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Introduction: This study investigated the impact of immunosuppressive drug concentrations on microvascular inflammation (MVI) in kidney transplant recipients with negative donor-specific antibodies (DSA) against human leukocyte antigen (HLA) and negative C4d deposition in peritubular capillaries., Methods: We analyzed data from 268 living kidney transplant recipients at the Department of Urology, University of Osaka, Japan. Patients received immunosuppressive therapy comprising extended-release tacrolimus, mycophenolate mofetil (MMF), and/or everolimus, with or without steroids. Graft biopsies were routinely performed at 3, 12, 36 and 60 months post-surgery., Results: No significant differences were observed between the MVI+DSA-C4d- and MVI-DSAC4d groups regarding graft survival rates (95.5% vs. 96.6%, p = 0.772) or patient survival rates (95.7% vs. 95.9%, p = 0.735). Lower tacrolimus and everolimus concentrations were significantly associated with an increased risk of MVI+DSA-C4d- (tacrolimus: OR, 0.169; 95% CI, 0.055-0.515; p = 0.002; everolimus: OR, 0.386; 95% CI, 0.171-0.874; p = 0.022). In contrast, MPA concentration was not significantly associated with MVI+DSA-C4d- (OR, 0.994; 95% CI, 0.554-1.780; p = 0.984). Steroid discontinuation did not significantly impact the risk of MVI+DSA-C4d- (OR, 1.980; 95% CI, 0.318-12.000; p = 0.470)., Conclusion: Lower trough levels of tacrolimus and everolimus correlated with a higher incidence of antibody-independent MVI, supporting the need for tailored immunosuppressive regimens in kidney transplantation., (© 2025 The Author(s). Clinical Transplantation published by Wiley Periodicals LLC.)

Complement C4 is a key component in the activation of classical and lectin complement pathways, which are observed in both animal tumor models and cancer patients. While its role in autoimmune disorders has been extensively studied, the functions of complement C4 and its activation in cancer have received inadequate consideration. Recent studies have detected C4 activation in animal tumor models and cancer patients, with its fragment C4d found in cancer tissues and lymph nodes. Elevated C4d levels could be a useful biomarker for detecting various cancers. This review aims to summarize recent developments on the role of complement C4 activation in promoting an immunosuppressive tumor microenvironment, thereby supporting tumor progression and metastasis; C4d as a biomarker; and its potential as a target for cancer immunotherapy. We also conduct a critical evaluation of methods used to measure complement C4 and its activation products, highlighting possible pitfalls and areas for improvement in existing research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

The role of the complement system in antibody mediated rejection (AMR) emerged in the last decades, and the demonstration of the presence of complement fragments in renal allograft biopsies is a consolidated diagnostic sign of AMR. However, antibodies against donor antigens may lead to microvascular inflammation and endothelial injury even in the absence of complement activation, and growing evidence suggests that complement-independent mechanisms may be prominent in late (i.e., occurring >6 months after transplantation) vs early AMR. Different donor specific antibodies (DSA) with different biological features and complement activation ability may be involved in late or early AMR. Downregulation of tissue complement inhibitors may happen early after transplantation, partially due to ischemia reperfusion injury, and could facilitate complement activation in early vs late AMR. Clinical and histological features of late AMR and C4d negative AMR seem to converge, and this narrative review analyzes the evidence that supports lower complement activation in late vs early AMR, including differential C4d staining prevalence based on the time after transplantation, differential response to anti-complement therapy and other direct and indirect signs of the complement system activation. The therapeutic approach in early vs late AMR should take into account possible differences in the pathophysiological mechanisms of microvascular inflammation and endothelial injury in early vs late AMR., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Microvascular inflammation (MVI) is a key diagnostic feature of antibody-mediated rejection (AMR); however, recipients without donor-specific antibodies (DSA) defy etiologic classification using C4d staining of peritubular capillaries (C4d ptc ) and conventional DSA assignment. We evaluated MVI ≥ 2 (Banff g + ptc ≥ 2) using Banff 2019 AMR (independent of MVI ≥ 2 but including C4d ptc ) with unconventional endothelial C4d staining of glomerular capillaries (C4d glom ) and - arterial endothelium and/or intima (C4d art ) using tissue immunoperoxidase, shared-eplet and subthreshold DSA (median fluorescence intensity, [MFI] 100-499), and capillary ultrastructure from 3398 kidney transplant samples for evidence of AMR. MVI ≥ 2 (n = 202 biopsies) from 149 kidneys (12.4% prevalence) correlated with DSA+, C4d ptc +, C4d glom +, Banff cg, i, t, ti scores, serum creatinine, proteinuria, and graft failure compared with 202 propensity score matched normal controls. The laboratory reported DSA- MVI ≥ 2 (MFI ≥500) occurred in 34.7%; however, subthreshold (28.6%), eplet-directed (51.4%), and/or misclassified anti-Human leukocyte antigen (HLA) DSA (12.9%) were identified in 67.1% by forensic reanalysis, with vascular C4d+ staining in 67.1%, and endothelial abnormalities in 57.1%, totaling 87.1%. Etiologic analysis attributed 62.9% to AMR (77.8% for MVI with negative reported DSA [DSA- MVI ≥2] with glomerulitis) and pure T cellular rejection in 37.1%. C4d ptc -DSA- MVI ≥ 2 was unrecognized AMR in 48.0%. Functional outcomes and graft survival were comparable to normal controls. We concluded that DSA- MVI ≥ 2 frequently signified a mild "borderline" phenotype of AMR which was recognizable using novel serologic and pathological techniques., Competing Interests: Declaration of competing interest The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. Apart from a travel bursary obtained from One Lambda (R.C.), the authors of this manuscript have no conflicts of interest to disclose., (Copyright © 2024 American Society of Transplantation & American Society of Transplant Surgeons. Published by Elsevier Inc. All rights reserved.)

Immunohistochemical staining with immunoglobulins and complements may aid the diagnosis of patients whose clinical and histological findings are consistent with autoimmune bullous dermatoses (AIBD). We aimed to investigate the diagnostic value of immunohistochemical markers in lesional biopsy and perilesional frozen samples in AIBD. We included 136 cases from whom lesional biopsies and perilesional samples for direct immunofluorescence (DIF) examination were collected with a preliminary diagnosis of AIBD between January 2019 and January 2023. All diagnoses were reconfirmed by evaluating the clinical, histopathological, and serological findings and DIF results (C3, IgG, IgA, or IgM positivity compatible with the clinical diagnosis) altogether, although DIF results were considered a priority. After confirming the diagnoses, the samples were categorized as AIBD or the others. The perilesional tissues obtained for DIF simultaneously with skin biopsy and stored at -80 °C were thawed, and FFPE tissues were prepared. We performed immunohistochemical staining (C4d, C3d, IgG, and IgG4) on FFPE tissues of both lesional and perilesional samples. Strong, linear, or granular staining patterns at the dermoepidermal junction or the intraepidermal blistering space were considered positive in line with the diagnosis of the case. Cases other than AIBD were used as negative control tissues to assess the specificity of immunohistochemical markers. Of the 136 cases, 52 were diagnosed with AIBD. In lesional samples, the sensitivity of C4d, C3d, IgG, and IgG4 was 80.6 %, 69.4 %, 75 %, and 5.7 % with corresponding specificity of 100 %, 98.7 %, 89.6 %, and 97.4 %, respectively in pemphigoid diseases compared to a sensitivity of 18.2 %, 9.1 %, 70 %, and 9.1 % and specificity of 98.7 %, 100 %, 89.6 %, and 97.4 %, respectively in pemphigus diseases. In frozen samples, we detected expression in a limited number of cases. The sensitivity of C4d, C3d, IgG, and IgG4 was 8.7 %, 2.2 %, 19.4 %, and 2.2 %, with corresponding specificity of 100 %, 100 %, 98.5 %, and 98.6, respectively. There was a none to slight concordance rate between the IHC results of lesional tissues and perilesional frozen samples. Kappa coefficients for C4d, C3d, IgG, and IgG4 were 0.120 (P = 0.029), 0.111 (P = 0.050), 0.203 (P = 0.003), and - 0.15 (P = 0.846), respectively. Immunohistochemical staining with C4d, C3d, IgG, and IgG4 on biopsy samples collected from lesions may guide the diagnosis of AIBD, thereby eliminating the need for an additional biopsy and accelerating the diagnostic process., Competing Interests: Declaration of competing interest The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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.

