Alteration of Gene Expression in Pathological Keratinization of the Ocular Surface.

Purpose: To investigate the molecular mechanism of pathological keratinization in the chronic phase of ocular surface (OS) diseases. Methods: In this study, a comprehensive gene expression analysis was performed using oligonucleotide microarrays on OS epithelial cells obtained from three patients with pathological keratinization (Stevens-Johnson syndrome [n = 1 patient], ocular cicatricial pemphigoid [n = 1 patient], and anterior staphyloma [n = 1 patient]). The controls were three patients with conjunctivochalasis. The expression in some transcripts was confirmed using quantitative real-time PCR. Results: Compared to the controls, 3118 genes were significantly upregulated by a factor of 2 or more than one-half in the pathological keratinized epithelial cells (analysis of variance P < 0.05). Genes involved in keratinization, lipid metabolism, and oxidoreductase were upregulated, while genes involved in cellular response, as well as known transcription factors (TFs), were downregulated. Those genes were further analyzed with respect to TFs and retinoic acid (RA) through gene ontology analysis and known reports. The expression of TFs MYBL2, FOXM1, and SREBF2, was upregulated, and the TF ELF3 was significantly downregulated. The expression of AKR1B15, RDH12, and CRABP2 (i.e., genes related to RA, which is known to suppress keratinization) was increased more than twentyfold, whereas the expression of genes RARB and RARRES3 was decreased by 1/50. CRABP2, RARB, and RARRES3 expression changes were also confirmed by qRT-PCR. Conclusions: In pathological keratinized ocular surfaces, common transcript changes, including abnormalities in vitamin A metabolism, are involved in the mechanism of pathological keratinization.

Conjunctival transcriptomics in ocular mucous membrane pemphigoid.

PURPOSE: Ocular Mucous Membrane Pemphigoid (OcMMP) is an orphan disease characterized by chronic autoimmune-driven conjunctival inflammation leading to progressive scarring, debilitating symptoms, and blinding sequelae. This feasibility study aims to demonstrate conjunctival genetic transcriptomic analyses as a putative tool for interrogation of pathogenic signaling pathways in OcMMP. METHODS: Conjunctival RNA profiling using the NanoString nCounter Human Fibrosis panel was undertaken on RNA extracted from conjunctival swabs obtained from 6 MMP patients (8 eyes; 4 M/2F; median age 78 [range 64-84] years); and 8 age-matched control participants (15 eyes; 3 M/5F; median age 69.5 [range 69-88] years). Data from 770 genes were analyzed with ROSALIND HyperScale architecture and stratified according to the level of clinically visible bulbar conjunctival inflammation. Normalization, fold-changes (≥+1.5-fold or ≤ -1.5-fold) and p-values adjustment (<0.05) using the Benjamini-Hochberg method were calculated. RESULTS: 93 differentially expressed genes (DEGs) were observed between OcMMP versus controls of which 48 were upregulated, and 45 downregulated. The top 4 upregulated DEGs represented fibrosis (COL3A1, COL1A1, FN1 and THBS1) while the key under-expressed genes (SCIN, HMGS2, XCL1/2) were indicative of ocular surface failure (goblet cell loss, keratinization, vulnerability to secondary infections). Forty-four pathways had a global significance score ≥2, the most significant being those related to extracellular matrix (ECM) remodeling, synthesis, and degradation. These pathways were accentuated in eyes with visible inflammation. CONCLUSIONS: NanoString methodology acquired via a simple conjunctival swab identifies profibrotic genes in OcMMP group and differentiates inflamed eyes. Longitudinal sampling and following investigative intervention will further mechanistic insight and development of novel biomarkers to monitor disease progression.

Genomewide association study identifies GALC as susceptibility gene for mucous membrane pemphigoid.

