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290 The role of S100B in the pathogenesis of vitiligo
ABSTRACTS | Inflammation, Immunity and Infection 286
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Diagnostic value of linear fluorescence of the basement membrane of sweat ducts IS Bagci, O Horva´th, T Ruzicka and M Sa´rdy Department of Dermatology and Allergology, Ludwig Maximilian University, Munich, Germany Direct immunofluorescence (DIF) is a specific and the most sensitive diagnostic test for bullous pemphigoid (BP). Immunofluorescence pattern of the adnexal basement membrane had been suggested to have a diagnostic importance, but no controlled studies have been performed. The aims of this study were to evaluate the fluorescence pattern and intensity of sweat ducts and other adnexal structures in BP and to compare it with a control group in order to assess their diagnostic performance. This is a single-centre, case-control study. DIF sections of 64 BP and 82 control patients were examined for the fluorescence pattern and intensity of IgG. Sweat ducts in papillary, mid and deep dermis, hair follicles and sebaceous glands were assessed separately. The intensity of the fluorescence was graded semi quantitatively between 0-4 (0: no fluorescence, 4: strongest fluorescence). All BP patients showed linear immunofluorescence along the epidermal basement membrane with IgG, whereas control skin samples did not. 58 (90.6%) BP and 44 (53.7%) control patients showed linear staining along the basement membrane of sweat ducts. Presence of positive sweat ducts in DIF was significantly different between two groups (P<0.0001). The sensitivity and specificity of positive sweat ducts for BP was 90.6% and 46.3%, respectively. Sweat ducts in papillary dermis with fluorescence intensities 3 and 4 showed the highest specificity (100%) to detect BP. The intensities of the fluorescence of sweat ducts in all layers of dermis, hair follicles and sebaceous glands were significantly different between BP and control patients (P<0.0001, P<0.0001, P¼0.0045). In conclusion, positive sweat ducts in DIF are highly sensitive for BP; however, only strong fluorescence has acceptable specificity. Evaluation of the location, fluorescence pattern and intensity of sweat ducts can be useful in the diagnosis of BP, but sweat duct fluorescence without linear fluorescence of the epidermal basement membrane cannot be recommended as the basis for diagnosis of BP if the signal is low.
The micro-fluidics reveals the role of vascular endothelium-specific B cells in cutaneous leukocytoclastic vasculitis A Yoshizaki1, K Nakamura1, S Ebata1, T Fukasawa1, R Saigusa1, T Taniguchi1, Y Asano1, K Mawatari2, T Kitamori2 and S Sato1 1 Dermatology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan and 2 Applied Chemistry, The University of Tokyo Graduate School of Engineering, Tokyo, Japan Vasculitis is an inflammatory disorder of unknown etiology, characterized by necrosis of blood vessels. Cutaneous leukocytoclastic vasculitis (CLV) is one of the vasculitis disorders that mainly shows the skin lesion and even systemic inflammation. Although oral corticosteroid and anticoagulation agents were commonly used, some resistant and recurrent cases need to be treated with strong immunosuppressive therapy, which we named severe inflammatory CLV (SICLV). Recent studies have shown that B cells play crucial roles in the pathogenesis of vasculitis. Especially, in anti-neutrophil cytoplasmic antibody associated vasculitis, antigen-specific B cells associate to progression of the disease. However, the population of antigen-specific B cells is so small that its function is not fully elucidated. In this study, we developed the original assay system utilizing micro-flow-channels and micro-fluidics regulating flow of micro channels that simulates the environment of blood vessels, by which the interaction of B cells with endothelial cells could be analyzed ex vivo. In addition, we utilized our original micro-enzyme-linked immunosorbent assay system that integrated immunoassay into a micro-channel in order to detect extremely small amounts of analytes. We found that the endothelium-specific B cells from SICLV bound to endothelium and release cytokines, such as IL-6, which can induce further immune cell activation and recruitment. These findings show that endothelium-specific B cells have important roles in SICLV whose pathophysiology is not fully understood to date. In addition, our original micro-fluidics methods can reveal new etiology of the inflammatory disease, which may lead the novel therapeutic strategies.
