Internalization of FITC-albumin in Human Adipose-derived Stem Cells: Involvement of Clathrin and Caveolin.

Adipose tissue-derived stem cells (AdSCs) are one of the most promising cell types for cell-based therapies. In addition, AdSCs systematically injected into the body have been reported to localize to damaged tissues and certain types of tumor. As an important part of establishing a potent drug delivery system with AdSCs, the mechanism and efficiency of uptake into AdSCs has drawn much research attention. However, this remains to be fully clarified. The aim of this study was to examine the characteristics of endocytosis-mediated uptake in human AdSCs. We used fluorescein isothiocyanate-labeled albumin (FITC-albumin) as a potent marker of endocytosis. FITC-albumin uptake was time- and temperature-dependent. Confocal microscopy showed punctate localization of fluorescence in the cytoplasm. FITC-albumin uptake was inhibited by human serum albumin in a concentration-dependent manner. FITC-albumin uptake was inhibited by a metabolic inhibitor (2,4-dinitrophenol), a microtubule polymerization inhibitor (colchicine), an actin polymerization inhibitor (cytochalasin D), endosomal acidification inhibitors (chloroquine and bafilomycin A1), clathrin-dependent endocytosis inhibitors (chloropromazine, phenylarsine oxide, and Pitstop2), and caveolin-dependent endocytosis inhibitors (nystatin and methyl-ß-cyclodextrin). Furthermore, the knockdown of the clathrin heavy chain and caveolin-1 significantly reduced FITC-albumin uptake. These findings suggest that AdSCs take up albumin via endocytic pathways in which clathrin and caveolin are involved.

Factors Associated With Efficacy and Nivolumab-Related Interstitial Pneumonia in Non-Small Cell Lung Cancer: A Retrospective Survey.

BACKGROUND AND OBJECTIVES: Immune-checitors have been established as a novel standard treatment for non-small cell lung cancer (NSCLC). The aim of this study was to identify factors associated with efficacy and nivolumab-related interstitial pneumonia in NSCLC by evaluating clinical data at the initiation of and during treatment. METHODS: We retrospectively reviewed the medical records of patients who underwent treatment with nivolumab between October 2015 and December 2017. Using pretreatment patient data, we investigated factors associated with overall survival (OS) and the onset of nivolumab-related pneumonitis. We investigated serum biochemistry during treatment to identify the determinants associated with progressive disease (PD) and the onset of nivolumab-related pneumonitis. RESULTS: A total of 94 patients were included. Eleven patients continued treatment, and 54 patients were diagnosed with progressive disease. Nivolumab-related pneumonitis occurred in 15 patients. A pretreatment Eastern Cooperative Oncology Group Performance Status (ECOG PS) = 0 was linked to significantly longer OS than ECOG PS = 1 (median: 20.1 vs. 6.5 months, respectively; p < 0.001). There was a higher incidence of nivolumab-related pneumonitis in patients with a history of interstitial pneumonia than in those without it (p = 0.008). During treatment, the level of albumin gradually decreased prior to PD and onset of nivolumab-related pneumonitis. CONCLUSION: These results suggest that the pretreatment ECOG PS is the determining factor that is associated with OS, whereas history of interstitial pneumonia is the factor associated with nivolumab-related pneumonitis. A decrease in albumin during treatment may be associated with both PD and nivolumab-related pneumonitis.

Fatty acids bound to albumin induce prostaglandin E2 production in human renal proximal tubular epithelial cell line HK-2.

Fatty acids bound to albumin have been reported to be involved in various responses in renal proximal tubular cells following albumin overload, leading to progression of tubulointerstitial damage in the kidneys. In addition, it has been reported that prostaglandin E2 (PGE2) plays an important role in nephrotoxicity. The aim of this study was to examine whether albumin-bound fatty acids induce PGE2 production in human renal proximal tubular epithelial cell line HK-2. Fatty acid-bearing human serum albumin increased PGE2 release in the culture medium in concentration-dependent and time-dependent manners, but fatty acid-depleted albumin had no effect on PGE2 production. Next, we investigated the effect of arachidonic acid, a precursor of eicosanoids, on PGE2 production. Arachidonic acid with fatty acid-free albumin significantly enhanced the release of PGE2 into the medium in a concentration-dependent manner. Furthermore, we examined the effect of arachidonic acid on mRNA expression of hypoxia inducible factor-1α (HIF-1α). Arachidonic acid increased HIF-1α mRNA expression in a concentration-dependent manner. These findings suggest that fatty acids, at least in part arachidonic acid, bound to albumin increase PGE2 production and expression of HIF-1α mRNA and protein, possibly resulting in various cell responses induced by albumin overload.

