Introdução: o transplante renal é o tratamento de escolha para uma significativa porção dos pacientes com perda crônica terminal da função renal. A rejeição aguda é uma importante complicação pós-transplante e entre outras disfunções agudas tem na biópsia do enxerto o padrão ouro para o seu diagnóstico. No entanto as biópsias apresentam uma série de limitações e riscos sendo necessário que se desenvolva biomarcadores não invasivos capazes de identificar disfunções do enxerto. Objetivos: analisar e quantificar a expressão dos microRNAs miR-142-3p, miR-155 e miR-210 em amostras de sangue periférico, urina e tecido renal coletadas de pacientes que submetidos à transplante renal que desenvolveram disfunção do enxerto. Métodos: estudo com delineamento transversal e executado no Laboratório de Biologia Molecular aplicado à Nefrologia (LABMAN), do Centro de Pesquisa Experimental do Hospital de Clínicas de Porto Alegre. As amostras são de pacientes submetidos a transplante renal que necessitaram de biópsia, por critério clínico. A expressão dos miRNAs miR-142-3p, miR-155 e miR-210 nos materiais biológicos (tecido renal, sangue periférico e células do sedimento urinário) foi avaliada através da técnica de reação em cadeia da polimerase quantitativo em tempo real. Resultados: foi encontrada, no sangue periférico uma diminuição estatisticamente significativa na expressão do miR-142-3p no grupo de pacientes com rejeição aguda (n=23) quando comparado ao grupo com outras causas de disfunção do enxerto (n=68) (P = 0,01). Não houve diferença entre os grupos na expressão do miR-155 e do miR-210, tampouco para o miR142-3p nos demais compartimentos. Conclusão: miR-142-3p mostra uma expressão diferenciada de rejeição aguda de enxertos renais, há um envolvimento deste marcador no grupo de biomarcadores moleculares em potencial para a disfunção do enxerto renal. / Background: kidney transplantation is the treatment of choice for a significant portion of patients with end-stage kidney disease. Acute rejection is a major post-transplant complication among other acute disorders and has on graft biopsy the gold standard for diagnosis. Biopsy, however it is an invasive and potentially harmful procedure so it is desirable to develop new noninvasive markers for diagnosing graft dysfunction. Objective: to analyze and quantify the expression of microRNAs miR-142-3p, miR-155 and miR-210 in the peripheral blood, urinary sediment and kidney tissue obtained from patients who developed graft dysfunction after kidney transplantation. Methods: crosssectional study performed at the Laboratory of Molecular Biology applied to Nephrology (Labman), Center of Experimental Research from Hospital de Clinicas de Porto Alegre. The samples are from kidney transplant patients who undertook indication biopsies as a part of investigation of graft dysfunction. Micro-RNAs expression was evaluated by quantitative real-time polymerase chain reaction. Results: it was found that in peripheral blood, a significant decrease in the expression of miR-142-3p occurred in patients with acute rejection (n = 23) as compared to the group of patients with other causes of graft dysfunction (n = 68), (P = 0.01). No other significant differences were found in gene expression of miR-155 and miR-210, neither for miR142-3p in the other urine or kidney tissue. Conclusion: miR-142-3p presents differential expression in the peripheral blood of patients with rejecting kidney grafts. The role of miRNAs as biomarkers for kidney graft dysfunction is worth be further explored.

Introdução: A sobrevivência em curto prazo dos transplantes renais tem melhorado notavelmente nas últimas duas décadas. No entanto, a sobrevivência em longo prazo de enxertos e pacientes ainda são muito inferiores ao desejado e a maioria dos enxertos são perdidos por falecimento dos receptores e deterioração crônica da função do enxerto. Acredita-se que a maior parte das lesões que resultam em encurtamento da sobrevida do enxerto se iniciam logo após o transplante e muitas vezes são subclínicas. O desenvolvimento de biomarcadores não invasivos para identificar com precisão as lesões sub-clínicas, sem a necessidade de biópsias de protocolo, seria um grande passo para a prática clínica de transplantes de órgãos, uma vez que permitiria o reconhecimento precoce de eventos de agressões ao enxerto e poderia levar a adequadas ações terapêuticas potencialmente propiciando sobrevida mais prolongada dos aloenxertos. Objetivos: O objetivo do presente estudo foi avaliar o potencial diagnóstico de agressões sub-clínicas da análise molecular não-invasiva da expressão gênica de leucócitos do sangue periférico em receptores de transplante renal com função estável em curto prazo. Métodos: Cento e trinta e seis pacientes renais com função estável do enxerto foram arrolados no estudo e realizaram biópsia protocolar no 3º mês póstransplante. Foram coletadas amostras de sangue periférico concomitantemente para realizarmos a quantificação da expressão gênica de perforina, TIM3, FOXP3, TGF-B, CTGF e CD138 através da metodologia de PCR em tempo real. Resultados: Trinta e nove pacientes foram diagnosticados com rejeição aguda (28,7%), sendo 33 destes com alterações borderline, 5 com rejeição aguda Banff IA e um paciente com rejeição Banff IB, vinte pacientes apresentaram fibrose intersticial e atrofia tubular (14,7%), sete apresentaram necrose tubular aguda (5,1%), três infecções pelo vírus do polioma (2,2%) e um caso de nefrotoxicidade aguda por inibidores da calcineurina (0,8%). A mensuração da expressão gênica foi realizada através de qPCR e os pacientes com disfunção do enxerto apresentaram expressões diminuídas de perforina, TIM3, FOXP3 e TGF-β em relação aos pacientes com rejeição aguda e histologia normal do enxerto. Outras análises demonstraram que a perforina, TIM3 e FOXP3 também são capazes de excluir o diagnóstico de rejeição aguda, com valores preditivos negativos (VPN) de 83%, 83% e 79,6%, respectivamente. Em uma análise combinada dos 3 genes associados o VPN para rejeição aguda foi de 86.4%. A avaliação do RNA mensageiro dos genes TGF-B e CTGF mostrou que eles estão hiperexpressos nos enxertos com fibrose intersticial e atrofia tubular. Conclusões: Existe uma elevada incidência de agressões sub-clínicas dos enxertos renais que podem ser detectadas por biópsias protocolares. A mensuração do RNA mensageiro, em amostras do sangue periférico, mostrou ser uma ferramenta de potencial utilidade em identificar essas agressões de forma não-invasiva. / Background: Short term survival of kidney transplants has improved remarkably over the last two decades. However, long term survival of grafts and patients are still much lower than desired and most of the grafts are lost by recipients’ death and chronic graft function deterioration. It is believed that most of the injuries that result in graft shortening survival are initiated early after transplantation and many times are subclinical. The development of noninvasive biomarkers to accurately identify sub-clinical injuries, without the need of protocol biopsies, would be a major step forward in the practice of clinical organ transplantation since it would allow the early recognition of graft insulting events and lead to proper therapeutic actions potentially leading to more prolonged allograft survivals Objective: The aim of the present study was to evaluate the diagnosis potential of the non-invasive molecular analyzes of peripheral blood leukocytes gene expression in stable kidney recipients in the short-term. Methods: One hundred and thirty-six patients were enrolled in this study and underwent protocol biopsies at 3 months after grafting. Peripheral blood samples were collected concomitantly for the gene expression quantitation of perforin, TIM3, FOXP3, TGF-B, CTGF and CD138 through qPCR methodology. Results: Thirty-nine patients were diagnosed as acute rejection (28.7%), being 33 with borderline histological changes, 5 Banff IA acute rejection and 1 patient with Banff IB acute rejection, twenty patients had interstitial fibrosis and tubular atrophy (14.7%), seven had acute tubular necrosis (5.1%), three had poliomavirus infection (2.2%) and one patient had calcineurin inhibitor toxicity (0.8%). Gene expression was measured through qPCR and patients with graft dysfunction presented lower expressions of perforin, TIM3, FOXP3 and TGF-β than patients with acute rejection and normal graft histology. Other analyzes showed that perforin, TIM3 and FOXP3 are also able to rule out acute rejection, with negative predictive values (NPV) of 83%, 83% and 79.6% respectively. In a combined analysis of the 3 genes associated the NPV was 86.4%. CTGF and TGF-B mRNA were overexpressed in grafts with interstitial fibrosis and tubular atrophy. Conclusions: An elevated incidence of sub-clinical injuries can be detected by protocol biopsies of stable grafts. The evaluation of mRNA in the peripheral blood has shown to be a potentially useful tool to uncover these injuries noninvasively.

