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Granzyme B-producing B cells have been reportedly reported to be an important regulatory B cell subset in the pathogenesis of many diseases. However, its role in liver transplant recipients with acute rejection has never been well elucidated. Seventeen liver transplant recipients with acute rejection and 25 controls with stable liver function were enrolled in this study to determine the function of granzyme B-producing B cells via flow cytometry. Liver transplant recipients with acute rejection had higher expression of granzyme B in CD19 + B cells when compared with controls. Following rejection, the granzyme B production was even elevated although comparison failed to reach significance. The percentages of CD27 + granzyme B + CD19 + B cells and CD138 + granzyme B + CD19 + B cells were lower than those of CD27 - granzyme B + CD19 + B cells and CD138 - granzyme B + CD19 + B cells in patients with acute rejection, respectively. While the production of CD27 and CD138 was not different between liver transplant recipients with and without acute rejection but increased expression of the two surface markers was observed following rejection. Furthermore, the frequency of granzyme B + CD19 + B cells correlated with the level of alanine aminotransferase instead of tacrolimus. CD19 + B cells could produce granzyme B when stimulated with IgG + IgM and CpG in the presence of the supernatant of activated CD4 + CD25 - T cells. In return, granzyme B + CD19 + B cells could suppress the proliferation of CD4 + CD25 - T cells in a granzyme B- and contact-dependent manner. The increased expression of granzyme B in CD19 + B cells from liver transplant recipients with acute rejection might function as a feedback loop for the activation of the CD4 + CD25 - T cells., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Abstract: Acute immune rejection is one of the most serious complications of heart transplantation, and its mechanism has always been a hot spot. Th17 cells and cytokine interleukin-17 (IL-17) have been proved to be involved in acute immune rejection, and the signaling pathway mechanism has attracted our interest. It has been confirmed that the Janus kinase 2-signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway is involved in the differentiation of CD4+ T cells, so we focus on whether the JAK2/STAT3 signaling pathway is involved in the occurrence of acute immune rejection by regulating the Th17/IL-17 axis. In this study, we used Bagg's Albino c mice and C57BL/6 mice to construct heterotopic heart transplantation models, which were divided into the acute rejection group and AG490-treated group (n = 5), and donor tissue and serum were collected in 3 experimental days from the recipient mice for H&E staining analysis of paraffin sections and ELISA, Western blot, flow cytometry, and real time-polymerase chain reaction. The results showed that the acute rejection rating of the heart decreased, and the expression of related factors decreased significantly after using the inhibitor AG490, suggesting that the JAK2/STAT3 signaling pathway regulates expression of the Th17/IL-17 axis in cardiac allograft rejection., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Chronic allograft dysfunction is a major cause of late graft failure after kidney transplantation. One of the histological changes is interstitial fibrosis, which is associated with epithelial-mesenchymal transition. Bortezomib has been reported to prevent the progression of fibrosis in organs. We used rat renal transplantation model and human kidney 2 cell line treated with tumor necrosis factor-α (TNF-α) to examine their response to bortezomib. To explore the mechanism behind it, we assessed the previously studied TNF-α/protein kinase B (Akt)/Smad ubiquitin regulatory factor 2 (Smurf2) signaling and performed RNA sequencing. Our results suggested that bortezomib could attenuate the TNF-α-induced epithelial-mesenchymal transition and renal allograft interstitial fibrosis in vitro and in vivo. In addition to blocking Akt/mammalian target of rapamycin (mTOR)/p70S6 kinase/Smurf2 signaling, bortezomib's effect on the epithelial-mesenchymal transition was associated with inhibition of nuclear factor kappa B (NF-κB) pathway by stabilizing inhibitor of NF-κB. The study highlighted the therapeutic potential of bortezomib on renal allograft interstitial fibrosis. Such an effect may result from inhibition of NF-κB/TNF-α/Akt/mTOR/p70S6 kinase/Smurf2 signaling via stabilizing protein of inhibitor of NF-κB., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Objectives: Antibody-mediated injury in chronic active antibody-mediated rejection, possibly with other effector T cells, may play a role in graft injury. The role of inflammatory cells in the inflammation and fibrosis and tubular atrophy region has been recently advocated in the progression of injury. Cytotoxic T cells play a prominent role in T-cell-mediated rejection; however, the possible role of cytotoxic T cells in circulation and the intragraft compartment in chronic active antibody-mediated rejection, a common immunological cause of long-term graft failure, has not been well-studied., Materials and Methods: We measured the frequency of circulating cytotoxic T cells with flow cytometry, serum granzyme B level by enzyme-linked immunosorbent assay and intragraft granzyme B+ cell, and mRNA by immunohistochemistry and real-time polymerase chain reaction in biopsy tissue from living donor renal allograft recipients with stable graft function and chronic active antibody-mediated rejection., Results: The frequency of CD3+ and CD3+CD8+ T cells was similar in both stable graft function patients and chronic active antibody-mediated rejection patients. The frequency of CD3+CD8+granzyme B+ cytotoxic T cells was significantly lower in peripheral blood. Serum granzyme B level and intragraft number of granzyme B+ cells (counts/mm²) were also significantly higher in the chronic active antibody-mediated rejection group compared with that of patients with stable graft function. The intragraft granzyme B+ T cell count was positively correlated with serum creatinine and 24-hour urine proteinuria but negatively correlated with estimated glomerular filtration rate., Conclusions: Granzyme B mediates covert graft injury in patients with chronic active antibody-mediated rejection in addition to antibody-mediated injury.

Background: Allograft vasculopathy (AV) is the primary limiting factor for long-term graft survival. An increased activity of matrix metalloproteinases (MMPs) contributes to neointima formation in AV and represents a potential therapeutic target. Adeno-associated virus (AAV)-mediated gene therapy comprises a potentially benign vector model for the long-term expression of MMP antagonists., Methods: Aortic allografts from DBA/2 mice were incubated with control buffer, AAV-enhanced green fluorescence protein (EGFP), or tissue inhibitor of metalloproteinases 1 (TIMP-1)-loaded AAV (AAV-TIMP-1) and transplanted into the infrarenal aorta of C57BL/6 mice. Cyclosporine A (10 mg/kg body weight) was administered daily. Explantation as well as histomorphometric and immunohistochemical evaluation was performed after 30 days. Matrix metalloproteinase (MMP) activity was visualized by gelatin in situ zymography., Results: Intima-to-media area ratio and neointima formation were significantly reduced in the AAV-TIMP-1 treatment group compared with those in the control group (by 40%; p < 0.001) and the AAV-EGFP group (by 38.2%; p < 0.001). TIMP-1 overexpression positively affected several pathomechanisms for the development of AV both in vitro and in vivo as compared to that in the control groups: endothelium integrity was preserved as shown by zona occludens 1 and occludin staining; MMP9 expression and activity were significantly reduced (p = 0.01); and smooth muscle cell migration was significantly reduced as smooth muscle actin positive cells predominantly remained in the aortic media in the treatment group (p = 0.001). Moreover, macrophage infiltration was markedly reduced by 49% in the AAV-TIMP-1 group (p < 0.001)., Conclusion: Immediate post-harvesting allograft incubation with AAV-TIMP-1 reduces neointima formation and macrophage infiltration, constituting a possible adjunct therapeutic strategy to preserve graft function after transplantation., (Copyright © 2020 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

The identification of risk factors for acute rejection (AR) may lead to strategies to improve success of kidney transplantation. Ectonucleotidases are ectoenzymes that hydrolyze extracellular nucleotides into nucleosides, modulating the purinergic signaling. Some members of the Ectonucleotidase family have been linked to transplant rejection processes. However, the association of Ectonucleotide Pyrophosphatase / Phosphodiesterase 1 (ENPP1) with AR has not yet been evaluated. The aim of this study was to evaluate the association between the K121Q polymorphism of ENPP1 gene and AR in kidney transplant patients. We analyzed 449 subjects without AR and 98 with AR from a retrospective cohort of kidney transplant patients from Southern Brazil. K121Q polymorphism was genotyped using allelic discrimination-real-time PCR. Cox regression analysis was used to evaluate freedom of AR in kidney transplant patients according to genotypes. Q allele frequency was 17.6% in recipients without AR and 21.9% in those with AR (P = 0.209). Genotype frequencies of the K121Q polymorphism were in Hardy-Weinberg equilibrium in non-AR patients (P = 0.70). The Q/Q genotype (recessive model) was associated with AR (HR = 2.83, 95% CI 1.08-7.45; P = 0.034) after adjusting for confounders factors. Our findings suggest a novel association between the ENPP1 121Q/Q genotype and AR in kidney transplant recipients., Competing Interests: The authors have declared that no competing interests exist.

The immunoproteasome is expressed in cells of hematopoietic origin and is induced during inflammation by IFN-γ. Targeting the immunoproteasome with selective inhibitors has been shown to be therapeutically effective in pre-clinical models for autoimmune diseases, colitis-associated cancer formation, and transplantation. Immunoproteasome inhibition prevents activation and proliferation of lymphocytes, lowers MHC class I cell surface expression, reduces the expression of cytokines of activated immune cells, and curtails  T helper 1 and 17 cell differentiation. This might explain the in vivo efficacy of immunoproteasome inhibition in different pre-clinical disease models for autoimmunity, cancer, and transplantation. In this review, we summarize the effect of immunoproteasome inhibition in different animal models for transplantation.

