Epidermal Antigen Research Articles

Identification, sequence and developmental expression of invertebrate flotillins from Drosophila melanogaster.

Caveolae are vesicular organelles that represent a sub-compartment of the plasma membrane. Caveolins (Cav-1, -2 and -3) and flotillins (FLO-1 and FLO-2 [also known as epidermal surface antigens (ESAs)] are two families of mammalian caveolae-associated integral membrane proteins. Although a caveolin gene family has recently been described in the invertebrate Caenorhabditis elegans, it remains unknown as to whether flotillin homologues exist in invertebrates. Here, we report the identification, cDNA sequence and embryonic expression pattern of the first invertebrate flotillin, i.e. flotillin from Drosophila melanogaster (FLODm). FLODm is most closely related to mammalian flotillin-1. Remarkably, the invertebrate FLODm protein behaves like mammalian flotillins and is targeted to the caveolae-enriched membrane fraction after transient expression in mammalian cells. Localization of the FLODm message in D. melanogaster embryos reveals that expression of FLODm is confined primarily to the developing nervous system. This is consistent with our previous observation that mammalian flotillin-1 mRNA and protein is expressed abundantly in brain tissue. Interestingly, the FLODm gene is localized to chromosomal region 52 B1-B2. In addition, we find that at least two flotillin-related genes are expressed in D. melanogaster. Our current results provide a starting point and systematic basis for dissecting the role of flotillin in caveolae and neuronal development using Drosophila as a genetic system.

Epidermal surface antigen (ESA) is distinct from ECS-1 antigen and confers a dendritic phenotype to cos cells

Epidermal surface antigen (ESA) is distinct from ECS-1 antigen and confers a dendritic phenotype to cos cells

Intracellular and extracellular epidermal antigens are recognized by different IgG subclass in autoimmune bullous skin diseases

Intracellular and extracellular epidermal antigens are recognized by different IgG subclass in autoimmune bullous skin diseases

IGE antibodies in sera from patients with bullous pemphigoid are autoantibodies exclusively directed against the 230kDa epidermal antigen (BP230)

IGE antibodies in sera from patients with bullous pemphigoid are autoantibodies exclusively directed against the 230kDa epidermal antigen (BP230)

IgE antibodies in sera from patients with bullous pemphigoid are autoantibodies preferentially directed against the 230-kDa epidermal antigen (BP230)

Bullous pemphigoid (BP) is unique among autoimmune skin diseases in which a high serum IgE level has been detected. We sought to determine the antigenic specificity of these IgE antibodies in 39 BP sera by immunofluorescence microscopy, immunoblot, and ELISA. The patient's sera contained IgG antibodies to 230-kDa (BP230) (n = 20), 180-kDa (BP180) (n = 9), and both BP230 and BP180 (n = 10) antigens. Serum IgE levels varied from 29 to 5000 kIU/L (mean +/- SD, 856 +/- 1426 kIU/L), among which sera containing IgG antibodies to BP230 had an IgE level on average 4.3 times higher than anti-BP180 sera. IgE antibodies in 18 sera were found to be autoantibodies reactive either with an epidermal component of basement membrane zone by immunofluorescence microscopy on 1 M NaCl-split skin or with a 230-kDa antigen by immunoblots of cultured human keratinocytes. The 230-kDa epidermal antigen recognized by IgE antibodies comigrated with the BP230 as labeled by a specific human monoclonal antibody. IgE anti-BP230 antibodies in patients' sera were always associated with IgG autoantibodies. No sera contained IgE antibodies to BP180 or to any other epidermal or dermal antigens as verified by immunoblot and ELISA. A good correlation was found between the presence of IgE circulating autoantibodies and the level of serum IgE (P < 0.004). IgE antibodies to BP230, like IgG autoantibodies, were mapped primarily to the C-terminal end of the protein, as they labeled rBP55, a BP230 recombinant protein encoded by a cDNA for the C-terminal end of BP230.