Prediction of Apparent Oral Clearance of Small-Molecule Inhibitors in Pediatric Patients.

The purpose of this study was to build regression models for the prediction of apparent oral clearance (CL/F) for small-molecule inhibitors in the pediatric population using data obtained from adults. Two approaches were taken; a simple allometric regression model which considers no interdrug or interindividual variability and an allometric regression model with mixed-effects modeling where some variability parameters are included in the model. Average CL/F values were obtained for 15 drugs at various dosages from 31 literatures (a total of 139 data sets) conducted in adults and for 15 drugs from 26 literatures (62 data sets) conducted in children. Data were randomly separated into the "modeling" or "validation" data set, and the 2 allometric regression models were applied to the modeling data set. The predictive ability of the models was examined by comparing the observed and model-predicted CL/F in children using the validation data set. The percentage root mean square error was 17.2% and 26.3% in the simple allometric regression model and the allometric regression model with mixed-effects modeling, respectively. The predictive ability of the 2 models seems acceptable, suggesting that they could be useful for predicting the CL/F of new small-molecule inhibitors and for determining adequate doses in clinical pharmacotherapy for children.

Generation of transgenic marmosets using a tetracyclin-inducible transgene expression system as a neurodegenerative disease model.

Controllable transgene expression systems are indispensable tools for the production of animal models of disease to investigate protein functions at defined periods. However, in nonhuman primates that share genetic, physiological, and morphological similarities with humans, genetic modification techniques have not been well established; therefore, the establishment of novel transgenic models with controllable transgene expression systems will be valuable tools to understand pathological mechanism of human disease. In the present study, we successfully generated transgenic marmosets using a tetracyclin-inducible transgene expression (tet-on) system as a neurodegenerative disease model. The mutant human ataxin 3 gene controlled by the tet-on system was introduced into marmoset embryos via lentiviral transduction, and 34 transgene-introduced embryos were transferred into the uteri of surrogate mothers. Seven live offspring (TET1-7) were obtained, of which four were transgenic. Fibroblasts from TET1 and 3 revealed that inducible transgene expression had occurred after treatment with 10 µg/mL of doxycycline, while treatment with doxycycline via drinking water resulted in 1.7- to 1.8-fold inducible transgene expression compared with before treatment. One transgenic second-generation offspring (TET3-3) was obtained from TET3, and doxycycline-inducible transgene expression in its fibroblasts showed that TET3-3 maintained a high transgene expression level that matched its parent. In conclusion, we established a novel transgenic marmoset line carrying the mutant human ataxin 3 gene controlled by the tet-on system. The development of nonhuman primate models with controllable transgene expression systems will be useful for the identification of disease biomarkers and evaluation of the efficacy and metabolic profiles of therapeutic candidates.

Live, full-term mouse pups from oocytes grown and matured in vitro with serum substitutes.

For in vitro growth and maturation of mouse oocytes (IVG-IVM), serum is added to media up to and including the stage of oocyte maturation; this subsequently supports oocytes through fertilization and early embryo development. However, problems may occur with sera, such as batch differences and issues of biosafety. The purpose of the present study was to determine the capacity for fertilization and pre- and post-implantation development of oocytes that underwent IVG-IVM with a serum substitute. Oocyte-granulosa cell complexes from preantral follicles were cultured in medium with either fetal bovine serum (FBS), Serum Substitute Supplement™ (SSS), or Knockout™ Serum Replacement (KSR) for 10days, and were then allowed to mature for 17 h. Subsequently, more than 90% of oocytes underwent germinal vesicle breakdown (GVBD) and more than 70% reached metaphase II, with no significant difference between the groups. A lower fertilization rate, presumably due to zona hardening, was found in the serum substitute groups. Nevertheless, more than 50% of the inseminated oocytes were fertilized and 35%-45% of them underwent first cleavage and developed to the blastocyst stage. Following embryo transfer, one and four live offspring were produced from the SSS and KSR groups, respectively. The present study demonstrated that murine IVG-IVM oocytes cultured in media with a serum substitute, achieved fertilization in vitro, pre- and post-implantation development, and the delivery of live pups, although the efficiency of the process is reduced compared to FBS supplementation.

