Production of rAAV by plasmid transfection induces antiviral and inflammatory responses in suspension HEK293 cells.

Recombinant adeno-associated virus (rAAV) is a clinically proven viral vector for delivery of therapeutic genes to treat rare diseases. Improving rAAV manufacturing productivity and vector quality is necessary to meet clinical and commercial demand. These goals will require an improved understanding of the cellular response to rAAV production, which is poorly defined. We interrogated the kinetic transcriptional response of HEK293 cells to rAAV production following transient plasmid transfection, under manufacturing-relevant conditions, using RNA-seq. Time-series analyses identified a robust cellular response to transfection and rAAV production, with 1,850 transcripts differentially expressed. Gene Ontology analysis determined upregulated pathways, including inflammatory and antiviral responses, with several interferon-stimulated cytokines and chemokines being upregulated at the protein level. Literature-based pathway prediction implicated multiple pathogen pattern sensors and signal transducers in up-regulation of inflammatory and antiviral responses in response to transfection and rAAV replication. Systematic analysis of the cellular transcriptional response to rAAV production indicates that host cells actively sense vector manufacture as an infectious insult. This dataset may therefore illuminate genes and pathways that influence rAAV production, thereby enabling the rational design of next-generation manufacturing platforms to support safe, effective, and affordable AAV-based gene therapies.

Comprehensive efficacy of ipragliflozin on various conditioned type 2 diabetes compared with dipeptidyl peptidase-4 inhibitors and with both agents, based on a real-world multicenter trial.

AIMS: The effects of ipragliflozin, the first sodium-glucose co-transporter 2 inhibitors (SGLT2i) launched in Japan in 2014, and with dipeptidyl peptidase-4 inhibitors (DPP-4i) on glycemic control and metabolic changes were investigated comprehensively on various conditioned type 2 diabetes (T2DM) by evaluating various clinical parameters in a real-world setting. MATERIALS AND METHODS: A total of 101 patients with T2DM aged 20-80 years with 7.0% ≤ HbA1c < 10.0% were followed in this 52-week, open-label, prospective, real-world, multicenter study. RESULTS: HbA1c decreased significantly in all groups. In ipragliflozin using groups, body weight, waist circumference, blood pressure, HOMA-IR, AST, ALT, γ-GTP, uric acid and leptin levels decreased, in contrast, HDL-cholesterol, total ketone bodies, blood urea nitrogen, creatinine, RBC, hemoglobin and hematocrit levels increased, however, in DPP-4i sole group, no significant trends were observed in these parameters. Change in leptin positively correlated with insulin, while change in total ketone bodies inversely correlated with ALT in ipragliflozin using groups. Fasting active gastric inhibitory polypeptide levels decreased in ipragliflozin sole group. Glucagon showed no changes. No significant safety concerns were observed in this study. CONCLUSIONS: Ipragliflozin is useful and safe, showing some contrastive effects on several clinical parameters which are not shown with DPP-4i, resulting several clinical benefits. The co-administration of ipragliflozin and a DPP-4i has a better clinical outcome than either single-agent therapy.

QuickIsoSeq for Isoform Quantification in Large-Scale RNA Sequencing.

RNA-sequencing (RNA-seq) is a powerful technology for transcriptome profiling. While most RNA-seq projects focus on gene-level quantification and analysis, there is growing evidence that most mammalian genes are alternatively spliced to generate different isoforms that can be subsequently translated to protein molecules with diverse or even opposing biological functions. Quantifying the expression levels of these isoforms is key to understanding the genes biological functions in healthy tissues and the progression of diseases. Among open source tools developed for isoform quantification, Salmon, Kallisto, and RSEM are recommended based upon previous systematic evaluation of these tools using both experimental and simulated RNA-seq datasets. However, isoform quantification in practical RNA-seq data analysis needs to deal with many QC issues, such as the abundance of rRNAs in mRNA-seq, the efficiency of globin RNA depletion in whole blood samples, and potential sample swapping. To overcome these practical challenges, QuickIsoSeq was developed for large-scale RNA-seq isoform quantification along with QC. In this chapter, we describe the pipeline and detailed the steps required to deploy and use it to analyze RNA-seq datasets in practice. The QuickIsoSeq package can be downloaded from https://github.com/shanrongzhao/QuickIsoSeq.

