Characterizing Foxp3+ and Foxp3- T cells in the homeostatic state and after allo-activation: resting CD4+Foxp3+ Tregs have molecular characteristics of activated T cells.

Due to the intracellular expression of Foxp3 it is impossible to purify viable Foxp3+ cells on the basis of Foxp3 staining. Consequently CD4+Foxp3+ regulatory T cells (Tregs) in mice have mostly been characterized using CD4+CD25+ T cells or GFP-Foxp3 reporter T cells. However, these two populations cannot faithfully represent Tregs as the expression of CD25 and Foxp3 does not completely overlap and GFP+Foxp3+ reporter T cells have been reported to be functionally altered. The aim of this study was to characterize normal Tregs without separating Foxp3+ and Foxp3- cells for the expression of the main functional molecules and proliferation behaviors by flow cytometry and to examine their gene expression characteristics through differential gene expression. Our data showed that the expressions of Foxp3, CD25, CTLA-4 (both intracellular and cell surface) and PD-1 was mostly confined to CD4+ T cells and the expression of Foxp3 did not completely overlap with the expression of CD25, CTLA-4 or PD-1. Despite higher levels of expression of the T cell inhibitory molecules CTLA-4 and PD-1, Tregs maintained higher levels of Ki-67 expression in the homeostatic state and had greater proliferation in vivo after allo-activation than Tconv. Differential gene expression analysis revealed that resting Tregs exhibited immune activation markers characteristic of activated Tconv. This is consistent with the flow data that the T cell activation markers CD25, CTLA-4, PD-1, and Ki-67 were much more strongly expressed by Tregs than Tconv in the homeostatic state.

Renal transplant anastomotic time-Every minute counts!

The impact of anastomotic time in renal transplant is under recognized and not well studied. It is one of the few controllable factors that affect the incidence of delayed graft function (DGF). Our study aimed at quantifying the impact of anastomotic time. We performed a retrospective review of 424 renal transplants between the years 2006 and 2020. A total of 247 deceased donor renal transplants formed the study cohort. Patients were divided into two groups based on the presence or absence of DGF. Variables with p < 0.3 were analyzed using the binary logistic regression test. The final analysis showed anastomotic time to be significantly associated with DGF with odds ratio of 1.04 per minute corresponding to 4% increase in DGF incidence with every minute increment in anastomotic time. Other variables that had significant impact on DGF were DCD donor (odds ratio - 8.7) and donor terminal creatinine. We concluded that anastomotic time had significant impact on the development of DGF and hence should be minimized.

Assessment of Restored Kidney Transplantation Including the Use of Wider Criteria for Accepting Renal Donors After Cancer Excision.

The transplantation of kidneys after cancer excision (restored kidney transplantation, RKT) warrants further evaluation as a source of kidneys for transplantation. We determined whether larger cancers can be safely transplanted, the risks of adverse events from RKT, and whether RKT confers a survival advantage for patients waiting for transplantation. METHODS: In a retrospective cohort study, 23 dialysis patients awaiting transplant underwent RKT at John Hunter Hospital, Australia between 2008 and 2015. Patients were >60 years old and accepted onto the National Organ Matching Service. This RKT Group was divided into donor renal cancers ≤30 mm and >30-≤50 mm. Adverse event profiles for RKT recipients were compared with 22 standard live donor recipients using logistic regression analyses. Recipient and transplant survivals for RKT were compared with 2050 controls from Australian New Zealand Dialysis Transplant Registry using Cox regression models. To increase statistical power for survival analyses, data from 25 RKT recipients from Princess Alexandra Hospital, Brisbane were added, thus creating 48 RKT recipients. RESULTS: There were no significant differences in mortality, transplant failure nor AEs between the 2 cancer Groups. RKT increased the risks of Adverse event profiles (odds ratio: 6.48 [2.92-15.44]; P < 0.001). RKT reduced mortality risk by 30% (hazard ratio [HR]: 0.70 [0.36-1.07]; P = 0.299) compared with those continuing on the transplant list who may or may not be transplanted. RKT significantly reduced mortality risk for those remaining on dialysis (HR: 2.86 [1.43-5.72]; P = 0.003). Transplant survival for RKT was reduced compared with control deceased donor (HR: 0.42 [0.21-0.83]; P = 0.013) and live donor transplants (HR: 0.33 [0.02-0.86]; P =0.023). CONCLUSIONS: The use of larger carefully selected cancer-resected kidneys for transplantation appears safe and effective. RKT confers a possible survival advantage compared with waiting for transplantation, an increased survival compared with those remaining on dialysis but reduced transplant survival.

