image_2.PDF

<p>Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera (N = 32, with positive reactivity with CXCR3) to healthy controls (N = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients (N = 31) from normal healthy controls (N = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients.</p

image_3_Therapeutic Effect of a Novel Phosphatidylinositol-3-Kinase δ Inhibitor in Experimental Epidermolysis Bullosa Acquisita.TIF

<p>Epidermolysis bullosa acquisita (EBA) is a rare, but prototypical, organ-specific autoimmune disease, characterized and caused by autoantibodies against type VII collagen (COL7). Mucocutaneous inflammation, blistering, and scarring are the clinical hallmarks of the disease. Treatment of EBA is difficult and mainly relies on general immunosuppression. Hence, novel treatment options are urgently needed. The phosphatidylinositol-3-kinase (PI3K) pathway is a putative target for the treatment of inflammatory diseases, including EBA. We recently discovered LAS191954, an orally available, selective PI3Kδ inhibitor. PI3Kδ has been shown to be involved in B cell and neutrophil cellular functions. Both cell types critically contribute to EBA pathogenesis, rendering LAS191954 a potential drug candidate for EBA treatment. We, here, demonstrate that LAS191954, when administered chronically, dose-dependently improved the clinical phenotype of mice harboring widespread skin lesions secondary to immunization-induced EBA. Direct comparison with high-dose corticosteroid treatment indicated superiority of LAS191954. Interestingly, levels of circulating autoantibodies were unaltered in all groups, indicating a mode of action independent of the inhibition of B cell function. In line with this, LAS191954 also hindered disease progression in antibody transfer-induced EBA, where disease develops dependent on myeloid, but independent of B cells. We further show that, in vitro, LAS191954 dose-dependently impaired activation of human myeloid cells by relevant disease stimuli. Specifically, immune complex-mediated and C5a-mediated ROS release were inhibited in a PI3Kδ-dependent manner. Accordingly, LAS191954 also modulated the dermal–epidermal separation induced in vitro by co-incubation of immune complexes with polymorph nuclear cells, thus pointing to an important role of PI3Kδ in EBA effector functions. Altogether, these results suggest a new potential mechanism for the treatment of EBA and potentially also other autoimmune bullous diseases.</p

image_1.PDF

<p>Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera (N = 32, with positive reactivity with CXCR3) to healthy controls (N = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients (N = 31) from normal healthy controls (N = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients.</p

image_4.PDF

<p>Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera (N = 32, with positive reactivity with CXCR3) to healthy controls (N = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients (N = 31) from normal healthy controls (N = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients.</p

Passive transfer of rabbit anti-mLAMC1-cterm IgG into adult mice is not pathogenic.

<p>Rabbit IgG against the murine laminin γ1 C-terminus (mLAMC1-cterm) was not pathogenic when passively transferred into adult C57BL/6 and BALB/c mice. Injection of 15 mg rabbit anti-mLAMC1-cterm IgG every second day for 10 days did not result in clinical or histopathological (a, e) lesions on day 12. Linear deposition of rabbit IgG at the dermal-epidermal junction (DEJ) was only observed in 2 of 5 C57BL/6 (b) and one of 5 BALB/c mice (f), while staining of murine C3 was negative in all mice (c, g). At day 12, in sera of all 10 mice, rabbit IgG labeled the basal keratinocytes at the DEJ of normal mouse skin (d, h) and reacted with recombinant mLAMC1-cterm by ELISA (i) and immunoblotting (j, C57BL/6, lanes 1; BALB/c, lane 2) and the 200 kDa p200 protein in extract of murine dermis (k, C57BL/6 lane 1; BALB/c, lane 2). Normal mouse sera (j and k, C57BL/6, lane 3; BALB/c, lane 4) were used as controls.</p

image_4_Therapeutic Effect of a Novel Phosphatidylinositol-3-Kinase δ Inhibitor in Experimental Epidermolysis Bullosa Acquisita.TIF

