Transient transfection assays of the human osteopontin promoter/luciferase construct which contains a 5'-flanking region between -1500 and +87 relative to the transcription start site demonstrate that either treatment with troglitazone or cotransfection of PPARgamma expression vector inhibits osteopontin promoter activity .


These data indicate that troglitazone reduces osteopontin gene expression at transcriptional level through PPARgamma activation , and suggest the role of troglitazone in inhibiting the ability of macrophages to produce extracellular matrix , which is particularly relevant to atherosclerotic plaque formation .


Transcription factor NF-kappa B regulates Ig lambda light chain gene rearrangement .


The tissue- and stage-specific assembly of Ig and TCR genes is mediated by a common V ( D ) J recombinase complex in precursor lymphocytes .


Directed alterations in the accessibility of V , D , and J gene segments target the recombinase to specific Ag receptor loci .


Accessibility within a given locus is regulated by the functional interaction of transcription factors with cognate enhancer elements and correlates with the transcriptional activity of unrearranged gene segments .


As demonstrated in our prior studies , rearrangement of the Igkappa locus is regulated by the inducible transcription factor NF-kappaB .


In contrast to the Igkappa locus , known transcriptional control elements in the Iglambda locus lack functional NF-kappaB binding sites .


Consistent with this observation , the expression of assembled Iglambda genes in mature B cells has been shown to be NF-kappaB independent .


Nonetheless , we now show that specific repression of NF-kappaB inhibits germline transcription and recombination of Iglambda gene segments in precursor B cells .


Molecular analyses indicate that the block in NF-kappaB impairs Iglambda rearrangement at the level of recombinase accessibility .


In contrast , the activities of known Iglambda promoter and enhancer elements are unaffected in the same cellular background .


These findings expand the range of NF-kappaB action in precursor B cells beyond Igkappa to include the control of recombinational accessibility at both L chain loci .


Moreover , our results strongly suggest the existence of a novel Iglambda regulatory element that is either directly or indirectly activated by NF-kappaB during the early stages of B cell development .


Transcription factor STAT5A is a substrate of Bruton 's tyrosine kinase in B cells .


STAT5A is a molecular regulator of proliferation , differentiation , and apoptosis in lymphohematopoietic cells .


Here we show that STAT5A can serve as a functional substrate of Bruton 's tyrosine kinase ( BTK ) .


Purified recombinant BTK was capable of directly binding purified recombinant STAT5A with high affinity ( K ( d ) = 44 nm ) , as determined by surface plasmon resonance using a BIAcore biosensor system .


BTK was also capable of tyrosine-phosphorylating ectopically expressed recombinant STAT5A on Tyr ( 694 ) both in vitro and in vivo in a Janus kinase 3 -independent fashion .


BTK phosphorylated the Y665F , Y668F , and Y682F , Y683F mutants but not the Y694F mutant of STAT5A .


STAT5A mutations in the Src homology 2 ( SH2 ) and SH3 domains did not alter the BTK -mediated tyrosine phosphorylation .


Recombinant BTK proteins with mutant pleckstrin homology , SH2 , or SH3 domains were capable of phosphorylating STAT5A , whereas recombinant BTK proteins with SH1/kinase domain mutations were not .


In pull-down experiments , only full-length BTK and its SH1/kinase domain ( but not the pleckstrin homology , SH2 , or SH3 domains ) were capable of binding STAT5A .


Ectopically expressed BTK kinase domain was capable of tyrosine-phosphorylating STAT5A both in vitro and in vivo .


BTK -mediated tyrosine phosphorylation of ectopically expressed wild type ( but not Tyr ( 694 ) mutant ) STAT5A enhanced its DNA binding activity .


In BTK -competent chicken B cells , anti-IgM-stimulated tyrosine phosphorylation of STAT5 protein was prevented by pretreatment with the BTK inhibitor LFM-A13 but not by pretreatment with the JAK3 inhibitor HI-P131 .


B cell antigen receptor ligation resulted in enhanced tyrosine phosphorylation of STAT5 in BTK -deficient chicken B cells reconstituted with wild type human BTK but not in BTK -deficient chicken B cells reconstituted with kinase-inactive mutant BTK .


Similarly , anti-IgM stimulation resulted in enhanced tyrosine phosphorylation of STAT5A in BTK -competent B cells from wild type mice but not in BTK -deficient B cells from XID mice .


In contrast to B cells from XID mice , B cells from JAK3 knockout mice showed a normal STAT5A phosphorylation response to anti-IgM stimulation .


These findings provide unprecedented experimental evidence that BTK plays a nonredundant and pivotal role in B cell antigen receptor-mediated STAT5A activation in B cells


Role of T-bet in commitment of TH1 cells before IL-12 -dependent selection .


How cytokines control differentiation of helper T ( TH ) cells is controversial .


We show that T-bet , without apparent assistance from interleukin 12 ( IL-12 ) / STAT4 , specifies TH1 effector fate by targeting chromatin remodeling to individual interferon-gamma ( IFN-gamma ) alleles and by inducing IL-12 receptor beta2 expression .


Subsequently , it appears that IL-12 / STAT4 serves two essential functions in the development of TH1 cells : as growth signal , inducing survival and cell division ; and as trans-activator , prolonging IFN-gamma synthesis through a genetic interaction with the coactivator , CREB-binding protein .


These results suggest that a cytokine does not simply induce TH fate choice but instead may act as an essential secondary stimulus that mediates selective survival of a lineage .


Stat6 is necessary and sufficient for IL-4 's role in Th2 differentiation and cell expansion .


IL-4 plays a critical role in the differentiation of T CR-stimulated naive CD4 T cells to the Th2 phenotype .


In response to IL-4 , the IL-4R activates a set of phosphotyrosine binding domain-containing proteins , including insulin receptor substrate 1/2 , Shc , and IL-4R interacting protein , as well as Stat6 .


Stat6 has been shown to be required for Th2 differentiation .


To determine the roles of the phosphotyrosine binding adaptors in Th2 differentiation , we prepared a retrovirus containing a mutant of the human ( h ) IL-4R alpha-chain , Y497F , which is unable to recruit these adaptors .


The mutant hIL-4Ralpha , as well as the wild-type ( WT ) hIL-4Ralpha , was introduced into naive CD4 T cells .


Upon hIL-4 stimulation , Y497F worked as well as the WT hIL-4Ralpha in driving Th2 differentiation , as measured by Gata3 up-regulation and IL-4 production .


Furthermore , IL-4-driven cell expansion was also normal in the cells infected with Y497F , although cells infected with Y497F were not capable of phosphorylating insulin receptor substrate 2 .


These results suggest that the signal pathway mediated by Y497 is dispensable for both IL-4 -driven Th2 differentiation and cell expansion .


Both WT and Y497F hIL-4Ralpha lose the ability to drive Th2 differentiation and cell expansion in Stat6-knockout CD4 T cells .


A constitutively activated form of Stat6 introduced into CD4 T cells resulted in both Th2 differentiation and enhanced cell expansion .


Thus , activated Stat6 is necessary and sufficient to mediate both IL-4 -driven Th2 differentiation and cell expansion in CD4 T cells .


The effect of HIV-1 regulatory proteins on cellular genes : derepression of the IL-2 promoter by Tat .


In HIV-infected individuals dysregulation of the immune system is characterized by severe disorders of the cytokine network .


Increase secretion of IL-2 , the major T cell growth and differentiation cytokine , may play a decisive role in sensitization of T cells for activation induced apoptosis and indirect death of activated T cells through augmented virus replication .


We investigated the cause of enhanced IL-2 secretion and found that the HIV Tat induces this effect .


We demonstrate that increased IL-2 secretion is due to Tat -enhanced IL-2 promoter activation .


Tat derepresses and activates the distal AP-1 site ( position -185 to -177 ) in the IL-2 promoter .


In nonstimulated T cells a repressor complex containing NF-IL6 , JunB , c-Fos and Fra-1 is formed on the AP-1 ( IL-2/d ) site and represses IL-2 promoter activity .


After T cell activation , a heterodimeric activator containing p65 and c-Jun binds to the AP-1 ( IL-2/d ) site .


HIV Tat enhances activation of NF-kappaB and consequently , activates the AP-1 ( IL-2/d ) site .


Our data provide evidence for a novel mechanism by which HIV Tat dysregulates IL-2 production and therefore may contribute to the HIV-1 infection in a way yet to be clarified .


Transforming growth factor-beta1 interferes with thrombopoietin-induced signal transduction in megakaryoblastic and erythroleukemic cells .


OBJECTIVE : Thrombopoietin ( TPO ) and transforming growth factor-beta ( 1 ) ( TGF-beta ( 1 ) ) have been shown to exert opposite effects on proliferation and megakaryocytic differentiation of hematopoietic cells .


To determine whether TGF-beta ( 1 ) interferes directly with TPO -induced signal transduction in hematopoietic cells , we compared the regulatory effects in the TPO -responsive cell lines Mo-7e and HEL .


MATERIALS AND METHODS : The cells were stimulated by 100 ng/mL TPO and/or 100 ng/mL TGF-beta1 and analyzed for proliferation ( 3H thymidine incorporation ) , viability ( trypan blue exclusion ) , and protein expression and phosphorylation ( Western blot ) .


RESULTS : TPO enhanced the proliferation of Mo-7e cells as determined by 3H-thymidine incorporation , whereas TGF-beta1 suppressed baseline cell growth and antagonized the proliferative effect of TPO .


TPO -induced proliferation also was reduced by a specific inhibitor of the mitogen-activated protein kinase ( MAPK ) pathway ( PD098059 ) , which inhibits activation of the MAPK extracellular signal-regulated kinases ( ERK ) ERK1 and ERK2 , and AG490 , an inhibitor of Janus kinase-2 , which completely blocked TPO -induced proliferation .


As demonstrated by Western blotting , TGF-beta1 reduced the TPO -stimulated ERK1 / ERK2 and STAT5 phosphorylation in Mo-7e and HEL cells .


This effect was completely reversed by preincubation with a tyrosine phosphatase inhibitor ( Na3VO4 ) , which suggests that TGF-beta1 activated a phosphatase .


Although STAT3 also was activated by TPO , STAT3 activation remained unaltered by TGF-beta1 .


CONCLUSION : Taken together , these data suggest that TGF-beta1 modulates TPO -mediated effects on megakaryocytic proliferation by interfering with TPO -induced signal transduction , particularly by reducing the activities of MAPK ERK1/ERK2 and STAT5 .


Invariant chain induces B cell maturation by activating a TAF ( II ) 105-NF-kappaB -dependent transcription program .


Early stages of B cell development occur in the bone marrow , resulting in formation of immature B cells .


From there these immature cells migrate to the spleen where they differentiate to mature cells .


This final maturation step is crucial for the B cells to become responsive to antigens and to participate in the immune response .


Recently , invariant chain ( Ii ) , a major histocompatibility complex class II chaperone , as well as the transcription factors c-Rel and p65/RelA , were found to play a role in the final antigen-independent differentiation stage of B cells in the spleen .


In this study , we investigated a possible link between Ii -dependent B cell maturation and the NF-kappaB pathway .


Our studies indicate that Ii -induced B cell maturation involves activation of transcription mediated by the NF-kappaB p65/RelA homodimer and requires the B cell-enriched coactivator TBP-associated factor ( II ) 105 .


Androgens indirectly accelerate thymocyte apoptosis .


Apoptotic processes , or the disturbance of the natural regulation of these processes , may be involved in the pathogenesis of autoimmune diseases ( AID ) .


Women are , in general , more susceptible than men to develop AID like rheumatoid arthritis .


Androgens and glucocorticoids , in contrast to oestrogens , have favourable effects in AID models as well as in human AID .