Transgenic Monkey Model of the Polyglutamine Diseases Recapitulating Progressive Neurological Symptoms.

Age-associated neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and the polyglutamine (polyQ) diseases, are becoming prevalent as a consequence of elongation of the human lifespan. Although various rodent models have been developed to study and overcome these diseases, they have limitations in their translational research utility owing to differences from humans in brain structure and function and in drug metabolism. Here, we generated a transgenic marmoset model of the polyQ diseases, showing progressive neurological symptoms including motor impairment. Seven transgenic marmosets were produced by lentiviral introduction of the human ataxin 3 gene with 120 CAG repeats encoding an expanded polyQ stretch. Although all offspring showed no neurological symptoms at birth, three marmosets with higher transgene expression developed neurological symptoms of varying degrees at 3-4 months after birth, followed by gradual decreases in body weight gain, spontaneous activity, and grip strength, indicating time-dependent disease progression. Pathological examinations revealed neurodegeneration and intranuclear polyQ protein inclusions accompanied by gliosis, which recapitulate the neuropathological features of polyQ disease patients. Consistent with neuronal loss in the cerebellum, brain MRI analyses in one living symptomatic marmoset detected enlargement of the fourth ventricle, which suggests cerebellar atrophy. Notably, successful germline transgene transmission was confirmed in the second-generation offspring derived from the symptomatic transgenic marmoset gamete. Because the accumulation of abnormal proteins is a shared pathomechanism among various neurodegenerative diseases, we suggest that this new marmoset model will contribute toward elucidating the pathomechanisms of and developing clinically applicable therapies for neurodegenerative diseases.

Significant effect of age on docetaxel pharmacokinetics in Japanese female breast cancer patients by using the population modeling approach.

PURPOSE: Docetaxel is frequently used in the treatment of a wide variety of solid tumors, including breast cancer. The aim of this study is to obtain the population pharmacokinetic parameters of docetaxel in Japanese female patients with breast cancer. METHODS: Blood samples from 24 patients were collected sequentially before and after docetaxel infusion. Genomic DNA was isolated from the peripheral blood and genotyped for the selected polymorphisms in the candidate genes of drug transporters and metabolizing enzymes. The influence of patient characteristics on the pharmacokinetics of docetaxel was evaluated using the nonlinear-mixed-effect modeling program, NONMEM. As a basis for comparison, the pharmacokinetics of another taxane paclitaxel in 41 separate female patients with breast cancer was calculated. RESULTS: A two-compartment model adequately described the pharmacokinetic profiles of docetaxel. The population mean estimates of the total body clearance for patients aged 58 years or less and the central volume of distribution for docetaxel were 32.6 L/h and 5.77 L, respectively. In patients over 58 years, the clearance was 24 % higher than that in the younger patients. No influences of the genotypes examined were noted on the clearance of docetaxel. The clearance of paclitaxel was not affected by patient age. CONCLUSIONS: Patients over the age of 58 years showed significantly higher clearance of docetaxel than that in patients aged 58 years or less. Since the clearance of paclitaxel was not affected by the age, it is possible that the pharmacokinetic mechanisms of docetaxel might be specifically affected by age in females.

Cryopreservation of ovaries from neonatal marmoset monkeys.

The ovary of neonatal nonhuman primates contains the highest number of immature oocytes, but its cryopreservation has not yet been sufficiently investigated in all life stages. In the current study, we investigated cryodamage after vitrification/warming of neonatal ovaries from a nonhuman primate, the common marmoset (Callithrix jacchus). A Cryotop was used for cryopreservation of whole ovaries. The morphology of the vitrified/warmed ovaries was found to be equivalent to that of fresh ovaries. No significant difference in the number of oocytes retaining normal morphology per unit area in histological sections was found between the two groups. In an analysis of dispersed cells from the ovaries, however, the cell viability of the vitrified/warmed group tended to be decreased. The results of a comet assay showed no significant differences in DNA damage. These results show that cryopreservation of neonatal marmoset ovaries using vitrification may be useful as a storage system for whole ovaries.

