Iscove's Modified Dulbecco's Medium Research Articles

Electrokinetic Manipulation of Biological Cells towards Biotechnology Applications.

The presented study demonstrates the capability of the template-based electrokinetic assembly (TEA) and guidance to manipulate and capture individual biological cells within a microfluidic platform. Specifically, dielectrophoretic (DEP) focusing of K-562 cells towards lithographically-defined "wells" on the microelectrodes and positioning singles cells withing these "wells" was demonstrated. K-562 lymphoblast cells, are widely used in immunology research. The DEP guidance, particularly involving positive DEP (pDEP), enables the controlled guidance and positioning of conductive and dielectric particles, including biological cells, opening new directions for the accurate and efficient microassembly of biological entities, which is crucial for single cell analysis and other applications in biotechnology. The investigation explores the use of glassy carbon and gold as electrode materials. It was established previously that undiluted physiological buffer is unsuitable for inducing positive DEP (pDEP); therefore, the change of media into a lower ionic concentration is necessary. After pDEP was observed, the cells are resubmerged in the Iscove's modified Dulbecco's medium (IMEM), a cell culturing media, and incubated. A dead/alive staining assay was performed on the cells to determine their survival in the diluted buffer for the period required to capture them. The staining assay confirmed the cells' survival after being immersed in the diluted biological buffer necessary for electrokinetic handling. The results indicate the promise of the proposed electrokinetic bio-sorting technology for applications in tissue engineering, lab-on-a-chip devices, and organ-on-a-chip models, as well as contributing to the advancement of single cell analysis.

BACH1 Inhibitor Hpp-D Mediates γ-Globin Gene Activation in Sickle Erythroid Progenitors

Introduction: Sickle cell disease (SCD) is one of the most common β-hemoglobinopathy caused by a point mutations in the adult β-globin gene. These individuals are affected with clinical symptoms such as anemia, pain, and progressive organ damage creating a great burden to annual healthcare costs. Induction of fetal hemoglobin (HbF) using pharmacologic agents is considered as one of the best therapeutic strategies to manage clinical symptoms of SCD. At present, hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD patients, however, it is not effective in all patients. In our previous studies we confirmed a role of NRF2 in γ-globin gene regulation and established molecular mechanisms involving competitive binding of the transcription factors NRF2 and BACH1 in the γ-globin gene promoter, antioxidant response elements (ARE) (Zhu X et al., Haematologica. 2017). Moreover, Attucks and colleagues, our collaborators at vTv Therapeutics LLC showed that HPP-D, a BACH1 inhibitor promotes nuclear export of BACH1 and translocation of NRF2 to regulate antioxidant gene expression (Attucks OC et al., PLoS One. 2014). Therefore, we tested the ability of the novel small molecule BACH1 inhibitor, HPP-D to induce HbF expression by increasing competitive binding of NRF2 in the γ-globin ARE in KU812 cells and human sickle erythroid progenitors. Methods: Human KU812 cells were cultured in Iscove's Modified Dulbecco's medium (IMDM) along with 10% fetal bovine serum, penicillin (100 unit/ml), streptomycin (0.1 mg/ml) and 4mM L-Glutamine at 37oC with 5% CO2. Human sickle cell erythroid progenitors generated from peripheral blood mononuclear cells isolated from SCD patients, were cultured using a two-phase erythroid culturing system (Li et al., Haematologica. 2018). Both cell types were treated with 0.5, 2.5, 5, 10, and 20µM of HPPD, 100µM HU or 50µM hemin for 48 hours. The HbF positive cells (F cells) were measured by flow cytometry analysis, γ-globin, β -globin gene expressions were quantified by Real Time - qPCR and BACH1, α-tubulin, and HbF protein levels were measured by Western blot analysis. ChIP assay was performed to investigate binding competition between Bach1 and NRF2 at the locus control region-hypersensitive site 2 (LCR-HS2) and γ-globin ARE in KU812 cells. The effects of HPPD on sickling of erythroid progenitors were measured by in vitro sickling assay at 1% oxygen for 6 hrs in a hypoxic chamber. Results: The KU812 cells treated with lower concentrations of HPPD (0.5, 2.5 and 5 µM) did not show toxicity to cell growth and viability. Moreover, we observed a significant increase in HbF positive cells (F-cells) by 2.5-fold (0.5μM) and 1.6-fold (5μM) when compared to the vehicle control, DMSO. In addition, Western blot analysis showed a maximal 2.7-fold induction of HbF (p<0.05) by 0.5μM HPP-D. While KU812 cells treated with 0.5µM HPP-D alone showed 20% F-cells increase, when combined with 100µM HU the F-cells increased to 46%; similar findings were observed for mean fluorescence intensity analysis by flow cytometry analysis. Simillarly, when quantifying HbF protein levels by Western blot, 0.5 μM HPP-D alone produced a 1.4-fold increase (p<0.05) of HbF and when combined with 100µM HU showed 1.7-fold HbF increase. Remarkably, enhanced binding of NRF2 were observed in the γ-globin ARE and LCR-HS2 while BACH1 binding affinity reduced in HPP-D treated cells. By contrast, no significant change was observed for NRF2 binding in the β-globin promoter by HPP-D treatment by ChIP assay. To confirm HbF induction by HPP-D, we performed similar experiments conducted at low concentrations (0.5μM and 2.5μM) in sickle erythroid progenitors without affecting cell growth and viability; HPP-D induced HbF levels by 1.6- and 1.9-fold (p<0.05) respectively, without affecting HbS protein level. Moreover, under hypoxic conditions HPP-D treatment reduced erythroid cells sickling by 50% when compared to DMSO control. Conclusions: Overall, our study findings support the ability of HPP-D to induce γ-globin protein as a single agent or in combination with HU through BACH1 silencing and enhancing NRF2 binding in the ARE region. Additionally, HPP-D induced HbF expression and mediated an anti-sickling effect in sickle erythroid progenitors. Future in vivo studies in the SCD mouse model will be conducted confirm our in vitro findings.

