CXCL9 as a key biomarker of vitiligo activity and prediction of the success of cultured melanocyte transplantation

This study aimed to investigate the potential biomarkers of vitiligo by evaluating the disease activity and curative effect of autologous cultured pure melanocyte transplantation (CMT) on patients. Altogether, 36patients with stable vitiligo were treated with CMT. Blister fluid samples were collected from patients with stable vitiligo. Patients with active vitiligo were matched with healthy controls. The chemokine levels in the serum and blister fluid samples were measured using Luminex. The curative effect on patients with stable vitiligo was evaluated 6 months after treatment. Treatment responses were defined according to the extent of repigmentation as effective (if 50% or more repigmentation was achieved) or ineffective (if less than 50% or worse repigmentation was achieved). Patients received re-transplantation if the initial treatment was ineffective. The levels of C-X-C motif chemokine ligand (CXCL)9 and CXCL10 in blister fluid samples were significantly lower in stable patients than in active participants. Receiver operating characteristic analysis revealed that the levels of CXCL9 and CXCL10 were sensitive and specific in diagnosing active vitiligo. Further, 65.6% (21/32) of patients who received CMT had effective treatment responses. The high CXCL9 level in the blister fluid was a significant predictor of ineffective treatment responses. The treatment response was significantly enhanced after treatment. Four patients with ineffective treatment responses received anti-inflammatory treatment and re-transplantation. The CXCL9 and CXCL10 levels in the blister fluid were related to the presence of active vitiligo. Also, the CXCL9 level was a predictor of the effectiveness of CMT in treating vitiligo.

P154 High-density proteomic analysis of skin blister fluid and plasma in systemic sclerosis identifies local and systemic differences for key proteins

