技术资料

The use of an interleukin beta$ antibody is currently being investigated in the clinic for the treatment of acne,a dermatological disorder affecting 650M persons globally. Inhibiting the protease responsible for the cleavage of inactive pro-IL1beta$ into active IL-1beta$,caspase-1,could be an alternative small molecule approach. This report describes the discovery of uracil 20,a potent (38 nM in THP1 cells assay) caspase-1 inhibitor for the topical treatment of inflammatory acne. The uracil series was designed according to a published caspase-1 pharmacophore model involving a reactive warhead in P1 for covalent reversible inhibition and an aryl moiety in P4 for selectivity against the apoptotic caspases. Reversibility was assessed in an enzymatic dilution assay or by using different substrate concentrations. In addition to classical structure-activity-relationship exploration,topical administration challenges such as phototoxicity,organic and aqueous solubility,chemical stability in solution,and skin metabolic stability are discussed and successfully resolved. View Publication

Store-operated calcium entry (SOCE) is a Calcium (Ca2+) influx pathway activated by depletion of intracellular stores that occurs in eukaryotic cells. In neurons,the presence and functions of SOCE are still in question. Here,we show evidences for the existence of SOCE in primary mouse cortical neurons. Endoplasmic reticulum (ER)-Ca2+ depletion using thapsigargin (Tg) triggered a maintained cytosolic Ca2+ increase,which rapidly returned to basal level in the presence of the SOCE blockers 2-Aminoethoxydiphenyl borate (2-APB) and YM-58483. Neural SOCE is also engaged by activation of metabotropic glutamate receptors (mGluRs) with (S)-3,5-dihydroxyphenylglycine (DHPG) (agonist of group I mGluRs),being an essential mechanism to maintain the mGluR-driven Ca2+ signal. Activation of group I of mGluRs triggers long-term depression (LTD) in many brain regions,but the underlying mechanism and,specifically,the necessity of Ca2+ increase in the postsynaptic neuron is controversial. In primary cortical neurons,we now show that the inhibition of Ca2+ influx through SOCE impaired DHPG-LTD,pointing out a key function of calcium and SOCE in synaptic plasticity. View Publication

The gastrointestinal tract is continuously exposed to many environmental factors that influence intestinal epithelial cells and the underlying mucosal immune system. In this article,we demonstrate that dietary fiber and short chain fatty acids (SCFAs) induced the expression of the vitamin A-converting enzyme RALDH1 in intestinal epithelial cells in vivo and in vitro,respectively. Furthermore,our data showed that the expression levels of RALDH1 in small intestinal epithelial cells correlated with the activity of vitamin A-converting enzymes in mesenteric lymph node dendritic cells,along with increased numbers of intestinal regulatory T cells and a higher production of luminal IgA. Moreover,we show that the consumption of dietary fiber can alter the composition of SCFA-producing microbiota and SCFA production in the small intestines. In conclusion,our data illustrate that dietary adjustments affect small intestinal epithelial cells and can be used to modulate the mucosal immune system. View Publication

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML,they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors,we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism,resulting in impaired production of the antioxidant factor glutathione,which was further impaired by ATM or G6PD inactivation. Moreover,FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo,revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML. View Publication

Bronchial epithelial cells (BECs) from healthy children inhibit human lung fibroblast (HLF) expression of collagen and fibroblast-to-myofibroblast transition (FMT),whereas asthmatic BECs do so less effectively,suggesting that diminished epithelial-derived regulatory factors contribute to airway remodeling. Preliminary data demonstrated that secretion of the activin A inhibitor follistatin-like 3 (FSTL3) by healthy BECs was greater than that by asthmatic BECs. We sought to determine the relative secretion of FSTL3 and activin A by asthmatic and healthy BECs,and whether FSTL3 inhibits FMT. To quantify the abundance of the total proteome FSTL3 and activin A in supernatants of differentiated BEC cultures from healthy children and children with asthma,we performed mass spectrometry and ELISA. HLFs were cocultured with primary BECs and then HLF expression of collagen I and alpha$-smooth muscle actin (alpha$-SMA) was quantified by qPCR,and FMT was quantified by flow cytometry. Loss-of-function studies were conducted using lentivirus-delivered shRNA. Using mass spectrometry and ELISA results from larger cohorts,we found that FSTL3 concentrations were greater in media conditioned by healthy BECs compared with asthmatic BECs (4,012 vs. 2,553 pg/ml; P = 0.002),and in media conditioned by asthmatic BECs from children with normal lung function relative to those with airflow obstruction (FEV1/FVC ratio {\textless} 0.8; n = 9; 3,026 vs. 1,922 pg/ml; P = 0.04). shRNA depletion of FSTL3 in BECs (n = 8) increased HLF collagen I expression by 92{\%} (P = 0.001) and alpha$-SMA expression by 88{\%} (P = 0.02),and increased FMT by flow cytometry in cocultured HLFs,whereas shRNA depletion of activin A (n = 6) resulted in decreased alpha$-SMA (22{\%}; P = 0.01) expression and decreased FMT. Together,these results indicate that deficient FSTL3 expression by asthmatic BECs impairs epithelial regulation of HLFs and FMT. View Publication