Complement receptor 1 (CR1) is a membrane glycoprotein with a highly duplicated domain structure able to bind multiple ligands such as C3b and C4b, the activated fragments of complement components C3 and C4, respectively. We have previously used our knowledge of this domain structure to identify CSL040, a soluble extracellular fragment of CR1 containing the long homologous repeat (LHR) domains A, B, and C. CSL040 retains the ability to bind both C3b and C4b but is also a more potent complement inhibitor than other recombinant CR1-based therapeutics. To generate soluble CR1 variants with increased inhibitory potential across all three complement pathways, or variants with activity skewed to specific pathways, we exploited the domain structure of CR1 further by generating LHR domain duplications. We identified LHR-ABCC, a soluble CR1 variant containing a duplicated C3b-binding C-terminal LHR-C domain that exhibited significantly enhanced alternative pathway inhibitory activity in vitro compared to CSL040. Another variant, LHR-BBCC, containing duplications of both LHR-B and LHR-C with four C3b binding sites, was shown to have reduced classical/lectin pathway inhibitory activity compared to CSL040, but comparable alternative pathway activity. Interestingly, multiplication of the C4b-binding LHR-A domain resulted in only minor increases in classical/lectin pathway inhibitory activity. The CR1 duplication variants characterized in these in vitro potency assays, as well as in affinity in solution C3b and C4b binding assays, not only provides an opportunity to identify new therapeutic molecules but also additional mechanistic insights to the multiple interactions between CR1 and C3b/C4b., Competing Interests: Conflict of interest S. W., A. B. M., T. R., and M. P. H. are listed as inventors on International Patent Publication number WO2019/218009. All authors are CSL shareholders., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: C4d is an activation product of lectin pathway of complement. Glomerular deposition of C4d is associated with poor prognosis in different types of immune-related glomerulonephritis. The present study was conducted to investigate expression level of C4d and its staining pattern in renal biopsy of patients with focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) by immunohistochemistry method., Materials and Methods: In this retrospective cross-sectional study, renal biopsy specimens from 46 samples of MCD, 47 samples of FSGS, and 15 samples without glomerular disease as the controls, were subjected to immunohistochemistry staining with C4d. Demographic characteristics and information obtained from light and electron microscopy (EM) of patients were also extracted from their files., Results: C4d positive staining was observed in 97.9 % of FSGS and 43.5 % of MCD samples, which showed a statistically significant difference (P < 0.001). The sensitivity and specificity of C4d expression for diagnosing FSGS were 97.9 % and 56.5 %, respectively. There was no significant correlation between C4d expression and any of the light and electron microscopy findings, including presence of foam cells, mesangial matrix expansion, interstitial fibrosis and tubular atrophy, and basement membrane changes in MCD patients. Also, no significant correlation was observed between C4d expression and clinical symptoms of proteinuria or prolonged high level of creatinine in patients with MCD., Discussion and Conclusion: The expression of C4d marker had a good sensitivity and negative predictive value in the diagnosis of FSGS., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

C4b-binding protein (C4BP) is a fluid-phase complement inhibitor that prevents uncontrolled activation of the classical and lectin complement pathways. As a complement inhibitor, C4BP also promotes apoptotic cell death and is hijacked by microbes and tumors for complement evasion. Although initially characterized for its role in complement inhibition, there is an emerging recognition that C4BP functions in a complement-independent manner to promote cell survival, protect against autoimmune damage, and modulate the virulence of microbial pathogens. In this Brief Review, we summarize the structure and functions of human C4BP, with a special focus on activities that extend beyond the canonical role of C4BP in complement inhibition., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Objective: It remains unclear if C4d staining is related to any peritubular and glomerular injury during antibody mediated rejection (ABMR). The goal of this study was to determine if myeloperoxidase (MPO) staining can highlight endothelial injury in peritubular capillaries (PTC) and glomeruli., Methods: The study included 12 native negative controls, 19 transplant biopsies with borderline changes (BC) as transplant controls, and one group of renal transplant biopsies with ABMR as the study group (acute/chronic, n=22). All three groups were stained for MPO immunohistochemically, and the MPO expressions in the endothelium of PTC and glomeruli were evaluated and correlated with serum creatinine (SCr). In addition, the ultrastructural layers of the PTC (an index for chronic allograft rejection) were correlated with MPO indices in PTC., Results: The negative control group and the transplant controls showed no MPO expression in the endothelium of glomeruli and PTC. However, in the biopsies with ABMR, there were MPO-positive stains in the endothelial cells of glomeruli (15/21 cases, 71.4 %) and PTC (16/22 cases, 72.7 %). There were significant correlations between the peritubular MPO staining versus SCr (r=0.355 and p =0.0106) and glomerular MPO staining versus SCr (r=0.365 and p =0.0092). Furthermore, the layers of PTC by electron microscopy were significantly correlated with MPO scores in PTC (r=0.696, p =0.0001)., Conclusion: Our data suggest that the MPO-positive endothelial injuries are most likely the cause leading to renal graft dysfunction following ABMR., (© 2023 by the Association of Clinical Scientists, Inc.)

Aims: To use experimental periodontitis models in rats to investigate the correlation between local expression of the complement components C3b and C4b in periodontal tissues and disease severity, and to assess the therapeutic effects of targeting C3b/C4b on inflammatory bone loss., Materials and Methods: The gingival expression of C3, C3b, and C4b in animal experimental periodontitis models were analysed immunohistochemically. The therapeutic effects of the C3b/C4b inhibitor (SB002) on ligation-induced experimental periodontitis was examined using biochemical, histological, and immunohistochemical analyses., Results: The gingival expression levels of C3, C3b, and C4b were positively correlated with the severity of periodontitis. Moreover, both single and multiple injections of the C3b/C4b inhibitor had preventive and therapeutic effects on alveolar bone loss in ligation-induced experimental periodontitis with no associated adverse consequences., Conclusions: The association between C3b/C4b and periodontitis may provide a basis for the development of novel therapeutic strategies for periodontitis and other inflammatory diseases., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: We aimed to investigate the immuno-histochemical expression of C4d, ADAM10 and WT1 in kidney biopsies of immunoglobulin A nephropathy (IgAN) patients and correlate the findings with clinical, laboratory and histopathologic features in the hope of defining new parameters to better understand the pathogenesis of the disease, and predict prognosis., Materials and Methods: Paraffin-embedded kidney biopsy samples of 128 IgAN patients were immuno-histochemically treated with C4d and ADAM10/WT1 dual stain. Results were evaluated according to Oxford classification parameters, epidemiologic features, laboratory findings at presentation and clinical follow-up., Results: We observed C4d positivity in 40.6% of our patients, 25% of which was mesangial/peri-mesangial (m/pm) staining. Only m/pmC4d positivity statistically correlated with progression to end-stage renal disease (ESRD). M/pmC4d positive patients had statistically significantly higher baseline proteinuria levels, presence of crescents and > 25% segmental sclerosis of glomeruli. There was cytoplasmic staining of WT1 in 11.2% of cases. Presence of cWT1 correlated with m/pmC4d positivity and progression to ESRD. There was no glomerular ADAM10 detected and tubular expression of this protein did not relate to amount of tubular damage or other parameters., Conclusion: This study is the first to show that cWT1is involved in IgAN and appears as an independent variable for worse prognosis., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Human Complement Receptor 1 (HuCR1) is a potent membrane-bound regulator of complement both in vitro and in vivo, acting via interaction with its ligands C3b and C4b. Soluble versions of HuCR1 have been described such as TP10, the recombinant full-length extracellular domain, and more recently CSL040, a truncated version lacking the C-terminal long homologous repeat domain D (LHR-D). However, the role of N-linked glycosylation in determining its pharmacokinetic (PK) and pharmacodynamic (PD) properties is only partly understood. We demonstrated a relationship between the asialo-N-glycan levels of CSL040 and its PK/PD properties in rats and non-human primates (NHPs), using recombinant CSL040 preparations with varying asialo-N-glycan levels. The clearance mechanism likely involves the asialoglycoprotein receptor (ASGR), as clearance of CSL040 with a high proportion of asialo-N-glycans was attenuated in vivo by co-administration of rats with asialofetuin, which saturates the ASGR. Biodistribution studies also showed CSL040 localization to the liver following systemic administration. Our studies uncovered differential PD effects by CSL040 on complement pathways, with extended inhibition in both rats and NHPs of the alternative pathway compared with the classical and lectin pathways that were not correlated with its PK profile. Further studies showed that this effect was dose dependent and observed with both CSL040 and the full-length extracellular domain of HuCR1. Taken together, our data suggests that sialylation optimization is an important consideration for developing HuCR1-based therapeutic candidates such as CSL040 with improved PK properties and shows that CSL040 has superior PK/PD responses compared with full-length soluble HuCR1., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

In the context of transplantation, complement activation is associated with poor prognosis and outcome. While complement activation in antibody-mediated rejection is well-known, less is known about complement activation in acute T cell-mediated rejection (TCMR). There is increasing evidence that complement contributes to the clearance of apoptotic debris and tissue repair. In this regard, we have analysed published human kidney biopsy transcriptome data clearly showing upregulated expression of complement factors in TCMR. To clarify whether and how the complement system is activated early during acute TCMR, experimental syngeneic and allogeneic renal transplantations were performed. Using an allogeneic rat renal transplant model, we also observed upregulation of complement factors in TCMR in contrast to healthy kidneys and isograft controls. While staining for C4d was positive, staining with a C3d antibody showed no C3d deposition. FACS analysis of blood showed the absence of alloantibodies that could have explained the C4d deposition. Gene expression pathway analysis showed upregulation of pro-apoptotic factors in TCMR, and apoptotic endothelial cells were detected by ultrastructural analysis. Monocytes/macrophages were found to bind to and phagocytise these apoptotic cells. Therefore, we conclude that early C4d deposition in TCMR may be relevant to the clearance of apoptotic cells.

