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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.

Background: The significance of humoral immune response for allograft survival after liver transplantation (LT) is still a matter of debate. The aim of this cross-sectional study was to assess immunological and clinical factors associated with advanced fibrosis (F3-F4) and chronic graft failure in LT recipients., Methods: Serum samples from 174 patients prospectively enrolled and followed up for 12 months were tested for anti-HLA antibodies and compared against donor HLA types. Immunohistochemical C4d staining was performed on formalin-fixed, paraffin-embedded liver tissue., Results: Mean time period from LT to enrollment was 66.9 ± 51.9 months. Independent predictive factors for graft failure included donor-positive cytomegalovirus serostatus (P = 0.02), donor-specific antibodies (DSA) against HLA class II (P = 0.03), donor age (P = 0.01), hepatitis C virus allograft reinfection (P = 0.0008), and biliary complications (P = 0.003). HLA class II DSA and HLA class I antibody positivity, hepatitis C virus reinfection, and mycophenolate mofetil-free regimens were significant risk factors for advanced fibrosis after LT. There was a significant association between C4d deposition on allograft endothelial cells and presence of class II DSA (P < 0.0001). Patients with C4d deposits had a 4.3 times higher risk of graft failure than those with negative staining and a significantly lower median time to graft failure (94.6 months [range, 3.6-158.9 months] vs 176.4 months [range, 9.4-217.8 months], P < 0.0001)., Conclusions: Screening for HLA DSA might be useful for early identification of LT recipients at increased risk of graft failure who could benefit from closer surveillance and tailored immunosuppressive regimens.

Background: Histology remains a cornerstone for antibody-mediated rejection (AMR) diagnosis. Little data exist supporting histology for assessing therapeutic responses. This study evaluates histologic components in assessing AMR therapeutic responses., Methods: Antibody-mediated rejection was diagnosed using Antibody Working Group criteria and Banff component scoring, and C4d staining data were analyzed. Statistics included independent and paired samples t test, χ(2), Fisher exact, or the Wilcoxon-signed rank test. Fifty-five AMR patients were analyzed. Early AMR was defined as occurring within 6 months after transplantation and treated with a single rituximab dose and 4 bortezomib doses preceded by plasmapheresis. Allograft biopsies were performed within 48 hours of treatment; repeat biopsy was performed 14 to 21 days later., Results: Early AMR demonstrated histologic improvement in mean scores for acute Banff components glomerulitis (g), C4d, g+ peritubular capillaritis (ptc) and acute composite score, but showed deterioration in chronic Banff components tubular atrophy and interstitial fibrosis. Late AMR showed improved mean scores for acute Banff components tubulitis, interstitial inflammation, g, ptc, g + ptc, C4d, and acute composite score, but chronic scores did not change. Significant changes in distribution of Banff scores after treatment were observed for g, C4d, tubular atrophy, and interstitial fibrosis scores in early AMR patients and tubulitis, interstitial inflammation, g, ptc, and C4d in late AMR., Conclusions: These results show that: (1) Banff component scoring provides insights into histologic responses to AMR therapy and may provide a potential endpoint for clinical AMR trials. (2) Early and late AMR demonstrate differences in acute and chronic Banff components at the time of the AMR diagnostic biopsy, as well as differential responses to AMR therapy.

Background: It is unclear if the category of acute rejection with intimal arteritis (ARV) is relevant to short- and long-term clinical outcomes and if the graft outcomes are affected by the severity of intimal arteritis., Methods: One hundred forty-eight ARV episodes were reviewed and categorized according to the 2013 Banff criteria of AMR: T cell-mediated rejection with intimal arteritis (v) lesion (TCMRV; n = 78), total antibody-mediated rejection with v lesion (AMRV), which were further divided into suspicious AMRV (n = 37) and AMRV (n = 33). The Banff scores of intimal arteritis (v1, v2 and v3) represented low, moderate, and high ARV severity., Results: The grafts with TCMRV, suspicious AMRV (sAMRV), and AMRV showed similar responses to antirejection therapy, whereas the grafts with v2- or v3-ARV responded significantly poorer compared to those with v1-ARV. The 8-year death-censored graft survival (DCGS) rate was 56.8% of TCMRV versus 34.1% of total AMRV (Log rank, P = 0.03), but the 1- and 5-year DCGS rates were comparable between the 2 groups; moreover, the 1-, 5-, and 8-year DCGS rates of v1-ARV were evidently higher than v2- and v3-ARV (each pairwise comparison to v1-AVR yields P < 0.01); in contrast, the DCGS rates were similar between sAMRV and AMRV. The existing donor-specific antibodies or moderate microvascular inflammation or C4d-positive staining or intensive tubulointerstitial inflammation played a less significant role on the long-term graft survival., Conclusions: Compared to the category, the ARV severity is more closely associated with the initial response to antirejection therapy and long-term graft failure. The sAMRV and AMRV might represent a spectrum of the same disorder.

