Targeted proteomics analysis of Cutaneous Lupus Erythematosus patient interstitial skin fluid and plasma (Neuro Exploratory panel data)
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ABSTRACT: We performed a targeted proteomics analysis of Cutaneous Lupus Erythematosus (CLE) patient blister biopsies and plasma using Olink Proteomics panels to validate the blister biopsy technique using previously identified biomarkers, to confirm protein-level expression of biomarkers previously identified at the RNA level, and to identify new biomarkers of disease.
Project description:We performed a targeted proteomics analysis of Cutaneous Lupus Erythematosus (CLE) patient blister biopsies and plasma using Olink Proteomics panels to validate the blister biopsy technique using previously identified biomarkers, to confirm protein-level expression of biomarkers previously identified at the RNA level, and to identify new biomarkers of disease.
Project description:Cutaneous lupus erythematosus (CLE) is an autoimmune disease that localizes to the skin and is known to contain elevated glycosaminoglycans (GAGs) on Hale’s stain of skin biopsy specimens. Recently, different GAG species have been shown to have distinct effects on the recruitment and activation of immune cells and stimulation of cytokine production (Taylor and Gallo, FASEB, 2006; 20: 9-22). Thus, we speculate that the elevated GAGs observed in CLE play a role in the local inflammatory process that produces skin lesions in these patients. In order to further investigate a molecular basis for the elevated expression of these GAGs in CLE skin lesions, we would like to determine the gene expression profiles of GAG synthesis, degradation, and modifier genes in lesional and non-lesional skin samples from CLE patients and compare to those from healthy controls. A microarray approach will give us a broader understanding of the genetic regulation of the expression of various GAG species in CLE skin. We will then be able to target future quantitative gene expression experiments by real-time RT-PCR to the genes that are shown to be involved in CLE. In order to accomplish our goal, we would like to examine the GAG gene expression profiles of DLE, TLE, and SCLE subtypes due to the differences in CS and HA staining that we found among these subtypes. Since HA and CS are elevated in DLE and HA in TLE, but not in SCLE, the SCLE samples will also serve as an internal control. We would like to examine both lesional and non-lesional skin biopsies to determine if CLE skin prior to developing a lesion is different at the genetic level from healthy control skin and how it changes once a lesion does develop. We will separate the dermis from the epidermis of the skin biopsies and extract RNA just from the dermis to enrich for dermal fibroblast RNA. We aim to submit four patient biopsies per subtype as well as four samples from healthy control skin for comparison. This number is necessary in order to account for the biologic variability among different patients. We would submit more samples per subtype but are limited by availability of patients in clinic. Thus, we will have a total of 28 samples to submit for microarray. This study design will allow us to analyze the GAG gene expression profiles among different CLE subtypes and enable us to identify which GAc
Project description:Cutaneous lupus erythematosus (CLE) is a photosensitive autoimmune disease characterized by a strong type-I-interferon (IFN) associated inflammation. Keratinocytes are known to determine the interface-dermatitis-pattern in CLE by production of proinflammatory cytokines in the lower epidermis. These cytokines drive a cytotoxic anti-epithelial immune response resulting in keratinocytic cell death and release of endogenous nucleic acids (eNA). We hypothesized that these eNA (RNA- and DNA-motifs) have the capacity to activate innate immune pathways in keratinocytes via pathogen-recognition-receptors (PRR). Gene expression analyses revealed an excessive activation of innate immune response pathways with strong expression of IFN-regulated cytokines in CLE skin lesions. Cultured keratinocytes produce large amounts of these cytokines in response to stimulation of PRR with eNA. UV-stimulation enhances the immunogenicity of eNA and induces CLE-like skin lesions in knockout mice lacking the cytosolic DNase TREX1. Our results provide evidence for a pathogenetic role of endogenous nucleic acids in CLE. They are released within the cytotoxic inflammation along the dermo-epidermal junction and have the capacity to drive the LE-typical inflammation. UV-irradiation supports this inflammation by generation of highly immunostimulatory DNA motifs (8-OHG). These findings explain the photosensitivity of lupus patients and identify pathways of the innate immune system as targets for future therapies.
Project description:Cutaneous lupus erythematosus (CLE) is an autoimmune disease that localizes to the skin and is known to contain elevated glycosaminoglycans (GAGs) on Hale’s stain of skin biopsy specimens. Recently, different GAG species have been shown to have distinct effects on the recruitment and activation of immune cells and stimulation of cytokine production (Taylor and Gallo, FASEB, 2006; 20: 9-22). Thus, we speculate that the elevated GAGs observed in CLE play a role in the local inflammatory process that produces skin lesions in these patients.
Project description:We performed targeted protein expression analysis on cultured Th0, Th1 and Th2 cells, and on T cells treated with known drug triggers of cutaneous lupus erythematosus (CLE). In tandem, we performed targeted protein expression analysis on serum from first vs flare Th2-injected mice in the CLE model.
Project description:Ticks (Acari: Ixodida) feed exclusively on blood which lacks essential nutrients such as vitamins and cofactors. These deficiencies are presumably complemented through specific symbiotic microorganisms such as Coxiella – Like Endosymbionts (CLEs) of Rhipicephalus ticks. CLE are localized in specialized host tissue cells within the Malpighian tubules (Mt) and the ovaries (Ov) from which they are maternally transmitted to developing oocytes. These two organs differ in function and cell types, and possibly also in activities performed by CLE occupants. To get insights into CLE functions within these two organs, we used comparative proteomics of CLEs in Mt and Ov of unfed R. sanguineus ticks