2-mm surgical margins are adequate for most basal cell carcinomas in Japanese: a retrospective multicentre study on 1000 basal cell carcinomas.

BACKGROUND: Surgery is the gold standard for basal cell carcinomas (BCC). Current recommended surgical margins for BCCs are determined from studies in Caucasian populations. However, the appropriate surgical margins for BCCs in non-white races are unclear. OBJECTIVES: To investigate the accuracy of preoperative determination of clinical tumour borders and appropriate surgical margins in Japanese patients with BCC. METHODS: The maximum calculated differences in distance between the preoperatively determined surgical margins and the actual histologic tumour side margins were considered as 'accuracy gaps' of clinical tumour borders. Estimated side margin positivity rates (ESMPRs) with narrower (2 and 3 mm) surgical margins were calculated on the basis of the accuracy gaps. RESULTS: Overall, 1000 surgically excised BCCs from 980 Japanese patients were included. The most frequent histologic subtype was nodular BCC (67%). The median accuracy gap was 0.3 mm [interquartile range (IQR): -0.5 to +1 mm]. The ESMPRs with 2- and 3-mm surgical margins were 3.8% and 1.4%, respectively. Only the ESMPRs between the well-defined (n = 921) and poorly defined clinical tumour border groups (n = 79) showed statistical difference [2-mm margin: 3.1% vs. 11.7%, OR: 3.89, 95% confidential interval (CI): 1.41-10.71, P <0.01; 3-mm margin: 0.97% vs. 6.3%, OR: 6.58, 95% CI: 1.67-25.99, P <0.01]. No significant differences in ESMPRs were noted in other subgroups including risk classifications. CONCLUSIONS: The determined clinical tumour border accuracy gaps in this Japanese cohort were negligible. Dermatologic surgeons may use narrower surgical margins with acceptable margin positivity rates. The clarity of clinical tumour borders could be an appropriate guide for selection of different surgical margins in the Japanese cohort.

Emerging role of interleukin-31 and interleukin-31 receptor in pruritus in atopic dermatitis.

Atopic dermatitis (AD) is a chronic or chronically relapsing, eczematous, severely pruritic skin disorder associated with skin barrier dysfunction. The lesional skin of AD exhibits T helper 2 (TH 2)-deviated immune reactions. Interleukin-31 (IL-31), preferentially produced from TH 2 cells, is a potent pruritogenic cytokine, and its systemic and local administration induces scratching behavior in rodents, dogs and monkeys. Recent clinical trials have revealed that administration of an anti-IL-31 receptor antibody significantly alleviates pruritus in patients with AD. In this review, we summarize recent topics related to IL-31 and its receptor with special references to atopic itch.

Influence of cerebral fluid drainage on the pharmacokinetics of vancomycin in neurosurgical patients.

The objective of this study was to retrospectively investigate the influence of cerebral fluid drainage on the serum concentrations and pharmacokinetic parameters of vancomycin (VCM). We analyzed 55 patients with normal renal function who had been hospitalized in the neurosurgical ward and received intravenous infusions of VCM. We compared the daily doses of VCM, serum VCM concentrations, serum concentration/dose ratio (C/D ratio), and pharmacokinetic parameters calculated using the Sawchuk-Zaske method between patients who underwent cerebral fluid drainage (drainage group) and controls (non-drainage group). The patients in the drainage group showed a significantly lower trough concentration of VCM (5.8 ± 3.3 µg/mL) than that shown by the non-drainage group (9.9 ± 5.4 µg/mL, p = 0.017). Further, the patients in the drainage group showed a significantly lower trough C/D ratio (0.32 ± 0.17) than that shown by the non-drainage group (0.50 ± 0.31, p = 0.047). In conclusion, cerebral fluid drainage may influence VCM pharmacokinetics. Our findings strongly suggest that a high dose of VCM is required to maintain optimal serum concentrations of VCM in patients managed with cerebral fluid drainage.