Objectives: The authors sought to evaluate cardiac activity of angiotensin-converting enzyme (ACE) and ACE2 after heart transplantation (HT) and its relation with acute rejection (AR) and chronic allograft vasculopathy (CAV)., Background: The renin-angiotensin system is altered in heart failure and HT. However, ACE and ACE2 activities in post-HT acute and chronic rejection have not been previously studied., Methods: HT patients (n = 45) were included when appropriate serial endomyocardial biopsies (EMB) and coronary angiography were available for analysis. In 21 patients, three post-HT time points were selected for CAV study in EMB tissue: basal (0-3 wks), second (2-3 months) and third (4-5 months). At 10 years post-HT, CAV was evaluated by coronary angiography (CA) and patients were grouped by degree of CAV: 0-1, non-CAV (n = 15) and 2-3, CAV (n = 6). For the AR study, 28 HT patients with evidence of one EMB rejection at grade 3 and two EMB grade 1A and/or 1B rejections were selected., Results: Post-HT, ACE2 activity was increased in the CAV group, compared to non-CAV. Patients with AR showed increased ACE, but not ACE2, activity., Conclusions: Our results suggest that early post-HT cardiac ACE2 activity may have an important role in CAV development. In contrast, ACE activity was increased in AR. The renin-angiotensin system seems to be altered after HT and strategies to balance the system may be useful., (Copyright © 2018. Published by Elsevier B.V.)

In kidney transplantation acute allograft rejection is the most common cause of late allograft loss. Changes in indoleamine 2,3 dioxygenase (IDO) activity, which catabolizes the degradation of tryptophan to kynurenine, may predict rejection. However, exogenous IDO is immunosuppressive in rodent kidney transplantation. Thus, the increase in IDO activity observed in acute allograft rejection is insufficient to prevent rejection. To address this question, we assessed the regulation of IDO and its role in acute rejection in a porcine model of kidney transplant. In tissue samples from rejecting kidney allografts, we showed a 13-fold increase in IDO gene transcription and 20-fold increase in IDO enzyme activity when compared with autotransplanted kidneys. Allografts also demonstrated an over fourfold increase in tissue interferon (IFN)-γ, with marked increases in tumor necrosis factor (TNF)-α, TNF-β and interleukin 1β. Gene transcription and protein levels of kynurenine 3-monooxygenase (KMO) were decreased. KMO generates the immunosuppressive kynurenine, 3-hydroxykynurenine. The results of these studies demonstrate a clear association between rejection and increased allograft IDO expression, likely driven in part by IFN-γ and facilitated by other cytokines of the allogeneic response. Moreover, the loss of downstream enzymatic activity in the IDO metabolic pathway may suggest novel mechanisms for the perpetuation of rejection., Competing Interests: Competing interests: None declared., (© American Federation for Medical Research 2018. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Hepatitis E virus (HEV) infection is a potential reason for elevated liver enzymes after liver transplantation (LT). Our aim was to analyze a real-world cohort of LT patients, who underwent liver biopsy for elevated transaminases and suspected acute rejection, to evaluate frequency of post-transplant HEV infection., Patients: Data from 160 liver biopsies were analyzed. Seventy-one patients were biopsied on schedule after LT without elevated liver enzymes. A subgroup of 25 patients with elevated liver enzymes and suspected rejection was chosen for further analysis. Patient demographics and data were retrieved from a clinical database, patients' charts, and reports., Results: Hepatitis E virus infection was diagnosed in five of 25 patients with suspected acute rejection (20%). HEV genotype 3 was detected in three of the five HEV-infected patients. Patients with HEV infection showed higher ALT levels (P = 0.014), lower De Ritis ratio (P = 0.021), and more frequent glucocorticoid therapy (P = 0.012) compared to HEV-negative patients., Conclusion: We found a rate of 20% HEV infections in LT patients undergoing liver biopsy for elevated liver enzymes and suspected acute rejection. These data indicate the necessity for HEV testing in all LT patients with elevated liver enzymes and suspected acute rejection., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Early damage to transplanted organs initiates excess inflammation that can cause ongoing injury, a leading cause for late graft loss. The endothelial glycocalyx modulates immune reactions and chemokine-mediated haptotaxis, potentially driving graft loss. In prior work, conditional deficiency of the glycocalyx-modifying enzyme N-deacetylase-N-sulfotransferase-1 (Ndst1 f/f TekCre + ) reduced aortic allograft inflammation. Here we investigated modification of heparan sulfate (HS) and chemokine interactions in whole-organ renal allografts. Conditional donor allograft Ndst1 deficiency (Ndst1 -/- ; C57Bl/6 background) was compared to systemic treatment with M-T7, a broad-spectrum chemokine-glycosaminoglycan (GAG) inhibitor. Early rejection was significantly reduced in Ndst1 -/- kidneys engrafted into wildtype BALB/c mice (Ndst1 +/+ ) and comparable to M-T7 treatment in C57Bl/6 allografts (P < 0.0081). M-T7 lost activity in Ndst1 -/- allografts , while M-T7 point mutants with modified GAG-chemokine binding displayed a range of anti-rejection activity. CD3+ T cells (P < 0.0001), HS (P < 0.005) and CXC chemokine staining (P < 0.012), gene expression in NFκB and JAK/STAT pathways, and HS and CS disaccharide content were significantly altered with reduced rejection. Transplant of donor allografts with conditional Ndst1 deficiency exhibit significantly reduced acute rejection, comparable to systemic chemokine-GAG inhibition. Modified disaccharides in engrafted organs correlate with reduced rejection. Altered disaccharides in engrafted organs provide markers for rejection with potential to guide new therapeutic approaches in allograft rejection.

Objectives: We aim to identify the key biomarker of acute rejection (AR) after kidney transplantation via bioinformatics methods., Methods: The gene expression data GSE75693 of 30 samples with stable kidney transplantation recipients and 15 AR samples were downloaded and analyzed by the limma package to identify differentially expressed genes (DEGs). Then, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were done to explore the biological functions and potential important pathways of DEGs. Finally, protein-protein interactions (PPIs) and literature mining were applied to construct the cocitation network and to select the hub protein., Results: A total of 437 upregulated genes and 353 downregulated genes were selected according to P < 0.01 and |log 2 (fold change)| > 1.0. DEGs of AR are mainly located on membranes and impact the activation of receptors in immune responses. In the PPI network, Src kinase, lymphocyte kinase (LCK), CD3G, B2M, interferon- γ , CD3D, tumor necrosis factor, VAV1, and CD3E in the T cell receptor signaling pathway were selected as important factors, and LCK was identified as the hub protein., Conclusion: LCK, via acting on T-cell receptor, might be a potential therapeutic target for AR after kidney transplantation.