Mucous membrane pemphigoid (MMP) is a rare, chronic and often aggressive subepidermal autoimmune blistering disease potentially affecting several mucous membranes with blisters and secondary erosions and scars. The pathogenesis of MMP is poorly understood, and the contribution of genetic predispositions, other than HLA class II allele variants to MMP, is unknown. The objective of this study is to identify susceptibility genes for MMP in a British cohort of MMP patients. A GWAS was conducted in a British cohort of 106 MMP patients. Publicly available genotypes of 2900 blood donors of the UK Blood Service and of 6740 individuals of the 1958 British Birth Cohort served as control. Subsequently, putative susceptibility genes were independently replicated in a German cohort of 42 MMP patients. The GWAS found 38 SNPs in 28 haploblocks with an odds ratio >2 reaching genomewide significance (P < 5.7 × 10-7 ). Replication confirmed an association of MMP with SNPs in rs17203398 (OR: 3.9), located intronically in the ß-galactocerebrosidase gene (GALC) on chromosome 14 and with recessive polymorphisms in rs9936045 (OR: 3.1) in the intergenic region between CASC16 and CHD9 on chromosome 16. The risk of developing MMP is partially genetically determined. SNPs in GALC enhance the risk for MMP, indicating that ß-galactocerebrosidase may be involved in the pathogenesis of MMP. Likewise, impacts of polymorphisms in the intergenic region between CASC16 and CHD9 on the activity of neighbouring genes may facilitate the emergence of MMP. The putative role of both polymorphisms requires functional studies in the future.

The molecular basis for the presence of two autoimmune diseases occurring simultaneously--preliminary observations based on computer analysis.

Specific Human Leukocyte Antigen Class II (HLA II) molecules associated with pemphigus vulgaris (PV), mucous membraine pemphigoid (MMP), and mixed connective tissue disease (MCTD) may react with multiple T cell epitopes within desmoglein 3 (Dsg 3), bullous pemphigoid antigen 2 (BPAG 2), and 70 kDa polypeptide small nuclear ribonucleoproteins (snRNP70) in autoantibody production. We report a group of patients with simultaneous occurrences of PV with MCTD, and MMP with MCTD. In one patient group, we performed serological studies to show presence of antibodies to Dsg 3, Dsg 1, and snRNP70 simultaneously. In the second group, we performed serological studies to show presence of antibodies to BPAG 1, BPAG 2, ß4 integrin, and snRNP70 simultaneously. In both groups, HLA II genes were analyzed and the observations were consistent with previously described associations with PV, MMP, and MCTD. It is possible that HLA-DQß1*0301 allele, present in 10 of 17 patients and DRß1*04 in some of the others, may have the ability to bind to several relevant T cell epitopes in the snRNP70 molecule. We have utilized a computer model to demonstrate that HLA II-restricted T cell epitopes present within the known autoantigens may be capable of eliciting an immune response. While other explanations and mechanisms exist, the authors suggest that epitope spreading may be one possible mechanism, amongst others, that may result in the simultaneous presence of two separate pathogenic autoantibodies.

Class II alleles HLA-DQB1* 0301 among a seven-membered Egyptian family of a child with oral pemphigoid.

OBJECTIVES: The presence of HLA-DBQ1* 0301 allele in an Egyptian patient with oral pemphigoid and six of his family members was investigated. BACKGROUND: Oral pemphigoid is a variant of vesiculobullous mucous membrane pemphigoid with an immunogenetic predisposition related to defects in major histocompatibility complex (OMIM 604305), HLA-DQB 1 alleles. METHODS: High resolution typing of HLA-DQB 1*0301 allele was carried out for the seven members of an Egyptian family including the child affected with oral pemphigoid. RESULTS: Both parents and three children including the proband were found to be carriers for the studied allele, while the other two children were not. CONCLUSION: Participation of HLA-DBQ1*0301 allele was not informative for both susceptibility and/or phenotypic expression of the disease within the studied family (Fig. 2, Ref. 31).

Role of MHC Class II genes in the pathogenesis of pemphigoid.