Cytotoxicity and Efficacy in Debris and Smear Layer Removal of HOCl-Based Irrigating Solution: An In Vitro Study.

In the present study we evaluated the cytotoxicity of super-oxidized water on human gingival fibroblasts and its efficacy in debris and smear layer removal from root canal walls. Cultured gingival fibroblasts were exposed to super-oxidized water (Sterilox), which was diluted in Iscove’s modified Dulbecco’s medium (IMDM) at 30%, 40%, 50%, 60% and 70% concentrations. The control group was maintained in IMDM. The cell viability was evaluated by means of an MTT assay after incubation periods of 1 h, 2 h, 24 h and 48 h. Pathological cellular changes were also observed under fluorescence and phase contrast microscopes. The efficacy in debris and smear layer removal was evaluated in comparison to the conventional application of sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid (EDTA). Forty maxillary premolars were randomly divided into two equal groups (n = 20) and shaped with ProTaper NEXT rotary instruments using Sterilox or NaOCl/EDTA for irrigation. Afterwards, roots were split longitudinally and examined under a scanning electron microscope. The results revealed that super-oxidized water and sterile distilled water have acceptable biological properties for endodontic applications at concentrations up to 50% (p > 0.05). Moreover, super-oxidized water is equally effective in debris and smear layer removal as compared to NaOCl/EDTA (p > 0.05).

Effect of fetal liver condition media derived cytokines (IL-6 and Flt-3) on human bone marrow stem cells colony formation