Objective: The objective of this study was to investigate the effect of nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) during acute antibody-mediated rejection (AMR)., Methods: NTPDase1 overexpression, NTPDase1 knockout, and wild-type nude mice skin graft models were used to induce acute AMR. NTPDase1 expression in B cells, NTPDase1 messenger RNA expression in skin grafts, extracellular adenosine diphosphate (ADP) concentration, B-cell volume and surface antigens expression, average platelet transport rate, and ultrastructure and apoptosis of skin graft cells were investigated., Results: During acute AMR in nude mice, higher NTPDase1 expression caused lower extracellular ADP concentration, smaller increase in B-cell volume, and major histocompatibility complex II surface antigen expression, suggesting a negative correlation between them; higher NTPDase1 expression also caused slower average platelet transport rate and less severe skin graft injury, suggesting a negative correlation between them. Pretreatment with high-dose exogenous NTPDase1 inhibited platelet activation and protected skin grafts, but it resulted in prolonged bleeding time (by 51.4%) and prolonged coagulation time (by 44.1%)., Conclusion: An NTPDase1-associated imbalance in extracellular ADP degradation may contribute to B-cell activation, platelet activation, and more severe skin graft injury in nude mice. Pretreatment with high-dose exogenous NTPDase1 effectively protected skin grafts in nude mice at 1 week, but it increased the risk of bleeding., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Regulatory T cells were recently proposed as the central actor in operational tolerance after renal transplantation. Tolerant patients harbor increased FoxP3hi memory Treg frequency and increased demethylation in the Foxp3 Treg-specific demethylated region when compared to stable kidney recipients and exhibit greater memory Treg suppressive capacities and higher expression of the ectonucleotidase CD39. However, in this particular and unique situation the mechanisms of action of Tregs were not identified. Thus, we analyzed the ability of memory Tregs to degrade extracellular ATP in tolerant patients, healthy volunteers, and patients with stable graft function under immunosuppression and determined the role of immunosuppressive drugs on this process. The conserved proportion of memory Tregs leads to the establishment of a pro-tolerogenic balance in operationally tolerant patients. Memory Tregs in tolerant patients display normal capacity to degrade extracellular ATP/ADP. In contrast, memory Tregs from patients with stable graft function do not have this ability. Finally, in vitro, immunosuppressive drugs may favor the lower proportion of memory Tregs in stable patients, but they have no effect on CD39-dependent ATP degradation and do not explain memory Treg lack of extracellular ATP/ADP degradation ability. Thus, intrinsic active regulatory mechanisms may act long after immunosuppressive drug arrest in operationally tolerant patients and may contribute to kidney allograft tolerance via the maintenance of CD39 Treg function., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Chronic antibody-mediated rejection is the major cause of fading allograft function and loss after renal transplantation. Currently, pharmacological agents for the suppression of chronic antibody-mediated rejection are lacking. Non-selective proteasome inhibitors suppress antibody-mediated allograft rejection. However, extensive adverse side effects of these inhibitors severely limit their application. In contrast, immunoproteasome inhibition is effective in preclinical models of autoimmune diseases and was applied over weeks without obvious adverse side effects. ONX 0914, an immunoproteasome subunit LMP7 (β5i)-selective inhibitor, impeded the chronic rejection of kidneys transplanted from Fischer to allogeneic Lewis rats. ONX 0914 inhibited immunoproteasome induction both in immune organs and renal allografts. Selective immunoproteasome inhibition reduced the numbers of B and plasma cells, and suppressed donor-specific alloantibody production. The infiltration of T cells, B cells and macrophages as well as interferon-γ, interleukin-17, IgG and complement deposition were reduced in renal allografts of ONX 0914-treated recipients. Chronic nephropathy was ameliorated and renal allograft function preserved, enabling long-term survival of recipients. Thus, our studies define a critical role of the immunoproteasome in chronic kidney allograft rejection and suggest immunoproteasome inhibition as a promising therapeutic approach to suppress chronic antibody-mediated rejection., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Despite advances in immunosuppressive therapies, the rate of chronic transplant loss remains substantial. Organ injury involves various forms of cell death including apoptosis and necrosis. We now recognize that early injury of cardiac transplants involves a newly described form of programmed necrotic cell death, termed necroptosis. Because this involves receptor-interacting protein (RIP) kinase 1/3, this study aimed to establish the role of RIP3 in chronic cardiac allograft rejection., Methods: We used major histocompatibility complex class II mismatched C57BL/6N (H-2; B6) or B6.RIP3 (H-2; RIP3) mice to B6.C-H-2 (H2-Ab1; bm12) mouse cardiac transplantation. Microvascular endothelial cells (MVEC) were developed from B6 and RIP3 cardiac grafts., Result: CD4 T cell-mediated cardiac graft rejection is inhibited using RIP3 deficient donor grafts, with reduced cellular infiltration and vasculopathy compared with wild type cardiac grafts. Alloreactive CD4 T cell-mediated MVEC death involves TNFα, Fas ligand (FasL) and granzyme B. Although necroptosis and release of danger molecule high-mobility group box 1 are eliminated by the absence of RIP3, CD4 T cells had attenuated MVEC death through granzyme B and FasL., Conclusions: CD4 T cell-mediated MVEC death involves in TNFα, FasL and granzyme B. Necroptotic cell death and release of the danger molecule may promote inflammatory responses and transplant rejection. Although loss of RIP3 does not eliminate alloimmune responses, chronic graft injury is reduced. RIP3 is an important therapeutic target but additional granzyme and caspases inhibition is required for sufficiently improving long-term graft survival.

Background: Glucocorticoid (GC)-refractory acute rejection (AR) is a risk factor for inferior renal allograft outcome. We investigated genetic predisposition to the response to steroid treatment of acute allograft rejection., Methods: Single nucleotide polymorphisms of genes involved in GC signaling (GR, GLCCI1) and drug metabolism and transport (CYP3A5, ABCB1, and PXR) were analyzed in kidney transplant recipients (1995-2005, Leiden cohort, n = 153) treated with methylprednisolone. Significant associations were verified in a second cohort (Berlin cohort, n = 66)., Results: Patients who received a CYP3A5*1 allele expressing allograft had a lower risk of resistance to methylprednisolone during AR (odds ratio, 0.29; 95% confidence interval, 0.11-0.79; P = 0.016 in combined cohorts analysis). No differences were observed for GC signaling or other drug metabolism/transport-related genes. Both before transplantation (n = 69) and at time of AR (n = 88), tissue CYP3A5 mRNA expression was significantly higher in CYP3A5*1 allele expressing donor kidneys than in CYP3A5*3/*3 allografts (P < 0.00001). Moreover, steroid-responsive patients (n = 64) expressed significantly higher intragraft CYP3A5 mRNA levels compared to steroid-refractory patients (n = 42) in AR (P = 0.006)., Conclusions: CYP3A5 protein expression was detected in tubular epithelial cells and inflammatory cells within the grafts. Our findings show that steroid resistance during AR is associated with donor genotype and intragraft expression levels of CYP3A5.

Background: Organ transplantation into sensitized patients with preexisting donor-specific antibodies (DSA) is very challenging. Spleen tyrosine kinase (Syk) promotes leukocyte recruitment and activation via signaling through various cell surface receptors. We investigated whether a selective Syk inhibitor (GS-492429) could suppress antibody-mediated rejection (AMR) in a rat model of AMR in sensitized recipients., Methods: Recipient Lewis rats (RT1) were immunized with donor (Dark Agouti, RT1) spleen cells (day -5). Recipients underwent bilateral nephrectomy and orthotopic renal transplantation (day 0). Cellular rejection was minimized by tacrolimus treatment from day -1. Groups received GS-492429 (30 mg/kg, twice a day) (n = 11) or vehicle (n = 12) from 1 hour before transplantation until being killed on day 3., Results: Vehicle-treated recipients developed graft dysfunction on day 1 which rapidly worsened by day 3. Histology showed severe damage (thrombosis, acute tubular injury, capillaritis) and infiltration of many Syk leukocytes. GS-492429 did not affect graft dysfunction on day 1, but treatment reduced allograft damage and prevented the rapid deterioration of graft function on day 3. GS-492429 reduced the prominent macrophage infiltrate and reduced the M1 proinflammatory response. Neutrophil and NK cell infiltration and capillary thrombosis were also significantly reduced by GS-492429 treatment. Serum DSA levels and the deposition of IgG and C4d in the allograft were equivalent in the 2 groups., Conclusions: Treatment with a Syk inhibitor significantly reduced renal allograft injury in a model of severe antibody-mediated damage in highly sensitized recipients. Further studies are warranted to determine whether Syk inhibition is a potential adjunctive treatment in clinical AMR.

Aim: To investigate whether bone marrow mesenchymal stem cells (BMMSCs) modified with the HO-1 and CXCR3 genes can augment the inhibitory effect of BMMSCs on small bowel transplant rejection., Methods: Lewis rat BMMSCs were cultured in vitro . Third-passage BMMSCs were transduced with the CXCR3/HO-1 genes or the HO-1 gene alone. The rats were divided into six groups and rats in the experimental group were pretreated with BMMSCs 7 d prior to small bowel transplant. Six time points (instant, 1 d, 3 d, 7 d, 10 d, and 14 d) ( n = 6) were chosen for each group. Hematoxylin-eosin staining was used to observe pathologic rejection, while immunohistochemistry and Western blot were used to detect protein expression. Flow cytometry was used to detect T lymphocytes and enzyme linked immunosorbent assay was used to detect cytokines., Results: The median survival time of BMMSCs from the CXCR3 / HO-1 modified group (53 d) was significantly longer than that of the HO-1 modified BMMSCs group (39 d), the BMMSCs group (26 d), and the NS group (control group) (16 d) ( P < 0.05). Compared with BMMSCs from the HO-1 modified BMMSCs, BMMSCs, and NS groups, rejection of the small bowel in the CXCR3 / HO-1 modified group was significantly reduced, while the weight of transplant recipients was also significantly decreased ( P < 0.05). Furthermore, IL-2, IL-6, IL-17, IFN-γ, and TNF-α levels were significantly decreased and the levels of IL-10 and TGF-β were significantly increased ( P < 0.05)., Conclusion: BMMSCs modified with the CXCR3 and HO-1 genes can abrogate the rejection of transplanted small bowel more effectively and significantly increase the survival time of rats that receive a small bowel transplant., Competing Interests: Conflict-of-interest statement: The authors declare no potential conflict of interest for this paper.

