Accurate Visualization of C4d Complement Fragment in Immunohistochemistry by C-Terminal Linear Neoepitope-Specific Antibodies.

C4d is the end degradation product of activated complement component C4b that appears during the early steps of the classical and lectin complement pathways. Within the primary sequence of C4d, there is a reactive thioester group that binds covalently to nearby surfaces, thus labeling the locations of complement activation. This feature makes C4d a target for immunohistochemical staining aimed to aid the diagnosis of, among others, the antibody-mediated rejection of transplanted organs, membranous glomerulonephritis, bullous pemphigoid, or inflammatory myopathies. However, the credibility of C4d immunostaining is debatable, as a high background in surrounding tissues and body fluids and diffused patterns of deposits in target structures are experienced with some of the available anti-C4d antibodies. Herein, we present an improved version of a rabbit anti-C4d antibody, originally raised against the C-terminal linear neoepitope of this complement fragment. Minor cross-reactivity with C4b and native C4 proteins, measured by ELISAs, as well as relatively low concentrations necessary for obtaining a specific signal in immunohistochemical analyses of formalin-fixed paraffin-embedded material, makes the improved antibody superior to commercially available rabbit monoclonal anti-C4d antibody SP91 dedicated to ex vivo diagnostics, as demonstrated by the staining of a panel of kidney transplant biopsies.

Contribution of tryptophan and its metabolites to transplant outcome: a mini-review.

Long-term tolerance in the absence of immunosuppressive drugs is a major goal in the transplantation field, not yet attained. Recent research on the role of commensal microbiota in the control of immunity has opened new avenues for the search of novel clinical interventions. Indeed, products of intestinal metabolism generated by both host cells and the microbiota have been identified as modulators of the immune response. Among these, tryptophan (Trp) and its derivatives are being investigated to understand their impact on alloimmunity and their potential usefulness as therapeutic targets to improve allograft survival. Here, we reviewed the latest findings on the contribution of Trp metabolic pathways to transplant outcomes.

FOS+ Macrophages Promote Chronic Rejection of Cardiac Transplantation.

OBJECTIVES: Chronic rejection remains the leading cause of progressive decline in graft function. Accumulating evidence indicates that macrophages participate in chronic rejection dependent on CD40-CD40L. The FOS family members are critical in inflammatory and immune responses. However, the mechanisms underlying the role of FOS family members in chronic rejection remain unclear. In this study, we aimed to elucidate the role and underlying mechanisms of FOS-positive macrophages regulated by CD40 that mediate chronic allograft rejection. MATERIALS AND METHODS: We downloaded publicly accessible chronic rejection kidney transplant single-cell sequencing datasets from the gene expression omnibus database. Differentially expressed genes between the CD40hi and CD40low macrophage chronic rejection groups were analyzed. We established a chronic rejection mouse model by using CTLA-4-Ig. We treated bone marrow-derived macrophages with an anti-CD40 antibody. We assessed expression of the FOS family by flow cytometry, real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. We identified altered signaling pathways by using RNA sequencing analysis. We detected DNA specifically bound to transcription factors by using ChIP-sequencing, with detection of the degree of graft fibrosis and survival. RESULTS: FOS was highly expressed on CD40hi macrophages in patients with chronic transplantrejection. Mechanistically, we showed that CD40 activated NF-κB2 translocation into the nucleus to upregulate c-Fos and FosB expression, thus promoting chronic rejection of cardiac transplant.We showed thatNF-κB2 regulated c-Fos and FosB expression by binding to the c-fos and fosb promoter regions. Inhibition of c-Fos/activator protein-1 decreased graft fibrosis and prolonged graft survival. CONCLUSIONS: CD40 may activate transcription factor NF-κB2 translocation into the nucleus of macrophages to upregulate c-Fos and FosB expression, thus promoting chronic rejection of cardiac transplant. Inhibition of c-Fos/activator protein-1 decreased grafts fibrosis and prolonged graft survival.

Kynurenine Pathway after Kidney Transplantation: Friend or Foe?