Background: Presence of circulating donor-specific antibodies (DSA) may be associated with worse clinical outcomes after intestine/multivisceral transplantation., Methods: In 79 intestine/multivisceral recipients, sera were prospectively screened for DSA by Luminex Single antigen test at 1, 3, 6, 9, 12, 18, 24, and 36 months after transplantation. Standard immunosuppression included thymoglobulin-rituximab induction and tacrolimus-prednisone maintenance. C4d staining was performed retrospectively on biopsies in patients that developed acute rejection (AR)., Results: Twenty-two (28%) patients developed de novo DSA at a median posttransplant period of 3 (1-36) months. De novo DSA were observed in 10 of 40 liver-including and 12 of 39 liver-excluding transplants (P = 0.57). Occurrence of AR was slightly higher in patients with de novo DSA (45% vs 33%, respectively; P = 0.41). Similarly, chronic rejection (14% vs 5%; P = 0.21) and graft loss due to AR (18% vs 7%; P = 0.14) were numerically higher in patients with de novo DSA. Only 35% patients experiencing AR had circulating de novo DSA at the time of AR. Antibody-mediated rejection was diagnosed in 6 patients based on C4d staining, of these 2 patients had circulating de novo DSA at the time of biopsy., Conclusions: De novo DSA formation, particularly early in the posttransplant course may be associated with trends toward worse outcomes. However, its significance in the pathophysiology of AR remains uncertain. Studies focusing mechanisms of DSA-related graft injury and intragraft DSA detection might provide further insight into this issue.

Background: Acute antibody-mediated rejection (AMR) is responsible for up to 20% to 30% of acute rejection after kidney transplantation. New therapeutic agents have recently emerged, such as eculizumab, an anticomplement protein-C5 monoclonal antibody. In the setting of renal transplantation, eculizumab has so far proved effective both for preventive and curative treatments of AMR in sensitized patients and patients diagnosed with severe AMR. Unsuccessful eculizumab treatment has only been reported once in the literature by Stegall et al. (Am J Transplant 2011; 11: 2405)., Methods and Results: We present two cases of AMR resistant to eculizumab after renal transplantation. One patient received the anti-C5 antibody curatively, and the other patient developed AMR while being treated with eculizumab after a relapse of atypical hemolytic uremic syndrome. The peculiarity of these two cases was the absence of C4d deposition in peritubular capillaries as well as the absence of C1q-binding donor-specific anti-human leukocyte antigen alloantibody, as determined retrospectively, suggesting that a complement-independent mechanism underlies the pathogenesis of these AMR., Conclusion: The use of eculizumab in C4d-negative or C1q-negative AMR does not seem effective.

