Comparison of desmoglein ELISA and indirect immunofluorescence using two substrates (monkey oesophagus and normal human skin) in the diagnosis of pemphigus

A prospective study was performed to assess the usefulness of desmoglein enzyme-linked immunosorbent assay testing compared with indirect immunofluorescence in the diagnosis of new cases of pemphigus, as well as to compare the relative sensitivities of monkey oesophagus and normal human skin as substrates for indirect immunofluorescence. These tests were performed on the sera of 29 consecutive new cases of pemphigus diagnosed over a 2-year period based on clinical, histological and direct immunofluorescence findings. Desmoglein enzyme-linked immunosorbent assay was positive in all patients whereas indirect immunofluorescence was positive in only 25 of 29 patients. All four patients with negative indirect immunofluorescence had positive antinuclear antibodies or cytoplasmic fluorescence that could have masked the anti-intercellular antibodies. Desmoglein enzyme-linked immunosorbent assay appeared to reflect the disease activity better than indirect immunofluorescence in a few patients who had active disease of recent onset. Monkey oesophagus was found to be superior or equal to human skin as a substrate for indirect immunofluorescence in both pemphigus vulgaris and foliaceus.     

Drug-related pemphigus and angiotensin converting enzyme inhibitors

A 105-year-old woman developed pemphigus foliaceus. She had been on fosinopril, an angiotensin-converting enzyme inhibitor (ACE inhibitor) for 4 years. Anti-intercellular cement substance antibodies were positive with titre > 160. She died during admission of an unrelated illness. A 57-year-old man developed pemphigus vulgaris after 11 months treatment with quinapril. At 14 months after developing pemphigus, this man continues on prednisone and azathioprine. We speculate that these are cases of ACE-inhibitor-related pemphigus and we review ACE-inhibitor-related pemphigus.     

Successful treatment of adolescent pemphigus vulgaris by immunoadsorption method.

A 15-year-old girl with pemphigus vulgaris did not respond to oral administration of prednisolone at 45 mg/day. The skin and oral mucous membrane lesions recurred after effective treatments with methylprednisolone pulse therapy and combination therapy with prednisolone and cyclosporine. The finally successful treatment involved eleven cycles of immunoadsorption using a tryptophan column and administration of a moderate dose of prednisolone. Serum gamma-globulin level and anti-intercellular antibody titer decreased from 1.08 g/dl to 0.5 g/dl and 1:320 to 1:20, respectively. She has been well controlled with 21.5 mg/day prednisolone for 8 months after the final adsorption. Considering the physical, mental and social situation of adolescent student patients, immunoadsorption is a highly preferable choice among a variety of treatment modalities for pemphigus vulgaris because it makes the term of hospitalization shorter and avoids undesirable side effects from initial high dose corticosteroids.     

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.     

Cutaneous pemphigus vulgaris with absence of desmoglein 1 autoantibodies. An example of the extended desmoglein compensation theory

A 79 year‐old male patient presented clinically as a typical case of pemphigus foliaceus. He displayed a seborrhoeic pattern of crusting and erosions with an absence of oral involvement. Surprisingly, a biopsy showed suprabasilar acantholysis and direct immunofluorescence consistent with pemphigus vulgaris. Indirect pemphigus antibodies were positive for desmoglein 3 (DSG‐3) but negative for DSG‐1 antibodies. Most cases of cutaneous pemphigus are reported to have both DSG‐1 and DSG‐3 antibodies. A rare cutaneous subtype of pemphigus vulgaris is reported in the literature that may present clinically as seen in our patient. Our patient is clinically similar to these previous reported cases but with a negative DSG‐1 antibody titre. The extended DSG compensation theory explains the differences of clinical expression in pemphigus based on the variable pathogenicity of DSG antibodies as well as the distribution of DSG‐1 and DSG‐3. We discuss this case, highlighting the theories of the pathogenesis of pemphigus and, in particular, the cutaneous pemphigus subtype.     

