Ptc Score Research Articles

Automated evaluation with deep learning of total interstitial inflammation and peritubular capillaritis on kidney biopsies.

Interstitial inflammation and peritubular capillaritis are observed in many diseases on native and transplant kidney biopsies. A precise and automated evaluation of these histological criteria could help stratify patients' kidney prognoses and facilitate therapeutic management. We used a convolutional neural network to evaluate those criteria on kidney biopsies. A total of 423 kidney samples from various diseases were included; 83 kidney samples were used for the neural network training, 106 for comparing manual annotations on limited areas to automated predictions, and 234 to compare automated and visual gradings. The precision, recall and F-score for leukocyte detection were, respectively, 81%, 71% and 76%. Regarding peritubular capillaries detection the precision, recall and F-score were, respectively, 82%, 83% and 82%. There was a strong correlation between the predicted and observed grading of total inflammation, as for the grading of capillaritis (r=0.89 and r=0.82, respectively, all P<.0001). The areas under the receiver operating characteristics curves for the prediction of pathologists' Banff total inflammation (ti) and peritubular capillaritis (ptc) scores were respectively all above 0.94 and 0.86. The kappa coefficients between the visual and the neural networks' scores were respectively 0.74, 0.78 and 0.68 for ti ≥1, ti ≥2 and ti ≥3, and 0.62, 0.64 and 0.79 for ptc ≥1, ptc ≥2 and ptc ≥3. In a subgroup of patients with immunoglobulin A nephropathy, the inflammation severity was highly correlated to kidney function at biopsy on univariate and multivariate analyses. We developed a tool using deep learning that scores the total inflammation and capillaritis, demonstrating the potential of artificial intelligence in kidney pathology.

Mesangiolysis in Pathological Chronic Active Antibody-Mediated Rejection in Kidney Transplant Biopsies

Introduction: This study aimed to determine if immune or nonimmune and acute or chronic lesions associated with mesangiolysis (MGLS) occurred in biopsy-proven pathological chronic active antibody-mediated rejection (P-CAABMR) in kidney transplant biopsies. Methods: We evaluated MGLS in 41 patients with biopsy findings of P-CAABMR from January 2016 to December 2019. Histological scoring was evaluated by Banff classification. Multivariate logistic regression analysis was performed using a forward selection method. Results: Fifteen of the 41 P-CAABMR biopsies (36.6%) cases showed MGLS. The estimated glomerular filtration rate (eGFR) was significantly lower in the MGLS-positive compared with the MGLS-negative group, and proteinuria was significantly higher in the MGLS-positive compared with the MGLS-negative group. In the clinical model, multivariate analysis was performed using covariates of eGFR and duration after transplantation significantly correlated with MGLS by simple analysis, in addition to type of calcineurin inhibitor use (tacrolimus or cyclosporine), donor-specific antibodies, diabetes, and hypertension grade defined by use of antihypertensive therapy or/and blood pressure level. Only hypertension grade was significantly correlated with MGLS. In the pathological model, multivariate analysis was performed using the presence of FSGS and the aah and cg scores significantly correlated with MGLS by simple analysis, in addition to g and ptc scores. The cg score was significantly correlated with hypertension grade, duration after transplantation, g, ah, and aah. Conclusion: Lower graft function and higher proteinuria was observed in MGLS of P-CAABMR. The Banff cg score was independently related to MGLS in multivariate analysis. Sustained glomerulitis, calcineurin inhibitor nephrotoxicity, and hypertension may cause Banff cg lesions, leading to MGLS in P-CAABMR.

A Banff-based histologic chronicity index is associated with graft loss in patients with a kidney transplant and antibody-mediated rejection

Antibody-mediated rejection (AMR) is the major cause of graft loss in kidney transplant recipients. The Banff classification defines two classes of AMR, active and chronic active but over time this classification has become increasingly complex. To simplify the approach to AMR, we developed activity and chronicity indices based on kidney transplant biopsy findings and examined their association with graft survival in 147 patients with active or chronic active AMR, all of whom had donor-specific antibodies and were treated for AMR. The activity index was determined as the sum of Banff glomerulitis (g), peritubular capillaritis (ptc), arteritis (v) and C4d scores, with a maximum score of 12. The chronicity index was the sum of interstitial fibrosis (ci), tubular atrophy (ct), chronic vasculopathy (cv), and chronic glomerulopathy (cg) scores, the latter doubled, with a maximum score of 15. While the activity index was generally not associated with graft loss, the chronicity index was significantly associated with graft loss with an optimal threshold value of 4 or greater for predicting graft loss. The association of the chronicity index of 4 or greater with graft loss was independent of other parameters associated with graft loss, including the estimated glomerular filtration rate at the time of biopsy, chronic active (versus active) AMR, AMR with de novo (versus persistent/rebound) donor-specific antibodies, Banff (g+ptc) scores, concurrent T cell-mediated rejection and donor-specific antibody reduction post-biopsy. The association of the chronicity index of 4 or greater with graft loss was confirmed in an independent cohort of 61 patients from Necker Hospital, Paris. Thus, our findings suggest that the chronicity index may be valuable as a simplified approach to decision-making in patients with AMR.

