Tag Archives: autoantibodies

IMPORTANCE A rare variant of mucous membrane pemphigoid (MMP) is characterized by circulating anti-laminin 332 (Lam332) autoantibodies and seems to be associated with concurrent malignant neoplasms.

OBJECTIVE To determine the prevalence and clinical significance of anti-Lam332 autoantibody detection from a large series of patients with MMP. DESIGN Multicenter retrospective study.

SETTING Four French national centers for autoimmune bullous diseases.

PARTICIPANTS One hundred fifty-four patients with MMP and 89 individuals serving as controls were included.

INTERVENTIONS Serum samples were analyzed by a new Lam332 enzyme-linked immunosorbent assay (ELISA); clinical and immunopathologic data were obtained from the patients’ medical records.

MAIN OUTCOME MEASURES The Lam332 ELISA scores were evaluated with respect to clinical characteristics, standard and salt-split indirect immunofluorescence, and bullous pemphigoid (BP) 230 and BP180-NC16A ELISAs.

RESULTS The Lam332 ELISA score was positive (≥9 U/mL) in 20.1% of serum samples from patients with MMP, 1 of 50 patients with bullous pemphigoid (BP), none of 7 with pemphigus, and 3 of 32 other controls. No relationship was evidenced between a positive ELISA Lam332 score and age; sex ratio; oral, ocular, genital, skin, or esophageal/laryngeal involvement; internal malignant neoplasm; or BP180 ELISA score. Salt-split skin indirect immunofluorescence and ELISA BP230 results were more frequently positive when Lam332 ELISA results were positive (P = .04 and .02, respectively). Patients with a positive Lam332 ELISA score frequently had more severe MMP (67.8% vs 47.2%; P = .04).

CONCLUSIONS AND RELEVANCE Results of this novel ELISA showed that serum anti-Lam332 autoantibodies are detected in 20.1% of patients with MMP. Anti-Lam332 autoantibodies are mainly detected in patients with severe MMP but not preferentially in those with a malignant neoplasm. The association between anti-Lam332 and anti-BP230 autoantibodies might arise from an epitope-spreading phenomenon.

JAMA dermatology (Chicago, Ill.)

Bullous pemphigoid is an autoimmune blistering skin disease characterized by the presence of circulating autoantibodies which recognize specific proteins of the epidermis and dermoepidermal junction. Diagnosis is based on clinical criteria and laboratory investigations, notably histology, direct and indirect immunofluorescence, and ELISA. This study describes a new immunofluorescence assay for parallel determination of anti-BP180 and anti-BP230 based on recombinant antigenic substrates. The aim of the study was to detect BP180 and BP230 autoantibodies by BIOCHIP technology using both a specially designed recombinant BP180-NC16A protein and cells expressing the BP230-gc antigen fragment. 18 patients with bullous pemphigoid were included in the study. Autoantibodies to BP180 were detected by the BIOCHIP technique in 83.33% of patients with clinical, serological, and immunohistological confirmed bullous pemphigoid while autoantibodies against BP230-gC were detected only in 39% of patients. The detection of anti-BP180-NC16A and anti-BP230-gC by a new biochip-based immunoassay is a suitable alternative to indirect immunofluorescence and ELISA. This method has the advantage of easily discriminating the different autoantibody specificities. The BIOCHIP method is faster, cheaper, and easy to use when compared with the ELISA approach. For this reason, the new method could be used as an initial screening test to identify patients with bullous pemphigoid, and doubtful results could then be confirmed by ELISA.

Full article (free) found here: http://www.hindawi.com/isrn/dermatology/2012/237802/

Senear-Usher syndrome or pemphigus erythematosus is a pathology that overlaps clinically and serologically with pemphigus foliaceus and lupus erythematosus. Skin biopsies of patients with pemphigus erythematosus reveal acantholysis and deposits of immunoglobulins in desmosomes, and they are positive in the lupus band test. In the present paper, we determined whether the autoantibodies associated with pemphigus erythematosus targeted a single antigen or multiple antigens as a result of the stimulation of independent B cell clones. Our present paper demonstrates that patients with pemphigus erythematosus produce both antiepithelial antibodies specific for desmoglein 1 and 3 and antinuclear antibodies specific for Ro, La, Sm, and double-stranded DNA antigens. After eluting specific anti-epithelial or anti-nuclear antibodies, which were recovered and tested using double-fluorescence assays, a lack of cross-reactivity was demonstrated between desmosomes and nuclear and cytoplasmic lupus antigens. This result suggests that autoantibodies in pemphigus erythematosus are directed against different antigens and that these autoantibodies are produced by independent clones. Given these clinical and serological data, we suggest that pemphigus erythematosus behaves as a multiple autoimmune disease.