Background: The correlation between histopathologic phenotypes and allograft outcomes among patients desensitized for donor-specific antibody (HLA-incompatible) is unknown., Methods: We analyzed 1-year biopsies from desensitized recipients transplanted between 1999 and 2010 and estimated graft survival for each histologic phenotype identified. Median time posttransplant for the 1-year biopsy was 367 days (interquartile range 357-388 days) and median follow-up of all patients post-1-year biopsy was 42 months (interquartile range 19.5-65 months)., Results: Transplant glomerulopathy was present in 25.0% of biopsies and resulted in worse graft survival (66.7% vs. 96.7%, P<0.001). C4d positivity and transplant glomerulopathy together portended exceptionally poor graft survival (33.3% vs. 97.2%, P<0.001). Microcirculation inflammation was prevalent, with glomerulitis and peritubular capillaritis found in 60.0% and 47.6% of 1-year biopsies, respectively. Glomerulitis was associated with worse graft survival (82.1% vs. 98.1%, P=0.004), whereas capillaritis was not (88.1% vs. 97.7% respectively, P=0.091). Among C4d-negative HLA-incompatible recipients (82.6% of biopsies), no difference in graft survival was observed between patients with or without microcirculation inflammation in contrast to previous reports by other investigators. Patients who had no C4d deposition, transplant glomerulopathy, or microcirculation inflammation had a 100.0% graft survival. On Cox regression analysis, no independent histopathological parameter was associated with graft survival., Conclusions: We have identified several histopathologic phenotypes in HLA-incompatible kidney recipients that correlate with allograft outcomes. Characterization of these phenotypes is the first step towards better understanding the pathophysiologic basis of chronic antibody-mediated allograft injury and individualizing therapeutic intervention.

Background: Positive crossmatch (CM) in liver transplantation (LT) is associated with worse outcomes. Role of induction immunosuppression in this setting remains to be studied., Methods: One thousand consecutive LT patients receiving rabbit antithymocyte globulin±rituximab induction were studied. Pretransplantation sera of 55 CM-positive (CM) patients were tested for C1q-fixing donor-specific antibodies (DSA). Diagnosis of antibody-mediated rejection required presence of diffuse vascular C4d expression on liver biopsies., Results: CM was positive in 112 (11%) recipients. Antibody-mediated rejection was observed in 3 (0.03%) patients, whereas acute cellular rejection (ACR) occurred in 31 (3%) patients. CM status was associated with a higher incidence of ACR (9% in CM vs. 2% in CM-negative [CM]; P<0.01) and chronic rejection (4% in CM vs. 1% in CM; P<0.01). Graft survival was slightly lower in CM patients (at 1 year; 85% in CM vs. 89% in CM; P=0.26). Patients with autoimmune hepatitis, primary sclerosing cholangitis, and primary biliary cirrhosis as a group had a tendency toward CM status as well as developing ACR. Upon multivariate analysis, CM status was the strongest predictor of ACR (B=1.14; P=0.02). Only half of CM patients harbored C1q-fixing DSA. Presence of C1q-fixing DSA was not associated with increased incidence of ACR., Conclusions: In LT, CM status is associated with an increased incidence of acute rejection, chronic rejection, and slightly worse graft survival. With the use of rabbit antithymocyte globulin±rituximab induction, overall low rejection rates can be achieved in CM LT.

Background: The mechanism of kidney injury in hematopoietic stem cell transplantation (HSCT)-associated thrombotic microangiopathy (TA-TMA) is not completely understood. Renal C4d staining is a marker of classic complement activation and endothelial injury and has been described in preliminary reports of HSCT recipients with TA-TMA. Our objective was to evaluate complement in the pathogenesis of small vessel injury in children receiving HSCT. We hypothesized that kidney tissue from children with TA-TMA would more frequently show C4d deposition compared with HSCT recipients without histologic TA-TMA., Methods: We reviewed kidney specimens (biopsy or autopsy) from children who had undergone HSCT at a single center. Using histologic criteria alone, subjects were divided into TA-TMA (n = 8) and non-TA-TMA (control) groups (n = 12). C4d staining was performed by immunohistochemistry and evaluated on arterioles, peritubular capillaries, glomeruli, and tubular basement membranes., Results: Diffuse or focal renal arteriolar C4d staining was more common in subjects with histologic TA-TMA (75%) compared with controls (8%). Rare peritubular capillary C4d staining was present in 50% of TA-TMA samples and was absent in controls. Glomerular C4d staining was seen at a similar frequency in cases and controls, whereas tubular basement membrane staining was less frequently observed and only in subjects with TA-TMA., Conclusions: Arteriolar C4d deposition may be a pathologic marker of TA-TMA, implicating localized complement fixation in HSCT recipients with kidney disease secondary to small vessel injury. Further studies to better characterize the preferential arteriolar C4d staining may identify a renal compartment of injury, possibly explaining the dramatic hypertension seen in TA-TMA.

