Organization and functions of glycolipid-enriched microdomains in phagocytes.

Populations of glycolipids change markedly during leukocyte differentiation, suggesting that these molecules are involved in biological functions. About 70% of the glycosphingolipids in human neutrophils are lactosylceramide, a molecule also expressed on monocytes and dendritic cells, but not on lymphocytes. In contrast, phosphatidylglucoside is mainly expressed on neutrophils. STED microscopic analysis showed that phosphatidylglucoside and lactosylceramide form different domains on plasma membranes of neutrophils, with phosphatidylglucoside preferentially expressed along the neutrophil differentiation pathway. Phosphatidylglucoside was found to mediate the differentiation of HL-60 cells into the neutrophilic lineage, and to be involved in FAS-dependent neutrophil apoptosis. In contrast, lactosylceramide was only expressed on mature neutrophils. Complexes of lactosylceramide and the Src family kinase Lyn form membrane microdomains. LacCer-enriched membrane microdomains mediate neutrophil innate immune responses; e.g. chemotaxis, phagocytosis, and superoxide generation. C24 fatty acid chains of LacCer are indispensable for the formation of LacCer-Lyn complexes and for LacCer-dependent functions. Moreover, Lyn-coupled LacCer-enriched microdomains serve as signal transduction platforms for αMß2 integrin-mediated phagocytosis. This review describes the organization and potential functions of glycolipids in phagocytes, as well as the roles of both phosphatidylglucoside and lactosylceramide in neutrophils. This article is part of a Special Issue entitled Linking transcription to physiology in lipidomics.

Possible Antipruritic Mechanism of Cyclosporine A in Atopic Dermatitis.

Cyclosporine A is an immunosuppressive agent that suppresses pruritus and is currently used in the treatment of patients with severe atopic dermatitis. The aim of this study was to elucidate the antipruritic mechanism of cyclosporine A using a mouse model of atopic dermatitis. Intraperitoneal injection of cyclosporine A (5 mg/kg) significantly reduced epidermal nerve density, number of scratching bouts, dermatitis scores, and transepidermal water loss, as well as decreasing the numbers of inflammatory cells in the dermis and decreasing epidermal thickness. Intraperitoneal injection of cyclosporine A dose-dependently inhibited increased itch-related receptor gene expression, such as interleukin-31 receptor A and neurokinin-1 receptor, in the dorsal root ganglion of atopic dermatitis model mice. Thus, the antipruritic efficacy of cyclosporine A may involve reduced epidermal nerve density and expression levels of itch-related receptor genes in the dorsal root ganglion, as well as improvement in acanthosis and reduction in cutaneous inflammatory cell number.

The novel neutrophil differentiation marker phosphatidylglucoside mediates neutrophil apoptosis.

A new type of glycolipid, phosphatidylglucoside (PtdGlc), was identified as a component of raft-like membrane domains of the human leukemia cell line HL-60. In this study, we show that PtdGlc forms functional domains that are different from those produced by lactosylceramide (LacCer)-enriched lipid rafts. These rafts initiate neutrophil apoptosis. Neutrophils are the only type of human peripheral blood leukocyte or monocyte-derived dendritic cell to express large amounts of PtdGlc on their cell surfaces. PtdGlc was not colocalized with LacCer. Anti-PtdGlc IgM DIM21 did not induce neutrophil chemotaxis or superoxide generation, whereas anti-LacCer IgM T5A7 induced these activities. DIM21, but not T5A7, significantly induced neutrophil apoptosis. DIM21-induced apoptosis was inhibited by specific inhibitors of cysteine-containing aspartate-specific proteases (caspases)-8, -9, and -3 but not by the Src family kinase inhibitor PP1, PIP(3) kinase inhibitor LY294002, NADPH oxidase inhibitor diphenyleneiodonium, superoxide dismutase, or catalase. PtdGlc was colocalized with Fas on the neutrophil plasma membrane. DIM21 and the agonist anti-Fas Ab DX2 induced the formation of large Fas-colocalized clusters of PtdGlc on the plasma membrane. Furthermore, the antagonistic anti-Fas Ab ZB4 significantly inhibited DIM21-induced neutrophil apoptosis. These results suggest that PtdGlc is specifically expressed on neutrophils and mediates apoptosis of these cells, and that the Fas-associated death signal may be involved in PtdGlc-mediated apoptosis.

[The significance of the combined usage of cytology and the HPV test in cervical cancer screening].

