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Background Malignancies in the urinary tract and the kidney graft are quite common after kidney transplantation. In some selected cases tumours develop from donor-derived tissue. Objectives We hypothesised that there is a clinical value to investigate donor/recipient origin in urologic malignancies in renal transplant recipients. Methods In this retrospective study, including patients transplanted between the years 1969 and 2014 at Uppsala University Hospital, Sweden, 11 patients with malignancies in urinary tract and 4 patients with malignancies in kidney transplants were investigated. Donor/recipient origin of tumour tissue was analysed by polymerase chain reaction (PCR) of human leucocyte antigen (HLA) genotypes or by fluorescence in situ hybridization (FISH analysis) of sex chromosomes. HLA genotype and sex chromosomes of the tumour were compared to the known HLA genotype and sex chromosomes of recipient and donor. Results Three of ten cancers in the urinary tract and three of four cancers in the kidney transplants were donor-derived. Conclusions We suggest that urologic malignancies in renal transplant recipients can be investigated for transplant origin. In addition to conventional therapy the allograft immune response against these tumours can be valuable to treat donor-derived cancers.

Background. Imlifidase is an immunoglobulin G (IgG)-specific protease conditionally approved in the EU for desensitization in highly sensitized crossmatch positive kidney transplant patients. Imlifidase efficiently cleaves both heavy chains of IgG in a 2-step process. However, low levels of the intermediate cleavage product, single-cleaved IgG (scIgG), may persist in the circulation. The study objective was to investigate Fc-mediated effector functions of scIgG and its potential impact on common clinical immunologic assays used to assess transplant eligibility. Methods. Imlifidase-generated scIgG, obtained by in vitro cleavage of HLA-sensitized patient serum or selected antibodies, was investigated in different complement- and Fc gamma R-dependent assays and models, including clinical tests used to evaluate HLA-specific antibodies. Results. ScIgG had significantly reduced Fc-mediated effector function compared with intact IgG, although some degree of activity in complement- and Fc gamma R-dependent models was still detectable. A preparation of concentrated scIgG generated from a highly HLA-sensitized individual gave rise to a positive signal in the anti-HLA IgG LABScreen, which uses anti-Fc detection, but was entirely negative in the C1qScreen. The same high-concentration HLA-binding scIgG preparation also generated positive complement-dependent cytotoxicity responses against 80%-100% of donor T and B cells, although follow-up titrations demonstrated a much lower intrinsic activity than for intact anti-HLA IgG. Conclusions. ScIgG has a significantly reduced capacity to mediate Fc-dependent effector functions. However, remaining HLA-reactive scIgG in plasma after imlifidase treatment can cause positive assay results equivalent to intact IgG in clinical assays. Therefore, complete IgG cleavage after imlifidase treatment is essential to allow correct decision-making in relation to transplant eligibility.

Imlifidase is a cysteine proteinase which specifically cleaves IgG, inhibiting Fc-mediated effector function within hours of administration. Imlifidase converts a positive crossmatch to a potential donor (T cell, B cell, or both), to negative, enabling transplantation to occur between previously HLA incompatible donor-recipient pairs. To date, 39 crossmatch positive patients received imlifidase prior to a kidney transplant in four single-arm, open-label, phase 2 studies. At 3 years, for patients who were AMR+ compared to AMR-, death-censored allograft survival was 93% vs 77%, patient survival was 85% vs 94%, and mean eGFR was 49 ml/min/1.73 m2 vs 61 ml/min/1.73 m2 , respectively. The incidence of AMR was 38% with most episodes occurring within the first month post-transplantation. Sub-analysis of patients deemed highly sensitized with cPRA ≥ 99.9%, and unlikely to be transplanted who received crossmatch-positive, deceased donor transplants had similar rates of patient survival, graft survival, and eGFR but a higher rate of AMR. These data demonstrate that outcomes and safety up to 3 years in recipients of imlifidase-enabled allografts is comparable to outcomes in other highly sensitized patients undergoing HLA-incompatible transplantation. Thus, imlifidase is a potent option to facilitate transplantation among patients who have a significant immunologic barrier to successful kidney transplantation. Clinical Trial: ClinicalTrials.gov (NCT02790437), EudraCT Number: 2016-002064-13.