Functional significance and risk factors for lymphocele formation after renal transplantation.

BACKGROUND: Lymphocele development following renal transplantation is a significant adverse event. It may cause acute graft dysfunction or venous obstruction. There are no consistent risk factors reported in literature. Perioperative fluid balance may lead to increased lymphocele formation and has never been studied. We aimed to analyse incidence and risk factors for lymphocele formation. We hypothesized that overhydration in perioperative period is a risk factor. METHODS: We analysed 250 consecutive renal transplant recipients from 2006 to 2014. All recipients had undergone protocol screening by computerized tomography and ultrasound scan at 3 months post-transplant. We analysed risk factors for lymphocele formation. Comparisons between lymphocele and no-lymphocele groups were made with binary logistic regression analyses. Renal function was compared between treated, untreated and no-lymphocele groups with linear regression analyses. RESULTS: Thirty-one of 250 (12.4%) transplant recipients developed lymphocele. Fourteen of 31 (45.4%) recipients required intervention due to symptoms (venous obstruction being the most common). Surgical drainage was done in all symptomatic patients (11 laparoscopic and three open). Two of 11 (18%) recipients had recurrence after laparoscopic drainage. There were no significant differences in risk factors between the lymphocele and no-lymphocele groups. Renal function was comparable between no-lymphocele and treated lymphocele groups. Untreated lymphocele group trended towards better graft function at 1 year (P = 0.051). CONCLUSION: Post-transplant lymphocele developed one in eight transplant recipients and tended to occur in those with good renal function. Around half of the recipients with lymphocele required intervention with good recovery of long-term renal function. No risk factor for lymphocele development was established.

Anti-PD-1-induced high-grade hepatitis associated with corticosteroid-resistant T cells: a case report.

Effective treatment or prevention of immune side effects associated with checkpoint inhibitor therapy of cancer is an important goal in this new era of immunotherapy. Hepatitis due to immunotherapy with antibodies against PD-1 is uncommon and generally of low severity. We present an unusually severe case arising in a melanoma patient after more than 6 months uncomplicated treatment with anti-PD-1 in an adjuvant setting. The hepatitis rapidly developed resistance to high-dose steroids, requiring anti-thymocyte globulin (ATG) to achieve control. Mass cytometry allowed comprehensive phenotyping of circulating lymphocytes and revealed that CD4+ T cells were profoundly depleted by ATG, while CD8+ T cells, B cells, NK cells and monocytes were relatively spared. Multiple abnormalities in CD4+ T cell phenotype were stably present in the patient before disease onset. These included a population of CCR4-CCR6- effector/memory CD4+ T cells expressing intermediate levels of the Th1-related chemokine receptor CXCR3 and abnormally high multi-drug resistance type 1 transporter (MDR1) activity as assessed by a rhodamine 123 excretion assay. Expression of MDR1 has been implicated in steroid resistance and may have contributed to the severity and lack of a sustained steroid response in this patient. The number of CD4+ rhodamine 123-excreting cells was reduced > 3.5-fold after steroid and ATG treatment. This case illustrates the need to consider this form of steroid resistance in patients failing treatment with corticosteroids. It also highlights the need for both better identification of patients at risk and the development of treatments that involve more specific immune suppression.

Interaction between castanospermine an immunosuppressant and cyclosporin A in rat cardiac transplantation.