Spectrum of autoantibodies other than anti‐desmoglein in pemphigus patients

Background Pemphigus is a life‐threatening autoimmune blistering disease mediated by autoantibodies against adhesion molecule of the skin. Its concurrence with systemic and organ‐specific autoimmune disease was described in case reports. Objectives To evaluate the presence of a broad spectrum of organ‐specific and non‐organ‐specific autoantibodies other than anti‐desmoglein antibodies in pemphigus patients. Patients and methods Serum samples were obtained from 105 pemphigus foliaceus (PF) patients, 51 pemphigus vulgaris (PV) patients and 50 controls. Both indirect immunofluorescence assay and ELISA were used to assess the presence of autoantibodies related to connective tissue diseases, autoimmune hepatitis, vasculitis, rheumatoid arthritis, coeliac disease, diabetes and thyroiditis. Results Significant difference was observed between the three groups for anti‐thyroglobulin antibodies in the pemphigus foliaceus group (18% vs. 4%, P = 0.03). A significantly higher occurrence of IgM anti‐cardiolipin (P = 0.03), IgG anti‐reticulin (P = 0.01) and IgG anti‐gliadin antibodies (P = 0.008) were observed in the PV group. Cases with more than four autoantibodies were frequently positives for both anti‐desmoglein 1 and anti‐desmoglein 3. Conclusion Autoantibodies other than anti‐desmoglein antibodies are not rare in pemphigus patients. Clinical and serological follow‐up of pemphigus patients with positive autoantibodies are needed to clarify their impact in disease evolution.     

The health impact of pemphigus vulgaris and pemphigus foliaceus assessed using the Medical Outcomes Study 36-item short form health survey questionnaire

BACKGROUND: Pemphigus vulgaris and pemphigus foliaceus are rare, potentially life-threatening, autoimmune disorders characterized by antibodies to epidermal adhesion molecules. Clinical characteristics are painful chronic erosions of mucous membranes and of the skin. There are only few published studies on the impact of the disease on the health status (HS) of patients with these conditions. OBJECTIVES: To assess the impact of disease on the HS of patients with pemphigus. METHODS: Fifty-eight patients enrolled at the Bullous Skin Diseases Unit of IDI-IRCCS in the period January-June 2006 were assessed for their HS using the Medical Outcomes Study 36-item short form health survey (SF-36) questionnaire and for anxiety and depression using the Institute for Personality and Ability Testing questionnaires. RESULTS: A compromised HS on both the physical and the psychosocial scales was observed, similar to other chronic dermatological diseases such as psoriasis. Patients with anxiety had severe disease as measured by the Physician's Global Assessment and the degree of mucocutaneous involvement, as well as those with a more recent disease onset. A better HS was observed in patients whose clinical condition was rated as nonsevere, and also in patients with pemphigus foliaceus. In pemphigus vulgaris, antidesmoglein 3 antibody levels directly correlated with clinical severity and with lower SF-36 scores. CONCLUSIONS: HS evaluated with the SF-36 can be a very useful additional outcome criterion in clinical studies with pemphigus. The management of the disease must take into account its impact on various aspects of life of the patient.     

Herpes simplex virus 1 and cytomegalovirus are associated with pemphigus vulgaris but not with pemphigus foliaceus disease

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are blistering autoimmune diseases that depend on interaction between genetic and environmental factors. Viral infections, like herpes simplex viruses 1 and 2 (HSV1/2), cytomegalovirus (CMV), Epstein‐Barr virus and dengue virus, could trigger or exacerbate pemphigus. IgM and IgG antibodies against these viruses in serum from PV and PF, their relatives and controls were determined. HSV1/2 expression was evaluated by direct immunofluorescence (DIF) and qPCR in affected or not oral mucosa from PV patients compared with uninjured PF mucosa. IgG anti‐HSV1 was higher in the PV group compared with all groups. IgG anti‐CMV resulted higher in PV group compared with PF patients and PV relatives. HSV1 was confirmed by DIF and qPCR on oral samples from patients with PV. Lack of HSV1 expression in the oral mucosa of patients with PF corroborate that immunosuppressive therapy cannot be the main cause for HSV1 replication in PV disease.     

