EndoS treatment of IgG decreases the binding of IC to neutrophils.

<p>Neutrophils were incubated at 4°C with suspended insoluble IC derived from untreated (a) or EndoS-treated anti-mCOL7 IgG (b) and cell-bound IC were detected by FITC-conjugated donkey-anti-rabbit IgG in flow cytometry Representative results (a, b) or mean ± s.d. of the percentage of IC-positive cells derived from 3 independent experiments (c) are shown. *indicates statistically significant differences (<i>p</i><0.001) between EndoS-treated and untreated IgG.</p

Effect of EndoS treatment of anti-mCOL7 IgG on tissue damage in an <i>ex vivo</i> model of EBA.

<p>Mouse skin cryosections were incubated with 0.2/ml rabbit-anti-mCOL7 IgG (a), or EndoS-treated 0.2 mg/ml rabbit-anti-mCOL7 IgG (b) or rabbit control IgG (c) for 1 hour at 37°C. Subsequently, specimen were exposed to freshly isolated human neutrophils. Sections of a representative experiment are shown. Arrows indicate the dermal-epidermal separation. Bar = 50 µm. Furthermore, skin separation was quantified as percentage of the length of epidermis detachment in relation to the length of the total dermal-epidermal zone (d). Data are presented as mean ± s.d. of 4 independent experiments. **indicates statistically significant differences (<i>p</i><0.01) between EndoS-treated and untreated IgG.</p

EndoS treatment of rabbit anti-mCOL7 IgG decreases neutrophil activation by immobilized immune complexes <i>in vitro</i>.

<p>Neutrophils were exposed for 1(control) or surfaces coated with IC generated from coated mCOL7 and rabbit anti-mCOL7 IgG (IC) or EndoS-treated rabbit anti-mCOL7 IgG (IC_EndoS). ROS release was quantified by chemiluminescence in the presence of luminol. Data of one representative experiment (a) are shown or given as mean ± s.d. of the integrals over one hour of 3 independent experiments (b). Neutrophil degranulation was determined by the amount of elastase (c) and lactoferrin released (d). Released proteins were determined in supernatants after 1 hour stimulation and given as percentage of their respective total amount. Data are presented as mean ± s.d. n = 3, with statically significant differences indicated by asterics (*<i>p</i><0.05 and **<i>p</i><0.001). Morphology of neutrophils (e–g) was analyzed by phase-contrast microscopy 1 hour after the stimulation. Data of one representative experiment out of 3are given.</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

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

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

Primer sequences for PCR amplification of cDNA fragments of human.

<p>F, forward primer; R, reverse primer.</p

Clinical picture, pedigree, and electropherogram of the Hailey-Hailey disease (HHD) family.

<p>Erythematous slightly scaling vesicles, papules, and plaques on the right breast, shoulder, and neck of HHD-affected 1 (<b>a</b>). Pedigree of the HHD family. Black squares represent affected males (aged 70, 39 and 35 years), white squares unaffected males (aged 4, 9 and 9 years), white circles represent unaffected women, icons with a crossing line represent family members that had passed away (<b>b</b>). ATP2C1 mutation of HHD-affected 2 shown by electropherogram at position 2355_2358 in exon 24 resulting in subsequent frameshift displayed by heterozygous mismatch (<b>c</b>) Electropherogram of the unaffected HHD- healthy 1displaying the reference allele (<b>d</b>).</p

Conservation scores and frequencies of SNPs in ATPase genes found in a Greek Family with atypical Hailey Hailey disease.

<p>Gene = Gene name for variant, ExonicFunc = synonymous, non-synonymous, indel, etc., SNV = single nucleotide variant, Aminoacid Change = variant change in nucleotide and protein format, ESP5400_ALL = MAF in Exome Sequencing Project dataset (5,400 exomes) for all populations, 1000genomes_ALL = MAF in 1000Genomes February 2012 release, dbSNP135 = RS# from the dbSNP database, AVSIFT = SIFT Pathogenicity score: closer to 0 is more damaging, LJB_PhyloP = Pathogenicity score from dbNSFP: conserved > 0.95, not conserved < 0.95, LJB_SIFT = Pathogenicity score from dbNSFP: tolerated < 0.95, deleterious > 0.95, LJB_PolyPhen2 = Pathogenicity score from dbNSFP: probably damaging > 0.85, possibly damaging 0.85–0.15, benign < 0.15, NA = not available.</p><p>Conservation scores and frequencies of SNPs in ATPase genes found in a Greek Family with atypical Hailey Hailey disease.</p

Serum autoantibody reactivity in anti-p200 pemphigoid patients with overlapping fragments of laminin γ1 covering the whole molecule.

<p>Serum autoantibody reactivity in anti-p200 pemphigoid patients with overlapping fragments of laminin γ1 covering the whole molecule.</p