To investigate clinical features of cutaneous sarcoidosis.A retrospective analysis was carried out based on the clinic data of 35 patients with cutaneous sarcoidosis who were hospitalized in Peking Union Medical College Hospital during 1980 - 2009. They were divided into two groups, the group without systemic involvement (skin group) and the group with systemic involvement (systemic group).(1) The ratio of men and women with cutaneous sarcoidosis was 1:3.38, and the average incident age was (47.5 ± 10.0) years old. The average incident age of skin group and systemic group were (41.8 ± 12.5) years old and (50.5 ± 7.1) years old, respectively. (2) The most common skin manifestation was subcutaneous nodule, followed by maculopapular and erythema nodosa. The most common involved sites were limbs. (3) The common involved systems extra-skin included the lung, joints and lymph nodes. The involvement rate of lung in cutaneous sarcoidosis of our present data was lower than those of foreign reports. However, the involvement rates of joints, lymph nodes, kidney, muscles and nervous system showed higher in our data. (4) The incidences of fatigue and weight loss in systemic group were higher than those in skin group (P < 0.05). The indexes of erythrocyte sedimentation rate, C-reactive protein and rheumatoid factor in systemic group were higher than those in skin group (P < 0.05). More patients in systemic group were treated with corticosteroid than that in skin group (95.7% vs 66.7%, P < 0.05).Subcutaneous nodules are the most common and the involvement rate of lung is lower in cutaneous sarcoidosis of our present data. Compared to the patients without systemic involvement, the average incident age of systemic ones is older, the indexes of inflammation markers and the usage of corticosteroid are higher.
The contribution and regulation of various CD4 + T cell lineages that occur with remitting vs progressive courses in sarcoidosis are poorly understood. We developed a multiparameter flow cytometry panel to sort these CD4 + T cell lineages followed by measurement of their functional potential using RNA-sequencing analysis at six-month intervals across multiple study sites. To obtain good quality RNA for sequencing, we relied on chemokine receptor expression to identify and sort lineages. To minimize gene expression changes induced by perturbations of T cells and avoid protein denaturation caused by freeze/thaw cycles, we optimized our protocols using freshly isolated samples at each study site. To accomplish this study, we had to overcome significant standardization challenges across multiple sites. Here, we detail standardization considerations for cell processing, flow staining, data acquisition, sorting parameters, and RNA quality control analysis that were performed as part of the NIH-sponsored, multi-center study, BRonchoscopy at Initial sarcoidosis diagnosis Targeting longitudinal Endpoints (BRITE). After several rounds of iterative optimization, we identified the following aspects as critical for successful standardization: 1) alignment of PMT voltages across sites using CS&T/rainbow bead technology; 2) a single template created in the cytometer program that was used by all sites to gate cell populations during data acquisition and cell sorting; 3) use of standardized lyophilized flow cytometry staining cocktails to reduce technical error during processing; 4) development and implementation of a standardized Manual of Procedures. After standardization of cell sorting, we were able to determine the minimum number of sorted cells necessary for next generation sequencing through analysis of RNA quality and quantity from sorted T cell populations. Overall, we found that implementing a multi-parameter cell sorting with RNA-seq analysis clinical study across multiple study sites requires iteratively tested standardized procedures to ensure comparable and high-quality results.
A subset of beryllium-exposed workers develop beryllium sensitisation (BeS) which precedes chronic beryllium disease (CBD). We conducted an in-depth analysis of differentially expressed candidate genes in CBD. We performed Affymetrix GeneChip 1.0 ST array analysis on peripheral blood mononuclear cells (PBMCs) from 10 CBD, 10 BeS and 10 beryllium-exposed, nondiseased controls stimulated with BeSO 4 or medium. The differentially expressed genes were validated by high-throughput real-time PCR in this group and in an additional group of cases and nonexposed controls. The functional roles of the top candidate genes in CBD were assessed using a pharmacological inhibitor. CBD gene expression data were compared with whole blood and lung tissue in sarcoidosis from the Gene Expression Omnibus. We confirmed almost 450 genes that were significantly differentially expressed between CBD and controls. The top enrichment of genes was for JAK (Janus kinase)–STAT (signal transducer and activator of transcription) signalling. A JAK2 inhibitor significantly decreased tumour necrosis factor-α and interferon-γ production. Furthermore, we found 287 differentially expressed genes overlapped in CBD/sarcoidosis. The top shared pathways included cytokine–cytokine receptor interactions, and Toll-like receptor, chemokine and JAK–STAT signalling pathways. We show that PBMCs demonstrate differentially expressed gene profiles relevant to the immunnopathogenesis of CBD. CBD and sarcoidosis share similar differential expression of pathogenic genes and pathways.
