RESUMO
Alternative 3-dimensional (3D) skin models that replicate in vivo human skin are required to investigate important events during wound healing, such as collective cell migration, epidermal layer formation, dermal substrate formation, re-epithelialisation and collagen production. In this study, a matched human 3D skin equivalent model (3D-SEM) was developed from human skin cells (fibroblast and keratinocytes), characterised using haematoxylin and eosin, immunofluorescence staining and microRNA profiling. The 3D-SEM was then functionally tested for its use in wound healing studies. Mesenchymal stem cells (MSCs) were isolated and characterised according to the criteria stipulated by the International Society for Cell Therapy. Cytokine and growth factor secretions were analysed by enzyme-linked immunosorbent assay. MSC-conditioned medium (MSC-CM) was then tested for wound healing capacity using the developed 3D-SEM at different timepoints i.e., at one, two and four weeks. The constructed 3D-SEM showed consistent development of skin-like structures composed of dermal layers and epidermal layers, with the ability to express epidermal differentiation markers and full stratification. They also showed prolonged longevity in culture media, retaining full differentiation and stratification within the four weeks. MicroRNA profiling revealed a strong correlation in microRNA expression between the developed 3D-SEM and the original native skin (p<0.001; R=0.64). Additionally, MSC-CM significantly enhanced migration, proliferation and differentiation of epidermal cells in the wounded models compared to control models at the different timepoints. In conclusion, in this study, the developed 3D-SEM mimicked native skin at the cellular and molecular levels, and clearly showed the important stages of skin regeneration during the healing process. MSC secretome contains growth factors that play a pivotal role in the healing process and could be used as a therapeutic option to accelerate skin healing.
Assuntos
Células-Tronco Mesenquimais , Cicatrização , Humanos , Meios de Cultivo Condicionados/farmacologia , Cicatrização/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Pele/lesões , Pele/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células CultivadasRESUMO
Extracellular vesicles (EV) are rich in small RNA; however, a frequent caveat can be low abundance of EV RNA content, especially in clinical studies. NanoString MicroRNA Assays allow for multiplexed profiling of n = 800 mature microRNAs and can be applied to assess EV microRNA cargo. Here, we describe a method to adapt NanoString nCounter microRNA profiling to assess mature microRNA expression in low-concentration RNA samples, including concentrating the RNA, quantifying the RNA, and performing the NanoString protocol. Twelve samples can be assessed at one time using this method.
Assuntos
Vesículas Extracelulares , MicroRNAs , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , MicroRNAs/genética , MicroRNAs/análise , Humanos , Perfilação da Expressão Gênica/métodosRESUMO
Osteoarthritis (OA) is a degenerative joint disease, causing impaired mobility. There are currently no effective therapies other than palliative treatment. Mesenchymal stromal cells (MSCs) and their secreted extracellular vesicles (MSC-EVs) have shown promise in attenuating OA progression, promoting chondral regeneration, and modulating joint inflammation. However, the precise molecular mechanism of action driving their beneficial effects has not been fully elucidated. In this study, we analyzed MSC-EV-treated human OA chondrocytes (OACs) to assess viability, proliferation, migration, cytokine and catabolic protein expression, and microRNA and mRNA profiles. We observed that MSC-EV-treated OACs displayed increased metabolic activity, proliferation, and migration compared to the controls. They produced decreased proinflammatory (Il-8 and IFN-γ) and increased anti-inflammatory (IL-13) cytokines, and lower levels of MMP13 protein coupled with reduced expression of MMP13 mRNA, as well as negative microRNA regulators of chondrogenesis (miR-145-5p and miR-21-5p). In 3D models, MSC-EV-treated OACs exhibited enhanced chondrogenesis-promoting features (elevated sGAG, ACAN, and aggrecan). MSC-EV treatment also reversed the pathological impact of IL-1ß on chondrogenic gene expression and extracellular matrix component (ECM) production. Finally, MSC-EV-treated OACs demonstrated the enhanced expression of genes associated with cartilage function, collagen biosynthesis, and ECM organization and exhibited a signature of 24 differentially expressed microRNAs, associated with chondrogenesis-associated pathways and ECM interactions. In conclusion, our data provide new insights on the potential mechanism of action of MSC-EVs as a treatment option for early-stage OA, including transcriptomic analysis of MSC-EV-treated OA, which may pave the way for more targeted novel therapeutics.
