The existence of Na+/K+-ATPase-immunoreactive cells in the pharyngeal villiform-papilla epithelium of the soft-shelled turtle, Trionyx sinensis japonicus

The pharyngeal villiform processes of the hibernating soft-shelled turtle, Trionyx sinensis japonicus, were studied by immunohistochemistry for Na+/K+-ATPase in combination with a mitochondrion staining. Mitochondria-rich cells were recognized in the epithelium constituting the distal part of most processes, and exclusively showed the Na+/K+-ATPase immunoreactivity. These cells tended to attract each other to form clusters. When considering the physiological and histological data previously obtained in corresponding cells in the fish gill epithelium, the mitochondria-rich cells in the hibernating turtle were suggested to be involved in the electrolyte (Na+) uptake from the aquatic habitat.     

In vivo Cisplatin Resistance Depending upon Canalicular Multispecific Organic Anion Transporter (cMOAT)

The in vitro sensitivities to cisplatin of AH66 and AH66F cells, a variant obtained from AH66 cells, were very similar, when assayed in a medium containing 5% fetal bovine serum (FBS), whereas in the in vivo experiments AH66F cells were sensitive and AH66 cells were highly resistant to cisplatin. In this study, we examined the mechanism of the in vivo cisplatin resistance of AH66 cells. The in vitro cisplatin sensitivity of AH66 cells was lowered by changing FBS to 5% ascites fluid (ASF) in the assay medium and the sensitivity in FBS by treatment with buthioninesulfoximine (BSO). The sensitivity of AH66F cells was not changed by these treatments. Moreover, after culture in 5% ASF for 48 h, the accumulation of cisplatin in AH66 cells was decreased and the efflux of cisplatin from the cells was accelerated. The accumulation of cisplatin in AH66 cells in ASF was increased by pretreatment with BSO, sodium azide or probenecid. Then, we examined the expression of the glutathione (GSH) conjugate efflux pump family. Among them, only the expression of canalicular multispecific organic anion transporter (cMOAT) in AH66 cells was decreased by culture in FBS and enhanced by ASF. These results suggest that some substances contained in ASF enhanced the expression of cMOAT in the plasma membrane of AH66 cells and this transporter actively extruded cisplatin-GSH conjugate from the cells. Consequently, AH66 cells afford a cisplatin-resistant tumor in the host.     

Difference in the distribution of tumor-infiltrating CD8+ T cells and FOXP3+ T cells between micronodular thymoma with lymphoid stroma and micronodular thymic carcinoma with lymphoid stroma

Micronodular thymoma with lymphoid stroma (MNT) is a rare thymic epithelial neoplasm subtype characterized by a micronodular tumor cell growth pattern and abundant lymphoid stroma. Micronodular thymic carcinoma with lymphoid stroma (MNCA) is considered as a malignant counterpart of MNT and exhibits a growth pattern similar to that of MNT but has histologic features reminiscent of thymic squamous cell carcinoma, such as cytologic atypia and CD5 and CD117 immunoexpression. Although both MNT and MNCA are characterized by abundant lymphoid stroma, it remains unknown whether there are differences in infiltrating lymphocytes between MNT and MNCA. We analyzed the immune microenvironment profile in eight MNT and three MNCA cases. The cell density of CD8-positive T cells was significantly higher in MNT than in MNCA, whereas that of FOXP3-positive T cells was significantly higher in MNCA than in MNT. There was no significant difference in the cell density of programmed death protein 1-positive T cells and programmed death ligand 1 expression between the MNT and MNCA cases. Our findings indicated that the immune microenvironment of MNCA differed from that of MNT and, compared with the T-cell profile of MNT, that of MNCA was more suppressive to patients′ antitumor immune response.     

Hydration of surfactant-modified and PEGylated cationic cholesterol-based liposomes and corresponding lipoplexes by monitoring a fluorescent probe and the dielectric relaxation time

For the optimization of plasmid DNA (pDNA)-cationic lipid complexes and lipoplex delivery, proper indexes of the physicochemical properties of lipoplexes are required. In general, the characteristics of lipoplexes are defined by particle size and zeta-potential at various mixing ratios of cationic liposomes and pDNA. In this study, we characterized the hydration level of surfactant-modified and PEGylated cationic cholesterol-based (OH-Chol) liposomes and their lipoplexes by monitoring both the fluorescent probe laurdan and the dielectric relaxation time. Fluorescence measurement using laurdan detected hydration of the headgroup of lipids in surfactant-modified liposomes and PEGylated DOTAP-liposomes, but hardly any fluorescence was detected in PEGylated OH-Chol-liposomes because the PEG layers may extend and cover the fluorescent maker. On the other hand, the measurement of dielectric relaxation time of water molecules revealed total hydration, including hydration of the PEG layer and the headgroup of cationic lipids. Furthermore, we found an inverse correlation between hydration level and cellular uptake of PEGylated lipoplexes (R=0.946). This finding indicated that the dielectric relaxation time of water molecules provides an important indicator of hydration of liposome and lipoplexes along with the fluorescence intensity of laurdan.     