Kidney transplantation significantly improves the survival of patients with end-stage kidney disease (ESKD) compared to other forms of kidney replacement therapy. However, kidney transplant recipients' outcomes are not fully satisfactory due to increased risk of cardiovascular diseases, infections, and malignancies. Immune-related complications remain the biggest challenge in the management of kidney graft recipients. Despite the broad spectrum of immunosuppressive agents available and more detailed methods used to monitor their effectiveness, chronic allograft nephropathy remains the most common cause of kidney graft rejection. The kynurenine (KYN) pathway is the main route of tryptophan (Trp) degradation, resulting in the production of a plethora of substances with ambiguous properties. Conversion of Trp to KYN by the enzyme indoleamine 2,3-dioxygenase (IDO) is the rate-limiting step determining the formation of the next agents from the KYN pathway. IDO activity, as well as the production of subsequent metabolites of the pathway, is highly dependent on the balance between pro- and anti-inflammatory conditions. Moreover, KYN pathway products themselves possess immunomodulating properties, e.g., modify the activity of IDO and control other immune-related processes. KYN metabolites were widely studied in neurological disorders but recently gained the attention of researchers in the context of immune-mediated diseases. Evidence that this route of Trp degradation may represent a peripheral tolerogenic pathway with significant implications for transplantation further fueled this interest. Our review aimed to present recent knowledge about the role of the KYN pathway in the pathogenesis, diagnosis, monitoring, and treatment of kidney transplant recipients' complications.

The ocular immunological alterations in the process of high-risk corneal transplantation rejection.

PURPOSE: This study aims to reveal the immunopathogenesis of the high-risk corneal transplantation using a comparative proteomic approach. METHODS: The immunological properties of ocular tissues (including corneal grafts, aqueous humour, and iris-ciliary body) were analysed using a high-risk rabbit corneal transplantation model employing a comparative proteomic approach. RESULTS: The corneal grafts revealed a dramatic increase in the immune response both at the early (postoperative day 7) and rejection stages, along with the appearance of transplantation stress-induced cellular senescence in the early stage. The aqueous humour (AH) displayed persistent pathological alterations, indicated by the significant enrichment of complement and coagulation cascades pathway in the early stage and interleukin (IL)-17 signalling pathway in the rejection stage. More surprisingly, the pronounced elevation of immune response was also observed in the iris-ciliary body (I-CB) tissues at the early and rejection stages. The enriched immune-related pathways were associated with antigen processing and presentation, complement and coagulation cascades, and IL-17 signalling pathway. Furthermore, proteomic analysis revealed that the implantation of Cyclosporine A drug delivery system (CsA-DDS) into the anterior chamber obviously mitigated corneal transplantation rejection by inhibiting immunoreaction both in the corneal grafts and I-CB tissues. CONCLUSION: The results highlighted the involvement of intraocular immunity both in the grafts and I-CB tissues during corneal transplantation rejection, further suggesting the anterior chamber as an optimal drug-delivery site for its treatment.

Salidroside ameliorates acute liver transplantation rejection in rats by inhibiting neutrophil extracellular trap formation.

