Serum autoantibody reactivity in bullous pemphigoid is associated with neuropsychiatric disorders and the use of antidiabetics and antipsychotics: a large, prospective cohort study.

BACKGROUND: Bullous pemphigoid (BP), the by far most frequent autoimmune blistering skin disease (AIBD), is immunopathologically characterized by autoantibodies against the two hemidesmosomal proteins BP180 (collagen type XVII) and BP230 (BPAG1 or dystonin). Several comorbidities and potentially disease-inducing medication have been described in BP, yet a systematic analysis of these clinically relevant findings and autoantibody reactivities has not been performed. OBJECTIVE: To determine associations of autoantibody reactivities with comorbidities and concomitant medication. METHODS: In this prospective multicenter study, 499 patients diagnosed with BP in 16 European referral centers were included. The relation between anti-BP180 NC16A and anti-BP230 IgG ELISA values at the time of diagnosis as well as comorbidities and concomitant medication collected by a standardized form were analysed. RESULTS: An association between higher serum anti-BP180 reactivity and neuropsychiatric but not atopic and metabolic disorders was observed as well as with the use of insulin or antipsychotics but not with dipeptidyl peptidase-4 (DPP4) inhibitors, inhibitors of platelet aggregation and L-thyroxine. The use of DPP4 inhibitors was associated with less anti-BP180 and anti-BP230 reactivity compared with BP patients without these drugs. This finding was even more pronounced when compared with diabetic BP patients without DPP4 inhibitors. Associations between anti-BP180 and anti-BP230 reactivities were also found in patients using insulin and antipsychotics, respectively, compared with patients without this medication, but not for the use of inhibitors of platelet aggregation, and L-thyroxine. CONCLUSION: Taken together, these data imply a relation between autoantibody reactivities at the time of diagnosis and both neuropsychiatric comorbidities as well as distinct concomitant medication suggesting a link between the pathological immune mechanisms and clinical conditions that precede the clinically overt AIBD.

Updated S2 K guidelines for the management of bullous pemphigoid initiated by the European Academy of Dermatology and Venereology (EADV).

BACKGROUND: Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease of the skin and mucous membranes. This disease typically affects the elderly and presents with itch and localized or, most frequently, generalized bullous lesions. A subset of patients only develops excoriations, prurigo-like lesions, and eczematous and/or urticarial erythematous lesions. The disease, which is significantly associated with neurological disorders, has high morbidity and severely impacts the quality of life. OBJECTIVES AND METHODOLOGY: The Autoimmune blistering diseases Task Force of the European Academy of Dermatology and Venereology sought to update the guidelines for the management of BP based on new clinical information, and new evidence on diagnostic tools and interventions. The recommendations are either evidence-based or rely on expert opinion. The degree of consent among all task force members was included. RESULTS: Treatment depends on the severity of BP and patients' comorbidities. High-potency topical corticosteroids are recommended as the mainstay of treatment whenever possible. Oral prednisone at a dose of 0.5 mg/kg/day is a recommended alternative. In case of contraindications or resistance to corticosteroids, immunosuppressive therapies, such as methotrexate, azathioprine, mycophenolate mofetil or mycophenolate acid, may be recommended. The use of doxycycline and dapsone is controversial. They may be recommended, in particular, in patients with contraindications to oral corticosteroids. B-cell-depleting therapy and intravenous immunoglobulins may be considered in treatment-resistant cases. Omalizumab and dupilumab have recently shown promising results. The final version of the guideline was consented to by several patient organizations. CONCLUSIONS: The guidelines for the management of BP were updated. They summarize evidence- and expert-based recommendations useful in clinical practice.

Warfarin-induced calciphylaxis in patients with normal renal function.

