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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.)

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.)

Pediatric renal transplant recipient survival continues to improve, but ABMR remains a significant contributor to graft loss. ABMR prognostic factors to guide treatment are lacking. C4d staining on biopsies, diagnostic of ABMR, is associated with graft failure. Persistent C4d+ on follow-up biopsies has unknown significance, but could be associated with worse outcomes. We evaluated a retrospective cohort of 17 pediatric renal transplant patients diagnosed with ABMR. Primary outcome at 12 months was a composite of ≥50% reduction in eGFR, transplant glomerulopathy, or graft failure. Secondary outcome was the UPCR at 12 months. We used logistic and linear regression modeling to determine whether persistent C4d+ on follow-up biopsy was associated with the outcomes. Forty-one percent reached the primary outcome at 12 months. Persistent C4d+ on follow-up biopsy occurred in 41% and was not significantly associated with the primary outcome, but was significantly associated with the secondary outcome (estimate 0.22, 95% CI 0.19-0.25, P < .001), after controlling for confounding factors. Persistent C4d+ on follow-up biopsies was associated with a higher UPCR at 12 months. Patients who remain C4d+ on follow-up biopsy may benefit from more aggressive or prolonged ABMR treatment., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The aim of this study was to evaluate whether asymptomatic recurrent (≥2) antibody-mediated rejection (pAMR 1+), defined as diffuse capillary C4d immunostaining (rAMR) on endomyocardial biopsies (EMBs), during the first year after heart transplantation impairs left ventricular (LV) function., Methods: Fifty-four consecutive heart transplant patients who survived well (New York Heart Association ≤2 and EF≥55%) the first month after transplantation were enrolled and prospectively underwent 490 echocardiographies and EMB. Asymptomatic rAMR without histopathologic findings was evaluated as a risk factor for deterioration of graft function. Primary endpoint, assessed 1 year after transplantation, was development of LV dysfunction and/or adverse remodeling according to pre-specified echo parameters., Results: During the first year from transplantation, rAMR occurred in five patients. Recurrent AMR was associated with a significant higher risk to develop LV concentric hypertrophy (OR 3.6, 95% CI: 1.8-7.0, P=.02) or reduced lateral S' peak velocity (OR 2.3, 95% CI: 1.5-3.6, P=.03). Patients with rAMR showed significative adverse graft remodeling (ΔLV end-diastolic volume: +16±12.3 vs -0.2±14.4 mL; P=.02) and deterioration of graft function (Δlateral S' peak velocity: -3.3±3 vs -0.4±2.9 cm/s; P=.03)., Conclusions: Recurrent asymptomatic diffuse capillary C4d immunostaining may play a role in the early development of cardiac allograft adverse remodeling and dysfunction., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: Complement C4d-containing fragments have been proposed as diagnostic markers for lung cancer. The purpose of this study was to evaluate the presence of C4d in oropharyngeal (OPSCC) and oral (OSCC) squamous cell carcinomas., Subjects and Methods: C4d staining was analyzed by immunohistochemistry in 244 OPSCC surgical specimens. C4d levels were quantified by ELISA in resting saliva samples from 48 patients with oral leukoplakia and 62 with OSCC. Plasma samples from 21 patients with leukoplakia and 30 with oral carcinoma were also studied., Results: C4d staining in OPSCC specimens was associated with nodal invasion (P = 0.001), histopathologic grade (P = 0.014), disease stage (P = 0.040), and focal-adhesion kinase expression (P < 0.001). No association was found between C4d and prognosis. Saliva C4d levels were higher in patients with oral cancer than in subjects with leukoplakia (0.07 ± 0.07 vs 0.04 ± 0.03 μg ml(-1) , P = 0.003). The area under the ROC curve was 0.63 (95%CI: 0.55-0.71). Salivary C4d levels in stage IV patients were higher than in patients with earlier stages (P = 0.028) and correlated with tumor size (P = 0.045). Plasma C4d levels also correlated with salivary C4d levels (P = 0.041), but differences between patients with oral cancer and subjects with leukoplakia were not significant (1.26 ± 0.59 vs 1.09 ± 0.39 μg ml(-1) , P = 0.232)., Conclusion: C4d-containing fragments are detected in oral primary tumors and are increased in saliva from patients with OSCC., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The C4d staining as a special tissue marker for humoral immunity has served criteria of pathological diagnosis for antibody-mediated rejection (ABMR) in Banff classification since 2003. However, the sensitivity and specificity of C4d staining have been questioned, and recently, C4d-negative ABMR has been more focused in renal allograft pathology. The aim of this study was to make certain of C4d staining for ABMR that was diagnosed by clinical and morphological findings. C4d staining was employed by immunofluorescence. This study included 14 patients with acute ABMR and 16 with chronic active ABMR. Eight of acute ABMR were ABO-blood-type-incompatible renal transplantation (ABOinRTx) pre-treated by DFPP and splenectomy or rituximub. In acute ABMR after ABOinRTx, C4d staining along peritubular capillary (PTC) was positive in five of them (62.5%). Only one graft biopsy of five acute ABMR with donor-specific antibody (DSA) showed C4d positive. We assembled 16 graft biopsies showing typical transplant glomerulopathy and thickened PTC basement membrane with peritubular capillaritis as a suspicious pathological chronic active ABMR. Four of eight DSA-positive patients were C4d negative in PTC; however, three of four DSA-positive and C4d-negative patients in PTC chronic active ABMR were C4d positive in only glomerular capillaries. C4d positivity could not come to a specific marker of ABMR diagnosing based on clinically and ordinary morphological findings., (© 2012 John Wiley & Sons A/S.)