Acute rejection is an important factor affecting the survival of recipients after liver transplantation. Salidroside has various properties, including anti-inflammatory, antioxidant, and hepatoprotective properties. This study aims to investigate whether salidroside can prevent acute rejection after liver transplantation and to examine the underlying mechanisms involved. An in vivo acute rejection model is established in rats that are pretreated with tacrolimus (1 mg/kg/d) or salidroside (10 or 20 mg/kg/d) for seven days after liver transplantation. In addition, an in vitro experiment is performed using neutrophils incubated with salidroside (1, 10, 50 or 100 µM). Hematoxylin-eosin staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, immunosorbent assays, immunofluorescence analysis, Evans blue staining, and western blot analysis are performed to examine the impact of salidroside on NET formation and acute rejection in vitro and in vivo. We find that Salidroside treatment reduces pathological liver damage, serum aminotransferase level, and serum levels of IL-1ß, IL-6, and TNF-α in vivo. The expressions of proteins associated with the HMGB1/TLR-4/MAPK signaling pathway (HMGB1, TLR-4, p-ERK1/2, p-JNK, p-P38, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, IL-1ß, TNF-α, and IL-6) are also decreased after salidroside treatment. In vitro experiments show that the release of HMGB1/TLR-4/MAPK signaling pathway-associated proteins from neutrophils treated with lipopolysaccharide is decreased by salidroside. Moreover, salidroside inhibits NETosis and protects against acute rejection by regulating the HMGB1/TLR-4/MAPK signaling pathway. Furthermore, salidroside combined with tacrolimus has a better effect than either of the other treatments alone. In summary, salidroside can prevent acute liver rejection after liver transplantation by reducing neutrophil extracellular trap development through the HMGB1/TLR-4/MAPK signaling pathway.

Cell-specific Extracellular Vesicles and Their miRNA Cargo Released Into the Organ Preservation Solution During Cold Ischemia Storage as Biomarkers for Liver Transplant Outcomes.

BACKGROUND: Liver transplantation (LT) is crucial for end-stage liver disease patients, but organ shortages persist. Donation after circulatory death (DCD) aims to broaden the donor pool but presents challenges. Complications like acute rejection, hepatic artery thrombosis, and biliary issues still impact posttransplant prognosis. Biomarkers, including extracellular vesicles (EVs) and microRNAs (miRNAs), show promise in understanding and monitoring posttransplant events. This study explores the role of EVs and their miRNA cargo in LT, including their potential as diagnostic tools. METHODS: EVs from intrahepatic end-ischemic organ preservation solution (eiOPS) in 79 donated livers were detected using different techniques (nanosight tracking analysis, transmission electron microscopy, and flow cytometry). EV-derived miRNAs were identified by quantitative real time-polymerase chain reaction. Bioinformatics analysis was performed using the R platform. RESULTS: Different-sized and origin-specific EVs were found in eiOPS, with significantly higher concentrations in DCD compared with donation after brain death organs. Additionally, several EV-associated miRNAs, including let-7d-5p , miR-28-5p , miR-200a-3p , miR-200b-3p , miR-200c-3p , and miR-429 , were overexpressed in DCD-derived eiOPS. These miRNAs also exhibited differential expression patterns in liver tissue biopsies. Pathway analysis revealed enrichment in signaling pathways involved in extracellular matrix organization and various cellular processes. Moreover, specific EVs and miRNAs correlated with clinical outcomes, including survival and early allograft dysfunction. A predictive model combining biomarkers and clinical variables showed promise in acute rejection detection after LT. CONCLUSIONS: These findings provide new insights into the use of EVs and miRNAs as biomarkers and their possible influence on posttransplantation outcomes, potentially contributing to improved diagnostic approaches and personalized treatment strategies in LT.

Cyclic guanosine monophosphate-adenosine monophosphate synthetase/stimulator of interferon genes signaling aggravated corneal allograft rejection through neutrophil extracellular traps.

The activation of innate immunity following transplantation has been identified as a crucial factor in allograft inflammation and rejection. However, the role of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)/stimulator of interferon genes (STING) signaling-mediated innate immunity in the pathogenesis of allograft rejection remains unclear. Utilizing a well-established murine model of corneal transplantation, we demonstrated increased expression of cGAS and STING in rejected-corneal allografts compared with syngeneic (Syn) and normal (Nor) corneas, along with significant activation of the cGAS/STING pathway, as evidenced by the enhanced phosphorylation of TANK-binding kinase 1and interferon regulatory factor 3. Pharmacological and genetic inhibition of cGAS/STING signaling markedly delayed corneal transplantation rejection, resulting in prolonged survival time and reduced inflammatory infiltration. Furthermore, we observed an increase in the formation of neutrophil extracellular traps (NETs) in rejected allografts, and the inhibition of NET formation through targeting peptidylarginine deiminase 4 and DNase I treatment significantly alleviated immune rejection and reduced cGAS/STING signaling activity. Conversely, subconjunctival injection of NETs accelerated corneal transplantation rejection and enhanced the activation of the cGAS/STING pathway. Collectively, these findings demonstrate that NETs contribute to the exacerbation of allograft rejection via cGAS/STING signaling, highlighting the targeting of the NETs/cGAS/STING signaling pathway as a potential strategy for prolonging allograft survival.