Regardless of central obesity, metabolic syndrome is a significant predictor of type 2 diabetes in Japanese Americans.

AIMS/INTRODUCTION: The impact of metabolic syndrome (MetS) on the development of type 2 diabetes has been reported in different ethnic populations. However, whether central obesity is an essential component as a diagnostic criterion for MetS remains a controversial topic. The aim of the present study was to investigate the association between MetS and the incidence of type 2 diabetes with or without central obesity in a Japanese American population. MATERIALS AND METHODS: We examined whether MetS predicts incident type 2 diabetes among 928 Japanese American participants who did not have diabetes enrolled in an ongoing medical survey between 1992 and 2007. MetS was defined on the basis of American Heart Association/National Heart, Lung, and Blood Institute criteria. The average follow-up period was approximately 6.8 years. RESULTS: During the follow-up period, 116 new cases of diabetes were diagnosed. Compared to the participants without MetS, the hazard ratio (HR) for incident type 2 diabetes was significantly higher in participants with MetS, after adjustment for sex, age and impaired glucose tolerance (HR 1.64, 95% CI 1.11-2.42). The risk of type 2 diabetes was found to be significantly higher in participants with MetS but without central obesity (HR 2.07, 95% CI 1.25-3.41), as well as in participants with MetS and with central obesity (HR 2.46, 95% CI 1.51-4.01) than in participants with neither MetS nor central obesity, after adjustment for sex, age and impaired glucose tolerance. CONCLUSIONS: These results show that the presence of MetS, with or without central obesity, could independently predict the development of type 2 diabetes in Japanese Americans.

Early depth assessment of local burns by videomicroscopy: 24 h after injury is a critical time point.

PURPOSE: Videomicroscopy has simple and prompt operability, and useful in the burn depth assessment in its early phase. A burn wound is, however, a dynamic environment in the first few days and the critical time to assess a burn wound by videomicroscopy has not been investigated. The aim of this study is to investigate the critical time point to assess the burn depth by videomicroscopy. METHODS: Forty one patients with 44 intermediate depth burns admitted within 7 days after injury were included. Accuracies were assessed by comparison with clinical outcome: healing within 21 days after injury or not with conservative treatment. We prospectively evaluated and compared the accuracy of the videomicroscopy measurements with the clinical assessments. All findings were serialized in order of time after injury and divided into three groups, and we compared the appreciation of burn depth by videomicroscopy findings among groups. RESULTS: The videomicroscopy measurements is significantly accurate compared with clinical assessments (p=0.001). The accuracy of videomicroscopy measurements was significantly lower in the post-injury <24 h group compared with post-injury ≥24 h group (p=0.004). CONCLUSION: Videomicroscopy is effective tool in assessment of early burn depth and the critical time point to assess the burn depth by videomicroscopy is 24 h after injury.

Association of genetic polymorphisms in the influx transporter SLCO1B3 and the efflux transporter ABCB1 with imatinib pharmacokinetics in patients with chronic myeloid leukemia.

This study explored the association of 14 single nucleotide polymorphisms in three genes coding for influx transporters (SLC22A1, SLCO1B1, and SLCO1B3), two genes coding for efflux transporters (ABCB1 and ABCG2), and four genes coding for enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A5) with the pharmacokinetics of imatinib in Japanese patients with chronic myeloid leukemia. Pharmacokinetic parameters were estimated by a population pharmacokinetic analysis based on 622 plasma samples from 34 patients at steady state. Approximately 4.6-fold variability in individual clearance was observed (range, 3.4-15.5 L/hr). The individual estimated clearance was significantly increased in patients with the SLCO1B3 334GG genotype (median value ± standard deviation, 9.5 ± 3.1 L/hr; n = 19) compared with SLCO1B3 334TT and TG genotypes (7.0 ± 3.1 L/hr; n = 15) (P = 0.019). Patients with the ABCB1 3435CC genotype had significantly higher imatinib clearance (12.7 ± 3.0 L/hr; n = 7) compared with patients with ABCB1 3435CT and TT genotypes (7.9 ± 2.7 L/hr; n = 27) (P = 0.035). In conclusion, the present study suggests that single nucleotide polymorphisms of the influx transporter SLCO1B3 and the efflux transporter ABCB1 were functionally associated with individual variability of imatinib pharmacokinetics in Japanese patients with chronic myeloid leukemia.

