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Background: The serum level of Krebs von den Lungen-6 (sKL-6) is a biomarker of interstitial pneumonia and has been reported to be elevated in patients with cancers. However, there have been few cases of gastric cancer (GC) with elevated sKL-6 that were treated by chemotherapy. We herein report two cases of GC with elevated sKL-6 that were treated with oxaliplatin plus S-1 (SOX) chemotherapy and discussed the resulting changes in sKL-6., Case Presentation: The first patient was a 79-year-old woman complaining of loss of appetite. Esophagogastroduodenoscopy (EGD) showed a type-3 tumor in the gastric antrum and biopsy specimens showed adenocarcinoma. Computed tomography (CT) showed multiple liver metastases. sKL-6 was elevated to 1,292 U/ml, but a CT revealed no obvious lesions of the lungs, including interstitial pneumonia. The tumor was diagnosed as GC with liver metastases and elevated sKL-6. Respiratory function data were normal. SOX therapy using oxaliplatin and S-1 was performed. After 3 courses of SOX therapy, CT showed reductions of the liver metastases as well as the primary tumor, and sKL-6 was decreased to 201 U/ml. After the 44 courses, sKL-6 was slightly elevated. Chest CT showed interstitial pneumonia and chemotherapy was stopped. The patient is still alive without any metastasis 72 months later. The second patient was a 69-year-old woman complaining of upper abdominal pain. EGD revealed a type-3 tumor in the gastric antrum showing adenocarcinoma with HER2-positive pathology. CT showed multiple node metastases around the abdominal aorta. sKL-6 was elevated to 2,239 U/ml, but a respiratory function test showed no abnormalities, and CT of the lungs showed no obvious lesions. The tumor was diagnosed as GC with distant node metastases and elevated sKL-6. The patient received SOX therapy combined with trastuzumab. After 6 courses, the size of the primary tumor and multiple node metastases were reduced, and sKL-6 was decreased to 284 U/ml., Conclusions: These two cases suggest that sKL-6 may be important not only as an indicator of interstitial pneumonia in chemotherapeutic courses, but also as a tumor marker in GC patients with multiple metastases., (© 2024. The Author(s).)

A 64-year-old woman presented with a fist-sized, severely painful lesion with scales, crusts, pustules, erythema with subcutaneous abscess, and hair loss on the left temporal region. Direct microscopic examination revealed a large number of spores around the hair, which indicated ectothrix hair invasion, and some hyphae were also found. Histopathological examination showed significant inflammatory cell infiltration from the dermis to the subcutaneous tissues and into the hair follicles, destruction of the hair follicles with granulomatous reactions, and fungal masses along the hair within the hair follicles. Microsporum canis was identified based on morphological features via culture method and molecular biological analysis of the internal transcribed spacer region DNA sequence. The patient was diagnosed with kerion celsi caused by M. canis. For treatment of kerion celsi, we chose an oral antifungal agent, fosravuconazole (FRVCZ), which has been available since 2018 only in Japan. Clinical symptoms were cured in 12 weeks without scarring. No side effects were observed during oral administration of FRVCZ. The results of our case and several previous reports suggest that FRVCZ is effective in treating various types of dermatomycoses.

Background: Many types of tumors can arise in the esophagogastric junction (EGJ). Squamous cell carcinoma (SCC) arising from the esophageal epithelia, adenocarcinoma arising from the gastric mucosa, or Barrett's esophageal mucosa are frequently observed in the EGJ. However, adenosquamous carcinoma (ASC) has been rarely observed in this area. We herein report a rare case of ASC of the EGJ., Case Presentation: An 81-year-old man visited our hospital complaining of dysphagia. Esophagogastroduodenoscopy detected an elevated tumor in the gastric cardia. Biopsy specimens taken from the tumor showed SCC. Computed tomography revealed a tumor located in the EGJ and node metastases surrounding the EGJ. The tumor was diagnosed as SCC, overhanging in the stomach, of the EGJ. The patient underwent a proximal gastrectomy with a lower esophagectomy and node dissection for the metastases surrounding the EGJ, and esophagogastrostomy in the lower mediastinum. Histopathologic examination showed the tumor consisted of SCC and adenocarcinoma. The adenocarcinoma consisted of nests scattered in the SCC. We observed adenocarcinoma component in 35% of the tumor and epithelial spread of SCC in the lower esophagus. Thus, we diagnosed the tumor as ASC of the EGJ. Eight metastatic nodes were dissected; both SCC and adenocarcinoma were observed in seven., Conclusions: In the present case, SCC may be originated from the squamous epithelia of the lower esophagus and grew into the stomach, and the adenocarcinoma may have differentiated from SCC through the infiltration., (© 2022. The Author(s).)

The patient was a 61-year-old woman who presented to the hospital with the chief complaints of anemia and thrombocytopenia. There was a mass in her left breast, and a needle biopsy with pathology revealed invasive ductal carcinoma, which was HR-positive and HER2-negative. A PET scan revealed multiple bone metastases, which were confirmed on bone marrow biopsy, leading to the diagnosis of bone marrow carcinomatosis. As the patient was in good general condition, an aromatase inhibitor(AI)therapy was selected. Rapid improvements in her hemoglobin level and platelet count were observed. At 19 months after the start of treatment, we were able to perform a left mastectomy with left axillary lymph node dissection. The histological evaluation of her response to treatment was Grade 2a, and severe lymph node metastasis was observed. The patient continued to receive the AI postoperatively. Thirty-two months after the start of treatment, there was no evidence of cancer on clinical imaging. Although it is rare for bone marrow carcinomatosis to occur, as in the present case, it is also notable that the patient had been in long-term remission with consistent AI therapy.

Onychomycosis with longitudinal spikes in the nail plate has been reported to be refractory to oral drugs as with dermatophytoma. We evaluated the efficacy of 10% efinaconazole solution in the treatment of onychomycosis with longitudinal spikes. Of the 223 subjects who were enrolled in a previous study, a post-hoc analysis of 82 subjects with longitudinal spikes was performed in this study. The opacity ratio of longitudinal spikes was decreased over time from 8.1 to 0.9 at the final assessment. In addition, the longitudinal spike disappearance rate increased early after the application to 81.7% at the final assessment. Therefore, 10% efinaconazole solution can be a first-line drug for longitudinal spikes, which have been regarded as refractory to oral drugs., (© 2021 The Authors. The Journal of Dermatology published by John Wiley & Sons Australia, Ltd on behalf of Japanese Dermatological Association.)

