Paraneoplastic Pemphigus Autoantibodies Against C-terminus of Desmoplakin Induced Acantholysis In Vitro and In Vivo.

Paraneoplastic pemphigus (PNP) is an autoimmune bullous disease associated with underlying neoplasms and characterized by antibodies against desmoglein 3 (Dsg 3) and plakins. Autoantibodies against desmoglein 3 in sera of patients with PNP have been proven to cause acantholysis in vivo in neonatal mice. As a member of the plakin family, autoantibodies against desmoplakin were detected frequently by immunoprecipitation in the sera of PNP. The recombinant C-terminus of desmoplakin was expressed and purified to adsorb the specific autoantibodies against the C-terminus of desmoplakin. In vitro dispase-dependent keratinocyte dissociation assay and in vivo IgG passive transfer into neonatal mice assay were performed, followed by the electronic microscopy examination and TUNEL assay. We found that anti-C terminus of desmoplakin autoantibodies caused blisters and acantholysis in mice skin at a dose-dependent manner. Moreover, dissociated fragments were observed after incubation with the purified IgG against desmoplakin, compared with normal human IgG (P-value =0.0207). The electronic microscopy examination showed the disconnection of keratin intermediate filaments from desmosomes. Lastly, apoptosis of keratinocytes in the TUNEL assay was all detected in the skins of neonatal mice after injection of the anti-C terminus of desmoplakin autoantibodies. Taken together, the study suggests that autoantibodies against the C-terminus of desmoplakin might be pathogenic in PNP.

Autoantibodies to full body vascular cell junctions colocalize with MYZAP, ARVCF, desmoplakins I and II and p0071 in endemic pemphigus in Colombia, South America.

BACKGROUND: We previously described a new variant of endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF). METHODS: Here we aimed to investigate disease autoreactivity to vessels in all body organs/systems. We compared 57 patients and 57 controls from the endemic area, matched by demographics, age, sex, and work activity. We performed immunofluorescence, immunohistochemistry, confocal microscopy, immunoblotting, indirect immune electron microscopy studies, and autometallographic studies. We performed ultrasonography on large patient arteries, investigating for vascular anomalies. In addition, we reviewed autopsies on seven patients who died affected by El Bagre-EPF. We immunoadsorbed any positive vessel immunofluorescence with desmoglein (Dsg1), investigating for new autoantigens. RESULTS: Overall, 57/57 patients affected by El Bagre-EPF displayed autoantibodies to vessels in all the organs/systems of the body via all methods (P < 0.01). The autoreactivity was polyclonal, and the patient's antibodies colocalized with commercial antibodies to desmoplakins I and II, p0071, ARVCF, and MYZAP (all from Progen Biotechnik, Germany; P < 0.01; all present at cell junctions). Immunoadsorption with Dsg1 on positive vessel immunofluorescence showed that the immune response against the vessels was directed against non-Dsg1 antigen(s). Autometallographic studies showed deposits of metals and metalloids in vessel cell junctions and in erythrocytes of 85% of patients (P < 0.01). CONCLUSIONS: Immune response to these vascular antigens is likely altering endothelial cells and vessel shapes, thus disturbing hemodynamic flow. The flow alterations likely lead to inflammation and may play a role in the atherogenesis often seen in these patients.

Patients with a new variant of endemic pemphigus foliaceus have autoantibodies against arrector pili muscle, colocalizing with MYZAP, p0071, desmoplakins 1 and 2 and ARVCF.