BACKGROUND: In the United States, the combined usage of cytology and the HPV test is employed in cervical cancer screening, but, in Japan, only cytology is performed in most cases. We conducted a comparative analysis of histopathological diagnoses by colposcopy, cytology, and the HPV test, and evaluated the effectiveness of cytology and the significance of its combined use with the HPV test. METHODS: We compared the detection rates using the test for high-risk HPV and cytology, which were conducted in cases with various histological diagnoses. The hybrid capture method was used in the HPV test. The blind test was conducted by 3 cytologists in 14 cases in which the cytological differed from the histological diagnosis by more than 2 classes. RESULTS: There were 89 cases above class IIIb or CIN 3, and 26 cases were below class IIIa. A total of 107 cases were positive on the HPV-DNA test, and 8 cases were negative. CONCLUSION: Cytology is essential as a morphological diagnostic method in cancer screening. In contrast, the HPV test detects the cause of cervical cancer. Therefore, the combined usage of cytology and the HPV test as initial screening for cervical cancer would facilitate the diagnosis of a premalignant lesion or early-stage cervical cancer more promptly and accurately.

[Correlation between human papilloma virus infection in cervical lesions and expression of p53, p21 proteins and Ki-67].

BACKGROUND: Human papilloma virus (HPV) is the most important factor in the oncogenic mechanism of cervical tumor. Furthermore, in a separate multi-stage process, abnormality in cell cycle kinetics has been demonstrated. In order to elucidate the oncogenic mechanism, we examined the relationship between cervical carcinoma and HPV infection, and also investigated the expression of p53 and p21 proteins as well as the cell proliferation capability by detecting Ki-67, and analyzed the correlations of these factors. MATERIALS AND METHODS: We studied the biopsy specimens from 107 patients of chronic cervicitis, cervical intraepithelial neoplasia and squamous cell carcinoma (SCC). HPV DNA was detected by the hybrid capture method. Immunostaining by LSAB procedures were performed using antibodies to p53 protein, p21 and MIB-1. The PCR-denaturing gradient gel electrophoresis (DGGE) method was used to search for mutation in exons 5, 6, 7 and 8 of p53. RESULTS: Of 107 cases studied, high-oncogenic HPV was detected in 80 cases (74.8%) with a particularly high prevalence in SCC. No correlation was observed between HPV infection and expression of p53, p21 or Ki-67. The degree of positivity of Ki-67 expression tended to be higher with disease progression. Cases strongly positive (2+) for p53 and p21 proteins were weakly positive for Ki-67, and cases positive (1+) or negative for p53 and p21 were strongly positive for Ki-67. CONCLUSION: In oncogenesis of cervical carcinoma, p53 protein, p21 protein and HPV may act separately as independent factors in some cases, and there is a strong possibility that other factors are involved.

[The relationship with human papilloma virus DNA in cervical adenocarcinoma].

Recently, cervical adenocarcinoma has been increasing especially among young women and account for 10-20% of cervical cancer. However, the detection rate of HPV-DNA was 35-85% and lower than that of squamous cell carcinoma. Furthermore, the relationship with HPV in cervical adenocarcinoma was not much investigated in Japan, so we studied HPV status in cervical adenocarcinoma by in situ PCR method using biotin-labeled DNA probes, because in situ PCR method possesses the advantages of both PCR and in situ hybridization in being highly sensitive and enabling visualization of the cellular localization of the DNA. HPV infection was analyzed in 60 cervical adenocarcinomas, including 1 adenocarcinoma in situ and 15 adenosquamous carcinomas. HPV-DNA was detected in 47 of all 60 cases(78%): 35 of 45(78%) in adenocarcinomas and 12 of 15(80%) in adenosquamous carcinomas. No significant correlation was found between the HPV-DNA detection rate and histological subtypes of adenocarcinoma. In conclusion, cervical adenocarcinoma demonstrates a high prevalence of HPV-DNA as well as other previous studies. Therefore, HPV infection plays a very important role in not only squamous cell carcinoma but also adenocarcinoma in uterine cervix.

Pseudomonas-derived ceramidase induces production of inflammatory mediators from human keratinocytes via sphingosine-1-phosphate.

Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P) stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed "3D keratinocytes"), which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i) 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii) S1P induces the production of TNF-α via S1P receptors, and (iii) released TNF-α stimulates the production of inflammatory mediators such as IL-8.

Membrane microdomains in immunity: glycosphingolipid-enriched domain-mediated innate immune responses.

