the CYP3A5 genotype to predict the appropriate initial dose of tacrolimus is the most promising option for individualization of drug therapy in organ transplantation.

Background: BK nephropathy (BKN) is an important cause of renal transplant dysfunction, believed to be associated with higher levels of immunosuppression. We assessed the experience of BKN in renal transplant patients in the London region., Methods: All six London transplant centers participated and case notes of patients with BKN in 2004 to 2005 were reviewed., Results: There were 17 cases of BKN, giving an incidence of 2.1%. Median time to diagnosis was 9 months. Median baseline creatinine rose from 150 to 196 mumol/L. At diagnosis, 16 patients were on tacrolimus, 15 on mycophenolate mofetil, and 10 on triple therapy with tacrolimus, mycophenolate mofetil, and prednisolone. Management of BKN involved reducing immunosuppression; cidofovir was used in two patients and methylprednisolone in five for acute rejection. Median follow-up time was 29.2 months. Creatinine returned to baseline in four patients, remained elevated in 12 and one patient lost his graft. The new median baseline creatinine was 216 mumol/L. Eight patients underwent repeat biopsies of which four became negative for BKV and three subsequently cleared the virus on blood and urine polymerase chain reaction and urine decoy cells. Overall, eight patients cleared the virus. None of age, sex, viral load, or biopsy characteristics (Banff ct score, Drachenberg grade, and number of BKV positive cells) were associated with poorer outcome when patients with increase in creatinine of less than 30% (n=7) or more than 30% (n=10) from baseline were compared., Conclusion: The incidence of BKN in this study is comparable with previous studies, with more favorable outcomes. It supports the association of BKN with potent immunosuppression.

Pharmacogenetics has the potential to complement therapeutic drug monitoring in achieving target blood concentrations of the immunosuppressive drugs during the critical early period after transplantation when adequate drug exposure is essential to prevent rejection. The most attractive candidate for a pharmacogenetic strategy is tacrolimus dosing based on the CYP3A5 genotype.

Existing immunosuppressive therapies used for solid-organ transplantation have narrow therapeutic indices, whereby underdosing is associated with acute immunological rejection of the transplanted organ and overdosing is associated with infections and malignancy, as well as organ-specific toxicities. There is significant inter-individual variation in the pharmacokinetics and pharmacodynamics of these drugs, an issue that has been addressed, in part, by therapeutic drug monitoring. Genetic polymorphisms in drug metabolising enzymes, drug efflux pumps and drug targets which may underly this heterogeneity have been identified and may provide a tool to guide prescribing. There are a number of associations between genotype and pharmacology, but as of now, only thiopurine-S-methyltransferase and cytochrome P450 3A5 have a sufficiently large influence to have potential in guiding therapy. Recent studies have also identified that donor genotype may play a significant role in immunosuppressive drug pharmacokinetics and pharmacodynamics.

The aim of this study was to determine whether plasma concentrations of the acyl (AcMPAG) and phenolic (MPAG) glucuronide metabolites of mycophenolic acid (MPA) were related to diarrhoea in renal transplant patients on mycophenolate mofetil (MMF) with cyclosporine (CsA) or tacrolimus (TCL). Blood samples (0, 30, 120 min) were taken at days 3, 10, week 4, months 3, 6 and 12 for determination of MPA, MPAG and AcMPAG. MPA-AUC was estimated using validated algorithms. Two hour AUCs were calculated for MPAG and AcMPAG. Immunosuppressive therapy consisted of CsA/MMF (n= 110) and of TCL/MMF (n= 180). In 70/290 (24%) patients 86 episodes of diarrhoea were recorded during 12 months. Significantly more patients on TCL (31.1%) suffered from diarrhea compared to CsA (12.7%). MMF dose, MPA-AUC and the 2 h AUCs of MPAG and AcMPAG did not differ between patients with and without diarrhoea. Plasma AcMPAG and MPAG concentrations were substantially higher in patients on CsA compared with TCL, while MPA-AUC was lower in the former group. These data support the concept that CsA inhibits the biliary excretion of MPAG and AcMPAG, thereby potentially reducing the risk of intestinal injury through enterohepatic recycling of MPA and its metabolites.

