Andreas Recke

Novel ELISA systems for antibodies to desmoglein 1 and 3: correlation of disease activity with serum autoantibody levels in individual pemphigus patients

Please cite this paper as: Novel ELISA systems for antibodies to desmoglein 1 and 3: correlation of disease activity with serum autoantibody levels in individual pemphigus patients. Experimental Dermatology 2010; 19: 458–463.

Incidence and causes of heparin-induced skin lesions

Background: Little is known about the incidence and causes of heparin-induced skin lesions. The 2 most commonly reported causes of heparin-induced skin lesions are immune-mediated heparin-induced thrombocytopenia and delayed-type hypersensitivity reactions. Methods: We prospectively examined consecutive patients who received subcutaneous heparin (most often enoxaparin or nadroparin) for the presence of heparin-induced skin lesions. If such lesions were identified, we performed a skin biopsy, platelet count measurements, and antiplatelet-factor 4 antibody and allergy testing. Results: We enrolled 320 patients. In total, 24 patients (7.5%, 95% confidence interval [CI] 4.7%–10.6%) had heparin-induced skin lesions. Delayed-type hypersensitivity reactions were identified as the cause in all 24 patients. One patient with histopathologic evidence of delayed-type hypersensitivity tested positive for antiplatelet-factor 4 antibodies. We identified the following risk factors for heparin-induced skin lesions: a body mass index greater than 25 (odds ratio [OR] 4.6, 95% CI 1.7–15.3), duration of heparin therapy longer than 9 days (OR 5.9, 95% CI 1.9–26.3) and female sex (OR 3.0, 95% CI 1.1–8.8). Interpretation: Heparin-induced skin lesions are relatively common, have identifiable risk factors and are commonly caused by a delayed-type hypersensitivity reaction (type IV allergic response). (ClinicalTrials.gov trial register no. NCT00510432.)

Development of ELISA for the specific determination of autoantibodies against envoplakin and periplakin in paraneoplastic pemphigus

INTRODUCTION In paraneoplastic pemphigus (PNP), indirect immunofluorescence microscopy using rat bladder sections is widely used to search for circulating autoantibodies which are predominantly directed against envoplakin and periplakin. A sensitive and specific detection system for envoplakin/periplakin-specific autoantibodies is not yet available. METHODS Overlapping fragments spanning envoplakin and periplakin, respectively, were analyzed for their ability to bind PNP-specific autoantibodies by immunoblotting and ELISA in sera from patients with PNP (n=31), pemphigus vulgaris (n=30), and bullous pemphigoid (n=50) as well as healthy volunteers (n=140). The results were compared with those obtained by immunoblotting of extract of cultured human keratinocytes. RESULTS Immunoblot analysis revealed that most sera contained antibodies against the N-termini of both plakins as well as against the C-terminus of envoplakin. By ELISA, reactivities against envoplakin(1-481), envoplakin(1626-2033), and periplakin(1-324) were found in 25 (80.6%), 25 (80.6%), and 23 (74.2%) PNP sera, and in 1 (1.2%), 3 (3.7%), and 2 (2.5%) control sera, respectively. CONCLUSIONS The new ELISA based on an N-terminal fragment of envoplakin shows a high diagnostic accuracy to detect circulating autoantibodies in PNP. It is easy to be setup and standardized and can help to differentiate PNP patients from those with pemphigus vulgaris.

Generation of antibodies of distinct subclasses and specificity is linked to H2s in an active mouse model of epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease, characterized by antibodies to type VII collagen (COL7). EBA can be induced in mice by immunization with a fragment of the non-collagenous 1 domain of murine COL7. Contrary to other autoimmune diseases, e.g., rheumatoid arthritis, little is known about the genetic susceptibility for EBA. We therefore used the EBA mouse model to address the hypothesis that disease induction depends on the major histocompatibility complex (MHC) haplotype. Mice from different inbred strains were immunized with recombinant murine COL7. Five distinct responses were observed: induction of (i) severe disease in SJL/J (H2s) and female MRL/MpJ (H2k), (ii) mild and transient disease in C57Bl/10.s (H2s), (iii) microscopic blistering in DBA/1J (H2q), (iv) only presence of non-pathogenic autoantibodies in C57Bl/6J (H2b), NZM2410/J (H2z), BXD2 (H2b), and male MRL/MpJ, and (v) complete resistance to EBA in NOD/ShiLtJ (H2g7) and C57Bl/10.q (H2q) mice. Overall, susceptibility to EBA was strongly associated with H2s. In addition, the diseased phenotype was associated with autoantibodies to specific regions of COL7. Our findings show that induction of antibodies with a distinct specificity is linked to the MHC haplotype in experimental EBA. Furthermore, our data are the basis for future studies with the goal of identifying non-MHC EBA susceptibility genes.