Urinary Exosomal Cystatin C and Lipopolysaccharide Binding Protein as Biomarkers for Antibody-Mediated Rejection after Kidney Transplantation.

We aimed to discover and validate urinary exosomal proteins as biomarkers for antibody−mediated rejection (ABMR) after kidney transplantation. Urine and for-cause biopsy samples from kidney transplant recipients were collected and categorized into the discovery cohort (n = 36) and a validation cohort (n = 65). Exosomes were isolated by stepwise ultra-centrifugation for proteomic analysis to discover biomarker candidates for ABMR (n = 12). Of 1820 exosomal proteins in the discovery cohort, four proteins were specifically associated with ABMR: cystatin C (CST3), serum paraoxonase/arylesterase 1, retinol-binding protein 4, and lipopolysaccharide−binding protein (LBP). In the validation cohort, the level of urinary exosomal LBP was significantly higher in the ABMR group (n = 25) compared with the T-cell-mediated rejection (TCMR) group and the no major abnormality (NOMOA) group. Urinary exosomal CST3 level was significantly higher in the ABMR group compared with the control and NOMOA groups. Immunohistochemical staining showed that LBP and CST3 in the glomerulus were more abundant in the ABMR group compared with other groups. The combined prediction probability of urinary exosomal LBP and CST3 was significantly correlated with summed LBP and CST3 intensity scores in the glomerulus and peritubular capillary as well as Banff g + ptc scores. Urinary exosomal CST3 and LBP could be potent biomarkers for ABMR after kidney transplantation.

401.3: Microvascular Inflammation in Renal Transplant Recipients Without Antibody Mediated Rejection

Purpose: Microvascular inflammation (MVI) defined by glomerulitis (g) and peritubular capillaritis (ptc) is a hallmark of ABMR in renal transplantation. The prevalence and significance of MVI in patients without ABMR is less understood. Methods: Here, we analyzed MVI in the 1st post-transplant year, its relationship to DSA, and its clinical significance in a large cohort of patients without ABMR. Along with any for-cause biopsy (fcBx), patients underwent 2 protocol biopsies (pBx, 3 & 12mos). Bx were categorized as early (0-4mos) or late (5-12mos). Serum was screened for DSA at 0, 1, 3, 6, 9 & 12mos post-transplant. Results: MVI Prevalence: 956/1187 patients transplanted between 2013-18 underwent 1950 Bx (67% protocol, 33% for-cause). Of these Bx, 64 had either ABMR or GN and were excluded. MVI was noted in 23% (g+ptc score 1-1.5 in 13% & ≥2 in 10%) of the remaining 1887 Bx with greater prevalence in fcBx (fcBx 14% vs. pBx 7%) and late Bx (late Bx 29% vs. early Bx 17%). Of note, the MVI prevalence and its severity increased with increasing grades of tubulointerstitial inflammation (TII) (Fig 1A). Of note, 65% of patients with allograft infections (pyelonephritis & BKVN) had MVI. MVI & DSA: DSA was detected concurrently in 12% of all the 1887 Bx without ABMR. Although, MVI was associated with concurrent DSA detection in these Bx (OR 1.8, 95% CI 1.3-2.5, p<0.,001), ~60% of the biopsies never had DSA (concurrent/historic or future). Furthermore, in biopsies stratified by the grade of TCMR, there was no significant difference in concurrent DSA detection between patients with and without MVI (Fig.1B). Outcomes: MVI in patients without ABMR was associated with decreased 7yr-graft survival (Fig 1C, p<0.001). Importantly, the combination of MVI and TII was associated with worse graft survival when compared to either no inflammation or TII alone (Fig1C, p<0.001). Further, in a select sub-group of patients with allograft infection, MVI was associated with a trend towards decreased graft survival compared to no MVI (76% vs. 88%). Finally, MVI was associated with poor graft survival independent of potential confounders including DSA (HR 2.5, 95% CI 1.6-3.4, p<0.001). Conclusion: MVI is common even in the absence of ABMR or DSA and is a marker for the severity of allograft inflammation and subsequent poor clinical outcomes.

Magnetic Resonance Elastography-derived Stiffness Predicts Renal Function Loss and Is Associated With Microvascular Inflammation in Kidney Transplant Recipients.