RESUMO
BACKGROUND: A genetic polymorphism, rs2204985, has been reported to be associated with the diversity of T-cell antigen receptor repertoire and TREC levels, reflecting the function of the thymus. As the thymus function can be assumed to be an important factor regulating the outcome of stem cell transplantation (SCT), it was of great interest that rs2204985 showed a genetic association to disease-free and overall survival in a German SCT donor cohort. Tools to predict the outcome of SCT more accurately would help in risk assessment and patient safety. OBJECTIVE: To evaluate the general validity of the original genetic association found in the German cohort, we determined genetic associations between rs2204985 and the outcome of SCT in 1,473 SCT donors from four different populations. STUDY DESIGN: Genetic associations between rs2204985 genotype AA versus AG/GG and overall survival (OS) and disease-free survival (DFS) in 1,473 adult, allogeneic SCT from Finland, the United Kingdom, Spain, and Poland were performed using the Kaplan-Meier analysis and log-rank tests. We adjusted the survival models with covariates using Cox regression. RESULTS: In unrelated SCT donors (N = 425), the OS of genotype AA versus AG/GG had a trend for a similar association (p = 0.049, log-rank test) as previously reported in the German cohort. The trend did not remain significant in the Cox regression analysis with covariates. No other associations were found. CONCLUSION: Weak support for the genetic association between rs2204985, previously also associated with thymus function, and the outcome of SCT could be found in a cohort from four populations.
Assuntos
Timo , Humanos , Adulto , Masculino , Feminino , Pessoa de Meia-Idade , Estudos de Coortes , Polimorfismo de Nucleotídeo Único , Genótipo , Doadores de Tecidos , Transplante de Células-Tronco , Idoso , Adulto Jovem , Adolescente , Polônia , Resultado do Tratamento , Espanha , Transplante de Células-Tronco Hematopoéticas , Reino UnidoRESUMO
Introduction: Data on genomic susceptibility for adverse outcomes after hematopoietic stem cell transplantation (HSCT) for recipients are scarce. Methods: We performed a genome wide association study (GWAS) to identify genes associated with survival/mortality, relapse, and severe graft-versus-host disease (sGvHD), fitting proportional hazard and subdistributional models to data of n=1,392 recipients of European ancestry from three centres. Results: The single nucleotide polymorphism (SNP) rs17154454, intronic to the neuronal growth guidant semaphorin 3C gene (SEMA3C), was genome-wide significantly associated with event-free survival (p=7.0x10-8) and sGvHD (p=7.5x10-8). Further associations were detected for SNPs in the Paxillin gene (PXN) with death without prior relapse or sGvHD, as well as for SNPs of the Plasmacytoma Variant Translocation 1 gene (PVT1, a long non-coding RNA gene), the Melanocortin 5 Receptor (MC5R) gene and the WW Domain Containing Oxidoreductase gene (WWOX), all associated with the occurrence of sGvHD. Functional considerations support the observed associations. Discussion: Thus, new genes were identified, potentially influencing the outcome of HSCT.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Estudo de Associação Genômica Ampla , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Doença Enxerto-Hospedeiro/genética , Genômica , RecidivaRESUMO
OBJECTIVES: Extracellular vesicles (EVs) are abundant in body fluids, contributing to intercellular signalling by transferring cargo that includes microRNAs (miRs) - themselves implicated in pathobiology. For the first time we evaluated the potential of EV miRs to contribute diagnostic information in early RA, predict methotrexate (MTX) efficacy or shed light on the drug's mechanism of action. METHODS: 798 miRs isolated from serum-derived EVs of 46 patients with untreated RA, 23 with untreated polymyalgia rheumatica (PMR; inflammatory disease control group) and 12 in whom significant inflammatory disease had been excluded (non-inflammatory controls; NICs) were profiled (Nanostring); the same measurements were made for RA patients after 6 months' MTX treatment. Analyses took multiple testing into account. RESULTS: 28 EV miRs were robustly differentially expressed between early RA (but not PMR) patients and NICs after correction for age and sex, suggesting discriminatory value. Cross-validated partial least squared-discriminant analysis also indicated the predictive potential of a distinct baseline EV miR signature with respect to MTX-induced remission at 6 months. The change in expression of 13 miRs over the course of MTX treatment differed significantly between responders and non-responders, and four of those exhibiting increased relative abundance amongst responders have known roles in regulating the pathogenic potential of synovial fibroblasts, namely miR-212-3p, miR-338-5p, miR-410-3p, and miR-537. CONCLUSION: Our data highlight the potential of serum EV miRs as diagnostic and therapeutic biomarkers, highlighting a novel potential mechanism via which MTX may exert its therapeutic effect in early RA that warrants further investigation.