Supplemental Digital Content is available in the text., Background. Highly HLA sensitized patients have limited access to life-saving kidney transplantation because of a paucity of immunologically suitable donors. Imlifidase is a cysteine protease that cleaves IgG leading to a rapid decrease in antibody level and inhibition of IgG-mediated injury. This study investigates the efficacy and safety of imlifidase in converting a positive crossmatch test to negative, allowing highly sensitized patients to be transplanted with a living or deceased donor kidney. Methods. This open-label, single-arm, phase 2 trial conducted at 5 transplant centers, evaluated the ability of imlifidase to create a negative crossmatch test within 24 h. Secondary endpoints included postimlifidase donor-specific antibody levels compared with predose levels, renal function, and pharmacokinetic/pharmacodynamic profiles. Safety endpoints included adverse events and immunogenicity profile. Results. Of the transplanted patients, 89.5% demonstrated conversion of baseline positive crossmatch to negative within 24 h after imlifidase treatment. Donor-specific antibodies most often rebounded 3–14 d postimlifidase dose, with substantial interpatient variability. Patient survival was 100% with graft survival of 88.9% at 6 mo. With this, 38.9% had early biopsy proven antibody–mediated rejection with onset 2–19 d posttransplantation. Serum IgG levels began to normalize after ~3–7 d posttransplantation. Antidrug antibody levels were consistent with previous studies. Seven adverse events in 6 patients were classified as possibly or probably related to treatment and were mild-moderate in severity. Conclusions. Imlifidase was well tolerated, converted positive crossmatches to negative, and enabled patients with a median calculated panel-reactive antibody of 99.83% to undergo kidney transplantation resulting in good kidney function and graft survival at 6 mo.

Safety, immunogenicity, pharmacokinetics, and efficacy of the IgG‐degrading enzyme of Streptococcus pyogenes (IdeS [imlifidase]) were assessed in a single‐center, open‐label ascending‐dose study in highly sensitized patients with chronic kidney disease. Eight patients with cytotoxic PRAs (median cytotoxic PRAs of 64%) at enrollment received 1 or 2 intravenous infusions of IdeS on consecutive days (0.12 mg/kg body weight ×2 [n = 3]; 0.25 mg/kg ×1 [n = 3], or 0.25 mg/kg ×2 [n = 2]). IgG degradation was observed in all subjects after IdeS treatment, with, In highly sensitized patients with chronic kidney disease, the immunoglobulin G–degrading enzyme of Streptococcus pyogenes (imlifidase) degrades plasma IgG efficiently, reduces HLA antibodies substantially, and abolishes C1q binding to anti‐HLA, thus enabling HLA‐incompatible kidney transplantation.

Background: Ischemia-reperfusion injury (IRI) is the most significant antigen-independent nonspecific insult, inevitably linked to organ transplantation. Endothelial glycocalyx is degraded during the earliest events in I/R. Previously we have been able to demonstrate the feasibility of coating the surface of the renal artery and renal tissue during hypothermic machine perfusion preservation (HMP) in a porcine model of brain death with a macromolecular heparin conjugate (CHC; Corline Systems AB, Uppsala, Sweden). The purpose of this study was to assess protective effects of coating the renal vessel walls with a heparin conjugate during hypothermic machine perfusion (HMP) against IRI and early kidney function.Methods: Brain death was achieved in male landrace pigs (n=6) by raising the intracranial pressure (ICP) through stepwise increasing the volume of an epidurally placed balloon to the point of exceeding the mean arterial pressure (MAP) creating a negative cerebral perfusion pressure (CPP). Both kidneys, (n = 6 in each group) were preserved for 18 h by hypothermic machine perfusion (HMP) in Lifeport® kidney transporters (Organ Recovery Systems, Chicago, IL, USA). In total 50 mg CHC was added to one of the HMP system (experimental group). An ex vivo normothermic perfusion (NP) system was developed with the purpose of evaluating kidneys function. Blood, urine and histological samples were collected during the subsequent 3 hours of NP, with an oxygenated autologous blood using cardiopulmonary bypass machine. The haemostatic dynamics of whole blood clotting and thrombin generation was evaluated by tromboelastography.Results: Serum creatinine was faster reduced in the experimental group compared to the control group (P=0.023). The total urine volume after 3h of warm perfusion was larger in the experimental group (239±71 mL) compared to the control group (172±42 mL) (Ρ=0.031). Histologically tubular changes were less frequent in the experimental group (P=0.045). No difference was seen between the groups regarding tromboelastography.Conclusions: Perfusion of porcine kidneys with CHC during HMP ameliorates early IRI and improves organ function close after reperfusion. No increased risk of bleed was seen with this treatment. This is a protective intervention strategy that potentially may improve the outcome of kidney transplantation in a clinical setting.

