Yoshio Ishibashi

Detection and Quantification of Specific IgE Antibodies against Eight Malassezia Species in Sera of Patients with Atopic Dermatitis by Using an Enzyme-Linked Immunosorbent Assay

The lipophilic yeast Malassezia, a member of the cutaneous microflora, is an exacerbating factor in atopic dermatitis (AD). Of the 11 currently recognized species, M. globosa and M. restricta are found to frequently colonize the skin of AD patients. In this study, we attempted to quantify specific IgE antibodies against eight Malassezia species, namely, M. dermatitis, M. furfur, M. globosa, M. obtusa, M. pachydermatis, M. slooffiae, M. sympodialis, and M. restricta, in sera from AD patients by using an enzyme‐linked immunosorbent assay (ELISA). The specific IgE value against M. restricta was greater than those against other Malassezia species. Competitive ELISA inhibition tests revealed that M. restricta contained species‐specific as well as shared antigens. Therefore, M. restricta could be considered as a candidate diagnostic antigen for detecting anti‐Malassezia IgE in sera from AD patients.

Role of nuclear factor-κB in the regulation of intercellular adhesion molecule 1 after infection of human bronchial epithelial cells by Bordetella pertussis

Previous work has demonstrated that infection of human bronchial epithelial cells by Bordetella pertussis up-regulates intercellular adhesion molecule-1 (ICAM-1) gene and protein expression. It has also been shown that interaction of the Arg-Gly-Asp (RGD) site of filamentous hemagglutinin (FHA) with host cell very late antigen (VLA)-5 (alpha 5 beta 1 integrin) is required for the up-regulation of epithelial ICAM-1 expression, and that pertussis toxin (PT) impairs this response. We therefore examined the molecular mechanisms leading to B. pertussis-induced ICAM-1 up-regulation in BEAS-2B human bronchial epithelial cells. A colorimetric nuclear factor kappa B (NF-kappa B) activation assay demonstrated that NF-kappa B was activated in response to infection of these cells with B. pertussis. This activation occurred in an FHA(RGD)-dependent manner, and was blocked by an antibody against VLA-5, implying that binding of the RGD to VLA-5 integrin is involved in NF-kappa B activation. Western blot analysis revealed that the activation of NF-kappa B by B. pertussis was preceded by degradation of I kappa B alpha, a major cytoplasmic inhibitor of NF-kappa B. Pretreatment of the BEAS-2B cells with the NF-kappa B inhibitors pyrrolidine dithiocarbamate (PDTC), MG-132, and SN50 resulted in a marked decrease in B. pertussis-induced ICAM-1 expression, implying the involvement of NF-kappa B in ICAM-1 expression. Purified PT abrogated both NF-kappa B activation and I kappa B alpha degradation. These results suggest that ligation of VLA-5 integrin by FHA induces RGD-dependent NF-kappa B activation, thus leading to the up-regulation of epithelial ICAM-1 expression, and that a PT-sensitive G protein may be involved in this signaling pathway.

Role of phosphatidylinositol 3‐kinase in the binding of Bordetella pertussis to human monocytes

Bordetella pertussis, the causative agent of whooping cough, adheres to human monocytes by means of filamentous haemagglutinin (FHA), a bacterial surface protein that is recognized by complement receptor type 3 (CR3, αMβ2 integrin). Previous work has shown that an FHA Arg‐Gly‐Asp (RGD, residues 1097–1099) site interacts with a complex composed of leucocyte response integrin (LRI, αvβ3 integrin) and integrin‐associated protein (IAP, CD47) on human monocytes, resulting in enhancement of CR3‐mediated bacterial binding. However, the pathway that mediates αvβ3‐αMβ2 integrin signalling remains to be characterized. Here we describe the involvement of phosphatidylinositol 3‐kinase (PI3‐K) in this pathway. Wortmannin and LY294002, inhibitors of PI3‐K, reduced αvβ3/IAP‐upregulated, CR3‐associated bacterial binding to human monocytes. B. pertussis infection of human monocytes resulted in a marked recruitment of cellular PI3‐K to the sites of B. pertussis contact. In contrast, cells infected with an isogenic strain carrying a G1098A mutation at the FHA RGD site did not show any recruitment of PI3‐K. We found that ligation of FHA by αvβ3/IAP induced RGD‐dependent tyrosine phosphorylation of a 60 kDa protein, which associated with IAP and PI3‐K in human monocytes. These results suggest that PI3‐K and a tyrosine phosphorylated 60 kDa protein may be involved in this biologically important integrin signalling pathway.