Abstract: Bullous pemphigoid (BP) is an autoimmune blistering disease that commonly affects elderly patients. Direct immunofluorescence (DIF) for immunoglobulin G (IgG) and C3c on frozen skin biopsies is the gold standard for the diagnosis of BP. In a minority of cases, IgG and/or C3c are found negative, and in these situations, there is a need for a more stable diagnostic marker of BP. C4d is biologically inactive, but has a long half-life, rendering it a long-lived marker for antibody-mediated complement activation. Previous studies already demonstrated that C4d was diagnostically useful in formalin-fixed paraffin-embedded skin biopsies of patients with BP. We hypothesized that C4d detected by DIF could also be a promising diagnostic marker for BP, particularly in IgG and/or C3c DIF-negative cases. In this single-center retrospective study, 69 cases of BP were analyzed for linear deposition of C4d; of the 69 cases, n = 26 were IgG+/C3c-, n = 10 IgG+/C3c+, and n = 33 IgG-/C3c-. Results were compared with n = 39 negative controls. Seven of the 26 (27%) IgG+/C3c- and 3 of the 33 (9%) IgG-/C3c- BP cases were positive for C4d. All 10 IgG+/C3c+ cases were also C4d positive. In the negative control group, 2 of the 39 (5%) were found positive for C4d. In conclusion, the current study shows that C4d is a more sensitive but not a 100% specific marker of BP. We conclude that C4d by DIF could be an interesting diagnostic adjunct for BP, particularly in IgG-/C3c- double negative cases., Competing Interests: B. Horvath reports fees from Janssen-Cilag (advisory boards, educational grants, consultations, and investigator initiative studies), AbbVie (advisory boards, educational grants, consultations, and investigator initiative studies), Novartis Pharma (advisory boards, consultations, and investigator initiative studies), UCB Pharma (advisory boards and consultations), Leo Pharma (consultations), Solenne B. V. (investigator initiative studies), Celgene (consultations and investigator initiative studies), Akari therapeutics (consultations and investigator initiative studies), Philips (consultation), Roche (consultation), Regeneron (consultation), and Sanofi (consultation), fees which were paid to the institution. The other authors declare no conflicts of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Antithymocyte globulin (ATG) consists of polyclonal antibodies directed primarily against human T lymphocytes but may contain antibodies with affinity for other tissues in the transplanted organ, resulting in complement (C4d) deposition. This phenomenon has been demonstrated in endomyocardial biopsies (EMBs) of adult cardiac transplants. We examined the relationship of induction immunosuppression with ATG and C4d deposition in EMB of pediatric cardiac transplants., Methods: Results of C4d immunohistochemistry were available from all EMB of patients transplanted at our center between June 2012 and April 2018 (n = 48) who received induction immunosuppression with either ATG (n = 20) or basiliximab (n = 28) as the standard of care., Results: C4d deposition in the first year post-heart transplant was more commonly seen among patients who received ATG induction (20% of EMBs in ATG group vs 1% of EMBs in basiliximab group; p < .0001). C4d deposition related to ATG was observed early post-transplant (50% ATG vs 0% basiliximab on first EMB; p < .0001 and 35% ATG vs 0% basiliximab on the second EMB; p = .0012). While this difference waned by the third EMB (5% ATG vs 0% basiliximab; p = .41), positive C4d staining persisted to the sixth EMB in the ATG group only (6%)., Conclusion: C4d deposition is common on EMB up to 1 year post-pediatric cardiac transplant following ATG induction. This high rate of positive C4d staining in the absence of histologic AMR after ATG induction therapy must be accounted for in making clinical decisions regarding cardiac allograft rejection diagnosis and treatment., (© 2021 Wiley Periodicals LLC.)

COVID-19, the disease caused by the SARS-CoV-2 virus, can progress to multisystem organ failure and viral sepsis characterized by respiratory failure, arrhythmias, thromboembolic complications, and shock with high mortality. Autopsy and preclinical evidence implicate aberrant complement activation in endothelial injury and organ failure. Erythrocytes express complement receptors and are capable of binding immune complexes; therefore, we investigated complement activation in patients with COVID-19 using erythrocytes as a tool to diagnose complement activation. We discovered enhanced C3b and C4d deposition on erythrocytes in COVID-19 sepsis patients and non-COVID sepsis patients compared with healthy controls, supporting the role of complement in sepsis-associated organ injury. Our data suggest that erythrocytes may contribute to a precision medicine approach to sepsis and have diagnostic value in monitoring complement dysregulation in COVID-19-sepsis and non-COVID sepsis and identifying patients who may benefit from complement targeted therapies.

Objectives: This study aimed to determine the prevalence and localization of complement factor C4d in renal biopsies from patients with lupus nephritis (LN), as well as its associations with the disease's clinico-pathological features. The correlation between arteriolar C4d deposition and renal microvascular lesions (RVLs) was further analyzed., Methods: A total of 325 biopsy-proven LN patients were enrolled, and their clinico-pathological data were collected. C4d staining of renal biopsies was performed by immunohistochemistry. The associations between C4d deposition and the clinico-pathological features were further analyzed., Results: C4d deposition was present in most (98.8%) renal specimens in our cohort. These deposits were localized in the glomeruli (98.2%), tubular basement membrane (TBM) (43.7%), arterioles (31.4%), and peritubular capillary (33.8%). Patients with TBM C4d staining had higher disease activity (measured with the Systemic Lupus Erythematous Disease Activity Index) and higher National Institutes of Health pathological activity and chronicity indices (all P < 0.01). Patients with arteriolar C4d deposition were more likely to develop RVLs (91.2%) compared to those with no arteriolar C4d deposition (78.0%; P = 0.004), especially with two or more types of RVLs ( P < 0.001). During the mean follow-up of 55.8 months, arteriolar C4d was related to worse renal outcomes [hazard ration (HR): 2.074, 95% confidence interval (CI) 1.056-4.075, P = 0.034]. Multivariate Cox hazard analysis showed that co-deposition of arteriolar C4d and C3c was an independent risk factor (HR: 3.681, 95% CI 1.519-8.921, P = 0.004) for predicting renal outcomes., Conclusions: C4d deposition was common in renal tissues from LN patients. TBM C4d deposition was related to the disease activity, and arteriolar C4d deposition was associated with RVLs and worse renal outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ding, Yu, Wu, Tan, Qu and Yu.)

Mesangial C4d deposits have been associated with worse outcomes in Western patients with IgA nephropathy (IgAN), but there is limited data in Asians. Previously, a high proportion of stained glomeruli was often required for the classification of C4d positive (C4d+ve). Positive staining in lower proportion of staining would be classified as C4d-ve. This retrospective study evaluated the prognostic value of C4d+ve using a less stringent definition (one C4d+ve glomerulus) in Thai patients with IgAN (n = 120). Baseline findings and outcomes were compared between those with more extensive C4d staining patterns and those with more restricted staining. Clinico-pathologic parameters and risk for kidney outcomes (kidney failure or decline GFR50%) were compared between C4d+ve versus C4d-ve, and between different patterns: Focal (< 50%) versus Diffuse (≥ 50% of glomeruli); or Global (≥ 50) versus Segmental (< 50% of mesangial area). The hazard ratios were estimated using Cox proportional hazard models for Model 1 (Oxford score+ C4d) and Model 2 (Model 1+ clinical factors). C4d+ve (n = 81) had lower eGFR, more global sclerosis, and interstitial fibrosis than C4d-ve at baseline. The 5-year kidney survival for C4d+ve was lower (53.7%) than C4d-ve (89.7%); P = 0.0255. By univariate analysis, T1, T2, C4d+ve, eGFR<60, proteinuria were predictors of kidney outcome. By multivariate analysis, proteinuria, T1, T2 and C4d+ve were independent predictors (Model 2 HR (95% CI) C4d+ve: 3.24 (1.09-9.58), p = 0.034). Segmental had lower eGFR, higher tubulointerstitial fibrosis, and segmental sclerosis compared to Global pattern. Clinicopathological parameters were not different between Focal and Diffuse patterns. Outcomes were similar between staining patterns. In conclusion, C4d staining may be a valuable marker of poor prognosis in Asian patients with IgAN. Less stringent criteria for C4d+ve should be considered as no differences in outcomes were observed between more extensive staining with less extensive patterns. More studies are needed to identify the optimum criteria for C4d+ve., Competing Interests: The authors have declared that no competing interests exist.