Liver transplantation is performed based on ABO blood type compatibility without dependence on crossmatch results. Combined liver-kidney transplantation (CLKT) is similarly performed without dependence of crossmatch results as the liver is thought to confer protection to the renal allograft against alloantibody. We report a case of CLKT in a sensitized patient with antibody-mediated rejection (AMR) of the renal allograft. AMR was confirmed with C4d staining and serial monitoring of donor-specific antibody (DSA). Despite intensive therapy directed against AMR and the presence of the liver allograft, the patient demonstrated increasing titers of alloantibody, never demonstrated adequate renal function, and ultimately expired after two months. This result demonstrates the potential for AMR of the renal allograft in sensitized recipients of CLKT., (© 2009 John Wiley & Sons A/S.)

Unlabelled: Histopathological change of acute vascular rejection (AVR) is characterized by intimal arteritis and transmural arteritis. In this report, we discuss the clinicopathological analysis of AVR cases after renal transplantation (RTX)., Patients: AVR was diagnosed in 17 patients from 17 renal transplant patients followed in our institute between January 2003 and September 2008. We retrospectively reviewed these 17 patients., Results: Among 17 cases of AVR, 10 cases were mild (v1 in Banff 07 classification), five were moderate (v2), and two were severe (v3). Interstitial inflammation (i1-i3) was present in all 17 biopsies. Moderate to severe tubulitis (t2-t3) was present in seven biopsies, and transplant glomerulitis (g1-g3) was present in 11, peritubular capillaritis (ptc1-ptc3) was in 15 of 17 biopsies. C4d deposition in peritubular capillary (PTC) was observed in 6 of 17 cases. By assaying with plastic beads coated with anti-human leukocyte antigen (HLA) antigen performed in 17 cases, the circulating ant-HLA alloantibody was detected in 10 patients, of which 5/10 were donor-specific antibodies (DSA). Acute antibody-mediated rejection (AAMR) was diagnosed in three cases. Many of v1 cases, steroid pulse therapy (SP) were effective. In v2 and v3 cases, six of seven were steroid-resistant rejection and were need more anti-rejection therapy (ART), such as muromonab CD3 (OKT3) injection, gusperimus (DSG) injection, plasmapheresis, intravenous immune globulin, and injection of rituximab. Ten of 17 patients recovered their renal allograft functions by ART, and 16 of 17 patients' grafts are functioning. Deterioration of renal allografts' function after biopsies was seen in seven patients with one of them lost their graft., Conclusions: In some cases, AVR might be provoked by anti-donor antibodies. The prognosis of the graft exhibiting AVR was relatively good in present immunosuppression and ART.

The clinical significance of C4d positivity in patients with stable graft function is undetermined. This study evaluated the clinical outcome of protocol biopsy-proven C4d-positive renal transplants with stable graft function in the early post-transplantation period. Protocol biopsies (n = 79) were performed on stable allografts on the 14th post-transplant day, and indication biopsies (n = 74) were performed on dysfunctioning allografts within one yr after transplantation. Clinical and histological findings, graft function and graft survival rates were compared between C4d-positive and C4d-negative grafts in each group. The incidence of C4d positivity was 5.1% in protocol biopsies and 9.5% in indication biopsies. In protocol biopsies, C4d-positive allografts showed minimal tubulointerstitial inflammation, and the graft function and graft survival rate did not differ from C4d-negative allografts. All C4d-positive allografts maintained stable graft function without anti-rejection therapy, and follow-up biopsies of two patients showed no C4d deposition or evidence of rejection. On the other hand, C4d-positive allografts in indication biopsies showed severe tissue injury, and the graft survival rate was significantly lower than C4d-negative allografts. In conclusion, C4d-positive allografts with stable graft function in the early post-transplantation period take an indolent course.

