RESUMO
OBJECTIVES: Four intrinsic molecular subsets (inflammatory, fibroproliferative, limited, normal-like) have previously been identified in SSc and are characterized by unique gene expression signatures and pathways. The intrinsic subsets have been linked to improvement with specific therapies. Here, we investigated associations between baseline demographics and intrinsic molecular subsets in a meta-analysis of published datasets. METHODS: Publicly available gene expression data from skin biopsies of 311 SSc patients measured by DNA microarray were classified into the intrinsic molecular subsets. RNA-sequencing data from 84 participants from the ASSET trial were used as a validation cohort. Baseline clinical demographics and intrinsic molecular subsets were tested for statistically significant associations. RESULTS: Males were more likely to be classified in the fibroproliferative subset (P = 0.0046). SSc patients who identified as African American/Black were 2.5 times more likely to be classified as fibroproliferative compared with White/Caucasian patients (P = 0.0378). ASSET participants sera positive for anti-RNA pol I and RNA pol III autoantibodies were enriched in the inflammatory subset (P = 5.8 × 10-5, P = 9.3 × 10-5, respectively), while anti-Scl-70 was enriched in the fibroproliferative subset. Mean modified Rodnan Skin Score (mRSS) was statistically higher in the inflammatory and fibroproliferative subsets compared with normal-like (P = 0.0027). The average disease duration for inflammatory subset was less than fibroproliferative and normal-like intrinsic subsets (P = 8.8 × 10-4). CONCLUSIONS: We identified multiple statistically significant differences in baseline demographics between the intrinsic subsets that may represent underlying features of disease pathogenesis (e.g. chronological stages of fibrosis) and have implications for treatments that are more likely to work in certain SSc populations.
Assuntos
Escleroderma Sistêmico , Masculino , Humanos , Escleroderma Sistêmico/patologia , Genômica , Transcriptoma , Análise de Sequência com Séries de Oligonucleotídeos , Pele/patologia , RNARESUMO
OBJECTIVE: We sought to determine histologic and gene expression features of clinical improvement in early diffuse cutaneous systemic sclerosis (dcSSc; scleroderma). METHODS: Fifty-eight forearm biopsies were evaluated from 26 individuals with dcSSc in two clinical trials. Histologic/immunophenotypic assessments of global severity, alpha-smooth muscle actin (aSMA), CD34, collagen, inflammatory infiltrate, follicles and thickness were compared with gene expression and clinical data. Support vector machine learning was performed using scleroderma gene expression subset (normal-like, fibroproliferative, inflammatory) as classifiers and histology scores as inputs. Comparison of w-vector mean absolute weights was used to identify histologic features most predictive of gene expression subset. We then tested for differential gene expression according to histologic severity and compared those with clinical improvement (according to the Combined Response Index in Systemic Sclerosis). RESULTS: aSMA was highest and CD34 lowest in samples with highest local Modified Rodnan Skin Score. CD34 and aSMA changed significantly from baseline to 52 weeks in clinical improvers. CD34 and aSMA were the strongest predictors of gene expression subset, with highest CD34 staining in the normal-like subset (p<0.001) and highest aSMA staining in the inflammatory subset (p=0.016). Analysis of gene expression according to CD34 and aSMA binarised scores identified a 47-gene fibroblast polarisation signature that decreases over time only in improvers (vs non-improvers). Pathway analysis of these genes identified gene expression signatures of inflammatory fibroblasts. CONCLUSION: CD34 and aSMA stains describe distinct fibroblast polarisation states, are associated with gene expression subsets and clinical assessments, and may be useful biomarkers of clinical severity and improvement in dcSSc.
Assuntos
Fibroblastos/fisiologia , Aprendizado de Máquina , Esclerodermia Difusa/genética , Índice de Gravidade de Doença , Actinas/metabolismo , Adulto , Antígenos CD34/metabolismo , Ensaios Clínicos como Assunto , Colágeno/metabolismo , Feminino , Antebraço , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Pele/metabolismoRESUMO
OBJECTIVE: The Scleroderma: Cyclophosphamide or Transplantation (SCOT) trial demonstrated clinical benefit of haematopoietic stem cell transplant (HSCT) compared with cyclophosphamide (CYC). We mapped PBC (peripheral blood cell) samples from the SCOT clinical trial to scleroderma intrinsic subsets and tested the hypothesis that they predict long-term response to HSCT. METHODS: We analysed gene expression from PBCs of SCOT participants to identify differential treatment response. PBC gene expression data were generated from 63 SCOT participants at baseline and follow-up timepoints. Participants who completed treatment protocol were stratified by intrinsic gene expression subsets at baseline, evaluated for event-free survival (EFS) and analysed for differentially expressed genes (DEGs). RESULTS: Participants from the fibroproliferative subset on HSCT experienced significant improvement in EFS compared with fibroproliferative participants on CYC (p=0.0091). In contrast, EFS did not significantly differ between CYC and HSCT arms for the participants from the normal-like subset (p=0.77) or the inflammatory subset (p=0.1). At each timepoint, we observed considerably more DEGs in HSCT arm compared with CYC arm with HSCT arm showing significant changes in immune response pathways. CONCLUSIONS: Participants from the fibroproliferative subset showed the most significant long-term benefit from HSCT compared with CYC. This study suggests that intrinsic subset stratification of patients may be used to identify patients with SSc who receive significant benefit from HSCT.