Full article can be viewed on: http://www.hindawi.com/journals/ad/2012/296214/

The molecular basis of disease heterogeneity in autoimmune conditions such as Pemphigus vulgaris is poorly understood. Although desmoglein 3 (Dsg3) has been well established as a primary target of immunoglobulin (Ig) autoantibodies in PV, there remain several questions regarding the overall distribution of anti-Dsg3 Ig subtypes among patient subsets and considerable controversy regarding whether an isotype switch can be observed between phases of disease activity. To systematically address the outstanding questions related to Ig-isotype specificity in PV, we analyzed IgA, IgM, IgG1, 2, 3 and 4 anti-Dsg3 levels by ELISA in 202 serum samples obtained from 92 patients with distinct clinical profiles based on a set of defined variable (activity, morphology, age, duration) and constant (HLA-type, gender, age of onset) clinical parameters, and 47 serum samples from HLA-matched and -unmatched controls. Our findings provide support for earlier studies identifying IgG4 and IgG1 as the predominant antibodies in PV with significantly higher levels in active than remittent patients. We do not see evidence for an isotype switch between phases of disease activity and remission, and both IgG4 and IgG1 subtypes remain elevated in remittent patients relative to controls. We do, however, find IgG4 to be the sole subtype that further distinguishes PV patient subgroups based on different disease morphologies, disease duration, and HLA-types. These data provide further insight into the immune mechanisms responsible for phenotypic expression of disease, and contribute to the broader effort to establish comprehensive immunoprofiles underlying disease heterogeneity to facilitate increasingly specific and individualized therapeutic interventions.

Full article available at: http://www.ncbi.nlm.nih.gov/pubmed/22779708

MedWire News: Researchers have identified the primary target of the autoantibodies found in the serum of patients with the blistering skin disorder pemphigus vulgaris (PV).

PV patients develop antibodies against the proteins desmoglein (DSG)1 and 3, which help epidermal cells stick together and maintain the integrity of the skin, causing painful blistering on the skin and mucus membranes.

Giovanna Zambruno (Istituto Dermopatico dell’Immacolata, Rome, Italy) and colleagues found that the cis-adhesive interface of the DSG3 extracellular domain (EC)1 is the main target of the PV autoantibody (A)224 generated in the serum of patients with PV.

Existing therapies for the condition target the whole immune system, but this can cause problems with side effects and can result in patients being vulnerable to infections.

To pinpoint the trigger of the autoantibody production in PV more specifically, Zambruno and team isolated 15 immunoglobulin (Ig)G antibodies specific for DSG3 from two patients with the disorder.

Of these, three disrupted layers of skin cells in the laboratory and two were pathogenic when expressed in a murine passive transfer model.

The epitopes recognized by the pathogenic PV antibodies were isolated to the DSG3 EC1 and EC2 subdomains and a specific serologic assay was used to pinpoint the target of the PVA224 as being the cis-adhesive interface on EC1.

The researchers suggest that the autoreactivity seen in PV is due to somatic mutations that are generated by an antigen other than DSG3, as binding to DSG3 disappeared when the somatic mutations reverted to the germline sequence.

“The identification of an immunodominant region targeted by pathogenic antibodies has implications for diagnosis of PV and opens new perspectives toward the establishment of therapeutic approaches for treatment of PV patients,” write Zambruno and team in the Journal of Clinical Investigation.

“Finally, the germlined version of the PV autoantibodies may lead to the identification of the antigens that eventually lead to development of this life-threatening disease.”

medwireNews (www.medwire-news.md) is an independent clinical news service provided by Springer Healthcare Limited. © Springer Healthcare Ltd; 2012

Read at: http://www.medwire-news.md/66/101414/Dermatology/Therapeutic_targets_for_pemphigus_vulgaris_discovered.html

Background  Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are potentially fatal blistering diseases caused by autoantibodies targeting desmoglein (Dsg) adhesion proteins. Previous studies have shown an IgG4 > IgG1 predominance of anti-Dsg antibodies in pemphigus; however, no studies have examined total serum IgG4 levels in pemphigus. IgG4 is induced by chronic antigen stimulation, which could occur with persistent skin blistering and potentially elevate the total serum IgG4 relative to other IgG subclasses in patients with pemphigus.

Objectives  The primary aim of the study was to quantitate total and Dsg-specific IgG subclasses in patients with pemphigus.

