Testicular teratomagenesis from primordial germ cells with overexpression of germinal center-associated nuclear protein.

Testicular teratomas are the major histologic type of testicular germ cell tumors and their incidence continues to grow. Moreover, teratomas can develop from undifferentiated cells in induced pluripotent stem (iPS) cell transplantation therapy, seriously hampering the progress of regenerative medicine. Germinal center-associated nuclear protein (GANP) is thought to be important to the biogenetic control of primordial germ cells and is among the genes susceptible to testicular germ cell tumors. Thus, we analyzed the expression of GANP in human testicular postpubertal-type teratomas and established a novel mouse model to reveal the association between GANP and teratomagenesis. We analyzed 31 cases of human testicular postpubertal-type teratomas and, in all cases, GANP was overexpressed. The aberrant expression was also detected in germ cell neoplasia in situ accompanied by the teratoma. GANP expression was particularly high in the epithelia of the epidermis, cutaneous appendages, and trachea-like ciliated epithelium. To further clarify the association between GANP and teratomagenesis, we established a novel teratomagenesis mouse model (CAG-ganpTg mice). In the GANP-teratoma mice, GANP-overexpressing teratomas were more frequent at the testes and the middle portion of the uterus than has been seen in the previously established mouse models. In conclusion, GANP is overexpressed in testicular postpubertal-type teratomas and is an essential teratomagenic factor. We also found that CAG-ganpTg mice are useful mouse models of teratomagenesis that mimics human midline teratomas and that teratomas may originate from the overexpression of GANP in primordial germ cells.

Increased chemosensitivity via BRCA2-independent DNA damage in DSS1- and PCID2-depleted breast carcinomas.

Breast cancer, the most common malignancy among women, is closely associated with mutations in the tumor suppressor gene BRCA. DSS1, a component of the TRanscription-EXport-2 (TREX-2) complex involved in transcription and mRNA nuclear export, stabilizes BRCA2 expression. DSS1 is also related to poor prognosis in patients with breast cancer owing to the induction of chemoresistance. Recently, BRCA2 was shown to be associated with the TREX-2 component PCID2, which prevents DNA:RNA hybrid R-loop formation and transcription-coupled DNA damage. This study aimed to elucidate the involvement of these TREX-2 components and BRCA2 in the chemosensitivity of breast carcinomas. Our results showed that compared with that in normal breast tissues, DSS1 expression was upregulated in human breast carcinoma, whereas PCID2 expression was comparable between normal and malignant tissues. We then compared patient survival time among groups divided by high or low expressions of DSS1, BRCA2, and PCID2. Increased DSS1 expression was significantly correlated with poor prognosis in recurrence-free survival time, whereas no differences were detected in the high and low BRCA2 and PCID2 expression groups. We performed in vitro analyses, including propidium iodide nuclear staining, single-cell gel electrophoresis, and clonogenic survival assays, using breast carcinoma cell lines. The results confirmed that DSS1 depletion significantly increased chemosensitivity, whereas overexpression conferred chemoresistance to breast cancer cell lines; however, BRCA2 expression did not affect chemosensitivity. Similar to DSS1, PCID2 expression was also inversely correlated with chemosensitivity. These results strongly suggest that DSS1 and PCID2 depletion is closely associated with increased chemosensitivity via BRCA2-independent DNA damage. Together with the finding that DSS1 is not highly expressed in normal breast tissues, these results demonstrate that DSS1 depletion confers a druggable trait and may contribute to the development of novel chemotherapeutic strategies to treat DSS1-depleted breast carcinomas independent of BRCA2 mutations.

Amphiphilic Polymeric Micelles Based on Deoxycholic Acid and Folic Acid Modified Chitosan for the Delivery of Paclitaxel.

