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INTRODUCTION The stem cell portion of the dental pulp derived cultures (DPSCs) showed a higher resistance to cytotoxic effect of restorative dental materials compared to pulpal fibroblasts (DPFs). Here,we aimed to compare the expression of some drug resistant genes between these cells. METHODS AND MATERIALS To separate DPSCs from DPFs,we used magnetic cell sorting technique based on CD146 expression. To assess the stem cell properties,the positive and negative portions underwent colony forming assays and were induced to be differentiated into the adipocytes,osteoblasts,hepatocytes,and neural cells. Cell surface antigen panels were checked using immune fluorescence and flow-cytometry techniques. The mRNA expression of 14 ABC transporters including ABCA2,ABCB1,ABCB11,ABCC1,ABCC2,ABCC3,ABCC4,ABCC5-2,ABCC5-4,ABCC5-13,ABCC6,ABCC10,ABCC11,and ABCG2 genes was assessed,using quantitative RT-PCR technique. RESULTS Only the CD146 positive portion could be differentiated into the desired fates,and they formed higher colonies (16.7 ± 3.32 vs. 1.7 ± 1.67,p {\textless} .001). The cell surface antigen panels were the same,except for CD146 and STRO-1 markers which were expressed only in the positive portion. Among the ABC transporter genes studied,the positive portion showed a higher expression (approximately two-fold) of ABCA2,ABCC5-13,and ABCC5-2 genes. CONCLUSION Dental pulp stem cells which can be separated from dental pulp fibroblasts based on CD146 expression,express higher levels of some drug resistance genes which probably accounts for their features of more resistance to cytotoxic effects of some dental materials. This needs to be more validated in future. View Publication

Activation of RIPK1 controls TNF-mediated apoptosis,necroptosis and inflammatory pathways1. Cleavage of human and mouse RIPK1 after residues D324 and D325,respectively,by caspase-8 separates the RIPK1 kinase domain from the intermediate and death domains. The D325A mutation in mouse RIPK1 leads to embryonic lethality during mouse development2,3. However,the functional importance of blocking caspase-8-mediated cleavage of RIPK1 on RIPK1 activation in humans is unknown. Here we identify two families with variants in RIPK1 (D324V and D324H) that lead to distinct symptoms of recurrent fevers and lymphadenopathy in an autosomal-dominant manner. Impaired cleavage of RIPK1 D324 variants by caspase-8 sensitized patients' peripheral blood mononuclear cells to RIPK1 activation,apoptosis and necroptosis induced by TNF. The patients showed strong RIPK1-dependent activation of inflammatory signalling pathways and overproduction of inflammatory cytokines and chemokines compared with unaffected controls. Furthermore,we show that expression of the RIPK1 mutants D325V or D325H in mouse embryonic fibroblasts confers not only increased sensitivity to RIPK1 activation-mediated apoptosis and necroptosis,but also induction of pro-inflammatory cytokines such as IL-6 and TNF. By contrast,patient-derived fibroblasts showed reduced expression of RIPK1 and downregulated production of reactive oxygen species,resulting in resistance to necroptosis and ferroptosis. Together,these data suggest that human non-cleavable RIPK1 variants promote activation of RIPK1,and lead to an autoinflammatory disease characterized by hypersensitivity to apoptosis and necroptosis and increased inflammatory response in peripheral blood mononuclear cells,as well as a compensatory mechanism to protect against several pro-death stimuli in fibroblasts. View Publication

Human-induced Pluripotent Stem Cell (hiPSC)-derived models have advanced the study of neurodegenerative diseases,including Parkinson's disease (PD). While age is the strongest risk factor for these disorders,hiPSC-derived models represent rejuvenated neurons. We developed hiPSC-derived Aged dopaminergic and cholinergic neurons to model PD and related synucleinopathies. Our new method induces aging through a `semi-natural' process,by passaging multiple times at the Neural Precursor Cell stage,prior to final differentiation. Characterization of isogenic hiPSC-derived neurons using heterochromatin and nuclear envelope markers,as well as DNA damage and global DNA methylation,validated our age-inducing method. Next,we compared neurons derived from a patient with SNCA-triplication (SNCA-Tri) and a Control. The SNCA-Tri neurons displayed exacerbated nuclear aging,showing advanced aging signatures already at the Juvenile stage. Noteworthy,the Aged SNCA-Tri neurons showed more $\alpha$-synuclein aggregates per cell versus the Juvenile. We suggest a link between the effects of aging and SNCA overexpression on neuronal nuclear architecture. View Publication

Human embryonic stem cells (hESCs) hold great value for future clinical applications. However,standard culture conditions maintain hESCs in a primed state,which bears heterogeneity in pluripotency and a tendency for spontaneous differentiation. To counter these drawbacks,primed hESCs have been converted to a naive state,but this has restricted the efficiency of existing directed differentiation protocols. In mouse,WNT inhibition by inhibitor of WNT production-2,together with a higher dose of fibroblast growth factor 2 (12 ng/mL) in DMEM/F12 basal medium (DhiFI),markedly improved derivation and maintenance of primed mouse epiblast stem cells. In this study,we show that DhiFI conditions similarly improved primed hESC traits,such as conferring a primed transcriptional signature with high levels of pluripotency markers and reduced levels of differentiation markers. When triggered to differentiate to neuronal and cardiac lineages,DhiFI hESCs and isogenic primed hESCs progressed similarly. Moreover,DhiFI conditions supported the derivation of hESC lines from a post-inner cell mass intermediate (PICMI). DhiFI-derived hESCs showed less spontaneous differentiation and expressed significantly lower levels of lineage-specific markers,compared to primed-derived lines from the same PICMI. Overall,DhiFI hESCs retained advantages of both primed and naive pluripotency and may ultimately represent a more favorable starting point for differentiation toward clinically desired cell types. View Publication