Observation of the spatial distribution of gravitationally bound quantum states of ultracold neutrons and its derivation using the Wigner function.

Ultracold neutrons (UCNs) can be bound by the potential of terrestrial gravity and a reflecting mirror. The wave function of the bound state has characteristic modulations. We carried out an experiment to observe the vertical distribution of the UCNs above such a mirror at the Institut Laue-Langevin in 2011. The observed modulation is in good agreement with that prediction by quantum mechanics using the Wigner function. The spatial resolution of the detector system is estimated to be 0.7 µm. This is the first observation of gravitationally bound states of UCNs with submicron spatial resolution.

Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase.

The hallmark of type 2 diabetes, the most common metabolic disorder, is a defect in insulin-stimulated glucose transport in peripheral tissues. Although a role for phosphoinositide-3-kinase (PI3K) activity in insulin-stimulated glucose transport and glucose transporter isoform 4 (Glut4) translocation has been suggested in vitro, its role in vivo and the molecular link between activation of PI3K and translocation has not yet been elucidated. To determine the role of PI3K in glucose homeostasis, we generated mice with a targeted disruption of the gene encoding the p85alpha regulatory subunit of PI3K (Pik3r1; refs 3-5). Pik3r1-/- mice showed increased insulin sensitivity and hypoglycaemia due to increased glucose transport in skeletal muscle and adipocytes. Insulin-stimulated PI3K activity associated with insulin receptor substrates (IRSs) was mediated via full-length p85 alpha in wild-type mice, but via the p50 alpha alternative splicing isoform of the same gene in Pik3r1-/- mice. This isoform switch was associated with an increase in insulin-induced generation of phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P3) in Pik3r1-/- adipocytes and facilitation of Glut4 translocation from the low-density microsome (LDM) fraction to the plasma membrane (PM). This mechanism seems to be responsible for the phenotype of Pik3r1-/- mice, namely increased glucose transport and hypoglycaemia. Our work provides the first direct evidence that PI3K and its regulatory subunit have a role in glucose homeostasis in vivo.

Topical application of PPARα (but not ß/δ or γ) suppresses atopic dermatitis in NC/Nga mice.

BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, which regulate not only adipogenesis and proliferation/differentiation but also the immune response of cells. Because topical application of the activators of some PPAR isoforms improved clinical symptoms in patients with atopic dermatitis (AD), we investigated the role of PPAR activators using a murine AD model in NC/Nga mice; to the best of our knowledge, this has not been previously reported. METHODS: Activators of three PPAR isoforms (α, ß/δ, γ) were topically applied on inflamed skin in a murine AD model that was developed by repeated topical application of mite antigen in NC/Nga mice. The efficacy of each topical PPAR activator was evaluated immunologically and serologically. RESULTS: Topical application of the PPARα activator, but not of the activators of PPARß/δ or PPARγ, improved clinical dermatitis, reduced inflammatory cell infiltration in the dermis, and alleviated the elevation of serum IgE levels. In addition, PPARα expression was downregulated in the epidermis in our murine AD model, as is seen in patients with AD. CONCLUSIONS: Topical application of PPARα activator could be a potent therapeutic agent for patients with AD and could take the place of topical steroid treatments.

Histo-blood group gene polymorphisms as potential genetic modifiers of the development of coronary artery lesions in patients with Kawasaki disease.

