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Increased prevalence of autoantibody Fab glycosylation has been demonstrated for several autoimmune diseases. To study whether elevated Fab glycosylation is a common feature of autoimmunity, this study investigated Fab glycosylation levels on serum IgG and its subclasses for autoantibodies associated with a range of different B cell–mediated autoimmune diseases, including rheumatoid arthritis, myasthenia gravis subtypes, pemphigus vulgaris, antineutrophil cytoplasmic antibody–associated vasculitis, systemic lupus erythematosus, anti–glomerular basement membrane glomerulonephritis, thrombotic thrombocytopenic purpura, and Guillain-Barré syndrome. The level of Fab glycosylated IgG antibodies was assessed by lectin affinity chromatography and autoantigen-specific immunoassays. In 6 of 10 autoantibody responses, in 5 of 8 diseases, the investigators found increased levels of Fab glycosylation on IgG autoantibodies that varied from 86% in rheumatoid arthritis to 26% in systemic lupus erythematosus. Elevated autoantibody Fab glycosylation was not restricted to IgG 4 , which is known to be prone to Fab glycosylation, but was also present in IgG 1. When autoimmune diseases with a chronic disease course were compared with more acute autoimmune illnesses, increased Fab glycosylation was restricted to the chronic diseases. As a proxy for chronic autoantigen exposure, the investigators determined Fab glycosylation levels on antibodies to common latent herpes viruses, as well as to glycoprotein 120 in individuals who are chronically HIV-1–infected. Immunity to these viral antigens was not associated with increased Fab glycosylation levels, indicating that chronic antigen-stimulation as such does not lead to increased Fab glycosylation levels. These data indicate that in chronic but not acute B cell–mediated autoimmune diseases, disease-specific autoantibodies are enriched for Fab glycans. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: The variable clinical severity of mucous membrane pemphigoid (MMP) often leads to diagnostic and therapeutic delays.Objective: To describe the characteristics of a large cohort of patients with MMP.Methods: A retrospective review of clinical and diagnostic characteristics as well as treatment responses in 145 patients with MMP.Results: Monosite involvement was seen in 41.4% and multisite involvement in 58.6% of the patients. The oral mucosa was affected in 86.9% of the patients, followed by the ocular mucosa (30.3%), skin (26.2%), genital mucosa (25.5%), nasal mucosa (23.4%), and pharyngeal and/or laryngeal mucosa (17.2%). Ocular disease developed during the disease course in 41.7% of patients with initially other mucosal site involvement. The malignancy rate was significantly higher in patients with autoantibodies against laminin-332 than in patients with MMP without laminin-332 autoantibodies (35.3% vs 10.9%, respectively; P = .007). Systemic immunosuppressive or immunomodulatory therapy was administered to 77.1% of the patients, mainly to patients with multisite (P < .001), ocular (P < .001), and pharyngeal and laryngeal involvement (P = .002). The remaining patients (22.9%) received topical therapy. Adverse events were frequently reported.Limitations: Retrospective design.Conclusion: Patients with MMP present with a heterogeneous clinical presentation, and new symptoms may develop during the disease course. Cancer screening should be considered for patients with MMP and, in particular, for those with autoantibodies against laminin-332. [ABSTRACT FROM AUTHOR]

Molecular biological techniques were used to investigate revertant mosaicism observed in a patient with generalized atrophic benign epidermolysis bullosa. The results confirmed that the clinical condition is caused by mitotic gene conversion of one of the two mutated COL17A1 alleles. Such reverse mutation was shown to be due to the nonreciprocal transfer of a part of one parental allele for the other.

Background: Nonbullous pemphigoid is an under-recognized phenotype of the autoimmune bullous disease pemphigoid, characterized by the absence of blisters. Several disease aspects have not been studied previously.Objective: To describe the characteristics of nonbullous pemphigoid.Methods: A retrospective review study of medical records. The diagnosis of pemphigoid was based on meeting 2 of the following 3 criteria: (1) pruritus, (2) positive direct immunofluorescence microscopy, or (3) positive indirect immunofluorescence microscopy on salt-split skin.Results: The review included 69 patients. The mean delay in diagnosis was 29 months. Skin examination most often showed pruritic papules/nodules (37%) or pruritus without primary skin lesions (22%). Histopathologic findings were mainly nonspecific. Results of direct and indirect immunofluorescence microscopy were positive in 60% and 69%, respectively. During follow-up, blisters formed in 17%, which was associated with a positive indirect immunofluorescence microscopy (P = .014) and a positive BP180 immunoblot result (P = .032). The Kaplan-Meier estimates of mortality at 1, 2, and 3 years were 14%, 34%, and 46%, respectively, with an 8.6-fold increased all-cause mortality risk.Limitations: The retrospective study design.Conclusions: Nonbullous pemphigoid presented with heterogeneous pruritic skin lesions, resulting in delayed diagnosis. Direct and indirect immunofluorescence microscopy are essential to diagnose nonbullous pemphigoid, in contrast to histopathology, mainly showing nonspecific findings. An increased all-cause mortality risk was observed during follow-up. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented regarding the structure of epidermis in patients with pemphigus foliaceus (PF) and found widening of intercellular between the desmosomes in Nikolsky-positive (N+) PF skin.