Introdução: A sobrevivência em curto prazo dos transplantes renais tem melhorado notavelmente nas últimas duas décadas. No entanto, a sobrevivência em longo prazo de enxertos e pacientes ainda são muito inferiores ao desejado e a maioria dos enxertos são perdidos por falecimento dos receptores e deterioração crônica da função do enxerto. Acredita-se que a maior parte das lesões que resultam em encurtamento da sobrevida do enxerto se iniciam logo após o transplante e muitas vezes são subclínicas. O desenvolvimento de biomarcadores não invasivos para identificar com precisão as lesões sub-clínicas, sem a necessidade de biópsias de protocolo, seria um grande passo para a prática clínica de transplantes de órgãos, uma vez que permitiria o reconhecimento precoce de eventos de agressões ao enxerto e poderia levar a adequadas ações terapêuticas potencialmente propiciando sobrevida mais prolongada dos aloenxertos. Objetivos: O objetivo do presente estudo foi avaliar o potencial diagnóstico de agressões sub-clínicas da análise molecular não-invasiva da expressão gênica de leucócitos do sangue periférico em receptores de transplante renal com função estável em curto prazo. Métodos: Cento e trinta e seis pacientes renais com função estável do enxerto foram arrolados no estudo e realizaram biópsia protocolar no 3º mês póstransplante. Foram coletadas amostras de sangue periférico concomitantemente para realizarmos a quantificação da expressão gênica de perforina, TIM3, FOXP3, TGF-B, CTGF e CD138 através da metodologia de PCR em tempo real. Resultados: Trinta e nove pacientes foram diagnosticados com rejeição aguda (28,7%), sendo 33 destes com alterações borderline, 5 com rejeição aguda Banff IA e um paciente com rejeição Banff IB, vinte pacientes apresentaram fibrose intersticial e atrofia tubular (14,7%), sete apresentaram necrose tubular aguda (5,1%), três infecções pelo vírus do polioma (2,2%) e um caso de nefrotoxicidade aguda por inibidores da calcineurina (0,8%). A mensuração da expressão gênica foi realizada através de qPCR e os pacientes com disfunção do enxerto apresentaram expressões diminuídas de perforina, TIM3, FOXP3 e TGF-β em relação aos pacientes com rejeição aguda e histologia normal do enxerto. Outras análises demonstraram que a perforina, TIM3 e FOXP3 também são capazes de excluir o diagnóstico de rejeição aguda, com valores preditivos negativos (VPN) de 83%, 83% e 79,6%, respectivamente. Em uma análise combinada dos 3 genes associados o VPN para rejeição aguda foi de 86.4%. A avaliação do RNA mensageiro dos genes TGF-B e CTGF mostrou que eles estão hiperexpressos nos enxertos com fibrose intersticial e atrofia tubular. Conclusões: Existe uma elevada incidência de agressões sub-clínicas dos enxertos renais que podem ser detectadas por biópsias protocolares. A mensuração do RNA mensageiro, em amostras do sangue periférico, mostrou ser uma ferramenta de potencial utilidade em identificar essas agressões de forma não-invasiva. / Background: Short term survival of kidney transplants has improved remarkably over the last two decades. However, long term survival of grafts and patients are still much lower than desired and most of the grafts are lost by recipients’ death and chronic graft function deterioration. It is believed that most of the injuries that result in graft shortening survival are initiated early after transplantation and many times are subclinical. The development of noninvasive biomarkers to accurately identify sub-clinical injuries, without the need of protocol biopsies, would be a major step forward in the practice of clinical organ transplantation since it would allow the early recognition of graft insulting events and lead to proper therapeutic actions potentially leading to more prolonged allograft survivals Objective: The aim of the present study was to evaluate the diagnosis potential of the non-invasive molecular analyzes of peripheral blood leukocytes gene expression in stable kidney recipients in the short-term. Methods: One hundred and thirty-six patients were enrolled in this study and underwent protocol biopsies at 3 months after grafting. Peripheral blood samples were collected concomitantly for the gene expression quantitation of perforin, TIM3, FOXP3, TGF-B, CTGF and CD138 through qPCR methodology. Results: Thirty-nine patients were diagnosed as acute rejection (28.7%), being 33 with borderline histological changes, 5 Banff IA acute rejection and 1 patient with Banff IB acute rejection, twenty patients had interstitial fibrosis and tubular atrophy (14.7%), seven had acute tubular necrosis (5.1%), three had poliomavirus infection (2.2%) and one patient had calcineurin inhibitor toxicity (0.8%). Gene expression was measured through qPCR and patients with graft dysfunction presented lower expressions of perforin, TIM3, FOXP3 and TGF-β than patients with acute rejection and normal graft histology. Other analyzes showed that perforin, TIM3 and FOXP3 are also able to rule out acute rejection, with negative predictive values (NPV) of 83%, 83% and 79.6% respectively. In a combined analysis of the 3 genes associated the NPV was 86.4%. CTGF and TGF-B mRNA were overexpressed in grafts with interstitial fibrosis and tubular atrophy. Conclusions: An elevated incidence of sub-clinical injuries can be detected by protocol biopsies of stable grafts. The evaluation of mRNA in the peripheral blood has shown to be a potentially useful tool to uncover these injuries noninvasively.

