Desensitization and treatment with APRIL/BLyS blockade in rodent kidney transplant model.

Alloantibody represents a significant barrier in kidney transplant through the sensitization of patients prior to transplant through antibody mediated rejection (ABMR). APRIL BLyS are critical survival factors for mature B lymphocytes plasma cells, the primary source of alloantibody. We examined the effect of APRIL/BLyS blockade via TACI-Ig (Transmembrane activator calcium modulator cyclophilin lig interactor-Immunoglobulin) in a preclinical rodent model as treatment for both desensitization ABMR. Lewis rats were sensitized with Brown Norway (BN) blood for 21 days. Following sensitization, animals were then sacrificed or romized into kidney transplant (G4, sensitized transplant control); desensitization with TACI-Ig followed by kidney transplant (G5, sensitized + pre-transplant TACI-Ig); kidney transplant with post-transplant TACI-Ig for 21 days (G6, sensitized + post-transplant TACI-Ig); desensitization with TACI-Ig followed by kidney transplant post-transplant TACI-Ig for 21 days (G7, sensitized + pre- post-transplant TACI-Ig). Animals were sacrificed on day 21 post-transplant tissues were analyzed using flow cytometry, IHC, ELISPOT, RT-PCR. Sensitized animals treated with APRIL/BLyS blockade demonstrated a significant decrease in marginal zone non-switched B lymphocyte populations (p<0.01). Antibody secreting cells were also significantly reduced in the sensitized APRIL/BLyS blockade treated group. Post-transplant APRIL/BLyS blockade treated animals were found to have significantly less C4d deposition less ABMR as defined by Banff classification when compared to groups receiving APRIL/BLyS blockade before transplant or both before after transplant (p<0.0001). The finding of worse ABMR in groups receiving APRIL/BLyS blockade before both before after transplant may indicate that B lymphocyte depletion in this setting also resulted in regulatory lymphocyte depletion resulting in a worse rejection. Data presented here demonstrates that the targeting of APRIL BLyS can significantly deplete mature B lymphocytes, antibody secreting cells, effectively decrease ABMR when given post-transplant in a sensitized animal model.

Complement System and C4d expression in cases of Membranous nephropathy / Sistema complemento e expressão de C4d em casos de Glomerulopatia Membranosa

Abstract Introduction: Membranous nephropathy (MN) is one of the major causes of nephrotic syndrome. The complement system plays a key role in the pathophysiology of MN. Objectives: To identify the complement pathway possibly activated in MN cases and correlate the presence of C4d with more severe clinical and histological markers. Methods: Sixty nine cases from renal biopsy with membranous nephropathy were investigated. The presence of C1q was analyzed by direct immunofluorescence; and expression of C4d by immunohistochemistry. Clinical and epidemiological data were obtained upon biopsy request. Results: The presence of focal segmental glomerulosclerosis, global glomerulosclerosis, vascular lesions and tubulointerstitial fibrosis were collected by anatomopathological report. C4d(+) was found in 58 (84%), and C1q(+) was found in 12 (17%) of the cases. Twelve patients had C4d(+)/C1q(+), 46 had C4d(+)/C1q(-), and 11 patients had C4d(-)/C1q(-), probably indicating the activation of the classical, lectin and alternative pathways, respectively. Conclusion: C4d was associated with increased interstitial fibrosis, but not with clinical markers of poor prognosis. Through the deposition of C4d and C1q we demonstrated that all complement pathways may be involved in MN, highlighting the lectin pathway. The presence of C4d has been associated with severe tubulointerstitial lesions, but not with clinical markers, or can be taken as a universal marker of all cases of MN.

