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In allergic contact dermatitis (ACD) and contact hypersensitivity (CHS), the healed skin shows greater swelling than the naïve skin in the same individual upon re-exposure to the same hapten. This \"local skin memory\" (LSM) in healed skin was maintained for a prolonged period of time and mediated by skin CD8 -resident memory T (T ) cells in C57BL/6 mice. However, the number of CD4 T cells is elevated in ACD-healed human skin, and the contribution of CD4 T cells to the formation of LSM currently remains unclear. We herein demonstrated that immediately after CHS subsided, the healed skin in BALB/c mice showed an accumulation of hapten-specific CD4 and CD8 T cells, with a predominance of CD4 T cells. The presence of CD4 or CD8 T cells in the healed skin was sufficient for the induction of a flare-up reaction upon a re-challenge. The CD4 and CD8 T cells both produced interferon-γ and tumor necrosis factor early after the re-challenge. Moreover, while CD8 T cells gradually decreased over time and were eventually lost from the healed skin at 40-51 weeks after the resolution of CHS, the CD4 T cell numbers remained elevated during this period. The present results indicate that the long-term maintenance of LSM is mediated by CD4 T cells, and thus CD4 T cells are an important target for the treatment of recurrent human ACD.

Glypican‐3 (GPC3) is an onco‐fetal antigen that is overexpressed in human hepatocellular carcinoma (HCC), and is only expressed in the placenta and embryonic liver among normal tissues. Previously, we identified an HLA‐A2‐restricted GPC3144–152 (FVGEFFTDV) peptide that can induce GPC3‐reactive CTLs without inducing autoimmunity in HLA‐A2 transgenic mice. In this study, we carried out a phase I clinical trial of HLA‐A2‐restricted GPC3144–152 peptide vaccine in 14 patients with advanced HCC. Immunological responses were analyzed by ex vivoγ‐interferon enzyme‐linked immunospot assay. The frequency of GPC3144–152 peptide‐specific CTLs after vaccination (mean, 96; range, 5–441) was significantly larger than that before vaccination (mean, 6.5; range, 0–43) (P < 0.01). An increase in the GPC3144–152 peptide‐specific CTL frequency was observed in 12 (86%) of 14 patients after vaccination. Additionally, there was a significant correlation between the maximum value of GPC3144–152 peptide‐specific CTLs after vaccination and the dose of the peptide injected (P = 0.0166, r = 0.665). Moreover, we established several GPC3144–152 peptide‐specific CTL clones from PBMCs of patients vaccinated with GPC3144–152 peptide by single cell sorting using Dextramer and CD107a antibody. These CTL clones had high avidity (the recognition efficiency showing 50% cytotoxicity was 10−10 or 10−11 M) and could recognize HCC cell lines expressing GPC3 in an HLA‐class I‐restricted manner. These results suggest that GPC3144–152 peptide vaccine can induce high avidity CTLs capable of killing HCC cells expressing GPC3. This trial was registered with University Hospital Medical Information Network number 000001395. (Cancer Sci 2011; 102: 918–925)

Carboxyl terminus of Hsc70-interacting protein (CHIP) is a ubiquitin ligase that induces ubiquitination and degradation of its target proteins including oncoproteins. We reported that its down-regulation is associated with tumor progression and metastasis of breast cancer. However, the mechanism through which CHIP gene affects cancer cells is unclear. We extracted RNA from 45 primary breast cancer samples and compared CHIP mRNA expression profiles, promoter DNA methylation status, and clinicopathological information. CHIP mRNA expression was significantly correlated with the tumor progression status. In several samples, a pinpoint CpG methylation in the CHIP gene promoter region was significantly correlated with CHIP mRNA expression. When this specific CpG was methylated in estrogen receptor (ER)-positive cases, a significant difference in 5-year recurrence was not found compared with ER-negative cases. CpG methylation contributes to the long-term prognosis of ER-positive breast cancer.