A letter to the editor on the role mutations of PLEC1 genes in adult-onset dilated cardiomyopathy in epidermolysis bullosa simplex with muscular dystrophy is presented.

This study characterized the high molecular mass BP180 complex that is observed when unheated sodium dodecyl sulfate extracts of human skin or keratinocytes are subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. In heated extracts BP180 is present as a monomer with a molecular weight of 180 kDa, in unheated extracts BP180 runs at a molecular weight position over 500 kDa. By preincubating the unheated extracts at temperatures between 31°C and 40°C, the high molecular weight complex could be “melted” down to monomeric BP180. Under the conditions employed the T1/2 of the dissociation process was between 35°C and 36°C. The temperature resistance of the high molecular weight complex was used to analyze its molecular composition by performing two-dimensional electrophoresis with a “low-temperature” first dimension step and a “high-temperature” second dimension step. Silver staining and immunoblotting of the two-dimensional gels revealed the high molecular weight complex to be composed of solely BP180, indicating that the complex is the nondissociated homotrimeric form of BP180. The 120 kDa linear IgA dermatosis antigen (LAD-1) is an collagenous anchoring filament protein with homology to the extracellular collagenous part of BP180. Two-dimensional immunoblotting showed that LAD-1, as BP180, is also present as a high molecular mass complex and does not form mixed complexes with BP180. [ABSTRACT FROM AUTHOR]

Circulating IgG from a large subset of bullous pemphigoid (BP) patients reacted on immunoblot with a 120-kDa protein in conditioned keratinocyte culture medium and in keratinocyte cell extracts. A protein with a similar molecular weight was recognized by circulating IgA from a subset of patients with linear IgA dermatosis (LAD). Both affinity-purified 120- kDa-specific BP IgG and 120-kDa-specific LAD IgA bound to the roof of salt-split skin. Both proteins recognized are collagenous glycoproteins. Deglycosylation with N-glycosidase F resulted in an identical reduction in molecular weight for both the BP-IgG- recognized protein and the LAD-IgA-recognized protein. Both proteins were equally susceptible to digestion with type VII collagenase. Furthermore, both proteins were absent from conditioned culture medium of keratinocytes from patients with BP180 deficient general atrophic benign epidermolysis bullosa (GABEB). Immunodepletion studies showed that the 120-kDa LAD antigen could be removed from conditioned culture medium by anti-120-kDa BP IgG. Thus these results indicate that these proteins are either highly related or, most probably, identical. A strong antigenic relationship between the 120-kDa protein and the 180-kDa bullous pemphigoid antigen (BP180) was detected by cross-reaction of affinity-purified anti-120- kDa BP patient antibodies to BP180 and cross-reaction of monoclonal anti-180-kDa antibodies to the 120-kDa protein. Notwithstanding this cross-reactivity, the 120- kDa protein also exhibits unique epitopes demonstrated by the nonreactivity of individual anti-120-kDa BP and LAD patient serum with the 180-kDa antigen. [ABSTRACT FROM AUTHOR]

Background: Direct immunofluorescence (DIF) microscopy of a skin biopsy specimen is the reference standard for the diagnosis of pemphigoid diseases (PDs). Serration pattern analysis enables the differentiation of epidermolysis bullosa acquisita (EBA) from other PDs using DIF microscopy alone. However, practice gaps need to be addressed in order to implement this technique in the routine diagnostic procedure.Objective: We sought to determine and optimize the technical requirements for serration pattern analysis of DIF microscopy and determine interrater conformity of serration pattern analysis.Methods: We compared serration pattern analysis of routine DIF microscopy from laboratories in Groningen, The Netherlands and Lübeck, Germany with 4 blinded observers. Skin biopsy specimens from 20 patients with EBA and other PDs were exchanged and analyzed. Various factors were evaluated, including section thickness, transport medium, and biopsy specimen processing.Results: The interrater conformity of our 4 observers was 95.7%. Recognition of serration patterns was comparable in samples transported in saline and in Michel's medium and with section thicknesses of 4, 6, and 8 μm.Limitations: Limitations include our small sample size and the availability of 20 samples that were compared retrospectively.Conclusion: DIF serration pattern analysis is not restricted by variation in laboratory procedures, transport medium, or experience of observers. This learnable technique can be implemented as a routine diagnostic method as an extension of DIF microscopy for subtyping PD. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented regarding the use of immunofluorescence antigen mapping of the skin which is a helpful technique in determining the level of blister formation and identifying the deficient protein.