Ischemia reperfusion injury is one of the major complications responsible for delayed graft function in kidney transplantation. Applications to reduce reperfusion injury are essential due to the widespread use of kidneys from deceased organ donors where the risk for delayed graft function is especially prominent. We have recently shown that coating of inflamed or damaged endothelial cells with a unique heparin conjugate reduces thrombosis and leukocyte recruitment. In this study we evaluated the binding capacity of the heparin conjugate to cultured human endothelial cells, to kidneys from brain-dead porcine donors, and to murine kidneys during static cold storage. The heparin conjugate was able to stably bind cultured endothelial cells with high avidity, and to the renal vasculature of explanted kidneys from pigs and mice. Treatment of murine kidneys prior to transplantation reduced platelet deposition and leukocyte infiltration 24 hours post-transplantation, and significantly improved graft function. The present study thus shows the benefits of enhanced protection of the renal vasculature during cold storage, whereby increasing the antithrombotic and anti-adhesive properties of the vascular endothelium yields improved renal function early after transplantation.

BACKGROUND Donor-specific antibodies create an immunologic barrier to transplantation. Current therapies to modify donor-specific antibodies are limited and ineffective in the most highly HLA-sensitized patients. The IgG-degrading enzyme derived from Streptococcus pyogenes (IdeS), an endopeptidase, cleaves human IgG into F(ab′)2 and Fc fragments inhibiting complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity, which suggests that IdeS might be useful for desensitization. We report on the combined experience of two independently performed open-label, phase 1–2 trials (conducted in Sweden and the United States) that assessed the efficacy of IdeS with regard to desensitization and transplantation of a kidney from an HLA-incompatible donor. METHODS We administered IdeS to 25 highly HLA-sensitized patients (11 patients in Uppsala or Stockholm, Sweden, and 14 in Los Angeles) before the transplantation of a kidney from an HLA-incompatible donor. Frequent monitoring for adverse events, outcomes, donor-specific antibodies, and renal function was performed, as were renal biopsies. Immunosuppression after transplantation consisted of tacrolimus, mycophenolate mofetil, and glucocorticoids. Patients in the U.S. study also received intravenous immune globulin and rituximab after transplantation to prevent antibody rebound. RESULTS Recipients in the U.S. study had a significantly longer cold ischemia time (the time elapsed between procurement of the organ and transplantation), a significantly higher rate of delayed graft function, and significantly higher levels of class I donor-specific antibodies than those in the Swedish study. A total of 38 serious adverse events occurred in 15 patients (5 events were adjudicated as being possibly related to IdeS). At transplantation, total IgG and HLA antibodies were eliminated. A total of 24 of 25 patients had perfusion of allografts after transplantation. Antibody-mediated rejection occurred in 10 patients (7 patients in the U.S. study and 3 in the Swedish study) at 2 weeks to 5 months after transplantation; all these patients had a response to treatment. One graft loss, mediated by non-HLA IgM and IgA antibodies, occurred. CONCLUSIONS IdeS reduced or eliminated donor-specific antibodies and permitted HLA-incompatible transplantation in 24 of 25 patients.

Summary Adoptive transfer of regulatory T cells (Tregs) has been proposed for use as a cellular therapy to induce transplantation tolerance. Preclinical data are encouraging, and clinical trials with Treg therapy are anticipated. In this study, we investigate different strategies for the isolation and expansion of CD4+CD25highCD127low Tregs from uraemic patients. We use allogeneic dendritic cells (DCs) as feeder cells for the expansion and compare Treg preparations isolated by either fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS) that have been expanded subsequently with either mature or tolerogenic DCs. Expanded Treg preparations have been characterized by their purity, cytokine production and in-vitro suppressive ability. The results show that Treg preparations can be isolated from uraemic patients by both FACS and MACS. Also, the type of feeder cells used in the expansion affects both the purity and the functional properties of the Treg preparations. In particular, FACS-sorted Treg preparations expanded with mature DCs secrete more interleukin (IL)-10 and granzyme B than FACS-sorted Treg preparations expanded with tolerogenic DCs. This is a direct comparison between different isolation techniques and expansion protocols with Tregs from uraemic patients that may guide future efforts to produce clinical-grade Tregs for use in kidney transplantation.