High dose oral prednisone vs. prednisone plus azathioprine for the treatment of oral pemphigus: a retrospective,bi‐centre,comparative study

Background Steroids are considered the cornerstone in the treatment of pemphigus vulgaris. However, subsequent morbidity of their use has led to the development of combined therapeutic protocols, using steroid sparing agents. Objectives The aim of this study was to compare tolerability and efficacy of high dose oral prednisone daily, vs. low dose oral prednisone on alternate days plus azathioprine every day (Lever’s mini treatment: LMT) in oral pemphigus. Patients and methods Data of 36 patients with oral pemphigus vulgaris, treated with either 1.5 mg/kg/day of oral prednisone daily, or LMT were re‐evaluated and statistically analysed. Primary endpoints were time required to control disease activity, prednisone dose required until the end of consolidation phase, cumulative prednisone dose and rates of remission. Secondary endpoints were time to complete (CR) or partial remission (PR) on/off therapy, treatment‐associated morbidity and days of hospitalization. Results Both treatments resulted in high rates of clinical response. The mean prednisone dose required until the end of consolidation phase, and until CR or PR, on/off minimal therapy, was significantly lower in LMT group. However, the mean time required until the control of disease activity and CR or PR, for the same group, was significantly higher. Adverse events were more frequent among patients under daily prednisone. Mean time of hospitalization was also longer in the latter group. Conclusions Both treatments seem efficacious. Rapidly progressive lesions necessitate high prednisone dose for early and adequate control of the disease. Patients with impaired physical status, especially those with relatively stable lesions, at baseline might safely and effectively be treated with LMT.     

The transition of pemphigus vulgaris into pemphigus foliaceus: a reflection of changing desmoglein 1 and 3 autoantibody levels in pemphigus vulgaris

The transition of pemphigus vulgaris (PV) into pemphigus foliaceus (PF) is rare and the immunological changes underlying this event are not well understood. We report a 44-year-old woman who presented with oral and cutaneous erosions typical of PV. Over a 9-year period, the clinical features evolved into those of PF. To examine whether quantitative changes in desmoglein (Dsg) antibodies were associated with this transition, Dsg1 and Dsg3 antibody levels were measured by enzyme-linked immunosorbent assay in 82 sequential serum samples collected over this period. At presentation, when the phenotype was PV with oral and cutaneous erosions, antibodies to both Dsg1 and Dsg3 were detected. The disappearance of oral involvement was associated with a decline in Dsg3 antibodies, which are now undetectable, while the development of more severe skin involvement was associated with rising Dsg1 antibody levels. These data strongly suggest that the change in clinical features is a reflection of qualitative and quantitative changes in antibody profile. It is not known whether the transition to PF is permanent or whether disease relapses in the future may be associated with the re-emergence of Dsg3 antibodies, oral ulceration and a PV phenotype.     

Direct immunofluorescence of the outer root sheath in anagen and telogen hair in pemphigus vulgaris and pemphigus foliaceus

Direct immunofluorescence of peri‐lesional skin is the gold standard in the diagnosis of pemphigus. A specific immunofluorescence pattern may also be demonstrated in the outer root sheath of anagen and telogen hair. We demonstrated an intercellular reticular deposition of immunoglobulin G in the outer root sheath of plucked anagen and telogen hair in all pemphigus vulgaris patients with active disease and for the first time in all patients with active pemphigus foliaceus. Moreover, we demonstrated for the first time that plucked hair samples may be kept at ?20°C for at least 2 weeks before immunofluorescent staining and analysis.     

