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 .


Non-Hodgkin 's lymphoma involving bilateral breasts [ see comments ]


We describe here two cases of diffuse large cell type non-Hodgkin 's lymphoma affecting the bilateral breasts .


The contralateral tumor in one case appeared 17 months after the first mastectomy , whereas the bilateral tumors occurred concurrently in the other patient who was pregnant and showed widespread dissemination at initial presentation .


Lymphoma cells from both cases showed the mature B-cell immunophenotype and had rearrangements of the BCL6 gene .


Both patients developed progressive disease despite chemo-radiotherapy and died of leukemic manifestations .


There were no apparent pathological features of lymphomas of mucosa-associated lymphoid tissue origin


Biochemical characterization of MIP-1 alpha nuclear protein .


A family of hematopoietic specific transcription factors , MIP-1 alpha nuclear protein ( MNP ) family , has recently been identified .


They are intimately involved in regulating the transcription of the huMIP-1 alpha gene in monocytes , T-cells , and transformed B-cells .


One member of the family ( MNP-1 ) is essential for promoter activity in monocytes and B-cells , while another ( MNP-2 ) is required for full promotor activity in T-cells .


A third member of the family ( MNP-3 ) is expressed in PMA induced HL60 cells and probably has a role in monocyte differentiation .


In this communication we demonstrate by two techniques that MNP-1 and MNP-2 are distinct but related factors , and we present further evidence to show that MNP-1 acts as a heterodimer .


Bcl-3 expression and nuclear translocation are induced by granulocyte-macrophage colony-stimulating factor and erythropoietin in proliferating human erythroid precursors .


Bcl-3 is a proto-oncogene involved in the chromosomal translocation t ( 14 ; 19 ) found in some patients with chronic lymphocytic leukemia .


It shares structural similarities with and is a member of the IkappaB family of proteins .


In this report , involvement of Bcl-3 in hematopoietic growth factor-stimulated erythroid proliferation and differentiation was examined .


In TF-1 cells , an erythroleukemia cell line , granulocyte-macrophage colony-stimulating factor ( GM-CSF ) and erythropoietin ( Epo ) greatly enhanced Bcl-3 expression at both the protein and mRNA levels in association with stimulation of proliferation .


Bcl-3 protein was also highly expressed in early burst-forming unit-erythroid ( BFU-E ) -derived erythroid precursors ( day 7 ) and decreased during maturation ( days 10 and 14 ) , suggesting that Bcl-3 is involved in normal erythroid proliferation .


In these hematopoietic cells , Bcl-3 was hyperphosphorylated .


GM-CSF and Epo modulated the subcellular localization of Bcl-3 .


Upon stimulation of TF-1 cells with GM-CSF or Epo , the nuclear translocation of Bcl-3 was dramatically enhanced .


Overexpression of Bcl-3 in TF-1 cells by transient transfection along with the NF-kappaB factors p50 or p52 resulted in significant induction of an human immunodeficiency virus-type 1 ( HIV-1 ) kappaB-TATA-luceriferase reporter plasmid , demonstrating that Bcl-3 has a positive role in transactivation of kappaB-containing genes in erythroid cells .


Stimulation with GM-CSF enhanced c-myb mRNA expression in these cells .


Bcl-3 in nuclear extracts of TF-1 cells bound to a kappaB enhancer in the c-myb promoter together with NF-kappaB2/p52 and this binding activity was enhanced by GM-CSF stimulation .


Furthermore , cotransfection of Bcl-3 with p52 or p50 in TF-1 cells resulted in significant activation of a c-myb kappaB-TATA-luceriferase reporter plasmid .


These findings suggest that Bcl-3 may participate in the transcriptional regulation of certain kappaB-containing genes involved in hematopoiesis , including c-myb .


Copyright 1998 by The American Society of Hematology .


5-Lipoxygenase compartmentalization in granulocytic cells is modulated by an internal bipartite nuclear localizing sequence and nuclear factor kappa B complex formation .