It is known that glucocorticoids ( GC ) , used for treatment of AID , increase apoptosis in the thymus resulting in decreased numbers of CD4+ CD8+ thymocytes .


It was asked whether androgens , in contrast to oestrogens , exert their favourable effects in the treatment of AID by a mechanism comparable to that described for GC by eliminating the apoptosis prone CD4+ CD8+ population in the thymus .


Although both androgens and oestrogens proved thymolytic , a significantly decreased percentage of CD4+ CD8+ thymocytes was observed by flow cytometry after treatment of mice with the androgen methyltestosterone , but not with the oestrogen ethinylestradiol .


To investigate whether the observed thymolytic effects were due to the presence of hormone receptors on thymocytes , cells were isolated from the thymus and incubated with androgens or oestrogens to measure apoptosis .


Several techniques were used to determine thymocyte apoptosis in vitro , but no enhanced apoptotic signal was observed .


Using the very sensitive TUNEL assay , no direct effect of androgens on thymocytes in vitro could be observed .


This is in sharp contrast to the high signal observed with GC .


Therefore , upon in vivo androgen treatment , other cells containing androgen receptors than thymocytes are probably involved in inducing the increase in thymic apoptosis .


To study the role of the androgen receptor on thymocyte apoptosis , androgen receptor mutant ( Tfm/Y ) mice were treated with androgens .


No alterations of thymocyte subpopulations were seen , suggesting that changes in the percentage of CD4+ CD8+ thymocytes after administration of androgens depend on the presence of functional androgen receptors .


Thus , it is concluded that androgens indirectly accelerate thymocyte apoptosis in vivo .


Inhibition of the transcription factors AP-1 and NF-kappaB in CD4 T cells by peroxisome proliferator-activated receptor gamma ligands .


The peroxisome proliferator-activated receptor gamma ( PPARgamma ) , a member of the nuclear hormone receptor superfamily , is essential for adipocyte differentiation and glucose homeostasis .


PPARgamma has been found recently to regulate macrophage activation in response to mitogens and inflammation .


Our study shows PPARgamma to be preferentially expressed in the nuclei of resting T cells and to increase upon activation of T cells by either anti-CD3 and anti-CD28 or phorbol myristyl acetate ( PMA ) .


We also found the PPARgamma ligand ciglitizone to attenuate the activation of T cells by inhibiting cytokine gene expression and anti-CD3 and anti-CD28 or PMA-induced proliferative responses .


Inhibition of both the proliferative response and inflammatory cytokine expression in CD4 T cells was correlated with suppression of the activated transcription factors AP1 and NF-kappaB .


PPARgamma ligands also strongly inhibited SEA-induced Vbeta3 T cell activation in vivo .


These results , together with previous findings of the inhibitory effect of PPARgamma ligands on activated macrophages , provide clear evidence for PPARgamma as a negative regulator of the inflammatory activation of both macrophage and T cells .


PPARgamma may thus be a potential therapeutic target for the treatment of autoimmunity .


A prominent role for activator protein-1 in the transcription of the human 2B4 ( CD244 ) gene in NK cells .


The cell surface glycoprotein 2B4 ( CD244 ) of the Ig superfamily is involved in the regulation of NK and T lymphocyte functions .


We have recently identified CD48 as the high affinity counterreceptor for 2B4 in both mice and humans .


The cytoplasmic domain of 2B4 associates with src homology 2 domain-containing protein or signaling lymphocyte activation molecule-associated protein , whose mutation is the underlying genetic defect in the X-linked lymphoproliferative syndrome .


In this study , we report the molecular cloning and characterization of the human 2B4 ( h2B4 ) promoter .


Through primer extension analysis , we found that the transcription of the h2B4 gene initiates at multiple start sites .


We isolated h2B4 genomic clones and PCR amplified the 5 ' untranslated region containing the promoter elements .


We have identified a functional AP-1 site that lies between ( -106 to -100 ) through transient transfection analysis in YT cells , a human NK cell line .


EMSAs with Abs specific for various protein factors of the AP-1 family revealed that multiple members of the Jun family are involved in the regulation of the h2B4 gene .


Mutation of the AP-1 site not only abolishes protein/DNA interactions but also promoter activity .


These results demonstrate a significant role for AP-1 in the transcriptional regulation of the h2B4 gene .


Functional correction of FA-C cells with FANCC suppresses the expression of interferon gamma-inducible genes .


Because hematopoietic cells derived from Fanconi anemia ( FA ) patients of the C-complementation group ( FA-C ) are hypersensitive to the inhibitory effects of interferon gamma ( IFNgamma ) , the products of certain IFNgamma-inducible genes known to influence hematopoietic cell survival were quantified .


High constitutive expression of the IFNgamma-inducible genes , IFN-stimulated gene factor 3 gamma subunit ( ISGF3gamma ) , IFN regulatory factor-1 ( IRF-1 ) , and the cyclin-dependent kinase inhibitor p21 ( WAF1 ) was found in FANCC mutant B lymphoblasts , low-density bone marrow cells , and murine embryonic fibroblasts .


Paradoxically , these cells do not activate signal transducer and activator of transcription ( STAT ) 1 properly .


In an attempt to clarify mechanisms by which FA-C cells overexpress IFNgamma-inducible genes in the face of defective STAT1 phosphorylation , it was reasoned that decreased levels of activated STAT1 might result in reduced expression of a hematopoietic IFNgamma-responsive protein that normally modulates expression of other IFNgamma-responsive genes .


Levels of the IFNgamma -inducible factor IFN consensus sequence binding protein ( ICSBP ) , a negative trans-acting regulator of some IFNgamma-inducible genes , were quantified .


ICSBP levels were reduced in FA-C B lymphoblasts and MEFs .


However , enforced expression of ICSBP failed to down-regulate IRF-1 , ISGF3gamma , and p21 ( WAF1 ) .


Thus , the FANCC protein functions to modulate expression of a family of genes that in normal cells are inducible only by specific environmental cues for apoptosis or mitogenic inhibition , but it does so independently of the classic IFN-STAT1 pathway and is not the direct result of reduced ICSBP expression .


A genetic investigation of E2A function in lymphocyte development .


Lymphocytes are derived from hematopoietic stem cells ( HSC ) following a series of regulated differentiation events .


Multipotent HSCs become committed to the B cell lineage in bone marrow and the T cell lineage in the thymus after receiving appropriate signals from the corresponding microenvironment .


These committed lymphoid cells must then undergo V ( D ) J recombination at the immunoglobulin gene or T cell receptor gene locus resulting in clonal production of functional B or T lymphocytes , respectively .


Lymphocyte commitment and differentiation are accompanied by programmed gene expression or repression events which are driven by lineage and stage specific transcription factors .


The basic-helix-loop-helix ( bHLH ) transcription factors encoded by the E2A gene are involved in several differentiation events during B and T cell development , including lineage commitment , initiation of V ( D ) J recombination , and antigen receptor mediated proliferation and differentiation .


Several recent reviews have provided a comprehensive discussion of biochemical , cellular , and genetic research on E2A function in lymphocyte development ( 1 , 2 ) .


Here , we only discuss some of the genetic approaches our laboratory ( except where it is noted ) has undertaken to investigate the molecular pathways mediated by E2A transcription factors in lymphocyte development


D609 inhibits ionizing radiation-induced oxidative damage by acting as a potent antioxidant .


Tricyclodecan-9-yl-xanthogenate ( D609 ) has been extensively studied in biological systems and exhibits a variety of biological functions , including antiviral , antitumor , and anti-inflammatory activities .


Most of these activities have been largely attributed to the inhibitory effect of D609 on phosphatidylcholine-specific phospholipase C .


However , as a xanthate derivative , D609 is a strong electrolyte and readily dissociates to xanthate anions and cations of alkali metals in solution .


Xanthate anions and protonated xanthic acid contain a free thiol moiety and are highly reductive .


This implies that D609 and other xanthate derivatives may function as potent antioxidants .


Indeed , we found that D609 inhibited the Fenton reaction-induced oxidation of dihydrorhodamine 123 in a dose-dependent manner similar to that of pyrrolidinedithiocarbamate , a well known antioxidant .


In addition , D609 inhibited the formation of the alpha-phenyl-tert-butylnitrone-free radical spin adducts and lipid peroxidation of synaptosomal membranes by the Fenton reagents .


Furthermore , preincubation of lymphocytes with D609 resulted in a significant diminution of ionizing radiation ( IR ) -induced 1 ) production of reactive oxygen species ; 2 ) decrease in intracellular reduced glutathione ; 3 ) oxidative damage to proteins and lipids ; and 4 ) activation of nuclear factor-kappaB .


Moreover , when D609 ( 50 mg/kg i.v. ) was administered to mice 10 min prior to total body IR ( 6.5 and 8.5 Gy ) , it protected the mice from IR-induced lethality .


Thus , these results indicate that D609 is a potent antioxidant and has the ability to inhibit IR-induced cellular oxidative stress .


Inhibition of AP-1 by the glucocorticoid-inducible protein GILZ .


The immunosuppressive effects of glucocorticoids arise largely by inhibition of cytokine gene expression , which has been ascribed to interference between the glucocorticoid receptor and transcription factors such as AP-1 and NF-kappa B as well as by competition for common coactivators .


Here we show that glucocorticoid-induced inhibition of interleukin-2 mRNA expression in activated normal T cells required new protein synthesis , suggesting that this phenomenon is secondary to expression of glucocorticoid-regulated genes .


One of the most prominent glucocorticoid-induced genes is glucocorticoid-induced leucine zipper ( GILZ ) , which has been reported to inhibit activation-induced up-regulation of Fas ligand ( FasL ) mRNA .


Indeed , transient expression of GILZ in Jurkat T cells blocked induction of a reporter construct driven by the FasL promoter .


This could be accounted for by GILZ -mediated inhibition of Egr-2 and Egr-3 , NFAT/AP-1 -inducible transcription factors that bind a regulatory element in the FasL promoter and up-regulate FasL expression .


GILZ also potently inhibited AP-1-driven and IL-2 promoter-driven reporter constructs , and recombinant GILZ specifically interacted with c-Fos and c-Jun in vitro and inhibited the binding of active AP-1 to its target DNA .


Whereas homodimerization of GILZ required the presence of its leucine zipper , the interaction with c-Fos and c-Jun occurred through the N-terminal 60-amino acid region of GILZ .


Thus , GILZ represents a glucocorticoid-induced gene product that can inhibit a variety of activation-induced events , at least in part by direct interference with AP-1 , and is therefore a candidate for a mediator of glucocorticoid-induced immunosuppression .


Pharmacokinetic differences between a T cell-tolerizing and a T cell-activating peptide .


Vaccination with a peptide representing a CTL epitope from the human papillomavirus ( HPV ) 16 E7 protein induces a specific CTL response that prevents the outgrowth of HPV16 E7-expressing tumors .


In contrast , vaccination with a peptide encoding an adenovirus type 5 ( Ad5 ) E1A CTL epitope results in CTL tolerance and enhanced growth of an Ad5 E1A-expressing tumor .


It is unclear why these peptides induce such opposite effects .


To determine whether a difference in pharmacokinetics can explain the functional contrasts , tritiated Ad5 E1A and HPV16 E7 peptides were injected into mice .


Results show that the tolerizing peptide spread through the body 16 times faster than the activating peptide and was cleared at least 2 times faster .


The HPV16 E7 peptide kinetics correlated with the kinetics of HPV16 E7 -specific CTL induction .


In contrast , Ad5 E1A peptide injection resulted in physical deletion of preexisting Ad5 E1A -specific CTLs within 24 h after injection .


This tolerization occurred at the time when the peptide reached its maximum peptide concentration in the organs .


