Human Bullous Pemphigoid Antigen Research Articles

Anti-BP230 Only Bullous Pemphigoid Constitutes a Distinct Disease Subgroup with Characteristic Serological and Clinical Features

Anti-BP230 Only Bullous Pemphigoid Constitutes a Distinct Disease Subgroup with Characteristic Serological and Clinical Features

Transcutaneous Gene Gun Delivery of hNC16A Induces BPAG2-Specific Tolerance

Immune recognition and rejection of tissues expressing transfected genes is a major complication of gene replacement therapy for inherited genetic disorders. Owing to the high immunogenicity of human bullous pemphigoid antigen 2 (hBPAG2), the induction and maintenance of tolerance to this neo-antigen is essential to deliver the gene product to patients with epidermolysis bullosa junctionalis. In a skin grafting mouse model, we used gene gun transfection with a construct encoding hNC16A, the immunodominant domain of hBPAG2, to induce antigen-specific immune tolerance. Eighty percent of wild-type mice transfected with hNC16A showed long-term survival of skin grafts expressing hBPAG2. Tolerance was stable and transferable by T cells but not by B cells of tolerant mice to naive hosts. A dense Foxp3(+) regulatory T-cell (T(reg)) infiltrate was noticed in grafts of tolerant mice and depletion of these cells resulted in a loss of tolerance. Taken together, we show that long-lasting hBPAG2-specific tolerance was induced with gene gun delivery of hNC16A through a T(reg)-dependent mechanism. This is of relevance to patients undergoing gene therapy and has broader implications for the treatment of antigen-specific autoimmune diseases.

Transient Anti-CD40L Co-Stimulation Blockade Prevents Immune Responses against Human Bullous Pemphigoid Antigen 2: Implications for Gene Therapy

Skin grafts from mice expressing human bullous pemphigoid antigen 2 (hBPAG2) in epidermal basement membrane elicit hBPAG2-specific IgG and graft loss in wild-type (Wt) recipients. Graft loss was dependent on CD4+ T cells and correlated with the production and tissue deposition of hBPAG2-specific IgG. To explore the role of CD40/CD40 ligand (CD40L) interaction in this model, Wt mice grafted with transgenic (Tg) skin were treated with hamster anti-CD40L mAb MR1. In contrast to grafted Wt mice treated with equivalent doses of control IgG, 22 of 23 MR1-treated Wt mice did not develop hBPAG2-specific IgG or graft loss for >or=60 days. MR1-treated mice also accepted a second Tg skin graft without durable production of hBPAG2-specific IgG or graft loss. Moreover, splenocytes and enriched CD4+ T cells from MR1-treated graft recipients transferred un- or hyporesponsiveness to hBPAG2 to other mice and demonstrated a dominant tolerant effect over cotransferred naive splenocytes following adoptive transfer to Rag2-/- mice. Successful inhibition of hBPAG2-specific IgG production and Tg graft loss following CD40:CD40L co-stimulatory blockade in this model provides opportunities to study mechanisms of peripheral tolerance and generate antigen-specific regulatory CD4+ cells-issues of relevance to patients with pemphigoid as well as individuals undergoing gene replacement therapy for epidermolyis bullosa.

Differential display and cloning of the hippocampal gene mRNAs in senescence accelerated mouse

Identification of genes that are specifically expressed in the hippocampus of senescence accelerated mouse (SAM) is important for understanding the molecular basis of the pathological changes in the brain and of the deterioration of learning and memory in SAM-prone/8 (SAMP8), a substrain of SAM. The differential display technique was applied to compare mRNAs expression between SAMP8 and SAM-resistance 1 (SAMR1), another substrain of SAM. Complementary DNA fragments corresponding to several apparently differentially expressed mRNAs were recovered and sequenced. Six differentially expressed cDNA bands were identified. Sequence analyses demonstrated that W4 and W5 cDNA fragments corresponded to unknown genes. W1 and W6 showed 66.1 and 62.3% homology to rat GTP-exchange protein (eIF-2B) and rat phospholipase D, respectively, while W2 and W3 showed 89.2 and 90.8% homology to human bullous pemphigoid antigen and human glucogen debranching enzyme isoform 1/2/3/4/6, respectively. The results suggested that these genes are closely related to the malfunction of the brain in SAM.

Childhood bullous pemphigoid: Report of a case with characterization of the targeted antigens

The clinical and immunopathologic features of children with acquired subepidermal blistering disorders show considerable overlap, and their classification frequently requires characterization of the targeted antigens. A 8-month-old boy developed a generalized subepidermal blistering disorder with striking palmoplantar involvement. The patient’s serum contained antibodies reacting against the epidermal side of 1 M sodium chloride separated normal human skin. Immunoblotting analysis demonstrated circulating IgG autoantibodies that reacted against a eukaryotic recombinant form of human bullous pemphigoid antigen 180 (BP180). In addition, the patient had circulating IgG autoantibodies that bound a protein of 120 kDa in skin basement membrane zone extracts, that might correspond to the linear IgA bullous disease (LABD) antigen. This study illustrates that a child with clinical and immunopathologic features considered characteristic of childhood bullous pemphigoid (BP) had circulating IgG antibodies that bound to an eukaryotic recombinant form of human BP180, and hence, fulfilled the diagnostic criteria of BP. Review of the literature disclosed only 10 cases of childhood BP, that were characterized on the basis of the targeted antigens. The concomitant presence of circulating IgG autoantibodies against BP180 and a 120 kDa protein may signify either coexistence of autoantibodies with distinct specificities or reflect antigenic cross-reactivity between BP180 and the 120/97 LABD antigen. (J Am Acad Dermatol 1999;40:338-44.)

