Shlokarth Balupuri

The trouble with kidneys derived from the non heart-beating donor: a single center 10-year experience.

BACKGROUND The demand for renal transplantation has increasingly outstripped the supply of donor organs especially over the past 10 years. Although related and unrelated live donation is being promoted as one option for increasing the donor pool, it is unlikely that this will in itself be able to bridge the gap. Non-heart beating donors (NHBD) can provide an alternative supply of organs, which should substantially increase the donor pool. METHODS In Newcastle, NHBD kidneys have been used for transplantation for a period of 10 years. In the early period (1988-1993) excellent results were obtained (90.5% success); however, these donors were controlled NHBD, Maastricht category III. In the second phase (1994-1998) increasing numbers of donors were obtained from the Accident and Emergency Department unit. These were failed resuscitation for cardiac arrest (category II). The rates of success in this period were poor (45.5% success) and the program was halted. The third phase of the program used machine perfusion of the kidneys and glutathione S transferase enzyme analysis to assess viability. RESULTS Using such approaches renal transplants from largely category II donors produced a success rate of 92.3% which was significantly better than the phase II period of the program (P=0.023, Fisher two-tail test). CONCLUSION Machine perfusion and viability assessment of NHB kidneys in phase III of the program has increased our donor pool as well as improved the graft survival. This is particularly relevant for the use of the category II NHB donor where the incidence of primary nonfunction was high, illustrated by phase II where machine perfusion/viability assessment was not used.

TGF-beta expression in renal transplant biopsies: a comparative study between cyclosporin-A and tacrolimus.

BACKGROUND Chronic rejection is a major cause of graft dysfunction after kidney transplantation. This fibroproliferative disease may be promoted by overproduction of transforming growth factor beta (TGF-beta). Previous studies have suggested that CsA might increase production of this growth factor. The current study was designed to measure the expression of TGF-beta(b) in renal transplant biopsy specimens from patients undergoing immunosuppressive therapy with either CsA or tacrolimus (FK506). METHOD Paraffin-embedded renal biopsy specimens were sectioned, dewaxed, and incubated with primary antibody against TGF-beta(b)1 latency-associated protein and active TGF-beta(b1). After washing, the sections were treated with secondary antibody conjugated with FITC. In each case, the sections were assessed by semi-quantitative scanning laser confocal microscopic method. RESULTS There was no significant difference in latent TGF-beta(b) expression between biopsy specimens from patients receiving CsA and patients receiving FK506. However, biopsy specimens from patients receiving CsA expressed significantly more active TGF-beta(b1) than biopsy specimens from patients receiving FK506 (P<0.0001, Mann-Whitney test). DISCUSSION The increased level of active TGF-beta1 expression in renal biopsy specimens of patients receiving CsA may indicate a mechanism of chronic rejection. However, these biopsies were performed to assess deranged renal function; therefore, the specimens may reflect events rather than differences in medication.

Assessment of non-heart-beating donor (NHBD) kidneys for viability on machine perfusion.

Abstract The shortage of organs has resulted in renewed interest in organs from non-heart-beating donors (NHBD). Viability assessment of such organs may reduce the incidence of delayed graft function and primary nonfunction. In Phase III of the NHBD programme, introduction of machine perfusion enabled the assessment of these marginal donors. Since then the graft survival has been 88.4% compared with the previous phase where machine perfusion or viability assessment was not done (45.5%). The parameters used were total glutathione S-transferase (GST) in the perfusate, the intrarenal vascular resistance (IRVR) and flow characteristics over time. Methods: All NHBD kidneys were machine perfused through a locally developed perfusion system. The viability was assessed by serial measurements of the above-mentioned parameters. Results: Forty-two local NHBD kidneys were retrieved and one kidney was imported, of which 19 donors (i.e. 38 kidneys) were of the uncontrolled (category II) donors. After viability assessment on machine perfusion; two kidneys were discarded due to positive tests for syphilis, four kidneys had high total GST levels, five kidneys due to high IRVR and poor flow characteristics and one did not flush on retrieval. Three kidneys were exported after viability tests. In 28 NHBD kidney recipients, immediate graft function was seen in two kidneys, 22 (84.6%) developed delayed graft function. One kidney had primary non-function, and two recipients lost their grafts, due to chronic rejection and renal vein thrombosis. There were two deaths, unrelated to transplantation. Graft survival was achieved in 88.4% (23/26 graft survival in phase III) of cases. Conclusion: Machine perfusion and assessment of NHBD kidneys has been successfully introduced to the Newcastle NHBD programme. This approach, using renal transplants from largely category II donors produced a success rate of 88.4% which was significantly better than the phase II period (45.5%) of the program p=0.023, Fisher 2 tail test).

