Project description:This SuperSeries is composed of the following subset Series: GSE3886: Scleroderma Morphea Normal Fibroblasts GSE3887: Scleroderma Architecture Cell Lines Abstract: We used DNA microarrays representing >12,000 human genes to characterize gene expression patterns in skin biopsies from individuals with a diagnosis of systemic sclerosis with diffuse scleroderma. We found consistent differences in the patterns of gene expression between skin biopsies from individuals with scleroderma and those from normal, unaffected individuals. The biopsies from affected individuals showed nearly indistinguishable patterns of gene expression in clinically affected and clinically unaffected tissue, even though these were clearly distinguishable from the patterns found in similar tissue from unaffected individuals. Genes characteristically expressed in endothelial cells, B lymphocytes, and fibroblasts showed differential expression between scleroderma and normal biopsies. Analysis of lymphocyte populations in scleroderma skin biopsies by immunohistochemistry suggest the B lymphocyte signature observed on our arrays is from CD20+ B cells. These results provide evidence that scleroderma has systemic manifestations that affect multiple cell types and suggests genes that could be used as potential markers for the disease Refer to individual Series
Project description:Fibroblast were isolated from either back or forearm punch biopsies taken by Dr. Kari Connolly(UCSF) from either normal control patients, patients with a diagnosis of Systemic Sclerosis with Diffuse Scleroderma and patients with a diagnosis of morphea. Scleroderma fibroblast were grown to confluence, media was changed and the RNA prepared from the cells 48 hrs later (to avoid serum responsive genes).
Project description:Fibroblast were isolated from either back or forearm punch biopsies taken by Dr. Kari Connolly(UCSF) from either normal control patients, patients with a diagnosis of Systemic Sclerosis with Diffuse Scleroderma and patients with a diagnosis of morphea. Scleroderma fibroblast were grown to confluence, media was changed and the RNA prepared from the cells 48 hrs later (to avoid serum responsive genes). A disease state experiment design type is where the state of some disease such as infection, pathology, syndrome, etc is studied. User Defined
Project description:Fibroblast were isolated from either back or forearm punch biopsies taken by Dr. Kari Connolly(UCSF) from either normal control patients, patients with a diagnosis of Systemic Sclerosis with Diffuse Scleroderma and patients with a diagnosis of morphea. Scleroderma fibroblast were grown to confluence, media was changed and the RNA prepared from the cells 48 hrs later (to avoid serum responsive genes). A disease state experiment design type is where the state of some disease such as infection, pathology, syndrome, etc is studied. Keywords: disease_state_design
Project description:We performed a comparative transcriptomics study of murine immune, fibrotic and skin gene expression profiles in subcutaneous bleomycin injection to determine whether this model mirrors human morphea (localized scleroderma).
Project description:In this study we compared 14 healthy and 14 LS samples evaluated by 10X Genomics single cell sequencing. Samples were processed and clustered for DEG analysis. LS = Localized Scleroderma, also called Morphea
Project description:The data from these cell lines is described in figure 2 of the mansucript "Systemic and cell-type specific gene expression patterns in scleroderam skin" by Whitfield et al. (2003) PNAS. Aortic smooth muscle cells and microvascular endothelial cells were obtained from Clonetics and cultured in the recommended media; Hs578T myofibroblast-like cells were grown in RPMI-1640 supplemented with phenol red, glutamine (2 mM) and 5% fetal calf serum; HeLa S3 epithelial cells were obtained from the American Type Culture Collection (ATCC) and cultured as described in Whitfield et al., MBC, (2002). Normal, morphea and scleroderma fibroblasts were derived from normal, morphea and scleroderma skin biopsies taken by Dr. Kari Connolly at UCSF. In all cases, RNA was prepared from cells 48 hrs after changing the culture media to avoid the induction of serum responsive genes. Poly(A) RNA from each cell line was reverse-transcribed into Cy5-dUTP (Amersham Pharmacia Biotech, Piscataway NJ) labeled cDNA and reference RNA reverse-transcribed into Cy3-dUTP (Amersham Pharmacia Biotech, Piscataway NJ) labeled cDNA using standard methods. Cells grown in culture were analyzed on cDNA microarrays containing 40,512 elements representing 28,384 UniGene clusters (UniGene Build No. 155, released 9-28-2002) manufactured in the Stanford Microarray Facility (http://www.microarray.org). Equal amounts of Cy5- and Cy3-labeled cDNA were hybridized to spotted cDNA microarrays and scanned using a GenePix 4000A Scanner (Axon Instruments, Union City CA). Detailed protocols are available at http://brownlab.stanford.edu/protocols.html/. Data were extracted by superimposing a grid over each array using GenePix 3.0 software (Axon Instruments, Union City CA). Spots of poor quality, determined by visual inspection, were excluded from further analysis. A cell type comparison design experiment design type compares cells of different type for example different cell lines. Computed
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Localized scleroderma (LoS), or morphea, refers to a group of rare autoimmune connective tissue diseases. Autologous fat grafting was able to correct volume loss in patients with LoS to improve facial disfigurement.However, whether it could exert a positive effect on reversing skin sclerosis remains unclear.