Mesenchymal cells arise from the neural crest (NC) or mesoderm. However, it is difficult to distinguish NC-derived cells from mesoderm-derived cells. Using double-transgenic mouse systems encoding P0-Cre, Wnt1-Cre, Mesp1-Cre, and Rosa26EYFP, which enabled us to trace NC-derived or mesoderm-derived cells as YFP-expressing cells, we demonstrated for the first time that both NC-derived (P0- or Wnt1-labeled) and mesoderm-derived (Mesp1-labeled) cells contribute to the development of dental, thymic, and bone marrow (BM) mesenchyme from the fetal stage to the adult stage. Irrespective of the tissues involved, NC-derived and mesoderm-derived cells contributed mainly to perivascular cells and endothelial cells, respectively. Dental and thymic mesenchyme were composed of either NC-derived or mesoderm-derived cells, whereas half of the BM mesenchyme was composed of cells that were not derived from the NC or mesoderm. However, a colony-forming unit-fibroblast (CFU-F) assay indicated that CFU-Fs in the dental pulp, thymus, and BM were composed of NC-derived and mesoderm-derived cells. Secondary CFU-F assays were used to estimate the self-renewal potential, which showed that CFU-Fs in the teeth, thymus, and BM were entirely NC-derived cells, entirely mesoderm-derived cells, and mostly NC-derived cells, respectively. Colony formation was inhibited drastically by the addition of anti-platelet-derived growth factor receptor-β antibody, regardless of the tissue and its origin. Furthermore, dental mesenchyme expressed genes encoding critical hematopoietic factors, such as interleukin-7, stem cell factor, and cysteine-X-cysteine (CXC) chemokine ligand 12, which supports the differentiation of B lymphocytes and osteoclasts. Therefore, the mesenchymal stem cells found in these tissues had different origins, but similar properties in each organ.

In this study, we investigated the relationships of pathological response after neoadjuvant chemo-endocrine therapy with alterations in the Ki67 labeling index (LI), expression of cyclin D1 (CCND1) and progesterone receptor (PgR), and estrogen receptor (ER) activity in breast cancer. A total of 43 Japanese post-menopausal ER-positive and human epidermal growth factor receptor 2-negative invasive breast cancer patients with tumors >2 cm or positive lymph nodes were enrolled. Exemestane alone was administered for 2 months. Neoadjuvant chemo-endocrine therapy included four cycles of doxorubicin plus paclitaxel followed by weekly paclitaxel. The immunohistochemical expression of Ki67 LI, CCND1, and PgR, and ER activity were evaluated using core needle biopsy samples at the pretreatment and post-exemestane alone stages. ER activity was evaluated through transfection of an adenovirus vector carrying an estrogen-response element-green fluorescent protein gene. In current study, marked pathological responses (including 4.7% with pathological complete response) were obtained in 34.9% of patients. Ki67 LI and PgR expression were significantly decreased after treatment. High Ki67 LI at pretreatment was a significant predictive factor of marked pathological response. At the stage of post-exemestane alone, Ki67 LI was not significantly associated with pathological response; however, high CCND1 expression was significantly correlated with high Ki67 LI. Moreover, low-level ER activity at the post-exemestane alone stage was significantly associated with marked pathological response. In conclusions, pretreatment Ki67 LI was a predictor of response to neoadjuvant chemo-endocrine therapy. CCND1 expression and ER activity at the post-endocrine therapy alone stage may be useful in determining further treatments.