WHAT IS KNOWN AND OBJECTIVE: Calciphylaxis is a rare and potentially life-threatening cause of skin necrosis and is poorly recognized by clinicians in non-uraemic patients. CASE DESCRIPTION: We report five cases of warfarin-induced calciphylaxis in patients with normal renal function. In four cases, sodium thiosulphate was successfully used as a treatment. No other predisposing factors besides obesity and warfarin were found in these patients. WHAT IS NEW AND CONCLUSION: Previously only few cases of solely warfarin-induced calciphylaxis have been described. Treatment with sodium thiosulphate has shown promising results, and there is thus a need to improve the recognition of calciphylaxis.

Increasing incidence of bullous pemphigoid in Northern Finland: a retrospective database study in Oulu University Hospital.

BACKGROUND: Bullous pemphigoid (BP) is an autoimmune blistering skin disease of elderly people. Some studies have suggested that the incidence of BP has increased, but the diagnostic accuracy and methodology of studies have varied considerably. OBJECTIVES: To examine the incidence of BP in Northern Finland, and whether the incidence has changed over time. METHODS: This was a retrospective database study of all BP cases diagnosed in the Oulu University Hospital, Finland between 1985 and 2009. The diagnostic criteria were clinical features characteristic of BP (all patients) and positive direct or indirect immunofluorescence in the skin biopsy. The age-standardized incidences were calculated by the direct standardization method. Incidence rate ratios (IRR) were estimated by the Poisson regression model. To derive adjusted IRRs, age and sex were used as potential confounding factors. RESULTS: The crude incidence of BP was 17 per 1 million person-years [95% confidence interval (CI) 15-20] between 1985 and 2009. Using the general European population as a reference, the age-standardized incidence was 14 per 1 million person-years (95% CI 12-17). The incidence of BP increased 1·8-fold (IRR 1·8, 95% CI 1·3-2·6; P < 0·001) in 2005-09 compared with the mean incidence of BP between 1985 and 2004, but after the adjustment for age and sex the increase was 1·4-fold (IRR 1·4, 95% CI 1·0-2·0; P = 0·043). CONCLUSIONS: This is the first study with immunohistologically verified BP diagnoses that reports the increase in the incidence of BP in age- and sex-adjusted populations.

Chlamydia trachomatis seroprevalence atlas of Finland 1983-2003.

OBJECTIVES: To study Chlamydia trachomatis seroprevalence trends and geographical distribution over time in Finland. MATERIALS AND METHODS: First pregnancy serum samples were retrieved from the Finnish Maternity Cohort serum bank for the subcohort of 8000 women stratified by calendar years (1983-1989, 1990-1996, 1997-2003) and age at time of sample withdrawal (14-22 and 23-28 years). C trachomatis antibodies were determined using standard major outer membrane protein peptide ELISA. The spatiotemporal variation of C trachomatis seroprevalence rates was visualised by a series of maps. RESULTS: A decreasing C trachomatis seroprevalence trend from 1983 to 2003 was seen for both women under 23 years of age (20.8% to 10.6%) and 23-28-year-old women (19.1% to 12.5%). Constant clusters were seen around the largest cities and in eastern Finland although seroprevalence rates were generally decreasing throughout the country. CONCLUSIONS: Only a few population-based serological studies have been undertaken on C trachomatis epidemiology over time. In Finland the seroprevalence of C trachomatis is decreasing all over the country, albeit with small clusters remaining.

Collagen XVII promotes integrin-mediated squamous cell carcinoma transmigration--a novel role for alphaIIb integrin and tirofiban.

The expression of collagen XVII (BP180), a transmembrane hemidesmosomal component, is upregulated in invasive areas of epithelial tumors. The collagenous ectodomain of collagen XVII is cleaved from the plasma membrane of keratinocytes and malignant epithelial cells. The released ectodomain is predicted to regulate cell detachment, differentiation, and motility. We report that the cell adhesion domain of collagen XVII, Col15, is able to chemotactically attract invasive HSC-3 SCC cells but not keratinocytes. Analysis of integrin expression revealed that HSC-3 cells, unlike keratinocytes, express alphaIIb integrin, a platelet-specific fibrinogen receptor. We show that this novel chemotactic function is mediated by the known Col15-binding integrins alpha5beta1 and alphav and the platelet integrin alphaIIb. Moreover, we report that tirofiban, a FDA-proved alphaIIb integrin-blocking drug widely used for the therapy of acute coronary ischaemic syndrome and the prevention of thrombotic complications, inhibits the Col15 chemotaxis of HSC-3 carcinoma cells. Together, these data demonstrate a novel interaction between collagen XVII and alphaIIb integrin and also suggest a possibility to use tirofiban to inhibit the invasion and progression of alphaIIb expressing SCC tumors.

