These data indicate that constitutively active Notch1 abrogates CD4+ and CD8+ maturation by interfering with TCR signal strength and provide an explanation for the physiological regulation of Notch expression during thymocyte development .


Expression of SART3 antigen and induction of CTLs by SART3-derived peptides in breast cancer patients .


We recently reported the SART3 tumour-rejection antigen as possessing tumour epitopes capable of inducing HLA-class I-restricted cytotoxic T lymphocytes ( CTLs ) .


This study investigated expression of the SART3 antigen in breast cancer to explore an appropriate molecule for use in specific immunotherapy of breast cancer patients .


The SART3 antigen was detected in all of the breast cancer cell lines tested , 30 of 40 ( 75 % ) breast cancer tissue samples , and 0 of 3 non-tumourous breast tissue samples .


SART3 derived peptides at positions 109-118 and 315-323 induced HLA-A24 restricted CTLs that reacted to breast cancer cells from the peripheral blood mononuclear cells ( PBMCs ) of breast cancer patients .


Therefore , the SART3 antigen and its peptides could be an appropriate molecule for use in specific immunotherapy of the majority of HLA-A24-positive breast cancer patients


Autostimulation of the Epstein-Barr virus BRLF1 promoter is mediated through consensus Sp1 and Sp3 binding sites .


As an essential step in the lytic cascade , the Rta homologues of gammaherpesviruses all activate their own expression .


Consistent with this biologic function , the Epstein-Barr virus ( EBV ) Rta protein powerfully stimulates the promoter of its own gene , Rp , in EBV-positive B cells in transient-transfection reporter-based assays .


We analyzed the activity of RpCAT in response to Rta by deletional and site-directed mutagenesis .


Two cognate Sp1 binding sites located at -279 and -45 relative to the transcriptional start site proved crucial for Rta-mediated activation .


Previously described binding sites for the cellular transcription factor Zif268 and the viral transactivator ZEBRA were found to be dispensable for activation of RpCAT by Rta .


Gel shift analysis , using extracts of B cells in latency or induced into the lytic cycle , identified Sp1 and Sp3 as the predominant cellular proteins bound to Rp near -45 .


During the lytic cycle , ZEBRA bound Rp near the Sp1/Sp3 site .


The binding of Sp1 and Sp3 to Rp correlated with the reporter activities in the mutagenesis study , establishing a direct link between transcriptional activation of Rp by Rta and DNA binding by Sp1 and/or Sp3 .


The relative abundance or functional state of the cellular Sp1 and Sp3 transcription factors may be altered in response to stimuli that induce the BRLF1 promoter and thereby contribute to the activation of the viral lytic cycle .


Gene transfer of antisense hypoxia inducible factor-1 alpha enhances the therapeutic efficacy of cancer immunotherapy .


Solid tumors meet their demands for nascent blood vessels and increased glycolysis , to combat hypoxia , by activating multiple genes involved in angiogenesis and glucose metabolism .


Hypoxia inducible factor-1 ( HIF-1 ) is a constitutively expressed basic helix-loop-helix transcription factor , formed by the assembly of HIF-1alpha and HIF-1beta ( Arnt ) , that is stablized in response to hypoxia , and rapidly degraded under normoxic conditions .


It activates the transcription of genes important for maintaining oxygen homeostasis .


Here , we demonstrate that engineered down-regulation of HIF-1alpha by intratumoral gene transfer of an antisense HIF-1alpha plasmid leads to the down-regulation of VEGF , and decreased tumor microvessel density .


Antisense HIF-1alpha monotherapy resulted in the complete and permanent rejection of small ( 0.1 cm in diameter ) EL-4 tumors , which is unusual for an anti-angiogenic agent where transient suppression of tumor growth is the norm .


It induced NK cell -dependent rejection of tumors , but failed to stimulate systemic T cell -mediated anti-tumor immunity , and synergized with B7-1-mediated immunotherapy to cause the NK cell and CD8 T cell -dependent rejection of larger EL-4 tumors ( 0.4 cm in diameter ) that were refractory to monotherapies .


Mice cured of their tumors by combination therapy resisted a rechallenge with parental tumor cells , indicating systemic antitumor immunity had been achieved .


In summary , whilst intensive investigations are in progress to target the many HIF-1 effectors , the results herein indicate that blocking hypoxia-inducible pathways and enhancing NK-mediated antitumor immunity by targeting HIF-1 itself may be advantageous , especially when combined with cancer immunotherapy .


Runx2 : a novel oncogenic effector revealed by in vivo complementation and retroviral tagging .


The Runx2 ( Cbfa1 , Pebp2alphaA , Aml3 ) gene was previously identified as a frequent target for transcriptional activation by proviral insertion in T-cell lymphomas of CD2-MYC transgenic mice .


We have recently shown that over-expression of the full-length , most highly expressed Runx2 isoform in the thymus perturbs T-cell development , leads to development of spontaneous lymphomas at low frequency and is strongly synergistic with Myc .


To gain further insight into the relationship of Runx2 to other lymphomagenic pathways , we tested the effect of combining the CD2-Runx2 transgene either with a Pim1 transgene ( E ( mu ) -Pim1 ) or with the p53 null genotype , as each of these displays independent synergy with Myc .


In both cases we observed synergistic tumour development .


However , Runx2 appeared to have a dominant effect on the tumour phenotype in each case , with most tumours conforming to the CD3 ( + ) , CD8 ( + ) , CD4 ( +/- ) phenotype seen in CD2-Runx2 mice .


Neonatal infection of CD2-Runx2 mice with Moloney murine leukaemia virus ( Moloney MLV ) also led to a dramatic acceleration of tumour onset .


Analysis of known Moloney MLV target genes in these lymphomas showed a high frequency of rearrangement at c-Myc or N-Myc ( 82 % ) , and a significant number at Pim1 or Pim2 ( 23 % ) , and at Pal1/Gfi1 ( 18 % ) .


These results indicate that Runx2 makes a distinct contribution to T-cell lymphoma development which does not coincide with any of the oncogene complementation groups previously identified by retroviral tagging .


The involvement of TNF-alpha -related apoptosis-inducing ligand in the enhanced cytotoxicity of IFN-beta -stimulated human dendritic cells to tumor cells .


TNF-alpha -related apoptosis-inducing ligand ( TRAIL ) is characterized by its preferential induction of apoptosis of tumor cells but not normal cells .


Dendritic cells ( DCs ) , besides their role as APCs , now have been demonstrated to exert cytotoxicity or cytostasis on some tumor cells .


Here , we report that both human CD34 ( + ) stem cell -derived DCs ( CD34DCs ) and human CD14 ( + ) monocyte -derived DCs ( MoDCs ) express TRAIL and exhibit cytotoxicity to some types of tumor cells partially through TRAIL .


Moderate expression of TRAIL appeared on CD34DCs from the 8th day of culture and was also seen on freshly isolated monocytes .


The level of TRAIL expression remained constant until DC maturation .


TRAIL expression on immature CD34DCs or MoDCs was greatly up-regulated after IFN-beta stimulation .


Moreover , IFN-beta could strikingly enhance the ability of CD34DCs or MoDCs to kill TRAIL-sensitive tumor cells , but LPS did not have such an effect .


The up-regulation of TRAIL on IFN-beta -stimulated DCs partially contributed to the increased cytotoxicity of DCS : Pretreatment of TRAIL-sensitive tumor cells with caspase-3 inhibitor could significantly increase their resistance to the cytotoxicity of IFN-beta -stimulated DCS : In contrast , NF-kappaB inhibitor could significantly increase the sensitivity of tumor cells to the killing by nonstimulated or LPS-stimulated DCS : Our studies demonstrate that IFN-beta -stimulated DCs are functionally cytotoxic .


Thus , an innate mechanism of DC-mediated antitumor immunity might exist in vivo in which DCs act as effectors to directly kill tumor cells partially via TRAIL .


Subsequently , DCs act as APCs involved in the uptake , processing , and presentation of apoptotic tumor Ags to cross-prime CD8 ( + ) CTL cells .


Tumor cells regulate the lytic activity of tumor-specific cytotoxic t lymphocytes by modulating the inhibitory natural killer receptor function .


Tumor-infiltrating p58+ T cells from a renal tumor were specifically expanded in response to tumor cell stimulation and cloned .


These p58+ T cells were found to express a memory phenotype and corresponded to clonal TCRBV3 T-cell expansion .


Functionally , p58 ( + ) CTLs displayed a low lytic activity for HLA-A2 tumor and normal cells .


However , this lytic activity was significantly increased after blockade of p58 with specific monoclonal antibodies .


Interestingly , we demonstrated that stimulation by tumor cells was required to trigger the inhibitory effect of p58 on the lytic activity of antigen-specific CTLs and that stimulation of the inhibitory function of p58 by tumor cells correlated with an inhibition of nuclear factor-kappaB activation in p58+ tumor-specific CTLS .


T-cell factor-1 expression during human natural killer cell development and in circulating CD56 ( + ) bright natural killer cells .


Transcription factors are essential to govern differentiation along the lymphoid lineage from uncommitted hematopoietic stem cells .


Although many of these transcription factors have putative roles based on murine knockout experiments , their function in human lymphoid development is less known and was studied further .


Transcription factor expression in fresh and cultured adult human bone marrow and umbilical cord blood progenitors was evaluated .


We found that fresh CD34 ( + ) Lin ( - ) cells that are human leukocyte antigen ( HLA ) -DR ( - ) or CD38 ( - ) constitutively express GATA-3 but not T-cell factor-1 ( TCF-1 ) or Id-3 .


Culture with the murine fetal liver cell line AFT024 and defined cytokines was capable of inducing TCF-1 mRNA .


However , no T-cell receptor gene rearrangement was identified in cultured progeny .


Id-3 , a basic helix loop helix factor with dominant negative function for T-cell differentiation transcription factors , also was upregulated and may explain unsuccessful T-cell maturation .


To better understand the developmental link between natural killer ( NK ) cells derived from progenitors , we studied NK cell subsets circulating in blood .


CD56 ( +bright ) , but not CD56 ( +dim ) , NK cells constitutively express TCF-1 by reverse transcriptase polymerase chain reaction and Western blot analysis .


The TCF-1 isoform found in CD56 ( +bright ) cells , which express lectin but not immunoglobulin class I recognizing inhibitory receptors , was identical to that induced in NK cell differentiation culture and was distinctly different from isoforms in T cells .


These results suggest that TCF-1 does not target human killer immunoglobulin receptor genes , TCF-1 is uniquely expressed in circulating CD56 ( +bright ) NK cells , and specific TCF-1 isoforms may play an important role in regulating NK differentiation from a common NK/T-cell progenitor .


Transcriptional activation by a matrix associating region-binding protein .


contextual requirements for the function of bright .


Bright ( B cell regulator of IgH transcription ) is a B cell -specific , matrix associating region-binding protein that transactivates gene expression from the IgH intronic enhancer ( E mu ) .


We show here that Bright has multiple contextual requirements to function as a transcriptional activator .


Bright can not transactivate via out of context , concatenated binding sites .


Transactivation is maximal on integrated substrates .


Two of the three previously identified binding sites in E mu are required for full Bright transactivation .


The Bright DNA binding domain defined a new family , which includes SWI1 , a component of the SWI.SNF complex shown to have high mobility group-like DNA binding characteristics .


Similar to one group of high mobility group box proteins , Bright distorts E mu binding site -containing DNA on binding , supporting the concept that it mediates E mu remodeling .


Transfection studies further implicate Bright in facilitating spatially separated promoter-enhancer interactions in both transient and stable assays .