A region of basic amino acids spanning residues 639-656 in the human 5-lipoxygenase sequence resembles a consensus bipartite nuclear localizing sequence .


A synthetic peptide consisting of the Kaposi fibroblast growth factor signal sequence fused to the 5-lipoxygenase639-656 bipartite nuclear localizing sequence has a prominent inhibitory effect on 5-lipoxygenase catalysis in granulocytic HL-60 cells activated by calcium ionophor A23187 .


Recombinant 5-lipoxygenase was not affected by the peptide .


The peptide also inhibited redistribution of 5-lipoxygenase from the cytosol to the nuclear membrane of HL-60 cells stimulated by A23187 .


5-Lipoxygenase protein was detected in nuclear factor kappaB ( NF-kappaB ) p65 subunit immunoprecipitate fractions prepared from HL-60 cell lysates .


The amount of 5-lipoxygenase protein coimmunoprecipitated by NF-kappaB antiserum was increased following A23187 stimulation .


In cells treated with agents that block 5-lipoxygenase translocation to the nucleus , 5-lipoxygenase protein appearing in the NF-kappaB immunoprecipitate was diminished .


Our results implicate an internal bipartite nuclear localizing sequence as a regulatory domain that modulates 5-lipoxygenase redistribution and catalysis in granulocytic cells .


Additionally , our results suggest that molecular determinants which govern 5-lipoxygenase and NF-kappaB redistribution to the nucleus may be coordinately controlled in granulocytic cells .


Inhibition of CD28/CD3-mediated costimulation of naive and memory human T lymphocytes by intracellular incorporation of polyclonal antibodies specific for the activator protein-1 transcriptional complex .


A number of indirect methods have been utilized in demonstrating activator protein-1 transcription factor function in IL-2 promoter activity .


However , there has been no direct demonstration that activator protein-1 is involved in CD28-dependent costimulation of IL-2 gene transcription in freshly isolated naive and memory human T lymphocytes .


To address this issue , the method of scrape loading was applied to purified peripheral blood T lymphocytes .


Since scrape loading relies on adherent cells , peripheral blood human T ( PB-T ) cells were immobilized on the nonspecific cell attachment factor poly-L-lysine .


Cells scraped off poly-L-lysine in the presence of Ig FITC efficiently incorporated Ig , with relatively uniform fluorescence .


T cells retained their physical parameters as measured by forward and side light scatter , and functional activity as measured by costimulation of proliferation and IL-2 production after being scraped off this substrate .


CD28/CD3-costimulated T cells produced intracellular IL-2 from all subsets measured ( CD4+ , CD4- , CD45RO+ , and CD45RO- ) .


IL-2 production and intracellular accumulation in nonscraped PB-T cells activated with CD28/CD3 coligation were skewed favoring CD45RO+ and CD4+ subsets , as was IL-2 production in scraped PB-T cells .


The intracellular incorporation of Abs specific for c-Fos and c-Jun family members by scrape loading inhibited the production and intracellular accumulation of IL-2 within 6 h of costimulation with PMA/ionomycin , or costimulation by CD28 and CD3 ligation .


Scrape loading thus provides an efficient mechanism for intracellular incorporation of macromolecules , and the first direct evidence that c-Fos and c-Jun are involved in transcription of the IL-2 gene within its correct chromosomal context , in resting human T lymphocyte subpopulations .


Differential expression of Nur77 family members in human T-lymphotropic virus type 1-infected cells : transactivation of the TR3/nur77 gene by Tax protein .


We analyzed the differential expression and regulation of three members of the Nur77 transcription factor family by the human T-lymphotropic virus type 1 ( HTLV-1 ) Tax protein .


We have demonstrated that in both HTLV-1-infected cells and Tax-expressing JPX-9 cells , TR3/nur77 is highly expressed , whereas neither NOR-1 nor NOT expression is detectable .


Transient transfection analysis further confirmed the Tax transactivation of the TR3/nur77 promoter but not the NOR-1 promoter in different cell types .