RESUMO
Haematopoietic stem cell transplantation (HSCT) is the treatment of choice for malignant haematological diseases. Despite continuous improvements in pre- and post-transplantation procedures, the applicability of allo-HSCT is limited by life-threatening complications such as graft-versus-host disease (GvHD), engraftment failure, and opportunistic infections. Extracorporeal photopheresis (ECP) is used to treat steroid resistant GvHD with significant success. However, the molecular mechanisms driving its immunomodulatory action, whilst preserving immune function, require further understanding. As ECP is safe to administer with few significant adverse effects, it has the potential for earlier use in the post-HSCT treatment of GvHD. Thus, further understanding the immunomodulatory mechanisms of ECP action may justify more timely use in clinical practice, as well as identify biomarkers for using ECP as first line or pre-emptive GvHD therapy. This review aims to discuss technical aspects and response to ECP, review ECP as an immunomodulatory treatment modality for chronic GvHD including the effect on regulatory T cells and circulating vs. tissue-resident immune cells and consider the importance of emerging biomarkers for ECP response.
Assuntos
Síndrome de Bronquiolite Obliterante , Doença Enxerto-Hospedeiro , Fotoferese , Humanos , Biomarcadores , ImunidadeRESUMO
BACKGROUND: Chronic graft-versus-host disease (cGvHD) is a complex disorder that typically manifests after allogeneic hematopoietic stem cell transplantation (HSCT). It is a major cause of non-relapse mortality, which makes finding biomarkers associated with its occurrence a priority. Recent studies increasingly indicate that microRNAs (miRNAs, short regulatory RNA molecules) can be used as biomarkers of various disorders. They can circulate in patients' bodies encapsulated within extracellular vesicles (EVs). OBJECTIVES: To identify miRNAs associated with the occurrence of cGvHD in EVs isolated from the plasma of patients after allogeneic HSCT. MATERIAL AND METHODS: We performed global miRNA expression profiling in a pilot cohort of 3 cGvHD cases and 4 non-cGvHD patients without disease symptoms 90 days after the transplantation (control group). RESULTS: The 2 groups were naturally clustered according to their miRNA profiles using unsupervised hierarchical clustering analysis. We identified 3 miRNAs that were differentially expressed in the cGvHD patients compared to the non-cGvHD patients. The levels of hsa-miR-630 and hsa-miR-374b-5p were lower in the cGvHD patients: 4.1-fold (p = 0.002) and 2.7-fold (p = 0.044), respectively. In contrast, the levels of hsa-miR-29c-3p were 5.8-fold higher (p = 0.004). CONCLUSIONS: Our results suggest that miRNA profiles from plasma EVs may act as markers of cGvHD onset.
Assuntos
Síndrome de Bronquiolite Obliterante , Vesículas Extracelulares , Doença Enxerto-Hospedeiro , MicroRNAs , Humanos , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/genética , Transplante Homólogo , MicroRNAs/genética , Biomarcadores , Vesículas Extracelulares/genética , Doença CrônicaRESUMO
Extracellular vesicles (EV) and the microRNAs that they contain are increasingly recognised as a rich source of informative biomarkers, reflecting pathological processes and fundamental biological pathways and responses. Their presence in biofluids makes them particularly attractive for biomarker identification. However, a frequent caveat in relation to clinical studies is low abundance of EV RNA content. In this study, we used NanoString nCounter technology to assess the microRNA profiles of n = 64 EV low concentration RNA samples (180-49125 pg), isolated from serum and cell culture media using precipitation reagent or sequential ultracentrifugation. Data was subjected to robust quality control parameters based on three levels of limit of detection stringency, and differential microRNA expression analysis was performed between biological subgroups. We report that RNA concentrations > 100 times lower than the current NanoString recommendations can be successfully profiled using nCounter microRNA assays, demonstrating acceptable output ranges for imaging parameters, binding density, positive/negative controls, ligation controls and normalisation quality control. Furthermore, despite low levels of input RNA, high-level differential expression analysis between biological subgroups identified microRNAs of biological relevance. Our results demonstrate that NanoString nCounter technology offers a sensitive approach for the detection and profiling of low abundance EV-derived microRNA, and may provide a solution for research studies that focus on limited sample material.
