Comparison between hangeshashinto and dexamethasone for IL-1α and ß-defensin 1 production by human oral keratinocytes.

OBJECTIVE: Human ß-defensin 1 (hBD-1) is a antimicrobial peptide that is constantly secreted by oral tissues. Hangeshashinto (HST), a traditional Japanese medicine, has been reported to be effective against stomatitis. This study aimed to clarify the profile of HST by comparing the system of production of interleukin-1α (IL-1α) and hBD-1 in human oral mucosal epithelial cells with dexamethasone (DEX), a steroid used for the treatment of stomatitis. METHODS: Human oral keratinocytes (HOK) were treated with HST, DEX, or HST components (baicalein, baicalin, berberine, and glycyrrhizin) for 24 h, and subsequently cultured for 24 h with or without Pam3CSK4 or lipopolysaccharide (LPS). The cell supernatants, total RNA, and intracellular proteins were collected, and changes in IL-1α and hBD-1 protein production and gene expression were evaluated using ELISA and RT-PCR. The phosphorylation of NF-kB and the cell proliferative ability of HOK were evaluated by western blotting and XTT assay, respectively. RESULTS: DEX (0.01-10 µM) significantly suppressed IL-1α and hBD-1 production induced by either Pam3CSK4 or LPS, and also decreased cell growth. In contrast, HST inhibited Pam3CSK4- and LPS-induced IL-1α production at a concentration range of 12.5-100 µg/mL without affecting the cell proliferative capacity and hBD-1 production of HOK. Baicalein and baicalin, which are flavonoid ingredients of HST, showed anti-IL-1α production. CONCLUSION: HST may be useful as a therapeutic agent for stomatitis and other inflammatory diseases of the oral cavity.

Branch-duct Intraductal Papillary Mucinous Neoplasm with Rapidly Developing Intracystic Xanthogranulomatous Nodules.

We herein report a case of a branch-duct intraductal papillary mucinous neoplasm (IPMN) with rapidly developing intracystic xanthogranulomatous nodules. A unilocular cystic lesion without a mural nodule was found in the pancreatic tail of a 69-year-old man. Ten months later, multiple mural nodules emerged unexpectedly within the cyst, and the patient underwent distal pancreatectomy. Based on immunohistochemical studies and a molecular analysis, we diagnosed him with branch-duct IPMN of the gastric immunophenotype. Fragility of the pancreatic duct mucosa and consequent exposure of the wall to pancreatic juice might have caused marked granulation nodule formation in the cyst lumen.

Multiplex Digital PCR Assay to Detect Multiple KRAS and GNAS Mutations Associated with Pancreatic Carcinogenesis from Minimal Specimen Amounts.

Digital PCR (dPCR) allows for highly sensitive quantification of low-frequency mutations and facilitates early detection of cancer. However, low-throughput targeting of single hotspots in dPCR hinders variant specification when multiple probes are used. We developed a dPCR method to simultaneously identify major variants related to pancreatic carcinogenesis. Using a two-dimensional plot of droplet fluorescence under the optimized concentration of two fluorescent probe pools, the absolute quantification of different KRAS and GNAS variants was determined. Successful detection of the multiple driver mutations was verified in 24 surgically resected tumor samples from 19 patients and 22 fine-needle aspiration samples from patients with pancreatic ductal adenocarcinoma. Precise quantification of the variant allele frequency was optimized by using template DNA at a concentration as low as 1 to 10 ng. Furthermore, amplicons targeting multiple hotspots were successfully enriched with fewer false-positive findings using high-fidelity polymerase, allowing for the detection of various KRAS and GNAS mutations with high probability in small amount of cell/tissue specimens. Using this target enrichment, mutations at a rate of 90% in small residual tissues, such as the fine-needle aspiration needle flush and microscopic lesions in resected specimens, were successfully identified. The proposed method allows for low-cost, accurate detection of driver mutations to diagnose cancers, even with minimal tissue collection.

GATA6 and CK5 Stratify the Survival of Patients With Pancreatic Cancer Undergoing Neoadjuvant Chemotherapy.

