Anti-HLA Donor-Specific IgG Subclasses and C1q-binding Evolution in Posttransplant Monitoring.

BACKGROUND: The identification of low-level antibodies by single-antigen bead methodology has brought advancements to risk evaluation of kidney transplant recipients. However, the use of mean fluorescence intensity (MFI) to quantify antibodies and to guide therapy is not enough. Notably, immunoglobulin G (IgG) subclass switching is hypothesized to follow a programmed sequence after an emergency signal from the germinal center. In transplantation this process is not clear yet. In the present study, we sequentially evaluate anti-HLA donor specific antibody (DSA) subclasses, their profile changes, and C1q-binding ability and the influence of those characteristics on antibody mediated rejection (AMR) occurrence and allograft function. METHODS: A total of 30 DSA-positive patients were tested for IgG subclass content and C1q-binding in sequential serum samples. RESULTS: Twenty-one patients were DSA-positive before transplant; patients sensitized only by transfusion or pregnancies had IgG1 and/or IgG3, and patients sensitized by both transfusion and pregnancies or previous transplant showed a broader range of IgG subclasses. C1q binding was detected in high MFI made up of IgG1 or multiple IgG subclasses. Only 4 patients were positive for C1q posttransplantation and 3 of these showed an increase in MFI, changes in subclasses patterns, AMR, and allograft dysfunction. CONCLUSIONS: Posttransplant evaluation of DSA subclasses and the ability to bind C1q may be informative for both AMR occurrence and allograft dysfunction. Monitoring these events may help to better define risk and interventional time points.

Complement-fixing donor-specific anti-HLA antibodies and kidney allograft failure.

Detection of donor-specific antibodies (DSA) has improved the risk classification and post-transplant evaluation of kidney recipients. Moreover, assessment of DSA C1q-binding ability has been shown to improve the individual risk classification of transplant patients for allograft loss, especially when detected after transplantation. The aim of this study was to evaluate the additional clinical impact of C1q-binding DSA detection in a population that was extensively monitored for DSA and MFI alterations. Forty-two kidney allograft recipients were followed-up at multiple time points for up to 5 years after transplantation for the presence of anti-HLA DSA-IgG total. The samples that were positive for these antibodies were retrospectively tested for the presence of complement-binding antibodies. Overall, 24 patients presented DSA, 29% (7) of which also produced complement-binding DSA. Compared to patients with non-C1q-binding DSA and non-sensitized patients, patients with C1q-binding DSA after transplantation had the lowest allograft survival rate at 5 years (p = 0.042) and showed a lower estimated glomerular filtration rate (based on the Modification of Diet in Renal Disease formula) during the post-transplant follow-up period (p = 0.01). Thus, post-transplant monitoring for complement-binding DSA is a useful tool for predicting individuals most at risk for allograft failure, and might also be beneficial for evaluation of immunosuppression regimens.

Avaliação da doença vascular do enxerto no transplante cardíaco: experiência de um centro brasileiro / Assessment of cardiac allograft vasculopathy in cardiac transplantation: experience of a Brazilian center

FUNDAMENTO: O transplante cardíaco continua sendo o tratamento de escolha para a insuficiência cardíaca refratária ao tratamento otimizado. Dois métodos diagnósticos apresentam elevada sensibilidade no diagnóstico de episódios de rejeição ao enxerto e Doença Vascular do Enxerto (DVE), causas importantes de mortalidade no pós-transplante. OBJETIVO: Avaliar a relação entre os resultados do ultrassom intracoronariano (USIV) e os laudos das biópsias endomiocárdicas (BX) no seguimento de pacientes submetidos a transplante cardíaco em um serviço de referência brasileiro. MÉTODOS: Foi realizado um ensaio epidemiológico retrospectivo observacional, com pacientes submetidos a transplante cardíaco ortotópico, no período de 2000 a 2009. Foram analisados os prontuários desses pacientes e os resultados dos USIV e BX realizados rotineiramente no seguimento clínico pós-transplante e terapêutica em uso. RESULTADOS: Dos 77 pacientes analisados, 63,63% são do sexo masculino, nas faixas etárias de 22 a 69 anos. Quanto aos resultados dos USIV, 33,96% foram classificados em Stanford classe I, e 32,08%, como Stanford IV. Dos 143 laudos das biópsias, 51,08% tiveram resultado 1R, 3R em 0,69% dos laudos, e 14,48% apresentaram a descrição de efeito Quilty. Todos usaram antiproliferativos, 80,51% usaram inibidores da calcineurina e 19,48% usaram inibidores do sinal de proliferação (ISP). CONCLUSÃO: A avaliação dos pacientes pós-transplante cardíaco por meio do USIV incorpora informações detalhadas para o diagnóstico precoce e sensível da DVE, que são complementadas pelas informações histológicas fornecidas pelas BX, estabelecendo uma possível relação causal entre a DVE e os episódios de rejeição humoral.