RESUMO
Hematopoietic allogeneic stem cell transplantation (allo-SCT) is a curative option for patients with hematological malignancies. However, due to disparities in major and minor histocompatibility antigens between donor and recipient, severe inflammatory complications can occur, among which chronic graft-versus-host disease (cGVHD) can be life-threatening. A classical therapeutic approach to the prevention and treatment of cGVHD has been broad immunosuppression, but more recently adjuvant immunotherapies have been tested. This review summarizes and discusses immunomodulatory approaches with T cells, including chimeric antigen receptor (CAR) and regulatory T cells, with natural killer (NK) cells and innate lymphoid cells (ILCs), and finally with mesenchymal stromal cells (MSC) and extracellular vesicles thereof. Clinical studies and pre-clinical research results are presented likewise.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Doença Enxerto-Hospedeiro/prevenção & controle , Imunidade Inata , Terapia Baseada em Transplante de Células e Tecidos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Células Matadoras NaturaisRESUMO
Children with B-cell non-Hodgkin lymphoma (B-NHL) have an excellent chance of survival, however, current clinical risk stratification places as many as half of patients in a high-risk group receiving very intensive chemo-immunotherapy. TP53 alterations are associated with adverse outcome in many malignancies; however, whilst common in paediatric B-NHL, their utility as a risk classifier is unknown. We evaluated the clinical significance of TP53 abnormalities (mutations, deletion and/or copy number neutral loss of heterozygosity) in a large UK paediatric B-NHL cohort and determined their impact on survival. TP53 abnormalities were present in 54.7% of cases and were independently associated with a significantly inferior survival compared to those without a TP53 abnormality (PFS 70.0% vs 100%, p < 0.001, OS 78.0% vs 100%, p = 0.002). Moreover, amongst patients clinically defined as high-risk (stage III with high LDH or stage IV), those without a TP53 abnormality have superior survival compared to those with TP53 abnormalities (PFS 100% vs 55.6%, p = 0.005, OS 100% vs 66.7%, p = 0.019). Biallelic TP53 abnormalities were either maintained from the presentation or acquired at progression in all paired diagnosis/progression Burkitt lymphoma cases. TP53 abnormalities thus define clinical risk groups within paediatric B-NHL and offer a novel molecular risk stratifier, allowing more personalised treatment protocols.
Assuntos
Linfoma de Células B/genética , Proteína Supressora de Tumor p53/genética , Adolescente , Criança , Pré-Escolar , Progressão da Doença , Feminino , Dosagem de Genes , Loci Gênicos , Humanos , Lactente , Linfoma de Células B/patologia , Masculino , MutaçãoRESUMO
Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) belong to the most common inflammatory rheumatic diseases. MicroRNAs (miRNAs) are small 18-22 RNA molecules that function as posttranscriptional regulators. They are abundantly present within extracellular vesicles (EVs), small intercellular communication vesicles that can be found in bodily fluids and that have key functions in pathological and physiological pathways. Recently, EVs have gained much interest because of their diagnostic and therapeutic potential. Using NanoString profiling technology, the miRNA repertoire of serum EVs was determined and compared in RA and AS patients before and after anti-TNF therapy to assess its potential use as a diagnostic and prognostic biomarker. Furthermore, possible functional effects of those miRNAs that were characterized by the most significant expression changes were evaluated using in silico prediction algorithms. The analysis revealed a unique profile of differentially expressed miRNAs in RA and AS patient serum EVs. We identified 12 miRNAs whose expression profiles enabled differentiation between RA and AS patients before induction of anti-TNF treatment, as well as 4 and 14 miRNAs whose repertoires were significantly changed during the treatment in RA and AS patients, respectively. In conclusion, our findings suggest that extracellular vesicle miRNAs could be used as potential biomarkers associated with RA and AS response to biological treatment.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Vesículas Extracelulares/genética , Espondilite Anquilosante/tratamento farmacológico , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Adulto , Artrite Reumatoide/genética , Vesículas Extracelulares/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , Espondilite Anquilosante/genética , Resultado do TratamentoRESUMO