Over the last 30 years, many studies have indicated that glycosphingolipids (GSLs) expressed on the cell surface may act as binding sites for microorganisms. Based on their physicochemical characteristics, GSLs form membrane microdomains with cholesterol, sphingomyelin, glycosylphosphatidylinositol (GPI)-anchored proteins, and various signaling molecules, and GSL-enriched domains have been shown to be involved in these defense responses. Among the GSLs, lactosylceramide (LacCer, CDw17) can bind to various microorganisms. LacCer is expressed at high levels on the plasma membrane of human neutrophils, and forms membrane microdomains associated with the Src family tyrosine kinase Lyn. LacCer-enriched membrane microdomains mediate superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. In contrast, several pathogens have developed infection mechanisms using membrane microdomains. In addition, some pathogens have the ability to avoid degradation by escaping from the vacuolar compartment or preventing phagosome maturation, utilizing membrane microdomains, such as LacCer-enriched domains, of host cells. The detailed molecular mechanisms of these membrane microdomain-associated host-pathogen interactions remain to be elucidated.

Human papillomavirus localization in cervical adenocarcinoma and adenosquamous carcinoma using in situ polymerase chain reaction: review of the literature of human papillomavirus detection in these carcinomas.

Many studies have suggested that human papillomavirus (HPV) infection plays an important role in the carcinogenesis of the cervical adenocarcinoma. However, the prevalence of HPV infection in cervical adenocarcinoma and adenosquamous carcinoma varies among the studies. Cervical adenocarcinoma (24 cases) and adenosquamous carcinoma (16 cases), including the underlying non-neoplastic epithelium were examined for HPV-DNA using in situ polymerase chain reaction (PCR), which enabled visualization of the localization on a glass slide. In adenocarcinoma, HPV-DNA was found in 13 cases (54%) and in eight cases in underlying non-neoplastic epithelium, resulting in a total of 21 positive cases (88%). In adenosquamous carcinoma, HPV-DNA was detected in 12 cases (75%) and and the HPV-DNA localization of each component was pure adenocarcinoma, 28.6%; mixed, 54.5%; and pure squamous cell carcinoma, 83.3%. In the underlying non-neoplastic epithelium, HPV-DNA was found more frequently in the squamous epithelium (73.3%) than the cervical glands (6.3%). In conclusion, HPV-DNA was detected in 54% of adenocarcinoma, and the rate was elevated by HPV localization in the underlying non-neoplastic epithelium. HPV infection in the underlying squamous epithelium might be related to the carcinogenesis, even in cervical adenocarcinoma. HPV-DNA localization was different in each component of adenosquamous carcinoma.

Prevalence of human papillomavirus infection and correlation with cervical lesions in Japanese women.

PURPOSE: To investigate the prevalence of human papillomavirus (HPV) infection in patients attending a gynecologic outpatient department, and to correlate the infection status with the presence or absence of uterine cervical lesions and the grades assessed by cytological or histological examinations. METHODS: Five hundred and seventy-two subjects were studied. In all subjects, HPV detection by the hybrid capture method and a cervical cytological examination were performed RESULTS: The HPV-positive rate in subjects with normal cytology was 12.3%. The detection rate was high (21.7%) in subjects aged in the twenties and low in the forties, and HPV was not detected in subjects aged in the sixties and seventies. When HPV-positive rates were examined according to cytological or histological grades, the rates were higher in subjects with abnormal cytology (P < 0.01) or cervical intraepithelial neoplasia (CIN) or squamous cell carcinoma compared with those with normal cytology. CONCLUSION: Diagnosis of HPV infection is also important for the prediction of progression to CIN and cervical cancer.

Secretory carcinoma of the breast: an immunohistochemical and ultrastructural study.

Abstract We report a rare case of secretory carcinoma of the breast in a 50-year-old Japanese woman. The patient had been aware of a right breast tumor for 8 years, but had left it untreated. The tumor enlarged in size and became painful, and she visited our hospital. Breast carcinoma was diagnosed, and mastectomy was performed. Histopathological examination revealed features of a secretory carcinoma characterized by prominent secretory activity in the glandular and microcystic spaces, with some areas showing a follicular pattern resembling the thyroid gland. The secretory material was PAS-positive and immunohistochemically alpha-lactalbumin-positive. Ultrastructurally, the tumor cell contained many secretory vacuoles in the cytoplasm. In addition, extracellular and intracytoplasmic lumina were conspicuous; these were lined by microvilli projection and contained secretory material. By flow cytometric analysis, the DNA index was 1.14, which was diploid, showing relatively low proliferative activity.