Relevant protein expression of GATA6, CK5, vimentin, and mucins using immunohistochemistry was assessed for predicting the prognosis of and chemotherapy efficacy in patients with pancreatic cancers (PCs). The protein expression was examined in 159 PCs resected after neoadjuvant chemotherapy (NAC-PCs) and compared with that of 120 matched biopsy specimens taken before NAC. KRAS mutations were assessed by digital PCR. NAC-PCs were classified by GATA6 expression initially and CK5 expression subsequently into 4 types: classical-type (n = 22) with GATA6-high (≥50%)/CK5-low (<10%) PCs; hybrid-type (n = 45) with GATA6-high/CK5-high (≥10%) PCs; basal-like-type (n = 53) with GATA6-low (<50%)/CK5-high (≥30%) PCs; and null-type (n = 39) with GATA6-low/CK5-low (<30%) PCs, which resulted in clear stratification of patient prognosis. The classical-type was associated with the most favorable prognosis, whereas the null-type was associated with the worst prognosis (multivariate hazard ratio: 3.56; 95% CI, 1.63-7.77; P = .0015). The hybrid and basal-like types correlated with in-between levels of prognosis. The risk of hepatic recurrence was lower in the classical-type than in null (multivariate odds ratio [mOR]: 0.18; 95% CI, 0.04-0.96; P = .0449) and basal-like (mOR: 0.24; 95% CI, 0.05-1.16; P =.0750) types. By contrast, the risk of locoregional recurrence was higher in the classical-type than in the basal-like-type (mOR: 5.03; 95% CI, 1.20-21.1; P = .0272). The hybrid-type was subclassified into transition and coexpression patterns with different gastric mucin expression levels. High levels of vimentin (≥10%, n = 30) in pre-NAC-PC tissues was associated with poor prognosis (P = .0256). Phenotypic transitions between pre-NAC and post-NAC-PCs were common (73/120; 61%). PCs with NAC regression grades 2 and 3 showed a transition to poorer prognostic phenotypes (P = .0497). KRAS mutations were not associated with these phenotypes. In conclusion, GATA6 and CK5 immunohistochemical expression phenotypes may stratify the survival of patients with NAC-PCs and reflect post-NAC phenotypic transitions associated with poor prognosis. Prompt evaluation of immunohistochemical phenotypes may contribute to designing a precision therapeutic strategy for patients with PCs.

Mutant GNAS limits tumor aggressiveness in established pancreatic cancer via antagonizing the KRAS-pathway.

BACKGROUND: Mutations in GNAS drive pancreatic tumorigenesis and frequently occur in intraductal papillary mucinous neoplasm (IPMN); however, their value as a therapeutic target is yet to be determined. This study aimed at evaluating the involvement of mutant GNAS in tumor aggressiveness in established pancreatic cancer. METHODS: CRISPR/Cas9-mediated GNAS R201H silencing was performed using human primary IPMN-associated pancreatic cancer cells. The role of oncogenic GNAS in tumor maintenance was evaluated by conducting cell culture and xenograft experiments, and western blotting and transcriptome analyses were performed to uncover GNAS-driven signatures. RESULTS: Xenografts of GNAS wild-type cells were characterized by a higher Ki-67 labeling index relative to GNAS-mutant cells. Phenotypic alterations in the GNAS wild-type tumors resulted in a significant reduction in mucin production accompanied by solid with massive stromal components. Transcriptional profiling suggested an apparent conflict of mutant GNAS with KRAS signaling. A significantly higher Notch intercellular domain (NICD) was observed in the nuclear fraction of GNAS wild-type cells. Meanwhile, inhibition of protein kinase A (PKA) induced NICD in GNAS-mutant IPMN cells, suggesting that NOTCH signaling is negatively regulated by the GNAS-PKA pathway. GNAS wild-type cells were characterized by a significant invasive property relative to GNAS-mutant cells, which was mediated through the NOTCH regulatory pathway. CONCLUSIONS: Oncogenic GNAS induces mucin production, not only via MUC2 but also via MUC5AC/B, which may enlarge cystic lesions in the pancreas. The mutation may also limit tumor aggressiveness by attenuating NOTCH signaling; therefore, such tumor-suppressing effects must be considered when therapeutically inhibiting the GNAS pathway.

Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR.

It is challenging to secure a cytopathologic diagnosis using minute amounts of tumor fluids and tissue fragments. Hence, we developed a rapid, accurate, low-cost method for detecting tumor cell-derived DNA from limited amounts of specimens and samples with a low tumor cellularity, to detect KRAS mutations in pancreatic ductal carcinomas (PDA) using digital PCR (dPCR). The core invention is based on the suspension of tumor samples in pure water, which causes an osmotic burst; the crude suspension could be directly subjected to emulsion PCR in the platform. We examined the feasibility of this process using needle aspirates from surgically resected pancreatic tumor specimens (n = 12). We successfully amplified and detected mutant KRAS in 11 of 12 tumor samples harboring the mutation; the positive mutation frequency was as low as 0.8%. We used residual specimens from fine-needle aspiration/biopsy and needle flush processes (n = 10) for method validation. In 9 of 10 oncogenic KRAS pancreatic tumor samples, the "water-burst" method resulted in a positive mutation call. We describe a dPCR-based, super-sensitive screening protocol for determining KRAS mutation availability using tiny needle aspirates from PDAs processed using simple steps. This method might enable pathologists to secure a more accurate, minimally invasive diagnosis using minute tissue fragments.

The relationship between twin language, twins' close ties, and social competence.

This study investigated the relationship between twin language, twins' close ties, and social competence in a prospective longitudinal study. We hypothesized that twins whose tie is close would be more likely to develop a twin language, and these twins would be less likely to develop social competence. In addition, we hypothesize that some environmental factors, such as having an older sibling, preschool attendance, zygosity, and sex are also related to twin language, twins' close ties, and social competence. At baseline in 1999 a mailed questionnaire survey was conducted, and a follow-up questionnaire was distributed in 2004 among 958 mothers. As a result, 516 respondents returned the questionnaire (53.9%). In this study, we used 261 twin pairs aged from 6 to 12 years (school-age children) for analysis, excluding those with missing values. In the present study, we found that zygosity and sex were associated with twins' close ties. Having an older sibling and preschool attendance did not affect the twins' close tie, twin language, or social competence. One of the most important findings was that social competence was not affected directly by twins' close tie, but was affected when a twin language was found.

[Social development of twins: the need for intervention to avoid adverse effects of twin language].

Social competence is one of the most important accomplishments of human development, and this skill in human relationships is learned through communication. Therefore, it is considered that delays in language development could be a barrier to building human relationships and social competence. Although it is well known that there are delays in language development in twins compared with that of singletons, little is known about how these linguistic delays affect the development of social competence. Because twin language is a language that is unique to each pair of twins and cannot be understood by either their mother or others, it may be assumed that the social competence of twins who have a twin language is less than that of twins who don't have a twin language. Therefore, in this prospective longitudinal study we also investigated the relationship between twin language and social competence. A mailed questionnaire survey was conducted in 958 mothers as a follow-up of a study conducted in 2004. As a result, 522 respondents returned the questionnaire (53.9%). In this study, we used only 256 twins aged 6- 12-years-old (school-age children) for analysis, excluding those with missing values. Multiple logistic regression analysis was employed. In the second child of twins, a low birth weight, the appearance of twin language and gestosis of the mother were positively related with social unbalance (OR = 1.846, 2.022 and 1.903). On the other hand, with the first child, however, there was no such link. The present results indicate that twin language might influence social competence in school-age children. It has been believed that linguistic intervention is unnecessary, because most twin language disappears spontaneously. However, early intervention, for example linguistic assistance by public health nurses or psychologists and early enrollment in a preschool may be necessary for twins with a twin language, to avoid adverse consequences in social competence at school-age.