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Staphylococcal lipoteichoic acid suppresses T cell function in vitro and in vivo Y Skabytska1, S Kaesler1, K Chen2 and T Biedermann1 1 Dermatology, Technical University, Munich, Germany and 2 Dermatology, University Tu¨bingen, Tu¨bingen, Germany The interplay between microbes and surface organs such as skin shapes a complex immune system. This interaction is crucial as microbes are believed to trigger inflammation as in atopic dermatitis (AD). Especially AD skin is characterized by a high susceptibility for colonization and infection with Staphylococcus aureus. Lipoteichoic acid (LTA) is part of the bacterial cell wall of S. aureus and bind Toll-like receptor (TLR) 2. It has been shown that the amount of LTA in dermatitis lesions positively correlates with AD aggravation. In the present work we aimed to investigate the properties of LTA in regard to T lymphocyte function. We found that LTA potently suppressed T lymphocyte activation in a TLR-independent manner. LTA-exposed T cells failed to proliferate and to produce cytokines. Importantly, these T cells remained completely viable and were susceptible to consecutive activation signals in the absence of LTA. Thus, LTA exposure of T cells resulted in temporary functional T cell paralysis. In vivo experiments revealed that T cell cytokine production and cutaneous recall responses were significantly suppressed by LTA. Thus, we identified a new mechanism of how bacterial compounds temporarily and indirectly modulate adaptive immune responses.
Clinical significance of serum soluble CD molecules to assess disease activity in vitiligo R Speeckaert and N van Geel Dermatology, Ghent University Hospital, Gent, Belgium It is difficult to determine disease activity in vitiligo due to the absence of inflammatory signs such as erythema or scaling. A biomarker which could confirm active disease and predict future disease progression would therefore be of considerable value. Our objective was to investigate if soluble CD27 (sCD27), CD25 (sCD25) or CD40L (sCD40L) could be valuable biomarkers to determine disease activity in vitiligo and predict future progression. Patients were recruited in this combined cross-sectional and prospective study between 2009 and 2015. This study was conducted at the department of Dermatology at Ghent University Hospital (Belgium). 93 vitiligo patients were enrolled, including 83 patients with nonsegmental vitiligo and 10 patients with segmental vitiligo. Blood sampling was performed and sCD25, sCD27 and sCD40L were measured in serum. The associations between soluble CD levels, disease activity and future progression were investigated. Both sCD27 (P ¼ 0.006) and sCD25 (P ¼ 0.002) were strongly associated with active disease. Moreover, a link with disease progression after 3-6 months was found for sCD27 (P ¼ 0.021) and to a lesser extent also for sCD25 (P ¼ 0.053). Further in vitro experiments showed a correlation between sCD25 and IFN-g, IL-10 and sCD27 secretion. No strong associations were found for sCD40L. This study demonstrates increased values of sCD27 and sCD25 in active vitiligo patients. Moreover, our results provide for the first time evidence that these markers have a predictive capacity on future disease progression. This supports their role as attractive biomarkers in vitiligo.
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The role of S100B in the pathogenesis of vitiligo R Speeckaert and N van Geel Department of Dermatology, Ghent University Hospital, Gent, Belgium S100B is a damage associated molecular pattern expressed by melanocytes. While its role as a biomarker in melanoma is well established, S100B has not yet been investigated in vitiligo. The active process of vitiligo involves melanocyte destruction. S100B could therefore be upregulated in patients with active vitiligo. As this protein has immunomodulatory capacities, S100B could play a role in the pathogenesis of vitiligo. We investigated the serum values of S100B in 101 vitiligo patients. Disease activity was assessed by both the patient and physician. In vitro experiments were performed on normal and vitiligo melanocytes using repeated freeze-thaw cycli. Significantly elevated S100B values were found in patients with clearly active non-segmental vitiligo based on follow-up pictures (p ¼ 0.001). Additionally, patientreported disease activity during the last 6 months was also linked to higher S100B levels (p ¼ 0.005). Interestingly, S100B levels correlated significantly with the affected BSA (p ¼ 0.001). Moreover, prospective follow-up showed also a predictive capacity of S100B levels on disease progression (p ¼ 0.0395). Additional in vitro experiments demonstrated a massive release of S100B after melanocyte necrosis and intracellular upregulation after freeze-thaw procedures. Importantly, the annexin V+/live death- melanocytes showed a strong release of S100B in the environment indicating that S100B is already released from early necrotic melanocytes. No association of S100B with soluble CD antigens was found indicating that the elevated S100B levels were derived from melanocyte destruction and not from an activated immune response. In conclusion, this is the first report that demonstrates the association of S100B with disease activity in vitiligo. While the differences in serum S100B concentrations are limited, S100B is released at the site of disease activity reaching locally very high levels. These data suggest that this protein could play a substantial role in the pathogenesis of vitiligo and may be a potential new target for treatment.