A phosphodiesterase type-5 inhibitor, sildenafil, induces sperm capacitation and penetration into porcine oocytes in a chemically defined medium.

The present study was undertaken to determine the effect of a phosphodiesterase (PDE) type-5 (cyclic guanosine monophosphate-specific) inhibitor, sildenafil, on capacitation and penetration of boar spermatozoa in a basic chemically defined medium (adenosine- and theophylline-free PGM-tac4). When ejaculated spermatozoa were cultured for 90 minutes in the absence or presence of sildenafil at 2.5 mM, the inhibitor significantly increased the percentage of capacitated/acrosome-reacted spermatozoa, as a result of the chlortetracycline assay. When fresh spermatozoa were co-cultured with oocytes in the presence of sildenafil at a different concentration (0, 2.5, 25, or 250 µM), higher sildenafil concentrations (25 and 250 µM) significantly resulted in higher sperm penetration rates. When oocytes matured in vitro were co-cultured with spermatozoa in the presence of 25 µM sildenafil or 25 mM caffeine benzoate for 8 hours, the incidence of penetrated oocytes did not differ between two groups, whereas the incidence of monospermic oocytes in penetrated one was significantly higher in the presence of sildenafil. Immunocytochemical analysis reported the presence of PDE type-5 on the acrosome region of boar spermatozoa. These results report that regulation of cyclic guanosine monophosphate-specific PDE type-5 by sildenafil somehow can increase the penetrability of boar spermatozoa in vitro.

Telaprevir-Induced Renal Adverse Events in Japanese Patients Reported in the PMDA Adverse Drug Reactions Reporting Database.

BACKGROUND: Telaprevir is a protease inhibitor currently used in the treatment of chronic hepatitis C virus (HCV) infection. One of its adverse effects is renal impairment. The Pharmaceutical and Medical Device Agency (PMDA) in Japan reported on telaprevir-related renal dysfunction in 2012. In this study, renal adverse events of telaprevir were investigated using the Japanese Adverse Drug Event Report database. Patient profiles with adverse events might provide useful information for HCV therapy. METHODS: We screened the case reports in Japanese Adverse Drug Event Report database (JADER) of the PMDA. The profiles of patients with renal adverse events were analyzed. RESULTS: The present results showed that reports of renal adverse events were most common in male patients between 60 and 69 years of age. Significant factors that affect the clinical outcomes of renal adverse events were not detected. However, it was suggested that anorexia is associated with renal adverse events. CONCLUSIONS: The number of reports of renal adverse events were highest in male patients 60 to 69 years of age treated with telaprevir. In addition, our findings suggested that anorexia is correlated with renal adverse events after telaprevir treatment. Further investigation is required to clarify the mechanism of renal impairments during triple therapy. Such knowledge might improve the safety of telaprevir therapy.

Valacyclovir-Induced Acute Kidney Injury in Japanese Patients Based on the PMDA Adverse Drug Reactions Reporting Database.

BACKGROUND: More than 250,000 reports of adverse drug events were included in the database of the Pharmaceuticals and Medical Devices Agency (PMDA) in Japan. However, these data have not been utilized sufficiently for analysis. While valacyclovir is the antiviral agent used worldwide, it is reported to induce nephrotoxicity. The aim of this study was to clarify the profiles of valacyclovir-induced adverse events using the PMDA database. METHODS: Case reports were screened in the PMDA adverse event database from 2004 to 2011. The profiles of patients with acute kidney injury (AKI) were analyzed by sex, age, diseases, concomitant suspected drugs, and outcomes. RESULTS: A total of 514 kidney-related adverse events were detected, and 344 were cases that included AKI. Of the AKI cases, 246 patients (71.5%) were female. There were 145 patients who were 70 to 79 years of age, which was the most affected of all age groups. Of the 344 patients, 183 patients had hypertension, and 65 had diabetes. Valacyclovir was the only drug used among 257 patients (74.1%). CONCLUSIONS: There were many reports of AKI involving valacyclovir and females, particularly in the 70- to 79-year age group in Japan. The results suggest that these patients were most likely to develop AKI after valacyclovir treatment.

Social factors of mental disorder and suicide in Japan-for understanding circumstance of suicides in each prefecture-.