Suppressive Role of Pigment Epithelium-derived Factor in a Rat Model of Corneal Allograft Rejection.

BACKGROUND: Immunological rejection is the most common reason for corneal transplantation failure. The importance of T cells in corneal allograft rejection is well demonstrated. Recent studies highlight that pigment epithelium-derived factor (PEDF) plays an immunoregulatory role in ocular diseases by enhancing the suppressive phenotype of regulatory T cells besides its other functions in neurotrophy and antiangiogenesis. METHODS: The effects of PEDF on immune rejection were examined in rat models of corneal transplantation using slit-lamp microscope observation, immunohistochemistry, flow cytometry, and Western blot. In vitro, we demonstrated PEDF reduced alloreactive T-cell activation using real-time polymerase chain reaction, flow cytometry, and Western blot. RESULTS: Topical administration of PEDF provided corneal transplantation rats with an improved graft survival rate of corneal allografts, reduced hemangiogenesis, and infiltration of immune cells in corneas, in particular, type 17 T helper cells while increased regulatory T cells. Moreover, nerve reinnervation within grafts was promoted in PEDF-treated recipient rats. In vitro, PEDF inhibited alloreactive T-cell activation via the c-Jun N-terminal kinase/c-Jun signaling pathway and upregulated the expressions of interleukin-10 and transforming growth factor-ß, emphasizing the suppressive role of PEDF on immune responses. CONCLUSIONS: Our results underscore the feasibility of PEDF in alleviating corneal allograft rejection and further illustrate its potential in managing immune-related diseases.

The CaMK Family Differentially Promotes Necroptosis and Mouse Cardiac Graft Injury and Rejection.

Organ transplantation is associated with various forms of programmed cell death which can accelerate transplant injury and rejection. Targeting cell death in donor organs may represent a novel strategy for preventing allograft injury. We have previously demonstrated that necroptosis plays a key role in promoting transplant injury. Recently, we have found that mitochondria function is linked to necroptosis. However, it remains unknown how necroptosis signaling pathways regulate mitochondrial function during necroptosis. In this study, we investigated the receptor-interacting protein kinase 3 (RIPK3) mediated mitochondrial dysfunction and necroptosis. We demonstrate that the calmodulin-dependent protein kinase (CaMK) family members CaMK1, 2, and 4 form a complex with RIPK3 in mouse cardiac endothelial cells, to promote trans-phosphorylation during necroptosis. CaMK1 and 4 directly activated the dynamin-related protein-1 (Drp1), while CaMK2 indirectly activated Drp1 via the phosphoglycerate mutase 5 (PGAM5). The inhibition of CaMKs restored mitochondrial function and effectively prevented endothelial cell death. CaMKs inhibition inhibited activation of CaMKs and Drp1, and cell death and heart tissue injury (n = 6/group, p < 0.01) in a murine model of cardiac transplantation. Importantly, the inhibition of CaMKs greatly prolonged heart graft survival (n = 8/group, p < 0.01). In conclusion, CaMK family members orchestrate cell death in two different pathways and may be potential therapeutic targets in preventing cell death and transplant injury.

Rho-kinase inhibitor alleviates CD4+T cell mediated corneal graft rejection by modulating its STAT3 and STAT5 activation.