BACKGROUND: We identified a new variant of endemic pemphigus foliaceus in El Bagre, Colombia, South America, which we term El Bagre-EPF, and observed reactivity to arrector pili muscle (APM), thus we tested for autoimmunity to APM. METHODS: We took skin biopsies from 30 patients with El Bagre-EPF and 30 healthy controls (HCs) matched by age, sex and occupation, who were all from the endemic area, and tested these using direct immunofluorescence (DIF), confocal microscopy, immunohistochemistry and immunoblotting (IB). RESULTS: Of the 30 patients with El Bagre-EPF, 27 had autoantibodies to APM that colocalized with commercial antibodies to myocardium-enriched zonula occludens-1-associated protein (MYZAP), desmoplakin (DP)1 and DP2, plakophilin 4, and Armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF) (P < 0.001, Fisher exact test). The positive staining also colocalized with Junctional Adhesion Molecule 1 (JAM-A), a control antibody for gap cell junctions. No HC samples were positive. In 27 of the 30 patients, serum that was APM-positive also displayed IB colocalization of their autoantibody molecular weights with the Progen antibodies (P < 0.001, Fisher exact test). CONCLUSIONS: Patients affected by El Bagre-EPF have autoantibodies to APM, colocalizing with the antibodies MYZAP, ARVCF, p0071, DP1 and DP2, suggesting that these molecules are El Bagre-EPF antigens. Further, all of these antigens represent components of cell junctions, indicating that the immune response is directed, at least partially, against cell junctions. The immune response in patients affected by El Bagre-EPF is polyclonal, and it includes B and T lymphocytes, mast cells, IgG, IgA, IgM, IgD, IgE, fibrinogen, albumin, complement/C1q, C3c and C4.

A Systems Vaccinology Approach Reveals Temporal Transcriptomic Changes of Immune Responses to the Yellow Fever 17D Vaccine.

In this study, we used a systems vaccinology approach to identify temporal changes in immune response signatures to the yellow fever (YF)-17D vaccine, with the aim of comprehensively characterizing immune responses associated with protective immunity. We conducted a cohort study in which 21 healthy subjects in China were administered one dose of the YF-17D vaccine; PBMCs were collected at 0 h and then at 4 h and days 1, 2, 3, 5, 7, 14, 28, 84, and 168 postvaccination, and analyzed by transcriptional profiling and immunological assays. At 4 h postvaccination, genes associated with innate cell differentiation and cytokine pathways were dramatically downregulated, whereas receptor genes were upregulated, compared with their baseline levels at 0 h. Immune response pathways were primarily upregulated on days 5 and 7, accompanied by the upregulation of the transcriptional factors JUP, STAT1, and EIF2AK2. We also observed robust activation of innate immunity within 2 d postvaccination and a durable adaptive response, as assessed by transcriptional profiling. Coexpression network analysis indicated that lysosome activity and lymphocyte proliferation were associated with dendritic cell (DC) and CD4+ T cell responses; FGL2, NFAM1, CCR1, and TNFSF13B were involved in these associations. Moreover, individuals who were baseline-seropositive for Abs against another flavivirus exhibited significantly impaired DC, NK cell, and T cell function in response to YF-17D vaccination. Overall, our findings indicate that YF-17D vaccination induces a prompt innate immune response and DC activation, a robust Ag-specific T cell response, and a persistent B cell/memory B cell response.

Evaluation of a Newly Available ELISA for Envoplakin Autoantibodies for the Diagnosis of Paraneoplastic Pemphigus.

IMPORTANCE: Paraneoplastic pemphigus (PNP) is routinely diagnosed by the presence of autoantibodies for desmoplakin by indirect immunofluorescence (IIF) on rat bladder epithelium (RBE). IIF on RBE has recently been found to be positive in select cases of other blistering disorders. A new ELISA that detects envoplakin autoantibodies has recently been developed for the diagnosis of PNP. In this study, we measure the specificity of IIF on RBE and compare it to the new ELISA. OBSERVATIONS: We measured the specificity of IIF on RBE to be 86% which is on the lower end of the previously reported specificity of 83% to 98.9%. The ELISA for envoplakin autoantibodies has a technical sensitivity of 100%, diagnostic sensitivity of 83%, and specificity of 91%. CONCLUSIONS AND RELEVANCE: This ELISA for envoplakin autoantibodies is now commercially available and technically easier to perform then the immunoblot. We recommend that this new ELISA serves as a confirmatory test in cases of a positive IIF on RBE given its higher specificity.

Circulating plakin autoantibodies in a patient with erythema multiforme major: are they pathogenic or a manifestation of epitope spreading?