Assuntos
Perfilação da Expressão Gênica/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Aprendizado de Máquina , Esclerodermia Difusa/classificação , Esclerodermia Difusa/terapia , Adulto , Ciclofosfamida/uso terapêutico , Feminino , Transplante de Células-Tronco Hematopoéticas/mortalidade , Humanos , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Esclerodermia Difusa/patologia , Transcriptoma , Resultado do TratamentoRESUMO
Systemic sclerosis (SSc) is a rare systemic autoimmune disease characterized by skin and organ fibrosis. The pathogenesis of SSc and its progression are poorly understood. The SSc intrinsic gene expression subsets (inflammatory, fibroproliferative, normal-like, and limited) are observed in multiple clinical cohorts of patients with SSc. Analysis of longitudinal skin biopsies suggests that a patient's subset assignment is stable over 6-12 months. Genetically, SSc is multi-factorial with many genetic risk loci for SSc generally and for specific clinical manifestations. Here we identify the genes consistently associated with the intrinsic subsets across three independent cohorts, show the relationship between these genes using a gene-gene interaction network, and place the genetic risk loci in the context of the intrinsic subsets. To identify gene expression modules common to three independent datasets from three different clinical centers, we developed a consensus clustering procedure based on mutual information of partitions, an information theory concept, and performed a meta-analysis of these genome-wide gene expression datasets. We created a gene-gene interaction network of the conserved molecular features across the intrinsic subsets and analyzed their connections with SSc-associated genetic polymorphisms. The network is composed of distinct, but interconnected, components related to interferon activation, M2 macrophages, adaptive immunity, extracellular matrix remodeling, and cell proliferation. The network shows extensive connections between the inflammatory- and fibroproliferative-specific genes. The network also shows connections between these subset-specific genes and 30 SSc-associated polymorphic genes including STAT4, BLK, IRF7, NOTCH4, PLAUR, CSK, IRAK1, and several human leukocyte antigen (HLA) genes. Our analyses suggest that the gene expression changes underlying the SSc subsets may be long-lived, but mechanistically interconnected and related to a patients underlying genetic risk.
Assuntos
Biologia Computacional/métodos , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/imunologia , Transcriptoma/genética , Idoso , Bases de Dados Genéticas , Matriz Extracelular/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação/genética , Masculino , Pessoa de Meia-Idade , Risco , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/fisiopatologiaRESUMO
OBJECTIVE: Pulmonary arterial hypertension (PAH), a common complication of limited cutaneous systemic sclerosis (lcSSc), is associated with alterations of markers of inflammation and vascular damage in peripheral blood mononuclear cells (PBMCs). Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been implicated in autoimmune and inflammatory diseases. The goal of this study was to assess whether markers of ER stress and the UPR are present in PBMCs from lcSSc patients with PAH. METHODS: PBMCs were purified from 36 healthy controls, 32 lcSSc patients with PAH, and 34 lcSSc patients without PAH. Gene expression in healthy control PBMCs stimulated with thapsigargin was analyzed by DNA microarray. Genes were validated by quantitative real-time reverse transcription-polymerase chain reaction in PBMCs from healthy controls and lcSSc patients. RESULTS: Several ER stress/UPR genes, including BiP, activating transcription factor 4 (ATF-4), ATF-6, and a spliced form of X-box binding protein 1, were up-regulated in PBMCs from lcSSc patients, with the highest levels in patients with PAH. Thapsigargin up-regulated heat-shock proteins (HSPs) and interferon (IFN)-regulated genes in PBMCs from healthy controls. Selected HSP genes (particularly DnaJB1) and IFN-related genes were also found at significantly elevated levels in PBMCs from lcSSc patients, while IFN regulatory factor 4 expression was significantly decreased. There was a positive correlation between DnaJB1 and severity of PAH (measured by pulmonary artery pressure) (r = 0.56, P < 0.05) and between ER stress markers and interleukin-6 levels (r = 0.53, P < 0.0001) in PBMCs from lcSSc patients. CONCLUSION: This study demonstrates an association between select ER stress/UPR markers and lcSSc with PAH, suggesting that ER stress and the UPR may contribute to the altered function of circulating immune cells in lcSSc.