Finally , we show that overexpression of Bright leads to enhanced DNase I sensitivity of the endogenous E mu matrix associating regions .


These data further suggest that Bright may contribute to increased gene expression by remodeling the immunoglobulin locus during B cell development .


Analysis of BCL-6 mutations in classic Hodgkin disease of the B- and T-cell type .


BCL-6 is essential for germinal center formation and thus for affinity maturation of immunoglobulin ( Ig ) genes by somatic mutations .


The 5'-noncoding region of the BCL-6 gene is even a target for the mutation machinery .


Translocations of the BCL-6 gene to heterologous promoters and mutations of its 5'-noncoding regulatory region were reported to be potential mechanisms for deregulating BCL-6 expression and for playing a role in the genesis of non-Hodgkin lymphoma .


In line with this hypothesis is the observation that B-cell lymphoma with somatic mutations , such as diffuse large B-cell lymphoma and follicular lymphoma , also carry BCL-6 mutations , some of which are recurrently detectable .


Classic Hodgkin disease ( cHD ) is also derived from B cells with high loads of somatic mutations and thus a further candidate for BCL-6 mutations .


To determine the presence and potential role of BCL-6 mutations in cHD , the 5'-noncoding BCL-6 proportion of single Hodgkin and Reed-Sternberg ( HRS ) cells from 6 cases of cHD and 6 cases of HD-derived cell lines was analyzed .


All B-cell-derived HD cases and cell lines harbored BCL-6 mutations .


In contrast , both T-cell-derived HD cases and cell lines were devoid of BCL-6 mutations .


With only one exception , there were no lymphoma-specific recurrent BCL-6 mutations detected , and BCL-6 protein was absent from the HRS cells of most cases .


In conclusion , ( 1 ) somatic BCL-6 mutations are restricted to cHD cases of B-cell origin , and ( 2 ) the BCL-6 mutations represent mostly irrelevant somatic base substitutions without consequences for BCL-6 protein expression and the pathogenesis of cHD .


Gadd45gamma is dispensable for normal mouse development and T-cell proliferation .


Gadd45gamma , a family member of the growth arrest and DNA damage-inducible gene family 45 ( Gadd45 ) , is strongly induced by interleukin-2 ( IL-2 ) in peripheral T cells .


While in most tissues all Gadd45 family members are expressed , Gadd45gamma is the only member that is induced by IL-2 .


Here we show that the IL-2 -induced expression of Gadd45gamma is dependent on a signaling pathway mediated by the tyrosine kinase Jak3 and the transcription factors Stat5a and Stat5b ( signal transducer and activator of transcription ) .


Previous studies with ectopically overexpressed Gadd45gamma in various cell lines implicated its function in negative growth control .


To analyze the physiological role of Gadd45gamma we used homologous recombination to generate mice lacking Gadd45gamma .


Gadd45gamma -deficient mice develop normally , are indistinguishable from their littermates , and are fertile .


Furthermore , hematopoiesis in mice lacking Gadd45gamma is not impaired and Gadd45gamma -deficient T lymphocytes show normal responses to IL-2 .


These data demonstrate that Gadd45gamma is not essential for normal mouse development and hematopoiesis , possibly due to functional redundancy among the Gadd45 family members .


Gadd45gamma is also dispensable for IL-2 -induced T-cell proliferation .


Decreased expression of c-myc family genes in thymuses from myasthenia gravis patients .


The thymus is a critical organ for the elimination of autoreactive T cells by apoptosis .


We studied the expression of apoptosis-associated genes , bcl-xL , bad , caspase-3 , and c-myc family genes in myasthenia gravis ( MG ) thymuses .


We observed that the mRNA levels of myc family genes , c-myc and max , were markedly reduced in MG thymuses .


These results indicate that c-myc -mediated signaling is abnormal in MG thymuses .


The levels of molecules whose expressions are associated with myc , such as STAM , prothymosin-alpha , and NFkappaB


Immunohistochemical detection of interferon-gamma-producing cells in dermatophytosis .


Skin lesions of dermatophytosis are thought to be a result of a T cell -dependent inflammatory response that is mediated by various cytokines .


We examined whether IFN-gamma-positive cells ( as expression of Th1 response ) were present in the skin lesions of dermatophytosis in situ by immunohistochemical techniques .


Mixtures of CD4-positive T cells and CD8-positive T cells were found to be present in the dermal infiltrates of the lesions .


Considerable numbers of CD1a-positive cells were detected in the upper dermis and epidermis .


A marked accumulation of CD68-positive cells was found in the upper dermis .


IFN-gamma-positive cells were present in the upper dermis of the lesions .


The pattern of IFN-gamma staining appeared to be intracellular in mononuclear lymphoid cells .


The staining was considered to be highly specific because it could be completely blocked by preabsorption with recombinant IFN-gamma .


Our data support the hypothesis that the skin lesions of dermatophytosis may be associated with a Th1 response .


Th1 response , which is characterized by IFN-gamma release , is thought to be involved in the host defense against dermatophytes and to reflect cutaneous reaction in dermatophytosis .


Regulation of the helix-loop-helix proteins , E2A and Id3 , by the Ras - ERK MAPK cascade .


Activation of mitogen-activated protein kinase ( MAPK ) pathways leads to cellular differentiation and/or proliferation in a wide variety of cell types , including developing thymocytes .


The basic helix-loop-helix ( bHLH ) proteins E12 and E47 and an inhibitor HLH protein , Id3 , play key roles in thymocyte differentiation .


We show here that E2A DNA binding is lowered in primary immature thymocytes consequent to T cell receptor ( TCR ) -mediated ligation .


Whereas expression of E2A mRNA and protein are unaltered , Id3 transcripts are rapidly induced upon signaling from the TCR .


Activation of Id3 transcription is regulated in a dose-dependent manner by the extracellular signal-regulated kinase ( ERK ) MAPK module .


These observations directly connect the ERK MAPK cascade and HLH proteins in a linear pathway .


Retinoic acid up-regulates myeloid ICAM-3 expression and function in a cell-specific fashion -- evidence for retinoid signaling pathways in the mast cell lineage .


Investigation of mast cell responsiveness toward retinoic acid ( RA ) revealed selective promotion of ICAM-3 expression in the human mast cell line HMC-1 .


This process was dose- and time-dependent and detectable by flow cytometry , Western blot analysis , ELISA , and Northern blot analysis .


ICAM-3 modulation was found to be cell-type dependent , detectable also for HL-60 cells and monocytes but not U-937 and only weakly for KU812 cells .


Terminally differentiated skin mast cells also failed to up-modulate their ICAM-3 , suggesting the requirement for some degree of immaturity for the process .


RA-mediated effects on ICAM-1 expression , studied in parallel , were clearly distinct from those on ICAM-3 .


Investigation of retinoid receptor expression , known to mediate intracellular RA signaling , revealed presence of RAR alpha , RAR gamma , RXR beta , and RXR gamma transcripts in all cell lines studied , and HMC-1 cells were the only line lacking RXR alpha .


RAR beta , not expressed at baseline , was induced by RA in a fashion obviously correlating with ICAM-3 up-regulation .


Increased ICAM-3 expression was of functional significance , such that processes stimulated or co-stimulated via ICAM-3 ( homotypic aggregation , IL-8 secretion ) were clearly enhanced upon RA pretreatment , suggesting that RA may contribute via hitherto unrecognized pathways to immune function and host defense .


Over the last decade the concept of T cell co-stimulation has emerged to take a central role in the process of T cell activation .


However , the exact definition of co-stimulation is still unclear .


In this review , we re-examine the concept of co-stimulation .


We suggest that while co-stimulation is important , there is little evidence to link co-stimulation with T cell anergy .


We then suggest a framework for studying co-stimulation .


Focusing on recent advances in our understanding of CD28 , we discuss four areas of T cell activation where co-stimulation may play a role .


Regulation of activator protein-1 and NF-kappa B in CD8+ T cells exposed to peripheral self-antigens .


The transcriptional events that control T cell tolerance to peripheral self Ags are still unknown .


In this study , we analyzed the regulation of AP-1 - and NF-kappa B -mediated transcription during in vivo induction of tolerance to a self Ag expressed exclusively on hepatocytes .


Naive CD8 ( + ) Desire ( Des ) ( + ) T cells isolated from the Des TCR-transgenic mice that are specific for the H-2K ( b ) class I Ag were transferred into Alb-K ( b ) -transgenic mice that express the H-2K ( b ) Ag on hepatocytes only .


We found that the self-reactive CD8 ( + ) Des ( + ) T cells were transiently activated , then became unresponsive and were further deleted .


In contrast to CD8 ( + ) Des ( + ) T cells activated in vivo with APCs , which express high AP-1 and high NF-kappa B transcriptional activity , the unresponsive CD8 ( + ) Des ( + ) T cells expressed no AP-1 and only weak NF-kappa B transcriptional activity .


The differences in NF-kappa B transcriptional activity correlated with the generation of distinct NF-kappa B complexes .


Indeed , in vivo primed T cells predominantly express p50/p50 and p65/p50 dimers , whereas these p50-containing complexes are barely detectable in tolerant T cells that express p65- and c-Rel-containing complexes .


These observations suggest that fine regulation of NF-kappa B complex formation may determine T cell fate .


Transcriptional regulation in lymphocytes .


Lymphocytes have been used to investigate many cellular processes , including lineage commitment , differentiation , proliferation and apoptosis .


The transcription factors that mediate these processes are often expressed broadly in many cell types .


The emerging theme is one of cell-type-specific regulation , affecting not only the functional activation of transcription factors but also their access to appropriate regions of DNA .


Existence of retinoic acid-receptor -independent retinoid X-receptor -dependent pathway in myeloid cell function .


We previously reported that ER-27191 ( 4- [ 4 , 5 , 7 , 8 , 9 , 10-hexahydro-7 , 7 , 10 , 10-tetramethyl-1- ( 3-pyridylmethyl ) anthra [ 1 , 2-b ] pyrrol-3-yl ] benzoic acid ) is a potent antagonist of retinoic acid receptor ( RAR ) , and ER-35795 ( ( 2E , 4E , 6E ) -7- [ 1- ( 1-methylethyl ) -8-chloro-1 , 2 , 3 , 4-tetrahydroquinolin-6-yl ] -6-fluoro-3-methyl-2 , 4 , 6-nonatrienoic acid ) is a novel retinoid X receptor ( RXR ) -specific agonist .


By using these compounds , we investigated whether distinct RAR -dependent and RXR -dependent pathways operate to mediate the diverse activities of retinoids , particularly , the effects of the RXR pathway on cellular function .


ER-27191 completely antagonized HL60 cell differentiation induced by all-trans-retinoic acid ( atRA ) .


However , the differentiation induced by the ER-35795 was not antagonized at all by the RAR antagonist , but was inhibited by an RXR homodimer antagonist ( LGD100754 , ( 2E , 4E , 6Z ) -7- ( 3-n-propoxy-5 , 6 , 7 , 8-tetrahydro-5 , 5 , 8 , 8-tetramethylnaphthalen-2-yl ) -3-methylocta-2 , 4 , 6-trienoic acid ) .


Its agonistic action on RXR/RAR heterodimer , on the other hand , was neutralized by the RAR antagonist .


During HL60 cell differentiation , atRA induced RARbeta mRNA , while the RXR had no effect .


Interestingly , a functional RXR -pathway was also seen in lipopolysaccharide-induced inhibition of mouse splenocyte proliferation .


These results strongly suggest the existence of a pharmacological RXR -dependent pathway that is activated by a ligand that can bind to RXR .