Anti-CD20 monoclonal antibody (rituximab) in the treatment of pemphigus

BACKGROUND: Pemphigus is a severe autoimmune blistering disorder caused by autoantibodies to desmoglein 1 and 3. The disease course is typically severe, thus requiring multiple immunosuppressive agents. The treatment is still challenging and in some patients with recalcitrant disease, therapies fail and therapeutic options are limited. OBJECTIVES: To investigate whether depletion of B lymphocytes that are thought to produce disease-causing autoantibodies shows a long-term benefit in pemphigus. METHODS: Five patients diagnosed as having pemphigus vulgaris and pemphigus foliaceus were treated with the monoclonal antibody rituximab. Rituximab was administered intravenously at a dosage of 375 mg m(-2) once weekly for 4 weeks. RESULTS: The treatment was well tolerated and all patients showed a good response over a follow-up period of up to 3 years, allowing immunosuppressive treatment to be reduced or terminated. B-cell depletion persisted for 6-12 months, and in one patient for almost 3 years. CONCLUSIONS: This study highlights the prolonged effect and disease control after one single course of rituximab and further extends the spectrum of treatments of bullous autoimmune disorders.     

The treatment of mild pemphigus vulgaris and pemphigus foliaceus with a topical corticosteroid

Seven patients with mild pemphigus vulgaris (n = 3) or pemphigus foliaceus (n = 4) were treated with a very potent topical corticosteroid alone. Clobetasol propionate 0.05% cream was applied to mucosal lesions and involved skin twice a day for at least 15 days, then progressively tapered. Pemphigus was considered to be controlled if healing of lesions was obtained, with a 75% decrease in the number of new lesions per week without addition of any systemic treatment. In all seven patients, the disease was controlled initially with healing of cutaneous lesions within 15 days, while healing of mucosal lesions took at least 1 month. In four patients, remission was maintained with topical corticosteroid alone for a mean 19-month follow-up. In three patients, relapse occurred after 2-11 months, requiring a systemic treatment.     

Treatment of pemphigus vulgaris and pemphigus foliaceus with mycophenolate mofetil

BACKGROUND: Mycophenolate mofetil is increasingly being used as a corticosteroid-sparing agent in immunosuppressive regimens. OBJECTIVE: To elucidate the effectiveness of mycophenolate as adjuvant therapy in the treatment of both pemphigus vulgaris and pemphigus foliaceus. DESIGN: Historical prospective study. SETTING: University hospital. PATIENTS: The study included 42 consecutive patients with pemphigus (31 with pemphigus vulgaris and 11 with pemphigus foliaceus) who had relapses during prednisone taper or had clinically significant adverse effects from previous drug therapy. RESULTS: Remission was achieved in 22 (71%) and 5 (45%) of patients with pemphigus vulgaris and pemphigus foliaceus, respectively. Partial remission was achieved in 1 (3%) and 4 (36%), respectively. The median time to achieve complete remission was 9 months (range, 1-13 months). The treatment was administered for a median of 22 months, and the median follow-up period was 22 months. Seventy-seven percent of patients had no adverse effect. Two patients had side effects severe enough to necessitate discontinuation of treatment, one because of symptomatic but reversible neutropenia and the other because of nausea. CONCLUSION: Mycophenolate is an effective and safe adjuvant in the treatment of both pemphigus vulgaris and pemphigus foliaceus.     

Oxidative stress in patients with endemic pemphigus foliaceus and healthy subjects with anti-desmoglein 1 antibodies

Background:Previous studies have shown oxidative stress in pemphigus vulgaris and pemphigus foliaceus, nevertheless, it remains unknown whether a similar response is characteristic of endemic pemphigus foliaceus in Peru.Objectives:To determine the oxidative stress response in endemic pemphigus foliaceus patients and subjects with positive for anti-desmoglein1 antibodies (anti-dsg1) from endemic areas of Peru.Subjects and methods:This is a cross-sectional study. The study population included 21 patients with Endemic Pemphigus foliaceus and 12 healthy subjects with anti-dsg1 antibodies from the Peruvian Amazon (Ucayali), as well as 30 healthy control subjects. Malondialdehyde, an indicator of lipid peroxidation by free radicals, was measured in serum.Results:We collected 21 cases of endemic pemphigus foliaceus, 15 of them with active chronic disease and 6 in clinical remission. Serum malondialdehyde values in patients with chronic active evolution and healthy subjects with anti-dsg1 antibodies were statistically higher than those of healthy controls (p<0.001). There was no significant difference between serum values of localized and generalized clinical forms.Study limitations:The main limitation of this present study is the small number of patients with endemic pemphigus and healthy subjects positive for desmoglein 1 antibodies.Conclusions:The increased serum levels of malondialdehyde in patients with chronic active endemic pemphigus foliaceus and healthy subjects from endemic areas with anti-dsg1 antibodies may suggest a contribution of systemic lipid peroxidation in the pathogenesis of endemic pemphigus foliaceus.     

