The binding of estrogen in preparations of human peripheral blood mononuclear cells , as well as by splenic and thymic cells is demonstrated by three different approaches ( Dextran-coated charcoal method , whole cell assay , and gel filtration on a sepharose 4B column ) .


Scatchard 's analysis of [ 3H ] -moxestrol ( R2858 ) and [ 3H ] -estradiol binding proves the existence of a single class of receptor sites having a dissociation constant of 0.18-2.4 X 10 ( -9 ) M .


Physicochemical properties of the binder , including binding capacity and steroid specificity , are quite similar to those reported for the thymus of small mammalian species or human thymoma .


Administration of fibroblast interferon to patients with advanced breast cancer : possible effects on skin metastasis and on hormone receptors .


Eleven patients with metastasized breast cancer received 8 intramuscular injections of 6 x 10 ( 6 ) units of human fibroblast interferon over a period of 40 days .


The injections did not cause local irritation or inflammation .


Fever occurred in only 1 of the 11 patients .


Although several types of metastases were monitored , only skin nodules consistently ( 10 out of 11 patients ) exhibited changes that were suggestive of a therapeutic effect of the treatment regimen : either a simple decrease in size of some nodules or central necrosis accompanied by an inflammatory reaction .


NK-activity of peripheral blood leukocytes was significantly increased after administration of the first dose ; the effect of subsequent injections was less clear .


Receptors for estrogens and progestogens were increased in the tumor biopsies of 2 out of 2 and 5 out of 6 patients tested respectively .


Decreased glucocorticoid receptor binding in adrenal insufficiency .


To examine the effect of glucocorticoid deficiency on the glucocorticoid receptor , we examine the binding of [ 3H ] dexamethasone to lymphocytes in normal subjects and patients with adrenal insufficiency before and after glucocorticoid replacement therapy .


Using a whole cell competitive binding assay , normal human lymphocytes had 5977 +/- 1487 ( mean +/- SD ) binding sites/cell and a dissociation constant of 10 +/- 2 nM .


Lymphocytes from patients with untreated adrenal insufficiency had fewer binding sites ( 3364 +/-322 ) and a 2-fold increase in binding affinity ( 5.4 +/- 0.9 mM ) .


The administration of conventional replacement doses of cortisone acetate for 6 months caused no change in receptor number , but was associated with a decrease in binding affinity toward normal .


After long term glucocorticoid replacement therapy , binding parameters were similar to those in patients before treatment .


The physiological implications of the decreased receptor number and increased binding affinity in adrenal insufficiency remain to be elucidated .


Glucocorticoid receptor concentrations and terminal transferase activity as indicators of prognosis in acute non-lymphocytic leukaemia .


Activity of terminal deoxynucleotidyl transferase ( TdT ) , adenosine deaminase , and 5'nucleotidase and the cellular concentration of glucocorticoid ( dexamethasone ) receptor were determined in 25 patients with acute non-lymphocytic leukaemia .


All patients were treated according to a common protocol .


Increased activity of TdT ( greater than 0.1 unit/microgram DNA ) was found in 11 patients .


This group of patients was shown to have higher remission and survival rates ( p = 0.06 ) compared with patients with low activity of TdT .


The glucocorticoid receptor concentration of the leukaemic blast cells ranged from 0 to 0.94 fmol/microgram DNA .


Thirteen patients had blast cells with a glucocorticoid receptor concentration over 0.22 fmol/microgram DNA .


These patients had significantly increased remission and survival rates ( p = 0.006 ) compared with those with a low receptor concentration .


This finding can not be explained by a difference in sensitivity to glucocorticoids since these were not used as therapeutic agents .


Adenosine deaminase and 5'nucleotidase activities both varied within two orders of magnitude .


No correlation could be found between activities of these enzymes and remission or survival rate .


These results show that measurements of TdT activity and the glucocorticoid receptor concentration yield valuable prognostic information in acute non-lymphocytic leukaemia


[ 3H ] cortivazol : a unique high affinity ligand for the glucocorticoid receptor .


Cortivazol ( CVZ ) and deacylcortivazol ( DAC ) are pyrazolosteroids with potent glucocorticoid activity .


In previous work we showed that DAC is 40-fold more potent than dexamethasone ( DEX ) in lysing leukemic lymphoblasts .


To assess the interaction between these atypical steroids and the glucocorticoid receptor , we examined the binding of [ 3H ] CVZ to cytosol from glucocorticoid-sensitive and -resistant variants of the human leukemic cell line CEM C7 .


In glucocorticoid-sensitive cells [ 3H ] CVZ causes a 2-fold induction of glutamine synthetase and binds to a protein in the 4.6 S region of high salt sucrose gradients .


On DEAE-cellulose chromatography , [ 3H ] CVZ-receptor complexes show a shift from high ( 0.25 M KP ) to low salt ( 0.09 M KP ) eluting forms upon activation .


CVZ competes for a 97 , 000-dalton protein labeled by [ 3H ] dexamethasone mesylate .


Scatchard analysis of the binding of [ 3H ] CVZ in glucocorticoid-sensitive cells revealed a curvilinear plot which resolved into high ( 0.4 nM ) and low ( 11 nM ) affinity components .


The receptor concentration of the low affinity site ( 0.30 pmol/mg protein ) was approximately twice that of the high affinity site ( 0.14 pmol/mg protein ) .


Dissociation experiments with dilution and/or excess unlabeled CVZ supported the presence of independent sites .


In contrast , the binding of [ 3H ] DEX to C7 cytosol revealed a single class of binding sites ( Kd = 1.9 nM ; receptor concentration , 0.46 pmol/mg protein ) .


Examination of the binding of [ 3H ] CVZ using 10 ( -5 ) M DEX as the competing ligand showed that DEX binds only to the low affinity site detected by [ 3H ] CVZ .


In cytosol from a glucocorticoid-resistant cell line with virtually no [ 3H ] DEX binding , [ 3H ] CVZ detected a single high affinity binding site that was similar in dissociation constant ( 0.8 nM ) and receptor concentration ( 0.13 pmol/mg protein ) to the high affinity site detected in the glucocorticoid-sensitive cell line C7 .


A controlled pore glass bead assay for the measurement of cytoplasmic and nuclear glucocorticoid receptors .


An assay for the quantitation of cytoplasmic and nuclear glucocorticoid receptors in lymphoid tissue has been developed using controlled pore glass ( CPG ) beads .


Soluble receptor -- 3H-steroid complex ( cytosol or nuclear extract ) is adsorbed quantitatively within the crevasses of porous glass beads .


Excess labeled steroid as well as most non-specifically bound steroid is easily washed away , leaving the hormone-receptor complex retained by the beads .


Bound 3H-steroid is eluted with ethanol and measured for radioactivity .


This procedure which is simple , rapid , and highly reproducible is carried out using frozen samples ( stable for many months ) containing as few as 1 X 10 ( 7 ) cells .


A comparison of the CPG assay to dextran coated charcoal and a whole cell assay demonstrates that CPG and dextran coated charcoal give equivalent measurements of cytosolic receptor concentration , while the CPG and whole cell assays provide equivalent values for total receptor content .


Plasmacytoid blast crisis in B-cell chronic lymphocytic leukemia : effect of estradiol on growth and differentiation in vitro .


Evolution of a case of chronic lymphocytic leukemia ( CLL ) into blast crisis was found to be characterized by three unusual features ( 1 ) the phenotype of the emerging blast cells was that of pre-plasmacytoid cells as shown by plasma cell morphology and an immunological phenotype corresponding partially with CLL- or intermediate B-cells , partially with plasma cells ( terminal transferase - , common acute lymphocytic leukemia antigen - , Ia+ , surface immunoglobulin heavy chains - , surface kappa light chains + , intracytoplasmic immunoglobulin A+ and G+ , BA-1+ , polyclonal gammaglobulin production ) ; ( 2 ) cytogenetic analysis of spontaneous metaphases revealed that in addition to the typical CLL abnormality , trisomy 12 , in all of the cells , an additional translocation between chromosomes 14 and 17 was present in 40 % with a presumptive breakpoint on chromosome 14 ( q12-3 ) never described before ( commonly q32 ) and ( 3 ) the progression of the disease was associated with a striking increase in the expression by the transformed cells of specific binding sites for estradiol ( E2 ) due to an actual increase in total cellular receptor proteins and not to a change in receptor affinity for E2 .


The functional status of the steroid receptors was confirmed by nuclear transfer of the cytoplasmic hormone-receptor complex upon temperature activation .


Since the rise in E2-receptor display paralleled a large increase in the proliferative activity of the cells as well as a change in their maturation status the question was raised as to whether the E2-receptor should be considered as a physiological marker of growth rate or of cellular differentiation .


Exposure of the patient 's blast cells to E2 in vitro resulted in cessation of cell growth following at least one mitosis after addition of the inducer as seen from the replacement of the large blasts by small CLL-like cells without definite signs of alteration of the differentiation status .


This suggests the association of E2-receptor expression with control of growth rather than cell maturation .


Multiple forms and fragments of cytosolic glucocorticoid receptors from human leukemic cells and normal lymphocytes .


Therapy with glucocorticoids is generally more effective in acute lymphoblastic leukemia than in other types of human leukemia .


Previous studies , however , have not revealed any consistent relationship between clinical responsiveness and the cellular or cytosolic concentration of glucocorticoid-binding sites .


The objectives of this study were to determine whether there are intrinsic structural differences among the glucocorticoid receptors in various types of leukemic cells and normal lymphocytes and to investigate the role of endogenous peptidases in receptor degradation .


Cytosols were prepared from fresh or rapidly frozen leukocytes from 6 healthy adults and 35 high-risk leukemia patients ( median white blood cell count , 150 , 000 cells/microliter ; median age , 13 years ) .


Receptors were labeled with [ 3H ] triamcinolone acetonide and quantitated by charcoal-dextran treatment or Sephadex LH-20 chromatography .


Mean and median cytosolic receptor concentrations in 12 acute lymphoblastic leukemia specimens lacking the standard B-cell or T-cell markers ( `` null cells '' ) were approximately 4-fold higher than in 23 other leukemic cell specimens .


No other consistent differences in receptor content were observed .


Agarose filtration and ultracentrifugation in hypotonic buffers containing 20 mM Na2MoO4 revealed complexes of similar size and shape in all clinical specimens tested and two established leukemic cell lines .


They had Stokes radii ( Rs ) of 8.1 +/- 0.5 ( S.D. ) nm ( n = 50 ) , sedimentation coefficients of 9.5 +/- 0.3S ( n = 40 ) , molecular weights of approximately 330 , 000 , and axial ratios ( a/b ) of approximately 12 .


In hypertonic , molybdate-free buffer , these oligomeric complexes were dissociated into subunits with Rs of 5.9 +/- 0.3 nm ( n = 12 ) and a/b of 11 to 12 , as observed previously for other receptors .


Fragmentation of the oligomer and the subunit was evident in some cytosols .


High activities of peptidases of various specificities were detected in leukemic cell cytosols , as in other cytosols , by fluorometric assays with derivatives of 7-amino-4-methylcoumarin .


Receptor cleavage by these and other endogenous enzymes may account for previous observations of `` abnormal '' receptors in cytosols from some leukemic specimens .


We conclude that intrinsic structural defects in the receptors are unlikely explanations for the unresponsiveness of some types of leukemia to steroid therapy .


Glucocorticoid receptor number and intracellular water space .


