Anti-desmocollin autoantibodies in nonclassical pemphigus.

BACKGROUND: Despite the established pathogenic role of anti-desmoglein (Dsg) antibodies in classical pemphigus, the significance of autoantibodies to another desmosomal cadherin, desmocollin (Dsc) is at present unknown. No consistent immunoassay for immunoglobulin (Ig) G autoantibodies to Dscs has been developed. OBJECTIVES: The aim of this study was to develop reliable assays to detect anti-Dsc autoantibodies. METHODS: We expressed soluble recombinant proteins (RPs) of human Dsc1-3 in mammalian cells and examined sera of various types of pemphigus, including 79 paraneoplastic pemphigus (PNP) sera, by novel enzyme-linked immunosorbent assays (ELISAs) using the RPs. We also performed ELISAs of Dsc baculoproteins and used the complementary DNA (cDNA) transfection method, and compared the results with those of mammalian ELISAs. RESULTS: Through mammalian ELISAs, IgG autoantibodies to Dsc1, Dsc2 and Dsc3 were detected in 16.5%, 36.7% and 59.5% of PNP sera, respectively, and considerable numbers of pemphigus herpetiformis (PH) and pemphigus vegetans (PVeg) sera reacted strongly with Dsc1 and Dsc3. Mammalian ELISAs were highly specific and more sensitive than baculoprotein ELISAs or the cDNA transfection method. Several Dsc-positive sera, particularly PH sera, showed no reactivity with Dsgs. The reactivity of PNP serum and PVeg serum with Dscs was not abolished by pre-absorption with Dsg RPs. CONCLUSIONS: The results of these novel ELISAs indicated that IgG anti-Dsc autoantibodies were frequently detected and potentially pathogenic in nonclassical pemphigus.

Clinical and immunological profiles in 17 Japanese patients with drug-induced pemphigus studied at Kurume University.

BACKGROUND: Drug-induced pemphigus (DIP) shows clinical, histopathological and immunological features of pemphigus. However, little is known about immunological profiles in DIP. OBJECTIVES: To characterize clinical and immunological profiles in patients with DIP. METHODS: We studied 17 Japanese patients with DIP who were treated at Kurume University Hospital or who consulted from other hospitals between 1997 and 2012. Complicated diseases, clinical and histopathological manifestations, responsible drugs and findings in immunofluorescence, enzyme-linked immunosorbent assays (ELISAs), immunoblotting (IB) and prognosis were analysed. RESULTS: Eight of the 17 patients with DIP showed pemphigus foliaceus-like appearance, three showed pemphigus herpetiformis-like appearance, and six showed atypical bullous lesions. Responsible drugs were thiol-containing drugs in 16 patients (bucillamine in nine cases, d-penicillamine in four cases, and cetapril, thiopronine and captopril in one patient each), and a nonthiol drug, sulfasalazine, in one patient. By ELISAs and/or IB analyses, nine patients reacted only with desmoglein 1 (Dsg1), four reacted with Dsg1 and Dsg3, and four showed no specific reactivity. By IB of normal human epidermal extracts, in addition to positive reactivity with Dsg1, four patients with no detectable malignancy showed paraneoplastic pemphigus-like reactivity with the 210-kDa envoplakin and the 190-kDa periplakin. Four cases showed anti-Dsg3 antibodies without mucosal lesions. While 11 cases recovered after discontinuation of the causative drugs, six patients had a very protracted or intractable disease course, and might develop true pemphigus. CONCLUSIONS: The present study indicated that the majority of the patients with DIP studied showed a pemphigus foliaceus-type phenotype with anti-Dsg1 autoantibodies, caused by thiol-containing drugs.

Sensitivity and specificity of recombinant omega-5 gliadin-specific IgE measurement for the diagnosis of wheat-dependent exercise-induced anaphylaxis.