Identification of genes differentially expressed in nasopharyngeal carcinoma by messenger RNA differential display.

We have applied the mRNA differential display method to compare and analyze mRNAs prepared from five normal nasopharyngeal epithelial cell cultures and five nasopharyngeal carcinoma cell lines. A total of 24 differential display experiments was performed using different combinations of PCR primers. Sixty-nine cDNA fragments differentially expressed in either normal or malignant nasopharyngeal epithelial cells were identified. Subsequent cloning and sequencing of these differentially expressed cDNA fragments resulted in the identification of seventeen distinct sequences. Seven of these sequences were shown to be novel cDNA sequences not previously reported. Ten of the remaining cDNA fragments showed sequence homology to previously reported genes. Differential expression of four of these seventeen cDNA fragments in normal nasopharyngeal epithelial cells was confirmed by reverse Northern hybridization. One of these cloned cDNA fragments is a novel cDNA sequence while the other three matched to previously reported cDNA sequences involved in cell growth and migration. Homologous sequences identified to be differentially expressed in normal nasopharyngeal epithelial cells in this study are: human 26 kDa cell surface protein (TAPA-1) mRNA, NF-E2 like basic leucine zipper transcriptional activator and the human bullous pemphigoid antigen. The mRNA differential display is a useful tool to identify candidate genes involved in the pathogenesis of nasopharyngeal carcinoma.

Human bullous pemphigoid antigen (BPAG1). Amino acid sequences deduced from cloned cDNAs predict biologically important peptide segments and protein domains.

Bullous pemphigoid antigens are defined as the autoantigens in a blistering skin disease, bullous pemphigoid. One of them, a 230-kDa protein (BPAG1), is associated with hemidesmosomes, attachment complexes at the basal keratinocyte-lamina lucida interface within the dermal-epidermal basement membrane zone. The precise functions and cellular compartmentalization of BPAG1 are unknown. In this study, a human keratinocyte lambda gt11 cDNA library was screened for clones corresponding to BPAG1. The composite of overlapping cDNAs delineated 8,930 base pairs of nucleotide sequences that contained an open reading frame encoding 2,649 amino acids. Analysis of the deduced amino acid sequences predicted a putative signal peptide of 43 amino acids and the presence of a membrane-associated sequence of 17 amino acids. Several potential sites for N-glycosylation, as well as for protein kinase C or cAMP- and cGMP-dependent protein kinase-mediated phosphorylation were identified. Three peptide segments were predicted to be highly antigenic, potentially serving as epitopes for the formation of autoantibodies. Eight repeat segments of 38 residues each with a high degree of homology with sequences in desmoplakin I, a component of desmosomal cytoplasmic plaques, were detected in the carboxyl-terminal end of the molecule. In addition, the presence of three subdomains characterized by heptad repeats predicted an alpha-helical coiled coil dimer structure in the central portion of the protein. These data suggest that BPAG1 may be a membrane-associated protein that plays a role in the attachment of basal keratinocytes to the underlying basement membrane.

Bullous Pemphigoid Antigen: cDNA Cloning, Cellular Expression, and Evidence for Polymorphism of the Human Gene

A human epidermal keratinocyte lambda gt11 recombinant cDNA library was screened with a 0.45-kb cDNA that was generated by polymerase chain reaction (PCR) amplification of a segment of human bullous pemphigoid (BP) antigen mRNA. The screen yielded five clones, the largest one, pcBPA-4, being 2.3 kb in size. The pcBPA-4 cDNA hybridized in Northern analyses with an approximately 9-kb mRNA from cultured keratinocytes, whereas no hybridization signal was detected with RNA from human skin fibroblast, fibrosarcoma HT-1080 cells, or amniotic epithelial WISH cell cultures. Nucleotide sequencing of pcBPA-4 revealed an open reading frame encoding a putative polypeptide of 447 amino acids. This polypeptide showed 88% homology with corresponding mouse BP antigen sequences, and a region of it was identical to a segment in previously published human BP antigen sequences. The 2.3-kb cDNA isolated here was different from a previously published human BP antigen cDNA, in that the open reading frame coded for 264 additional amino acids at the carboxyl end of the putative polypeptide. Known human BP antigen amino acid sequences, compared with mouse sequences, were predicted to be moderately divergent with a unit of evolutionary period (UEP) of 4.5 millions of years (MY). Southern hybridizations suggested that the BP antigen gene (BPAG1) is present as a single copy in the human genome. Southern analyses also revealed the presence of a StuI restriction fragment length polymorphism that can be used for linkage analyses to study the inheritance of a particular BPAG1 allele and a heritable cutaneous disorder affecting the basement membrane zone, such as epidermolysis bullosa.