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In addition to antigen-driven signals,T cells need co-stimulatory signals for robust activation. Several receptors,including members of the tumor necrosis factor receptor superfamily (TNFRSF),can deliver co-stimulatory signals to T cells. Thioredoxin interacting protein (TXNIP) is an important inhibitor of glucose uptake and cell proliferation,but it is unknown how TXNIP is regulated in T cells. The aim of this study was to determine expression levels and regulation of TXNIP in human T cells. We found that na{\{i}}ve T cells express high levels of TXNIP and that treatment of blood samples with TNF results in rapid down-regulation of TXNIP in the T cells. TNF-induced TXNIP down-regulation correlated with increased glucose uptake. Furthermore we found that density gradient centrifugation (DGC) induced down-regulation of TXNIP. We demonstrate that DGC induced TNF production that paralleled the TXNIP down-regulation. Treatment of blood with toll-like receptor (TLR) ligands induced TNF production and TXNIP down-regulation suggesting that damage-associated molecular patterns (DAMPs) such as endogenous TLR ligands released during DGC play a role in DGC-induced TXNIP down-regulation. Finally we demonstrate that TNF-induced TXNIP down-regulation is dependent on caspase activity and is caused by caspase-mediated cleavage of TXNIP." View Publication

BACKGROUND Upregulation of RNA polymerase (Pol) III products,including tRNAs and 5S rRNA,in tumor cells leads to enhanced protein synthesis and tumor formation,making it a potential target for cancer treatment. In this study,we evaluated the inhibition of Pol III transcription by triptolide and the anti-cancer effect of this drug in colorectal tumorigenesis. METHODS The effect of triptolide on colorectal cancer development was assessed in colorectal cancer mouse models,3D organoids,and cultured cells. Colorectal cancer cells were treated with triptolide. Pol III transcription was measured by real-time quantitative polymerase chain reaction (PCR). The formation of TFIIIB,a multi-subunit transcription factor for Pol III,was determined by chromatin immunoprecipitation (ChIP),co-immunoprecipitation (Co-IP),and fluorescence resonance energy transfer (FRET). RESULTS Triptolide reduced both tumor number and tumor size in adenomatous polyposis coli (Apc) mutated (ApcMin/+) mice as well as AOM/DSS-induced mice. Moreover,triptolide effectively inhibited colorectal cancer cell proliferation,colony formation,and organoid growth in vitro,which was associated with decreased Pol III target genes. Mechanistically,triptolide treatment blocked TBP/Brf1interaction,leading to the reduced formation of TFIIIB at the promoters of tRNAs and 5S rRNA. CONCLUSIONS Together,our data suggest that inhibition of Pol III transcription with existing drugs such as triptolide provides a new avenue for developing novel therapies for colorectal cancer. View Publication

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner,thereby determining their degradation or inhibiting translation. They are involved in processes such as proliferation,differentiation and apoptosis by fine-tuning the expression of genes underlying such events. The expression of specific miRNAs is involved in hematopoietic differentiation and their deregulation contributes to the development of hematopoietic malignancies such as acute myeloid leukemia (AML). miR-130a is over-expressed in AML. Here we show that miR-130a is physiologically expressed in myeloblasts and down-regulated during monocyte differentiation. Gain- and loss-of-function experiments performed on CD34+ human hematopoietic stem cells confirmed that expression of miR-130a inhibits monocyte differentiation by interfering with the expression of key transcription factors HOXA10,IRF8,KLF4,MAFB and PU-1. The data obtained in this study highlight that the correct modulation of miR-130a is necessary for normal differentiation to occur and confirming that deregulation of this miRNA might underlie the differentiation block occurring in AML. View Publication

Cyclic dinucleotides are second messengers in the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway,which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and inhibitor of nuclear factor-$\kappa$B (I$\kappa$B) kinase (IKK)/nuclear factor-$\kappa$B (NF$\kappa$B) signaling cascades. In this work,we describe an enzymatic preparation of 2'-5',3'-5'-cyclic dinucleotides (2'3'CDNs) with use of cyclic GMP-AMP synthases (cGAS) from human,mouse,and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide-5'-triphosphate (NTP) analogues and use them to prepare 33 2'3'CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell-based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next,we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells (PBMCs) and evaluate their cytotoxic effect on monocytes. Additionally,we report X-ray crystal structures of two new CDNs bound to STING protein and discuss structure-activity relationship by using quantum and molecular mechanical (QM/MM) computational modeling. View Publication