Pemphigoid (Pg) is an autoimmune subepidermal blistering disease that affects the elderly population. The phenotype can be Bullous Pemphigoid (BP), which primarily involves the skin, or Mucous Membrane Pemphigoid (MMP), which primarily involves mucus membranes. Ocular Cicatricial Pemphigoid (OCP) and Oral Pemphigoid (OP) are subsets of MMP. The known antigens in BP are Bullous Pemphigoid Antigen 1 (BPAG1, also known as BP230), Bullous Pemphigoid Antigen 2 (BPAG2, also known as BP180), and subunits of human integrins α6 and ß4. The Human Leukocyte Antigen (HLA) allele HLA-DQß1*0301 has been reported to be associated with enhanced susceptibility to all of these subsets. Sera of patients with the four subsets are characterized by the presence of anti-Basement Membrane Zone (anti-BMZ) antibodies. In this manuscript, we present a model in which relevant portions of the four different antigens involved in pemphigoid have potential sites that could be presented by an antigen presenting cell (APC) in conjunction with DQß1*0301 to a T cell receptor to initiate the process that results in anti-BMZ antibody production. Thus, this model provides a hypothetical computer-based mechanism to explain how a single HLA allele can be associated with the production of antibodies to four different antigens that result in four different subsets of a disease with four different clinical profiles and prognoses.

Relationship between target antigens and major histocompatibility complex (MHC) class II genes in producing two pathogenic antibodies simultaneously.

In this report,we present 15 patients with histological and immunopathologically proven pemphigus vulgaris (PV). After a mean of 80 months since the onset of disease, when evaluated serologically, they had antibodies typical of PV and pemphigoid (Pg). Similarly, 18 patients with bullous pemphigoid (BP) and mucous membrane pemphigoid (MMP) were diagnosed on the basis of histology and immunopathology.After a mean of 60 months since the onset of disease, when their sera were evaluated they were found to have Pg and PV autoantibodies. In both groups of patients the diseases were characterized by a chronic course, which included several relapses and recurrences and were non-responsive to conventional therapy. The major histocompatibility complex class II (MHC II) genes were studied in both groups of patients and phenotypes associated typically with them were observed. Hence, in 33 patients, two different pathogenic autoantibodies were detected simultaneously. The authors provide a computer model to show that each MHC II gene has relevant epitopes that recognize the antigens associated with both diseases. Using the databases in these computer models, the authors present the hypothesis that these two autoantibodies are produced simultaneously due to the phenomena of epitope spreading.

Bullous pemphigoid antigen II (BP180) and its soluble extracellular domains are major autoantigens in mucous membrane pemphigoid: the pathogenic relevance to HLA class II alleles and disease severity.

BACKGROUND: Mucous membrane pemphigoid (MMP), a chronic autoimmune subepithelial blistering disease, is associated with circulating IgG and/or IgA autoantibodies against several basement membrane zone antigens. The heterogeneity of clinical presentation and diversity of target autoantigens have contributed to difficulties in characterizing this condition immunologically. OBJECTIVES: To analyse serum autoantibody profile and HLA class II alleles in MMP patients and to correlate this with the clinical presentation of disease. METHODS: Well-defined subgroups consisting of 124 patients with MMP were examined for IgG and IgA reactivity with immunoblotting using human epidermal, dermal and placental amnion proteins. The results were further analysed on the basis of detailed clinical (sites of involvement and disease severity) and immunopathological criteria (immunofluorescence study and HLA class II alleles). RESULTS: Immunoblot assay revealed that the majority of MMP patients had IgG (93 of 124, 75%) and/or IgA autoantibodies (63 of 124, 51%) to BP180 (including its soluble ectodomains, 120-kDa LAD-1 and 97-kDa LABD97 antigens). Other antigens targeted predominantly by IgG autoantibodies included: BP230 in 34 (27%), beta4 integrin in 26 (21%), and laminin 5 in three (2%). All the BP230+ sera and 23 (88%) beta4 integrin+ sera also reacted with at least one of the BP180 antigens. Over 85% of patients with reactivity to beta4 integrin had ocular involvement. In most cases of MMP, more severe clinical features were associated with antibody reactivity to multiple basement membrane zone antigens, as well as reactivity to multiple BP180 component antigens. Dual BP180/LAD-1 reactivity with IgG and IgA was associated with a more severe phenotype. In addition, the subset-dependent autoantibody reactivity correlated well with specific HLA class II alleles, DQB1*0301, DRB1*04 and DRB1*11. CONCLUSIONS: Our results confirmed that BP180 is a major autoantigen targeted by the sera of patients with MMP. The disease-prevalent HLA class II alleles and humoral autoimmune response against the particular subsets of antigenic epitope(s) within BP180 ectodomain may contribute to the clinicopathological significance and disease severity of MMP.

