Francesco L. Ierino

Thymic Transplantation in Miniature Swine. II. Induction of Tolerance by Transplantation of Composite Thymokidneys to Thymectomized Recipients

Previous studies in our laboratory have demonstrated that the presence of the thymus is essential for rapid and stable tolerance induction in allotransplant models. We now report an attempt to induce tolerance to kidney allografts by transplanting donor thymic grafts simultaneously with the kidney in thymectomized recipients. Recipients were thymectomized 3 wk before receiving an organ and/or tissues from a class I-mismatched donor. Recipients received 1) a kidney allograft alone, 2) a composite allogeneic thymokidney (kidney with vascularized autologous thymic tissue under its capsule), or 3) separate kidney and thymic grafts from the same donor. All recipients received a 12-day course of cyclosporine. Thymectomized animals receiving a kidney allograft alone or receiving separate thymic and kidney grafts had unstable renal function due to severe rejection with the persistence of anti-donor cytotoxic T cell reactivity. In contrast, recipients of composite thymokidney grafts had stable renal function with no evidence of rejection histologically and donor-specific unresponsiveness. By postoperative day 14, the thymic tissue in the thymokidney contained recipient-type dendritic cells. By postoperative day 60, recipient-type class I positive thymocytes appeared in the thymic medulla, indicating thymopoiesis. T cells were both recipient and donor MHC-restricted. These data demonstrate that the presence of vascularized-donor thymic tissue induces rapid and stable tolerance to class I-disparate kidney allografts in thymectomized recipients. To our knowledge, this is the first evidence of functional vascularized thymic grafts permitting transplantation tolerance to be induced in a large animal model.

Thymic transplantation in miniature swine. I. Development and function of the "thymokidney".

BACKGROUND Previous studies in our laboratory have demonstrated the importance of the thymus for rapid and stable tolerance induction in an allotransplant model. The focus of the present study was to explore the feasibility of autologous thymic transplantation to produce a new transplantable organ (thymokidney) and to examine the function of subsequent vascularized thymokidney transplants in T cell development. MATERIALS AND METHODS Eight juvenile swine received autologous thymic grafts under the renal capsule. Thymic tissue was obtained through a partial (n=6) or complete (n=2) thymectomy, and growth of the autologous thymic graft was compared between partially and completely thymectomized animals. Two of the partially thymectomized animals received irradiated (1000 cGy) as well as non-irradiated autologous thymic grafts. Graft survival, growth and evidence of thymocyte development was determined by (a) macroscopic examination of the implanted tissue, (b) histological examination, and (c) flow cytometry. Naive CD4 SP T cells were identified by CD45RA-expression. RESULTS Growth of transplanted thymic tissue was demonstrated in all thymic graft recipients. No difference was seen between partially and completely thymectomized animals. By POD 60, the thymic grafts exhibited normal macroscopic and microscopic structure, and normal thymocyte composition. Irradiated thymic tissue displayed a similar pattern of development, but growth was markedly delayed. To evaluate thymic function of the graft, a composite thymokidney was transplanted into a recipient which had previously been thymectomized, had few circulating CD4-single positive cells and had lost MLR reactivity. The number of CD4+/CD45RA+ cells in this animal increased steadily from POD 30 to POD 150, indicating that the thymus of the composite thymokidney allograft was functional; in addition, MLR assays demonstrated that the recipient recovered immunocompetence. CONCLUSIONS The establishment of a thymokidney by thymic autografting to the renal subcapsular space results in normal thymic growth and function, and may provide a valuable tool for studying the role of the thymus in tolerance induction. As far as we are aware, we provide the first evidence of functional vascularized thymic graft reconstituting T cells and leading to a return of a immunocompetence in a large animal model.

Secular trends in cardiovascular mortality rates of patients receiving dialysis compared with the general population.