Earlier research from our laboratory demonstrated the presence of stimulatory activity of different growth factors in the fetal liver (FL) extracts when collected in a medium known as fetal liver conditioned medium (FLCM) using Enzyme-linked Immunosorbent Assay (ELISA).In the present study, we have assessed two other cytokines viz. IL-6 and FMS like tyrosine kinase-3 (Flt-3) with the help of bioneutralization assay. FLCM was prepared by incubating fetal liver cells with Iscove’s Modified Dulbecco’s Medium (IMDM) containing 10% fetal bovine serum (FBS) and 10% Phytohemagglutinin and collected after 24hrs, 48hrs, 72 hrs. and on the 7th day of incubation. Clonal cultures were established for 1 X 105 normal bone marrow (BM) mononuclear cells (NBM MNC) per plate with methylcellulose medium containing cytokines SCF and EPO. Mean Colony forming units-granulocytes, erythrocytes, macrophages, megakaryocytes (CFU-GEMM) were assessed with and without the addition of FLCM. It was found that FLCM enhanced the number of colonies made by NBM MNCs. Further, cytokines IL-6 and Flt-3, present in FLCM, were bioneutralized with respective anti-cytokine antibodies. Neutralized FLCM was evaluated for the colony-forming potential of CFU-GEMM colonies. The maximum reduction of 42% was seen with 20 ng/ml of anti-IL-6 antibody. Maximum suppression up to 20% was observed with 0.7 ng/ml of anti Flt-3 antibody for CFU-GEMM colonies. Presence of cytokines IL-6 and Flt-3 in FL extracts and their colony stimulatory activity suggests that fetal liver infusion (FLI) may be a valuable alternative for managing BM recovery in certain clinical conditions such as AA.

The Effect of Platelet Lysate on Expansion and Differentiation Megakaryocyte Progenitor Cells from Cord Blood CD34+ enriched Cells

Background: Umbilical cord blood hematopoietic stem cells (UCB-HSCs) are an attractive source for transplantation. The generation of megakaryocyte-committed cells could lead to shorten period of thrombocytopenia after HSCs transplantation. Platelet lysate (PL) unlike fetal bovine serum (FBS) can prevent immune problems as well as avert transmission of certain diseases to the recipient. In this study, the authors aimed to assess the effect of PL on UCB CD34+ cells expansion and megakaryocyte differentiation. Materials and Methods: In this experimental study, PL prepared and the subsequent isolation of UCB CD34+ cells were done by magnetic cell sorting. The isolated cells were cultivated in Iscove’s Modified Dulbecco’s medium (IMDM) supplemented with PL or FBS. Cell expansion was evaluated using Trypan blue. Furthermore, Flow cytometry using monoclonal antibodies (CD41-FITC and CD42b-PE) and the expression of specific genes including GATA1, GATA2, FLI1, NFE2, and RUNX1 via real-time PCR were performed to evaluate the megakaryocyte differentiation. Results: The results showed that PL insignificantly enhanced UCB CD34+ cell expansion (32.83± 8.47 fold in FBS and 41.67± 10.31 fold in PL containing media). Besides, flow cytometry results showed that expression of CD41 was increased markedly (37.81± 4.78 fold in FBS and 45.78 ± 7.37 in PL containing media, P-value &lt;0.05) but the elevation of CD42b (10.53 ± 2.13 and 13.20 ± 2.06 in FBS and PL containing media, respectively) was not significant (P-value = 0.051). The results of real-time PCR demonstrated a notable increase in GATA binding protein 1 (1.58, P-value &lt;0.01), GATA binding protein 2 (2.45, P-value &lt;0.001), RUNX family transcription factor 1 (1.60, P-value &lt;0.01), Fli-1 proto-oncogene (1.87, P-value &lt;0.001) in PL supplemented media, however, the increase of Nuclear Factor-Erythroid 2 gene expression was not significant in PL supplemented media (P-value = 0.11). Conclusion: PL improved UCB CD34+ cells expansion and megakaryocyte differentiation compared to FBS.

Retraction

Background Small interfering RNAs (siRNAs) specifically target mRNAs and induce silencing of protein-coding genes. MicroRNAs are single-stranded RNA molecules that can target several genes. This study evaluated microRNA expression following the inhibition of γ to β-globin gene switching and examined the increased expression of γ genes through the effect of siRNAs on the B-cell lymphoma/leukemia 11A (BCL11A) gene. Methods Stem cells were isolated from peripheral blood mononuclear cells using the Ficoll-Paque method. The cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) for differentiation into erythroid cells over 28 days and stored under optimal conditions. On the 21st day, siRNAs were inserted into BCL11A. The expression levels of miR-15a, miR-16, miR-23a, miR-27a, and miR-486 were evaluated before and after siRNA treatment. The comparative CT method (2-ΔΔCT) was used to compare miRNA expression. For data analysis, Student's t-tests were performed, with P<0.05 considered statistically significant. Results The five miRNAs in this study showed significant differences (P<0.01). The relative expression of BCL11A after siRNA transmission decreased, while the expression of γ gene showed an increasing trend (1.2, 2.1, and 2.3, respectively). Conclusions The expression levels of miR-15a, miR-16, miR-23a, miR-27a, and miR-486 changed before and after γ-to-β gene switching. Therefore, this may be an effective intervention for controlling the switching and synthesis of fetal hemoglobin.