The present investigation aimed to develop a tumor-targeting drug delivery system for paclitaxel (PTX). The hydrophobic deoxycholic acid (DA) and active targeting ligand folic acid (FA) were used to modify water-soluble chitosan (CS). As an amphiphilic polymer, the conjugate FA-CS-DA was synthesized and characterized by Proton nuclear magnetic resonance (¹H-NMR) and Fourier-transform infrared spectroscopy (FTIR) analysis. The degree of substitutions of DA and FA were calculated as 15.8% and 8.0%, respectively. In aqueous medium, the conjugate could self-assemble into micelles with the critical micelle concentration of 6.6 × 10-3 mg/mL. Under a transmission electron microscope (TEM), the PTX-loaded micelles exhibited a spherical shape. The particle size determined by dynamic light scattering was 126 nm, and the zeta potential was +19.3 mV. The drug loading efficiency and entrapment efficiency were 9.1% and 81.2%, respectively. X-Ray Diffraction (XRD) analysis showed that the PTX was encapsulated in the micelles in a molecular or amorphous state. In vitro and in vivo antitumor evaluations demonstrated the excellent antitumor activity of PTX-loaded micelles. It was suggested that FA-CS-DA was a safe and effective carrier for the intravenous delivery of paclitaxel.

Morphological study of efficacy of clarithromycin-loaded nanocarriers for treatment of biofilm infection disease.

In this study, we developed a drug delivery system (DDS) using polymeric nanocarriers for the treatment of biofilm infection disease. Clarithromycin (CAM)-encapsulated and chitosan (CS) modified polymeric nanoparticles (NPs) were prepared using a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus®) (Sol) and poly-(DL-lactide-co-glycolide), respectively. To understand the availability of the prepared NPs, we made morphological observations of the antibacterial activity derived from the NPs toward the bacterial cells within the biofilm using scanning electron microscopy and transmission electron microscopy measurements. These results revealed different antibacterial activities for the two types of drug carriers. In the case of CAM-encapsulated + CS-modified Sol micelles treatment, NPs can exert their antibacterial activity not only by the surfactant, CAM and CS effects but also by intrusion into the bacterial cells. Thereby, CAM-encapsulated + CS-modified Sol micelles had a higher antibacterial activity. The morphological information is useful to design suitable NPs for the treatment against biofilm infections.

Multifunctional Composite Microcapsules for Oral Delivery of Insulin.

In this study, we designed and developed a new drug delivery system of multifunctional composite microcapsules for oral administration of insulin. Firstly, in order to enhance the encapsulation efficiency, insulin was complexed with functional sodium deoxycholate to form insulin-sodium deoxycholate complex using hydrophobic ion pairing method. Then the complex was encapsulated into poly(lactide-co-glycolide) (PLGA) nanoparticles by emulsion solvent diffusion method. The PLGA nanoparticles have a mean size of 168 nm and a zeta potential of -29.2 mV. The encapsulation efficiency was increased to 94.2% for the complex. In order to deliver insulin to specific gastrointestinal regions and reduce the burst release of insulin from PLGA nanoparticles, hence enhancing the bioavailability of insulin, enteric targeting multifunctional composite microcapsules were further prepared by encapsulating PLGA nanoparticles into pH-sensitive hydroxypropyl methyl cellulose phthalate (HP55) using organic spray-drying method. A pH-dependent insulin release profile was observed for this drug delivery system in vitro. All these strategies help to enhance the encapsulation efficiency, control the drug release, and protect insulin from degradation. In diabetic fasted rats, administration of the composite microcapsules produced a great enhancement in the relative bioavailability, which illustrated that this formulation was an effective candidate for oral insulin delivery.

[A Case of Disseminated Peritoneal Leiomyomatosis with Several Recurrences].

A 60's woman had undergone abdominal total hysterectomy due to uterine leiomyoma 17 years previously. She underwent resection of multiple intra-abdominal tumors 9 years previously, and was diagnosed with disseminated peritoneal leiomyomatosis. Because of several recurrences, she was referred to our hospital and treated with GnRH agonist therapy. However, the tumors enlarged gradually and she underwent further resection for multiple intra-abdominal tumors. After surgery, we performed several operations. This is an extremely rare disease and we present this case with a discussion of the literature.

Electron microscopy of Staphylococcus epidermidis fibril and biofilm formation using image-enhancing ionic liquid.