In order to elucidate the relationship between cell water content and number of glucocorticoid receptors , eleven normal and malignant lymphoid or myelomonocytic cell types originating from mouse , rat and man were investigated .


The cellular water space was measured with 3H2O , and glucocorticoid receptor number was measured in a whole-cell binding assay with [ 3H ] dexamethasone at 30 and 37 degrees C .


The intracellular water phase concentration of glucocorticoid receptors ( around 40 nmol/l cell water ) , and the dependence of receptor affinity on temperature were similar in normal and malignant rodent and human cells .


It is concluded that comparisons of glucocorticoid receptor levels are best made on the basis of intracellular receptor concentrations .


Covalent labeling of rat thymocyte and human lymphoid glucocorticoid receptor .


Lymphoid cells contain specific receptors for glucocorticoids .


We have used [ 3H ] dexamethasone-21-mesylate to label covalently glucocorticoid receptors in rat thymic lymphocytes and in neoplastic cells obtained from patients with acute lymphoblastic leukemia and malignant lymphoma .


The covalently labeled glucocorticoid receptors were identified by polyacrylamide gel electrophoresis ( in the presence of 0.1 % sodium dodecyl sulfate ) .


In cytosolic fractions prepared from rat thymic lymphocytes , [ 3H ] -dexamethasone-21-mesylate labels a protein ( Mr approximately equal to 95 , 000 ) which was identified as the glucocorticoid receptor by the following criteria : ( a ) labeling of this moiety is inhibited by treatment with a 100-fold molar excess of glucocorticoids , such as dexamethasone and triamcinolone acetonide ; and ( b ) the covalently labeled Mr approximately equal to 95 , 000 protein is activated ( by heating at 20 degrees for 30 min ) to a form that binds to DNA-cellulose .


When intact thymocytes are treated with [ 3H ] dexamethasone-21-mesylate , an Mr approximately equal to 95 , 000 moiety is also labeled covalently .


Approximately 35 % of the glucocorticoid receptors can be labeled covalently when intact thymocytes are treated with 100 nM [ 3H ] dexamethasone-21-mesylate for 30 min at 4 degrees .


Neoplastic cells from acute lymphoblastic leukemia and malignant lymphoma were treated with [ 3H ] dexamethasone-21-mesylate .


In all samples , an Mr approximately equal to 95 , 000 moiety was labeled covalently ; labeling was inhibited by excess glucocorticoid .


Smaller moieties were also identified by competition experiments ; these may represent proteolytic fragments of the Mr approximately equal to 95 , 000 receptor .


Thus , in rat and human lymphoid cells , [ 3H ] dexamethasone-21-mesylate can be used to label covalently the glucocorticoid receptor .


The association of cytosol oestrogen and progesterone receptors with histological features of breast cancer and early recurrence of disease .


Two hundred and eighty-eight primary breast tumours were examined for the presence or absence of oestrogen ( REc ) and progesterone ( RPc ) receptors .


Analysis has shown a relative interdependence between the steroid receptor status of primary breast cancer and other prognostic variables such as histological grade , lymphocytic infiltration and tumour elastosis .


There were significant associations between epithelial cellularity , stromal fibrosis and the value of REc in those tumours in which the receptor was present .


Cellularity and fibrosis were unrelated to the presence or absence of oestrogen receptor .


By contrast , neither the presence or absence nor the value of RPc could be related to cellularity or fibrosis .


The value of REc and RPc analysis as an indicator of prognosis was examined in a sub-group of 175 patients receiving no additional treatment following mastectomy .


Overall relapse-free survival ( RFS ) was no different for those patients with receptors compared to those without them ( REc P = 0.11 , RPc P = 0.7 ) .


There was no difference in RFS of receptor positive and negative tumours when the axillary node status was taken into account .


Clinical implications of glucocorticoid receptors in human leukemia .


Normal lymphoid cells contain glucocorticoid receptor .


A variety of stimuli that activate these cells also induce increases in receptor concentration .


Similar glucocorticoid receptors can be detected in lymphoid cells from patients with acute lymphoblastic leukemia ( ALL ) .


Absence of the glucocorticoid receptor ( usually found in treated patients ) predicts lack of glucocorticoid responsiveness .


Furthermore , in our hands , glucocorticoid receptor levels correlate with the duration of complete remission in ALL ( though not in other forms of leukemia ) .


This association is independent of cell type , age , sex , or initial leukocyte count .


The level of receptor shows a negative correlation with increasing aggressiveness of the tumor ( null-cell leukemia greater than T-cell leukemia greater than Burkitt 's lymphoma ) .


Corticosteroid-mediated immunoregulation in man .


Glucocorticoids have profound and complex effects on the human immune response .


However , the precise mechanisms of the corticosteroid-induced immunoregulation in man have not been precisely defined .


Intracytoplasmic corticosteroid-specific receptors appear to be an important common pathway for steroid-induced changes , but variations of receptor parameters do not account for the multifaceted effects on the immune system .


Human circulating mononuclear cells redistribute out of the intravascular compartment following treatment with corticosteroids .


Although certain components at this redistribution phenomenon have been well-characterized , the importance of this compartmental cellular shift with respect to the mechanisms of corticosteroid-induced immunoregulation are less well-defined .


Recent observations that activated lymphocytes may be sensitive to the lytic effects of glucocorticoids suggest that under certain situations the elimination of selected subsets of cells may be a relevant mechanism of corticosteroid-mediated immunoregulation in man .


Corticosteroid-mediated effects on monocyte function may be an important mechanism of drug-induced immunoregulation in monocyte-dependent responses .


In some experimental conditions , corticosteroids inhibit Interleukin 1 production by monocytes .


The immunoregulatory effects of corticosteroids on lymphocyte immune responses are complex .


In vitro corticosteroids appear to selectively affect early immunoregulatory events as opposed to altering an established response .


Multiple sites of steroid-induced modulations of human B cell responses have been defined .


Human lymphoid cell lines and glucocorticoids : II .


Whole cell and cytoplasmic binding properties of lymphoblastoid , leukaemia and lymphoma lines .


The glucocorticoid binding properties of 18 human lymphoid cell lines ( HLCL ) have been investigated .


The specificity of steroid binding was confirmed with various glucocorticoid agonists and antagonists .


A gradation in whole cell and cytoplasmic glucocorticoid binding capacity was observed in the different cell line types : lymphoblastoid greater than lymphoma greater than leukaemia .


The cytoplasmic receptors of leukaemia and lymphoblastoid lines appeared to contain both proteinaceous and phospholipid components .


Cytoplasmic steroid-receptor complexes exhibited a wide range of sedimentation coefficients ( 8.5-11.3S ) in low ionic strength buffer but there was no correlation with cell line type or glucocorticoid sensitivity .


Activation of these complexes by heat ( 37 degrees C ) or exposure to high ionic strength buffer ( 0.3 M NaCl ) induced nuclear binding of steroid but only complexes in high ionic strength buffer manifested changes in sedimentation coefficient .


No correlation was observed between the level or nature of glucocorticoid binding and the cytolethal or cytostatic responsiveness of HLCL to glucocorticoid treatment in vitro .


The resistance to cytolethal effects can not be ascribed to a failure of cells to take up and bind steroid or to significant differences in the molecular species of cytoplasmic receptors present .


The molecular mechanisms by which glucocorticoids achieve cytolethal responses in human lymphoid cells


CD28 -mediated activation in CD45RA+ and CD45RO+ T cells : enhanced levels of reactive oxygen intermediates and c-Rel nuclear translocation in CD45RA+ cells .


We have analyzed the effect of complete T cell activation ( anti-CD3 plus anti-CD28 ) on the activation of NF-kappaB in CD45RA+ ( naive ) and CD45RO+ ( memory/effector ) T cells .


Long exposure ( 24 h ) induced stronger NF-kappaB DNA binding in CD45RA+ cells than in CD45RO+ cells .


Analysis of the nuclear c-Rel protein indicated that after anti-CD3+anti-CD28 stimulation the level of c-Rel was higher in CD45RA+ cells .


Analysis of the cytoplasmic inhibitor IkappaBalpha indicated that anti-CD3+anti-CD28 stimulation induced a long-lasting degradation in CD45RA+ cells but in CD45RO+ cells the degradation process was more rapid .


Because the CD28 costimulus is known to induce the production of reactive oxygen intermediates ( ROIs ) , the intracellular ROI levels in CD45RA+ and CD45RO+ cells were compared by flow cytometry .


ROIs were produced in both cell types , but more strongly in CD45RA+ cells .


The data presented in this study further emphasize the differences between CD45RA+ and CD45RO+ T lymphocytes in ROI-dependent signaling pathways .


Ikaros in hemopoietic lineage determination and homeostasis .


Studies on the molecular mechanisms that control hemopoietic differentiation have focused on signaling cascades and nuclear effectors that drive this complex developmental system in a regulated fashion .


Here we review the role of Ikaros , the founding member of a unique family of zinc finger transcription factors in this developmental process .


Studies on an Ikaros null mutation have revealed an essential role for this factor in lymphoid cell fate determination and at subsequent branch points of the T cell differentiation pathway .


Differences in the phenotypes of a null and a dominant negative ( DN ) Ikaros mutation provide insight into a regulatory network through which Ikaros proteins exert their effects in development .


In addition a comparative analysis of the hemopoietic stem cell and precursor compartment resulting from the two Ikaros mutations reveals a profound yet not absolute requirement for Ikaros in the production and differentiation of these populations .


Overexpression of p65 and c-Jun substitutes for B7-1 costimulation by targeting the CD28RE within the IL-2 promoter .


The role of Rel and activation protein-1 ( AP-1 ) in IL-2 promoter activity in B7-1- and leukocyte function-associated Ag-3 ( LFA. 3 ) -costimulated T cells has been evaluated .


We demonstrate that overexpression of c-Jun but not c-Fos increases IL-2 promoter activity in both B7-1- and LFA-3-costimulated Jurkat T cells .


Cotransfection of both c-Jun and c-Fos substitutes for B7-1 costimulation in driving an activation protein-1 response element but not for the IL-2 promoter .


Overexpression of Rel proteins demonstrated that p65-expressing Jurkat cells transcribed equally well a nuclear factor kappabeta reporter construct when costimulated with B7-1 or LFA-3 , but transcription of IL-2 promoter or CD28 response element ( CD28RE ) -driven reporters was superior in B7-1-costimulated cells .


Combined expression of c-Jun and p65 induced vigorous transcription of IL-2 promoter- and CD28RE-driven reporter constructs in both LFA-3- and B7-1-costimulated Jurkat cells .


Mutating the CD28RE but not the upstream nuclear factor kappabeta-binding site in the IL-2 promoter reduced B7-1 -driven transcription > 90 % .


The results implicates a major role of the CD28RE in the integration of p65 / c-Jun -mediated transcription within the IL-2 promoter .


We suggest that the transition from an autocrine LFA-3 -driven immune response to a B7 -- induced paracrine immune response involves the activation of c-Jun and p65 , which target the CD28RE region of the IL-2 promoter .


Defects in actin-cap formation in Vav-deficient mice implicate an actin requirement for lymphocyte signal transduction .


BACKGROUND : Antigen-receptor interactions on lymphocytes result in local clustering of actin , receptors and signaling molecules into an asymmetric membrane structure termed a cap .


Although actin polymerization is known to be required , the mechanisms underlying cap formation are unclear .


We have studied the events underlying cap formation using mice bearing a null mutation in vav ( vav -/- ) , a gene that encodes a guanine-nucleotide exchange factor for the GTPase Rac .


