Vascularized composite allotransplantation faces significant challenges, including limited preservation time and high rates of acute rejection because of the ischemia susceptibility of muscle and immunogenicity of skin. However, no studies have investigated whether normothermic machine perfusion (NMP) has an effect on acute rejection compared with static cold storage (SCS). Heterotopic hindlimb transplants were performed in a full-mismatch model after 6 h of SCS or NMP. Postoperative assessments included clinical scoring, histological examination, cytokine profiling, and flow cytometry of splenocytes and lymphocytes at the end of the study. Clinical assessment revealed significantly lower rejection scores in the NMP group on postoperative day (POD) 2 (P = 0.03). Notably, histology indicated delayed rejection, with Banff scores showing severe rejection from POD2 in the SCS group and from POD6 in the NMP group. Cytokine profiling demonstrated early upregulation of anti-inflammatory interleukin-10 in the NMP group at POD1, and flow cytometry showed a reduction in B cell populations. While NMP is not a cure-all, it is associated with a delayed onset of acute rejection compared with SCS in a full-mismatch rodent vascularized composite allotransplantation model. Future studies should focus on analysis earlier in the rejection process and on long-term graft survival to further elucidate the mechanism of the effect of NMP on the immune response.

Deceased-donor kidneys experience cellular stress before undergoing transplantation. To alleviate this, preservation techniques were developed, including normothermic machine perfusion (NMP). Here, we performed kidney NMP on discarded human kidneys for up to 24 h. Volume management was regulated either by urine recirculation (UR) or urine replacement (NUR) with Ringer's lactate. Notably, UR led to longer perfusion times compared with NUR. To investigate kidney NMP metabolic traits with or without UR over time, we performed longitudinal metabolomics analyses of perfusates of 8 NMP kidneys by 2-dimensional gas chromatography mass spectrometry (GCxGC-MS). Over 600 metabolic features were profiled, from which 74 were identified and 54 consistently quantified across 26 perfusate samples. Most notably, elevated levels of disaccharides (different isomers), hydroxy-purines, urea, glutamate, and amino acids are associated with the perfusion factor UR. Moreover, donor estimated glomerular filtration rate correlated significantly with the accumulation of lactate and gluconate. Most strikingly, lactate levels seemed to be more balanced in UR-NMP perfusate, which otherwise accumulated rapidly within the first 6 h. Kidney preservation by NMP was previously limited to hours. UR-NMP affected kidney energy homeostasis, carbohydrate and purine metabolism, and the urea and citric acid cycles. These insights add value to explain how urine-driven adaptations contribute to prolonged kidney function under NMP.

Donor-derived cell-free DNA (dd-cfDNA) has emerged as a promising noninvasive biomarker for detecting allograft rejection in solid organ transplantation. However, its diagnostic utility in pancreas transplantation, including in multiorgan recipients, remains underexplored. We conducted a retrospective analysis of 42 pancreas transplant recipients (38 simultaneous pancreas-kidney, 4 pancreas transplant alone) who underwent for-cause organ biopsy and dd-cfDNA testing between April 2020 and December 2024. dd-cfDNA was measured using the Prospera assay, with a threshold of ≥1% considered high risk and <1% low-risk. Biopsy results were correlated with dd-cfDNA to assess test performance. A total of 50 dd-cfDNA tests and 45 organ biopsies were analyzed. Among simultaneous pancreas-kidney recipients, dd-cfDNA demonstrated a sensitivity of 86.7%, specificity of 52.2%, positive predictive value (PPV) of 54.2%, and negative predictive value (NPV) of 85.7% for rejection. In pancreas transplant alone recipients, all high-risk dd-cfDNA results corresponded to biopsy-proven rejection, yielding 100% sensitivity, specificity, PPV, and NPV. When combining both cohorts, dd-cfDNA testing achieved an overall sensitivity of 88.2%, specificity of 57.7%, PPV of 57.7%, and NPV of 88.2%. Additionally, dd-cfDNA kinetics suggested its potential utility as a surveillance tool posttreatment, potentially reducing the need for closure biopsies. dd-cfDNA is a valuable noninvasive adjunct for monitoring of rejection in pancreas transplant recipients. Further prospective studies are needed to validate these findings and define the optimal role of dd-cfDNA in posttransplant care.