A patient with recessive epidermolysis bullosa simplex due to a previously described homozygous KRT14 1842–2A→C splice-site mutation was re-examined, because we unexpectedly found signs of revertant mosaicism. The germline mutation resulted in different aberrant transcripts containing premature termination codons, all leading to truncated keratin 14 proteins. Basal keratinocytes in skin and in culture completely lacked keratin 14 and intermediate filaments. From this keratin 14–/– patient we started cultures from a new skin biopsy and here, we serendipitously found keratinocytes that spontaneously expressed keratin 14. This biopsy had been taken from an area of skin that was clinically affected, because blisters could simply be evoked by gentle rubbing. Immunofluorescence and electron microscopy of additional biopsies from this skin area revealed a mosaic expression of keratin 14 and reappearance of intermediate filaments in basal keratinocytes. Immunoblotting showed a revertant keratin 14 polypeptide with seemingly normal molecular weight. DNA analysis of exon 2 and its flanking intron borders showed no additional mutations in the genomic KRT14 sequence. Analysis of mRNA isolated from mosaic skin keratinocytes revealed an additional in-frame transcript (1844T→G, 1845Δ6) that codes for an abnormal keratin 14 polypeptide with a two residue deletion and one amino acid change. The re-expression of a revertant, albeit abnormal, keratin 14 polypeptide, so-called partial revertant mosaicism, accounts for the antibody staining pattern and for the reappearance of intermediate filaments, which however, are semifunctional and not able to revert the clinical phenotype. The combination of a keratin 14-positive and a keratin 14-negative cell population in epidermis as well as in cultured keratinocytes suggests that the cellular reversion might be caused by an endogenous factor. We hypothesize that a second somatic modulating... [ABSTRACT FROM AUTHOR]

We studied a kindred with recessive epidermolysis bullosa simplex in which the affected members lacked expression of the basal cell keratin 14. The patients had severe generalized skin blistering that improved slightly with age. The basal cells of the patients did not express keratin 14 and contained no keratin intermediate filaments. The expression of keratin 5, the obligate copolymer of keratin 14, was slightly reduced. The expression of keratin 15, the alternative basal cell keratin, was increased, suggesting upregulation or stabilization to compensate for the lack of keratin 14. The expression of keratin 16, keratin 17, and keratin 19 in the patient's skin was not different from controls. Immunoelectron microscopy showed a loose network of keratin 5/keratin 15 protofilaments in the basal cells. Keratin 15 filaments did not aggregate into higher order bundles. Sequence analysis of genomic DNA revealed a homozygous mutation in the 3'-acceptor splice site of intron 1 (1840 A → C) in the affected individuals. This mutation led to the skipping of exon 2 in 24% of the KRT14 transcripts and to the use of a cryptic splice site in 76% of the transcripts. Premature termination codons were generated in all transcripts (codons 175+1 or 175+29), leading to a truncated keratin 14 protein within the helical 1B rod domain. The disorder was associated with Circumscribed hyperkeratotic lesions with the histology of epidermolytic hyperkeratosis. The prognosis of keratin 14 ablation is much better in the human than in the mouse. [ABSTRACT FROM AUTHOR]