RESULTS : Lymphocytes from vav -/- mice failed to form T-cell receptor caps following activation and had a defective actin cytoskeleton .


The vav-/- T cells were deficient in interleukin-2 ( IL-2 ) production and proliferation , and the peak of Ca2+ mobilization was reduced although of normal duration .


Activation of Jun N-terminal kinase or stress-activated kinase ( JNK or SAPK ) and mitogen-activated protein kinase ( MAPK ) and the induction of the transcription factor NF-ATc1 and egr-1 genes was normal .


Despite the reduced Ca2+ mobilization , translocation of cytoplasmic NF-ATc to the nucleus was normal , reflecting that the lower levels of Ca2+ in vav-/- cells were still sufficient to activate calcineurin .


Treatment of lymphocytes with cytochalasin D , which blocks actin polymerization , inhibited cap formation and produced defects in signaling and IL-2 transcriptional induction in response to antigen-receptor signaling that were nearly identical to those seen in vav-/- cells .


In transfection studies , either constitutively active Vav or Rac could complement constitutively active calcineurin to activate NF-AT-dependent transcription .


CONCLUSIONS : These results indicate that Vav is required for cap formation in lymphocytes .


Furthermore , the correlation between cap formation , IL-2 production and proliferation supports the hypothesis that an actin-dependent pathway is a source of specialized growth regulatory signals .


CD14 -dependent activation of human endothelial cells by Bacteroides fragilis outer membrane .


We studied the capacity of isolated Bacteriodes fragilis outer membrane , B. fragilis NCTC9343 lipopolysaccharide ( LPS ; endotoxin ) , and B. fragilis NCTC9343 capsular polysaccharides to activate human umbilical vein endothelial cell ( HUVEC ) monolayers .


To assess HUVEC activation , E-selectin expression was measured by enzyme-linked immunosorbent assay ( ELISA ) , Northern blot analysis for E-selectin-specific mRNA , and electrophoretic gel mobility shift assay ( EMSA ) for NF-kappa B , a transcription factor necessary for E-selectin gene activation .


Exposure of HUVECs to B. fragilis outer membrane fractions , separated from other components of the B. fragilis cell wall by isopycnic , sucrose gradient centrifugation , significantly increased surface expression of E-selectin and induced functional endothelial cell -dependent leukocyte adhesion .


B. fragilis outer membranes induced translocation of NF-kappa B to HUVEC nuclei and accumulation of E-selectin mRNA in HUVEC cytoplasm .


E-selectin expression induced by B. fragilis outer membranes was not blocked by polymixin B .


In contrast , E-selectin expression induced by outer membrane fractions purified from E. coli was competitively inhibited by polymixin B .


Neither purified B. fragilis LPS , a prominent constituent of the outer membrane , nor purified B. fragilis capsular polysaccharides induced HUVEC activation .


Two different monoclonal antibodies directed against human CD14 completely inhibited B. fragilis outer membrane-induced NF-kappa B activation , E-selectin transcription , and E-selectin surface expression .


We conclude that the outer membrane component of the B. fragilis cell wall contains a proinflammatory factor ( s ) , that is not LPS , which induces human endothelial cell activation by a soluble CD14 -dependent mechanism .


NF-kappaB protects HIV-1-infected myeloid cells from apoptosis .


HIV-1 infection of primary monocytic cells and myeloid cell lines results in sustained NF-kappaB activation .


Recently , NF-kappaB induction has been shown to play a role in protecting cells from programmed cell death .


In the present study , we sought to investigate whether constitutive NF-kappaB activity in chronically HIV-1-infected promonocytic U937 ( U9-IIIB ) and myeloblastic PLB-985 ( PLB-IIIB ) cells affects apoptotic signaling .


TNFalpha and cycloheximide caused infected cells to undergo apoptosis more rapidly than parental U937 and PLB-985 cells .


Inhibition of TNFalpha -induced NF-kappaB activation using the antioxidant N-acetylcysteine ( NAC ) resulted in increased apoptosis in both U937 and U9-IIIB cells , while preactivation of NF-kappaB with the non-apoptotic inducer IL-1beta caused a relative decrease in apoptosis .


Inhibition of constitutive NF-kappaB activity in U9-IIIB and PLB-IIIB cells also induced apoptosis , suggesting that NF-kappaB protects cells from a persistent apoptotic signal .


TNFalpha plus NAC treatment resulted in a marked decrease in Bcl-2 protein levels in HIV-1-infected cells , coupled with an increase in Bax protein compared to uninfected cells , suggesting that the difference in susceptibility to TNFalpha -induced apoptosis may relate to the differences in relative levels of Bcl-2 and Bax .


The protective role of NF-kappaB in blocking TNFalpha- and HIV-1-induced apoptosis was supported by studies in Jurkat T cells engineered to express IkappaB alpha repressor mutants ( TD-IkappaB ) under the control of a tetracycline-responsive promoter .


Cells underwent apoptosis in response to TNFalpha only when NF-kappaB activation was inhibited by TD-IkappaB expression .


As was observed for the U9-IIIB cells , TNFalpha treatment also induced a marked decrease in Bcl-2 protein levels in TD-IkappaB expressing cells .


These experiments demonstrate that apoptotic signaling is perturbed in HIV-1-infected U9-IIIB cells and indicate that NF-kappaB activation may play an additional protective role against HIV-1-induced apoptosis in myeloid cells .


Human normal peripheral blood B-lymphocytes are deficient in DNA-dependent protein kinase activity due to the expression of a variant form of the Ku86 protein .


The heterodimeric Ku protein , which comprises a 86 kDa ( Ku86 ) amd a 70 kDa ( Ku70 ) subunits , is an abundant nuclear DNA-binding protein which binds in vitro to DNA termini without sequence specificity .


Ku is the DNA-targeting component of the large catalytic sub-unit of the DNA-dependent protein kinase complex ( DNA-PK [ CS ] ) , that plays a critical role in mammalian double-strand break repair and lymphoid V ( D ) J recombination .


By using electrophoretic mobility shift assays , we demonstrated that in addition to the major Ku x DNA complex usually detected in cell line extracts , a second complex with faster electrophoretic mobility was observed in normal peripheral blood lymphocytes ( PBL ) extracts .


The presence of this faster migrating complex was restricted to B cells among the circulating lymphocyte population .


Western blot analysis revealed that B cells express a variant form of the Ku86 protein with an apparent molecular weight of 69 kDa , and not the 86 kDa- full-length protein .


Although the heterodimer Ku70/variant-Ku86 binds to DNA-ends , this altered form of the Ku heterodimer has a decreased ability to recruit the catalytic component of the complex , DNA-PK ( CS ) , which contributes to an absence of detectable DNA-PK activity in B cells .


These data provide a molecular basis for the increased sensitivity of B cells to ionizing radiation and identify a new mechanism of regulation of DNA-PK activity that operates in vivo .


Co-stimulation of human peripheral blood mononuclear cells with IL-2 and anti-CD3 monoclonal antibodies induces phosphorylation of CREB .


Phosphorylation of the cAMP-response element binding protein CREB within 1 h of CD2 but not CD3 cross-linking of human PBMC was recently demonstrated .


The absence of P-CREB following CD3 cross-linking was unexpected , as other laboratories reported increased phosphorylation of CREB following CD3 cross-linking of the Jurkat lymphocyte cell line .


Due to Jurkat T-cells being IL-2-independent , it was postulated that IL-2 might provide a necessary co-stimulus for phosphorylation of CREB in primary lymphocytes .


Therefore , P-CREB was evaluated following co-stimulation of human PBMC through the IL-2 and CD2 or CD3 receptors .


IL-2 did not further augment phosphorylation of CREB following CD2 cross-linking .


However , while neither IL-2 nor CD3 cross-linking alone induced P-CREB , a 4.5-fold increase in phosphorylation of CREB within 1 h of IL-2/CD3 co-stimulation was observed .


Phosphorylation was not associated with the induction of cAMP , and inhibition of PKA signaling had no effect on P-CREB .


Consistent with signal transduction through p56lck or p59fyn , inhibition of PTK signaling reduced phosphorylation 50 % .


Interestingly , inhibiting PKC signaling with calphostin C further increased P-CREB levels 3-fold over that observed in IL-2/CD3 co-stimulated cells not pretreated with a PKC inhibitor .


In contrast to previous studies performed in the absence of exogenous IL-2 , no increase in binding of CREB to a 32P-labeled oligonucleotide probe was observed by electrophoretic mobility shift assay .


These data suggest that the IL-2 and CD3 signaling pathways provide a necessary and co-operative stimulus promoting phosphorylation of CREB following receptor cross-linking .


HIV-1 infection induces a selective reduction in STAT5 protein expression .


HIV-1 infection is accompanied by qualitative and quantitative defects in CD4+ T lymphocytes .


Loss of immune function in HIV patients is usually associated with a profound dysregulation of cytokine production .


To investigate whether cytokine signaling defects occur during HIV infection , PHA blasts from healthy human donors were infected with two strains of HIV-1 and screened for the expression of STAT proteins used in cytokine signaling .


A selective decrease in STAT5B was seen 8 days after infection with the BZ167 dual-tropic HIV isolate , but not with the Ba-L , M-tropic strain .


Based on these findings , purified T cells from HIV-infected patients in different stages of disease were also tested for STAT expression ; decreases in STAT5A , STAT5B , and STAT1alpha were observed in all patients .


The reduction in STATs seen in vivo and in vitro after HIV infection may contribute to the loss of T cell function in HIV disease .


Transcription factors that regulate monocyte / macrophage differentiation .


Although all the cells in an organism contain the same genetic information , differences in the cell phenotype arise from the expression of lineage-specific genes .


During myelopoiesis , external differentiating signals regulate the expression of a set of transcription factors .


The combined action of these transcription factors subsequently determines the expression of myeloid-specific genes and the generation of monocytes and macrophages .


In particular , the transcription factor PU.1 has a critical role in this process .


We review the contribution of several transcription factors to the control of macrophage development


Transcription factor LKLF is sufficient to program T cell quiescence via a c-Myc -- dependent pathway .


T lymphocytes circulate in a quiescent state until they encounter cognate antigen bound to the surface of an antigen-presenting cell .


The molecular pathways that regulate T cell quiescence remain largely unknown .


Here we show that forced expression of the lung Kruppel-like transcription factor ( LKLF ) in Jurkat T cells is sufficient to program a quiescent phenotype characterized by decreased proliferation , reduced cell size and protein synthesis and decreased surface expression of activation markers .


Conversely , LKLF-deficient peripheral T cells produced by gene targeting showed increased proliferation , increased cell size and enhanced expression of surface activation markers in vivo .


LKLF appeared to function , at least in part , by decreasing expression of the proto-oncogene encoding c-Myc .


Forced expression of LKLF was associated with markedly decreased c-Myc expression .


In addition , many effects of LKLF expression were mimicked by expression of the dominant-negative MadMyc protein and rescued by overexpression of c-Myc .


Thus , LKLF is both necessary and sufficient to program quiescence in T cells and functions , in part , by negatively regulating a c-Myc -- dependent pathway .


HTLV-1 p12 ( I ) protein enhances STAT5 activation and decreases the interleukin-2 requirement for proliferation of primary human peripheral blood mononuclear cells .


The p12 ( I ) protein , encoded by the pX open reading frame I of the human T-lymphotropic virus type 1 ( HTLV-1 ) , is a hydrophobic protein that localizes to the endoplasmic reticulum and the Golgi .


