Wholly vascularized millimeter-sized engineered tissues by cell-sized microscaffolds.

The in vitro fabrication of wholly vascularized millimeter-sized engineered tissues is still a key challenge in the tissue engineering field. Recently we reported a unique approach 'sedimentary culture' using a collagen microfiber (CMF) to fabricate large-scale engineered tissues. The millimeter-sized tissues with high extracellular matrix (ECM) density were easily obtained by centrifugation of cells and CMFs and subsequent cultivation because the CMFs acted as a micrometer-sized scaffold. However, cell distribution in the obtained tissues was not homogeneous because of the different sedimentation velocity of the cells and CMFs because of their size difference. Here we report the fabrication of wholly vascularized millimeter-sized engineered tissues using cell-sized CMFs. To avoid dissolving, vacuum drying was performed at 200 °C for 24 h for thermal crosslinking of primary amine groups of type I collagen. The 200- and 20-µm-sized CMFs (CMF-200 and CMF-20) were obtained by homogenization and subsequent sonication of the crosslinked collagen. Interestingly, the CMF-20 indicated a similar sedimentation velocity with cells because of their same size range, thus uniform millimeter-sized tissue with homogeneous cell distribution was fabricated by the sedimentary culture method. To form a whole blood capillary structure in the tissues, fibronectin (FN) was adsorbed on the surface of CMF-20 to stimulate endothelial cell migration. The distribution of the blood capillary network in 1.6-mm-sized tissues was markedly improved by FN-adsorbed CMF-20 (FN-CMF-20). Sedimentary culture using FN-CMF-20 will create new opportunities in tissue engineering for the in vitro fabrication of wholly vascularized millimeter-sized engineered tissues.

Experience of Quatro-Therapy With Everolimus to Minimize Calcineurin Inhibitor for Kidney Transplant Recipients.

BACKGROUND: This study was divided into three phases, on the occasion of the introduction of everolimus (EVR) in our hospital. METHODS: In the first phase, a study group of six maintenance patients (three living related donors, three deceased donors) who had a history of malignant disease with less than 500 mg/day of proteinuria were enrolled; a high serum creatinine and upper limit of duration after kidney transplant operation was not considered. EVR was discontinued in four of the six patients because of side effects or worsening renal function. The second phase comprised a study group of 12 maintenance patients (12 living related donors) who were more than 5 years after kidney transplant operation with serum creatinine <3 ng/mL and proteinuria <500 mg/day. In two patients, EVR was discontinued because of a skin rash or general fatigue, but EVR was continued in 10 cases. Calcineurin inhibitor (CNI) dosage was reduced and renal function improved, and mean estimated glomerular filtration rate recovered from 42.3 mL/min to 44.8 mL/min, with no rejections occurring. In the third phase, a study group of eight de novo transplant patients who were 2 to 3 weeks after transplant operation were examined. In one case, EVR was discontinued because of proteinuria but was restarted with a stepwise increasing method after 4 months and was continued without any side effects. RESULTS: Our study indicates that EVR was a useful drug for the maintenance of kidney transplant recipients for the optimal patients. CONCLUSIONS: In de novo cases, EVR plus a high dose of mizoribine and low CNI protocol was a useful regimen without serious adverse effects.

Single-dose new insulin glargine 300 U/ml provides prolonged, stable glycaemic control in Japanese and European people with type 1 diabetes.

AIMS: Two single-dose studies were conducted in Japan and Europe to compare the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of new insulin glargine 300 U/ml (Gla-300) and insulin glargine 100 U/ml (Gla-100) in people with type 1 diabetes mellitus. METHODS: In two double-blind, randomized, crossover studies, 18 Japanese participants (aged 20-65 years) and 24 European participants (aged 18-65 years) with glycated haemoglobin levels ≤9.0% (≤75 mmol/mol) received single subcutaneous doses of Gla-300, 0.4, 0.6 and 0.9 U/kg (0.9 U/kg in the European study only), and Gla-100, 0.4 U/kg. A 36-h euglycaemic clamp procedure was performed after each dosing. RESULTS: The serum insulin glargine concentration (INS) and glucose infusion rate (GIR) developed more gradually into more constant and prolonged profiles with Gla-300 than with Gla-100. In support of this, the times to 50% of glargine exposure and insulin activity were longer for all Gla-300 doses than for Gla-100 during the 36-h clamp period, indicating a more evenly distributed exposure and metabolic effect beyond 24 h. Exposure to insulin glargine and glucose utilization were lower with the 0.4 and 0.6 U/ml Gla-300 doses in both studies compared with the 0.4 U/ml Gla-100 dose. Glucose-lowering activity was detected for up to 36 h with all doses of Gla-300. CONCLUSIONS: Single-dose injections of Gla-300 present more constant and prolonged PK and PD profiles compared with Gla-100, maintaining blood glucose control for up to 36 h in euglycaemic clamp settings in Japanese and European participants with type 1 diabetes.