Expression of fetal cytokeratins in epidermai cells and colloid bodies in lichen planus*

Clusters of immunoglobulin (Ig)-coated colloid bodies (CBs) in the dermo-epidermal zone are a typical immunohistochemical feature in lichen planus (LP)-lesions. They are considered to represent dyskeratotic basal keratinocytes, yet their composition has not been completely elucidated. In the present study, skin biopsies of 10 LP-lesions, 3 other dermatoses, and 10 biopsies of normal skin were studied immunohistochemically using monoclonal antibodies (MAbs) against fetal and differentiated epidermal antigens. CBs were identified by FITC-anti-Ig. Binding of MAb was visualized by double staining technique. Cytokeratin (CK) 10/11, a marker of epidermal differentiation, was consistently detected in suprabasal keratinocytes and also in up to 95% of Ig-positive CBs in LP. CK10/11 was additionally detected in basal keratinocytes in 9 LP-lesions, but not in normal skin. The basal cell-specific MAb BL7 stained basal layer keratinocytes in all biopsies. In contrast to normal skin, in LP scattered suprabasal keratinocytes and CBs were also positive for BL7 in 10 and 7 cases, respectively. While fetal cytokeratins (CK13 and CK8/18) were completely absent in control skin specimens, both cytokeratins were detected in various numbers of keratinocytes and CBs in all LP-lesions. Our results support the hypothesis of an epidermal origin of CBs. The cytokeratin profile seems to be severely disturbed in LP. This includes both accelerated differentiation by the expression of suprabasal CK10/11 in basal keratinocytes and dedifferentiation by the expression of fetal epidermal antigens (CK13 and CK8/18). It is tempting to speculate that the observed alterations may trigger T-cell activation and inflammatory onset in LP.

Developmental roles of nuclear complex factors released during oocyte maturation in the ascidians Halocynthia roretzi and Boltenia villosa.

The developmental roles of factors associated with the nuclear complex of Halocynthia roretzi and Boltenia villosa oocytes were investigated by cutting mature oocytes into animal and vegetal merogons before and during GVBD. Animal and vegetal merogons were cultured in sea water until the GV cytoplasm had dispersed within the cytoplasm of control oocytes and then they were cross-fertilized and scored for their ability to undergo normal development. Halocynthia oocyte fragments produced from the animal region of oocytes containing intact GVs exhibited a low frequency of polyspermy, a high frequency of fertilization and cleavage, and a high frequency of expressing an epidermal antigen, Epi-2. In contrast, merogons produced from the vegetal region of Halocynthia oocytes in which GVs were intact exhibited a high frequency of polyspermy, did not undergo cell division, and expressed a high frequency of Epi-2 expression. When vegetal fragments were produced after the dispersal of approximately 50-70% of the GV nucleoplasm, these merogons exhibited a low frequency of polyspermy, high frequencies of cell division (including the formation of epidermal layer), and in most cases expressed Epi-2. Vegetal Boltenia fragments produced during GVBD in some cases developed into larvae. These results suggest that the ascidian GV nucleoplasm may contain factors required for fertilization and cell division and that epidermal determinants reside in the oocyte cytoplasm.

Drug‐induced pemphigus. Does a relationship exist between inducing drug and lesion topography?

AbstractPemphigus subclasses differ both in target epidermal antigen of the autoantibodies and in predilection sites of lesions. The patterns of lesions are well documented and often lead to diagnosis of the subtype of the disease: but the causative factors have never been studied. We present a case of pemphigus exacerbated by two unrelated drugs, with distinct exacerbation‐specific patterns following each drug consumption. The patient is regarded as exemplifying a fixed drug eruption‐like phenomenon, with specificity of drug to anatomic distribution of lesions.