BACKGROUND Cardiovascular mortality rates in the general population have decreased over time. We hypothesized that cardiovascular mortality rates in dialysis patients, which are higher than in the general population, have not decreased as much as those in the general population. STUDY DESIGN Comparison of registry data with population data. SETTING & PARTICIPANTS Data for prevalent Australian patients for whom dialysis was the first renal replacement therapy were obtained from the Australia and New Zealand Dialysis and Transplant (ANZDATA) Registry for 1992-2005. Data for a comparable Australian general population were obtained from the Australian Bureau of Statistics. OUTCOME Cardiovascular mortality rates per 100 person-years were calculated from ANZDATA Registry data, and age-specific relative risks were calculated relative to cardiovascular mortality rates in the general population. RESULTS Included in this cohort were 34,741 dialysis patients with 93,112 person-years of follow-up and 7,267 cardiovascular deaths. Cardiovascular mortality rates decreased over time in the general population and in dialysis patients older than 55 years. In patients aged 55-64 years, cardiovascular mortality rates were 9.0 (95% CI, 7.8-10.3) per 100 person-years in 1992-1994 and 6.4 (95% CI, 5.5-7.3) in 2004-2005; corresponding relative risks were 32.4 (95% CI, 28.2-37.2) and 52.0 (95% CI, 45.2-59.9), respectively. The corresponding cardiovascular mortality rates for dialysis patients aged 65-74 years were 11.6 (95% CI, 10.4-13.0) and 8.3 (95% CI, 7.4-9.3); relative risks were 12.9 (95% CI, 11.6-14.5) and 20.8 (95% CI, 18.7-23.2). Using negative binomial regression, the relative risk associated with dialysis compared with the general population increased over time (P for interaction = 0.001). LIMITATIONS Causes of death used to define cardiovascular mortality were not coded using identical systems in the ANZDATA Registry and the Australian population. CONCLUSIONS Despite decreasing cardiovascular mortality rates in some dialysis patients, the excess cardiovascular risk compared with the general population is increasing.

Approaching the promise of operational tolerance in clinical transplantation.

Long-term acceptance of transplanted organs without requirement for indefinite immunosuppression remains the ultimate goal of transplant clinicians and scientists. This clinical state of allograft acceptance termed “operational tolerance” has been elusive in routine practice. However, there are published reports of recipients where immunosuppression has been discontinued, by intention or patient noncompliance, in which the outcome is a nondestructive immune response and normal function. The question now arises how clinical operational tolerance might be achieved in the majority of recipients. This review provides an overview of current approaches to achieve operational tolerance, including the use of donor bone marrow and depletion of recipient T cells and the resistance of liver transplants to rejection. It also describes the key role of clinical immune monitoring and future approaches to tolerance induction including inhibition of T-cell signaling, manipulation of costimulatory pathways, and expansion of regulatory T cells. The principles of these experimental approaches may ultimately be extended to provide safe and effective control of transplant rejection and induction of clinical operational tolerance.

Adenovirus Tubulointerstitial Nephritis Presenting As a Renal Allograft Space Occupying Lesion

This report describes a case of adenovirus infection in a renal allograft 36 days after transplantation that presented with transient macroscopic hematuria, prominent systemic features and acute renal dysfunction. The patient had persistent high fevers despite broad antibiotic cover. A CT scan demonstrated a new discrete space occupying lesion in the allograft, which was devoid of blood flow on Doppler sonography. A targeted renal biopsy showed florid and focal necrotizing interstitial nephritis with intranuclear tubular viral inclusions. Treatment with ganciclovir and reduction in immunosuppression resulted in a rapid improvement. Immunohistochemistry and electron microscopy confirmed adenovirus infection. This case demonstrates an uncommon presentation of necrotizing adenoviral nephropathy, which should be considered in cases of renal allograft mass lesions.