Background: Antibody-mediated rejection (AMR) is associated with allograft loss. Identification of factors associated with poor outcome has not been extensively studied., Methods: We retrospectively studied 469 patients who received a negative crossmatch renal transplant with alemtuzumab induction. Forty-eight of 469 (10.2%) patients were treated for AMR. Thirty of 48 (62.5%) of the cases fulfilled the Banff criteria for definite AMR, whereas 18 of 48 (37.5%) were categorized as suspicious for AMR (tissue injury with C4d staining or donor-specific antibodies [DSAbs]). Sensitization, high human leukocyte antigen, and -DR mismatch were risk factors for the development of AMR (P = 0.0016, 0.001, and 0.012, respectively)., Results: Allograft survival was inferior in the AMR group (70.2%) compared with the nonrejector group (97.0%) (P<0.001). Forty-two of 48 (87.5%) of patients with acute AMR had DSAbs. Patients with CII DSAbs at the time of AMR, whether alone or in combination with CI DSAbs had the worst allograft survival (P = 0.014). Both the mean cumulative and immunodominant mean fluorescence index were higher in those patients who subsequently lost their grafts (P<0.001). Patients with diffuse C4d staining had inferior allograft survival than those with focal C4d or no staining (P = 0.02). There was no significant difference in survival by histological grade but a trend to inferior outcomes in those with vascular involvement (P = 0.06). Those patients who met the full Banff criteria had worse survival than those with suspicion for AMR only (P = 0.04)., Conclusion: This study identifies patients at risk of graft failure from AMR. These patients may benefit from newer therapeutic strategies including the use of eculizumab or bortezomib.

Background: Acute rejection after renal transplantation has been shown to be negatively associated with long-term graft survival. Identifying donor factors that are associated with acute rejection in the recipient could help to a better understanding of the relevant underlying processes that lead to graft injury. Complement activation has been shown to be an important mediator of renal transplant related injury. In this study, we analyzed the effect of systemic complement activation in deceased donors before transplantation of their kidneys on posttransplant outcome in the recipient., Methods: Plasma from 232 deceased brain-dead and deceased cardiac-dead donors were analyzed for the complement activation markers C5b-9, C4d, Bb, and complement component mannan binding lectin by ELISA. The association of these parameters with posttransplant outcome in recipients was analyzed in a multivariate regression model., Results: It was found that C5b-9 level in donor plasma is associated with biopsy-proven acute rejection in the recipient during the first year after renal transplantation (P = 0.035). Both in deceased brain-dead and deceased cardiac-dead donors increased complement activation was found., Conclusions: In conclusion, we found C5b-9 in the donor to be associated with acute rejection of renal transplants in the recipient. Whether targeting complement activation in the donor may ameliorate acute rejection in the recipient needs to be studied.

Background: Human leukocyte antigen (HLA) antibodies, especially those that fix complement, are associated with antibody-mediated rejection and graft failure. The C1q assay on single antigen beads detects a subset of HLA antibodies that can fix complement and precede C4d deposition. The aim of this study was to determine whether C1q-fixing antibodies distinguish de novo donor-specific antibodies (DSA) that are clinically relevant and harmful., Methods: We retrospectively studied 31 of 274 kidney transplant recipients who had pretransplant and concurrent biopsy and serum specimens, 13 with C4d-positive and 18 with C4d-negative staining. We measured IgG and C1q DSA pretransplant and at the time of biopsy using single antigen bead assays. We identified 13 recipients who developed de novo DSA by IgG or C1q and examined associations with C4d deposition, transplant glomerulopathy, and graft failure., Results: Testing for DSA by IgG is more sensitive for C4d deposition (IgG: 100%, 95% confidence interval [CI] 0.60-1; C1q: 75%, 95% CI 0.36-0.96). Testing for DSA by C1q is more specific for transplant glomerulopathy (C1q: 81%, 95% CI 0.57-0.94; IgG: 67%, 95% CI 0.43-0.85) and graft loss (C1q: 79%, 95% CI 0.54-0.93; IgG: 63%, 95% CI 0.39-0.83). Absence of de novo DSA by IgG and C1q has a high negative predictive value for the absence of C4d deposition (IgG: 100%, 95% CI 0.73-1; C1q: 88%, 95% CI 0.62-0.98), transplant glomerulopathy (IgG: 100%, 95% CI 0.73-1; C1q: 100%, 95% CI 0.77-1), and graft failure (IgG: 86%, 95% CI 0.56-0.97; C1q: 88%, 95% CI 0.62-0.98)., Conclusion: Monitoring patients with the C1q assay, which detects antibodies that fix complement, offers a minimally invasive means of identifying patients at risk for transplant glomerulopathy and graft loss.