Background.Organ stiffening can be caused by inflammation and fibrosis, processes that are common causes of transplant kidney dysfunction. Magnetic resonance elastography (MRE) is a contrast-free, noninvasive imaging modality that measures kidney stiffness. The objective of this study was to assess the ability of MRE to serve as a prognostic factor for renal outcomes.Methods.Patients were recruited from the St Michael’s Hospital Kidney Transplant Clinic. Relevant baseline demographic, clinical, and Banff histologic information, along with follow-up estimated glomerular filtration rate (eGFR) data, were recorded. Two-dimensional gradient-echo MRE imaging was performed to obtain kidney “stiffness” maps. Binary logistic regression analyses were performed to examine for relationships between stiffness and microvascular inflammation score. Linear mixed-effects modeling was used to assess the relationship between stiffness and eGFR change over time controlling for other baseline variables. A G2-likelihood ratio Chi-squared test was performed to compare between the baseline models with and without “stiffness.”Results.Sixty-eight transplant kidneys were scanned in 66 patients (mean age 56 ± 12 y, 24 females), with 38 allografts undergoing a contemporaneous biopsy. Mean transplant vintage was 7.0 ± 6.8 y. In biopsied allografts, MRE-derived allograft stiffness was associated only with microvascular inflammation (Banff g + ptc score, Spearman ρ = 0.43, P = 0.01), but no other histologic parameters. Stiffness was negatively associated with eGFR change over time (Stiffness × Time interaction β = –0.80, P < 0.0001), a finding that remained significant even when adjusted for biopsy status and baseline variables (Stiffness × Time interaction β = –0.46, P = 0.04). Conversely, the clinical models including “stiffness” showed significantly better fit (P = 0.04) compared with the baseline clinical models without “stiffness.”Conclusions.MRE-derived renal stiffness provides important prognostic information regarding renal function loss for patients with allograft dysfunction, over and above what is provided by current clinical variables.

Relationship of training load and wellness status with player’s in-game basketball performance: An automated exploratory data analysis case

The purpose of the study was to examine the relationships of different training load variables and wellness responses, with in-game basketball performance across playing positions (backcourt and frontcourt players). External load variables (e.g., total distance, accelerations, etc), internal responses (e.g., rate of perceived exertion [RPE]) and wellness status (e.g., Hooper index) were monitored during 7 consecutive in-season months on 15 professional male basketball players. Besides, game-related individual statistics (performance index rating [PIR], and player total contribution [PTC]) were used to assess the performance during competition. Although no positive relationship was found between training load variables and basketball game performance, some wellness questionnaire parameters were significantly associated to game-related individual statistics. In particular, we found that only competing against direct rivals, players that reported stable values of stress stability achieved significantly higher PTC and PIR scores than players with high variability in stress values (8.53 IQR [6.09, 14.8] vs 0.00 IQR [-0.46, 0.84], and 0.47 ± 0.40 vs 0.10 ± 0.50; respectively; P &lt; 0.05). Similarly, players with variable values of stress managed to maximize PIR scores during losses compared to players that presented high variability in stress levels (0.42 IQR [0.27, 0.55] vs 0.00 IQR [-0.12, 0.37], P &lt; 0.05). Regarding playing positions, backcourt players showed higher PTC scores compared to frontcourt players when the fatigue levels are stable during the microcycle (8.27 ± 5.75 vs 4.77 ± 4.42; P &lt; 0.05). Because basketball teams tend to accumulate more backcourt players that frontcourt players, it would be advisable to control training load maintaining it stable and avoiding load spikes during microcycle to allow team performance optimization. In conclusion, the results suggest that the best performances during official competition are not associated with higher training external and internal loads. Nevertheless, the wellness status could provide useful information when assessing player’s training responses and approaching possible peak performance during basketball competition. This shows the necessity to assess basketball performance from a holistic approach and consider more than just physical and physiological parameters, such as decision-making and psychological capacities, to better understand player’s performance during basketball competition.

FC 108: Gene Expression Profiles of Peritubular Capillaritis in Chronic Antibody-Mediated Rejection

Abstract BACKGROUND AND AIMS Chronic antibody-mediated rejection (ABMR) is one of the most significant contributors to late allograft loss. Hallmark characteristics of ABMR include microvascular injury (MVI) lesions such as peritubular capillaritis (ptc) and glomerulitis (g). Diffuse ptc (extent &amp;gt; 50%) was associated with worse graft survival independent from the ptc score. Nevertheless, current ptc thresholds are arbitrarily defined and may not reflect pathophysiological phenotypes accurately enough. The Banff peritubular capillaritis working group has been re-established to study the diagnostic and prognostic relevance of the ptc extent in different scenarios. We hypothesize that the re-assessment of chronic ABMR specimens using Nanostring NCounter based gene expression analysis allows the definition of novel thresholds of ptc extent, reflecting molecular ABMR phenotypes more accurately. METHOD We retrospectively analysed 25 patients with historical diagnosis of ABMR/chronic ABMR and presence of donor specific antibodies (including 44 biopsies). Patients were treated at two different Austrian centres (Medical University of Vienna and Ordensklinikum—Elisabethinnen Linz). Peritubular capillaritis was re-evaluated by an experienced external nephropathologist (M.M.) and included the ptc score as well as the ptc extent (focal ptc: 10–50% capillaries involved, diffuse ptc: &amp;gt;50%). Nanostring nCounter Gene expression analysis was performed with a customized gene set corresponding to the recommendations of molecular ABMR phenotypes in Banff 2017 guidelines (including over 200 genes). To test the correlation of gene expression levels and histological scores, gene expression levels of all 44 patients were divided into quartiles. Gene expressions above the first quartile (ABMRQ&amp;gt;1) were considered as positive values for ROC analysis. RESULTS Ptc was categorized as followed: no ptc in 13, focal ptc in 23 and diffuse ptc in seven biopsies [median ptc extent 25/0–40% (median/IQR)]. ABMR was diagnosed in 27 (67.5%) biopsies, mixed rejection in five (12.5%) and borderline TCMR in three (7.5%). In biopsies with diffuse ptc significant higher gene expressions were found in the ABMR gene set [63/55–83 versus 32/27–52; (median/IQR); P = 0.012], the ABMR exhaust gene set (390/245–609 versus 245/128–358; P = 0.022), the Eculizumab gene set (180/143–339 versus 65/57–133; P = 0.0027) and the TCMR gene set (48/40–75 versus 25/19–35; P = 0.001). Sensitivity analysis revealed improved AUCs for predicting biopsies with ABMR gene expressions over the first quartile with a ptc cutoff of 35% compared to ptc cut-off of 50% [ptc&amp;gt;35: AUC 0.76/0.61–0.90 (95% confidence interval); P = 0.013; ptc&amp;gt;50: AUC: 0.71/0.54–0.88; P = 0.039]. The new ptc&amp;gt;35% cutoff also provided higher AUCs for the prediction of gene expressions over the 25th percentile in all other analysed rejection-associated gene sets. CONCLUSION With the application of gene expression-based Nanostring platform, we were able to identify a new threshold of ptc extent. The newly proposed cut off &amp;gt;35% may reflect molecular phenotypes of ABMR more accurate than the current one and could improve early diagnosis of ABMR.