Immunoglobulin A nephropathy (IgAN) is the most common primary glomerular disease. The main challenge in this disease is the evaluation of prognostic factors for end-stage renal disease (ESRD). The aim of our study was to assess the clinical and prognostic implications of C4d staining in primary IgAN. This was a retrospective study, including adults with primary IgAN. The study was conducted over a period of 10 years. Renal biopsies were scored according to the Oxford classification. C4d immunohistochemical staining was performed. We included 44 patients with a sex ratio of 2.6. The average age was 35.1 ± 11 years. Twenty-two patients (57%) had hypertension (HTN). The median proteinuria was 1.92 g/day. The median of the glomerular filtration rate was 47.66 mL/min/1.73 m 2 . According to the Oxford classification, mesangial proliferation, endocapillary proliferation, glomerulosclerosis, interstitial fibrosis and/or tubular atrophy and crescents were present in 41%, 36%, 86%, 34%, and 25 % of cases, respectively. We found positive glomerular C4d staining in 25 renal biopsies (57%). Age at diagnosis, mean arterial pressure, HTN, and baseline glomerular filtration rate were not correlated with C4d staining. On the other hand, proteinuria was significantly higher in patients with C4d-positive renal biopsy. The median follow-up duration was 30.5 months. Ten patients (23%) reached ESRD. At univariate analysis, positive C4d staining in more than 25% of glomeruli in patients without C1q deposition in the immunofluorescent study was associated with ESRD. Our study confirms the prognostic value of C4d staining in primary IgAN.

In order to study the mechanisms of COVID-19 damage following the complement activation phase occurring during the innate immune response to SARS-CoV-2, CR1 (the regulating complement activation factor, CD35, the C3b/C4b receptor), C4d deposits on Erythrocytes (E), and the products of complement activation C3b/C3bi, were assessed in 52 COVID-19 patients undergoing O 2 therapy or assisted ventilation in ICU units in Rheims France. An acquired decrease of CR1 density on E from COVID-19 patients was observed (Mean = 418, SD = 162, N = 52) versus healthy individuals (Mean = 592, SD = 287, N = 400), Student's t-test p < 10 -6 , particularly among fatal cases, and in parallel with several parameters of clinical severity. Large deposits of C4d on E in patients were well above values observed in normal individuals, mostly without concomitant C3 deposits, in more than 80% of the patients. This finding is reminiscent of the increased C4d deposits on E previously observed to correlate with sub endothelial pericapillary deposits in organ transplant rejection, and with clinical SLE flares. Conversely, significant C3 deposits on E were only observed among ¼ of the patients. The decrease of CR1/E density, deposits of C4 fragments on E and previously reported detection of virus spikes or C3 on E among COVID-19 patients, suggest that the handling and clearance of immune complex or complement fragment coated cell debris may play an important role in the pathophysiology of SARS-CoV-2. Measurement of C4d deposits on E might represent a surrogate marker for assessing inflammation and complement activation occurring in organ capillaries and CR1/E decrease might represent a cumulative index of complement activation in COVID-19 patients. Taken together, these original findings highlight the participation of complement regulatory proteins and indicate that E are important in immune pathophysiology of COVID-19 patients. Besides a potential role for monitoring the course of disease, these observations suggest that novel therapies such as the use of CR1, or CR1-like molecules, in order to down regulate complement activation and inflammation, should be considered., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Objectives: Distinguishing Kikuchi disease (KD) from lupus lymphadenitis (LL) histologically is nearly impossible. We applied C4d immunohistochemical (IHC) stain to develop diagnostic tools., Methods: We retrospectively investigated clinicopathological features and C4d IHC staining in an LL-enriched development cohort (19 LL and 81 KD specimens), proposed risk stratification criteria and trained machine learning models, and validated them in an external cohort (2 LL and 55 KD specimens)., Results: Clinically, we observed that LL was associated with an older average age (33 vs 25 years; P=0.005), higher proportion of biopsy sites other than the neck [4/19 (21%) vs 1/81 (1%); P=0.004], and higher proportion of generalized lymphadenopathy compared with KD [9/16 (56%) vs 7/31 (23%); P=0.028]. Histologically, LL involved a larger tissue area than KD did (P=0.006). LL specimens exhibited more frequent interfollicular pattern [5/19 (26%) vs 3/81 (4%); P=0.001] and plasma cell infiltrates (P=0.002), and less frequent histiocytic infiltrates in the necrotic area (P=0.030). Xanthomatous infiltrates were noted in 6/19 (32%) LL specimens. Immunohistochemically, C4d endothelial staining in the necrotic area [11/17 (65%) vs 2/62 (3%); P<10-7], and capillaries/venules [5/19 (26%) vs 7/81 (9%); P=0.048] and trabecular/hilar vessels [11/18 (61%) vs 8/81 (10%); P<10-4] in the viable area was more common in LL. During validation, both the risk stratification criteria and machine learning models were superior to conventional histological criteria., Conclusions: Integrating clinicopathological and C4d findings could distinguish LL from KD., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Chlamydia trachomatis (C. trachomatis) is the leading cause of sexually transmitted bacterial infections worldwide, with over 120 million annual cases. C. trachomatis infections are associated with severe reproductive complications in women such as extrauterine pregnancy and tubal infertility. The infections are often long lasting, associated with immunopathology, and fail to elicit protective immunity which makes recurrent infections common. The immunological mechanisms involved in C. trachomatis infections are only partially understood. Murine infection models suggest that the complement system plays a significant role in both protective immunity and immunopathology during primary Chlamydia infections. However, only limited structural and mechanistic evidence exists on complement-mediated immunity against C. trachomatis. To expand our current knowledge on this topic, we analyzed global complement deposition on C. trachomatis using comprehensive in-depth mass spectrometry-based proteomics. We show that factor B, properdin, and C4b bind to C. trachomatis demonstrating that C. trachomatis-induced complement activation proceeds through at least two activation pathways. Complement activation leads to cleavage and deposition of C3 and C5 activation products, causing initiation of the terminal complement pathway and deposition of C5b, C6, C7, C8, C9 on C. trachomatis. Interestingly, using immunoelectron microscopy, we show that C5b-9 deposition occurred sporadically and only in rare cases formed complete lytic terminal complexes, possibly caused by the presence of the negative regulators vitronectin and clusterin. Finally, cleavage analysis of C3 demonstrated that deposited C3b is degraded to the opsonins iC3b and C3dg and that this complement opsonization facilitates C. trachomatis binding to human B-cells.

In recent years, the utility of vascular complement factor 4d (C4d) deposition as diagnostic tool for antibody mediated rejection (AMR) after lung transplantation, has become a controversial issue. We aimed to pinpoint the problematic nature of C4d as biomarker with a simple experiment. We quantified C4d in broncho-alveolar lavage (BAL) of lung transplant patients with diverse post-transplant complications in 3 different settings of clinically clear cases of: 1/ chronic lung allograft dysfunction (CLAD); 2/ acute complications acute rejection (AR), lymphocytic bronchiolitis (LB), antibody-mediated rejection (AMR) and respiratory infection (INF); 3/ patients with parallel C4d immunostaining and Anti-HLA. All groups were compared to BAL of stable patients. C4d was measured via standard ELISA. C4d was increased in CLAD, predominantly in RAS (p = 0.0026) but not in BOS (p = 0.89). C4d was increased in all acute events, AR (p = 0.0025), LB (p < 0.0001), AMR (p = 0.0034), infections (p < 0.0001). In patients with parallel C4d immunostaining and serum HLA antibodies, C4d was increased in C4d-/HLA- (p = 0.0011); C4d-/HLA+ (p = 0.013); HLA+/C4d + (p = 0.0081). A correlation of systemic C-reactive protein (CRP) with C4d was found in all patients (r = 0.49; p < 0.0001). We hypothesize that free C4d in BAL may only be representative of a general immune response in the transplanted lung., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In immunoglobulin A (IgA) nephropathy, activation of lectin pathway leads to severe renal damage and more pronounced histological damage. As C4d is a marker of lectin pathway activation, the presence of mesangial C4d positivity will help in identifying those patients at risk. The study was conducted to study the prevalence of mesangial C4d positivity in patients with primary IgA nephropathy and to compare the clinical and histopathological features with C4d-positive and C4d-negative cases. It is a retrospective study conducted for four years. The inclusion criterion was IgA nephropathy with a minimum of four viable glomeruli. Biopsies with >25% of nonsclerotic glomeruli with mesangial positivity will be considered as positive for C4d. Seventy-six patients of IgA nephropathy were included of which mesangial C4d positivity was noted in 33 patients (43%). The mean age was 35 years. The male:female ratio was 2.3:1. The most common presentation was edema (56%) followed by microscopic hematuria (28%). Fifty-four patients were hypertensive. Among the clinical and laboratory parameters, absence of hematuria (P = 0.04) and presence of proteinuria (P = 0.02) showed a significant association with C4d positivity. The histological parameters in Oxford classification which had significant association with C4d positivity were segmental sclerosis (P = 0.01) and tubular atrophy (P = 0.001). Among 45 patients on follow-up with a maximum duration of 51 months, 10 developed end-stage renal disease of which four had C4d expression (0.05%) in the biopsy. Nearly half of IgA nephropathy patients have mesangial C4d positivity. Elevated creatinine with chronicity changes is more common in C4d-positive patients. Hence, C4d can be used as a marker for poor prognosis.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease mediated by pathogenic autoantibodies. While complement protein C4 is associated with SLE, its isoforms (C4A and C4B) are not equal in their impact. Despite being 99% homologous, genetic studies identified C4A as more protective than C4B. By generating gene-edited mouse strains expressing either human C4A or C4B and crossing these with the 564lgi lupus strain, we show that, overall, C4A-like 564Igi mice develop less humoral autoimmunity than C4B-like 564Igi mice. This includes a decrease in the number of GCs, autoreactive B cells, autoantibodies, and memory B cells. The higher efficiency of C4A in inducing self-antigen clearance is associated with the follicular exclusion of autoreactive B cells. These results explain how the C4A isoform is protective in lupus and suggest C4A as a possible replacement therapy in lupus., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale & Objective: Glomerular C4d (C4dG) as an indicator of the lectin pathway of complement activation in immunoglobulin A nephropathy (IgAN) has been associated with more severe kidney damage. Recent studies have suggested that vascular lesions in IgAN biopsy specimens with complement deposition are also associated with disease progression. We aimed to study the clinical significance of arteriolar C4d (C4dA) in IgAN kidney biopsy tissue., Study Design: Retrospective cohort study., Setting & Participants: Kidney biopsy specimens from 126 adults with IgAN diagnosed by Oxford classification criteria were stained using immunohistochemistry and classified according to C4dG and C4dA deposition. Additionally, vascular lesions including acute and chronic microangiopathy, arteriolar hyalinosis, and arterial intima fibrosis were characterized., Predictor: C4dA., Outcome: Progressive kidney disease, defined as a decline in estimated glomerular filtration rate by≥50% or occurrence of kidney failure., Analytical Approach: The association of C4dA and C4dG with baseline clinical and histologic characteristics, as well as progressive kidney disease, were assessed with survival analysis using multivariable Cox regression analysis., Results: C4dA was identified in 21 (17%) patients and was associated with mean arterial pressure, arterial intima fibrosis, and chronic microangiopathy. C4dA was also significantly associated with C4dG and both were associated with progressive kidney disease. In regression analysis, C4dA remained significantly associated with progressive kidney disease after adjusting for other significant predictors, including baseline estimated glomerular filtration rate, mean arterial pressure, and the presence of crescents., Limitations: Findings based on the retrospective evaluation of a single center's experience, limited number of events, a small number of patients with a broad range of kidney disease stages, and use of immunohistochemistry rather than immunofluorescence to detect C4d., Conclusions: C4dA is a potential biomarker for disease progression in IgAN. It should be further investigated in larger cohorts to determine the value of C4dA in improving prediction of IgAN disease progression., (Copyright © 2020 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