We established an optimized biofilm observation method using a hydrophilic ionic liquid (IL), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]). In the present study, a biofilm was formed by Staphylococcus epidermidis. Using field emission (FE) scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the colonization of assemblages formed by microbial cells was observed as a function of the cultivation time. FE-TEM analysis revealed that the fibril comprises three types of protein. In addition, the ultrastructure of each protein monomer was visualized. It was expected that the curly-structured protein plays an important role in extension during fibril formation. Compared to the conventional sample preparation method for electron microscopy, a fine structure was easily obtained by the present method using IL. This observation technique can provide valuable information to characterize the ultrastructure of the fibril and biofilm that has not been revealed till date. Furthermore, these findings of the molecular architecture of the fibril and the colonization behavior of microbial cells during biofilm formation are useful for the development of antibacterial drugs and microbial utilization.

Quetiapine free base complexed with cyclodextrins to improve solubility for parenteral use.

Quetiapine, an antipsychotic drug used for schizophrenia treatment, is poorly water soluble, and therefore, administration of the more water-soluble quetiapine fumarate is preferred. Absorption of quetiapine through biological membranes may be improved by enhancing the solubility of the quetiapine base, the non-ionic form. In this study, the currently used salt form was converted into the free base (oily material). We employed cyclodextrins (CDs) as pharmaceutical additives to improve the solubility of the quetiapine base. The formation of quetiapine-ß-cyclodextrin (ß-CD) complexes was studied by phase solubility studies, continuous variation method, NMR spectroscopy, and powder X-ray diffraction. The formation of a poorly water-soluble complex was confirmed by the phase solubility study, and the interaction between quetiapine and ß-CD in water was confirmed by NMR spectroscopy. In addition, the effects of ß-CD derivatives (glucosyl-ß-CD, maltosyl-ß-CD, 2-hydroxypropyl-ß-CD, dimethyl-ß-CD, and trimethyl-ß-CD) on the solubility of the quetiapine base were studied. The findings indicated that the aforementioned hydrophilic ß-CD derivatives could be used as pharmaceutical additives of quetiapine for parenteral formulations as a result of the improved solubility of the quetiapine base because of inclusion complexation. Therefore, converting the currently used salt form into the free base, investigating the free base as a candidate for CD inclusion, and converting the oily material such as the free base into a powder by forming an inclusion complex that is easy to deal with is considered a worthwhile approach that may lead to novel formulations of the drug in question.

Endocytosis-like uptake of surface-modified drug nanocarriers into giant unilamellar vesicles.

We had previously developed surface-modified poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) for use as a cellular drug delivery system. The cellular uptake of PLGA-NPs was mediated predominantly by endocytosis, and this uptake was increased by surface modifications with polymers, such as chitosan (CS) and polysorbate 80 (P80). In the present study, we prepared a cell-sized giant unilamellar vesicle (GUV) that mimics a cell membrane to investigate the interaction between cell membranes and NPs. Endocytosis-like uptake of NPs into a GUV was observed when the NPs were modified with nonionic surfactant P80 probably due to change in viscoelasticity and enhanced fusion activity of the membrane induced by P80. In contrast, unmodified NPs and those modified with CS were not internalized into a GUV. These results suggest that surface properties of PLGA-NPs are an important formulation parameter for their interaction with lipid membranes.

Effects of food intake on the mucoadhesive and gastroretentive properties of submicron-sized chitosan-coated liposomes.

The gastrointestinal transition of mucoadhesive drug carriers may be affected by food intake, since food changes the physiological conditions of the gastrointestinal tract, and the food content itself is a physical obstruction for the drug carriers. Here we investigated the effects of food intake on the gastrointestinal transition and mucoadhesive function of submicron-sized chitosan-coated liposomes (ssCS-Lip). The stomach and small intestine were removed after oral administration of ssCS-Lip and non-coated liposomes (ssLip) containing fluorescent dye to fasted or fed rats, and retentive properties were quantitatively confirmed by measuring the amount of dye in each part of the gastrointestinal tract. Both types of liposome were retained in the stomach at approx. 40% in the fed rats at 1 h after oral administration, whereas transitions in the intestine were reduced compared to the fasted rats. However, the transition of ssCS-Lip in intestine was prolonged compared to ssLip even, in the fed state. The mucoadhesive behavior of ssCS-Lip was evaluated by confocal laser scanning microscopy. The ssCS-Lip tended to penetrate into the mucosal part of the intestine, and in addition, ssCS-Lip was detected in the basolateral side in both conditions, and therefore the mucopenetrative function was confirmed in the fed condition. Based on these results, we confirmed that ssCS-Lip shows a predominant gastrointestinal transition and mucopenetration, even after food intake.