Introdução: o transplante renal é o tratamento de escolha para uma significativa porção dos pacientes com perda crônica terminal da função renal. A rejeição aguda é uma importante complicação pós-transplante e entre outras disfunções agudas tem na biópsia do enxerto o padrão ouro para o seu diagnóstico. No entanto as biópsias apresentam uma série de limitações e riscos sendo necessário que se desenvolva biomarcadores não invasivos capazes de identificar disfunções do enxerto. Objetivos: analisar e quantificar a expressão dos microRNAs miR-142-3p, miR-155 e miR-210 em amostras de sangue periférico, urina e tecido renal coletadas de pacientes que submetidos à transplante renal que desenvolveram disfunção do enxerto. Métodos: estudo com delineamento transversal e executado no Laboratório de Biologia Molecular aplicado à Nefrologia (LABMAN), do Centro de Pesquisa Experimental do Hospital de Clínicas de Porto Alegre. As amostras são de pacientes submetidos a transplante renal que necessitaram de biópsia, por critério clínico. A expressão dos miRNAs miR-142-3p, miR-155 e miR-210 nos materiais biológicos (tecido renal, sangue periférico e células do sedimento urinário) foi avaliada através da técnica de reação em cadeia da polimerase quantitativo em tempo real. Resultados: foi encontrada, no sangue periférico uma diminuição estatisticamente significativa na expressão do miR-142-3p no grupo de pacientes com rejeição aguda (n=23) quando comparado ao grupo com outras causas de disfunção do enxerto (n=68) (P = 0,01). Não houve diferença entre os grupos na expressão do miR-155 e do miR-210, tampouco para o miR142-3p nos demais compartimentos. Conclusão: miR-142-3p mostra uma expressão diferenciada de rejeição aguda de enxertos renais, há um envolvimento deste marcador no grupo de biomarcadores moleculares em potencial para a disfunção do enxerto renal. / Background: kidney transplantation is the treatment of choice for a significant portion of patients with end-stage kidney disease. Acute rejection is a major post-transplant complication among other acute disorders and has on graft biopsy the gold standard for diagnosis. Biopsy, however it is an invasive and potentially harmful procedure so it is desirable to develop new noninvasive markers for diagnosing graft dysfunction. Objective: to analyze and quantify the expression of microRNAs miR-142-3p, miR-155 and miR-210 in the peripheral blood, urinary sediment and kidney tissue obtained from patients who developed graft dysfunction after kidney transplantation. Methods: crosssectional study performed at the Laboratory of Molecular Biology applied to Nephrology (Labman), Center of Experimental Research from Hospital de Clinicas de Porto Alegre. The samples are from kidney transplant patients who undertook indication biopsies as a part of investigation of graft dysfunction. Micro-RNAs expression was evaluated by quantitative real-time polymerase chain reaction. Results: it was found that in peripheral blood, a significant decrease in the expression of miR-142-3p occurred in patients with acute rejection (n = 23) as compared to the group of patients with other causes of graft dysfunction (n = 68), (P = 0.01). No other significant differences were found in gene expression of miR-155 and miR-210, neither for miR142-3p in the other urine or kidney tissue. Conclusion: miR-142-3p presents differential expression in the peripheral blood of patients with rejecting kidney grafts. The role of miRNAs as biomarkers for kidney graft dysfunction is worth be further explored.

Introdução: A relevância clínica da presença de anticorpos anti-HLA após o transplante renal tem sido foco de recente atenção para os estudiosos da histocompatibilidade. Pacientes que possuem anticorpos anti-HLA no póstransplante apresentam maior incidência de rejeição aguda (RA) e de nefropatia crônica do enxerto (NCE). Como conseqüência, alguns perdem o órgão transplantado ou sofrem com as reações imunopatológicas correspondentes. Entretanto, existem algumas controvérsias sobre o grau de valorização da presença desses anticorpos na etiopatogenia da RA e da NCE, pois nem todos os pacientes com anticorpos evoluem mal. Objetivo: Avaliar a presença de anticorpos anti-HLA no primeiro ano do transplante renal e verificar sua associação com a ocorrência de RA e NCE. Pacientes e Método: Este estudo incluiu consecutivamente 88 pacientes submetidos a transplante renal no Serviço de Nefrologia do Hospital de Clínicas de Porto Alegre, entre outubro de 2002 a outubro de 2004. Amostras de sangue foram colhidas no 1º, 3º, 6º e 12º meses pós-transplante renal, visando à pesquisa de anticorpos IgG anti-HLA de classes I e II. Nos pacientes que consentiram, biópsias renais de protocolo foram realizadas entre o 2º e o 3º mês e no 12º mês póstransplante. A detecção dos anticorpos foi realizada através de ensaio ELISA (LATM e LAT-1240, One Lambda Inc., USA). Rejeição aguda e a NCE foram diagnosticadas por critérios clínicos, laboratoriais e histopatológicos. Resultados: Oitenta e oito pacientes foram avaliados, sendo 40 (45,5%) do sexo feminino e setenta e dois (81,8%) de etnia caucasóide. Setenta e um (80,6%) receberam rins de doador falecido. Foi detectada a presença de anticorpos anti-HLA em vinte pacientes (22,7%). Desses, somente 3 (4,4%) desenvolveram anticorpos anti-HLA (classe I) no período pós-transplante; os demais (17) já os apresentavam no período prétransplante. No seguimento até um ano, 23 pacientes (26,1%) apresentaram RA e 43 (51,2%) desenvolveram NCE. Nove (45%) pacientes com anticorpos no póstransplante desenvolveram RA contra 14 (20,6%) dos sem anticorpos (P=0,058). Entre os pacientes com anticorpos no pós-transplante, 11 (64,7%) desenvolveram NCE contra 32 (47,8%) dos sem anticorpos (P=0,329). Na análise histológica, os anticorpos anti-HLA foram associados à RA IIA (P=0,001) e à NCE grau II (P= 0,012). As variáveis preditoras para a RA e NCE foram, respectivamente, presença de anticorpos anti-HLA de classe I no 1º mês pós-transplante (OR= 4,30; IC 95%= 1,32-14,1; P= 0,016) e transplante com órgão de doador-limítrofe (OR= 4,81; IC 95%= 1,18-20,3; P= 0,028). Setenta por cento (70%) dos pacientes com RA desenvolveram NCE, contra 45,3% dos pacientes sem RA (P= 0,054). Conclusão: Os anticorpos anti-HLA presentes no primeiro ano do transplante renal foram associados a RA e NCE. A pesquisa de anticorpos anti-HLA no pós-transplante renal realizada por outros pesquisadores e aqui também avaliada, se adotada como rotina, possibilitaria a identificação de casos de mau prognóstico e a escolha de planos terapêuticos mais adequados. A correlação entre anticorpos anti-HLA e rejeição deverá se tornar mais evidente com o passar dos anos, sendo que nossos resultados fortalecem a convicção da necessidade de continuidade desses estudos. / Introduction: The clinical relevance of the presence of anti-HLA antibodies following kidney transplant has been the recent focus of attention of histocompatibility researchers. Patients who present anti-HLA