Long-term Renal Transplant Recipients Research Articles

Dampened Inflammatory Signalling and Myeloid-Derived Suppressor-Like Cell Accumulation Reduces Circulating Monocytic HLA-DR Density and May Associate With Malignancy Risk in Long-Term Renal Transplant Recipients

BackgroundMalignancy is a major cause of morbidity and mortality in transplant recipients. Identification of those at highest risk could facilitate pre-emptive intervention such as reduction of immunosuppression. Reduced circulating monocytic HLA-DR density is a marker of immune depression in the general population and associates with poorer outcome in critical illness. It has recently been used as a safety marker in adoptive cell therapy trials in renal transplantation. Despite its potential as a marker of dampened immune responses, factors that impact upon monocytic HLA-DR density and the long-term clinical sequelae of this have not been assessed in transplant recipients.MethodsA cohort study of stable long-term renal transplant recipients was undertaken. Serial circulating monocytic HLA-DR density and other leucocyte populations were quantified by flow cytometry. Gene expression of monocytes was performed using the Nanostring nCounter platform, and 13-plex cytokine bead array used to quantify serum concentrations. The primary outcome was malignancy development during one-year follow-up. Risk of malignancy was calculated by univariate and multivariate proportionate hazards modelling with and without adjustment for competing risks.ResultsMonocytic HLA-DR density was stable in long-term renal transplant recipients (n=135) and similar to non-immunosuppressed controls (n=29), though was suppressed in recipients receiving prednisolone. Decreased mHLA-DRd was associated with accumulation of CD14+CD11b+CD33+HLA-DRlo monocytic myeloid-derived suppressor-like cells. Pathway analysis revealed downregulation of pathways relating to cytokine and chemokine signalling in monocytes with low HLA-DR density; however serum concentrations of major cytokines did not differ between these groups. There was an independent increase in malignancy risk during follow-up with decreased HLA-DR density.ConclusionsDampened chemokine and cytokine signalling drives a stable reduction in monocytic HLA-DR density in long-term transplant recipients and associates with subsequent malignancy risk. This may function as a novel marker of excess immunosuppression. Further study is needed to understand the mechanism behind this association.

Transitional immature regulatory B cells and regulatory cytokines can discriminate chronic antibody-mediated rejection from stable graft function

BackgroundThe balance between inflammatory and anti-inflammatory responses of the immune system has been demonstrated to determine the fate of transplanted allografts. Here we analyzed CD19+CD24hiCD38hi immature transitional regulatory B (TRB) cells, as well as the gene and protein levels of interleukin (IL)-10 and transforming growth factor (TGF)-β in the three separate groups, include of stable transplanted subjects, chronic antibody-mediated rejection (cAMR) patients, and healthy individuals. MethodPeripheral blood mononuclear cells (PBMCs) from stable subjects (n = 36), cAMR patients (n = 36) and healthy controls (n = 18) were isolated. Flowcytometry was performed for CD19, CD24, and CD38 surface markers. ELISA and quantitative real-time PCR were performed for IL-10 and TGF-β cytokines. ResultThe percentages of immature TRB cells were significantly decrease in cAMR patients (0.98%) versus stable recipients (2.81%) and healthy subjects (4.03%) (P = 0.001 and P < 0.001, respectively). Total lymphocytes, circulating B cells, memory and mature subsets of B cells did not show any significant difference between the groups. TGF-β mRNA was 3-fold upregulated in the cAMR group compared to stable patients (P < 0.001.), but without significant alteration at the protein level. Also, long-term survival renal transplant recipients had a higher protein but not mRNA levels of IL-10 than short-term survival renal transplant recipients. ConclusionIt seems that immature TRB cell subpopulation might be a crucial regulator of immune system response and plays an important role in determining the transplantation outcome. Furthermore, immunosuppressive IL-10 and TGF-β cytokines might act as a double sword and can exhibit either pathogenic or protective effects against allograft.

Serum Magnesium and Related Factors in Long-Term Renal Transplant Recipients: An Observational Study

BackgroundLow serum magnesium (MgS) is a known risk factor for cardiovascular and mineral bone disease. In renal transplant recipients (RTRs), low MgS levels have been related to higher glomerular filtration rates (GFR) and with calcineurin inhibitors, particularly tacrolimus. We aimed to evaluate MgS in renal transplant recipients with over 1 year of follow-up to establish related risk factors and the impact of the use of cyclosporine versus tacrolimus. MethodsCross-sectional study of 94 RTRs with more than 12 months of follow-up. Hypomagnesemia was defined as serum magnesium level <1.5 mg/dL. ResultsHypomagnesemia was found in 5.3% of patients. MgS showed a negative correlation with creatinine clearance. A positive correlation between MgS with urinary magnesium and phosphorus was found. Cyclosporine versus tacrolimus analysis did not show a significant difference regarding MgS when considering all the population and the subgroup of patients with GFR >45 mL/min/1.73 m2. On the subgroup with GFR <45 mL/min/1.73 m2, those on tacrolimus had lower MgS than those on cyclosporine, but those same patients presented with significantly different GFR, higher in the tacrolimus subgroup. ConclusionsHypomagnesemia has a low prevalence in RTRs with more than 1 year of follow-up. MgS levels evidenced a strong correlation with GFR. A significant difference on MgS levels between patients on tacrolimus and cyclosporine was found only when considering GFR <45 mL/min/1.73 m2, in which patients on tacrolimus had significantly higher GFR than patients on cyclosporine, which may explain these results.