The lectin pathway (LP) of complement activation is believed to contribute to brain inflammation. The study aims to identify the key components of the LP contributing to TBI outcome as possible novel pharmacological targets. We compared the long-term neurological deficits and neuropathology of wild-type mice (WT) to that of mice carrying gene deletions of key LP components after experimental TBI. WT or MASP-2 (Masp2 -/- ), ficolin-A (Fcna -/- ), CL-11 (Colec11 -/- ), MASP-1/3 (Masp1 -/- ), MBL-C (Mbl2 -/- ), MBL-A (Mbl1 -/- ) or MBL -/- (Mbl1 -/- /Mbl2 -/- ) deficient male C57BL/6J mice were used. Mice underwent sham surgery or TBI by controlled cortical impact. The sensorimotor response was evaluated by neuroscore and beam walk tests weekly for 4 weeks. To obtain a comparative analysis of the functional outcome each transgenic line was rated according to a health score calculated on sensorimotor performance. For selected genotypes, brains were harvested 6 weeks after injury for histopathological analysis. MASP-2 -/- , MBL -/- and FCN-A -/- mice had better outcome scores compared to WT. Of these, MASP-2 -/- mice had the best recovery after TBI, showing reduced sensorimotor deficits (by 33% at 3 weeks and by 36% at 4 weeks). They also showed higher neuronal density in the lesioned cortex with a 31.5% increase compared to WT. Measurement of LP functional activity in plasma from MASP-2 -/- mice revealed the absence of LP functional activity using a C4b deposition assay. The LP critically contributes to the post-traumatic inflammatory pathology following TBI with the highest degree of protection achieved through the absence of the LP key enzyme MASP-2, underlining a therapeutic utility of MASP-2 targeting in TBI.

The classical and lectin pathways of the complement system are important for the elimination of pathogens and apoptotic cells and stimulation of the adaptive immune system. Upon activation of these pathways, complement component C4 is proteolytically cleaved, and the major product C4b is deposited on the activator, enabling assembly of a C3 convertase and downstream alternative pathway amplification. Although excessive activation of the lectin and classical pathways contributes to multiple autoimmune and inflammatory diseases and overexpression of a C4 isoform has recently been linked to schizophrenia, a C4 inhibitor and structural characterization of the convertase formed by C4b is lacking. In this study, we present the nanobody hC4Nb8 that binds with picomolar affinity to human C4b and potently inhibits in vitro complement C3 deposition through the classical and lectin pathways in human serum and in mouse serum. The crystal structure of the C4b:hC4Nb8 complex and a three-dimensional reconstruction of the C4bC2 proconvertase obtained by electron microscopy together rationalize how hC4Nb8 prevents proconvertase assembly through recognition of a neoepitope exposed in C4b and reveals a unique C2 conformation compared with the alternative pathway proconvertase. On human induced pluripotent stem cell-derived neurons, the nanobody prevents C3 deposition through the classical pathway. Furthermore, hC4Nb8 inhibits the classical pathway-mediated immune complex delivery to follicular dendritic cells in vivo. The hC4Nb8 represents a novel ultrahigh-affinity inhibitor of the classical and lectin pathways of the complement cascade under both in vitro and in vivo conditions., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Urticarial vasculitis (UV) is a clinicopathologic entity characterized by persistent urticarial lesions with biopsy features of vasculitis. Currently, only certain clinical features such as arthralgia and serum complement concentrations are used to identify UV patients at risk for an underlying systemic disease. Hypocomplementemic urticarial vasculitis (HUV) is in contrast to normocomplementemic urticarial vasculitis (NUV), strongly associated with underlying systemic disease, especially systemic lupus erythematosus (SLE). The aim of this study was to find specific histopathological features associated with HUV and underlying systemic disease in UV. In addition, the use of complement C4d deposition in skin biopsies was evaluated as a diagnostic adjunct for HUV- and UV-associated systemic disease. In this retrospective study, the clinical, histopathological, and immunohistological (C4d) features of 43 patients with UV were compared between HUV and NUV and analyzed for association with UV-associated systemic disease. Eight of 43 patients with UV (19%) had hypocomplementemia. Patients with HUV showed a significantly higher number of perivascular neutrophils and lower number of eosinophils compared to NUV. Of all histopathological features, alignment of neutrophils along the dermal-epidermal junction (DEJ) and dermal granular C4d deposition were found to be strongly associated with HUV and underlying SLE. This study shows that both the alignment of neutrophils along the DEJ and dermal C4d deposition are strongly associated with HUV and SLE. Therefore, these (immuno)histopathological features can be used as an easy diagnostic adjunct for early detection of underlying systemic disease in UV.

Background: The aim of this study is to evaluate the efficacy of a treatment protocol for the management of acute antibody-mediated rejection (AAMR) and mixed-type rejection (MTR), both histopathologically and clinically., Materials and Methods: Of the 362 cases undergoing kidney transplantation from January 2010 to January 2018, patients with AAMR or MTR in the first 3 months after transplantation were included. All patients had follow-up biopsy (Bx) after treatment., Results: 33 (9.1%) patients had diagnosis of AMR. Mean follow-up was 35 ± 20 months. 28.5% of patients had poor clinical progression at the last follow-up. Of the 25 patients with functional grafts, the mean serum creatinine was 1.6 ± 0.6 mg/dL for live and 2.1 ± 1.0 mg/dL for deceased transplant recipients. In diagnostic biopsies, there were 61% MTR. In follow-up Bx, after treatment, both C4d positivity and the severity of rejection had decreased while the mean chronic allograft damage index (CADI) score and transplant glomerulopathy showed an increase., Conclusion: With effective antibody mediated rejection (AMR) therapy, renal function parameters were significantly improved. Histologically, improvement in tubulointerstitial inflammation may be responsible for this process. However, progressive chronic changes, particularly in the glomeruli, are noteworthy.