Effectiveness of submicronized chitosan-coated liposomes in oral absorption of indomethacin.

The plasma profile of indomethacin (IMC) after oral administration of IMC-loaded submicronized chitosan-coated liposomes (ssCS-Lip) was evaluated to reveal the effectiveness of the mucoadhesive function for improving the absorption of this poorly absorbable drug. The stomach and small intestine were removed from rats after 1, 2, and 4 hours of oral administration of submicron-sized liposomes (ssLip) or ssCS-Lip containing fluorescent dye, and the retentive properties were confirmed by measuring the amount of dye in each part of the gastrointestinal (GI) tract. Results showed that ssCS-Lip tended to be better retained in the upper part of the GI tract, compared with ssLip, at 1, 2, and 4 hours after administration, and was significantly better retained in the small intestine at 4 hours. The plasma profile and bioavailability of IMC after oral administration of both types of liposomes were improved, compared with oral administration of IMC solution. The maximum residence time of ssCS-Lip was significantly longer than those of ssLip. The extended plasma profile of ssCS-Lip was attributed to its prolonged retention in the upper region of the GI tract, and its delayed migration to the lower part of the intestine, the neutral pH of which is more soluble for IMC, an acidic drug. Therefore, the chitosan-coated ssLip, with its higher retention in the GI tract, is a promising drug carrier for the oral administration of poorly absorbed compounds.

Nanomedical system for nucleic acid drugs created with the biodegradable nanoparticle platform.

Nanomedical applications of biodegradable poly(DL-lactide-co-glycolide) (PLGA) nanoparticles (NPs) developed are discussed in this review. A surface-functionalized PLGA NP platform for drug delivery was established to encapsulate a number of macromolecular drugs such as peptides and nucleic acids as well as low-molecular-weight drugs by the emulsion solvent diffusion method. The interaction of PLGA NPs with cells and tissues could be controlled by changing the surface properties of NPs, suggesting their potential utility for the intracellular drug delivery of nucleic acid-based drugs. Furthermore, orally administered NF-κB decoy oligonucleotide-loaded CS-PLGA NPs are also useful in treating experimental colitis. These approaches using surface-modified PLGA NPs could be able to open new possibilities for nucleic acid-based drug delivery via noninvasive administration method.

Histology of Cardiac Sarcoidosis with Novel Considerations Arranged upon a Pathologic Basis.

Sarcoidosis is a rare disease of isolated or diffuse granulomatous inflammation. Although any organs can be affected by sarcoidosis, cardiac sarcoidosis is a fatal disorder, and it is crucial to accurately diagnose it to prevent sudden death due to dysrhythmia. Although endomyocardial biopsy is invasive and has limited sensitivity for identifying granulomas, it is the only modality that yields a definitive diagnosis of cardiac sarcoidosis. It is imperative to develop novel pathological approaches for the precise diagnosis of cardiac sarcoidosis. Here, we aimed to discuss commonly used diagnostic criteria for cardiac sarcoidosis and to summarize useful and novel histopathologic criteria of cardiac sarcoidosis. While classical histologic observations including noncaseating granulomas and multinucleated giant cells (typically Langhans type) are the most important findings, others such as microgranulomas, CD68+ CD163- pro-inflammatory (M1) macrophage accumulation, CD4/CD8 T-cell ratio, Cutibacterium acnes components, lymphangiogenesis, confluent fibrosis, and fatty infiltration may help to improve the sensitivity of endomyocardial biopsy for detecting cardiac sarcoidosis. These novel histologic findings are based on the pathology of cardiac sarcoidosis. We also discussed the principal histologic differential diagnoses of cardiac sarcoidosis, such as tuberculosis myocarditis, fungal myocarditis, giant cell myocarditis, and dilated cardiomyopathy.