Altered expression of collagen XVII in ameloblastomas and basal cell carcinomas.

BACKGROUND: Collagen XVII (BP180) is an epithelial transmembrane protein, which presumably plays a role in cell migration and differentiation under both physiological and pathological conditions. Ameloblastoma, the most common odontogenic neoplasm, and basal cell carcinoma (BCC) of the skin exhibit similar growth patterns and share histological features. METHODS: Here, we examined the distribution and expression of collagen XVII in ameloblastomas and BCCs using immunohistochemistry and non-radioactive in situ hybridization. In both tumors, the distribution of collagen XVII varied in different parts of the lesions. RESULTS: In ameloblastomas, immunostaining for collagen XVII was usually localized in the basal and suprabasal cells of the tumor nests, although in some tumors, a diffuse intracellular staining was detected in the central cells of the neoplastic islands. In BCCs, collagen XVII was mostly seen as diffuse cytoplasmic staining in some central and peripheral cells of the tumor islands and also at the cell membranes in the basal keratinocytes of the epidermis overlying the tumor nests. Double immunostaining with antibody against gamma2 chain of laminin-5 showed that these two components of the keratinocyte adhesion complex are usually co-localized in ameloblastomas and BCCs. In both tumors, collagen XVII mRNA was found in the basal epithelial cells and in some central and peripheral cells of the tumor islands, while the stromal cells were negative. CONCLUSIONS: These findings indicate that the expression of collagen XVII may be differentially regulated in various parts of the tumor. Diffuse intracellular distribution of collagen XVII and a consequent loss of critical cellular attachments may contribute to the infiltrative and progressive growing potential of tumors.

The cell adhesion domain of type XVII collagen promotes integrin-mediated cell spreading by a novel mechanism.

Type XVII collagen (BP180) is a keratinocyte transmembrane protein that exists as the full-length protein in hemidesmosomes and as a 120-kDa shed ectodomain in the extracellular matrix. The largest collagenous domain of type XVII collagen, COL15, has been described previously as a cell adhesion domain (Tasanen, K., Eble, J. A., Aumailley, M., Schumann, H., Baetge, J, Tu, H., Bruckner, P., and Bruckner-Tuderman, L. (2000) J. Biol. Chem. 275, 3093-3099). In the present work, the integrin binding of triple helical, human recombinant COL15 was tested. Solid phase binding assays using recombinant integrin alpha(1)I, alpha(2)I, and alpha(10)I domains and cell spreading assays with alpha(1)beta(1)- and alpha(2)beta(1)-expressing Chinese hamster ovary cells showed that, unlike other collagens, COL15 was not recognized by the collagen receptors. Denaturation of the COL15 domain increased the spreading of human HaCaT keratinocytes, which could migrate on the denatured COL15 domain as effectively as on fibronectin. Spreading of HaCaT cells on the COL15 domain was mediated by alpha(5)beta(1) and alpha(V)beta(1) integrins, and it could be blocked by RGD peptides. The collagen alpha-chains in the COL15 domain do not contain RGD motifs but, instead, contain 12 closely related KGD motifs, four in each of the three alpha-chains. Twenty-two overlapping, synthetic peptides corresponding to the entire COL15 domain were tested; three peptides, all containing the KGD motif, inhibited the spreading of HaCaT cells on denatured COL15 domain. Furthermore, this effect was lost by mutation from D to E (KGE instead of KGD). We suggest that the COL15 domain of type XVII collagen represents a specific collagenous structure, unable to interact with the cellular receptors for other collagens. After being shed from the cell surface, it may support keratinocyte spreading and migration.