Osteoarthritis (OA) is a heterogeneous disease in which the cross-talk between the cells from different tissues within the joint is affected as the disease progresses. Extracellular vesicles (EVs) are known to have a crucial role in cell-cell communication by means of cargo transfer. Subchondral bone (SB) resident cells and its microenvironment are increasingly recognised to have a major role in OA pathogenesis. The aim of this study was to investigate the EV production from OA SB mesenchymal stromal cells (MSCs) and their possible influence on OA chondrocytes. Small EVs were isolated from OA-MSCs, characterized and cocultured with chondrocytes for viability and gene expression analysis, and compared to small EVs from MSCs of healthy donors (H-EVs). OA-EVs enhanced viability of chondrocytes and the expression of chondrogenesis-related genes, although the effect was marginally lower compared to that of the H-EVs. miRNA profiling followed by unsupervised hierarchical clustering analysis revealed distinct microRNA sets in OA-EVs as compared to their parental MSCs or H-EVs. Pathway analysis of OA-EV miRNAs showed the enrichment of miRNAs implicated in chondrogenesis, stem cells, or other pathways related to cartilage and OA. In conclusion, OA SB MSCs were capable of producing EVs that could support chondrocyte viability and chondrogenic gene expression and contained microRNAs implicated in chondrogenesis support. These EVs could therefore mediate the cross-talk between the SB and cartilage in OA potentially modulating chondrocyte viability and endogenous cartilage regeneration.
RESUMO
Introduction: Acute graft vs. host disease (aGvHD) is a frequent complication following allogeneic haematopoeitic transplantation (HSCT). Despite recent advances, there are no universally accepted biomarkers to determine development of aGvHD. MicroRNAs miR-146a and miR-155 have been previously associated with aGvHD and show promise as clinically translatable biomarkers. In this study, we performed comprehensive expression profiling of miR-146a, miR-155, and miR-155* expression in aGvHD target tissue and biofluids and relate expression to post-HSCT outcomes. Materials and Methods: MicroRNA expression was assessed by qRT-PCR in gastrointestinal (n = 31) and skin (n = 31) biopsies as well as serum (exploratory cohort n = 34, verification cohort n = 81, diagnostic cohort n = 65) and urine (exploratory cohort n = 30, verification cohort n = 56, diagnostic cohort n = 20) biofluids, including extracellular vesicle (EV) cohorts (serum EV n = 15, urine EV n = 30). Expression was related to aGvHD incidence, severity and overall survival. Results: In GI samples, expression of miR-155 (p = 0.03) and miR-146a (p = 0.03) was higher at aGvHD onset compared to patients with no GvHD. In skin biopsies, expression of miR-155 (p = 0.004) was upregulated in aGvHD patients compared to normal control skin. Expression of miR-146a was higher in aGvHD compared to no aGvHD biopsies (p = 0.002). In serum, miR-155 (p = 0.03) and miR-146a (p = 0.02) expression was higher at day 14 (D14), while in urine expression was elevated at D7 post-HSCT in patients who developed aGvHD compared to those disease-free. This was verified in an independent serum (miR-155 p = 0.005, miR-146a p = 0.003) and urine (miR-155 p = 0.02, miR-146a p = 0.04) cohort, where both microRNAs were also associated with aGvHD by ROC analysis. In serum and urine samples taken at the time of aGvHD symptoms, expression of miR-155 and miR-146a was also elevated (serum miR-155 p = 0.03, miR-146a p < 0.001; urine miR-155 p = 0.02, miR-146a p = 0.02). In contrast, miR-146a and miR-155 were downregulated at D14 in serum EVs and at D7 in urine EVs in patients who developed aGvHD compared to those that remained disease-free, in both an exploratory (serum miR-155 p = 0.02, miR-146a p = 0.06; urine miR-155 p = 0.02, miR-146a p = 0.07) and an independent cohort (serum miR-155 p = 0.01, miR-146a p = 0.02). Conclusions: These results further support a role for miR-155 and miR-146a as non-invasive, clinically relevant biomarkers for aGvHD. However, the link between their involvement in generalized inflammation and in specific pathophysiology requires further investigation at a systemic level.