We analyzed rates of both SCR and CR in children receiving SB at three months post-transplant to determine if SCR predisposed patients to acute CR. Acute rejection was defined according to Banff criteria to include borderline classification or higher. All cases of SCR and CR were treated with anti-rejection protocols. Between October 2004 and July 2008, 89 SB were performed at three months post-transplant. Twenty-six cases of SCR were detected (29%). Sixteen patients experienced 22 episodes of biopsy-proven CR occurring after SB, including seven episodes following SCR and 15 after normal SB. The onset of CR varied from one to 27 months after SB and occurred at similar intervals for cases with SCR and normal SB. The percentage of patients remaining free of CR at 30 months post-transplant was similar in patients with SCR and normal SB. Renal function and graft survival at 30 months also were no different between patients with SCR and those with normal SB. Early-SCR, when treated with rejection protocols, is not a prognostic indicator for subsequent CR episodes.

Background: In the early post-transplant period, renal allograft rejection with diffuse peritubular capillary (PTC) C4d deposition predicts poor graft survival. In the late post-transplant setting, that is, one or more yr after transplantation, the implication of diffuse PTC C4d deposition is still a topic of debate. The purpose of our study was to see if diffuse PTC C4d deposition, in late acute rejection (LAR), occurring more than one yr post-transplant, has any impact on graft survival and function., Methods: We selected cases, both cadaveric as well as living donor renal transplant recipients, in whom acute rejection with PTC C4d deposition was first detected after the first year post-transplant. Recipients with multiple acute rejection episodes during the first year post-transplant were excluded from the study. The first biopsy diagnosed with LAR was considered the index biopsy (n = 40). We formed two groups: group 1, C4d-positive LAR (n = 20), and group 2, C4d-negative LAR (n = 20). Groups were matched for maintenance and post-rejection immunosuppressive therapy, baseline serum creatinine levels before the time of the index biopsy, time from transplant to index biopsy, as well as chronic allograft damage index (CADI) score in the index biopsies. We compared the rate of graft loss, and the graft function of the surviving grafts at the end of the study period, as well as histologic parameters in the index biopsy specimens between the two groups. The mean follow-up period was 20 months., Results: No significant differences in the rate of graft loss or graft function were found between groups 1 and 2 at the end of the follow-up period. Histologically, PTC margination and transplant glomerulopathy were more common in the C4d-positive group, and this difference was statistically significant. There was no statistically significant difference in the degree of plasma cell infiltrates., Conclusions: Unlike in the acute setting, the presence or absence of PTC C4d staining in renal allografts with LAR may not have a predictive value regarding graft outcome.

Background: Immunological staining of the transplanted kidney for C4d in peritubular capillaries (C4d(PTC)) has emerged as a useful method to detect antibody-mediated rejection in situ. In this retrospective study, we evaluated the prevalence of C4d(PTC) deposition in allograft renal biopsies diagnosed of IgA nephropathy (IgAN) and analysed its clinical significance., Method: Sixty-six biopsy specimens of post-transplant IgAN, which were obtained to evaluate azotemia and/or heavy proteinuria, were examined by immunohistochemical staining of the paraffin sections with polyclonal antibody for C4d., Results: C4d was stained positively in peritubular capillaries in 16 (24%) of the 66 cases. The C4d(PTC)-negative (n=50) and C4d(PTC)-positive groups (n=16) were not different in recipient gender, age, donor age, type of donor (living vs. cadaveric), interval from transplantation to graft biopsy (41.6+/- 21.8 vs. 48.3+/-26.1 months) and post-biopsy follow-up period (60.3+/-23.3 vs. 56.9+/-25.4 months). During the follow-up period, 12 of 50 (24%) although the incidence of graft failure was not different by the C4d deposition in peritubular capillaries, intervals from renal biopsy to graft failure tended to be shorter in C4d(PTC)-positive cases than C4d(PTC)-negative cases. In Kaplan-Meier analysis, the renal allograft function of the C4d(PTC)-positive group deteriorated more rapidly than that of the C4d(PTC)-negative group (p<0.05). Histologically, the C4d(PTC)-positive group had findings suggestive of acute cellular rejection more commonly than the C4d(PTC)-negative group (p<0.01)., Conclusions: Evidence of humoral rejection, as demonstrated by C4d(PTC) deposition, was concurrently present in significant portions of post-transplant IgAN biopsy specimens and was associated with more rapid deterioration of renal function. These results suggest that C4d(PTC) positivity needs to be determined at the time of biopsy even in cases of post-transplant glomerulonephritis and immunosuppression may need to be modified accordingly.