S210 Journal of Investigative Dermatology (2016), Volume 136
Effect of rituximab therapy on total CD25+ and CD4+CD25+cells of pemphigus vulgaris patients B ELZawahry, D Bassiouny, R Hegazy, H Gawdat, S Shalaby and MA Saleh Dermatology, Cairo University, Cairo, Egypt B-cell depleting therapy using anti-CD20 monoclonal antibody, rituximab is considered a successful way to treat pemphigus vulgaris (PV) patients. In this work we aimed to assess the effect of rituximab therapy on total CD25+ cells and CD4+CD25+cells extracted from the peripheral blood mononuclear cells of PV patients by flow cytometry. Fourteen PV cases were included in this study (Six males and 8 females) and were followed up for 12 months. Three patients were followed up for 24 months. The patients’ ages ranged from 29 - 57 years (median¼ 44 years). Their disease duration ranged from 2 months - 15 years (median¼ 28 months). The patients were treated with 1000mg rituximab in a 2-week interval +daily oral systemic steroids + azathioprine. The percentage of the CD4, CD8, CD20 and CD25 to the total lymphocytes was determined at 0, 3, 6 and 12 months after therapy. The baseline CD20, CD8, CD4 and CD25 ranged from 7-27.7 (mean¼ 13.7), 19.9-40.6 (mean¼ 29.4), 18.4-66.4 (mean¼ 42.2) and 0.1-3.5 (mean¼ 1.3) respectively. The percentage of the baseline CD4+CD25+cells ranged from 1.5-4.3 (mean¼2.6). Nine cases (64%) were in clinical remission and 5 (36%) were in partial remission. Seven cases developed relapse. At the end of the 12 months follow up CD20, CD8, CD4 and CD25 ranged from 0.1-13.1 (mean¼ 1.8), 23.5-47.5 (mean¼ 3.24), 21.9-62.7 (mean¼ 43.6) and 0.5-9.1 (mean¼ 2.3) respectively. There was significant decrease in CD20+ cells, p value was 0.0002 and there was significant increase in CD25+ cells at the end of the follow up, p value was 0.018. However CD4+ and CD8+ cells did not change. There was significant decrease in the CD4+CD25+cells after therapy, p value was 0.041. The relapse of the patients was paralleled by increase in the antidesmoglein level, increase in the CD20 cells and increase in the CD4+CD25+cells. In conclusion CD4+CD25+cells are decreased in pemphigus vulgaris patients and they decrease more after rituximab therapy. This suggests that the effect rituximab on CD4+CD25+cells of pemphigus patients is different from other autoimmune diseases.
287
Diagnostic value of linear fluorescence of the basement membrane of sweat ducts IS Bagci, O Horva´th, T Ruzicka and M Sa´rdy Department of Dermatology and Allergology, Ludwig Maximilian University, Munich, Germany Direct immunofluorescence (DIF) is a specific and the most sensitive diagnostic test for bullous pemphigoid (BP). Immunofluorescence pattern of the adnexal basement membrane had been suggested to have a diagnostic importance, but no controlled studies have been performed. The aims of this study were to evaluate the fluorescence pattern and intensity of sweat ducts and other adnexal structures in BP and to compare it with a control group in order to assess their diagnostic performance. This is a single-centre, case-control study. DIF sections of 64 BP and 82 control patients were examined for the fluorescence pattern and intensity of IgG. Sweat ducts in papillary, mid and deep dermis, hair follicles and sebaceous glands were assessed separately. The intensity of the fluorescence was graded semi quantitatively between 0-4 (0: no fluorescence, 4: strongest fluorescence). All BP patients showed linear immunofluorescence along the epidermal basement membrane with IgG, whereas control skin samples did not. 58 (90.6%) BP and 44 (53.7%) control patients showed linear staining along the basement membrane of sweat ducts. Presence of positive sweat ducts in DIF was significantly different between two groups (P<0.0001). The sensitivity and specificity of positive sweat ducts for BP was 90.6% and 46.3%, respectively. Sweat ducts in papillary dermis with fluorescence intensities 3 and 4 showed the highest specificity (100%) to detect BP. The intensities of the fluorescence of sweat ducts in all layers of dermis, hair follicles and sebaceous glands were significantly different between BP and control patients (P<0.0001, P<0.0001, P¼0.0045). In conclusion, positive sweat ducts in DIF are highly sensitive for BP; however, only strong fluorescence has acceptable specificity. Evaluation of the location, fluorescence pattern and intensity of sweat ducts can be useful in the diagnosis of BP, but sweat duct fluorescence without linear fluorescence of the epidermal basement membrane cannot be recommended as the basis for diagnosis of BP if the signal is low.