Association of peripheral NK cell counts with Helios+ IFN-γ- Tregs in patients with good long-term renal allograft function.

Little is known about a possible interaction of natural killer (NK) cells with regulatory T cells (Treg ) in long-term stable kidney transplant recipients. Absolute counts of lymphocyte and Treg subsets were studied in whole blood samples of 136 long-term stable renal transplant recipients and 52 healthy controls using eight-colour fluorescence flow cytometry. Patients were 1946 ± 2201 days (153-10268 days) post-transplant and showed a serum creatinine of 1·7 ± 0·7 mg/dl. Renal transplant recipients investigated >1·5 years post-transplant showed higher total NK cell counts than recipients studied <1·5 years after transplantation (P = 0·006). High NK cells were associated with high glomerular filtration rate (P = 0·002) and low serum creatinine (P = 0·005). Interestingly, high NK cells were associated with high CD4+ CD25+ CD127- forkhead box protein 3 (FoxP3+ ) Treg that co-express the phenotype Helios+ interferon (IFN)-γ- and appear to have stable FoxP3 expression and originate from the thymus. Furthermore, high total NK cells were associated with Treg that co-express the phenotypes interleukin (IL)-10- transforming growth factor (TGF)-β+ (P = 0·013), CD183+ CD62L- (P = 0·003), CD183+ CD62+ (P = 0·001), CD183- CD62L+ (P = 0·002), CD252- CD152+ (P < 0·001), CD28+ human leucocyte antigen D-related (HLA-DR- ) (P = 0·002), CD28+ HLA-DR+ (P < 0·001), CD95+ CD178- (P < 0·001) and CD279- CD152+ (P < 0·001), suggesting that these activated Treg home in peripheral tissues and suppress effector cells via TGF-β and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). The higher numbers of NK and Treg cell counts in patients with long-term good allograft function and the statistical association of these two lymphocyte subsets with each other suggest a direct or indirect (via DC) interaction of these cell subpopulations that contributes to good long-term allograft acceptance. Moreover, we speculate that regulatory NK cells are formed late post-transplant that are able to inhibit graft-reactive effector cells.

Impact of seasonality on the dynamics of native Vitamin D repletion in long-term renal transplant patients.

Background: Renal transplant recipients (RTRs) are often Vitamin D (VitD) depleted as a result of both chronic kidney disease and mandated sun avoidance behaviours. Repleting VitD may be warranted, but how, and for how long, is unknown, as is the impact of seasonality on the success of repletion. We investigated the impact of seasonality on VitD status following VitD repletion in a large cohort of stable, long-term RTRs. Methods: Serum 25-hydroxyvitamin D [25(OH)D] concentrations and bone biochemistry parameters were analysed from 102 VitD repletion courses in 98 RTRs that had undergone VitD repletion. Repletion was delivered over 6 months with either 240 000 IU colecalciferol if pre-repletion serum VitD was between 20 and 50 nmol/L, or with 360 000 IU if VitD was <20 nmol/L. Twelve months post-repletion 25(OH)D and parathyroid hormone (PTH) were available for 75 patients. Results: At baseline, 25(OH)D was 20.1 ± 1.0 nmol/L, increasing to 65.4 ± 1.8 nmol/L following repletion (+7.55 nmol/L/month, P < 0.0001). Twelve months post-repletion and after no further VitD administration, 25(OH)D fell to 35.4 ± 1.8 nmol/L (14.2 ± 0.7 ng/mL; −2.50 nmol/L/month, P < 0.0001). PTH followed the opposite trend with baseline, repletion-end and post-repletion values being 144.2 ± 12.0, 109.6 ± 7.5 and 129.2 ± 11.4 ng/L, respectively. VitD repletion during the summer was associated with significantly higher at repletion-end 25(OH)D compared with any other time of year [summer 80.9 ± 4.0, autumn 64.1 ± 3.0 (P = 0.002), winter 48.9 ± 3.0 (P <0.001), spring 63.8 ± 2.5 nmol/L (P <0.001)]. There was no hypercalcaemia during repletion and renal transplant function remained stable without any evidence of allograft rejection. Conclusions: VitD repletion can safely and effectively be achieved in the majority of chronic stable RTRs using a 6-month bolus intermediate-dose schedule. Winter repletion is associated with an inadequate response in 25(OH)D; however, all patients experience a post-repletion fall towards deficiency in the absence of maintenance supplementation, irrespective of the season of repletion.

CD8+ Immunosenescence Predicts Post-Transplant Cutaneous Squamous Cell Carcinoma in High-Risk Patients.