Erythema multiforme is a well-recognised entity but its pathogenesis remains elusive. Theories hypothesise a cell-mediated immune pathogenesis, however recent case reports have observed autoantibodies to the plakin family of proteins, suggesting a role for the humoral immune system. We present a case of erythema multiforme major with circulating desmoplakin autoantibodies in a 36-year old woman who was previously diagnosed with pemphigoid gestationis. The close correlation between the concentration of these autoantibodies and the severity of clinical disease strongly suggests a pathogenic role in her disease. As previous case reports have proposed, these autoantibodies may be directly pathogenic. Alternatively, the epiphenomenon of epitope spreading must be considered in the subset of patients with erythema multiforme major.

Antibody phage display assisted identification of junction plakoglobin as a potential biomarker for atherosclerosis.

To date, no plaque-derived blood biomarker is available to allow diagnosis, prognosis or monitoring of atherosclerotic vascular diseases. In this study, specimens of thrombendarterectomy material from carotid and iliac arteries were incubated in protein-free medium to obtain plaque and control secretomes for subsequent subtractive phage display. The selection of nine plaque secretome-specific antibodies and the analysis of their immunopurified antigens by mass spectrometry led to the identification of 22 proteins. One of them, junction plakoglobin (JUP-81) and its smaller isoforms (referred to as JUP-63, JUP-55 and JUP-30 by molecular weight) were confirmed by immunohistochemistry and immunoblotting with independent antibodies to be present in atherosclerotic plaques and their secretomes, coronary thrombi of patients with acute coronary syndrome (ACS) and macrophages differentiated from peripheral blood monocytes as well as macrophage-like cells differentiated from THP1 cells. Plasma of patients with stable coronary artery disease (CAD) (n = 15) and ACS (n = 11) contained JUP-81 at more than 2- and 14-fold higher median concentrations, respectively, than plasma of CAD-free individuals (n = 13). In conclusion, this proof of principle study identified and verified JUP isoforms as potential plasma biomarkers for atherosclerosis. Clinical validation studies are needed to determine its diagnostic efficacy and clinical utility as a biomarker for diagnosis, prognosis or monitoring of atherosclerotic vascular diseases.

Prognostic factors of paraneoplastic pemphigus.

OBJECTIVE: To identify the prognostic factors of overall survival in a series of patients with paraneoplastic pemphigus (PNP). DESIGN: Multicenter retrospective cohort study. SETTING: Twenty-seven dermatology departments in France. PATIENTS: A total of 53 patients (31 men and 22 women; median age, 59 years; age range, 30-88 years) were diagnosed as having PNP between 1992 and 2010. MAIN OUTCOME MEASURES: Overall Kaplan-Meier survival rates were estimated, and features associated with survival were assessed using univariate (log-rank test) and multivariate (Cox regression) analyses. RESULTS: The study included 53 patients with PNP. Thirty-six patients (68%) died during the study. The 1-, 3-, and 5-year overall survival rates were 49%, 41%, and 38%, respectively. The main causes of death were infections (n=21) and evolution of neoplasia (n=6). In univariate analysis, the main detrimental prognostic factors identified were erythema multiforme­like skin lesions (P=.05) and histologic keratinocyte necrosis (P=.03). None of the 5 patients with Castleman disease died during the study. After adjustment for age and sex in multivariate analysis, erythema multiforme­like skin lesions remained predictive of fatal outcome, with a 2-fold increase in death rate (hazard ratio [HR], 2.3; 95% CI, 1.05-5.03; P=.04). The prognosis of patients with PNP was even poorer when erythema multiforme­like skin lesions were associated with severe skin or mucosal involvement at presentation (HR of death, 3.0; 95% CI, 1.01-8.92; P=.049). CONCLUSION: Patients with PNP with erythema multiforme­like skin lesions and histologic keratinocyte necrosis, especially when associated with extensive lesions at presentation, are likely to have a more severe and rapid fatal outcome and should be managed very carefully.