Assuntos
Estresse do Retículo Endoplasmático/genética , Hipertensão Pulmonar/genética , Leucócitos Mononucleares/metabolismo , Esclerodermia Limitada/genética , Resposta a Proteínas não Dobradas/genética , Hipertensão Pulmonar Primária Familiar , Regulação da Expressão Gênica , Humanos , Hipertensão Pulmonar/sangue , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/fisiopatologia , Leucócitos Mononucleares/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Esclerodermia Limitada/sangue , Esclerodermia Limitada/complicações , Esclerodermia Limitada/fisiopatologia , Índice de Gravidade de Doença , Tapsigargina/farmacologia , Regulação para CimaRESUMO
OBJECTIVE: Among individuals with systemic sclerosis (SSc) randomized to cyclophosphamide (CYC) (n = 34) or hematopoietic stem cell transplantation (HSCT) (n = 33), we examined longitudinal trends of clinical, pulmonary function, and quality of life measures while accounting for the influence of early failures on treatment comparisons. METHODS: Assuming that data were missing at random, mixed-effects regression models were used to estimate longitudinal trends for clinical measures when comparing treatment groups. Results were compared to observed means and to longitudinal trends estimated from shared parameter models, assuming that data were missing not at random. Longitudinal trends for SSc intrinsic molecular subsets defined by baseline gene expression signatures (normal-like, inflammatory, and fibroproliferative signatures) were also studied. RESULTS: Available observed means for pulmonary function tests appeared to improve over time in both arms. However, after accounting for participant loss, forced vital capacity in HSCT recipients increased by 0.77 percentage points/year but worsened by -3.70/year for CYC (P = 0.004). Similar results were found for diffusing capacity for carbon monoxide and quality of life indicators. Results for both analytic models were consistent. HSCT recipients in the inflammatory (n = 20) and fibroproliferative (n = 20) subsets had superior long-term trends compared to CYC for pulmonary and quality of life measures. HSCT was also superior for modified Rodnan skin thickness scores in the fibroproliferative subset. For the normal-like subset (n = 22), superiority of HSCT was less apparent. CONCLUSION: Longitudinal trends estimated from 2 statistical models affirm the efficacy of HSCT over CYC in severe SSc. Failure to account for early loss of participants may distort estimated clinical trends over the long term.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Esclerodermia Localizada , Escleroderma Sistêmico , Humanos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Imunossupressores/uso terapêutico , Qualidade de Vida , Transplante Autólogo , Ciclofosfamida/uso terapêutico , Escleroderma Sistêmico/diagnóstico , Escleroderma Sistêmico/tratamento farmacológico , Esclerodermia Localizada/tratamento farmacológico , Resultado do TratamentoRESUMO
Eosinophilia-myalgia syndrome (EMS) is characterized by subacute onset of myalgias and peripheral eosinophilia, followed by chronic neuropathy and skin induration. An epidemic of EMS in 1989 was linked to consumption of L-tryptophan that had originated from a single source. Following the ban by the Food and Drug Administration (FDA) on the sale of L-tryptophan, the incidence of EMS declined rapidly. Moreover, no new cases have been described since the FDA ban was lifted in 2005. We report the clinical, histopathologic, and immunogenetic features of a new case of L-tryptophan-associated EMS, along with evidence of activated transforming growth factor ß and interleukin-4 signaling in the lesional skin.
Assuntos
Síndrome de Eosinofilia-Mialgia/induzido quimicamente , Triptofano/efeitos adversos , Adulto , Feminino , HumanosRESUMO
BACKGROUND: Our objective was to determine the growth kinetics of bacteria in leukoreduced apheresis platelets (LR-AP) in a platelet (PLT) additive solution (PAS; InterSol, Fenwal, Inc.) compared to LR-AP stored in plasma. STUDY DESIGN AND METHODS: Hyperconcentrated, double-dose LR-AP were collected from healthy donors with a separator (AMICUS, Fenwal, Inc.). LR-AP were evenly divided, InterSol was added to half (65% InterSol:35% plasma [PAS]), and PLTs in autologous plasma were used for a paired control (PL). Bacteria were inoculated into each LR-AP PAS/PL pair (0.5-1.6 colony-forming units [CFUs]/mL), and bacterial growth was followed for up to 7 days. Time to the end of the lag phase, doubling times, maximum concentration (conc-max), and time to maximum concentration (time-max) were estimated. RESULTS: Streptococcus viridans did not grow to detectable levels in either PAS or PL units. The other bacteria had no significant overall difference in the conc-max (p = 0.47) or time-max (p = 0.7) between PL and PAS LR-AP; PL had a 0.14 hours faster doubling rate (p = 0.023); and PAS had a 4.7 hours shorter lag time (p = 0.016). CONCLUSION: We observed that five index organisms will grow in LR-AP stored in a 35%:65% ratio of plasma to InterSol where initial bacterial concentrations are 0.5 to 1.6 CFUs/mL. The more rapid initiation of log-phase growth for bacteria within a PAS storage environment resulted in a bacterial concentration up to 4 logs higher in the PAS units compared to the plasma units at 24 hours, but with no difference in the conc-max. This may present an early bacterial detection advantage for PAS-stored PLTs.