Abnormal immunological responses to certain microbial agents may play a crucial role in the pathogenesis of Kawasaki disease (KD). The association studies between histo-blood group genes (Lewis and ABO blood types) and various types of infectious diseases or vasculopathy have been carried out based on the fact that glycosylated antigens could directly mediate microbial infections. We attempted to clarify the role of blood type antigens in the development of KD and coronary artery lesions in KD patients. The subjects included 164 KD patients enrolled from 1998 to 2003 (1st group), 232 patients from 2004 to 2009 (2nd group), and 223 healthy children and 118 patients with growth hormone deficiency as controls. The genotyping of the FUT2 and FUT3 genes, and ABO genotypes, was determined with the TaqMan SNP assay and allele-specific polymerase chain reaction. No significant differences were observed in the genotypes and allele frequencies of the FUT2 and FUT3 genes between the groups. The frequency of the BB blood genotype was significantly higher in KD patients with coronary artery lesions in the 1st and 2nd groups than in the controls (17% and 14% vs. 5%, P = 0.0020). This is the first report to investigate the roles of ABO and Lewis blood types in the development of KD, and in the formation of coronary artery lesions in KD patients. These data suggest that the ABO blood type may play a role in the development of coronary artery lesions in KD patients.

Recurrent atrial fibrillation after high-dose methylprednisolone therapy in a girl with lupus-associated hemophagocytic syndrome.

Hemophagocytic syndrome (HPS) is a serious complication of systemic lupus erythematosus (SLE). A 15-year-old female with lupus-nephritis developed HPS. Bone marrow study showed florid thrombophagocytosis. There was no associated infection. High-dose methylprednisolone therapy ameliorated HPS. However, atrial fibrillation (Af) repeated after the infusion and required direct-current cardioversion. No underlying diseases were found in the heart and endocrine system. Chest roentgenogram and echocardiography were normal. Electrocardiogram showed slightly prolonged PR interval in sinus rhythm. Af occurred at high circulating levels of interferon-γ and interleukin (IL)-10, but not IL-6, IL-2, tumor necrosis factor-α, C-reactive protein or catecholamines. This is the first observation that high-dose corticosteroid induced Af in a case of lupus-HPS. Af is unusual in SLE children without cardiac disease, while conduction defect occurs associated with lupus-myocarditis. Lupus-HPS may be an aggressive SLE subset with cardiac involvement. High-dose corticosteroid infusion controls lupus activity, but could disclose the cardiac stress in lupus-HPS patients.

Gene expression profiling: identification of genes with altered expression in Ayu17-449 knockout mice.

Ayu17-449, a novel gene in mice, has been identified as a tumor-suppressor gene in myeloid malignancy; its product catalyzes the conversion of 5-methylcytosine of DNA to 5-hydroxymethylcytosine. However, in vivo, its functional target genes and biological function have remained unclear. Based on the assumption that alterations in the expression of the Ayu17-449 gene affect the expression of other related genes, we screened a microarray of altered gene expression in Ayu17-449(-/-) and Ayu17-449(+/+) mice. We identified 4049 genes with altered expression, including 1296 up-regulated (fold change ≥2) and 2753 down-regulated (fold change ≤0.5) genes in knockout mice compared with control mice. We then used qRT-PCR and RT-PCR to validate the chip data. Gene ontology and pathway analysis were performed on these altered genes. We found that these altered genes are functional genes in the complement and coagulation cascades, metabolism, biosynthesis, transcriptional regulation, proteolysis, and intracellular signaling pathways, such as the peroxisome proliferator-activated-receptor signaling pathway, the TNF-α-NF-κB pathway, the Notch signaling pathway, the MAPK signaling pathway, and the insulin signaling pathway. The results of our genome-wide comprehensive study could be helpful for comprehending the underlying functional mechanisms of the Ayu17-449 gene in mammals.

Prevention of systemic lupus erythematosus in autoimmune BXSB mice by a transgene encoding I-E alpha chain.

