Ricardo C. Gehrau

MicroRNAs as Biomarkers in Solid Organ Transplantation

Important progress has been made in improving short‐term outcomes in solid organ transplantation. However, long‐term outcomes have not improved during the last decades. There is a critical need for biomarkers of donor quality, early diagnosis of graft injury and treatment response. MicroRNAs (miRNAs) are a class of small single‐stranded noncoding RNAs that function through translational repression of specific target mRNAs. MiRNA expression has been associated with different diseases and physiological conditions. Moreover, miRNAs have been detected in different biological fluids and these circulating miRNAs can distinguish diseased individuals from healthy controls. The noninvasive nature of circulating miRNA detection, their disease specificity and the availability of accurate techniques for detecting and monitoring these molecules has encouraged a pursuit of miRNA biomarker research and the evaluation of specific applications in the transplant field. miRNA expression might develop as excellent biomarkers of allograft injury and function. In this minireview, we summarize the main accomplishments of recently published reports focused on the identification of miRNAs as biomarkers in organ quality, ischemia‐reperfusion injury, acute rejection, tolerance and chronic allograft dysfunction emphasizing their mechanistic and clinical potential applications and describing their methodological limitations.

The urine microRNA profile may help monitor post-transplant renal graft function.

Non-invasive, cost-effective biomarkers that allow accurate monitoring of graft function are needed in kidney transplantation. Since microRNAs (miRNAs) have emerged as promising disease biomarkers we sought to establish an miRNA signature in urinary cell pellets comparing kidney transplant patients diagnosed with chronic allograft dysfunction (CAD) with interstitial fibrosis and tubular atrophy and those recipients with normal graft function. Overall, we evaluated 191 samples from 125 deceased donor primary kidney transplant recipients in the discovery, initial validation and the longitudinal validation studies for non-invasive monitoring of graft function. Of 1,733 mature miRNAs studied using microarrays, 22 were found to be differentially expressed between groups. Ontology and pathway analyses showed inflammation as the principal biological function associated with these miRNAs. Twelve selected miRNAs were longitudinally evaluated in urine samples of an independent set of 66 patients, at two time-points post-kidney transplant. A subset of these miRNAs was found to be differentially expressed between groups early post-kidney transplant before histological allograft injury was evident. Thus, a panel of urine miRNAs was identified as potential biomarkers for monitoring graft function and anticipating progression to CAD in kidney transplant patients.

Biology of Krüppel-like factor 6 transcriptional regulator in cell life and death

An essential role for the Krüppel‐like transcription factor family has been determined in the regulation of remarkable processes including cell proliferation, differentiation, signal transduction, oncogenesis, and cell death. A member of this group, Krüppel‐like factor 6 (KLF6), identified on the basis of its ability to regulate a group of genes belonging to the carcinoembryonic antigen gene family, has been involved in human carcinogenesis. Early studies proposed a tumor suppressor function for KLF6 because of its ability to reduce cell proliferation through several biochemical mechanisms including regulation of cell cycle components, oncogene products, and apoptosis. Mutations within the klf6 gene, decreased expression and/or loss‐of‐heterozygosity were associated with the development of different human malignancies, and, hence, further supporting the tumor suppressor function of KLF6. This view has been challenged by other studies in distinct types of human cancers describing infrequent genetic alterations of klf6 gene or even enhanced expression in some tumors. The scenario about KLF6 function became still more complex as the description of oncogenic KLF6 splice variant 1 (SV1) with dominant negative activity against the wild type KLF6 (wtKLF6) protein. Additionally, increased evidence is suggesting that KLF6 is a bonafide target of several signaling cascades, which ultimate regulatory effect on this protein could drive decisions of cell life and death, facing the dilemma about how wtKLF6 could be involved in both processes. These apparently conflicting situations, emerged by apparently opposite effects mediated by wtKLF6, may be related, at least in part, to the biological cross‐talk with the c‐Jun oncoprotein. Depending on the stimulus received by the cell, wtKLF6 interaction with c‐Jun determines different cell outcomes such as proliferation control or apoptosis. Thus, KLF6 responsiveness represents a kind of cell warning signal on receiving different stimuli, including oncogenic activation and microbial infections, orchestrating the implementation of proliferation and apoptotic programs.