Most morbidity associated with malignancy in long-term renal transplant recipients is due to cutaneous squamous cell carcinoma (SCC). Previously identified measures to stratify SCC risk have limited use, however. We hypothesized that an increased proportion of senescent, terminally differentiated CD8(+) T cells would identify renal transplant recipients at elevated SCC risk. Peripheral blood lymphocytes were isolated from 117 stable transplant recipients at high risk of SCC and analyzed phenotypically by flow cytometry. Participants were followed up prospectively for SCC development. The predictive value of variables was assessed using Cox regression. Age at transplant and enrollment, dialysis duration, and previous disease were predictive of SCC development during follow-up. Previously published clinical phenotype-based risk scores lost predictive value with the removal of age as a covariate. The percentage of CD57-expressing CD8(+) T cells was the strongest immunologic predictor of future SCC and correlated with increasing CD8(+) T cell differentiation. We dichotomized participants into those with a majority (CD57hi) and a minority (CD57lo) of CD8(+) T cells expressing CD57; CD57hi participants were more likely to develop SCC during follow-up (hazard ratio, 2.9; 95% confidence interval, 1.0 to 8.0), independent of potential confounders, and tended to develop earlier recurrence. The CD57hi phenotype was stable with time and associated with increasing age and cytomegalovirus seropositivity. Our results show that the CD57hi phenotype is a strong predictor of SCC development and recurrence in this cohort of long-term, high-risk renal transplant recipients. This information may allow identification of recipients who may benefit from intensive dermatologic screening and immunosuppression reduction.

Ibandronate in stable renal transplant recipients with low bone mineral density on long-term follow-up.

Bone mineral density (BMD) has been reported to increase without specific treatment in long-term renal transplant recipients. The aim of this study was to evaluate the effect of ibandronate on BMD and kidney function in long-term renal transplant recipients as compared to a control group. Furthermore, we searched for a gender-specific treatment effect of ibandronate on BMD. In a retrospective, matched case-control study 60 stable renal transplant recipients were included on long-term follow-up. The patient cohort was divided into two groups. The control group (n = 30) comprised patients with close-to-normal bone mineral density who did not receive ibandronate treatment and the treatment group (n = 30) comprised patients with reduced bone mineral density who received ibandronate treatment. The groups were matched for sex, age at the time of renal transplantation, use of steroids, renal transplant function and time lag between the dual-energy X-ray absorptiometry (DEXA) measurements and renal transplantation. Patients of the treatment group were treated with 12.0 ± 6.7 g ibandronate. Treatment cycles lasted 19.3 ± 11.0 months. The first bone mineral density testing was performed 55.3 ± 60.2 months after renal transplantation followed by a second measurement 26.8 ± 12.1 months later. Both groups did not differ in absolute (g/cm(2)) or relative (%) changes in BMD at the lumbar spine (0.033 ± 0.079 vs. 0.055 ± 0.066 g/cm(2), p = 0.217 and 3.6 ± 7.8 vs. 6.4 ± 8.1 %, p = 0.124) or femoral neck (0.013 ± 0.106 vs. 0.025 ± 0.077 g/cm(2), p = 0.647 and 3.2 ± 13.6 vs. 5.0 ± 13.1 %, p = 0.544) over the study period. There was no correlation of ibandronate dosages with changes in BMD (LS: r = -0.089; p = 0.639 and FN: r =+0.288; p = 0.445). We could neither determine a negative effect of ibandronate on renal transplant function over the study period, estimated via the CKD-EPI formula (-2.9 ± 7.6 vs. -2.7 ± 10.6 mL/min/1.73 m(2), p = 0.900) nor a gender-specific action of ibandronate on bone mass changes. Ibandronate treatment was safe with respect to renal transplant function but did not result in a significant additive improvement in bone mineral density as compared to the untreated control group. A gender-specific action of ibandronate on BMD at the LS or FN could not be determined either.

Nutritional Status in Japanese Renal Transplant Recipients With Long-term Graft Survival

BackgroundNutritional status affects clinical outcomes in patients with chronic renal failure. Glucose intolerance, dyslipidemia, obesity, hypertension, and a calcium–phosphorus–vitamin D imbalance are the major nutritional and metabolic problems that occur in posttransplant patients. In this study, we assessed the daily intake in long-term renal transplant recipients to determine whether they have sufficient nutrients based on the Japanese nutrition recommendations (recommended dietary allowances [RDA] in Japan 2010). Subjects and MethodsThirty-one renal allograft recipients followed for >10 years (median, 16.3) were recruited. The median serum creatinine level was 1.2 g/dL (95% CI, 0.6–3.4). We estimated the intake of nutrients, including protein and salt, using a simple food frequency questionnaire. ResultsThe median body mass index was 20.1 kg/m2. The median total energy intake was 1566 kcal/d (95% CI, 892–2556). The daily intake of protein and salt was 65.1 and 9.1 g/d, respectively. The calcium, iron, vitamin D, and vitamin K intakes were 423 mg, 7.0 mg/d, 9.7 μg/d, and 197 μg/d, respectively. Patients with dyslipidemia displayed greater amounts of lipid and calcium than those with normal lipid levels. DiscussionOur findings suggest that long-term renal transplant recipients in Japan seem to restrict caloric intake, while maintaining appropriate intake of protein, lipids, carbohydrates, and vitamins A, D, and K. However, daily calcium and iron intake were insufficient; salt intake was greater than the recommended dietary allowances in all subjects. In patients with dyslipidemia, calcium intake was lower than those in patients without dyslipidemia, although their intake of lipids was also lower than those without dyslipidemia. ConclusionNutritional guidance beginning during the early posttransplant phase helps to foster a healthy body mass index and nutritional balances for long-term renal transplant recipients. However, greater salt restriction was needed, and additional nutritional guidance aiming to prevent osteoporosis seems to be considered.