Experiences of high-dose mizoribine as antimetabolite immunosuppressants for kidney transplantation.

We have used low doses of mizoribine (MZ) or mycophenolate mofetil (MMF) as induction and maintenance immunosuppressants, but since 2009 have employed a high dose of MZ. We reviewed the efficacy and side effects of MZ compared with MMF. It is difficult to compare graft survivals between these periods because of different patient demographics, though the high dose of MZ cohort showed no significant difference from MMF. High doses of MZ serum to prevent acute rejection episodes as the induction and maintenance therapy. MZ controlled with blood concentrations showed less side effects, suggesting that high MZ doses could be safely used for an induction and maintenance antimetabolite.

Evaluation of the 24-hour profiles of physiological insulin, glucose, and C-peptide in healthy Japanese volunteers.

BACKGROUND: Some studies have demonstrated the benefit of blood glucose control as close as possible to physiological conditions. Not enough reports have investigated in detail the 24-h plasma profiles of insulin/glucose/C-peptide. Here we investigated the 24-h plasma profiles of physiological insulin/glucose/C-peptide in healthy Japanese adults. METHODS: In order to evaluate the 24-h profiles of physiological insulin/glucose/C-peptide profiles, 42 blood samples were taken from each subject in our group of healthy Japanese volunteers to measure the 24-h profile with three standardized meals. RESULTS: Plasma glucose and insulin increased rapidly followed by a rapid decrease after each meal with little variation at night. The average peak values of insulin after each meal were as follows: 426.20 pmol/L (breakfast), 373.75 pmol/L (lunch), and 410.28 pmol/L (dinner). The average times to peak insulin were 0.651 h (breakfast), 0.677 h (lunch), and 0.689 h (dinner). The corresponding average maximum postprandial plasma glucose levels were 8.39 mmol/L (breakfast), 8.77 mmol/L (lunch), and 8.74 mmol/L (dinner). The average times to peak glucose were 0.738 h (breakfast), 0.650 h (lunch), and 0.625 h (dinner). The average maximum postprandial C-peptide levels were 2.64 nmol/L (breakfast), 2.55 nmol/L (lunch), and 2.67 nmol/L (dinner). No major differences were found in these parameters between the Caucasian and Japanese populations. CONCLUSIONS: This is the first investigation to measure the 24-h profiles of insulin/glucose/C-peptide in healthy Japanese volunteers with standardized meals. It is hoped this information will provide useful reference for future research and clinical management.

Detection of Human papillomavirus and cellular regulators p16INK4a, p53, and NF-kappaB in penile cancer cases in Kenya.

Human papillomavirus (HPV) E6 and E7 gene products play a central role in the induction of benign proliferation and malignant transformation by interacting with several cellular regulatory proteins such as p53, p16(INK4a), and nuclear factor kappaB (NF-kappaB). In this study, HPV DNA was detected by in situ hybridization (ISH) and p53, p16(INK4a), and NF-kappaB by immunochemistry in 22 penile cancer cases in Kenya. HPV DNA was found in 68.2% of the cases. There was no difference in the p53- and p16(INK4a)-positivities in HPV DNA-positive and HPV DNA-negative cases. In HPV DNA-positive cases, the NF-kappaB positivity in the nucleus, cytoplasm, and nucleus and/or cytoplasm amounted to 73.3%, 93.3%, and 100%, respectively, while in HPV DNA-negative cases, a 28.7% NF-kappaB positivity of in the nucleus and/or cytoplasm was observed. It is concluded that NF-kappaB in penile cancer is expressed more frequently in the presence of HPV infection than in its absence.

Clock gene expression in peripheral leucocytes of patients with type 2 diabetes.