Early results of a non-heartbeating donor (NHBD) programme with machine perfusion.

Abstract Freeman Hospital, Newcastle upon Tyne restarted their non‐heartbeating donor (NHBD) programme in September 1998 using machine perfusion, due to early poor results with conventional cold storage (45% graft survival, phase II). Since then, 15 NHBD kidneys have been transplanted. The retrieval protocol consisted of in situ perfusion with a double balloon triple lumen cannula in Maastricht category II male donors age range 13‐59 years. Mean primary warm ischaemic time was 24.8 min (range 10‐44). All kidneys were machine perfused through a locally developed perfusion system. The viability was assessed by serial measurements of total GST (maximum acceptable limit of 200 units/l) and intrarenal vascular resistance (IRVR) was recorded. Fifteen of the 22 kidneys (68.62%) were transplanted. Delayed graft function (DGF) was seen in ten recipients (66.6%), two kidneys had immediate function (IF), one organ was exported, two recipients died of unrelated causes and a further seven kidneys were discarded (two had high tGST, two were infected and three had poor flow characteristics). In phase III, a success rate of 91.7% was thus achieved, which was better than the phase II period (P = 0.027, Fisher 2‐tail test). Machine perfusion has been successfully introduced in phase III to the Newcastle NHBD programme and facilitates viability assessment of NHBD kidneys.

Granular cell tumor as an unusual cause of obstruction at the hepatic hilum: report of a case.

A diagnosis of malignancy is reasonably assumed when a lesion is found at the hilum or bile ducts in a patient with jaundice who has never undergone biliary surgery. Although benign tumors occasionally develop in this location, preoperative recognition is difficult and most are treated as malignant lesions. We illustrate this clinical scenario in this case report of a granular cell tumor (GCT) that developed at the biliary bifurcation, necessitating right hemi-hepatectomy with extrahepatic biliary tree excision. We describe the clinical presentation, imaging findings, treatment, and histological findings of this tumor. Although rare, a GCT can develop at the hilum and mimic a malignant lesion such as cholangiocarcinoma (CC) radiologically. To our knowledge, this is the fourth report of a GCT at the hilum of the liver. However, the possibility of this tumor should be considered in the differential diagnosis of a lesion in this location.

Comparison of proteolytic enzymes and glutathione S-transferase levels in non-heart-beating donors' (NHBD) kidney perfusates.

Abstract In order to identify biochemical markers of kidney damage prior to transplantation, we determined the levels of activity of a range of proteolytic enzymes in kidney perfusate samples from non-heart-beating donor (NHBD) cases. Urinary protease activities have been described as indices of kidney damage in renal disease; their potential as markers of tissue damage in kidneys before transplantation has not been assessed. In an attempt to identify additional/ improved biochemical markers, the present study compared the levels of total glutathione S-transferase (GST) with corresponding levels of several proteolytic enzymes in perfusate fluid from machine perfused NHBD kidneys. Proteases were selected to represent factors that may influence enzyme efflux, such as intracellular localization or molecular size. Methods: Perfusate samples were obtained over an 8-hour period from machine-preserved NHBD kidneys. Protease activities in these samples were determined by fluorometric assays and comparison made with total GST activity. Individual proteases were analysed in the transplanted and non-transplanted kidney groups (discarded on the basis of other viability parameters). Results: A correlation between protease activity and total GST was obtained for only leucyl- and pyroglutamyl aminopeptidase. Furthermore, in the transplanted group, it was possible to set nominal upper limits of activity for alanyl-arginyl- and dipeptidyl IV-aminopeptidase (AP). In the non-transplanted kidney group protease levels were increased above “normal” upper limits for the same enzyme types. By the use of alanyl AP it was possible to discriminate 75% of unsuitable kidneys discarded by the use of other criteria. Conclusion: The lack of correlation between total GST and protease activity for most of the enzymes investigated and alanyl AP levels in perfusate samples could be related to differences in cellular localisation, suggesting that assays of alanyl AP may give complimentary biochemical information relating to kidney tissue damage. Quantification of alanyl AP in machine perfusate samples may be a valuable additional independent biomarker of tissue damage.

En bloc paediatric renal donors into adult recipients: the Newcastle technique

Whilst debate still continues about the best use of kidneys from small donors, the techniques used have been varied because of the high vascular thromboses rates and ureteric leak rates. The method described here employs a vessel transposition as described by two German series, but it is combined with an extraperitoneal approach. It is now the method of choice in our unit for such en bloc transplants.