Deletion of the Natural Killer Cell Receptor NKG2C Encoding KLR2C Gene and Kidney Transplant Outcome.

Natural killer (NK) cells may contribute to antibody-mediated rejection (ABMR) of renal allografts. The role of distinct NK cell subsets in this specific context, such as NK cells expressing the activating receptor NKG2C, is unknown. Our aim was to investigate whether KLRC2 gene deletion variants which determine NKG2C expression affect the pathogenicity of donor-specific antibodies (DSA) and, if so, influence long-term graft survival. We genotyped the KLRC2 wt/del variants for two distinct kidney transplant cohorts, (i) a cross-sectional cohort of 86 recipients who, on the basis of a positive post-transplant DSA result, all underwent allograft biopsies, and (ii) 1,860 recipients of a deceased donor renal allograft randomly selected from the Collaborative Transplant Study (CTS) database. In the DSA+ patient cohort, KLRC2 wt/wt (80%) was associated with antibody-mediated rejection (ABMR; 65% versus 29% among KLRC2 wt/del subjects; P=0.012), microvascular inflammation [MVI; median g+ptc score: 2 (interquartile range: 0-4) versus 0 (0-1), P=0.002], a molecular classifier of ABMR [0.41 (0.14-0.72) versus 0.10 (0.07-0.27), P=0.001], and elevated NK cell-related transcripts (P=0.017). In combined analyses of KLRC2 variants and a functional polymorphism in the Fc gamma receptor IIIA gene (FCGR3A-V/F158), ABMR rates and activity gradually increased with the number of risk genotypes. In DSA+ and CTS cohorts, however, the KLRC2 wt/wt variant did not impact long-term death-censored graft survival, also when combined with the FCGR3A-V158 risk variant. KLRC2 wt/wt may be associated with DSA-triggered MVI and ABMR-associated gene expression patterns, but the findings observed in a highly selected cohort of DSA+ patients did not translate into meaningful graft survival differences in a large multicenter kidney transplant cohort not selected for HLA sensitization.

Evaluation of Clazakizumab (Anti–Interleukin-6) in Patients With Treatment-Resistant Chronic Active Antibody-Mediated Rejection of Kidney Allografts

IntroductionInterleukin-6 (IL-6) is an important mediator of inflammation and activation of T cells, B cells, and plasma cells. Excessive IL-6 production is linked to human diseases characterized by unregulated antibody production, including alloimmunity, where persistence of donor-specific antibodies (DSAs), chronic active antibody-mediated rejection (cAMR), and graft loss are noted. Here, we report our experience investigating clazakizumab, a novel IL-6 inhibitor, in treating human leukocyte antigen (HLA)-sensitized patients with cAMR.MethodsBetween February 2018 and January 2019, 10 adults with biopsy-proven cAMR were enrolled in a phase 2, single-center, open-label study. Patients received clazakizumab 25 mg subcutaneously (s.c.) monthly for 12 months, with a 6-month protocol biopsy. Primary end points included patient survival, graft survival, estimated glomerular filtration rate (eGFR), and safety. Secondary end points assessed immune markers (DSAs, IgG, T-regulatory [Treg] cells). At 12 months, stable patients entered a long-term extension (LTE).ResultsLTE patients received clazakizumab for >2.5 years. Mean eGFRs showed significant declines from −24 months to study initiation (0 months) (52.8 ± 14.6 to 38.11 ± 12.23 ml/min per 1.73 m2, P = 0.03). However, after initiation of clazakizumab, eGFR stabilized at (41.6 ± 14.2 and 38.1 ± 20.3 ml/min per 1.73 m2, at 12 and 24 months, respectively). Banff 2017 analysis of pre- and post-treatment biopsies showed reductions in g+ptc and C4d scores. DSA reductions were seen in most patients. Adverse events (AEs) were minimal, and 2 graft losses occurred, both in patients who discontinued clazakizumab therapy at 6 months and 12 months after study initiation.ConclusionIn this small cohort of patients with cAMR, clazakizumab treatment showed a trend toward stabilization of eGFR and reductions in DSA and graft inflammation. No significant safety issues were observed. A randomized, placebo-controlled clinical trial (IMAGINE) of clazakizumab in cAMR treatment is underway (NCT03744910).