Nuclear Expression of KLF6 Tumor Suppressor Factor Is Highly Associated with Overexpression of ERBB2 Oncoprotein in Ductal Breast Carcinomas

Background Krüppel-like factor 6 (KLF6) is an evolutionarily conserved and ubiquitously expressed protein that belongs to the mammalian Sp1/KLF family of transcriptional regulators. Though KLF6 is a transcription factor and harbors a nuclear localization signal it is not systematically located in the nucleus but it was detected in the cytoplasm of several tissues and cell lines. Hence, it is still not fully settled whether the tumor suppressor function of KLF6 is directly associated with its ability to regulate target genes. Methodology/Principal Findings In this study we analyzed KLF6 expression and sub-cellular distribution by immunohistochemistry in several normal and tumor tissues in a microarray format representing fifteen human organs. Results indicate that while both nuclear and cytoplasmic distribution of KLF6 is detected in normal breast tissues, breast carcinomas express KLF6 mainly detected in the cytoplasm. Expression of KLF6 was further analyzed in breast cancer tissues overexpressing ERBB2 oncoprotein, which is associated with poor disease prognosis and patients survival. The analysis of 48 ductal carcinomas revealed a significant population expressing KLF6 predominantly in the nuclear compartment (X2 p = 0.005; Fisher p = 0.003). Moreover, this expression pattern correlates directly with early stage and small ductal breast tumors and linked to metastatic events in lymph nodes. Conclusions/Significance Data are consistent with a preferential localization of KLF6 in the nuclear compartment of early stage and small HER2-ERBB2 overexpressing ductal breast tumor cells, also presenting lymph node metastatic events. Thus, KLF6 tumor suppressor could represent a new molecular marker candidate for tumor prognosis and/or a potential target for therapy strategies.

Molecular pathways differentiate hepatitis C virus (HCV) recurrence from acute cellular rejection in HCV liver recipients.

Acute cellular rejection (ACR) and hepatitis C virus (HCV) recurrence (HCVrec) are common complications after liver transplantation (LT) in HCV patients, who share common clinical and histological features, making a differential diagnosis difficult. Fifty-three liver allograft samples from unique HCV LT recipients were studied using microarrays, including a training set (n = 32) and a validation set (n = 19). Two no-HCV-ACR samples from LT recipients were also included. Probe set intensity values were obtained using the robust multiarray average method (RMA) method. Analysis of variance identified statistically differentially expressed genes (P ≤ 0.005). The limma package was used to fit the mixed-effects models using a restricted maximum likelihood procedure. The last absolute shrinkage and selection operator (LASSO) model was fit with HCVrec versus ACR as the dependent variable predicted. N-fold cross-validation was performed to provide an unbiased estimate of generalization error. A total of 179 probe sets were differentially expressed among groups, with 71 exclusive genes between HCVrec and HCV-ACR. No differences were found within ACR group (HCV-ACR vs. no-HCV-ACR). Supervised clustering analysis displayed two clearly independent groups, and no-HCV-ACR clustered within HCV-ACR. HCVrec-related genes were associated with a cytotoxic T-cell profile, and HCV-ACR-related genes were associated with the inflammatory response. The best-fitting LASSO model classifier accuracy, including 15 genes, has an accuracy of 100% in the training set. N-fold cross-validation accuracy was 78.1%, and sensitivity, specificity and positive and negative predictive values were 50.0%, 90.9%, 71.4% and 80.0%, respectively. Arginase type II (ARG2), ethylmalonic encephalopathy 1 (ETHE1), transmembrane protein 176A (TMEM176A) and TMEM176B genes were significantly confirmed in the validation set. A molecular signature capable of distinguishing HCVrec and ACR in HCV LT recipients was identified and validated.