AIM/HYPOTHESIS: Recent studies have demonstrated relationships between circadian clock function and the development of metabolic diseases such as type 2 diabetes. We investigated whether the peripheral circadian clock is impaired in patients with type 2 diabetes. METHODS: Peripheral leucocytes were obtained from eight patients with diabetes and six comparatively young non-diabetic volunteers at 09:00, 15:00, 21:00 and 03:00 hours (study 1) and from 12 male patients with diabetes and 14 age-matched men at 09:00 hours (study 2). Transcript levels of clock genes (CLOCK, BMAL1 [also known as ARNTL], PER1, PER2, PER3 and CRY1) were determined by real-time quantitative PCR. RESULTS: In study 1, mRNA expression patterns of BMAL1, PER1, PER2 and PER3 exhibited 24 h rhythmicity in the leucocytes of all 14 individuals. The expression levels of these mRNAs were significantly (p < 0.05) lower in patients with diabetes than in non-diabetic individuals at one or more time points. Moreover, the amplitudes of mRNA expression rhythms of PER1 and PER3 genes tended to diminish in patients with diabetes. In study 2, leucocytes obtained from patients with diabetes expressed significantly (p < 0.05) lower transcript levels of BMAL1, PER1 and PER3 compared with leucocytes from control individuals, and transcript expression was inversely correlated with HbA(1c) levels (rho = -0.47 to -0.55, p < 0.05). CONCLUSIONS/INTERPRETATION: These results suggest that rhythmic mRNA expression of clock genes is dampened in peripheral leucocytes of patients with type 2 diabetes. The impairment of the circadian clock appears to be closely associated with the pathophysiology of type 2 diabetes in humans.

Tolerability, pharmacokinetics and pharmacodynamics of the once-daily human GLP-1 analog liraglutide in Japanese healthy subjects: a randomized, double-blind, placebo-controlled dose-escalation study.

OBJECTIVES: Liraglutide is a once-daily human GLP-1 analog being developed as a Type 2 diabetes therapy. A dose-finding study in Japanese patients with Type 2 diabetes showed liraglutide to produce dose-dependent decreases in HbA(1C). Studies have also shown that, with stepped dose titration, liraglutide is well tolerated. This double-blind trial in 24 healthy Japanese men assessed the safety, tolerability, pharmacokinetics and pharmacodynamics of once-daily subcutaneous (s.c.) liraglutide using doses exceeding those previously studied, and using the stepped titration approach. MATERIALS AND METHODS: Subjects were randomized to three groups in each of which 6 received liraglutide, and 2 placebo for 35 consecutive days. The daily dose of liraglutide was stepped from 5 microg/kg (s.c. abdomen, morning) to 10 and then 15 microg/kg at 7-day intervals. One group remained at this dose, the others titrating further to 20 and 25 microg/kg, respectively. Subjects remained at the study site from Day 21 until the end of the trial, with standard meals served during inhouse periods. RESULTS: No safety issues, hypoglycemia, gastrointestinal or any other adverse events were observed. Liraglutide showed dose-dependent increases in the pharmacokinetic parameters of AUC0-24 h, C(max) and C(trough), while t(max), t(1/2) and V(d/F) were constant. Mean plasma glucose concentrations were similar across all treatment groups at baseline, but dose-dependent decreases in mean and postprandial plasma glucose were seen with liraglutide, although all values remained within normal ranges. There was a tendency for weight to decrease with liraglutide in comparison to placebo. CONCLUSIONS: Liraglutide appears to be well tolerated at doses of up to 25 microg/kg in Japanese subjects. Despite clear pharmacodynamic effects in this euglycemic cohort, a low risk for hypoglycemia was suggested together with good gastrointestinal tolerability.

Pharmacogenetic characterization of sulfasalazine disposition based on NAT2 and ABCG2 (BCRP) gene polymorphisms in humans.

The role of breast cancer resistance protein (BCRP), an efflux ABC transporter, in the pharmacokinetics of substrate drugs in humans is unknown. We investigated the impact of genetic polymorphisms of ABCG2 (421C>A) and NAT2 on the pharmacokinetics of sulfasalazine (SASP), a dual substrate, in 37 healthy volunteers, taking 2,000 mg of conventional SASP tablets. In ABCG2, SASP AUC(0-48) of C/C, C/A, and A/A subjects was 171 +/- 85, 330 +/- 194, and 592 +/- 275 microg h/ml, respectively, with significant differences among groups. In contrast, AUC(0-48) of sulfapyridine (SP) tended to be lower in subjects with the ABCG2-A allele as homozygosity. In NAT2, AUC(AcSP)/AUC(SP) was significantly higher in rapid than in intermediate and slow acetylator (SA) genotypes. We successfully described the pharmacokinetics of SASP, SP, and N -acetylsulfapyridine (AcSP) simultaneously by nonlinear mixed-effects modeling (NONMEM) analysis with regard to both gene polymorphisms. The data indicate that SASP is a candidate probe of BCRP, particularly in its role in intestinal absorption.

Serum ghrelin levels are higher in Caucasian men than Japanese men aged 40-49 years.