Background: Chronic allograft nephropathy (CAN), associated with late-allograft dysfunction is caused by alloantigen-dependent and -independent mechanisms, and eventually leads to interstitial fibrosis (ci). Activation of complement cascade is considered to be a poor prognostic marker of graft survival. This study was designed to examine the relationship between the expression of C4d and heat-shock protein 47 (HSP47, a collagen-specific chaperone) in the development of interstitial fibroproliferative lesions in CAN., Methods: Sixty-three renal allograft biopsy specimens, obtained from 48 patients, were examined for the expression of C4d, HSP47, CD68 and alpha-smooth muscle actin (alpha-SMA) by immunohistochemistry. Double-staining was performed to determine the colocalization of C4d and HSP47. The relationship of between the expression of C4d, HSP47, CD68 and alpha-SMA and the clinical and histopathological parameters were statistically analysed., Results: No expression of C4d was noted in the tubulointerstitium including peritubular capillary (PTC) of the control kidney. C4d was expressed in PTC in one-third of allograft renal tissues with morphological evidences of CAN. The interstitial cells around the fibrotic areas of the PTC of CAN were positive for the expression of HSP47. The deposition of C4d in PTC correlated with interstitial expression of HSP47 around the PTC. Most HSP47 expressing cells were phenotypically altered myofibroblasts, as determined by the dual staining of alpha-SMA., Conclusions: The increased expression of HSP47 positively correlated with the expression of C4d in PTC, and might contribute to the progression of interstitial ci in CAN.

The effects of antibody-mediated rejection on long-term graft survival have not been fully investigated. The aim of this study is to clarify the influence on long-term survival of deposition of the complement split product C4d in allografts using polyclonal anti-C4d antibody. Inclusion criteria were recipients who underwent graft biopsy during acute deterioration of graft function within the first 2 yr after transplantation. Patients whose graft did not survive more than 1 yr and who received graft from an human leucocyte antigen (HLA)-identical sibling or an ABO-incompatible donor were excluded. Among the 92 recipients investigated, 22 (23.9%) had peritubular capillary C4d deposition, 15 (16.3%) had glomerular capillary C4d deposition and seven (7.6%) had both peritubular and glomerular capillary C4d deposition. Twenty of these 22 patients revealed acute cellular rejection, including borderline changes. There was no significant relationship between pathological severity of acute rejection and presence or absence of peritubular capillary C4d deposition. Graft survival was inferior in patients with peritubular capillary C4d deposition to that in patients without C4d deposition (p = 0.0419). Graft survival in patients with glomerular C4d deposition did not differ from that in patients without C4d deposition. In conclusion, C4d deposition in peritubular capillaries has a substantial impact on long-term graft survival.

A 41-year-old Japanese male patient with end-stage renal disease received ABO compatible living related kidney transplantation from his sister on April 2003. The kidney functioned immediately after kidney transplantation. Protocol allograft biopsy at 1 yr after kidney transplantation was performed on April 2004. His serological data was not particular and he did not suffer with chronic inflammation. The allograft biopsy specimen revealed moderate accumulations of polymorphonuclear leukocytes in peritubular capillaries (PTCs), dilatation of PTCs and moderate infiltrations of polymorphonuclear and/or mononuclear cell in glomeruli (Transplant glomerulitis, moderate). Immunofluorescent study (IF) of a frozen section of the allograft biopsy specimen showed a strong, diffusely distributed endothelial-staining pattern in PTCs for C4d. The C4d was also strongly detected in a linear glomerular basement membrane (GBM) pattern. And widespread moderate C3c deposits, weak IgM, and IgG deposits were also seen in PTCs. Immunofluorescent study also showed granularly peripheral and mesangial deposits of strong IgM, C1q, and moderate IgG in glomeruli, IgA and C3c were faintly positive. The panel reactive antibody, which had been negative before transplantation, was positive for both HLA classes I and II at that time. We diagnosed as acute humoral rejection (AHR) and he was treated with course of steroid pulses and 5 d of gusperimus (DSG); and a total of three times Plasma exchange (PE) treatment was added. The level of serum creatinine, once increased to 1.7 mg/dL, decreased gradually to 1.4 mg/dL. He has a stable graft function. This is the only case of various depositions of immunoglobulins and complements in PTC and/or glomerular capillaries during AHR.