Cellular and molecular immune profiles in renal transplant recipients after conversion from tacrolimus to sirolimus

Tacrolimus and Sirolimus are commonly used maintenance immunesuppressants in kidney transplantation. Since their effects on immune cells and allograft molecular profiles have not been elucidated, we characterized the effects of Tacrolimus to Sirolimus conversion on frequency and function of T cells, and on graft molecular profiles. Samples from renal transplant patients in a randomized trial of 18 patients with late Sirolimus conversion and 12 on Tacrolimus maintenance were utilized. Peripheral blood was collected at 0, 6, 12 and 24-months post-randomization with T cell subpopulations analyzed by flow cytometry and T cell alloreactivity tested by IFN-γ ELISPOT. Graft biopsy samples obtained 24-months post-randomization were used for gene expression analysis. Sirolimus conversion led to an increase in CD4+25+++Foxp3+ regulatory T cells. While Tacrolimus-maintained patients showed a decrease in indirect alloreactivity over time post-transplant, Sirolimus conversion increased indirect alloreactive T cell frequencies compared to Tacrolimus-maintained patients. No histological differences were found in graft biopsies, but molecular profiles showed activation of the antigen presentation, IL-12 signaling, oxidative stress, macrophage-derived production pathways, and increased inflammatory and immune response in Sirolimus-converted patients. Thus, chronic immune alterations are induced after Sirolimus conversion. Despite the molecular profile being favorable to calcineurin inhibitor-based regimen, there was no impact in renal function over 30 months of follow-up.

MicroRNA Signature at the Time of Clinical HCV Recurrence Associates With Aggressive Fibrosis Progression Post-Liver Transplantation

Diagnosis and prediction of the severity of hepatitis C virus recurrence (HCVrec) after liver transplantation (LT) remain a challenge. MicroRNAs have been recently recognized as potential disease biomarkers. Archival liver biopsy samples from 43 HCV+ LT recipients were collected at clinical HCVrec time and at 3 years post‐LT. Patients were classified as progressors (P = F0/F1) or nonprogressors (NP = F3/F4) according to the severity of fibrosis on the 3‐year biopsy. Training (n = 27) and validation (n = 16) sets were defined. RNA was isolated from all biopsies at clinical HCVrec time, labeled and hybridized to miRNA‐arrays. Progressors versus nonprogressors were compared using the two‐sample t‐test. A p‐value ≤0.01 was considered significant. The ingenuity pathway analysis tool was used for microRNA and miRNA:mRNA ontology data integration. Nine microRNAs were differentially expressed between groups. A supervised cluster analysis separated samples in two well‐defined groups (progressors vs. nonprogressors). Pathway analysis associated those microRNAs with hepatitis, steatosis, fibrosis, cirrhosis and T cell‐related immune response. Data integration identified 17 genes from a previous genomic study as 9‐microRNAs signature targets. Seven microRNAs were successfully validated in the validation set using QPCR. We have identified a 9‐microRNA signature able to identify early post‐LT patients at high risk of severe HCVrec during long‐term follow‐up.

Donor Hepatic Steatosis Induce Exacerbated Ischemia-Reperfusion Injury Through Activation of Innate Immune Response Molecular Pathways.

Background Severe liver steatosis is a known risk factor for increased ischemia-reperfusion injury (IRI) and poor outcomes after liver transplantation (LT). This study aimed to identify steatosis-related molecular mechanisms associated with IRI exacerbation after LT. Methods Paired graft biopsies (n = 60) were collected before implantation (L1) and 90 minutes after reperfusion (L2). The LT recipients (n = 30) were classified by graft macrosteatosis: without steatosis (WS) of 5% or less (n = 13) and with steatosis (S) of 25% or greater (n = 17). Plasma samples were collected at L1, L2, and 1 day after LT (postoperative [POD]1) for cytokines evaluation. Tissue RNA was isolated for gene expression microarrays. Probeset summaries were obtained using robust multiarray average algorithm. Pairwise comparisons were fit using 2-sample t test. P values 0.01 or less were significant (false discovery rate <5%). Molecular pathway analyses were conducted using Ingenuity Pathway Analysis tool. Results Significantly differentially expressed genes were identified for WS and S grafts after reperfusion. Comprehensive comparison analysis of molecular profiles revealed significant association of S grafts molecular profile with innate immune response activation, macrophage production of nitric oxide and reactive oxygen species, IL-6, IL-8, IL-10 signaling activation, recruitment of granulocytes, and accumulation of myeloid cells. Postreperfusion histological patterns of S grafts revealed neutrophilic infiltration surrounding fat accumulation. Circulating proinflammatory cytokines after reperfusion and 24 hours after LT concurred with intragraft-deregulated molecular pathways. All tested cytokines were significantly increased in plasma of S grafts recipients after reperfusion when compared with WS group at same time. Conclusions Increases of graft steatosis exacerbate IRI by exacerbation of innate immune response after LT. Preemptive strategies should consider it for safety usage of steatotic livers.