Renal Biopsy and Clinical Outcomes in Patients With ABO-Incompatible Renal Transplant: Experience From a Tertiary Care Hospital.

Renal transplant with ABO-incompatible donors expands the donor pool. Earlier studies have focused the use of protocol biopsies in ABO-incompatible transplant patients. Our study described outcomes of indication (for cause) renal biopsies and clinical outcomes in patients with ABO-incompatible renal transplant. This retrospective study included 164 patients from January 2012 to June 2019. Biochemical parameters, serial immunoglobulin G anti-ABO titers, and class I and II donor-specific antibody findings were obtained from hospital records, and renal graft biopsies were reviewed according to the Banff 2017 update. We analyzed the results of 65 biopsies from 54 patients. Biopsy-proven acute antibody-mediated rejection (12.8%) was found to be more prevalent than acute cellular rejection (1.8%). Patients with antibodymediated rejection all had microvascular inflammation (g+ptc score of 2 or more, where g+ptc is the sum of the glomerulitis and peritubular capillaritis scores) and were positive for C4d. Acute tubular injury per se was seen in 10.3% of patients; 65% of these patients had C4d positivity in peritubular capillaries, and only 1 patient developed chronic active antibody-mediated rejection on follow-up. Patient and death-censored graft survival rates were 92% and 98% at 1 year after transplant and 88% and 91% at 3 years, respectively. Patients with an episode of antibody-mediated rejection had lower rates of patient (76.5%) and deathcensored graft survival (84.6%) at 1 year. The microvascular inflammation score (g+ptc score of 2 or higher) is more reliable than diffuse C4d positivity to determine antibody-mediated rejection in ABO-incompatible transplants because diffuse C4d positivity may also be seen in etiologies unrelated to antibody-mediated rejection. Acute tubular injury with C4d positivity without microvascular injury does not confirm antibody-mediated rejection. We suggest that Banff classification be updated in ABO-incompatible transplants to include diagnostic criteria for the diagnosis of antibody-mediated rejection.

Organ Preservation Solutions in Transplantation: A Literature Review.

Renal transplant with ABO-incompatible donors expands the donor pool. Earlier studies have focused the use of protocol biopsies in ABOincompatible transplant patients. Our study described outcomes of indication (for cause) renal biopsies and clinical outcomes in patients with ABO-incompatible renal transplant. This retrospective study included 164 patients from January 2012 to June 2019. Biochemical parameters, serial immunoglobulin G anti-ABO titers, and class I and II donor-specific antibody findings were obtained from hospital records, and renal graft biopsies were reviewed according to the Banff 2017 update. We analyzed the results of 65 biopsies from 54 patients. Biopsy-proven acute antibody-mediated rejection (12.8%) was found to be more prevalent than acute cellular rejection (1.8%). Patients with antibodymediated rejection all had microvascular inflammation (g+ptc score of 2 or more, where g+ptc is the sum of the glomerulitis and peritubular capillaritis scores) and were positive for C4d. Acute tubular injury per se was seen in 10.3% of patients; 65% of these patients had C4d positivity in peritubular capillaries, and only 1 patient developed chronic active antibody-mediated rejection on follow-up. Patient and death-censored graft survival rates were 92% and 98% at 1 year after transplant and 88% and 91% at 3 years, respectively. Patients with an episode of antibody-mediated rejection had lower rates of patient (76.5%) and deathcensored graft survival (84.6%) at 1 year. The microvascular inflammation score (g+ptc score of 2 or higher) is more reliable than diffuse C4d positivity to determine antibody-mediated rejection in ABO-incompatible transplants because diffuse C4d positivity may also be seen in etiologies unrelated to antibody-mediated rejection. Acute tubular injury with C4d positivity without microvascular injury does not confirm antibody-mediated rejection. We suggest that Banff classification be updated in ABOincompatible transplants to include diagnostic criteria for the diagnosis of antibody-mediated rejection.