Males from the BXSB murine strain (H-2b) spontaneously develop an autoimmune syndrome with features of systemic lupus erythematosus (SLE), which results in part from the action of a mutant gene (Yaa) located on the Y chromosome. Like other H-2b mice, the BXSB strain does not express the class II major histocompatibility complex antigen, I-E. Here we report that the expression of I-E (E alpha dE beta b) in BXSB males bearing an E alpha d transgene prevents hypergammaglobulinemia, autoantibody production, and subsequent autoimmune glomerulonephritis. These transgenic mice bear on the majority of their B cells not only I-E molecules, but also an I-E alpha chain-derived peptide presented by a higher number of I-Ab molecules, as recognized by the Y-Ae monoclonal antibody. The I-E+ B cells appear less activated in vivo than the I-E- B cells, a minor population. This limited activation of the I-E+ B cells does not reflect a functional deficiency of this cell population, since it can be stimulated to IgM production in vitro by lipopolysaccharides at an even higher level than the I-E- B cell population. The development of the autoimmune syndrome in the transgenic and nontransgenic bone marrow chimeric mice argues against the possibility that the induction of regulatory T cells or clonal deletion of potential autoreactive T cells as a result of I-E expression is a mechanism of the protection conferred by the E alpha d transgene. We propose a novel mechanism by which the E alpha d transgene protects BXSB mice against SLE: overexpression of I-E alpha chains results in the generation of excessive amounts of a peptide displaying a high affinity to the I-Ab molecule, thereby competing with pathogenic autoantigen-derived peptides for presentation by B lymphocytes and preventing their excessive stimulation.

Strain dependency of B and T lymphoma development in immunoglobulin heavy chain enhancer (E mu)-myc transgenic mice.

The transgenic mice were produced by injecting eggs of B6 and C3H/HeJ mice with the human E mu-myc gene. Preferential development of B lymphomas was observed in the B6 transgenic mice, whereas the C3H/HeJ transgenic mice developed mostly T lymphomas. The phenotypic activation of B lineage cells but not of T lineage cells was detected in the prelymphomatous transgenic mice of both strains. The transgene was similarly expressed in B and T cells of the transgenic mice of both strains. These results suggest that a high incidence of T lymphomas in the C3H/HeJ transgenic mice may not be due to the preferential activation of or the preferential E mu-myc expression in T lymphocytes. When the bone marrow or fetal liver cells from the prelymphomatous transgenic mice of both strains were transferred into irradiated normal C3H/HeJ mice, most of the recipients developed T lymphomas. Moreover, even when irradiated B6 mice received the hematopoietic stem cells from the prelymphomatous B6 transgenic mice, the incidence of T lymphoma increased up to 50%. These findings suggest that B6 and C3H/HeJ mice might provide the environment that supports the development or growth of B and T lymphomas, respectively, and that such an environment could be modified by irradiation of the mice.

Multimeric forms of Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide enhance the inhibition of tumor growth and metastasis.

The Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide derived from the laminin B1 chain has been shown to decrease tumor growth and metastasis. Utilizing the multimeric antigen peptide system assembled on a branched lysine core, we synthesized several sizes of multimeric YIGSR, (CH3CO-Tyr-Ile-Gly-Ser-Arg-Gly)16-Lys8-Lys4-Lys2 -Lys-Gly [(Ac-YIGSRG)16 K8K4K2KG] (designated Ac-Y16), (Ac-YIGSRG)8K4K2KG (Ac-Y8), and (Ac-YIGSRG)4K2KG (Ac-Y4), and related peptides, Ac-(YIGSRG)4-NH2 (Ac-Y4L) and Ac-YIGSR-NH2 (Ac-Y1) and evaluated their biological activities in inhibiting tumor growth and metastasis. Coinjection of 0.2 mg/mouse of Ac-Y16 i.v. with B16-F10 mouse melanoma cells inhibited lung colony formation by 97%, whereas 0.2 mg/mouse of Ac-Y1 inhibited by 50%. The larger the peptide (Ac-Y16 > Ac-Y8 > Ac-Y4 > Ac-Y1), the more inhibitory effect there was on lung metastasis. Ac-Y16 also inhibited the growth of s.c.-injected B16-F10 tumors. These data demonstrate that the multimeric YIGSR peptides strongly enhanced the activity of YIGSR in inhibiting tumor growth and metastasis and suggest that these compounds are potentially useful for clinical applications.

Melanoma cells selected for adhesion to laminin peptides have different malignant properties.