Background: Although C4d deposition in peritubular capillaries has been identified as a strong risk factor for subsequent renal allograft loss, the optimal cutoff for the fraction of peritubular capillaries needed to establish a positive stain in formalin-fixed, paraffin-embedded material has not been defined systematically. The objective of this study was to establish the threshold for positive staining that best predicts renal outcome in renal biopsies in a multicenter study in which local and central pathologic conditions were compared., Methods: Unstained renal biopsy slides were obtained from 296 patients. The percentage of peritubular capillaries staining positively for C4d was detected by immunoperoxidase staining., Results: The percentage C4d deposition ranged from 0% to 90% with 44% (129/296) having a positive percentage of C4d staining. The median for positive cases was 25%. Local C4d+ results were reported qualitatively, with 28% recorded as positive for C4d. Using a centrally determined cutoff of 10%, tests for agreement of local and central C4d staining were fair (κ 0.40, 95% confidence interval 0.29-0.51). Raising the centrally determined cutoff to 25% or 50% did not change the κ values (0.44 and 0.41, respectively). By Cox proportional hazards model, C4d positivity (centrally determined assessment) using a cutoff of 10% was the strongest predictor of time to graft loss (hazard ratio 2.66, 95% confidence interval 1.68-4.21). Centrally determined C4d positivity correlated with Banff scores indicative of acute inflammation but not with scores indicative of fibrosis/atrophy or transplant glomerulopathy., Conclusions: Our findings indicate that C4d positivity, defined as more than or equal to 10% by immunoperoxidase, is a strong predictor of graft loss.

Background: Transplant glomerulopathy (TG) is a renal allograft disease defined by glomerular basement membrane duplication with peritubular capillary basement membrane multilayering (PTCML), and associated with anti-human leukocyte antigen antibodies and C4d. Outcome in TG is poor but variable, and prognostic factors, particularly those affecting long-term outcome, are not well known. We investigated several potentially prognostic clinical and pathologic factors in TG and evaluated estimated glomerular filtration rate (eGFR) slopes to assess graft function and early decline., Methods: We examined all cases of TG from 2001 to 2005 with at least 4-year follow-up after biopsy, excluding those with a second confounding diagnosis., Results: Among 36 cases of pure TG, mean graft age at biopsy was 8.8±6 years. C4d stain was positive in 11 (33%) cases. Clinical characteristics at biopsy were not different based on C4d. C4d was associated with greater PTCML (P=0.03), peritubular capillaritis (P=0.04), and glomerulitis (P=0.03). Death-censored graft survival was significantly associated with interstitial fibrosis (P=0.001), PTCML (P=0.001), and arteriolar hyalinosis (P=0.007), and it showed a trend with proteinuria (P=0.07) and C4d positivity (P=0.08). C4d-positive cases also showed a trend toward rapid graft loss. Analysis of eGFR slopes showed a pattern of preserved, slightly negative slope from transplant until approximately 1 year before biopsy, at which point the slope became significantly more negative (P<0.001)., Conclusion: Interstitial fibrosis, PTCML, and arteriolar hyalinosis were significant predictors of graft survival in TG. C4d positivity was associated with a more rapid rate of function decline. eGFR slope data showed significant deterioration in graft function well before the diagnostic biopsy.