Objectives: This review focuses on the current limited evidence of graft function and graft survival in various immunosuppressive regimens involving mammalian target of rapamycin inhibitors with or without calcineurin inhibitors., Materials and Methods: We evaluated the current literature for describing the role of mammalian target of rapamycin inhibitors as an alternative to calcineurin inhibitors by searching the PubMed, EMBASE, Cochrane, Crossref, and Scopus databases using medical subject heading terms., Results: Our detailed analyses of all relevant literature showed use of mammalian target of rapamycin inhibitor-based de novo regimens, early calcineurin inhibitor withdrawal with subsequent introduction of mammalian target of rapamycin inhibitor-based regimens, and late conversion from a calcineurin inhibitor-based regimen to mammalian target of rapamycin inhibitor-based regimens. Notably, early calcineurin inhibitor withdrawal with subsequent introduction of mammalian target of rapamycin inhibitor-based regimen seemed to be a more practical and realistic approach toward immunosuppressive treatment of renal transplant recipients. However, in view of the high rejection rate observed in these studies, it is advisable not to offer these regimens to patients with moderate to high immunologic risk., Conclusions: The present evidences suggest that treatment with mammalian target of rapamycin inhibitors allows early and substantial calcineurin inhibitor minimization. The mammalian target of rapamycin inhibitors everolimus and sirolimus are preferred due to their complementary mechanisms of action and favorable nephrotoxicity profile, which have opened the way for calcineurin inhibitor reduction/withdrawal in the early posttransplant period.

Background: Serum lactate dehydrogenase (LDH) levels may help to distinguish ischemic acute tubular necrosis (ATN) from acute rejection after kidney transplantation., Methods: All kidney biopsies performed in the years 2010 to 2012 were reviewed. Serum LDH, creatinine level, clinical variables, and presence of donor-specific antibodies were recorded before the biopsy., Results: Overall 150 biopsies were included. Ischemic ATN was diagnosed in 45 biopsies and acute cellular-mediated rejection and/or antibody-mediated rejection in 59 biopsies, 38 of which were accompanied by ATN. Serum LDH was elevated in 23 (51%) of 45 cases with ischemic ATN versus 15 (14%) of 105 cases with other diagnoses ( P < .0001). Median serum LDH was 478 U/L (range 277-2018) for ischemic ATN and 372 U/L (range 191-748) for all other diagnoses ( P < .001). When delayed graft function or primary nonfunctioning grafts were caused by ischemic ATN, serum LDH was elevated in 58% of cases, but when caused by acute rejection, LDH was normal in 88% of cases ( P = .02)., Conclusions: There is a strong association between elevated serum LDH 1 to 3 days before performing kidney biopsy and the diagnosis of ischemic ATN after kidney transplantation, especially at the immediate posttransplantation period. Normal serum LDH at this period should raise a suspicion of acute rejection.

Immunosuppression after liver transplantation (LT) is presently based on use of calcineurin inhibitors (CNI), although they are associated with an increased incidence of renal dysfunction, cardiovascular complications, and de novo and recurrent malignancies. Over the past decade, mammalian target of rapamycin inhibitors have received considerable attention as immunosuppressants because they are associated with a more favorable renal profile versus CNI, as well as antiproliferative activity in clinical studies. Comprehensive guidelines on use of everolimus (EVR) in LT are still lacking. In Italy, a project, named Everolimus: the road to long-term functioning, was initiated to collect the experience on EVR after LT with the aim of providing guidance for transplant clinicians. Herein, recommendations by this national consensus group, based on Delphi methodology, are presented. Consensus was reached on 20 of the 23 statements proposed, and their level of evidence, grade of recommendation, and percent of agreement are reported. Statements are grouped into 4 areas: (A) renal function; (B) time of EVR introduction, CNI reduction and elimination, and risk for graft rejection; (C) antiproliferative effects of EVR; and (D) management of EVR-related adverse events. The high level of consensus shows that there is good agreement on the routine use of EVR in predefined clinical scenarios, especially in light of posttransplant nephrotoxicity and other adverse events associated with long-term administration of CNIs., Competing Interests: P.D.S. has served as advisor and has received speaker’s fees from Novartis. The other authors declare no conflicts of interest.

Malignancy is the second most common single cause of death observed in organ transplant recipients. The excess cancer risk is related to intensity and duration of immunosuppressive therapy and inversely to recipient age. Immunodeficiency and (chronic/oncogenic) viral infections together constitute a major risk. Nonmelanoma skin cancer, Kaposi sarcoma, and posttransplant lymphoproliferative disease have standardized incidence ratios exceeding 10- or 50-fold. The mammalian target of rapamycin (mTOR) inhibitors, sirolimus and everolimus, are increasingly used after organ transplantation with potential advantages in virus-associated posttransplant malignancies as well as anti-cancer properties. Despite a seemingly clear mechanism of action and solid rationale for their use in cancer therapy, mTORis have met only modest success rates in clinical trials with advanced malignancies except for specific tumors, such as Kaposi sarcoma and mantle cell lymphoma. Because mTORis are primarily cytostatic, not cytotoxic, the observed clinical efficacy is a reflection of disease stabilization rather than tumor regression. Nonmelanoma skin cancers, in particular cutaneous squamous cell carcinoma, have the highest standardized incidence ratios in transplant recipients. Recent meta-analyses and randomized trials on secondary prevention of squamous cell carcinoma observed a reduction in cumulative tumor load, suggesting most benefit to be gained by early conversion to an mTOR inhibitor-based maintenance regime. There is ongoing debate on the mechanisms involved including withdrawal of the carcinogenic effects of calcineurin inhibitors and/or their impact on chronic (oncogenic) viral infections. At present, there is, however, insufficient evidence for the primary use of mTORis as protective agents against most other cancer types.

The profound involvement of cytokines in allograft rejection makes the molecules that control their actions, members of the Jak-Stat pathway, ideal targets for pharmacological intervention. Numerous studies have demonstrated that Jak3 is widely involved in the activation cascade and function of most immune cells. Tofacitinib, an oral Janus kinase inhibitor that targets Jak1/Jak3 dependent Stat activation, has been assessed as a substitute for calcineurin inhibitor therapy after low-to-moderate risk kidney transplantation in 3 randomized trials. Results using fixed-dose regimens showed a low incidence of rejection and better renal function with less interstitial fibrosis/tubular atrophy versus calcineurin inhibitor therapy. However, the safety profile of tofacitinib was poor, including increased incidences of cytomegalovirus disease, herpes zoster, BK virus, and nephropathy, which led to the discontinuation of its development for transplantation. High tofacitinib concentrations were independently associated with serious infection. Dosing according to exposure levels, coupled with pharmacodynamic monitoring based on phosphorylation of Stat5, could improve safety compared to the early fixed-dose regimens. Future studies could assess individualized dosing based on pharmacokinetic and pharmacodynamic monitoring. Additionally, because the increase of viral infections under tofacitinib may have been influenced by overlapping toxicity with concomitant mycophenolic acid, exploration of alternative adjunctive therapies (eg, a mammalian target of rapamycin inhibitor or belatacept) may demonstrate a better efficacy/safety profile. We believe that Jak inhibitors are a good and useful addition to the immunosuppressive armentarium for kidney transplant patients, and that new studies with personalized drug dosing, improved immune monitoring, and better patient selection should be performed.

Background: De novo alloantibodies (donor-specific antibody) contribute to antibody-mediated rejection and poor long-term graft survival. Because the development of donor-specific antibody is associated with early graft loss of cell transplants and reduced long-term survival of solid organ transplants, we hypothesized that conventional immunosuppressives, calcineurin inhibitors (CNi), and mammalian target of rapamycin inhibitors (mTORi), may not be as effective for suppression of humoral alloimmunity as for cell-mediated immunity., Methods: Wild-type or CD8-depleted mice were transplanted with allogeneic hepatocytes. Recipients were treated with mTORi and/or CNi and serially monitored for alloantibody and graft survival. The direct effect of mTORi and CNi on alloprimed B cell function was investigated in Rag1 mice adoptively transferred with alloprimed IgG1 B cells. The efficacy of mTORi and/or CNi to suppress CD8-mediated cytotoxicity of IgG1 B cells was evaluated in in vitro and in vivo cytotoxicity assays., Results: Mammalian target of rapamycin inhibitors, but not CNi, reduced alloantibody production in transplant recipients, directly suppressed alloantibody production by alloprimed IgG1 B cells and delayed graft rejection in both low and high alloantibody producers. Combination treatment with mTORi and CNi resulted in loss of the inhibitory effect observed for mTORi monotherapy in part due to CNi suppression of CD8 T cells which downregulate alloantibody production (CD8 TAb-supp cells)., Conclusions: Our data support that mTORi is a potent inhibitor of humoral immunity through suppression of alloprimed B cells and preservation of CD8 TAb-supp cells. In contrast, alloantibody is readily detected in CNi-treated recipients because CNi does not suppress alloprimed B cells and interferes with downregulatory CD8 TAb-supp cells., Competing Interests: The authors of this manuscript have no conflicts of interest to disclose.