antibodies in the post-transplant period have shown higher incidence of acute rejection (AR) and of chronic allograft nephropathy (CAN). As a result, some lose the transplanted organ or suffer from the corresponding immunopathological reactions. However, there has been some controversy as to the importance of the presence of these antibodies in the ethiopathology of AR and CAN, since not all patients who have these antibodies present the same outcome. Objective: To evaluate the presence of anti-HLA antibodies during the first year of kidney transplantation and to check its association with the occurrence of AR and CAN. Patients and Method: This research included consecutively 88 patients who had undergone kidney transplants in the Hospital de Clínicas de Porto Alegre Nephrology Service between October 2002 and October 2004. Blood samples were taken during the 1st, 3rd, 6th and 12th months post kidney transplant, aiming at researching for Class I and II IgG anti-HLA antibodies. In consenting patients, protocol kidney biopsies were carried out between the 2nd and 3rd months and in the 12th month after the transplant. Detection of antibodies was done through ELISA test (LAT-M and LAT-1240, One Lambda Inc., USA). Acute rejection and CAN were diagnosed through clinical, laboratorial and histopathological criteria. Results: Eighty-eight patients were evaluated, among which 40 (45.5%) were female and seventy-two (81.8%) were Caucasian. Seventy-one (80.6%) received kidneys from deceased donors. The presence of anti-HLA antibodies was found in 20 patients (22.7%). Among these, only 3 (4.4%) developed anti-HLA antibodies (class I) during the post-transplant period; the remaining (17) already presented these antibodies during the pre-transplant period. In the follow-up up to one year, 23 patients (26.1%) presented AR and 43 (51.2%) developed CAN. Nine patients (45%) with antibodies in the post-transplant period developed AR as opposed to 14 (20.6%) patients without antibodies (P=0.058). Among the patients with antibodies in the post-transplant period, 11 (64.7%) developed CAN as opposed to 32 (47.8%) of those without antibodies (P=0.329). In the histological analysis, the anti-HLA antibodies were associated to AR IIA (P=0.001) and to CAN degree II (P= 0.012). The predictive variables for AR and CAN were, respectively, the presence of Class I anti-HLA antibodies in the first month post-transplant (OR= 4.30; IC 95%= 1.32-14.1; P= 0.016) and transplant with expanded criteria donors (OR= 4.81; IC 95%= 1.18-20.3; P= 0.028). Seventy per cent of the patients presenting AR developed CAN, as opposed to 45.3% of the patients without AR (P= 0.054). Conclusion: The anti-HLA antibodies present in the first year of the kidney transplant were associated to AR and CAN. The research of anti-HLA antibodies in the kidney post-transplant period carried by other researchers, as well as in this study, if done routinely, would allow the identification of cases with a poor prognosis and the choice of more adequate treatments. The correlation of anti-HLA antibodies and rejection will become more evident with time, and our results reinforce the certainty that these studies must continue.

Introdução: A relevância clínica da presença de anticorpos anti-HLA após o transplante renal tem sido foco de recente atenção para os estudiosos da histocompatibilidade. Pacientes que possuem anticorpos anti-HLA no póstransplante apresentam maior incidência de rejeição aguda (RA) e de nefropatia crônica do enxerto (NCE). Como conseqüência, alguns perdem o órgão transplantado ou sofrem com as reações imunopatológicas correspondentes. Entretanto, existem algumas controvérsias sobre o grau de valorização da presença desses anticorpos na etiopatogenia da RA e da NCE, pois nem todos os pacientes com anticorpos evoluem mal. Objetivo: Avaliar a presença de anticorpos anti-HLA no primeiro ano do transplante renal e verificar sua associação com a ocorrência de RA e NCE. Pacientes e Método: Este estudo incluiu consecutivamente 88 pacientes submetidos a transplante renal no Serviço de Nefrologia do Hospital de Clínicas de Porto Alegre, entre outubro de 2002 a outubro de 2004. Amostras de sangue foram colhidas no 1º, 3º, 6º e 12º meses pós-transplante renal, visando à pesquisa de anticorpos IgG anti-HLA de classes I e II. Nos pacientes que consentiram, biópsias renais de protocolo foram realizadas entre o 2º e o 3º mês e no 12º mês póstransplante. A detecção dos anticorpos foi realizada através de ensaio ELISA (LATM e LAT-1240, One Lambda Inc., USA). Rejeição aguda e a NCE foram diagnosticadas por critérios clínicos, laboratoriais e histopatológicos. Resultados: Oitenta e oito pacientes foram avaliados, sendo 40 (45,5%) do sexo feminino e setenta e dois (81,8%) de etnia caucasóide. Setenta e um (80,6%) receberam rins de doador falecido. Foi detectada a presença de anticorpos anti-HLA em vinte pacientes (22,7%). Desses, somente 3 (4,4%) desenvolveram anticorpos anti-HLA (classe I) no período pós-transplante; os demais (17) já os apresentavam no período prétransplante. No seguimento até um ano, 23 pacientes (26,1%) apresentaram RA e 43 (51,2%) desenvolveram NCE. Nove (45%) pacientes com anticorpos no póstransplante desenvolveram RA contra 14 (20,6%) dos sem anticorpos (P=0,058). Entre os pacientes com anticorpos no pós-transplante, 11 (64,7%) desenvolveram NCE contra 32 (47,8%) dos sem anticorpos (P=0,329). Na análise histológica, os anticorpos anti-HLA foram associados à RA IIA (P=0,001) e à NCE grau II (P= 0,012). As variáveis preditoras para a RA e NCE foram, respectivamente, presença de anticorpos anti-HLA de classe I no 1º mês pós-transplante (OR= 4,30; IC 95%= 1,32-14,1; P= 0,016) e transplante com órgão de doador-limítrofe (OR= 4,81; IC 95%= 1,18-20,3; P= 0,028). Setenta por cento (70%) dos pacientes com RA desenvolveram NCE, contra 45,3% dos pacientes sem RA (P= 0,054). Conclusão: Os anticorpos anti-HLA presentes no primeiro ano do transplante renal foram associados a RA e NCE. A pesquisa de anticorpos anti-HLA no pós-transplante renal realizada por outros pesquisadores e aqui também avaliada, se adotada como rotina, possibilitaria a identificação de casos de mau prognóstico e a escolha de planos terapêuticos mais adequados. A correlação entre anticorpos anti-HLA e rejeição deverá se tornar mais evidente com o passar dos anos, sendo que nossos resultados fortalecem a convicção da necessidade de continuidade desses estudos. / Introduction: The clinical relevance of the presence of anti-HLA antibodies following kidney transplant has been the recent focus of attention of histocompatibility researchers. Patients who present anti-HLA antibodies in the post-transplant period have shown higher incidence of acute rejection (AR) and of chronic allograft nephropathy (CAN). As a result, some lose the transplanted organ or suffer from the corresponding immunopathological reactions. However, there has been some controversy as to the importance of the presence of these antibodies