AIM: To investigate the interaction between castanospermine and cyclosporin A (CsA) and to provide an explanation for it. METHODS: The alkaloid castanospermine was prepared from the seeds of Castanospermum austral consistently achieving purity. Rat heterotopic cardiac transplantation and mixed lymphocyte reactivity were done using genetically inbred strains of PVG (donor) and DA (recipient). For the mixed lymphocyte reaction stimulator cells were irradiated with 3000 rads using a linear accelerator. Cyclosporin A was administered by gavage and venous blood collected 2 h later (C2). The blood levels of CsA (Neoral) were measured by immunoassay which consisted of a homogeneous enzyme assay (EMIT) on Cobas Mira. Statistical analyses of interactions were done by an accelerated failure time model with Weibull distribution for allograft survival and logistic regression for the mixed lymphocyte reactivity. RESULTS: Castanospermine prolonged transplant survival times as a function of dose even at relatively low doses. Cyclosporin A also prolonged transplant survival times as a function of dose particularly at doses above 2 mg/kg. There were synergistic interactions between castanospermine and CsA in the prolongation of cardiac allograft survival for dose ranges of CsA by castanospermine of (0 to 2) mg/kg by (0 to 200) mg/kg (HR = 0.986; 95%CI: 0.981-0.992; P < 0.001) and (0 to 3) mg/kg by (0 to 100) mg/kg (HR = 0.986; 95%CI: 0.981-0.992; P < 0.001) respectively. The addition of castanospermine did not significantly increase the levels of cyclosporin A on day 3 or day 6 for all doses of CsA. On the contrary, cessation of castanospermine in the presence of CsA at 2 mg/kg significantly increased the CsA level (P = 0.002). Castanospermine inhibited mixed lymphocyte reactivity in a dose dependent manner but without synergistic interaction. CONCLUSION: There is synergistic interaction between castanospermine and CsA in rat cardiac transplantation. Neither the mixed lymphocyte reaction nor the metabolism of CsA provides an explanation.

Effect of immunosuppression for primary renal disease on the risk of cancer in subsequent renal transplantation: a population-based retrospective cohort study.

BACKGROUND: To measure the risk of cancer in renal transplantation for recipients who had previously been treated with immunosuppressive agents for primary renal disease. METHODS: A retrospective population-based cohort study of 5970 renal transplant recipients in Australia registered on the Australia and New Zealand Dialysis and Transplant Registry between 1982 and 1997 and followed until 2007. Data about the incidence of a range of cancer types from this Registry were compared with cancer incidence data for the general population matched for cancer type, year of incidence, age, and gender derived from national cancer records. Outcome measures for each cancer group with or without pretransplantation immunosuppression were cancer-specific standardized incidence ratios and a multivariate hazard ratio (HR) standardized to 1. RESULTS: For those treated with pretransplantation immunosuppression, the risks for four cancer groups during renal transplantation were significantly increased: anogenital cancer (HR, 3.13; confidence interval [CI], 1.92-5.11; P<0.0001), non-Hodgkin's lymphoma (HR, 2.37; CI, 1.53-3.68; P=0.0001), breast cancer (HR, 2.52; CI, 1.13-5.61; P=0.024), and urinary tract cancer (excluding kidney) (HR, 1.84; CI, 1.13-3.01; P=0.015). However, the risks of cancer in the oral cavity and pharynx, kidney, thyroid, colon, leukemia, lung, melanoma, prostate, and stomach were not significantly increased. CONCLUSIONS: Pretransplantation immunosuppression for primary renal disease increases the risks of four cancer types in renal transplantation while sparing the others. Patients in whom this treatment is being considered should be informed of these risks.

The effects of Castanospermine, an oligosaccharide processing inhibitor, on mononuclear/endothelial cell binding and the expression of cell adhesion molecules.