BACKGROUND: Ghrelin, a 28-amino-acid gastric peptide hormone, has an appetite-stimulating effect and controls the energy balance. Serum ghrelin levels inversely correlate with body mass index. Recently, several papers reported the ethnic difference in the ghrelin levels. To our knowledge, however, no studies have compared the serum ghrelin levels between Caucasians in the USA and the Japanese in Japan. METHODS: We conducted a cross-sectional study of 189 men 40-49 years of age (91 US Caucasians in the U.S. and 98 Japanese in Japan) to examine serum ghrelin levels and metabolic and other factors. RESULTS: Serum ghrelin levels correlated with waist circumferences and lipid profiles among Caucasian Americans and the Japanese. Serum ghrelin levels were significantly higher among Caucasian Americans than among the Japanese (904.5 (632.0, 1132.0) pg/mL, 508.0 (399.0, 1378.3) pg/mL (median and 95% confidence interval), respectively, P < 0.01), although Caucasian Americans were much more obese (BMI: 26.9 +/- 3.3 kg/m(2) versus 23.3 +/- 3.1 kg/m(2) respectively, P < 0.01). The ethnic difference remained after adjusting for metabolic factors, smoking status, and other factors (P < 0.01). CONCLUSIONS: We have shown in our population-based study that serum ghrelin levels among men aged 40-49 are significantly higher in Caucasian Americans than in the Japanese in Japan. Reasons for the ethnic difference in the ghrelin levels are largely unknown and warrant further investigation.

SLCO1B1 (OATP1B1, an uptake transporter) and ABCG2 (BCRP, an efflux transporter) variant alleles and pharmacokinetics of pitavastatin in healthy volunteers.

To investigate the contribution of genetic polymorphisms of SLCO1B1 and ABCG2 to the pharmacokinetics of a dual substrate, pitavastatin, 2 mg of pitavastatin was administered to 38 healthy volunteers and pharmacokinetic parameters were compared among the following groups: 421C/C(*)1b/(*)1b (group 1), 421C/C(*)1b/(*)15 (group 2), 421C/C(*)15/(*)15 and 421C/A(*)15/(*)15 (group 3), 421C/A(*)1b/(*)1b (group 4), 421A/A(*)1b/(*)1b (group 5), and 421C/A(*)1b/(*)15 (group 6). In SLCO1B1, pitavastatin area under plasma concentration-time curve from 0 to 24 h (AUC(0-24)) for groups 1, 2, and 3 was 81.1+/-18.1, 144+/-32, and 250+/-57 ng h/ml, respectively, with significant differences among all three groups. In contrast to SLCO1B1, AUC(0-24) in groups 1, 4, and 5 was 81.1+/-18.1, 96.7+/-35.4, and 78.2+/-8.2 ng h/ml, respectively. Although the SLCO1B1 polymorphism was found to have a significant effect on the pharmacokinetics of pitavastatin, a nonsynonymous ABCG2 variant, 421C>A, did not appear to be associated with the altered pharmacokinetics of pitavastatin.

Novel real-time monitoring system for human cytomegalovirus-infected cells in vitro that uses a green fluorescent protein-PML-expressing cell line.

Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for "immediate-early 1") protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

Microtubule network facilitates nuclear targeting of human cytomegalovirus capsid.

We assessed the requirement of the host cytoskeleton for the intracytosolic transport of incoming human cytomegalovirus (HCMV) capsids. Treatments with microtubule (MT)-depolymerizing drugs nocodazole and colchicine led to a drastic decrease in levels of IE1 antigen, whereas cytochalasin B had no effect on the level of IE1 as determined by Western blot analyses. Sequential treatment including nocodazole washout and removal of cell surface virion revealed that HCMV entry into the cells occurred normally in the absence of the MT network. This finding was also supported by data obtained by monitoring pUL83 signals with an immunofluorescent assay (IFA). Furthermore, we demonstrated a close association of incoming HCMV capsids with MTs by IFA and ultrastructural analyses. In the absence of the MT network, the capsids which had entered the cytoplasm did not move to close proximity of the nucleus. These data suggest that HCMV capsids associate with the MT network to facilitate their own movement to the nucleus before the onset of immediate-early (IE) gene expression and that this association is required to start efficient IE gene expression.

An endoplasmic reticulum protein, p180, is highly expressed in human cytomegalovirus-permissive cells and interacts with the tegument protein encoded by UL48.