To assess whether a panel of serum pemphigoid autoantibody tests could be used to confirm an immunopathologic diagnosis of mucous membrane pemphigoid (MMP) in direct immunofluorescent negative (DIF–) MMP patients. Prospective cross-sectional study. Seventy-six patients with multisite MMP with 45 matched control participants. Enzyme-linked immunosorbent assays (ELISAs) for BP180 and BP230 (MBL International), immunoglobulin A (IgA) A and immunoglobulin G indirect immunofluorescence (IIF) on human salt-split skin and the keratinocyte footprint assay for anti–laminin 332 antibodies. Sensitivity and specificity of autoantibody detection and significant differences for individual tests and test combinations for MMP involving different sites. All DIF– patients (24/73 [31.8%]) had either ocular-only disease or ocular involvement in multisite disease. Serum pemphigoid autoantibodies were detected in 29 of 76 MMP patients (38.2%) compared with 3 of 45 control participants (6.7%). Autoantibody reactivity detected by any 1 or more of the tests was present in 6 of 24 DIF– patients (25%) compared with 22 of 49 DIF positive (DIF+) patients (44.9%). Ocular-only MMP serum reactivity was not significantly different for any test or test combination compared with control participants, whereas DIF– multisite ocular MMP differed for 1 ELISA and 3 of 7 test combinations. By contrast, for DIF+ nonocular MMP patients, all the individual tests, apart from IgA IIF, and all test combinations were significantly different compared with those for control participants. For the entire MMP cohort, the sensitivity of all individual tests was low, having a maximum of 21.05% for BP180 reactivity but increasing to 38.16% for an optimal test combination. Disease activity was associated strongly with positive serologic findings. Pemphigoid serum autoantibody tests did not provide immunopathologic evidence of MMP in ocular-only MMP patients but showed limited value in DIF– multisite ocular MMP patients. The requirement for immunopathologic confirmation of MMP by autoantibody detection is inappropriate for DIF– ocular-only MMP patients, resulting in missed diagnoses, delayed therapy, and poor outcomes. Alternative diagnostic criteria for ocular-only MMP are required to exclude the other causes of scarring conjunctivitis until more sensitive and specific immunopathologic tests become available. [ABSTRACT FROM AUTHOR]

Large-scale electron microscopy ('nanotomy') allows straight forward ultrastructural examination of tissue, cells, organelles, and macromolecules in a single data set. Such data set equals thousands of conventional electron microscopy images and is freely accessible (www.nanotomy.org). The software allows zooming in and out of the image from total overview to nanometer scale resolution in a 'Google Earth' approach. We studied the life-threatening human autoimmune blistering disease pemphigus, using nanotomy. The pathomechanism of cell-cell separation (acantholysis) that underlies the blistering is poorly understood. Ultrastructural examination of pemphigus tissue revealed previously unreported findings: (i) the presence of double-membrane structures between cells in all pemphigus types; (ii) the absence of desmosomes around spontaneous blisters in pemphigus foliaceus (PF); (iii) lower level blistering in PF when force induced; and (iv) intercellular widening at non-acantholytic cell layers. Thus, nanotomy delivers open-source electron microscopic maps of patient tissue, which can be analyzed for additional anomalies from any computer by experts from different fields. [ABSTRACT FROM AUTHOR]

Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease of the skin and mucous membranes, characterized by autoantibodies against type VII collagen (COL7), a major component of anchoring fibrils. Different clinical EBA phenotypes are described, including mechanobullous and inflammatory variants. Most EBA patients' sera react with epitopes located within the non-collagenous 1 (NC1) domain of human COL7. However, it has remained unclear whether antibody binding to these different epitopes is pathogenically relevant. To address this issue, we generated recombinant proteins covering the entire NC1 domain. IgG reactivity with these proteins was analyzed in sera of 69 EBA patients. Most recognized clusters of epitopes throughout the NC1 domain. No correlation was detected between antibody specificity and clinical phenotype. To study the pathogenicity of antibodies specific to different NC1 subdomains, rabbit antibodies were generated. All these antibodies caused dermal-epidermal separation ex vivo. Antibodies against two of these subdomains were injected into mice carrying null mutations of mouse COL7 and the human COL7 transgene and induced subepidermal blisters. We here document that autoantibodies to COL7, independent of the targeted epitopes, induce blisters both ex vivo and in vivo. In addition, using COL7-humanized mice, we provide in vivo evidence of pathogenicity of autoantibodies binding to human COL7. [ABSTRACT FROM AUTHOR]

The article offers information on a research related to revertant cell therapy for the long term survival of type XVII collagen revertant cells. Topics of discussion included were presence of revertant mosaicism in genetic disorders like epidermolysis bullosa (EB), case of revertant mosaicism in skin in a Dutch patient 026-01 with junctional EB caused by mutations in COL17A1 and use of naturally corrected keratinocytes expressing type XVII collagen for autologous cell therapy.

A letter to the editor is presented which discusses a study on the differential immunoglobulin G (IgG) recognition of desmoglein 3 in paraneoplastic pemphigus (PNP) patients.