Development of a simple enzyme-linked immunosorbent assay for the detection of autoantibodies in anti-p200 pemphigoid.

Background  Anti‐p200 pemphigoid is a subepidermal blistering skin disease characterized by autoantibodies against a 200‐kDa protein (p200) of the dermal–epidermal junction. The laminin γ1 chain has recently been identified as target antigen in this disease and the C‐terminus was described as an immunodominant region of laminin γ1. Diagnosis of anti‐p200 pemphigoid requires detection of serum IgG at the dermal side of 1 mol L−1 salt‐split skin by indirect immunofluorescence microscopy and labelling of a 200‐kDa protein by Western blotting of dermal extract. However, preparation of dermal extract is not widely available, limiting the possibility of diagnosing this disease to a few laboratories.

Repetitive Immunization Breaks Tolerance to Type XVII Collagen and Leads to Bullous Pemphigoid in Mice

Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease of the elderly associated with considerable morbidity and mortality. As unspecific immunosuppressants are still the mainstay of BP therapy, several animal models, based on the passive transfer of autoantibodies or immune cells, have been developed to obtain a better understanding of the pathogenesis of BP and evaluate novel therapeutic interventions. We describe in this study an experimental model inducing BP by immunization of immunocompetent mice with a recombinant form of the immunodominant 15th noncollagenous domain of murine BP180 (type XVII collagen). The homologous noncollagenous 16A domain of human BP180 has previously been identified as an immunodominant region in human BP. Immunization of female SJL/J mice with the murine peptide led to clinical disease within 14 wk in 56% of mice. In contrast, none of the other strains developed blisters despite the presence of autoantibodies. The clinical disease manifested for at least 8 wk without further manipulation. This novel immunization-induced model reflects key immunopathological characteristics of human BP, including binding of complement-fixing autoantibodies along the dermal–epidermal junction, elevated total IgE serum levels, and infiltration of skin lesions with eosinophilic granulocytes. The use of immunocompetent mice and the induction of sustained clinical disease not requiring additional interventions make this immunization-induced mouse model most suitable to further explore the pathogenesis of BP and novel therapeutic interventions for this and other autoantibody-mediated diseases.

Catheter-directed Foam Sclerotherapy of Great Saphenous Veins in Combination with Pre-treatment Reduction of the Diameter Employing the Principals of Perivenous Tumescent Local Anesthesia

OBJECTIVES The aim of this study was to evaluate occlusion rates of great saphenous veins (GSV) with a diameter between 5-10 mm that received a pre-treatment size reduction via perivenous tumescent application (TA) followed by catheter-directed foam sclerotherapy (CDFS). METHODS A prospective blinded randomized clinical trial comparing the occlusion rates of GSV at 1-, 6-, and 12-month follow-up. Fifty patients were included and randomized into two groups. CDFS was performed accessing the GSV at knee level and applying 8 mL of 2% polidocanol-foam (EasyFoam) while the catheter was withdrawn. Strictly perivenous TA was performed in group 1 before applying the sclerosant agent. Occlusion rates and clinical scores were assessed by blinded examiners. RESULTS After 12 months in group 1 full occlusion was achieved in 73.9%, partial occlusion in 8.7%, and 17.4% were classified as treatment failure. In group 2, 75% of the targeted GSV were fully occluded, 20% were partially occluded, and 5% were diagnosed as treatment failure. Both groups showed a significant reduction of the vein diameter. Patients tolerance and satisfaction with the treatment was high in both groups. CONCLUSION No benefit could be found using additional TA to reduce the vein diameter before the treatment.