In recent years there have been over 30000 suicides annually in Japan. This is one of the most serious problems for Japanese society. Because mental disorder is closely associated with suicide, factors related to the increase in mental disorders and suicides should be clarified. In this study, various data regarding social factors were evaluated to assess the correlation of the number of patients with mental disorders and suicides among the 47 prefectures of Japan. Various data regarding social factors, such as income, savings, or rate of divorce, were obtained from the database of the Ministry of Health, Labour and Welfare of Japan. Among the factors, the annual income and the amount of savings were significantly correlated with the number of patients with mental disorder. On the other hand, while the annual income did not have a significant correlation with suicides, the amount of savings had a significant correlation with suicides. In conclusion, the annual income and amount of savings may both be one of the important factors involved in mental disorders, and the savings may also be a factor affecting suicides. These analyses are valuable in helping to clarify the causes of mental disease, and can hopefully contribute to the health and welfare of Japanese.

Effect of the size of zona pellucida opening on hatching in the common marmoset monkey (Callithrix jacchus) embryo.

The use of the common marmoset monkey in biomedical research has increased recently, and further attention has been devoted to this model after the successful production of transgenic marmosets. To extend genetic engineering approaches to widespread biomedical research fields, efficient prolonged in vitro culturing of embryo development is necessary. We aimed to evaluate the effects of the size of the zona pellucida opening on promoting the hatching process in the marmoset embryo. Piezo-microdrilling of a 6-µm opening in eight embryos resulted in four partially hatched embryos and one hatched embryo after 5 days of culture. Piezo-microdrilling a 20-µm opening in 11 embryos resulted in nine partial hatchings and no hatched embryos. Piezo-scraping an 80-µm opening in six embryos resulted in no partially hatched embryos and five hatched embryos. These results suggest that an 80-µm opening, rather than 6-µm or 20-µm openings, is suitable to complete the hatching process in the marmoset embryo.

Ultrasound-guided non-surgical embryo collection in the common marmoset.

Experimental primate embryology has been hampered by limited access to embryos. In addition to surgical techniques, the less stressful non-surgical technique of uterine flushing has been developed but has had only limitedly used in recovering pre-implantation embryos from marmoset monkeys. In this study, we introduce the use of ultrasonography during marmoset non-surgical uterine flushing to make the cannulation easier, to further reduce stress, and to ensure thorough uterine flushing. We were able to cannulate in 99% of the transcervical cannulation attempts, repeat the flushing up to 17 times with the same animal, and recover up to 90% of the ovulation products. We also found that 8-cell or earlier stage embryos could be frequently obtained by non-surgical uterine flushing at 4 or 5 days after ovulation. The easiness and effectiveness of this novel ultrasound-guided technique will enable more research groups to study marmoset embryology and facilitate progress in this field.

Precise comparison of protein localization among OCT, OAT, and MATE in human kidney.

Organic anion transporters (OATs) and organic cation transporters (OCT) play pivotal roles in the uptake of drugs into epithelial cells at the basolateral membranes, and multidrug and toxin extrusion (MATE) mediates drug secretion into urine at the brush-border membranes. In this study, the expression and distribution of apical MATE1 and MATE2-K, and basolateral OAT1, OAT3, and OCT2 were compared using serial sections of human kidney cortex. First, mRNA expression in the proximal tubules was evaluated using laser microdissection. Levels of OAT, OCT2, and MATE mRNA in the proximal tubules were greatly higher compared with glomerulus. The results quantitatively indicated that these transporters were localized to proximal tubules in the renal cortex. Second, MATE1 and MATE2-K protein were detected in proximal epithelial cells in which OCT2 protein was expressed at the basolateral membranes. In addition, MATE1 was expressed at the brush-border membranes of tubular epithelial cells in which OAT1 and OAT3 were expressed. The results confirmed that OAT1, OAT3, OCT2, MATE1, and MATE2-K were coexpressed in tubular epithelial cells. The cooperation among OAT, OCT, and MATE in renal drug secretion was consistent with their distribution.

Multidrug and toxin extrusion family SLC47: physiological, pharmacokinetic and toxicokinetic importance of MATE1 and MATE2-K.