These data suggest that ubiquitous expression of the tolerizing Ad5 E1A peptide within a short period of time causes activation-induced cell death of Ad5 E1A -specific CTLs .


Therefore , information on the pharmacokinetics of peptides is vital for the safety and efficacy of peptide-based vaccines .


Smad3 and Smad4 mediate transforming growth factor-beta1 -induced IgA expression in murine B lymphocytes .


Transforming growth factor ( TGF ) -beta1 is well established as a critical IgA isotype switching factor and Smad molecules have been reported to act as transducers and transcriptional factors in the expression of TGF-beta1 -targeted genes .


We examined the involvement of Smad proteins in TGF-beta1 -induced IgA expression .


First , we found that TGF-beta1 significantly increases endogenous germ-line ( GL ) alpha transcripts by LPS-stimulated CH12.LX.4933 ( mu ( + ) ) B lymphoma cells .


To investigate its signaling mechanisms , the lymphoma cell line was transfected with pFL3 that contains the TGF-beta-responsive element of the GLalpha promoter , and stimulated with TGF-beta1 .


Similar to endogenous GLalpha transcripts , TGF-beta1 induces GLalpha promoter activity and overexpression of Smad3 markedly enhances the promoter activity .


This activity is further augmented by cotransfected Smad4 .


On the other hand , Smad7 substantially abrogates the synergistic effect of Smad3/4 on GLalpha promoter activity .


In addition , overexpression of Smad3/4 enhances TGF-beta1 -induced endogenous GLalpha transcripts in normal spleen B cell s .


Finally , in the presence of TGF-beta1 , overexpression of Smad3/4 selectively increases both surface IgA expression and IgA production .


The results from the present study indicate that Smad3 , Smad4 , and Smad7 , at least in part , serve as mediators linking TGF-beta1 to transcriptional regulation of IgA switching related gene and regulation of IgA class switching .


The translesion DNA polymerase zeta plays a major role in Ig and bcl-6 somatic hypermutation .


Ig somatic mutations would be introduced by a polymerase ( pol ) while repairing DNA outside main DNA replication .


We show that human B cells constitutively express the translesion pol zeta , which effectively extends DNA past mismatched bases ( mispair extender ) , and pol eta , which bypasses DNA lesions in an error-free fashion .


Upon B cell receptor ( BCR ) engagement and coculture with activated CD4+ T cells , these lymphocytes upregulated pol zeta , downregulated pol eta , and mutated the Ig and bcl-6 genes .


Inhibition of the pol zeta REV3 catalytic subunit by specific phosphorothioate-modified oligonucleotides impaired Ig and bcl-6 hypermutation and UV damage-induced DNA mutagenesis , without affecting cell cycle or viability .


Thus , pol zeta plays a critical role in Ig and bcl-6 hypermutation , perhaps facilitated by the downregulation of pol eta .


Molecular mechanism of cell cycle progression induced by the oncogene product Tax of human T-cell leukemia virus type I .


The trans-activator protein Tax of human T-cell leukemia virus type I ( HTLV-I ) plays an important role in the development of adult T-cell leukemia through , at least in part , its ability to stimulate cell growth .


We previously reported that Tax induced cell cycle progression from G0/G1 phase to S and G2/M phases in human T-cell line Kit 225 cells .


To elucidate molecular mechanism of Tax -induced cell cycle progression , we systematically examined the effects of Tax on biochemical events associated with cell cycle progression .


Introduction of Tax into resting Kit 225 cells induced activation of the G1/S transition regulation cascade consisting of activation of cyclin dependent kinase 2 ( CDK2 ) and CDK4 , phosphorylation of the Rb family proteins and an increase in free E2F .


The kinase activation was found to result from Tax -induced expression of genes for cell cycle regulatory molecules including cyclin D2 , cyclin E , E2F1 , CDK2 , CDK4 and CDK6 , and Tax -induced reduction of CDK inhibitors p19 ( INK4d ) and p27 ( Kip1 ) .


These modulations by Tax always paralleled the ability of Tax to activate the NF-kappaB transcription pathway .


These results indicate the important role of Tax -mediated trans-activation of the genes for cell cycle regulatory molecules in Tax -induced cell cycle progression .


Cot kinase induces cyclooxygenase-2 expression in T cells through activation of the nuclear factor of activated T cells .


Cyclooxygenase-2 ( COX-2 ) is induced in human T lymphocytes upon T cell receptor triggering .


Here we report that Cot kinase , a mitogen-activated protein kinase kinase kinase involved in T cell activation , up-regulates COX-2 gene expression in Jurkat T cells .


Induction of COX-2 promoter activity by Cot kinase occurred mainly through activation of the nuclear factor of activated T cells ( NFAT ) .


Mutation of the distal ( -105/-97 ) and proximal ( -76/-61 ) NFAT response elements in the COX-2 promoter abolished the activation induced by Cot kinase .


Even more , coexpression of a dominant negative version of NFAT inhibited Cot kinase -mediated COX-2 promoter activation , whereas cotransfection of a constitutively active version of the calcium-dependent phosphatase calcineurin synergizes with Cot kinase in the up-regulation of COX-2 promoter -driven transcription .


Strikingly , Cot kinase increased transactivation mediated by a GAL4-NFAT fusion protein containing the N-terminal transactivation domain of NFATp .


In contrast to phorbol ester plus calcium ionophore A23187 , Cot kinase increases both COX-2 promoter activity and NFAT -mediated transactivation in a cyclosporin A-independent manner .


These data indicate that Cot kinase up-regulates COX-2 promoter -driven transcription through the NFAT response elements , being the Cot kinase -induced NFAT -dependent transactivation presumably implicated in this up-regulation .


Positive and negative roles of the trans-acting T cell factor-1 for the acquisition of distinct Ly-49 MHC class I receptors by NK cells .


Members of the Ly-49 gene family code for class I MHC-specific receptors that regulate NK cell function .


Due to a combinatorial distribution of Ly-49 receptors , NK cells display considerable clonal heterogeneity .


The acquisition of one Ly-49 receptor , Ly-49A is strictly dependent on the transcriptional trans-acting factor T cell-specific factor-1 ( TCF-1 ) .


Indeed , TCF-1 binds to two sites in the Ly-49a promoter and regulates its activity , suggesting that the Ly-49a gene is a direct TCF-1 target .


TCF-1 deficiency resulted in the altered usage of additional Ly-49 receptors .


We show in this study , using TCF-1 beta ( 2 ) -microglobulin double-deficient mice , that these repertoire alterations are not due to Ly-49/MHC class I interactions .


Our findings rather suggest a TCF-1 -dependent , cell autonomous effect on the acquisition of multiple Ly-49 receptors .


Besides reduced receptor usage ( Ly-49A and D ) , we also observed no effect ( Ly-49C ) and significantly expanded ( Ly-49G and I ) receptor usage in the absence of TCF-1 .


These effects did not in all cases correlate with the presence of TCF binding sites in the respective proximal promoter .


Therefore , besides TCF-1 binding to the proximal promoter , Ly-49 acquisition may also be regulated by TCF-1 binding to more distant cis-acting elements and/or by regulating the expression of additional trans-acting factors .


Consistent with the observed differential , positive or negative role of TCF-1 for Ly-49 receptor acquisition , reporter gene assays revealed the presence of an inducing as well as a repressing TCF site in certain proximal Ly-49 promoters .


These findings reveal an important role of TCF-1 for the formation of the NK cell receptor repertoire .


Ligation of CD11b and CD11c beta ( 2 ) integrins by antibodies or soluble CD23 induces macrophage inflammatory protein 1alpha ( MIP-1alpha ) and MIP-1beta production in primary human monocytes through a pathway dependent on nuclear factor-kappaB .


Chemokines and adhesion molecules such as integrins play a major part in the trafficking , extravasation , and recruitment of leukocytes to inflammatory sites .


This study investigated the effects of beta ( 2 ) integrin engagement on chemokine production by freshly isolated human monocytes .


We found that ligation of CD11b or CD11c but not CD11a alpha chains of beta ( 2 ) integrins by antibodies or soluble CD23 ( sCD23 ) fusion proteins rapidly induced transcription and secretion of interleukin 8 , macrophage inflammatory protein ( MIP ) 1alpha , and MIP-1beta .


Because the promoters of these chemokine genes contain kappaB binding sites , we assessed the possible role of nuclear factor-kappaB ( NF-kappaB ) in controlling induction of the genes through beta ( 2 ) integrin engagement .


Electrophoretic mobility shift assays showed that sCD23 or antibodies to CD11b or to CD11c up-regulated DNA-binding activity of NF-kappaB .


Activation of NF-kappaB was accompanied by degradation of its cytosolic inhibitor IkappaB-alpha .


Blockade of depletion of IkappaB-alpha by proteasome inhibitors ( proteasome inhibitor I or acetyl-leucinyl-leucinyl-norleucinal ) led to concomitant inhibition of NF-kappaB DNA-binding activity and expression of MIP-1alpha and MIP-1beta messenger RNA induced by beta ( 2 ) integrin ligation .


These results suggest that triggering of CD11b or CD11c beta ( 2 ) integrin on primary human monocytes provides activation signals leading to nuclear translocation of NF-kappaB and subsequent secretion of MIP-1alpha and MIP-1beta that may have an important role in recruitment of other inflammatory cells during initiation of an inflammatory response


Synergistic transcriptional activation of human Acyl-coenzyme A : cholesterol acyltransterase-1 gene by interferon-gamma and all-trans-retinoic acid THP-1 cells .


Acyl-coenzyme A : cholesterol acyltransferase ( ACAT ) is an intracellular enzyme involved in cellular cholesterol homeostasis and in atherosclerotic foam cell formation .


Human ACAT-1 gene contains two promoters ( P1 and P7 ) , each located in a different chromosome ( 1 and 7 ) ( Li , B. L. , Li , X. L. , Duan , Z. J. , Lee , O. , Lin , S. , Ma , Z. M. , Chang , C. C. , Yang , X. Y. , Park , J. P. , Mohandas , T. K. , Noll , W. , Chan , L. , and Chang , T. Y. ( 1999 ) J. Biol Chem. 274 , 11060-11071 ) .


Interferon-gamma ( IFN-gamma ) , a cytokine that exerts many pro-atherosclerotic effects in vivo , causes up-regulation of ACAT-1 mRNA in human blood monocyte-derived macrophages and macrophage-like cells but not in other cell types .


To examine the molecular nature of this observation , we identified within the ACAT-1 P1 promoter a 159-base pair core region .


This region contains 4 Sp1 elements and an IFN-gamma activated sequence ( GAS ) that overlaps with the second Sp1 element .


In the monocytic cell line THP-1 cell , the combination of IFN-gamma and all-trans-retinoic acid ( a known differentiation agent ) enhances the ACAT-1 P1 promoter but not the P7 promoter .


Additional experiments showed that all-trans-retinoic acid causes large induction of the transcription factor STAT1 , while IFN-gamma causes activation of STAT1 such that it binds to the GAS/Sp1 site in the ACAT-1 P1 promoter .


Our work provides a molecular mechanism to account for the effect of IFN-gamma in causing transcriptional activation of ACAT-1 in macrophage-like cells .


Inhaled nitric oxide down-regulates intrapulmonary nitric oxide production in lipopolysaccharide-induced acute lung injury .


OBJECTIVE : To examine whether inhaled nitric oxide ( NO ) affected the intrapulmonary production of NO , reactive oxygen species , and nuclear factor-kappaB in a lipopolysaccharide ( LPS ) -induced model of acute lung injury .


DESIGN : Prospective , randomized , laboratory study .


SETTING : Experimental laboratory at a biomedical institute .


SUBJECTS : Twenty male rabbits weighing 2.5-3.5 kg .