The intestinal efflux pump P-glycoprotein (P-gp), the product of the multi-drug resistance-1 (MDR-1) gene, significantly influences the pharmacokinetics of several drugs. Ciclosporin is a substrate for P-gp and is metabolized by cytochrome P450 (CYP) 3A enzymes. P-gp activity is affected by several known single nucleotide polymorphisms (SNPs) and haplotypes. MDR-1 genotypes of SNPs C1236T, G2677T/A and C3435T, as well as haplotypes C-G-C and T-T-T and CYP3A5*1 genotype (predictive of CYP3A5 expression), were related to ciclosporin blood concentrations measured at both 0 and 2 h after drug dosing in 197 stable renal transplant patients. Significant differences (of a magnitude unlikely to be relevant clinically) in dose-normalized blood ciclosporin concentrations were found only between MDR-1 genotypes of the C1236T SNP and between haplotype groups C-G-C and T-T-T in patients that were expressers of CYP3A5. MDR-1 SNPs and haplotypes and also CYP3A5*1 genotype, do not appear to have a major influence on ciclosporin pharmacokinetics.

P-glycoprotein (P-gp) and the drug metabolizing enzymes have major pharmacokinetic effects. Variability in tacrolimus absorption is influenced by P-gp activity which, in turn, is affected by single nucleotide polymorphisms (SNPs) within the multidrug resistance-1 gene (MDR-1). Tacrolimus dose requirements of 206 stable renal transplant patients were related to MDR-1 genotypes of SNPs C1236T, G2677T/A and C3435T, as well as haplotypes: C-G-C and T-T-T. Lower dose-normalized blood tacrolimus concentrations were achieved for: 2677-GG genotype patients, as compared to 2677-TT, and for 3435-CC patients as compared to 3435-TT patients. There was a small, but significant, difference in dose requirements between haplotypes C-G-C and T-T-T patients, which was not significant when patients were subclassified as producers and non-producers of cytochrome P450 3A5 (CYP3A5). The activities of CYP3A5 and P-gp have been shown to influence bioavailability of several drugs. Our data suggest that MDR-1 haplotypes have a relatively minor association with tacrolimus pharmacokinetics.

Mercuric chloride (HgCl2)-induced autoimmunity in Brown Norway rats is a spontaneously resolving autoimmune response driven by the activation of T helper type 2 lymphocytes (Th2 cells). Treatment with antibody to OX40-ligand (OX40-L) from the time of the first HgCl2 injection for 12 days had little effect. Delayed treatment commenced 8 days after the first HgCl2 injection significantly suppressed immunoglobulin E production, splenomegaly, weight loss and mortality. This makes OX40/OX40-L signalling an attractive therapeutic target for Th2-driven autoimmune diseases. Intravenous administration of the murine antibody to OX-40-L elicited a vigorous anti-mouse immunoglobulin antibody response that was significantly enhanced compared to the response to control immunoglobulin. It is likely that this response significantly reduced the plasma half-life of the anti-OX40-L antibody and this observation has clear implications for the interpretation of data from experiments where anti-OX40-L is used in vivo.

The immunosuppressive drugs used in organ transplantation have a narrow therapeutic index, with rejection occurring as a consequence of underdosing and infection, malignancy and a number of drug-specific side effects with excessive dosing. Significant heterogeneity in the dose of drug required to achieve therapeutic blood concentrations adds to the complexity of the problem, which has been partly resolved by therapeutic drug monitoring. Single nucleotide polymorphisms have been identified in genes encoding metabolic enzymes, drug efflux pumps and drug targets for most of the drugs in widespread use. A pharmacogenetic approach to immunosuppressive drug prescribing remains to be tested. Based on current evidence, the most promising strategy would be use of the cytochrome P450 3A5 expressor genotype to guide initial dosing with tacrolimus.

Cytochrome P450 3A5 (CYP3A5) is involved in the biotransformation of many orally administered drugs, some of which are dose optimized using therapeutic drug monitoring. The CYP3A5 gene exhibits variable inter-individual expression, which is related to a single-nucleotide polymorphism. Producers of the enzyme possess at least one CYP3A5*1 allele. Knowledge of patients' CYP3A5 genotype, in conjunction with therapeutic drug monitoring (TDM), may aid patient management. Real-time polymerase chain reaction (PCR) was used to genotype the A6986G mutation of the CYP3A5 gene. Specific primers were employed to generate a DNA product, co-amplified with two internal hybridization probes, using a LightCycler. DNA melt curve analysis readily identified the genotypes CYP3A5*1/*1, CYP3A5*1/*3 and CYP3A5*3/*3. Results were confirmed using DNA sequencing with 100% correlation. Genotypes were determined from 263 individuals and compared with the genotypes of a pseudogene CYP3AP1, which is in disequilibrium with CYP3A5. This is a rapid and reliable method for genotyping the CYP3A5 polymorphism which may be used as an important adjunct to the TDM service offered by laboratories to optimize drug prescription.