Assuntos
Biomarcadores/sangue , Doença Enxerto-Hospedeiro/imunologia , Inflamação/imunologia , Intestinos/fisiologia , MicroRNAs/sangue , Adolescente , Adulto , Idoso , Biomarcadores/urina , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , MicroRNAs/urina , Pessoa de Meia-Idade , Fenômenos Fisiológicos da Pele , Adulto JovemRESUMO
Chronic graft-versus-host disease (cGvHD) is one of the major complications of allogeneic stem cell transplantation (HSCT). cGvHD is an autoimmune-like disorder affecting multiple organs and involves a dermatological rash, tissue inflammation and fibrosis. The incidence of cGvHD has been reported to be as high as 30% to 60% and there are currently no reliable tools for predicting the occurrence of cGvHD. There is therefore an important unmet clinical need for predictive biomarkers. The present review summarizes the state of the art for genetic variation as a predictive biomarker for cGvHD. We discuss three different modes of action for genetic variation in transplantation: genetic associations, genetic matching, and pharmacogenetics. The results indicate that currently, there are no genetic polymorphisms or genetic tools that can be reliably used as validated biomarkers for predicting cGvHD. A number of recommendations for future studies can be drawn. The majority of studies to date have been under-powered and included too few patients and genetic markers. Like in all complex multifactorial diseases, large collaborative genome-level studies are now needed to achieve reliable and unbiased results. Some of the candidate genes, in particular, CTLA4, HSPE, IL1R1, CCR6, FGFR1OP, and IL10, and some non-HLA variants in the HLA gene region have been replicated to be associated with cGvHD risk in independent studies. These associations should now be confirmed in large well-characterized cohorts with fine mapping. Some patients develop cGvHD despite very extensive immunosuppression and other treatments, indicating that the current therapeutic regimens may not always be effective enough. Hence, more studies on pharmacogenetics are also required. Moreover, all of these studies should be adjusted for diagnostic and clinical features of cGvHD. We conclude that future studies should focus on modern genome-level tools, such as machine learning, polygenic risk scores and genome-wide association study-transcription meta-analyses, instead of focusing on just single variants. The risk of cGvHD may be related to the summary level of immunogenetic differences, or whole genome histocompatibility between each donor-recipient pair. As the number of genome-wide analyses in HSCT is increasing, we are approaching an era where there will be sufficient data to incorporate these approaches in the near future.
Assuntos
Doença Enxerto-Hospedeiro/genética , Antígenos HLA/genética , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Histocompatibilidade/genética , Variantes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , Animais , Doença Crônica , Marcadores Genéticos , Predisposição Genética para Doença , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Antígenos HLA/imunologia , Humanos , Imunossupressores/uso terapêutico , Medição de Risco , Fatores de Risco , Transplante Homólogo/efeitos adversos , Resultado do TratamentoRESUMO
Prognostic, diagnostic or predictive biomarkers are urgently needed for assessment of chronic graft-versus-host disease (cGvHD), a major risk for patients undergoing allogeneic hematopoietic stem cell transplantation. The main goal of this review generated within the COST Action EUROGRAFT "Integrated European Network on Chronic Graft Versus Host Disease" was to identify potential novel biomarkers for cGvHD besides the widely accepted molecular and cellular biomarkers. Thus, the focus was on cellular biomarkers, alloantibodies, glycomics, endothelial derived particles, extracellular vesicles, microbiome, epigenetic and neurologic changes in cGvHD patients. Both host-reactive antibodies in general, and particularly alloantibodies have been associated with cGvHD and require further consideration. Glycans attached to IgG modulate its activity and represent a promising predictive and/or stratification biomarker for cGVHD. Furthermore, epigenetic changes such as microRNAs and DNA methylation represent potential biomarkers for monitoring cGvHD patients and novel targets for developing new treatment approaches. Finally, the microbiome likely affects the pathophysiology of cGvHD; bacterial strains as well as microbial metabolites could display potential biomarkers for dysbiosis and risk for the development of cGvHD. In summary, although there are no validated biomarkers currently available for clinical use to better inform on the diagnosis, prognosis or prediction of outcome for cGvHD, many novel sources of potential markers have shown promise and warrant further investigation using well characterized, multi-center patient cohorts.