Skin Cancer and Immunosuppression Type in Long-Term Renal Transplant Recipients: A Case-Controlled Analysis.

Introduction: Renal transplant recipients are 3 times more likely to develop skin cancer (SC) when compared to age-matched general populations, and this is associated with increased morbidity and mortality. The reason underpinning the increased prevalence in this group most probably relates to treatment with immunosuppression (IS), however the association is not yet fully understood. Objectives: To determine the prevalence of SC in our long-term kidney transplant recipients. To assess whether SC prevalence is related to the type of IS taken by this patient cohort. Methods: We collected data from a retrospective cohort of all (n= 335) long-term (> 8 years) kidney recipients attending our annual review transplant clinic between 2010-2013. We documented the total number of SCs, time to development of first SC post transplantation and IS treatment. For the analysis of IS as risk factor, patients with SC were matched to an equal number of controls in age, total years from first transplant and skin type criteria. McNemar's test was used to test the effect of IS on risk of SC. Results: 67 (21%) patients had at least one SC. In these a total of 281 basal cell carcinomas, 157 squamous cell carcinomas, 4 melanomas and 1 Kaposi's sarcoma were diagnosed. The average time to development of a SC was 13 years (range 2-32 years) after transplant. There were no statistically significant differences between case and control patients in the proportion of patients taking any of the IS drugs one year before the first SC. However the difference in the proportion of patients who developed SC after taking azathioprine, compared to controls, is borderline significant.Table: No Caption available.Conclusion: We have identified a high prevalence of SC in our long-term kidney transplant patients. In our analysis we found that prevalence was not significantly related to the type of IS taken, although preliminary results point to azathioprine as a risk factor, and tacrolimus as protective. Further work to determine the relevance of cumulative IS dose is warranted.

The Impact of Interleukin 12B (1188A>C), Interleukin 16 (-295T>C), and Interleukin 18 (607C>A, 137G>C) Gene Polymorphisms on Long-Term Renal Transplant Function and Recipient Outcomes

BackgroundInflammatory mediators play an important role in kidney graft outcome. The cytokine and chemokine gene polymorphisms are associated with variable production, activity, expression, or ligand-receptor affinity. Genetic variation in the DNA sequence of the interleukin 12B (IL12B), interleukin 16 (IL16), and interleukin 18 (IL18) genes may lead to altered cytokine production and activity. These variations can lead to changes in individual patient outcomes after kidney transplantation. It is known that polymorphisms of interleukins have an influence on inflammatory diseases, eg, Crohn's disease, diabetes, and asthma. AimThe aim of this study was to evaluate the correlation between IL12B, IL16, and IL18 gene polymorphisms with delayed graft function (DGF), acute rejection episodes (AR), and chronic rejection episodes (CR). Materials and MethodsA total of 267 (38.6% women, 61.4% men) recipients were included in the study. Cadaveric kidney transplantations were performed at the Department of General Surgery and Transplantation. Polymerase chain reaction was used to determine gene polymorphisms of IL12B (rs3212227), IL16 (4778889), and IL18 (rs1946518, rs187238) in 2 mL of serum. Statistical significance (P < .05) was analyzed by logit regression, ANOVA and odds ratio (OR) of χ2 with Yates correction (95% confidence interval). ResultsRegression analysis revealed no significance between AR/DGF/CR and IL-2B, IL16, IL18rs1946518, and IL18-rs187238 (P > .05). The CR group, AA vs CC genotype of IL18 (rs1946518), had an OR = 2.35 (P = .04). AR and DGF groups had no significance in OR. ConclusionsThere was no statistical significance between IL12B, IL16, and IL18 (rs187238) gene polymorphisms and kidney graft outcome after transplantation. Presence of AA genotype (IL18–rs1946518) is connected with a 2.35 times higher risk of CR occurrence.

Short-term efficacy and safety of sitagliptin treatment in long-term stable renal recipients with new-onset diabetes after transplantation