Evidence exists that sex steroids such as estrogens affect epithelial ovarian cancer. The expression profiles of the estrogen receptors (ER) and ERβ in particular have not been fully described. Therefore, in our present study, we examined the methylation status of the promoters 0K and 0N, and the expression of ERβ isoforms in human epithelial ovarian carcinoma. We then correlated methylation status with ER expression status. Twelve ovarian carcinoma cell lines, six primary cultures of ovarian surface epithelial cells (OSE), and 64 cases of ovarian carcinoma tissues were examined. Bisulfite sequencing and quantitative reverse transcription–polymerase chain reaction were used to evaluate methylation status and expression of ERβ isoforms. The relative abundance of exon 0N, ERβ1, ERβ2, and ERβ4 mRNA was significantly lower in ovarian cancer cell lines and tissues than in their corresponding normal counterparts. However, ERβ5 mRNA level was relatively higher in the cancers, in clear cell adenocarcinoma in particular, than in the normal ovary. Bisulfite sequencing analysis demonstrated that the two promoters of the ERβ gene exhibited distinct methylation patterns. Promoter 0N was unmethylated in OSE, rarely methylated in normal ovarian tissues, and extensively methylated in ovarian cancer cell lines and tissues (11/15 cell lines and 18/32 cancer tissues were extensively methylated). The promoter 0K was, however, unmethylated in both normal and malignant ovarian cells and tissues. A significant correlation between promoter 0N hypermethylation and the loss of exon 0N, ERβ1, ERβ2, and ERβ4 mRNA expression was detected in ovarian carcinoma cells and tissues. Treatment of ovarian carcinoma cells with 5‐aza‐2′ deoxycytidine resulted in reexpression of the ERβ gene. The results of our present study suggest that ERβ is inactivated mainly through aberrant DNA methylation. This process may play an important role in the pathogenesis of epithelial ovarian cancer. (Cancer Sci 2008; 99: 2365–2372)

Aromatase inhibitors (AIs) effectively treat hormone receptor-positive postmenopausal breast cancer, but some patients do not respond to treatment or experience recurrence. Mechanisms of AI resistance include ligand-independent activation of the estrogen receptor (ER) and signaling via other growth factor receptors; however, these do not account for all forms of resistance. Here we present an alternative mechanism of AI resistance. We ectopically expressed aromatase in MCF-7 cells expressing green fluorescent protein as an index of ER activity. Aromatase-overexpressing MCF-7 cells were cultured in estrogen-depleted medium supplemented with testosterone and the AI, letrozole, to establish letrozole-resistant (LR) cell lines. Compared with parental cells, LR cells had higher mRNA levels of steroid sulfatase (STS), which converts estrone sulfate (E1S) to estrone, and the organic anion transporter peptides (OATPs), which mediate the uptake of E1S into cells. LR cells proliferated more in E1S-supplemented medium than did parental cells, and LR proliferation was effectively inhibited by an STS inhibitor in combination with letrozole and by ER-targeting drugs. Analysis of ER-positive primary breast cancer tissues showed a significant correlation between the increases in the mRNA levels of STS and the OATPs in the LR cell lines, which supports the validity of this AI-resistant model. This is the first study to demonstrate the contribution of STS and OATPs in E1S metabolism to the proliferation of AI-resistant breast cancer cells. We suggest that E1S metabolism represents a new target in AI-resistant breast cancer treatment.

Histone deacetylase (HDAC) 6 is a subtype of the HDAC family; it deacetylates α -tubulin and increases cell motility. Here, we investigate the impact of an alteration of HDAC6 expression in estrogen receptor α (ER)-positive breast cancer MCF-7 cells, as we identified that HDAC6 is a novel estrogen-regulated gene. MCF-7 treated with estradiol showed increased expression of HDAC6 mRNA and protein and a four-fold increase in cell motility in a migration assay. Cell motility was increased to the same degree by stably transfecting the HDAC6 expression vector into MCF-7 cells. In both cases, the cells changed in appearance from their original round shape to an axon-extended shape, like a neuronal cell. This HDAC6 accumulation caused the deacetylation of α -tubulin. Either the selective estrogen receptor modulator tamoxifen (TAM) or the pure antiestrogen ICI 182,780 prevented estradiol-induced HDAC6 accumulation and deacetylation of α -tubulin, leading to reduced cell motility. Tubacin, an inhibitory molecule that binds to the tubulin deacetylation domain of HDAC6, also prevented estradiol-stimulated cell migration. Finally, we evaluated HDAC6 protein expression in 139 consecutively archived human breast cancer tissues by immunohistochemical staining. The prognostic analyses for these patients revealed no significant differences based on HDAC6 expression. However, subset analysis of ER-positive patients who received adjuvant treatment with TAM ( n =67) showed a statistically significant difference in relapse-free survival and overall survival in favor of the HDAC6-positive group ( P <0.02 and P <0.05, respectively). HDAC6 expression was an independent prognostic indicator by multivariate analysis (odds ratio=2.82, P =0.047). These results indicate the biological significance of HDAC6 regulation via estrogen signaling.