Traumatic brain injury (TBI) is soon predicted to become the third leading cause of death and disability worldwide. After the primary injury,a complex set of secondary injuries develops hours and days later with prolonged neuroinflammation playing a key role. TBI and other inflammatory conditions are currently being treated in preclinical and clinical trials by a number of cellular therapies. Mesenchymal stem cells (MSC) are of great interest due to their widespread usage,safety,and relative ease to isolate and culture. However,there has been a wide range in efficacy reported using MSC clinically and in preclinical models,likely due to differences in cell preparations and a significant amount of donor variability. In this study,we seek to find a correlation between in vitro activity and in vivo efficacy. We designed assays to explore the responsiveness of MSC to immunological cues to address the immunomodulatory properties of MSC,one of their primary modes of therapeutic activity in TBI. Our results showed intrinsic differences in the immunomodulatory capacity of MSC preparations from different bone marrow and amniotic fluid donors. This difference mirrored the therapeutic capacity of the MSC in an experimental model of TBI,an effect confirmed using siRNA knockdown of COX2 followed by overexpressing COX2. Among the immunomodulatory factors assessed,the therapeutic benefit correlated with the secretion of prostaglandin E2 (PGE2 ) by MSC prior to treatment,suggesting that measurement of PGE2 could be a very useful potency marker to create an index of predicted efficacy for preparations of MSC to treat TBI. Stem Cells 2017;35:1416-1430. View Publication

Targeted cancer immunotherapy with irradiated,granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting,allogeneic cancer cell lines has been an effective approach to reduce tumor burden in several patients. It is generally assumed that to be effective,these cell lines need to express immunogenic antigens coexpressed in patient tumor cells,and antigen-presenting cells need to take up such antigens then present them to patient T cells. We have previously reported that,in a phase I pilot study (ClinicalTrials.gov NCT00095862),a subject with stage IV breast cancer experienced substantial regression of breast,lung,and brain lesions following inoculation with clinical formulations of SV-BR-1-GM,a GM-CSF-secreting breast tumor cell line. To identify diagnostic features permitting the prospective identification of patients likely to benefit from SV-BR-1-GM,we conducted a molecular analysis of the SV-BR-1-GM cell line and of patient-derived blood,as well as a tumor specimen. Compared to normal human breast cells,SV-BR-1-GM cells overexpress genes encoding tumor-associated antigens (TAAs) such as PRAME,a cancer/testis antigen. Curiously,despite its presumptive breast epithelial origin,the cell line expresses major histocompatibility complex (MHC) class II genes (HLA-DRA,HLA-DRB3,HLA-DMA,HLA-DMB),in addition to several other factors known to play immunostimulatory roles. These factors include MHC class I components (B2M,HLA-A,HLA-B),ADA (encoding adenosine deaminase),ADGRE5 (CD97),CD58 (LFA3),CD74 (encoding invariant chain and CLIP),CD83,CXCL8 (IL8),CXCL16,HLA-F,IL6,IL18,and KITLG. Moreover,both SV-BR-1-GM cells and the responding study subject carried an HLA-DRB3*02:02 allele,raising the question of whether SV-BR-1-GM cells can directly present endogenous antigens to T cells,thereby inducing a tumor-directed immune response. In support of this,SV-BR-1-GM cells (which also carry the HLA-DRB3*01:01 allele) treated with yellow fever virus (YFV) envelope (Env) 43-59 peptides reactivated YFV-DRB3*01:01-specific CD4+ T cells. Thus,the partial HLA allele match between SV-BR-1-GM and the clinical responder might have enabled patient T lymphocytes to directly recognize SV-BR-1-GM TAAs as presented on SV-BR-1-GM MHCs. Taken together,our findings are consistent with a potentially unique mechanism of action by which SV-BR-1-GM cells can act as APCs for previously primed CD4+ T cells. View Publication

Expression of miR-143 and miR-145 is reduced in hematopoietic stem/progenitor cells (HSPCs) of myelodysplastic syndrome patients with a deletion in the long arm of chromosome 5. Here we show that mice lacking miR-143/145 have impaired HSPC activity with depletion of functional hematopoietic stem cells (HSCs),but activation of progenitor cells (HPCs). We identify components of the transforming growth factor beta$ (TGFbeta$) pathway as key targets of miR-143/145. Enforced expression of the TGFbeta$ adaptor protein and miR-145 target,Disabled-2 (DAB2),recapitulates the HSC defect seen in miR-143/145-/- mice. Despite reduced HSC activity,older miR-143/145-/- and DAB2-expressing mice show elevated leukocyte counts associated with increased HPC activity. A subset of mice develop a serially transplantable myeloid malignancy,associated with expansion of HPC. Thus,miR-143/145 play a cell context-dependent role in HSPC function through regulation of TGFbeta$/DAB2 activation,and loss of these miRNAs creates a preleukemic state. View Publication