Although p12 ( I ) contains 4 minimal proline-rich , src homology 3-binding motifs ( PXXP ) , a characteristic commonly found in proteins involved in signaling pathways , it has not been known whether p12 ( I ) has a role in modulating intracellular signaling pathways .


This study demonstrated that p12 ( I ) binds to the cytoplasmic domain of the interleukin-2 receptor ( IL-2R ) beta chain that is involved in the recruitment of the Jak1 and Jak3 kinases .


As a result of this interaction , p12 ( I ) increases signal transducers and activators of transcription 5 ( STAT5 ) DNA binding and transcriptional activity and this effect depends on the presence of both IL-2R beta and gamma ( c ) chains and Jak3 .


Transduction of primary human peripheral blood mononuclear cells ( PBMCs ) with a human immunodeficiency virus type 1-based retroviral vector expressing p12 ( I ) also resulted in increased STAT5 phosphorylation and DNA binding .


However , p12 ( I ) could increase proliferation of human PBMCs only after stimulation of T-cell receptors by treatment of cells with low concentrations of alphaCD3 and alphaCD28 antibodies .


In addition , the proliferative advantage of p12 ( I ) -transduced PBMCs was evident mainly at low concentrations of IL-2 .


Together , these data indicate that p12 ( I ) may confer a proliferative advantage on HTLV-1-infected cells in the presence of suboptimal antigen stimulation and that this event may account for the clonal proliferation of infected T cells in vivo .


Single dose intranasal administration of retinal autoantigen generates a rapid accumulation and cell activation in draining lymph node and spleen : implications for tolerance therapy .


BACKGROUND/AIMS : A single intranasal delivery of retinal autoantigen suppresses effectively experimental autoimmune uveoretinitis ( EAU ) .


To further unravel underlying mechanisms the authors wished to determine , firstly , the kinetics of antigen delivery and , secondly , the early cellular responses involved in the initial stages of nasal mucosal tolerance induction .


METHODS : Flow cytometry , cell proliferation assays , and microscopy were used to track antigen following a single , intranasal dose of Alexa-488 labelled retinal antigen .


RESULTS : A rapid accumulation of antigen within both superficial cervical lymph nodes ( SCLN ) and spleen was observed after 30 minutes .


Significant proliferative responses to IRBP were elicited by 48 hours indicating that systemic priming of naive T cells to retinal antigen had occurred .


Cell activation was further confirmed by immunoprecipitation studies , which demonstrated phosphorylation of STAT4 but not STAT6 in both lymph nodes and spleen .


However , at 24 hours , STAT4 heterodimerisation with STAT 3 was only observed in spleen .


CONCLUSIONS : The results provide novel evidence that following a single intranasal application rapid transfer of antigen occurs .


Resulting T cell proliferation develops consequent to differential cell signalling in SCLN and spleen .


Further understanding of these underlying cellular mechanisms , in particular as is inferred by the results the contribution of local versus systemic tolerance induction , may assist in strategies to clinically apply mucosal tolerance therapy successfully .


Regulation of interleukin ( IL ) -18 receptor alpha chain expression on CD4 ( + ) T cells during T helper ( Th ) 1/Th2 differentiation .


Interleukin ( IL ) -18 has been well characterized as a costimulatory factor for the induction of IL-12-mediated interferon ( IFN ) -gamma production by T helper ( Th ) 1 cells , but also can induce IL-4 production and thus facilitate the differentiation of Th2 cells .


To determine the mechanisms by which IL-18 might regulate these diametrically distinct immune responses , we have analyzed the role of cytokines in the regulation of IL-18 receptor alpha chain ( IL-18Ralpha ) expression .


The majority of peripheral CD4 ( + ) T cells constitutively expressed the IL-18Ralpha .


Upon antigen stimulation in the presence of IL-12 , marked enhancement of IL-18Ralpha expression was observed .


IL-12 -mediated upregulation of IL-18Ralpha required IFN-gamma .


Activated CD4 ( + ) T cells that expressed low levels of IL-18Ralpha could produce IFN-gamma when stimulated with the combination of IL-12 and IL-18 , while CD4 ( + ) cells which expressed high levels of IL-18Ralpha could respond to IL-18 alone .


In contrast , T cell stimulation in the presence of IL-4 resulted in a downregulation of IL-18Ralpha expression .


Both IL-4 ( -/ ) - and signal transducer and activator of transcription ( Stat ) 6 ( -/ ) - T cells expressed higher levels of IL-18Ralpha after TCR stimulation .


Furthermore , activated T cells from Stat6 ( -/ ) - mice produced more IFN-gamma in response to IL-18 than wild-type controls .


Thus , positive/negative regulation of the IL-18Ralpha by the major inductive cytokines ( IL-12 and IL-4 ) determines the capacity of IL-18 to polarize an immune response .


Glucocorticoid-regulated transcription factors .


Glucocorticoids are the most effective antiinflammatory drugs used in the treatment of asthma .


They act by binding to a specific receptor ( GR ) that , upon activation , translocates to the nucleus and either increases ( transactivates ) or decreases ( transrepresses ) gene expression .


Inhibition of pro-inflammatory transcription factors such as activator protein ( AP ) -1 , signal transducers and activators of transcription ( STATs ) , nuclear factor of activated T cells ( NFAT ) and nuclear factor ( NF ) -kappa B is thought to be a major action of glucocorticoids .


Acetylation of histones allows unwinding of the local DNA structure and enables RNA polymerase II to enhance gene transcription .


Histone acetylation is regulated by a balance between the activity of histone acetyltransferases ( HATs ) and histone deacetylases ( HDACs ) .


GR acts as a direct inhibitor of NF-kappa B-induced HAT activity and also by recruiting HDAC2 to the NF-kappa B/HAT complex .


A sub-group of patients with glucocorticoid-insensitive asthma have an inability to induce histone acetylation in response to dexamethasone suggesting reduced expression of a GR-specific HAT .


This suggests that pharmacological manipulation of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases .


Identification of the precise mechanism by which activated GR recruits HDAC2 may reveal new targets for the development of drugs that may dissociate the antiinflammatory actions of glucocorticoids from their side effects that are largely due to gene induction .


Differential ultraviolet-B-induced immunomodulation in XPA , XPC , and CSB DNA repair-deficient mice .


Ultraviolet B irradiation has serious consequences for cellular immunity and can suppress the rejection of skin tumors and the resistance to infectious diseases .


DNA damage plays a crucial role in these immunomodulatory effects of ultraviolet B , as impaired repair of ultraviolet-B-induced DNA damage has been shown to cause suppression of cellular immunity .


Ultraviolet-B-induced DNA damage is repaired by the nucleotide excision repair mechanism very efficiently .


Nucleotide excision repair comprises two subpathways : transcription-coupled and global genome repair .


In this study the immunologic consequences of specific nucleotide excision repair defects in three mouse models , XPA , XPC , and CSB mutant mice , were investigated .


XPA mice carry a total nucleotide excision repair defect , whereas XPC and CSB mice only lack global genome and transcription-coupled nucleotide excision repair , respectively .


Our data demonstrate that cellular immune parameters in XPA , XPC , and CSB mice are normal compared with their wild-type ( control ) littermates .


This may indicate that the reported altered cellular responses in xeroderma pigmentosum patients are not constitutive but could be due to external factors , such as ultraviolet B .


Upon exposure to ultraviolet B , only XPA mice are very sensitive to ultraviolet-B-induced inhibition of Th1-mediated contact hypersensitivity responses and interferon-gamma production in skin draining lymph nodes .


Lipopolysaccharide-stimulated tumor necrosis factor alpha and interleukin-10 production are significantly augmented in both XPA and CSB mice after ultraviolet B exposure .


Lymph node cell numbers were increased very significantly in XPA , mildly increased in CSB , and not in XPC mice .


In general XPC mice do not exhibit any indication of enhanced ultraviolet B susceptibility with regard to the immune parameters analyzed .


These data suggest that both global genome repair and transcription-coupled repair are needed to prevent immunomodulation by ultraviolet B , whereas transcription-coupled repair is the major DNA repair subpathway of nucleotide excision repair that prevents the acute ultraviolet-B-induced effects such as erythema .


Biomechanical strain induces class a scavenger receptor expression in human monocyte/macrophages and THP-1 cells : a potential mechanism of increased atherosclerosis in hypertension .


BACKGROUND : Although hypertension is an important risk factor for the development of atherosclerosis , the mechanisms for this interaction are incompletely described .


Previous studies have suggested that biomechanical strain regulates macrophage phenotype .


We tested the hypothesis that biomechanical strain can induce expression of the class A scavenger receptor ( SRA ) , an important lipoprotein receptor in atherogenesis .


METHODS AND RESULTS : Human monocyte/macrophages or THP-1 cells were cultured in a device that imposes uniform biaxial cyclic 1-Hz strains of 0 % , 1 % , 2 % , or 3 % , and SRA expression was analyzed .


Mechanical strains induced SRA mRNA ( 3.5+/-0.6-fold at 3 % strain for 48 hours , P < 0.01 ) and SRA protein in THP-1 cells in an amplitude-dependent manner .


This induction was accompanied by augmented expression of the class B scavenger receptor CD36 ( 2.8+/-0.3-fold , P < 0.001 ) but not by increased peroxisome proliferator-activated receptor-gamma expression .


To evaluate this effect in vivo , apolipoprotein E ( -/- ) mice were randomly assigned to receive standard chow , a high-cholesterol diet , or a high-cholesterol diet with hypertension induced by angiotensin II infusion for 8 weeks .


Immunohistochemistry revealed that among macrophages in atherosclerotic lesions of the aorta , the proportion of macrophages with SRA expression was highest in hypertensive animals on a high-cholesterol diet ( 43.9+/-0.7 % , versus 12.0+/-2.0 % for normotensive animals on a high-cholesterol diet and 4.7+/-4.7 % for animals on standard chow ; P < 0.001 ) .


CONCLUSIONS : Biomechanical strain induces SRA expression by monocyte/macrophages , suggesting a novel mechanism for promotion of atherosclerosis in hypertensive patients .


High glucose induces MCP-1 expression partly via tyrosine kinase-AP-1 pathway in peritoneal mesothelial cells .


BACKGROUND : High glucose in peritoneal dialysis solutions has been implicated in the pathogenesis of peritoneal fibrosis in chronic ambulatory peritoneal dialysis ( CAPD ) patients .


However , the mechanisms are not very clear .


Peritoneal macrophages seem to participate in the process of peritoneal fibrosis and monocyte chemoattractant protein-1 ( MCP-1 ) plays a key role in the recruitment of monocytes toward the peritoneal cavity .


However , little is known about the effect of high glucose on MCP-1 expression and its signal transduction pathway in human peritoneal mesothelial cells .


METHODS : Mesothelial cells were cultured with glucose ( 5 to 100 mmol/L ) or mannitol chronically for up to seven days .


MCP-1 expression of mRNA and protein was measured by Northern blot analysis and enzyme-linked immunosorbent assay ( ELISA ) .


Chemotactic activity of high-glucose-conditioned culture supernatant was measured by chemotactic assay .


To examine the roles of the transcription factors activator protein-1 ( AP-1 ) and nuclear factor-kappaB ( NF-kappaB ) , electrophoretic mobility shift assay ( EMSA ) was performed .


RESULTS : Glucose induced MCP-1 mRNA expression in a time- and dose-dependent manner .