Using pigs as tissue donors may eliminate the shortage of replacement organs. As in allotransplantation, antidonor antibodies cause xenotransplant failure. Many antibodies target products of the major histocompatibility complex. Pigs contain a novel major histocompatibility complex gene, swine leukocyte antigen-12 (SLA-12), which has been proposed to behave as a classical class I SLA protein. Though predicted to bind B2m and short peptides, the functionality of the SLA-12 protein remains uncertain. In addition, its similarity to classical class I SLA molecules suggests that the SLA-12 could be a xenoantigen. Here we tested these predictions. Protein modeling was used to compare SLA-12 and classical class I proteins SLA-1, SLA-2, and SLA-3. cDNA encoding an isoform of SLA-12 was introduced into a cell line devoid of HLA proteins. B2m binding of SLA-12 at the surface of this cell line was evaluated. In addition, expression of SLA-12 molecules was examined in these cells after disrupting the genes encoding the transporters of antigenic peptides for class I proteins. Serum samples collected from transplant patients were also tested for the presence of SLA-12-specific IgG and IgM. Predictive algorithms indicate SLA-12 and classical class I SLA proteins have very similar structures. We observed strong biochemical similarities as well in that SLA-12 binds to B2m and peptides to become a stable cell surface protein. Finally, we observed that SLA-12-expressing cells bind more serum IgG and IgM from some transplant patients, indicating that it can be a xenoantigen. SLA-12 has marked structural and biochemical similarities with classical class I SLA proteins and may be another target of human anti-pig antibodies. Similar approaches may be successful in mitigating the antigenic issues of SLA-12, SLA-1, SLA-2, and SLA-3.

BK polyomavirus (BKPyV) is a common opportunistic infection in kidney transplant recipients, typically reactivating in the context of immunosuppression. Although asymptomatic in immunocompetent individuals, reactivation in transplant recipients can cause BKPyV-associated nephropathy (BKPyVAN), a leading cause of graft dysfunction and loss. BKPyV viremia affects approximately 10%-15% of transplant recipients, and once BKPyVAN is established, the risk of graft failure can exceed 50%. The current standard of care involves immunosuppression reduction to restore virus-specific immunity, but this approach increases the risk of acute and chronic rejection. Intravenous immunoglobulin (IVIg), which contains antiviral and immunomodulatory antibodies, has been proposed as an adjunctive therapy, although evidence supporting its efficacy remains limited to small observational studies. The BEAT-BK trial is a multicenter, adaptive, randomized controlled trial comparing standard immunosuppression reduction with or without IVIg in kidney and simultaneous pancreas-kidney transplant recipients with BKPyV viremia or biopsy-confirmed BKPyVAN. The trial will enroll participants aged 2 y and older, randomizing them 1:1 to receive standard care alone or in combination with a total IVIg dose of 2 g/kg for 8 wk. An adaptive Bayesian design will permit interim analyses with the potential for early trial success or futility. The primary endpoint is a composite ordinal outcome at 12 wk, incorporating death, graft loss, estimated glomerular filtration rate decline, acute rejection, viral load, and degree of immunosuppression reduction. Secondary outcomes include viral clearance, development of BKPyVAN and donor-specific antibodies, infections, malignancy, quality of life, and cost-effectiveness. This trial will provide the first high-quality evidence on the role of IVIg in BKPyV management and inform clinical decision-making in posttransplant care.

