The Second Study of Clinical and Immunological Findings in Anti-laminin 332-Type Mucous Membrane Pemphigoid Examined at Kurume University-Diagnosis Criteria Suggested by Summary of 133 Cases.

Background: Recently, we published an article retrospectively summarizing the results in 55 anti-laminin 332 (LM332)-type mucous membrane pemphigoid (MMP) cases examined at Kurume University, which were diagnosed by strict inclusion criteria, including positive reactivity in direct immunofluorescence and absence of antibodies to non-LM332 autoantigens. However, indirect immunofluorescence using 1M-NaCl-split normal human skin (ssIIF) is also valuable for diagnosis of anti-LM332-type MMP. Methods: In this second study, we selected 133 anti-LM332-type MMP cases, which were diagnosed by our different inclusion criteria: (i) immunoglobulin G (IgG) deposition to basement membrane zone (BMZ) by direct immunofluorescence or IgG reactivity with dermal side of split skin by ssIIF, (ii) positivity for at least one of the three subunits of LM332 by immunoblotting of purified human LM332, and (iii) the presence of mucosal lesions. Clinical, histopathological, and immunological findings were summarized and analyzed statistically. Although these cases included the 55 previous cases, the more detailed study for larger scale of patients was conducted for further characterization. Results: Clinically, among the 133 patients, 89% and 43% patients had oral and ocular mucosal lesions, respectively, 71% had cutaneous lesions, and 17% had associated malignancies. Histopathologically, 93% patients showed subepidermal blisters. The sensitivities of ssIIF and direct immunofluorescence are similar but are significantly higher than indirect immunofluorescence using non-split human skin (both p < 0.001). In immunoblotting of purified LM332, patient IgG antibodies most frequently reacted with LMγ2 subunit (58%), followed by LMα3 (49%) and LMß3 (36%). Thirty-four percent patients recognized additional non-LM332 autoantigens. Statistical analysis revealed that autoantibodies against non-LM332 autoantigens might stimulate the production of anti-LMγ2 antibodies. Conclusions: This retrospective study further characterized in more detail the clinical and immunological features of 133 cases of anti-LM332-type MMP, in which the new diagnostic criteria without positive direct immunofluorescence reactivity were useful for the diagnosis. Higher frequency with anti-LMγ2 antibodies suggested more significant pathogenic role of this subunit. Additional autoantibodies to non-LM332 autoantigens detected in one-third of the patients may contribute to complexity in anti-LM332-type MMP, including the induction of anti-LMγ2 antibodies.

Comparison of Two Diagnostic Assays for Anti-Laminin 332 Mucous Membrane Pemphigoid.

Anti-laminin 332 mucous membrane pemphigoid (MMP) is an autoimmune blistering disease characterized by predominant mucosal lesions and autoantibodies against laminin 332. The exact diagnosis of anti-laminin 332 MMP is important since nearly 30% of patients develop solid cancers. This study compared two independently developed diagnostic indirect immunofluorescence (IF) tests based on recombinant laminin 332 expressed in HEK239 cells (biochip mosaic assay) and the migration trails of cultured keratinocytes rich in laminin 332 (footprint assay). The sera of 54 anti-laminin 332 MMP, 35 non-anti-laminin 332 MMP, and 30 pemphigus vulgaris patients as well as 20 healthy blood donors were analyzed blindly and independently. Fifty-two of 54 and 54/54 anti-laminin 332 MMP sera were positive in the biochip mosaic and the footprint assay, respectively. In the 35 non-anti-laminin 332 MMP sera, 3 were positive in both tests and 4 others showed weak reactivity in the footprint assay. In conclusion, both assays are easy to perform, highly sensitive, and specific, which will further facilitate the diagnosis of anti-laminin 332 MMP.

Circulating bullous pemphigoid autoantibodies in the setting of negative direct immunofluorescence findings for bullous pemphigoid: A single-center retrospective review.