Methods  IgG subclasses and Dsg-specific IgG1 and IgG4 were quantitated in patients with PV and PF, and in sera from age-matched controls using a subclass enzyme-linked immunosorbent assay. The effectiveness of IgG4 depletion in blocking IgG pathogenicity in PV was determined using a keratinocyte dissociation assay.

Results  Dsg-specific antibodies comprised a median of 7·1% and 4·2% of total IgG4 in patients with PV and PF, respectively, with eightfold and fourfold enrichment in IgG4 vs. IgG1. Total serum IgG4, but not other IgG subclasses, was enriched in patients with PV and PF compared with age-matched controls (P = 0·004 and P = 0·005, respectively). IgG4 depletion of PV sera reduced pathogenicity in a keratinocyte dissociation assay and showed that affinity-purified IgG4 is more pathogenic than other serum IgG fractions.

Conclusions  Dsg-specific autoantibodies are significantly enriched in IgG4, which may explain the enrichment of total serum IgG4 in some patients with pemphigus. By preferentially targeting autoimmune rather than beneficial immune antibodies, IgG4-targeted therapies may offer safer treatment options for pemphigus.

Full article available at: http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2133.2012.11144.x/abstract

Pemphigus vulgaris (PV) is an autoimmune blistering disease of skin and mucous membranes caused by autoantibodies to the desmoglein (DSG) family proteins DSG3 and DSG1, leading to loss of keratinocyte cell adhesion. To learn more about pathogenic PV autoantibodies, we isolated 15 IgG antibodies specific for DSG3 from 2 PV patients. Three antibodies disrupted keratinocyte monolayers in vitro, and 2 were pathogenic in a passive transfer model in neonatal mice. The epitopes recognized by the pathogenic antibodies were mapped to the DSG3 extracellular 1 (EC1) and EC2 subdomains, regions involved in cis-adhesive interactions. Using a site-specific serological assay, we found that the cis-adhesive interface on EC1 recognized by the pathogenic antibody PVA224 is the primary target of the autoantibodies present in the serum of PV patients. The autoantibodies isolated used different heavy- and light-chain variable region genes and carried high levels of somatic mutations in complementary-determining regions, consistent with antigenic selection. Remarkably, binding to DSG3 was lost when somatic mutations were reverted to the germline sequence. These findings identify the cis-adhesive interface of DSG3 as the immunodominant region targeted by pathogenic antibodies in PV and indicate that autoreactivity relies on somatic mutations generated in the response to an antigen unrelated to DSG3.

Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering disease of skin and mucous membranes caused by autoantibodies that bind to the cadherin-type cell-cell adhesion molecules desmoglein 3 (DSG3) and DSG1, the main constituents of desmosomes, and cause the loss of keratinocyte cell adhesion. The critical role of autoantibodies in PV pathogenesis is supported by the observations that the disease activity correlates with anti-DSG3 antibody titers , that newborns of mothers with active PV exhibit blisters caused by the placental transfer of maternal antibodies, and that pemphigus-like lesions are induced in neonatal mice by passive transfer of anti-DSG3 IgG from PV patients.

In the skin, DSG3 is mainly expressed in the basal and suprabasal layers, while DSG1 is predominantly expressed in the upper epidermal layers. In contrast, in noncornified stratified epithelia, such as the oral mucosa, DSG3 is highly expressed throughout the epithelium, while DSG1 is expressed at a much lower level. The differential expression pattern of DSG1 and DSG3 is responsible for clinical variants of pemphigus: antibodies to DSG3 are present in the mucosal form, while antibodies to both DSG3 and DSG1 are associated with mucocutaneous lesions.

DSG3 is a calcium-binding membrane glycoprotein with an extracellular domain comprising 5 distinct subdomains (EC1–EC5), and it is synthesized as proprotein, which is processed in the Golgi apparatus by removal of a propeptide before transporting to the cell surface. The cleavage of the propeptide occurs upstream of a conserved tryptophan residue in the EC1 subdomain, unmasking residues critical for the formation of homophilic interactions with DSG3 on opposing cells. Several studies have shown that polyclonal antibodies in PV serum react primarily with the aminoterminus of DSG3 in the EC1 and EC2 subdomains (amino acids 1–161).

