Pigmentary demarcation lines associated with pregnancy.

We report three cases of pigmentary demarcation lines associated with pregnancy. In addition, we reviewed 19 cases including our 3 cases, which were reported in Japan. Most cases occurred during the latter period of pregnancy (after the seventh month), and the pigmentation faded spontaneously or disappeared a few months after delivery in all cases except one. Pigmentary demarcation lines are classified into five groups (types A-E). Of the 19 cases we reviewed, 2 cases showed lines of both types A and B, whereas all the other cases showed type B lines. Although there have only been 29 cases of pigmentary demarcation lines associated with pregnancy reported to date, before ours, we experienced 3 cases within 3 months, therefore it is possible that many such cases are overlooked. Pigmentary demarcation lines are mainly a cosmetic problem. Two of our three cases presented to obstetricians initially. We suggest that dermatologists should be aware that pigmentary demarcation lines may be associated with pregnancy.

Efficient synthesis of O-linked glycopeptide by a transglycosylation using endo alpha-N-acetylgalactosaminidase from Streptomyces sp.

Gal beta-(1-->3)-GalNAc-linked hexapeptide was synthesized by a transglycosylation using Gal beta-(1-->3)-GalNAc beta-pNP as a donor and a serine-containing hexapeptide as an acceptor using endo GalNAc-ase from Streptomyces sp.. The Gal beta-(1-->3)-GalNAc residue was transferred to the hydroxyl group of the serine residue of the peptide. The total yield of the glycopeptide via this process was better than that of the chemoenzymatic method. This process was confirmed to be a versatile method for the synthesis of O-linked glycopeptides.

Linkage of sugar chains to a fragment peptide of FGF-5S by a chemoenzymatic strategy and changes in the rate of proteolytic hydrolysis.

Various O-linked and N-linked sugar chains were linked enzymatically to a fragment peptide (Leu-Ser-Gln(or Asn)-Val-His-Arg) of FGF-5S. First, galactose was linked with beta-(1-->3)-linkage to GalNAc-linked peptide by a transglycosylation using beta-galactosidase from Bacillus circulans (recombinant). Then sialic acid was linked with the aid of sialyltransferase from rat liver (recombinant) to give NeuAcalpha-(2-->3)-Galbeta-(1-->3)-GalNAc-linked hexapeptide. Further, a sialylated 2-chain biantennary sugar chain was linked by a transglycosylation using endo N-acetyl-beta-D-glucosaminidase from Mucor hiemalis (endo M, recombinant). The activity of DNA synthesis in a fibroblast cell line was increased by this glycosylation. The resistance of the obtained glycopeptides towards proteolytic hydrolysis by rat serum and by five proteases was compared with that of original peptide. The resistance was remarkably enhanced by the glycosylation.

Efficient synthesis of a sialyl T-antigen-linked glycopeptide by the chemoenzymatic method.

A sialyl T-antigen-linked tetrapeptide was prepared by the combined method of chemical synthesis and enzymatic synthesis. The GalNAc-linked peptide was first obtained by using a commercial peptide synthesizer, and then a galactose residue was attached with beta-(1-->3)-linkage by transglycosylating with a recombinant beta-galactosidase from Bacillus circulans. The sialic acid residue was then combined by alpha-(2-->3)-linkage with sialytransferase from rat liver.

Immunobead and density gradient purification of Paget cells: in vitro studies of proliferation.