Evaluation of the Levels of Specific IgE against Cryptococcus diffluens and Cryptococcus liquefaciens in Patients with Atopic Dermatitis

Cryptococcus diffluens and Cryptococcus liquefaciens, 2 basidiomycetous yeasts, frequently colonize the skin of patients with atopic dermatitis (AD). In this study, we investigated the presence of specific IgE antibodies against C. diffluens and C. liquefaciens in the sera of AD patients by using an enzyme immunoassay. Of the 122 AD serum samples tested, 43 (35.2%) and 50 (41.0%) were positive for specific IgE antibodies against C. diffluens and C. liquefaciens, respectively. The levels of specific IgE against the C diffluens antigen and that against the C. liquefaciens antigen were strongly correlated (r = 0.96). In contrast, no remarkable correlation was observed between the levels of specific IgE against the 2 Cryptococcus species and that of specific IgE against Malassezia restricta. Competitive enzyme‐linked immunosorbent assay (ELISA) inhibition tests revealed that C. diffluens and C. liquefaciens shared common antigens. This finding was consistent with the IgE immunoblotting data which demonstrated that several IgE‐binding proteins with molecular masses of 77, 54, and 30 kDa were recognized in both C. diffluens and C. liquefaciens antigens. These results suggest that fungal components from C. diffluens and C. liquefaciens may act as allergens and play a role in the pathogenesis of AD.

Secretion of thymic stromal lymphopoietin from human keratinocytes in response to Malassezia yeasts

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Candida albicans abrogates the expression of interferon‐γ‐inducible protein‐10 in human keratinocytes

Candida albicans is the predominant causative agent of human cutaneous candidiasis. Epidermal keratinocytes play an important role in the cutaneous immune response through the production of cytokines and chemokines, including IFN-gamma-inducible protein 10 (IP-10). Here, we investigated the influence of C. albicans infection on IP-10 production by normal human epidermal keratinocytes (NHEK) in vitro. Our results showed that IFN-gamma-stimulated NHEK showed enhanced IP-10 mRNA and protein expression; this expression was downregulated by C. albicans infection. Candida tropicalis also impaired IFN-gamma-induced IP-10 expression, but Candida glabrata did not. Heat-killed C. albicans did not impair IFN-gamma-induced IP-10 expression. We found that coincubation of NHEK with live C. albicans without cell-to-fungi contact impaired IFN-gamma-induced IP-10 mRNA and protein expression in NHEK, suggesting the role of soluble factors derived from live C. albicans in this impairment. Enzyme-linked immunosorbent assay analysis revealed that C. albicans and C. tropicalis could produce marked levels of prostaglandin (PG) E(2), while C. glabrata produced low levels of this prostaglandin. Treatment with E-series prostaglandin receptor antagonists, AH6809 and AH23848, restored IFN-gamma-induced IP-10 expression in C. albicans-infected NHEK. Thus, Candida-derived PGE(2) may impair IFN-gamma-induced IP-10 expression in human keratinocytes and may play a role in the pathogenesis of cutaneous candidiasis.

Mutant of Salmonella typhimurium lacking the inhibitory function for phagosome-lysosome fusion in murine macrophages

It has recently been described that Salmonella typhimurium is capable of inhibiting phagosome-lysosome fusion in murine macrophages after ingestion. We selected a mutant of S. typhimurium lacking the phagosome-lysosome fusion inhibitory function from a collection of Tn5-insertion mutants and examined its relevance to the pathogenesis in mice. The Tn5 insertion mutant which has a defect in fusion inhibitory function was found to be significantly sensitive to the intracellular killing by murine macrophages in vitro. However, the loss of the fusion inhibitory function did not reduce the level of virulence for mice in vivo. These results demonstrated that fusion inhibition did not play a critical role in the pathogenesis of S. typhimurium although it might contribute to at least a part of the resistance against macrophage killing mechanisms.