BACKGROUND: A recent study has shown that the measurement of specific IgE antibodies to B-cell epitope peptides of wheat omega-5 gliadin (Pep A) and high molecular weight glutenin subunit (Pep B) are useful to diagnose wheat-dependent exercise-induced anaphylaxis (WDEIA). AIMS OF THE STUDY: We sought to compare the sensitivity and specificity of the in vitro tests for measuring the specific IgE antibodies to recombinant omega-5 gliadin (romega-5 gliadin) with those for wheat, gluten, Pep A, and Pep B in identification of patients with WDEIA. METHODS: Fifty patients with WDEIA, 25 healthy subjects and 25 patients with atopic dermatitis with specific IgE antibodies to wheat but without experience of allergic reactions after ingestion of wheat products were enrolled in this study. The concentrations of specific IgE antibodies were measured using ImmunoCAP. The empirical receiver operating characteristics curves (ROC) for each test were prepared and the areas under the ROC curve (AUC) were compared. RESULTS: In patients with WDEIA, the sensitivities of the allergen-specific IgE tests for wheat, gluten, Pep A, Pep B and romega-5 gliadin were 48%, 56%, 76%, 22%, and 80%, respectively. The seven of 10 WDEIA patients with no specific IgE antibodies to romega-5 gliadin had specific IgE antibodies to Pep B. The highest AUC (0.850) was observed in the test for romega-5 gliadin. CONCLUSIONS: Measuring the concentration of specific IgE antibodies to romega-5 gliadin is more useful than to wheat, gluten, or Pep A in the identification of patients with WDEIA.

Mutational analysis of copper binding by human tyrosinase.

Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme that catalyzes several reactions in the biosynthesis of melanin pigments and is deficient in patients with type I oculocutaneous albinism (OCA1). Tyrosinase is thought to bind two copper ions, one at each of two conserved sequence motifs, termed CuA and CuB, but to date this has been directly proved only for the Neurospora and mushroom enzyme. Here, we demonstrate that mammalian tyrosinase directly binds copper, and that the CuA and CuB sites are both required for copper binding and for catalytic activity. We show that in human tyrosinase, copper binding by the CuB site is most likely coordinated by residues His363, His367, and His389, and that copper binding may be cooperative, with copper binding at one site facilitating copper binding by the other site. Furthermore, correct folding of the tyrosinase polypeptide appears to be necessary for copper binding, and a number of human OCA1 mutations disrupt copper binding and thus catalytic function of tyrosinase.

Melanosomal proteins as melanoma-specific immune targets.

Pigmentation of our skin, hair and eyes is essential for photoprotection, embryological development, detoxification and protective/cosmetic coloration. A number of proteins important to the production of melanin within melanosomes have now been identified including enzymatic and structural proteins encoded at the murine albino, brown, pinkeyed-dilution, MART1, slaty and silver loci. Interestingly, many of those melanosomal proteins (including epitopes derived from tyrosinase, TRP1/gp75, silver/gp100 and MART1/melan-A) function in vivo as targets of humoral and cellular autoimmune responses directed specifically against normal or transformed melanocytes. These findings have provided new impetus to research on immune responses to melanoma and, perhaps more importantly, examining why they are insufficient to provide protection against tumour growth and what type of immune therapy can be designed to correct that. The melanosome must now be considered beyond its function in pigmentation, and assumes the role of a valuable source for specific immune targets for malignant melanoma.

Epidermolysis bullosa herpetiformis (Dowling-Meara type) exhibits ultrastructural derangement of tonofilaments and desmosomes.

