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.

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.

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.

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 æg/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

Involvement of ITF2 in the transcriptional regulation of melanogenic genes.

In response to agouti signal protein, melanocytes switch from producing eumelanin to pheomelanin concomitant with the down-regulation of melanogenic gene transcription. We previously reported that a ubiquitous basic helix-loop-helix transcription factor, known as ITF2, is up-regulated during this switch, and we now report that treatment of melanocytes with melanocyte-stimulating hormone down-regulates expression of ITF2. To more fully characterize the involvement of ITF2 in regulating melanogenic gene transcription, ITF2 sense or antisense constructs were introduced into melan-a melanocytes. Gene and protein expression analyses and luciferase reporter assays using promoters from melanogenic genes showed that up-regulation of ITF2 suppressed melanogenic gene expression as well as the expression of Mitf, a melanocyte-specific transcription factor. In addition, stable ITF2 sense transfectants had significant reductions in pigmentation and a less dendritic phenotype compared with mock transfectants. In contrast, ITF2 antisense-transfected melanocytes were more pigmented and more dendritic. These results demonstrate that up-regulation of ITF2 during the pheomelanin switch is functionally significant and reveal that differential expression of a ubiquitous basic helix-loop-helix transcription factor can modulate expression of melanogenic genes and the differentiation of melanocytes.

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.

The melanocortin 1 receptor is the principal mediator of the effects of agouti signaling protein on mammalian melanocytes.

The agouti gene codes for agouti signaling protein (ASP), which is temporally expressed in wild-type mouse follicular melanocytes where it induces pheomelanin synthesis. Studies using purified full-length agouti signaling protein has shown that it competes with (&agr;)-melanocyte stimulating hormone for binding to the melanocortin 1 receptor. We have investigated whether ASP binds exclusively to the melanocortin 1 receptor expressed on mouse melanocytes in primary culture, or additionally activates a receptor that has not been identified yet. We have compared the responses of congenic mouse melanocytes derived from C57 BL/6J-E(+)/E(+), e/e, or E(so)/E(so) mice to (alpha)-MSH and/or ASP. E(+)/E(+) melanocytes express the wild-type melanocortin 1 receptor, e/e melanocytes express a loss-of-function mutation in the melanocortin 1 receptor that results in a yellow coat color, and E(so)/E(so) is a mutation that causes constitutive activation of the melanocortin 1 receptor and renders melanocytes unresponsive to (alpha)-melanocyte stimulating hormone. Mouse E(+)/E(+) melanocytes, but not e/e or E(so)/E(so) melanocytes, respond to agouti signaling protein with decreased basal tyrosinase activity, and reduction in levels of tyrosinase and tyrosinase-related proteins 1 and 2. Only in E(+)/E(+) melanocytes does agouti signaling protein abrogate the stimulatory effects of (alpha)-melanocyte stimulating hormone on cAMP formation and tyrosinase activity. These results indicate that a functional melanocortin 1 receptor is obligatory for the response of mammalian melanocytes to agouti signaling protein.

Transcription factor hierarchy in Waardenburg syndrome: regulation of MITF expression by SOX10 and PAX3.

Waardenburg syndrome (WS) is associated with neural crest-derived melanocyte deficiency caused by mutations in either one of three transcription factors: MITF, PAX3, and SOX10. However, the hierarchical relationship of these transcription factors is largely unknown. We show that SOX10 is capable of transactivating the MITF promoter 100-fold, and that this transactivation is further stimulated by PAX3. Promoter deletion and mutational analyses indicate that SOX10 can activate MITF expression through binding to a region that is evolutionarily conserved between the mouse and human MITF promoters. A SOX10 mutant that models C-terminal truncations in WS can reduce wild-type SOX10 induction of MITF, suggesting these mutations may act in a dominant-negative fashion. Our data support a model in which the hypopigmentation in WS, of which these factors have been implicated, results from a disruption in function of the central melanocyte transcription factor MITF.

Bioactive motifs of agouti signal protein.