Laminin is an important promoter of cell-matrix interactions. A number of active laminin domains have been defined by use of synthetic peptides. The Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence on the B1 chain in laminin can decrease tumor growth and metastasis, whereas another sequence containing Ser-Ile-Lys-Val-Ala-Val (SIKVAV) on the A chain can increase tumor growth and metastasis. Here, we selected B16-F10 melanoma cells by adherence or nonadherence to either YIGSR- or SIKVAV-coated dishes and established 3 B16-F10 variants: YIGSR-adherent cells (Y+), YIGSR-nonadherent cells (Y-), and SIKVAV-nonadherent cells (S-). SIKVAV-adherent cells were not selected because most of F10 cells attached to the SIKVAV-coated dish. These cell lines proliferated at the same rate as the parent F10 cells and attached equally to laminin, collagen IV, and fibronectin. Y+ cells produced rapidly growing tumors after s.c. injection and twice as many lung colonies as the parental F10 cells after i.v. injection. In contrast, Y- cells produced more slowly growing tumors after s.c. injection and produced one-third of the lung colonies relative to the parent cells after i.v. injection. S- cells produced slowly growing tumors after s.c. injection and yielded similar numbers but smaller colonies in the lung than the parental B16-F10 cells after i.v. injection. These data suggest that interactions of melanoma cells with the YIGSR site on laminin are probably important for both colony formation in a target organ (lung) and subsequent tumor growth, while the SIKVAV-containing site on laminin may be more important for tumor growth.

Accelerated disappearance of melanocytes in bcl-2-deficient mice.

Follicular melanocytes in bcl-2(-/-) mice have been reported to turn gray during the second hair cycle. Light microscopic analysis revealed that about half of bcl-2(-/-) mouse hair shafts had no detectable melanin granules after the second hair follicle cycle, but the remaining hair appeared to be pigmented normally. After depilation to induce new anagen hair, more than 97% of the hair shafts did not have visible melanin granules in bcl-2(-/-) mice, whereas 100% of the hair shafts in bcl-2(+/+) mice were pigmented. In bcl-2(+/+) mice, dopa-positive melanocytes appeared on day 4 after depilation, whereas bcl-2(-/-) mice developed few dopa-positive melanocytes after depilation, as assessed by light and electron microscopic observation. bcl-2(-/-) mouse hair in the second hair cycle contained about 60-70% less melanin than normal mouse hair, and newly generated bcl-2(-/-) mouse hair after depilation contained a level of melanin as low as that of albino mouse hair. These observations suggest that the expression of bcl-2 might be essential for melanocyte maintenance after the second hair cycle.

Use of the hemagglutinating virus of Japan (HVJ)-liposome method for analysis of infiltrating lymphocytes induced by hepatitis B virus gene expression in liver tissue.

We previously developed a method for introducing foreign genes into liver tissue using liposomes with incorporated hemagglutinating virus of Japan (HVJ, Sendai virus), and found that liver cells transfected with the E. coli beta-galactosidase gene or the gene for hepatitis B virus (HBV) surface protein (HBsAg) expressed these proteins in vivo. Here, we analyzed cellular reactions leading to hepatitis in the liver by expressing the genes of HBV in vivo. Lymphocytes were eluted directly from liver transfected with the HBsAg genes and shown to be cytotoxic only to cells expressing HBsAg in vitro. These lymphocytes were identified as cytotoxic T lymphocytes with the CD4- CD8+ phenotype. Transfer of these lymphocytes to transgenic mice with the whole HBV genome led to elevation of the serum glutamic-pyruvic transaminase (SGPT) level, indicating the induction of hepatitis due to the cytotoxic T lymphocytes in vivo. Similarly, direct transfer of the gene for the HBV secretory core protein (HBeAg) induced expression of HBeAg in hepatocytes and the appearance of antibody against HBeAg in the serum. However, using this system, we found that the lymphocytes infiltrating the transfected liver showed no cytotoxicity specific for HBeAg. These results indicate that expression of HBsAg, one of the components of virions, in animal liver induced hepatitis efficiently through generation of specific cytotoxic T lymphocytes (CTL) without any expression of the other viral components. This in vivo experimental system should be useful for evaluating how expression of a given gene induces cellular reactions and intrinsic functions in the living body.