Antigen-independent expansion of T cells from psoriatic skin lesions: phenotypic characterization and antigen reactivity

The pathogenesis of psoriasis appears to depend on T cells, which have been proposed to mediate the disease through an autoimmune process. To test this hypothesis we have propagated four T-cell lines from biopsies of psoriatic skin lesions by antigen-independent methods. Flow cytometric immunophenotyping showed the lines to be composed mainly of CD4-positive, alpha beta T-cell receptor (TCR)-positive cells, which secreted a cytokine profile suggestive of predominant T-helper type 1 (Th1) status. Analysis of TCR variable region (V beta) usage revealed two- to eight-fold increases in the expression of certain V beta species in lesional lines as compared with autologous peripheral blood mononuclear cells (PBMC), with the increased V beta species being expressed on more than 5% of cells in two of the lines. Lines were also used to test for responses to a range of epidermal antigen preparations in the presence of irradiated autologous PBMC as antigen-presenting cells. The lines failed--to proliferate in response to psoriatic lesional stratum corneum extracts, dispase-separated normal human epidermal extracts, and an epidermal keratin preparation before and after trypsinization, in spite of good proliferative responses to anti-CD3 which indicated that the lines were not anergic. In addition, the lines and PBMC from normal volunteers and the patients with psoriasis gave little or no response to recombinant streptococcal M protein. Thus, in spite of accumulating evidence for selective expansion of certain V beta-expressing T cells in psoriatic lesions, epidermal autoantigens have not been identified by using a bioassay which depended largely on the proliferation of lesional CD4-positive cells. The role of streptococcal M protein, which bears some homology with epidermal keratin is also open to question, at least in chronic plaque psoriasis. Further work is therefore required to obtain direct evidence that autoimmune processes are important in the pathogenesis of chronic plaque psoriasis.

Drug-induced pemphigus. Does a relationship exist between inducing drug and lesion topography?

Pemphigus subclasses differ both in target epidermal antigen of the autoantibodies and in predilection sites of lesions. The patterns of lesions are well documented and often lead to diagnosis of the subtype of the disease; but the causative factors have never been studied. We present a case of pemphigus exacerbated by two unrelated drugs, with distinct exacerbation-specific patterns following each drug consumption. The patient is regarded as exemplifying a fixed drug eruption-like phenomenon, with specificity of drug to anatomic distribution of lesions.

Linear IgA Bullous Dermatosis with IgA Antibodies Exclusively Directed against the 180- or 230-kDa Epidermal Antigens

This study describes the presence in sera from patients with linear IgA bullous dermatosis (LABD) of IgA antibodies specific for 230- or 180-kDa epidermal antigens. Of 11 patients' sera with IgA antibodies reactive with the epidermal antigens obtained from cultured keratinocytes, 6 sera recognized the 230-kDa antigen and co-migrated with the polypeptide recognized by a human monoclonal antibody against the 230-kDa bullous pemphigoid antigen (BPAgl). Five sera recognized the 180-kDa antigen and co-migrated with the polypeptide stained by a polyclonal antibody to the 180-kDa bullous pemphigoid antigen (BPAg2). None of these LABD sera contained IgG antibodies reactive with the basement membrane zone antigens and none labeled a 97-kDa epidermal antigen or a 290-kDa dermal antigen. Immunoaffinity-purified IgA antibodies from the 230 kDa band further reacted with the epidermal side of the skin basement membrane zone. Epitope mapping with rBP55, a fusion protein containing the C-terminal end of BPAg1, suggested that the major antigenic epitopes for LABD and BP antibodies on the 230-kDa antigen are different. Only one serum with IgA antibodies was found to label rBP55, contrasting with nine of ten BP sera reacting with this protein. Our study demonstrates the presence of an exclusive IgA response against the 230- or 180-kDa antigens in a subset of patients with LABD.

Flotillin and Epidermal Surface Antigen Define a New Family of Caveolae-associated Integral Membrane Proteins

Caveolae are plasmalemmal microdomains that are involved in vesicular trafficking and signal transduction. We have sought to identify novel integral membrane proteins of caveolae. Here we describe the identification and molecular cloning of flotillin. By several independent methods, flotillin behaves as a resident integral membrane protein component of caveolae. Furthermore, we have identified epidermal surface antigen both as a flotillin homologue and as a resident caveolar protein. Significantly, flotillin is a marker for the Triton-insoluble, buoyant membrane fraction in brain, where to date mRNA species for known caveolin gene family members have not been detected.