INTERVENTIONS : Saline or LPS ( 5 mg/kg of body weight ) was administered intravenously with or without NO inhalation ( 10 ppm ) in each group of five rabbits .


MEASUREMENTS AND MAIN RESULTS : LPS increased the lung leak index , the neutrophils and NO levels in bronchoalveolar lavage fluid , and NO levels produced by resting and stimulated alveolar macrophages .


Inhaled NO decreased the lung leak index , the neutrophils and NO levels as measured by nitrite levels in the lavage fluid , and NO produced by the resting and stimulated alveolar macrophages .


Inhaled NO also blocked the activities of reactive oxygen species and nuclear factor-kappaB binding to DNA in lavage cells and in alveolar macrophages .


CONCLUSION : Inhaled NO attenuates LPS-induced acute lung injury , possibly by decreasing NO production in the lungs .


The mechanism of reducing NO production resulting from inhaled NO may involve , in part , the activities of reactive oxygen species and/or nuclear factor-kappaB .


Treatment of allergic airway inflammation and hyperresponsiveness by antisense-induced local blockade of GATA-3 expression .


Recent studies in transgenic mice have revealed that expression of a dominant negative form of the transcription factor GATA-3 in T cells can prevent T helper cell type 2 ( Th2 ) -mediated allergic airway inflammation in mice .


However , it remains unclear whether GATA-3 plays a role in the effector phase of allergic airway inflammation and whether antagonizing the expression and/or function of GATA-3 can be used for the therapy of allergic airway inflammation and hyperresponsiveness .


Here , we analyzed the effects of locally antagonizing GATA-3 function in a murine model of asthma .


We could suppress GATA-3 expression in interleukin ( IL ) -4 -producing T cells in vitro and in vivo by an antisense phosphorothioate oligonucleotide overlapping the translation start site of GATA-3 , whereas nonsense control oligonucleotides were virtually inactive .


In a murine model of asthma associated with allergic pulmonary inflammation and hyperresponsiveness in ovalbumin ( OVA ) -sensitized mice , local intranasal administration of fluorescein isothiocyanate-labeled GATA-3 antisense oligonucleotides led to DNA uptake in lung cells associated with a reduction of intracellular GATA-3 expression .


Such intrapulmonary blockade of GATA-3 expression caused an abrogation of signs of lung inflammation including infiltration of eosinophils and Th2 cytokine production .


Furthermore , treatment with antisense but not nonsense oligonucleotides induced a significant reduction of airway hyperresponsiveness in OVA-sensitized mice to levels comparable to saline-treated control mice , as assessed by both enhanced pause ( PenH ) responses and pulmonary resistance determined by body plethysmography .


These data indicate a critical role for GATA-3 in the effector phase of a murine asthma model and suggest that local delivery of GATA-3 antisense oligonucleotides may be a novel approach for the treatment of airway hyperresponsiveness such as in asthma .


This approach has the potential advantage of suppressing the expression of various proinflammatory Th2 cytokines simultaneously rather than suppressing the activity of a single cytokine .


T helper-cell phenotype regulates atherosclerosis in mice under conditions of mild hypercholesterolemia .


BACKGROUND : T cells are implicated in atherosclerosis , but little is known about the genetic control or molecular pathways , especially under conditions of mild hypercholesterolemia .


METHODS AND RESULTS : BALB/c mice , making a CD4+ Th2 ( IL-4+ ) cell response , express both MHC class II antigens ( IA ( d ) , IE ( d ) ) and are atherosclerosis-resistant .


C57Bl/6 mice produce a CD4+ Th1 ( interferon [ IFN ] gamma+ ) response , express IA ( b ) but no IE , and are atherosclerosis-prone .


To evaluate T helper-cell phenotype in fatty streak formation , wild-type C57Bl/6 mice ( IA ( b ) +IE- ) and transgenic mice , either AB ( o ) , IA ( b ) -IE- ; ABEalpha , IA-IE ( k ) + ; or BL : TG : Ealpha , IA ( b ) +IE ( k ) + , were fed a high-cholesterol diet for 16 weeks and evaluated histomorphometrically for aortic lesions .


Lesion size in AB ( o ) , ABEalpha , and BL : TG : Ealpha strains was decreased by 54 % , 79 % , and 82 % , respectively , compared with wild-type , correlating with decreased Th1 and increased Th2 expression and suggesting that T helper-cell phenotype is important in fatty lesion development .


Decreasing Th1 cells by antibodies ( alpha-CD4 ) or cytokines ( IL-4 ) also caused > /=80 % reductions in lesion size .


Immunohistology revealed IFN-gamma , but not IL-4 , colocalized with activated macrophages .


Confirming these findings in a different mouse strain , BALB/c Stat 6 knockout mice ( Th2 cell-deficient ) developed aortic lesions comparable to C57Bl/6 mice on the same diet .


CONCLUSIONS : In mildly hypercholesterolemic C57Bl/6 mice , presence of IA ( b ) and absence of IE regulated CD4+ T helper-cell phenotype ; fatty lesions were proportional to IFNgamma+ Th1 cells in both C57Bl/6 and BALB/c strains .


IFN-gamma may participate through macrophage activation , whereas IL-4 may act to limit Th1-cell response .


Requirement for p38 and p44/p42 mitogen-activated protein kinases in RAGE -mediated nuclear factor-kappaB transcriptional activation and cytokine secretion .


Advanced glycation end product ( AGE ) activation of the signal-transducing receptor for AGE ( RAGE ) has been linked to a proinflammatory phenotypic change within cells .


However , the precise intracellular signaling pathways involved have not been elucidated .


We demonstrate here that human serum albumin modified with N ( varepsilon ) - ( carboxymethyl ) lysine ( CML ) , a major AGE adduct that progressively accumulates with aging , diabetes , and renal failure , induced nuclear factor ( NF ) -kappaB -driven reporter gene expression in human monocytic THP-1 cells .


The NF-kappaB response was blocked with a synthetic peptide corresponding to the putative ligand-binding domain of RAGE , with anti- RAGE antiserum , and by coexpression of truncated receptors lacking the intracellular domain .


Signal transduction from RAGE to NF-kappaB involved the generation of reactive oxygen species , since reporter gene expression was blocked with the antioxidant N-acetyl-L-cysteine .


CML-modified albumin produced rapid transient activation of tyrosine phosphorylation , extracellular signal-regulated kinase 1 and 2 , and p38 mitogen-activated protein kinase ( MAPK ) , but not c-Jun NH ( 2 ) -terminal kinase .


RAGE -mediated NF-kappaB activation was suppressed by the selective p38 MAPK inhibitor SB203580 and by coexpression of a kinase-dead p38 dominant-negative mutant .


Activation of NF-kappaB by CML-modified albumin increased secretion of proinflammatory cytokines ( tumor necrosis factor-alpha , interleukin-1beta , and monocyte chemoattractant protein-1 ) severalfold , and inhibition of p38 MAPK blocked these increases .


These results indicate that p38 MAPK activation mediates RAGE -induced NF-kappaB -dependent secretion of proinflammatory cytokines and suggest that accelerated inflammation may be a consequence of cellular activation induced by this receptor .


Antigen-receptor cross-linking and lipopolysaccharide trigger distinct phosphoinositide 3-kinase -dependent pathways to NF-kappa B activation in primary B cells .


The NF-kappaB/Rel transcription factors play an important role in the expression of genes involved in B cell development , differentiation and function .


Nuclear NF-kappaB is induced in B cells by engagement of either the BCR or CD40 or by stimulation with lipopolysaccharide ( LPS ) .


Despite the importance of NF-kappaB to B cell function , little is known about the signaling pathways leading to NF-kappaB activation .


In this report we address the role of phosphoinositide 3'-kinase ( PI 3-kinase ) in BCR - and LPS-induced NF-kappaB activation using populations of primary murine resting B cells .


Using the specific pharmacological inhibitors of PI 3-kinase , Wortmannin and LY294002 , we demonstrate that PI 3-kinase activity is vital for BCR -induced NF-kappaB DNA-binding activity .


Furthermore , we show that this is achieved via protein kinase C -dependent degradation of IkappaBalpha .


Similar analyses reveal that PI 3-kinase is also critical in triggering NF-kappaB DNA-binding activity and IkappaBalpha degradation following LPS stimulation .


Interestingly , a PKC inhibitor which blocked the BCR -induced IkappaBalpha degradation had no effect on the degradation of IkappaBalpha after LPS stimulation .


Taken together , our results indicate the involvement of PI 3-kinase in at least two distinct signaling pathways leading to activation of NF-kappaB in B cells .


Tetramer-guided epitope mapping : rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens .


T cell responses to Ags involve recognition of selected peptide epitopes contained within the antigenic protein .


In this report , we describe a new approach for direct identification of CD4+ T cell epitopes of complex Ags that uses human class II tetramers to identify reactive cells .


With a panel of 60 overlapping peptides covering the entire sequence of the VP16 protein , a major Ag for HSV-2 , we generated a panel of class II MHC tetramers loaded with peptide pools that were used to stain peripheral lymphocytes of an HSV-2 infected individual .


With this approach , we identified four new DRA1*0101/DRB1*0401- and two DRA1*0101/DRB1*0404-restricted , VP16-specific epitopes .


By using tetramers to sort individual cells , we easily obtained a large number of clones specific to these epitopes .


Although DRA1*0101/DRB1*0401 and DRA1*0101/DRB1*0404 are structurally very similar , nonoverlapping VP16 epitopes were identified , illustrating high selectivity of individual allele polymorphisms within common MHC variants .


This rapid approach to detecting CD4+ T cell epitopes from complex Ags can be applied to any known Ag that gives a T cell response .


Localized pancreatic NF-kappaB activation and inflammatory response in taurocholate-induced pancreatitis .


Transcription factor nuclear factor-kappaB ( NF-kappaB ) is activated in cerulein pancreatitis and mediates cytokine expression .


The role of transcription factor activation in other models of pancreatitis has not been established .


Here we report upregulation of NF-kappaB and inflammatory molecules , and their correlation with local pancreatic injury , in a model of severe pancreatitis .


Rats received intraductal infusion of taurocholate or saline , and the pancreatic head and tail were analyzed separately .


NF-kappaB and activator protein-1 ( AP-1 ) activation were assessed by gel shift assay , and mRNA expression of interleukin-6 , tumor necrosis factor-alpha , KC , monocyte chemoattractant protein-1 , and inducible nitric oxide synthase was assessed by semiquantitative RT-PCR .


Morphological damage and trypsin activation were much greater in the pancreatic head than tail , in parallel with a stronger activation of NF-kappaB and cytokine mRNA .


Saline infusion mildly affected these parameters .


AP-1 was strongly activated in both pancreatic segments after either taurocholate or saline infusion .


NF-kappaB inhibition with N-acetylcysteine ameliorated the local inflammatory response .


Correlation between localized NF-kappaB activation , cytokine upregulation , and tissue damage suggests a key role for NF-kappaB in the development of the inflammatory response of acute pancreatitis .


CD45 tyrosine phosphatase controls common gamma-chain cytokine -mediated STAT and extracellular signal-related kinase phosphorylation in activated human lymphoblasts : inhibition of proliferation without induction of apoptosis .


The objective of this study was to test whether CD45 signals can influence signaling processes in activated human lymphoblasts .


To this end , we generated lymphoblasts which proliferate in response to common gamma-chain cytokines , but readily undergo apoptosis after cytokine withdrawal .


In experiments with the CD45R0 mAb UCHL-1 , but not control CD45 mAbs , we found significant inhibition of proliferation .


Interestingly , the pan-CD45 mAb GAP8.3 , which is most effective in inhibition of OKT-3-mediated proliferation in quiescent lymphocytes , was ineffective in lymphoblasts .