MCP-1 protein in cell culture supernant was also increased .


Equivalent concentrations of mannitol had no significant effect .


High-glucose-conditioned supernatant possessed an increased chemotactic activity for monocytes , which was neutralized by anti-MCP-1 antibody .


EMSA revealed that glucose increased the AP-1 binding activity in a time- and dose-dependent manner , but not NF-kappaB .


Curcumin , an inhibitor of AP-1 , dose-dependently suppressed the induction of MCP-1 mRNA by high glucose .


Tyrosine kinase inhibitors such as genistein ( 12.5 to 50 micromol/L ) and herbimycin A ( 0.1 to 1 micromol/L ) inhibited the high-glucose-induced MCP-1 mRNA expression in a dose-dependent manner , and also suppressed the high-glucose-induced AP-1 binding activity .


CONCLUSIONS : : High glucose induced mesothelial MCP-1 expression partly via the tyrosine kinase- AP-1 pathway .


Signal thresholds and modular synergy during expression of costimulatory molecules in B lymphocytes .


We analyzed intracellular pathways modulating surface densities of CD80 and CD86 in B cells activated through ligation of the Ag receptor , and the adhesion molecule CD54 .


Whereas B cell Ag receptor ( BCR ) cross-linking alone stimulated increased expression of CD86 , up-regulation of CD80 required dual stimulation with anti-IgM and anti-CD54 .


The principal downstream component contributed by BCR signaling , toward both CD80 and CD86 induction , was the elevated concentration of free cytoplasmic Ca ( 2+ ) , recruited by way of capacitative influx .


This alone was sufficient to generate an increase in CD86 levels .


However , CD80 enhancement required the concerted action of both intracellular Ca ( 2+ ) concentration and CD54-initiated pathways .


The nexus between anti-IgM and anti-CD54 stimulation , in the context of CD80 regulation , was identified to involve a self-propagating process of sequential synergy .


The first step involved amplified accumulation of intracellular cAMP , as a result of cross-talk between BCR -mobilized Ca ( 2+ ) and CD54-derived signals .


This then facilitated a second synergistic interaction between Ca ( 2+ ) and cAMP , culminating in CD80 expression .


Our findings of distinct signal transducer requirements , with the added consequences of cross-talk , offers an explanation for variable modulation of costimulatory molecule expression in response to diverse physiological stimuli .


Importantly , these results also reveal how concentration threshold barriers for recruitment of individual second messengers can be overcome by constructive convergence of signaling modules .


The role of Epstein-Barr virus in neoplastic transformation .


In this review , we focus on new data from basic , translational and clinical research relating to the Epstein-Barr virus ( EBV ) .


Beside its well-known tropism for B lymphocytes and epithelial cells , EBV also infects T lymphocytes , monocytes and granulocytes .


After primary infection , EBV persists throughout the life span in resting memory B cells , from where it is reactivated upon breakdown of cellular immunity .


In the process of neoplastic transformation , the EBV-encoded latent membrane protein 1 ( LMP1 ) oncogene represents the major driving force .


LMP1 acts like a constitutively activated receptor of the tumor necrosis factor receptor family and allows the amplification or bypassing of physiological regulatory signals through direct and indirect interactions with proteins of the tumor necrosis factor receptor-associated factor ( TRAF ) family .


TRAF2 -mediated NF-kappaB activation , AP-1 induction and JAK3 / STAT activation may result in sustained proliferation leading to lymphoma .


The ability of LMP1 to suppress germinal center formation and its capacity to mediate its own transcriptional activation shed new light on the pathogenesis of EBV-associated latency type II lymphoproliferations like Hodgkin 's disease and angioimmunoblastic lymphadenopathy .


The carboxy terminus of LMP1 is also a reliable marker for individual EBV strain identification and thus offers new possibilities in tracing the molecular events leading to posttransplant lymphoproliferative disorders ( PTLDs ) .


Cytotoxic T lymphocytes directed against well-characterized epitopes of EBV latency genes represent an already successful and promising therapeutic approach to EBV-associated lymphomas , in particular PTLDs


Interferon-alpha drives T cell-mediated immunopathology in the intestine .


The ability of interferon ( IFN ) -alpha to induce autoimmunity and exacerbate Th1 diseases is well known .


We have recently described enhanced expression of IFN-alpha in the mucosa of patients with celiac disease ( CD ) , a gluten-sensitive Th1-mediated enteropathy , characterized by villous atrophy and crypt cell hyperplasia .


Previous studies from this laboratory have shown that T cell activation in explant cultures of human fetal gut can also result in villous atrophy and crypt cell hyperplasia .


We have , therefore , examined changes that take place in explant cultures of human fetal gut after activation of T cells with anti-CD3 and/or IFN-alpha .


We show that activation of T cells with anti-CD3 alone elicits a small IFN-gamma and TNF-alpha response with no tissue injury .


Similarly , no changes are seen in explants cultured with IFN-alpha alone .


However , addition of IFN-alpha with anti-CD3 results in enhanced Th1 response and crypt cell hyperplasia .


This is associated with enhanced phosphorylation of STAT1 , STAT3 , and Fyn , a Src homology tyrosine kinase , which interacts with both TCR and IFN-alpha signal components .


Together these data indicate that IFN-alpha can facilitate activation of Th1-reactive cells in the gut and drive immunopathology .


Suppression of tumor necrosis factor alpha production by cAMP in human monocytes : dissociation with mRNA level and independent of interleukin-10 .


BACKGROUND : Elevation of cellular cAMP inhibits lipopolysaccharide ( LPS ) -stimulated tumor necrosis factor alpha ( TNF-alpha ) production and increases the expression of interleukin ( IL ) -10 in mononuclear cells .


TNF-alpha gene expression obligates activation of the transcription factor nuclear factor kappaB ( NF-kappaB ) .


Exogenous IL-10 inhibits NF-kappaB in monocytes and thus attenuates TNF-alpha production .


We examined the role of endogenous IL-10 in the regulation of NF-kappaB activation and TNF-alpha production in human monocytes by cAMP .


METHODS : Human monocytes were stimulated with Escherichia coli LPS ( 100 ng/ml ) with and without forskolin ( FSK , 50 microM ) or dibutyryl cyclic AMP ( dbcAMP , 100 microM ) .


Cytokine ( TNF-alpha and IL-10 ) release was measured by immunoassay .


TNF-alpha mRNA was measured by reverse transcription polymerase chain reaction , and NF-kappaB DNA binding activity was assessed by gel mobility shift assay .


RESULTS : cAMP-elevating agents inhibited LPS-stimulated TNF-alpha release ( 0.77 +/- 0.13 ng/10 ( 6 ) cells in LPS + dbcAMP and 0.68 +/- 0.19 ng/10 ( 6 ) cells in LPS + FSK , both P < 0.05 vs 1.61 +/- 0.34 ng/10 ( 6 ) cells in LPS alone ) .


Conversely , cAMP enhanced LPS-stimulated IL-10 release ( 100 +/- 21.5 pg/10 ( 6 ) cells in LPS + dbcAMP and 110 +/- 25.2 pg/10 ( 6 ) cells in LPS + FSK , both P < 0.05 vs 53.3 +/- 12.8 pg/10 ( 6 ) cells in LPS alone ) .


Neither TNF-alpha mRNA expression nor NF-kappaB activation stimulated by LPS was inhibited by the cAMP-elevating agents .


Neutralization of IL-10 with a specific antibody did not attenuate the effect of cAMP-elevating agents on TNF-alpha production .


CONCLUSION : The results indicate that cAMP inhibits LPS-stimulated TNF-alpha production through a posttranscriptional mechanism that is independent of endogenous IL-10 .


Activation of the p21 ( CIP1/WAF1 ) promoter by bone morphogenetic protein-2 in mouse B lineage cells .


BMPs exert a negative growth effect on various types of cells .


We have previously reported that BMP-2 inhibited the growth of HS-72 mouse hybridoma cells by inducing p21 ( CIP1/WAF1 ) expression .


In the present study , we demonstrated that BMP-2 activated the mouse p21 ( CIP1/WAF1 ) promoter in HS-72 cells , and that a 29-base pair ( b ) region of the promoter ( -1928/-1900 relative to the TATA box ) , conserved between mice and humans , was responsive to BMP-2 as well as expression of Smad1 , Smad4 , and constitutively active mutants of BMP type I receptors .


Furthermore , an oligonucleotide containing the 29-b region was found to be associated with Smad4 and phosphorylated Smad1 in the nuclear extract of BMP-2 -stimulated HS-72 cells .


These results suggested that BMP-2 might activate p21 ( CIP1/WAF1 ) transcription by inducing a binding of Smad4 and Smad1 to the 29-b region in HS-72 cells .


Dendritic cell development from common myeloid progenitors .


Dendritic cells ( DCs ) are professional antigen-presenting cells which both initiate adaptive immune responses and control tolerance to self-antigens .


It has been suggested that these different effects on responder cells depend on subsets of DCs arising from either myeloid or lymphoid hematopoietic origins .


In this model , CD8 alpha+ Mac-1- DCs are supposed to be of lymphoid while CD8 alpha- Mac-1+ DCs are supposed to be of myeloid origin .


Here we summarize our findings that both CD8 alpha+ and CD8 alpha- DCs can arise from clonogenic common myeloid progenitors ( CMPs ) in both thymus and spleen .


Therefore CD8 alpha expression DCs does not indicate a lymphoid origin and differences among CD8 alpha+ and CD8 alpha- DCs might rather reflect maturation status than ontogeny .


On the basis of transplantation studies , it seems likely that most of the DCs in secondary lymphoid organs and a substantial fraction of thymic DCs are myeloid-derived .


Macrophage stimulation with Murabutide , an HIV-suppressive muramyl peptide derivative , selectively activates extracellular signal-regulated kinases 1 and 2 , C/EBPbeta and STAT1 : role of CD14 and Toll-like receptors 2 and 4 .


The smallest unit of bacterial peptidoglycans known to be endowed with biological activities is muramyl dipeptide ( MDP ) .


A clinically acceptable synthetic derivative of MDP , namely murabutide ( MB ) , has been found to present interesting pharmacological properties and to suppress HIV-1 replication in monocyte-derived macrophages ( MDM ) .


We have addressed the signaling events activated in MDM following stimulation with either MB or the potent immunostimulant LPS .


We also examined whether signaling by muramyl peptides involves the use of cell surface receptors , including CD14 and Toll-like receptor 2 ( TLR2 ) or TLR4 that are known to be signal-transducing receptors for other bacterial cell wall components .


We demonstrate that , unlike LPS , the safe immunomodulator MB selectively activates extracellular signal-regulated kinases ( Erk ) 1/2 , in the absence of detectable Jun N-terminal kinase ( JNK ) or p38 mitogen-activated kinase activation .


Furthermore , STAT1 activation but weak or no activation of STAT3 or STAT5 respectively , could be detected in MB-stimulated MDM .


Using MonoMac6 cells , we observed high C/EBPbeta and AP-1 but weaker and transient NF-kappaB activation by MB .


Moreover , the truncated form of C/EBPbeta , known to repress HIV-1 transcription , was detected in extracts from MB-treated THP-1 cells .


Surprisingly , neither MB nor MDP were able to transduce signals via CD14 and TLR2 or 4 .


These findings present major differences in the early cell activation process between LPS and muramyl peptides , and strongly argue for the implication of co-receptors other than TLR2 and TLR4 in mediating the signaling events induced by defined subunits of bacterial peptidoglycans .