Improvements in transfection efficiency with chitosan modified poly(DL-lactide-co-glycolide) nanospheres prepared by the emulsion solvent diffusion method, for gene delivery.

This study sought to evaluate the in vitro transfection efficiency of plasmid DNA (pDNA)-loaded chitosan-modified poly(DL-lactide-co-glycolide) nanospheres (CS-PLGA NS) in a gene-delivery system. Using the emulsion solvent diffusion (ESD) method, pDNA-loaded PLGA NS was prepared and the surface of the PLGA NS was modified by binding to CS. Gene transfection ability of CS-PLGA NS was examined in A549 cells. The luciferase gene was used as a reporter gene. The pattern of luciferase activity by pDNA-loaded CS-PLGA NS was initially weak, but gradually grew stronger before decreasing activity. These phenomena should be in accordance with the sustained-release profile of pDNA from PLGA NS in the cytosol and the pDNA protection against DNase. Positively charged CS-PLGA NS was found, by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, not to exhibit cytotoxicity on A549 cells. These results suggest that CS-PLGA NS are potential contributors to efficient pDNA delivery due to their increased interactions with cells and lack of cytotoxic effects.

Intracellular drug delivery using polysorbate 80-modified poly(D,L-lactide-co-glycolide) nanospheres to glioblastoma cells.

We investigated doxorubicin (DOX)-loaded nanospheres (NS) formulated using a biodegradable polymer, poly(D,L-lactide-co-glycolide) (PLGA), for targeted chemotherapy of brain tumours. A nonionic surfactant, polysorbate 80 (P80), was used to modify the surfaces of PLGA NS to improve cellular drug delivery. DOX-loaded PLGA NS were formulated by emulsion solvent diffusion and characterised for DOX encapsulation and in vitro release. The effectiveness of DOX-loaded P80-PLGA NS was investigated in A172 human glioblastoma cells. The drug release pattern was dependent on the pH of the medium. Quantitatively, the cellular uptake of NS was significantly increased by P80 surface modification compared with unmodified NS. Confocal laser scanning microscopy studies revealed that DOX was released from NS following accumulation in the cell nuclei. DOX-loaded P80-PLGA NS could significantly inhibit both DOX efflux from the cells and cell proliferation compared with a DOX solution.

Inclusion complex based on N-acetyl-L-cysteine and arginine modified hydroxypropyl-ß-cyclodextrin for oral insulin delivery.

Insulin is the most effective drug in the treatment of diabetes mellitus. At present, subcutaneous injection is still the common way for insulin delivery. However, oral delivery is considered as the most preferred way for its high patient compliance and the minimal invasiveness. In this study, a novel N-acetyl-L-cysteine and arginine modified hydroxypropyl-ß-cyclodextrin (NAC-HP-ß-CD-Arg) was successfully synthesized and characterized. The polymer was used as a carrier for oral delivery of insulin by forming NAC-HP-ß-CD-Arg@insulin complex. Enzymatic degradation study indicated that the NAC-HP-ß-CD-Arg could protect insulin from enzymolysis. Moreover, the polymer exhibited strong binding ability with mucin. The transportation efficiency of NAC-HP-ß-CD-Arg@insulin across the Caco-2 cell monolayer was much greater than free insulin. The in vivo study demonstrated that the orally administered NAC-HP-ß-CD-Arg@insulin exhibited an excellent and sustained hypoglycemic effect in diabetic rats. It can be concluded that the NAC-HP-ß-CD-Arg is a potential carrier for oral delivery of insulin.

Therapeutic neovascularization by nanotechnology-mediated cell-selective delivery of pitavastatin into the vascular endothelium.