Baseline expression and effect of TGF-beta 1 on type I and III collagen mRNA and protein synthesis in human odontoblasts and pulp cells in vitro.

Since growth factors have been suggested to regulate dentin collagen formation in response to external irritation, we investigated the effect of TGF-beta 1 on pro alpha 1 (I) collagen mRNA expression in cultured mature human odontoblasts and pulpal fibroblasts, as well as cultured human pulp tissue, using quantitative PCR. Cultured gingival fibroblasts (GF) and osteoblasts (OB) served as controls. Also, type I collagen synthesis in cultured odontoblasts and pulp tissue, as well as type III collagen synthesis in odontoblasts, were studied by measuring respective procollagen (PINP and PIIINP) secretion into culture media with radio-immunoassay (RIA). Odontoblasts expressed significantly higher basic level of type I collagen mRNA than pulp tissue or pulp fibroblasts in culture, but markedly lower level than GF and OB cells. TGF-beta 1 (10 ng/ml) had negligible effects on type I collagen mRNA expression or PINP synthesis in cultured odontoblasts and pulp tissue, and PIIINP synthesis in the odontoblasts. In PF cells, the effect of TGF-beta 1 depended on culturing conditions; a 6-fold increase in mRNA expression was observed using serum-free medium but no effect was seen in the cells cultured with 10% FBS. In contrast, GF cells serving as controls were not markedly affected by the culture conditions, with 2-3-fold increase in mRNA expression by TGF-beta 1. These experiments demonstrate that mature human odontoblasts are capable of synthesizing type III collagen protein, and that TGF-beta 1 has negligible effect on mature human odontoblast and pulp tissue collagen expression.

Baseline expression and effect of TGF-ß1 on Type I and III collagen mRNA and protein synthesis in human odontoblasts and pulp cellsIn Vitro.

Since growth factors have been suggested to regulate dentin collagen formation in response to external irritation, we investigated the effect of TGF-ß1 on proα1(I) collagen mRNA expression in cultured mature human odontoblasts and pulpal fibroblasts, as well as cultured human pulp tissue, using quantitative PCR. Cultured gingival fibroblasts (GF) and osteoblasts (OB) served as controls. Also, type I collagen synthesis in cultured odontoblasts and pulp tissue, as well as type III collagen synthesis in odontoblasts, were studied by measuring respective procollagen (PINP and PIIINP) secretion into culture media with radioimmunoassay (RIA). Odontoblasts expressed significantly higher basic level of type I collagen mRNA than pulp tissue or pulp fibroblasts in culture, but markedly lower level than GF and OB cells. TGF-ß1 (10 ng/ml) had negligible effects on type I collagen mRNA expression or PINP synthesis in cultured odontoblasts and pulp tissue, and PIIINP synthesis in the odontoblasts. In PF cells, the effect of TGF-ß1 depended on culturing conditions; a 6-fold increase in mRNA expression was observed using serum-free medium but no effect was seen in the cells cultured with 10% FBS. In contrast, GF cells serving as controls were not markedly affected by the culture conditions, with 2-3-fold increase in mRNA expression by TGF-ß1. These experiments demonstrate that mature human odontoblasts are capable of synthesizing type III collagen protein, and that TGF-ß1 has negligible effect on mature human odontoblast and pulp tissue collagen expression.

Hemizygosity for a glycine substitution in collagen XVII: unfolding and degradation of the ectodomain.