Biological effects of narrow-band (311 nm TL01) UVB irradiation: a review

The narrow-band UVB (TL01) lamp (311 nm emission) was developed for use in phototherapy, as an alternative to a broad-band UVB source and to photochemotherapy, both of which have significant side effects and carry a risk of carcinogenesis. This new lamp has proved to be particularly effective at clearing psoriasis. It is now acknowledged that the TL01 lamp is probably 2–3 times more carcinogenic per minimum erythema dose than broad-band UVB, but the cumulative dose required in therapy is considerably less than when using broad-band UVB sources. In terms of irradiation dose, the TL01 lamp is about 5–10-fold less potent than broad-band UVB for erythema induction, hyperplasia, oedema, sunburn cell formation and Langerhans cell depletion from skin. However, the broad-band UVB to TL01 potency ratio for cis-urocanic acid formation in the skin is approximately unity. In addition, the TL01 lamp, as used in phototherapy, has relatively more suppressive effects than broad-band UVB on systemic immune responses as judged by natural killer cell activity, lymphoproliferation and cytokine responses. However, the TL01 lamp is less effective at reducing epidermal antigen presentation, inducing dendritic cell migration to lymph nodes draining irradiated sites and suppressing contact hypersensitivity at the doses tested. Therefore the use of the TL01 lamp in phototherapy should be considered carefully after weighing up its diverse effects on the skin and immune system.

Disparate Locations of the 52- and 60-kDa Ro/SS-A Antigens in Cultured Human Keratinocytes

Anti-52- and anti-60-kDa Ro/SS-A (Ro) autoantibodies are produced by most patients with subacute cutaneous lupus erythematosus and neonatal lupus erythematosus and are thought to be pathogenic in these two disorders. To learn more about the epidermal antigens targeted by Ro autoantibodies, a panel of anti-52 and anti-60-kDa Ro antibodies was purified from human autoimmune sera and rabbit antisera and then used to: (i) determine the expression and location of the Ro antigens in human keratinocytes; (ii) clarify discrepancies in previous localization studies; and (iii) verify the existence of Ro autoantibodies that cross-react with the 52- and 60-kDa Ro antigens, as previously reported. By immunoblot analysis these antibodies demonstrate that 52- and 60-kDa Ro proteins are expressed in normal human skin and cultured keratinocytes. By indirect immunofluorescence studies with cultured human cells, the anti-52-kDa Ro antibodies produce fine granular cytoplasmic fluorescence and less intense nuclear fluorescence, with apparent nucleolar sparing. The anti-60-kDa Ro autoantibodies produce weak cytoplasmic fluorescence and intense coarse granular nuclear fluorescence with apparent nucleolar sparing. We found distinct differences in the intracellular localization of the 52- and 60-kDa Ro autoantigens. This difference suggests that the 52-and 60-kDa Ro antigens may have independent cellular functions. Finding 60-kDa Ro antigen predominantly in the nucleus challenges the notion that the majority of the intracellular 60-kDa Ro antigen is complexed with the cytoplasmic hY RNA. Additionally, our failure to find a cross-reactive epitope on these two proteins indicates that the 52-kDa Ro antigen is probably a true immunogen and not merely a protein that cross-reacts with anti-60-kDa Ro autoantibodies, as others have suggested.

Identification of a 450-kDa Human Epidermal Autoantigen as a New Member of the Plectin Family

The serum from an individual with a subepidermal blistering disease was previously shown to recognized a 450-kDa epidermal autoantigen. The molecular structure of this antigen was investigated by screening a human keratinocyte cDNA library with the patient's serum. One clone, with a 276-bp cDNA insert, that encoded an epitope recognized by the serum was isolated. Rabbit polyclonal antibodies that were prepared against the corresponding fusion protein recognized the 450-kDa epidermal antigen and stained the basal keratinocytes in human epidermis. This clone was used for further screening of the original keratinocyte and HeLa cell cDNA libraries. Two different, but closely related, 0.8- and 2.0-kb cDNAs were isolated, and their deduced amino acid sequences indicated that the encoded proteins belonged to the plectin family. Northern blot analysis of total RNA from human keratinocytes with these cDNA inserts as probes detected RNAs of approximately 12-13 kb. The 0.8-kb cDNA hybridized to polyadenylated RNA species from human skeletal muscle, heart, lung, and kidney, whereas the 2.0-kb cDNA hybridized to transcripts present only in kidney and lung. Southern blot analysis of genomic DNA from the human placenta revealed similar, but not identical, patterns of hybridization with the 0.8- and 2.0-kb cDNAs. Data suggest that the 0.8- and 2.0-kb cDNAs encode two different proteins but are derived from the same gene.