Nuclear peroxisome proliferator-activated receptors alpha and gamma have opposing effects on monocyte chemotaxis in endometriosis .


The peroxisome proliferator-activated receptors ( PPARs ) alpha and gamma are nuclear receptors that play important roles in inflammatory diseases like ulcerative colitis and arthritis .


In this study , we examined the possible role of PPARs in macrophage attraction into the peritoneal cavity of patients with endometriosis .


We identified PPAR-alpha and -gamma messenger RNA by RT-PCR and protein by immunoblotting of lysates of peritoneal macrophages and monocytic U937 cells .


Using immunocytochemistry , we localized PPAR-alpha and -gamma within the nuclei of both cell types .


Monocyte chemotactic activity of peritoneal fluid from patients with endometriosis was quantified in Boyden chambers .


Migration of U937 cells was increased by WY 14643 and reduced by rosiglitazone .


Peritoneal fluid from patients with endometriosis activated U937 cells transiently transfected with a PPAR-alpha/GAL4 luciferase reporter .


By contrast , peritoneal fluid did not cause significant activation of PPAR-gamma/GAL4 constructs .


The U937 cells transiently transfected with a PPAR response element luciferase reporter showed disease stage-dependent up-regulation when treated with peritoneal fluid from patients with endometriosis .


Treatment with peritoneal fluid from healthy controls down-regulated PPAR response element transactivation .


We conclude that peritoneal fluid of endometriosis patients contains activators of PPAR-alpha that stimulate macrophage chemotaxis .


Inhibitors of PPAR-alpha or activators of PPAR-gamma could be developed for the treatment of inflammation associated with endometriosis .


Long-term-impaired expression of nuclear factor-kappa B and I kappa B alpha in peripheral blood mononuclear cells of trauma patients .


Nuclear factor ( NF ) -kappa B expression and dimer characteristics were studied in peripheral blood mononuclear cells ( PBMCs ) of major-trauma patients and healthy controls .


Analysis of PBMCs on days 1 , 3 , 5 , and 10 after trauma revealed that expression of both p65p50 heterodimers and p50p50 homodimers was significantly reduced compared with that in controls .


In vitro lipopolysaccharide ( LPS ) stimulation of PBMCs induced NF-kappa B translocation .


However , throughout the survey , p65p50 activation remained significantly lower in trauma patients than in controls .


After LPS stimulation in vitro , the p65p50/p50p50 ratio was significantly lower in PBMCs from trauma patients than from healthy controls .


The ex vivo expression of I kappa B alpha was higher in PBMCs of controls than of trauma patients .


LPS did not induce I kappa B expression in PBMCs from trauma patients , but strong induction was obtained with staphylococci , suggesting that this defect is not universal and depends on the nature of the activating signal .


Although no direct correlation was found between levels of interleukin-10 or transforming growth factor-beta and NF-kappa B , these immunosuppressive cytokines were significantly elevated in trauma patients by 10 days after admission .


The long-term low-basal and LPS-induced nuclear translocation of NF-kappa B recalled long-term immunoparalysis observed in patients with severe inflammatory stress such as trauma .


BACKGROUND : The morbidity associated with liver transplantation and major hepatic resections is partly a result of ischemia-reperfusion injury .


DATA SOURCES : The entire world literature on the subject was searched via Medline .


Keywords included reperfusion injury , transplantation , liver resection , nitric oxide , endothelin , cytokines , Kupffer cells , ischemic/ischaemic preconditioning , and nuclear factor-kappa B .


CONCLUSIONS : An imbalance between endothelin and nitric oxide levels results in failure of the hepatic microcirculation at the onset of reperfusion .


Activation of nuclear factor-kappa B in the liver promotes proinflammatory cytokine and adhesion molecule synthesis .


These result in oxygen-derived free radical production and neutrophil recruitment , further contributing to cellular injury .


Various therapeutic modalities acting on the above mediators have been successfully used to attenuate reperfusion injury in animal models of hepatic transplantation and resection .


Application of the knowledge gained from animal models of hepatic ischemia-reperfusion to the clinical setting will improve the outcome of hepatic surgery .


Down-regulation of IL-12 p40 gene in Plasmodium berghei-infected mice .


We analyzed the mechanism that causes suppression of IL-12 p40 gene induction during Plasmodium berghei infection .


Although IL-12 together with IFN-gamma plays an important role in protection against pathogenic infection , the IL-12 p70 protein production of infected macrophages is lower than that by the uninfected macrophages .


We showed in the present study that the induction of IL-12 p40 gene but not IL-12 p35 gene in macrophages of P. berghei-infected mice was profoundly inhibited .


The inhibition was induced by interaction with macrophages that had contacted with P. berghei-infected erythrocytes and was mediated by a soluble factor , IL-10 .


There was comparable activation of NF-kappaB in uninfected and infected cells .


The induction of IFN-regulatory factor-1 gene was comparable in transcription level in uninfected and infected cells , while the unidentified complex formation of IFN-regulatory factor-1 was observed in infected cells .


Therefore , the inhibition of the IL-12 p40 gene induction appeared to be regulated at transcriptional regulation level of the gene .


The Friend of GATA proteins U-shaped , FOG-1 , and FOG-2 function as negative regulators of blood , heart , and eye development in Drosophila .


Friend of GATA ( FOG ) proteins regulate GATA factor-activated gene transcription .


During vertebrate hematopoiesis , FOG and GATA proteins cooperate to promote erythrocyte and megakaryocyte differentiation .


The Drosophila FOG homologue U-shaped ( Ush ) is expressed similarly in the blood cell anlage during embryogenesis .


During hematopoiesis , the acute myeloid leukemia 1 homologue Lozenge and Glial cells missing are required for the production of crystal cells and plasmatocytes , respectively .


However , additional factors have been predicted to control crystal cell proliferation .


In this report , we show that Ush is expressed in hemocyte precursors and plasmatocytes throughout embryogenesis and larval development , and the GATA factor Serpent is essential for Ush embryonic expression .


Furthermore , loss of ush function results in an overproduction of crystal cells , whereas forced expression of Ush reduces this cell population .


Murine FOG-1 and FOG-2 also can repress crystal cell production , but a mutant version of FOG-2 lacking a conserved motif that binds the corepressor C-terminal binding protein fails to affect the cell lineage .


The GATA factor Pannier ( Pnr ) is required for eye and heart development in Drosophila .


When Ush , FOG-1 , FOG-2 , or mutant FOG-2 is coexpressed with Pnr during these developmental processes , severe eye and heart phenotypes result , consistent with a conserved negative regulation of Pnr function .


These results indicate that the fly and mouse FOG proteins function similarly in three distinct cellular contexts in Drosophila , but may use different mechanisms to regulate genetic events in blood vs . cardial or eye cell lineages


The RING finger protein Siah-1 regulates the level of the transcriptional coactivator OBF-1 .


The transcriptional coactivator OBF-1 , which interacts with Oct-1 and Oct-2 and the octamer site DNA , has been shown to be critical for development of a normal immune response and the formation of germinal centers in secondary lymphoid organs .


Here we have identified the RING finger protein Siah-1 as a protein interacting specifically with OBF-1 .


This interaction is mediated by the C-terminal part of Siah-1 and by residues in the N-terminus of OBF-1 , partly distinct from the residues required for formation of a complex with the Oct POU domains and the DNA .


Interaction between Siah-1 and OBF-1 leads to downregulation of OBF-1 protein level but not mRNA , and to a corresponding reduction in octamer site-dependent transcription activation .


Inhibition of the ubiquitin-proteasome pathway in B cells leads to elevated levels of OBF-1 protein .


Furthermore , in immunized mice , OBF-1 protein amounts are dramatically increased in primary activated B cells , without concomitant increase in OBF-1 mRNA .


These data suggest that Siah-1 is part of a novel regulatory loop controlling the level of OBF-1 protein in B cells .


The beta-catenin -- TCF-1 pathway ensures CD4 ( + ) CD8 ( + ) thymocyte survival .


The association of trans-acting T cell factors ( TCFs ) or lymphoid enhancer factor 1 ( LEF-1 ) with their coactivator beta-catenin mediates transient transcriptional responses to extracellular Wnt signals .


We show here that T cell maturation depends on the presence of the beta-catenin -- binding domain in TCF-1 .


This domain is necessary to mediate the survival of immature CD4 ( + ) CD8 ( + ) double-positive ( DP ) thymocytes .


Accelerated spontaneous thymocyte death in the absence of TCF-1 correlates with aberrantly low expression of the anti-apoptotic protein Bcl-x ( L ) .


Increasing anti-apoptotic effectors in thymocytes by the use of a Bcl-2 transgene rescued TCF-1-deficient DP thymocytes from apoptosis .


Thus , TCF-1 , upon association with beta-catenin , transiently ensures the survival of immature T cells , which enables them to generate and edit T cell receptor ( TCR ) alpha chains and attempt TCR -mediated positive selection .


TRAIL/Apo2L ligand selectively induces apoptosis and overcomes drug resistance in multiple myeloma : therapeutic applications .


Multiple myeloma ( MM ) remains incurable and novel treatments are urgently needed .


Preclinical in vitro and in vivo evaluations were performed to assess the potential therapeutic applications of human recombinant tumor necrosis factor ( TNF ) -related apoptosis-inducing ligand/Apo2 ligand ( TRAIL/Apo2L ) in MM .


TRAIL/Apo2L potently induced apoptosis of MM cells from patients and the majority of MM cell lines , including cells sensitive or resistant to dexamethasone ( Dex ) , doxorubicin ( Dox ) , melphalan , and mitoxantrone .


TRAIL/Apo2L also overcame the survival effect of interleukin 6 on MM cells and did not affect the survival of peripheral blood and bone marrow mononuclear cells and purified B cells from healthy donors .


The status of the TRAIL receptors ( assessed by immunoblotting and flow cytometry ) could not predict TRAIL sensitivity of MM cells .


The anti-MM activity of TRAIL/Apo2L was confirmed in nu/xid/bg mice xenografted with human MM cells ; TRAIL ( 500 microg intraperitoneally daily for 14 days ) was well tolerated and significantly suppressed the growth of plasmacytomas .


Dox up-regulated the expression of the TRAIL receptor death receptor 5 ( DR5 ) and synergistically enhanced the effect of TRAIL not only against MM cells sensitive to , but also against those resistant to , Dex- or Dox-induced apoptosis .


Nuclear factor ( NF ) -kappaB inhibitors , such as SN50 ( a cell-permeable inhibitor of the nuclear translocation and transcriptional activity of NF-kappaB ) or the proteasome inhibitor PS-341 , enhanced the proapoptotic activity of TRAIL/Apo2L against TRAIL-sensitive MM cells , whereas SN50 reversed the TRAIL resistance of ARH-77 and IM-9 MM cells .


Importantly , normal B lymphocytes were not sensitized to TRAIL by either Dox , SN50 , or PS-341 .


These preclinical studies suggest that TRAIL/Apo2L can overcome conventional drug resistance and provide the basis for clinical trials of TRAIL -based treatment regimens to improve outcome in patients with MM .


CD28 costimulation is required not only to induce IL-12 receptor but also to render janus kinases/STAT4 responsive to IL-12 stimulation in TCR-triggered T cells .


The activation of resting T cells for the acquisition of various functions depends on whether CD28 costimulatory signals are provided upon T cell receptor stimulation .


Here , we investigated how CD28 costimulation functions to allow TCR -triggered resting T cells to acquire IL-12 responsiveness .