IgG/IgA pemphigus in a patient with a history of pemphigus vulgaris: An example of epitope spreading?

We report a case of IgG/IgA pemphigus presenting as pemphigus foliaceus following diagnosis and treatment of classic IgG‐mediated pemphigus vulgaris. The dual presentation of IgG and IgA positivity on direct immunofluorescence (DIF) constitutes a rare form of pemphigus with a wide variety of clinicopathologic manifestations. The progression of pemphigus vulgaris is commonly because of epitope spreading. However, the patient's disease was partially refractory to rituximab and showed a change in the DIF with dual staining for IgG and IgA. This indicates that class‐switching may have occurred with epitope spreading or that there was autoreactive IgA at the onset of disease below the threshold of detection by DIF. Our case indicates that in progressive autoimmune disease refractory to treatment, re‐evaluation of the patient for antibody isotypes absent on initial diagnosis may offer key information in better identifying the cause of progression as well as in directing the necessary treatment.     

Drug-induced pemphigus: autoantibodies directed against the pemphigus antigen complexes are present in penicillamine and captopril-induced pemphigus

Pemphigus is an autoimmune blistering disease characterized by circulating autoantibodies directed against the keratinocyte cell surface. The two variants, pemphigus foliaceus and pemphigus vulgaris, can be distinguished at the molecular level by immunochemical studies. The large majority of patients with pemphigus develop the disease spontaneously; however, there is a small group of patients who develop pemphigus after treatment with certain medications, of which penicillamine and captopril are the best documented. Most patients with drug-induced pemphigus have circulating and/or tissue bound epidermal cell surface autoantibodies; however, the molecular specificity of these autoantibodies has not been studied. We performed immunoprecipitation studies utilizing extracts of 125I-labeled suction blister epidermis and the sera of three patients with drug-induced pemphigus foliaceus (two due to penicillamine and one due to captopril) and one patient with captopril-induced pemphigus vulgaris. We found that the three patients with drug-induced pemphigus foliaceus had circulating autoantibodies that are directed against the pemphigus foliaceus antigen complex and that the one patient with drug-induced pemphigus vulgaris had circulating autoantibodies that are directed against the pemphigus vulgaris antigen complex. This study demonstrates that autoantibodies from drug-induced pemphigus patients have the same antigenic specificity, on a molecular level, as do autoantibodies from other pemphigus patients.     

Therapeutic effect of mizoribine on pemphigus vulgaris and pemphigus foliaceus

We evaluated the effectiveness of mizoribine, a newly developed immunosuppressive agent, as an adjuvant therapy in the treatment of both pemphigus vulgaris and pemphigus foliaceus. Eleven pemphigus patients (eight pemphigus vulgaris and three pemphigus foliaceus) received the combination therapy of prednisolone and mizoribine. Complete remission was observed in three of the eight patients with pemphigus vulgaris and in one of the three patients with pemphigus foliaceus. The four patients with complete remission had a rapid clinical response and achieved remission at a median of 11.8 months. Partial remission was achieved in two of the three patients with pemphigus foliaceus. The median time to achieve partial remission was 16.0 months. Six (55.6%) of the 11 patients with pemphigus had complete or partial remission and were able to taper their prednisolone. The cumulative probability of having a complete remission was 64.3% at 19 months of follow-up using Kaplan-Meier analysis. The effectiveness of the additional mizoribine therapy could be attributed to its corticosteroid-sparing properties as well as its immunosuppressive effects. The serum concentration titer of mizoribine was around 1.0?μg/mL 2 hours after administration. Patients who were not improved by the additional mizoribine might require a continuously higher dose of mizoribine to achieve effective therapy.     