Abstract: Background: The current classification of recessive dystrophic epidermolysis bullosa (RDEB) comprises two major subtypes: ‘severe generalized RDEB’ (RDEB-sev gen) with early-onset, extensive, generalized blistering and scarring, complete absence of type VII collagen, and bi-allelic COL7A1 null mutations; milder ‘generalized other RDEB’ (RDEB-O) with reduced-to-normal type VII collagen expression, and non-null genotypes. Objective: To search for previously unrecognized phenotype–genotype correlations in 33 Dutch RDEB families. Methods: We analyzed extensive clinical follow-up data, available for all patients up to 19 years, detailed type VII collagen immunostaining and genotypes, and correlated clinical phenotype to molecular phenotype and genotype. Results: We identified 20 novel COL7A1 mutations. In 14 of 15 RDEB-sev gen patients type VII collagen was completely absent, one had strongly reduced type VII collagen, and all carried bi-allelic null mutations. Five of 11 RDEB-O patients developed pseudosyndactyly of the fingers preceded by skin atrophy and flexion contractures later in childhood and adolescence. All five had esophageal involvement and growth retardation. Type VII collagen immunostaining ranged from strongly reduced to slightly reduced in RDEB-O patients with pseudosyndactyly, whereas RDEB-O patients without pseudosyndactyly had slightly reduced to normal type VII collagen staining. There was no difference in genotypes between both groups, although we unexpectedly found bi-allelic null mutations in two of five RDEB-O patients with pseudosyndactyly. Conclusion: Pseudosyndactyly occurs in approximately half of RDEB-O patients when type VII collagen is strongly reduced. The prognosis in RDEB cannot always be simply predicted from the COL7A1 genotype. [Copyright &y& Elsevier]

Summary: Background: Type XVII collagen is an important structural component of keratinocyte hemidesmosomes and its functional loss in genetic or autoimmune disease results in blistering of the skin. In neoplastic tissue aberrant expression is seen dependent on the stage of the tumor. While the sequence of the type XVII collagen encoding gene –COL17A1 – is now completely elucidated, the sequence of the 5′-untranslated region (UTR) of the mRNA is still unknown. Since UTRs can modulate translation efficiency, the determination of the UTR sequence is indispensable for understanding the regulation of translation of type XVII collagen mRNA. Objective: To resolve the sequence of the 5′UTR of type XVII collagen mRNA and to analyse the promoter region for transcription motifs. Methods: 5′ Rapid amplification of cDNA ends (RACE) followed by sequence analysis and ribonuclease protection assays (RPA) were performed. Results: RACE and sequence analysis revealed the presence of six different 5′UTRs for the type XVII collagen mRNA. The start points of these six transcripts differ but no alternative exons are used. The longest 5′UTR starts 220 nucleotides before the open reading frame, whereas the shortest UTR is only 89 nucleotides in length. RPA confirmed the RACE results and furthermore demonstrated that the 5′UTRs with lengths of 102 and 220 nucleotides are the two major transcripts. Transcription motif analysis of the 5′ region of the COL17A gene demonstrated several binding sites for transcription factors including the Sp1 and activating protein-1 (AP-1) families. Conclusion: Type XVII collagen mRNA is alternatively transcribed, which may result in complex regulation of type XVII collagen. [Copyright &y& Elsevier]

The anchoring filament protein LAD-1 has been recently identified as the target of autoantibodies in the acquired blistering disorder linear IgA bullous dermatosis. Because this protein appears to be involved in the process of dermal-epidermal cohesion, this study sought to determine the involvement of LAD-i in the pathology of junctional epidermolysis bullosa (JEB). To this end, 44 patients with a variety of subtypes of JEB were analyzed by indirect immunofluorescence microscopy with antibodies to LAD-1, BP180, and laminin-5. We found that only patients with generalized atrophic benign epidermolysis bullosa (GABEB) contained LAD-1 defects. Of the 16 GABEB patients studied, 13 showed absent or greatly reduced expression of LAD-1 (including 2 patients with a peculiar interrupted staining pattern) and 3 patients showed defects of laminin-5 expression with normal LAD-1 expression. Patients who showed LAD-1 defects also showed abnormal expression of BP180. Keratinocytes were cultured from the skin of two GABEB patients and analyzed by indirect immunofluorescent microscopy. One culture demonstrated defects of BP180 and LAD-1 expression (which was also verified by radioimmunoprecipitation assay), and one culture showed decreased laminin-5 expression but normal BP150 and LAD-1 expression. Thus, these studies demonstrate that: (i) LAD-1 and BP180 are normally expressed in all subtypes of JEB except GABEB, (ii) the majority of GABEB patients show absent or near absent expression of both LAD-1 and BP 180 but normal expression of laminin-5, and (iii) a smaller subset of GABEB patients show normal LAD-1 and BP180 expression but express persistent but reduced levels of laminin-5. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented which discusses the presence of apoptic cells in pemphigus acantholysis.