BAFF Mediates Splenic B Cell Response and Antibody Production in Experimental Chagas Disease

Background B cells and antibodies are involved not only in controlling the spread of blood circulating Trypanosoma cruzi, but also in the autoreactive manifestations observed in Chagas disease. Acute infection results in polyclonal B cell activation associated with hypergammaglobulinemia, delayed specific humoral immunity and high levels of non-parasite specific antibodies. Since TNF superfamily B lymphocyte Stimulator (BAFF) mediates polyclonal B cell response in vitro triggered by T. cruzi antigens, and BAFF-Tg mice show similar signs to T. cruzi infected mice, we hypothesized that BAFF can mediate polyclonal B cell response in experimental Chagas disease. Methodology/Principal Findings BAFF is produced early and persists throughout the infection. To analyze BAFF role in experimental Chagas disease, Balb/c infected mice were injected with BR3:Fc, a soluble receptor of BAFF, to block BAFF activity. By BAFF blockade we observed that this cytokine mediates the mature B cell response and the production of non-parasite specific IgM and IgG. BAFF also influences the development of antinuclear IgG and parasite-specific IgM response, not affecting T. cruzi-specific IgG and parasitemia. Interestingly, BAFF inhibition favors the parasitism in heart. Conclusions/Significance Our results demonstrate, for the first time, an active role for BAFF in shaping the mature B cell repertoire in a parasite infection.

Ex Vivo Perfusion With Adenosine A2A Receptor Agonist Enhances Rehabilitation of Murine Donor Lungs After Circulatory Death.

Background Ex vivo lung perfusion (EVLP) enables assessment and rehabilitation of marginal donor lungs before transplantation. We previously demonstrated that adenosine A2A receptor (A2AR) agonism attenuates lung ischemia-reperfusion injury. The current study utilizes a novel murine EVLP model to test the hypothesis that A2AR agonist enhances EVLP-mediated rehabilitation of donation after circulatory death (DCD) lungs. Methods Mice underwent euthanasia and 60 minutes warm ischemia, and lungs were flushed with Perfadex and underwent cold static preservation (CSP, 60 minutes). Three groups were studied: no EVLP (CSP), EVLP with Steen solution for 60 minutes (EVLP), and EVLP with Steen solution supplemented with ATL1223, a selective A2AR agonist (EVLP + ATL1223). Lung function, wet/dry weight, cytokines and neutrophil numbers were measured. Microarrays were performed using the Affymetrix GeneChip Mouse Genome 430A 2.0 Array. Results Ex vivo lung perfusion significantly improved lung function versus CSP, which was further, significantly improved by EVLP + ATL1223. Lung edema, cytokines, and neutrophil counts were reduced after EVLP and further, significantly reduced after EVLP + ATL1223. Gene array analysis revealed differential expression of 1594 genes after EVLP, which comprise canonical pathways involved in inflammation and innate immunity including IL-1, IL-8, IL-6, and IL-17 signaling. Several pathways were uniquely regulated by EVLP + ATL1223 including the downregulation of genes involved in IL-1 signaling, such as ADCY9, ECSIT, IRAK1, MAPK12, and TOLLIP. Conclusions Ex vivo lung perfusion modulates proinflammatory genes and reduces pulmonary dysfunction, edema, and inflammation in DCD lungs, which are further reduced by A2AR agonism. This murine EVLP model provides a novel platform to study rehabilitative mechanisms of DCD lungs.