Association of SLCO1B3 polymorphism with intracellular accumulation of imatinib in leukocytes in patients with chronic myeloid leukemia.

Intracellular concentration of imatinib in leukemic cells is thought to affect the clinical efficacy of this drug in patients with chronic myeloid leukemia (CML); however, there is no report that directly indicates the relationship between intracellular concentration and clinical outcome and/or, plasma concentration. In addition, the impacts of genetic variations of drug transporters, which mediate leukocyte concentration of imatinib, are unknown. In the present study, we investigated the correlation between intracellular imatinib concentrations in leukocytes, plasma imatinib levels, and genotypes of drug transporters, including ATP binding cassette B1 (ABCB), ABCG2, solute carrier 22A1 (SLC22A1), solute carrier organic anion transporter family members 1B1 (SLCO1B1) and SLCO1B3. The imatinib levels in leukocytes were determined using HPLC in 15 patients with chronic phase CML. No significant correlation between intracellular and plasma concentrations of imatinib was observed. The intracellular concentration was comparable in both patients with or without complete cytogenetic response. The intracellular imatinib concentration was significantly higher in patients with SLCO1B3 334TT than in those with 334TG/GG (p=0.0188). Plasma concentrations were similar in both SLCO1B3 genotypes (p=0.860), thereby resulting in the intracellular to plasma concentration ratio being higher in patients with SLCO1B3 334TT than those with 334 TG/GG (p=0.0502). These results suggested that the SLCO1B3 334T>G polymorphism could have a significant impact on the intracellular concentration of imatinib in leukocytes as a promising biomarker for personalized treatment of CML patients.

Improvement of hypercortisolism by ß-blocker therapy in subclinical Cushing's syndrome associated with ACTH-independent macronodular adrenocortical hyperplasia.

A 61-year-old man with hypertension and diabetes was referred for the evaluation of multiple bilateral adrenal tumors. While Cushingoid features were not apparent, an elevated cortisol level in response to a low-dose dexamethasone suppression test (187.7 nmol/l), an elevated urinary cortisol level (170.9 nmol/day), and a weak response to a cosyntropin-releasing hormone (CRH) provocation test were observed. Furthermore, the serum cortisol level increased in response to a posture test or isoproterenol infusion. Accordingly, the patient was diagnosed as having ACTH-independent macronodular adrenal hyperplasia (AIMAH) with subclinical Cushing's syndrome associated with the aberrant expression of ß-adrenergic receptors. After 2 months of propranolol therapy, the serum cortisol responses to a posture test and isoproterenol infusion, the cortisol level in response to a low-dose dexamethasone suppression test (102.1 nmol/l), and the urinary cortisol level (165.9 nmol/day) all normalized. While the suppression of cortisol secretion was sustained for 24 months, glucose metabolism and adrenal size were unaffected. To our knowledge, this is the first report of AIMAH accompanied by subclinical Cushing's syndrome associated with the aberrant expression of ß-adrenergic receptors. Furthermore, propranolol inhibited cortisol hypersecretion in the present case. Additional cases or controlled studies are needed to determine the potential effect of propranolol on metabolic disorders and adrenal size in patients with AIMAH.

Relationship between an effective dose of imatinib, body surface area, and trough drug levels in patients with chronic myeloid leukemia.