New-onset diabetes after transplantation (NODAT) is a common complication after renal transplantation. There are limited available oral drugs to treat hyperglycaemia in this population owing to reduced renal function, potential interactions with immunosuppressive drugs and adverse effects such as hypoglycaemic events that may increase the cardiovascular risk. This study was initiated to investigate efficacy and safety of sitagliptin treatment that may represent a novel alternative in renal transplant recipients. Nineteen long-term stable renal transplant recipients with NODAT were included in a controlled, cross-over study and randomized to first receive either sitagliptin 50-100 mg/day or a sitagliptin-free period of 4 weeks. Median age (interquartile range, IQR) was 67 (62-72) years (12 males/7 females), all studied 1 (1-3) year after transplantation. The immunosuppressive regimen was a triple calcineurin inhibitor-based therapy. Oral glucose tolerance test (OGTT) with insulin and C-peptide responses and laser Doppler (LD) flowmetry assessment of endothelial function were performed at baseline and after each treatment period. Home measurements of plasma glucose were performed daily during the study. The median (IQR) first- and second-phase insulin secretion responses increased significantly by 56.3% (45.2-112.6%, P = 0.005) and 39.3% (26.5-81.0%, P = 0.006), respectively, following sitagliptin treatment as compared with no sitagliptin treatment. Fasting and 2-h plasma glucose concentrations fell significantly {0.9 mmol/L [0.5-1.7 mmol/L (16.2 mg/dL), P = 0.003] and 2.9 mmol/L [0.5-6.4 mmol/L (52.3 mg/dL), P = 0.004], respectively}, as did also home measurements of plasma glucose. Endothelial function and plasma markers of cardiovascular risk were unaffected. No serious adverse events were observed. Two mild and asymptomatic hypoglycaemic episodes were observed in combination with glipizide. Sitagliptin increases insulin secretion and reduces fasting and postprandial plasma glucose in renal transplant recipients with NODAT. The short-term treatment was well tolerated, and sitagliptin seems safe in this population.

The impact of CTLA4 and PTPN22 genes polymorphisms on long-term renal allograft function and transplant outcomes

Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) downregulates the immune system. Lymphoid tyrosine phosphatase (Lyp)—PTPN22 protein—is suggested to be negative regulator of T-cell reaction. There are several polymorphisms in the CTLA4 and PTPN22 genes, which can influence the immune response and allograft function after kidney transplantation. The aim of this study was to examine the impact of CTLA4 and PTPN22 genes polymorphisms on the long-term renal transplant function and recipients’ outcomes during a 5-year follow-up observation. The study enrolled 268 Caucasian renal transplant recipients. Genotyping of the rs231775 (+49AG) CTLA4 gene polymorphism was performed using real-time PCR and rs2476601 (C1858T) PTPN22 gene polymorphism using PCR-RFLP method. The 5-year graft survival rate was 81.7%. Dialysis was necessary in 22 (8%) patients, 7 (2.6%) patients died and 20 (7.4%) switched to another transplantation center. We found no association between studied polymorphisms and graft loss/dialysis. Comparison of the distribution of the +49AG CTLA4 and C1858T PTPN22 genes polymorphisms genotypes among dead and living patients showed no statistically significant differences. Above results suggest that the rs231775 (+49AG) CTLA4 and rs2476601 (C1858T) PTPN22 genes polymorphisms are not associated with long-term allograft failure, graft loss and mortality after transplantation.

The impact of ICAM1 and VCAM1 gene polymorphisms on long-term renal transplant function and recipient outcomes

Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are implicated in endothelial cell-leukocyte adhesion. Several functional polymorphisms that affect the expression of ICAM-1 and VCAM-1, and therefore post-transplant immunological response, have been found in genes of these molecules. The aim of this study was to examine the impact of ICAM1 and VCAM1 gene polymorphisms on long-term renal transplant function and recipient outcome during a 5-year follow-up period. The study enrolled 269 Caucasian renal transplant recipients (165 males, 104 females, mean age 47.58 ± 12.96 years) remaining under observation for 5 years. Genotyping of the c.1405A>G (Lys469Glu, rs5498) ICAM1, T(-1592)C (rs1041163), and T(-833)C (rs3170794) VCAM1 genes polymorphisms was performed using real-time PCR. The rs5498 ICAM1 GG genotype was an independent risk factor associated with increased creatinine concentration at 12, 24, 36, 48, and 60 months after transplantation (p=0.0004, p=0.02, p=0.01, p=0.01 and p=0.04, respectively). Creatinine concentrations 36 and 48 months after transplantation were higher among CC homozygotes of the rs1041163 VCAM1 (TT+CT vs. CC, p=0.049 and p=0.04, respectively). The C allele of the rs1041163 VCAM1 gene polymorphism was identified as a risk factor for dialysis after transplantation (HR=3.323, 95%CI=1.44-7.67, p=0.005). The C allele of the rs1041163 VCAM1 gene was at the border of statistical significance as a risk factor for death after transplantation (p=0.06). These results suggest that the rs5498 ICAM1 GG genotype is correlated with early and long-term allograft failure. The C allele of the rs1041163 VCAM1 gene may be associated with long-term allograft failure and graft loss, as well as increased morbidity after transplantation.

Higher Sensitivity of Peripheral Blood Lymphocytes to Endogenous Glucocorticoid in Renal Transplant Recipients Treated with Tacrolimus, as Compared to Those Treated with Cyclosporine