Background: Current transplant immunosuppression regimens have numerous limitations. Recent evidence suggests histone deacetylase inhibitors (HDACis) may represent a class of drug with immunosuppressive properties. This study compares cyclosporin A (CyA) with the pan-HDACi suberoylanilide hydroxamic acid (SAHA) and a novel HDAC6-specific inhibitor (KA1010) in models of alloreactivity., Methods: Proliferation and mixed lymphocyte reaction (MLR)-based assays were used to determine the immunosuppressive effect of compounds, and a murine model of allogeneic skin transplantation was adopted to assess the in vivo effects of HDAC6 inhibition., Results: KA1010 displayed superior inhibitory effects on the activation of peripheral mononuclear cells using in vitro models of transplantation. In a 1-way MLR, KA1010 (5 μΜ) reduced parent cell proliferation from 92% to 64% (P = 0.001). A 2-way MLR, adopting IFN-γ production as a marker of alloresponse, resulting in up to 91% reduction. Dose-response curves revealed dose-dependent profiles with greater potency of HDACis over CyA (IC50 values of 82.0 nM and 13.4 nM for KA1010 and SAHA).Mice treated with KA1010 displayed no significant features of skin allograft rejection upon histological analysis at 70 days and graft survival of 80% in subjects treated with 160 mg/kg. Immunological assessment, revealed a significant increase in CD4CD25forkhead box P3 regulatory T cells (from 18% to 25%, P = 0.0002) and a corresponding reduction in CD4 T cells (from 58% to 42%, P = 0.0009)., Conclusions: HDAC6 may represent an optimal target for future immunosuppressant therapeutics with a particular role in transplantation. In this article, we have demonstrated a superior immunosuppressive effect of KA1010 over both CyA and SAHA, in the models of allotransplantation adopted.

Objective: The transplant vasculopathy as a sign of chronic graft rejection affects both the epicardial and the intramyocardial arteries of the graft. This is at least partially mediated by NO synthases. The aim of this study was to assess possible protective effects of cyclosporine A (CsA), tacrolimus (FK506), and mycophenolate mofetil (MMF) on the expression of NO synthases in an experimental transplant rat model., Aims: Heart transplantation was performed in 322 rats. These were randomly assigned to four equal groups (control, CsA, FK506, MMF). Recipients were monitored up to 60 days after transplantation, while transplanted hearts were recovered at certain time points for analysis. Expression and staining intensity for endothelial nitric oxide synthases (e-nos) and inducible nitric oxide synthases (i-nos) were analyzed in epicardial and intramyocardial vessels in each group., Results: All employed drugs led to a significant reduction of expression or staining intensity of i-nos and e-nos. MMF was most effective in reduction in expression of both NO synthases., Conclusions: These results imply that all described drugs prevent endothelial impairment induced by toxicity of NO and thereby prevent transplant vasculopathy. MMF seems to be the most effective drug., (© 2016 John Wiley & Sons Ltd.)

Objectives: Cytomegalovirus infection and disease remain an issue in solid-organ transplant. Universal prophylaxis is more cost-effective than a preemptive strategy and is associated with significantly less Cytomegalovirus resistance after kidney transplant, especially in Cytomegalovirus-seropositive donors and Cytomegalovirus-seronegative recipients., Materials and Methods: Registry data and meta-analyses have shown that mammalian target of rapamycin inhibitors (sirolimus- and everolimus-based immunosuppression) are associated with significantly less Cytomegalovirus events in de novo kidney transplant patients than in patients who are treated with calcineurin inhibitors plus mycophenolate-based immunosuppression., Results: Recent pooled analyses of 3 randomized controlled trials in de novo kidney transplant patients, where immunosuppression was based on cyclosporine with either mycophenolate or everolimus, showed that patients who received everolimus had significantly less Cytomegalovirus events (Cytomegalovirus viremia, Cytomegalovirus infection/disease) than those who received mycophenolate, with or without cytomegalovirus as prophylaxis. An even more recent prospective randomized controlled study on de novo kidney transplant patients with no anticytomegalovirus prophylaxis demonstrated that everolimus-based immunosuppression plus low-dose tacrolimus was associated with significantly less Cytomegalovirus infection than standard-dose tacrolimus plus mycophenolate., Conclusions: The potential benefits are not fully known of such a therapeutic strategy to limit the long-term indirect effects mediated by Cytomegalovirus infections.

Objectives: Polymorphisms of the endothelial nitric oxide synthase gene have been associated with altered endothelial nitric oxide synthase activity. The purpose of this study was to investigate the relation between endothelial nitric oxide synthase -786T/C and 894G/T polymorphism and their haplotypes on the occurrence of acute rejection episodes in liver transplant recipients., Materials and Methods: We conducted a case control study in which 100 liver transplant recipients and 100 healthy controls were recruited from Shiraz Transplant Center. The patients used triple therapy including tacrolimus, mycophenolate mofetil, and prednisolone for immunosuppression maintenance. DNA was extracted from peripheral blood and endothelial nitric oxide synthase polymorphisms were determined by polymerase chain reaction and restriction fragment length polymorphism., Results: Patients included 60 men and 40 women (mean age, 32.35 ± 10.2 y). There was a significant association of endothelial nitric oxide synthase 894G/T and acute rejection episode. The GT* gen-otype and acute rejection episodes had a significant association (odds ratio, 2.42; 95% confidence interval, 0.97-6.15; P = .03). The GG and GT* genotype and T* allele frequency were significantly different between patients and control subjects (P = .001). Haplotype TT* was higher in recipients than control subjects (odds ratio, 2.17; 95% confidence interval, 1.12-4.25; P = .01). Haplotype TG was higher in the control group (odds ratio, 0.62; 95% confidence interval, 0.40-0.96; P = .02)., Conclusions: Our results suggest a relation between different endothelial nitric oxide synthase geno-types and risk of acute rejection episodes. However, further study is necessary to determine genetic susceptibility for transplant patients.

Background: Hypodermin A (HA) is a serine esterase that degrades complement, a key element of the innate immune system. Immunosuppressive properties of HA have previously been studied in vitro. However, such properties have not been fully demonstrated in vivo. The aim of this study was to evaluate the effect of HA in inhibiting allograft rejection in an HA transgenic mouse model., Methods: FVB (HA transgenic mice or wild-type mice) to BALB/c mice skin transplantation model were used. Skin grafts were analyzed by histology, immunohistochemistry, and Western blotting., Results: HA overexpression resulted in significantly prolonged skin allograft survival. Histologic changes in the skin allografts paralleled the gross appearance of rejection. ELISA and Western blotting showed that HA significantly reduced the content of complement C3 and C9 in HA skin allografts. The expressions of CD4, B7-2, and MHC class II were all significantly suppressed in HA skin allografts compared with the control group., Conclusions: These findings suggest that HA effectively prolongs skin allograft survival. The study results provide insight into a promising strategy to improve the survival of grafts in humans., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Current immunosuppressive medications used after transplantation have significant toxicities. Foxp3(+) T-regulatory cells can prevent allograft rejection without compromising protective host immunity. Interestingly, inhibiting the class III histone/protein deacetylase Sirtuin-1 can augment Foxp3(+) T-regulatory suppressive function through increasing Foxp3 acetylation. Here we determined whether Sirtuin-1 targeting can stabilize biological allograft function. BALB/c kidney allografts were transplanted into C57BL/6 recipients with a CD4-conditional deletion of Sirtuin-1 (Sirt1(fl/fl)CD4(cre)) or mice treated with a Sirtuin-1-specific inhibitor (EX-527), and the native kidneys removed. Blood chemistries and hematocrit were followed weekly. Sirt1(fl/fl)CD4(cre) recipients showed markedly longer survival and improved kidney function. Sirt1(fl/fl)CD4(cre) recipients exhibited donor-specific tolerance, accepted BALB/c, but rejected third-party C3H cardiac allografts. C57BL/6 recipients of BALB/c renal allografts that were treated with EX-527 showed improved survival and renal function at 1, but not 10 mg/kg/day. Pharmacologic inhibition of Sirtuin-1 also improved renal allograft survival and function with dosing effects having relevance to outcome. Thus, inhibiting Sirtuin-1 can be a useful asset in controlling T-cell-mediated rejection. However, effects on non-T cells that could adversely affect allograft survival and function merit consideration., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Expression of both platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) is increased during the development of chronic rejection which remains the major reason for late allograft loss in clinical kidney transplantation. Sunitinib is a tyrosine kinase inhibitor which inhibits both VEGF and PDGF receptors. Here we investigated its effect on the development of chronic rejection., Methods: Rat aortic denudation model was used to define sunitinib dose. In vitro studies were done to investigate the effect of sunitinib on smooth muscle cell proliferation and migration. Kidney transplantations were performed from dark agouti rat strain (DA) to Wistar furth rat strain rats and syngenic DA-DA grafts were used as controls. Allografts were immunosuppressed either with cyclosporine or with cyclosporine and sunitinib. Grafts were harvested at 5 and 90 days for histology and immunohistochemistry. Serum creatinine levels were measured weekly to monitor graft function., Results: Sunitinib decreased neointimal formation and smooth muscle cell proliferation and migration in a dose-dependent manner. Sunitinib was well tolerated and almost completely prevented chronic rejection changes and preserved significantly better renal graft function after transplantation. Sunitinib also inhibited chronic PDGF-A and -B and VEGF-A and -B expressions., Conclusions: These results demonstrate that combined inhibition of PGDF and VEGF with sunitinib prevents chronic rejection changes in experimental kidney transplantation which indicates that sunitinib could be a potential intervention also in clinical kidney transplantation.

Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation., (Copyright © 2015, American Association for the Advancement of Science.)

Mammalian target of rapamycin inhibitors, such as rapamycin and more recently everolimus, have substituted calcineurin inhibitors in many minimization strategies. Despite their acclaimed renal safety profile, several lines of evidence are emerging on their potential nephrotoxic effect. Predisposing conditions for nephrotoxicity involve a complex interplay between several environmental and genetic factors in the donor-recipient pair. Renal injury may be enhanced by pharmacodynamic interactions when combined with other drugs such as calcineurin inhibitors or nutrients that are predominantly related to an increase in local tissue exposure. These toxic interactions may occur within adequate doses and therapeutic blood levels. This explains the occurrence of nephrotoxicity in some but not all cases. Here, we postulated that activity of a low permeability glycoprotein efflux pump related to low protein expression and/or inhibition enhanced immunosuppressive drug entry in different cells. A rise in intracellular drug concentration increases bioactivity, leading to greater immunosuppression and more immune-related, nonrenal adverse events in the recipient and increased nephrotoxicity in the kidney graft. Under specific isolated or combined environmental and/or genetic conditions in both the recipient and donor affecting the glycoprotein efflux pump and/or the mammalian target of rapamycin pathway, these renal injuries may be aggravated by heightened drug tissue concentrations despite adherence to therapeutic drug and blood levels. Mammalian target of rapamycin inhibitors may induce predominantly a dose-dependent renal epithelial cell injury affecting either the glomerular or the renal tubular epithelial cells, leading to cell death and apoptosis. Epithelial mesenchymal transition mediated interstitial fibrosis and tubular atrophy observed with these drugs may be the result of a cumulative toxic renal tubular injury induced by the direct insult of the drug itself and/or podocytopathy-associated proteinuria. The resulting glomerular tubular damage will ultimately lead to graft failure and loss, if exposure persists.

Background: The immunoregulatory properties of mesenchymal stem cells (MSCs) have been well characterized in vitro. However, the role of MSCs in organ transplantation remains unclear. The purpose of this study was to examine the role of indoleamine 2, 3-dioxgenase (IDO)-transfected MSCs in immunoregulation both in vitro and in vivo., Methods: Wild-type (WT) MSCs, empty lentivirus-transfected MSCs (Lenti-MSCs) or IDO-lentivirus-transfected MSCs (IDO-MSCs) were cocultured with peripheral blood mononuclear cells (PBMCs) or CD4CD25 regulatory T (Treg) cells to examine the impact of IDO on the immunoregulatory properties of MSCs in vitro. WT-MSCs, Lenti-MSCs or IDO-MSCs (2 × 10/kg) were intravenously injected into rabbit renal transplant recipients immediately after surgery to examine the role of IDO-MSCs in tolerance induction in vivo., Results: Lentivirus infection of MSCs resulted in stable expression of IDO. The IDO-MSCs inhibited the proliferation of CD4CD25 effector T cells to a greater extent than WT-MSCs. Coculture of PBMCs and IDO-MSCs induced a higher percentage of CD4CD25Foxp3 Treg cells in PBMCs. Additionally, the antigen-specific suppressive function of these CD4CD25 Treg cells was increased. The IDO-MSCs-treated Treg cells showed upregulated expression of cytotoxic T-lymphocyte-associated antigen 4 and increased secretion of IL-10 and TGF-β. Low doses of IDO-MSCs prolonged graft survival and induced tolerance by inducing antigen-specific CD4CD25 Treg cells, as evidenced by the finding that IDO-MSCs-treated kidney transplant recipients accepted donor-specific skin grafts but rejected third-party grafts., Conclusions: The IDO increased the direct immunoregulatory properties of MSCs. The IDO-MSCs enhanced the expression and function of CD4CD25 Foxp3 Treg cells and induced allograft tolerance.

Background: There is a need for new immunosuppression strategies to minimize calcineurin inhibitor (CNI) toxicity while effectively preventing antibody-mediated rejection (AMR)., Methods: We tested the efficacy of an investigational proteasome inhibitor, ixazomib, alone and in a CNI minimization strategy in a rat kidney transplant model of transfusion-elicited acute AMR. Nonsensitized (naïve) and sensitized allograft recipients were randomized into 4 treatment groups (8 groups total, n = 3 to 6 in each group) and treated for 1 week. Groups included: no treatment, full dose cyclosporine (CsA, 10 mg/kg per day), ixazomib (0.25 mg/kg on days -5, -2 and +2) alone, and half dose CsA (5 mg/kg per day) + ixazomib., Results: Compared to untreated animals, ixazomib alone or in combination with ½ dose CsA reduced donor-specific antibody, intragraft transcripts for chemokines CCL-21 and CXCL-13, and CD19 expression in both sensitized and naïve transplants. Compared to full dose CsA, the CNI minimization strategy with ixazomib inhibited AMR and allograft injury as evidenced by reduced C4d staining in peritubular capillaries, microcirculation inflammation, splenic plasma cells, circulating B cell activating factor, and intragraft transcripts for major histocompatibility complex class II, Toll-like receptors (TLR-1, TLR-10, and TLR-12) and CCL-21 and CXCL-13 in sensitized animals, indicating downregulation of B cell activation, antigen presentation and T-cell and B-cell signaling., Conclusions: These studies suggest that CNI minimization strategies including ixazomib are effective to prevent AMR including in sensitized kidney allograft recipients. Clinical studies are needed to determine the role of novel proteasome inhibitors for the prevention and treatment of AMR.

Background: Antibody-mediated rejection is caused in part by increasing circulation/production of donor-specific antibody (DSA). Activation-induced cytidine deaminase (AID) is a key regulator of class switch recombination and somatic hypermutation of immunoglobulin in B cells, yet its role in antibody-mediated transplant rejection remains unclear. We show here that AID deficiency in mice enables suppression of allograft vasculopathy (AV) after aorta transplantation, a DSA-mediated process., Methods: Splenocytes from C57BL/6 J (B6) AID(−/−) mice were used for determining in vitro proliferation responses, alloreactivity, cell surface marker expression, and antibody production. BALB/c mouse aortas were transplanted into B6 AID(−/−) mice with or without FK506 treatment. Blood and aorta grafts were harvested on day 30 after transplantation and were subjected to DSA, histological, and immunohistological analyses., Results: The AID(−/−) splenocytes were comparable to wild type splenocytes in proliferation responses, alloreactivity, and expression of cell surface markers in vitro. However, they completely failed to produce immunoglobulin G, although they were not impaired in immunoglobulin M production relative to controls. Furthermore, BALB/c aorta grafts from B6 AID(−/−) recipient mice on day 30 after transplantation showed reduced signs of AV compared to the grafts from B6 wild type recipient mice which had severe vascular intimal hyperplasia, interstitial fibrosis, and inflammation. Treatment with FK506 produced a synergistic effect in the grafts from AID(−/−) recipients with further reduction of intimal hyperplasia and fibrosis scores., Conclusions: The AID deficiency inhibits DSA-mediated AV after aorta transplantation in mice. We propose that AID could be a novel molecular target for controlling antibody-mediated rejection in organ transplantation.

This study was designed to explore the clinical significance of dynamically monitoring the serum level of matrix metalloproteinase 9 (MMP-9) before and after renal transplantation. Before transplantation and 1, 3, 5, 7, 10, 15, and 20 days after transplantation, the peripheral blood was collected from 102 renal transplant recipients, including 8 with acute rejection (ARs) and 94 non-ARs. The serum MMP-9 level was detected by Luminex 200 analyzer (Luminex Corporation, Austin, TX, USA). By day 3 post-transplantation, the serum MMP-9 level in non-ARs had significantly reduced as compared to the pretransplantation level, and reached the lowest value on day 20 post-transplantation. In contrast, the serum MMP-9 level in ARs had significantly increased by day 3, reached the highest value on day 7, and remained significantly higher on day 20 as compared to the pretransplantation level. The receiver operating characteristic curve was plotted to evaluate the power of serum MMP-9 level on day 20 post-transplantation to differentiate the non-AR and AR groups. Our data revealed that with a threshold of 8473.26 pg/mL, the area under the curve was 0.758 (0.661, 0.856); the sensitivity and specificity of the diagnostic were 78.40% and 61.30%, respectively; the positive and the negative predictive values were 74.60% and 66.67%, respectively; and the accuracy rate was up to 71.57%. Taken together, the results indicated that dynamically monitoring serum MMP-9 levels in renal allograft recipients might be a convenient and safe method to diagnose ARs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: The protein tyrosine phosphatase nonreceptor 22 gene (PTPN22) encodes a strong T-cell regulator called lymphoid protein tyrosine phosphatase. Previously, PTPN22 was described as a susceptibility gene for autoimmunity because it contains single nucleotide polymorphisms (SNPs) associated with several autoimmune diseases. One SNP (rs2476601; 1858G>A) has emerged as a particularly potent risk factor for autoimmunity. We address the question whether PTPN22 polymorphisms are also associated with acute rejection after liver transplantation., Methods: We investigated the influence of six PTPN22 SNPs on the susceptibility to acute liver allograft rejection. Consequently, we carried out a retrospective study genotyping 345 German liver recipients at six SNP loci, which include rs2488457 (-1123G>C), rs33996649 (788C>T), rs2476601 (1858G>A), rs1310182 (-852A>G), rs1217388 (-2200G>A), rs3789604 (64434T>G). Our study enrolled 165 recipients who did not develop rejection, 123 who showed one rejection episode, and 57 patients who suffered from multiple acute rejections after transplantation., Results: The 1858A allele containing genotypes (GA+AA) and the 1858A allele had a significantly higher frequency in the group of patients with multiple rejection episodes (35.1% and 18.4%) compared to rejection-free patients (15.8% and 7.9%; P=0.022 and 0.023). In contrast, we could not detect any association between rejection and the other tested SNPs. Additionally, we identified one haplotype contributing to risk of multiple rejections, however, exhibiting no stronger impact than the 1858A allele alone., Conclusion: We conclude that the 1858G>A SNP may confer susceptibility to multiple acute liver transplant rejections in the German population.