BACKGROUND: Cardiac transplantation continues to be the treatment of choice for heart failure refractory to optimized treatment. Two methods have high sensitivity for diagnosing allograft rejection episodes and cardiac allograft vasculopathy (CAV), important causes of mortality after transplantation. OBJECTIVE: To assess the relationship between intravascular ultrasound (IVUS) results and endomyocardial biopsy (BX) reports in the follow-up of patients undergoing cardiac transplantation in a Brazilian reference service. METHODS: A retrospective epidemiological observational study was carried out with patients undergoing orthotopic cardiac transplantation from 2000 to 2009. The study assessed the medical records of those patients and the results of the IVUS and BX routinely performed in the clinical post-transplant follow-up, as well as the therapy used. RESULTS: Of the 77 patients assessed, 63.63% were males, their ages ranging from 22 to 69 years. Regarding the IVUS results, 33.96% of the patients were classified as Stanford class I, and 32.08%, as Stanford class IV. Of the 143 BX reports, 51.08% were 1R, and 0.69%, 3R. The Quilty effect was described in 14.48% of the BX reports. All patients used antiproliferative agents, 80.51% used calcineurin inhibitors, and 19.48% used proliferation signal inhibitors. CONCLUSION: The assessment of cardiac transplant patients by use of IVUS provides detailed information for the early and sensitive diagnosis of CAV, which is complemented by histological data derived from BX, establishing a possible causal relationship between CAV and humoral rejection episodes.

Assessment of cardiac allograft vasculopathy in cardiac transplantation: experience of a Brazilian center.

BACKGROUND: Cardiac transplantation continues to be the treatment of choice for heart failure refractory to optimized treatment. Two methods have high sensitivity for diagnosing allograft rejection episodes and cardiac allograft vasculopathy (CAV), important causes of mortality after transplantation. OBJECTIVE: To assess the relationship between intravascular ultrasound (IVUS) results and endomyocardial biopsy (BX) reports in the follow-up of patients undergoing cardiac transplantation in a Brazilian reference service. METHODS: A retrospective epidemiological observational study was carried out with patients undergoing orthotopic cardiac transplantation from 2000 to 2009. The study assessed the medical records of those patients and the results of the IVUS and BX routinely performed in the clinical post-transplant follow-up, as well as the therapy used. RESULTS: Of the 77 patients assessed, 63.63% were males, their ages ranging from 22 to 69 years. Regarding the IVUS results, 33.96% of the patients were classified as Stanford class I, and 32.08%, as Stanford class IV. Of the 143 BX reports, 51.08% were 1R, and 0.69%, 3R. The Quilty effect was described in 14.48% of the BX reports. All patients used antiproliferative agents, 80.51% used calcineurin inhibitors, and 19.48% used proliferation signal inhibitors. CONCLUSION: The assessment of cardiac transplant patients by use of IVUS provides detailed information for the early and sensitive diagnosis of CAV, which is complemented by histological data derived from BX, establishing a possible causal relationship between CAV and humoral rejection episodes.

GroES/GroEL and DnaK/DnaJ have distinct roles in stress responses and during cell cycle progression in Caulobacter crescentus.