Background: Steroids in maintenance immunosuppressive drugs produce severe adverse events, and therefore, the use of steroids is withdrawn for many renal transplant recipients. However, steroids need to be restarted to prevent the worsening of renal function because of rejection episodes in some patients or in patients who experience steroid withdrawal syndrome. Currently, there are no indicators to predict which patients can sustain steroid withdrawal. Lymphocyte sensitivity to endogenous glucocorticoid cortisol could be a biological marker for safety reduction and steroid withdrawal. We examined relationship between lymphocyte sensitivity to cortisol in vitro and clinical outcome after steroid reduction or withdrawal in the renal transplant recipients. We also compared peripheral lymphocyte sensitivity to cortisol between transplant recipients treated with tacrolimus (Tac) and those treated with cyclosporine. Methods: The suppressive efficacies of cortisol against T-cell mitogen stimulated proliferation of peripheral lymphocytes was investigated in totally 41 patients with renal tramsplant patients, who were reducedor withdrawnsteroid. Nineteen out of the 41 patients were treated with Tac and the other 22 patients with cyclosporine A (CyA). Relationship between lymphocyte sensitivity to cortisol invitro and clinical outcome after steroid reduction or withdrawal was evaluated in 30 out of 41 renal transplant recipients. The lymphocyte sensitivity to cortisol was compared between 19 patients treated with Tac and 22 patients treated with CyA. Results: The cortisol IC50 values in the Tac and CyA groups were 0.09 ± 0.12 and 14.2 ± 12.7ng/ml, respectively. Lymphocyte sensitivity to cortisol in the Tac-treated group was significantly higher than that in the CyA treated group (p =0.0283). On the other hand, incidences of steroid withdrawal syndrome and increase in serum creatinine concentration were not significantly different between the Tac and CyA groups. Lymphocyte sensitivity to cortisol and its relationship with the clinical outcome after steroid reduction and withdrawal was investigated in 30 long stable renal transplant recipients. Serum creatinine levels increased in a significantly higher number of patients with lymphocytes hyposensitive to cortisol (IC50 ≥ 10000 ng/mL) than in normally sensitive patients (IC50 < 10000 ng/mL) (P < 0.005). The incidences of steroid withdrawal syndrome (P< 0.05) and necessity for increasing steroid dose or restarting steroid administration (P< 0.005) were also higher in the patients hyposensitive to cortisol than in normally sensitive patients. The patients in whom the lymphocyte proliferation rate was less than 60% did not show increase in S-Cr levels, experience steroid withdrawal symptoms, or require an increase in the steroid dose or restart of steroid administration. Conclusion: We considered from these observations that the lymphocyte sensitivity to cortisol would be useful for selecting patients for whom steroids should be withdrawn, and as a biomarker to safely reduce and withdraw steroids. Lymphocyte sensitivity to cortisol was higher in the Tac treated patients than that in the CyA treated ones. Since the cortisol sensitivity of peripheral lymphocytes is suggested to be a predictive marker for safe steroid withdrawal as described above, Tac administration shows promise in aiding successful withdrawal of steroid in long-term renal transplant recipients.

Interferon-Gamma Production to Non-Polymorphic HLA Class 2 Derived Peptides Correlates with Those to HLA Class 1 Derived Peptides in Long-Term Renal Transplant Patients

Introduction: In long-term renal transplant patients we have shown a response to non-polymorphic peptides derived from the HLA Class 1 α3-domain: cryptic self-epitopes (AJT 2007). These responses were more common in patients with evidence of deteriorating graft function suggesting that measures of immune function based on responses to these peptides may be used as a basis for an assay of T cell responses to HLA that can be standardized across the population. We hypothesized that long-term renal transplant recipients would also respond to non-polymorphic HLA class 2 derived peptides and these responses would provide an opportunity to refine an assay that could be tested prospectively. Methods: An ‘in silico’ approach was used to design potential peptide epitopes (13-19 amino acids long) derived from the distal β1 and β2 domain of HLA-DR (sequences with significant homology to other HLA class 2 molecules). This used an EpiMatrix algorithm for epitope prediction and calculated the grand average of hydropathicity index to exclude peptides that were extremely insoluble. In contrast to our approach with class 1 peptides we did not then test for binding of these peptides to class 2 molecules in vitro but used them directly to test PBMC responses in transplant recipients. Peptides were synthesized using Fmoc Chemistry (GL Biochem, Shanghai, China). Recruits were at least 18 months post transplantation, recruited as previously described; interferon ▪ ELISPOT was performed and positive responses defined as previously described. The responses to class 2 peptides were compared to those previously documented against single or multiple class 1 peptides (Transplantation 2011). Results: The mean values for responses to HLA class 1 and class 2 peptides in healthy controls was 2.86 ± 0.35 (n=23) and 2.30 ± 0.13 (n=19) respectively but for transplant patients was 5.54 ± 0.288 (p< 10-3) and 4.45 ± 0.19 (p=0.02) respectively. The response to HLA class 2 derived peptides correlated with responses to class 1 derived peptides, tested singly or as mixes (p< 10-4). The numbers discordant between the two groups however, preclude any meaningful interpretation of clinical correlates of differential responses.Table: [Reponses to HLA Class 1 & Class 2 Derived Peptides]Conclusion: These data demonstrate a significant correlation between responses to non-polymorphic HLA class 1 and class 2 peptides. They imply that indirect alloresponses most frequently arise to both class 1 and class 2 peptides, although it cannot be proved that these particular responses are pathogenic. They suggest that a limited peptide set is likely to be sufficient to identify patients who response to such peptides; the clinical significance of which requires testing in a prospective manner.