Assuntos
Biomarcadores/metabolismo , Doença Enxerto-Hospedeiro/metabolismo , Animais , Bactérias/metabolismo , Micropartículas Derivadas de Células/metabolismo , Doença Crônica , Tomada de Decisão Clínica , Vesículas Extracelulares/metabolismo , Microbioma Gastrointestinal , Marcadores Genéticos , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/microbiologia , Humanos , Isoanticorpos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Valor Preditivo dos Testes , PrognósticoRESUMO
Type 1 diabetes (T1DM) is associated with increased cardiovascular disease (CVD) and reduced life expectancy. We thus hypothesized that anti-angiogenic miRs are increased in T1DM, and the cardioprotective effect of metformin is mediated via reducing those miRs. In an open label, case-controlled study, 23 T1DM patients without CVD were treated with metformin for eight weeks (TG), matched with nine T1DM patients on standard treatment (SG) and 23 controls (CG). Plasma miR-222, miR-195, miR-21a and miR-126 were assayed by real-time RT-qPCR. The results were correlated with: endothelial function (RHI), circulating endothelial progenitor cells (cEPCs) (vascular repair marker, CD45dimCD34âºVEGFR2⺠cells) and circulating endothelial cells (cECs) (vascular injury marker, CD45dimCD34âºCD133â»CD144⺠cells). miR-222, miR-195 and miR-21a were higher in T1DM than CG; p = 0.009, p < 0.0001, p = 0.0001, respectively. There was an inverse correlation between logmiR-222 and logRHI (p < 0.05) and a direct correlation between logmiR-222 and logCD34⺠(p < 0.05) in TG. Metformin reduced miR-222, miR-195 and miR-21a levels in TG; p = 0.007, p = 0.002 p = 0.0012, respectively. miRs remained unchanged in SG. miR-126 was similar in all groups. There was a positive association between changes in logmiR-222 and logcECs after metformin in TG (p < 0.05). Anti-angiogenic miRs are increased in T1DM. Metformin has cardioprotective effects through downregulating miR-222, miR-195 and miR-21a, beyond improving glycemic control.
Assuntos
Diabetes Mellitus Tipo 1/sangue , Angiopatias Diabéticas/prevenção & controle , Hipoglicemiantes/uso terapêutico , Metformina/uso terapêutico , MicroRNAs/sangue , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/tratamento farmacológico , Angiopatias Diabéticas/sangue , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Allogeneic hematopoietic stem cell transplantation is a curative treatment for numerous hematological malignancies. However, acute graft-versus-host disease (aGvHD) is a major complication affecting 40-70% of all transplant patients, whereby the earliest and most frequent presentation is in the skin. MicroRNAs play a role in varied biological process and have been reported as potential biomarkers for aGvHD. More recently, microRNAs have received added attention as circulatory biomarkers that can be detected in biofluids. In this study, we performed global microRNA expression profiling using a discovery cohort of diagnostic cutaneous aGvHD biopsies (n = 5, stages 1-3) and healthy volunteers (n = 4), in order to identify a signature list of microRNAs that could be used as diagnostic biomarkers for cutaneous aGvHD. Candidate microRNAs (n = 8) were then further investigated in a validation cohort of post-HSCT skin biopsies (n = 17), pre-HSCT skin biopsies (n = 6) and normal controls (n = 6) for their association with aGvHD. Expression of let-7c (p = 0.014), miR-503-5p (p = 0.003), miR-365a-3p (p = 0.02), miR-34a-5p (p < 0.001) and miR-34a-3p (p = 0.006) were significantly differentially expressed between groups and significantly associated with survival outcome in post-HSCT patients (miR-503-5p ROC AUC = 0.83 p = 0.021, Log Rank p = 0.003; miR-34a-3p ROC AUC = 0.93, p = 0.003, Log Rank p = 0.004). There was no association with relapse. A statistical interaction between miR-34a-3p and miR-503-5p (p = 0.016) was diagnostic for aGvHD. Expression levels of the miR-34a-5p protein target p53 were assessed in the epidermis of the skin, and an inverse correlation was identified (r2 = 0.44, p = 0.039). Expression of the validated candidate microRNAs was also assessed at day 28 post-HSCT in the sera of transplant recipients, in order to investigate their potential as circulatory microRNA biomarkers. Expression of miR-503-5p (p = 0.001), miR-34a-5p (p = 0.005), and miR-34a-3p (p = 0.004) was significantly elevated in the sera of patients who developed aGvHD versus no-aGvHD (n = 30) and miR-503-5p was associated with overall survival (OS) (ROC AUC = 0.80, p = 0.04, Log Rank p = 0.041). In conclusion, this investigation reports that microRNA expression levels in clinical skin biopsies, obtained at the time of cutaneous aGvHD onset, show potential as diagnostic biomarkers for aGvHD and as predictive biomarkers for OS. In addition, the same microRNAs can be detected in the circulation and show predictive association with post-HSCT outcomes.