Targeting Src homology 2 domain-containing tyrosine phosphatase ( SHP-1 ) into lipid rafts inhibits CD3 -induced T cell activation .


To study the mechanism by which protein tyrosine phosphatases ( PTPs ) regulate CD3 -induced tyrosine phosphorylation , we investigated the distribution of PTPs in subdomains of plasma membrane .


We report here that the bulk PTP activity associated with T cell membrane is present outside the lipid rafts , as determined by sucrose density gradient sedimentation .


In Jurkat T cells , approximately 5 -- 10 % of Src homology 2 domain-containing tyrosine phosphatase ( SHP-1 ) is constitutively associated with plasma membrane , and nearly 50 % of SHP-2 is translocated to plasma membrane after vanadate treatment .


Similar to transmembrane PTP , CD45 , the membrane-associated populations of SHP-1 and SHP-2 are essentially excluded from lipid rafts , where other signaling molecules such as Lck , linker for activation of T cells , and CD3 zeta are enriched .


We further demonstrated that CD3 -induced tyrosine phosphorylation of these substrates is largely restricted to lipid rafts , unless PTPs are inhibited .


It suggests that a restricted partition of PTPs among membrane subdomains may regulate protein tyrosine phosphorylation in T cell membrane .


To test this hypothesis , we targeted SHP-1 into lipid rafts by using the N-terminal region of Lck ( residues 1 -- 14 ) .


The results indicate that the expression of Lck/SHP-1 chimera inside lipid rafts profoundly inhibits CD3 -induced tyrosine phosphorylation of CD3 zeta/epsilon , IL-2 generation , and nuclear mobilization of NF-AT .


Collectively , these results suggest that the exclusion of PTPs from lipid rafts may be a mechanism that potentiates TCR / CD3 activation


An instructive component in T helper cell type 2 ( Th2 ) development mediated by GATA-3 .


Although interleukin ( IL ) -12 and IL-4 polarize naive CD4 ( + ) T cells toward T helper cell type 1 ( Th1 ) or Th2 phenotypes , it is not known whether cytokines instruct the developmental fate in uncommitted progenitors or select for outgrowth of cells that have stochastically committed to a particular fate .


To distinguish these instructive and selective models , we used surface affinity matrix technology to isolate committed progenitors based on cytokine secretion phenotype and developed retroviral-based tagging approaches to directly monitor individual progenitor fate decisions at the clonal and population levels .


We observe IL-4 -dependent redirection of phenotype in cells that have already committed to a non-IL-4-producing fate , inconsistent with predictions of the selective model .


Further , retroviral tagging of naive progenitors with the Th2-specific transcription factor GATA-3 provided direct evidence for instructive differentiation , and no evidence for the selective outgrowth of cells committed to either the Th1 or Th2 fate .


These data would seem to exclude selection as an exclusive mechanism in Th1 / Th2 differentiation , and support an instructive model of cytokine-driven transcriptional programming of cell fate decisions .


In vitro-activated human lupus T cells express normal estrogen receptor proteins which bind to the estrogen response element .


We have shown that estrogen receptor ( ERalpha , ERbeta ) transcripts are expressed in SLE and normal T cells .


In this study , T cell nuclear extracts from female lupus patients and normal donors were tested for biologically active ER proteins capable of binding to the human estrogen response element ( hERE ) by electrophoretic mobility shift assays .


When peripheral blood T cells were stimulated with 17beta-estradiol ( E2 ) , PMA and ionomycin , two major retarded bands in T cell nuclear extracts exhibited a migration pattern similar to slow migrating protein-ERE complexes in human breast cancer cell extracts .


T cells cultured only with E2 did not have these complexes .


The formation of the complexes was inhibited by competition with the hERE cold oligonucleotide and partially with anti-ERalpha antibodies .


There was no notable difference in the migration pattern of ERE-binding proteins between the SLE and normal T cell extracts .


Together , these results suggest that activated human T cells , whether lupus-derived or normal-derived , contain biologically active ERalpha proteins .


Other factors may be responsible for differential sensitivity of lupus T cells to estrogen .


Mechanism of the inhibitory effect of protease inhibitor on tumor necrosis factor alpha production of monocytes .


If the inflammatory response becomes excessive or uncontrolled by some stimuli , inappropriate inflammatory responses occur .


Monocytes are extremely important cells for regulating the cytokine network and tumor necrosis factor alpha ( TNFalpha ) and interleukin- ( IL ) 10 , which are mainly synthesized by monocytes , are representative cytokines that play a central role in the cytokine network .


Protease inhibitors such as gabexate mesilate ( GM ) and ulinastatin ( UTI ) have been shown to have various beneficial effects by inhibiting the activation of leukocytes , but the mechanism for this has yet to be fully elucidated .


In this study we investigated the mechanism of the inhibitory effect of protease inhibitors on the proinflammatory cytokine production of lipopolysaccharide- ( LPS ) stimulated monocytes .


LPS-stimulated monocytes were treated with GM or UTI .


The value of TNFalpha and IL-10 in the culture medium of monocytes was measured and each mRNA expression was assayed .


The inhibitory effect of protease inhibitors on the activity of intracellular signal transduction pathways such as protein kinase C ( PKC ) and nuclear factor kappa B ( NFkappaB ) were also evaluated .


GM decreased the TNFalpha production of LPS-stimulated monocytes as shown by the inhibition of mRNA expression and increased the IL-10 production of LPS-stimulated monocytes .


GM also suppressed the NFkappaB activity of LPS-stimulated monocytes .


UTI decreased the TNFalpha production of LPS-stimulated monocytes , but did not inhibit the TNFalpha mRNA expression .


The present study shows that the inhibitory effect of GM on the TNFalpha production of activated human monocytes is mediated by the suppression of NFkappaB activation , while the mechanism of UTI inhibiting TNFalpha production of human monocytes may be due to the inhibition of either the translation or secretion of TNFalpha .


Homocysteine stimulates the expression of monocyte chemoattractant protein-1 in endothelial cells leading to enhanced monocyte chemotaxis .


Hyperhomocysteinemia has been identified as an independent risk factor for atherosclerosis .


The infiltration of monocytes into the arterial wall is one of the key events during atherogenesis .


Monocyte chemoattractant protein-1 ( MCP-1 ) is a potent chemokine that stimulates the migration of monocytes into the intima of the arterial wall .


The mechanism by which increased monocyte infiltration occurs in atherosclerotic lesions in patients with hyperhomocysteinemia has not been delineated .


The objective of the present study was to investigate the effect of homocysteine on MCP-1 production in endothelial cells .


Cells were incubated with homocysteine .


The secretion of MCP-1 protein was significantly increased ( 195 % as compared to the control ) in cells treated with pathological concentrations of homocysteine .


Such effect was accompanied by an increased expression of MCP-1 mRNA ( 176 % as compared to the control ) in endothelial cells which resulted in enhanced monocyte chemotaxis .


The p38 MAP kinase as well as other members of the p38 MAP kinase pathway , including MKK3 , MKK6 , ATF-2 and Elk-1 , were activated in homocysteine-treated cells .


Homocysteine-induced MCP-1 expression and subsequent monocyte chemotaxis were blocked by a p38 MAP kinase inhibitor ( SB203580 ) suggesting that the p38 MAP kinase pathway might be involved in homocysteine-induced MCP-1 expression in endothelial cells .


In contrast , staurosporine , a protein kinase C inhibitor , had no effect on homocysteine-induced MCP-1 expression .


In conclusion , our results indicate that homocysteine stimulates MCP-1 expression in endothelial cells leading to enhanced monocyte chemotaxis .


Inducible resistance to Fas-mediated apoptosis in B cells .


Apoptosis produced in B cells through Fas ( APO-1 , CD95 ) triggering is regulated by signals derived from other surface receptors : CD40 engagement produces upregulation of Fas expression and marked susceptibility to Fas -induced cell death , whereas antigen receptor engagement , or IL-4R engagement , inhibits Fas killing and in so doing induces a state of Fas -resistance , even in otherwise sensitive , CD40 -stimulated targets .


Surface immunoglobulin and IL-4R utilize at least partially distinct pathways to produce Fas -resistance that differentially depend on PKC and STAT6 , respectively .


Further , surface immunoglobulin signaling for inducible Fas -resistance bypasses Btk , requires NF-kappaB , and entails new macromolecular synthesis .


Terminal effectors of B cell Fas -resistance include the known anti-apoptotic gene products , Bcl-xL and FLIP , and a novel anti-apoptotic gene that encodes FAIM ( Fas Apoptosis Inhibitory Molecule ) .


faim was identified by differential display and exists in two alternatively spliced forms ; faim-S is broadly expressed , but faim-L expression is tissue-specific .


The FAIM sequence is highly evolu- tionarily conserved , suggesting an important role for this molecule throughout phylogeny .


Inducible resistance to Fas killing is hypothesized to protect foreign antigen-specific B cells during potentially hazardous interactions with FasL-bearing T cells , whereas autoreactive B cells fail to become Fas -resistant and are deleted via Fas -dependent cytotoxicity .


Inadvertent or aberrant acquisition of Fas -resistance may permit autoreactive B cells to escape Fas deletion , and malignant lymphocytes to impede anti-tumor immunity .


Stromal cell-derived factor 1 alpha -induced chemotaxis in T cells is mediated by nitric oxide signaling pathways .


Stromal cell-derived factor 1 alpha ( SDF1 alpha ) and its cognate chemokine receptor CXCR4 act as potent chemoattractants and regulate trafficking and homing of hematopoietic progenitor cells and lymphocytes .


However , the molecular mechanisms regulating SDF1 alpha -driven cell migration are not well defined .


In this study , we have explored the roles of the second messenger NO and the transcription factor NF-kappa B in SDF1 alpha -induced T cell migration .


SDF1 alpha treatment of Jurkat T cells increased the activity of NO synthase , which catalyzes the generation of NO .


We observed that pretreatment of Jurkat cells or activated PBLs with several NO donors significantly enhanced the SDF1 alpha -induced migration , whereas various inhibitors of NO synthase markedly abrogated the chemotactic response in a concentration-dependent manner .


Furthermore , we observed that inhibitors of the transcription factor NF-kappa B , which is linked to NO signaling pathways , also significantly blocked the SDF1 alpha -induced chemotactic response .


However , these compounds did not have a significant effect on SDF1 alpha -induced mitogen-activated protein kinase activity .


In addition , the MAP/Erk kinase kinase inhibitor PD98059 did not abrogate SDF1 alpha -induced chemotaxis .


AKT , which has been shown to mediate NO production , was also phosphorylated upon SDF1 alpha stimulation .


These studies suggest that NO-related signaling pathways may mediate SDF1 alpha -induced chemotaxis , but not mitogen-activated protein kinase activation .


The lack of NF-kappa B transactivation and PKC epsilon expression in CD4 ( + ) CD8 ( + ) thymocytes correlates with negative selection .


Deletion of autoreactive thymocytes at the DP stage is the basis for tolerance to thymus-expressed self antigens .


In this study we investigated whether distinct signalling pathways are induced in DP thymocytes as compared to mature T cells upon stimulation with antigen .


Using triple transgenic mice expressing a TCR transgene , dominant negative ras/Mek proteins and a reporter gene construct with AP-1 or NF-kappa B binding sites , we showed a complete lack of transcriptional activity of NF-kappa B but not AP-1 in DP thymocytes , whereas both were transcriptionally active in mature T cells after antigenic stimulation .


Lack of NF-kappa B induction correlated with increased death in response to antigen .


AP-1 induction was dependent on the integrity of the ras/Mek pathway indicating that this pathway was activated in the DP thymocytes .