The isolation of pathogenic mAbs is instrumental for addressing questions as to the mechanism that induces the autoreactive response and drives blister formation in PV patients. Amagai and coworkers isolated from an active mouse model of PV a pathogenic antibody, AK23, which causes loss of cell adhesion by binding to the EC1 subdomain of DSG3 that is involved in the formation of the trans-adhesive interface. A number of human anti-DSG pathogenic and nonpathogenic mAbs were isolated as single-chain variable-region fragments (scFvs) from a PV patient . Similarly to the AK23 mAb, the pathogenic activity of these human antibodies was mapped to the aminoterminal region of EC1, which is masked by the propeptide . Taken together, the human and mouse data suggest that pathogenic antibodies bind primarily to EC1 and disrupt the keratinocyte adhesion by interfering with the trans-adhesive interface of DSG3.

In this study, we isolated from 2 PV patients several IgG autoantibodies that bind DSG3. These antibodies carried high levels of somatic mutations that were required for binding to DSG3. The epitopes recognized by 3 pathogenic antibodies were mapped to the EC1 and EC2 subdomains in regions that are expected to be involved in cis-adhesive interactions. This region was found to be the primary target of serum autoantibodies in PV patients. These results identify the cis-adhesive interface as the immunodominant region targeted by pathogenic antibodies in PV and suggest that autoreactivity relies on somatic mutations triggered by an unrelated antigen.

Full article Available at: http://www.jci.org/articles/view/64413

MADAM, Autoantibodies in pemphigus target preferentially desmoglein 1 (Dsg1) and Dsg3, and rarely desmocollins 1-3 (Dsc1-3). Pemphigus herpetiformis (PH) is one of pemphigus subtypes and characterized by pruritic annular erythemas with vesicles in the periphery, rarity of mucosal involvement and histopathological change of eosinophilic spongiosis. Recently, IgG anti-Dsc3 autoantibodies were suggested to cause skin lesion in a case of pemphigus vulgaris. In this study, we report the first case of concurrent bullous pemphigoid (BP) and PH with IgG antibodies to both Dsgs and Dscs.

from: http://onlinelibrary.wiley.com/doi/10.1111/bjd.12019/abstract

We read with interest the study by Koga H et al1 and we believe that in light of recent observations including our data (Table 1) the “desmoglein compensation theory” as a explanation for localization of blisters should be revisited 2,3,4. Although the disruption of desmoglein-dependent cell adhesion by autoantibodies is the basic pathophysiology underlying blister formation in pemphigus 2−4, the clinical spectrum does not always mirror this pathogenic process. Three clinical types of pemphigus have been described, the mucosal dominant, cutaneous and mucocutaneous type 2,,3,4 .
http://onlinelibrary.wiley.com/doi/10.1111/bjd.12012/abstract

Bullous pemphigoid (BP) is an autoimmune blistering skin disease. Autoantibodies to BP180 and BP230 can be detected by indirect immunofluorescence (IIF) on different substrates (oesophagus, salt-split-skin, BP180-antigen dots, BP230-transfected cells) and ELISA. Here, we compared test characteristics of these test systems. We analysed sera from BP patients (n=60) in whom the clinical diagnosis had been confirmed histopathologically. The control cohort comprised sera from patients with other autoimmune-associated (n=22) or inflammatory (n=35) skin diseases. All samples were tested by IIF (EUROIMMUN™ Dermatology Mosaic) and ELISA (EUROIMMUN and MBL). Anti-BP180 is best detected with BP180-antigen dots by IIF (sensitivity: 88%; specificity: 97%). As

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compared to IIF, the differences with both BP180 ELISA techniques are small though. Likelihood ratios (LRs) for positive and negative test results are >10 and between 0.1 and 0.2, respectively, for all test systems. Detection of anti-BP230 is highly variable (sensitivity range 38-60%; specificity range 83-98%). Only the IIF test reveals a LR for positive test results >10. Since the LRs for a negative test are all ~0.5, negative test results for anti-BP230 antibodies do not help to exclude BP. In conclusion, the multi-parameter IIF test reveals a good diagnostic performance in BP. Since this test simultaneously allows for the detection of anti-Dsg1 and anti-Dsg3 antibodies, involved in pemphigus foliaceus and vulgaris, a single test-incubation may be sufficient to differentiate between the most frequent autoimmune blistering diseases.

In conclusion, the multi-parameter IIF test reveals a good diagnostic performance in BP. Since this test simultaneously allows for the detection of anti-Dsg1 and anti-Dsg3 antibodies, involved in pemphigus foliaceus and vulgaris, a single test-incubation may be sufficient to differentiate between the most frequent autoimmune blistering diseases. PMID: 22580378 [PubMed – in process] (Source: Journal of Immunological Methods)
from MedWorm: Pemphigus http://www.medworm.com/index.php?rid=6304089&cid=c_297_3_f&fid=33859&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2FPubMed%2F22580378%3Fdopt%3DAbstract