Addition of CD3 mAb OKT-3 had no influence on IL-2-mediated proliferation ( with or without UCHL-1 ) .


In contrast , after addition of OKT-3 to IL-4 - and IL-7 -stimulated proliferation assays , UCHL-1 signals could not significantly alter cellular proliferation .


We did not find induction of apoptosis following CD45R0 signaling .


In Western blots using mAbs detecting phosphorylated STAT-3 , STAT-5 , STAT-6 , or extracellular signal-related kinase 1/2 , we found that CD45R0 signaling could effectively diminish phosphorylation of these intracellular signaling components .


Using RT-PCR , we found that CD45R0 signaling inhibited IL-2 mRNA production without major influence on IL-13 , IL-5 , or IFN-gamma mRNA levels .


Costimulation with OKT-3 and IL-2 optimally induced secretion of IFN-gamma , TNF-alpha , and IL-5 , which was not decreased by CD45 signals .


In conclusion , we illustrate that CD45R0 signals control early cytokine receptor-associated signaling processes and mRNA and DNA synthesis in activated human lymphoblasts .


Furthermore , we show the existence of CD45 epitopes ( GAP8.3 ) , which are active and critical for signaling in quiescent lymphocytes , but are nonfunctional in activated human lymphoblasts .


Pax5 determines the identity of B cells from the beginning to the end of B-lymphopoiesis .


Despite being one of the most intensively studied cell types , the molecular basis of B cell specification is largely unknown .


The Pax5 gene encoding the transcription factor BSAP is required for progression of B-lymphopoiesis beyond the pro-B cell stage .


Pax5-deficient pro-B cells are , however , not yet committed to the B-lymphoid lineage , but instead have a broad lymphomyeloid developmental potential .


Pax5 appears to mediate B-lineage commitment by repressing the transcription of non-B-lymphoid genes and by simultaneously activating the expression of B-lineage-specific genes .


Pax5 thus functions both as a transcriptional repressor and activator , depending on its interactions with corepressors of the Groucho protein family or with positive regulators such as the TATA-binding protein .


Once committed to the B-lineage , B cells require Pax5 function to maintain their B-lymphoid identity throughout B cell development


Partners in transcription : NFAT and AP-1 .


Combinatorial regulation is a powerful mechanism that enables tight control of gene expression , via integration of multiple signaling pathways that induce different transcription factors required for enhanceosome assembly .


The four calcium-regulated transcription factors of the NFAT family act synergistically with AP-1 ( Fos/Jun ) proteins on composite DNA elements which contain adjacent NFAT and AP-1 binding sites , where they form highly stable ternary complexes to regulate the expression of diverse inducible genes .


Concomitant induction of NFAT and AP-1 requires concerted activation of two different signaling pathways : calcium/calcineurin , which promotes NFAT dephosphorylation , nuclear translocation and activation ; and protein kinase C ( PKC ) /Ras , which promotes the synthesis , phosphorylation and activation of members of the Fos and Jun families of transcription factors .


A fifth member of the NFAT family , NFAT5 , controls the cellular response to osmotic stress , by a mechanism that requires dimer formation and is independent of calcineurin or of interaction with AP-1 .


Pharmacological interference with theNFAT : AP-1 interaction may be useful in selective manipulation of the immune response .


Balanced activation of NFAT and AP-1 is known to be required for productive immune responses , but the role of NFAT : AP-1 interactions in other cell types and biological processes remains to be understood .


Cytokine production by Vgamma ( + ) -T-cell subsets is an important factor determining CD4 ( + ) -Th-cell phenotype and susceptibility of BALB/c mice to coxsackievirus B3-induced myocarditis .


Two coxsackievirus B3 ( CVB3 ) variants ( H3 and H310A1 ) differ by a single amino acid mutation in the VP2 capsid protein .


H3 induces severe myocarditis in BALB/c mice , but H310A1 is amyocarditic .


Infection with H3 , but not H310A1 , preferentially activates Vgamma4 Vdelta4 cells , which are strongly positive for gamma interferon ( IFN-gamma ) , whereas Vgamma1 Vdelta4 cells are increased in both H3 and H310A1 virus-infected animals .


Depletion of Vgamma1 ( + ) cells using monoclonal anti-Vgamma1 antibody enhanced myocarditis and CD4 ( + ) - , IFN-gamma ( + ) -cell responses in both H3- and H310A1-infected mice yet decreased the CD4 ( + ) - , IL-4 ( + ) -cell response .


Depleting Vgamma4 ( + ) cells suppressed myocarditis and reduced CD4 ( + ) IFN-gamma ( + ) cells but increased CD4 ( + ) IL-4 ( + ) T cells .


The role of cytokine production by Vgamma1 ( + ) and Vgamma4 ( + ) T cells was investigated by adoptively transferring these cells isolated from H3-infected BALB/c Stat4 knockout ( Stat4ko ) ( defective in IFN-gamma expression ) or BALB/c Stat6ko ( defective in IL-4 expression ) mice into H3 virus-infected wild-type BALB/c recipients .


Vgamma4 and Vgamma1 ( + ) T cells from Stat4ko mice expressed IL-4 but no or minimal IFN-gamma , whereas these cell populations derived from Stat6ko mice expressed IFN-gamma but no IL-4 .


Stat4ko Vgamma1 ( + ) cells ( IL-4 ( + ) ) suppress myocarditis .


Stat6ko Vgamma1 ( + ) cells ( IFN-gamma ( + ) ) were not inhibitory .


Stat6ko Vgamma4 ( + ) cells ( IFN-gamma ( + ) ) significantly enhanced myocarditis .


Stat4ko Vgamma4 ( + ) cells ( IL-4 ( + ) ) neither inhibited nor enhanced disease .


These results show that distinct gammadelta-T-cell subsets control myocarditis susceptibility and bias the CD4 ( + ) -Th-cell response .


The cytokines produced by the Vgamma subpopulation have a significant influence on the CD4 ( + ) -Th-cell phenotype .


Plasmin -induced expression of cytokines and tissue factor in human monocytes involves AP-1 and IKKbeta -mediated NF-kappaB activation .


It was previously shown that plasmin activates human peripheral monocytes in terms of lipid mediator release and chemotactic migration .


Here it is demonstrated that plasmin induces proinflammatory cytokine release and tissue factor ( TF ) expression by monocytes .


Plasmin 0.043 to 1.43 CTA U/mL , but not active site-blocked plasmin , triggered concentration-dependent expression of mRNA for interleukin-1alpha ( IL-1alpha ) , IL-1beta , tumor necrosis factor-alpha ( TNF-alpha ) , and TF with maximum responses after 4 hours .


Plasmin -mediated mRNA expression was inhibited in a concentration-dependent manner by the lysine analogue trans-4- ( aminomethyl ) cyclohexane-1-carboxylic acid ( t-AMCA ) .


Increases in mRNA levels were followed by concentration- and time-dependent release of IL-1alpha , IL-1beta and TNF-alpha and by TF expression on monocyte surfaces .


Neither cytokines nor TF could be detected when monocytes were preincubated with actinomycin D or cycloheximide .


Electrophoretic mobility shift assays indicated plasmin -induced activation of NF-kappaB ; DNA-binding complexes were composed of p50 , p65 , and c-Rel , as shown by supershift experiments .


Nuclear translocation of NF-kappaB/Rel proteins coincided with IkappaBalpha degradation .


At variance with endotoxic lipopolysaccharide , plasmin elicited the rapid degradation of another cytoplasmic NF-kappaB inhibitor , p105 .


Proteolysis of NF-kappaB inhibitors was apparently due to transient activation of IkappaB kinase ( IKK ) beta that reached maximum activity at 1 hour after plasmin stimulation .


In addition , AP-1 binding was increased in plasmin -treated monocytes , with most complexes composed of JunD , c-Fos , and FosB .


These findings further substantiate the role of plasmin as a proinflammatory activator of human monocytes and reveal an important new link between the plasminogen-plasmin system and inflammation .


STAT3 is constitutively active in some patients with Polycythemia rubra vera .


OBJECTIVE : Polycythemia vera is a clonal stem cell disorder characterized by hyperproliferation of the erythroid , myeloid , and megakaryocytic lineages .


While it has been shown that progenitor cells of P. vera patients are hypersensitive to several growth factors including erythropoietin , insulin-like growth factor-1 , thrombopoietin , interleukin-3 , and granulocyte/monocyte colony-stimulating factor , the molecular pathogenesis of this disease remains unknown .


Growth factor hypersensitivity could be mediated by changes in signal transduction pathways .


We therefore investigated a common downstream effector of cytokines , the signal transducers and activators of transcription ( STATs ) .


A constitutive activation of STAT factors could explain the increased proliferation of P. vera cells even in the absence of growth factor stimulation .


METHODS : Peripheral granulocytes from patients with P. vera and from healthy volunteers were assayed for STAT1 , 3 , and 5 DNA binding by electrophoretic mobility shift assay .


RESULTS : Four of 14 P. vera patients analyzed showed constitutive STAT3 DNA binding in unstimulated peripheral granulocytes , while none of the 17 healthy volunteers tested did .


of the subjects showed constitutive STAT1 or STAT5 activity .


Western blotting demonstrated that , in the three patients , STAT3 is constitutively phosphorylated on Tyr 705 , whereas it is unphosphorylated in the other patients and in controls .


Interestingly , constitutive STAT3 activity did not correlate with the duration of disease or the treatment regimen .


It was observed in a recently diagnosed patient and in two patients treated only with phlebotomy .


CONCLUSION : Our data suggest that constitutive phosphorylation and activation of STAT3 is not a secondary event induced by mutagenizing agents or by prolonged hyperproliferation of hematopoietic cells , but rather represents a primary molecular aberration .


Constitutively active STAT3 may contribute to the growth factor hypersensitivity of P. vera cells .


Identification of phosphorylation sites for Bruton 's tyrosine kinase within the transcriptional regulator BAP/TFII-I .


Bruton 's tyrosine kinase ( Btk ) , a member of the Tec family of cytosolic kinases , is essential for B cell development and function .


BAP/TFII-I , a protein implicated in transcriptional regulation , is associated with Btk in B cells and is transiently phosphorylated on tyrosine following B cell receptor engagement .


BAP/TFII-I is a substrate for Btk in vitro and is hyperphosphorylated on tyrosine upon coexpression with Btk in mammalian cells .


In an effort to understand the physiologic consequences of BAP/TFII-I tyrosine phosphorylation following B cell receptor stimulation , site-directed mutagenesis and phosphopeptide mapping were used to locate the predominant sites of BAP/TFII-I phosphorylation by Btk in vitro .


These residues , Tyr248 , Tyr357 , and Tyr462 , were also found to be the major sites for Btk -dependent phosphorylation of BAP/TFII-I in vivo .


Residues Tyr357 and Tyr462 are contained within the loop regions of adjacent helix-loop-helix-like repeats within BAP/TFII-I .


Mutation of either Tyr248 , Tyr357 , or Tyr462 to phenylalanine reduced transcription from a c-fos promoter relative to wild-type BAP/TFII-I in transfected COS-7 cells , consistent with the interpretation that phosphorylation at these sites contributes to transcriptional activation .


Phosphorylation of BAP/TFII-I by Btk may link engagement of receptors such as surface immunoglobulin to modulation of gene expression .


Expression of interferon consensus sequence binding protein induces potent immunity against BCR/ABL-induced leukemia .


Mice deficient in the interferon consensus sequence binding protein ( ICSBP ) develop a disease resembling chronic myeloid leukemia ( CML ) , which in humans is caused by the BCR/ABL oncoprotein .


Interferon-alpha ( IFN-alpha ) induces ICSBP expression and is an effective therapy for CML .