RESUMO
Acute graft-versus-host disease (aGvHD) is a major cause of adverse outcome in hematopoietic stem cell transplantation (HSCT), with a high incidence (20-50%). A novel, non-invasive diagnostic test to predict for prevalence and severity would enable improved prophylaxis and reduce morbidity. Circulatory microRNAs (miRNAs) miR-423, miR-199, miR-93*, and miR-377 have previously been associated with aGvHD in post-HSCT patient plasma, but validation is lacking and their expression within extracellular vesicles (EVs) has not been explored. This study replicated elevated serum expression of miR-423 (p < 0.001), miR-199 (p = 0.04), miR-93* (p < 0.001), and miR-377 (p = 0.03) in aGvHD, using a prognostic cohort of day 14 (D14) post-HSCT patient samples (n = 81). Expression also associated with disease severity. Further analysis at aGvHD diagnosis in an independent cohort (n = 65) confirmed high miR-423 (p = 0.02), miR-199 (p = 0.007), and miR-93* (p = 0.004) expression at disease onset. Investigation of expression patterns during early HSCT sequential timepoints (pre-HSCT to D28) identified elevated miRNAs at D7 post-HSCT in all transplant patients. In a novel investigation of miRNA expression in serum EVs (n = 15), miR-423 (p = 0.09), miR-199 (p = 0.008), and miR-93* (p = 0.001) levels were lower at D14 in patients who later developed aGvHD, and this was replicated for miR-423 (p = 0.02) and miR-199 (p = 0.04) (n = 47). Comparing serum to circulating EVs, at D14 patients remaining aGvHD-free had higher expression of miR-423 (p = 0.03), miR-199 (p = 0.009), and miR-93* (p = 0.002) in the EV fraction. Results verify the capacity for circulating miR-423, miR-199, and miR-93* as diagnostic and prognostic aGvHD biomarkers. The novel finding of their differential expression in EVs suggests a potential role in aGvHD etiology.
RESUMO
The outcome of hematopoietic stem cell transplantation (HSCT) is controlled by genetic factors among which the leukocyte antigen human leukocyte antigen (HLA) matching is most important. In addition, minor histocompatibility antigens and non-HLA gene polymorphisms in genes controlling immune responses are known to contribute to the risks associated with HSCT. Besides single-nucleotide polymorphisms (SNPs) in protein coding genes, SNPs in regulatory elements such as microRNAs (miRNAs) contribute to these genetic risks. However, genetic risks require for their realization the expression of the respective gene or miRNA. Thus, gene and miRNA expression studies may help to identify genes and SNPs that indeed affect the outcome of HSCT. In this review, we summarize gene expression profiling studies that were performed in recent years in both patients and animal models to identify genes regulated during HSCT. We discuss SNP-mRNA-miRNA regulatory networks and their contribution to the risks associated with HSCT in specific examples, including forkheadbox protein 3 and regulatory T cells, the role of the miR-155 and miR-146a regulatory network for graft-versus-host disease, and the function of MICA and its receptor NKG2D for the outcome of HSCT. These examples demonstrate how SNPs affect expression or function of proteins that modulate the alloimmune response and influence the outcome of HSCT. Specific miRNAs targeting these genes and directly affecting expression of mRNAs are identified. It might be valuable in the future to determine SNPs and to analyze miRNA and mRNA expression in parallel in cohorts of HSCT patients to further elucidate genetic risks of HSCT.