Penetrating keratoplasty remains the most common treatment to restore vision for corneal diseases. Immune rejection after corneal transplantation is one of the major causes of graft failure. In recent years, Rho-associated protein kinase (ROCK) inhibitors have been found to be associated with the activation of the STATs pathway and are widely studied in autoimmune diseases. Therefore, it may be possible that the ROCK inhibitors also participate in the local and systemic immune regulation in corneal transplantation through activation of the STATs pathway and affect the CD4+ T cell differentiation. This study aimed to explore the role of ROCK-STATs pathway in the occurrence of immune rejection in corneal transplantation by applying Y27632, a ROCK inhibitor, to the recipient mice and peripheral CD4+ T cells. We found that Y27632 significantly up-regulated the phosphorylation level of STAT5 in both spleen and lymph nodes, down-regulated the phosphorylation level of STAT3 in the CD4+ T cells in the spleen. It also increased the proportion of CD4+CD25+Foxp3+Helios+ Tregs while decreased CD4+IL17A+ -Th17 cells. Moreover, Y27632 also reduced the proportion of dendritic cells in both spleen and lymph nodes, as well as the expression level of CD86 on their surfaces in the spleen, while the proportion of macrophages was not affected. The expression levels of ROCK1, ROCK2, CD11c and IL-17A mRNA were also found to be low in the graft tissue while the expression of Helios was upregulated. Rho-kinase inhibitor can modulate the balance of Tregs/Th17 by regulating the phosphorylation levels of both STAT3 and STAT5, thereby inhibiting the occurrence of immune rejection in allogeneic corneal transplantation.

Inflammatory and hypoxic stress-induced islet exosomes released during isolation are associated with poor transplant outcomes in islet autotransplantation.

Islets experience enormous stress during the isolation process, leading to suboptimal endocrine function after total pancreatectomy with islet autotransplantation (TPIAT). Our investigation focused on inducing isolation stress in islets ex vivo, where proinflammatory cytokines and hypoxia prompted the release of stress exosomes (exoS) sized between 50 and 200 nm. Mass spectrometry analysis revealed 3 distinct subgroups of immunogenic proteins within these exoS: damage-associated molecular patterns (DAMPs), chaperones, and autoantigens. The involvement of endosomal-sorting complex required for transport proteins including ras-associated binding proteins7A, ras-associated binding protein GGTA, vacuolar protein sorting associated protein 45, vacuolar protein sorting associated protein 26B, and the tetraspanins CD9 and CD63, in exoS biogenesis was confirmed through immunoblotting. Next, we isolated similar exoS from the islet infusion bags of TPIAT recipients (N = 20). The exosomes from infusion bags exhibited higher DAMP (heat shock protein family A [Hsp70] member 1B and histone H2B) levels, particularly in the insulin-dependent TPIAT group. Additionally, elevated DAMP protein levels in islet infusion bag exosomes correlated with increased insulin requirements (P = .010) and higher hemoglobin A1c levels 1-year posttransplant. A deeper exploration into exoS functionality revealed their potential to activate monocytes via the toll-like receptor 3/7: DAMP axis. This stimulation resulted in the induction of inflammatory phenotypes marked by increased levels of CD68, CD80, inducible nitric oxide synthase, and cyclooxygenase-2. This activation mechanism may impact the successful engraftment of transplanted islets.

MEK1/2-PKM2 Pathway Modulates the Immunometabolic Reprogramming of Proinflammatory Allograft-infiltrating Macrophages During Heart Transplant Rejection.