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here,we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1{\&}2),a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS,for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1{\&}2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1{\&}2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore,PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway,and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1{\&}2 reversed arthritis in mice,providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies. View Publication

Rabies is a zoonotic neurological infection caused by lyssavirus that continues to result in devastating loss of human life. Many aspects of rabies pathogenesis in human neurons are not well understood. Lack of appropriate ex-vivo models for studying rabies infection in human neurons has contributed to this knowledge gap. In this study,we utilize advances in stem cell technology to characterize rabies infection in human stem cell-derived neurons. We show key cellular features of rabies infection in our human neural cultures,including upregulation of inflammatory chemokines,lack of neuronal apoptosis,and axonal transmission of viruses in neuronal networks. In addition,we highlight specific differences in cellular pathogenesis between laboratory-adapted and field strain lyssavirus. This study therefore defines the first stem cell-derived ex-vivo model system to study rabies pathogenesis in human neurons. This new model system demonstrates the potential for enabling an increased understanding of molecular mechanisms in human rabies,which could lead to improved control methods. View Publication

NF-$\kappa$B is a key regulator of inflammation and cancer progression,with an important role in leukemogenesis. Despite its therapeutic potential,targeting NF-$\kappa$B using pharmacologic inhibitors has proven challenging. Here,we describe a myeloid cell-selective NF-$\kappa$B inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a,C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6),thereby blocking activation of NF-$\kappa$B in target cells. IV injections of C-miR146a mimic to miR-146a-deficient mice prevented excessive NF-$\kappa$B activation in myeloid cells,and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly,C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions,miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set,we found an inverse correlation of miR-146a levels with NF-$\kappa$B-related genes and with patient survival. Correspondingly,C-miR146a induced cytotoxic effects in human MDSL,HL-60,and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-$\kappa$B target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell-targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders. View Publication

R-2-hydroxyglutarate (R-2HG),produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes,was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically,R-2HG inhibits fat mass and obesity-associated protein (FTO) activity,thereby increasing global N6-methyladenosine (m6A) RNA modification in R-2HG-sensitive leukemia cells,which in turn decreases the stability of MYC/CEBPA transcripts,leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG,whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively,while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation,our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m6A/MYC/CEBPA signaling. View Publication

Leukocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in host defense and immune regulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD),characterized by recurrent bacterial and fungal infections and aberrant inflammation. Key drivers of hyper-inflammation induced by fungal cell walls in CGD are still incompletely defined. Here,we found that CGD (CYBB-null) neutrophils produced higher amounts of leukotriene B4 (LTB4) in vitro following activation with zymosan or Immune complexes,as compared to wild type (WT) neutrophils. This correlated with increased calcium influx in CGD neutrophils,which is restrained in WT neutrophils by the electrogenic activity of the NADPH oxidase. Increased LTB4 generation by CGD neutrophils was also augmented by paracrine cross-talk with the LTB4 receptor BLT1. CGD neutrophils formed more numerous and larger clusters in the presence of zymosan in vitro compared to WT,which was also LTB4- and BLT1-dependent. In zymosan-induced lung inflammation,focal neutrophil infiltrates were increased in CGD compared to WT mice and associated with higher LTB4 levels. Inhibiting LTB4 synthesis or antagonizing the BLT1 receptor following zymosan challenge reduced lung neutrophil recruitment in CGD to WT levels. Thus,LTB4 was the major driver of excessive neutrophilic lung inflammation in CGD mice in the early response to fungal cell walls,likely by a dysregulated feed-forward loop involving amplified neutrophil production of LTB4. This study identifies neutrophil LTB4 generation as a target of NADPH oxidase regulation,which could potentially be exploited therapeutically to reduce excessive inflammation in CGD. View Publication

Immune surveillance against pathogens and tumours in the central nervous system is thought to be limited owing to the lack of lymphatic drainage. However,the characterization of the meningeal lymphatic network has shed light on previously unappreciated ways that an immune response can be elicited to antigens that are expressed in the brain1-3. Despite progress in our understanding of the development and structure of the meningeal lymphatic system,the contribution of this network in evoking a protective antigen-specific immune response in the brain remains unclear. Here,using a mouse model of glioblastoma,we show that the meningeal lymphatic vasculature can be manipulated to mount better immune responses against brain tumours. The immunity that is mediated by CD8 T cells to the glioblastoma antigen is very limited when the tumour is confined to the central nervous system,resulting in uncontrolled tumour growth. However,ectopic expression of vascular endothelial growth factor C (VEGF-C) promotes enhanced priming of CD8 T cells in the draining deep cervical lymph nodes,migration of CD8 T cells into the tumour,rapid clearance of the glioblastoma and a long-lasting antitumour memory response. Furthermore,transfection of an mRNA construct that expresses VEGF-C works synergistically with checkpoint blockade therapy to eradicate existing glioblastoma. These results reveal the capacity of VEGF-C to promote immune surveillance of tumours,and suggest a new therapeutic approach to treat brain tumours. View Publication