Fosravuconazole L-lysine ethanolate (F-RVCZ), a ravuconazole prodrug, is a newly available agent with high expectations for efficacy in the treatment of onychomycosis. However, clinical data regarding the efficacy of F-RVCZ are limited because the drug was launched only in Japan in 2018. Therefore, we analyzed the outcome of F-RVCZ therapy in the treatment of onychomycosis at outpatient dermatology clinics in Japan. We examined data for 109 patients (68 male, 41 female) with varying clinical type, including total dystrophic onychomycosis and dermatophytoma, and a wide range of age groups, including the elderly. The complete cure rate at 12 weeks was 6.4% (7/109) and 67.9% (74/109) at the last visit (mean time to last visit: 32 ± 14.2 weeks). Mean rate of improvement in the affected nail area was 49.1 ± 23.3% at 12 weeks and 86.8 ± 22.4% at the last visit. Efficacy at 12 weeks and the last visit, respectively, was as follows: none, 4 cases and 1 case; slight, 35 cases and 4 cases; moderate, 51 cases and 21 cases; significant, 12 cases and 9 cases; complete cure, 7 cases and 74 cases. There were no serious adverse events. This retrospective survey was the first large-scale analysis of actual clinical practice outcomes and had minimal exclusions. Compared to previous reports, our results demonstrated excellent efficacy of F-RVCZ therapy in a variety of patients. Considering our results and the ease of oral administration (1 capsule/day for 12 weeks) and few adverse events, F-RVCZ therapy appears to be a useful option for the treatment of onychomycosis.

The "Guidelines for the management of dermatomycosis" of the Japanese Dermatological Association were first published in Japanese in 2009 and the Guidelines Committee of the Japanese Dermatological Association revised it in 2019. The first guidelines was prepared according to the opinions of the Guidelines Committee members and it was of educational value. The revised version is composed of introductory descriptions of the disease concepts, diagnosis, medical mycology and recent advances in treatment, along with clinical questions (CQ), which is intended to help in general practice for dermatologists. The CQ are limited to those involved in therapy but include some of the recently launched antifungal agents. The level of evidence and the degree of recommendation for each item were reviewed by the committee based on clinical studies published by 2018. For rare dermatomycoses, recommendations by the committee are described in the guidelines. In this field, there are still few good quality studies on treatment. Periodic revision in line with new evidence is necessary., (© 2020 Japanese Dermatological Association.)

Nannizzia gypsea is a geophilic dermatophyte, previously known as Microsporum gypseum before renaming under the new taxonomy. This organism is distributed all over the world and is considered to be involved in keratin degradation in the soil. Generally, human infection involves direct contact with fertile soil. Tinea caused by geophilic dermatophytes is much rarer than that caused by anthropophilic dermatophytes. According to the latest survey in Japan, dermatophytosis due to N. gypsea accounted for only 0.4% of cases. Clinical presentations vary and may mimic other inflammatory dermatitis, leading to incorrect diagnosis and delayed treatment. According to that past report, distal parts of the upper and lower extremities were more commonly affected, followed by the trunk, face and scalp, and rarely the nail plate. A 38-year-old woman presented with an approximately 3-week history of an itchy, solitary erythematous lesion on the left medial angle of the eyelid. Direct microscopic examination of scales revealed fungal elements, and the causative agents was identified as N. gypsea by morphological and molecular biological diagnoses. The eruption improved with systemic itraconazole treatment at 100 mg/day for 8 weeks. No recurrence has been seen for a year. However, she had no history of contact with any infectious source. Herein, we report a case of tinea faciei due to N. gypsea with an uncommon site and route of infection.

In Japan, an epidemiological survey of onychomycosis pathogens was performed using culture methods; however, the positive culture rate was 40% or less. As part of an epidemiological survey of dermatomycoses in Japan, we overcame this low positive rate by employing a real-time polymerase chain reaction (PCR) assay that allowed rapid and accurate detection and identification. In 2011, nail specimens were collected from patients at nine institutes in various prefectures in Japan and diagnosed as onychomycosis. For the detection and identification of the main pathogens causing onychomycosis, we performed real-time PCR using specific TaqMan® MGB probes and primer sets. Of the 496 onychomycosis samples, real-time PCR detected 382 cases (77.0%) caused by Trichophyton rubrum; 74 cases (15.0%) caused by Trichophyton interdigitale; and eight cases (1.6%) caused by Candida albicans. The real-time PCR positive rate was 96.2%. The most frequent pathogen was T. rubrum throughout life, with the number of patients affected peaking in the range of 60 to 69 years of age and no significant differences in the composition of causative pathogens by sex. We were able to detect and identify pathogens from almost all specimens and succeeded in analyzing the pathogens involved in onychomycosis cases in Japan. These data confirmed that our real-time PCR method was effective for detecting and identifying the main fungal pathogens from onychomycosis specimens., (© The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

We evaluated the efficacy of efinaconazole 10% topical solution in long-term use, for up to 72 weeks, for onychomycosis, including severe cases. Among 605 participants, 219 patients diagnosed as having onychomycosis were evaluated for the efficacy of efinaconazole. The treatment success rate (<10% clinical involvement of the target toenail) at the final assessment time point was 56.6%, the complete cure rate was 31.1% and the mycological cure rate was 61.6%, all of which increased over time, demonstrating that continuous application contributed to the improvement of cure rate. Even in severe cases, reduction of the affected nail area was observed, showing the potential efficacy of the treatment. Responses to a quality of life questionnaire among patients with onychomycosis, OnyCOE-t, suggested that efinaconazole treatment improved the patients' quality of life. The incidence of adverse drug reaction in the patients eligible for the assessment was 6.3%, and this developed only in the administration site in all cases. No systemic adverse event was observed. In addition, no increase in the incidence of adverse drug reaction due to long-term use was found. Efinaconazole therapy was proved to exhibit excellent balance between efficacy and safety, and thus may serve as a useful treatment option for onychomycosis., (© 2019 The Authors. The Journal of Dermatology published by John Wiley & Sons Australia, Ltd on behalf of Japanese Dermatological Association.)