In contrast , we found a complete lack of constitutive expression of the epsilon isoform of Protein Kinase C ( PKC ) in DP thymocytes , although it was present in mature thymocytes and peripheral T cells .


Taken together the results suggest that the lack of PKC epsilon in DP thymocytes could lead to the absence of NF-kappa B activity after antigenic stimulation contributing to negative selection .


Cell Death and Differentiation ( 2000 ) 7 , 1253 - 1262 .


CD2 stimulation leads to the delayed and prolonged activation of STAT1 in T cells but not NK cells .


OBJECTIVE : T lymphocytes can be activated by soluble factors such as cytokines or through direct cell-cell interactions .


Although cytokine receptors are known to signal through STAT family transcription factors , the mechanisms by which other cell-surface molecules , such as CD2 , transduce signals is unclear .


The goal of this study was to determine whether stimulation of T cells through CD2 recapitulates aspects of cytokine -induced T-cell activation by use of STAT transcription factors .


MATERIALS AND METHODS : T cells were treated with anti-CD2 antibodies or cells bearing the natural CD2 ligand CD58 , after which signaling through STAT transcription factors was assessed .


RESULTS : Stimulation of CD2 on primary T lymphocytes leads to the tyrosine phosphorylation , nuclear translocation , and DNA binding of STAT1 .


In contrast to stimulation by cytokines , the activation of STAT1 in response to CD2 ligation is delayed and does not involve Jak kinases .


Furthermore , while STAT phosphorylation induced by cytokines is generally transient , STAT1 phosphorylation following CD2 stimulation persists for a period of days .


Transcription of key target genes such as IRF1 and c-fos proceeds with delayed kinetics following CD2 stimulation , suggesting that this unique pattern of STAT activation may lead to a distinct cellular response following CD2 ligation .


This pathway appears to be restricted to T cells , as stimulation of CD2 on NK cells does not lead to STAT1 activation .


CONCLUSION : Stimulation of T cells through cell-surface molecules such as CD2 involves activation of STAT transcription factors , thus recapitulating elements of cytokine signaling .


NFATc1 and NFATc2 together control both T and B cell activation and differentiation .


NFAT transcription factors play critical roles in gene transcription during immune responses .


To investigate further the two most prominent NFAT family members , NFATc1 and NFATc2 , we generated mice bearing lymphoid systems devoid of both .


Doubly deficient T cells displayed cell surface markers of activation yet were significantly deficient in the development of multiple effector functions , including Th cytokine production , surface effector molecule expression , and cytolytic activity .


Nevertheless , doubly deficient B cells were hyperactivated , as evidenced by extremely elevated serum IgG1 and IgE , as well as plasma cell expansion and infiltration of end organs .


Thus , in T cells , NFATc1 and NFATc2 are dispensable for inflammatory reactivity but are required for effector differentiation , while in B cells , NFATs regulate both normal homeostasis and differentiation .


Epstein-barr virus immediate-early protein BZLF1 is SUMO-1 modified and disrupts promyelocytic leukemia bodies .


Although the immediate-early proteins of both herpes simplex virus ( HSV ) and cytomegalovirus ( CMV ) are known to modify promyelocytic leukemia ( PML ) ( ND10 ) bodies in the nucleus of the host cell , it has been unclear whether lytic infection with gamma herpesviruses induces a similar effect .


The PML protein is induced by interferon , involved in major histocompatibility complex class I presentation , and necessary for certain types of apoptosis .


Therefore , it is likely that PML bodies function in an antiviral capacity .


SUMO-1 modification of PML is known to be required for the formation of PML bodies .


To examine whether Epstein-Barr virus ( EBV ) lytic replication interferes with PML bodies , we expressed the EBV immediate-early genes BZLF1 ( Z ) and BRLF1 ( R ) in EBV-positive cell lines and examined PML localization .


Both Z and R expression resulted in PML dispersion in EBV-positive cells .


Z but not R expression is sufficient to disrupt PML bodies in EBV-negative cell lines .


We show that dispersion of PML bodies by Z requires a portion of the transcriptional activation domain of Z but not the DNA-binding function .


As was previously reported for the HSV-1 ICP0 and CMV IE1 proteins , Z reduces the amount of SUMO-1-modified PML .


We also found that Z itself is SUMO-1 modified ( through amino acid 12 ) and that Z competes with PML for limiting amounts of SUMO-1 .


These results suggest that disruption of PML bodies is important for efficient lytic replication of EBV .


Furthermore , Z may potentially alter the function of a variety of cellular proteins by inhibiting SUMO-1 modification


Suppression of nuclear factor-kappaB and stimulation of inhibitor kappaB by troglitazone : evidence for an anti-inflammatory effect and a potential antiatherosclerotic effect in the obese .


To elucidate whether troglitazone exerts an antiinflammatory effect in humans , in vivo , we investigated the suppression of nuclear factor kappaB ( NFkappaB ) in mononuclear cells ( MNC ) by this drug .


We measured intranuclear NFkappaB , total cellular NFkappaB , inhibitor kappaB ( IkappaB ) alpha , reactive oxygen species ( ROS ) generation , and p47 ( phox ) subunit ( a key component protein of nicotinamide adenine dinucleotide phosphate oxidase ) in MNC .


Plasma tumor necrosis factor ( TNF ) -alpha , soluble intercellular adhesion molecule-1 ( sICAM-1 ) , monocyte chemoattractant protein-1 ( MCP-1 ) , plasminogen activator inhibitor type 1 ( PAI-1 ) , C-reactive protein ( CRP ) , and interleukin ( IL ) -10 ( antiinflammatory cytokine ) concentrations were also measured as mediators of inflammatory activity that are regulated by the proinflammatory transcription factor NFkappaB .


Seven nondiabetic obese patients were given 400 mg troglitazone daily for 4 weeks .


Blood samples were collected before and at weekly intervals thereafter .


MNC were separated ; and the levels of intranuclear NFkappaB , total cellular NFkappaB , IkappaBalpha , and p47 ( phox ) subunit and ROS generation were determined .


Plasma was used to measure insulin glucose , TNFalpha , sICAM , MCP-1 , PAI-1 , CRP , and IL-10 .


Plasma insulin concentrations fell significantly at week 1 , from 31.2 +/- 29.1 to 14.2 +/- 11.4 mU/L ( P < 0.01 ) and remained low throughout 4 weeks .


Plasma glucose concentrations did not alter significantly .


There was a fall in intranuclear NFkappaB , total cellular NFkappaB , and p47 ( phox ) subunit , with an increase in cellular IkappaBalpha at week 2 , which persisted until week 4 .


There was a parallel fall in ROS generation by MNC at week 1 ; this progressed and persisted until week 4 ( P < 0.001 ) .


Plasma TNF-alpha , sICAM-1 , MCP-1 , and PAI-1 concentrations fell significantly at week 4 .


Plasma IL-10 concentration increased significantly , whereas plasma CRP concentrations decreased .


We conclude that troglitazone has an antiinflammatory action that may contribute to its putative antiatherosclerotic effects .


Tyrosine phosphorylation-dependent activation of NF-kappa B .


Requirement for p56 LCK and ZAP-70 protein tyrosine kinases .


Phosphorylation of the N-terminal domain of I kappa B inhibitory subunits induces activation of the transcription factor NF-kappa B .


Although serine phosphorylation has been shown to induce ubiquitination and subsequent proteasome-mediated degradation of I kappa B-alpha , little is known about the mechanisms that lead to release of active NF-kappa B in T cells as a consequence of tyrosine phosphorylation of I kappa B-alpha [ Imbert , V. , Rupec , R.A. , Livolsi , A. , Pahl , H.L. , Traenckner , B.M. , Mueller-Dieckmann , C. , Farahifar , D. , Rossi , B. , Auberger , P. , Baeuerle , P. & Peyron , J.F. ( 1996 ) Cell 86 , 787 -- 798 ] .


The involvement of the tyrosine kinases p56 ( lck ) and ZAP-70 in this reaction is demonstrated here using specific pharmacological inhibitors and Jurkat mutants unable to express these kinases .


Although the inhibitors prevented both pervanadate-induced phosphorylation of I kappa B-alpha on Tyr42 and NF-kappa B activation , we observed that , in p56 ( lck ) -deficient Jurkat mutants , NF-kappa B could still associate with I kappa B-alpha despite phosphorylation on Tyr42 .


Furthermore , the SH2 domain of p56 ( lck ) appeared to be required for pervanadate-induced NF-kappa B activation but not for Tyr42 phosphorylation .


These results show that p56 ( lck ) and ZAP-70 are key components of the signaling pathway that leads to phosphotyrosine-dependent NF-kappa B activation in T cells and confirm that tyrosine kinases must control at least two different steps to induce activation of NF-kappa B .


Finally , we show that H ( 2 ) O ( 2 ) , which stimulates p56 ( lck ) and ZAP-70 in T cells , is an activator of NF-kappa B through tyrosine phosphorylation of I kappa B-alpha .


Specific missense mutations in NEMO result in hyper-IgM syndrome with hypohydrotic ectodermal dysplasia .


The gene that encodes nuclear factor kappaB ( NF-kappaB ) essential modulator ( or NEMO , also known as IKKgamma ) is required for activation of the transcription factor NF-kappaB .


We describe mutations in the putative zinc-finger domain of NEMO that result in an X-linked primary immunodeficiency characterized by hyper-IgM syndrome and hypohydrotic ectodermal dysplasia ( XHM-ED ) .


These mutations prevent CD40 ligand ( CD40L ) -mediated degradation of inhibitor of NF-kappaB alpha ( IkappaB-alpha ) and account for the following observations : B cells from XHM-ED patients are unable to undergo immunoglobulin class-switch recombination and antigen-presenting cells ( APCs ) are unable to synthesize the NF-kappaB-regulated cytokines interleukin 12 ( IL-12 ) or tumor necrosis factor alpha ( TNF-alpha ) when stimulated with CD40L .


Nevertheless , innate immunity is preserved in XHM-ED patients because APCs retain the capacity to respond to stimulation by lipopolysaccharide or Staphylococcus aureus Cowan 's antigen ( SAC ) .


Overall , the phenotype observed in XHM-ED patients shows that the putative zinc-finger domain of NEMO has a regulatory function and demonstrates the definite requirement of CD40 -mediated NF-kappaB activation for B cell immunoglobulin class-switching .


Granulocytic differentiation of human NB4 promyelocytic leukemia cells induced by all-trans retinoic acid metabolites .


The metabolism of all-trans retinoic acid ( ATRA ) has been reported to be partly responsible for the in vivo resistance to ATRA seen in the treatment of human acute promyelocytic leukemia ( APL ) .


However , ATRA metabolism appears to be involved in the growth inhibition of several cancer cell lines in vitro .


The purpose of this study was to evaluate the in vitro activity of the principal metabolites of ATRA [ 4-hydroxy-retinoic acid ( 4-OH-RA ) , 18-hydroxy-retinoic acid ( 18-OH-RA ) , 4-oxo-retinoic acid ( 4-oxo-RA ) , and 5 , 6-epoxy-retinoic acid ( 5 , 6-epoxy-RA ) ] in NB4 , a human promyelocytic leukemia cell line that exhibits the APL diagnostic t ( 15 ; 17 ) chromosomal translocation and expresses the PML-RAR alpha fusion protein .


We established that the four ATRA metabolites were indeed formed by the NB4 cells in vitro .


NB4 cell growth was inhibited ( 69-78 % at 120 h ) and cell cycle progression in the G1 phase ( 82-85 % at 120 h ) was blocked by ATRA and all of the metabolites at 1 microM concentration .