BACKGROUND: Emerging evidence has highlighted the role of macrophages in heart transplant rejection (HTR). However, the molecular signals modulating the immunometabolic phenotype of allograft-infiltrating macrophages (AIMs) during HTR remain unknown. METHODS: We analyzed single-cell RNA sequencing data from cardiac graft-infiltrating immunocytes to characterize the activation patterns and metabolic features of AIMs. We used flow cytometry to determine iNOS and PKM2 expression and MEK/ERK signaling activation levels in AIMs. We then generated macrophage-specific Mek1/2 knockout mice to determine the role of the MEK1/2-PKM2 pathway in the proinflammatory phenotype and glycolytic capacity of AIMs during HTR. RESULTS: Single-cell RNA sequencing analysis showed that AIMs had a significantly elevated proinflammatory and glycolytic phenotype. Flow cytometry analysis verified that iNOS and PKM2 expressions were significantly upregulated in AIMs. Moreover, MEK/ERK signaling was activated in AIMs and positively correlated with proinflammatory and glycolytic signatures. Macrophage-specific Mek1/2 deletion significantly protected chronic cardiac allograft rejection and inhibited the proinflammatory phenotype and glycolytic capacity of AIMs. Mek1/2 ablation also reduced the proinflammatory phenotype and glycolytic capacity of lipopolysaccharides + interferon-γ-stimulated macrophages. Mek1/2 ablation impaired nuclear translocation and PKM2 expression in macrophages. PKM2 overexpression partially restored the proinflammatory phenotype and glycolytic capacity of Mek1/2 -deficient macrophages. Moreover, trametinib, an Food and Drug Administration-approved MEK1/2 inhibitor, ameliorated chronic cardiac allograft rejection. CONCLUSIONS: These findings suggest that the MEK1/2-PKM2 pathway is essential for immunometabolic reprogramming of proinflammatory AIMs, implying that it may be a promising therapeutic target in clinical heart transplantation.

The Sertoli Cell Complement Signature: A Suspected Mechanism in Xenograft Survival.

The complement system is an important component of transplant rejection. Sertoli cells, an immune regulatory testicular cell, survive long-term when transplanted across immunological barriers; thus, understanding the mechanisms behind this unique survival would be of great benefit to the transplantation field. This study focused on Sertoli cell inhibition of complement as relevant in xenotransplantation. Neonatal pig Sertoli cells (NPSCs) survived activated human complement in vitro while neonatal pig islet (NPI) aggregates and pig aortic endothelial cell (PAEC) survival were diminished to about 65% and 12%, respectively. PAECs cultured in NPSC-conditioned media and human complement demonstrated a 200% increase in survival suggesting that NPSCs secrete complement-inhibiting substances that confer protection. Bioinformatic and molecular analyses identified 21 complement inhibitors expressed by NPSCs with several significantly increased in NPSCs compared to NPIs or PAECs. Lastly, RNA sequencing revealed that NPSCs express 25 other complement factors including cascade components and receptors. Overall, this study identified the most comprehensive Sertoli cell complement signature to date and indicates that the expression of a variety of complement inhibitors ensures a proper regulation of complement through redundant inhibition points. Understanding the regulation of the complement system should be further investigated for extending xenograft viability.

Proteomics: Its Promise and Pitfalls in Shaping Precision Medicine in Solid Organ Transplantation.

Solid organ transplantation is an established treatment of choice for end-stage organ failure. However, all transplant patients are at risk of developing complications, including allograft rejection and death. Histological analysis of graft biopsy is still the gold standard for evaluation of allograft injury, but it is an invasive procedure and prone to sampling errors. The past decade has seen an increased number of efforts to develop minimally invasive procedures for monitoring allograft injury. Despite the recent progress, limitations such as the complexity of proteomics-based technology, the lack of standardization, and the heterogeneity of populations that have been included in different studies have hindered proteomic tools from reaching clinical transplantation. This review focuses on the role of proteomics-based platforms in biomarker discovery and validation in solid organ transplantation. We also emphasize the value of biomarkers that provide potential mechanistic insights into the pathophysiology of allograft injury, dysfunction, or rejection. Additionally, we forecast that the growth of publicly available data sets, combined with computational methods that effectively integrate them, will facilitate a generation of more informed hypotheses for potential subsequent evaluation in preclinical and clinical studies. Finally, we illustrate the value of combining data sets through the integration of 2 independent data sets that pinpointed hub proteins in antibody-mediated rejection.

Soluble HLA-G levels in heart transplant recipients: Dynamics and correlation with clinical outcomes.