Background/Aims Simultaneous analysis of multiple proteins in biological fluids offers insight into the pathogenesis of SSc. Here, we report a proteomic analysis of plasma and dermal interstitial fluid in SSc compared with healthy controls (HC). Methods The prospectively collected BIOPSY cohort recruited 52 SSc patients (21 early dcSSc, 15 established dcSSc,16 lcSSc) and 16 HC. Mean baseline skin score (MRSS) for early dcSSc was 21 (sd 11.2). This analysis utilised forearm skin blister fluid obtained using the dermal suction blister method and paired simultaneous plasma samples from early dcSSc and HC at baseline. These were assayed using the Olink antibody platform(www.olink.com) and reported as normalised protein expression (NPX), corresponding to log2 (expression). T-test with FDR correction (p < 0.05) assessed statistical significance. Pathway analysis was conducted by STRING consortium 2020. Results 1,196 proteins were analysed in paired blister/plasma samples from 14 early dcSSc patients and 16 HC. 447 proteins were significantly different in the blister fluid of early dcSSc patients compared with HC (Table 1), whereas only 183 proteins in plasma. Of these, 130 proteins were simultaneously different in both blister fluid and plasma of early dcSSc including key cytokines associated with fibrosis and vasculopathy such as IL-6,VEGF-1, MCP-1, COL4A1, COMP, Thy1 and THBS4. No correlation was seen between these proteins and MRSS. 310 proteins were significantly elevated in blister fluid alone in early dcSSc patients compared to HC. These included cytokines (IL7,IL18, OSM), chemokines (CCL7,CCL18, CCL3), matricellular proteins (CYR61 and osteopontin). KEGG pathway analysis of the significantly elevated proteins in blister fluid in early dcSSc compared to HC highlighted pathways including cytokine-cytokine receptor interaction, cell adhesion molecules, MAPK signalling pathway and PI3K-AKT signalling pathway (FDR<0.01) P154 Table 1:Protein symbolRaw mean NPXFold ChangeAdjusted p valueDetails of proteinHCdcSScKLK42.69815.9260.004Kallikrein-related peptidase 4IL616.13815.1070.008Interleukin 6NT-proBNP33.60712.8570.018N-terminal pro-brain natriuretic peptideAREG12.74711.9780.017AmphiregulinLTBP24.59311.7990.015Latent-TGF beta-binding protein 2SFRP181.10710.4040.025Secreted Frizzled Related Protein 1TNC18.0128.7280.010Tenascin CCPXM12.1477.4650.002Probable carboxypeptidase X1CYR6110.4936.1060.011Cysteine-rich angiogenic inducer 61 (CCN1)PAPPA10.2675.4800.008Pregnancy-associated plasma protein AEDA2R11.4795.4070.008Ectodysplasin-A2 rceptorNOV61.6385.1190.003Nephroblastoma overexpressed protein (CCN3)GDF-1520.8074.5400.002growth/differentiation factor 15SCARF211.8684.3770.008Scavenger Receptor Class F Member 2CXCL1071.6204.1960.028interferon-γ inducible protein 10THBS4110.6824.1630.008Thrombospondin 4CXCL1324.2564.0470.011C-X-C Motif Chemokine Ligand 13MAPT1.6614.0410.015Microtubule-associated protein tauCOL4A116.7054.0090.003Collagen type IV alpha 1MCP-11128.943.6510.016Monocyte chemoattractant protein-1Top 20 most upregulated proteins in skin blister fluid for early dcSSc (n = 14) compared with healthy control (HC, n = 16). Conclusion Numerous dysregulated proteins were identified in dermal blister fluid and plasma of early dcSSc patients. Substantially more were identified in dermal blister fluid, highlighting its potential for providing detailed information on local pathologic processes. A subset of proteins were dysregulated in both plasma and blister fluid, suggesting these may reflect systemic abnormalities. Further work will utilise this cohort to integrate gene and protein expression across the full spectrum of early dcSSc, established dcSSc and lcSSc Disclosure K.E. Clark: None. C. Campochiaro: None. E. Csomor: Corporate appointments; employee of GSK. A. Taylor: Corporate appointments; employee of GSK. K. Nevin: Corporate appointments; employee of GSK. M.A. Morse: Corporate appointments; employee of GSK. V.H. Ong: None. E. Derrett-Smith: None. N. Wisniacki: Corporate appointments; employee of GSK. S. Flint: Corporate appointments; employee of GSK. C.P. Denton: Consultancies; Actelion, GlaxoSmithKline, Bayer, Sanofi, lnventiva, Boehringer Ingelheim, Roche, Bristol Myers Squibb, CSL Behring, UCB, Leadiant Biosciences, Corbus, Servier, Arxx Therapeutics.

Preliminary Assessment of Burn Depth by Paper-Based ELISA for the Detection of Angiogenin in Burn Blister Fluid—A Proof of Concept

Rapid assessment of burn depth is important for burn wound management. Superficial partial-thickness burn (SPTB) wounds heal without scars, but deep partial-thickness burn (DPTB) wounds require a longer healing time and have a higher risk of scar formation. We previously found that DPTB blister fluid displayed a higher angiogenin level than SPTB blister fluid by conventional ELISA. In this study, we developed a paper-based ELISA (P-ELISA) technique for rapid assessment of angiogenin concentration in burn blister fluid. We collected six samples of SPTB blister fluid, six samples of DPTB blister fluid, and seven normal healthy serum samples for analysis. We again chose ELISA to measure and compare angiogenin levels across all of our samples, but we developed a P-ELISA tool and compared sample results from that tool to the results from conventional ELISA. As with conventional ELISA, DPTB blister fluid displayed higher angiogenin levels than SPTB in P-ELISA. Furthermore, our P-ELISA results showed a moderate correlation with conventional ELISA results. This new diagnostic technique facilitates rapid and convenient assessment of burn depth by evaluating a key molecule in burn blister fluid. It presents a novel and easy-to-learn approach that may be suitable for clinically determining burn depth with diagnostic precision.