Assuntos
Plaquetas/microbiologia , Preservação de Sangue/métodos , Plaquetoferese , Infecções Estafilocócicas/sangue , Staphylococcus epidermidis/crescimento & desenvolvimento , Preservação de Sangue/efeitos adversos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/sangue , Humanos , Infecções por Klebsiella/sangue , Klebsiella oxytoca/crescimento & desenvolvimento , Klebsiella oxytoca/isolamento & purificação , Plasma/microbiologia , Infecções por Serratia/sangue , Serratia marcescens/crescimento & desenvolvimento , Serratia marcescens/isolamento & purificação , Soluções , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/isolamento & purificação , Staphylococcus epidermidis/isolamento & purificação , Infecções Estreptocócicas/sangue , Estreptococos Viridans/crescimento & desenvolvimento , Estreptococos Viridans/isolamento & purificaçãoRESUMO
Systemic sclerosis (SSc) is a heterogeneous autoimmune disorder that results in skin fibrosis, autoantibody production, and internal organ dysfunction. We previously identified 4 "intrinsic" subsets of SSc based upon skin gene expression that are found across organ systems. Gene expression regulators that underlie the SSc-intrinsic subsets, or are associated with clinical covariates, have not been systematically characterized. Here, we present a computational framework to calculate the activity scores of gene expression regulators and identify their associations with SSc clinical outcomes. We found that regulator activity scores can reproduce the intrinsic molecular subsets, with distinct sets of regulators identified for inflammatory, fibroproliferative, limited, and normal-like samples. Regulators most highly correlated with modified Rodnan skin score (MRSS) also varied by intrinsic subset. We identified subgroups of patients with fibroproliferative and inflammatory SSc with more severe pathophenotypes, such as higher MRSS and increased likelihood of interstitial lung disease (ILD). Using an independent cohort, we show that the group with more severe ILD was more likely to show forced vital capacity decline over a period of 36-54 months. Our results demonstrate an association among the activation of regulators, gene expression subsets, and clinical variables that can identify patients with SSc with more severe disease.
Assuntos
Biologia Computacional/métodos , Redes Reguladoras de Genes , Fibrose Pulmonar/genética , Escleroderma Sistêmico/genética , Algoritmos , Biomarcadores/metabolismo , Humanos , Fibrose Pulmonar/complicações , Fibrose Pulmonar/patologia , Escleroderma Sistêmico/complicações , Escleroderma Sistêmico/patologia , TranscriptomaRESUMO
OBJECTIVE: High-throughput gene expression profiling of tissue samples from patients with systemic sclerosis (SSc) has identified 4 "intrinsic" gene expression subsets: inflammatory, fibroproliferative, normal-like, and limited. Prior methods required agglomerative clustering of many samples. In order to classify individual patients in clinical trials or for diagnostic purposes, supervised methods that can assign single samples to molecular subsets are required. We undertook this study to introduce a novel machine learning classifier as a robust accurate intrinsic subset predictor. METHODS: Three independent gene expression cohorts were curated and merged to create a data set covering 297 skin biopsy samples from 102 unique patients and controls, which was used to train a machine learning algorithm. We performed external validation using 3 independent SSc cohorts, including a gene expression data set generated by an independent laboratory on a different microarray platform. In total, 413 skin biopsy samples from 213 individuals were analyzed in the training and testing cohorts. RESULTS: Repeated cross-fold validation identified consistent and discriminative markers using multinomial elastic net, performing with an average classification accuracy of 87.1% with high sensitivity and specificity. In external validation, the classifier achieved an average accuracy of 85.4%. Reanalyzing data from a previous study, we identified subsets of patients that represent the canonical inflammatory, fibroproliferative, and normal-like subsets. CONCLUSION: We developed a highly accurate classifier for SSc molecular subsets for individual patient samples. The method can be used in SSc clinical trials to identify an intrinsic subset on individual samples. Our method provides a robust data-driven approach to aid clinical decision-making and interpretation of heterogeneous molecular information in SSc patients.
Assuntos
Escleroderma Sistêmico/classificação , Aprendizado de Máquina Supervisionado , Transcriptoma , Adulto , Idoso , Algoritmos , Conjuntos de Dados como Assunto , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , Escleroderma Sistêmico/genética , Adulto JovemRESUMO
BACKGROUND: Infectious agents have long been postulated to be disease triggers for systemic sclerosis (SSc), but a definitive link has not been found. Metagenomic analyses of high-throughput data allows for the unbiased identification of potential microbiome pathogens in skin biopsies of SSc patients and allows insight into the relationship with host gene expression. METHODS: We examined skin biopsies from a diverse cohort of 23 SSc patients (including lesional forearm and non-lesional back samples) by RNA-seq. Metagenomic filtering and annotation was performed using the Integrated Metagenomic Sequencing Analysis (IMSA). Associations between microbiome composition and gene expression were analyzed using single-sample gene set enrichment analysis (ssGSEA). RESULTS: We find the skin of SSc patients exhibits substantial changes in microbial composition relative to controls, characterized by sharp decreases in lipophilic taxa, such as Propionibacterium, combined with increases in a wide range of gram-negative taxa, including Burkholderia, Citrobacter, and Vibrio. CONCLUSIONS: Microbiome dysbiosis is associated with disease duration and increased inflammatory gene expression. These data provide a comprehensive portrait of the SSc skin microbiome and its association with local gene expression, which mirrors the molecular changes in lesional skin.