Resumo Introdução: A Glomerulopatia membranosa (GM) é uma das principais causas da síndrome nefrótica. O sistema do complemento desempenha um papel chave na fisiopatologia do GM. Objetivos: Identificar a via do complemento possivelmente ativada nos casos de GM e correlacionar a presença de C4d com marcadores clínicos e histológicos mais graves. Métodos: Foram investigados 69 casos de biópsia renal com GM. A presença de C1q foi analisada por imunofluorescência direta e a expressão de C4d por imunohistoquímica. Dados clínicos e epidemiológicos foram obtidos mediante solicitação de biópsia renal. Resultados: A presença de glomerulosclerose segmentar focal, glomeruloesclerose global, lesões vasculares e fibrose tubulointersticial foi coletada por relato anatomopatológico. C4d (+) foi encontrado em 58 (84%), e C1q (+) foi encontrado em 12 (17%) casos. Doze pacientes tinham C4d (+)/C1q (+), 46 tinham C4d (+)/C1q (-) e 11 pacientes tinham C4d (-)/C1q (-), indicando provavelmente a ativação da via clássica, da lectina e da alternativa, respectivamente. Conclusão: O C4d foi associado ao aumento da fibrose intersticial, mas não com marcador clínico de mau prognóstico. Através da deposição de C4d e C1q, demonstrou-se que todas as vias do complemento podem estar envolvidas em GM, destacando a via da lectina. A presença de C4d tem sido associada a lesões tubulointersticiais graves, mas não com marcadores clínicos, ou pode ser tomada como um marcador universal de todos os casos de GM.

Complement System and C4d expression in cases of Membranous nephropathy.

INTRODUCTION: Membranous nephropathy (MN) is one of the major causes of nephrotic syndrome. The complement system plays a key role in the pathophysiology of MN. OBJECTIVES: To identify the complement pathway possibly activated in MN cases and correlate the presence of C4d with more severe clinical and histological markers. METHODS: Sixty nine cases from renal biopsy with membranous nephropathy were investigated. The presence of C1q was analyzed by direct immunofluorescence; and expression of C4d by immunohistochemistry. Clinical and epidemiological data were obtained upon biopsy request. RESULTS: The presence of focal segmental glomerulosclerosis, global glomerulosclerosis, vascular lesions and tubulointerstitial fibrosis were collected by anatomopathological report. C4d(+) was found in 58 (84%), and C1q(+) was found in 12 (17%) of the cases. Twelve patients had C4d(+)/C1q(+), 46 had C4d(+)/C1q(-), and 11 patients had C4d(-)/C1q(-), probably indicating the activation of the classical, lectin and alternative pathways, respectively. CONCLUSION: C4d was associated with increased interstitial fibrosis, but not with clinical markers of poor prognosis. Through the deposition of C4d and C1q we demonstrated that all complement pathways may be involved in MN, highlighting the lectin pathway. The presence of C4d has been associated with severe tubulointerstitial lesions, but not with clinical markers, or can be taken as a universal marker of all cases of MN.

Plasma cell densities and glomerular filtration rates predict renal allograft outcomes following acute rejection.

The contribution of T cells and graft-reactive antibodies to acute allograft rejection is widely accepted, but the role of graft-infiltrating B and plasma cells is controversial. We examined 56 consecutive human renal transplant biopsies classified by Banff schema into T-cell-mediated (N = 21), antibody-mediated (N = 18), and mixed (N = 17) acute rejection, using standard immunohistochemistry for CD3, CD20, CD138, and CD45. In a predominantly African-American population (75%), neither Banff classification nor C4d deposition predicted the return to dialysis. Immunohistochemical analysis revealed CD3(+) T cells as the dominant cell type, followed by CD20(+) B cells and CD138(+) plasma cells in all acute rejection types. Using univariate Cox Proportional Hazard analysis, plasma cell density significantly predicted graft failure while B-cell density trended toward significance. Surprisingly T-cell density did not predict graft failure. The estimated glomerular filtration rate (eGFR) at diagnosis of acute rejection also predicted graft failure, while baseline eGFR ≥6 months prior to biopsy did not. Using multivariate analysis, a model including eGFR at biopsy and plasma cell density was most predictive of graft loss. These observations suggest that plasma cells may be a critical mediator and/or an independently sensitive marker of steroid-resistant acute rejection.