Many clinicians prefer to treat onychomycosis systemically. However, systemic therapy may not be suitable for all onychomycosis patients due to drug interactions, side effects of oral medications, or comorbidities. Two topical agents (efinaconazole 10% in 2014 and luliconazole 5% in 2016) have recently been approved for treatment of onychomycosis in Japan. We investigated the efficacy of these topical agents at Teikyo University Mizonokuchi Hospital, Kanagawa, Japan. We conducted a retrospective survey among patients diagnosed with onychomycosis at our outpatient clinic and had been treated with either efinaconazole 10% solution or luliconazole 5% solution. Prior to commencement of treatment, the disease severity was evaluated using the Scoring Clinical Index for Onychomycosis (SCIO). Furthermore, the efficacies of these agents were evaluated using turbidity scoring at each visit to our outpatient clinic. Sixty-two patients (33 men, 29 women) applied efinaconazole 10% solution, and 72 patients (35 men, 37 women) applied luliconazole 5% solution. The mean SCIO scores were 18.1 and 17.4, respectively, and the mean 5-grade evaluation scores were 3.5 and 3.4, respectively. Complete cure rates were 40.3% (25/62) and 33.3% (24/72), respectively. The mean durations of treatment were 15.4 months and 11.9 months, respectively. There were no serious side effects in either treatment group. There were no significant differences between the two agents in improvement scores as assessed by the Tukey's test. Thus, efinaconazole 10% and luliconazole 5% topical solutions were effective for the treatment of onychomycosis. These topical agents may become important treatment options for this indication.

We report here the results of the 2016 epidemiological survey of dermatomycosis in Japan. In total, 6,776 cases were analyzed as follows: dermatophytosis, 5,772 cases (85.2%); candidiasis, 757 cases (11.2%); Malassezia infection, 235 cases (3.5%); and other fungal infections, 11 cases (0.2%). In dermatophytosis, tinea pedis was the most frequent (3,314 cases: male, 1,705; female, 1,609), followed by tinea unguium (1,634 cases: male, 766; female, 868), tinea corporis (423 cases: male, 241; female, 182); tinea cruris (316 cases: male, 242; female, 74); tinea manuum (58 cases: male, 29; female, 29); tinea capitus, Celsus' kerion (26 cases: male, 19; female, 7); and tinea barbae (1 case: male, 1). The most frequent pathogen was Trichophyton rubrum. In candidiasis, candidal intertrigo was the most frequent (181 cases: male, 98; female, 83), followed by oral candidiasis (165 cases: male, 84; female, 81), genital candidiasis (119 cases: male, 45; female, 74), diaper candidiasis (113 cases: male, 49; female, 64), erosio interdigitalis (63 cases: male, 13; female, 50), onychomycosis (41 cases: male, 17; female, 24), onychia et paronychia (28 cases: male, 2; female, 26), and angular cheilitis (23 cases: male, 6; female, 17). Although the number of cases varied depending on the role of each cooperating medical institution in the area and on population composition, no significant differences in the frequencies of clinical types were observed.

A 41-year-old man visited our dermatology clinic because an eruption, which was resistant to steroid ointment treatment, had appeared on his right forearm. An oval, soybean-sized erythematous infiltrated lesion with scales and crusts was located in the central part of the extensor surface of the right forearm and showed partial erosion with attached yellow crusts. The lesion had an impetigo-like appearance. Fungal elements were confirmed from the scales by KOH examination and the fungus was identified as Trichophyton tonsurans by fungal culture and molecular method. Clinical features of T. tonsurans infection vary, wherein some patients have strong inflammatory manifestations, while others remain as asymptomatic carriers. Especially at the early stage of the infection, diagnosis is difficult because it is often misdiagnosed as eczema. We report a case of T. tonsurans infection that had impetigo-like appearance. We also studied the mechanism of the disease.

Topical or systemic antifungal therapy was administered to patients diagnosed with Malassezia folliculitis during the 5-year period between March 2007 and October 2013. The diagnosis of Malassezia folliculitis was established on the basis of characteristic clinical features and direct microscopic findings (10 or more yeast-like fungi per follicle). Treatment consisted of topical application of 2% ketoconazole cream or 100 mg oral itraconazole based on symptom severity and patients' preferences. Treatment was given until papules flattened, and flat papules were examined to determine whether the patient's clinical condition had "improved" and the treatment had been "effective". The subjects were 44 patients (35 men, 9 women), with a mean disease period of 25±15 days. In regard to the lesion site, the frontal portion of the chest was the most common, accounting for 60% of all patients. The mean period required for improvement was 27±16 days in 37 patients receiving the topical antifungal agent and 14±4 days in the 7 patients receiving the systemic antifungal agent. The results were "improved" and the treatment was "effective" in all patients. Neither treatment resulted in any adverse reactions. Although administration of oral agents has been recommended for the treatment of Malassezia folliculitis, this study revealed that beneficial results are safely obtained with topical antifungal therapy alone, similar to those of systemic antifungal agents.

Poorly-differentiated colorectal cancers (PD-CRC) show high metastatic potential and poor prognosis. However, molecular characteristics of PD-CRC remain unknown to date, particularly in molecular targeting therapy for patients with PD-CRC. In this study, we examined the expression of EGFR, HER2 and HER3 in PD-CRC by immunohistochemical analysis of archived clinical specimens of primary tumors and investigated the sensitivity of PD-CRC cell lines to gefitinib, a tyrosine kinase inhibitor for EGFR in vitro. We found that HER3 expression of PD-CRC among members of the HER family was significantly lower than that of well to moderately differentiated CRC (WMD-CRC) and 37% of the PD cases showed a EGFR+/HER2+/HER3- expression pattern. COLM-5 cells, a PD-CRC-derived cell line, which exhibits EGFR+/HER2+/HER3- expression pattern and recapitulates the typical histology of PD-CRC in xenografted tumors, showed high gefitinib sensitivity both in vitro and in vivo, compared with WMD-CRC cell line (COLM-2). Treatment with gefitinib resulted in the upregulation of p27Kip1 expression and induction of G1 cell cycle arrest, concomitantly associated with inactivation of PI3K/Akt signaling in COLM-5 cells and marked inhibition of xenografted tumors in nude mice, but not evident in COLM-2 cells. Treatment with sodium butyrate, an HDAC inhibitor that induces differentiation, upregulated the expression of HER3 associated with enhancement of the PI3K/Akt signaling, attenuated gefitinib-mediated p27Kip1 upregulation and reduced gefitinib sensitivity in COLM-5 cells in vitro. Furthermore, enforced expression of HER3 in COLM-5 cells resulted in significant resistance to gefitinib treatment both in vitro and in vivo. These findings suggest that deficient HER3 expression plays an important role in gefitinib sensitivity and that a malignant subset of PD with EGFR+/HER2+/HER3- phenotype is a potential candidate for a target of anti-EGFR molecular therapy such as gefitinib.