Abstract Background Most phenotyping paradigms in sarcoidosis are based on expert opinion; however, no paradigm has been widely adopted because of the subjectivity in classification. We hypothesized that cluster analysis could be performed on common clinical variables to define more objective sarcoidosis phenotypes. Methods Model-based clustering was performed using the VarSelLCM R package to identify distinct phenotypes of sarcoidosis based on 29 clinical features. The Integrated Completed Likelihood (ICL) criteria were used to estimate number of clusters. To identify features associated with cluster membership, features were ranked based on variable importance scores from the VarSelLCM model, and additional univariate tests (Fisher’s exact test and one-way ANOVA) were performed using q-values correcting for multiple testing. The Wasfi severity score was also compared between clusters. Results Cluster analysis resulted in 6 sarcoidosis phenotypes. Salient characteristics for each cluster are as follows: Phenotype 1) supranormal lung function and majority Scadding stage 2/3; phenotype 2) supranormal lung function and majority Scadding stage 0/1; phenotype 3) normal lung function and split Scadding stages between 0/1 and 2/3; phenotype 4) obstructive lung function and majority Scadding stage 2/3; phenotype 5) restrictive lung function and majority Scadding stage 2/3; phenotype 6) mixed obstructive and restrictive lung function and mostly Scadding stage 4. Clusters 4,5,6 were significantly more likely to have ever been on immunosuppressive treatment and had higher Wasfi disease severity scores. Conclusions Cluster analysis produced 6 sarcoidosis phenotypes that demonstrated non-severe and severe phenotypes. Phenotypes 1,2,3 have less lung function abnormalities, a lower percentage on immunosuppressive treatment and lower Wasfi severity scores. Phenotypes 4,5,6 were characterized by lung function abnormalities, more parenchymal abnormalities, an increased percentage on immunosuppressive treatment and higher Wasfi severity scores. These data support using cluster analysis as an objective and clinically useful way to phenotype sarcoidosis subjects.
Abstract Introduction Sarcoidosis is a heterogeneous, granulomatous disease that can prove difficult to diagnose, with no accurate biomarkers of disease progression. Therefore, we profiled and integrated the DNA methylome, mRNAs, and microRNAs to identify molecular changes associated with sarcoidosis and disease progression that might illuminate underlying mechanisms of disease and potential genomic biomarkers. Methods Bronchoalveolar lavage cells from 64 sarcoidosis subjects and 16 healthy controls were used. DNA methylation was profiled on Illumina HumanMethylationEPIC arrays, mRNA by RNA-sequencing, and miRNAs by small RNA-sequencing. Linear models were fit to test for effect of diagnosis and phenotype, adjusting for age, sex, and smoking. We built a supervised multi-omics model using a subset of features from each dataset. Results We identified 46,812 CpGs, 1,842 mRNAs, and 5 miRNAs associated with sarcoidosis versus controls and 1 mRNA, SEPP1 - a protein that supplies selenium to cells, associated with disease progression. Our integrated model emphasized the prominence of the PI3K/AKT1 pathway in sarcoidosis, which is important in T cell and mTOR function. Novel immune related genes and miRNAs including LYST, RGS14, SLFN12L , and hsa-miR-199b-5p, distinguished sarcoidosis from controls. Our integrated model also demonstrated differential expression/methylation of IL20RB, ABCC11, SFSWAP, AGBL4 , miR-146a-3p, and miR-378b between non-progressive and progressive sarcoidosis. Conclusions Leveraging the DNA methylome, transcriptome, and miRNA-sequencing in sarcoidosis BAL cells, we detected widespread molecular changes associated with disease, many which are involved in immune response. These molecules may serve as diagnostic/prognostic biomarkers and/or drug targets, although future testing will be required for confirmation.
Chronic beryllium disease is a granulomatous lung disease, characterized by the accumulation of macrophages and beryllium-specific CD4+ T-cells that proliferate and produce T-helper cell type 1 cytokines. Previous studies indicate that beryllium-mediated oxidative stress enhances cytokine response in chronic beryllium disease CD4+ T-cells. 5-Aminosalicylic acid (5-ASA) is currently used to treat inflammatory bowel disease and has both antioxidant and antiinflammatory actions. We hypothesized that 5-ASA therapy may be a beneficial therapy in chronic beryllium disease. An investigator-initiated, randomized, double-blind, placebo-controlled 5-ASA trial was undertaken. Patients with chronic beryllium disease were randomized 3:1 to receive 5-ASA (500-mg capsules) four times daily (n = 13) or placebo capsules (n = 5), for 6 weeks. Primary study endpoints included changes in beryllium lymphocyte proliferation test responses in peripheral blood mononuclear cells and bronchoalveolar lavage cells. Secondary endpoints included changes in bronchoalveolar lavage fluid cells, serum, and peripheral blood mononuclear cell glutathione levels, bronchoalveolar lavage cell–stimulated tumor necrosis factor-α levels, lung function, and quality of life as measured by the Short Form 36 questionnaire. 5-ASA decreased bronchoalveolar lavage cell beryllium lymphocyte proliferation test responses by 20% within the 5-ASA treatment group (P = 0.06), although this was not seen for blood beryllium lymphocyte proliferation test responses. No significant changes were observed in serum, peripheral blood mononuclear cells, bronchoalveolar lavage fluid, or bronchoalveolar lavage cell glutathione levels in either the 5-ASA or placebo treatment group. 5-ASA treatment decreased ex vivo beryllium-stimulated bronchoalveolar lavage cell tumor necrosis factor-α levels within the 5-ASA group (P = 0.06) and when compared with placebo (P = 0.04). Significant improvements were noted in quality of life measurements with 5-ASA treatment. The ability of 5-ASA to decrease bronchoalveolar lavage cell beryllium lymphocyte proliferation test responses and diminish beryllium-stimulated bronchoalveolar lavage cell tumor necrosis factor-α levels suggests that 5-ASA may impact the beryllium-specific immune response in chronic beryllium disease.Clinical trial registered with www.clinicaltrials.gov (NCT01088243).
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