Assuntos
Disbiose/genética , Inflamação/genética , Microbiota/genética , Escleroderma Sistêmico/genética , Pele/metabolismo , Adulto , Idoso , Bactérias/classificação , Bactérias/genética , Biópsia , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação/microbiologia , Inflamação/patologia , Masculino , Metagenômica/métodos , Pessoa de Meia-Idade , Dinâmica Populacional , Escleroderma Sistêmico/microbiologia , Escleroderma Sistêmico/patologia , Pele/microbiologia , Pele/patologia , Fatores de TempoRESUMO
BACKGROUND: The current prevalence of hospital-acquired infections and evolving amplification of bacterial resistance are major public health concerns. A heightened awareness of intraoperative transmission of potentially pathogenic bacterial organisms may lead to implementation of effective preventative measures. METHODS: Sixty-one operative suites were randomly selected for analysis. Sterile intravenous stopcock sets and two sites on the anesthesia machine were decontaminated and cultured aseptically at baseline and at case completion. The primary outcome was the presence of a positive culture on the previously sterile patient stopcock set. Secondary outcomes were the number of colonies per surface area sampled on the anesthesia machine, species identification, and antibiotic susceptibility of isolated organisms. RESULTS: Bacterial contamination of the anesthesia work area increased significantly at the case conclusion, with a mean difference of 115 colonies per surface area sampled (95% confidence interval [CI], 62-169; P < 0.001). Transmission of bacterial organisms, including vancomycin-resistant enterococcus, to intravenous stopcock sets occurred in 32% (95% CI, 20.6-44.9%) of cases. Highly contaminated work areas increased the odds of stopcock contamination by 4.7 (95% CI, 1.42-15.42; P = 0.011). Contaminated intravenous tubing was associated with a trend toward increased nosocomial infection rates (odds ratio, 3.08; 95% CI, 0.56-17.5; P = 0.11) and with an increase in mortality (95% CI odds ratio, 1.11-infinity; P = 0.0395). CONCLUSION: Potentially pathogenic, multidrug-resistant bacterial organisms are transmitted during the practice of general anesthesia to both the anesthesia work area and intravenous stopcock sets. Implementation of infection control measures in this area may help to reduce both the evolving problem of increasing bacterial resistance and the development of life-threatening infectious complications.
Assuntos
Anestesia Geral/instrumentação , Bactérias/isolamento & purificação , Infecções Bacterianas/transmissão , Infecção Hospitalar/microbiologia , Contaminação de Equipamentos , Salas Cirúrgicas , Adulto , Idoso , Anestesia Geral/efeitos adversos , Anestesia Intravenosa/efeitos adversos , Anestesia Intravenosa/instrumentação , Contagem de Colônia Microbiana , Infecção Hospitalar/diagnóstico , Infecção Hospitalar/mortalidade , Eletroforese em Gel de Campo Pulsado , Contaminação de Equipamentos/estatística & dados numéricos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Projetos Piloto , Medição de RiscoRESUMO
OBJECTIVE: To assess the safety and efficacy of treatment with belimumab in patients with early diffuse cutaneous systemic sclerosis (dcSSc) treated with background mycophenolate mofetil (MMF). METHODS: In this 52-week, investigator-initiated, single-center, double-blind, placebo-controlled, pilot study, 20 patients with dcSSc recently started on MMF were randomized 1:1 to additionally receive belimumab at 10 mg/kg intravenously or placebo. We assessed safety, efficacy, and differential gene expression. RESULTS: In the belimumab group, the median modified Rodnan skin thickness score (MRSS) decreased from 27 (interquartile range [IQR] 26.5, 31) to 18 (IQR 11, 23) (P = 0.039). In the placebo group, the median MRSS decreased from 28 (IQR 22, 28) to 21 (IQR 14, 25) (P = 0.023). The median change in MRSS was -10 (IQR -13, -9) in the belimumab group and -3.0 (IQR -15, -1) in the placebo group (P = 0.411). There were no significant differences between the groups in the number of adverse events (AEs). A significant decrease in expression of B cell signaling and profibrotic genes and pathways was observed in patients with improved MRSS in the belimumab group but not in the placebo group. CONCLUSION: Patients in both treatment groups experienced significant improvements in MRSS. The median difference was greater in the belimumab group but did not achieve statistical significance in this small pilot study. AEs were similar between the groups. Changes in gene expression were consistent with mechanism of action and showed that clinical response to treatment with belimumab is associated with a significant decrease in profibrotic genes and pathways. Additional studies are needed to determine the role of belimumab in the treatment of dcSSc.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Imunossupressores/uso terapêutico , Esclerodermia Difusa/tratamento farmacológico , Adulto , Idoso , Anticorpos Monoclonais Humanizados/efeitos adversos , Método Duplo-Cego , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Imunossupressores/efeitos adversos , Masculino , Pessoa de Meia-Idade , Ácido Micofenólico/uso terapêutico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Projetos Piloto , Esclerodermia Difusa/genética , Índice de Gravidade de Doença , Pele/patologia , Resultado do Tratamento , Escala Visual AnalógicaRESUMO
Fewer than half of patients with systemic sclerosis demonstrate modified Rodnan skin score improvement during mycophenolate mofetil (MMF) treatment. To understand the molecular basis for this observation, we extended our prior studies and characterized molecular and cellular changes in skin biopsies from subjects with systemic sclerosis treated with MMF. Eleven subjects completed ≥24 months of MMF therapy. Two distinct skin gene expression trajectories were observed across six of these subjects. Three of the six subjects showed attenuation of the inflammatory signature by 24 months, paralleling reductions in CCL2 mRNA expression in skin and reduced numbers of macrophages and myeloid dendritic cells in skin biopsies. MMF cessation at 24 months resulted in an increased inflammatory score, increased CCL2 mRNA and protein levels, modified Rodnan skin score rebound, and increased numbers of skin myeloid cells in these subjects. In contrast, three other subjects remained on MMF >24 months and showed a persistent decrease in inflammatory score, decreasing or stable modified Rodnan skin score, CCL2 mRNA reductions, sera CCL2 protein levels trending downward, reduction in monocyte migration, and no increase in skin myeloid cell numbers. These data summarize molecular changes during MMF therapy that suggest reduction of innate immune cell numbers, possibly by attenuating expression of chemokines, including CCL2.