Clinicopathological Analysis of Acute/Active Antibody-Mediated Rejection in Renal Allografts According to the Banff 2013 Classification

Aim: This study evaluated the clinicopathological findings of acute/active antibody-mediated rejection (AABMR) according to the Banff 2013 classification. Methods: We analyzed 345 biopsies of 269 kidney transplant recipients. Pathological AABMR (PAABMR) was defined as histological evidence of acute tissue injury and endothelial injury by light microscopy regardless of donor-specific antibodies (DSAs). Results: Among the 345 biopsies, 29 (8.4%) were diagnosed as PAABMR. The mean g score was 1.17 ± 0.60, the mean ptc score was 1.97 ± 1.32, and DSA positivity was found in 69% of PAABMR. The mean duration after transplantation was 22.9 ± 26.7 months. Among 3 groups (DSA-high, mean fluorescence intensity (MFI) ≥ 5,000; DSA-low, MFI < 5,000 to ≥1,000; below cutoff), ABO incompatibility in DSA-high was significantly lower and second transplantation in DSA-high was significantly higher. We found 83% of PAABMR by the protocol biopsy (subclinical AABMR [SAABMR]). The short-term clinical and light microscopical changes in 8 cases of SAABMR did not show worsening during follow-up period (9–24 months). However, ultrastructural finding, including glomerular endothelial swelling, subendothelial electron-lucent widening, and early glomerular basement duplication, were found by electron microscopy (EM) in the first biopsies, and half of the SAABMR cases developed de novo circular peritubular capillary multilayering in the follow-up biopsies. Conclusion: PAABMR was mainly found by the protocol biopsy. The short-term follow-up of SAABMR patients did not show worsening clinically and light microscopically, but ultrastructural examination by EM was useful to detect early lesions of endothelial injury and progression of glomerular and peritubular capillary basement membrane alterations.

Lack of Histological and Molecular Signature Response to Tocilizumab in Kidney Transplants with Chronic Active Antibody Mediated Rejection: A Case Series.

Traditional therapies for caAbMR have unclear efficacy with significant side effects in recipients of kidney transplants (KTs). A recent single-center case series suggested tocilizumab (TCZ) could stabilize renal function and improve microvascular inflammation. Here we report our findings of the use of TCZ in patients with caAbMR. Ten adult recipients of KTs with biopsy-proven caAbMR were treated with TCZ at 8 mg/kg per month. Patients were monitored for adverse events, and therapy was interrupted in the setting of serious infections. Six patients (60%) underwent post-treatment biopsies. Patients (mean age of 43 years) were initiated on TCZ at a median of 36 months post-KT. A majority of patients were black (70%), underwent regrafts (40%), and were sensitized (mean cPRA=41%). Patients received a median of six doses of TCZ (range=3-10). At a median follow-up of 12 months (range=8-24 months), renal function did not show improvement (mean eGFR, 42±18 ml/min per 1.73 m2 to 37±24 ml/min per 1.73 m2; P=0.27). The slope of decline in eGFR remained unchanged (-0.14±0.9 to -0.33±1.1; P=0.25). There was no improvement in mean MVI (g+ptc) (4.8±1.4 to 4.2±2.0; P=0.39) scores or Molecular Microscope Diagnostic System (MMDx) AbMR scores (0.79±0.17 to 0.78±0.26; P=0.86). There was a numeric worsening of chronicity (ci+ct) scores (2.5±0.8 to 3.3±1.7; P=0.38) and MMDx atrophy fibrosis scores (0.36±0.24 to 0.58±0.15; P=0.21). Patient survival was 90%, with one patient death due to complications from a hip infection. Overall death-censored graft survival was 80%, with two graft losses in patients who had recurrent infections requiring hospitalization. In this early experience, we report a lack of efficacy and toxicity with the use of TCZ for caAbMR. Prospective clinical trials are needed to clarify the role of IL-6 blockade and the possibility of increased incidence of infections in patients with caAbMR who are treated with TCZ.

Immunoadsorption Combined with Membrane Filtration to Counteract Early Treatment-Refractory Antibody-Mediated Rejection

Introduction: Immunoadsorption (IA) represents a therapeutic option for acute antibody-mediated rejection (ABMR) after kidney transplantation. The addition of membrane filtration (MF) to enhance elimination of macromolecular components that potentially contribute to rejection, such as key complement component C1q and alloreactive IgM, may be an effective strategy to further improve its therapeutic efficiency. Results: Here we present 4 consecutive patients with episodes of HLA donor-specific antibody-positive ABMR nonresponsive to cycles of 6–16 sessions of IA treatment. Rejection episodes were characterized by severe microvascular injury (high-grade microcirculation inflammation and/or signs of thrombotic microangiopathy) and evidence of intense complement activation in peritubular capillaries (diffuse C4d-positivity). IA combined with MF led to substantial morphologic improvement (follow-up biopsies: g + ptc and C4d scores ≤1) and stabilization of allograft function. Conclusions: Our findings provide evidence for an effect of combination of IA + MF in refractory early acute/active ABMR in kidney transplant recipients.

Microvascular inflammation in renal allograft biopsies assessed by endothelial and leukocyte co-immunostain: a retrospective study on reproducibility and clinical/prognostic correlates.