ATRA and its metabolites could induce NB4 cells differentiation with similar activity , as evaluated by cell morphology , by the nitroblue tetrazolium reduction test ( 82-88 % at 120 h ) or by the expression of the maturation specific cell surface marker CD11c .


In addition , nuclear body reorganization to macropunctated structures , as well as the degradation of PML-RAR alpha , was found to be similar for ATRA and all of its metabolites .


Comparison of the relative potency of the retinoids using the nitroblue tetrazolium reduction test showed effective concentrations required to differentiate 50 % of cells in 72 h as follows : ATRA , 15.8 +/- 1.7 nM ; 4-oxo-RA , 38.3 +/- 1.3 nM ; 18-OH-RA , 55.5 +/- 1.8 nM ; 4-OH-RA , 79.8 +/- 1.8 nM ; and 5 , 6-epoxy-RA , 99.5 +/- 1.5 nM .


The ATRA metabolites were found to exert their differentiation effects via the RAR alpha nuclear receptors , because the RAR alpha-specific antagonist BMS614 blocked metabolite-induced CD11c expression in NB4 cells .


These data demonstrate that the principal ATRA Phase 1 metabolites can elicit leukemia cell growth inhibition and differentiation in vitro through the RAR alpha signaling pathway , and they suggest that these metabolites may play a role in ATRA antileukemic activity in vivo .


Expression of oestrogen and progesterone receptors by mast cells alone , but not lymphocytes , macrophages or other immune cells in human upper airways .


BACKGROUND : Nasal polyposis often coexists with asthma in airway inflammatory conditions characterised by the infiltration of a range of immune cells .


A potentially important role for ovarian hormones has been implicated in airway inflammation but the cellular target for such action is not known .


METHODS : Expression of oestrogen receptors ( ER ) and progesterone receptors ( PR ) was examined using immunohistochemistry in formalin fixed nasal polyp tissues from 47 subjects .


The cells positive for ER or PR were confirmed by spatial location , dual immunolabelling , and histochemical staining .


RESULTS : Consistent with the known features of nasal polyps , CD4+ ( T helper/inducer ) , CD8+ ( cytotoxic/suppressor ) , CD68+ ( macrophages ) , mast cells , eosinophils and neutrophils were all clearly detected by their relevant monoclonal antibodies or appropriate histochemical staining , but only mast cells tested positive for ER /PR labelling with their polyclonal and monoclonal antibodies .


The frequencies for expression were 61.7 % for ER positive and 59.6 % for PR positive cells .


The expression of ER / PR was independent of patient sex and age but was highly correlated with the numbers of mast cells ( r = 0.973 , p < 0.001 for ER ; r = 0.955 , p < 0.001 for PR ) .


Fewer than 5 % of mast cells were found to be negative for ER / PR expression .


CONCLUSIONS : Mast cells alone , but not lymphocytes , macrophages , or other immune cells , express ER / PR in human upper airways .


Numerous ER/PR positive mast cells exist in nasal polyps , indicating that this may be a major route for the involvement of sex hormones in airway inflammation when exposed to the higher and varying concentration of oestrogen and progesterone characteristic of females .


NF kappa b signaling in posthypoxic endothelial cells : relevance to E-selectin expression and neutrophil adhesion .


Our previous studies have implicated the nuclear transcription factor kappa B ( NF kappa B ) in the regulation of adhesion molecule expression in endothelial cells exposed to anoxia-reoxygenation ( A/R ) or a redox imbalance .


The objectives of this study were ( 1 ) to define the kinetics of NF kappa B activation by examining I kappa B alpha degradation and the nuclear translocation of p65 in response to A/R or redox imbalance ( induced by treatment of cells with diamide and buthionine sulfoximine ) and ( 2 ) to determine whether the signal for I kappa B alpha degradation , nuclear translocation of p65 , and E-selectin -mediated neutrophil adhesion is related to the activity of protein tyrosine kinase ( PTK ) , protein tyrosine phosphatase ( PTPase ) and/or protein kinase C ( PKC ) .


The results demonstrate that both A/R and redox imbalance led to I kappa B alpha degradation within 30 min and the concomitant appearance of p65 in the nucleus , consistent with rapid cytosolic activation of NF kappa B and subsequent nuclear translocation of the activated p65 subunit .


Inhibition of PKC blocked I kappa B alpha degradation and p65 translocation in A/R-challenged , but not redox-altered , endothelial cells .


However , both A/R- and redox-induced NF kappa B activation was blocked by inhibition of PTK .


Similarly , A/R-induced E-selectin expression and neutrophil-endothelial cell adhesion were blocked by inhibition of PKC or PTK , while only PTK inhibited the redox-induced adhesion response .


Pretreatment of cells with N-acetyl cysteine effectively blocked A/R- or redox-induced I kappa B degradation and significantly attenuated the respective neutrophil adhesion responses .


Collectively , these findings indicate that A/R-induced E-selectin expression and neutrophil-endothelial cell adhesion are mediated by both PKC and PTK , which signal rapid activation of NF kappa B .


This A/R-induced NF kappa B signaling response appears to be mediated , at least in part , by intracellular redox imbalance .


Induction of apoptosis in human lymphocytes by the herbicide 2 , 4-dichlorophenoxyacetic acid .


Dimethylammonium salt of 2 , 4-dichlorophenoxyacetic acid ( DMA-2 , 4-D ) is a widely used herbicide that is considered moderately toxic .


In the present study we found that DMA-2 , 4-D is able to cause apoptosis in peripheral blood lymphocytes of healthy individuals and Jurkat T cells .


Apoptosis induced by DMA-2 , 4-D was dose and time dependent , independent of Fas , TNF receptor 1 or the aromatic hydrocarbon receptor , and involved disruption of the mitochondrial transmembrane potential and activation of caspase-9 .


ZVAD-FMK , a broad-spectrum inhibitor of caspases , blocked DMA-2 , 4-D-induced apoptosis completely .


While an inhibitor of caspase-9 , as well as caspase-9 and caspase-3 inhibitors in combination , strongly blocked DMA-2 , 4-D-induced apoptosis , an inhibitor of caspase-3 had a moderate inhibitory effect .


Unlike Fas -mediated apoptosis , the initiator caspase , caspase-8 , was not involved in DMA-2 , 4-D-induced apoptosis .


Transfection of Jurkat cells with Bcl-2 prevented DMA-2 , 4-D-induced disruption of the mitochondrial transmembrane potential and led to a complete blockage of apoptosis .


Our data indicate that DMA-2 , 4-D kills human lymphocytes by initiating apoptosis via a direct effect on mitochondria .


The activation of caspases occurs downstream of mitochondrial damage , and the dysfunction of mitochondria appears to be sufficient for triggering all downstream events leading to apoptosis .


Inhibition of Th1 differentiation by IL-6 is mediated by SOCS1 .


Interleukin 6 ( IL-6 ) is a cytokine produced by immune and nonimmune cells and exhibits functional pleiotropy and redundancy .


IL-6 plays an important role in the differentiation of several cell types .


Here , we describe a novel function of IL-6 : the negative regulation of CD4 + Th1 cell differentiation .


While IL-6 -directed CD4 + Th2 differentiation is mediated by IL-4 , inhibition of Th1 differentiation by IL-6 is independent of IL-4 .


IL-6 upregulates suppressor of cytokine signaling 1 ( SOCS1 ) expression in activated CD4+ T cells , thereby interfering with signal transducer and activator of transcription 1 ( STAT1 ) phosphorylation induced by interferon gamma ( IFNgamma ) .


Inhibition of IFNgamma receptor-mediated signals by IL-6 prevents autoregulation of IFNgamma gene expression by IFNgamma during CD4+ T cell activation , thereby preventing Th1 differentiation .


Thus , IL-6 promotes CD4 + Th2 differentiation and inhibits Th1 differentiation by two independent molecular mechanisms .


Interaction between CCAAT/enhancer binding protein and cyclic AMP response element binding protein 1 regulates human immunodeficiency virus type 1 transcription in cells of the monocyte/macrophage lineage .


Recent observations have shown two CCAAT/enhancer binding protein ( C/EBP ) binding sites to be critically important for efficient human immunodeficiency virus type 1 ( HIV-1 ) replication within cells of the monocyte/macrophage lineage , a cell type likely involved in transport of the virus to the brain .


Additionally , sequence variation at C/EBP site I , which lies immediately upstream of the distal nuclear factor kappa B site and immediately downstream of a binding site for activating transcription factor ( ATF ) / cyclic AMP response element binding protein ( CREB ) , has been shown to affect HIV-1 long terminal repeat ( LTR ) activity .


Given that C/EBP proteins have been shown to interact with many other transcription factors including members of the ATF/CREB family , we proceeded to determine whether an adjacent ATF/CREB binding site could affect C/EBP protein binding to C/EBP site I .


Electrophoretic mobility shift analyses indicated that selected ATF/CREB site variants assisted in the recruitment of C/EBP proteins to an adjacent , naturally occurring , low-affinity C/EBP site .


This biophysical interaction appears to occur via at least two mechanisms .


First , low amounts of CREB-1 and C/EBP appear to heterodimerize and bind to a site consisting of a half site from both the ATF/CREB and C/EBP binding sites .


In addition , CREB-1 homodimers bind to the ATF/CREB site and recruit C/EBP dimers to their cognate weak binding sites .


This interaction is reciprocal , since C/EBP dimer binding to a strong C/EBP site leads to enhanced CREB-1 recruitment to ATF/CREB sites that are weakly bound by CREB .


Sequence variation at both C/EBP and ATF/CREB sites affects the molecular interactions involved in mediating both of these mechanisms .


Most importantly , sequence variation at the ATF/CREB binding site affected basal LTR activity as well as LTR function following interleukin-6 stimulation , a treatment that leads to increases in C/EBP activation .


Thus , HIV-1 LTR ATF/CREB binding site sequence variation may modulate cellular signaling at the viral promoter through the C/EBP pathway


Selective inhibition of interleukin-4 gene expression in human T cells by aspirin .


Previous studies indicated that aspirin ( acetylsalicylic acid [ ASA ] ) can have profound immunomodulatory effects by regulating cytokine gene expression in several types of cells .


This study is the first in which concentrations of ASA in the therapeutic range were found to significantly reduce interleukin ( IL ) -4 secretion and RNA expression in freshly isolated and mitogen-primed human CD4+ T cells .


In contrast , ASA did not affect IL-13 , interferon-gamma , and IL-2 expression .


ASA inhibited IL-4 , but not IL-2 , promoter-driven chloramphenicol acetyltransferase expression in transiently transfected Jurkat T cells .


The structurally unrelated nonsteroidal anti-inflammatory drugs indomethacin and flurbiprofen did not affect cytokine gene expression in T cells , whereas the weak cyclo-oxygenase inhibitor salicylic acid was at least as effective as ASA in inhibiting IL-4 expression and promoter activity .


The inhibitory effect of ASA on IL-4 transcription was not mediated by decreased nuclear expression of the known salicylate target nuclear factor ( NF ) -kappaB and was accompanied by reduced binding of an inducible factor to an IL-4 promoter region upstream of , but not overlapping , the NF of activated T cells- and NF-kappaB-binding P1 element .


It is concluded that anti-inflammatory salicylates , by means of a previously unrecognized mechanism of action , can influence the nature of adaptive immune responses by selectively inhibiting the expression of IL-4 , a critical effector of these responses , in CD4+ T cells .


Molecular and cellular mediators of interleukin-1 -dependent acute inflammatory arthritis .