Previously, we demonstrated that the dose-normalized tacrolimus blood concentration after renal transplantation was associated with a single nucleotide polymorphism (SNP) in the CYP3AP1 gene, probably through linkage with an SNP in the CYP3A5 gene. Individuals with at least one CYP3A5*1 allele synthesize CYP3A5 and CYP3A5*3/*3 homozygotes do not. We now present results with direct typing of the CYP3A5 genotype for this group of 180 kidney-only transplant recipients from a single center. South Asian and white patients with at least one CYP3A5*1 allele achieved twofold lower dose-normalized tacrolimus blood concentrations compared with CYP3A5*3/*3 homozygotes, confirming our previous findings for the CYP3AP1 SNP. There was a significant delay in achieving target blood concentrations in those with at least one CYP3A5*1 allele. Determination of the CYP3A5*1/*3 genotype could be used to predict the tacrolimus dose requirement and, given incomplete linkage, would be better than determination of the CYP3AP1 genotype.

Previously, we reported that, at 3 months after renal transplantation, individuals with CYP3AP1 genotype CYP3AP1*1 (linked to CYP3A5*1 and strongly associated with expression of CYP3A5) required twofold higher doses of tacrolimus to achieve target blood concentrations than individuals with the genotype CYP3AP1*3/*3 (CYP3A5 nonexpressors). This study assesses the relationship between concentration-controlled dosing during the early period after transplantation, the time to achieve target concentrations and genotype in 178 renal transplant recipients (CYP3AP1*1/*3 or *1/*1: n = 53, CYP3AP1*3/*3: n = 125). Patients with CYP3AP1*1/*3 or *1/*1 had lower mean tacrolimus concentrations during the first week (Median 13.5 vs. 18.5 microg/L, p < 0.0001) with significant delay in achieving target concentrations (15-20 microg/L during week 1, then 10-15 microg/L). More CYP3AP1*3/*3 patients had tacrolimus concentrations above target during the first week (73.6% vs. 35.8%, p = 0.003). There was no difference in the rate of biopsy-confirmed acute rejection, but rejection occurred earlier in the CYP3AP1*1/*3 or *1/*1 group (median 7 d vs. 13 d, p = 0.005). In conclusion, an initial dosing regimen for tacrolimus based on knowledge of the CYP3AP1 genotype and subsequently guided by concentration measurements has the potential to increase the proportion of patients achieving target blood concentrations early after transplantation.

Helper T-lymphocytes have been shown to differentiate into two mutually regulatory subsets. Cells primarily secreting interleukin-2 (IL-2) and interferon-gamma are known as Th1 cells and mediate classical cell-mediated immune responses such as delayed-type hypersensitivity. Cells secreting interleukin-4 (IL-4) are known as Th2 cells and promote humoral immune responses, in particular the production of IgE and IgG4 (human) or IgG1 (rodents). Over-activity of either cell type can result in tissue-damaging autoimmune disease. A number of human diseases including asthma and some kidney diseases are thought to be caused by a Th-2 type autoimmune response. Study of an animal model of Th2-driven autoimmunity (mercuric chloride-induced autoimmunity in Brown Norway rats) has yielded insights into a possible role for oxidant stress in the generation of Th-2 driven autoimmune responses. Mercuric chloride probably causes oxidant stress by the generation of free-radicals, activating NK-kappaB, a transcription factor for the IL-4 gene. Treatment with the antioxidants N-acetlcysteine and desferrioxamine has been shown to suppress vasculitis and IgE production in this model. These findings suggest a possible clinical role for antioxidants in the therapy of human autoimmune disease.