in the ethiopathology of AR and CAN, since not all patients who have these antibodies present the same outcome. Objective: To evaluate the presence of anti-HLA antibodies during the first year of kidney transplantation and to check its association with the occurrence of AR and CAN. Patients and Method: This research included consecutively 88 patients who had undergone kidney transplants in the Hospital de Clínicas de Porto Alegre Nephrology Service between October 2002 and October 2004. Blood samples were taken during the 1st, 3rd, 6th and 12th months post kidney transplant, aiming at researching for Class I and II IgG anti-HLA antibodies. In consenting patients, protocol kidney biopsies were carried out between the 2nd and 3rd months and in the 12th month after the transplant. Detection of antibodies was done through ELISA test (LAT-M and LAT-1240, One Lambda Inc., USA). Acute rejection and CAN were diagnosed through clinical, laboratorial and histopathological criteria. Results: Eighty-eight patients were evaluated, among which 40 (45.5%) were female and seventy-two (81.8%) were Caucasian. Seventy-one (80.6%) received kidneys from deceased donors. The presence of anti-HLA antibodies was found in 20 patients (22.7%). Among these, only 3 (4.4%) developed anti-HLA antibodies (class I) during the post-transplant period; the remaining (17) already presented these antibodies during the pre-transplant period. In the follow-up up to one year, 23 patients (26.1%) presented AR and 43 (51.2%) developed CAN. Nine patients (45%) with antibodies in the post-transplant period developed AR as opposed to 14 (20.6%) patients without antibodies (P=0.058). Among the patients with antibodies in the post-transplant period, 11 (64.7%) developed CAN as opposed to 32 (47.8%) of those without antibodies (P=0.329). In the histological analysis, the anti-HLA antibodies were associated to AR IIA (P=0.001) and to CAN degree II (P= 0.012). The predictive variables for AR and CAN were, respectively, the presence of Class I anti-HLA antibodies in the first month post-transplant (OR= 4.30; IC 95%= 1.32-14.1; P= 0.016) and transplant with expanded criteria donors (OR= 4.81; IC 95%= 1.18-20.3; P= 0.028). Seventy per cent of the patients presenting AR developed CAN, as opposed to 45.3% of the patients without AR (P= 0.054). Conclusion: The anti-HLA antibodies present in the first year of the kidney transplant were associated to AR and CAN. The research of anti-HLA antibodies in the kidney post-transplant period carried by other researchers, as well as in this study, if done routinely, would allow the identification of cases with a poor prognosis and the choice of more adequate treatments. The correlation of anti-HLA antibodies and rejection will become more evident with time, and our results reinforce the certainty that these studies must continue.

Introdução: A relevância clínica da presença de anticorpos anti-HLA após o transplante renal tem sido foco de recente atenção para os estudiosos da histocompatibilidade. Pacientes que possuem anticorpos anti-HLA no póstransplante apresentam maior incidência de rejeição aguda (RA) e de nefropatia crônica do enxerto (NCE). Como conseqüência, alguns perdem o órgão transplantado ou sofrem com as reações imunopatológicas correspondentes. Entretanto, existem algumas controvérsias sobre o grau de valorização da presença desses anticorpos na etiopatogenia da RA e da NCE, pois nem todos os pacientes com anticorpos evoluem mal. Objetivo: Avaliar a presença de anticorpos anti-HLA no primeiro ano do transplante renal e verificar sua associação com a ocorrência de RA e NCE. Pacientes e Método: Este estudo incluiu consecutivamente 88 pacientes submetidos a transplante renal no Serviço de Nefrologia do Hospital de Clínicas de Porto Alegre, entre outubro de 2002 a outubro de 2004. Amostras de sangue foram colhidas no 1º, 3º, 6º e 12º meses pós-transplante renal, visando à pesquisa de anticorpos IgG anti-HLA de classes I e II. Nos pacientes que consentiram, biópsias renais de protocolo foram realizadas entre o 2º e o 3º mês e no 12º mês póstransplante. A detecção dos anticorpos foi realizada através de ensaio ELISA (LATM e LAT-1240, One Lambda Inc., USA). Rejeição aguda e a NCE foram diagnosticadas por critérios clínicos, laboratoriais e histopatológicos. Resultados: Oitenta e oito pacientes foram avaliados, sendo 40 (45,5%) do sexo feminino e setenta e dois (81,8%) de etnia caucasóide. Setenta e um (80,6%) receberam rins de doador falecido. Foi detectada a presença de anticorpos anti-HLA em vinte pacientes (22,7%). Desses, somente 3 (4,4%) desenvolveram anticorpos anti-HLA (classe I) no período pós-transplante; os demais (17) já os apresentavam no período prétransplante. No seguimento até um ano, 23 pacientes (26,1%) apresentaram RA e 43 (51,2%) desenvolveram NCE. Nove (45%) pacientes com anticorpos no póstransplante desenvolveram RA contra 14 (20,6%) dos sem anticorpos (P=0,058). Entre os pacientes com anticorpos no pós-transplante, 11 (64,7%) desenvolveram NCE contra 32 (47,8%) dos sem anticorpos (P=0,329). Na análise histológica, os anticorpos anti-HLA foram associados à RA IIA (P=0,001) e à NCE grau II (P= 0,012). As variáveis preditoras para a RA e NCE foram, respectivamente, presença de anticorpos anti-HLA de classe I no 1º mês pós-transplante (OR= 4,30; IC 95%= 1,32-14,1; P= 0,016) e transplante com órgão de doador-limítrofe (OR= 4,81; IC 95%= 1,18-20,3; P= 0,028). Setenta por cento (70%) dos pacientes com RA desenvolveram NCE, contra 45,3% dos pacientes sem RA (P= 0,054). Conclusão: Os anticorpos anti-HLA presentes no primeiro ano do transplante renal foram associados a RA e NCE. A pesquisa de anticorpos anti-HLA no pós-transplante renal realizada por outros pesquisadores e aqui também avaliada, se adotada como rotina, possibilitaria a identificação de casos de mau prognóstico e a escolha de planos terapêuticos mais adequados. A correlação entre anticorpos anti-HLA e rejeição deverá se tornar mais evidente com o passar dos anos, sendo que nossos resultados fortalecem a convicção da necessidade de continuidade desses estudos. / Introduction: The clinical relevance of the presence of anti-HLA antibodies following kidney transplant has been the recent focus of attention of histocompatibility researchers. Patients who present anti-HLA antibodies in the post-transplant period have shown higher incidence of acute rejection (AR) and of chronic allograft nephropathy (CAN). As a result, some lose the transplanted organ or suffer from the corresponding immunopathological reactions. However, there has been some controversy as to the importance of the presence of these antibodies in the ethiopathology of AR and CAN, since not all patients who have these antibodies present the same outcome. Objective: To evaluate the presence of anti-HLA antibodies during the first year of kidney transplantation and to check its association with the occurrence of AR and CAN. Patients and Method: This research included consecutively 88 