IL-4, IL-5, TGF-beta1 and IFN-gamma mRNAs detected by a new in situ amplification system in cicatricial pemphigoid.

The process that induces chronic progressive scarring in cicatricial pemphigoid (CP), a rare group of autoimmune mucocutaneous blistering diseases, is still under investigation. The tendency to heal with scar formation observed in CP could be due to the specific localization of the antigen in the basement membrane zone or could depend on the frequent recurrence of the disease in a localized area. The release of soluble fibrogenic factors by inflammatory infiltrating cells has also been considered as pathogenetically relevant. The aim of this study is to evaluate the expression of mRNA for IL-4, IL-5, TGF-beta1, IFN-gamma in patients with CP, and investigate the role of the cytokine profile as a possible cause of the clinical features and course of the disease. Fourteen patients (3 male, 11 female; age range 40-72 years) with oral (n = 10), preputial (n = 3) and cutaneous (n = 1) CP were studied. The formalin-fixed and paraffin-embedded biopsies were examined by in situ hybridization performing a new amplification system based on biotinyl-tyramide. As a control, 4 patients (2 male, 2 female; age range 58-73 years) affected by bullous pemphigoid (BP), the most common autoimmune subepidermal blistering disease, were also examined. In CP, IL-4 mRNA expression was present in 4 out of the 14 cases analysed. IL-5 was detected in 12 CP biopsies. TGF-beta1 and IFN-gamma mRNAs were identified in 9 and 11 CP cases, respectively. In BP, an IL-4 hybridization signal could not be observed in any of the cases. By contrast IL-5, TGF-beta1 and IFN-gamma mRNA analyses were positive in all four BP cases. Our results suggest the presence of a T-cell population with a mixed cytokine pattern in the cellular infiltrate of both blistering diseases, with a corresponding increase of Th2-like activity in fully developed lesions, irrespective of the different sites involved. In addition, on the basis of the constant presence of TGF-beta1 mRNA in the different lesional phases of CP, and its overlapping expression in BP, we hypothesize that the involvement of additional factors is responsible for the scarring course typical of CP.

HLA-DQB1 alleles in Italian patients with mucous membrane pemphigoid predominantly affecting the oral cavity.

BACKGROUND: Mucous membrane pemphigoid (MMP) used to be considered as a single entity but it is now evident that a range of variants exists. Among them, pure ocular cicatricial pemphigoid (OCP) and pure oral pemphigoid (OP) appear to be very different subsets. Previous immunogenetics studies have found increased occurrence of the DQB1*0301 allele mainly in patients with OCP whereas in patients with OP the data are more open to doubt. OBJECTIVES: To analyse HLA predisposition in a group of Italian patients with MMP predominantly affecting the oral cavity. METHODS: We carried out high-resolution typing of HLA-DQB1 alleles in 28 patients with MMP predominantly affecting the oral cavity and in 97 geographically matched, healthy controls. All were Italian caucasians. RESULTS: The frequency of HLA-DQB1*0301 was significantly increased in the MMP patients compared with the controls (96% vs. 48%; corrected P, Pc = 0.001; relative risk, RR = 28.73). A strong association with DQB1*0301 was also evident in patients with OP compared with the controls (95% vs. 48%; Pc = 0.01; RR = 20.21). There was no significant difference in DQB1*0301 frequency between patients with OP and with MMP not restricted to the oral cavity. Patients with MMP were more frequently homozygous for DQB1*0301 than the controls (43% vs. 8%; Pc < 0.001; RR = 8.34). CONCLUSIONS: Our data suggest that Italian patients with MMP lesions predominantly affecting the oral cavity present the same genetic predisposition linked to HLA-DQB1*0301 previously reported mainly in patients with OCP.