Cystatin C for estimation of glomerular filtration rate in patients with spinal cord injury

Background: Serum creatinine is not a satisfactory marker of glomerular filtration rate (GFR) in patients with spinal cord injury (SCI) who have varying degrees of muscle atrophy. In contrast to serum creatinine, serum cystatin C, a 13-kDa protein, is not affected by muscle mass and is therefore potentially a useful marker of GFR in patients with SCI. In addition, cystatin C is not dependent on sex or age and is not secreted by the renal tubule. Aim: We assessed serum cystatin C as a surrogate marker of GFR in SCI patients. Methods: Cystatin C was analysed using a particle-enhanced immunonephelometric assay (Dade Behring) in serum samples sent for routine measurement of creatinine (64 patients) and creatinine clearance (27 patients) from patients in the Spinal Unit of the Austin Health. We compared these results with serum cystatin C of 57 non-SCI patients who had had a creatinine clearance measurement during the study period. Results: In patients with SCI, the reciprocal of cystatin C had a stronger correlation (r = 0·48, P<0·01) with creatinine clearance than the reciprocal of serum creatinine (r = 0·25, P<0·19). Further, the value of serum creatinine was much lower for a given creatinine clearance in SCI patients than in non-SCI patients; the serum cystatin C concentrations were equivalent. Conclusion: The serum cystatin C is a convenient and more reliable surrogate marker of GFR than serum creatinine and will enable early detection of renal impairment. We need to confirm this finding with a larger study, including comparison with an accepted gold standard for GFR.

Role of the thymus in transplantation tolerance in miniature swine: II. Effect of steroids and age on the induction of tolerance to class I mismatched renal allografts.

BACKGROUND Recent studies in young (5-7 months) miniature swine have demonstrated that the thymus is involved in the rapid induction of stable tolerance to class I mismatched renal allografts after a 12-day course of Cyclosporine (CyA). Because both steroids and age are known to influence the structure and function of the thymus, we have now studied the effects of these two parameters on tolerance induction in this model. MATERIALS AND METHODS In young swine, the administration of methylprednisolone (MP) during the standard tolerance-inducing regimen (a 12-day course of CyA) produced severe renal dysfunction and acute cellular rejection histologically. However, the renal allografts recovered and were accepted for >100 days with histological evidence of chronic rejection. To test the effect of age, two relatively old swine (55 and 71 months) received transplants of class I mismatched renal allografts and the standard 12-day course of CyA. One animal rejected the allograft acutely on postoperative day 22, and the second also rejected, but more slowly, with manifestations of chronic rejection. CONCLUSION These findings suggest that both MP and old age interfere with the induction of stable tolerance in a fashion similar to the previously described effect of thymectomy. These results may have important implications for the mechanism of thymic-dependent tolerance, for the use of steroids in clinical protocols for the induction of allograft tolerance, and for the application of such protocols to adult patients.

Role of the thymus in transplantation tolerance in miniature swine. III. Surgical manipulation of the thymus interferes with stable induction of tolerance to class I-mismatched renal allografts.

BACKGROUND Previous studies have demonstrated that long-term tolerance of class I mismatched renal allografts in miniature swine is induced by a short course of cyclosporine (CyA), and that a total thymectomy 21 days before transplantation abrogates the induction of stable tolerance. We have now examined the effects of surgical manipulation of the thymus, with or without a reduction in the thymic volume, on the induction of tolerance. MATERIALS AND METHODS Miniature swine receiving a transplant of a class I-mismatched renal allograft and 12 days of CyA underwent either (1) a partial thymectomy 21 days before kidney transplantation (day -21), (2) serial thymic biopsies (to evaluate the effect of surgical trauma and reduction in volume of the thymus) or serial incisions of the thymus thymus (to evaluate the effect of surgical trauma without changes in thymic volume), (3) a sham thymectomy on day -21, or serial sham thymic surgery on the same POD as the thymic biopsies and incisions (control animals). RESULTS Control animals had a stable plasma creatinine, had donor-specific unresponsiveness in cell-mediated lympholysis (CML) assays, had absence of rejection in kidney biopsy specimens, and did not develop anti-donor class I immunoglobulin (Ig)G alloantibodies. Animals undergoing a partial thymectomy on day -21 or serial thymic biopsies showed severe renal dysfunction, histological evidence of rejection in kidney biopsy specimens and anti-donor reactivity in CML assays; all but one animal developed anti-donor class I IgG alloantibodies. Serial incisions of the thymus induced an increase in plasma creatinine and histological rejection in 1 of 3 animals and anti-donor cytotoxic T cells in vitro in all 3 animals. CONCLUSIONS A partial thymectomy or serial thymic biopsies markedly interfere with the induction of tolerance to renal allografts. Serial thymic incisions also interfere with the induction of tolerance, but to a lesser degree. These studies may have implications for tolerance-inducing protocols that involve thymic manipulation.