Peripheral Nerve Regeneration Using a Cytokine Cocktail Secreted by Skeletal Muscle-Derived Stem Cells in a Mouse Model

Severe peripheral nerve injury, which does not promise natural healing, inevitably requires clinical treatment. Here, we demonstrated the facilitation effect of peripheral nerve regeneration using a cytokine cocktail secreted by skeletal muscle-derived stem cells (Sk-MSCs). Mouse sciatic nerve was transected with a 6 mm gap and bridged collagen tube, and the culture supernatant of Sk-MSCs with 20% adult mouse serum (AMS)/Iscove’s modified Dulbecco’s medium (IMDM) was administered into the tube immediately after the operation, followed by an injection once a week for six weeks through the skin to the surrounding tube of the cytokine (CT) group. Similarly, 20% AMS/IMDM without cytokines was administered to the non-cytokine control (NT) group. Tension recovery in the plantar flexor muscles via electrical stimulation at the upper portion of the damaged nerve site, as well as the numerical recovery of axons and myelinated fibers at the damaged site, were evaluated as an index of nerve regeneration. Specific cytokines secreted by Sk-MSCs were compared with damaged sciatic nerve-derived cytokines. Six weeks after operation, significantly higher tension output and numerical recovery of the axon and myelinated fibers were consistently observed in the CT group, showing that the present cytokine cocktail may be a useful nerve regeneration acceleration agent. We also determined 17 candidate factors, which are likely included in the cocktail.

Abstract 17093: Human Neonatal Cardiac Mesenchymal Stem Cells Demonstrates Superior Cardiac Regenerative Abilities Compared to Other Stem Cells

Stem cells transplantation is being explored as an effective therapy for heart diseases. However, majority of stem cell therapies for adult patients with myocardial infarction (MI) had mixed and inconsistent results implying chronological age may influence the effectiveness of regenerative therapies. Therefore, herein, we performed a head-to-head comparison between different, well-studied stem cell types to identify the superior regenerative cell type using rodent MI model.After our standard characterization for each stem cell type (FACS for cell surface markers), 1 million neonatal Cardiac Mesenchymal Stem cells (nMSCs), adult MSCs (aMSCs), adult derived cardiosphere derived cells (aCDCs), umbilical cord derived cells (UCBCs), Bone Marrow derived Mesenchymal Stem cells (BM-MSCs), or cell-free Iscove Modified Dulbecco Medium (IMDM as placebo control) were injected into athymic rat myocardial infarct model. Although all the tested groups significantly improved ejection fraction, nMSCs outperformed other stem cells in cardiac functional recovery. Additionally, nMSCs also showed significant increased cardiac functional recovery compared to aMSCs in wild type rat MI model. Mason trichrome staining with heart sections revealed that decreased fibrosis was evident on nMSCs injection compared to aMSCs in both athymic and wild type rat MI model. Myocardial sections from rats received nMSCs showed significantly reduced M1 macrophages (inflammatory) and increased M2 macrophages (anti-inflammatory) compared with sections from rats having received aMSCs and IMDM control. Pro and anti-inflammatory cytokines analyzed on sera collected on day 2 and 7 revealed that anti-inflammatory cytokine (IL10) was significantly increased and inflammatory cytokines (IL4 and IL12) reduced in nMSCs compared to aMSCs transplanted MI rat model.In conclusion, nMSCs demonstrated superior functional abilities, reduced fibrosis, inflammatory cells and cytokines compared to all the other cell types and with aMSCs demonstrating that nMSCs is an ideal stem cell type for therapeutic application in myocardial infarction.

Multiplexed Functional Assessment of Genetic Variants in CARD11

SummaryGenetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a “cloning-free” saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR.

In kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor‐specific antibody development. Eplets are theoretically defined configurations of polymorphic amino acids and require experimental verification to establish whether they can be bound by alloantibodies. Human HLA‐specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we isolated single HLA‐DR‐specific memory B cells from peripheral blood of immunized individuals (n = 3) using HLA class II tetramers to generate recombinant human HLA‐DR antigen‐reactive mAbs (n = 5). Comparison of the amino acid composition of the reactive HLA alleles in relation to the antibody reactivity patterns led to identification of 3 configurations, 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognized, respectively, by HLA‐DRB1*01:01, HLA‐DRB1*04:01, and HLA‐DRB1*07:01 antigen‐reactive mAbs. The first 2 correspond to eplets 70QA and 31FYY and can now be considered antibody verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody verified. Generation and reactivity analysis of human HLA‐DR mAbs allowed for identification of amino acid configurations corresponding to known eplets, whereas the other patterns may be used to redefine eplets with similar, but not identical predicted amino acid composition.

Endogenous Fluorescence Dissimilarity Assessment of Four Potential Biomarkers of Early Liver Fibrosis by Preservation Media Effect.

Build-up of extracellular matrix in liver fibrosis results in changes on endogenous molecules expression that may be studied through the fluorescence characterization of ex vivo liver samples. To the best of our knowledge, no investigations have provided in-depth evidence and discussion on the changes of the endogenous fluorescence in ex vivo tissue due to the effects of the preservation media. In this work, we contrast and analyze the endogenous fluorescence from tryptophan, vitamin A, hydroxyproline and elastin cross-links potential biomarkers of the liver fibrosis, in in vivo measurements and liver samples preserved on formaldehyde, and two standard preservation media. As it is known, chemical changes in tissue, caused by formaldehyde fixation, alter the endogenous fluorescence spectra. We propose the use of phosphate-buffered saline (PBS), and Iscove's Modified Dulbecco's Medium (IMDM) to elude the fluorescence changes. PBS and IMDM showed to maintain the endogenous fluorescence characteristics similar to in vivo conditions. The results of this work point the way for a more reliable assessment of endogenous fluorescence in ex vivo hepatic studies.

Leukemic Stem Cell Phenotype Is Associated with Mutational Profile in Acute Myeloid Leukemia

