Rapid and Sensitive Assessment of Globin Chains for Gene and Cell Therapy of Hemoglobinopathies

The β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous β-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer from long run times, high sample requirements, or inability to separate murine from human β-globin chains. The latter point is problematic for in vivo studies with gene-addition vectors in murine disease models and mouse/human chimeras. This study demonstrates HPLC-based measurements of globin expression (1) after differentiation of the commonly applied human umbilical cord blood-derived erythroid progenitor-2 cell line, (2) in erythroid progeny of CD34+ cells for the analysis of clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of the globin regulator BCL11A, and (3) of transgenic mice holding the human β-globin locus. At run times of 8 min for separation of murine and human β-globin chains as well as of human γ-globin chains, and with routine measurement of globin-chain ratios for 12 nL of blood (tested for down to 0.75 nL) or of 300,000 in vitro differentiated cells, the methods presented here and any variant-specific adaptations thereof will greatly facilitate evaluation of novel therapy applications for β-hemoglobinopathies

Normal growth and development in mice over-expressing the CCN family member WISP3

Loss-of-function mutations in the gene WISP3 cause the autosomal recessive human skeletal disease Progressive Pseudorheumatoid Dysplasia, whereas mice with knockout mutations of Wisp3 have no phenotype. The lack of a phenotype in the Wisp3 knockout mice has constrained studies of the protein’s in vivo function. Over-expression experiments in zebrafish indicated that WISP3 may function as a BMP and Wnt signaling modulator. To determine whether these biologic activities are retained in mice, we created two strains of transgenic mice that over-express WISP3 in a broad array of tissues. Despite strong and persistent protein over-expression, the transgenic mice remained phenotypically indistinguishable from their non-transgenic littermates. Surprisingly, WISP3 contained in conditioned medium recovered from transgenic mouse primary kidney cell cultures was able to bind BMP and to inhibit BMP signaling in vitro. Factors that account for the difference between the in vitro and in vivo activities of WISP3 remain unknown. At present, the mouse remains a challenging model organism in which to explore the biologic function of WISP3

CVIT expert consensus document on primary percutaneous coronary intervention (PCI) for acute myocardial infarction (AMI) in 2018

While primary percutaneous coronary intervention (PCI) has significantly contributed to improve the mortality in patients with ST segment elevation myocardial infarction even in cardiogenic shock, primary PCI is a standard of care in most of Japanese institutions. Whereas there are high numbers of available facilities providing primary PCI in Japan, there are no clear guidelines focusing on procedural aspect of the standardized care. Whilst updated guidelines for the management of acute myocardial infarction were recently published by European Society of Cardiology, the following major changes are indicated; (1) radial access and drug-eluting stent over bare metal stent were recommended as Class I indication, and (2) complete revascularization before hospital discharge (either immediate or staged) is now considered as Class IIa recommendation. Although the primary PCI is consistently recommended in recent and previous guidelines, the device lag from Europe, the frequent usage of coronary imaging modalities in Japan, and the difference in available medical therapy or mechanical support may prevent direct application of European guidelines to Japanese population. The Task Force on Primary Percutaneous Coronary Intervention of the Japanese Association of Cardiovascular Intervention and Therapeutics (CVIT) has now proposed the expert consensus document for the management of acute myocardial infarction focusing on procedural aspect of primary PCI

PLGA-Nanoparticles for Intracellular Delivery of the CRISPR-Complex to Elevate Fetal Globin Expression in Erythroid Cells

Ex vivo gene editing of CD34+ hematopoietic stem and progenitor cells (HSPCs) offers great opportunities to develop new treatments for a number of malignant and non-malignant diseases. Efficient gene-editing in HSPCs has been achieved using electroporation and/or viral transduction to deliver the CRISPR-complex, but cellular toxicity is a drawback of currently used methods. Nanoparticle (NP)-based gene-editing strategies can further enhance the gene-editing potential of HSPCs and provide a delivery system for in vivo application. Here, we developed CRISPR/Cas9-PLGA-NPs efficiently encapsulating Cas9 protein, single gRNA and a fluorescent probe. The initial ‘burst’ of Cas9 and gRNA release was followed by a sustained release pattern. CRISPR/Cas9-PLGA-NPs were taken up and processed by human HSPCs, without inducing cellular cytotoxicity. Upon escape from the lysosomal compartment, CRISPR/Cas9-PLGA-NPs-mediated gene editing of the γ-globin gene locus resulted in elevated expression of fetal hemoglobin (HbF) in primary erythroid cells. The development of CRISPR/Cas9-PLGA-NPs provides an attractive tool for the delivery of the CRISPR components to target HSPCs, and could provide the basis for in vivo treatment of hemoglobinopathies and other genetic diseases

