Project description:Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis; (TEN) are life-threatening adverse drug reactions characterized; by massive epidermal necrosis, in which the specific danger; signals involved remain unclear. Here we show that blister; cells from skin lesions of SJS-TEN primarily consist of cytotoxic; T lymphocytes (CTLs) and natural killer (NK) cells, and both; blister fluids and cells were cytotoxic. Gene expression profiling; identified granulysin as the most highly expressed cytotoxic; molecule, confirmed by quantitative PCR and immunohistochemistry. Granulysin concentrations in the blister fluids; were two to four orders of magnitude higher than perforin,; granzyme B or soluble Fas ligand concentrations, and depleting; granulysin reduced the cytotoxicity. Granulysin in the blister; fluids was a 15-kDa secretory form, and injection of it into; mouse skin resulted in features mimicking SJS-TEN. Our; findings demonstrate that secretory granulysin is a key molecule; responsible for the disseminated keratinocyte death in SJS-TEN; and highlight a mechanism for CTL- or NK cellâmediated; cytotoxicity that does not require direct cellular contact. Experiment Overall Design: Blood samples were obtained from 5 different patients with SJS/TEN. The peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood samples by Ficoll-Isopaque (Pharmacia Fine Chemicals) density gradient centrifugation. Total RNA from PBMC was isolated using the RNeasy kit (Qiagen). The 28S and 18S ribosomal RNA peak ratios were determined using microfluidics technology (Agilent). RNA was subjected to reverse transcription using the Superscript II kit (Invitrogen), and the cleaned cRNA was then hybridized to an Affymetrix human genome U133 plus 2.0 array.
Project description:Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening adverse drug reactions characterized by massive epidermal necrosis, in which the specific danger signals involved remain unclear. Here we show that blister cells from skin lesions of SJS-TEN primarily consist of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, and both blister fluids and cells were cytotoxic. Gene expression profiling identified granulysin as the most highly expressed cytotoxic molecule, confirmed by quantitative PCR and immunohistochemistry. Granulysin concentrations in the blister fluids were two to four orders of magnitude higher than perforin, granzyme B or soluble Fas ligand concentrations, and depleting granulysin reduced the cytotoxicity. Granulysin in the blister fluids was a 15-kDa secretory form, and injection of it into mouse skin resulted in features mimicking SJS-TEN. Our findings demonstrate that secretory granulysin is a key molecule responsible for the disseminated keratinocyte death in SJS-TEN and highlight a mechanism for CTL- or NK cell—mediated cytotoxicity that does not require direct cellular contact.
Project description:Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening adverse drug reactions characterized by massive epidermal necrosis, in which the specific danger signals involved remain unclear. Here we show that blister cells from skin lesions of SJS-TEN primarily consist of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, and both blister fluids and cells were cytotoxic. Gene expression profiling identified granulysin as the most highly expressed cytotoxic molecule, confirmed by quantitative PCR and immunohistochemistry. Granulysin concentrations in the blister fluids were two to four orders of magnitude higher than perforin, granzyme B or soluble Fas ligand concentrations, and depleting granulysin reduced the cytotoxicity. Granulysin in the blister fluids was a 15-kDa secretory form, and injection of it into mouse skin resulted in features mimicking SJS-TEN. Our findings demonstrate that secretory granulysin is a key molecule responsible for the disseminated keratinocyte death in SJS-TEN and highlight a mechanism for CTL- or NK cell—mediated cytotoxicity that does not require direct cellular contact.