Background: Understanding leukemic stem cell (LSC) is important for acute myeloid leukemia (AML) treatment. As such, understanding the relationship between LSC and genetically defined sub-clones can, in turn, help to understand the heterogeneity of AML. However, to date, there are only a few reports specifically focusing on this topic. To this end, we conducted this study to (1) examine the phenotypic diversity of AML-LSC, (2) explore the association between AML-LSC phenotypes and gene mutations, and (3) investigate the prognostic implications of AML-LSCs. Methods: Mononuclear cells (MNCs) were isolated from the patient's bone marrow aspirates by ficoll gradient centrifugation and cryopreserved in serum-free medium. Stored cells were thawed to Iscove's Modified Dulbecco's Medium (IMDM) and washed with fluorescence-activated cell sorting (FACS) buffer [1% FBS, Dulbecco's Phosphate-Buffered Saline (DPBS)]. Cells were stained with following anti-human monoclonal antibodies: CD45-APC/cy7, CD34-APC, CD38-BV421, CD90-PE, CD123-PE/Cy7, CD45RA-PerCP/Cy5.5. Analyses were performed on a FACSCanto II (HTS) (BD Bioscience) and FlowJo V 10.0 (BD Bioscience) program. For sequencing, the DNA capture probes for 76 target genes were designed using the Agilent SureDesign web-based application. The target regions included protein coding exons with 10 bp intron flanking regions and hot spot regions of the 20 genes involved in recurrent translocations. DNA was extracted on a Chemagic 360 instrument (Perkin Elmer, Baesweiler, Germany). The genomic DNA was sheared using Covaris S220 focused‐ultrasonicator (Covaris, Woburn, MA). We used 50ng of total input genomic DNA. A library preparation was performed according to Agilent's SureSelectQXT Target Enrichment protocol. Paired-end 150-bp sequencing was using NextSeq 550 Dx platform (Illumina, San Diego, CA). Targeted sequencing raw data was obtained in FASTQ format. Results: In secondary AML patients, MPP-like LSC was significantly higher than de-novo AML (p=0.0037), and was higher in MPN-AML than in MDS-AML (p=0.0485). There was no correlation between age and LSC phenotype, though CD34+CD38- subpopulation was enriched in younger patients (&lt;65 yrs). Mutations of KRAS and NRAS were frequently observed in MPP-like LSC dominant patients (3/14 and 4/14), TP53 and ASXL1 mutations in LMPP-like LSC dominant patients (4/12 and 4/12) , and CEBPA, DNMT3A and IDH1 (6/12, 4/12, and 3/12) mutations in GMP-like LSC dominant patients. Furthermore, as shown in Figure, KRAS mutation was significantly associated with the percentage of MPP-like LSC phenotype (p=0.0540), and TP53 mutation with the percentage of LMPP-like LSC phenotype (p=0.0276). When the patients were separated according to the combined risk including next generation sequencing data, the poorer the prognosis, the higher the LMPP-like LSC expression (p=0.0052). The importance of our study lies in that we showed for a given AML patients there is a dominant LSC phenotype and LSCs are associated with clinical outcomes, supporting the significance of cancer stem cell model for human AML. First of all, based on detailed characterization of the surface immunophenotype of AML LSCs we found that AML show evidence of a hierarchical cellular organization. We also recognized that the composition of LSC phenotypes is associated with AML phenotypes. For example, secondary AML patients showed higher fraction of MPP-like LSCs compared to de novo AML patients. In this regard, the higher expression of MPP-like LSCs could explain the poor response to standard treatments traditionally associated with secondary AML. Furthermore, the higher expression of MPP-like LSCs in post-MPN AML compared to post-MDS AML could explain the dismal prognosis associated with post-MPN AML, despite the relative indolent clinical course in their chronic phase and the presence of druggable target. Conclusion: In conclusion, our findings provide better insights into the characteristics and clinical implications of LSC. Although in a small scale, we provide evidence that specific LSC phenotypes are associated with certain mutations thus should be in the AML risk stratification process. Figure Disclosures Yoon: Janssen: Consultancy; Kyowa Hako Kirin: Research Funding; Genentech, Inc.: Research Funding; Yuhan Pharma: Research Funding; MSD: Consultancy; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Curcumin prevents experimental autoimmune encephalomyelitis by inhibiting proliferation and effector CD4+ T cell activation.

Multiple sclerosis (MS) has affected over 2 million people worldwide and it is thought to be initiated by the activated central nervous system (CNS). Reactive CD4+ T cells (TH1, TH17, and Treg phenotypes) are crucial to MS. The TH1 phenotype can promote major histocompatibility complex-II expression and TH17 can induce inflammatory gene expression. Curcumin, a yellow pigment, is found in turmeric rhizomes and has been reported to have various activities, such as anti-proliferative and anti-inflammatory activity. Curcumin has great potential in MS treatment. Little is known about the effect of curcumin on MS. Therefore, we investigated the effect of curcumin on MS, especially on CD4+ T cells. CD4+ T cells (TH1, TH17, and Treg cells) were cultured in Iscove's Modified Dulbecco's Medium (IMDM) medium. Cell proliferation was evaluated by MTT assay. The ability of individual CD4+ T cells to aggregate into viable colony clusters was assessed by clonogenic survival assay. Apoptosis of CD4+ T cells was determined by flow cytometry. The expression of Bcl-2, Bax, and active caspase-3 was detected by Western blotting. The effect of curcumin on the activation molecule was also evaluated by flow cytometry. MTT assay showed that curcumin significantly inhibited CD4+ T cell viability. Furthermore, TH1, TH17, and Treg all showed a dose-dependent but not time-dependent. The results of clonogenic survival assay revealed that curcumin markedly decreased the colony formation ability of CD4+ T cells. Flow cytometry results indicated that curcumin-induced remarkable apoptosis in TH1, TH17, and Treg cells. After treatment with curcumin, the expression of Bcl-2 was decreased and that of Bax and active caspase-3 was increased. Western blotting results also showed that curcumin-induced apoptosis in CD4+ T cells. Hence, our results demonstrated that curcumin inhibited CD4+ T cell proliferation via inducing apoptosis in CD4+ T cells. Meanwhile, flow cytometry results also showed that curcumin directly inhibited CD4+ T cell activation. Curcumin could inhibit CD4+ T cell proliferation and effector cell activation.