Expansion of donor pool: lack of function predictors.

Sir: The review article by Dafoe and Alfrey [l] focuses on the current debate over the use of marginal kidneys (especially older kidneys) for expansion of the donor pool. Whilst we applaud this first review on dual kidney transplants, it raises the issue of assessing kidneys prior to retrieval. Kidneys harvested from such marginal donors, if transplanted together, offer the potential of increasing the donor pool by using organs that would normally be discarded. However, if kidneys are incorrectly labelled as being marginal when, in fact, they have more potential, then the dual renal transplant program would reduce the donor pool. According to the authors, the identification of donor kidneys ideal for dual kidney transplantation is based on donor serum creatinine clearance. This approach relies totally on the use of serum creatinine as a marker of glomerular filtration rate (GFR). Transplant centers have evolved individual policies and scoring systems for dual kidney transplantation. In addition to including donor creatinine, they also consider donor age, percent of glomerulosclerosis, and kidney weight [Prof. N. Senninger, Munster, Germany, personal communication]. The daily variation in creatinine excretion, even in normal subjects, makes the measuring of endogenous creatinine clearance unreliable [3]. Even a 24-h urine collection for creatinine clearance is notoriously unreliable, as its reproducibility is rather low; therefore, this parameter of kidney function can only be used as a screening tool [3]. This is attributed to the unpredictable patterns of change in the rate of tubular transport of creatinine [5]. Repeated measurements of serum creatinine clearance have been shown to be unreliable, as the variation in serial creatinine clearance is high and potentially misleading [ 5 ] . The change in GFR suggests progression of disease whilst creatinine clearance does not necessarily follow this [6]. In addition, many donors with raised intracranial pressure have diabetes insipidus. This causes their creatinine to be abnormally low, affecting the creatinine clearance. The Cockfort and Gault formula used by the authors reduces the variability of serum creatinine estimates of glomerular filtration [7]. However, this formula does not take into account the differences in creatinine production between individuals of the same age and sex or the variation in an individual over time [2]. The use of this criterion for the selection of dual kidney donors will not be feasible for non-heart-beating donors (NHBD), who constitute a major group of marginal donors. This source has, in recent years, increased the donor pool, providing 40 % more kidneys in some centers, as reported by the Maastricht group [4]. Consequently, serum creatinine clearance can, at best, function as an indicator of donor GFR in stable, marginal donors who have not suffered from large fluid shifts. We feel that, at present, there is no reliable indicator of donor kidney function that can be used to accurately predict whether or not a dual or single graft is more appropriate. This issue needs further evaluation before the dual transplant program using marginal donors can be allowed to flourish freely.

Significance of the duration of delayed graft function in DCD kidney transplantation