Mucous membrane pemphigoid: HLA-DQB1*0301 is associated with all clinical sites of involvement and may be linked to antibasement membrane IgG production.

BACKGROUND: Class I human leucocyte antigens (HLA) -A, -B, -Cw and class II HLA-DRB1, -DQB1 alleles were determined in 131 British Caucasian patients with mucous membrane pemphigoid (MMP) using serological and DNA-based methods. OBJECTIVES: To analyse the class I and II alleles expressed in well-defined clinical and immunopathological subgroups of MMP, in order to establish whether specific alleles or haplotypes might in part explain disease susceptibility, clinical sites of involvement or disease severity. METHODS: Subgroups of patients were analysed according to the following clinical criteria: age of onset, sex, sites of clinical involvement (oral, ocular, skin, nasal, genital, pharyngeal, oesophageal, laryngeal, perianal), disease severity and history of autoimmune disease. Subgroups were also analysed according to the following immunopathological criteria: autoantibody profile, the presence of circulating antibasement membrane IgG or IgA antibodies and the detection of target basement membrane zone (BMZ) antigens (BP230 and BP180) by IgG autoantibodies. RESULTS: Class I HLA typing showed no significant disease or subgroup associations. Class II DRB1 typing showed a significantly increased allelic frequency in MMP vs. controls for DRB1*11 (RR = 2.08, Pc < 0.0000056). For DQB1, MMP vs. controls, there was a significantly increased allelic frequency for DQB1*0301 (Pc < 0.00000028) in both males and females; all clinical sites of involvement, with the exception of laryngeal, oesophageal and perianal sites and in patients with detectable circulating anti-BMZ IgG compared with those negative for IgG (P < 0.0096, Pc < 0.019). A positive trend was noted in patients with ocular involvement compared with no ocular involvement and in patients with a clinical score > or = 10 compared with < 10. We found no difference in DQB1*0301 allele frequency between subgroups with or without BP180 or BP230 target antigens. Haplotype frequencies showed an increase in DRB1*04, DQB1*0301 (Pc < 0.000066) and DRB1*11, DQB1*0301 (Pc < 0.000002) among patients compared with controls. CONCLUSIONS: The DQB1*0301 allele confers a predisposition to all subgroups of MMP and may have a role in T-cell recognition of basement membrane antigens, resulting in the production of anti-BMZ IgG autoantibodies. The positive trend between increased allelic expression of DQB1*0301 in patients with ocular disease and in those with a higher clinical score, further suggests a role for this allele in disease severity.

CTLA4 exon 1 dimorphism in bullous and cicatricial pemphigoid.

The human cytotoxic T-lymphocyte antigen 4 (CTLA4) gene encodes proteins regulating the immune response. The polymorphism of this gene is associated with some autoimmune diseases. In this study, we analysed the distribution of the dimorphisms of exon 1 (+ 49 A/G) in bullous pemphigoid (BP) and cicatricial pemphigoid (CP), two types of autoimmune bullous skin diseases that occur in elderly people. The frequency of the exon 1 A-G genotype was marginally decreased in patients (36.4%; n = 55) compared with controls (52.8%, n = 53), but the results were not statistically significant (P = 0.09).

Three different autoimmune bullous diseases in one family: is there a common genetic base?

We report an unusual familial occurrence of autoimmune bullous diseases. Three members of a family suffered from three different autoimmune bullous diseases: pemphigus vulgaris (PV), linear IgA disease (LAD) and cicatricial pemphigoid (CP). The HLA type was determined in five family members: all were positive for HLA-DQ5/DR6, which is reported to be associated with susceptibility to PV. The CP patient was DQ7(3) positive, which is in concordance with enhanced susceptibility to ocular CP and CP. The LAD patient was B8 and DR3 negative but positive for HLA-A1. Our study supports the hypothesis that there is a genetically transmitted susceptibility to autoimmune bullous diseases but that additional factors seem necessary actually to develop a particular disease.