Ultrastructural and immunohistochemical studies of clinically intact skin obtained from three severe neonatal cases of epidermolysis bullosa herpetiformis (Dowling-Meara type) demonstrated disorders in the assembly of keratin intermediate filaments and desmosomes of the keratinocytes. During mitosis, K5- and K14-positive and K1- and K10-negative tonofilaments were disrupted and formed spherical bodies associated with intracytoplasmic desmosomes by invagination of the desmosomes and the adjacent plasma membrane. During the invagination process, destructive changes in the internalized membrane were noted. These were accompanied by gradual loss of reactivity with a monoclonal antibody ZK31, which detected plasma membrane adjacent to the attachment plaques of desmosomes. However, the reactivity of the attachment plaques of the internalized desmosomes for desmoplakins and desmoglein did not decline during the process of internalization. In the suprabasal layers of the epidermis, filamentous substructures and K1 and K10 appeared at the periphery of the spherical bodies. Simultaneously, the desmosomes that were sparsely located in the lower epidermis, increased in number as cell differentiation progressed. Thus, the keratinocytes attained an almost normal appearance with respect to tonofilaments and desmosomes by the time they reached the upper layer of the epidermis. These findings may be relevant to the mechanism responsible for the clinical appearance of the herpetiform blisters in epidermolysis bullosa herpetiformis, which are also characterized by spontaneous involution during childhood or when exposed to high ambient temperatures.

Identification of a cell layer containing alpha-smooth muscle actin in the connective tissue sheath of human anagen hair.

Immunohistochemical and immunoelectron microscopy studies revealed the presence of alpha-smooth muscle (alpha-SM) actin in fibroblasts located in the connective tissue sheath (CTS) of human anagen hair follicles. Immunostaining was positive from the base of the bulb to the upper part of the lower portion of the mature anagen hair follicles. The late catagen hair follicles did not stain. Ultrastructurally, alpha-SM actin was detected only in the fibroblasts located in the innermost layer of the transverse collagenous fibres. Since alpha-SM actin is located in cells with contractile potential, this newly identified layer may play an important role in the morphological changes of the lower portion of the hair follicle during the hair growth cycle.

Biological activity of Serratia marcescens cytotoxin.

Serratia marcescens cytotoxin was purified to homogeneity by ion-exchange chromatography on a DEAE Sepharose Fast Flow column, followed by gel filtration chromatography on a Sephadex G100 column. The molecular mass of the cytotoxin was estimated to be about 50 kDa. Some biological properties of the cytotoxin were analyzed and compared with well-characterized toxins, such as VT1, VT2 and CNF from Escherichia coli and hemolysin produced by S. marcescens. The sensitivity of the cell lines CHO, HeLa, HEp-2, Vero, BHK-21, MA 104 and J774 to the cytotoxin was determined by the cell viability assay using neutral red. CHO and HEp-2 were highly sensitive, with massive cellular death after 1 h of treatment, followed by BHK-21, HeLa, Vero and J774 cells, while MA 104 was insensitive to the toxin. Cytotoxin induced morphological changes such as cell rounding with cytoplasmic retraction and nuclear compactation which were evident 15 min after the addition of cytotoxin. The cytotoxic assays show that 15 min of treatment with the cytotoxin induced irreversible intoxication of the cells, determined by loss of cell viability. Concentrations of 2 CD50 (0.56 g/ml) of purified cytotoxin did not present any hemolytic activity, showing that the cytotoxin is distinct from S. marcescens hemolysin. Antisera prepared against S. marcescens cytotoxin did not neutralize the cytotoxic activity of VT1, VT2 or CNF toxin, indicating that these toxins do not share antigenic determinants with cytotoxin. Moreover, we did not detect gene sequences for any of these toxins in S. marcescens by PCR assay. These results suggest that S. marcescens cytotoxin is not related to any of these toxins from E. coli.

Induction of apoptosis in Caco-2 and HT-29 human intestinal epithelial cells by enterohemolysin produced by classic enteropathogenic Escherichia coli.