Background: Decay-accelerating factor (CD55) accelerates the decay of C3 and C5 convertases, participating in classical and alternative complement activation pathways. Complement activation plays a major role in antibody-mediated rejection of allografts (AMR); C4d is used as a marker of AMR. Emerging evidence suggests an important role of CD55 in the pathogenesis of AMR. The aim of this study was to investigate the expression of CD55 in renal allografts and to correlate it with the expression of C4d, allograft survival, changes in serum creatinine (SC)., Methods: More than 200 renal allograft biopsies, performed for allograft dysfunction, were assessed for peritubular capillary (PTC) C4d and CD55 expression., Results: We found significant correlation between changes in SC and PTC CD55 staining pattern in patients with no PTC C4d staining. In these patients, SC increased from baseline by 2.2+0.34, 1.7+0.36, and 0.93+0.24 mg/dL in negative, focal, and diffuse PTC CD55 staining subgroups, respectively. Survival of renal allografts was better in diffuse PTC CD55 staining subgroup than in negative PTC CD55 staining subgroup., Conclusions: These data suggest that CD55 expression has a protective effect on PTC C4d negative renal allografts, and the pattern of PTC CD55 expression may be used as a potential marker of renal allograft survival in patients with no evidence of AMR.

Background: Chronic antibody-mediated rejection (CAMR) of renal allografts has recently been recognized as a defined nosologic entity. The outcome of CAMR is poor; there is no established treatment protocol for this condition. We therefore initiated a pilot study on treatment of CAMR with an antihumoral regimen consisting of high-dose intravenous immunoglobulin (IVIG) and the chimeric anti-CD20 antibody rituximab., Methods: Six pediatric renal transplant recipients with CAMR received four weekly doses of IVIG (1 g/kg body weight per dose), followed by a single dose of rituximab (375 mg/m2 body surface area) 1 week after the last IVIG infusion. Renal allograft biopsies were evaluated using the Banff '05 classification. Human leukocyte antigen-specific antibodies were detected by panel-reactive lymphocytotoxicity and solid phase ELISA assays., Results: Median glomerular filtration rate during 6 months before intervention dropped by 25 (range, 11-26) mL/min/1.73 m2 (P<0.05) and increased in response to antihumoral therapy by 21 (-14 to +30) 6 months (P<0.05) and by 19 (-14 to +23) mL/min/1.73 m2 12 months (P=0.063) after start of treatment. Glomerular filtration rate improved or stabilized in 4 patients; the two nonresponders had the highest degree of transplant glomerulopathy, the highest degree of C4d deposition in peritubular capillaries and pronounced interstitial inflammation. The treatment regimen was well tolerated., Conclusion: This pilot study demonstrates that CAMR in pediatric renal transplant recipients can be treated successfully and safely with a combination of IVIG and rituximab. This observation should encourage more extensive studies to evaluate this new treatment strategy.

Background: Chronic rejection (CR) is an important cause of kidney graft loss. Some studies have suggested the role of antibodies mediating chronic graft dysfunction. In this context, C4d identification is an important tool to evaluate antibody-mediated rejection., Method: This is a retrospective study that analyzed 80 patients with histological diagnosis of chronic allograft nephropathy (CAN) according Banff 97 and no evidence of transplant glomerulopathy. These patients had renal biopsies available for C4d immunoperoxidase staining at the time of diagnosis. Cases were reclassified by the presence of C4d in peritubular capillaries., Results: C4d was negative in 30 cases (37.5%) and positive in 50 (62.5%). C4d+ group had more female and highly sensitized patients (PRA) at transplant. All variables were similar between C4d- and C4d+ cases at diagnosis time, but more C4d+ patients presented proteinuria (>0.3 g/L). Patients were submitted to various immunosuppression regimens after the CAN diagnosis. Four years after the diagnosis, death-censored graft survival was 87% for C4d- and 50% for C4d+ (P=0.002). In the multivariate Cox regression analysis, C4d+, PRA>10%, and vascular intimal proliferation were the variables that present higher relative risk for graft loss., Conclusion: These data indicate that C4d positive chronic rejection is very common, associated with proteinuria, and has a poor outcome. A larger study is warranted to identify which immunosuppressive regimen may modify the poor course of this entity.