Transplantation has transformed the treatment of patients with organ failure in a number of clinical settings, and immunosuppressive drug therapy is fundamental to its success. However, all the drugs in current use have a narrow therapeutic index. Under-dosing can lead to rejection, while over-dosing increases the risks of infection, malignant disease, and serious drug-specific adverse effects, including diabetes mellitus, nephrotoxicity, hypertension, and hyperlipidemia. Heterogeneity in the pharmacokinetics of these drugs makes initial dose determination difficult, as there is a poor correlation between dose and blood concentration. This results in difficulties in achieving target blood concentrations early after transplantation, which are important for reducing the rate of immunological rejection. This problem is compounded by the observation that neither drug dose nor drug blood concentration accurately predict clinical efficacy or toxicity. The main determinant of heterogeneity in dose requirements is intestinal absorption of the active drug. The oxidative enzymes, cytochrome P450 (CYP) 3A4 and CYP3A5, and the drug efflux pump P-glycoprotein (P-gp) in enterocytes regulate this process. Most substrates for the P-gp pump are also substrates for the CYP3A enzymes. An efficient barrier to xenobiotic absorption is formed by the CYP enzymes and P-gp, and by the two systems working synergistically. Genetic polymorphisms have been reported for the genes associated with the expression of the CYP3A enzymes and P-gp. Genotyping patients for CYP3A genes has the potential to aid the establishment of optimal dosage regimens for transplant patients. Genetic polymorphism of the multiple drug resistance gene-1 (MDR1, also known as ABCB1) [3435C/T] and the CYP3A5 genes (CYP3A5*1, CYP3AP1*1) have the greatest potential to influence the pharmacokinetics of immunosuppressants. Homozygosity of the T allele of the MDR1 3435C/T polymorphism has been associated with reduced enterocyte expression of P-gp resulting in increased drug absorption. The presence of the CYP3A5*1 allele is necessary for the production of a fully catalytic CYP3A5 protein, and also influences the ratio of CYP3A4 : CYP3A5 as well as the overall CYP3A catalytic activity. The CYP3A4 : CYP3A5 ratio may, in turn, influence the pattern of drug metabolites formed. Heterogeneity in the production of active and inactive metabolites has implications for both the pharmacokinetics and pharmacodynamics of these drugs.Gene frequencies and drug dose requirements differ between ethnic groups. Ethnic differences in dose requirements for immunosuppressants have been discussed widely. However, ethnicity is a rather crude marker for genotype. Pharmacogenetic typing offers the possibility of significant improvement in the individualization of immunosuppressive drug prescribing with reduced rates of rejection and toxicity.

Background: There is marked heterogeneity in blood concentrations of tacrolimus following standard body-weight-based dosing. This is most apparent in black patients, who have a higher dose requirement when compared with other ethnic groups. Differences in intestinal P-glycoprotein and hepatic and intestinal cytochrome P4503A activity have been postulated as contributing to this problem., Methods: The dose-normalized blood concentrations of tacrolimus at 3 months after renal transplantation were related to CYP3AP1 and multiple drug resistance (MDR)-1 genotypes determined by polymerase chain reaction followed by restriction fragment length polymorphism analysis., Results: We found that a single nucleotide polymorphism in the CYP3AP1 pseudogene (A/G(44)) that previously has been noted to be more common in African Americans and strongly associated with hepatic CYP3A5 activity correlated well with the tacrolimus dose requirement. A weaker association was found for a polymorphism in the MDR-1 gene, which influences intestinal P-glycoprotein expression., Conclusions: The CYP3AP1 genotype is a major factor in determining the dose requirement for tacrolimus, and genotyping may be of value in planning patient-specific drug dosing.

Mercuric chloride (HgCl2)-induced autoimmunity in Brown Norway (BN) rats is a highly polarized polyclonal Th2-driven autoimmune response with increased IgE production, lymphoproliferation, vasculitis and proteinuria. The increase in serum IgE concentration is clearly measurable by day 4 after the first HgCl2 injection and peaks between days 15 and 20. Treatment with CD80 and CD86 antibodies prior to administration of HgCl2 completely suppresses the autoimmune process. To determine whether interruption of CD28 signalling after initial stimulation of the Th2-response would be suppressive, antibody treatment was delayed. BN rats were given 5 doses of HgCl2 subcutaneously on alternate days. CD80 and CD86 antibodies, or an isotype control, were given daily for 3 days and then on alternate days until day 12 commencing either on the day of the first HgCl2 injection (day 0) or on days 4 or 8. Treatment from day 0 reduced serum IgE concentrations to below baseline (median 9.34 microg/ml on day 0 versus 4.6 microg/ml, on day 5, P = 0.03) suggesting that ongoing costimulation via CD28 is required to maintain basal serum IgE production. Delaying treatment until day 4 or day 8 after the first HgCl2 injection resulted in significant inhibition of IgE secretion, lymphoproliferation, and vasculitis, although less markedly than when treatment was commenced on day 0. These data indicate that CD28-mediated costimulation is not only required for the initiation of the Th2-response but is required for maintenance of a maximal response, making this an attractive therapeutic target for antibody-mediated autoimmune diseases.