The standard dose of imatinib for the treatment of chronic-phase chronic myeloid leukemia (CML) is 400 mg/day. Some patients receive reduced doses of imatinib because of serious adverse effects. Recently, the effective plasma threshold for trough imatinib levels was demonstrated to be 1,002 ng/mL. In this study, we evaluated the association of an imatinib dose with trough plasma concentrations and clinical outcomes in 31 patients with chronic-phase CML who were treated at Kumamoto University Hospital. Twenty-seven patients were optimally treated with various doses of imatinib. The mean (+/-SD) trough plasma concentrations of imatinib were 1.40 +/- 0.57 microg/mL in 13 patients receiving 400 mg/day and 1.15 +/- 0.44 microg/mL in 9 patients receiving 300 mg/day as an effective dose. Mean trough levels of the two groups were not significantly different and exceeded the effective plasma threshold. Body surface area (BSA) was significantly smaller in patients receiving the reduced dose compared with those receiving the standard dose (p = 0.001). The effective imatinib dose was associated with age and gender as well as BSA. A reduced dose of 300 mg/day of imatinib may be sufficient for the treatment of CML patients with smaller body size, particularly when intolerability arises.

Is there any association between subcutaneous adipose tissue area and plasma total and high molecular weight adiponectin levels?

This study was conducted for the purpose of clarifying the correlations between the subcutaneous adipose tissue area and plasma total and high-molecular weight (HMW) adiponectin levels. The subjects of this study comprised 359 men and 142 women who underwent general health examinations from October 2005 to December 2006. The abdominal subcutaneous and visceral adipose tissue areas were measured using low-dose x-ray computed tomography. Total and HMW adiponectin levels were measured using the enzyme-linked immunosorbent assay system based on a monoclonal antibody to humans. There were negative correlations between the plasma total and HMW adiponectin levels and visceral and subcutaneous adipose tissue areas using simple correlation analysis. Multiple linear regression analysis clearly indicated that the subcutaneous adipose tissue area was independently correlated with the HMW adiponectin levels in men and closely related in women. Many studies reported that only the visceral adipose tissue area showed a significant correlation with metabolic syndrome. However, these results clearly indicate that it is also important to consider the subcutaneous adipose tissue area in metabolic syndrome.

Constitutive overexpression of P-glycoprotein, rather than breast cancer resistance protein or organic cation transporter 1, contributes to acquisition of imatinib-resistance in K562 cells.

PURPOSE: The purpose of this study was to investigate the contribution of drug transporters in acquired imatinib-resistance. Specifically, we focused on the efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), and an influx transporter, organic cation transporter 1 (OCT1). MATERIALS AND METHODS: We established imatinib-resistant K562 cells (K562/IM). Real-time PCR or Western blot analyses were performed to examine the mRNA or protein levels. Alamar blue method was used in the cytotoxicity assay. The transport activities and intracellular imatinib levels were measured by flow cytometry and HPLC, respectively. RESULTS: K562/IM displayed a 47-fold increase in resistance to imatinib over the parent K562 cells. Both P-gp and BCRP were overexpressed in K562/IM relative to K562. Furthermore, the intracellular imatinib level was markedly reduced in K562/IM. Interestingly, cyclosporin A, a P-gp inhibitor, but not fumitremorgin C, a BCRP inhibitor, restored both imatinib-sensitivity and the intracellular imatinib level. By contrast, no significant difference in the expression and function of OCT1 was observed between K562/IM and K562. CONCLUSIONS: P-gp, rather than BCRP or OCT1, is partially responsible for the development of imatinib-resistance due to constitutive and functional overexpression, leading to reduced intracellular accumulation of imatinib in K562/IM.

Fenofibrate increases high molecular weight adiponectin in subjects with hypertriglyceridemia.