This study examined whether enforced expression of ICSBP might antagonize BCR/ABL-induced leukemia ; results demonstrated that ICSBP-modified cells generated a protective CD8 ( + ) cytotoxic T-cell response against BCR/ABL-transformed BaF3 cells in a murine leukemia model .


ICSBP expression represents a novel means of stimulating a host immune response to BCR/ABL ( + ) leukemia cells and a potential strategy for immunotherapy of CML .


A transcriptional block in the IL-2 promoter at the -150 AP-1 site in effector CD8+ T cells .


Both CD4+ and CD8+ T cells that produce IL-2 in response to Ag recognition have been isolated .


However , most effector CD8+ T cells recovered after exposure to Ag do not produce sufficient IL-2 to sustain growth , and depend on CD4+ T helper cells for this obligate growth factor .


IL-2 expression in CD4+ T cells is primarily controlled at the level of transcription , but mechanisms restricting IL-2 production in CD8+ T cells have not been elucidated .


To evaluate transcriptional regulation of the IL-2 gene in CD8+ T cells , we stably transfected reporter genes into Ag -specific CD8+ T cell clones .


CD28+ CD8 ( + ) T cells unable to transcribe the IL-2 gene in response to antigenic stimulation had a block in transactivation of the -150 CD28 response element ( CD28RE ) /AP-1 site of the IL-2 promoter , but did transactivate the composite NFAT/AP-1 and OCT/AP-1 sites , and a consensus AP-1 motif .


Mutation of the nonconsensus -150 AP-1 site to a consensus AP-1 site , or insertion of a CD28RE/AP-1 consensus site upstream of the native -150 CD28RE/AP-1 site restored transactivation of the altered promoter .


These results suggest that the defect at the -150 site may reflect the absence or inactivity of a required factor rather than repression of the IL-2 promoter .


Stem cell factor and interleukin-3 induce stepwise generation of erythroid precursor cells from a basic fibroblast growth factor -dependent hematopoietic stem cell line , A-6 .


A m ultipotent immature myeloid cell population was produced from a basic fibroblast growth factor ( bFGF ) -dependent hematopoietic stem cell line , A-6 , when cultured with stem cell factor ( SCF ) replacing bFGF .


Those cells were positive for stem cell markers , c-kit and CD34 , and a myeloid cell marker , F4/80 .


Some cell fractions were also positive for Mac-1 , a macrophage marker or Gr-1 , a granulocytic maker , but negative for an erythroid marker TER119 .


They also showed the expression of mRNA for the myeloid-specific PU.1 but did not that for the erythroid-specific GATA-1 .


Among various cytokines , interleukin-3 ( IL-3 ) induced erythroid precursor cells that expressed the erythroid-specific GATA-1 and beta-major globin .


The quantitative analysis showed that erythroid precursor cells were newly produced from the immature myeloid cells by cultivation with IL-3 .


SCF and IL-3 induced stepwise generation of erythroid precursor cells from an A-6 hematopoietic stem cell line .


Distinct BMI-1 and EZH2 expression patterns in thymocytes and mature T cells suggest a role for Polycomb genes in human T cell differentiation .


BMI-1 and EZH2 Polycomb-group ( PcG ) proteins belong to two distinct protein complexes involved in the regulation of hematopoiesis .


Using unique PcG-specific antisera and triple immunofluorescence , we found that mature resting peripheral T cells expressed BMI-1 , whereas dividing blasts were EZH2 ( + ) .


By contrast , subcapsular immature double-negative ( DN ) ( CD4 ( - ) /CD8 ( - ) ) T cells in the thymus coexpressed BMI-1 and EZH2 or were BMI-1 single positive .


Their descendants , double-positive ( DP ; CD4 ( + ) /CD8 ( + ) ) cortical thymocytes , expressed EZH2 without BMI-1 .


Most EZH2 ( + ) DN and DP thymocytes were dividing , while DN BMI-1 ( + ) /EZH2 ( - ) thymocytes were resting and proliferation was occasionally noted in DN BMI-1 ( + ) /EZH2 ( + ) cells .


Maturation of DP cortical thymocytes to single-positive ( CD4 ( + ) /CD8 ( - ) or CD8 ( + ) /CD4 ( - ) ) medullar thymocytes correlated with decreased detectability of EZH2 and continued relative absence of BMI-1 .


Our data show that BMI-1 and EZH2 expression in mature peripheral T cells is mutually exclusive and linked to proliferation status , and that this pattern is not yet established in thymocytes of the cortex and medulla .


T cell stage-specific PcG expression profiles suggest that PcG genes contribute to regulation of T cell differentiation .


They probably reflect stabilization of cell type-specific gene expression and irreversibility of lineage choice .


The difference in PcG expression between medullar thymocytes and mature interfollicular T cells indicates that additional maturation processes occur after thymocyte transportation from the thymus .


Stepwise lineage restriction of progenitors in lympho-myelopoiesis .


It has long been controversial whether hematopoiesis progresses through ordered stages of determination as in embryonic development .


This is due to the absence of a methodology capable of exactly determining the developmental potential of hematopoietic stem/progenitor cells .


The multilineage progenitor ( MLP ) assay enabled us to discriminate among seven types of hematopoietic progenitors , which are multipotent progenitor p-MTB ( capable of generating myeloid , T and B cells ) , bipotent progenitors p-MT , p-MB and p-TB , and unipotent progenitors p-M , p-T and p-B .


Among these seven types , the p-TB type progenitor was found to be absent .


These findings indicate that the process of lineage commitment proceeds through an ordered but not random process .


By extending the area of investigation to include the erythroid lineage , more convincing evidence for the ordered process was obtained .


Detailed and exact illustration of the process of hematopoiesis will provide an opportunity to revive hematopoiesis as one of the most fascinating targets of research in developmental biology


Epstein-Barr Virus and its glycoprotein-350 upregulate IL-6 in human B-lymphocytes via CD21 , involving activation of NF-kappaB and different signaling pathways .


Epstein-Barr virus ( EBV ) is a ubiquitous and highly immunotropic gamma herpesvirus that infects more than 90 % of humans worldwide .


Its pathogenicity leads to a number of diseases including tumors that result from EBV 's ability to readily transform B-lymphocytes and , to a lesser extent , epithelial cells .


EBV utilizes CD21/CR2 as its receptor on B cells to initiate the infection process .


EBV binds to CR2 through its major envelope glycoprotein-350 ( gp350 ) and is also a remarkable immunomodulating agent .


We had previously shown that EBV is capable of modulating the synthesis of a number of cytokines .


We now show that while both purified recombinant gp350 ( rgp350 ) and EBV upregulate IL-6 mRNA synthesis in B cells , EBV-induced IL-6 gene activation occurs for a significantly longer period of time ( i.e. 12 hours for EBV as compared to 6 hours for rgp350 ) .


Moreover , the half-life of EBV-induced IL-6 mRNA was also significantly longer ( 10 hours ) than that of mRNA induced by rgp350 ( about 6 hours ) .


Both EBV and gp350 enhance the binding of the NF-kappaB transcription factor , as determined by band-shift and augment NF-kappaB -mediated activation of a CAT reporter plasmid .


Furthermore , we demonstrate that while the activation of IL-6 gene expression by gp350 is mediated primarily by the protein kinase C pathway , EBV can mediate its effects through multiple signaling pathways .


To our knowledge this is the first report showing that the binding of a herpesvirus envelope glycoprotein to CR2 on human B cells results in the activation of the NF-kappaB transcription factor leading to the upregulation of IL-6 gene expression in these lymphocytes .


Gene- and tissue-specificity of mutation in Big Blue rats treated with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene .


In a previous study , we found that treating transgenic Big Blue rats with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene ( N-OH-AAF ) produced the same major DNA adduct in the target liver and the nontarget spleen lymphocytes and bone marrow cells , induced lacI mutants in the liver , and induced much lower frequencies of l acI and hprt mutants in spleen lymphocytes .


In the present study , sequence analysis was conducted on lacI DNA and hprt cDNA from the mutants , to determine the mutational specificity of N-OH-AAF in the rat .


All the mutation spectra from N-OH-AAF-treated rats differed significantly from corresponding mutation profiles from untreated animals ( P = 0.02 to P < 0.0001 ) .


Although there were similarities among the mutational patterns derived from N-OH-AAF-treated rats ( e.g. , G : C -- > T : A transversion was the most common mutation in all mutation sets ) , there were significant differences in the patterns of basepair substitution and frameshift mutation between the liver and spleen lymphocyte lacI mutants ( P = 0.02 ) and between the spleen lymphocyte lacI and hprt mutants ( P = 0.04 ) .


Also , multiplex PCR analysis of genomic DNA from the hprt mutants indicated that 12 % of mutants from treated rats had major deletions in the hprt gene ; no corresponding incidence of large deletions was evident among lacI mutations .


All the mutation profiles reflect the general mutational specificity of the major DNA adduct formed by N-OH-AAF .


The differences between N-OH-AAF mutation in the endogenous gene and transgene can be partially explained by the structures of the two genes .


The tissue-specificity of the mutation spectra may contribute to targeting tumor formation to the liver .


Caspase -dependent cleavage of the hematopoietic specific adaptor protein Gads alters signalling from the T cell receptor .


Gads is a SH2 and SH3 domain -containing , hematopoietic-specific adaptor protein that functions in signalling from the T cell receptor .


Gads acts by linking SLP-76 , bound by the carboxy-terminal Gads SH3 domain , to tyrosine phosphorylated LAT which contains binding sites for the Gads SH2 domain .


Gads is distinguished from Grb2 and the closely related Grap protein by the presence of a 120 amino acid unique region between the SH2 domain and the carboxy terminal SH3 domain .


Here we demonstrate that the unique region of Gads contains a capase cleavage site .


Induction of apoptosis in lymphocytes results in detectable Gads cleavage by 60 min .


Gads cleavage is blocked in vivo by treating cells with a caspase 3 inhibitor .


A putative caspase 3 cleavage site was identified within the unique region and mutation of this site prevented Gads cleavage in vitro , and in vivo .


The Gads cleavage products retained the predicted binding specificity for SLP-76 and LAT .


Expression of the Gads cleavage products in Jurkat T cells inhibited NFAT activation following TCR cross linking .


These findings indicate that cleavage of Gads in vivo could function to alter signalling downstream of the T cell receptor by disrupting cross talk between SLP-76 and LAT .


Targeting of p300 to the interleukin-2 promoter via CREB-Rel cross-talk during mitogen and oncogenic molecular signaling in activated T-cells .


In this report , we explore the mechanisms of targeting of p300 to the interleukin-2 ( IL-2 ) promoter in response to mitogenic and oncogenic molecular signals .


Recruitment of p300 by cAMP-responsive element-binding protein-Rel cross-talk at the composite CD28 response element ( CD28RE ) - TRE element of the IL-2 promoter is essential for promoter inducibility during T-cell activation , and CD28RE-TRE is the exclusive target of the human T-cell lymphotropic virus type I oncoprotein Tax .


The intrinsic histone acetyltransferase activity of p300 is dispensable for activation of the IL-2 promoter , and the N-terminal 743 residues contain the minimal structural requirements for synergistic transactivation of the CD28RE-TRE , the IL-2 promoter , and endogenous IL-2 gene expression .


Mutational analysis of p300 reveals differential structural requirements for the N-terminal p300 module by individual cis-elements within the IL-2 promoter .


These findings provide evidence that p300 assembles at the IL-2 promoter to form an enhanceosome-like signal transduction target that is centrally integrated at the CD28RE-TRE element of the IL-2 promoter through specific protein module-targeted associations in activated T-cells .