patients who had undergone kidney transplants in the Hospital de Clínicas de Porto Alegre Nephrology Service between October 2002 and October 2004. Blood samples were taken during the 1st, 3rd, 6th and 12th months post kidney transplant, aiming at researching for Class I and II IgG anti-HLA antibodies. In consenting patients, protocol kidney biopsies were carried out between the 2nd and 3rd months and in the 12th month after the transplant. Detection of antibodies was done through ELISA test (LAT-M and LAT-1240, One Lambda Inc., USA). Acute rejection and CAN were diagnosed through clinical, laboratorial and histopathological criteria. Results: Eighty-eight patients were evaluated, among which 40 (45.5%) were female and seventy-two (81.8%) were Caucasian. Seventy-one (80.6%) received kidneys from deceased donors. The presence of anti-HLA antibodies was found in 20 patients (22.7%). Among these, only 3 (4.4%) developed anti-HLA antibodies (class I) during the post-transplant period; the remaining (17) already presented these antibodies during the pre-transplant period. In the follow-up up to one year, 23 patients (26.1%) presented AR and 43 (51.2%) developed CAN. Nine patients (45%) with antibodies in the post-transplant period developed AR as opposed to 14 (20.6%) patients without antibodies (P=0.058). Among the patients with antibodies in the post-transplant period, 11 (64.7%) developed CAN as opposed to 32 (47.8%) of those without antibodies (P=0.329). In the histological analysis, the anti-HLA antibodies were associated to AR IIA (P=0.001) and to CAN degree II (P= 0.012). The predictive variables for AR and CAN were, respectively, the presence of Class I anti-HLA antibodies in the first month post-transplant (OR= 4.30; IC 95%= 1.32-14.1; P= 0.016) and transplant with expanded criteria donors (OR= 4.81; IC 95%= 1.18-20.3; P= 0.028). Seventy per cent of the patients presenting AR developed CAN, as opposed to 45.3% of the patients without AR (P= 0.054). Conclusion: The anti-HLA antibodies present in the first year of the kidney transplant were associated to AR and CAN. The research of anti-HLA antibodies in the kidney post-transplant period carried by other researchers, as well as in this study, if done routinely, would allow the identification of cases with a poor prognosis and the choice of more adequate treatments. The correlation of anti-HLA antibodies and rejection will become more evident with time, and our results reinforce the certainty that these studies must continue.

No description available.

La transplantation d'organes est souvent la meilleure approche thérapeutique pour l'insuffisance organique au stade terminal. Le rejet de greffe est le principal obstacle pour une transplantation réussie, car cette dernière est le plus fréquemment réalisée entre individus génétiquement distincts. Les tissus ou organes transplantés sont généralement reconnus par le système immunitaire comme corps étrangers et sont rapidement détruits. Une série d'approches a été réalisée en clinique pour augmenter l'acceptation de la transplantation d’organes. Les immunosuppresseurs ont un rôle clé dans le combat contre le rejet de greffe. Actuellement, les résultats à court terme de la transplantation d'organes ont été considérablement améliorés avec l'émergence des inhibiteurs de la calcineurine (ICN), mais les résultats à long terme sont encore insatisfaisants. L'une des principales raisons est que les médicaments immunosuppresseurs actuels manquent de spécificité. Ces agents, en particulier ICN, sont considérés comme les facteurs de risque pour la perte tardive du greffon et les décès avec un greffon fonctionnel en raison de la toxicité des médicaments, de la sur-immunosuppression et d'autres effets secondaires. Ainsi, il y a un besoin urgent de rechercher de nouvelles thérapies idéales. Les lymphocytes T jouent un rôle central dans l'initiation du rejet des allogreffes. La pleine activation des cellules T nécessite au moins deux signaux combinés, mais distincts. En plus du signal généré par l'interaction entre le récepteur des cellules T (TCR) et les complexes CMH-peptides, le second signal, appelé signal de costimulation qui est délivré par l'engagement du récepteur de costimulation avec son ligand, est essentiel. L'engagement du TCR en l'absence de signaux de costimulation peut donner lieu à l'anergie des cellules T, un état d'absence de réponse immunitaire. Les molécules costimulatrices acquièrent ainsi l'attention en tant que cibles thérapeutiques potentielles. Du fait que ces molécules soient largement limitées à des cellules T et/ou des cellules présentatrices d'antigène, cibler la voie de signalisation de costimulation offre la possibilité de moduler le système immunitaire d'une manière plus sélective par rapport à des agents immunosuppresseurs conventionnels. À ce jour, de nombreuses molécules costimulatrices ont été identifiées et certaines ont été testées en tant que cibles thérapeutiques dans des modèles de transplantation d'organes. Les axes CD28–CD80/86 et CD40–CD40L sont importants et les deux voies de signalisation de costimulation les mieux connues. Bélatacept est un variant de l'antigène 4 des lymphocytes T cytotoxiques-immunoglobuline G (CTLA4-Ig) qui bloque la voie de signalisation CD28–CD80/86. C'est le seul bloqueur de la costimulation à être approuvé pour utilisation clinique en transplantation d'organes. Par rapport à la thérapie basée sur les ICN pour les receveurs de transplantation rénale, les thérapies à base de bélatacept montrent un taux similaire de survie, une fonction supérieure du greffon et l'amélioration du profil de risque cardiovasculaire. Cependant, bélatacept est également associée à des taux plus élevés de rejet aigu et de syndrome lymphoprolifératif post-greffe (SLPG). Dans notre étude, l'efficacité d'ASKP1240, un nouvel AcM anti-CD40 complètement humain qui bloque la voie de costimulation CD40–CD40L, a été évaluée sur la prévention du rejet d'allogreffe avec le modèle de transplantation rénale chez le singe cynomolgus. Quand ASKP1240 a été administré seul, il a réduit l'incidence du rejet aigu et a prolongé la survie de l'allogreffe rénale dépendamment de la dose administrée. L'acceptation de l'allogreffe rénale a été encore améliorée chez des singes qui ont reçus des traitements d'ASKP1240 combiné avec le tacrolimus (dose sub-thérapeutique) ou le mycophénolate mofétil (MMF). Le rejet aigu d'allogreffe a été totalement éliminé chez ces animaux et la médiane de survie du greffon rénal de ces groupes était significativement plus longue que ceux des groupes avec un traitement monothérapie. ASKP1240 a été bien toléré pour un traitement allant jusqu'à 180 jours. Il n'y avait pas d'effets secondaires évidents, y compris les complications thromboémboliques liées au médicament. L'administration d'ASKP1240 n'a pas induite de libération de cytokines. Ensuite, nous avons étudié les effets d'ASP2409 sur le rejet de l'allogreffe rénale et la survie chez des singes cynomolgus. ASP2409 est une nouvelle