When T cells are stimulated with low doses of anti-CD3 mAb , CD28 costimulation was required for the optimal levels of IL-12 receptor ( IL-12R ) expression .


However , stimulation of T cells with high doses of anti-CD3 alone induced comparable levels of IL-12R expression to those induced upon CD28 costimulation .


Nevertheless , there was a substantial difference in IL-12 responsiveness between these two groups of T cells : compared to anti-CD28-costimulated T cells , T cells that were not costimulated with anti-CD28 exhibited decreased levels of Janus kinases ( JAK ) JAK2/TYK2 and STAT4 phosphorylation and IFN-y production following IL-12 stimulation .


Importantly , STAT6 phosphorylation following IL-4 stimulation was not decreased in anti-CD28-uncostimulated T cells .


These resutls indicate that CD28 costimulation not only contributes to up-regulating IL-12R expression but is also required to render JAKs/STAT4 responsive to IL-12 stimulation .


Constitutively activated Akt-1 is vital for the survival of human monocyte-differentiated macrophages .


Role of Mcl-1 , independent of nuclear factor ( NF ) -kappaB , Bad , or caspase activation .


Recent data from mice deficient for phosphatase and tensin homologue deleted from chromosome 10 or src homology 2 domain-containing 5 ' inositol phosphatase , phosphatases that negatively regulate the phosphatidylinositol 3-kinase ( PI3K ) pathway , revealed an increased number of macrophages in these animals , suggesting an essential role for the PI3K pathway for macro-phage survival .


Here , we focused on the role of the PI3K-regulated serine/threonine kinase Akt-1 in modulating macrophage survival .


Akt-1 was constitutively activated in human macrophages and addition of the PI3K inhibitor , LY294002 , suppressed the activation of Akt-1 and induced cell death .


Furthermore , suppression of Akt-1 by inhibition of PI3K or a dominant negative ( DN ) Akt-1 resulted in loss of mitochondrial transmembrane potential , activation of caspases-9 and -3 , and DNA fragmentation .


The effects of PI3K inhibition were reversed by the ectopic expression of constitutively activated Akt-1 or Bcl-x ( L ) .


Inhibition of PI3K /Akt-1 pathway either by LY294002 or DN Akt-1 had no effect on the constitutive or inducible activation of nuclear factor ( NF ) -kappaB in human macrophages .


However , after inhibition of the PI3K / Akt-1 pathway , a marked decrease in the expression of the antiapoptotic molecule Mcl-1 , but not other Bcl-2 family members was observed , and Mcl-1 rescued macrophages from LY294002-induced cell death .


Further , inhibition of Mcl-1 by antisense oligonucleotides , also resulted in macrophage apoptosis .


Thus , our findings demonstrate that the constitutive activation of Akt-1 regulates macrophage survival through Mcl-1 , which is independent of caspases , NF-kappaB , or Bad .


GRbeta expression in nasal polyp inflammatory cells and its relationship to the anti-inflammatory effects of intranasal fluticasone .


BACKGROUND : Nasal polyposis disease is an inflammatory disorder with intense eosinophilic infiltration of respiratory mucosa that is often difficult to control with topical steroids .


Recent evidence suggests that overexpression of the glucocorticoid receptor splice variant GRbeta in inflammatory cells might contribute to steroid insensitivity in diseases such as asthma .


OBJECTIVE : The purposes of this investigation were to determine whether nasal polyp ( NP ) inflammatory cells overexpress GRbeta and to examine whether GRbeta overexpression is associated with insensitivity to the potent topical steroid fluticasone propionate ( FP ) .


METHODS : Biopsies were obtained from 10 subjects with NPs before and 4 weeks after treatment with intranasal FP .


Middle turbinates biopsies from 6 healthy , nonallergic subjects served as normal controls .


Biopsies were immunostained for inflammatory cell markers as well as GRbeta and probed for various cytokine mRNA .


The anti-inflammatory response to FP was examined in relation to pretreatment levels of GRbeta expression .


RESULTS : The total numbers of inflammatory cells were increased in NPs .


The percentage of inflammatory cells expressing GRbeta was also increased ( 40.5 % +/- 19.2 % vs 16.1 % +/- 4.0 % , P =.009 ) .


GRbeta expression in NPs was almost exclusive to T lymphocytes , eosinophils , and macrophages .


An inverse correlation was observed between the baseline inflammatory cell GRbeta expression and the reduction after FP treatment in EG2-positive eosinophils , CD4-positive T lymphocytes , endothelial VCAM-1 expression , and IL-4 mRNA-positive cells .


NPs that were `` FP-insensitive '' in terms of suppression of eosinophil numbers ( major basic protein -positive ) had a significantly greater percentage of GRbeta-positive inflammatory cells , a higher ratio of GRbeta-positive/GRalpha-positive cells , and increased numbers of GRbeta-positive eosinophils and macrophages in comparison with those that were `` FP-sensitive. ''


`` FP-insensitive '' NPs also demonstrated a higher percentage of IL-5-positive inflammatory cells expressing GRbeta before and after FP treatment .


CONCLUSION : GRbeta expression appears to be a marker of steroid insensitivity in NPs .


Expression of GRbeta by NP inflammatory cells , particularly T cells and eosinophils , might render them resistant to suppression by topical steroids and thereby contribute to persistent NP inflammation .


Transcriptional regulation of galectin-10 ( eosinophil Charcot-Leyden crystal protein ) : a GC box ( -44 to -50 ) controls butyric acid induction of gene expression .


Galectin-10 ( gal-10 , also known as Charcot-Leyden crystal protein ) is a member of the galectin family of beta-galactoside binding proteins that is expressed uniquely in eosinophilic and basophilic leukocytes .


To gain a better understanding of galectin gene expression , we present an analysis of the transcriptional regulation of the gene encoding gal-10 .


Analysis of the minimal promoter revealed nine consensus-binding sites for transcription factors , including several that are also found in the minimal promoters of galectins -1 , -2 , and -3 .


The decrease in gal-10 promoter activity after disruption of either the GC box ( -44 to -50 ) or the Oct site ( -255 to -261 ) suggests that these sites , along with the previously characterized GATA and EoTF sites , are necessary for full promoter activity .


By supershift analysis , we demonstrate binding of the transcription factors Sp1 and Oct1 to the consensus GC box and the Oct site , respectively .


Similar to gal-1 , gal-10 expression is induced by butyric acid , an effect that is lost upon ablation of the GC box .


Additionally , we demonstrate AML3 binding to the consensus AML site and YY1 binding to the Inr sequence , both elements functioning as silencers in the gal-10 promoter .


Role of the stroma in mammary development .


Mammary development depends on branching morphogenesis , namely the bifurcation and extension of ductal growth points ( end buds ) and secretory lobules into a more or less fatty stroma .


Because breast carcinomas are overwhelmingly ductal in origin , this review focuses on stromal influences guiding postnatal ductal development and there is only the briefest account of the role of embryonic stroma ( mesenchyme ) .


The stroma as the necessary target for endocrine mammogens and the source of stimulatory growth factors is described and the importance of mammary epithelium-induced modifications of the periductal stroma is emphasized .


Evidence is presented that if they are to grow , end buds must condition proximal fatty stroma by recruiting white blood cells as well as inducing stromal cell division and , possibly , estrogen receptors .


The induction of a fibrous stromal tunic around the end bud is described and its likely role as a complex ductal morphogen is discussed ; a possible role in growth inhibition is also considered .


Although the signals governing fibrotic induction , ductal morphogenesis , and growth inhibition are unknown , a role for transforming growth factor-beta is highly likely and is discussed .


Finally , a need for new conceptual and experimental approaches to understanding stromal-epithelial signaling is discussed .


Adipophilin is a sensitive marker for lipid loading in human blood monocytes .


Adipophilin , a marker of lipid accumulation initially described in adipocytes , was recently shown to be induced in macrophage foam cells .


We found that even freshly isolated blood monocytes express adipophilin and that the amount of adipophilin protein is variable in monocytes from different healthy individuals .


However , the physiological expression of adipophilin does not correlate with the levels of free fatty acids , cholesterylesters or free cholesterol .


Enzymatically modified low-density lipoprotein ( E-LDL ) induces rapid foam cell formation in monocytes and upregulates adipophilin mRNA and protein within 2 h of incubation .


This rapid induction of adipophilin is accompanied by a significant increase of free fatty acids in monocytes incubated with E-LDL .


Adipophilin facilitates the uptake of free fatty acids , and here we demonstrate that free fatty acids increase is related to the early upregulation of adipophilin expression in blood monocytes .


Fatty acids are ligands for peroxisome proliferator-activated receptor-gamma ( PPARgamma ) , and the upregulation of adipophilin mRNA by PPARgamma agonists like 15d-PGJ ( 2 ) and ciglitazone indicates that PPARgamma may mediate the induction of adipophilin expression in human blood monocytes .


Constitutive expression of MHC class II genes in melanoma cell lines results from the transcription of class II transactivator abnormally initiated from its B cell-specific promoter .


In melanoma cell lines , two different patterns of MHC class II expression have been described , either an IFN gamma-inducible expression of HLA-DR and HLA-DP , with a faint or null expression of HLA-DQ , resembling that described for melanocytes , or a constitutive expression , i.e. , IFN-gamma independent , of all three HLA-D isotypes .


As this latter phenotype has been associated with a more rapid progression of melanoma tumors , we have analyzed in different melanoma cell lines the molecular mechanisms leading to this abnormal pattern of MHC class II expression .


In agreement with the evidence of a coordinate transcription of the HLA-D genes in these cell lines , we have shown the constitutive expression of CIITA ( class II transactivator ) transcripts , CIITA being known as the master switch of MHC class II expression .


Unexpectedly , these transcripts initiate from promoter III of the CIITA gene , a promoter that is mainly used constitutively in B lymphocytes .


This expression was further shown to occur through factor ( s ) acting on the enhancer located upstream of CIITA promoter III , which was previously described in epithelioid cells as an IFN-gamma-response sequence .


The hypothesis of a general abnormality of the IFN-gamma transduction pathway was dismissed .


Constitutive transcription of CIITA from promoter III having been observed in unrelated melanoma cell lines , we propose the hypothesis that this phenomenon might not be a random event , but could be linked to the neoplasic state of the melanoma cells


Caspase -mediated calcineurin activation contributes to IL-2 release during T cell activation .


Calcineurin , a Ca ( 2+ ) /calmodulin-dependent Ser/Thr phosphatase ( protein phosphatase 2B ) , plays a critical role in IL-2 production during T cell activation .


It has been previously reported that IL-2 release in activated Jurkat T requires caspase-like activity ( Posmantur et al. ( 1998 ) Exp. Cell. Res. 244 , 302-309 ) .


We report here that the 60-kDa catalytic subunit of calcineurin A ( Cn A ) was partially cleaved to a 45-kDa form in phytohemagglutinin A ( PHA ) or phorbol ester + ionomycin ( P + I ) -activated Jurkat cells .


In parallel , proteolytic activation of upstream caspases ( caspase-8 and -9 ) as well as effector caspase-3 was also observed .


Cn A cleavage was caspase mediated , since it was inhibitable by pan-caspase inhibitor Cbz-Asp-CH ( 2 ) OC ( O ) -2 , 6-dichlorobenzene ( Z-D-DCB ) .


Cn A cleavage was also observed when purified calcineurin was digested in vitro with caspase-3 .


Truncated Cn A was associated with enhanced phosphatase activity and reduced calmodulin sensitivity .