Pesquisa de fração c4d e imunoglobulina para tratamento da rejeição aguda mediada por anticorpos no transplante renal / Search of c4d fraction and immunoglobulin for treatment of acute rejection mediated by antibodies in the Kidney transplantation

A rejeição aguda mediada por anticorpos, diagnosticada pela deposição de C4d na biópsia do enxerto, é uma complicação grave do transplante renal, com taxas históricas de perda do órgão transplantado da ordem de 30% -50%. Ainda que não existam ensaios clínicos adequadamente conduzidos para avaliar o papel da imunoglobulina no tratamento da RMA, a evidência proveniente de séries de casos publicadas por centros especializados aponta para uma taxa de preservação do enxerto em um ano de até cerca de 90% em com o uso da imunoglobulina humana intravenosa, sobretudo quando associada à plasmaférese, resultado esse muito superior ao controle histórico sem uso de imunoglobulina. Diante disso, recomenda-se a inclusão da imunoglobulina humana intravenosa no CEAF para o tratamento da rejeição aguda mediada por anticorpos comprovada por biópsia (presença de depósitos de C4d) pós-transplante renal, refratária a corticoide e OKT3. Não há evidências suficientes para recomendar um regime preferencial de administração de imunoglobulina em relação aos demais. Recomenda-se o regime mais estudado, constituído de imunoglobulina humana 500 mg/Kg/dia por até sete dias, quando utilizada isoladamente, ou 500 mg/Kg/dia em dias alternados, quando associada à plasmaférese. Recomendação da CONITEC: Os membros da CONITEC presentes na 7ª reunião ordinária do dia 02/08/2012 recomendaram a incorporação da pesquisa de fração C4d e da imunoglobulina para o tratamento da rejeição aguda mediada por anticorpos no transplante renal, conforme PCDT a ser elaborado pelo Ministério da Saúde. A Portaria SCTIE/MS Nº 36, de 27 de setembro de 2012 - Torna pública a decisão de incorporar a imunoglobulina para tratamento da rejeição aguda mediada por anticorpos no transplante renal e o exame de pesquisa da fração C4d no Sistema Único de Saúde.

Clinical and pathological correlations of C4d immunostaining and its influence on the outcome of kidney transplant recipients.

INTRODUCTION: C4d is a marker of antibody-mediated rejection (ABMR) in kidney allografts, although cellular rejection also have C4d deposits. OBJECTIVE: To correlate C4d expression with clinico-pathological parameters and graft outcomes at three years. METHODS: One hundred forty six renal transplantation recipients with graft biopsies by indication were included. C4d staining was performed by paraffin-immunohistochemistry. Graft function and survival were measured, and predictive variables of the outcome were determined by multivariate Cox regression. RESULTS: C4d staining was detected in 48 (31%) biopsies, of which 23 (14.7%) had diffuse and 25 (16%) focal distribution. Pre-transplantation panel reactive antibodies (%PRA) class I and II were significantly higher in C4d positive patients as compared to those C4d negative. Both glomerulitis and pericapillaritis were associated to C4d (p = 0.002 and p < 0.001, respectively). The presence of C4d in biopsies diagnosed as no rejection (NR), acute cellular rejection (ACR) or interstitial fibrosis/ tubular atrophy (IF/TA) did not impact graft function or survival. Compared to NR, ACR and IF/TA C4d⁻, patients with ABMR C4d⁺ had the worst graft survival over 3 years (p = 0.034), but there was no difference between ABMR versus NR, ACR and IF/TA that were C4d positive (p = 0.10). In Cox regression, graft function at biopsy and high %PRA levels were predictors of graft loss. CONCLUSIONS: This study confirmed that C4d staining in kidney graft biopsies is a clinically useful marker of ABMR, with well defined clinical and pathological correlations. The impact of C4d deposition in other histologic diagnoses deserves further investigation.

Humoral heart rejection (severe allograft dysfunction with no signs of cellular rejection or ischemia): incidence, management, and the value of C4d for diagnosis.