Dermatose bulleuse à IgA linéaire de l'enfant avec autoanticorps dirigés contre l'antigène 180 kDa de la pemphigoïde

Background.— Linear IgA bullous dermatosis (LABD) is an autoimmune subepidermal blistering disease defined on the basis of direct immunofluorescence findings. Case report.— An 18 month-old girl suffering from LABD was studied by indirect immunofluorescence on salt-split skin and by Western blot in an attempt to characterize the involved autoantigen. Direct immunofluorescence showed an exclusive linear IgA deposit at the dermal-epidermal junction. Indirect immunofluorescence revealed circulating autoantibodies that reacted with the epidermal side of salt-split skin; they reacted by Western blot with a 180 kDe epidermal antigen, as in bullous pemphigoid. Conclusion.— This dermatosis fulfilling the clinical features and direct immunofluorescence criteria for childhood LABD seems to represent a case of IgA bullous pemphigoid. It further underscores the nosologic heterogeneity of LABD, which probably includes, apart from bullous pemphigoid, epidermolysis bullosa acquisita and cicatricial pemphigoid.

Cicatricial Pemphigoid Autoantibodies React with Multiple Sites on the BP180 Extracellular Domain

Cicatricial pemphigoid (CP) is an autoimmune blistering disease that primarily affects mucosal tissues. Autoantibodies to laminin-5 have previously been detected in certain patients with a CP-like disease; however, individuals that exhibit this reactivity profile apparently represent a small subset of CP patients. In the present investigation, 0 of 18 CP sera showed reactivity with laminin-5 by immunoblotting. In contrast, 18 of 23 CP sera (78%) recognized a 180-kDa epidermal antigen that, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, co-migrated with BP180, a hemidesmosomal glycoprotein associated with two other autoimmune blistering diseases, bullous pemphigoid and herpes gestationis. To investigate further the CP autoimmune response, various segments of human BP180 were expressed as bacterial fusion proteins and assayed by immunoblotting for reactivity with CP patients' sera. The results of this investigation demonstrated that the BP180 autoantigen is indeed a major target of CP autoantibodies. Further, two distinct CP-reactive sites were identified on the extracellular domain of the BP180 protein, one located in the non-collagenous (NC) 16A domain (at or near the previously defined autoantibody-reactive site recognized by bullous pemphigoid and herpes gestationis sera) and the other in the carboxy-terminal region of this protein. Sixteen of 23 CP sera (70%) reacted with one or both of these antigenic sites of BP180. Other immunologic data suggested that BP180 may harbor additional CP-reactive sites. In conclusion, there are now three bullous diseases, bullous pemphigoid, herpes gestationis, and cicatricial pemphigoid, that are known to be associated with an autoimmune response against the extracellular domain of the BP180 antigen.

Immunohistochemical localization of epidermal growth factor receptor and epithelial antigen in tumors of the human conjunctiva, eyelid, lacrimal gland, and orbit