Furthermore , in PHA or P + I-activated Jurkat cells , dephosphorylation of calcineurin substrate NFATc ( a transcription factor known to be involved in transactivation of the IL-2 gene ) , was also suppressed by Z-D-DCB .


Taken together , our results suggest that caspase-mediated cleavage of Cn A contributes to IL-2 production during T cell activation .


Maturation of human dendritic cells as sulfasalazine target .


AIM : Sulfasalazine , a nonsteroidal anti-inflammatory drug , is effective in treating some autoimmune diseases , but its mechanism of action is unclear .


To determine whether dendritic cells could be a possible target of the drug , we studied the effects of sulfasalazine and its metabolites , aminosalicylate and sulfapyridine , on in vitro maturation ( terminal differentiation ) of human myeloid dendritic cells .


METHODS : We prepared immature dendritic cells by incubating CD14-positive cells in the presence of granulocyte- macrophage colony-stimulating factor and interleukin ( IL ) -4 .


The cells were matured by addition of tumor necrosis factor ( TNF ) -a , IL-1 beta , and prostaglandin E2 in the presence of sulfasalazine or its metabolites -- aminosalicylate and sulfapyridine , or their combinations .


We quantified the effect of drugs on the dendritic cell characteristics , such as stimulation of autologous and allogeneic pan-T cell proliferation , surface marker phenotype , IL-12 p40 subunit secretion , and activation of nuclear transcription factor ( NF ) -kappa B .


RESULTS : Dendritic cells treated with sulfasalazine ( 1.25 micromol/L or 2.5 micromol/L ) could not stimulate T cells ( p < 0.028 , two-sided paired t-test ) .


In distinction to drug-free maturing dendritic cells , 2.5 micromol/L sulfasalazine upregulated the levels of CD14 and CD68 and downregulated the levels of CD40 , CD80 , and CD83 ( for all CD markers , p < 0.03 for difference between measurements in the absence and the presence of sulfasalazine ) .


From concentration-dependent changes in CD83 expression , we found an apparent ID50 > > 1.5 micromol/L sulfasalazine .


The apparent ID50 value for aminosalicylate-inhibited maturation was 4 micromol/L .


At 1.25 micromol/L , sulfasalazine largely inhibited NF-kB activation in dendritic cells .


CONCLUSION : Maturing human dendritic cells are hundred-fold more sensitive to sulfasalazine than T cells and NK cells and the most sensitive human cells described so far .


Thus , dendritic cell maturation is an important target of sulfasalazine .


Because of the role of dendritic cells in ( auto ) immunity , inhibition of their maturation might provide a target for further optimization of sulfasalazine therapy .


Defective function of the proteasome in autoimmunity : involvement of impaired NF-kappaB activation .


Type 1 diabetes ( also known as insulin-dependent diabetes mellitus or juvenile-onset diabetes ) is usually caused by T cell-mediated autoimmunity , with a prediabetic state characterized by the production of autoantibodies specific for proteins expressed by pancreatic beta cells .


The nonobese patient with diabetes ( NOD ) mouse is a spontaneous model of type 1 diabetes with a strong genetic component that maps to the major histocompatibility complex ( MHC ) region of the genome .


A specific proteasome defect has been identified in NOD mouse in select lymphocytic and monocytic lineages that results from down-regulation of expression of the proteasome subunit LMP2 , which is encoded by a gene in the MHC genomic region .


This defect prevents the proteolytic processing required for the production and activation of the transcription factor nuclear factor-kappaB ( NF-kappaB ) , which plays important roles in immune and inflammatory responses , as well as increases the susceptibility of the affected cells to apoptosis induced by tumor necrosis factor-alpha ( TNF-alpha ) .


The novel role of the proteasome in dysfunction in autoimmunity is presented and documented to be both tissue and developmental stage specific .


We propose a role of the proteasome as a step in disease pathogenesis and tissue targeting .


Down-regulation of TDT transcription in CD4 ( + ) CD8 ( + ) thymocytes by Ikaros proteins in direct competition with an Ets activator .


Ikaros is a unique regulator of lymphopoiesis that associates with pericentromeric heterochromatin and has been implicated in heritable gene inactivation .


Binding and competition experiments demonstrate that Ikaros dimers compete with an Ets activator for occupancy of the lymphocyte-specific TdT promoter .


Mutations that selectively disrupt Ikaros binding to an integrated TdT promoter had no effect on promoter function in a CD4 ( + ) CD8 ( + ) thymocyte line .


However , these mutations abolished down-regulation on differentiation , providing evidence that Ikaros plays a direct role in repression .


Reduced access to restriction enzyme cleavage suggested that chromatin alterations accompany down-regulation .


The Ikaros-dependent down-regulation event and the observed chromatin alterations appear to precede pericentromeric repositioning .


Current models propose that the functions of Ikaros should be disrupted by a small isoform that retains the dimerization domain and lacks the DNA-binding domain .


Surprisingly , in the CD4 ( + ) CD8 ( + ) thymocyte line , overexpression of a small Ikaros isoform had no effect on differentiation or on the pericentromeric targeting and DNA-binding properties of Ikaros .


Rather , the small isoform assembled into multimeric complexes with DNA-bound Ikaros at the pericentromeric foci .


The capacity for in vivo multimer formation suggests that interactions between Ikaros dimers bound to the TdT promoter and those bound to pericentromeric repeat sequences may contribute to the pericentromeric repositioning of the inactive gene .


Type I interferons and IL-12 : convergence and cross-regulation among mediators of cellular immunity .


Therapeutic use of type I IFN ( IFN-alpha/beta ) has become common .


Many of the diverse diseases targeted are marked by pathogenetic abnormalities in cell-mediated immunity ( CMI ) , these cellular immune responses either causing injury to the host , lacking sufficient vigor for virus or tumor clearance , or both .


In general , therapeutic efficacy is limited .


It is thus notable that the pleiotropic effects of type I IFN on CMI remain poorly understood .


We characterized the effects of type I IFN on the production of IL-12 , the central immunoregulatory cytokine of the CD4 ( + ) T cell arm of CMI .


We show that type I IFN are potent inhibitors of IL-12 production by human monocytes/macrophages .


The underlying mechanism involves transcriptional inhibition of the IL-12p40 gene , marked by down-regulation of PU.1 binding activity at the upstream Ets site of the IL-12p40 promoter .


Type I IFN have previously been shown to be able to substitute for IL-12 in driving IFN-gamma production from T and NK cells .


The ability of IFN-alpha/beta to suppress IL-12 production while up-regulating IFN-gamma production suggests a possible mechanistic basis for the difficulties of employing these cytokines in diseases involving abnormalities of CMI .


Nuclear factor-kappaB suppressive and inhibitor-kappaB stimulatory effects of troglitazone in obese patients with type 2 diabetes : evidence of an antiinflammatory action ?


It has been shown recently that troglitazone exerts an anti-inflammatory effect , in vitro , and in experimental animals .


To test these properties in humans , we investigated the effect of troglitazone on the proinflammatory transcription factor nuclear factor-kappaB and its inhibitory protein IkappaB in mononuclear cells ( MNC ) and plasma soluble intracellular adhesion molecule-1 , monocyte chemoattractant protein-1 , plasminogen activator inhibitor-1 , and C-reactive protein .


We also examined the effect of troglitazone on reactive oxygen species generation , p47 ( phox ) subunit expression , 9-hydroxyoctadecadienoic acid ( 9-HODE ) , 13-HODE , o-tyrosine , and m-tyrosine in obese patients with type 2 diabetes .


Seven obese patients with type 2 diabetes were treated with troglitazone ( 400 mg/day ) for 4 weeks .


Blood samples were obtained at weekly intervals .


Nuclear factor-kappaB binding activity in MNC nuclear extracts was significantly inhibited after troglitazone treatment at week 1 and continued to be inhibited up to week 4 .


On the other hand , IkappaB protein levels increased significantly after troglitazone treatment at week 1 , and this increase persisted throughout the study .


Plasma monocyte chemoattractant protein-1 and soluble intracellular adhesion molecule-1 concentrations did not decrease significantly after troglitazone treatment , although there was a trend toward inhibition .


Reactive oxygen species generation by polymorphonuclear cells and MNC , p47 ( phox ) subunit protein quantities , plasminogen activator inhibitor-1 , and C-reactive protein levels decreased significantly after troglitazone intake .


13-HODE/linoleic acid and 9-HODE/linoleic acid ratios also decreased after troglitazone intake .


However , o-tyrosine/phenylalanine and m-tyrosine/phenylalanine ratios did not change significantly .


These data show that troglitazone has profound antiinflammatory effects in addition to antioxidant effects in obese type 2 diabetics ; these effects may be relevant to the recently described beneficial antiatherosclerotic effects of troglitazone at the vascular level .


OX40 stimulation by gp34/OX40 ligand enhances productive human immunodeficiency virus type 1 infection .


OX40 is a member of the tumor necrosis factor ( TNF ) receptor superfamily and known to be an important costimulatory molecule expressed on activated T cells .


To investigate the role of costimulation of OX40 in human immunodeficiency virus type 1 ( HIV-1 ) infection by its natural ligand , gp34 , the OX40-transfected ACH-2 cell line , ACH-2/OX40 , chronically infected with HIV-1 , was cocultured with paraformaldehyde ( PFA ) -fixed gp34-transfected mouse cell line , SV-T2/gp34 .


The results showed that HIV-1 production was strongly induced .


This was followed by apparent apoptosis , and both processes were specifically inhibited by the gp34 -specific neutralizing monoclonal antibody 5A8 .


Endogenous TNF alpha ( TNF-alpha ) and TNF-beta production were not involved in the enhanced HIV-1 production .


Furthermore , enhanced HIV-1 transcription in gp34 -stimulated ACH-2/OX40 cells was dependent on the kappa B site of the HIV-1 long terminal repeat , and the OX40-gp34 interaction activated NF-kappa B consisting of p50 and p65 subunits .


When primary activated CD4 ( + ) T cells acutely infected with HIV-1 ( NL4-3 ) ( CXCR4-using T-cell-line-tropic ) were cocultured with PFA-fixed gp34 ( + ) human T-cell leukemia virus type 1-bearing MT-2 cells or SV-T2/gp34 cells , HIV-1 production was also markedly enhanced .


The enhancement was again significantly inhibited by 5A8 .


The present study first shows that OX40-gp34 interaction stimulates HIV-1 expression and suggests that OX40 triggering by gp34 may play an important role in enhancing HIV-1 production in both acutely and latently infected CD4 ( + ) T cells in vivo .


Troglitazone , a PPARgamma ligand , inhibits osteopontin gene expression in human monocytes/macrophage THP-1 cells .


Peroxizome proliferator-activated receptor-gamma ( PPARgamma ) is a member of the nuclear receptor family of transcription factors that regulate adipocyte differentiation .


Recent studies indicate that liganded PPARgamma not only promotes differentiation but also inhibits the activation of macrophages .


Osteopontin , a component of extracellular matrix , is synthesized by macrophages in atherosclerotic plaques .


In this study , we examined whether PPARgamma ligand regulates osteopontin gene expression in THP-1 cells , a cell line derived from human monocytic leukemia cells which can differentiate to macrophage upon stimulation with phorbol ester PMA .


Northern blot analysis showed that osteopontin expression is markedly induced in response to PMA .


Troglitazone , a PPARgamma ligand , dramatically attenuated the PMA-induced osteopontin expression .