Environmental factors contribute to Type 1 diabetes (T1D) susceptibility. The gut microbiome,which includes bacteria,viruses,and fungi,contributes to this environmental influence,and can induce immunological changes. The gut viral component of the microbiome,related to T1D has mostly focused on coxsackieviruses and rotavirus. The role of norovirus,another common enteric virus,in susceptibility to T1D was hitherto unknown. Norovirus is highly infectious and encountered by many children. We studied the mouse norovirus 4 (MNV4),related to human noroviruses,in the Non-obese diabetic (NOD) mouse model,to determine its role in influencing susceptibility to T1D. We infected MNV-free NOD mice with MNV4 by exposing the mice to MNV4-positive bedding from an endemically-infected mouse colony to mimic a natural infection. Control MNV-free NOD mice were exposed to MNV-free bedding from the same colony. Interestingly,MNV4 infection protected NOD mice from the development of T1D and was associated with an expansion of Tregs and reduced proinflammatory T cells. We also found MNV4 significantly modified the gut commensal bacteria composition,promoting increased $\alpha$-diversity and Firmicutes/Bacteroidetes ratio. To elucidate whether T1D protection was directly related to MNV4,or indirectly through modulating gut microbiota,we colonized germ-free (GF) NOD mice with the MNV4-containing or non-MNV4-containing viral filtrate,isolated from filtered fecal material. We found that MNV4 induced significant changes in mucosal immunity,including altered Tuft cell markers,cytokine secretion,antiviral immune signaling markers,and the concentration of mucosal antibodies. Systemically,MNV4-infection altered the immune cells including B cell subsets,macrophages and T cells,and especially induced an increase in Treg number and function. Furthermore,in vitro primary exposure of the norovirus filtrate to na{\{i}}ve splenocytes identified significant increases in the proportion of activated and CTLA4-expressing Tregs. Our data provide novel knowledge that norovirus can protect NOD mice from T1D development by inducing the expansion of Tregs and reducing inflammatory T cells. Our study also highlights the importance of distinguishing the mucosal immunity mediated by bacteria from that by enteric viruses." View Publication

AG-490 is a member of the tyrphostin family of tyrosine kinase inhibitors. While AG-490 has been considered to be a Janus kinase (JAK)2-specific inhibitor,these conclusions were primarily drawn from acute lymphoblastic leukemia cells that lack readily detectable levels of JAK3. In the present study,evidence is provided that clearly demonstrates AG-490 potently suppresses IL-2-induced T cell proliferation,a non-JAK2-dependent signal,in a dose-dependent manner in T cell lines D10 and CTLL-2. AG-490 blocked JAK3 activation and phosphorylation of its downstream counterpart substrates,STATs. Inhibition of JAK3 by AG-490 also compromised the Shc/Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways as measured by phosphorylation of Shc and extracellular signal-related kinase 1 and 2 (ERK1/2). AG-490 effectively inhibited tyrosine phosphorylation and DNA binding activities of several transcription factors including STAT1,-3,-5a,and -5b and activating protein-1 (AP-1) as judged by Western blot analysis and electrophoretic mobility shift assay. These data suggest that AG-490 is a potent inhibitor of the JAK3/STAT,JAK3/AP-1,and JAK3/MAPK pathways and their cellular consequences. Taken together,these findings support the notion that AG-490 possesses previously unrecognized clinical potential as an immunotherapeutic drug due to its inhibitory effects on T cell-derived signaling pathways. View Publication

Analysis of the National Cancer Institute Human Tumor Cell Line Anti-Cancer Drug Screen data using the COMPARE algorithm to detect similarities in the pattern of compound action to flavopiridol,a known inhibitor of cyclin-dependent kinases (CDKs),has suggested several possible novel CDK inhibitors. 9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one,NSC-664704 (kenpaullone),is reported here to be a potent inhibitor of CDK1/cyclin B (IC50,0.4 microM). This compound also inhibited CDK2/cyclin A (IC50,0.68 microM),CDK2/cyclin E (IC50,7.5 microM),and CDK5/p25 (IC50,0.85 microM) but had much less effect on other kinases; only c-src (IC50,15 microM),casein kinase 2 (IC50,20 microM),erk 1 (IC50,20 microM),and erk 2 (IC50,9 microM) were inhibited with IC50s less than 35 microM. Kenpaullone acts by competitive inhibition of ATP binding. Molecular modeling indicates that kenpaullone can bind in the ATP binding site of CDK2 with residue contacts similar to those observed in the crystal structures of other CDK2-bound inhibitors. Analogues of kenpaullone,in particular 10-bromopaullone (NSC-672234),also inhibited various protein kinases including CDKs. Cells exposed to kenpaullone and 10-bromopaullone display delayed cell cycle progression. Kenpaullone represents a novel chemotype for compounds that preferentially inhibit CDKs. View Publication