BACKGROUND: Bullous pemphigoid (BP) autoantibody levels are generally elevated in patients with BP but can be present nonspecifically in patients without BP. OBJECTIVE: To analyze the clinical findings of patients with elevated BP180 or BP230 autoantibody levels and negative direct immunofluorescence (DIF) study findings. METHODS: We retrospectively reviewed records of patients seen at our institution during January 1, 2005-December 31, 2015, who were positive for BP180 or BP230 autoantibodies and had a negative DIF study finding. These patients' demographic characteristics and BP180 and BP230 levels were compared with those of a BP control group who were positive for BP180 or BP230 autoantibodies and had positive DIF study findings. RESULTS: We identified 208 patients with BP autoantibodies but without positive DIF study findings. These patients' mean age and enzyme-linked immunosorbent assay values were significantly lower than those of the control group. Dermatitis was the most common final clinical diagnosis. Of the 208 patients, 41 (19.7%) had at least 2 years' follow-up. Four patients had positive DIF results upon repeating the test and ultimately received pemphigoid diagnoses. LIMITATIONS: Retrospective design with limited follow-up. CONCLUSION: Patients might harbor serum BP autoantibodies in the context of a wide range of dermatoses. Low positive BP180 and BP230 autoantibody levels should not be overinterpreted as evidence for BP in the setting of a negative DIF.

A sensitive and specific assay for the serological diagnosis of antilaminin 332 mucous membrane pemphigoid.

BACKGROUND: Antilaminin 332 mucous membrane pemphigoid (MMP) is an autoimmune subepidermal blistering disease with predominant mucosal involvement and autoantibodies against laminin 332. Malignancies have been associated with this disease; however, no standardized detection system for antilaminin 332 serum antibodies is widely available. OBJECTIVES: Development of a sensitive and specific assay for the detection of antilaminin 332 antibodies. METHODS: An indirect immunofluorescence (IF) assay using recombinant laminin 332 was developed and probed with a large number of antilaminin 332 MMP patient sera (n = 93), as well as sera from patients with antilaminin 332-negative MMP (n = 153), bullous pemphigoid (n = 20), pemphigus vulgaris (n = 20) and noninflammatory dermatoses (n = 22), and healthy blood donors (n = 100). RESULTS: In the novel IF assay, sensitivities with the laminin 332 heterotrimer and the individual α3, ß3 and γ2 chains were 77%, 43%, 41% and 13%, respectively, with specificities of 100% for each substrate. The sensitivity for the heterotrimer increased when an anti-IgG4 enriched antitotal IgG conjugate was applied. Antilaminin 332 reactivity paralleled disease activity and was associated with malignancies in 25% of patients with antilaminin 332 MMP. CONCLUSIONS: The novel IF-based assay will facilitate the serological diagnosis of antilaminin 332 MMP and may help to identify patients at risk of a malignancy.

Multiple and repeated sampling increases the sensitivity of direct immunofluorescence testing for the diagnosis of mucous membrane pemphigoid.

BACKGROUND: Mucous membrane pemphigoid (MMP) is an autoimmune disease characterized by the predominant blistering of mucosal surfaces and the linear deposition of complement, IgG, or IgA along the basement membrane detected by direct immunofluorescence (DIF) test. OBJECTIVE: To assess the impact of multiple and repeated DIF sampling on establishing the diagnosis of MMP. METHODS: We reviewed the results of DIF studies in 136 nonlesional biopsies from 78 patients who were immunologically confirmed to have MMP. RESULTS: Thirty-six of 52 patients (69%) who underwent only 1 biopsy at the first workup were positive. In 13 cases, the initial single biopsy was negative, and later biopsies were positive. Twenty-two of 26 patients (85%) who underwent multiple biopsies at the initial workup showed ≥1 positive DIF test result. Simultaneously obtained biopsies yielded discordant positive and negative findings in 11 patients. Overall, 74 of 78 patients (95%) had ≥1 positive result by DIF test. In the remaining 4 cases, the diagnosis was confirmed by the detection of circulating autoantibodies against BP180. LIMITATIONS: This is a retrospective, single-center study. CONCLUSION: Our data demonstrate that multiple and repeated biopsies increase the sensitivity of the DIF test for MMP diagnosis. Negative DIF test findings in cases clinically suggestive of MMP should prompt repeat biopsies.

Integrin ß4 is a major target antigen in pure ocular mucous membrane pemphigoid.