Background Even for pediatric patients, the use of laparoscopic appendectomy has been widely accepted, and three trocars usually are necessary to perform a laparoscopic appendectomy. However, single-port appendectomy for children represents an attractive alternative. To reduce the number of incisions and trocars, the authors have adopted a transumbilical laparoscopically assisted single-port appendectomy (TULAA) approach. This study aimed to evaluate the results of their single-channel, single-port appendectomy. Methods A retrospective study of TULAA was performed during 12 years with 500 children ages 2–16 years (median, 10.2 years). The TULAA approach is a single-channel surgery using a 12 mm conventional single-port. The vertical incision through the umbilicus is used for laparoscopic access. Two laparoscopic instruments, a 5 mm telescope and a 5 mm grasper, are inserted simultaneously into the single-channel. The grasper holds the base of the appendix, and the appendix is exteriorized through the umbilical incision. Thereafter, a conventional appendectomy is performed extracorporeally. Results The TULAA procedure was successful for 416 patients (83.2%). These successful TULAA procedures required a mean surgery time of 44.5 min. The pathologic diagnosis of the appendix was acute for 59 patients, phlegmonous for 203 patients, gangrenous for 152 patients, and not detected for two patients. Complications occurred for 47 of these patients (11.3%). Most of the complications were associated with severe intraabdominal inflammation. Two patients needed reoperation under general anesthesia. Conversion to multitrocar surgery or open appendectomy was performed for 84 of the patients (16.8%). Conclusions The TULAA procedure is a preferable operation for acute appendicitis in children because it is simple and provides good cosmetic results.

Estrogens play an important role in the pathobiology of breast cancer. In postmenopausal women, peripheral synthesis of estrogens from adrenal/ovarian androgens, dehydroepiandrosterone (DHEA) or androstenedione (Adione), by estrogen‐metabolizing enzymes is important. Besides estrone (E1) and estradiol (E2), androgen metabolites, such as androstene‐3β, 17β‐diol (Aenediol) or 5α‐androstane‐3β, 17β‐diol (Aanediol), are known to have estrogenic functions, although they have been studied much less in breast cancer. To precisely elucidate steroid metabolism in breast cancer patients and to identify the pathobiological role of estrogenic androgen metabolites, concentrations of DHEA, Adione, Aenediol, Aanediol, E1, and E2 in pairs of serum and tumor tissue from patients with primary breast cancer were measured by liquid chromatography‐tandem mass spectrometry. Cell proliferation assays using Aenediol were performed for four breast cancer cell lines. Serous E2 concentration was extremely low in postmenopausal women; however, a marked increase in tumor tissue was observed in hormone receptor‐positive cases. E1 concentration, in contrast, was sustained at a higher level, even in postmenopausal serum, and did not increase in tumor tissue irrespective of the hormone receptor status. Dehydroepiandrosterone was most abundant in all samples, and exhibited a similar pattern as Adione and Aenediol. 5α‐Androstane‐3β, 17β‐diol was undetectable in most samples. Androstene‐3β, 17β‐diol proliferated estrogen receptor‐α‐positive breast cancer cells in the absence of E2. The intratumoral increase of E2, but not E1, in hormone receptor‐positive postmenopausal breast cancer tissue, as well as the proliferative role of Aenediol, was elucidated. (Cancer Sci 2011; 102: 1848–1854)