Project description:Activated B cells play a key role in the pathogenesis of Sjögren’s syndrome (SjS) through the production of autoantibodies and the development of ectopic germinal centers in the salivary glands and other affected sites. Around 5-10% of SjS patients develop B-cell lymphoma, usually extranodal marginal zone lymphomas (eMZL) of the mucosa-associated lymphoid tissue (MALT). The aim of the current study is to screen for the eMZL clonotype in prediagnostic blood and tissue biopsies of SjS patients. We studied prediagnostic tissue biopsies of 3 SjS patients diagnosed with eMZL through immunoglobulin (IG) gene repertoire sequencing. In all 3 cases, we observed the eMZL clonotype in prediagnostic tissue biopsies. To evaluate if skewing in the IGH repertoire may also be detected in the peripheral blood, we sequenced a peripheral blood mononuclear cell (PBMC) sample drawn at eMZL diagnosis and 2 years prior to eMZL relapse in two SjS patients. The eMZL clonotype was detected in the peripheral blood prior to diagnosis for both cases. Next, we selected 3 SjS patients who developed eMZL lymphoma and 3 additional SjS patients who remained cancer-free. We sequenced the IG heavy chain (IGH) gene repertoire in PBMC samples taken a median of 3 years before eMZL diagnosis. In 2 out of 3 eMZL patients, the dominant clonotype in the prediagnostic PBMC samples matched the eMZL clonotype in the diagnostic biopsy. The eMZL clonotypes observed utilized stereotypic IGHV gene combinations (IGHV1-69/IGHJ4 and IGHV4-59/IGHJ5) associated with rheumatoid factor activity, a previously reported feature of eMZL in SjS. Notably, no significant abnormalities were observed in the IGH gene repertoire of the controls. In conclusion, our results indicate that immunogenetic sequencing of the IGH gene repertoire may be an effective method of early detection of eMZL in SjS patients.
Project description:The present study was aimed to identify aberrantly expressed lncRNAs involved in the progression of SjS and explore their potential functions. Labial salivary gland of 4 SjS patients and 4 healthy controls was collected. LncRNA expression profile in labial salivary gland was analyzed by LncRNA microarray.
Project description:(Objectives) The goal of this study is to investigate paracrine effect of peripheral blood mononuclear cells (PBMCs) on ovarian cancer cell lines. (Methods) Transcription profiles of PBMC (control) and PBMC co-cultured with ovarian cancer cell lines (treatment) were generated by deep sequencing, in triplicate, using Illumina HiSeq X Ten. (Results and Conclusions) Transcriptome analysis of PBMCs has revealed the expression change when co-cultured with EOC cell lines with significantly in up-regulated genes; CDKN1B, GIMAP8 and SNN. The qRT-PCR validation have indicated that GIMAP8 was high expressed in EOC patients when compared with healthy female. We summarized that PBMCs were changed their expression as a result of paracrine signals from ovarian cancer cells.
Project description:Recently, we reported the development of the C57BL/6.NOD-Aec1Aec2 mouse that carries two genetic intervals derived from the NOD mouse capable of conferring Sjogren's syndrome (SjS)-like disease in SjS-non-susceptible C57BL/6 mice. In an attempt to define the molecular bases underlying onset of stomatitis sicca (xerostomia) in this C57BL/6.NOD-Aec1Aec2 mouse model, we have carried out a study utilizing genomic microarray technology. The present study was designed to define the changing gene expression profiles within the Lacrimal glands of C57BL/6.NOD-Aec1Aec2 mice at five time points representing a pre-disease stage (4 weeks), the early pre-clinical stage (8 weeks), the initial influx of leukocytes into the Lacrimal glands (12 weeks), the early clinical phase of autoimmunity (16 weeks), and the early onset of clinical SjS-like disease characterized by secretory dysfunction (20 weeks). The C57BL/6.NOD-Aec1Aec2 mouse is a model of primary SjS in which the Idd3 region of chromosome 3 and the Idd5 region of chromosome 1 derived from the NOD mouse were bred into the non-autoimmune C57BL/6 mouse, resulting in a SjS-like disease susceptibility that mimics both the pathophysiological characteristics and reduced secretory responses observed with NOD mice during development and onset of disease. This SjS-susceptible strain was designated C57BL/6.NOD-Aec1Aec2, where Aec1 corresponds to Idd3 (of chromosome 3) and Aec2 corresponds to Idd5 (of chromosome 1).
Project description:Blister fluid (BF) is a novel and viable research matrix for burn injury study, which can reflect both systemic and local micro-environmental responses. The protein abundance in BF from different burn severities were initially observed using a 2D SDS-PAGE approach. Subsequently, a quantitative data independent acquisition (DIA) method – SWATHTM was employed to characterize the proteome of pediatric burn blister fluid. More than 600 proteins were quantitatively profiled in 87 BF samples from different pediatric burn patients.