Previous studies using primary monolayer cultures of epithelial cells from the involved epidermis of patients with mammary and extramamary Paget's disease investigated whether Paget cells proliferate as other malignant cells do. Although epithelial monolayers from the involved skin were maintained for approximately 45 days, no permanent cell lines were established. The proportion of carcinoembryonic antigen (CEA)-positive cells did not increase in the long-term cultures. Herein, we report studies of whether there is a real reduction of Paget cell numbers or if this is merely a decrease in the expression of CEA by the cells. Furthermore, we investigated whether Paget cells survive longer when cultured free from any potential inhibitory keratinocytes or other epidermal cells. Skin samples were obtained from one patient with mammary Paget's disease and three with extramammary Paget's disease; epidermal cells were cultured in vitro. An enrichment of Paget cells was carried out from the cultured epidermal cells by combining an antiepithelial membrane antigen monoclonal antibody, binding to immunobeads, and density gradient centrifugation in Nycodenz. The separated cells were re-cultured in Keratinocyte-SFM serum-free media. The proportion of CEA-positive cells did not increase in the culture, and the purified cells did not show any increase in survival times compared to the non-purified cultured cells. These results suggest that the decrease of CEA-positive cells noted during culture results from a decline in expression of CEA in the Paget cells. Paget cells in the involved epidermis do not proliferate significantly and thus differ from many other malignant cells.

An alpha-L-fucosidase from Penicillium multicolor as a candidate enzyme for the synthesis of alpha (1-->3)-linked fucosyl oligosaccharides by transglycosylation.

A new alpha-L-fucosidase was partially purified from the culture broth of Penicillium multicolor, which was available commercially as a freeze dried powder by the name of Lactase-P. This enzyme catalysed the transglycosylation of fucose residue of p-nitrophenyl-alpha-L-fucopyranoside to give alpha-L-Fuc-(1-->3)-D-Glc or alpha-L-Fuc-(1-->3)-D-GlcNAc regioselectively. This enzyme was more stable in the organic co-solvents than the alpha-fucosidase from Aspergillus niger, which was also proposed previously by us as an enzyme to produce fucosyl oligosaccharides.

Purification and properties of recombinant beta-galactosidase from Bacillus circulans.

A gene encoding beta-galactosidase from Bacillus circulans which had hydrolysis specificity for the beta1-3 linkage was expressed in Escherichia coli. The beta-galactosidase was purified from crude cell lysates of E. coli by column chromatographies on Resource Q and Sephacryl S-200 HR. The enzyme released galactose with high selectivity from oligosaccharides which had terminal beta1-3 linked galactose residues. However it did not hydrolyse beta1-4 linked galactooligosaccharides. Moreover, Galbeta1-3GlcNAc, Galbeta1-3GalNAc, and their p-nitrophenyl glycosides were regioselectively synthesized in 10-46% yield by the transglycosylation reaction using this enzyme.

Human desmocollin 1 (Dsc1) is an autoantigen for the subcorneal pustular dermatosis type of IgA pemphigus.

IgA pemphigus showing IgA anti-keratinocyte cell surface autoantibodies is divided into subcorneal pustular dermatosis (SPD) and intraepidermal neutrophilic IgA dermatosis (IEN) types. We previously showed by immunoblotting that IgA from some IgA pemphigus patients reacted with bovine desmocollins (Dsc), but not human Dsc. To determine the antigen for IgA pemphigus, we focused on conformation-dependent epitopes of Dsc, because sera of patients with classical pemphigus recognize conformation-sensitive epitopes of desmogleins. We constructed mammalian expression vectors containing the entire coding sequences of human Dsc1, Dsc2, and Dsc3 and transiently transfected them into COS7 cells by lipofection. Immunofluorescence of COS7 cells transfected with single human Dscs showed that IgA antibodies of all six SPD-type IgA pemphigus cases reacted with the surface of cells expressing Dsc1, but not with cells expressing Dsc2 or Dsc3. In contrast, none of seven IEN-type IgA pemphigus cases reacted with cells transfected with any Dscs. These results convincingly indicate that human Dsc1 is an autoantigen for SPD-type IgA pemphigus, suggesting the possibility of an important role for Dsc1 in the pathogenesis of this disease. This study shows that a Dsc can be an autoimmune target in human skin disease.

Serum levels of eosinophil cationic protein reflect the state of in vitro degranulation of blood hypodense eosinophils in atopic dermatitis.