Bombyx mori nucleopolyhedrovirus ORF54, a viral desmoplakin gene, is associated with the infectivity of budded virions.

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF54 (Bm54), a member of the viral desmoplakin N-terminus superfamily, is homologous to Autographa californica nucleopolyhedrovirus (AcMNPV) ORF66, which is required for the efficient egress of nucleocapsids from the nucleus and occlusion body formation. In this paper, we generated a bacmid with the Bm54 gene deleted via homologous recombination in Escherichia coli and characterized the mutant virus using a transfection-infection assay and transmission electron microscopy analysis. Our results demonstrated that the cells transfected with viral DNA lacking Bm54 produced non-infectious budded viruses (BVs). Electron microscopy showed that although the deletion of Bm54 did not affect assembly and release of nucleocapsids, it severely affected polyhedron formation. In conclusion, deletion of Bm54 resulted in non-infectious BV and defective polyhedra. Although the sequences of Bm54 and Ac66 are very similar, the two genes function quite differently in the regulation of viral life cycle.

Cardiac autoantibodies from patients affected by a new variant of endemic pemphigus foliaceus in Colombia, South America.

Several patients affected by a new variant of endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF) have experienced a sudden death syndrome, including persons below the age of 50. El Bagre-EPF patients share several autoantigens with paraneoplastic pemphigus patients, such as reactivity to plakins. Further, paraneoplastic pemphigus patients have autoantibodies to the heart. Therefore, we tested 15 El Bagre-EPF patients and 15 controls from the endemic area for autoreactivity to heart tissue using direct and indirect immunofluorescence, confocal microscopy, immunohistochemistry, immunoblotting, and immunoelectron microscopy utilizing heart extracts as antigens. We found that 7 of 15 El Bagre patients exhibited a polyclonal immune response to several cell junctions of the heart, often colocalizing with known markers. These colocalizing markers included those for the area composita of the heart, such as anti-desmoplakins I and II; markers for gap junctions, such as connexin 43; markers for tight junctions, such as ezrin and junctional adhesion molecule A; and adherens junctions, such pan-cadherin. We also detected colocalization of the patient antibodies within blood vessels, Purkinje fibers, and cardiac sarcomeres. We conclude that El Bagre-EPF patients display autoreactivity to multiple cardiac epitopes, that this disease may resemble what is found in patients with rheumatic carditis, and further, that the cardiac pathophysiology of this disorder warrants further evaluation.

Antibodies to pilosebaceous units along their neurovascular supply routes in a new variant of endemic pemphigus foliaceus in Colombia, South America.

Senear Usher syndrome is a variant of pemphigus foliaceus, confined to seborrheic sites and considered to be a clinical overlap syndrome, with features of both pemphigus foliaceus and lupus erythematosus. We recently described autoantibodies to skin eyelid meibomian glands in patients with a new variant of endemic pemphigus foliaceus (El Bagre EPF) in South America. We tested for El Bagre EPF patient sera autoreactivity to pilosebaceous units utilizing direct and indirect immunofluorescence, confocal microscopy, immunohistochemistry and immunoelectron microscopy. Hematoxylin and eosin staining of skin biopsies revealed that one third of the patients affected by El Bagre-EPF demonstrated some histologic alteration of the pilosebaceous units. By immunohistochemistry, most El Bagre EPF biopsies demonstrated evidence of an autoimmune response along the neural and vascular supply routes of the pilosebaceous units. An active immune response was seen with antibodies such as anti-human mast cell tryptase, myeloid/histoid antigen, CD8, CD20, CD68, CD117/c-kit, ZAP-70 and vimentin. Immunoelectron microscopy demonstrated autoantibodies within the hair follicle and at the basement membrane area of the sebaceous glands. El Bagre-EPF patients have autoantibodies to pilosebaceous units and to their surrounding neurovascular packages. Our results warrant further characterization and may explain the loss of hair described in severe endemic pemphigus foliaceus before the therapeutic steroid era.

Anti-desmoplakin antibodies in erythema multiforme and Stevens-Johnson syndrome sera: pathogenic or epiphenomenon?