Assuntos
Imunossupressores/uso terapêutico , Ácido Micofenólico/uso terapêutico , Células Mieloides/efeitos dos fármacos , Escleroderma Sistêmico/tratamento farmacológico , Adulto , Biópsia , Estudos de Casos e Controles , Contagem de Células , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Imunossupressores/farmacologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Ácido Micofenólico/farmacologia , Células Mieloides/imunologia , Estudos Prospectivos , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/patologia , Pele/citologia , Pele/efeitos dos fármacos , Pele/imunologia , Pele/patologia , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologia , Resultado do TratamentoRESUMO
BACKGROUND: Systemic sclerosis (SSc) is a multi-organ autoimmune disease characterized by skin fibrosis. Internal organ involvement is heterogeneous. It is unknown whether disease mechanisms are common across all involved affected tissues or if each manifestation has a distinct underlying pathology. METHODS: We used consensus clustering to compare gene expression profiles of biopsies from four SSc-affected tissues (skin, lung, esophagus, and peripheral blood) from patients with SSc, and the related conditions pulmonary fibrosis (PF) and pulmonary arterial hypertension, and derived a consensus disease-associate signature across all tissues. We used this signature to query tissue-specific functional genomic networks. We performed novel network analyses to contrast the skin and lung microenvironments and to assess the functional role of the inflammatory and fibrotic genes in each organ. Lastly, we tested the expression of macrophage activation state-associated gene sets for enrichment in skin and lung using a Wilcoxon rank sum test. RESULTS: We identified a common pathogenic gene expression signature-an immune-fibrotic axis-indicative of pro-fibrotic macrophages (MØs) in multiple tissues (skin, lung, esophagus, and peripheral blood mononuclear cells) affected by SSc. While the co-expression of these genes is common to all tissues, the functional consequences of this upregulation differ by organ. We used this disease-associated signature to query tissue-specific functional genomic networks to identify common and tissue-specific pathologies of SSc and related conditions. In contrast to skin, in the lung-specific functional network we identify a distinct lung-resident MØ signature associated with lipid stimulation and alternative activation. In keeping with our network results, we find distinct MØ alternative activation transcriptional programs in SSc-associated PF lung and in the skin of patients with an "inflammatory" SSc gene expression signature. CONCLUSIONS: Our results suggest that the innate immune system is central to SSc disease processes but that subtle distinctions exist between tissues. Our approach provides a framework for examining molecular signatures of disease in fibrosis and autoimmune diseases and for leveraging publicly available data to understand common and tissue-specific disease processes in complex human diseases.
Assuntos
Redes Reguladoras de Genes , Escleroderma Sistêmico/genética , Transcriptoma , Biópsia , Esôfago/metabolismo , Fibrose , Humanos , Leucócitos Mononucleares/metabolismo , Pulmão/metabolismo , Especificidade de Órgãos , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Pele/metabolismoRESUMO
BACKGROUND: Systemic sclerosis (SSc; scleroderma) is an uncommon autoimmune rheumatic disease characterised by autoimmunity, vasculopathy and fibrosis. Gene expression profiling distinguishes scleroderma from normal skin, and can detect different subsets of disease, with potential to identify prognostic biomarkers of organ involvement or response to therapy. We have performed gene expression profiling in skin samples from patients with limited cutaneous SSc (lcSSc). METHODS: Total RNA was extracted from clinically uninvolved skin biopsies of 15 patients with lcSSc and 8 healthy controls (HC). Gene expression profiling was performed on a DNA oligonucleotide microarray chip. Differentially expressed genes (DEG) were identified using significance analysis of microarrays (SAM). Functional enrichment analysis of gene signatures was done via g:Profiler. RESULTS: There were 218 DEG between lcSSc and HC samples (false discovery rate <10%): 181/218 DEG were upregulated in lcSSc samples. Hierarchical clustering of DEG suggested the presence of two separate groups of lcSSc samples: "limited 1" and "limited 2". The limited-1 group (13 samples, 10 unique patients) showed upregulation of genes involved in cell adhesion, cardiovascular system (CVS) development, extracellular matrix and immune and inflammatory response. The CVS development signature was of particular interest as its genes showed very strong enrichment in response to wounding, response to transforming growth factor (TGF)-ß and kinase cascade. Neither limited-2 samples (six samples, five unique patients) nor HC samples showed functional enrichment. There were no significant differences in demographic or clinical parameters between these two groups. These results were confirmed using a second independent cohort. CONCLUSIONS: Our study suggests the presence of molecular subsets in lcSSc based on gene expression profiling of biopsies from uninvolved skin. This may reflect important differences in pathogenesis within these patient groups. We identify differential expression of a subset of genes that relate to CVS and are enriched in fibrotic signalling. This may shed light on mechanisms of vascular disease in SSc. The enrichment in profibrotic profile suggests that dysregulated gene expression may contribute to vasculopathy and fibrosis in different disease subsets.