Functional flow cytometry of monocytes for routine diagnosis of innate primary immunodeficiencies

Functional flow cytometry of monocytes for routine diagnosis of innate primary immunodeficiencies

Recurrent inflammatory disease caused by a heterozygous mutation in CD48

Recurrent inflammatory disease caused by a heterozygous mutation in CD48

De novo generation of a functional human thymus from induced pluripotent stem cells

A proper functional thymus is required for generation of T cell–mediated immunity. This is dramatically illustrated by patients lacking a thymus, such as children with complete DiGeorge syndrome, which is fatal if left untreated.1-3 Therapeutic options for such patients are limited and confined to transplantation of small fragments from allogeneic neonatal thymi.

Evaluation of in vitro culture systems for the maintenance of microfilariae and infective larvae of Loa loa

BackgroundSuitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa.MethodsIn vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco’s modified Eagle’s medium (DMEM) and Iscove’s modified Dulbecco’s medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval.ResultsOf the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T90 of 16.4–19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture with LLC-MK2, and IMDM + BSA (1.5%), DMEM + FBS (10%) and DMEM + NCS (5%) without feeder cells. DMEM + 1% BSA in co-culture scored the highest moulting rate of 57 of 81 (70.37%). The factors that promoted L. loa mf viability included feeder cells (β = 0.490), both IMDM (β = 0.256) and DMEM (β = 0.198) media and the protein supplements NCS (β = 0.052) and FBS (β = 0.022); while for L. loa L3, in addition to feeder cells (β = 0.259) and both IMDM (β = 0.401) and DMEM (β = 0.385) media, the protein supplements BSA (β = 0.029) were found important in maintaining the worm motility.ConclusionsThe findings from this work display a range of culture requirements for the maintenance of Loa loa stages, which are suitable for developing an effective platform for drug screening.

Abstract 4823: Characterization of newly established WM-1-SNU cell line harboring negative for MYD88 L265P derived from Waldenstrom macroglobulinemia patient's peripheral blood using xenograft model

Abstract Waldenstrom macroglobulinemia (WM) is a rare hematologic malignancy that is a lymphoplasmacytic lymphoma a type of B-cell disease. The common characteristics of WM cells are expression of B cell markers, CD19 and CD20 and secretion of monoclonal immunoglobulin (Ig) M. The other critical feature of WM is MYD88 L265P that is found almost 90% patients. MYD88 gene is related with immune response and inflammatory signaling. Many researches are underway about MYD88 L265P role in WM. However, there is a lack of functional studies in the negative for MYD88 L265P in WM due to the absence of the MYD88 L265P negative cell line. Each cell lines represent the heterogeneity and molecular diversity by genetic features, it is important to establish the MYD88 L265P negative WM cell line for elucidating the biological characteristics of WM. Mononuclear cells obtained from patient’s peripheral blood by ficoll separation and were injected to NRG (JAX, USA) mouse via tail vein. After 24 days, mouse sacrificed and extracted bone marrow (BM) cells from mouse tibia by syringe flushing. Mouse BM cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 20% heat-inactivated FBS, Penicillin-Streptomycin (10,000 U/mL) (Gibco, USA). G-banding was used to confirm the human chromosomes. Sanger sequencing conducted for detecting the MYD88 L265P mutation. Flow cytometry was used for screening cell surface markers and ELISA was used for the detection of immunoglobulin level. For confirm the tumorigenicity of the cell line, we re-injected the cell line to NSG (JAX, USA) mouse by subcutaneously. After 3 weeks, tumor mass and metastatic tissues were cultured in conditioned medium. Tumor cells have had adherent phenotype and human chromosomes were identified by G-banding. MYD88 L265P mutation that genetic signature of WM was not detected both patient’s peripheral blood and established cell line. CD19 and CD20 expressions were not detected, but CD138 highly expression was confirmed about 90%. Lambda and Kappa secretion were not detected in cell culture supernatant. We confirmed the tumorigenicity through re-injection the cell line by subcutaneously. Tumor mass was formed with a 100% probability and metastasis to lung and bone marrow occurred in about 66%. Tumor mass and metastatic tissues were cultured in in vitro and confirmed the reproducibility. In this study, we describe the newly established human WM cell line derived from peripheral blood of the WM patient who negative for MYD88 L265P using xenograft model having unique biological characteristics comparing to MYD88 L265P positive cells that high tumor formation ability and high expression of CD138 and does not secrete IgM. Citation Format: Hyejoo Park, Youngil Koh, Kwang-Sung Ahn, Hyo Jung Kim, Sung-Soo Yoon. Characterization of newly established WM-1-SNU cell line harboring negative for MYD88 L265P derived from Waldenstrom macroglobulinemia patient's peripheral blood using xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4823. doi:10.1158/1538-7445.AM2017-4823