AIMS: Detect the cytotoxic effects of the Enterohemolysin from enteropathogenic Escherichia coli C3888 (O 26: H-) on Caco 2 and HT-29-human epithelial intestinal cells. METHODS AND RESULTS: The Caco 2 and HT-29 cells, which were treated with Enterohemolysin (EHly) within 10-15 min, became round, lost attachment to substrate, showed extensive surface blebbing, nucleus shrank, and the chromatin became more compact. After 10 min of exposure to the EHly, the cells showed lactate dehydrogenase (LDH) leakage and reduction of mitochondrial activity. The cells showed disorganization of the actin fibers at 15 min. The death of these human epithelial intestinal cells by apoptosis was confirmed by annexin V. CONCLUSIONS: Enterohemolysin induced apoptosis on human epithelial intestinal cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding of EHly cytotoxic activity suggests the involvement of this hemolysin in the (Enteropathogenic Escherichia coli) EPEC infection mechanism and may facilitate the understanding of the diarrhea caused by EPEC.

Cytotoxic activity of clinical Stenotrophomonas maltophilia.

AIMS: To determine the potential virulence factors produced by culture supernatants of clinical isolates of Stenotrophomonas maltophilia. METHODS AND RESULTS: Culture supernatants of clinical isolates of S. maltophilia were assayed for haemolytic, enzymatic (lipase, protease and phospholipase) and cytotoxic activity. Cytotoxic activity was assayed in Vero (African green monkey), HeLa (human cervix) and HEp-2 (human larynx epidermoid carcinoma) cells. Microscopic analyses revealed intensive rounding, loss of intercellular junctions and membrane alterations (blebbing) followed by death of HEp-2 cells. In Vero and HeLa cells, the cytotoxic effects were characterized by vigorous endocytosis and cell aggregation. The viability of cultured mammalian cells was determined with neutral red and demonstrated that the sensitivity among the cells was different. This activity was inactivated by heating at 56 degrees C for 15 min and protease inhibitors did not inhibit cytotoxic activity. The clinical S. maltophilia presented a cell-free haemolytic activity similar to the 'hot-cold' haemolysins. CONCLUSIONS: S. maltophilia culture supernatants caused vigorous endocytosis and cell aggregation in HeLa and Vero cells, produced haemolytic and enzymatic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Stenotrophomonas maltophilia strains.

Actin bundles in human hair follicles as revealed by confocal laser microscopy.

A confocal laser microscope was used to examine the distribution pattern of actin bundles in whole-mounts of human hair follicles stained with fluorescently labeled phalloidin. Actin bundles were found exclusively in the epithelial outer root sheath of the lower and middle portions of the follicle. In the growth stage, the lower follicle was characterized by well-developed actin bundles arranged circumferentially in the innermost and outermost cell layers of the outer root sheath. Actin bundles in the innermost cells were aligned end-to-end so that they formed complete circular bands surrounding the inner root sheath. In the outermost cells, actin bundles ran underneath the basal plasma membrane to which they attached at both ends. In contrast, in the quiescent stage, actin bundles in the lower follicle were disposed radially toward the follicle surface where they terminated perpendicular to the basal plasma membrane. In the middle follicle, circumferential actin bundles were found only in the intermediate layer of the outer root sheath throughout the hair cycle. Immunofluorescent anti-myosin and anti-alpha-actinin staining showed a striated pattern along actin bundles. Vinculin was localized at both ends of actin bundles, corresponding to the cell-to-cell or cell-to-substrate adherens junctions. Glycerinated follicles changed in shape on the addition of MgATP, suggesting a contraction of actin bundles. From these observations, we conclude that actin bundles in the hair follicle are comparable to stress fibers and that they serve as a tensile scaffold for the growth and integrity of the follicle.

Deposition of basic fibroblast growth factor on surface of epidermal melanocytes suggesting the stromal control of epidermal pigmentation.