In patients with atopic dermatitis (AD), serum levels of eosinophil cationic protein (ECP) have been shown to be a good reflector of disease severity. To elucidate what serum levels of ECP actually reflect, ECP levels in serum and plasma and cytological aspects of blood eosinophils were examined in AD patients (n = 27) and compared to healthy subjects (n = 12). Significantly elevated levels of serum ECP were noted in AD patients, while plasma ECP were uniformly recorded at nadir levels in both AD patients and normal subjects. In addition to blood eosinophilia, AD patients had significantly increased numbers of hypodense eosinophils (HEo) with morphological characteristics consistent with an activated state. Serum ECP levels strongly correlated with HEo numbers rather than with total eosinophil counts. These results indicate that elevated levels of serum ECP may be a consequence of in vitro degranulation of "activated" HEo, not of ECP supplementation from lesional skin. In addition, the dynamic correlations of eosinophil-associated parameters (total eosinophil counts, HEo numbers, and serum ECP levels) with AD severity suggest that inflammatory events in lesional skin may be involved in causing not only eosinophilopoiesis in bone marrow, but also development of HEo in the periphery, whose degree in turn may be mirrored in the levels of serum ECP in vitro.

Purification of human blood eosinophils by a combination method using anti-CD16 monoclonal antibody, immunobeads, and Nycodenz density gradient.

A simple method is described for the procurement of human blood eosinophil phenotypes by combining an anti-CD16 monoclonal antibody, immunobeads, and a non-toxic and non-ionic density gradient medium, Nycodenz. The purification depends on the removal of mononuclear cells using a 1.076/1.102 g/ml Nycodenz density gradient, partial removal of neutrophils based on different binding to plastic dishes, interaction of residual neutrophils with immunobeads via an anti-CD16 monoclonal antibody and, finally, extraction of eosinophil phenotypes by sifting the immunobeads-loaded neutrophils through an 1.080/1.102 g/ml Nycodenz density gradient. This method permits simultaneous preparation of highly purified normodense (> 1.080 g/ml) and hypodense eosinophils (< 1.080 g/ml) with reasonable chemiluminescence responses to opsonized zymosans and helminthotoxic activity to opsonized schistosomula corresponding to their own immunocytological properties.

Eosinophilic cellulitis (Wells' syndrome) associated with ascariasis.

A case of eosinophilic cellulitis (Wells' syndrome) in association with ascariasis is described. The clinical and histopathologic features of the patient responded well to an oral anthelminthic drug. According to our search, this association has not previously been reported.

Eosinophil chemiluminescence response to cytokines and opsonized zymosans in atopic dermatitis.

Chemiluminescence (CL) responsiveness of eosinophils (Eos) to zymosan particles coated with either IgG, C3 or both and eosinophilopoiesis-modulating cytokines was investigated in patients with atopic dermatitis (AD), and an attempt was made to correlate these CL responses with serum levels of eosinophil cationic protein (ECP) which increased in response to the extent of AD severity, but not blood eosinophil counts. A high degree of positive correlation existed between serum levels of ECP and either IgG containing opsonized zymosan- or interleukin-5 (IL-5)-induced CLs. The CL values induced by these two stimuli appeared to be directly correlated with the intensity of each ligand-linked receptor expression. These results suggested that Fc gamma RII-and/or IL-5R-mediated eosinophil activation at the site of lesional skins might implicate in deterioration of cutaneous inflammation, and consequently cause an elevation of serum levels of ECP in AD.

[Eosinophil granule proteins (MBP, ECP, EPX/EDN, EPO)--a possible process of eosinophil activation and degranulation].

From current information, a brief review was made on the basic properties of a possible process of eosinophil activation and degranulation. The "activated" eosinophils show the following characteristics: diminished cell density, morphologic alterations, increased surface receptors, heightened parasite killing, increased metabolic activity and prolonged survival. Immune complexes (secretory IgA, IgG, IgE) are known as potent triggering stimuli of eosinophil degranulation as well as complement fragments (C3b, C3bi). Cytokines (IL-5, GM-CSF), PAF and peptides (substance P) act both as weak degranulation inducer and degranulation enhancer. Synergism between the two pathways, Ca2+ and protein kinase C, is now recognized as a common feature of control of secretion in eosinophils.