HLA-DRB1 and HLA-DQB1 genes in susceptibility and resistance to cicatricial pemphigoid in French Caucasians.

Cicatricial pemphigoid (CP) is a chronic, autoimmune, subepithelial blistering disease, characterized by the presence of antibasement membrane antibodies (BMZ) against anchoring filaments components. An association between the HLA-DQB1*0301 allele and ocular cicatricial pemphigoid has been previously reported in North American Caucasians. In this study, we compared high resolution typing HLA-DRB1 and -DQB1 alleles in 25 CP patients (50 haplotypes) with 106 geographically matched, healthy controls (212 haplotypes), who were all French Caucasians. As in American Caucasians, we confirmed a positive association of CP with the HLA-DQB1*0301 allele which was present in 54% of CP haplotypes (27/50); by contrast 21.7% (46/212) of the matched normal individuals carried the DQB1*0301allele (Pc = 7 x 10(-5); RR = 4.23). HLA-DQB1*0301 is in linkage disequilibrium with several DRB1 alleles. Only the DRB1*1101 DQB1*0301 haplotype frequency was significantly increased (24.0% in CP, 6.6% in control, Pc = 0.002). Furthermore, we observed a decrease of the frequency of the DQB1*02 allele (6% vs 25% in the control group, Pc = 0.05; RR = 0.19). The negative association corresponds to a significant decreased frequency of the DRB1*0701 DQB1*0202 haplotype (0% in CP vs 12.7%, Pc = 0.07 PC) and a non-significant decrease of DRB1*0301 DQB1*0201 (4% in CP vs 12.3%). HLA-DQB1*0301 encodes a negative charge at position DQ 57 (Asp), a critical position in peptide binding to the HLA-DQ molecule groove and therefore we speculate that this molecule may play a role in the selection of BMZ peptides in CP.

Eosinophil granule proteins expressed in ocular cicatricial pemphigoid.

BACKGROUND: Blister formation and tissue damage in bullous pemphigoid have been attributed to the release of eosinophil granule proteins--namely, to eosinophil derived cationic protein (ECP) and major basic protein (MBP). In the present investigation these eosinophil granule proteins were studied in the conjunctiva of patients with ocular cicatricial pemphigoid (OCP). METHODS: Conjunctival biopsy specimens obtained from patients with subacute (n = 8) or chronic conjunctival disease (n = 13) were analysed histologically and immunohistochemically using antibodies directed against EG1 (stored and secreted ECP), EG2 (secreted ECP), MBP, CD45 (common leucocyte antigen), CD3 (pan T cell marker), and HLA-DR (class II antigen). RESULTS: Subepithelial mononuclear cells, mast cells, and neutrophils were detected in all specimens. The number of mononuclear cells, neutrophils, CD45+ cells, CD3+ cells, and the HLA-DR expression were significantly higher in the subacute than in the chronic disease group. Some eosinophils were found in specimens from five of eight patients with subacute OCP, but in none of the patients with chronic disease. The eosinophil granule proteins (ECP and MBP) were found in the epithelium and substantia propria in patients with subacute conjunctivitis. CONCLUSIONS: Subepithelial cell infiltration in the conjunctiva greatly differs between subacute and chronic ocular cicatricial pemphigoid specimens. The findings suggest that eosinophil granule proteins may participate in tissue damage in acute phase of inflammation in OCP.

Significantly increased occurrence of HLA-DQB1*0301 allele in patients with ocular cicatricial pemphigoid.

