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Mucous membrane pemphigoid (MMP) is a rare mucocutaneous immunobullous disorder associated with significant morbidity. Early diagnosis is paramount to reduce scarring complications and to optimise therapy. Preliminary work in our laboratory has demonstrated the potential benefit of saliva as an alternative to serum for diagnosis. However, the relationship between target antigens and clinical phenotypes is unclear. Identification of patients at higher clinical risk might be improved through identification of potential biomarkers. Aims: To investigate 1) the association of clinical phenotypes with specific target antigens and epitopes in MMP that may explain the spectrum of clinical presentation and disease severity. 2) serum and salivary biomarkers in the analysis of disease activity and therapeutic responses in MMP. 3) the diagnostic and disease-monitoring capacity of saliva in MMP. Methodology: Sera were collected from 117 MMP patients, 50 healthy controls (HC) and 16 disease controls (DC). Matched whole saliva samples were collected from 63/117 MMP patients. Parotid saliva was taken from 16 MMP patients, 11 DC and 4 HC. Basement membrane zone autoantibody binding patterns were noted. Reactivity with laminin 332 (L332) and bullous pemphigoid antigen 2 (BP180) were determined by enzyme-linked immunosorbent assays (ELISA). Disease severity was assessed using the Guy's Oral Disease Severity Score (ODSS). Sequential samples were analysed over 9 months in 11 MMP patients. A detailed clinical presentation and severity scoring was undertaken and correlated with immunopathological findings. Results: Among the MMP patients (n=117), 101 were biopsied with 95/101 (94%) showing positive DIF. IIF was performed on 112 patients and 54 (48.2%) were positive on salt split skin (SSS), of these 50 (92.5%) were epidermal and only 4 (7.4%) showed dermal binding pattern on SSS. Serum IgG antibodies to BP180 NC16a polypeptide were detected in 29/55(52.7%) MMP patients and serum IgA in 10/55(18%) whereas salivary IgG and IgA antibodies were present in 12/28(42.8%) and 9/28 (32%), respectively. Serum IgG antibodies to selected peptides from BP180 midportion were detected in 26/55 (47.2%) MMP and serum IgA in 17/55(31%) while salivary IgG and IgA were in 14/28 (50%) and 7 /28(25%), respectively. Serum IgG to the C-terminal (4575 epitope) and adjacent peptides was present in 15 /55 (27.2%) patients and IgA in 12(21.8%); salivary IgG was in 11/28 (39.2%) and salivary IgA in 5(17.8%) and 40% of MMP were negative to NC16a but positive to either midportion or the C-terminal region of BP180. Combined use of serum and saliva increased detection of specific antibodies by 39% for NC16a and by 28% for 4575 domains. Antibodies to Laminin 332 were detected in 48/117(41%) MMP patient sera.; IgG 40/117 (34%), IgA 11/117 (9.4%) and both 3/117 (2.5%). In whole saliva samples, IgG to L332 was detected in 13/63 (20.6%), IgA 17/63 (26.9%), both 5/63(8%) and in parotid saliva secretory IgA antibodies in 8/16 (50%). Combined use of serum and saliva increased detection of specific antibodies by 27%. A significant association was found between serum IgA antibodies against L332 and the presence of ocular lesions (χ2 = 6.56 p = 0.038). There was a positive relation between the titre of IgG antibody in serum and ODSS in MMP (Spearman r =0.26 p = < 0.01). Sequential samples showed that there was a positive correlation between the titre of serum IgG antibody to L332 and ODSS at baseline (Spearman r =0.63 p = <0.04), but neither serum IgA nor saliva IgA and/or IgG anti-L332 antibodies were found to be a significantly correlated with the ODSS. Mean serum and salivary IgG and IgA anti-L332 antibody titres showed statistically significant decreases over the 9-month period (p = <0.01). Conclusion: Our data has showed that 1) demonstration of a relationship between serum and saliva L332 or BP180 specific antibodies in defined clinical subgroups and oral disease severity. 2 ) the presence of serum and salivary IgG and IgA antibodies to L332 in a predominantly epidermal binding cohort of MMP patients. 3) the novel finding of salivary IgG and secretory IgA antibodies to L332 and/or BP180 and when combined results with serum increase diagnostic sensitivity. 4) the novel epitopes within the BP180 which can recognise both antibody isotypes . 5) preliminary evidence to support the potential association between L332 reactive patients and an underlying malignancy. These new findings demonstrate that serum and salivary IgG and IgA antibodies to the multiple extracellular epitopes of BP180 and L332 may be useful in the diagnosis of MMP, and that saliva is useful for diagnosing and monitoring MMP and furthering the understanding of disease pathogenesis.

Mucous membrane pemphigoid (MMP) and pemphigus vulgaris (PV) are uncommon mucocutaneous immunobullous disorders associated with significant morbidity. The aims were to: 1) Investigate the potential of saliva for diagnosis and disease-monitoring 2) Utilize serum and salivary biomarkers in the analysis of disease severity and therapeutic responses 3) Correlate phenotypic subgroups with target antigens to potentially explain the spectrum of clinical presentation and disease severity in MMP. Methodology: Matched serum and whole saliva samples were taken from a total of 100 MMP patients, 26 PV patients, 50 healthy controls (HC) and 16 Lichen planus (LP) patients. All LP and PV patients, 40 MMP and 6 HC provided parotid saliva. Reactivity with the NC16a epitope on bullous pemphigoid antigen 2 (BP180), the alpha 6 beta 4 integrin (α6β4) and desmoglein 3 (Dsg3) was determined by Enzyme-linked immunosorbent assays (ELISA). Disease severity was assessed using the Guy’s Oral Disease Severity Score. Results: In MMP, IgG and/or IgA antibody to BP180-NC16a (44%) in whole saliva showed a similar sensitivity to serum (48%). In parotid saliva IgA to BP180-NC16a was positive in 45% and was also secretory component positive (r =0.9141). A change in serum antibodies (IgG and IgA) to BP180-NC16a was significantly related to a change in severity scores (p =0.048 and 0.033, respectively). A positive association was found between 1) the presence of serum IgG to BP180-NC16a and the presence of lesions in multisite disease (p =0.003) and 2) the presence of IgA to BP180-NC16a in whole saliva and the presence of oral and ocular lesions (p =0.016). IgG antibody to α6β4 integrin was positive in 36% of serum and 18% of saliva. Regarding PV, anti-Dsg3 IgG antibody was positive in serum (74%) and whole saliva (61%) with a positive correlation (r =0.9044). Serum IgA anti-Dsg3 antibody was positive in 61% (with a combined IgG positivity of 78% in PV patients). A significant relationship between anti-Dsg3 IgG antibody in whole saliva and therapeutic responses was found (p =0.004). Conclusions: The results show 1) the value of salivary anti-Dsg3 IgG antibodies in monitoring PV patients and confirm the sensitivity and specificity of IgG anti-Dsg3 serum and saliva antibodies in PV diagnosis. The high positivity of serum IgA anti-Dsg3 antibodies requires further elucidation in disease pathogenesis. The salivary biomarkers, IgG and IgA antibodies to BP180-NC16a and α6β4 may be useful in the diagnosis of MMP. 2) IgG and IgA antibodies to BP180-NC16a in serum can be used for disease monitoring and therapeutic responses in MMP. 3) Clinical phenotypes did not appear to be explained by epitope or isotype specificity. However, the novel finding of locally produced antibodies to BP180-NC16a may provide further insight into the pathogenesis of the oral disease. These new findings demonstrate the value of saliva in the diagnosis and monitoring of MMP and for furthering the understanding of disease pathogenesis.