Background.Transplantation of isolated human islets is a valuable therapy for individuals with severe type 1 diabetes. A shortage of suitable pancreata and islets hinders access to islet transplantation (IT). Organ donation after circulatory death (DCD) islets are believed inferior and not appropriate for clinical IT. We readdressed this and compared outcomes between DCD and standard-of-care donation after brain death (DBD) islets.Methods.The data sample comprised 801 human islet isolations >2 decades from a single IT center. Islets from DCD and DBD donor organs were compared for islet outcome and quality. In some instances, a noninferior equivalency analysis was performed.Results.Across the study interval, islet yield, viability, and function remained high despite a deterioration in donor health biometrics. Noninferior analysis demonstrated that DCD islets were equivalent to DBD islets in islet yield post-culture and in murine diabetes reversion. In fact, in vivo islet function trended to more diabetes reversal in mice receiving DCD islets versus animals given DBD islets (75% versus 67%). As well, no significant differences were observed between groups in age, body mass index, pancreas cold ischemia time, digestion switch time, islet yield (success >250 000 islet equivalent), purity (>80%), recovery (>75%), post-culture viability, transplantable preparations, or insulin stimulation index. The median days of hospitalization and warm ischemia time were significantly greater in DCD versus the DBD group. Predictably, elevated donor hemoglobin A1c negatively impacted islet quality regardless of donation death status.Conclusions.Results from islet isolations >2 decades from a single center are presented. DCD islets were found to be equivalent to DBD islets on several relevant metrics. These findings encourage wider use of DCD donor islets for clinical IT.

There is a paucity of data to guide the management of posttransplant secondary antibody deficiency (SAD). We aimed to evaluate the role of IVIG in solid organ recipients with SAD. A phase 2, randomized, multicenter, open-label study of solid organ recipients with severe infection and SAD defined as serum IgG <600 mg/dL. The study evaluated the efficacy of adding a fixed protocol of IVIG early after detection of SAD to standard of care (SOC) for the prevention of infection occurring on-study and the treatment of infections, versus SOC. We also assessed reconstitution of humoral immunity in a substudy. Forty-four transplant recipients were randomized in 6 transplant centers in Spain. The IVIG protocol comprised 2 doses of 15 g (interval 7-15 d) followed by 3 doses of 20 g (interval 15-30 d). The primary endpoint was infection occurring on-study defined as a new severe infection after randomization. Forty-two patients were included in the intention-to-treat analysis (IVIG arm, n = 21 versus non-IVIG arm, n = 21). The rate of infection occurring on-study was lower in patients randomized to receive IVIG versus SOC (23.8% versus 66.7%, Fisher exact test, P = 0.012). The median number of infections occurring on-study was lower in the IVIG group. The median total number of new admissions considered to be related to an infection occurring on-study was significantly lower in IVIG-treated patients. A significant increase in specific anti-cytomegalovirus IgG, anti-varicella zoster IgG, anti-Clostridium difficile toxins A and B IgG, and anti-tetanus toxoid IgG and IgG1 was demonstrated in the IVIG arm. In solid organ recipients with severe infection and SAD, IVIG may reduce infection occurring on-study occurrence versus SOC.

Background.In circulatory death transplantation, time to death (TTD) following withdrawal of life-sustaining treatment is unpredictable. Concerns persist that prolonged TTD may cause ischemic injury, resulting in organ nonuse. We assessed the impact of donor TTD on liver transplant outcomes and utilization.Methods.We used Organ Procurement and Transplantation Network data on adult donors after circulatory death transplants (2010–2025). Multivariable nonlinear (restricted cubic splines) regression models were used to analyze associations. Simulation studies estimated potential increases in liver acceptance rates.Results.In 8489 recipients, short donor TTD was associated with inferior outcome, contrary to popular belief. Prolonged TTD did not show inferior posttransplant outcomes, irrespective of whether normothermic regional perfusion (NRP) was used. Nonlinear modeling (n = 37 447) revealed a sharp decline in utilization once TTD exceeded just 15 min. Given prolonged TTD did not impact outcomes, these declines represent a missed opportunity for organ use. Simulation studies revealed that if surgeons assess organ offers from patients with TTD of 15–30 min identically to those with TTD <10 min, there would have been a 17.1% (95% confidence interval, 15.0%-19.3%) relative increase in utilization, with potentially better outcomes than current practice. In the setting of NRP, short TTD was associated with increased organ nonuse, potentially because of failed viability criteria in damaged livers.Conclusions.Short TTD was associated with inferior posttransplant outcome, challenging the dogma that shorter is better. Prolonged TTD did not negatively impact posttransplant outcomes, irrespective of NRP use. These findings support expanded use of liver donors with prolonged TTD, especially in the era of NRP and advanced perfusion, where viability assessment provides an additional safeguard.