Kidney allografts induce strong T-cell and antibody responses which mediate acute rejection. Spleen tyrosine kinase (Syk) is expressed by most leucocytes, except mature T cells, and is involved in intracellular signalling following activation of the Fcγ-receptor, B-cell receptor and some integrins. A role for Syk signalling has been established in antibody-dependent native kidney disease, but little is known of Syk in acute renal allograft rejection. Sprague-Dawley rats underwent bilateral nephrectomy and received an orthotopic Wistar renal allograft. Recipient rats were treated with a Syk inhibitor (CC0482417, 30 mg/kg/bid), or vehicle, from 1 h before surgery until being killed 5 days later. Vehicle-treated recipients developed severe allograft failure with marked histologic damage in association with dense leucocyte infiltration (T cells, macrophages, neutrophils and NK cells) and deposition of IgM, IgG and C3. Immunostaining identified Syk expression by many infiltrating leucocytes. CC0482417 treatment significantly improved allograft function and reduced histologic damage, although allograft injury was still clearly evident. CC0482417 failed to prevent T-cell infiltration and activation within the allograft. However, CC0482417 significantly attenuated acute tubular necrosis, infiltration of macrophages and neutrophils and thrombosis of peritubular capillaries. In conclusion, this study identifies a role for Syk in acute renal allograft rejection. Syk inhibition may be a useful addition to T-cell-based immunotherapy in renal transplantation., (© 2014 The Authors. International Journal of Experimental Pathology © 2014 International Journal of Experimental Pathology.)

Background: Production of reactive oxygen species (ROS) and thereby induction of oxidative stress seem to be one of the major mediators of inflammatory adverse outcomes after renal transplantation. p22(phox) is a polymorphic subunit of NAD(P)H-oxidase that is critical for activation and stabilization of the enzyme. This enzyme is involved in the production of superoxide that triggers inflammatory injuries to the kidney. So in this study, the association between donors and recipients' C242T polymorphism of p22(phox) and acute rejection (AR), delayed graft function (DGF), creatinine clearance (CrCl), and blood pressure in renal-allograft recipients was studied., Methods: One hundred ninety six donor-recipient pairs were studied. The C242T polymorphism of p22(phox) was determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). According to p22 genotype, the subjects were divided in wild-type (CC) and T allele carriers (CT+TT). Transplantation outcomes were determined using acute rejection and delayed graft function criteria. The mean arterial pressure was also measured monthly after transplantation., Results: There was a significant association between the recipients' p22(phox) polymorphism and DGF occurrence (OR=2.5, CI: 1.2-4.9, p=0.0009). No significant association was detected between donors' p22(phox) polymorphism and AR and DGF events. CrCl during the six months follow-up after transplantation was lower in the patients who received allograft from donors carrying 242T allele (B=-12.8, CI: -22.9-12.8 (-22.9 to -2.6)). Changes in the blood pressure were not different among the patients having different genotypes of p22(phox)., Conclusion: These results suggest that the recipients' p22(phox) C242T polymorphism may be a major risk factor for DGF in renal transplantation. Moreover, the donors' 242T allele seems to affect the rate of CrCl in the renal allograft recipients., (Copyright © 2014. Published by Elsevier B.V.)

Acute rejection is a major risk factor for chronic allograft injury (CAI). Blood leukocytes interacting with allograft endothelial cells during acute rejection were suggested to contribute to the still enigmatic pathogenesis of CAI. We hypothesize that tissue transglutaminase (Tgm2), a multifunctional protein and established marker of M2 macrophages, is involved in acute and chronic graft rejection. We focus on leukocytes accumulating in blood vessels of rat renal allografts (Fischer-344 to Lewis), an established model for reversible acute rejection and CAI. Monocytes in graft blood vessels overexpress Tgm2 when acute rejection peaks on day 9 after transplantation. Concomitantly, caspase-3 is activated, suggesting that Tgm2 expression is linked to apoptosis. After resolution of acute rejection on day 42, leukocytic Tgm2 levels are lower and activated caspase-3 does not differ among isografts and allografts. Cystamine was applied for 4 weeks after transplantation to inhibit extracellular transglutaminase activity, which did, however, not reduce CAI in the long run. In conclusion, this is the first report on Tgm2 expression by monocytes in vivo. Tgm2 may be involved in leukocytic apoptosis and thus in reversion of acute rejection. However, our data do not support a role of extracellular transglutaminase activity as a factor triggering CAI during self-limiting acute rejection.

Aim: The purpose of this study was to develop a population pharmacokinetic and pharmacodynamic (PK-PD) model for mycophenolic acid (MPA) in paediatric renal transplant recipients in the early post-transplant period., Methods: A total of 214 MPA plasma concentrations-time data points from 24 patients were available for PK model development. In 17 out of a total of 24 patients, inosine monophosphate dehydrogenase (IMPDH) enzyme activity measurements (n = 97) in peripheral blood mononuclear cells were available for PK-PD modelling. The PK-PD model was developed using non-linear mixed effects modelling sequentially by 1) developing a population PK model and 2) incorporating IMPDH activity into a PK-PD model using post hoc Bayesian PK parameter estimates. Covariate analysis included patient demographics, co-medication and clinical laboratory data. Non-parametric bootstrapping and prediction-corrected visual predictive checks were performed to evaluate the final models., Results: A two compartment model with a transit compartment absorption best described MPA PK. A non-linear relationship between dose and MPA exposure was observed and was described by a power function in the model. The final population PK parameter estimates (and their 95% confidence intervals) were CL/F, 22 (14.8, 25.2) l h(-1) 70 kg(-1) ; Vc /F, 45.4 (29.6, 55.6) l; Vp /F, 411 (152.6, 1472.6)l; Q/F, 22.4 (16.0, 32.5) l h(-1) ; Ka , 2.5 (1.45, 4.93) h(-1) . Covariate analysis in the PK study identified body weight to be significantly correlated with CL/F. A simplified inhibitory Emax model adequately described the relationship between MPA concentration and IMPDH activity. The final population PK-PD parameter estimates (and their 95% confidence intervals) were: E0 , 3.45 (2.61, 4.56) nmol h(-1)  mg(-1) protein and EC50 , 1.73 (1.16, 3.01) mg l(-1) . Emax was fixed to 0. There were two African-American patients in our study cohorts and both had low IMPDH baseline activities (E0 ) compared with Caucasian patients (mean value 2.13 mg l(-1) vs. 3.86 mg l(-1) )., Conclusion: An integrated population PK-PD model of MPA has been developed in paediatric renal transplant recipients. The current model provides information that will facilitate future studies and may be implemented in a Bayesian algorithm to allow a PK-PD guided therapeutic drug monitoring strategy., (© 2014 The British Pharmacological Society.)

Mammalian target of rapamycin inhibitors (mTOR-I), everolimus and sirolimus, are immunosuppressive drugs extensively used in renal transplantation. Their main mechanism of action is the inhibition of cell signaling through the PI3 K/Akt/mTOR pathway. This interesting mechanism of action confers to these medications both great immunosuppressive potential and important anti-neoplastic properties. Although the clinical utility of this drug category, as with other antineoplastic/immunosuppressants, is clear, the use of mTOR-I commonly results in the development of several complications. In particular, these agents may determine severe renal toxicity that, as recent studies report, seems clearly correlated to dose and duration of drug use. The mTOR-I-induced renal allograft spectrum of toxicity includes the enhanced incidence of delayed graft function, nephrotoxicity in particular when co-administered with calcineurin inhibitors (CNI) and onset of proteinuria. The latter effect appears highly frequent in patients undergoing mTOR-I treatment and significantly associated with a rapid graft lost. The damage leading to this complication interests both the glomerular and tubular area. mTOR-I cause an inhibition of proliferation in podocytes and the epithelial-to-mesenchymal transition in tubular cells. Interestingly, all these side effects are mostly reversible and dose related. Therefore, it is unquestionable that these particular drugs should be administered at the lowest dose able to maintain relatively low trough levels, in order to maximize their important and specific therapeutic effects while minimizing or avoiding drug toxicities. Utilization of low dosages of mTOR-I should be encouraged not only in CNI-combined schemas, but also when administered alone in a CNI-free immunosuppressive protocol.