Effects of genetic and nutritional factors on bone mineral density in young adults

To estimate the genetic and dietary factors influencing bone mineral density (BMD) in young adults, a total of 53 healthy volunteers (HV) (age 20.89+/-1.34), from whom informed consent was obtained, answered a questionnaire on dietary factors and had DNA from peripheral blood mononuclear cells analyzed for single nucleotide polymorphisms (SNPs) for vitamin (Vit) D receptor (VDR), estrogen receptor alpha (ERalpha), interleukin 1 receptor antagonist (IL1RA), and apolipoprotein E (ApoE) genes. Daily intakes of Vit C, fiber, soybean and related foods, and green and yellow vegetables showed a correlation with % BMD. In addition, Vit B2 as well as Vit C, and vegetables were identified as important factors for BMD by Stepwise regression analysis. Among the SNPs analyzed, the B+ type of the VDR gene tended to be associated with a lower BMD, and pp type of the ER gene digested by the PvuII enzyme in females indicated a significantly lower BMD than that in males. In addition, these SNPs were also identified by factor analysis to be associated with BMD. These results suggested that a complex array of genetic factors, such as two or more SNPs or SNPs and gender, may be important to BMD.     

Treatment of intrabony periodontal defects with enamel matrix derivative in private practice: a long-term retrospective study

The present study describes treatment of intrabony periodontal defects with enamel matrix derivative (EMD) in private practice. Ten patients with clinical diagnosis of chronic periodontitis were subjected to data analysis. A total of 18 teeth with various osseous defects received regenerative therapy with EMD, and were followed for a minimum of 2 years. Treatment of the intrabony defects with EMD led to a statistically significant improvement in the mean value of probing depth at 1-year when compared with at the baseline (p<0.01). Reduction in probing depth was achieved with minimal recession of the gingival margin, and was maintained over the 2-year observation period with no significant change. Mean values of attachment gain at 1 and 2 years were of clinical significance: 3.39+/-1.46 mm and 3.22+/-1.40 mm, respectively. Although one tooth was extracted because of subsequent loss of attachment and bone, most teeth treated have been successfully maintained for 2 to 7 years with no significant signs of disease progression. In conclusion, EMD treatment of intrabony osseous defects yielded clinically favorable responses. The gain in clinical attachment can be longitudinally maintained in a private practice setting. Further controlled studies are needed to elucidate the clinical significance of EMD treatment.     

Use of universal and type-specific primers in the polymerase chain reaction for the detection and typing of genital human papillomaviruses.

By using polymerase chain reaction (PCR), we have developed a system for type-specific as well as universal detection of genital human papillomaviruses (HPVs). Primers and probes for specific detection of HPV-16, -18 and -33 were synthesized from the E7 open reading frame (ORF). They were capable of detecting corresponding HPV types with high specificity and sensitivity. Primers for detection of a broad spectrum of HPV (universal primers) were synthesized from the L1 ORF. The universal primers were shown to be capable of amplifying HPV-6b, -11, -16, -18, -33, -52b and -58. The system was applied to various cervical tissue specimens from Japanese patients. They consisted of 26 normal specimens, 18 from cervical dysplasias and 29 from cervical carcinomas. HPV was detected in none of the normal specimens. On the other hand, many of the specimens from cervical dysplasias and carcinomas were found to be positive for HPV, especially HPV-16. Except for one, all the specimens which were positive with the type-specific PCRs were also positive with the universal PCR. Furthermore, substantial numbers of specimens were found to be positive only with the universal PCR. Cloning and sequencing of DNA segments amplified by the universal primers were undertaken to characterize some of the unknown HPVs. Our PCR system may thus be useful for the specific detection of the three major types of oncogenic HPVs and also for the detection of a broad spectrum of HPVs including possibly novel HPV types.     

Enhanced antitumor effect of camptothecin loaded in long-circulating polymeric micelles.

A water-insoluble antitumor agent, camptothecin (CPT) was successfully incorporated into polymeric micelles formed from poly(ethylene glycol)-poly(benzyl aspartate) block copolymers (CPT-loaded polymeric micelles). Antitumor effects and biodistribution of CPT-loaded micelles were evaluated in mice subcutaneously transplanted by colon 26 tumor cells. Tumor growth was significantly inhibited after a single i.v. injection of CPT-loaded polymeric micelles at doses of either 15 or 30 mg/kg. Efficacy of a single high-dose injection was comparable to low dose multiple injections. CPT loaded in polymeric micelles showed prolonged blood circulation and higher accumulation in tumors compared with CPT in solution. Polymeric micelle systems offer a stable and effective platform for cancer chemotherapy with CPT.