Post-transplant malignancies cause significant morbidity and mortality. In this thesis we investigated malignancies in renal transplanted patients from a clinical viewpoint. The use of regional tumour registries considerably improved identification of pre- and post-transplant malignancies, which are generally underreported in transplant registries.Despite previously adequate cancer treatments with favourable prognosis, patients with pre-transplant malignancies showed higher incidence of post-transplant cancer and reduced survival compared to a 1:3 ratio matched control group of patients without a previous cancer from the Collaborative Transplant Study in Europe. A careful oncological surveillance pre-transplant and post-transplant is recommended.A multidisciplinary team evaluated the immunosuppressive and oncological treatment in a clinical prospective observational study of 120 renal transplanted patients with post-transplant malignancies. In two-thirds of the patients immunosuppression was possible to change to mTOR inhibitors with anti-tumour effects. Oncological treatment was adjusted in 50% of patients with solid or haematological tumours. MDT assessments are essential for optimizing treatment of post-transplant malignancies.Number of previous cutaneous squamous cell carcinoma (SCC) posed the most significant risk variable in predicting subsequent SCCs during a two-years study of 73 transplanted patients with at least one SCC.Incidence of transplant-derived tumours is 5 times higher than anticipated. Three of eleven cancers in urinary tract and two of four cancers in the transplants were transplant-derived. Five of eleven cancers of the urinary tract were BK-virus positive. Allograft immune response against these tumours offer new options for cancer treatment such as immunomodulatory or anti-viral treatment in combination with modified immunosuppression.

The immunosuppressive properties of regulatory T cells have emerged as an attractive tool for the development of immunotherapies in various disease contexts, e.g. to treat transplantation induced immune reactions. This paper focuses on the process of obtaining and functionally characterizing CD4+CD25+FoxP3+ regulatory T cells and Tr1 cells from uremic patients awaiting kidney transplantation.From October 2010 to March 2011 uremic patients awaiting living donor kidney transplantation, and their corresponding kidney donors, were enrolled in the study. A total of seven pairs were included. Isolation of CD4+CD25+FoxP3+ regulatory T cells was performed by magnetic activated cell sorting of peripheral blood mononuclear cells obtained from the uremic patients. Donor specific Tr1 cells were differentiated by repetitive stimulation of immature CD4+ T cells with immature dendritic cells, with the T cells coming from the future kidney recipients and the dendritic cells from the corresponding kidney donors. Cells were then expanded and functionally characterized by the one-way mixed leukocyte reaction and assessment of IL-10 production. Phenotypic analysis was performed by flow cytometry.The fraction of CD4+CD25+FoxP3+ regulatory T cells after expansion varied from 39.1 to 50.4% and the cells retained their ability to substantially suppress the mixed leukocyte reaction in all but one patient (3.8-19.2% of the baseline stimulated leukocyte activity, p0.05). Tr1 cells were successfully differentiated from all but one patient and produced high levels of IL-10 when stimulated with immature dendritic cells (1,275-11,038% of the baseline IL-10 secretion, p0.05).It is practically feasible to obtain and subsequently expand CD4+CD25+FoxP3+ regulatory T cells and Tr1 cells from uremic patients without loss of function as assessed by in vitro analyses. This forms a base for adoptive regulatory T cell therapy in the setting of living donor kidney transplantation.

Utilization of Small Pediatric Donors Including Infants for Pancreas and Kidney Transplantation : Exemplification of the Surgical Technique and the Surveillance