To determine the immunogenetic characteristics of patients with immune-mediated subepithelial blistering diseases of mucous membranes, we performed HLA-typing for the class II MHC gene DQB1*0301 allele using a direct method. Genomic DNA extracted from Caucasian patients was amplified by polymerase chain reaction using primer pairs specific for the DQB loci followed by Southern blotting with a peroxidase-conjugated sequence-specific oligonucleotide probe. Seventy-six percent (16/21) of patients with ocular mucosal disease (with or without oral mucosal and skin diseases) carried the DQB1*0301 allele; by contrast, only 33% (14/42) of race-, age-, and geography-matched normal individuals carried the DQB1*0301 allele (p < 0.005). The relative risk for ocular disease if DQB1*0301 allele is present is 6.4, similar to the relative risk of 8 for patients with ocular but no oral disease (pure ocular cicatricial pemphigoid, p < 0.025). In patients with oral mucosal disease (with or without ocular mucosal and skin diseases), 68% (15/22) carried the DQB1*0301 allele (p < 0.025). When patients with ocular disease were excluded, however, the increased occurrence of the DQB1*0301 allele in patients with oral disease was not statistically significant (64%, 7/11, p < 0.25). In patients with subepidermal blistering skin disease but no oral or ocular disease, there was no increase in the occurrence of the DQB1*0301 allele (38%, 5/13, p > 0.5). The significantly increased occurrence of the DQB1*0301 allele in patients with ocular mucosal disease may point to a distinct immunogenetic factor that predisposes patients to develop an ocular scarring process.

Differences in the anti-basement membrane zone antibodies in ocular and pseudo-ocular cicatricial pemphigoid.

PURPOSE: Ocular cicatricial pemphigoid (OCP) is a chronic autoimmune cicatrizing disease which affects the conjunctiva and other squamous epithelium, resulting in a scarring process. A similar process, limited only to the conjunctiva, observed in some patients using eye drops for the treatment of glaucoma, is called pseudo-ocular cicatricial pemphigoid (P-OCP). Immunofluorescence studies demonstrate deposition of immunoglobulins and complement components in the basement membrane zone (BMZ) of the conjunctiva and an anti-basement membrane zone antibody in the serum of patients. A striking association between OCP and MHC class II gene DQB1*0301 has been observed. The purpose of this study was to determine some of the differences in the binding of OCP and P-OCP sera to different lysate in an immunoblot assay, in an attempt to partially characterize the OCP and P-OCP antigens. Furthermore, we wanted to determine if the MHC class II gene association of P-OCP is similar to that of OCP. METHODS: We studied sera from 11 patients with active ocular cicatricial pemphigoid and seven patients with pseudo-ocular cicatricial pemphigoid and controls. Indirect immunofluorescence (IIF) studies were done using monkey esophagus and salt split normal human skin as substrate. A sensitive immunoblot assay (IBA) was developed using normal human epidermis, dermis and conjunctiva as substrate. Typing for MHC class II genes was performed on eight pseudo-ocular cicatricial pemphigoid patients by dot-blot analysis and compared to 38 matched controls. RESULTS: Weak staining of the basement membrane zone was observed in nine of ten ocular cicatricial pemphigoid sera and five of seven pseudo-ocular cicatricial pemphigoid sera in the IIF assay using monkey esophagus. Using salt split human skin as substrate, ten of eleven ocular cicatricial pemphigoid sera demonstrated low titer weak binding to the epidermal side of the split. No consistent pattern of staining was seen with pseudo-ocular cicatricial pemphigoid sera. Ten of the 11 ocular cicatricial pemphigoid sera demonstrated binding to 230, 205, 160 and 85 kDa proteins in the IBA using normal human epidermis and conjunctiva lysates. When the lysates were first reacted with BP sera and then immunoblotted with ocular cicatricial pemphigoid sera, the 230, 160, and 86 kDa bands disappeared, and only the 205 kDa band persisted. The sera of five of seven pseudo-ocular cicatricial pemphigoid patients bound to 290, 230, 205, 180, 97, and 85 kDa proteins in the epidermis and conjunctiva. However, the 230, 205, 180, and 85 kDa proteins are depleted when the lysates are first reacted with BP and ocular cicatricial pemphigoid sera. In the dermal lysate, the pseudo-ocular cicatricial pemphigoid sera recognize 400, 290, 150 and 45 kDa proteins. None of these are absorbed by BP, ocular cicatricial pemphigoid or pemphigus vulgaris or epidermolysis bullosa acquisita sera. The 290 kDa proteins identified in the dermis and epidermis are distinct from each other. No binding was seen with control sera with the 3 lysates. Statistically, dot-blot analysis did not demonstrate a significant increase in the frequency of the MHC DQB1*0301 gene. CONCLUSIONS: Patients with ocular cicatricial pemphigoid and pseudo-ocular cicatricial pemphigoid produce several autoantibodies. However, there are similarities and differences between them. The MHC class II genes associated with pseudo-ocular cicatricial pemphigoid are different from those with ocular cicatricial pemphigoid. This provides a new model system to study the immune abnormalities in idiopathic and drug-related organ specific autoimmunity.