<p>Epidermolysis bullosa acquisita (EBA) is a rare, but prototypical, organ-specific autoimmune disease, characterized and caused by autoantibodies against type VII collagen (COL7). Mucocutaneous inflammation, blistering, and scarring are the clinical hallmarks of the disease. Treatment of EBA is difficult and mainly relies on general immunosuppression. Hence, novel treatment options are urgently needed. The phosphatidylinositol-3-kinase (PI3K) pathway is a putative target for the treatment of inflammatory diseases, including EBA. We recently discovered LAS191954, an orally available, selective PI3Kδ inhibitor. PI3Kδ has been shown to be involved in B cell and neutrophil cellular functions. Both cell types critically contribute to EBA pathogenesis, rendering LAS191954 a potential drug candidate for EBA treatment. We, here, demonstrate that LAS191954, when administered chronically, dose-dependently improved the clinical phenotype of mice harboring widespread skin lesions secondary to immunization-induced EBA. Direct comparison with high-dose corticosteroid treatment indicated superiority of LAS191954. Interestingly, levels of circulating autoantibodies were unaltered in all groups, indicating a mode of action independent of the inhibition of B cell function. In line with this, LAS191954 also hindered disease progression in antibody transfer-induced EBA, where disease develops dependent on myeloid, but independent of B cells. We further show that, in vitro, LAS191954 dose-dependently impaired activation of human myeloid cells by relevant disease stimuli. Specifically, immune complex-mediated and C5a-mediated ROS release were inhibited in a PI3Kδ-dependent manner. Accordingly, LAS191954 also modulated the dermal–epidermal separation induced in vitro by co-incubation of immune complexes with polymorph nuclear cells, thus pointing to an important role of PI3Kδ in EBA effector functions. Altogether, these results suggest a new potential mechanism for the treatment of EBA and potentially also other autoimmune bullous diseases.</p

Passive transfer of rabbit anti-mLAMC1-cterm IgG into neonatal mice does not reproduce the human disease.

<p>Rabbit IgG against the murine laminin γ1 C-terminus (mLAMC1-cterm) was not pathogenic when passively transferred into neonatal C57BL/6 mice. Injection of rabbit anti-mLAMC1-cterm IgG at a concentration of 10 mg/g body weight every second day for 10 days did not <u>induce</u> histopathological lesions on day 12 (<u>a</u>). Linear deposition of rabbit IgG at the DEJ was only observed in 2 of 8 mice (b), while staining of murine C3 was always negative (c). At day 12, in sera of all mice, rabbit IgG stained the basal keratinocytes at the DEJ of normal mouse skin (d), reacted with recombinant mLAMC1-cterm by ELISA (e) and immunoblotting (f, lanes 2–6) and with the 200 kDa p200 protein in extract of murine dermis (g, lanes 2–6). Polyclonal rabbit antibody H-190 against mLAMC1 (f, g, lane 1) and normal mouse serum (f, g, lane 7) was used as controls.</p

Human IgG specific to laminin γ1 is not pathogenic ex vivo.

<p>Using recombinant forms of the C-terminus of laminin γ1 (hLAMC1-cterm) and full length laminin γ1 (LAMC1-FL) IgG specific for hLAMC1-cterm (a, c; lane 3) and LAMC1-FL (b, lane 3; c lane 5 ) was generated from anti-p200 pemphigoid serum (a, b, c; lane 2), as well as serum depleted from anti-hLAMC1-cterm (a, c, lane 4) and LAMC1-FL reactivity (b, lane 4; c lane 6) reactivity, respectively, as shown by immunoblotting with recombinant hLAMC1-cterm (a), LAMC1-FL (b), and extract of human dermis<u>-</u>(c). Interestingly, serum depleted from anti-hLAMC1-cterm and LAMC1-FL reactivity, respectively, (a, b; lane 4) still labeled the p200 protein in dermal extract (c, lane 4, lane 6). Monoclonal antibody against LAMC1 (a, b, c; lane 1) and serum from a healthy volunteer (a, b; lane 5; c lane 7) were used as controls. Arrows indicate the positions of the proteins and bars the molecular weight markers (a, 34 kD and 26 kD; b, 200 kD; c, 200 kD). hLAMC1-cterm-specific (h, i) and LAMC1-FL-specific patients IgG (l, m) and the monoclonal anti-LAMC1 antibody (d, e) labeled the dermal-epidermal junction (DEJ) by indirect immunofluorescence (IF) microscopy but did not induce dermal-epidermal separation (DES). In contrast, serum depleted from reactivity against hLAMC1-cterm (j, k), and LAMC1-FL (n, o), respectively, as well as patient serum (f, g) resulted in DES (black triangles mark base of the split). While untouched patient serum (f) as well as serum depleted from anti-hLAMC1-cterm (j) and LAMC1-FL reactivity (n), respectively, stained the DEJ of human skin in a linear pattern, the monoclonal anti-hLAMC1-antibody (d), hLAMC1-cterm-specific patient<u>-</u>IgG (h), and hLAMC1-FL-specific patient IgG showed an additional staining of basal keratinocytes. Serum from a healthy volunteer was used as control (p, q). Magnification: x200.</p

image_3.PDF

<p>Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera (N = 32, with positive reactivity with CXCR3) to healthy controls (N = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients (N = 31) from normal healthy controls (N = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients.</p

data_sheet_2.xlsx

<p>Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera (N = 32, with positive reactivity with CXCR3) to healthy controls (N = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients (N = 31) from normal healthy controls (N = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients.</p