Background: Trastuzumab (Tmab) resistance is a major clinical problem to be resolved in patients with HER2-positive gastric cancers. However, in contrast to the situation for HER2-positive breast cancer lines, the Tmab-resistant gastric cancer preclinical models that are needed to develop a new therapy to overcome this problem are not yet available., Methods: We developed three new cell lines from HER2 gene-amplified gastric cancer cell lines (GLM-1, GLM-4, NCI N-87) by a new in vivo selection method consisting of the repeated culture of small residual peritoneal metastasis but not subcutaneous tumor after Tmab treatment. We then evaluated the anti-tumor efficacy of lapatinib for these Tmab-resistant cells., Results: We successfully isolated two Tmab-resistant cell lines (GLM1-HerR2(3), GLM4-HerR2) among the three tested cell lines. These resistant cells differed from the parental cells in their flat morphology and rapid growth in vitro, but HER2, P95HER2 expression, and Tmab binding were essentially the same for the parental and resistant cells. MUC4 expression was up- or downregulated depending on the cell line. These resistant cells were still sensitive to lapatinib, similar to the parental cells, in vitro. This growth inhibition of the Tmab-resistant cells by lapatinib was due to both G1 cell-cycle arrest and apoptosis induction via effective blockade of the PI3K/Akt and MAPK pathways. A preclinical study confirmed that the Tmab-resistant tumors are significantly susceptible to lapatinib., Conclusion: These results suggest that lapatinib has antitumor activity against the Tmab-resistant gastric cancer cell lines, and that these cell lines are useful for understanding the mechanism of Tmab resistance and for developing a new molecular therapy for Tmab-resistant HER2-positive gastric cancers.

Background: Intraperitoneal (i.p.) chemotherapy with paclitaxel is a potential therapeutic modality for patients with peritoneal metastasis of gastric cancer. To overcome paclitaxel resistance, which is a major clinical problem with this modality, prediction of i.p. paclitaxel resistance is critically important., Materials and Methods: We developed three new i.p. paclitaxel-resistant cell lines from parental gastric cancer cell lines by an in vivo selection method using i.p. paclitaxel chemotherapy. With these cell lines, we performed gene expression profiling analysis to select up-regulated genes in i.p. paclitaxel-resistant cells and validated the genes with clinical samples., Results: We successfully isolated nine up-regulated genes in i.p. paclitaxel-resistant cell lines compared with parental cells by microarray analysis, followed by confirmation with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Among these, we identified four genes, namely kinesin family member-23 (KIF23), ERBB2 interacting protein (ERBB2IP), ATPase family, AAA domain containing-2 (ATAD2) and PHD finger protein (PHF19) as candidate genes for paclitaxel resistance after validation with clinical samples derived from responders and non-responders to paclitaxel treatment., Conclusion: These i.p. paclitaxel-resistant cell lines are ideal models for understanding the mechanism of resistance to i.p. paclitaxel and development of a new therapeutic modality. Four up-regulated genes may be potential new predictive markers for resistance to i.p. paclitaxel in patients with peritoneal metastasis of gastric cancer.

Background: Poorly differentiated signet-ring cell carcinoma (SRCC) and non signet-ring cell carcinoma (NSRCC) are prevalent histological subtypes of gastric cancers with distinct morphological features. To date, however, the molecular basis of their growth, differentiation, and metastasis still remains unclear, because of the limitation of available cell lines., Methods: In the present study, we established novel SRCC and NSRCC cell lines (designated GPM-2 and GPM-1) derived from the ascites of two individual gastric cancer patients with peritoneal metastasis., Results: Immunohistochemical and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that GPM-2 cells showed both gastric and intestinal differentiation phenotypes (E-cadherin+/MUC5AC+/MUC6+/Villin+), and formed xenografted tumors with typical SRCC histology in nude mice. In contrast, GPM-1 cells only weakly expressed differentiation markers, showing a phenotype of E-cadherin(low)+/MUC2-/MUC5AC-/Villin(low)+. Characteristically, GPM-2 cells were found to highly express both membrane-bound mucin (MUC1/MUC4) and secreted mucin glycoproteins (MUC5AC/MUC6), whose expression is regulated by an epigenetic mechanism such as histone acetylation. GPM-2 cells also secreted a large amount of sTn antigen into the culture medium. These mucin profiles of GPM-2 cells are distinct from those of conventional SRCC cell lines (KATO III and HSC-39), which preferentially express intestinal MUC2/MUC4 as well as sLe(x) and sLe(A) antigens. In addition, GPM-2 cells showed a slow growth rate, and a lower metastatic potential than GPM-1 cells., Conclusions: These results indicate that the cells of the new SRCC line, GPM-2 cells, are more differentiated and less aggressive than NSRCC-type GPM-1 cells, and would thus offer an excellent model for understanding the molecular mechanism underlying the growth, differentiation, and mucin production of an SRCC gastric cancer cell line.