OBJECTIVE: Recent clinical studies of therapeutic neovascularization using angiogenic growth factors demonstrated smaller therapeutic effects than those reported in animal experiments. We hypothesized that nanoparticle (NP)-mediated cell-selective delivery of statins to vascular endothelium would more effectively and integratively induce therapeutic neovascularization. METHODS AND RESULTS: In a murine hindlimb ischemia model, intramuscular injection of biodegradable polymeric NP resulted in cell-selective delivery of NP into the capillary and arteriolar endothelium of ischemic muscles for up to 2 weeks postinjection. NP-mediated statin delivery significantly enhanced recovery of blood perfusion to the ischemic limb, increased angiogenesis and arteriogenesis, and promoted expression of the protein kinase Akt, endothelial nitric oxide synthase (eNOS), and angiogenic growth factors. These effects were blocked in mice administered a nitric oxide synthase inhibitor, or in eNOS-deficient mice. CONCLUSIONS: NP-mediated cell-selective statin delivery may be a more effective and integrative strategy for therapeutic neovascularization in patients with severe organ ischemia.

Insulin-S.O (sodium oleate) complex-loaded PLGA nanoparticles: formulation, characterization and in vivo evaluation.

S.O (sodium oleate) is an anionic surfactant, which is able to forman ionic complex with positively charged insulin at suitable pH. In a previous study, the insulin-S.O (Ins-S.O) complex was prepared by a hydrophobic ion pairing (HIP) method to improve the apparent liposolubility of insulin. The formation of the complex was further confirmed by Zeta potential and X-ray method. Based on the preliminary study, poly(lactide-co-glycolide) (PLGA) nanoparticles harbouring Ins-S.O complex was prepared via an emulsion solvent diffusion method. The effects of key parameters such as concentration of PVA, concentration of PLGA and initial-loaded drug on the properties of the nanoparticles were investigated. The insulin encapsulation efficiency (EE(%)) reached up to 91.2% and mean diameter of the nanoparticles was sized approximately 160 nm under optimal conditions. The pharmacological effects of the nanoparticles made of PLGA (75/25, Av Mw 15,000) were further evaluated to confirm their potential suitability for oral delivery. In order to evaluate hyperglycaemic effect of the nanoparticles for oral administration, Ins-S.O complex-loaded PLGA nanoparticles (20 IU/Kg) were administered orally by force-feeding to diabetic rats. In the case of the nanoparticles, the plasma glucose level reduced to 23.85% from the initial one 12 h post-administration and this continued for 24 h. The results showed that the use of Ins-S.O complex-loaded PLGA nanoparticles is an effective method of reducing plasma glucose levels. The insulin nanoparticles also improved the glycaemic response to an oral glucose challenge.

A Chitosan-Based Micellar System as Nanocarrier For the Delivery of Paclitaxel.

In this study, a redox-sensitive chitosan derivative with modifications by cholesterol, sulfhydryl, and mPEG (mPEG-CS(SH)-CHO) was successfully synthesized and characterized. Due to its amphiphilicity, the conjugate could spontaneously form micelles in an aqueous environment. The optimized paclitaxel (PTX)-loaded mPEG-CS(SH)-CHO micelles, with a mean diameter of 158 nm, zeta potential of +26.9 mV, drug loading of 11.7%, and entrapment efficiency of 88.3%, were successfully prepared. The results of an XRD study demonstrated that PTX was loaded in the core of the micelles in a non-crystalline state. Inspiringly, the PTX-loaded micelles possessed excellent anticancer effect but low toxicity to the body. It can be concluded that the mPEG-CS(SH)-CHO micellar system is a promising drug delivery carrier for the controlled release of PTX.

Crystallographic evaluation of the conformation of quetiapine included in ß-cyclodextrin.

Single-crystal X-ray diffraction and theoretical calculations were conducted for insights into the ß-cyclodextrin (ß-CD)-quetiapine inclusion complex structure. ß-CD and quetiapine form a host-guest inclusion complex at a ratio of 2:1 in which the ß-CD molecules form head-to-head dimers with their secondary hydroxyl groups linked by multiple hydrogen bonds. Quetiapine is totally contained within the ß-CD cavity and exhibits two kinds of disorder (parts 1 and 2) in opposite directions in the ß-CD complex. To clarify the mobility of the guest molecule in the ß-CD cavity, theoretical molecular conformational calculations, crystal optimization and crystal energy calculations were conducted using CONFLEX software. The results of theoretical molecular conformation calculations showed that the mobility of quetiapine is restricted because its tricyclic structure is covered by ß-CD. The results of crystal energy calculations indicated that the conformation of disorder part 1, which has high occupancy, was more stable.