PURPOSE: To describe the evolution of the serum levels of soluble HLA-G (s-HLA-G) during the first 12 months after heart transplantation (HT) and to correlate it with clinical outcomes. METHODS: Observational study based in a single-center cohort of 59 patients who underwent HT between December-2003 and March-2010. Soluble HLA-G levels were measured from serum samples extracted before HT, and 1, 3, 6 and 12 months after HT. The cumulative burden of s-HLA-G expression during the first post-transplant year was assessed by means of the area under the curve (AUC) of s-HLA-G levels over time and correlated with the acute rejection burden -as assessed by a rejection score-, the presence of coronary allograft vasculopathy (CAV) grade ≥ 1 and infections during the first post-transplant year; as well as with long-term patient and graft survival. Mean follow-up was 12.4 years. RESULTS: Soluble HLA-G levels decreased over the first post-transplant year (p = 0.020). The AUC of s-HLA-G levels during the first post-transplant year was higher among patients with infections vs. those without infections (p = 0.006). No association was found between the AUC of s-HLA-G levels and the burden of acute rejection or the development of CAV. Overall long-term survival, long-term survival free of late graft failure and cancer-free survival were not significantly different in patients with an AUC of s-HLA-G levels higher or lower than the median of the study population. CONCLUSIONS: Soluble HLA-G levels decreased over the first year after HT. Higher HLA-G expression was associated with a higher frequency of infections, but not with the burden of acute rejection or the development of CAV, neither with long-term patient or graft survival.

Investigation of Circulating MicroRNA Levels in Antibody-Mediated Rejection After Kidney Transplantation.

BACKGROUND: One of the most important possible complications determining long-term graft survival after kidney transplant is antibody-mediated rejection (ABMR). The criterion standard approach to recognize ABMR is currently the kidney biopsy with histopathologic analysis. However, this test has limitations because of difficulties in timing of sampling, the evaluability of histology because of the questionable representativeness of specimens, and the limited number of this intervention. Hence, new reliable, noninvasive biomarkers are required to detect the development of ABMR in time. METHODS: In this study, we analyzed the clinical data of 45 kidney transplant patients (mean age of 44.51 years, 20 male and 25 female subjects). These participants were recruited into 5 subcohorts based on their clinical status, histologic findings, and level of donor-specific anti-HLA antibodies. Circulating microRNAs (miR-21, miR-181b, miR-146a, miR-223, miR-155, miR-150) in plasma samples were quantified by quantitative polymerase chain reaction and their levels were correlated with the clinical characteristics in different subgroups. RESULTS: The relative expression of plasma miR-155 (P = .0003), miR-223 (P = .0316), and miR-21 (P = .0147) were significantly higher in patients who had subsequent histology-approved ABMR with donor-specific anti-HLA antibody positivity (n = 10) than in the "triple negative" group (n = 21), and miR-155 showed the highest sensitivity (90%) and specificity (81%) to indicate ABMR development based on receiver operating characteristic analysis. CONCLUSIONS: According to our preliminary data, plasma miR-155, miR-21, and miR-223 can indicate the development of ABMR after kidney transplant in correlation with classic clinical parameters. However, future studies with larger number of participants are necessary to further evaluate the diagnostic properties of blood miRNAs in prediction of this life-threatening condition.

Changes and Significance of IL-17 in Acute Renal Allograft Rejection in Rats.

BACKGROUND: To investigate the changes and significance of interleukin-17 (IL-17) in acute rejection following rat kidney transplantation. METHODS: Using inbred Sprague Dawley rats as donors and Wistar rats as recipients, an acute rejection model of kidney transplantation was established to evaluate the effects of IL-17. Reverse transcription polymerase chain reaction and immunohistochemistry were used to detect IL-17. RESULTS: Compared with those in the normal control group, the rats in the allogeneic transplantation (ATX) group had different degrees of acute rejection 3, 5, and 7 days after operation, and the expression of IL-17 mRNA in the transplanted kidney was significantly increased (P < .05). In the ATX group, acute rejection was observed 7 days after operation, and the integrated optical density (IOD) value of IL-17 was significantly increased (P < .05). Compared with the normal control group, acute rejection occurred in varying degrees at 3, 5, and 7 days after operation in the ATX group, and the IOD value of IL-17 significantly increased (P < .05). CONCLUSIONS: IL-17 expression is increased in acute rejection after renal transplantation in rats. Other surgical factors in addition to acute rejection had no effect on IL-17 expression in rat kidney transplants. The immunosuppressant cyclosporin A was used to prevent the expression of IL-17 in rats with acute rejection.