Misfolding and aggregation of protein molecules are major threats to all living organisms. Therefore, cells have evolved quality control systems for proteins consisting of molecular chaperones and proteases, which prevent protein aggregation by either refolding or degrading misfolded proteins. DnaK/DnaJ and GroES/GroEL are the best-characterized molecular chaperone systems in bacteria. In Caulobacter crescentus these chaperone machines are the products of essential genes, which are both induced by heat shock and cell cycle regulated. In this work, we characterized the viabilities of conditional dnaKJ and groESL mutants under different types of environmental stress, as well as under normal physiological conditions. We observed that C. crescentus cells with GroES/EL depleted are quite resistant to heat shock, ethanol, and freezing but are sensitive to oxidative, saline, and osmotic stresses. In contrast, cells with DnaK/J depleted are not affected by the presence of high concentrations of hydrogen peroxide, NaCl, and sucrose but have a lower survival rate after heat shock, exposure to ethanol, and freezing and are unable to acquire thermotolerance. Cells lacking these chaperones also have morphological defects under normal growth conditions. The absence of GroE proteins results in long, pinched filamentous cells with several Z-rings, whereas cells lacking DnaK/J are only somewhat more elongated than normal predivisional cells, and most of them do not have Z-rings. These findings indicate that there is cell division arrest, which occurs at different stages depending on the chaperone machine affected. Thus, the two chaperone systems have distinct roles in stress responses and during cell cycle progression in C. crescentus.

Cells lacking ClpB display a prolonged shutoff phase of the heat shock response in Caulobacter crescentus.

The heat shock response in Caulobacter crescentus was previously shown to be positively regulated by the alternative sigma factor of RNA polymerase (RNAP) sigma(32), and negatively modulated by DnaK during the induction phase of the heat shock response but not during the recovery phase. In the present work we have investigated the involvement of the chaperone ClpB in the control of the heat shock response in C. crescentus. Data obtained indicated a role of ClpB in downregulation of heat shock protein (HSP) synthesis, as cells lacking this chaperone showed a prolonged shutoff phase of the heat shock response. In Escherichia coli, it has been proposed that the DnaK chaperone system switches transcription back to constitutively expressed genes through simultaneous reactivation of heat-aggregated sigma(70), as well as sequestration of sigma(32) away from RNAP. In C. crescentus, results obtained with a clpB null mutant indicate that ClpB could be involved in the reactivation of the major sigma factor sigma(73). In support of this hypothesis, we showed that transcription directed from sigma(73)-dependent promoters is not switched back in the clpB null mutant during the recovery phase. Furthermore, we observed that resolubilization of heat-aggregated sigma(73) is dependent on the presence of ClpB. Our findings also indicated that the absence of ClpB made cells more sensitive to heat shock and ethanol but not to other stresses, and unable to acquire thermotolerance.

Functional and structural analysis of HrcA repressor protein from Caulobacter crescentus.

A large number of bacteria regulate chaperone gene expression during heat shock by the HrcA-CIRCE system, in which the DNA element called CIRCE serves as binding site for the repressor protein HrcA under nonstress conditions. In Caulobacter crescentus, the groESL operon presents a dual type of control. Heat shock induction is controlled by a sigma32-dependent promoter and the HrcA-CIRCE system plays a role in regulation of groESL expression under physiological temperatures. To study the activity of HrcA in vitro, we purified a histidine-tagged version of the protein, and specific binding to the CIRCE element was obtained by gel shift assays. The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that the GroE chaperonin machine modulates HrcA activity. Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C. crescentus cells, producing different amounts of GroES/GroEL. In addition, we identified the putative DNA-binding domain of HrcA through extensive protein sequence comparison and constructed various HrcA mutant proteins containing single amino acid substitutions in or near this region. In vitro and in vivo experiments with these mutated proteins indicated several amino acids important for repressor activity.

Downregulation of the heat shock response is independent of DnaK and sigma32 levels in Caulobacter crescentus.

Expression of heat shock genes in Gram-negative proteobacteria is positively modulated by the transcriptional regulator RpoH, the sigma(32) subunit of RNA polymerase (RNAP). In this study we investigated the chaperones DnaK/DnaJ and GroES/GroEL as possible modulators of the heat response in Caulobacter crescentus. We have shown that cells overexpressing DnaK show poor induction of heat shock protein (HSP) synthesis, even though sigma(32) levels present a normal transient increase upon heat stress. On the other hand, depletion of DnaK led to higher levels of sigma(32) and increased transcription of HSP genes, at normal growth temperature. In contrast, changes in the amount of GroES/EL had little effect on sigma(32) levels and HSP gene transcription. Despite the strong effect of DnaK levels on the induction phase of the heat shock response, downregulation of HSP synthesis was not affected by changes in the amount this chaperone. Thus, we propose that competition between sigma(32) and sigma(73), the major sigma factor, for the core RNAP could be the most important factor controlling the shut-off of HSP synthesis during recovery phase. In agreement with this hypothesis, we have shown that expression of sigma(73) gene is heat shock inducible.