Regulation of cytokine production in T-cell responses to inhalant allergen : GATA-3 expression distinguishes between Th1- and Th2-polarized immunity .


BACKGROUND : The precise nature of allergen-specific cytokine responses in atopics versus non-atopics , in particular the 'Th1 polarity ' of responses in non-atopics , remains controversial .


This is due in part to the relative insensitivity of cytokine detection systems , and associated variations in kinetics of cytokine production and catabolism in in vitro culture systems .


As an alternative to cytokine measurement , this study focuses on expression of the transcription factor GATA-3 for analysis of allergen-specific Th cell responses .


METHODS : Cord blood mononuclear cells were Th1- or Th2-polarized by culture in IL-12- or IL-4-employing established methods ; PBMC from house dust mite ( HDM ) -sensitive atopics and controls were stimulated overnight with HDM ; cytokine production was measured by ELISA and GATA-3 mRNA expression by PCR .


RESULTS : Cytokine -driven Th2 polarization of naive T cells is associated with marked upregulation of GATA-3 expression , whereas a reciprocal expression pattern accompanies differentiation towards the Th1 cytokine phenotype .


In T cells from HDM skin prick test-positive ( HDM-SPT+/HDM-IgE+ ) volunteers , overnight stimulation results in marked upregulation of GATA-3 expression , compared to an equally marked downregulation of expression in T cells from SPT-/IgE- subjects .


In subjects who are HDM-SPT+ but IgE- , GATA-3 expression levels remained relatively stable during culture with HDM .


CONCLUSIONS : Upregulation of GATA-3 expression in PBMC is a hallmark of the early phase of Th2 recall responses to specific allergen in atopics .


The reciprocal expression pattern observed in HDM-specific recall responses of non-atopics provides independent confirmation of the presence of underlying Th1-like immunity in these subjects .


The parallel findings in neonatal T cells suggest that the same approach may be utilized for monitoring the progress of allergen-specific Th1/Th2 memory development during early childhood , and hence in assessment of risk for future allergic disease .


The heat shock response reduces myelin oligodendrocyte glycoprotein -induced experimental autoimmune encephalomyelitis in mice .


The stress response ( SR ) can block inflammatory gene expression by preventing activation of transcription factor nuclear factor-kappa B ( NF-kappaB ) .


As inflammatory gene expression contributes to the pathogenesis of demyelinating diseases , we tested the effects of the SR on the progression of the demyelinating disease experimental autoimmune encephalomyelitis ( EAE ) .


EAE was actively induced in C57BL/6 mice using an encephalitogenic myelin oligodendrocyte glycoprotein ( MOG ( 35-55 ) ) peptide .


Whole body hyperthermia was used to induce a heat shock response ( HSR ) in immunized mice 2 days after the booster MOG ( 35-55 ) peptide injection .


The HSR reduced the incidence of EAE by 70 % , delayed disease onset by 6 days , and attenuated disease severity .


The HSR attenuated leukocyte infiltration into CNS assessed by quantitation of perivascular infiltrates , and by reduced staining for CD4 and CD25 immunopositive T-cells .


T-cell activation , assessed by the production of interferon gamma ( IFNgamma ) in response to MOG ( 35-55 ) , was also decreased by the HSR .


The HSR reduced inflammatory gene expression in the brain that normally occurs during EAE , including the early increase in RANTES ( regulated on activation of normal T-cell expressed and secreted ) expression , and the later expression of the inducible form of nitric oxide synthase .


The early activation of transcription factor NF-kappaB was also blocked by the HSR .


The finding that the SR reduces inflammation in the brain and the clinical severity of EAE opens a novel therapeutic approach for prevention of autoimmune diseases .


In vivo detection of intracellular signaling pathways in developing thymocytes .


Information regarding the intracellular signaling processes that occur during the development of T cells has largely been obtained with the use of transgenic mouse models , which although providing invaluable information are time consuming and costly .


To this end , we have developed a novel system that facilitates the in vivo analysis of signal transduction pathways during T-lymphocyte development .


This approach uses reporter-plasmids for the detection of intracellular signals mediated by the mitogen-activated protein kinase or cyclic AMP-dependent protein kinase .


Reporter-plasmids are transfected into thymocytes in fetal thymic organ culture by accelerated DNA/particle bombardment ( gene gun ) , and the activation of a signaling pathway is determined in the form of a standard luciferase assay .


Importantly , this powerful technique preserves the structural integrity of the thymus , and will provide an invaluable tool to study how thymocytes respond to normal environmental stimuli encountered during differentiation within the thymic milieu .


Thus , this method allows for the monitoring of signals that occur in a biological time frame , such as during differentiation , and within the natural environment of differentiating cells .


Core-binding factor beta ( CBFbeta ) , but not CBFbeta-smooth muscle myosin heavy chain , rescues definitive hematopoiesis in CBFbeta -deficient embryonic stem cells .


Core-binding factor beta ( CBFbeta ) is the non-DNA-binding subunit of the heterodimeric CBFs .


Genes encoding CBFbeta ( CBFB ) , and one of the DNA-binding CBFalpha subunits , Runx1 ( also known as CBFalpha2 , AML1 , and PEBP2alphaB ) , are required for normal hematopoiesis and are also frequent targets of chromosomal translocations in acute leukemias in humans .


Homozygous disruption of either the Runx1 or Cbfb gene in mice results in embryonic lethality at midgestation due to hemorrhaging in the central nervous system , and severely impairs fetal liver hematopoiesis .


Results of this study show that Cbfb-deficient mouse embryonic stem ( ES ) cells can differentiate into primitive erythroid colonies in vitro , but are impaired in their ability to produce definitive erythroid and myeloid colonies , mimicking the in vivo defect .


Definitive hematopoiesis is restored by ectopic expression of full-length Cbfb transgenes , as well as by a transgene encoding only the heterodimerization domain of CBFbeta .


In contrast , the CBFbeta - smooth muscle myosin heavy chain ( SMMHC ) fusion protein generated by the inv ( 16 ) associated with acute myeloid leukemias ( M4Eo ) can not rescue definitive hematopoiesis by Cbfb-deficient ES cells .


Sequences responsible for the inability of CBFbeta -SMMHC to rescue definitive hematopoiesis reside in the SMMHC portion of the fusion protein .


Results also show that the CBFbeta-SMMHC fusion protein transdominantly inhibits definitive hematopoiesis , but not to the same extent as homozygous loss of Runx1 or Cbfb .


CBFbeta-SMMHC preferentially inhibits the differentiation of myeloid lineage cells , while increasing the number of blastlike cells in culture .


The latency pattern of Epstein-Barr virus infection and viral IL-10 expression in cutaneous natural killer/T-cell lymphomas .


The nasal type , extranodal natural killer or T ( NK/T ) -cell lymphoma is usually associated with latent Epstein-Barr virus ( EBV ) infection .


In order to elucidate the EBV gene expression patterns in vivo , we examined eight patients with cutaneous EBV-related NK/T-cell lymphomas , including six patients with a NK-cell phenotype and two patients with a T-cell phenotype .


The implication of EBV in the skin lesions was determined by the presence of EBV-DNA , EBV-encoded nuclear RNA ( EBER ) and a clonality of EBV-DNA fragments containing the terminal repeats .


Transcripts of EBV-encoded genes were screened by reverse transcription- polymerase chain reaction ( RT-PCR ) , and confirmed by Southern blot hybridization .


The expression of EBV-related antigens was examined by immunostaining using paraffin-embedded tissue sections and cell pellets of EBV-positive cell lines .


Our study demonstrated that all samples from the patients contained EBV nuclear antigen ( EBNA ) -1 mRNA which was transcribed using the Q promoter , whereas both the Q promoter and another upstream promoter ( Cp/Wp ) were used in EBV-positive cell lines , B95.8 , Raji and Jiyoye .


Latent membrane protein-1 ( LMP-1 ) mRNA was detected in seven of eight patients and all cell lines , whereas EBNA-2 transcripts were found only in the cell lines .


Immunostaining showed no LMP-1 , EBNA-2 or ZEBRA antigens in the paraffin-embedded tissue sections , although they were positive in the cell line cells .


Latent BHRF1 transcripts encoding bcl-2 homologue and BCRF1 transcripts encoding viral interleukin ( vIL ) -10 were detected in one and two of eight patients , respectively .


A patient with NK-cell lymphoma expressing both transcripts died of rapid progression of the illness .


Our results indicate that the restricted expression of the latency-associated EBV genes and the production of vIL-10 and bcl-2 homologue may favour tumour growth , evading the host immune surveillance .


Copyright 2001 Cancer Research Campaign .


Oxidized alkyl phospholipids are specific , high affinity peroxisome proliferator-activated receptor gamma ligands and agonists .


Synthetic high affinity peroxisome proliferator-activated receptor ( PPAR ) agonists are known , but biologic ligands are of low affinity .


Oxidized low density lipoprotein ( oxLDL ) is inflammatory and signals through PPARs .


We showed , by phospholipase A ( 1 ) digestion , that PPARgamma agonists in oxLDL arise from the small pool of alkyl phosphatidylcholines in LDL .


We identified an abundant oxidatively fragmented alkyl phospholipid in oxLDL , hexadecyl azelaoyl phosphatidylcholine ( azPC ) , as a high affinity ligand and agonist for PPARgamma .


[ ( 3 ) H ] azPC bound recombinant PPARgamma with an affinity ( K ( d ) ( ( app ) ) approximately 40 nm ) that was equivalent to rosiglitazone ( BRL49653 ) , and competition with rosiglitazone showed that binding occurred in the ligand-binding pocket .


azPC induced PPRE reporter gene expression , as did rosiglitazone , with a half-maximal effect at 100 nm .


Overexpression of PPARalpha or PPARgamma revealed that azPC was a specific PPARgamma agonist .


The scavenger receptor CD36 is encoded by a PPRE-responsive gene , and azPC enhanced expression of CD36 in primary human monocytes .


We found that anti-CD36 inhibited azPC uptake , and it inhibited PPRE reporter induction .


Results with a small molecule phospholipid flippase mimetic suggest azPC acts intracellularly and that cellular azPC accumulation was efficient .


Thus , certain alkyl phospholipid oxidation products in oxLDL are specific , high affinity extracellular ligands and agonists for PPARgamma that induce PPAR-responsive genes


Regulation of the human MAT2B gene encoding the regulatory beta subunit of methionine adenosyltransferase , MAT II .


Methionine adenosyltransferase ( MAT ) catalyzes the biosynthesis of S-adenosylmethionine ( AdoMet ) , a key molecule in transmethylation reactions and polyamine biosynthesis .


The MAT II isozyme consists of a catalytic alpha2 and a regulatory beta subunit .


Down-regulation of the MAT II beta subunit expression causes a 6-10-fold increase in intracellular AdoMet levels .


To understand the mechanism by which the beta subunit expression is regulated , we cloned the MAT2B gene , determined its organization , characterized its 5'-flanking sequences , and elucidated the in vitro and in vivo regulation of its promoter .


Transcription of the MAT2B gene initiates at position -203 relative to the translation start site .


Promoter deletion analysis defined a minimal promoter between positions +52 and +93 base pairs , a GC-rich region .


Inclusion of the sequences between -4 and +52 enhanced promoter activity ; this was primarily because of an Sp1 recognition site at +9/+15 .


The inclusion of sequences up to position -115 provided full activity ; this was attributed to a TATA at -32 .


The Sp1 site at position +9 was key for the formation of protein.DNA complexes .


Mutation of both the Sp1 site at +9 and the TATA at -32 reduced promoter activity to its minimal level .


Supershift assays showed no effect of the anti-Sp1 antibody on complex formation , whereas the anti-Sp3 antibody had a strong effect on protein.DNA complex formation , suggesting that Sp3 is one of the main factors binding to this Sp1 site .