Defects of collagen XVII, a keratinocyte adhesion protein, are associated with epidermal detachment in junctional epidermolysis bullosa. Although some missense mutations in the collagen XVII gene COL17A1 have been described, the molecular mechanisms leading to disease have remained elusive in these cases. Here we assessed the biologic consequences of a missense mutation by studying the folding and stability of wild-type and mutated recombinant collagen XVII domains. The mutation occurred in a junctional epidermolysis bullosa patient who was compound heterozygous for the novel glycine substitution mutation G633D and the novel nonsense mutation R145X. Collagen XVII mRNA was significantly reduced, indicating nonsense-mediated mRNA degradation and hemizygosity of the patient for the G633D substitution. As glycine residues within the collagen triple helices are important for stable conformation, the thermal stability of the wild-type and mutated eukaryotic recombinant Col15 domain of collagen XVII was assessed. The stability of the mutated fragment was clearly reduced. The midpoint of the helix-to-coil transition, Tm, was 5 degrees C lower than that of wild-type rCol15, indicating abnormal triple-helix folding and susceptibility to proteolysis. Consistently, immunoassays demonstrated reduced amounts of the full-length collagen XVII and absence of the soluble ectodomain in keratinocyte cultures, and lack of the ectodomain from the junctional epidermolysis bullosa skin. These observations show that the glycine substitution G633D in collagen XVII causes abnormal folding and susceptibility to degradation, and thus perturbs the physiologic adhesive functions of collagen XVII in the skin.

Collagen XVII is destabilized by a glycine substitution mutation in the cell adhesion domain Col15.

Collagen XVII is a hemidesmosomal transmembrane molecule important for epithelial adhesion in the skin. It exists in two forms, as a full-length protein and as a soluble ectodomain that is shed from the keratinocyte surface by furin-mediated proteolysis. To obtain information on the conformation and the functions of this unusual collagen, its largest collagenous domain, Col15, was expressed in a eukaryotic episomal expression system and purified by DEAE and fast protein liquid- Mono S chromatography. The protein was triple-helical (T(m) of 26.5 degrees C) when produced in cultures containing ascorbic acid. When the vitamin supply was limited, the 4-hydroxyproline content was reduced from 74 to 9%, which, in turn, resulted in a drastic reduction of the stability of the triple helix. The glycine substitution mutation G627V associated with junctional epidermolysis bullosa, a human blistering skin disease, also had a striking effect on thermal stability of rCol15 causing partial unfolding already at 4 degrees C. Col15 promoted cell adhesion of epithelial and fibroblastic cell lines with a beta1 integrin-mediated mechanism. In concert with this, in acquired autoimmune blistering skin diseases, circulating IgG and IgA autoantibodies were found to target rCol15r.

The shed ectodomain of collagen XVII/BP180 is targeted by autoantibodies in different blistering skin diseases.

Collagen XVII/BP180, an epidermal adhesion molecule, exists as a full-length transmembrane protein and as a soluble 120-kd ectodomain that is shed from the keratinocyte surface by furin-mediated proteolysis. Despite a number of studies on autoantibody targets in blistering skin diseases, it has remained unclear whether the physiologically shed ectodomain of collagen XVII plays a role as an autoantigen. Here we isolated the authentic, soluble form of human collagen XVII and showed that it is an autoantigen recognized by IgG and IgA autoantibodies in different blistering skin diseases and is, in some cases, the preferential target. The ectodomain was isolated from the epidermis, keratinocyte media, amniotic fluid, and pemphigoid blister fluid, and autoantibodies affinity-purified with this ectodomain bound to the proximal surface of the epidermis in normal skin but not in collagen XVII-deficient skin. The antibody reactivity was not dependent on the native conformation or the N-glycosylation of the soluble ectodomain, but was abolished by collagenase treatment. Sera of 81 patients with a clinically active blistering skin disease were reacted with full-length collagen XVII, the authentic soluble ectodomain, and recombinant fragments. In bullous and cicatricial pemphigoid, IgG reactive with full-length collagen XVII also recognized the soluble ectodomain. In linear IgA dermatosis and chronic bullous dermatosis of childhood, IgA targeted the soluble ectodomain more efficiently than the full-length protein. The use of recombinant fragments demonstrated that epitopes were present in several noncollagenous and collagenous subdomains of the molecule, and that a significant portion of the sera targeted Col15 domain, a hitherto unrecognized epitope region.