CD86-sélective variante de CTLA4-Ig, qui possède une affinité de liaison au CD86 14 fois plus élevée que le bélatacept in vitro et une amélioration de la puissance immunosuppressive. Une haute dose d'ASP2409 en monothérapie a montré des résultats supérieurs dans la réduction de rejet aigu et la prolongation de la survie de l'allogreffe rénale en comparaison avec une faible dose d'ASP2409 en monothérapie. Une faible dose d'ASP2409 en combinaison avec tacrolimus (dose sub-thérapeutique) inhibe complètement le rejet aigu d'allogreffe et prolonge significativement la survie de l'allogreffe rénale par rapport à une monothérapie avec une faible dose d'ASP2409 ou une dose sub-thérapeutique de tacrolimus. La médiane de survie de l'allogreffe des animaux recevant un traitement à base d'une dose élevée d'ASP2409, bélatacept, ou une dose thérapeutique de tacrolimus étaient identiques (> 91 jours). Les traitements à base d'une dose élevée d'ASP2409 présentaient de meilleurs résultats histopathologiques que le traitement à base de bélatacept. En outre, la fréquence des cellules FoxP3+ dans les allogreffes rénales a été observée plus haute dans les traitements à base d'ASP2409 et de bélatacept comparés aux traitements à base de tacrolimus. L'étude a également montré que ASP2409 est sans danger pour les animaux pour les doses qui ont été testées. Nous n'avons pas trouvé de graves effets secondaires liés à ASP2409 au cours des 91 jours d'étude. Collectivement, ces résultats suggèrent que la modulation sélective de la costimulation des cellules T avec des bloqueurs de la costimulation est une stratégie réalisable pour la prévention et le traitement du rejet d'allogreffe. ASKP1240 et ASP2409 sont tous deux des agents immunosuppresseurs prometteurs pour les régimes d'évitement ou de réduction des inhibiteurs de la calcineurine. / Organ transplantation is often the best therapeutic approach for end-stage organ failure. Graft rejection is the major obstacle to successful transplantation because transplantation is most frequently carried out between genetically distinct individuals. Transplanted tissues or organs are usually recognized by the immune system as foreign and are rapidly destructed without immune interventions. A series of approaches have thus been applied in clinic to inhibit the allogenic immune responses and in turn increase organ transplant acceptance. Immunosuppressive drugs are the key players in the "war" against immune cell-mediated rejection of allogenic transplants. Currently, the use of calcineurin inhibitors (CNIs) has dramatically decreased the risk of acute transplant rejection and improved the short-term outcomes of organ transplantation, but the long-term outcomes are still unsatisfied. One of the main reasons causing unsatisfied long-term outcomes is that current immunosuppressive drugs do not specifically target immune cells that cause transplant rejection. These immunosuppressive agents, especially CNIs, are the risk factors for late graft loss and death with functioning graft (DWFG) due to drug toxicity, over-immunosuppression, and other side-effects. Thus there is an urgent need for seeking novel therapies that can selectively eliminate the alloreactive immune responses while preserving the integrity of the remainder of the host immune system. It has been known that T cells play a central role in initiating allograft rejection. Full activation of T cells requires at least two collaborative but distinct signals. The first signal is generated by the interaction between T cell receptor (TCR) and MHC-peptide complex. The second signal, termed costimulatory signal, is delivered through the engagement of costimulatory receptors by their ligands. Importantly, TCR engagement in the absence of costimulatory signals can result in T cell anergy, a state of T cell unresponsiveness. Costimulatory molecules thus gain attention as potential therapeutic targets. Because these molecules are primary expressed on T cells and/or antigen-presenting cells, targeting costimulatory signaling pathway offers the oppertumities to modulate immune system in a more selective way relative to conventional immunosuppressive agents. To date, numerous costimulatory molecules have been identified and some have been tested as therapeutic targets in organ transplant models. CD28–CD80/86 and CD40–CD40L axis are two important and the most well known costimulatory signaling pathways. Belatacept, a variant of cytotoxic T lymphocyte antigen 4-immunoglobulin G (CTLA4-Ig) that blocks CD28–CD80/86 signaling pathway, is the only costimulation blocker to be approved for clinical use in organ transplantation. Compared to CNI-based regimen for kidney transplant recipients, belatacept-based regimens show similar patient and graft survival rate, superior graft function, and improved cardiovascular risk profile. However, belatacept is also associated with higher rates of acute rejection and posttransplant lymphoproliferative disorder (PTLD). Disruption of CD40–CD40L interaction with anti-CD40L mAbs has also been demonstrated to be a reliable approach for preventing acute rejection and for prolonging allograft survival. Unfortunately, unexpected thromboembolic complications in preclinical studies and clinical trials discontinued the development of anti-CD40L mAbs. The main objective of this thesis is to identify the optimal T cell costimulation blockers that can show improved safety and non-inferior efficacy in promoting allograft acceptance relative to current costimulatory blocking agents. Anti-CD40 mAbs are an alternative approach to block CD40–CD40L costimulatory pathway. CD40 is not involved in the stability of platelet thrombi and anti-CD40 mAbs are expected to not cause thromboembolic events. ASKP1240 is a novel fully human anti-CD40 mAb. In this study, the efficacy of ASKP1240 in the prevention of renal allograft rejection was evaluated in cynomolgus monkey kidney transplantation model. When ASKP1240 was administered alone, it dose-dependently reduced the incidence of acute rejection and prolonged renal allograft survival. Renal allograft acceptance was further improved in the monkey which received regiments using ASKP1240 combined with conventional immunosuppressive drugs tacrolimus (sub-therapeutic dose) or mycophenolate mofetil (MMF). Acute allograft rejection was totally eliminated in these animals and the kidney allograft median survival times (MST) of these groups were significantly longer than those of monotherapy groups. ASKP1240 administration did not induce cytokine release. This agent was well tolerated in monkey kidney transplant recipients during the 180-day treatment period. There were no obvious side effects including drug-related thromboembolic complications. Next, we tested the hypothesis that a CD86-selective variant of CTLA4-Ig might show non-inferior efficacy on the prevention of allograft rejection and prolongation of graft survival in comparison with belatacept. CD86 is the dominating ligand between the two natural ligands