The most prominent histologic lesion in antibody-mediated rejection is microvascular inflammation (MVI); however, its recognition and scoring can be challenging and poorly reproducible between pathologists. We developed a dual immunohistochemical (IHC)-stain (anti-CD34/anti-CD45 for endothelium/leukocytes) as ancillary tool to improve on the semi-quantitative Banff scores and allow quantification of MVI. We examined the relationship between CD34-CD45 IHC-based quantitative MVI score (the inflamed peritubular capillary ratio, iptcr) and renal-graft failure or donor-specific antibodies (DSA) strength at the time of biopsy. Quantitative iptcr score was significantly associated with renal graft failure (hazard ratio 1.81, per 1 SD-unit [0.13 points] of iptcr-increase; P=0.026) and predicted the presence and strength of DSA (ordinal odds ratio: 2.42; P=0.005; 75 biopsies/60 kidney transplant recipients; 30 HLA- and/or ABO-incompatible). Next, we assessed inter-pathologist agreement for ptc score and ptc extent (focal/diffuse) using CD34-CD45 IHC as compared to conventional stain. Compared to conventional stain, CD34-CD45 IHC significantly increased inter-pathologist agreement on ptc score severity and extent (κ-coefficient from 0.52-0.80 and 0.46-0.68, respectively, P<0.001). Our findings show that CD34-CD45 IHC improves reproducibility of MVI scoring and facilitates MVI quantification and introduction of a dual anti-CD34/CD45 has the potential to improve recognition of MVI ahead of DSA results.

An integrative approach for the assessment of peritubular capillaritis extent and score in low-grade microvascular inflammation-associations with transplant glomerulopathy and graft loss.

Peritubular capillaritis (ptc), reported by the ptc score, is a major feature of kidney allograft rejection and microvascular inflammation (MVI). MVI sum scores (ptc + glomerulitis score ≥2) are accepted diagnostic surrogates of human leucocyte antigen (HLA)-antibody interaction. However, low-grade inflammation is common and ptc scores (number of leucocytes/capillary) may not mirror all aspects of ptc morphology. Recently we observed a relationship of the diffuse extent of ptc (inflammation of >50% of the renal cortex) with graft loss and significantly higher donor-specific antibody levels, suggesting potential inclusion of diffuse ptc as an additional surrogate of antibody-antigen interaction. We sought to assess how a combination of ptc score and extent in low-grade inflammation (ptc1) affects transplant glomerulopathy (TG) and graft loss risk. Patients (n = 616) were assessed for MVI in first indication biopsies. Cases with a ptc score of 1 but diffuse extent (ptc1diffuse, g-score = 0, n = 26) were considered additional surrogates of HLA-antibody interaction and compared with MVI ≥2 and MVI <2. The ptc1diffuse and MVI score ≥2 subjects had worse graft survival (42% and 59%) compared with an MVI score <2 (70%) (P = 0.002). The incorporation of ptc1diffuse in the MVI score ≥2 increased the receiver operating characteristics curve for TG [area under the curve (AUC) 0.602; P = 0.008] compared with a Banff MVI score ≥2 (AUC 0.56; P = 0.12); cases with baseline TG were excluded. In multivariate analysis, ptc1diffuse remained independently related to TG (odds ratio 3.89; P = 0.008) and graft loss (hazard ratio 2.64; P = 0.001) even after inclusion of all rejection episodes. An integrated view of ptc morphology including diffuse ptc in MVI is superior for TG and graft loss risk assessment.

An Increase in Low-Grade Spot Urine Protein Excretion During the First Year after Kidney Transplantation Associates with Subclinical De-Novo Donor-Specific Antibodies and Microvascular Inflammation at 1-Year Surveillance Biopsies