Objectives: Podoplanin, a transmembrane sialomucin-like glycoprotein, is known to express at high frequency in oral squamous cell carcinomas (OSCC) and possess metastasis-promoting activity such as increased invasion and platelet-aggregating activity. However, the regulatory mechanism of podoplanin expression in OSCC remains unknown., Materials and Methods: In the present study, we investigated the podoplanin expression in both clinical specimens from total 80 patients (50 OSCC and 30 pharyngeal SCC) and in 4 OSCC cell lines in vitro., Results: Immunohistochemical analysis of surgically resected specimens of OSCC revealed podoplanin expression in 70% of OSCC cases with localization primarily in the basal layer of squamous cancer nest and the expression was inversely correlated with squamous cell differentiation. In vitro analysis of OSCC cell lines revealed 36 that podoplanin expression was decreased in response to the squamous cell differentiation (Cytokeratin 10 expression as a marker) induced by treatment with histone deacetylase (HDAC) inhibitors such as sodium butyrate and trichostatin. Furthermore, transforming growth factor-β (TGF-β) significantly enhanced podoplanin expression in OSCC cell lines in line with increased phosphorylation of Smad2. A TGF-β type I receptor inhibitor (SB431542) significantly inhibited such induction of podoplanin expression by TGF-β at both the protein and mRNA level. However, in a subset of OSCC cell line, its expression was only weakly dependent on TGF-β and squamous differentiation., Conclusion: These results suggest that regulation of podoplanin is not simple, but in the majority of OSCC cell lines, its expression is positively and negatively regulated by TGF-β receptor/Smad signaling pathway and epigenetic mechanism leading to squamous differentiation, respectively., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Altered patterns of DNA methylation associated with Helicobacter pylori (HP) infection of gastric epithelial cells are thought to contribute to gastric cancer risk. However, it is unclear whether this increased risk reflects an infection-associated inflammatory response or the infection itself. In this study, we sought to clarify mechanisms in a gerbil model of gastric cancer where we showed that HP infection is causally involved in induction of aberrant DNA methylation. By genome-wide screening, CpG islands that were aberrantly methylated in gerbil gastric cancer cell lines were isolated, and 10 islands were shown to be specifically methylated only in gastric mucosae infected with HP. By temporal analysis, methylation levels in gastric epithelial cells started to increase at 5 to 10 weeks after infection and reached high levels by 50 weeks. When HP was eradicated, methylation levels markedly decreased 10 and 20 weeks later, but they remained higher than those in gerbils that were not infected by HP. Expression levels of several inflammation-related genes (CXCL2, IL-1beta, NOS2, and TNF-alpha) paralleled the temporal changes of methylation levels. Significantly suppressing inflammation with the immunosuppressive drug cyclosporin A did not affect colonization by HP but blocked the induction of altered DNA methylation. Our findings argue that DNA methylation alterations that occur in gastric mucosae after HP infection are composed of transient components and permanent components, and that it is the infection-associated inflammatory response, rather than HP itself, which is responsible for inducing the altered DNA methylation.

Oxidative stress is linked to gastric carcinogenesis because of its ability to damage DNA. Here we examined antioxidative and anti-inflammatory effects of 4-vinyl-2,6-dimethoxyphenol (canolol), a recently identified potent antioxidative compound obtained from crude canola oil, on Helicobacter (H.) pylori-induced gastritis and gastric carcinogenesis using a Mongolian gerbil model. The animals were allocated to H. pylori-infection alone (12 weeks) or H.pylori + N-methyl-N-nitrosourea (MNU) administration (52 weeks). After oral inoculation of H. pylori, they were fed for 10 and 44 weeks with or without 0.1% canolol. H. pylori-induced gastritis, 5'-bromo-2'-deoxyuridine (BrdU) labeling and scores for cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) immunohistochemistry were attenuated in the canolol-treated groups. Expression of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), COX-2 and iNOS mRNA in the gastric mucosa, and serum 8-hydroxy-2'-deoxyguanosine (8-OHdG), anti-H. pylori IgG and gastrin levels were also significantly lower in canolol-treated groups. Furthermore, the incidence of gastric adenocarcinomas was markedly reduced in the H. pylori + MNU + canolol-treated group [15.0% (6/40)] compared to the control group [39.4% (13/33)] (p < 0.05). These data indicate canolol to be effective for suppressing inflammation, gastric epithelial cell proliferation and gastric carcinogenesis in H. pylori-infected Mongolian gerbils. Interestingly, the viable H. pylori count was not changed by the canolol containing diet. Thus, the data point to the level of inflammation because of H. pylori rather than the existence of the bacteria as the determining factor. Importantly, canolol appears to suppress induction of mRNAs for inflammatory cytokines., ((c) 2007 Wiley-Liss, Inc.)

Spasmolytic polypeptide (TFF2)-expressing metaplasia (SPEM) is observed in mucosa adjacent to human gastric cancer and in fundic glands showing oxyntic atrophy in Helicobacter felis-infected mice. Mongolian gerbils infected with Helicobacter pylori (Hp) develop goblet cell intestinal metaplasia and adenocarcinoma, but the presence of SPEM has not been studied in gerbils. We therefore have sought to examine the development of metaplastic mucosal changes in Hp-infected Mongolian gerbils. Mongolian gerbils were assigned to either uninfected controls or infected with Hp at 17 weeks of age. The animals were killed at 17, 20, 26, 31, 41 and 56 weeks of age. Stomach sections were stained using antibodies for TFF2, intrinsic factor, H/K-ATPase, BrdU and MUC2. Dual immunofluorescence staining for TFF2 with intrinsic factor and for TFF2 with MUC2 was performed. In uninfected animals, no SPEM or intestinal metaplasia was observed. Infected gerbils developed SPEM initially in the intermediate zone along the lesser curvature and subsequently spread out towards the greater curvature. In the earlier stages of infection, SPEM glands demonstrated TFF2 and intrinsic factor double staining cells. However, after 35 weeks of infection, the number of double staining SPEM cells decreased. While early in infection SPEM organized in straight glands, in the later stages of infections, SPEM glands became distorted or dilated along with the development of gastritis cystica profunda that was TFF2 positive. Goblet cell intestinal metaplasia developed only late in the infection. Dual staining for TFF2 and MUC2 showed glands containing both SPEM- and MUC2-positive goblet cell intestinal metaplasia. SPEM develops early in Hp infection in Mongolian gerbils, and alterations in gland morphology arise from SPEM glands during the course of gastric infection with goblet cell intestinal metaplasia developing subsequent to SPEM.