Severe allograft dysfunction after heart transplant (HT), without ischemia or evidence of cellular rejection upon endomyocardial biopsy (EMB), is a rare but potentially fatal condition that suggests humoral rejection (HR). Its incidence, and the methods of choice for its diagnosis and management, remain uncertain. We retrospectively studied 445 HT patients (April 1991-December 2003) to determine incidence of HR diagnosed by clinical and conventional histopathological criteria. We used immunofluorescence (IF) techniques to test archived frozen EMB issue for IgM, IgG, C1q, C3, fibrin and C4d. Twelve patients (2.7%) fulfilled the criteria for HR after a mean time post-HT of 21.3 +/- 24.7 months (range: 2-72 months). Patients were treated with high doses of steroids and plasmapheresis, with successful recovery in 11 cases. IF studies using classical markers were mainly negative for the six patients with enough EMB tissue for testing. All six patients showed positivity for C4d during the HR episode but not before or after. Humoral rejection was observed in less than 3% of HT patients. Plasmapheresis treatment was highly effective. Classical IF tests were not useful for diagnosis, but C4d appears to be useful both for confirmation of diagnosis and for monitoring response to treatment.

Ultra-late antibody-mediated rejection 30 years after a living-related renal allograft.

Antibody-mediated renal allograft rejection has become increasingly recognized and more clearly defined through the use of flow cytometry cross-matching and the deposition of C4d in renal allograft biopsies. All of the cases reported thus far have developed an antibody within 10 years of transplantation, and many lacked HLA and/or donor specificity. The present patient developed an anti-HLA donor-specific antibody between the 22nd and 30th year after a living-related renal transplant. At the 30th year post-transplantation, she experienced a rise in the serum creatinine from 0.7 to 1.9 mg/dL associated with transplant biopsy C4d deposition in peritubular capillaries and glomeruli. After the replacement of azathioprine with mycophenolate mofetil, and six apheresis treatments followed by two infusions of IVIG, the renal function stabilized at 1.9 mg/dL, 33 years after transplantation. Antibody-mediated rejection must be considered as a possible cause or renal allograft dysfunction at all time periods after transplantation.

Association of CD20+ infiltrates with poorer clinical outcomes in acute cellular rejection of renal allografts.

We undertook a study to ascertain the relationship between the presence of CD20-positive B-lymphocytes in renal allografts undergoing acute cellular rejection and graft survival. We identified 27 patients transplanted between January 1, 1998 and December 31, 2001, with biopsy-proven Banff 1-A or Banff 1-B rejection in the first year after transplantation, and stained the specimens for CD20 and C4d. At least 4 years of follow-up data were available for each patient studied. Six patients had CD20-positive B-cell clusters in the interstitium, and 21 patients were negative for CD20 infiltrates. The CD20-positive group was significantly more likely to have steroid-resistant rejection and reduced graft survival compared to CD20-negative controls. This study supports prospective identification of CD20-positive B-cell clusters in biopsy-proven rejection and offers a therapeutic rationale for a trial of monoclonal anti-CD20 antibody in such patients.

Acute humoral rejection of human lung allografts and elevation of C4d in bronchoalveolar lavage fluid.

Antibody-mediated rejection is well established for renal allografts but remains controversial for lung allografts. Cardinal features of antibody-mediated rejection in renal allografts include antibodies to donor human leukocyte antigen (HLA) and evidence for antibody action, such as complement activation demonstrated by C4d deposition. We report a lung allograft recipient with circulating antibodies to donor HLA who failed treatment for acute cellular rejection but responded to therapy for humoral rejection. To address the second criteria for antibody-mediated rejection, we determined whether complement activation could be detected by measuring C4d in bronchoalveolar lavage fluid (BALF) by ELISA. Airway allergen challenge of asthmatics activates the complement pathway; therefore, we used BALF from asthmatics pre- and post-allergen challenge to measure C4d. These controls demonstrated that ELISA could detect increases in C4d after allergen challenge. BALF from the index patient had elevated C4d concomitant with graft dysfunction and anti-donor HLA in the absence of infection. Analysis of BALF from 25 additional lung allograft recipients showed that C4d concentrations >100 ng/mL were correlated with anti-HLA antibodies (p = 0.006), but were also observed with infection and in asyptomatic patients. The findings support the occurrence of anti-HLA-mediated lung allograft rejection and suggest that C4d measurement in BALF may be useful in diagnosis.