Low Frequency and Variability of FLT3 Mutations in Korean Patients with Acute Myeloid Leukemia

FLT3 mutations are common genetic changes, and are reported to have prognostic significance in acute myeloid leukemia (AML). The FLT3 internal tandem duplication (ITD) and the D835 activating mutation in the tyrosine kinase domain (TKD) were analyzed by polymerase chain reaction (PCR) in the genomic DNA of Korean patients with AML at diagnosis and during follow-up. There were 226 patients with AML enrolled between March 1996 and August 2005. The incidence of ITD and TKD at diagnosis was 13% (29/226) and 3% (6/226). When compared to Western and other Asian patients with AML, Korean patients had a lower frequency by about two-thirds of ITD and TKD. Among the non-M3 cases (N=203), the patients with an ITD had a significantly shorter event-free survival when compared with those without an ITD (p=0.0079). Among 54 relapsed patients, 9 patients had the FLT3 ITD at diagnosis. Six patients demonstrated a reappearance of the ITD and 3 patients remained negative at relapse. One patient, among 45 patients who relapsed, had a negative baseline ITD but acquired a de novo ITD at relapse. There were 101 samples from 93 patients in remission; they were all negative for an ITD. Among 34 patients who failed to achieve a remission, five patients had a persistent ITD and one patient had a de novo ITD. These results support the concept of resistance of FLT3 ITD leukemic clones to chemotherapy. Therefore, effective therapy with FLT3 targeting agents may improve the prognosis of non-M3 AML patients with the FLT3 mutation

High-throughput Metabolism-induced Toxicity Assays Demonstrated on a 384-pillar Plate

The US Environmental Protection Agency (EPA) launched the Transform Tox Testing Challenge in 2016 with the goal of developing practical methods that can be integrated into conventional high-throughput screening (HTS) assays to better predict the toxicity of parent compounds and their metabolites in vivo. In response to this need and to retrofit existing HTS assays for assessing metabolism-induced toxicity of compounds, we have developed a 384-pillar plate that is complementary to traditional 384-well plates and ideally suited for culturing human cells in three dimensions at a microscale. Briefly, human embryonic kidney (HEK) 293 cells in a mixture of alginate and Matrigel were printed on the 384-pillar plates using a microarray spotter, which were coupled with 384-well plates containing nine model compounds provided by the EPA, five representative Phase I and II drug metabolizing enzymes (DMEs), and one no enzyme control. Viability and membrane integrity of HEK 293 cells were measured with the calcein AM and CellTiter-Glo (R) kit to determine the IC50 values of the nine parent compounds and DME-generated metabolites. The Z\u27 factors and the coefficient of variation measured were above 0.6 and below 14%, respectively, indicating that the assays established on the 384-pillar plate are robust and reproducible. Out of nine compounds tested, six compounds showed augmented toxicity with DMEs and one compound showed detoxification with a Phase II DME. This result indicates that the 384-pillar plate platform can be used to measure metabolism-induced toxicity of compounds in high-throughput with individual DMEs. As xenobiotics metabolism is a complex process with a variety of DMEs involved, the predictivity of our approach could be further improved with mixtures of DMEs

High-Content Imaging of 3D-Cultured Neural Stem Cells on a 384-Pillar Plate for the Assessment of Cytotoxicity

© 2020 Elsevier Ltd The assessment of neurotoxicity has been performed traditionally with animals. However, in vivo studies are highly expensive and time-consuming, and often do not correlate to human outcomes. Thus, there is a need for cost-effective, high-throughput, highly predictive alternative in vitro test methods based on early markers of mechanisms of toxicity. High-content imaging (HCI) assays performed on three-dimensionally (3D) cultured cells could provide better understanding of the mechanism of toxicity needed to predict neurotoxicity in humans. However, current 3D cell culture systems lack the throughput required for screening neurotoxicity against a large number of chemicals. Therefore, we have developed miniature 3D neural stem cell (NSC) culture on a unique 384-pillar plate, which is complementary to conventional 384-well plates. Mitochondrial membrane impairment, intracellular glutathione level, cell membrane integrity, DNA damage, and apoptosis have been tested against 3D-cultured ReNcell VM on the 384-pillar plate with four model compounds rotenone, 4-aminopyridine, digoxin, and topotecan. The HCI assays performed in 3D-cultured ReNcell VM on the 384-pillar plates were highly robust and reproducible as indicated by the average Z′ factor of 0.6 and CV values around 12%. From concentration-response curves and IC50 values, mitochondrial membrane impairment appears to be the early stage marker of cell death by the compounds

Suppression of the antitumoral activity of natural killer cells under indirect coculture with cancer-associated fibroblasts in a pancreatic TIME-on-chip model