Positive Regulation by GABABR1 Subunit of Leptin Expression through Gene Transactivation in Adipocytes

Background: The view that c-aminobutyric acid (GABA) plays a functional role in non-neuronal tissues, in addition to an inhibitory neurotransmitter role in the mammalian central nervous system, is prevailing, while little attention has been paid to GABAergic signaling machineries expressed by adipocytes to date. In this study, we attempted to demonstrate the possible functional expression of GABAergic signaling machineries by adipocytes. Methodology/Principal Findings: GABAB receptor 1 (GABABR1) subunit was constitutively expressed by mouse embryonic fibroblasts differentiated into adipocytes and adipocytic 3T3-L1 cells in culture, as well as mouse white adipose tissue, with no responsiveness to GABA BR ligands. However, no prominent expression was seen with mRNA for GABA BR2 subunit required for heteromeric orchestration of the functional GABABR by any adipocytic cells and tissues. Leptin mRNA expression was significantly and selectively decreased in adipose tissue and embryonic fibroblasts, along with drastically reduced plasma leptin levels, in GABA BR1-null mice than in wild-type mice. Knockdown by siRNA of GABA BR1 subunit led to significant decreases in leptin promoter activity and leptin mRNA levels in 3T3-L1 cells. Conclusions/Significance: Our results indicate that GABABR1 subunit is constitutively expressed by adipocytes to primarily regulate leptin expression at the transcriptional level through a mechanism not relevant to the function as a partner o

A20 (TNFAIP3) Deletion in Epstein-Barr Virus-Associated Lymphoproliferative Disorders/Lymphomas

A negative regulator of the nuclear factor (NF)-kappa B pathway, A20 (TNFAIP3), is inactivated in several types of lymphomas; particularly in diffuse large B-cell lymphoma (DLBCL), classical Hodgkin's lymphoma, and extranodal marginal zone lymphoma of the mucosa-associated lymphoid tissue. These findings suggest that the NF-kappa B activation is related to A20 inactivation. Recently, A20 inactivation has also been observed in Epstein-Barr virus (EBV)-related lymphomas; however, this occurrence has not been well investigated. Moreover, NF-kappa B is a key molecule in activated B-cell-like (ABC)-type DLBCL; EBV-associated DLBCL is of the ABC type. Therefore, we focused on A20 deletions in EBV-associated lymphoproliferative disorders/lymphomas. Using fluorescent in situ hybridization analysis, A20 deletions were identified in 4 of 13 samples from patients with pyothorax-associated lymphoma (PAL) (31%), 3 of 20 samples from nasal-type NK/T cell lymphomas (NKTLs) (15%), 1 of 8 samples of EBV-positive DLBCL of the elderly (DLBCL-e) (13%), but not in any of the 11 samples from individuals with methotrexate-related lymphoproliferative disorder (MTX-LPD) (0%). Among the samples with A20 deletions, EBV latent membrane protein 1 (LMP-1) expression was detected in all 4 of the PAL samples with A20 deletions and in the DLBCL-e sample with an A20 deletion, but not in any of the 3 NKTL samples. This finding indicated that A20 deletions were not directly related to the EBV latency pattern of lymphomas, although such deletions might be related to the diagnostic category. Immunohistologically, the A20 protein was absent in 2 (15%) of the13 PAL samples, 1 (9%) of 11 MTX-LPD samples, and in none of the 20 NKTL (0%) or 8 DLBCL-e samples. In conclusion, A20 deletion and/or dysfunctional expression are frequently associated with PALs, and A20 abnormalities may be related to the pathogenesis of PAL

Extension of the operational regime of the LHD towards a deuterium experiment

As the finalization of a hydrogen experiment towards the deuterium phase, the exploration of the best performance of hydrogen plasma was intensively performed in the large helical device. High ion and electron temperatures, Ti and Te, of more than 6 keV were simultaneously achieved by superimposing high-power electron cyclotron resonance heating onneutral beam injection (NBI) heated plasma. Although flattening of the ion temperature profile in the core region was observed during the discharges, one could avoid degradation by increasing the electron density. Another key parameter to present plasma performance is an averaged beta value $\left\langle \beta \right\rangle$ . The high $\left\langle \beta \right\rangle$ regime around 4% was extended to an order of magnitude lower than the earlier collisional regime. Impurity behaviour in hydrogen discharges with NBI heating was also classified with a wide range of edge plasma parameters. The existence of a no impurity accumulation regime, where the high performance plasma is maintained with high power heating  >10 MW, was identified. Wide parameter scan experiments suggest that the toroidal rotation and the turbulence are the candidates for expelling impurities from the core region

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)