Background Solid organ transplant recipients are at increased risk of developing malignancies. The objective of this prospective, observational, one-armed study was to study the feasibility to add a mammalian target of rapamycin (mTOR) inhibitor to the immunosuppressive regimen in transplanted patients with post-transplant malignancies. During the trial the need to improve identification of post-transplant malignancies and to reassure adequate oncological treatment of these patients became evident. Multidisciplinary team (MDT) evaluation of oncological and immunosuppressive treatments was implemented for all patients with malignancies after renal or combined renal and pancreas transplantation because of the trial. Material and methods At Uppsala University Hospital, Sweden, a MDT consisting of transplant surgeons, nephrologists, oncologists and dermatologists evaluated 120 renal or combined renal and pancreas-transplanted recipients diagnosed with malignancies from September 2006 to July 2012. To identify all malignancies, the population was linked to the Regional Tumor Registry (RTR). We recorded to which extent a switch to mTOR inhibitors was possible and how often the originally planned oncological managements were adjusted. All patients were followed for three years. (ClinicalTrials.gov: NCT02241564). Results In 76 of 120 patients (63%) a switch to mTOR inhibitors was possible. Immunosuppression was interrupted in seven patients (6%), reduced in three patients (2%) and remained unchanged in 34 of 120 patients (28%). Identification of post-transplant malignancies increased significantly after linkage to RTR (p = 0.015). The initially recommended oncological treatment was adjusted in 23 of 44 patients (52%) with solid or hematological malignancies; 36 of these patients (82%) were treated according to national guidelines. Conclusion In two thirds of the patients the immunosuppressive treatment could be changed to an mTOR inhibitor with anti-tumor effects in transplanted patients with post-transplant malignancies. The use of regional tumor registers considerably improved the identification of patients with post-transplant malignancies indicating that post-transplant malignancies might be timely underreported in transplant registers.

Background: Prolonged cold ischemia is frequently associated with a greater risk of delayed graft function and enhanced graft failure. We hypothesized that media, combining a high oxygen-dissolving capacity with specific qualities of organ preservation solutions, would be more efficient in reducing immediate ischemia-reperfusion injury from organs stored long term compared with standard preservation media. Methods: Kidneys retrieved from brain-dead pigs were flushed using either cold histidine-tryptophan-ketoglutarate (HTK) or oxygen-precharged emulsion composed of 75% HTK and 25% perfluorohexyloctane. After 18 h of cold ischemia the kidneys were transplanted into allogeneic recipients and assessed for adenosine triphosphate content, morphology, and expression of genes related to hypoxia, environmental stress, inflammation, and apoptosis. Results: Compared with HTK-flushed kidneys, organs preserved using oxygen-precharged HTK-perfluorohexyloctane emulsion had increased elevated adenosine triphosphate content and a significantly lower gene expression of hypoxia inducible factor-1α, vascular endothelial growth factor, interleukin-1α, tumor necrosis factor-α, interferon-α, JNK-1, p38, cytochrome-c, Bax, caspase-8, and caspase-3 at all time points assessed. In contrast, the mRNA expression of Bcl-2 was significantly increased. Conclusions: The present study has demonstrated that in brain-dead pigs the perfusion of kidneys with oxygen-precharged HTK-perfluorohexyloctane emulsion results in significantly reduced inflammation, hypoxic injury, and apoptosis and cellular integrity and energy content are well maintained. Histologic examination revealed less tubular, vascular, and glomerular changes in the emulsion-perfused tissue compared with the HTK-perfused counterparts. The concept of perfusing organs with oxygen-precharged emulsion based on organ preservation media represents an efficient alternative for improved organ preservation. © 2012 Elsevier Inc. All rights reserved.

Morphine-based analgesia is effective but can compromise donor safety. We investigated whether continuous infusion of local anesthetics (CILA) can provide sufficient pain control and reduce morbidity related to opiate analgesics after hand-assisted retroperitoneoscopic (HARS) live donor nephrectomy. Forty consecutive live kidney donors underwent HARS and were treated with the ON-Q system providing CILA with 0.5% ropivacaine through two SilvaGard catheters placed in the retroperitoneal cavity and the rectus sheath, respectively. The case control group consisted of 40 donors matched with regard to sex, age, BMI and surgical technique. All donors were maintained on standardized multimodal analgesia combining nurse-controlled oxycodone treatment and acetaminophen. CILA donors had lower median cumulative consumption of morphine equivalents (CCME) (7 mg [0-56] vs. 42 mg [15-127]; p < 0.0000001), lower incidence of nausea (18 [45%] vs. 35 [87.5%] donors; p < 0.001), shorter time in postoperative care unit (160 vs. 242.5 min; p < 0.001) and shorter hospital stay (4 [4-7] vs. 6 [4-11] days; p < 0.001). In 32.5% of CILA donors the CCME was 0 mg (0% in matched control group, p < 0.001). CILA with 0.5% ropivacaine provides effective postoperative pain relief, reduces the need for opioid treatment and promotes postoperative recovery.