Increased numbers of epidermal growth factor (EGF) receptors are observed in squamous cell carcinomas of human lung, head, neck, and cervix. We studied the presence of EGF receptors and epithelial antigen in some ophthalmic lesions. Immunohistochemical staining for EGF receptors was assessed in tumors of human conjunctiva, eyelid, lacrimal glands, and orbit with monoclonal antibodies (EGF-R1 and clone 29.1). Reactivity of Ber-EP4, which recognizes epithelial antigen, was also examined. Strong staining of EGF-R1 and clone 29.1 and weak to moderate staining of Ber-EP4 were demonstrated in conjunctival squamous cell carcinomas. Cell membranes of conjunctival papilloma were moderately or strongly stained with these antibodies. Ductal components in sebaceous gland adenoma of the eyelid and pleomorphic adenoma of the lacrimal gland were positively stained. The antibodies did not bind to reactive lymphoid hyperplasia of the orbit and Wegener's granulomatosis. Relatively good correlation for immunostaining reaction was observed among EGF-R1, clone 29.1, and Ber-EP4 in each tumor. Immunostaining using EGF-R1, clone 29.1, and Ber-EP4 may be useful in differentiating epithelial tumors from non-epithelial lesions. Strong immunostaining for EGF receptor may be the hallmark of epidermoid malignancy.

Oncogene proteins and proliferation antigens in thymomas: increased expression of epidermal growth factor receptor and Ki67 antigen.

To examine thymomas for proteins encoded by oncogenes and to determine whether their presence correlates with tumour growth and associated myasthenia gravis. Sections of 24 thymomas were incubated with anti-EGF receptor (EGF-R), anti-Ki67 antigen, anti-p53, and anti-bcl-2 antibodies, and then stained using the alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique. Cell suspensions and epithelial cell cultures from some of the tumours were also studied. Whereas EGF-R expression was not detected in any of the controls (but only in a 20 week old fetus), it was detected in neoplastic epithelial cells of all thymomas, and was most strongly expressed in metastases and in samples from donors with severe myasthenia gravis. Ki67 labelling was also increased, especially in the larger thymomas. Epithelial expression of both of these markers was confirmed in fresh cell suspensions and monolayer cultures from the five available cases. In contrast, p53 and bcl-2 were not detected in the neoplastic cells, but bcl-2 was present in the intermingling thymocytes. Neoplastic thymoma cells express EGF-R and Ki67, but there is no concomitant increase in the expression of p53 and bcl-2 proteins. Increased EGF-R expression may result in increased proliferation of neoplastic cells and also in myasthenia gravis. Measurement of EGF-R concentrations may be of prognostic value. The bcl-2 staining pattern in T lymphocytes illustrates the broad spectrum of maturational stages in thymoma lymphocytes.

Expression of Several Biologic Markers as Prognostic Markers in Non-Small Cell Lung Cancers

Background: Despite modern diagnostic, staging, and therapeutic advances, esp. with molecular biologic techniques, the 5-year survival rate of all cases of lung cancer does not exceed 15%. Also, the incidence of lung cancer of both sex in Korea is increasing year by year and the lung cancer is one of the leading causes of cancer death. Therefore, it is strongly needed to develop the new combination of treatment modalities including neoadjuvant chemotherapy and to identify tumor specific characteristics with staging or prognostic markers. Here we present the clinical significance of several biologic tumor markers to use as a prognostic markers in patients with non-small cell lung cancers. Method: The survival has correlated with the expressibility of proliferative cell nuclear antigen (PCNA), epidermal growth factor receptor(EGFR), p53 and/or blood group antigen A(BGAA) using immunohistochemistry in 46 patients with non-small cell lung cancers. Results: 1) The expression rates of PCNA, EGFR, p53 and BGAA were 80.6%, 61.3%, 45.9% and 64.3%, respectively and those were not correlated to cell types or clinical stges. 2) The expression of BGAA was correlated with better survival in median survival and in 2-year survival rate and that of PCNA was correlated with worse survival in median survival and 2-year survival rate. 3) The expression of EGFR or p53 was not valuable to predict prognosis in non-small cell lung cancers. 4) With simultaneous applications of PCNA, EGFR and p53 immunostain, the patients with 2 or more negative expressions showed better prognosis than the patients with 2 or more positive expressions. Conclusion: It is suggested that the expression of blood group antigen may be a positive prognostic factor and that of PCNA may be a negative prognostic factor. Also, the combination of expressions of PCNA, EGFR and p53 may be used as a negative prognostic factor.