Beneficial effects of peroxisome proliferator-activated receptor alpha (PPAR alpha) agonists have been reported in improving insulin sensitivity and raising serum total adiponectin. High molecular weight (HMW) adiponectin, which is secreted from adipocytes, and visfatin, which is also expressed in adipose tissue, is related to glucose metabolism. In view of the additive effects of PPAR alpha agonists on these adipocytokines and glucose metabolism, we investigated male hypertriglyceridemic subjects who were treated with fenofibrate. Eleven male subjects with hypertriglyceridemia were treated with fenofibrate and serum total cholesterol (T-cho), triglyceride, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), fasting glucose, fasting insulin, total and HMW adiponectin, and serum visfatin levels were determined before and 3 months after treatment. Fenofibrate treatment significantly lowered T-cho, triglyceride, and LDL-C levels. There was a statistically significant increase of HDL-C. No differences in insulin sensitivity indices (G/I ratio and HOMA-IR) were observed between before and after treatment with fenofibrate. The treatment did not alter the levels of serum total adiponectin and visfatin in the hypertriglyceridemic patients, while serum HMW adiponectin increased significantly. This study demonstrates that fenofibrate increases serum HMW adiponectin levels, whereas visfatin is not regulated by fenofibrate in hypertriglyceridemic subjects. Further investigations are warranted to determine whether the elevation of HMW adiponectin caused by fenofibrate might improve insulin sensitivity.

Serum leptin and adiponectin are positively associated with bone mineral density at the distal radius in patients with type 2 diabetes mellitus.

There have been several reports about associations of serum leptin or adiponectin with bone mineral density and biochemical markers of bone turnover. However, the precise roles of adipocytokines in bone metabolism have not been fully elucidated. We investigated the associations of serum level of leptin or adiponectin with bone mineral density, serum osteocalcin, and urinary N-terminal telopeptide of type I collagen (NTX) in 40 Japanese patients with type 2 diabetes mellitus. Bone mineral density was measured by using dual-energy x-ray absorptiometry at different sites (distal radius, femoral neck, and lumbar spine) and was expressed as z score. Multiple regression analysis revealed that there were significant positive correlations between serum leptin or adiponectin level and z score at the distal radius, but not at the femoral neck or the lumbar spine. Although no correlation was observed between serum leptin and serum osteocalcin, there was a significant negative correlation between serum leptin and urinary NTX, a marker of bone resorption. No correlation was observed between serum adiponectin and serum osteocalcin or urinary NTX. These results indicate that leptin and adiponectin may have a protective effect on bone metabolism in patients with type 2 diabetes mellitus.

Decreased total and high molecular weight adiponectin are independent risk factors for the development of type 2 diabetes in Japanese-Americans.

CONTEXT: Adiponectin is a hormone secreted by adipocytes that acts as an antidiabetic adipokine. Adiponectin exists as multimers in plasma, and high molecular weight (HMW) adiponectin is particularly thought to be the active form of the protein. OBJECTIVE: The aim of the study was to assess whether decreased total and HMW adiponectin are independent risk factors for the development of type 2 diabetes. DESIGN: Study subjects were Japanese-Americans enrolled in the Hawaii-Los Angeles-Hiroshima study between 1992 and 2002. Duration of follow-up was an average of 5.4 yr. PARTICIPANTS: We investigated 321 men and 445 women who were nondiabetic Japanese-Americans. Glucose tolerance was evaluated according to 1997 American Diabetes Association criteria, and 112 subjects developed type 2 diabetes during the follow-up period. MAIN OUTCOME MEASURE: The influence of baseline total and HMW adiponectin on the development of type 2 diabetes was the main outcome measure. RESULTS: Subjects who developed type 2 diabetes had significantly decreased plasma total and HMW adiponectin compared with those who did not develop the disease (P < 0.001, respectively). In a Cox proportional hazards model, both decreased total and HMW adiponectin levels were independent risk factors for the progression to type 2 diabetes after adjusting for sex, age, body mass index, waist-to-hip ratio, homeostasis model assessment, and classification of 75-g glucose tolerance test (hazards ratio: total, 0.600, P = 0.018; HMW, 0.614, P = 0.001, respectively). Dividing tertiles of adiponectin, hazards ratios in the lowest vs. highest tertile were total, 1.787 (95% confidence interval, 1.006-3.173); and HMW, 2.493 (95% confidence interval, 1.342-4.632), after similar adjustments. CONCLUSIONS: Decreased total adiponectin is an independent risk factor for the progression to type 2 diabetes in Japanese-Americans. Moreover, HMW adiponectin more closely associates with the progression to type 2 diabetes when compared with total adiponectin.