The kidney plays an important role in the secretion of organic compounds including drugs, toxins and endogeneous metabolites. The renal elimination process of organic cations is mediated by two distinct transport systems expressed on the apical and basolateral membrane of proximal epithelial cells. In 2005, mammalian multidrug and toxin extrusion 1 (MATE1)/SLC47A1 was identified as an orthologue of bacterial NorM. MATE1 is the H(+)/organic cation antiporter at the apical membrane, which mediates the secretion of organic cations. Kidney-specific MATE2-K was isolated from human kidney and localized at the brush-border membrane of proximal tubules. Like MATE1, MATE2-K mediates the secretion of organic cations into urine. MATE1 and MATE2-K are involved in the excretion of important medications and the disruption of these transporters can cause severe pharmacological problems. Recent findings regarding the MATE/SLC47 family are summarized in this review.

Efficient embryo transfer in the common marmoset monkey (Callithrix jacchus) with a reduced transfer volume: a non-surgical approach with cryopreserved late-stage embryos.

Among primates, the common marmoset is suitable for primate embryology research. Its small body size, however, has delayed the technical development of efficient embryo transfer. Furthermore, three factors have been determined to adversely affect the performance of marmoset embryo transfer: nonsurgical approaches, the use of cryopreserved embryos, and the use of late-stage embryos. Here we performed embryo transfer under conditions that included the above three factors and using either a small (1 µl or less) or a large volume (2-3 µl) of medium. The pregnancy and birth rates were 50% (5/10) and 27% (3/11), respectively, when using the large volume, and 80% (8/10) and 75% (9/12), respectively, when using the small volume. The latter scores exceed those of previous reports using comparable conditions. Thus, it appears that these three previously considered factors could be overcome, and we propose that reducing the transfer volume to 1 µl or less is essential for successful marmoset embryo transfer.

Organic cation transporter OCTs (SLC22) and MATEs (SLC47) in the human kidney.

In the kidney, human organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) are the major transporters for the secretion of cationic drugs into the urine. In the human kidney, OCT2 mediates the uptake of drugs from the blood at the basolateral membrane of tubular epithelial cells, and MATE1 and MATE2-K secrete drugs from cells into the lumen of proximal tubules. However, the expression of these transporters depends on the species of the animal. In the rodent kidney, OCT1 and OCT2 are expressed at the basolateral membrane, and MATE1 localizes at the brush-border membrane. Together, these transporters recognize various compounds and have overlapping, but somewhat different, substrate specificities. OCTs and MATEs can transport important drugs, such as metformin and cisplatin. Therefore, functional variation in OCTs and MATEs, including genetic polymorphisms or inter-individual variation, may seriously affect the pharmacokinetics and/or pharmacodynamics of cationic drugs. In this review, we summarize the recent findings and clinical importance of these transporters.

Developmental expression of renal organic anion transporters in rat kidney and its effect on renal secretion of phenolsulfonphthalein.

Organic anion transporters (OAT1 and OAT3) and multidrug resistance-associated proteins (MRP2 and MRP4) play important roles in anionic drug secretion in renal proximal tubules. Changes in the expression of such transporters are considered to affect the tubular secretion of anionic drugs. The purpose of this study was to elucidate the developmental changes in the expression of OAT1, OAT3, MRP2, and MRP4 and their effects on the tubular secretion of drugs. The mRNA level of each transporter was measured by real-time PCR, and the protein expression was evaluated by Western blotting and immunohistochemical analysis. In addition, the tubular secretion of phenolsulfonphthalein (PSP) in infant (postnatal day 14) and adult rats was estimated based on in vivo clearance study. The protein expression of organic anion transporters were very low at postnatal day 0 and gradually increased with age. In postnatal day 14 rats, the expression of OAT1 and OAT3 seemed to be at almost mature levels, while MRP2 and MRP4 seemed to be at immature levels. Immunohistochemical analysis in the kidney of postnatal day 0 rats revealed OATs on the basolateral membrane and MRPs on the brush-border membrane. At postnatal day 0, the distribution of these transporters was restricted to the inner cortical region, while after postnatal day 14, it was identical to that in adult kidney. An in vivo clearance study revealed that the tubular secretion of PSP was significantly lower in postnatal day 14 rats than adult rats. These results indicate that age-dependent changes in organic anion transporter expression affect the tubular secretion of anionic drugs in pediatric patients.