Eosinophil phenotypes in bullous pemphigoid.

Eosinophil phenotypes were investigated in peripheral blood and skin lesions from eight patients with bullous pemphigoid (BP). By Nycodenz density gradients fractionation, blood eosinophils were divided into two phenotypes; normodense (greater than 1.080 g/ml) and hypodense (less than or equal to 1.080 g/ml). Increased numbers of hypodense eosinophils were observed in the blood from all patients with BP. Immunocytochemical observations, using an EG2 monoclonal antibody to react with the secretion form of eosinophil cationic protein (ECP), revealed that EG2 was expressed in 86 +/- 3% of hypodense phenotypes and 3 +/- 2% of normodense phenotypes. Ultrastructurally, hypodense eosinophils were characterized by numerous spheroidal granules, each with a lytic crystalloid core. These indicate that the hypodense phenotype represents a cell in an activated state. Only eosinophils with immunocytochemical and morphological characteristics similar to hypodense phenotypes infiltrated around the basement membrane zone in involved skin of BP. Furthermore, direct adherence of eosinophils associated with degranulation into basal keratinocytes was seen at the sites of blistering lesions. Bullous fluids contained higher concentrations of ECP than sera as determined by a radioimmunosorbent assay; thus hypodense (activated) eosinophils may directly damage the basal keratinocytes by releasing their granule proteins, subsequently leading to dermo-epidermal separation.

Eosinophil involvement in atopic dermatitis as reflected by elevated serum levels of eosinophil cationic protein.

Eosinophil cationic protein (ECP), one of the eosinophil granule proteins, is released during allergic reactions. We investigated the possibility of correlations among the serum levels of ECP, clinical activity, and eosinophil number in patients with atopic dermatitis (AD). Forty-four patients with AD and 25 normal, non-atopic subjects were studied. ECP was quantitated by a double antibody radioimmunoassay. The levels of serum ECP correlate with the grading of severity of clinical evaluations in AD. The patients with severe and moderate AD had significantly higher ECP concentrations than normal controls (p less than 0.001); mild AD had levels identical with those of control groups. A positive correlation was observed between the number of peripheral blood eosinophils and serum ECP levels in the severe cases (r = 0.67, p less than 0.05). Furthermore, these ECP levels significantly decreased in response to either improvement of clinical severity of AD or decreased numbers of blood hypodense eosinophils in anti-allergic drug-treated patients. No coefficient of correlation was observed between serum ECP and IgE levels. These findings indicate that eosinophils may release their granular contents, including ECP, into the peripheral circulation and/or inflammatory skin lesions and subsequently provoke a clinical exacerbation by stimulating allergic reactions.

Alteration in the density, morphology, and biological properties of eosinophils produced by bullous pemphigoid blister fluid.

Bullous pemphigoid blister fluid (BP-BF) was examined for its effects on the density, morphology, and biological properties of eosinophils. Normodense eosinophils (NEo) were prepared from guinea pig peritoneal exudates by Nycodenz density gradient centrifugation. After culturing with BP-BF, NEo were converted into hypodense eosinophils (HEo) in a time-dependent manner. HEo were morphologically different from NEo in that HEo had spheroidal granules each with a lytic crystalloid core and a significantly increased cell volume. These HEo showed an enhanced antibody- and/or complement-dependent helminthotoxic activity to Schistosoma mansoni larvae, amplified chemiluminescence response to opsonized zymosan, and augmented expression of both FcR+ and CR+. These results suggest that BP-BF contains an activity that may not only induce an eosinophil hypodensity as a consequence of increasing cell volume, but simultaneously enhance an eosinophil cytotoxic potential through augmenting cell-surface receptors and receptor-linked oxidative metabolism. In addition, observed tissue accumulation of this activity suggests that eosinophils may be regulated by their phenotypic change in the skin lesions of bullous pemphigoid and be involved in blister formation.