The presence of microvascular inflammation (MVI) in kidney transplant biopsies often relates to occurrence of donor-specific antibodies (DSA) and antibody-mediated rejection. Here we investigated whether an increase in spot urine protein excretion during the first year after kidney transplantation associates with subclinical de-novo DSA and different Banff MVI scores at 1-year surveillance biopsies. Patients and Methods This prospective observational study included 79 consecutive non-sensitized patients who received a deceased donor kidney transplant between Dec 2013 and Jan 2016. All patients received basiliximab induction and tacrolimus-based immunosuppression. Estimated protein excretion rate (ePER) was calculated monthly from spot urine protein-to-creatinine ratios. At 1 year, all recipients underwent surveillance graft biopsy and were screened for de-novo DSA. Screening-positive sera were subjected to single antigen bead (SAB) testing (LABScreen SAB assays; One Lambda). The presence of subclinical de-novo DSA was determined on the basis of SAB reactivity patterns using a mean fluorescence intensity threshold >1000 and stable graft function within the first year (variability in serum creatinine <25% from baseline). Biopsies were read by a single pathologist and presence of MVI (any glomerulitis [g] and/or peritubular capillaritis [ptc] score >0) was graded in accordance with Banff 2013 criteria. Results Among the 79 study patients, 10 (13%) developed de-novo DSA at 1 year after transplant (5 patients class I, 5 patients class II). At 1-year surveillance biopsy, 12 patients (15%) had histologic evidence of MVI with g and/or ptc score >0. Among them, 7 patients had mild MVI with [g+ptc] <2, and 5 patients had moderate MVI with [g+ptc] ≥2 and g ≥1. There was no evidence of transplant glomerulopathy or peritubular capillary basement membrane multilayering in electron microscopy. At 1 year, patients with de-novo DSA had significantly greater mean ePER than those without DSA (870, 95% CI 770–1040 mg/day/1.73m2 versus 192, 95% CI 178–206 mg/day/1.73m2; P<0.001). Compared with absence of MVI, mild and moderate MVI were associated with a progressive increase in mean ePER at 1 year (no MVI: 190, 95% CI 178–202 mg/day/1.73m2 versus mild MVI: 740, 95% CI 578–926 mg/day/1.73m2 versus moderate MVI: 1022, 95% CI 884–1226 mg/day/1.73m2; P<0.001). Linear mixed effect regression analyses demonstrated a progressive increase and a significant difference in slope of ePER during the first year between patients with no DSA and de-novo DSA at 1 year (P<0.001), and between patients with no MVI, mild MVI, and moderate MVI at 1-year surveillance biopsies (P<0.001) [Figure].Conclusions An increase in low-grade ePER during the first year after kidney transplantation associates with subclinical de-novo DSA and the degree of MVI at 1-year surveillance biopsies and may be used as a noninvasive clinical biomarker of endothelial cell injury. The study was financially supported by the research project of the Slovenian Research Agency (Project ID L3-7582).

Functional Fc gamma receptor gene polymorphisms and donor-specific antibody-triggered microcirculation inflammation.

Fc-dependent effector mechanisms may contribute to antibody-mediated rejection (ABMR), and distinct gene polymorphisms modifying the function of Fc gamma receptors (FcγRs) may influence the capability of donor-specific antibodies (DSAs) to trigger inflammation. To evaluate the relevance of functional FcγR variants in late ABMR, 85 DSA-positive kidney allograft recipients, who were recruited upon antibody screening of 741 prevalent patients, were genotyped for polymorphisms in FcγRIIA (FCGR2A-H/R131 ; rs1801274), FcγRIIIA (FCGR3A-V/F158 ; rs396991), and FcγRIIIB (FCGR3B-neutrophil antigen 1 ([NA1]/NA2; rs35139848). Individuals with high-affinity FCGR3A-V158 alleles (V/V158 or V/F158 ) showed a higher rate (and extent) of peritubular capillaritis (ptc) in protocol biopsies than homozygous carriers of the lower-affinity allele (ptc score ≥1: 53.6% vs 25.9%; P=.018). Associations were independent of C1q-binding to DSA or capillary C4d. In parallel, there was a trend toward increased macrophage- and injury-repair response-associated transcript subsets. Kidney function over 24months, however, was not different. In support of a functional role of FcγRIIIA polymorphism, NK92 cells expressing FCGR3A-V158 produced >2 times as much interferon gamma upon incubation with HLA antibody-coated cells as those expressing FCGR3A-F158 . FcγRIIA and FcγRIIIB polymorphisms were not associated with allograft morphology. Our data suggest that the presence of high-affinity FcγRIIIA variants may favor DSA-triggered microcirculation inflammation.

Diffuse Extent of Peritubular Capillaritis in Late Antibody-Mediated Rejection

Recently, diffuse peritubular capillaritis (ptc) has been suggested to independently predict chronic transplant injury and loss, and although the ptc score is a diagnostic criterion for antibody-mediated rejection, the utility of diffuse ptc is under debate. We evaluated the diagnostic value of ptc characteristics in this cross-sectional study including 85 biopsies of patients with donor-specific antibodies (DSA). Biopsies were reevaluated for the extent (diffuse vs focal), score and leukocytic composition in relation to DSA binding strength (mean fluorescence intensity [MFI]_max). Chronic allograft injury (transplant chronic glomerulopathy [cg] or chronic lesion score CLS]) were associated with ptc features. Peritubular capillaritis was detected in 50% (76% mononuclear ptc). Peritubular capillaritis scores 1, 2, and 3 were present in 36%, 55%, and 9%, and focal or diffuse ptc in 36% or 64%. Diffuse ptc was associated with DSA MFI_max (median: 4407 vs 2419 [focal ptc; P = 0.04] or 1946 [no ptc; P = 0.004]), cg (58% vs no ptc 24% [P = 0.02]), and higher CLS (mean: 6.81 vs 4.67 [focal ptc, P = 0.01] or 5.18 [no ptc, P = 0.001]), respectively. The association of ptc score of 2 or greater with cg was slightly better than with diffuse ptc. Diffuse ptc and ptc score of 2 or greater remained independently related to cg after adjusting for DSA_MFI_max, C4d, or previous rejection episodes, however lost their independent relation after adjusting for total microcirculation scores. Diffuse ptc was the only ptc characteristic independently related to CLS. Our results emphasize the clinical relevance of reporting diffuse ptc, which may relate to DSA binding strength and potentially to chronic graft injury.