Recent epidemiological studies have demonstrated that consumption of certain natural products can lower cancer risk in humans. For example, plant-derived lignans have been shown to exert chemopreventive effects against cancer in vitro and in vivo. In the present study, the effects of three such lignans, termed arctiin, arctigenin, and nordihydroguaiaretic acid (NDGA), on the proliferation of Helicobacter pylori and the prevention of H. pylori-associated gastric cancer were investigated in Mongolian gerbils. To examine the effects of arctigenin and NDGA on stomach carcinogenesis, specific pathogen-free male, 5-week-old gerbils were infected with H. pylori, administered 10 p.p.m. N-methyl-N-nitrosourea in their drinking water and fed diets containing various concentrations of lignans until they were killed after 52 weeks. At a dietary level of 0.25%, NDGA significantly decreased the incidence of gastric adenocarcinomas. Arctigenin, in contrast, failed to attenuate neoplasia at a level of 0.1%. Both NDGA and arctigenin significantly reduced serum 8-hydroxy-2'-deoxyguanosine levels at doses of 0.25 and 0.05% (NDGA), and 0.1% (arctigenin). Administration of 0.25% NDGA significantly suppressed the formation of intestinal metaplasia both in the antrum and the corpus. Although all three lignans dose-dependently inhibited the in vitro proliferation of H. pylori, there were no differences in the titers of anti-H. pylori antibodies or the amount of the H. pylori-specific urease A gene among all H. pylori-infected groups. These results suggest that NDGA might be effective for prevention of gastric carcinogenesis. The possible mechanisms appear to be related to inhibitory effects on progression of gastritis and antioxidative activity rather than direct antimicrobial influence.

Helicobacter pylori (H. pylori) infection causes chronic gastritis and is also related to gastric carcinoma. The present study focused on severity of H. pylori-induced gastritis as a determinant of carcinogenesis. Seven-week-old male Mongolian gerbils were inoculated with H. pylori at experimental weeks 0, 12, or 18, then given N-methyl-N-nitorosourea (MNU) from weeks 20-40. At week 70, stomachs were then excised for histological examination 70, 58, or 52 weeks after H. pylori inoculation, respectively (Groups A, B, and C for long-, middle-, and short-term). The respective incidences of glandular stomach adenocarcinomas were 65.0% (13/20), 20.0% (2/10), and 23.0% (3/13) (P<0.05). Higher scores of infiltration of inflammatory cells, hyperplasia, intestinal metaplasia and mucosal bromodeoxyuridine (BrdU) labeling index in antrum and corpus mucosa, were seen in group A than B or C (P<0.05) and serum anti-H. pylori IgG titer and gastrin levels were also significantly higher, along with mRNA levels for mucosal interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). The results demonstrated the term and severity of H. pylori infection to play important roles in gastric carcinogenesis, with essential involvement of chronic inflammation, especially increased rates of cell proliferation, in H. pylori-associated carcinogenesis.

Intake of salt and salty food is known as a risk factor for gastric carcinogenesis. To examine the dose-dependence and the mechanisms underlying enhancing effects, Mongolian gerbils were treated with N-methyl-N-nitrosourea (MNU), Helicobacter pylori and food containing various concentrations of salt, and were sacrificed after 50 weeks. Among gerbils treated with MNU and H. pylori, the incidences of glandular stomach cancers were 15% in the normal diet group and 33%, 36% and 63% in the 2.5%, 5% and 10% NaCl diet groups, showing dose-dependent increase (p < 0.01). Intermittent intragastric injection of saturated NaCl solution, in contrast, did not promote gastric carcinogenesis. In gerbils infected with H. pylori, a high salt diet was associated with elevation of anti-H. pylori antibody titers, serum gastrin levels and inflammatory cell infiltration in a dose-dependent fashion. Ten percent NaCl diet upregulated the amount of surface mucous cell mucin (p < 0.05), suitable for H. pylori colonization, despite no increment of MUC5AC mRNA, while H. pylori infection itself had an opposing effect, stimulating transcription of MUC6 and increasing the amount of gland mucous cell mucin (GMCM). High salt diet, in turn, decreased the amount of GMCM, which acts against H. pylori infection. In conclusion, the present study demonstrated dose-dependent enhancing effects of salt in gastric chemical carcinogenesis in H. pylori-infected Mongolian gerbils associated with alteration of the mucous microenvironment. Reduction of salt intake could thus be one of the most important chemopreventive methods for human gastric carcinogenesis., (Copyright 2006 Wiley-Liss, Inc.)

A methanol extract from unripe Japanese apricot showed inhibitory activity of Helicobacter pylori motility. Inhibitory compound 1 was isolated and identified as (+)-syringaresinol (1) by spectoroscopic means. (+)-Syringaresinol (1) inhibited >90% of the H. pylori motility at a concentration of 500 microg/ml and the IC50 value was 50 microg/ml.

The incomplete intestinal metaplasia (IM) that is reported to be a risk factor for gastric carcinogenesis in man usually features sulfomucin production and thus is considered of colonic type. To cast light on the underlying mechanisms, we here examined the proportions of colonic and small intestinal phenotypes in IM by immunohistochemistry and real-time reverse transcription-polymerase chain reaction at the single isolated gland level. Carbonic anhydrase 1 (CA1) is a specific marker of colonic epithelial cells, whereas sucrase is specific to absorptive cells of the small intestine. Totals of 139 (23.5%) and 452 (76.5%) IM glands were judged to be CA1 positive and CA1 negative, respectively, in resected pyloric mucosa from cancer patients. The average score for MUC5AC in CA1-positive IMs was significantly lower than in CA1-negative counterpart tissue (P<0.0001), whereas the opposite was the case for sucrase (P<0.0001). High iron diamine-Alcian blue staining revealed CA1 expression to coincide with type I complete IM. The expression of CA1 mRNA strongly correlated with that of sucrase-isomaltase, and inversely with that of MUC5AC in isolated IM glands. In conclusion, CA1 could be colocalized with small intestinal proteins such as sucrase, but only rarely with the gastric mucin, MUC5AC. Its expression warrants further study, with the focus on stimulation and/or suppression mechanisms by gastric and intestinal transcription factors.