The pathophysiology of erythema multiforme (EM), Stevens-Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN) is unclear. Whether autoantibodies against desmoplakin (Dp) I and II play a pathogenic role or result from an epitope spreading phenomenon is uncertain. Our aim was to characterize the keratinocyte antigens recognized in EM, TEN and SJS. Of 33 patients studied, 2 had TEN, 1 SJS, 9 EM major and 21 EM minor, according to Roujeau's criteria. All sera were studied by indirect immunofluorescence (IIF), immunoblotting and immunoprecipitation. Twenty normal sera were used as controls. 10/33 sera reacted with polypeptides of 215 and/or 250-kDa molecular mass, which co-migrate with Dp I and II as assessed by an anti-Dp I and II monoclonal antibody on IB. In IP, none of the anti-Dp I and -Dp II 10 patient sera immunoprecipitated Dp I and/or II from radiolabeled keratinocyte extracts. Two of 10 patient sera (SJS, EM minor) reacted with DpI and II when denaturated by the IB procedure. The reactivity against intracellular antigens DpI and II as denaturated proteins may result from the epidermal damage produced by aggressive autoreactive T cells, playing therefore only a secondary role in the pathogenesis of the disease.

Desmoplakin and KIF20B as target antigens in patients with paroxysmal nocturnal haemoglobinuria.

At least two hypotheses have been proposed to explain the mechanism of clonal expansion of mutant cells in paroxysmal nocturnal haemoglobinuria (PNH). One hypothesis assumes an immune escape mechanism and another proposes an intrinsic second mutational event within clonal cells. We hypothesised that autoantibodies detected in PNH patients could identify antigens that might play a role in the pathophysiology of this disease and screened a human fetal liver cDNA library for serological reactivity against haematopoietic stem/progenitor cells antigens using the SEREX approach. Two antigens were identified that are constitutively expressed in CD34(+) cells. Three and four of 10 PNH patients showed antibody responses against kinesin family member 20B (KIF20B) (previously termed M-phase phosphoprotein 1, MPP1) and desmoplakin (DSP) respectively. We also found an antibody response in one of 20 healthy volunteers against desmoplakin, yet at a much lower titre than in PNH patients. No response to KIF20B or desmoplakin was detected in five patients with aplastic anaemia without a glycosyl phosphatidyl inositol -deficient clone. We conclude that KIF20B and desmoplakin have been shown to be the first known auto-antigens to be recognised by the immune system of patients with PNH. The analysis of the mechanisms underlying the autoimmune response might contribute to our understanding of the clonal expansion in PNH.

Paraneoplastic pemphigus with negative direct immunofluorescence in epidermis or mucosa but positive findings in adnexal structures.

INTRODUCTION: Paraneoplastic pemphigus (PNP) is considered an autoimmune, multiorgan disease caused by antiplakin antibodies. We present three PNP patients who had negative epithelial direct immunofluorescence (DIF) findings in one or more biopsies. PATIENTS: An early lip biopsy of uninvolved oral epithelia in patient 1 was negative. A later biopsy from foreskin showed intense intercellular immunoglobulin G (IgG) deposits in the epithelia. In the early phase of the disease in patient 2, the intercellular fluorescence was negative in the epidermis, while intercellular IgG and C3 were observed in the sweat ducts. A later biopsy showed weak intercellular epidermal IgG and C3 fluorescence. Patient 3 showed intercellular IgG and/or C3 in follicular, sebaceous and sweat duct structures in several biopsies. No intercellular IgG or C3 was observed in the epithelia. DISCUSSION: The presence of immunoreactants in adnexal structures suggests that desmoplakins can be more strongly expressed in adnexa than in the epidermis, facilitating visualization of antibody deposits. CONCLUSIONS: Negative DIF findings in epithelia do not rule out the diagnosis of PNP, and the presence of IgG and/or C3 at the intercellular level of adnexal structures can help establish this diagnosis.

The area composita of adhering junctions connecting heart muscle cells of vertebrates. VI. Different precursor structures in non-mammalian species.