1 A Novel siRNA-Containing Solution Protecting Donor Organs in Heart Transplantation. Xiufen Zheng,1 Dameng Lian,1 Mu Li,1 Xusheng Zhang,1 Mahdieh Khoshniat,3 Hongtao Sun,1 Weihua Liu,1 Jifu Jiang,1 Dong Chen,1 Costin Vladau,1 Motohiko Suzuki,1 James C. Lacefi eld,3 Bertha Carcia,1,2 Anthony M. Jevnikar,1,2,3 Wei-Ping Min.1,2,3 1Department of Surgery, Pathology, and Microbiology & Immunology, University of Western Ontario, London, ON, Canada; 2Multi-Organ Transplant Program, London Health Sciences Centre, London, ON, Canada; 3Robarts Research Institute, London, ON, Canada. Background: Ischemia-reperfusion (I/R) injury occurs in organ transplantation. We hypothesize that siRNA can effectively suppress infl ammatory, apoptosis and complement genes which contribute to I/R injury. This study was designed to develop a novel siRNA-containing preservation solution, which offers an alternative for protection of donor organs. Methods: Multiple siRNAs that specifi cally target TNFα, Fas, and C3 genes were prepared using our newly-developed bio-synthesis method. Heart grafts from BALB/c mice were preserved in UW solution (control) or siRNA-containing UW solution (siRNA solution) at 4°C for 48 hrs, and subsequently transplanted into syngeneic BALB/c mice. Cardiac functions were quantitatively assessed by High Frequency Ultrasound. The I/R injury was assessed by immunohistochemistry. Results: After 48 hrs preservation in the siRNA solution, the expressions of TNFα, Fas, and C3 genes in the grafts were inhibited at mRNA and protein levels detected by qPCR and immunoblot. Using siRNA solution preserved organ as donor, the graft survival was signifi cantly prolonged in heart transplantation. While siRNA solution-treated heart grafts retained strong heartbeat up to the end point of observation (>70 days), the control grafts lost function within 7 days. In addition, an improved cardiac function was observed in the graft preserved in siRNA solution. Grafts treated by siRNA solution displayed normal function, whereas grafts preserved in control solution showed dysfunction as detected by ultrasound scanning. The protection of graft by siRNA solution is associated with prevention of I/R injury. siRNA solution-treated organs exhibited almost normal histological structures, less neutrophil and lymphocyte infi ltration as compared with control solution-treated organs. Furthermore, siRNA solution prevented apoptosis and necrosis of cardiac tissues. Conclusions: This is the fi rst demonstration of a novel siRNA solution that can prevent cardiac I/R injury, protect cardiac function, and prolong graft survival in heart transplantation, implying a signifi cant and potential application in clinic. Abstract# 2 The Long-Term Evaluation of HLA-Mismatched Combined Kidney and Bone Marrow Transplant Recipients after Complete Withdrawal of Immunosuppression. Tatsuo Kawai,1 David H. Sachs,2 Thomas Spitzer,3 Nina Tolkoff-Rubin,1 Susan Saidman,4 Winfred Williams,1 Bimalangshu Dey,3 Nelson Goes,1 Dicken Ko,1 Waichi Wong,1 Robert B. Colvin,4 Megan Sykes,2 A. Benedict Cosimi.1 1Transplantation Unit, Massachusetts General Hospital, Boston, MA; 2Trasnplant Biology Research Center, MGH, Charlestown, MA; 3Bone Marrow Transplant Unit, MGH, Boston, MA; 4Pathology, MGH, Boston, MA; 5this Study was Performed in Partnership with Immune Tolerance Network. BACKGROUND: Induction of allograft tolerance has been the ultimate goal in organ transplantation. METHODS: Five subjects with ESRD received combined kidney and bone marrow transplantation (CKBMT) from HLA mismatched donors. The preparative regimen consisted of cyclophosphamide, thymic irradiation, anti-CD2 mAb and a 9-14 months course of a calcineurin inhibitor. Two subjects also received two doses of antiCD20 mAb prior to transplant. RESULTS: All recipients developed transient mixed chimerism without GVHD.Complete withdrawal of immunosuppressive medication was achieved in 4/5 recipients with current immunosuppression-free survival exceeding 3.2, 2 The Long-Term Evaluation of HLA-Mismatched Combined Kidney and Bone Marrow Transplant Recipients after Complete Withdrawal of Immunosuppression. Tatsuo Kawai,1 David H. Sachs,2 Thomas Spitzer,3 Nina Tolkoff-Rubin,1 Susan Saidman,4 Winfred Williams,1 Bimalangshu Dey,3 Nelson Goes,1 Dicken Ko,1 Waichi Wong,1 Robert B. Colvin,4 Megan Sykes,2 A. Benedict Cosimi.1 1Transplantation Unit, Massachusetts General Hospital, Boston, MA; 2Trasnplant Biology Research Center, MGH, Charlestown, MA; 3Bone Marrow Transplant Unit, MGH, Boston, MA; 4Pathology, MGH, Boston, MA; 5this Study was Performed in Partnership with Immune Tolerance Network. BACKGROUND: Induction of allograft tolerance has been the ultimate goal in organ transplantation. METHODS: Five subjects with ESRD received combined kidney and bone marrow transplantation (CKBMT) from HLA mismatched donors. The preparative regimen consisted of cyclophosphamide, thymic irradiation, anti-CD2 mAb and a 9-14 months course of a