Single and serial measurements of cardiac troponin I in asymptomatic patients on chronic hemodialysis.

AIMS Coronary artery disease is the major cause of death in patients with end-stage renal failure on dialysis. This study aimed to assess the predictive value of a single cardiac troponin I (cTnI), and also the kinetics of serial values. METHODS Since cTnI is a potential biomarker of cardiac outcome, the present study examined single cTnI measurements (n = 88 patients) and its predictive value for future cardiac events, and a kinetic substudy of serial weekly cTnI measured for 8 weeks (n = 57) in a group of patients on hemodialysis. RESULTS Single cTnI measurements: 9 patients (10.2%) had a detectable cTnI at baseline and 79 patients (89.8%) had a negative baseline cTnI. There were no significant differences in age, sex, history of ischemic heart disease, diabetes, smoking or dyslipidemia between patients with detectable and negative cTnI. At the end of 9 months, the rate of combined primary endpoints, which included myocardial infarction, cardiac death and cardiac revascularization, was significantly higher in the patients with a detectable baseline cTnI (55.6%), compared to patients with a negative cTnI (6.3%) (p = 0.0007). Serial weekly cTnI measurements: significant fluctuations in cTnI were noted over time; 27% of patients with an undetectable cTnI measured at baseline had subsequent detectable levels in the serial follow-up. CONCLUSION A single detectable cTnI in asymptomatic patients on hemodialysis defines patients at high risk of future cardiac events. However, the incidence of detectable cTnI levels is markedly increased when serial weekly measurements are performed. The clinical significance of detectable serial measurements of cTnI is the focus of ongoing studies.

B-type Natriuretic Peptides Strongly Predict Mortality in Patients Who Are Treated with Long-Term Dialysis

BACKGROUND AND OBJECTIVES Left ventricular abnormalities contribute to cardiovascular disease in patients with chronic kidney disease and may be detected by measurement of B-type natriuretic peptide in serum. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS In a prospective cohort study of predialysis patients, patients who were on dialysis, and kidney transplant recipients, serum was collected and assayed for both B-type natriuretic peptide and its N-terminal fragment. Median levels were compared using nonparametric tests, and predictors of B-type natriuretic peptide were determined by linear regression. Survival analysis and Cox regression were performed to examine the association of levels of B-type natriuretic peptide with cardiovascular events and death. RESULTS Levels of B-type natriuretic peptide were highest in patients who were on dialysis. Patients who were receiving dialysis and had known cardiovascular disease, were not on the waiting list for kidney transplantation, or had left ventricular systolic dysfunction on echocardiography had significantly higher levels of B-type natriuretic peptide than patients without these characteristics. Glomerular filtration rate was an important predictor of B-type natriuretic peptide levels for patients who were not on dialysis (predialysis and renal transplant recipients). Left ventricular systolic dysfunction predicted B-type natriuretic peptide levels in patients who were on dialysis. Both forms of B-type natriuretic peptide were associated with a two- to three-fold increased risk for death in patients who were on dialysis. CONCLUSIONS Levels of B-type natriuretic peptide are greatest in patients who are on dialysis and have cardiovascular comorbidities and are strong predictors of death.