for CD28 in alloimmune response. Improvements in CD86 binding affinity of CTLA4-Igs confer increased immunosuppressive potency. A CD86-selective CTLA4-Ig variant may reach therapeutic levels of CD86 occupancy while occupies substantially less CD80 ligand than non-CD86-selective CTLA4-Igs. Preservation of CD80 signaling may be favoring to retain protective immunity and to improve safety of CTLA4-Ig therapy. This thesis investigated the effects of a novel CD86-selective CTLA4-Ig variant, ASP2409, on renal allograft rejection and survival in cynomolgus monkeys. ASP2409 possesses 14-fold higher in vitro CD86 binding affinity than belatacept and improved immunosuppressive potency. Compared to no-treatment control, low-dose (0.3 mg/kg) ASP2409 monotherapy significantly prolonged renal allograft survival (MST = 5 and 26 days, respectively) but did not decrease the incidence of acute rejection (8/8). The rate of acute renal allograft rejection was reduced in the group of high-dose ASP2409 monotherapy (2/7) and, correspondingly, a much longer MST (>91 days) was shown in this group. Combination therapy of low-dose ASP2409 and tacrolimus (sub-therapeutic dose) completely eliminated acute allograft rejection and notably prolonged renal allograft survival (MST >91 days). The MSTs of renal allografts in the animals receiving high-dose ASP2409-, belatacept-, and therapeutic dose tacrolimus-based immunosuppressive regimens were same (>91 days). High-dose ASP2409-based regimens exhibited better histopathological results than belatacept-based regimen. Furthermore, higher frequencies of FoxP3+ Tregs in renal allografts were observed in ASP2409- and belatacept-based regimens compared to tacrolimus-based regimen. ASP2409 was also demonstrated to be safe for animals with the doses to be tested. There were no serious side effects related to ASP2409 to be found in term of 91-day study. This study demonstrates that ASKP1240 is as effective as anti-CD40L mAbs on inhibition of acute rejection and prolongation of renal allograft survival, and do not cause thromboembolic complications. Previous studies indicated that CNIs could diminish the effects of anti-CD40L mAbs. In our study, ASKP1240 in combination with tacrolimus or MMF shows improved efficiency. Furthermore, the effects of ASP2409 on promoting renal allograft acceptance are not inferior to belatacept. These results collectively suggest that ASKP1240 and ASP2409 both are the promising immunosuppressive agents for solid organ transplantation.

Introdução: O fragmento C4d e os anticorpos anti-HLA doador específicos (DSA) são marcadores de resposta humoral em enxertos renais com rejeição, mas o papel das células B nesse processo ainda não é claro. Neste estudo foi avaliada a correlação entre C4d, DSA e células B de enxertos com disfunção e sua associação com aspectos morfológicos, função e sobrevida do rim transplantado. Material e Métodos: A marcação para C4d, células B CD20+ e plasmócitos CD138+ foi realizada por imunoperoxidase em biópsias por indicação de 110 receptores de transplante renal. Positividade para CD20 e CD138 foi definida por curva ROC (≥5 céls./mm2). O soro coletado concomitante a biópsia foi testado para DSA classe I e classe II. Estes marcadores foram correlacionados com dados clínicos e do transplante, a histopatologia de Banff e a evolução do rim transplantado. Resultados: Depósitos de C4d e DSA circulantes foram detectados em 100% e 70% dos pacientes com rejeição mediada por anticorpos (RMA) respectivamente, e nos casos de rejeição aguda celular (RAC) em 42% (p<0,001, vs. RMA) e 28% (p=0,003, vs. RMA). Estes dois marcadores correlacionaram-se positivamente (r=0,31, p=0,016). Houve correlação significativa entre DSA e plasmócitos CD138+ (r=0.32 p=0,006), mas as células CD20 e CD138 não se correlacionaram entre si. As células CD138+ predominaram na RMA, associadas com maior painel pré-transplante e DSA, mas não a C4d, e as células CD20+ predominaram na RAC e nas biópsias com fibrose intersticial/atrofia tubular, associadas a maior incompatibilidade HLA e a retransplantes. Pacientes com C4d+ tiveram pior função e sobrevida do enxerto em três anos de transplante, e aqueles com DSA+ uma pior 7 sobrevida do enxerto. Positividade para CD20 ou CD138 não foi preditiva da função ou sobrevida do enxerto. Na análise multivariada, somente o C4d foi fator de risco para perda do enxerto. Conclusões: Esses resultados confirmam o valor prognóstico do C4d e dos DSA para uma pior evolução do enxerto renal, e sugerem uma associação das células B CD20+ com parâmetros de rejeição celular e dos plasmócitos CD138+ com marcadores de resposta humoral. Entretanto, nesse estudo o infiltrado de células B na biópsia do enxerto não foi preditivo de uma pior evolução do rim transplantado. / Introduction: The fragment C4d and the donor specific anti-HLA antibodies (DSA) are markers of the humoral response in rejecting kidney grafts, but the role of B cells in this process is still unclear. In this study we evaluated the correlation between C4d, DSA and B cells in dysfunctional grafts, and their association with morphological features, and graft function and survival. Material and Methods: The staining for C4d, CD20+ B cells and CD138+ plasmocytes were done by immunoperoxidase in 110 kidney graft biopsies for cause. Positivity for CD20 and CD138 were established by ROC curve (≥5 cells/mm2). Serum collected at biopsy were tested for anti-HLA class I and II antibodies. These markers were correlated with clinical and transplant characteristics, Banff histopathology and graft outcomes. Results: C4d deposits and circulating DSA were detected in 100% and 70% of the patients with antibody-mediated rejection (AMR) respectively, and in cases with acute cellular rejection (ACR) in 42% (p<0.001, vs. AMR) and 28% (p=0.003, vs. AMR), respectively. Both markers were positively correlated (r=0.31, p=0.016), and there was also a significant correlation between DSA and plasmocytes CD138+ (r=0.32 p=0.006). CD20 and C138 cells were not siginificantly correlated. Plasmocytes CD138+ predominated in AMR, and were associated with higher pre transplant PRA and DSA positivity, but not with C4d. CD20+ B cells were highly expressed in ACR and in biopsies with interstitial fibrosis and tubular atrophy, in association with more HLA mismatches and re-transplants. Patients with C4d had poorer graft function and survival, and those 9 with DSA + also had a worse graft survival in three years of transplant. CD20 or CD138 cells were not predictive of graft outcomes. In multivariated analysis, only C4d remained a risk factor for graft loss. Conclusion: These results confirm the prognostic value of C4d and circulating DSA for a worse kidney graft outcome, and suggest an association of CD20+ B cells with parameters of cellular rejection whereas CD138+ plasmocytes correlated with markers of the humoral response. However, in this study the B cell infiltrate in graft biopsy was not predictive of adverse outcomes to the transplanted kidney.