Common human leukocyte antigen alleles in pemphigus vulgaris and pemphigus foliaceus Italian patients.

Pemphigus refers to a group of autoimmune blistering skin diseases, mainly identified as pemphigus vulgaris and pemphigus foliaceus, both characterized by the presence of autoantibodies against keratinocyte adhesion molecules, leading to loss of cell-cell adhesion with consequent blister formation. Pemphigus vulgaris is reported to be associated with human leukocyte antigen DR4 and/or DR6 whereas no data are available on pemphigus foliaceus, except for the endemic Brazilian form (fogo selvagem), which is reported to be associated with DR1 and DR4. We here report human leukocyte antigen molecular typing on a total of 87 patients, 61 with pemphigus vulgaris and 26 with pemphigus foliaceus, versus 128 healthy matched controls. Generic typing showed an increase of DRB1*04 and DRB1*14 and a decrease of DRB1*07 in both pemphigus vulgaris and pemphigus foliaceus patients. Molecular subtyping of DR4+ and DR14+ subjects showed a highly significant association between the DRB1*1401 and both pemphigus vulgaris (p < 0.0001) and pemphigus foliaceus patients (p < 0.0001) together with a significant increase of the linked DQB1*0503 (pemphigus vulgaris p < 0.0001; pemphigus foliaceus p < 0.0001). Moreover, whereas the association between DRB1*0402 and pemphigus vulgaris (p < 0.0001) has been confirmed, no significant association between a specific allele of the DR4 group and pemphigus foliaceus, has been found. Therefore, at least in Italian patients, pemphigus vulgaris and pemphigus foliaceus share DRB1*1401 and DQB1*0503, as susceptible human leukocyte antigen alleles, whereas DRB1*0402 is only found associated with pemphigus vulgaris. The observation that both diseases, pemphigus vulgaris and pemphigus foliaceus, carry the same susceptible human leukocyte antigen alleles has been interpreted as a common genetic background predisposing to pemphigus as, like in other autoimmune disorders, it is not sufficient to explain the onset of the disease on the basis of the sole aforementioned alleles. Other linked genes and/or environmental factors should play a facilitating role in the outbreak of pemphigus, either pemphigus vulgaris or pemphigus foliaceus.     

The anti-desmoglein 1 autoantibodies in pemphigus vulgaris sera are pathogenic

Pemphigus vulgaris and pemphigus foliaceus are two closely related, but clinically and histologically distinct, autoimmune skin diseases. The autoantigens for pemphigus vulgaris and pemphigus foliaceus are desmoglein 3 and desmoglein 1, respectively. The anti-desmoglein 1 antibodies in pemphigus foliaceus and anti-desmoglein 3 antibodies in pemphigus vulgaris are pathogenic as determined by immunoglobulin G passive transfer animal models. More than 50% of pemphigus vulgaris sera also contain anti-desmoglein 1 autoantibodies; however, the pathogenicity of the anti-desmoglein 1 autoantibodies in pemphigus vulgaris remains unknown. In this study, we used soluble recombinant extracellular domains of desmoglein 1 and desmoglein 3 to obtain affinity-purified anti-desmoglein 1 and anti-desmoglein 3 autoantibodies from pemphigus vulgaris sera and examined the pathogenicity of each fraction separately using the passive transfer mouse model. By immunoprecipitation, the purified anti-desmoglein 1 and anti-desmoglein 3 showed no cross-reactivity. The anti-desmoglein 1 autoantibodies in pemphigus vulgaris induced typical pemphigus foliaceus lesions in neonatal mice, whereas the anti-desmoglein 3 fraction induced pemphigus vulgaris-like lesions. In addition, the pathogenic anti-desmoglein 1 and anti-desmoglein 3 autoantibodies in pemphigus vulgaris had predominant IgG4 subclass specificity. These findings suggest that the anti-desmoglein 1 antibodies in pemphigus vulgaris are pathogenic.