Assuntos
Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/genética , Perfilação da Expressão Gênica/métodos , Escleroderma Sistêmico/diagnóstico , Escleroderma Sistêmico/genética , Pele/patologia , Adulto , Idoso , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: Accumulation of myofibroblasts in fibrotic skin is a hallmark of systemic sclerosis (SSc; scleroderma), but the origins of these cells remain unknown. Because loss of intradermal adipose tissue is a consistent feature of cutaneous fibrosis, we sought to examine the hypothesis that myofibroblasts populating fibrotic dermis derive from adipocytic progenitors. METHODS: We performed genetic fate mapping studies to investigate the loss of intradermal adipose tissue and its potential role in fibrosis in mice with bleomycin-induced scleroderma. Modulation of adipocytic phenotypes ex vivo was investigated in adipose tissue-derived cells in culture. RESULTS: A striking loss of intradermal adipose tissue and its replacement with fibrous tissue were consistently observed in mice with bleomycin-induced fibrosis. Loss of adipose tissue and a decline in the expression of canonical adipogenic markers in lesional skin preceded the onset of dermal fibrosis and expression of fibrogenic markers. Ex vivo, subcutaneous adipocytes were driven by transforming growth factor ß to preferentially undergo fibrogenic differentiation. Cell fate mapping studies in mice with the adiponectin promoter-driven Cre recombinase transgenic construct indicated that adiponectin-positive progenitors that are normally confined to the intradermal adipose tissue compartment were distributed throughout the lesional dermis over time, lost their adipocytic markers, and expressed myofibroblast markers in bleomycin-treated mice. CONCLUSION: These observations establish a novel link between intradermal adipose tissue loss and dermal fibrosis and demonstrate that adiponectin-positive intradermal progenitors give rise to dermal myofibroblasts. Adipose tissue loss and adipocyte-myofibroblast transition might be primary events in the pathogenesis of cutaneous fibrosis that represent novel potential targets for therapeutic intervention.
Assuntos
Adiponectina/metabolismo , Linhagem da Célula , Miofibroblastos/patologia , Escleroderma Sistêmico/patologia , Dermatopatias/patologia , Pele/patologia , Adipócitos/metabolismo , Adipócitos/patologia , Animais , Bleomicina , Modelos Animais de Doenças , Fibrose/metabolismo , Fibrose/patologia , Camundongos , Miofibroblastos/metabolismo , Escleroderma Sistêmico/induzido quimicamente , Escleroderma Sistêmico/metabolismo , Pele/metabolismo , Dermatopatias/metabolismoRESUMO
INTRODUCTION: Tyrosine kinase inhibitors (TKI) are medications of interest in the treatment of Systemic Sclerosis (SSc) because of their ability to inhibit pathways involved in fibrosis. In this open-label pilot trial, our objectives were to assess the safety, efficacy, and molecular change associated with treatment of patients with diffuse cutaneous (dc)SSc with the TKI nilotinib (Tasigna™). METHODS: Ten adult patients with early dcSSc were treated with nilotinib. Primary endpoints were safety and change in modified Rodnan Skin Score (MRSS) after 6 months. Lesional skin biopsies at baseline, 6 and 12 months of treatment were assessed by histopathology, immunohistochemistry, and DNA microarray. RESULTS: Patients had early and active dcSSc with median disease duration of 0.7 years (range 0.5, 1.7) and increasing MRSS in the month prior to baseline (mean +2.9, p=0.02). Seven out of ten patients completed 6 and 12 months of treatment. Seventy-one adverse events (AEs) including 2 serious AEs were observed, and 92 % of AEs were grade 1-2. Two patients discontinued the medication due to mild QTc prolongation. MRSS improved by a mean of 4.2 points (16 %) at 6 months and by 6.3 points (23 %) at 12 months in the 7 completers, p=0.02 and 0.01, respectively. Patients with a decrease in MRSS >20 % from baseline at 12 months (classified as improvers) had significantly higher expression of transforming growth factor beta receptor (TGFBR) and platelet-derived growth factor receptor beta (PDGFRB) signaling genes at baseline than non-improvers, and the expression of these genes significantly decreased in improvers post-treatment. CONCLUSION: Nilotinib was well tolerated by the majority of patients in this study, with tolerability limited primarily by mild QTc-prolongation. Significant MRSS improvement was observed in these early, active patients, but is not conclusive of treatment effect given the open-label study-design and small number of patients in this pilot study. Improvers had higher levels of expression of genes associated with TGFBR and PDGFRB signaling at baseline, and a significant decrease in the expression of these genes occurred only in patients with higher MRSS improvement. The findings of this pilot study warrant more conclusive evaluation. TRIAL REGISTRATION: Clinicaltrials.gov NCT01166139 , July 1, 2010.