Objective: To evaluate the frequency of cell-bound complement activation products (CB-CAPs) as a marker of complement activation in patients with suspected systemic lupus erythematosus (SLE) and the usefulness of this biomarker as a predictor of the evolution of probable SLE into SLE as classified by the American College of Rheumatology (ACR) criteria., Methods: Patients in whom SLE was suspected by lupus experts and who fulfilled 3 ACR classification criteria for SLE (probable SLE) were enrolled, along with patients with established SLE as classified by both the ACR and the Systemic Lupus International Collaborating Clinics (SLICC) criteria, patients with primary Sjögren's syndrome (SS), and patients with other rheumatic diseases. Individual CB-CAPs were measured by flow cytometry, and positivity rates were compared to those of commonly assessed biomarkers, including serum complement proteins (C3 and C4) and autoantibodies. The frequency of a positive multianalyte assay panel (MAP), which includes CB-CAPs, was also evaluated. Probable SLE cases were followed up prospectively., Results: The 92 patients with probable SLE were diagnosed more recently than the 53 patients with established SLE, and their use of antirheumatic medications was lower. At the enrollment visit, more patients with probable SLE were positive for CB-CAPs (28%) or MAP (40%) than had low complement levels (9%) (P = 0.0001 for each). In probable SLE, MAP scores of >0.8 at enrollment predicted fulfillment of a fourth ACR criterion within 18 months (hazard ratio 3.11, P < 0.01)., Conclusion: Complement activation occurs in some patients with probable SLE and can be detected with higher frequency by evaluating CB-CAPs and MAP than by assessing traditional serum complement protein levels. A MAP score above 0.8 predicts transition to classifiable SLE according to ACR criteria., (© 2019, The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology.)

Background: Immunoglobulin A nephropathy (IgAN) is a major cause of end-stage renal disease. Complement activation via the lectin pathway influences outcomes in IgAN. We examined the association of glomerular C4d deposition with clinicopathological severity at diagnosis and the disappearance of proteinuria in Japanese pediatric IgAN patients., Methods: We retrospectively analyzed 25 children newly diagnosed with IgAN at Hokkaido University Hospital. We evaluated glomerular C4d immunofluorescent staining at diagnosis. We compared clinical findings, pathological findings (based on Oxford classification), and the disappearance of proteinuria within 24 months after renal biopsy between C4d-positive and C4d-negative patients., Results: Glomerular C4d staining was observed in 14 patients (56.0%). C4d-positive patients had significantly higher proteinuria at diagnosis than C4d-negative patients (2.03 g/gCr vs 0.78 g/gCr; P = 0.005). The number of glomeruli with segmental glomerulosclerosis or adhesion (8.0% vs 0.0%; P = 0.046) and the extent of tubular atrophy/interstitial fibrosis (9.46% vs 2.86%; P = 0.031) were significantly increased in C4d-positive patients compared with C4d-negative patients. Further, the proportion of patients with modified T1 (>10%) was significantly higher in the C4d-positive group than the C4d-negative group. There was no significant difference, however, in the disappearance rate of proteinuria at 24 months after renal biopsy between groups (64% vs 82%; P = 0.149)., Conclusions: Glomerular C4d deposition was associated with clinicopathological severity at diagnosis in Japanese pediatric patients with IgAN. Glomerular C4d deposition, however, was not a predictor of the disappearance of proteinuria within 24 months after diagnosis in Japanese pediatric patients with IgAN., (© 2019 Japan Pediatric Society.)

Complement split products (CSPs), such as the fragments C4d and C3d, which are generated as a consequence of complement regulatory processes, are established markers for disease activity in autoimmunity or antibody-mediated graft rejection. Since immunoglobulin-like transcript 4 (ILT4) was previously shown to interact with soluble CSPs, but not with CSPs covalently-bound to target surfaces following classical complement activation, the present study aimed to identify novel cellular receptors interacting with covalently-deposited CSPs. By applying an unbiased screening approach using a cDNA mammalian expression library generated from human monocyte-derived dendritic cells and probed with recombinant human C4d, we identified neuropilin-1 (NRP1) as a novel receptor for C4d, C3d, and iC3b. NRP1, a highly conserved type 1 transmembrane protein, plays important roles in the development of the nervous and cardiovascular system as well as in tumorigenesis through interaction with its established binding partners, such as vascular endothelial growth factor (VEGF) and semaphorin 3A (Sema3A). NRP1 is also expressed on immune cells and serves as a marker for murine Tregs. Although NRP1 contains domains homologous to ones found in some complement proteins, it has not been linked to the complement system. We demonstrate that binding of C4d to NRP1 expressing cells was dose-dependent and saturable, and had a K D value of 0.71 μM. Importantly, and in contrast to ILT4, NRP1 interacted with CSPs that were covalently bound to target surfaces in the course of complement activation, therefore representing a classical complement receptor. The binding site of CSPs was mapped to the b1 domain of the coagulation factor V/VIII homology domain of NRP1. Taken together, our results demonstrate a novel role for NRP1 as a receptor for CSPs deposited on surfaces during complement activation. Further work is required to elucidate the functional consequences of the NRP1-CSP interactions in immunity., (Copyright © 2019 Battin, De Sousa Linhares, Paster, Isenman, Wahrmann, Leitner, Zlabinger, Steinberger and Hofer.)

Among the innate immune sentinels, the complement system is a formidable first line of defense against pathogens, including viruses. Chandipura virus (CHPV), a neurotropic vesiculovirus of the family Rhabdoviridae , is a deadly human pathogen known to cause fatal encephalitis, especially among children. The nature of interaction and the effect of human complement on CHPV are unknown. Here, we report that CHPV is a potent activator of complement and, thus, is highly sensitive to complement proteins in normal human serum (NHS). Utilizing a panel of specific complement component depleted/reconstituted human serum, we have demonstrated that CHPV neutralization is C3, C4, and C1q dependent and independent of factor B, suggesting the importance of the classical pathway in limiting CHPV. Employing a range of biochemical approaches, we showed (i) a direct association of C1q to CHPV, (ii) deposition of complement proteins C3b, C4b, and C1q on CHPV, and (iii) virus aggregation. Depletion of C8, an important component of the pore-forming complex of complement, had no effect on CHPV, further supporting the finding that aggregation and not virolysis is the mechanism of virus neutralization. With no approved vaccines or treatment modalities in place against CHPV, insights into such interactions can be exploited to develop potent vaccines or therapeutics targeting CHPV. IMPORTANCE Chandipura virus is a clinically important human pathogen of the Indian subcontinent. The rapidity of death associated with CHPV infection in addition to the absence of an effective vaccine or therapeutics results in poor clinical prognosis. The biology of the virus and its interaction with the host immune system, including the complement system, are understudied. Our investigation reveals the susceptibility of CHPV to fluid phase complement and also dissects the pathway involved and the mechanism of virus neutralization. Direct binding of C1q, an important upstream component of the classical pathway of complement to CHPV, and the strong dependency on C1q for virus neutralization highlight the significance of identifying such interactions to better understand CHPV pathogenesis and devise strategies to target this deadly pathogen., (Copyright © 2019 American Society for Microbiology.)

Recent studies suggest that complement plays a role in the pathogenesis of focal segmental glomerulosclerosis (FSGS). Moreover, co-localization of IgM and C3 deposits with FSGS lesions has frequently been reported. Here, we investigated whether glomerular complement deposition precedes the development of FSGS and whether it represents local complement activation. Renal biopsies from 40 patients with primary FSGS, 84 patients with minimal change disease, and 10 healthy individuals were stained for C4d, C1q, and mannose-binding lectin. C4d deposits were also measured in renal allograft biopsies from 34 patients with native primary FSGS, 18 of whom subsequently developed recurrent FSGS. Lastly, we measured C4d deposits in the Munich Wistar Frömter rat model of FSGS. The prevalence of C4d-positive glomeruli was significantly higher among patients with FSGS (73%) compared to patients with minimal change disease (21%) and healthy individuals (10%). Moreover, segmental sclerosis was absent in 42% of C4d-positive glomeruli. Glomerular C1q was significantly more prevalent in FSGS compared to minimal change disease or healthy individuals, while mannose-binding lectin was infrequently observed. C4d deposition was significantly more prevalent in recurrent FSGS (72%) before the development of sclerotic lesions compared to control transplant samples (27%). Finally, at the onset of albuminuria but before the development of FSGS lesions, Munich Wistar Frömter rats had a significantly higher percentage of C4d-positive glomeruli (31%) compared to control rats (4%). Thus, glomerular C4d deposition can precede the development of FSGS, suggesting that complement activation may play a pathogenic role in the development of FSGS., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Aims: Antibody-mediated rejection (AMR) is an emerging and challenging issue in transplantation. Endothelial deposition of C4d and microvascular inflammation (MI) are reliable markers of AMR in renal and cardiac transplantation, but remain controversial in the lung. Our aim was to assess C4d immunohistochemistry and histological patterns for the diagnosis of lung AMR., Methods and Results: We reviewed 158 transbronchial biopsies (TBBs) (n = 85 clinically indicated, and n = 73 surveillance TBBs) from 48 recipients, blinded to clinical and serological data. C4d was scored as 0, 1+ (<10%), 2+ (10-50%) or 3+ (>50%). TBBs were reassessed for MI and acute lung injury (ALI). Donor-specific antibodies (DSAs), acute clinical graft dysfunction and chronic lung allograft graft dysfunction (CLAD) were recorded. C4d3+, C4d2+, C4d1+ and C4d0 occurred respectively in four (2.5%), six (3.8%), 28 (17.7%) and 120 (75.9%) TBBs. MI and ALI were rare but more frequent in C4d1-3+ TBBs than in the absence of C4d. C4d2+ was frequently observed with concomitant infection. Among the surveillance TBBs, only two (2.7%) showed MI. Neither ALI nor C4d3+ was diagnosed on surveillance TBBs. No significant association was found between histopathological findings and DSAs. All four patients with C4d3+ could retrospectively be diagnosed with AMR and developed CLAD., Conclusion: Although rare, diffuse C4d deposition appears to be a strong indication of acute clinical AMR in lung transplant patients, whereas intermediate C4d2+ requires more investigations. In stable patients, histopathology and C4d may lack the sensitivity to diagnose subclinical AMR. This emphasises the need for a multidisciplinary evaluation of each suspected AMR case, and the need for complementary diagnostic tools., (© 2019 John Wiley & Sons Ltd.)