The clinicopathological significance of colonic and small-intestinal phenotypes has hitherto remained unclear in gastric cancers. The purpose of the present study was therefore to examine 86 gastric carcinomas histologically and phenotypically using several phenotypic markers, including colon-specific carbonic anhydrase 1 (CA1) and sucrase as small-intestine specific marker. Of 86 gastric cancers, sucrase and CA1 expression was observed in 12 (14.0%) and only in two cases (2.3%), respectively, associated with other intestinal markers such as villin and mucin core protein (MUC)2. In the sucrase cases, expression appeared independent of the stage. However, CA1 expression was observed only in two advanced cases. No association was observed between colonic and small-intestinal phenotypes, and lymph node metastasis and postoperative survival in the advanced gastric cancer cases with intestinal phenotypic expression. Cdx2 appeared to be linked to upregulation of both CA1 and sucrase. In conclusion, the data suggest that colonic phenotype occurs rarely in gastric carcinogenesis. Colonic and small-intestinal phenotypes appear with expression of several intestinal phenotypic markers under the control of Cdx2 and presumably other related transcription factors.

Helicobacter pylori (Hp) infection is an important factor in human gastric disorders, including chronic active gastritis, peptic ulcers, intestinal metaplasia and cancer. Since epidemiologic studies overwhelmingly agree on a protective influence of fruits and vegetables in reducing the risk of gastric neoplasia and processed foods made from Prunus mume Sieb. et Zucc. (Japanese apricot or "Ume" in Japanese) are traditionally known for their miscellaneous medical effects, in the present study we investigated the efficacy of a fruit-juice concentrate of Japanese apricot (CJA) in the glandular stomach of Hp-infected Mongolian gerbils. Hp-inoculated gerbils were given CJA in their drinking water at concentrations of 1 and 3% for 10 weeks. The microscopic scores for gastritis and mucosal hyperplasia in the CJA groups were significantly lower than in the Hp-inoculated control group, with dose-dependence. Real-time PCR was performed to quantitate Hp by demonstrating urease A gene amount using gerbils glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal control. Average relative urease A gene dosage in the glandular stomach in the 1 and 3% CJA and Hp-inoculated control groups was 26.6 +/- 11.6% (average +/- SE), 30.3 +/- 10.5%, 100 +/- 40.9%, respectively, the fruit-juice concentrate causing significant lowering (P<0.01 and P<0.05, respectively, with 1 and 3%). These findings suggest that suppressive effects on gastric cancer development might also be expected as a result of decreased numbers of Hp and improvement of Hp-induced chronic active gastritis on administration of CJA.

Mongolian gerbils are an ideal animal model to explore the role of H. pylori on cancer development. However, there have been no established adenocarcinoma cell lines from this model animal. In the present study, we have established cancer cell lines from a primary gastric cancer tissue of a Mongolian gerbil. The derived cells could be stably attached with H. pylori, revealed under a scanning electron microscope, and easily transplanted to the nude mice. Rapid phosphorylation of IkappaB, Erk1/2, and AKT of these cells was observed by Interleukin-1 beta stimulation, and luciferase reporter gene assay on transcriptional activation of Nuclear Factor kappa B after challenging with either H. pylori NCTC11637 or its isogenic cagE-knockout mutant, H. pylori revealed the cagE-dependent NF-kappaB transcriptional activation. The newly established cancer cell lines from the in vivo gastric carcinogenesis model animal, the Mongolian gerbil, can be used to develop effective therapeutic strategies against gastric cancer, especially in exploring the effect of H. pylori, and thus might greatly contribute to gastric cancer prevention and treatment in humans.

We have proposed to divide intestinal metaplasia (IM) into two categories, i.e., a mixed gastric and intestinal (GI) type, and a solely intestinal (I) type, based on the residual gastric phenotype cells. The GI-mixed-type IM can be identified by the presence of both cells with either gastric or intestinal phenotypes in a single gland. This study is conducted to elucidate whether cells in the GI-mixed-type IM glands can simultaneously present both gastric and intestinal phenotypes. MUC5AC, MUC2, CD10 and villin expressions were investigated in 20 samples from five gastric cancer cases, directly using either AlexaFluor 488- or 568-labeled specific monoclonal antibodies and observed by fluorescent microscopy and confocal laser-scanning microscopy. GI-mixed IM glands comprise a population expressing MUC5AC and MUC2, MUC5AC and villin, and MUC5AC and CD10. MUC2 and villin expressions were reciprocally increased with decreasing MUC5AC expression, while CD10 expression was limited to cells with only a residual MUC5AC expression or no expression. These results suggest that a heterogeneous cell population with both gastric and intestinal phenotypes would develop into a single intestinal phenotype, as reflected in the progression of intestinal metaplasia from GI-mixed-type- to I-type IM-type glands.

The goal of this study was to elucidate whether beta-catenin gene mutations might contribute to glandular stomach carcinogenesis in Helicobacter pylori (H.pylori)-infected Mongolian gerbils. Firstly, exon 3 of gerbil beta-catenin cDNA, a mutation hot spot, was cloned and sequenced and found to have 89.3% homology with the human form and 95.5% with the rat and mouse forms. Peptide sequence in this region was shown to be 100% conserved in these mammals. Then, 45 stomach adenocarcinomas induced with N-methyl-N-nitrosourea (MNU) plus H. pylori infection and 7 induced with MNU alone were examined for beta-catenin expression by immunohistochemistry and for DNA mutations using a combination of microdissection and PCR-single strand conformation polymorphism analysis. One gastric cancer in the MNU + H. pylori group (2.2%) displayed nuclear (N) beta-catenin localization, 3 (6.7%) showed cytoplasmic (C) distribution in local regions, and 41 (91.1%) demonstrated cell membrane (M) localization. Tumors induced by MNU alone showed only membranous beta-catenin localization (7/7). Analysis of exon 3 of the beta-catenin gene dem-onstrated all tumors with membrane or cytoplasmic staining as well as surrounding normal mucosa (S) to feature wild-type beta-catenin. In contrast, the lesion with nuclear staining had a missense mutation at codon 34 [GAC (Gly) --> GAA (Glu)] in exon 3 (1/1 = 100%, N vs. M, P < 0.05; and N vs. S, P < 0.05). In conclusion, these results suggest that beta-catenin may not be a frequent target for mutation in stomach carcinogenesis in MNU + H. pylori-treated gerbils.