Background.We previously reported a porcine model of vascularized composite allograft (VCA) transplantation with concurrent hematopoietic cell transplantation (HCT). Tolerance developed when major histocompatibility complex (MHC) class I was matched in the absence of class II matching but not when class II was matched without class I matching. We investigated whether rejection resulted from a mismatch between class I and class II.Methods.Recipient pigs were either mismatched from donors by a single class I haplotype or matched by a single class II haplotype. They received CD3 immunotoxin and total-body irradiation followed by HCT and VCA transplantation from the same donor. Cyclosporine immunosuppression was discontinued after 45 d.Results.Pigs mismatched for only 1 MHC class I haplotype maintained peripheral chimerism and accepted the VCA graft, except for 1 animal that rejected the epidermis. Pigs matched for only 1 MHC class II haplotype lost bone marrow engraftment and rejected the VCA graft.Conclusions.When combined with our prior experiments, our results suggest that some degree of MHC class I matching is necessary for tolerance induction to HCT/VCA, but this is dependent on the degree of class II matching. Full class I matching reliably generates VCA tolerance, regardless of the degree of class II matching. VCA tolerance fails to develop reliably when MHC class I is fully mismatched, regardless of the degree of class II matching. When a single class I haplotype is matched, VCA tolerance depends on the degree of class II matching.

Background. Ischemia and reperfusion (IR) injury is a major complication in solid organ transplantation, necessitating new therapeutic strategies to suppress IR injury and thereby reduce early allograft dysfunction and delayed graft function. In kidney transplantation, IR injury is associated with disruption of mitochondrial homeostasis, including depletion of nicotinamide adenine dinucleotide (NAD + ) leading to diminished ATP levels, which cells need for survival. Here, we describe a novel approach to attenuate IR injury in a mouse kidney model using a NAD + -loaded nanoparticle, capable of direct intracellular delivery of NAD + . Methods. C57BL/6NCrl mice underwent a laparotomy and occlusion of the left renal pedicle for 30 min to induce IR injury. Right nephrectomy was performed during the injury incubation. Mice received the test agents by 2 different techniques: direct kidney injection via the renal artery and systemically via intravenous (IV) injection into the vena cava. Mice received a single dose of the NAD + -loaded nanoparticle at 3 different concentrations. Blood and kidney tissue were collected for analysis 24 h after IR injury. Measurement of blood creatinine levels and histological analysis was performed to assess the protective effectiveness of the NAD + -loaded nanoparticle. Results. A single dose of NAD + -loaded nanoparticles resulted in significantly reduced creatinine levels in both the direct kidney-injected animals ( P &lt; 0.0001) and the IV-injected animals ( P &lt; 0.01) compared with empty nanoparticle, free NAD + , or saline controls. IR-induced renal tubular injury scores were markedly reduced for both IV delivery ( P &lt; 0.01) and direct injection ( P &lt; 0.05) compared with control treatments. Conclusions. These results justify further development of this novel NAD + -loaded nanoparticle in the realm of organ transplantation to ameliorate IR injury and potentially increase usage of expanded criteria organs and reduce instances of delayed graft function.