Chromatin immunoprecipitation assays supported the involvement of both Sp1 and Sp3 in complexes formed on the MAT2B promoter .


The data show that the 5'-untranslated sequences play an important role in regulating the MAT2B gene and identifies the Sp1 site at +9 as a potential target for modulating MAT2B expression , a process that can have a major effect on intracellular AdoMet levels .


Molecular pathogenesis of influenza A virus infection and virus-induced regulation of cytokine gene expression .


Despite vaccines and antiviral substances influenza still causes significant morbidity and mortality world wide .


Better understanding of the molecular mechanisms of influenza virus replication , pathogenesis and host immune responses is required for the development of more efficient means of prevention and treatment of influenza .


Influenza A virus , which replicates in epithelial cells and leukocytes , regulates host cell transcriptional and translational systems and activates , as well as downregulates apoptotic pathways .


Influenza A virus infection results in the production of chemotactic ( RANTES , MIP-1 alpha , MCP-1 , MCP-3 , and IP-10 ) , pro-inflammatory ( IL-1 beta , IL-6 , IL-18 , and TNF-alpha ) , and antiviral ( IFN-alpha/beta ) cytokines .


Cytokine gene expression is associated with the activation of NF-kappa B , AP-1 , STAT and IRF signal transducing molecules in influenza A virus-infected cells .


In addition of upregulating cytokine gene expression , influenza A virus infection activates caspase-1 enzyme , which is involved in the proteolytic processing of proIL-1 beta and proIL-18 into their biologically active forms .


Influenza A virus-induced IFN-alpha/beta is essential in host 's antiviral defence by activating the expression of antiviral Mx , PKR and oligoadenylate synthetase genes .


IFN-alpha/beta also prolongs T cell survival , upregulates IL-12 and IL-18 receptor gene expression and together with IL-18 stimulates NK and T cell IFN-gamma production and the development of Th1-type immune response .


Comparison of hprt and lacI mutant frequency with DNA adduct formation in N-hydroxy-2-acetylaminofluorene-treated Big Blue rats .


N-Hydroxy-2-acetylaminofluorene ( N-OH-AAF ) is the proximate carcinogenic metabolite of the powerful rat liver carcinogen 2-acetylaminofluorene .


In this study , transgenic Big Blue ( R ) rats were used to examine the relationship between in vivo mutagenicity and DNA adduct formation by N-OH-AAF in the target liver compared with that in nontarget tissues .


Male rats were given one , two , or four doses of 25 mg N-OH-AAF/kg body weight by i.p. injection at 4-day intervals , and groups of treated and control rats were euthanized up to 10 weeks after beginning the dosing .


Mutant frequencies were measured in the spleen lymphocyte hprt gene , and lacI mutant frequencies were determined in the liver and spleen lymphocytes .


At 6 weeks after beginning the dosing , the hprt mutant frequency in spleen lymphocytes from the four-dose group was 16.5 x 10 ( -6 ) compared with 3.2 x 10 ( -6 ) in control animals .


Also at 6 weeks , rats given one , two , or four doses of N-OH-AAF had lacI mutant frequencies in the liver of 97.6 , 155.6 , and 406.8 x 10 ( -6 ) , respectively , compared with a control frequency of 25.7 x 10 ( -6 ) ; rats given four doses had lacI mutant frequencies in spleen lymphocytes of 55.8 x 10 ( -6 ) compared with a control frequency of 20.4 x 10 ( -6 ) .


Additional rats were evaluated for DNA adduct formation in the liver , spleen lymphocytes , and bone marrow by ( 32 ) P-postlabeling .


Adduct analysis was conducted 1 day after one , two , and four treatments with N-OH-AAF , 5 days after one treatment , and 9 days after two treatments .


N- ( Deoxyguanosin-8-yl ) -2-aminofluorene was the major DNA adduct identified in all the tissues examined .


Adduct concentrations increased with total dose to maximum values in samples taken 1 day after two doses , and remained essentially the same after four doses .


In samples taken after four doses , adduct levels were 103 , 28 , and 7 fmol/microg of DNA in liver , spleen lymphocytes , and bone marrow , respectively .


The results indicate that the extent of both DNA adduct formation and mutant induction correlates with the organ specificity for N-OH-AAF carcinogenesis in the rat .


Expression of Mad1 in T cells leads to reduced thymic cellularity and impaired mitogen-induced proliferation .


To investigate Mad1 function in vivo , transgenic mice were generated that express a Mad1 transgene in T lineage cells under the control of the proximal lck promoter .


Thymus size in lck-Mad1 transgenic mice is drastically reduced although representation of the various thymocyte sub populations appears normal .


To investigate more closely any effects of Mad1 expression on thymocytes , we examined thymic selection using MHC class I-restricted H-Y-TCR transgenic mice .


Mad1 expression in vivo reduces the efficiency of positive selection .


Furthermore , thymocytes and splenic T cells from lck-Mad1 transgenic mice display a profound proliferative defect in response to activation with either PMA/Ionomycin or immobilized anti-CD3/CD28 antibody .


This proliferative defect is not reversed by addition of exogenous IL-2 and is p53-independent .


The growth inhibition caused by Mad1 is overcome by expression of active c-Myc .


Differential requirement for the transcription factor PU.1 in the generation of natural killer cells versus B and T cells .


PU.1 is a member of the Ets family of transcription factors required for the development of various lymphoid and myeloid cell lineages , but its role in natural killer ( NK ) cell development is not known .


The study shows that PU.1 is expressed in NK cells and that , on cell transfer into alymphoid Rag2/gammac ( -/- ) mice , hematopoietic progenitors of PU.1 ( -/- ) fetal liver cells could generate functional NK cells but not B or T cells .


Nevertheless , the numbers of bone marrow NK cell precursors and splenic mature NK cells were reduced compared to controls .


Moreover , PU.1 ( -/- ) NK cells displayed reduced expression of the receptors for stem cell factor and interleukin ( IL ) -7 , suggesting a nonredundant role for PU.1 in regulating the expression of these cytokine receptor genes during NK cell development .


PU.1 ( -/- ) NK cells also showed defective expression of inhibitory and activating members of the Ly49 family and failed to proliferate in response to IL-2 and IL-12 .


Thus , despite the less stringent requirement for PU.1 in NK cell development compared to B and T cells , PU.1 regulates NK cell differentiation and homeostasis .


Benzene-extracted components are important for the major activity of diesel exhaust particles : effect on interleukin-8 gene expression in human bronchial epithelial cells .


Epidemiologic and experimental studies suggest that diesel exhaust particles ( DEPs ) may be related to increasing respiratory mortality and morbidity .


We have shown that DEPs augmented the production of inflammatory cytokines by human airway epithelial cells in vitro .


To better understand the mechanisms of their proinflammatory activities , we studied the effects of several components extracted from DEPs on interleukin ( IL ) -8 expression in human bronchial epithelial cell line BEAS-2B and normal human airway epithelial cells obtained from very peripheral airways by an ultrathin bronchoscope .


We used several agents active on signal transduction pathways in cytokine expression , such as the protein kinase C inhibitor staurosporin , antioxidant agents including N-acetyl cysteine ( NAC ) and pyrrolidine dithiocarbamate ( PDTC ) , and p38 mitogen-activated protein kinase ( MAPK ) inhibitor SB203580 .


Benzene-extracted components showed effects mimicking DEPs on IL-8 gene expression , release of several cytokines ( IL-8 ; granulocyte macrophage colony-stimulating factor ; and regulated on activation , normal T cells expressed and secreted ) and nuclear factor ( NF ) -kappa B activation .


We also found that NAC , PDTC , and SB203580 suppressed the activities of DEPs and their benzene extracts , suggesting the roles of oxidants-mediated NF-kappa B activation and p38MAPK pathways .


Finally , benzo [ a ] pyrene , one of the important compounds included in the benzene component , replicated the activities shown by DEPs .


The nuclear receptor PPAR gamma is expressed by mouse T lymphocytes and PPAR gamma agonists induce apoptosis .


Peroxisome proliferator-activated receptor ( PPAR ) -gamma is a nuclear hormone receptor that serves as a trans factor to regulate lipid metabolism .


Intense interest is focused on PPAR-gamma and its ligands owing to its putative role in adipocyte differentiation .


Little is known , however , about the functions of PPAR-gamma in the immune system , especially in T lymphocytes .


We demonstrate that both naive and activated ovalbumin-specific T cells from DO11.10-transgenic mice express PPAR-gamma mRNA and protein .


In order to determine the function of PPAR-gamma , T cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin or antigen and antigen-presenting cells .


Simultaneous exposure to PPAR-gamma ligands ( e.g. 15-deoxy-Delta ( 12 , 14 ) -prostaglandin J ( 2 ) , troglitazone ) showed drastic inhibition of proliferation and significant decreases in cell viability .


The decrease in cell viability was due to apoptosis of the T lymphocytes , and occurred only when cells were treated with PPAR-gamma , and not PPAR-alpha agonists , revealing specificity of this response for PPAR-gamma .


These observations suggest that PPAR-gamma agonists play an important role in regulating T cell-mediated immune responses by inducing apoptosis .


T cell death via PPAR-gamma ligation may act as a potent anti-inflammatory signal in the immune system , and ligands could possibly be used to control disorders in which excessive inflammation occurs .


Suppression of lung inflammation in rats by prevention of NF-kappaB activation in the liver .


Activation of NF-kappaB and production of NF-kappaB -dependent chemokines are thought to be involved in the pathogenesis of neutrophilic lung inflammation .


Calpain-1 inhibitor ( CI-1 ) blocks activation of NF-kappaB by preventing proteolysis of the inhibitory protein IkappaB-alpha by the ubiquitin/proteasome pathway .


We hypothesized that inhibition of proteasome function with CI-1 would block NF-kappaB activation in vivo after intraperitoneal ( i.p. ) treatment with bacterial lipopolysaccharide ( LPS ) , and that NF-kappaB inhibition would be associated with suppression of chemokine gene expression and attenuation of neutrophilic alveolitis .


We treated rats with a single i.p. injection of CI-1 ( 10 mg/kg ) two hours prior to i.p. LPS ( 7 mg/kg ) .


Treatment with Cl-1 prevented degradation of IkappaB-alpha and activation of NF-kappaB in the liver in response to LPS ; however , Cl-1 treatment had no detected effect on NF-kappaB activation in lung tissue .


CI-1 treatment prior to LPS resulted in 40 % lower MIP-2 concentration in lung lavage fluid compared to rats treated with vehicle prior to LPS ( 502 +/- 112 pg/ml vs. 859 +/-144 pg/ml , P < 0.05 ) .


In addition , CI-1 treatment substantially inhibited LPS-induced neutrophilic alveolitis ( 2.7+ /- 1.2 x 10 ( 5 ) vs. 43.7 +/- 12.2 x 10 ( 5 ) lung lavage neutrophils , P < 0.01 ) .


These data indicate that NF-kappaB inhibition in the liver can alter lung inflammation induced by systemic LPS treatment and suggest that a liver-lung interaction contributes to the inflammatory response of the lung .


Notch1 regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength .


Notch signaling regulates cell fate decisions in multiple lineages .


We demonstrate in this report that retroviral expression of activated Notch1 in mouse thymocytes abrogates differentiation of immature CD4+CD8+ thymocytes into both CD4 and CD8 mature single-positive T cells .


The ability of Notch1 to inhibit T cell development was observed in vitro and in vivo with both normal and TCR transgenic thymocytes .


Notch1 -mediated developmental arrest was dose dependent and was associated with impaired thymocyte responses to TCR stimulation .


Notch1 also inhibited TCR-mediated signaling in Jurkat T cells .