Some data suggest that the interaction between CD28 and CD80 (B7.1) stimulates Th1-responses and that CD28 and CD86 (B7.2) stimulates Th2-responses, however this is controversial. We addressed this issue by using mercuric chloride (HgCl2)-induced autoimmunity in Brown Norway (BN) rats as a highly polarized Th2 model and experimental autoimmune encephalomyelitis (EAE) in Lewis rats as a highly polarized Th1 model. Monoclonal antibodies (MoAbs) to CD80 and CD86, given singly, had little effect in either model, however when given together they almost completely suppressed the HgCl2-induced autoimmunity: the peak immunoglobulin (Ig)E concentration was 3.25 microg/ml in treated animals versus 2770 microg/ml in controls (P < 0.0001); caecal vasculitis was suppressed with a median vasculitis score of 0 in treated animals versus 6 in controls (P < 0.0001); and new germinal centre formation was significantly suppressed. A combination of the antibodies also markedly reduced the severity of clinical EAE; from a median aggregate clinical score of 9 to 3 (P = 0.02) and delayed the onset from a median of 12.5 days to 16 days after immunization (P = 0.006). We have demonstrated profound suppression of both Th1 and Th2-driven autoimmunity in rats by a combination of anti-CD80 and CD86, but have been unable to demonstrate any clear differential effects.

Background: Mycophenolate mofetil (MMF) acts as a prodrug for the immunosuppressive drug mycophenolic acid (MPA). It is rapidly converted to MPA following oral ingestion. MPA is metabolized to MPA glucuronide (MPAG), which is renally excreted. This study examines the pharmacokinetics of MPA and MPAG in patients with end-stage renal failure who were on hemodialysis (N = 10) or peritoneal dialysis (N = 10) treatment., Methods: After an overnight fast, a single oral dose of 1 g MMF was given. Plasma concentrations of MPA and MPAG were measured from 0 (predose) to 36 hours after administration, using high-performance liquid chromatography (HPLC). The area under the concentration time curve (AUC) from 0 to 36 hours was calculated using the trapezoidal rule., Results: Mean (+/- SD) AUC for MPA was 55.7 +/- 32.6 mg/L.h for hemodialysis patients and 44.7 +/- 14.7 mg/L.h for peritoneal dialysis patients, which is similar to expected values for subjects with normal renal function. The mean (+/- SD) maximum plasma concentration (Cmax) for MPA was lower than would be expected for subjects with normal renal function (16.01 +/- 10.61 mg/L for hemodialysis, 11.48 +/- 4.98 mg/L for peritoneal dialysis). MPAG clearance was prolonged with AUC approximately five times what would be expected in subjects with normal renal function (1565 +/- 596 mg/L.h for hemodialysis, 1386 +/- 410 mg/L.h for peritoneal dialysis). There was no significant difference for any of the pharmacokinetic parameters between subjects on hemodialysis and those on peritoneal dialysis. Plasma concentrations of MPA and MPAG did not fall significantly during hemodialysis. No MPA was detectable in hemodialysis or peritoneal dialysis fluid, but small amounts of MPAG were detected in hemodialysis fluid in 1 out of 10 subjects and in peritoneal dialysis fluid in 3 out of 10 subjects., Conclusions: The accumulation of MPAG may be responsible for the poor gastrointestinal tolerance of this drug in dialysis patients and probably limits the maximum dose of MMF that can be tolerated.

Injection of Brown-Norway rats with mercuric chloride (HgCl2) activates a T helper type 2 (Th2) autoimmune response, with production of a number of autoantibodies and vasculitis primarily affecting the gut. Glucocorticoids have been shown to suppress Th1 and to promote the development of Th2-type responses. Conversely dehydroepiandrosterone (DHEA) promotes Th1 responses with suppression of Th2 responses. This study set out to define the role of these hormones in this animal model. Rats were adrenalectomized (Adx) with no steroid replacement (n = 11), Adx with basal steroid replacement given by a 25 mg corticosterone pellet inserted subcutaneously (n = 13), or sham-Adx (n = 14) prior to administration of HgCl2. In both groups of Adx animals there was a delay in the production of immunoglobulin E (IgE) and serum concentrations on day 9 were marginally lower (P = 0.035, repeated measures ANOVA). All of the animals Adx with no steroid replacement and two Adx animals with steroid replacement died between 10 and 14 days after HgCl2 challenge. There was no difference in the severity of caecal vasculitis between the groups. A significant increase in adrenal size was noted following administration of HgCl2. Administration of subcutaneous DHEA implants (100 mg and 200 mg) had no significant effect on IgE concentrations or severity of vasculitis. These observations do not support the hypothesis that corticosterone and DHEA play a central role in setting the Th1/Th2 balance in this experimental Th2-mediated autoimmune disease; in contrast with the Th1-mediated autoimmune disease experimental allergic encephalomyelitis where corticosterone plays a key role in immunoregulation.