Atopic Dermatitis and Skin Fungal Microorganisms

A wide variety of bacteria and fungi are found on the human skin. Although some skin microorganisms produce antibacterial peptides that inhibit invasion by pathogens or promote the integrity of cutaneous defenses by eliciting host immune responses, the normal microbiome can also cause several skin diseases. Atopic dermatitis (AD) is a chronic disease that causes pruritus and involves cycles of remission and deterioration. AD is the result of dry hypersensitive skin. When the skin is dry, the protective barrier function of the cutaneous surface horny layer is compromised, and the skin readily develops dermatitis in response to various external stimuli, including skin microorganisms. Serum from almost all AD patients contains IgE antibodies against some skin microorganisms. For example, staphylococcal superantigen-specific IgE is present in the serum of AD patients, but not in the serum of healthy individuals. Normally, the weakly acidic condition of healthy skin prevents colonization by Staphylococcus aureus. However, in patients with AD, the skin pH is shifted toward neutrality, allowing S. aureus to grow and exacerbate AD. In the cutaneous fungal microbiome, lipophilic yeasts of the genus Malassezia are the predominant species on human skin. As Malassezia species require lipids for growth, they preferentially colonize sebum-rich areas such as the head, face, and neck, as opposed to the limbs or trunk. Specific IgE antibody against Malassezia species is found in the serum of AD patients. Antifungal therapy improves the symptoms of AD by decreasing the level of Malassezia colonization, suggesting that these microorganisms also exacerbate AD. Malassezia species, unlike S. aureus, colonize both AD patients and healthy subjects. Currently, the genus Malassezia consists of 14 species. Of these, M. globosa and M. restricta have been detected in almost all AD patients, suggesting that these two Malassezia species play a significant role in AD. The level of specific IgE antibody against both species is greater than that against other Malassezia species. This chapter discusses cutaneous fungi as an exacerbating factor in AD, focusing on: the fungal microbiome in patients with AD. immunological aspects of fungal colonization, and treatment with antifungal agents.

Comparative Analysis of Extracellular Polymeric Substances from Cryptococcus gattii VGIIa Strain Isolated for the First Time in Japan

Cryptococcus gattii and C. neoformans are pathogenic yeasts that cause meningoencephalitis. C. gattii has four molecular types: VGI, VGII, VGIII, and VGIV. Furthermore, three genotypes have been reported for VGII, and a high pathogenicity of the VGIIa genotype has been proposed. The VGIIa strain has been isolated from a patient in Japan, but little is known about the characteristics of the polysaccharides in this strain. In this study we examined the induction of interleukin-8（IL-8）transcriptional activation and compared the nuclear magnetic resonance（NMR）spectra of extracellular polymeric substances（EPSs）, mainly polysaccharides, from the VGIIa, VGIIb, and VGIIc genotypes. The induction of IL-8 by C. gattii EPSs was weaker than that by C. neoformans EPSs. The anomeric proton signals in the NMR spectra of EPSs obtained from VGII isolates were similar, and the polysaccharides were mainly mannose, xylose, galactose, and glucuronic acid. These results suggest that the extracellular polysaccharides from the VGIIa strain isolated in Japan are almost the same as those from other VGII strains.

A Human Monoclonal Antibody Cocktail for Experimental Mouse Infection with Clinically Isolated Strains of Pseudomonas aeruginosa

A human IgG monoclonal antibody cocktail against Pseudomonas aeruginosa serotypes A, B, E, G, I, and M (MCA5) was tested for its therapeutic effect on the infected mice with clinically isolated P. aeruginosa strains. More than 80% of the collected strains were found to belong to the above six serotypes. Direct agglutination with MCA5 gave slight reduced rates, but over 70% of the strains were agglutinated with MCA5. Fifty percent lethal doses of the representative strains to mice (LD50) were estimated, and 50% protective doses of MCA5 to the infected mice with 5 LD50 of the strains (ED50) were determined. MCA5 was found not to be protective to all the infected mice with above six serotypes but protective only to the mice infected with the agglutinated strains. Confirmation of the agglutination of the causative organism with MCA5 is required for the treatment by MCA5. ED50 for a clinically isolated strain and the virulent stock strain were determined under the various challenge doses. It was found that ED50 increased in parallel with the increase of challenged doses but did not change depending on the virulence of the infected strains. These results indicated that this cocktail worked as opsonin. Opsogenic anti-O IgG antibody could be most effective to protect the opportunistic infections caused by the bacteria which have not so powerful exotoxins.