Increased Frequency of Pretransplant MLC-Induced CD4+CD25+Foxp3+IFNg+ Treg Associated with Poor Long-Term Graft Outcome

Recently, a new subset of induced regulatory T-cells (iTreg) was detected. IFNg-producing CD3+CD4+CD25+Foxp3+ PBL were shown to have an immunoregulatory role in long-term renal transplant recipients. This iTreg subset is more frequently detectable in the blood of patients with good than in the blood of patients with impaired long-term kidney graft function. In-vitro, separated CD3+CD4+CD25+Foxp3+IFNg+ PBL from primary MLCs of healthy controls suppressed secondary MLCs antigen-unspecifically. In the present study we tried to find indications of antigen-specific MLC suppression mediated by separated CD4+CD25+IFNg+ PBL. PBL of a healthy control (HC1) were stimulated in 6-day-MLC with irradiated PBL of 2 HLA class-I/II incompatible healthy controls (HC2, HC3). CD4+CD25+IFNg+ PBL were separated from primary MLC and added to secondary 6-day-MLC. Secondary MLC HC1 versus HC2 showed 9.3% blasts, secondary MLC HC1 versus HC2 co-cultured with separated CD4+CD25+IFNg+ PBL from primary MLC HC1 versus HC3 exhibited 5.0% blasts, whereas secondary MLC HC1 versus HC2 cocultured with separated CD4+CD25+IFNg+ PBL from primary MLC HC1 versus HC2 exhibited only 1.7% blasts. Similar findings were observed for secondary MLC HC1 versus HC3 co-cultured with corresponding CD4+CD25+IFNg+ PBL from primary MLC: 7.0% blast formation in primary MLC, 2.1% blasts in third-party, and 2.0% blasts in autologous secondary MLC. These data suggest that primary MLC-derived CD4+CD25+IFNg+ PBL inhibit the allogeneic immune response in secondary MLC unspecifically. However, the strongest suppression was observed in antigen-specific MLC/iTreg combinations, suggesting antigen-specific suppression by a subset of separated CD4+CD25+IFNg+ PBL. Next, we studied whether patients with good long-term graft outcome (n=3; current serum creatinine 0.9-1.3 mg/dl) might show a higher frequency of iTreg in MLC than patients with intermediate (n=3, current serum creatinine 1.8-2.7 mg/dl) or poor long-term graft function (n=2, current serum creatinine 11 and 14 mg/dl). All patients were studied >15 years posttransplant (range: 18 - 29 years). Recipients shared a maximum of 3 HLA class I/II mismatches with the original graft donor. PBL obtained prior to transplantation and stored in liquid nitrogen were used. Recipient PBL were stimulated with either donor or pooled third-party PBL for 6 days. Then, CD4+CD25+ PBL co-expressing Foxp3+IFN-g+, GARP+Foxp3+, or GARP+IFNg+ were determined using four-color fluorescence flow-cytometry. Cell proliferation was measured using CFSE-staining of responder cells. Patients with impaired long-term graft outcome showed a stronger anti-donor response than patients with good long-term graft outcome. They exhibited stronger cell proliferation in recipient/donor MLC and increased iTreg induction in recipient/donor as well as recipient/third-party MLC. Thus, strong iTreg and blast induction in pretransplant MLCs with donor cells was associated with impaired long-term graft outcome. We conclude that CD4+CD25+Foxp3+IFNg+ PBL represent a heterogenous population containing iTreg that suppress allogeneic T-cell responses and may be involved in inhibition of the specific posttransplant alloresponse. As published, CD4+CD25+Foxp3+IFNg+ iTreg express IFNg receptors, are induced by IFNg and represent the first line of iTreg during an initiating immune response. Therefore, increased MLC responses are associated with increased induction of IFNg-sensitive CD4+CD25+Foxp3+IFNg+ iTreg in-vitro, thus explaining our finding that high pretransplant iTreg frequencies in-vitro are associated with poor long-term graft outcome.

Decreased Numbers of Total CD19+ B Lymphocytes and the Presence of Monoclonal Proteins Are Frequent in Ultra-Long (30-43 Year) Renal Transplant Recipients: Implications for Allograft and Patient Survival