Ocular cicatricial pemphigoid. A case report of monozygotic twins discordant for the disease.

OBJECTIVE: To identify the major histocompatibility complex markers and the autoantibody associated with ocular cicatricial pemphigoid (OCP) in a proband, her unaffected cotwin, and the children of the cotwin. Ocular cicatricial pemphigoid is a chronic autoimmune disorder that affects the conjunctiva and other squamous epithelium. It is associated with the major histocompatibility complex class II alleles that are presumed to provide enhanced susceptibility to the disease. We encountered a pair of monozygotic female twins, one of whom has OCP. In addition to totally identical physical appearances since birth, the two sisters have identical blood groups. METHODS: The following studies were performed on the patient, her unaffected cotwin sister, and her children: (1) polymorphism of major histocompatibility complex class II genes by DNA typing, (2) sequence analysis of DQ beta gene second and third exons, and (3) serologic evaluation for the presence of anti-basement membrane zone autoantibodies specific for OCP by Western immunoblot with the use of skin and conjunctiva lysates as substrates. RESULT: Both monozygotic twins had the same HLA haplotypes. The sequence analysis of the second and third exons of DQ beta genes revealed no significant differences between the proband and her unaffected cotwin. Autoantibodies specific to OCP were detected only in the patient's serum. The serum of the unaffected cotwin and the other relatives did not demonstrate the presence of the OCP autoantibody. CONCLUSION: This isolated family study does not support a single-gene theory for the development of OCP. It is most likely due to a multigene effect and associated with environmental factors.

Common major histocompatibility complex class II markers in clinical variants of cicatricial pemphigoid.

Cicatricial pemphigoid (CP) is a chronic autoimmune blistering disease affecting multiple mucous membranes derived from stratified squamous epithelium and occasionally the skin. CP has a wide spectrum of disease manifestations. Patients with oral pemphigoid (OP) have a benign self-limited disease in which pathological changes are restricted to the oral mucosa. On the other hand, patients with ocular cicatricial pemphigoid (OCP), a chronic condition marked with relapses and remissions, have ocular involvement and also perhaps involvement of other mucous membranes. All clinical subsets are characterized by the presence of a similar anti-basement zone autoantibody. The factors that determine the development of one form of CP or the other are not known. In a previous study, we described the association between OCP and the DQB1*0301 allele (P = 0.006). In this study, we have analyzed 22 Caucasian patients with OP and their family members for major histocompatibility complex DRB generic, DQA1, and DQB1 allele associations by PCR-sequence-specific oligonucleotide probe hybridization. The results were compared to those obtained from 17 Caucasian patients with OCP and to control Caucasian alleles and haplotypes. The DQB1*0301 allele frequency was 38.6% in OP, 52.9% in OCP, and 17.8% in controls. Statistically significant associations were detected between the DQB1*0301 allele and both OP (P = 0.0047) and OCP (P < 0.0001). In addition, DRB1*04 showed a statistically significant association (P = 0.005) with OCP when compared to controls. Analysis of major histocompatibility complex class II haplotypes showed significant statistical associations between both OCP and OP and the HLA-DRB1*04, DRB4*0101, DQA1*03, DQB1*0301 haplotype (P < 0.0001 and P = 0.0012, respectively). Our results indicate that DQB1*0301 is a marker of both oral and ocular forms of CP. The analysis of the amino acid sequence of the DQB1 alleles present in both OP and OCP suggested that amino acid residues at position 57 and positions 71-77 may also be markers of CP.