OBJECTIVE : To examine the molecular and cellular mechanisms in a model of acute inflammatory monarticular arthritis induced by methylated bovine serum albumin ( mBSA ) and interleukin-1 ( IL-1 ) .


METHODS : Mice were injected intraarticularly with mBSA on day 0 and subcutaneously with recombinant human IL-1beta on days 0-2 .


At day 7 , knee joints were removed and assessed histologically .


Flow cytometry and RNase protection were used to analyze IL-1 -dependent events .


RESULTS : C57BL/6 ( B6 ) , 129/Sv , and ( B6 x 129/ Sv ) F1 hybrid mice , all H-2b strains , were susceptible to mBSA /IL-1-induced arthritis , whereas C3H/HeJ ( H-2k ) mice were not .


B6 mice lacking T and B cells ( RAG1-/- ) or major histocompatibility complex ( MHC ) class II antigens ( MHCII-/- ) , and B6 mice treated with a CD4+ T cell-depleting monoclonal antibody , were resistant to disease .


In contrast , B cell-deficient ( muMT/ muMT ) mice developed arthritis at an incidence and severity similar to that of controls .


RelB-deficient ( RelB-/- ) bone marrow chimeric mice had arthritis that was significantly reduced in incidence and severity .


In B6 mice , flow cytometry demonstrated an IL-1-dependent leukocyte infiltration into the synovial compartment and RNase protection assays revealed induction of messenger RNA ( mRNA ) for the chemokines monocyte chemoattractant protein 1 , macrophage inhibitory protein 2 ( MIP-2 ) , RANTES , MIP-1alpha , and MIP-1beta , in vivo and in vitro .


CONCLUSION : Arthritis induced by mBSA / IL-1 is strain specific and dependent on CD4+ T lymphocytes and at least partially on RelB , but not on B lymphocytes or antibody .


IL-1 contributes to leukocyte recruitment to the synovium and directly induces chemokine mRNA production by synovial cells .


This model of acute monarticular arthritis is particularly suitable for further investigations into cell-mediated immunity in arthritis and the role of IL-1 .


Positive and negative regulation of granulopoiesis by endogenous RARalpha .


Acute promyelocytic leukemia ( APL ) is always associated with chromosomal translocations that disrupt the retinoic acid receptor alpha ( RARalpha ) gene .


Whether these translocations relate to a role for endogenous RARalpha in normal granulopoiesis remains uncertain because most studies addressing this question have used non-physiological overexpression systems .


Granulocyte differentiation in cells derived from RARalpha-deficient ( RARalpha ( -/- ) ) mice was studied and evaluated in the context of agonist-bound and ligand-free RARalpha .


Our results demonstrate that RARalpha is dispensable for granulopoiesis , as RARalpha ( -/- ) mice have a normal granulocyte population despite an impaired ability to respond to retinoids .


However , although it is not absolutely required , RARalpha can bidirectionally modulate granulopoiesis .


RARalpha stimulates differentiation in response to exogenous retinoic acid .


Furthermore , endogenous retinoids control granulopoiesis in vivo , as either vitamin A-deficient mice or animals treated with an RAR antagonist accumulate more immature granulocytes in their bone marrow .


Conversely , RARalpha acts to limit differentiation in the absence of ligand because granulocyte precursors from RARalpha ( -/- ) mice differentiate earlier in culture .


Thus , the block in granulopoiesis exerted by RARalpha fusion proteins expressed in APL cells may correspond to an amplification of a normal function of unliganded RARalpha .


Expression and function of a stem cell promoter for the murine CBFalpha2 gene : distinct roles and regulation in natural killer and T cell development .


The Runt family transcription factor CBFalpha2 ( AML1 , PEBP2alphaB , or Runx1 ) is required by hematopoietic stem cells and expressed at high levels in T-lineage cells .


In human T cells CBFalpha2 is usually transcribed from a different promoter ( distal promoter ) than in myeloid cells ( proximal promoter ) , but the developmental and functional significance of this promoter switch has not been known .


Here , we report that both coding and noncoding sequences of the distal 5 ' end are highly conserved between the human and the murine genes , and the distal promoter is responsible for the overwhelming majority of CBFalpha2 expression in murine hematopoietic stem cells as well as in T cells .


Distal promoter activity is maintained throughout T cell development and at lower levels in B cell development , but downregulated in natural killer cell development .


The distal N-terminal isoform binds to functionally important regulatory sites from known target genes with two- to threefold higher affinity than the proximal N-terminal isoform .


Neither full-length isoform alters growth of a myeloid cell line under nondifferentiating conditions , but the proximal isoform selectively delays mitotic arrest of the cell line under differentiating conditions , resulting in the generation of greater numbers of neutrophils .


Constitutive PI3-K activity is essential for proliferation , but not survival , of Theileria parva-transformed B cells .


Theileria is an intracellular parasite that causes lymphoproliferative disorders in cattle , and infection of leucocytes induces a transformed phenotype similar to tumour cells , but the mechanisms by which the parasite induces this phenotype are not understood .


Here , we show that infected B lymphocytes display constitutive phosphoinositide 3-kinase ( PI3-K ) activity , which appears to be necessary for proliferation , but not survival .


Importantly , we demonstrate that one mechanism by which PI3-K mediates the proliferation of infected B lymphocytes is through the induction of a granulocyte-monocyte colony-stimulating factor ( GM-CSF ) -dependent autocrine loop .


PI3-K induction of GM-CSF appears to be at the transcriptional level and , consistently , we demonstrate that PI3-K is also involved in the constitutive induction of AP-1 and NF-kappaB , which characterizes Theileria-infected leucocytes .


Taken together , our results highlight a novel strategy exploited by the intracellular parasite Theileria to induce continued proliferation of its host leucocyte .


Lymphokine dependence of STAT3 activation produced by surface immunoglobulin cross-linking and by phorbol ester plus calcium ionophore treatment in B cells .


Stimulation of B cells by surface immunoglobulin ( sIg ) triggering , or through the mitogenic combination of phorbol ester and calcium ionophore , is accompanied by activation of STAT transcription factors .


The mechanism responsible for the delayed nuclear accumulation of phosphorylated STAT3 was examined in detail , focusing on the role of B cell-derived lymphokines .


sIg -induced activation of STAT3 was partially inhibited in B cells obtained from IL-6- or IL-10-deficient mice , and was partially blocked by neutralizing antibodies directed against either of these lymphokines .


sIg -induced STAT3 activation was completely inhibited by combining IL-6- and IL-10-specific neutralizing antibodies , or by adding individual neutralizing antibodies to B cells obtained from lymphokine-deficient animals .


In contrast , IL-10 alone appeared to account for STAT3 activation resulting from B cell stimulation with phorbol ester and calcium ionophore .


In keeping with these results , soluble IL-6 and IL-10 were found in supernatant fluid obtained from stimulated B cells .


This work indicates that a lymphokine pathway is responsible for STAT3 activation that occurs late after B cell stimulation , and points out differences in B cell activation that result from stimulation through the antigen receptor and through pharmacological mimicry of signaling mediators .


Transcription factor AP-4 is a ligand for immunoglobulin-kappa promoter E-box elements .


Immunoglobulin ( Ig ) -kappa promoters from humans and mice share conserved sequences .


The octamer element is common to all Ig promoters and pivotal for their function .


However , other conserved sequence motifs , that differ between Ig variable gene families , are required for normal promoter function .


These conserved motifs do not stimulate transcription in the absence of an octamer .


One example is an E-box of the E47/E12 type ( 5'-CAGCTG-3 ' ) , which is found in all promoters of the human and murine Ig-kappa gene subgroups/families , with the exception of subgroups II and VI and their related murine families .


In the present study we show that the ubiquitously expressed transcription factor AP-4 , and not E47 , interacts specifically with the kappa promoter E-boxes when tested in electrophoretic mobility-shift assays using nuclear extracts derived from human and murine B-cell lines .


Furthermore , AP-4 , unlike E47 , did not act as a transactivator , which is in agreement with previous studies on intact kappa promoters , showing that transcription is absent when the octamer element has been mutated .


Based on these data , and the conservation of the 5'-CAGCTG-3 ' motif among human and murine kappa promoters , we propose that AP-4 is the major ligand for Ig-kappa promoter E-boxes .


A 16-mer peptide ( RQIKIWFQNRRMKWKK ) from antennapedia preferentially targets the Class I pathway .


Translocation of antigenic peptides into the cytosol of antigen presenting cells facilitates proteosomal processing and loading into Class I molecules for MHC presentation on the cell surface .


The DNA binding domain of the Drosophila transcription factor ( Antennapedia ) , a 60 amino acid protein , is rapidly taken up by cells and has been fused to selected antigens to enhance their immunogenicity .


We now demonstrate that a 16 amino acid peptide from antennapedia can facilitate the cytoplasmic uptake of CTL epitope 9-mer peptides .


Synthetic peptides were made containing the 16-mer antennapedia peptide linked in tandem to the ovalbumin SIINFEKL CTL peptide .


The peptide complex was shown to rapidly internalise into APCs by confocal microscopy .


This peptide induced CTL in C57BL/6 mice and protected them against growth of an ovalbumin expressing tumour cell line ( E.G7-OVA ) .


The ability of the hybrid peptide to be processed and presented by APCs was similar , whether the SIINFEKL sequence was appended at the C-terminus or N-terminus of the Antennapedia peptide .


The production of synthetic peptides containing other CTL peptide epitopes may be useful for priming CTLs in vitro and in vivo


Bone marrow cells promote TH2 polarization and inhibit virus-specific CTL generation .


This laboratory recently reported that human bone marrow cells ( BMC ) inhibit the generation of virus-specific CTL in culture .


The culture supernatants contained increased levels of prostaglandin E ( 2 ) ( PGE ( 2 ) ) ( shown to favor TH2 cell development ) and also inhibited EBV-CTL effector cell development .


In this study , we obtained PBL from Epstein-Barr virus ( EBV ) IgG antibody positive kidney transplant recipients ( R ) and their living-related donors ( LRD ) one year after renal transplantation .


EBV-specific CTL were then generated in vitro by stimulating PBL with autologous EBV-transformed B cells ( EBV-B ) in the presence or absence of autologous BMC .


The addition of BMC to the EBV-CTL generation cultures increased the intracellular expression in CD3+ cells of IL-4 , -5 , -6 , -10 , and -13 .


These CD3+ cells also expressed increased levels of the TH2 associated receptor CCR3 .


Inhibition was even observed by preparing EBV-CTL generating cultures in trans-wells that separated the autologous BMC from the PBL + EBV-B .


It was then observed that CD3+ cells obtained after 7 days of culture in the presence of autologous BMC could be used as inhibitors of EBV-CTL generation .


Protein Kinase A ( PKA ) , a cAMP kinase that is involved in the upregulation of TH2 cytokine activity , was increased in EBV-CTL cultures by the presence of BMC .


Additionally , IL-4-mediated signal transduction and activation of transcription ( STAT-6 ) phosphorylation was slightly increased .


These results show that the BMC inhibition is mediated by soluble factors ( cytokines ) and that cell-cell contact in this autologous system is not required , so that BMC ( at least partially , via cytokine production ) promote TH2 polarization in culture .


Moreover , TH2 cells induced by culturing with autologous BMC directly inhibit EBV-CTL generation , and TH2 associated PKA , CCR3 , and STAT-6 phosphorylation are enhanced by BMC .


Reduction in DNA binding activity of the transcription factor Pax-5a in B lymphocytes of aged mice .


Aging has been associated with intrinsic changes of the humoral immune response , which may lead to an increased occurrence of autoimmune disorders and pathogenic susceptibility .