Background: To evaluate renal expression of C4d, a complement component in the classical/mannose binding lectin (MBL) pathway, in patients with primary Sjögren's syndrome (pSS)-associated renal impairments., Methods: We retrospectively reviewed the clinical and pathological data from 39 patients with pSS presenting with renal impairments. C4d was examined in paraffin-embedded biopsy tissues using immunohistochemistry. Glomerular C4d positive was defined when > 75% glomeruli were globally stained. Tubulointerstitial C4d (TI-C4d) were scored semi-quantitatively as 0 (absent), 1 (spotty or weak), 2 (patchy) and 3 (diffuse). A TI-C4d score ≥ 2 was considered TI-C4d positive and included in the TI-C4d + group and vice versa. Peritubular capillary (PTC) C4d was scored as 0 (absent), 1 (0~10%, minimal), 2 (10%~ 50%, focal), and 3 (> 50%, diffuse)., Results: Glomerular C4d deposition was observed in all 8 patients with pSS-related membranous nephropathy (MN) without obvious C1q deposition. Two of 5 patients with mesangial proliferative glomerulonephritis and 1 of 2 patients with IgA nephropathy had mild mesangial C4d deposition. Sixteen patients (6 glomerular dominant and 10 tubulointerstitial dominant) presented TI-C4d score ≥ 2. Patients in the TI-C4d + group exhibited a higher serum creatinine level at the time of renal biopsy (TI-C4d + 132.5 [89.7, 165.5] vs. TI-C4d - 83.0 [70.7, 102.0] μmol/L, P = 0.008). PTC C4d was observed in 12 patients, with each of minimal, focal and diffuse staining being noted in 4 patients., Conclusions: The MBL pathway of complement activation was potentially involved in pSS-related MN. Tubulointerstitial C4d might be a pathological marker of severe renal injury in patients with pSS-related renal impairments.

B cell activating factor (BAFF) belonging to TNF family is a cytokine that enhances B-cell proliferation and differentiation. Recently, It has been suggested that BAFF might be a potential therapeutic target for treating autoimmune disease. However, the relationship between BAFF and allograft rejection is controversial, and the clinical significance of BAFF in predicting allograft rejection need to be further explored. We conducted 6-month follow-up study to confirm the hypothesis that BAFF might be a risk factor for predicting acute rejection in kidney transplant recipients. At the end of the study, a total of 155 kidney transplant recipients were recruited from October 2015 to October 2017, and classified into acute rejection group ( n = 34) and stable renal function group ( n = 121) according to their clinical course. We demonstrate that the serum BAFF levels when acute rejection occurred was significantly higher than that in the stable renal function group (2426.19 ± 892.19 vs. 988.17 ± 485.63 pg/mL, P < 0.05). BAFF expression was significantly enhanced in the membrane and cytoplasm of renal tubule epithelial cells in the transplant kidney tissue with acute rejection, and a positive correlation between BAFF and C4d expression was also observed ( r = 0.880, P = 0.001). ROC analyses highlight the superiority of serum BAFF level before transplant over those on other post-transplant days in prediction of acute rejection episodes. The sensitivity, specificity and AUC (area under curve) were 83.3, 89.5, and 0.886%, respectively. Kaplan-Meier survival analysis showed that recipients with higher pretransplant BAFF levels had higher acute rejection incidence ( P = 0.003). In conclusion, we have identified that BAFF levels are associated with the acute rejection and could be a promising biomarker to predict kidney transplant rejection risks.

The DeKAF study was developed to better understand the causes of late allograft loss. Preliminary findings from the DeKAF cross-sectional cohort (with follow-up < 20 months) have been published. Herein, we present long-term outcomes in those recipients (mean follow-up ± SD, 6.6 ± 0.7 years). Eligibility included being transplanted prior to October 1, 2005; serum creatinine ≤ 2.0 mg/dL on January 1, 2006; and subsequently developing new-onset graft dysfunction leading to a biopsy. Mean time from transplant to biopsy was 7.5 ± 6.1 years. Histologic findings and DSA were studied in relation to postbiopsy outcomes. Long-term follow-up confirms and expands the preliminary results of each of 3 studies: (1) increasing inflammation in area of atrophy (irrespective of inflammation in nonscarred areas [Banff i]) was associated with increasingly worse postbiopsy death-censored graft survival; (2) hierarchical analysis based on Banff scores defined clusters (entities) that differed in long-term death-censored graft survival; and (3) C4d-/DSA- recipients had significantly better (and C4d+/DSA+ worse) death-censored graft survival than other groups. C4d+/DSA- and C4d-/DSA+ had similar intermediate death-censored graft survival. Clinical and histologic findings at the time of new-onset graft dysfunction define high- vs low-risk groups for long-term death-censored graft survival, even years posttransplant. These findings can help differentiate groups for potential intervention studies., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: C4d deposition is defined as the footprint of immune injury and it is associated with unfavorable renal outcomes in patients with IgA nephropathy. We searched whether mesangial C4d deposition is associated with poor renal survival in patients with primary focal segmental glomerulosclerosis (FSGS)., Methods: Biopsy specimens were stained with anti-C4d antibody. Patients were classified based on mesangial C4d deposition as C4d-negative and C4d-positive. Groups were compared according to baseline and follow-up clinical variables. Factors that predict renal progression and treatment failure were determined using Cox-regression and multivariate logistic regression models, respectively., Results: Forty-one FSGS patients were followed for a mean of 67.7 ± 40.8 months. C4d-positive group included 18 patients while remaining 23 patients were C4d-negative. Urinary protein excretion and serum creatinine levels at baseline were comparable between groups. Fifteen patients reached the composite primary endpoint which included serum creatinine increasing > 30% from the baseline and reaching > 1.5 mg/dl, and/or evolution to end-stage renal disease (36.6%). In multivariate regression analysis, baseline eGFR (OR 0.71, 95% CI 0.53-0.94; p = 0.016) and mesangial C4d deposition (OR 10.5, 95% CI 1.51-73.18; p = 0.018) were independently associated with treatment failure rates. Mesangial C4d deposition was independently associated with the progression to the primary endpoint (HR 6.54, 95% CI 1.49-28.7, p = 0.013)., Conclusion: We showed for the first time that mesangial C4d deposition is an independent predictor of disease progression and treatment failure in patients with primary FSGS.

Objective To detect the levels of cell-bound complement activation product (C4d) and immunoglobulins (IgG and IgM) on T and B lymphocytes in patients with systemic lupus erythematosus (SLE), and to evaluate the relationships between C4d and immunoglobulins (Ig) on lymphocytes, and finally to evaluate their diagnostic value for SLE. Methods A cross-sectional study including 142 SLE patients, 144 patients with other non-SLE autoimmune diseases and 100 healthy individuals was conducted. C4d and immunoglobulins on peripheral T and B lymphocytes (T-C4d, B-C4d, T-Ig and B-Ig) were measured by flow cytometry. In vitro immunobinding experiment was performed to characterize serum Ig from SLE patients to generate T-C4d, B-C4d, T-Ig and B-Ig. ANA, anti-dsDNA, anti-Smith antibody and other biomarkers were measured. Area under the receiver operating characteristic curve (AUC), nonparametric tests, partial correlation analysis, sensitivity and specificity were used for statistical analysis. Results Levels of T-C4d, B-C4d, T-Ig and B-Ig were found to be highest in SLE patients. Levels of T-C4d, B-C4d, T-Ig and B-Ig in non-SLE patients were also significantly higher than those in healthy individuals. T-C4d was positively correlated with T-Ig (r=0.587), as well as B-C4d vs B-Ig (r=0.734). Purified Ig from SLE plasma generated higher levels of T-C4d, B-C4d, T-Ig and B-Ig than Ig-removed SLE plasma. AUC of T-C4d, B-C4d, T-Ig and B-Ig were 0.733, 0.834, 0.707 and 0.825, respectively. Compared with anti-dsDNA antibody (38.7%), the sensitivity for SLE diagnosis increased significantly when the combination of T-C4d, B-C4d, T-Ig and B-Ig (85.2%) was made. Conclusion The autoantibody is a key factor for cell-bound C4d formation, and T-C4d, B-C4d, T-Ig and B-Ig may be reliable indicators for the diagnosis of SLE.