Concentration-dependent differential effects of N-acetyl-L-cysteine on the expression of HSP70 and metallothionein genes induced by cadmium in human amniotic cells.

Cadmium induces the expression of the 70 kDa heat shock protein (HSP70) and metallothionein (MT), both of which are considered to be associated with intracellular glutathione (GSH) metabolism in the cellular protection mechanism against cadmium-induced cellular injury. We determined the effects of N-acetyl-L-cysteine (NAC), which increases the intracellular GSH levels, on the induction of HSP70 and MT gene expression in a cultured cell line of human amniotic cells (WISH) exposed to CdCl2. The mRNA level of MT-II, a major isoform of MT genes, was more prominently increased than that of HSP70 when WISH cells were exposed to CdCl2 (5-15 microM, for 6 h). The treatment of WISH cells with 1.5 and 30 mM NAC for 2 h increased the intracellular GSH levels by 1.4- and 3.1-fold, respectively. Pretreatment of cells with 30 mM NAC significantly reduced both HSP70 and MT-II mRNA levels in the cells exposed to 50 microM CdCl2. This concentration of NAC also efficiently suppressed the cadmium-induced lethality. On the contrary, pretreatment with 1.5 mM NAC suppressed only the induction of HSP70 gene expression in the 50 microM CdCl2-treated cells, and did not inhibit the metal toxicity. However, this low concentration of NAC efficiently suppressed lipid peroxidation which was increased by 50 microM CdCl2. Furthermore, this low concentration of NAC also decreased the CdCl2-induced gene expression of HSP32 which represents a general response to oxidative stress. Taken together, NAC seems to have at least two concentration-dependent functions in WISH cells exposed to CdCl2; the low concentration of NAC can suppress the induction of HSP70 gene expression as well as the increase of lipid peroxidation via an antioxidant pathway, while the high concentration of NAC can suppress the induction of MT-II mRNA as well as cadmium-induced cell death. Our present data suggest that changes in intracellular redox status, as reflected by GSH concentration, have more important effects on the induction of HSP70 mRNA rather than that of MT-II mRNA in human amniotic cells exposed to cadmium.

Importance of ornithine transcarbamylase (OTC) deficiency in small intestine for urinary orotic acid excretion: analysis of OTC-deficient spf-ash mice with OTC transgene.

We report the effect of the ornithine transcarbamylase (OTC) transgene composed of 1.3 kb of the 5' flanking region of the rat OTC gene fused to rat OTC cDNA on urinary orotic acid excretion in OTC-deficient spf-ash (sparse-fur with abnormal skin and hair) mice during overnight-starvation and nitrogen loading. During starvation, spf-ash mice with about 6% and 2% of control levels of OTC activity in the liver and small intestine excreted a large amount of orotic acid in the urine. Transgenic spf-ash mice with about 10% and 30% of the control OTC activities in the liver and small intestine did not excrete more than the normal level of orotic acid. Accidental parasitization of transgenic spf-ash mice with ticks (Myocoptes musculinus) resulted in decrease of the OTC activities in the liver and small intestine to the levels in spf-ash mice, and increased excretion of orotic acid. During extermination of the ticks, the mice showed varied levels of OTC activity and orotic acid excretion. On nitrogen loading, transgenic spf-ash mice as well as spf-ash mice excreted larger amounts of orotic acid, while control mice showed no increase in its excretion. The levels of urinary orotic acid were inversely correlated to the logarithms of the OTC activities in the liver and small intestine, the correlation being significantly higher with intestinal OTC than with hepatic OTC activity. These results suggest that the level of OTC activity in the small intestine is important for production of orotic acid.