Scanning electron microscopy with immunogold labeling was used to demonstrate the in vivo distribution of molecules of basic fibroblast growth factor (bFGF) that were expressed and/or present on the surface of the cells of the normal epidermis and dermal connective tissue of humans. We found that molecules of bFGF, seen as deposits of gold particles, were present densely on the surfaces of the melanocytes but not the epidermal keratinocytes. In connective tissue, these molecules were present exclusively on the surfaces of the fibroblasts, macrophages, vascular endothelial cells, and the basement membrane surrounding the endothelial tube. The selective deposition of bFGF molecules on the melanocytes suggests that the dermal connective tissue may be involved in controlling the proliferation of melanocytes by means of bFGF molecules in vivo, since these melanocytes require bFGF to proliferate in vitro. The latter is synthesized and stored exclusively in the connective tissue.

Three neonatal cases of epidermolysis bullosa herpetiformis (Dowling-Meara type) with severe erosive skin lesions.

We describe three neonates who had large eroded areas of skin on their extremities. The clinical course and ultrastructural findings were consistent with a diagnosis of epidermolysis bullosa herpetiformis (Dowling-Meara type). In each case blisters developed around eroded areas after birth and enlarged centrifugally in a herpetiform fashion. One patient died of sepsis at 8 days of age. In the two survivors blister formation subsided gradually within 1 year. Ultrastructural studies confirmed intraepidermal blister formation associated with spheric aggregates of tonofilaments in the lower epidermis. Spheric aggregates were also found in clinically uninvolved skin.

Differential localization of ICAM-1 and HLA-DR expression on epidermal basal surface in mycosis fungoides and lichenoid reaction.

Scanning electron microscopy with immunogold labeling revealed that epidermal keratinocytes expressed ICAM-1 (intercellular adhesion molecule-1) and HLA-DR molecules on their surfaces in patterns that differed in mycosis fungoides (MF) and lichenoid reaction (LR). ICAM-1 molecules, visualized as deposits of gold particle, were visualized as clusters adjacent to the junctions interconnecting the keratinocytes of MF lesions. LFA-1 (leukocyte function-associated antigen-1) molecules were seen as granules on the surfaces of all infiltrates, most of which also expressed ICAM-1. HLA-DR molecules were seen continuously along the borders of the individual keratinocytes, thus producing a cobblestone appearance on the epidermal undersurface. In contrast, ICAM-1 and HLA-DR were found only sparsely on the undersurface of the epidermis from LR. These findings may help to explain the differing histological features of MF and LR: ICAM-1 molecules present on the intercellular junctions of MF epidermis lead the LFA-1-bearing cells to migrate into the interspaces, thus producing epidermotropism. These cells aggregate by means of co-expressed ICAM-1 to thus produce Pautrier's microabscess. In LR, the minimal expression of ICAm-1 on the epidermal undersurface leaves most infiltrates within the dermis, thus producing a band-like infiltrate.

Quantitation and visualization of ultraviolet-induced DNA damage using specific antibodies: application to pigment cell biology.

The major types of DNA damage induced by sunlight in the skin are DNA photoproducts, such as cyclobutane pyrimidine dimers (CPDs), (6-4)photoproducts (6-4PPs) and Dewar isomers of 6-4PPs. A sensitive method for quantitating and visualizing each type of DNA photoproduct induced by biologically relevant doses of ultraviolet (UV) or sunlight is essential to characterize DNA photoproducts and their biological effects. We have established monoclonal antibodies specific for CPDs, 6-4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured cells or from the skin epidermis using an enzyme-linked immunosorbent assay. One can also use those specific antibodies with in situ laser cytometry to visualize and measure DNA photoproducts in cultured cells or in the skin, using indirect immunofluorescence and a laser-scanning confocal microscope. This latter method allows us to reconstruct three-dimensional images of nuclei containing DNA photoproducts and to simultaneously examine DNA photoproducts and histology in multilayered epidermis. Using those techniques, one can determine the induction and repair of these three distinct types of DNA photoproducts in cultured cells and in the skin exposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techniques and antibodies, we describe the DNA repair kinetics following irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured pigmented cells and in human epidermis.