The transcription factor Pax-5 is a key regulator of B cell development .


Pax-5a/B cell-specific activator protein and an alternatively spliced isoform , Pax-5d , may have opposing functions in transcriptional regulation due to the lack of a transactivation domain in Pax-5d .


To study B cell -specific changes that occur during the aging process , we investigated expression patterns of Pax-5a and 5d in mature B cells of young and aged mice .


RNase protection assays showed a similar transcriptional pattern for both age groups that indicates that aging has no affect on transcription initiation or alternative splicing for either isoform .


In contrast , a significant reduction in the DNA binding activity of Pax-5a but not Pax-5d protein was observed in aged B cells in vitro , while Western blot analyses showed that similar levels of Pax-5a and 5d proteins were present in both age groups .


The observed decrease in Pax-5a binding activity correlated with changes in expression of two Pax-5 target genes in aged B cells .


Expression of the Ig J chain and the secreted form of Ig mu , which are both known to be suppressed by Pax-5a in mature B cells , were increased in B cells of aged mice .


Together , our studies suggest that changes associated with the aging phenotype cause posttranslational modification ( s ) of Pax-5a but not Pax-5d , which may lead to an abnormal B cell phenotype in aged mice , associated with elevated levels of J chain , and secretion of IgM


NF-kappa B/Rel participation in the lymphokine-dependent proliferation of T lymphoid cells .


Proliferative responses of lymphoid cells to IL-2 and IL-4 depend on activation of the cells , but the mechanism ( s ) by which activation enhances cellular competence to respond to cytokines is not fully understood .


The NF-kappaB/Rel family represents one signal transduction pathway induced during such activation .


We show in this study that inhibition of NF-kappaB through the expression of an IkappaBalpha ( inhibitory protein that dissociates from NF-kappaB ) mutant refractory to signal-induced degradation ( IkappaBalpha ( DeltaN ) ) interfered with the acquisition of competence to proliferate in response to IL-4 as well as IL-2 .


Thymocytes and T cells from IkappaBalpha ( DeltaN ) transgenic mice expressed normal levels of IL-2R subunits .


However , transgenic cells exhibited a dramatic defect in Stat5A activation treatment with IL-2 , and a similar defect was observed for IL-4 -induced Stat5 .


In contrast , T lymphoid cells with inhibition of NF-kappaB showed normal insulin receptor substrate-2 phosphorylation and only a modest decrease in Stat6 activation and insulin receptor substrate-1 phosphorylation after IL-4 stimulation .


These results indicate that the NF-kappaB/Rel/IkappaBalpha system can regulate cytokine receptor capacitation through effects on the induction of downstream signaling by the Stat transcription factor family .


BMS-189453 , a novel retinoid receptor antagonist , is a potent testicular toxin .


BMS-189453 is a synthetic retinoid that acts as an antagonist at retinoic acid receptors alpha , beta , and gamma .


In Sprague Dawley rats at daily oral doses of 15 , 60 , or 240 mg/kg for 1 month , BMS-189453 produced increases in leukocyte counts , alkaline phosphatase and alanine aminotransferase levels , and marked testicular degeneration and atrophy at all doses .


Significant overt signs of toxicity and deaths occurred at 240 mg/kg , whereas body-weight and food-consumption decreases occurred at 60 and 240 mg/kg .


When BMS-189453 was administered to male rats at daily doses ranging from 12.5 to 100 mg/kg for 1 week , only minimal testicular changes occurred at all doses , shortly after the dosing period .


However , after a 1-month drug-free observation period , marked testicular atrophy was evident at all doses .


BMS-189453 was then administered at doses of 2 , 10 , or 50 mg/kg to male rats for 1 , 3 , or 7 consecutive days .


Dose- and duration-dependent testicular toxicity that occurred after a 1-month observation period did not recover , and , in some cases , was more severe 4 months after the last dose .


In rabbits administered BMS-189453 at oral doses of 2 , 10 , or 50 mg/kg for 1 week , testicular degeneration and atrophy were evident in the high-dose group at 1 month following treatment .


These studies indicate that retinoid antagonists can selectively produce progressive and prolonged testicular toxicity after single or repeated oral doses that are otherwise well tolerated .


Sequential involvement of NFAT and Egr transcription factors in FasL regulation .


The critical function of NFAT proteins in maintaining lymphoid homeostasis was revealed in mice lacking both NFATp and NFAT4 ( DKO ) .


DKO mice exhibit increased lymphoproliferation , decreased activation-induced cell death , and impaired induction of FasL .


The transcription factors Egr2 and Egr3 are potent activators of FasL expression .


Here we find that Egr2 and Egr3 are NFAT target genes .


Activation of FasL occurs via the NFAT -dependent induction of Egr3 , as demonstrated by the ability of exogenously provided NFATp to restore Egr -dependent FasL promoter activity in DKO lymph node cells .


Further , Egr3 expression is enriched in Th1 cells , suggesting a molecular basis for the known preferential expression of FasL in the Th1 versus Th2 subset .


Mechanisms and clinical relevance of nongenomic glucocorticoid actions .


Glucocorticoids have profound anti-inflammatory and immunosuppressive actions when used therapeutically .


The therapeutic dose is quite variable and depends on the disease , but ranges from very low to extremely high .


The rationale for the use of various dosage regimens for specific clinical indications is the existence of three distinct , therapeutically relevant effects : genomic , specific nongenomic and unspecific nongenomic .


Genomic effects are mediated by cytosolic receptors that alter expression of specific genes .


Specific nongenomic effects occur within a few minutes and are mediated by steroid-selective membrane receptors .


Unspecific nongenomic effects occur within seconds , but only at high glucocorticoid dosages , and seem to result from direct interactions with biological membranes .


For unspecific nongenomic effects , methylprednisolone and other glucocorticoids have been shown to inhibit cation cycling across the plasma membrane , but to have little effect on protein synthesis .


Thus , glucocorticoids could diminish or prevent the acute immune response by interfering with processes such as the rise in intracellular Ca2+ concentration .


It is proposed that the additional therapeutic benefit of higher doses is obtained via these nongenomic effects .


High glucose-induced intercellular adhesion molecule-1 ( ICAM-1 ) expression through an osmotic effect in rat mesangial cells is PKC-NF-kappa B-dependent .


AIMS/HYPOTHESIS : Infiltration of mononuclear cells and glomerular enlargement accompanied by glomerular cell proliferation are very early characteristics of the pathophysiology of diabetes .


To clarify the mechanism of early diabetic nephropathy , we measured [ 3H ] -thymidine incorporation and cell numbers to show the influence of a high ambient glucose concentration and the osmotic effect on rat mesangial cell proliferation .


We also measured the effect of high glucose on the expression of intercellular adhesion molecule-1 and vascular adhesion molecule-1 by flow cytometry and semiquantitative RT-PCR in mesangial cells and the adhesion of leukocytes to mesangial cells .


METHODS/RESULTS : Cells exposed to high D-glucose ( 30 mmol/l ) caused an increase in [ 3H ] -thymidine incorporation and cell numbers at 24 and 48 h and normalized at 72 h ( p < 0.05 ) , whereas these changes were not found in high mannitol ( 30 mmol/l ) , IL-1 beta , or TNF alpha -stimulated mesangial cells .


Cells exposed to high-glucose ( 15 , 30 , or 60 mmol/l ) or osmotic agents ( L-glucose , raffinose and mannitol ) showed that intercellular adhesion molecule-1 expression began to increase after 24 h , reached its maximum at 24 and 48 h and gradually decreased afterwards .


The stimulatory effects of high glucose and high mannitol on mRNA expression were observed as early as 6 h and reached its maximum at 12 h .


Up-regulation of ICAM-1 protein and mRNA was also found in IL-1-beta and TNF-alpha -stimulated mesangial cells .


Neither vascular adhesion molecule-1 protein nor mRNA expression was , however , affected by high glucose and high mannitol .


Notably , the protein kinase C inhibitors calphostin C and staurosporine reduced high glucose- or high mannitol-induced intercellular adhesion molecule-1 mRNA expression and high glucose-induced proliferation .


Furthermore , the NF-kappa B inhibitor N-tosyl-L-phenylalanine chloromethyl ketone reduced high glucose- or high mannitol-induced intercellular adhesion molecule-1 mRNA expression and high glucose-induced proliferation .


Results showed that high glucose ( 15 , 30 mmol/l ) or high concentrations of osmotic agents remarkably increased the number of adherent leukocytes to mesangial cells ( p < 0.01 ) compared with control cells ( 5 mmol/l D-glucose ) .


Functional blocking of intercellular adhesion molecule-1 on mesangial cells with rat intercellular adhesion molecule-1 monoclonal antibody , calphostin C , staurosporine , or N-tosyl-L-phenylalanine chloromethyl ketone significantly inhibited high glucose- or high mannitol-induced increase in leukocyte adhesion ( p < < 0.05 ) .


CONCLUSION/INTERPRETATION : These results suggest that high glucose can upregulate intercellular adhesion molecule-1 protein and mRNA expression but not vascular adhesion molecule-1 expression in mesangial cells and promote leukocyte adhesion through up-regulation of intercellular adhesion molecule-1 through osmotic effect , possibly depending on the protein kinase C nuclear factor-kappa B ( PKC - NF-kappa B ) pathway .


High glucose itself can also promote mesangial cell proliferation through the PKC-NF-kappa B pathways .


We conclude that hyperglycaemia in itself seems to be an important factor in the development of early diabetic nephropathy .


Combined corticosteroid/ granulocyte colony-stimulating factor ( G-CSF ) therapy in the treatment of severe congenital neutropenia unresponsive to G-CSF : Activated glucocorticoid receptors synergize with G-CSF signals .


More than 90 % of patients with severe congenital neutropenia ( SCN ) respond to granulocyte colony-stimulating factor ( G-CSF ) therapy .


The basis for the refractory state in the remaining patients is unknown .


To address this issue , we studied a child with SCN who was totally unresponsive to G-CSF and had a novel point mutation in the extracellular domain of the G-CSF receptor ( GCSF-R ) .


Marrow stromal support of granulopoiesis was evaluated by plating CD34 ( + ) cells on preformed stromal layers .


Nonadherent cells were harvested and assayed in clonogenic assays for granulocytic colony production .


The in vitro effect of G-CSF and corticosteroids on granulopoiesis was evaluated in clonogenic assays of marrow mononuclear cells , by proliferation studies of the murine myeloid cell line 32D expressing the patient 's mutated G-CSFR , and by measuring STAT5 activation in nuclear extracts from stimulated cells .


Patient 's stroma supported granulopoiesis derived from control marrow CD34 ( + ) cells in a normal manner .


Normal stroma , however , failed to induce granulopoiesis from patient 's CD34 ( + ) cells .


Clonogenic assays of the patient 's marrow mononuclear cells incorporating either G-CSF or hydrocortisone produced little neutrophil growth .


In contrast , inclusion of both G-CSF and hydrocortisone in the cytokine `` cocktail '' markedly increased the neutrophil numbers .


Proliferation of 32D cells expressing the mutated receptor and STAT5 activation were improved by a combination of G-CSF and dexamethasone .


When small daily doses of oral prednisone were then administered to the patient with conventional doses of subcutaneous G-CSF , the patient responded with increased neutrophil numbers and with a complete reversal of the infectious problems .


These data provide insight into SCN unresponsive to standard G-CSF treatment and to the potential corrective action of combined treatment with G-CSF and corticosteroids through synergistic activation of STAT5 .


Human renal mesangial cells are a target for the anti-inflammatory action of 9-cis retinoic acid .