Previous studies of ocular mucous membrane pemphigoid (OMMP) have identified several components of the basement membrane zone to be autoantigens, including integrin ß4. However, there are no extensive or definitive reported studies that address this, particularly in pure OMMP. To clarify the major autoantigens in pure OMMP. In this study, we examined sera from 43 pure OMMP patients for both IgG and IgA antibodies using newly developed immunoblotting analyses with a hemidesmosome-rich fraction and various recombinant proteins of integrin α6ß4, in addition to our routine immune-serological tests. Using a hemidesmosome-rich fraction, sera from patients with pure OMMP demonstrated reactivity of IgG and/or IgA antibodies to integrin ß4, BP180 and laminin-332. The reactivity of pure OMMP sera to integrin ß4 was further confirmed by immunoblotting using integrin ß4 recombinant proteins. Using concentrated supernatant of HaCaT cells, only one serum sample showed positive IgG and IgA reactivity to LAD-1, the ectodomain of BP180. None of the pure OMMP sera reacted with any autoantigens on immunoblotting using normal human epidermal or dermal extracts, or purified human laminin-332. Integrin ß4 was considered to be the major and specific autoantigen for pure OMMP. The new methods established in this study are useful for detection of various autoantigens, particularly integrin ß4.

Diagnosis of oral mucous membrane pemphigoid by means of combined serologic testing.

OBJECTIVE: Mucous membrane pemphigoid (MMP) is a rare autoimmune bullous disease caused by various autoantibodies. This study aimed to evaluate the diagnostic value of MMP-specific autoantibodies in patient sera. STUDY DESIGN: We analyzed sera from 30 MMP-suspected patients with intractable oral mucosal lesions using a combination of indirect immunofluorescence with 1M NaCl-split skin, immunoblot analysis, and ELISAs. We also analyzed clinical features among different types of MMP. RESULTS: Seventeen, 4, and 3 patients were diagnosed with anti-BP180-type MMP, anti-laminin-332-type MMP, and combined anti-BP180/anti-laminin-332-type MMP, respectively. CONCLUSIONS: Our results indicated that a combination of immunologic testing for circulating autoantibodies is useful for the diagnosis of MMP.

Prevalence and clinical significance of anti-laminin 332 autoantibodies detected by a novel enzyme-linked immunosorbent assay in mucous membrane pemphigoid.

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.

Mucous membrane pemphigoid with antibodies against ß3 subunit of laminin-332: first report from India.

Mucous membrane pemphigoid (MMP) is a chronic, recurrent, progressive, subepidermal blistering disorder, mainly affecting the mucous membranes. Anti-laminin 332 MMP is a distinct subset of MMP with antibodies, mainly targeted against α3 or γ2 subunit. Antibodies exclusively against ß3 subunit are rarely seen. An internal malignancy is frequently associated with anti-laminin 332 MMP. This disorder usually responds poorly to treatment, requiring multidisciplinary approach. Herein, we describe the first case of anti-laminin-332 MMP from India, which showed antibodies exclusively to ß3 subunit.

Pentoxifylline (anti-tumor necrosis factor drug): effective adjuvant therapy in the control of ocular cicatricial pemphigoid.

PURPOSE: The detection of tumor necrosis factor-a (TNF-a) in conjunctiva affected by ocular cicatricial pemphigoid (OCP) may indicate that this cytokine plays an important role in its pathogenesis. The purpose of this randomized, controlled, comparative, blinded study was to evaluate the effectiveness of adding pentoxifylline as an anti-TNF-a drug to the well-documented therapy of steroids and cyclophosphamide in controlling OCP. METHODS: Thirty patients with different grades of OCP were included. They were randomly divided into 2 equal groups. Group A patients received pulse steroid and cyclophosphamide therapy; in addition, group B patients received intravenous pentoxifylline. Patients were evaluated before and after therapy clinically, histopathologically, and serologically (serum level of TNF-a). Twenty controls were included to compare their serum TNF-a level with that measured in patients with OCP. RESULTS: Group B patients showed a more significant improvement in their clinical and histopathologic evaluation. The serum TNF-a was significantly higher in OCP cases prior to therapy compared to the control group (p = 0.0001). Following therapy, serum TNF-a showed a more significant reduction in group B patients (77.4 ± 26.1 to 19.2 ± 15.6) compared to group A patients (50.3 ± 14.3 to 36.2 ± 18.3). CONCLUSIONS: The significantly increased level of serum TNF-a in OCP as compared to controls proves that TNF-a has an important role in the pathogenesis of this disease. The study illustrates that the addition of pentoxifylline to pulse steroid cyclophosphamide therapy is an effective, safe, and economical method in controlling OCP through directly reducing TNF-a levels, with long periods of remission as detected in our 18-month follow-up period.