Acute allograft rejection remains a major problem in solid organ transplantation. The enzyme α-enolase has been shown to induce an immune response in cardiac transplantation. In this study, we investigated the role of α-enolase in acute allograft rejection in a rat model of heart transplantation. Hearts from either (WF: RT1(u) ) or (Lew: RT1(1) ) rats were transplanted into (Lew: RT1(1) ) rats. No rejection occurred in the isograft group, for which the median survival time was >168 days, whereas the median survival time of the allograft group was significantly less at 10 ± 2.1 days (n = 8 per group, p < 0.001). Increased inflammation was observed in allografts, including increased α-enolase expression and increased numbers of infiltrating CD4(+) T cells (p < 0.05). By immunohistochemical staining, we confirmed that α-enolase was expressed not only in myocardial cells but also in the infiltrating lymphocytes. However, on the fifth day after transplantation, α-enolase expression was no longer observed in the lymphocytes (n = 3, p < 0.001). In contrast, no lymphocytes were found in isografts after transplantation (n = 3, p < 0.001). α-enolase expression was increased in lymphocytes, which are implicated in the acute rejection of cardiac transplants. Intragraft α-enolase inhibition may be useful as an adjuvant therapy to systemic immunosuppression in heart transplantation., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: To evaluate the effects and mechanism of indoleamine 2, 3-dioxygenase (IDO) modified rat bone marrow mesenchymal stem cells (BMSCs) in composite tissue allograft rejection., Methods: BMSCs isolated from Brown Norway (BN) rats (aged, 4-6 weeks) were infected by IDO [green fluorescent protein (GFP)]-lentivirus. The high expression target gene and biological activity cell line (IDO-BMSCs) were screened. IDO mRNA and protein expressions were detected by RT-PCR and Western blot. The biological activity of IDO in supernatant was detected by measuring the amount of kynurenine generation. In mixed lymphocyte reaction system, different numbers of IDO-BMSCs mixed with responding cells (peripheral blood mononuclear cell isolated from 4-6-week-old LEWIS rats, as recipient) and stimulating cells (peripheral blood mononuclear cell isolated from BN rats, as donor), with the cells ratios of 1:5:5, 1:10:10, 1:50:50, and 1:100:100 (as experimental groups 1, 2, 3, and 4, respectively). Each reaction system was blocked by 1 mmol/L 1-methyl-tryptophan (1-MT) (IDO specific inhibitor): IDO-BMSCs mixed with responding cells (1:5) as the negative control group, responding cells mixed with stimulating cells (1:1) as positive control group; and IDO-BMSCs were cultured in RPM11640 medium alone as blank control group. MTT assay was used to detect the T lymphocytes proliferation at 5 days. Furthermore, GFP-BMSCs (group A), IDO-BMSCs (group B), and normal saline (group C) were infused via the tail vein of allogeneic limb transplantation rats, and graft survival time and rejection were observed in each group., Results: The IDO expression of BMSCs after genetic modification was higher than that before genetic modification. IDO-BMSCs could significantly improved kynurenine concentration in culture medium supernatant when compared with GFP-BMSCs (P < 0.05). Before adding 1-MT, with the ratio of IDO-BMSCs to responding cells decreased, T lymphocytes proliferation rate increased in experimental groups 1, 2, and 3, showing significant differences between groups (P < 0.05); there was no significant difference between experimental group 4 and the positive control group (F > 0.05). After adding 1-MT, T lymphocytes proliferation rate was significantly higher than that before adding 1-MT in the other experimental groups (P < 0.05) except experimental group 4 (F > 0.05). In vivo, IDO-BMSCs could promote colonization in allograft, inhibit transplantation rejection, and prolong survival time of composite tissue allograft; the survival time of composite tissue allograft was (11.5 ± 0.6) days in group A, (14.5 ± 0.8) days in group B, and (9.0 ± 0.3) days in group C, and it was significantly longer in group B than in groups A and C, and in group A than in group C (P < 0.05)., Conclusion: JDO-BMSCs can promote the survival of allogeneic composite tissue grafts in rats, and its mechanism may involve in inhibition of T lymphocytes proliferation and promotion their own colonization in allograft.

Background: Cells of the monocyte/macrophage lineage have been implicated as effectors in acute allograft rejection based on short-term depletion studies. However, the therapeutic potential of targeting monocyte/macrophages in acute rejection is unknown. We investigated the potential of a c-fms kinase inhibitor (fms-I) in acute renal allograft rejection., Methods: Lewis rats underwent bilateral nephrectomy and received an orthotopic Dark Agouti renal allograft. Recipients received fms-I or vehicle from the time of transplantation until being killed on day 5., Results: Vehicle-treated rats developed severe allograft rejection with massive macrophage and T-cell infiltration. In contrast, fms-I substantially inhibited renal allograft dysfunction and structural damage with abrogation of macrophage and dendritic cell infiltration but had only a minor effect on the T-cell infiltrate. However, fms-I suppressed T-cell activation within the allograft, whereas systemic T- and B-cell activation was not affected. In a longer-term study to assess therapeutic potential, fms-I-treated rats developed severe antibody-mediated rejection on day 8 after transplantation. These transplants exhibited features of antibody-mediated rejection including capillaritis with thrombosis, acute tubular injury, IgG and C4d deposition, and neutrophil infiltration and activation. Interestingly, T-cell activation within these rejecting allografts remained suppressed, indicating separation of T-cell and antibody-mediated rejection., Conclusion: This study demonstrates the ability of c-fms kinase blockade to selectively deplete monocyte/macrophages in acute allograft rejection, although this did not result in significant prolongation of allograft survival. Furthermore, we identify contrasting roles for macrophages in cellular and humoral mechanisms of acute renal allograft rejection.

Background: The efficiency of islet graft survival after intraportal implantation is compromised by host innate immune responses and the production of proinflammatory cytokines that cause acute cellular injury. This reaction activates intraislet nuclear factor-κB (NF-κB), causing production of gene products that have detrimental effects on β-cell survival and function. We hypothesized that small interfering RNA targeting of IKKβ, a crucial kinase in the NF-κB activation pathway, in islets before transplantation would ameliorate the detrimental effects of cytokines and improve islet survival after transplantation., Methods: To test this hypothesis, we prepared small interfering RNA-based spherical nucleic acid nanoparticle conjugates targeting IKKβ IKKβ SNA-NCs). We treated isolated islets with IKKβ SNA-NCs and assessed the functional consequences of IKKβ knockdown in vitro and after intraportal transplantation in mice., Results: Treatment of freshly isolated mouse islets with IKKβ SNA-NCs reduced constitutive IKKβ expression and protected against proinflammatory cytokine-induced NF-κB activation, resulting in improved cell viability and decreased expression of gene products associated with β-cell dysfunction. Intraportal transplantation of a marginal mass (50 islets) of syngeneic islets treated with nanoparticle conjugates targeting IKKβ resulted in reversion to normoglycemia in 50% of streptozotocin-induced diabetic recipients (n=12) compared with 0% of controls (n=12). Histologic analyses showed reduced CD11b(+) cellular infiltration and decreased islet apoptosis., Conclusions: These results are consistent with the hypothesis that inhibition of intraislet NF-κB activation ameliorates the detrimental effects of host cytokines and demonstrates that preconditioning freshly isolated islets in culture with IKKβ SNA-NCs may be a promising therapy to enhance islet graft function and survival after transplantation.

Background: Both acute rejection (AR) and major infection events (MIE) can reduce long-term allograft survival. We assessed the simultaneous efficacy of serum and urine biomarker indoleamine 2,3-dioxygenase (IDO) enzyme activity and peripheral blood CD4-ATP levels for AR and MIE association, respectively., Methods: We prospectively tested 217 blood and 167 urine serial samples, collected monthly for 12 months after transplantation from 29 consecutive children receiving a kidney transplant. The indoleamine 2,3-dioxygenase activity was assessed by mass spectrometry assays using the ratio of product L-kynurenine (kyn) to substrate tryptophan (trp). Kyn/trp ratios and blood CD4 T-cell ATP levels were correlated with AR, MIE, or stable group (no events) in the next 30 days., Results: Using absolute cutoffs and allocating to samples to AR, MIE, or stable group, mean serum kyn/trp ratios were significantly elevated in the group that experienced AR (P=0.0007). Similarly, peripheral blood CD4-ATP levels were significantly lower in the group experiencing MIE (P=0.0351). Urine kyn/trp ratios and blood tacrolimus levels were not different between AR and stable groups. Within-subject analyses, accounting for repeated measures in subjects, also showed that, over time, serum kyn/trp ratios were higher before AR (P=0.031) and blood CD4-ATP levels were lower before MIE (P=0.008)., Conclusions: These results from our pilot discovery group suggest that a panel of biomarkers together can predict overimmunosuppression or underimmunosuppression. Further independent validation in a multicenter cohort is suggested.