Background: Since the introduction of cyclosporine (CsA), 1-year renal allograft survival has improved, but concern persists about the long-term adverse effects of CsA, especially with respect to renal function and blood pressure. This randomized controlled trial was set up to establish whether withdrawal of CsA would alter long-term outcome., Methods: Adult patients who, at 1 year after renal transplantation, had a stable serum creatinine of less than 300 micromol/L and who had not had acute rejection within the last 6 months were eligible for entry. Patients were randomized either to continue on CsA (n=114) or to stop CsA and start azathioprine (Aza, n=102). All patients remained on prednisolone. Median follow-up was 93 months after transplantation (range: 52-133 months)., Results: There was no significant difference in actuarial 10-year patient or graft survival (Kaplan-Meier), despite an increased incidence of acute rejection within the first few months after conversion. Median serum creatinine was lower in the Aza group (Aza: 119 micromol/L; CsA. 153 micromol/L at 5 years after randomization, P=0.0002). The requirement for antihypertensive treatment was also reduced after conversion to Aza; 75% of patients required antihypertensive treatment at the start of the study, decreasing to 55% from 1 year after randomization in the Aza group and increasing to >80% in the CsA group (55% (Aza) and 84% (CsA) at 5 years after randomization, P<0.005)., Conclusions: Conversion from CsA to Aza at 1 year after renal transplantation results in improvement in both blood pressure control and renal allograft function, and is not associated with significant adverse effects on long-term patient or graft survival.

Experimentally induced and naturally occurring inflammatory diseases of the central nervous system (CNS) are often associated with a breakdown of the blood-brain barrier and edema within the CNS itself. CD4+ T cells are now clearly implicated in the pathogenesis of the induced CNS disease, experimental autoimmune encephalomyelitis, and previous in vivo experiments had indicated that these cells may be capable of directly damaging the CNS vasculature. To assess the capacity of CD4+ T cells to damage brain vascular endothelial cells (EC) in vitro, two lines with specificity for myelin basic protein and OVA were prepared and added to cultures of EC. We show here that both lines, when added in a resting state, severely disrupt the EC monolayers in an Ag-specific manner. The interaction is dependent on the recognition of Ag in the context of MHC class II and is blocked in the presence of mAb specific for CD4. Addition of T cell lines preactivated on irradiated thymocyte APC caused a high level of Ag nonspecific damage to the EC, which was not blocked by the addition of anti-CD4 mAb. Supernatants derived from these latter cells did not alone damage the EC monolayers despite the presence of TNF activity suggesting that T cell-EC contact may be required for these cell lines to mediate their effector function. Both resting and preactivated lines adhered strongly to the EC in the absence of Ag. The capacity of CD4+ T cells to strongly adhere to, and disrupt the integrity of, brain vascular EC may be important in the early stages of CNS disease mediated by this cell type.

Lewis rats immunized with myelin basic protein (MBP) in Freund's complete adjuvant (FCA) suffer from a single episode of paralysis from which they recover spontaneously. Subsequent to recovery, further episodes of paralysis cannot normally be induced by reimmunization with MBP in FCA. It is well established that serum, obtained from rats in the refractory state, can suppress the induction of experimental allergic encephalomyelitis (EAE) when given to animals from the time of immunization with MBP in FCA. Here it is shown that treatment with some such sera from Day 7 after immunization also suppressed the disease. However, not all convalescent sera were suppressive, indicating that rats immunized with MBP in FCA could become refractory to EAE without assayable levels of suppressive activity in their sera. In the context of this result it was notable that a correlation was found between the level of antibody specific for the encephalitogenic peptide in sera and the ability to suppress EAE. An inverse relationship was also shown between the amount of anti-encephalitogenic peptide antibody produced after immunization and the severity of EAE induced. Spleen cells from animals treated with Lewis anti-MBP serum after immunization with MBP in FCA could be activated to transfer EAE by in vitro culture with MBP despite the absence of any clinical signs in the donor animals, i.e. the serum inhibited the expansion or differentiation of these cells rather than preventing their priming or bringing about clonal deletion.