MiR-17-92 represses PTPROt and PP2A phosphatases and amplifies tonic BCR signaling in DLBCL cells.

B-cell receptor (BCR) signaling plays a pivotal role in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and targeting the BCR pathway is a highly promising therapeutic strategy in this malignancy. The oncogenic microRNA miR-17-92 modulates multiple cellular processes such as survival, proliferation, apoptosis, angiogenesis, and BCR signaling. In the present study, we identified new targets of miR-17-92, PTPROt (protein phosphatase, receptor type O, truncated) and PP2A (protein phosphatase 2A) phosphatases, which regulate the activity of spleen tyrosine kinase (SYK) and AKT, critical components of BCR signal transduction in DLBCL cells. Introduction of miR-17-92 into DLBCL cells dampened the expression of the PTPROt and PP2A regulatory subunits PPP2R2A (protein phosphatase 2, regulatory subunit B, alpha) and PPP2R5E (protein phosphatase 2, regulatory subunit B, epsilon isoform) and increased the magnitude of SYK and AKT phosphorylation upon BCR ligation. Finally, we found that miR-17-92 expression modulates response to inhibitors of BCR signaling because downregulation of miR-17-92 increased SYK inhibitor-mediated toxicity in DLBCL cells. Our study reveals novel posttranscriptional regulatory pathways that contribute to the deregulation of BCR signaling and modulate SYK inhibitor activity in DLBCL.

Xeno-free culture condition for human bone marrow and umbilical cord matrix-derived mesenchymal stem/stromal cells using human umbilical cord blood serum

Fetal bovine serum (FBS) is widely used in cell culture laboratories, risk of zoonotic infections and allergic side effects create obstacles for its use in clinical trials. Therefore, an alternative supplement with proper inherent growth-promoting activities is demanded. To find FBS substitute, we tested human umbilical cord blood serum (hUCS) for proliferation of human umbilical cord matrix derived mesenchymal stem cells (hUC-MSCs) and human bone marrow-derived mesenchymal cells (hBM-MSCs). Umbilical cord blood of healthy neonates, delivered by Caesarian section, was collected and the serum was separated. hUC-MSCs and hBM-MSCs were isolated and characterized by assessment of cell surface antigens by flow cytometry, alkaline phosphatase activity and osteogenic/adipogenic differentiation potential. The cells were then cultured in Iscove's Modified Dulbecco's Medium (IMDM) by conventional methods in three preparations: 1- with hUCS, 2- with FBS, and 3- without serum supplements. Cell proliferation was measured using WST-1 assay, and cell viability was assessed by trypan blue staining. The cells cultured in hUCS and FBS exhibited similar morphology and mesenchymal stem cells properties. WST-1 proliferation assay data showed no significant difference between the proliferation rate of either cells following hUCS and FBS supplementation. Trypan blue exclusion dye test also revealed no significant difference for viability between hUCS and FBS groups. A significant difference was detected between the proliferation rate of stem cells cultured in serum-supplemented medium compared with serum-free medium. Our results indicate that human umbilical cord serum can effectively support proliferation of hBM-MSCS and hUC-MSCs in vitro and can be used as an appropriate substitute for FBS, especially in clinical studies.