Background: Most humoral rejection (HR) episodes occur early after cardiac transplantation and are associated with hemodynamic compromise and poor prognosis. Late cases of HR (>6 months after transplant) have been reported. We examined the differences in clinical characteristics and outcomes in patients presenting with HR in the early (<6 months) and late transplant periods., Methods: A retrospective chart review was performed of all cases of HR at a single large transplant center from January 1, 1995 to March 1, 2006., Results: A total of 37 adult transplants had biopsy-proven HR; 13 patients had early HR and 24 patients had HR a mean of 5 yr after transplantation (range, 7 months to 17 yrs). Treatment for HR included plasmapheresis, cyclophosphamide, and rituximab. The age of the early and late humoral rejecters was similar (58+/-14 vs. 50+/-14 yrs; P=0.12). There was a trend toward more women in the early HR group (54% vs. 33%). Use of left ventricular assist devices was similar (38% vs. 33%). Early rejecters were more likely to have positive cross-matches (46% vs. 8%; P<0.01). Patients with late HR had a coexistent diagnosis of malignancy, or significant recent infection in 50% vs. 8% for early HR, suggesting an activation of a nonhuman leukocyte antigen antibody-mediated immune response to an acute illness. One-year survival after the diagnosis of HR was 78% for the both groups (P=NS)., Conclusions: Humoral rejection occurs now more frequently in patients with remote transplants and is commonly associated with the presence of malignancy or infection.

Background: Peritubular capillary deposition of C4d (C4d(PTC)) is a marker of antibody-mediated alloresponse and is associated with poor graft survival in adults. C3d(PTC) has received less attention; its significance is unclear. To date no information has been gained in children., Methods: The prevalence of C4d(PTC) and C3d(PTC) in pediatric renal allograft biopsies (n=77, 31 cadaveric kidneys) was analyzed retrospectively. Associations with histology, donor-specific antibodies (DSAs), and outcome were investigated., Results: The overall prevalence of C4d(PTC) and C3d(PTC) was 52% and 48%, respectively. C3d(PTC) was associated with C4d(PTC) (P<0.0001). Thirty-six percent of acute rejections were cellular, 28% were humoral, and 36% were combined cellular and humoral. C3d(PTC) was found in 57% of acute rejection biopsies. C4d(PTC), but not C3d(PTC), was associated with accumulation of polymorphonuclear cells in peritubular capillaries (P=0.02). Fifty-one percent of late biopsies (>6 months posttransplantation) had features of chronic allograft nephropathy: 50% were C4d(PTC&#95; positive, and 50% were C3d(PTC) positive. C4d(PTC) positive chronic allograft nephropathy biopsies had more transplant glomerulopathy (P=0.020) and mesangial matrix increase (P=0.026). C3d(PTC) tended to be associated with transplant glomerulopathy (P=0.06), but not with mesangial matrix increase. C4d(PTC) was correlated with DSA (P=0.011). Excluding early nonrejection graft losses, more grafts were lost in the C4d(PTC) positive group (P=0.019). C3d(PTC) was not associated with DSA or graft outcome., Conclusions: Our results support C4d(PTC) being a hallmark of humoral rejection in pediatric renal transplantation; its presence was associated with DSA and poorer immunologic graft outcome. In contrast, C3d(PTC), although highly associated with C4d(PTC), did not correlate with DSA or outcome.

Background: Human C4b-binding protein (C4bp) functions as a cofactor for factor I in the degradation of C4b and C3b and, in addition, accelerates the rate of decay of the C4b2a complex., Methods: In this study, we constructed a surface-bound form of human C4b-binding protein (C4bp-PI) consisting of a short consensus repeat 1-8 of the alpha-chain of C4bp and a glycosyl phosphatidylinositol (GPI) of the decay-accelerating factor (CD55) and established stable swine endothelial cell (SEC) lines expressing C4bp-PI by transfection of cDNA. Amelioration of complement-mediated lysis by the transfectant molecules was tested as an in vitro hyperacute rejection model of swine to human discordant xenograft, using the lactate dehydrogenase assay., Results: Flow cytometric profiles of the stable SEC lines with C4bp-PI showed a high level of expression of this molecule. The cell lysate of the SEC line with C4bp-PI showed strong cofactor activity in not only C4b but also C3b, whereas the activity of plasma C4bp to bind to C3 was very weak. Approximately 150 x 10(4) molecules of C4bp-PI per SEC blocked human complement-mediated cell lysis by approximately 75%., Conclusions: The results suggest that the surface-bound form of C4bp will be very useful in clinical xenotransplantation.