Purpose: The molecular mechanisms underlying the development of intestinal metaplasia (IM) of the human stomach have yet to be clarified in detail. Besides ectopic expression of intestinal transcription factors, Cdx1 and Cdx2, little information is available regarding other regulatory factors. Hence, we here analyzed Sox2, a human homolog of a chicken gastric transcription factor, with reference to our new classification for gastric/intestinal (GI)-mixed type IM., Methods: Twenty specimens of surgically resected antral mucosa were subjected to a gland isolation technique. Isolated glands were classified into gastric (G), GI-mixed, and solely intestinal (I) types according to Alcian blue and paradoxical concanavalin A staining and were quantified for mRNA levels of gastrointestinal markers., Results: MUC5AC and MUC6 transcripts decreased with the progression of IM, while MUC2 and villin-1 were inversely correlated. Sox2 showed a gradual decrease from G, through GI, to the I type (G vs GI and GI vs I, P<0.01 and P<0.005, respectively). On the other hand, Cdx1 (G vs GI and GI vs I, P<0.0001 and P=0.337, respectively) and Cdx2 (G vs GI and GI vs I, P<0.0001 and P<0.05, respectively) appeared in IM. Immunohistochemical study confirmed decreased expression of Sox2 and ectopic emergence of Cdx2 protein in IM glands., Conclusion: Down-regulation of Sox2, besides ectopic expression of Cdx genes, may be important factors for the development of IM.

Helicobacter pylori (Hp) infection and gastric carcinogenesis are known to have a close relationship, but the effect of eradication of Hp on Hp-related gastric carcinogenesis has not been fully studied experimentally. To evaluate the effect of eradication in gastric carcinogenesis, an experimental model with eradication in the early, middle or late period was studied using Hp-infected and N-methyl-N-nitrosourea (MNU)-treated Mongolian gerbils. The animals were divided into seven groups: group A (MNU + Hp + eradication at 15 w), group B (MNU + Hp + eradication at 35 w), group C (MNU + Hp + eradication at 55 w), group D (MNU + Hp), group E (MNU), group F (Hp) and group G (control). The tumor incidences at week 75 in the early (group A), middle (group B) and late (group C) eradicated groups were 6.7%, 27.3% and 38.2%, respectively. The incidence of 56.3% in the non-eradicated group was the highest (group D). Incidences in group E, group F and group G were 6.3%, 0.0% and 0.0%, respectively. The tumor incidences were related to the period of inflammatory status induced by Hp infection. Hp infection strongly enhanced gastric carcinogenesis initiated with a chemical carcinogen, and following eradication at an early period this enhancing effect was effectively reduced. Eradication at an early stage of inflammation might be effective in preventing Hp-related gastric carcinogenesis.

Helicobacter pylori (H. pylori) is now well known to be associated with stomach cancer, with infection during childhood rather than as an adult considered to be more important for carcinogenesis. To evaluate the difference in susceptibility to stomach carcinogenesis in relation to age of acquisition of H. pylori infection, we designed an experiment involving inoculation of H. pylori ATCC43504 followed by N-methyl-N-nitrosourea (MNU) treatment at different ages. Four-week-old male Mongolian gerbils (MGs) were divided into twelve groups. H. pylori was inoculated at 4, 18 and 32 weeks of age, as representatives of early, middle and late infection, respectively. Two weeks later, the animals were treated with MNU. Groups without H. pylori and/or MNU were included as controls. The incidences of adenocarcinomas at 52 weeks after the inoculation in the early (H. pylori+MNU), middle (H. pylori+MNU), and late (H. pylori+MNU) group were 60% (12/20), 18.4% (2/11), and 10% (2/20), respectively. The corresponding figures were 14.8% (4/27), 0% (0/11), and 0% (0/21) in the MNU-alone groups. A higher titer of serum IgG for H. pylori and higher gastrin level were seen in the early-infected compared to the middle and the late groups (P<0.01). The results clearly demonstrated that early acquisition of H. pylori significantly increases gastric chemical carcinogenesis with MNU, as compared to the case with later infection, possibly because of differences in host gastric mucosal factors and immunologic responses.

We present a clinical analysis of 6 cases of pericardial cysts. There were two males and four females. Only one case of them had complaint of persistent cough. Four of 6 were observed in right costo-phrenic angle, and 2 were in left costo-phrenic angle. Surgical excision was performed in 6 cases, and all patients recovered good health. We suggest that in cases of pericardial cysts, surgical treatment should be performed, even if patients would not have remarkable complaint.

The Technicon H6000 system simultaneously determines blood cell and leucocyte differential counts automatically using cytochemistry and cell size measurements. Part of the information provided by this instrument is a plot of peroxidase staining intensity against leucocyte cell size called 'the leucocyte peroxidase distribution display'. Recently, an abnormal leucocyte peroxidase distribution display was observed, on a sample from a post-operative lung cancer patient, in which eosinophilic leucocytes did not react with peroxidase. Neutrophils reacted in a normal way with this sample. The eosinophils were further examined by electron microscopy and manual peroxidase staining. Some peroxidase positive granules were observed in the eosinophilic leucocytes. The abnormal leucocyte peroxidase distribution display was found to be due to a separate group of eosinophilic leucocytes with lowered peroxidase activity.

A 59-year-old man, who had had right middle and lower lobectomy for pulmonary tuberculosis, admitted for the treatment of empyema with fistula. Closure of empyema space with free rectus abdominis myocutaneous flap was performed following open window thoracotomy and thoracoplasty. As he previously underwent two major operation, lobectomy by posterolateral approach and gastrectomy for gastric ulcer, free rectus abdominis flap was chosen instead of omental flap or latissimus dorsi myocutaneous flap. Postoperative CT film showed that this flap was filled up in all interstices of the empyema cavity. The pedicle vessels to this flap are large enough to provide long stalks, so microsurgical anastomosis can be accomplished safely. The use of free rectus abdominis myocutaneous flap is one of a useful maneuver for chronic empyema with fistula.