The mounted alloimmunity of the iris-ciliary body devotes a hotbed of immune cells for corneal transplantation rejection.

Graft rejection is still the major obstacle causing corneal transplantation failure. However, the underlying pathogenesis remains largely unclear. The iris-ciliary body (I-C) is enriched with blood vessels and various immune cell populations, presumably predisposed to be involved in corneal transplantation rejection. After penetrating keratoplasty, compared to the normal (Nor) and syngeneic (Syn) groups, I-C tissues in the allogeneic (Allo) group displayed stronger alloimmune responses, with more infiltrations of CD45+ inflammatory cells and CD3+ lymphocytes, increased transcriptional levels of pro-inflammatory cytokines, and elevated NF-κB activity. This histopathology was similar to the pathological alterations of corneal allografts. Angiography analysis revealed the abnormal vasculature in the iris during allograft rejection, characterized by vasodilatation, increased vessel density, and vascular permeability. While, immunofluorescence staining showed the intact tight junction of the posterior iris epithelium. In vitro, human microvascular endothelial cells (HMECs) stimulated by tumor necrosis factor-α (TNF-α) showed an increased Evans blue (EB)-albumin leakage, with lower expression of zonula occludens-1 (ZO-1) and Occludin. The increased EB-albumin leakage, up-regulated NF-κB activity, and reduced expression of ZO-1 and Occludin could be partially reversed after cyclosporine A (CsA) administration. In contrast, the barrier function in primary mouse iris pigment epithelial cells (IPEs) after TNF-α treatment remained largely unchanged. These findings revealed the vigorous alloimmunity in I-C tissues, characterized with impaired vascularization but intact posterior epithelial barrier in the iris, which allowed proteins and immune cells to be exudated from the front surface of I-C tissues, and facilitated immune reaction in the anterior chamber, thereby contributing to aggravated corneal transplantation rejection.

Donor CYP3A5 Expression Decreases Renal Transplantation Outcomes in White Renal Transplant Recipients.

BACKGROUND After renal transplantation, immunosuppressants should be administered to prevent organ rejection and prolong graft survival. One of them is tacrolimus, which is metabolized by the CYP3A enzyme family. The variability of the CYP3A5 gene in renal transplant recipients has been previously studied for its correlation with acute rejection and allogeneic kidney function. CYP3A5 enzyme is also present in the renal tissue, and its relevance has not yet been extensively investigated. This study aimed to evaluate the effect of donor and recipient CYP3A5 expression status on early and long-term transplant outcomes. MATERIAL AND METHODS Single-nucleotide polymorphism in CYP3A5 (rs776746) was analyzed in 95 kidney transplant recipients and their grafts. The effect of donor and recipient genotypes on the primary endpoint, which was the loss of the renal graft over 5-year follow-up, was assessed. The secondary endpoints were biopsy-proven acute rejection, proteinuria, delayed graft function, and renal function. RESULTS Patients who received a CYP3A5*1 allele-carrying kidney (n=16) were at greater risk of graft loss (adjusted hazard ratio, 95% CI: 10.61, 2.28-49.42, P=.003) than those with the CYP3A5*3/*3 genotype (n=79). Renal CYP3A5 expression was also a predictor of acute rejection between the 2nd and 12th post-transplant months (adjusted odds ratio, 95% CI: 4.36; 1.08-17.6, P=.038) and proteinuria at different time intervals. No effect of the recipient CYP3A5 genotype was observed. CONCLUSIONS The donor CYP3A5 genotype is associated with inferior transplantation outcomes. Local renal tacrolimus metabolism is a potential target for improving long-term transplantation outcomes.