INTRODUCTION: In this study we aimed to determine whether Castanospermine, a transplant immunosuppressive agent, impaired mononuclear/endothelial cell binding and expression of their cell adhesion molecules. METHODS: The binding of human umbilical vein endothelial cells with peripheral blood mononuclear cells was measured by a binding assay using Chromium 51 label; the membrane expression of cell adhesion molecules was measured by flow cytometry expressed as mean fluorescence intensity ratios. RESULTS: Castanospermine decreased mononuclear/endothelial cell binding if and only if both cell types were treated with Castanospermine: this impairment occurred if endothelial cells were treated with a range of doses of Castanospermine and mononuclear cells were treated with a constant dose of Castanospermine (p<0.001 versus untreated p=0.978) or vice versa (p=0.004 versus untreated p=0.582). Upon human umbilical vein endothelial cells Castanospermine reduced the mean fluorescence intensity ratios of E-selectin (p=0.003), ICAM-1 (p<0.001), ICAM-2 (p=0.004) and PECAM-1 (p<0.001) but increased it for P-selectin (p<0.001). Upon peripheral blood mononuclear cells Castanospermine reduced the mean fluorescence intensity ratios of L-selectin (P<0.001), LFA-1α (p<0.001), VLA-4 (p<0.001), Mac-1 (P<0.001) and CR4 (p<0.001) but increased the mean fluorescence intensity ratios of PSGL-1 (p<0.001) and PECAM-1 (p=0.001). Similar changes in mean fluorescence intensity ratios were found in the subset of lymphocytes and monocytes but the reductions in LFA-1α and VLA-4 on lymphocytes and Mac-1 and CR4 on monocytes were greater. CONCLUSIONS: The reduction in mononuclear/endothelial cell binding mediated by CAST and the reduction in expression of multiple cell adhesion molecules on these cell types help to explain the mechanism of its established immunosuppressive effect.

Assessment of the bioequivalence of a generic cyclosporine A by a randomized controlled trial in stable renal recipients.

BACKGROUND: The aim of this study was to determine the bioequivalence of Cysporin, a generic cyclosporine A, compared with Neoral in stable renal transplant recipients. METHODS: Study design consisted of an open label, two-way crossover, randomized controlled trial of Cysporin versus Neoral in stable renal transplant recipients. In all, 33 patients were enrolled; 31 were randomized and 28 were evaluable. AUCs(0-12) were done on day 14 and 28; C(0) and C(2) were done on days 0, 7, 21 and 35. Dose conversion was 1:1. Outcome measures for serum cyclosporin A concentrations expressed as the mean+/-SD were AUC(0-12) (microg x hr/L), C(max) (microg/L), C(2) (microg/L), T(max) (hr) and T(1/2) (hr). Mean and 90% CI of the ratio Cysporin/Neoral of log-transformed data were calculated using a general linear model. RESULTS: The main pharmacokinetic features were: AUC(0-12): Cysporin 3495+/-1319, Neoral 3853+/-1378 (P<0.05); C(max): Cysporin 755+/-301, Neoral 881+/-368 (P<0.05); C(2): Cysporin 613+/-235, Neoral 672+/-255 (P>0.05); T(max): Cysporin 1.9+/-0.8, Neoral 1.4+/-0.6 (P<0.005); and T1/2: Cysporin 8.8+/-4.3, Neoral 8.7+/-6.2 (P>0.05). Estimated ratios of Cysporin/Neoral were: AUC 0.93 (90% CI 0.88-0.98; P<0.05); C(max) 0.88 (90% CI 0.80-0.97; P<0.05); and T(max) 1.32 (90% CI 1.14-1.53; P<0.005). CONCLUSIONS: Both the extent and rate of absorption of Cysporin are significantly less than those of Neoral. The 90% CI for the ratios of Cysporin/Neoral for AUC and C(max) lie within 0.80-1.25. Hence in this clinical context Cysporin is pharmacologically bioequivalent with Neoral. This study illustrates the importance of testing bioequivalence of generic cyclosporine A products in transplant recipients not healthy volunteers.