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) grown in engineered heart tissue (EHT) can be used for drug screening,disease modeling,and heart repair. However,the immaturity of hiPSC-CMs currently limits their use. Because mechanical loading increases during development and facilitates cardiac maturation,we hypothesized that afterload would promote maturation of EHTs. To test this we developed a system in which EHTs are suspended between a rigid post and a flexible one,whose resistance to contraction can be modulated by applying braces of varying length. These braces allow us to adjust afterload conditions over two orders of magnitude by increasing the flexible post resistance from 0.09 up to 9.2 mu$N/mu$m. After three weeks in culture,optical tracking of post deflections revealed that auxotonic twitch forces increased in correlation with the degree of afterload,whereas twitch velocities decreased with afterload. Consequently,the power and work of the EHTs were maximal under intermediate afterloads. When studied isometrically,the inotropy of EHTs increased with afterload up to an intermediate resistance (0.45 mu$N/mu$m) and then plateaued. Applied afterload increased sarcomere length,cardiomyocyte area and elongation,which are hallmarks of maturation. Furthermore,progressively increasing the level of afterload led to improved calcium handling,increased expression of several key markers of cardiac maturation,including a shift from fetal to adult ventricular myosin heavy chain isoforms. However,at the highest afterload condition,markers of pathological hypertrophy and fibrosis were also upregulated,although the bulk tissue stiffness remained the same for all levels of applied afterload tested. Together,our results indicate that application of moderate afterloads can substantially improve the maturation of hiPSC-CMs in EHTs,while high afterload conditions may mimic certain aspects of human cardiac pathology resulting from elevated mechanical overload. View Publication

Protein glycosylation provides proteomic diversity in regulating protein localization,stability,and activity; it remains largely unknown whether the sugar moiety contributes to immunosuppression. In the study of immune receptor glycosylation,we showed that EGF induces programmed death ligand 1 (PD-L1) and receptor programmed cell death protein 1 (PD-1) interaction,requiring beta$-1,3-N-acetylglucosaminyl transferase (B3GNT3) expression in triple-negative breast cancer. Downregulation of B3GNT3 enhances cytotoxic T cell-mediated anti-tumor immunity. A monoclonal antibody targeting glycosylated PD-L1 (gPD-L1) blocks PD-L1/PD-1 interaction and promotes PD-L1 internalization and degradation. In addition to immune reactivation,drug-conjugated gPD-L1 antibody induces a potent cell-killing effect as well as a bystander-killing effect on adjacent cancer cells lacking PD-L1 expression without any detectable toxicity. Our work suggests targeting protein glycosylation as a potential strategy to enhance immune checkpoint therapy. View Publication

CD34 is a transmembrane phosphoglycoprotein used to selectively enrich bone marrow in hematopoietic stem cells for transplantation. Treating rats with CD34+ cells derived from human umbilical cord blood before or after heat stroke has been shown to promote survival. We investigated whether CD34- human placenta-derived stem cells (PDMSCs) could improve survival following heat stroke in rats. Rats were subjected to heat stress (42°C for 98 min) to induce heat stroke. Intravenous administration of PDMSCs 1 day before or immediately after the onset of heat stroke improved survival by 60{\%} and 20{\%},respectively. Pre-treatment with CD34- PDMSCs protected against heat stroke injury more effectively than that treatment after injury. PDMSCs treatment attenuated cerebrovascular dysfunction,the inflammatory response,and lipid peroxidation. These data suggest human PDMSCs protect against heat stroke injury in rats. Moreover,these effects do not require the presence of CD34+ cells. View Publication

Nonhuman primate (NHP) models are more predictive than rodent models for developing induced pluripotent stem cell (iPSC)-based cell therapy,but robust and reproducible NHP iPSC-cardiomyocyte differentiation protocols are lacking for cardiomyopathies research. We developed a method to differentiate integration-free rhesus macaque iPSCs (RhiPSCs) into cardiomyocytes with {\textgreater}85{\%} purity in 10 days,using fully chemically defined conditions. To enable visualization of intracellular calcium flux in beating cardiomyocytes,we used CRISPR/Cas9 to stably knock-in genetically encoded calcium indicators at the rhesus AAVS1 safe harbor locus. Rhesus cardiomyocytes derived by our stepwise differentiation method express signature cardiac markers and show normal electrochemical coupling. They are responsive to cardiorelevant drugs and can be successfully engrafted in a mouse myocardial infarction model. Our approach provides a powerful tool for generation of NHP iPSC-derived cardiomyocytes amenable to utilization in basic research and preclinical studies,including in vivo tissue regeneration models and drug screening. View Publication