OBJECTIVE To investigate the inhibition of matrix metalloproteinase 1 (MMP-1),MMP-8,and MMP-13 by doxycycline,and to determine whether the variable hemopexin-like domain of each MMP was responsible for the differences in susceptibility to doxycycline inhibition among these collagenases. METHODS Recombinant human MMP-1 (collagenase 1),MMP-8 (collagenase 2),and MMP-13 (collagenase 3),truncated forms of MMP-8 and MMP-13 lacking the hemopexin-like domain,and a mutant form of truncated MMP-13 were used in these studies. The activity of the full-length MMP in the presence of doxycycline was tested against type II collagen,a natural substrate for the enzymes. A small peptolide substrate was used to determine which structural features of the MMPs were related to sensitivity to doxycycline inhibition. RESULTS The activity of MMP-13 and MMP-8 against type II collagen was inhibited by 50-60% by 30 microM doxycycline,while that of MMP-1 was inhibited only 18% by 50 microM doxycycline. In contrast,in experiments with the peptolide substrate,neither full-length nor truncated MMP-13 was inhibited until the concentration of the drug exceeded 90 microM. MMP-8 and truncated MMP-8 were sensitive to inhibition by 30 microM doxycycline,while MMP-1 was slightly inhibited (14%) by 90 microM doxycycline. For MMP-8,inhibition was reversible upon dilution and was independent of the order in which the reagents were added. Kinetic analysis of the inhibition constant (K(i)) of MMP-8 (K(i) = 36 microM) and truncated MMP-8 (K(i) = 77 microM) indicated that inhibition was noncompetitive. CONCLUSION Significant inhibition of MMP-13 and MMP-8 activity against collagen occurred in vitro at concentrations that were near the concentrations achieved in serum after oral dosing. Studies with truncated enzymes and 2 substrates suggest that doxycycline disrupts the conformation of the hemopexin-like domain of MMP-13 and the catalytic domain of MMP-8. View Publication

Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Because these effects are in part determined by p53-mediated apoptosis,temporary suppression of p53 has been suggested as a therapeutic strategy to prevent damage of normal tissues during treatment of p53-deficient tumors. To test this possibility,a small molecule was isolated for its ability to reversibly block p53-dependent transcriptional activation and apoptosis. This compound,pifithrin-alpha,protected mice from the lethal genotoxic stress associated with anticancer treatment without promoting the formation of tumors. Thus,inhibitors of p53 may be useful drugs for reducing the side effects of cancer therapy and other types of stress associated with p53 induction. View Publication

We have previously identified multipotent neuroepithelial (NEP) stem cells and lineage-restricted,self-renewing precursor cells termed NRPs (neuron-restricted precursors) and GRPs (glial-restricted precursors) present in the developing rat spinal cord (A. Kalyani,K. Hobson,and M. S. Rao,1997,Dev. Biol. 186,202-223; M. S. Rao and M. Mayer-Proschel,1997,Dev. Biol. 188,48-63; M. Mayer-Proschel,A. J. Kalyani,T. Mujtaba,and M. S. Rao,1997,Neuron 19,773-785). We now show that cells identical to rat NEPs,NRPs,and GRPs are present in mouse neural tubes and that immunoselection against cell surface markers E-NCAM and A2B5 can be used to isolate NRPs and GRPs,respectively. Restricted precursors similar to NRPs and GRPs can also be isolated from mouse embryonic stem cells (ES cells). ES cell-derived NRPs are E-NCAM immunoreactive,undergo self-renewal in defined medium,and differentiate into multiple neuronal phenotypes in mass culture. ES cells also generate A2B5-immunoreactive cells that are similar to E9 NEP-cell-derived GRPs and can differentiate into oligodendrocytes and astrocytes. Thus,lineage restricted precursors can be generated in vitro from cultured ES cells and these restricted precursors resemble those derived from mouse neural tubes. These results demonstrate the utility of using ES cells as a source of late embryonic precursor cells. View Publication

We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells,up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking,however,decreased to 0.1% or lower within 2 weeks. In contrast,EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks,the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis,and EGFP expression was observed in CD4(+),CD8(+),CD20(+),and CD16/56(+) lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP(+) myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol. (Blood. 2000;95:445-452) View Publication

Stable cell lines that individually express the eight known human prostanoid receptors (EP(1),EP(2),EP(3),EP(4),DP,FP,IP and TP) have been established using human embryonic kidney (HEK) 293(EBNA) cells. These recombinant cell lines have been employed in radioligand binding assays to determine the equilibrium inhibitor constants of known prostanoid receptor ligands at these eight receptors. This has allowed,for the first time,an assessment of the affinity and selectivity of several novel compounds at the individual human prostanoid receptors. This information should facilitate interpretation of pharmacological studies that employ these ligands as tools to study human tissues and cell lines and should,therefore,result in a greater understanding of prostanoid receptor biology. View Publication