The micro-fluidics reveals the role of vascular endothelium-specific B cells in cutaneous leukocytoclastic vasculitis A Yoshizaki1, K Nakamura1, S Ebata1, T Fukasawa1, R Saigusa1, T Taniguchi1, Y Asano1, K Mawatari2, T Kitamori2 and S Sato1 1 Dermatology, The University of Tokyo Graduate School of Medicine, Tokyo, Japan and 2 Applied Chemistry, The University of Tokyo Graduate School of Engineering, Tokyo, Japan Vasculitis is an inflammatory disorder of unknown etiology, characterized by necrosis of blood vessels. Cutaneous leukocytoclastic vasculitis (CLV) is one of the vasculitis disorders that mainly shows the skin lesion and even systemic inflammation. Although oral corticosteroid and anticoagulation agents were commonly used, some resistant and recurrent cases need to be treated with strong immunosuppressive therapy, which we named severe inflammatory CLV (SICLV). Recent studies have shown that B cells play crucial roles in the pathogenesis of vasculitis. Especially, in anti-neutrophil cytoplasmic antibody associated vasculitis, antigen-specific B cells associate to progression of the disease. However, the population of antigen-specific B cells is so small that its function is not fully elucidated. In this study, we developed the original assay system utilizing micro-flow-channels and micro-fluidics regulating flow of micro channels that simulates the environment of blood vessels, by which the interaction of B cells with endothelial cells could be analyzed ex vivo. In addition, we utilized our original micro-enzyme-linked immunosorbent assay system that integrated immunoassay into a micro-channel in order to detect extremely small amounts of analytes. We found that the endothelium-specific B cells from SICLV bound to endothelium and release cytokines, such as IL-6, which can induce further immune cell activation and recruitment. These findings show that endothelium-specific B cells have important roles in SICLV whose pathophysiology is not fully understood to date. In addition, our original micro-fluidics methods can reveal new etiology of the inflammatory disease, which may lead the novel therapeutic strategies.
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Staphylococcal lipoteichoic acid suppresses T cell function in vitro and in vivo Y Skabytska1, S Kaesler1, K Chen2 and T Biedermann1 1 Dermatology, Technical University, Munich, Germany and 2 Dermatology, University Tu¨bingen, Tu¨bingen, Germany The interplay between microbes and surface organs such as skin shapes a complex immune system. This interaction is crucial as microbes are believed to trigger inflammation as in atopic dermatitis (AD). Especially AD skin is characterized by a high susceptibility for colonization and infection with Staphylococcus aureus. Lipoteichoic acid (LTA) is part of the bacterial cell wall of S. aureus and bind Toll-like receptor (TLR) 2. It has been shown that the amount of LTA in dermatitis lesions positively correlates with AD aggravation. In the present work we aimed to investigate the properties of LTA in regard to T lymphocyte function. We found that LTA potently suppressed T lymphocyte activation in a TLR-independent manner. LTA-exposed T cells failed to proliferate and to produce cytokines. Importantly, these T cells remained completely viable and were susceptible to consecutive activation signals in the absence of LTA. Thus, LTA exposure of T cells resulted in temporary functional T cell paralysis. In vivo experiments revealed that T cell cytokine production and cutaneous recall responses were significantly suppressed by LTA. Thus, we identified a new mechanism of how bacterial compounds temporarily and indirectly modulate adaptive immune responses.
Clinical significance of serum soluble CD molecules to assess disease activity in vitiligo R Speeckaert and N van Geel Dermatology, Ghent University Hospital, Gent, Belgium It is difficult to determine disease activity in vitiligo due to the absence of inflammatory signs such as erythema or scaling. A biomarker which could confirm active disease and predict future disease progression would therefore be of considerable value. Our objective was to investigate if soluble CD27 (sCD27), CD25 (sCD25) or CD40L (sCD40L) could be valuable biomarkers to determine disease activity in vitiligo and predict future progression. Patients were recruited in this combined cross-sectional and prospective study between 2009 and 2015. This study was conducted at the department of Dermatology at Ghent University Hospital (Belgium). 93 vitiligo patients were enrolled, including 83 patients with nonsegmental vitiligo and 10 patients with segmental vitiligo. Blood sampling was performed and sCD25, sCD27 and sCD40L were measured in serum. The associations between soluble CD levels, disease activity and future progression were investigated. Both sCD27 (P ¼ 0.006) and sCD25 (P ¼ 0.002) were strongly associated with active disease. Moreover, a link with disease progression after 3-6 months was found for sCD27 (P ¼ 0.021) and to a lesser extent also for sCD25 (P ¼ 0.053). Further in vitro experiments showed a correlation between sCD25 and IFN-g, IL-10 and sCD27 secretion. No strong associations were found for sCD40L. This study demonstrates increased values of sCD27 and sCD25 in active vitiligo patients. Moreover, our results provide for the first time evidence that these markers have a predictive capacity on future disease progression. This supports their role as attractive biomarkers in vitiligo.