A 44-year-old man who received allogenic hematopoietic stem cell transplantation after being diagnosed with acute myeloid leukemia developed nephrosis when the dose of tacrolimus was tapered. A renal biopsy showed the granular deposition of immunoglobulin G in the glomerular basement membrane and subepithelial electron-dense deposits, crescent formation, C4d-positive staining of the peritubular capillary, and subendothelial swelling, suggesting that the main pathological diagnosis was membranous nephropathy and that chronic graft-versus-host disease played a role in the etiology of nephrosis. We herein report a case of membranous nephropathy with various pathological findings. C4d deposition suggests complement activation and the involvement of humoral factors.

C4d positive glomerulopathies with pseudolinear capillary wall deposits caused by basement membrane (GBM) remodeling have sporadically been reported in renal transplants. Here we describe the case of a hypertensive 60 year-old male with a 5 month history of nephrotic range proteinuria in the setting of normal serum creatinine, complement and ANA levels. Work-up showed MGUS (IgG/kappa restricted). A diagnostic renal biopsy to search for monoclonal gammopathy of renal significance demonstrated thickened glomerular capillary walls with strong pseudolinear complement factor C4d deposits by immunofluorescence microscopy (IF); all other IF studies including stains for Col4A3 were unrevealing with only minor abnormalities seen for Col4A5 . The strong and unusual C4d staining of undetermined direct diagnostic significance triggered additional electron microscopic studies uncovering marked structural GBM changes suggestive of a hereditary nephropathy. Further genetic testing revealed a very rare X-linked single missense mutation in the NC1 domain of Col4A5 (exon 51) with a single amino acid substitution ( COL4A5 p.A1581S) that has thus far not been reported in hereditary nephropathies. Our case provides further support for pseudolinear glomerular C4d deposits as general markers of GBM remodeling, in our case an unexpected hereditary nephropathy in an older male. Pseudolinear C4d: a general signpost for architectural GBM disturbance and a stimulus for in-depth studies including electron microscopy.

Objective: Ischemia-reperfusion (I/R) injury is a major clinical problem linked to vascular surgery. Currently, no drugs to prevent or to treat I/R injury are approved for clinical use. C1 inhibitor (C1 INH) is known to reduce activation of the plasma cascade systems that are involved in the pathophysiologic process of I/R injury. The aim of this study was therefore to investigate the effect of C1 INH on complement deposition and endothelial cell activation in a rat model of hind limb I/R injury., Methods: Male Wistar rats (wild type, bred at the central animal facility, University of Bern), weighing 250 to 320 g, were used. The rats underwent 2-hour ischemia and 24-hour reperfusion by unilateral clamping of the femoral artery and additional use of a tourniquet. Five groups were divided according to intravenous treatment 5 minutes before ischemia: 50 IU/kg C1 INH (n = 5); 100 IU/kg C1 INH (n = 7); vehicle control (n = 5); nontreated control (n = 7); and normal, healthy control without intervention (n = 4). At the end, muscle edema, tissue viability, and histologic features were assessed. Deposition of immunoglobulin M, C1r, C4d, and fibrin and expression of plasminogen activator inhibitor 1, heparan sulfate (HS), E-selectin, and vascular cell adhesion molecule 1 were evaluated by fluorescence staining. In addition, high-mobility group box 1 protein was measured in plasma., Results: Edema formation was reduced by C1 INH at two dosages, mirrored by improved histologic injury scores and preserved muscle viability. Deposition of immunoglobulin M, C4d, and fibrin was significantly decreased by 100 IU/kg C1 INH compared with nontreated controls. Pretreatment with 100 IU/kg C1 INH also significantly reduced HS shedding and expression of plasminogen activator inhibitor 1 as well as plasma levels of high-mobility group box 1 protein., Conclusions: Pretreatment with both 50 and 100 IU/kg C1 INH attenuated reperfusion injury of rat hind limbs. Pretreatment with 100 IU/kg also preserved the endothelial HS layer as well as the natural, profibrinolytic phenotype of the endothelium. Prevention of endothelial cell activation by C1 INH may therefore be a promising strategy to prevent I/R injury in the clinical setting of peripheral vascular diseases and elective surgery on extremities., (Copyright © 2017 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Introduction: Few studies have reported the association between the complement cascade and pediatric immunoglobulin A nephropathy (IgAN). This study aimed to investigate the association between C4d staining positivity and the clinical/histopathological characteristics of pediatric patients with IgAN., Methods: Children diagnosed with IgAN through renal biopsy were retrospectively reviewed. Renal biopsy specimens were stained using C4d immunohistochemistry., Results: Among the 56 patients, 31 (55.4%) showed positive mesangial or peripheral capillary C4d staining in glomeruli. Urine protein-to-creatinine ratio was significantly higher in C4d-positive patients (p = 0.001). The severity of mesangial proliferation according to the Haas and Oxford classification was positively associated with positive C4d staining (p < 0.001)., Conclusion: Positive C4d staining was found to be significantly associated with the clinical/histopathological severity of IgAN.

The diagnostic criteria for antibody-mediated rejection (ABMR) after small bowel transplantation (SBT) are not clearly defined, although the presence of donor-specific antibodies (DSAs) has been reported to be deleterious for graft survival. We aimed to determine the incidence and prognostic value of DSAs and C4d in pediatric SBT and to identify the histopathologic features associated with C4d positivity. We studied all intestinal biopsies (IBx) obtained in the first year posttransplantation (N = 345) in a prospective cohort of 23 children. DSAs and their capacity to fix C1q were identified by using Luminex technology. Eighteen patients (78%) had DSAs, and 9 had the capacity to fix C1q. Seventy-eight IBx (22.6%) were C4d positive. The independent determinants of C4d positivity were capillaritis grades 2 and 3 (odds ratio [OR] 4.02, P = .047 and OR 5.17, P = .003, respectively), mucosal erosion/ulceration (OR 2.8, P = .019), lamina propria inflammation grades 1 and 2/3 (OR 1.95, P = .043 and OR 3.1, P = .016, respectively), and chorion edema (OR 2.16, P = .028). Complement-fixing DSAs and repeated C4d-positive IBx were associated with poor outcome (P = .021 and P = .001, respectively). Our results support that capillaritis should be considered as a feature of ABMR in SBT and identify C1q-fixing DSAs and repeated C4d positivity as potential markers of poor outcome., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: The progression from acute to chronic antibody-mediated rejection in kidney transplant recipients is usually not prevented by current therapeutic options. Here, we investigated whether the use of tofacitinib (TOFA), a Janus kinase 3 inhibitor, was capable of preventing the progression of allograft dysfunction in a Fisher-to-Lewis rat model of kidney transplantation., Methods: Rats were treated from the third week after transplantation to allow the development of rejection. Treatment was based on cyclosporin A, rapamycin or TOFA. Renal function was assessed at 1, 4, 8, and 12 weeks after transplantation, whereas rat survival, histological lesions, and infiltrating lymphocytes were analyzed at 12 weeks., Results: Tofacitinib prolonged graft survival, preserved tubular and glomerular structures and reduced humoral damage characterized by C4d deposition. Tofacitinib was able to reduce donor-specific antibodies. In addition, T and natural killer cell graft infiltration was reduced in TOFA-treated rats. Although rapamycin-treated rats also showed prolonged graft survival, glomerular structures were more affected. Moreover, only TOFA treatment reduced the presence of T, B and natural killer cells in splenic parenchyma., Conclusions: Tofacitinib is able to reduce the immune response generated in a rat model of kidney graft rejection, providing prolonged graft and recipient survival, better graft function, and less histological lesions.

Purpose of Review: To review changes in the Banff schema for antibody-mediated renal allograft rejection over the past decade, including key revisions agreed upon during and immediately subsequent to the 2017 Banff Conference on Allograft Pathology., Recent Findings: The original Banff schema for diagnosis of acute and chronic active antibody-mediated rejection (ABMR) in renal allografts was formulated at the 2001 and 2007 Banff Conferences, and required histologic (primarily microvascular inflammation and transplant glomerulopathy), immunohistologic (C4d in peritubular capillaries), and serologic [circulating donor-specific antibodies (DSA)] evidence for a definitive diagnosis of ABMR. This schema was updated at the 2013 Banff Conference, recognizing C4d-negative ABMR, intimal arteritis as a potential manifestation of ABMR, and revising definitions and thresholds for glomerulitis and transplant glomerulopathy to improve interobserver agreement and correlation with clinical, molecular, and serologic data. Compared with the 2007 criteria, Banff 2013 improved the sensitivity of the classification for diagnosing ABMR and the correlation of ABMR diagnosis with graft outcomes. At the 2017 Banff Conference, new modifications to the classification were discussed and have subsequently been agreed upon, accepting C4d and thoroughly validated molecular classifiers as surrogate markers for DSA., Summary: From a consensus reached at the 2017 Banff Conference, updated criteria for diagnosis of active and chronic active ABMR have been developed that recognize C4d and molecular classifiers as surrogate markers for DSA. In addition, specific recommendations for the use of molecular diagnostics in the diagnosis of ABMR were developed.