Abstract Background Recently, natural killer (NK) cells emerged as a treatment option for various solid tumors. However, the immunosuppressive tumor immune microenvironment (TIME) can reduce the cytotoxic ability of NK cells in pancreatic ductal adenocarcinoma. Cancer-associated fibroblasts within the tumor stroma can suppress immune surveillance by dysregulating factors involved in the cellular activity of NK cells. Herein, the effect of activated pancreatic stellate cells (aPSCs) on NK cell-mediated anticancer efficacy under three-dimensional (3D) coculture conditions was investigated. Methods 3D cocultures of PANC-1 tumor spheroids (TSs) with aPSCs and NK-92 cells in a collagen matrix were optimized to identify the occurring cellular interactions and differential cytokine profiles in conditioned media using microchannel chips. PANC-1 TSs and aPSCs were indirectly cocultured, whereas NK-92 cells were allowed to infiltrate the TS channel using convective medium flow. Results Coculture with aPSCs promoted PANC-1 TSs growth and suppressed the antitumor cytotoxic effects of NK-92 cells. Mutual inhibition of cellular activity without compromising migration ability was observed between aPSCs and NK-92 cells. Moreover, the reduced killing activity of NK-92 cells was found to be related with reduced granzyme B expression in NK cells. Conclusions Herein, a novel TIME-on-chip model based on the coculture of PANC-1 TSs, aPSCs, and NK-92 cells was described. This model may be useful for studying the detailed mechanisms underlying NK cells dysregulation and for exploring future therapeutic interventions to restore NK cell activity in the tumor microenvironment

3D-Cultured Neural Stem Cell Microarrays on a Micropillar Chip for High-Throughput Developmental Neurotoxicology

© 2018 Numerous chemicals including environmental toxicants and drugs have not been fully evaluated for developmental neurotoxicity. A key gap exists in the ability to predict accurately and robustly in vivo outcomes based on in vitro assays. This is particularly the case for predicting the toxicity of chemicals on the developing human brain. A critical need for such in vitro assays is choice of a suitable model cell type. To that end, we have performed high-throughput in vitro assessment of proliferation and differentiation of human neural stem cells (hNSCs). Conventional in vitro assays typically use immunofluorescence staining to quantify changes in cell morphology and expression of neural cell-specific biomarkers, which is often time-consuming and subject to variable specificities of available antibodies. To alleviate these limitations, we developed a miniaturized, three-dimensional (3D) hNSC culture with ReNcell VM on microarray chip platforms and established a high-throughput promoter-reporter assay system using recombinant lentiviruses on hNSC spheroids to assess cell viability, self-renewal, and differentiation. Optimum cell viability and spheroid formation of 3D ReNcell VM culture were observed on a micropillar chip over a period of 9 days in a mixture of 0.75% (w/v) alginate and 1 mg/mL growth factor reduced (GFR) Matrigel with 25 mM CaCl2 as a crosslinker for alginate. In addition, 3D ReNcell VM culture exhibited self-renewal and differentiation on the microarray chip platform, which was efficiently monitored by enhanced green fluorescent protein (EGFP) expression of four NSC-specific biomarkers including sex determining region Y-box 2 (SOX2), glial fibrillary acidic protein (GFAP), synapsin1, and myelin basic protein (MBP) with the promoter-reporter assay system

Susceptibility and trailing growth of Candida albicans to fluconazole: results of a Korean multicentre study

The work reported here is the first nationwide, multicenter surveillance study conducted in Korea to obtain data on fluconazole susceptibility of Candida albicans (C. albicans) isolates. A total of 1137 isolates of C. albicans obtained from 17 university hospitals in South Korea during the 6-month period, July through December 2004, were tested. No resistant strains were observed in any of the isolates. Only five of the 1137 isolates (0.44%) of C. albicans were found to be susceptible dose dependent, with all remaining strains (99.56%) susceptible to fluconazole. Trailing growth at 48 h was found in only four isolates (0.35%)

Guidelines for Mobile Laboratories for Molecular Diagnostic Testing of COVID-19

With the rapid spread of the coronavirus disease (COVID-19), the need for rapid testing and diagnosis and consequently, the demand for mobile laboratories have increased. Despite this need, there are no clear guidelines for the operation, maintenance, or quality control of mobile laboratories. We provide guidelines for the operation, management, and quality control of mobile laboratories, and specifically for the implementation and execution of COVID-19 molecular diagnostic testing. These practical guidelines are primarily based on expert opinions and a laboratory accreditation inspection checklist. The scope of these guidelines includes the facility, preoperative evaluation, PCR testing, internal and external quality control, sample handling, reporting, laboratory personnel, biosafety level, and laboratory safety management. These guidelines are useful for the maintenance and operation of mobile laboratories not only in normal circumstances but also during public health crises and emergencies.N