Autoantibody-induced intestinal inflammation and weight loss in experimental epidermolysis bullosa acquisita.

Type VII collagen (COL7) is a major constituent of the cutaneous basement membrane. Loss of tolerance to COL7 leads to the blistering skin disease epidermolysis bullosa acquisita (EBA). Antibodies to COL7 have also been detected in patients with inflammatory bowel disease (IBD), yet reports on the expression of COL7 in the gut are controversial and a pathogenic relevance of anti‐COL7 autoantibodies in IBD has not been demonstrated. We therefore characterized the expression patterns of COL7 in murine gastrointestinal organs and investigated if anti‐COL7 antibodies induce an inflammatory response in the gut. COL7 expression was analysed on the mRNA and protein levels. Mice were injected with rabbit anti‐murine COL7 IgG (passive EBA) or immunized with a fragment of murine COL7 (active EBA). COL7 was found to be expressed in buccal mucosa, oesophagus, stomach, small intestine, and colon. In addition to skin blistering, in both passive and active EBA, autoantibodies bound to the gastrointestinal basement membrane, fixed complement, and led to recruitment of leukocytes. Furthermore, blister formation was observed in the oesophagus (40%/38% of mice in passive/active model), stomach (40%/63%), small intestine (20%/13%), and colon (20%/13%). Compared to control animals, we found a significantly reduced body weight in diseased mice, suggesting that autoantibody‐induced gastrointestinal inflammation is clinically relevant. Those observations may help us to understand the co‐incidence of IBD with EBA, and vice versa: The inflammatory response in IBD might expose novel antigens (COL7), which leads to the formation of anti‐COL7 antibodies. On the contrary, anti‐COL7 antibody‐induced gastrointestinal inflammation might pave the way for IBD pathogenesis. In summary, our results provide strong evidence that COL7 is expressed in different portions of the gut and that anti‐COL7 antibodies induce distinct gastrointestinal tissue damage. Copyright

Methylprednisolone Blocks Autoantibody-Induced Tissue Damage in Experimental Models of Bullous Pemphigoid and Epidermolysis Bullosa Acquisita through Inhibition of Neutrophil Activation

Corticosteroids are regularly used to treat autoimmune diseases, such as bullous pemphigoid (BP). In BP, autoantibodies bind to type XVII collagen (COL17), located at the dermal-epidermal junction. A crucial role of neutrophils in experimental BP has been established. Specifically, reactive oxygen species and proteolytic granule enzymes mediate tissue injury. Therefore, we investigated the effects of methylprednisolone (MP) on neutrophils, which are likely to be affected by topical treatment. First, MP inhibited dermal-epidermal separation ex vivo in cryosections of the human skin induced by co-incubation of BP autoantibodies with neutrophils from healthy volunteers. Next, MP inhibited neutrophil activation in vitro induced by immune complexes (ICs) of COL17 and autoantibodies. This neutrophil activation was associated with phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and Akt. In turn, inhibition of ERK1/2, p38 MAPK, or Akt phosphorylation inhibited neutrophil activation by IC in vitro and dermal-epidermal separation ex vivo. In addition, we observed an increase of p38 MAPK phosphorylation in dermal infiltrates of BP patients. Treatment of mice with either MP or inhibitors of p38-MAPK or ERK1/2 phosphorylation impaired induction of autoantibody- or irritant-induced neutrophil-dependent inflammation. We here identify the inhibition of Akt, ERK1/2, and p38 MAPK phosphorylation as molecular mechanisms to promote MPs therapeutic effects.

IgE autoantibodies against the intracellular domain of BP180

Background  Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by autoantibodies against the hemidesmosomal proteins BP180 (type XVII collagen) and BP230. BP not only involves IgG‐mediated neutrophil activation, leading to blistering, but also IgE‐dependent activation of mast cells and basophils. While IgG and IgE autoantibodies target the extracellular noncollagenous (NC) 16A domain of BP180, little is known whether other BP180 regions are targeted by these antibody classes.