Mesangial cells play an active role in the inflammatory response to glomerular injury .


We have studied in cultured human mesangial cells ( CHMC ) several effects of 9-cis retinoic acid ( 9-cRA ) , an activator of both retinoic acid receptors ( RARs ) and retinoid X receptors ( RXRs ) .


9-cRA inhibited foetal calf serum-induced CHMC proliferation .


It also prevented CHMC death induced by the inflammatory mediator H ( 2 ) O ( 2 ) .


This preventive effect was not due to any increase in H ( 2 ) O ( 2 ) catabolism and it persisted even when both catalase and glutathione synthesis were inhibited .


Finally , 9-cRA diminished monocyte adhesion to FCS-stimulated CHMC .


Interestingly , the retinoid also inhibited in FCS-stimulated cells the protein expression of two mesangial adhesion molecules , fibronectin and osteopontin , but it did not modify the protein expression of intercellular adhesion molecule-1 and vascular adhesion molecule-1 .


All major RARs and RXRs isotypes were expressed in CHMC regardless of the presence or absence of 9-cRA .


Transcripts to RAR-alpha , RAR-beta and RXR-alpha increased after incubation with 9-cRA whereas RXR-gamma was inhibited , suggesting a major role for RARs and RXRs in 9-cRA-anti-inflammatory effects .


9-cRA was toxic only at 50 microM ( a concentration 50 - 5000 times higher than required for the effects above ) .


Cell death occurred by apoptosis , whose onset was associated with a pronounced increase in catalase activity and reduced glutathione content , being more effectively induced by all-trans retinoic acid .


Modulation of the oxidant/antioxidant balance failed to inhibit apoptosis .


We conclude that mesangial cells might be a target for the treatment of inflammatory glomerulopathies with 9-cRA .


Expression of mammalian defensin genes .


Antimicrobial peptides are a prevalent mechanism of host defense found throughout nature .


In mammals , defensins are among the most abundant of these broad-spectrum antibiotics , and are expressed in epithelial and hematopoietic cells .


The defensin peptides are especially abundant in neutrophils ; however , gene expression is limited to the promyelocyte stage .


In epithelial cells , defensin genes are found as both constitutively expressed and inducible .


Induction has been observed in vitro by stimulation with bacterial lipopolysaccharide as well as inflammatory mediators .


In vivo , up-regulation of several defensin genes occurs in both infectious and inflammatory states .


Gene regulation occurs via signal transduction pathways common to other innate immune responses , utilizing transcription factors such as nuclear factor ( NF ) -kappaB and NF interleukin-6 .


Together , the data suggest a broad-based innate host defense whereby potent antimicrobial peptides are present to prevent initial colonization by pathogenic microorganisms .


In addition , the recognition of bacteria coupled with a nascent inflammatory response can bolster this defense by a coordinated up-regulation of the peptides .


Multiple signals required for cyclic AMP-responsive element binding protein ( CREB ) binding protein interaction induced by CD3/CD28 costimulation .


The optimal activation of cAMP-responsive element binding protein ( CREB ) , similar to the full activation of T lymphocytes , requires the stimulation of both CD3 and CD28 .


Using a reporter system to detect interaction of CREB and CREB-binding protein ( CBP ) , in this study we found that CREB binds to CBP only by engagement of both CD3 and CD28 .


CD3/CD28 -promoted CREB - CBP interaction was dependent on p38 mitogen-activated protein kinase ( MAPK ) and calcium/calmodulin-dependent protein kinase ( CaMK ) IV in addition to the previously identified extracellular signal-regulated kinase pathway .


Extracellular signal-regulated kinase , CaMKIV , and p38 MAPK were also the kinases involved in CREB Ser ( 133 ) phosphorylation induced by CD3/CD28 .


A reconstitution experiment illustrated that optimum CREB - CBP interaction and CREB trans-activation were attained when these three kinase pathways were simultaneously activated in T cells .


Our results demonstrate that coordinated activation of different kinases leads to full activation of CREB .


Notably , CD28 ligation activated p38 MAPK and CaMKIV , the kinases stimulated by CD3 engagement , suggesting that CD28 acts by increasing the activation extent of p38 MAPK and CaMKIV .


These results support the model of a minimum activation threshold for CREB - CBP interaction that can be reached only when both CD3 and CD28 are stimulated .


The murine IL-2 promoter contains distal regulatory elements responsive to the Ah receptor , a member of the evolutionarily conserved bHLH-PAS transcription factor family .


Signaling through the TCR and costimulatory signals primarily control transcription of the IL-2 gene in naive T cells .


The minimal promoter necessary for this expression lies proximal , between -300 and the transcription start site .


We had previously shown that activation of the arylhydrocarbon receptor ( AHR ) , a member of the bHLH-PAS family of transcription factors , leads to increased mRNA expression of IL-2 in murine fetal thymocytes .


The AHR is abundant in the thymus and may play a role for the development of the immune system .


Moreover , its overactivation by chemicals such as dioxins leads to immunosuppression and thymic involution .


Binding motifs for the liganded AHR can be identified in the distal region -1300 to -800 of the mouse IL-2 promoter .


We show here that these DNA motifs , the so-called dioxin response elements , after binding to the liganded AHR are sufficient to transactivate luciferase expression in a reporter gene system .


The IL-2 gene can be induced by the AHR also in thymocytes in vivo after injection of 2 , 3 , 7 , 8-tetrachlorodibenzo-p-dioxin , a potent ligand of the AHR .


The AHR mediates the IL-2 induction as shown with AHR -deficient mice .


However , in spleen cells in vitro costimulation via the TCR is necessary for optimal IL-2 gene induction .


Thus , the IL-2 promoter region contains novel distal regulatory elements that can be addressed by the AHR to induce IL-2 and can cooperate with the proximal promoter


Visualization of Syk-antigen receptor interactions using green fluorescent protein : differential roles for Syk and Lyn in the regulation of receptor capping and internalization .


The cross-linking of the B cell Ag receptor ( BCR ) is coupled to the stimulation of multiple intracellular signal transduction cascades via receptor-associated , protein tyrosine kinases of both the Src and Syk families .


To monitor changes in the subcellular distribution of Syk in B cells responding to BCR cross-linking , we expressed in Syk -deficient DT40 B cells a fusion protein consisting of Syk coupled to green fluorescent protein .


Treatment of these cells with anti-IgM Abs leads to the recruitment of the kinase from cytoplasmic and nuclear compartments to the site of the cross-linked receptor at the plasma membrane .


The Syk-receptor complexes aggregate into membrane patches that redistribute to form a cap at one pole of the cell .


Syk is not demonstrably associated with the internalized receptor .


Catalytically active Syk promotes and stabilizes the formation of tightly capped BCR complexes at the plasma membrane .


Lyn is not required for the recruitment of Syk to the cross-linked receptor , but is required for the internalization of the clustered BCR complexes .


In the absence of Lyn , receptor-Syk complexes at the plasma membrane are long lived , and the receptor-mediated activation of the NF-AT transcription factor is enhanced .


Thus , Lyn appears to function to negatively regulate aspects of BCR -dependent signaling by stimulating receptor internalization and down-regulation .


Renal interstitial fibrosis is reduced in angiotensin II type 1a receptor -deficient mice .


Unilateral ureteral obstruction ( UUO ) results in tubulointerstitial fibrosis of the affected kidney by stimulating the renin-angiotensin system .


This study established a UUO model in angiotensin type 1a receptor ( AT1a ) deficient ( mutant ) mice to elucidate the role of angiotensin II through AT1a on the fibrosis of the obstructed kidney ( OBK ) .


The relative volume of the tubulointerstitium was measured by an image analyzer ; deposition of collagen types III and IV and monocyte / macrophage infiltration were histologically examined using specific antibodies .


Also determined were the mRNA levels of transforming growth factor-beta by Northern blot analysis .


Nuclear factor-kappaB activity was assessed by gel shift assay .


UUO in wild mice resulted in a marked expansion of relative volume of the tubulointerstitium , together with increased deposition of collagen types III and IV and number of infiltrated monocytes / macrophages in the interstitium , relative to sham-operated mice .


In comparison , these changes were significantly lower in mutant mice with UUO .


The mRNA level of transforming growth factor-beta was significantly higher in the OBK of wild mice with UUO compared with sham-operated mice .


In contrast , the increase in mRNA level in the OBK of mutant mice was significantly less than in wild mice .


Finally , UUO resulted in activation of nuclear factor-kappaB in wild mice but was inhibited in the OBK of mutant mice .


The results provide direct evidence that angiotensin II acting via the AT1a plays a pivotal role in the development of tubulointerstitial fibrosis in UUO .


HLA-DQ tetramers identify epitope-specific T cells in peripheral blood of herpes simplex virus type 2-infected individuals : direct detection of immunodominant antigen-responsive cells .


Ag-specific CD4+ T cells are present in peripheral blood in low frequency , where they undergo recruitment and expansion during immune responses and in the pathogenesis of numerous autoimmune diseases .


MHC tetramers , which constitute a labeled MHC-peptide ligand suitable for binding to the Ag-specific receptor on T cells , provide a novel approach for the detection and characterization of such rare cells .


In this study , we utilized this technology to identify HLA DQ -restricted Ag-specific T cells in the peripheral blood of human subjects and to identify immunodominant epitopes associated with viral infection .


Peptides representing potential epitope regions of the VP16 protein from HSV-2 were loaded onto recombinant DQ0602 molecules to generate a panel of Ag-specific DQ0602 tetramers .


VP16 Ag -specific DQ-restricted T cells were identified and expanded from the peripheral blood of HSV-2-infected individuals , representing two predominant epitope specificities .


Although the VP16 369-380 peptide has a lower binding affinity for DQ0602 molecules than the VP16 33-52 peptide , T cells that recognized the VP16 369-380 peptide occurred at a much higher frequency than those that were specific for the VP16 33-52 peptide .


Down-regulation of BOB.1/OBF.1 and Oct2 in classical Hodgkin disease but not in lymphocyte predominant Hodgkin disease correlates with immunoglobulin transcription .


In contrast to the tumor cells ( L & H cells ) of lymphocyte predominant Hodgkin disease ( LPHD ) , Hodgkin and Reed-Sternberg ( HRS ) cells of classical Hodgkin disease ( cHD ) are unable to transcribe immunoglobulin , despite the presence of rearranged immunoglobulin genes .


Although initial studies have suggested crippling immunoglobulin gene mutations to be the cause of absent immunoglobulin expression in cHD , recent work of our group has demonstrated an impaired activation of the immunoglobulin promoter as a superior mechanism .


As immunoglobulin transcription is mainly regulated by the B-cell transcription factors Oct2 and BOB.1/OBF.1 , we analyzed 35 cases of LPHD , 32 cases of cHD , and 2 Hodgkin disease cell lines for the expression of these transcription factors and also in parallel for immunoglobulin expression .


Our results demonstrate an absence of Oct2 and/or BOB.1/OBF.1 in cHD and a striking overexpression of Oct2 in LPHD .


Immunoglobulin expression was lacking in cHD but present in LPHD .


Furthermore , the reintroduction of BOB.1/OBF.1 and Oct2 into cultured HRS cells restored the activity of cotransduced immunoglobulin promoter constructs .


Our findings dismiss the concept that the different immunoglobulin expression in cHD and LPHD is due to disrupting mutations of immunoglobulin V genes in cHD but is most likely due to a down-regulation of Oct2 and/or BOB.1/OBF.1 .


This study further revealed Oct2 as a new and valuable marker for the identification of L & H cells and their distinction from HRS cells .