We have used a virus overlay assay to detect cellular proteins associated with human cytomegalovirus (HCMV) particles. The radiolabeled HCMV particles specifically bound to two host proteins with molecular sizes of 150 and 180 kDa. By a micro-amino-acid sequencing technique, the 180-kDa protein was identified as a human homologue of the ES130/p180 ribosome receptor (p180), which is an integral endoplasmic reticulum (ER) membrane protein possessing a very unique tandem repeat domain at its N-terminal region. The virus overlay assay using truncated p180 polypeptides revealed that HCMV binding to human p180 occurred through the N-terminal region. In HCMV-permissive cells the high level of expression of the human p180 protein was clearly observed regardless of cell type. Furthermore, we showed that p180 binds to the UL48 gene product, which is one of the predominant tegument proteins of HCMV and which is considered to be tightly associated with the capsid. The interaction between the two proteins was assumed to be specific and was observed both in vitro and in vivo. During the late phase of infection, the unique relocation of human p180 was observed, that is, to the juxtanuclear region, which appeared to be in the vicinity of the area where naked virions were frequently observed in an electron-microscopic study. Thus our data suggest that p180 interacts with the HCMV tegument, at least through pUL48, during the HCMV replication process. We discuss the possible role of the interaction between p180 and pUL48 in the intracellular transport of HCMV virions.

A test model of hip brace for prevention of dislocation after total hip arthroplasty (Zetton Band).

Hip dislocations remain an intractable problem in patients with soft tissue impairment, particularly in those with muscle weakness around the hip, such as those who have undergone revision total hip arthroplasty (THA). At the authors' hospital, postoperative dislocations were observed in 10 of 154 hips between January 1985 and June 1988. Five hips required re-replacement. Conventional measures to prevent or treat post-THA dislocations have been anti-dislocation pants for soft fixation and a cast or abduction-forcing braces for firm fixation. However, the anti-dislocation pants for soft fixation were not as effective as indicated by the above 10 postoperative dislocations. The firm fixation techniques are considered to cause a reduction in muscle strength, causing psychological stress and poor activity of daily living (ADL). The authors devised a soft brace for easy application and prepared its test model to prevent muscle weakening, allow stability of the hip during rotation and avoid restrictions in ADL. This brace was applied to a patient who had 3 dislocations in a short period after being discharged who sustained a postoperative dislocation and achieved good results.

Electrophoretic characterization of ribosomal subunits and proteins in apoptosis: specific downregulation of S11 in staurosporine-treated human breast carcinoma cells.

Stimulation of death receptors (Fas on human T-cell leukemia Jurkat cells and tumor necrosis factor receptor-1 on human monoblastic leukemia U937 cells) triggers the specific degradation of 28S ribosomal RNA, and this process may contribute to cell death through the inhibition of protein synthesis. We have developed an analytical method using a polyacrylamide-agarose composite gel to evaluate ribosomal subunits in apoptotic cells (human breast carcinoma MCF-7 cells treated with staurosporine and human 293T cells irradiated with ultraviolet light were used in addition to the two apoptosis systems described above). No alterations were detected by this method, suggesting that apoptosis, including the process of ribosomal RNA degradation, does not cause fragmentation or extensive conformational changes in the ribosome. We also examined the status of 21 different ribosomal proteins in apoptotic cells by immunoblotting with polyclonal antibodies. S11 was specifically downregulated in apoptotic MCF-7 cells and in other apoptotic breast carcinoma cells. Previous studies have shown that S11 is heterogeneously expressed in cancer cells. Taken together, it appears that particular intracellular environments regulate the expression of S11 protein. However, the mechanism by which this process is modulated is as yet unknown. Furthermore, we have demonstrated that our composite gel electrophoresis system can efficiently detect ubiquitination of ribosomal subunits.

Identification of two Fas-associated phosphatase-1 (FAP-1) promoters in human cancer cells.

Fas-associated phosphatase-1 (FAP-1) has been reported as a negative regulator of Fas-mediated signal transduction in human cancer cells. To obtain insights into the potential carcinogenesis of the FAP-1 gene, we investigated its transcriptional regulation in normal and cancerous cells. To identify the FAP-1 promoter sequences, we first isolated P1 and cosmid clones that contained the regulatory region upstream from the FAP-1 gene by using the PCR products of 5' rapid amplification of cDNA end (5'-RACE) as probes. Genomic analysis of positive clones revealed that the major FAP-1 mRNA was transcribed from its proximal promoter (pPRM) in all human cancer cell lines tested, but 1 additional large transcript derived from its distal promoter (dPRM) was found in the human colon cancer cell line DLD-1. This suggests that the FAP-1 gene may be aberrantly dysregulated in some types of human cancers, including colon carcinoma. Sequence analysis of the region upstream from the FAP-1 gene strongly suggests that the transcript of the FAP-1 gene may be controlled by a variety of transcriptional regulatory elements, including NF-kappa B, NF-IL6, and p53 in its 2 promoters. These results imply that the FAP-1 gene may be a target gene under the control of important apoptosis-related nuclear factors in human cancers.