calcineurin inhibitor. Two subjects also received two doses of antiCD20 mAb prior to transplant. RESULTS: All recipients developed transient mixed chimerism without GVHD.Complete withdrawal of immunosuppressive medication was achieved in 4/5 recipients with current immunosuppression-free survival exceeding 3.2, 2.5, 1.1 and 0.2 years. A recipient rejected his kidney allograft on day 10. (1) The most recent creatinine/GFR of these 4 subjects were 1.2/61, 1.6/75, 1.6/96 (by renogram), 1.8/71 ml/min, respectively. (2)Although Subjects 2 and 5 initially developed anti-donor antibodies (ADA), both recipients lost ADA by 10th and 3rd month, respectively. Subject 4 developed post-transplant de novo anti-class II ADA one month after discontinuation of his immunosuppression, but without any functional derangements. (3) Subjects 1, 2 and 4 showed donor specifi c hyporesponsiveness in CML and MLR after nine months. (4) Allograft biopsies taken at 3 years from Subjects 1 and 2 revealed normal glomerulus, no tubulitis and no vasculopathy by light and electron microscopic examination with no C4d staining. Biopsy taken at 1.5 years from Subject 4 showed positive C4d deposition with no changes diagnostic of rejection. A most recent biopsy taken from Subject 5 on day 243 showed no rejection. (5) In Subject 1, extensive warts due to human polyoma virus existed before transplantation, but it disappeared after discontinuation of immunosuppression. All four patients show no hypertension, no DM, no abnormal lipid profi les. CONCLUSIONS: Renal allograft function of CKBMT recipients has been stable up to 3 years after complete withdrawal of their immunosuppression. Abstract# 3 A Novel Human Anti-CD40 Monoclonal Antibody, 4D11, for Kidney Transplantation in Cynomolgus Monkeys, Effect of Induction and Maintenance Therapy. Takeshi Aoyagi,1 Tomomi Suzuki,1 Atsushi Sugitani,2 Atsushi Imai,1 Motohiro Uno,1 Ryoichi Goto,1 Kenichiro Yamashita,1 Toru Miura,3 Nobuaki Takahashi,3 Tomoo Itoh,4 Hiroyuki Furukawa,1 Satoru Todo.1 11st Dept of Surgery, Hokkaido Univ., Sapporo, Hokkaido, Japan; 21st Dept of Surgery, Kyusyu Univ., Hakata, Fukuoka, Japan; 3Kirin Brewery Co., Ltd, Takasaki, Gunma, Japan; 4Dept of Surgical Pathology, Hokkaido Univ. Hospital, Sapporo, Hokkaido, Japan. Background/Aim: We have previously demonstrated that a novel human anti-CD40 monoclonal antibody (mAb), 4D11, markedly prolongs renal allograft survival in cynomolgus monkeys. In this study, we evaluated the effect of induction and maintenance therapy using 4D11. Methods: Twenty-four monkey renal transplants were divided into three groups: no treatment (Control), induction (Group A) and maintenance (Group B) treatments. Groups A and B were further subdivided according to the dose of 4D11 (n=3 each). The 4D11 was given intravenously on days 0, 4, 7, 11 and 14 for the induction therapy. In the group B, a half of the initial dose was given weekly thereafter until day 180. Graft survival, graft histology, anti-4D11 and anti-donor antibodies were examined. Results: Treatment Group 4D11 (mg/kg) Graft survival (days) Mean (days) Cause of death No treatment Control 5, 6, 7 6.0 AR, AR, AR Induction A-I 5 >96, 79, 75 83.3 Alive, AR, AR A-II 10 >200, 107, 92 133.0 Alive, CAN, AR A-III 20 169, 105, 90 121.3 AR, AR, AR Maintenance B-I 1 80, 79, 10 56.3 AR, AR, AR B-II 5 >202, 98, 44 114.3 Alive, AR, AR B-III 10 374, 253, 27 218.3 AR, AR, UTI B-IV 20 216, 169, 153 179.3 CAN, AR, CAN AR: Acute rejection, CAN: Chronic allograft nephropathy, UTI: Urinary tract infection No adverse events were observed. Graft survival was markedly prolonged by the induction treatment, which was further extended by the maintenance treatment. While graft biopsies revealed grade II or III rejection in the control, majority of those from groups A and B at 1 month post-transplantation showed only a borderline change. Anti 4D11-Ab was detected only in group A-III after drug cessation. In addition, animals receiving induction treatment and those given maintenance treatment with high doses of 4D11 did not develop anti-donor Ab unless the treatment was discontinued. Conclusion: 4D11 exhibited potent immunosuppressive effect on renal allograft survival both by induction and maintenance treatments. Besides, anti-drug and anti-donor antibody productions were effectively suppressed by 4D11. Abstract# 4 Pre-Transplant (Tx) Donor-Specifi c B Cells in Highly Sensitized Patients (HS), Detected by Intracellular Cytokine Flow Cytometry (CFC) Predict Antibody Mediated Rejection (AMR). Mieko Toyoda,1 Andy Pao,1 Ashley Vo,1 Marina Lukovsky,1 Raju Radha,1 Tetsu Sado,1 Lara Baden,1 Anna Petrosyan,1 Stanley C. Jordan.1 1Transplant Immunology Laboratory and Comprehensive Transplant Center, Cedars-Sinai Medical Center, Los Angeles, CA. Background: Intravenous immunoglobulin treatment (IVIG-Rx) reduces panel reactive antibodies and crossmatch (CMX) positivity, facilitating CMX(-) kidney Tx in HS. However, high rates of allograft rejec