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The role of S100B in the pathogenesis of vitiligo R Speeckaert and N van Geel Department of Dermatology, Ghent University Hospital, Gent, Belgium S100B is a damage associated molecular pattern expressed by melanocytes. While its role as a biomarker in melanoma is well established, S100B has not yet been investigated in vitiligo. The active process of vitiligo involves melanocyte destruction. S100B could therefore be upregulated in patients with active vitiligo. As this protein has immunomodulatory capacities, S100B could play a role in the pathogenesis of vitiligo. We investigated the serum values of S100B in 101 vitiligo patients. Disease activity was assessed by both the patient and physician. In vitro experiments were performed on normal and vitiligo melanocytes using repeated freeze-thaw cycli. Significantly elevated S100B values were found in patients with clearly active non-segmental vitiligo based on follow-up pictures (p ¼ 0.001). Additionally, patientreported disease activity during the last 6 months was also linked to higher S100B levels (p ¼ 0.005). Interestingly, S100B levels correlated significantly with the affected BSA (p ¼ 0.001). Moreover, prospective follow-up showed also a predictive capacity of S100B levels on disease progression (p ¼ 0.0395). Additional in vitro experiments demonstrated a massive release of S100B after melanocyte necrosis and intracellular upregulation after freeze-thaw procedures. Importantly, the annexin V+/live death- melanocytes showed a strong release of S100B in the environment indicating that S100B is already released from early necrotic melanocytes. No association of S100B with soluble CD antigens was found indicating that the elevated S100B levels were derived from melanocyte destruction and not from an activated immune response. In conclusion, this is the first report that demonstrates the association of S100B with disease activity in vitiligo. While the differences in serum S100B concentrations are limited, S100B is released at the site of disease activity reaching locally very high levels. These data suggest that this protein could play a substantial role in the pathogenesis of vitiligo and may be a potential new target for treatment.
S210 Journal of Investigative Dermatology (2016), Volume 136
Effect of rituximab therapy on total CD25+ and CD4+CD25+cells of pemphigus vulgaris patients B ELZawahry, D Bassiouny, R Hegazy, H Gawdat, S Shalaby and MA Saleh Dermatology, Cairo University, Cairo, Egypt B-cell depleting therapy using anti-CD20 monoclonal antibody, rituximab is considered a successful way to treat pemphigus vulgaris (PV) patients. In this work we aimed to assess the effect of rituximab therapy on total CD25+ cells and CD4+CD25+cells extracted from the peripheral blood mononuclear cells of PV patients by flow cytometry. Fourteen PV cases were included in this study (Six males and 8 females) and were followed up for 12 months. Three patients were followed up for 24 months. The patients’ ages ranged from 29 - 57 years (median¼ 44 years). Their disease duration ranged from 2 months - 15 years (median¼ 28 months). The patients were treated with 1000mg rituximab in a 2-week interval +daily oral systemic steroids + azathioprine. The percentage of the CD4, CD8, CD20 and CD25 to the total lymphocytes was determined at 0, 3, 6 and 12 months after therapy. The baseline CD20, CD8, CD4 and CD25 ranged from 7-27.7 (mean¼ 13.7), 19.9-40.6 (mean¼ 29.4), 18.4-66.4 (mean¼ 42.2) and 0.1-3.5 (mean¼ 1.3) respectively. The percentage of the baseline CD4+CD25+cells ranged from 1.5-4.3 (mean¼2.6). Nine cases (64%) were in clinical remission and 5 (36%) were in partial remission. Seven cases developed relapse. At the end of the 12 months follow up CD20, CD8, CD4 and CD25 ranged from 0.1-13.1 (mean¼ 1.8), 23.5-47.5 (mean¼ 3.24), 21.9-62.7 (mean¼ 43.6) and 0.5-9.1 (mean¼ 2.3) respectively. There was significant decrease in CD20+ cells, p value was 0.0002 and there was significant increase in CD25+ cells at the end of the follow up, p value was 0.018. However CD4+ and CD8+ cells did not change. There was significant decrease in the CD4+CD25+cells after therapy, p value was 0.041. The relapse of the patients was paralleled by increase in the antidesmoglein level, increase in the CD20 cells and increase in the CD4+CD25+cells. In conclusion CD4+CD25+cells are decreased in pemphigus vulgaris patients and they decrease more after rituximab therapy. This suggests that the effect rituximab on CD4+CD25+cells of pemphigus patients is different from other autoimmune diseases.