The switch between the synthesis of eu- and pheomelanins is modulated by the interaction of two paracrine signaling molecules, alpha-melanocyte stimulating hormone (MSH) and agouti signal protein (ASP), which interact with melanocytes via the MSH receptor (MC1R). Comparison of the primary sequence of ASP with the known MSH pharmacophore provides no suggestion about the putative bioactive domain(s) of ASP. To identify such bioactive motif(s), we synthesized 15-mer peptides that spanned the primary sequence of ASP and determined their effects on the melanogenic activities of murine melanocytes. Northern and Western blotting were used, together with chemical analysis of melanins and enzymatic assays, to identify three distinct bioactive regions of ASP that down-regulate eumelanogenesis. The decrease in eumelanin production was mediated by down-regulation of mRNA levels for tyrosinase and other melanogenic enzymes, as occurs in vivo, and these effects were comparable to those elicited by intact recombinant ASP. Shorter peptides in those motifs were synthesized and their effects on melanogenesis were further investigated. The amino acid arginine, which is present in the MSH peptide pharmacophore (HFRW), is also in the most active domain of ASP (KVARP). Our data suggest that lysines and an arginine (in motifs such as KxxxxKxxR or KxxRxxxxK) are important for the bioactivity of ASP. Identification of the specific ASP epitope that interacts with the MC1R has potential pharmacological applications in treating dysfunctions of skin pigmentation.

In situ localization of agouti signal protein in murine skin using immunohistochemistry with an ASP-specific antibody.

Switching between production of eumelanin or pheomelanin in follicular melanocytes is responsible for hair color in mammals; in mice, this switch is controlled by the agouti locus, which encodes agouti signal protein (ASP) through the action of melanocortin receptor 1. To study expression and processing patterns of ASP in the skin and its regulation of pigment production in hair follicles, we have generated a rabbit antibody (termed alphaPEP16) against a synthetic peptide that corresponds to the carboxyl terminus of ASP. The specificity of that antibody was measured by ELISA and was confirmed by Western blot analysis. Using immunohistochemistry, we characterized the expression of ASP in the skin of newborn mice at 3, 6, and 9 days postnatally. Expression in nonagouti (a/a) black mouse skin was negative at all times examined, as expected, and high expression of ASP was observed in 6 day newborn agouti (A/+) and in 6 and 9 day newborn lethal yellow (A(y)/a) mouse skin. In lethal yellow (pheomelanogenic) mice, ASP expression increased day by day as the hair color became more yellow. These expression patterns suggest that ASP is delivered quickly and efficiently to melanocytes and to hair matrix cells in the hair bulbs where it regulates melanin production.

Possible involvement of proteolytic degradation of tyrosinase in the regulatory effect of fatty acids on melanogenesis.

The purpose of this study was to investigate the mechanism of fatty acid-induced regulation of melanogenesis. An apparent regulatory effect on melanogenesis was observed when cultured B16F10 melanoma cells were incubated with fatty acids, i.e., linoleic acid (unsaturated, C18:2) decreased melanin synthesis while palmitic acid (saturated, C16:0) increased it. However, mRNA levels of the melanogenic enzymes, tyrosinase, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2), were not altered. Regarding protein levels of these enzymes, the amount of tyrosinase was decreased by linoleic acid and increased by palmitic acid, whereas the amounts of TRP1 and TRP2 did not change after incubation with fatty acids. Pulse-chase assay by [35S]methionine metabolic labeling revealed that neither linoleic acid nor palmitic acid altered the synthesis of tyrosinase. Further, it was shown that linoleic acid accelerated, while palmitic acid decelerated, the proteolytic degradation of tyrosinase. These results suggest that modification of proteolytic degradation of tyrosinase is involved in regulatory effects of fatty acids on melanogenesis in cultured melanoma cells.

Cysteine transport in melanosomes from murine melanocytes.

The synthesis of pheomelanin requires the incorporation of thiol-containing compound(s) during the process of mammalian melanogenesis. Since melanins are produced only in specialized, membrane-bound organelles, known as melanosomes, such thiol donor(s) must cross the membrane barrier from the cytosol to the melanosome interior. Cysteine and/or glutathione (GSH) were proposed as suitable thiol donors, although uptake of these compounds into melanosomes was not previously characterized. In this study, we show that cysteine is transported, in a temperature- and concentration-dependent manner, across membranes of melanosomes derived from murine melanocytes. Additional proof that cysteine uptake results from a carrier-mediated process and is not due to simple diffusion or to a membrane channel, was obtained in countertransport experiments, in which melanosomes preloaded with cysteine methyl ester took up significantly more [35S]cysteine than did unloaded controls. In contrast, we were unable to detect any significant uptake of [35S]GSH over a wide concentration range, in the presence or in the absence of reducing agent. This study is the first demonstration of melanosomal membrane transport of cysteine, and it strongly suggests that free cysteine is the thiol source utilized for pheomelanin synthesis in mammalian melanocytes.