A child with localized vulval pemphigoid and IgG autoantibodies targeting the C-terminus of collagen XVII/BP180.

Localized vulval pemphigoid of childhood (LVPC) has previously been reported in six girls. Clinical features and immunopathological data have suggested it to be a morphological variant of bullous pemphigoid. Epitope targets of the autoantibodies of these patients have not been defined in detail. We describe a 9-year-old girl with possible cicatricial LVPC and circulating IgG antibodies directed against native collagen XVII/BP180, its 120-kDa soluble ectodomain and against the C-terminus of collagen XVII/BP180. No reactivity was detected towards the NC16A domain of collagen XVII/BP180. Linear IgG and C3 deposits were found along the cutaneous basement membrane zone. On 1 mol/L salt-split skin, IgG autoantibodies were shown to bind to the epidermis, and the HLA type II allele DQB1*0301, a marker with significantly increased occurrence in patients with ocular and oral cicatricial pemphigoid, was identified in this patient. Our data suggest that LVPC is a variant of bullous pemphigoid in which direct immunofluorescence microscopy combined with immunoblot analysis can deliver valuable diagnostic information for differential diagnosis. However, differentiation between the scarring and non-scarring course of the disease cannot be made with the present diagnostic markers and therefore careful follow-up of patients with LVPC is required.

Demonstration of increased levels of type I collagen mRNA using quantitative polymerase chain reaction in fibrotic and granulomatous skin diseases.

Collagen changes occur in localized scleroderma, scleredema and sarcoidosis. Previous biochemical, immunohistochemical and in situ hybridization studies have revealed increased collagen synthesis in these diseases. In the present study, we measured by pro alpha 1 (I) collagen and beta-actin mRNA levels in skin punch biopsy specimens from lesional and healthy skin using a quantitative polymerase chain reaction (PCR). In this method, the targeted mRNA and a synthetic RNA as a internal standard are co-amplified together with the same primers. The amount of pro alpha 1 (I) collagen mRNA in cutaneous sarcoidosis lesions was found to be increased about two- to threefold compared with the values obtained for the healthy skin of the same two patients. In lesional skin of three patients with localized scleroderma the number of pro alpha 1 (I) collagen molecules was increased about two-fold. The beta-actin mRNA values were at the same level in the affected and unaffected skin of all the patients studied. In conclusion, a marked increase in type I collagen gene expression was seen in localized scleroderma and scleredema, leading to fibrosis of the skin, and in a granulomatous skin disease, cutaneous sarcoidosis.

The molecular basis of glucocorticoid-induced skin atrophy: topical glucocorticoid apparently decreases both collagen synthesis and the corresponding collagen mRNA level in human skin in vivo.

The effects of topical betamethasone-17-valerate on collagen propeptide levels, collagen mRNA level, lysyl oxidase mRNA and matrix metalloproteinase (MMP)-1 and MMP-2 mRNA levels were studied in human skin. Three days' treatment of healthy skin with topical betamethasone caused a 70-80% decrease in type I and III collagen propeptides in suction blister fluid. A similar decrease was found in type I collagen mRNA when assayed by either slot-blot hybridization or a quantitative polymerase chain reaction method, indicating that the decrease in collagen synthesis after topical glucocorticoid treatment is apparently due to a decrease in corresponding mRNA. mRNA of lysyl oxidase, which is an important enzyme catalysing the cross-linking of collagen chains, and collagen-degrading enzyme MMP-1 and MMP-2 mRNAs were not decreased in the same skin samples. This suggests that in vivo, glucocorticoids modulate variably the genes involved in collagen synthesis and degradation. Our study provides a solid molecular basis for glucocorticoid-induced dermal atrophy, which results from the decrease in functional collagen mRNA in the skin.