Recent studies on the formation and molecular organization of the mammalian heart have emphasized the architectural and functional importance of the adhering junctions (AJs), which are densely clustered in the bipolar end regions (intercalated disks, IDs) connecting the elongated cardiomyocytes of the adult heart. Moreover, we learned from genetic studies of mutated AJ proteins that desmosomal proteins, which for the most part are integral components of ID-specific composite AJs (areae compositae, AC), are essential in heart development and function. Developmental studies have shown that the bipolar concentration of cardiomyocyte AJs in IDs is a rather late process and only completed postnatally. Here we report that in the adult hearts of diverse lower vertebrates (fishes, amphibia, birds) most AJs remain separate and distinct in molecular character, representing either fasciae adhaerentes, maculae adhaerentes (desmosomes) or--less frequently--some form of AC. In the mature hearts of the amphibian and fish species examined a large proportion of the AJs connecting cardiomyocytes is not clustered in the IDs but remains located on the lateral surfaces where they appear either as puncta adhaerentia or as desmosomes. In many places, these puncta connect parallel cardiomyocytes in spectacular ladder-like regular arrays (scalae adhaerentes) correlated with--and connected by--electron-dense plaque-like material to sarcomeric Z-bands. In the avian hearts, on the other hand, most AJs are clustered in the IDs but only a small proportion of the desmosomes appears as AC, compared to the dominance of distinct fasciae adhaerentes. We conclude that the fusion and amalgamation of AJs and desmosomes to ACs is a late process both in ontogenesis and in evolution. The significance and possible functional implications of the specific junctional structures in vertebrate evolution and the class-specific requirements of architectural and molecular assembly adaptation during regeneration processes are discussed.

Detection of autoantibodies to desmoplakin in a patient with oral erythema multiforme.

Anti-desmoplakin (DP) I and II are detected in patients with paraneoplastic pemphigus. However, these autoantibodies have also been detected in patients with other disorders. A 73-year-old woman presented with a 20-year history of erosions and ulcers of the tongue and oral mucosa. Biopsy specimens of the oral mucosa showed several necrotic keratinocytes in the mucosal epithelium. The patient's serum was negative for anti-desmoglein 1 and anti-desmoglein 3 antibodies by ELISA, although anti-keratinocyte cell surface antibodies were detected by indirect immunofluorescence. On immunoblotting using protein extracts of normal human epidermis, the patient's serum was found to contain autoantibodies to 250 kDa and 210 kDa proteins, indicating the presence of autoantibodies to DP I and II. Based on these results, the diagnosis of erythema multiforme was made. An immunofluorescence and immunoblotting are crucial for the differential diagnosis between an erythema multiforme which is positive for anti-DP I and II antibodies and other autoimmune bullous diseases.

Antidesmoplakin antibodies in pemphigus vulgaris.

BACKGROUND: Besides being present in paraneoplastic pemphigus (PNP), circulating antidesmoplakin (DP) antibodies have been found anecdotally in other bullous diseases, including pemphigus foliaceus and pemphigus vulgaris. OBJECTIVES: To verify how frequent anti-DP antibodies are in pemphigus vulgaris. METHODS: We studied 48 sera from patients with proven pemphigus vulgaris (29 mucosal dominant pemphigus and 19 mucocutaneous pemphigus) by indirect immunofluorescence (IIF) with rat bladder epithelium (RBE) as a substrate and by immunoblotting (IB) on human keratinocyte cultures enriched in DP. RESULTS: Ten sera (21%) were positive in IIF on RBE. By IB, eight sera proved to have antibodies to both DP I (250 kDa) and DP II (210 kDa), one serum had antibodies directed to DP I only, and two sera to DP II only. CONCLUSIONS: Our data confirm that RBE is not a specific IIF substrate for the serological diagnosis of PNP. It remains a sensitive and specific substrate for the detection of anti-DP antibodies, which, in patients with pemphigus vulgaris, are probably caused by an epitope-spreading phenomenon.