Assuntos
Pirimidinas/uso terapêutico , Esclerodermia Difusa/tratamento farmacológico , Pele/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Adolescente , Adulto , Idoso , Anemia/induzido quimicamente , Doenças Cardiovasculares/induzido quimicamente , Feminino , Cefaleia/induzido quimicamente , Humanos , Masculino , Pessoa de Meia-Idade , Náusea/induzido quimicamente , Análise de Sequência com Séries de Oligonucleotídeos , Projetos Piloto , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/efeitos adversos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Esclerodermia Difusa/genética , Esclerodermia Difusa/patologia , Transdução de Sinais/genética , Pele/metabolismo , Pele/patologia , Transcriptoma/genética , Resultado do Tratamento , Adulto JovemRESUMO
INTRODUCTION: Systemic sclerosis is an autoimmune disease characterized by inflammation and fibrosis of the skin and internal organs. We sought to assess the clinical and molecular effects associated with response to intravenous abatacept in patients with diffuse cutaneous systemic. METHODS: Adult diffuse cutaneous systemic sclerosis patients were randomized in a 2:1 double-blinded fashion to receive abatacept or placebo over 24 weeks. Primary outcomes were safety and the change in modified Rodnan Skin Score (mRSS) at week 24 compared with baseline. Improvers were defined as patients with a decrease in mRSS of ≥30% post-treatment compared to baseline. Skin biopsies were obtained for differential gene expression and pathway enrichment analyses and intrinsic gene expression subset assignment. RESULTS: Ten subjects were randomized to abatacept (n = 7) or placebo (n = 3). Disease duration from first non-Raynaud's symptom was significantly longer (8.8 ± 3.8 years vs. 2.4 ± 1.6 years, p = 0.004) and median mRSS was higher (30 vs. 22, p = 0.05) in the placebo compared to abatacept group. Adverse events were similar in the two groups. Five out of seven patients (71%) randomized to abatacept and one out of three patients (33%) randomized to placebo experienced ≥30% improvement in skin score. Subjects receiving abatacept showed a trend toward improvement in mRSS at week 24 (-8.6 ± 7.5, p = 0.0625) while those in the placebo group did not (-2.3 ± 15, p = 0.75). After adjusting for disease duration, mRSS significantly improved in the abatacept compared with the placebo group (abatacept vs. placebo mRSS decrease estimate -9.8, 95% confidence interval -16.7 to -3.0, p = 0.0114). In the abatacept group, the patients in the inflammatory intrinsic subset showed a trend toward greater improvement in skin score at 24 weeks compared with the patients in the normal-like intrinsic subset (-13.5 ± 3.1 vs. -4.5 ± 6.4, p = 0.067). Abatacept resulted in decreased CD28 co-stimulatory gene expression in improvers consistent with its mechanism of action. Improvers mapped to the inflammatory intrinsic subset and showed decreased gene expression in inflammatory pathways, while non-improver and placebos showed stable or reverse gene expression over 24 weeks. CONCLUSIONS: Clinical improvement following abatacept therapy was associated with modulation of inflammatory pathways in skin. TRIAL REGISTRATION: ClinicalTrials.gov NCT00442611. Registered 1 March 2007.
Assuntos
Abatacepte/uso terapêutico , Antígenos CD28/biossíntese , Expressão Gênica/efeitos dos fármacos , Imunossupressores/uso terapêutico , Esclerodermia Difusa/tratamento farmacológico , Adulto , Antígenos CD28/efeitos dos fármacos , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de OligonucleotídeosRESUMO
INTRODUCTION: Esophageal involvement in patients with systemic sclerosis (SSc) is common, but tissue-specific pathological mechanisms are poorly understood. There are no animal scleroderma esophagus models and esophageal smooth muscle cells dedifferentiate in culture prohibiting in vitro studies. Esophageal fibrosis is thought to disrupt smooth muscle function and lead to esophageal dilatation, but autopsy studies demonstrate esophageal smooth muscle atrophy and the absence of fibrosis in the majority of SSc cases. Herein, we perform a detailed characterization of SSc esophageal histopathology and molecular signatures at the level of gene expression. METHODS: Esophageal biopsies were prospectively obtained during esophagogastroduodenoscopy in 16 consecutive SSc patients and 7 subjects without SSc. Upper and lower esophageal biopsies were evaluated for histopathology and gene expression. RESULTS: Individual patient's upper and lower esophageal biopsies showed nearly identical patterns of gene expression. Similar to skin, inflammatory and proliferative gene expression signatures were identified suggesting that molecular subsets are a universal feature of SSc end-target organ pathology. The inflammatory signature was present in biopsies without high numbers of infiltrating lymphocytes. Molecular classification of esophageal biopsies was independent of SSc skin subtype, serum autoantibodies and esophagitis. CONCLUSIONS: Proliferative and inflammatory molecular gene expression subsets in tissues from patients with SSc may be a conserved, reproducible component of SSc pathogenesis. The inflammatory signature is observed in biopsies that lack large inflammatory infiltrates suggesting that immune activation is a major driver of SSc esophageal pathogenesis.