Introduction: The B-cell signature of tolerance in kidney transplant recipients on no immunosuppression includes an increase in total CD19+ B cells (mean 287 cells/μL) (Newell et al. J Clin Invest. 2010;120:1836). The question is whether long-term renal transplant recipients on minimal immunosuppression have similar findings. Methods: Lymphocyte subsets were analyzed by flow cytometry. Monoclonal proteins were identified by immunofixation. Results: Among 44 renal transplant recipients whose primary immunosuppression was minimal daily doses of prednisone (≤10 mg) and/or azathioprine (≤100 mg) and whose initial graft functioned for 30-43 years, there was the opposite finding of low total CD19+ B lymphocyte numbers (< 60 cells/μL) in 65.9% (29/44). Mean B cell counts were 52.0 ± 65.2 cells/μL for all 44 recipients. Low CD4+ or CD8+ T cell counts (< 490 and < 265 cells/μL, respectively) occurred in 13.6% (6/44) and 20.5% (9/44), respectively, of these recipients. CD8+CD38+ T cells were low (< 192 cells/μL) in 59.1% (26/44) of patients. Monoclonal proteins (MP) were identified in 45.5% (20/44) of these same recipients. The mean age of the patients with and without MP was similar at 53.4 and 53.6 years, respectively, as was the time post-transplant at 28.7 and 28.0 years, respectively. The donor sources for patients with and without MP were: LRD (14 and 19, respectively, including HLA-Id 9 and 5, respectively) and DD (5 and 6, respectively). There were 15 patients with a single monoclonal gammopathy (SMG), primarily IgG lambda (7), and 5 patients with a double monoclonal gammopathy (DMG) who also had varying degrees of free light chains. In the 26 recipients with good/excellent renal function (eGFR >45 ml/min) (Group1), 12 had SMG, 13 no MP, and 1 DMG. Conversely, in the 18 patients with fair/failed function (eGFR < 40ml/min in 8 or ESRD after 30 years in 10) (Group 2), 3 had transient SMG or free light chains only, 11 no MG, and 4 DMG) (p=0.047). No myeloma has developed with MP follow-up to 10 years. Allograft biopsies in 5 patients with MP did not show changes of light chain or monoclonal pathology; amyloid staining was negative. Although speculative, it is possible that in recipients receiving minimal immunosuppression usually including azathioprine for more than 30 years a SMP could have, or be a marker for, a protective renal allograft effect, represent an epiphenomenon for another event, or simply be a toxic effect of long-term azathioprine. Neither depressed B cell number nor MP was significantly associated with mortality. Conclusion: Ultra-long renal transplant function for 30-43 years with minimal immunosuppression is associated with uncharacterized total low CD19+ B cell numbers in nearly 2/3 of patients, opposite to what has been reported in B-cell tolerance in patients on no immunosuppression. Preservation of renal function was associated with a single monoclonal gammopathy in certain patients. These findings warrant further investigation.

Skin Cancer Prevention: Recent Evidence from Randomized Controlled Trials

Despite the billions of health care dollars spent each year on treating skin cancer, there is a dearth of randomized controlled trials (RCTs) that have evaluated skin cancer prevention. RCTs published in the last 3 years that have directly assessed skin cancer prevention as their primary aim suggest that regular use of sunscreen is cost effective, but prolonged use of topical therapies such as tretinoin and 5-fluorouracil may not be. Sirolimus-based immunosuppression for secondary skin cancer prevention in long-term renal transplant recipients appears effective, but benefits may be offset by the adverse effects. Many RCTs using pre-invasive actinic keratoses (AKs) as endpoints are too small and/or too short to provide evidence on skin cancer prevention. Another stumbling block is the difficulty in reproducibly diagnosing and counting AKs in response to preventive agents. Longer term and better surveillance methods are urgently required to improve the quality of evidence from future RCTs.

Higher Sensitivity of Peripheral Blood Lymphocytes to Endogenous Glucocorticoid in Renal Transplant Recipients Treated With Tacrolimus, as Compared to Those Treated With Cyclosporine.

Lymphocyte sensitivity to endogenous glucocorticoid cortisol could be a biological marker for safe reduction and withdrawal of steroids in renal transplant recipients. We compared peripheral lymphocyte sensitivity with cortisol between transplant recipients treated with tacrolimus (Tac) and those treated with cyclosporine. The suppressive efficacies of cortisol against T-cell mitogen-stimulated proliferation of peripheral lymphocytes were investigated in 44 renal transplant patients, who either had reduced or been withdrawn from steroid treatment. Twenty of the 44 patients were treated with Tac, and the other 24 patients were treated with cyclosporine A (CyA). The lymphocyte sensitivity to cortisol was compared between these two patient groups. The cortisol IC50 values in the Tac and CyA groups were 0.09 ± 0.12 and 14.2 ± 12.7 ng/ml, respectively. Lymphocyte sensitivity to cortisol in the Tac-treated group was significantly higher than that in the CyA-treated group (p = 0.0283). On the other hand, incidences of steroid withdrawal syndrome and increases in serum creatinine concentration were not significantly different between the Tac and CyA groups. Lymphocyte sensitivity to cortisol was higher in the Tac-treated patients than that in the CyA-treated ones. Since the cortisol sensitivity of peripheral lymphocytes is suggested to be a predictive marker for safe steroid withdrawal, Tac administration shows promise in aiding successful withdrawal of steroid treatment in long-term renal transplant recipients.

Management of Chronic Hepatitis B in Special Populations: Immunosuppressed Patients and Chronic Kidney Disease

Hepatitis B virus (HBV) infection remains an important cause of liver disease in the population with chronic kidney disease, including patients on long-term dialysis and renal transplant (RT) recipients. Diminished survival due to hepatitis B has been observed after RT. A thorough evaluation, including liver biopsy as well as assessment of serum markers of HBV replication (ie, hepatitis B e antigen and/or HBV DNA) is required before transplantation. Tolerance to interferon is poor both in dialysis patients and after renal transplant. Oral antiviral therapy now permits safe and potent antiviral treatment of HBV-related liver disease in chronic kidney disease patients with prevention of progressive liver disease. Preliminary evidence shows an improved survival of HBsAg positive renal allograft recipients on antiviral therapy. However, numerous issues concerning the treatment of hepatitis B in the population with chronic kidney disease remain unclear and further clinical trials are needed.