Furthermore , expression of a luciferase reporter gene driven by the NGFI-B ( rat homolog of TR3/Nur77 ) response element ( NBRE ) provided evidence that Tax -mediated transactivation resulted in the induction of a functional protein .


Cotransfection assays with the TR3/nur77 promoter sequence or the NBRE binding motif together with a series of Tax mutants have shown that Tax -induced TR3/nur77 expression is mediated by CREB/ATF-related transcription factors .


Negative regulation of the heat shock transcriptional response by HSBP1 .


In response to stress , heat shock factor 1 ( HSF1 ) acquires rapid DNA binding and transient transcriptional activity while undergoing conformational transition from an inert non-DNA-binding monomer to active functional trimers .


Attenuation of the inducible transcriptional response occurs during heat shock or upon recovery at non-stress conditions and involves dissociation of the HSF1 trimer and loss of activity .


We have used the hydrophobic repeats of the HSF1 trimerization domain in the yeast two-hybrid protein interaction assay to identify heat shock factor binding protein 1 ( HSBP1 ) , a novel , conserved , 76-amino-acid protein that contains two extended arrays of hydrophobic repeats that interact with the HSF1 heptad repeats .


HSBP1 is nuclear-localized and interacts in vivo with the active trimeric state of HSF1 that appears during heat shock .


During attenuation of HSF1 to the inert monomer , HSBP1 associates with Hsp70 .


HSBP1 negatively affects HSF1 DNA-binding activity , and overexpression of HSBP1 in mammalian cells represses the transactivation activity of HSF1 .


To establish a biological role for HSBP1 , the homologous Caenorhabditis elegans protein was overexpressed in body wall muscle cells and was shown to block activation of the heat shock response from a heat shock promoter-reporter construct .


Alteration in the level of HSBP1 expression in C. elegans has severe effects on survival of the animals after thermal and chemical stress , consistent with a role for HSBP1 as a negative regulator of the heat shock response .


Long-range transcriptional regulation of cytokine gene expression .


Most studies on the control of cytokine gene expression have involved the functional analysis of proximal promoters .


Recent work has identified distal elements that mediate long-range cytokine gene regulation and has implicated chromatin reorganization in regulation of cytokine gene loci .


These studies have begun to elucidate the basis for cell-specificity and high-level expression of cytokine genes .


A signaling complex of Ca2+-calmodulin-dependent protein kinase IV and protein phosphatase 2A [ see comments ]


Stimulation of T lymphocytes results in a rapid increase in intracellular calcium concentration ( [ Ca2+ ] i ) that parallels the activation of Ca2+-calmodulin-dependent protein kinase IV ( CaMKIV ) , a nuclear enzyme that can phosphorylate and activate the cyclic adenosine monophosphate ( cAMP ) response element-binding protein ( CREB ) .


However , inactivation of CaMKIV occurs despite the sustained increase in [ Ca2+ ] i that is required for T cell activation .


A stable and stoichiometric complex of CaMKIV with protein serine-threonine phosphatase 2A ( PP2A ) was identified in which PP2A dephosphorylates CaMKIV and functions as a negative regulator of CaMKIV signaling .


In Jurkat T cells , inhibition of PP2A activity by small t antigen enhanced activation of CREB -mediated transcription by CaMKIV .


These findings reveal an intracellular signaling mechanism whereby a protein serine-threonine kinase ( CaMKIV ) is regulated by a tightly associated protein serine-threonine phosphatase ( PP2A ) .


Hypoxia down-regulates MCP-1 expression : implications for macrophage distribution in tumors .


Monocyte chemoattractant protein 1 ( MCP-1 ) is likely to contribute to the macrophage infiltrate in human ovarian carcinomas .


Although MCP-1 is predominantly expressed by the tumor parenchyma , macrophages accumulate at highest density in necrotic regions , which are associated with low oxygen tensions .


Tumor necrosis factor alpha ( TNF-alpha ) can stimulate MCP-1 production and is also present within ovarian carcinomas .


We have investigated the effect of hypoxia both on MCP-1 expression in ovarian cancer cell lines and monocyte migration .


Hypoxia down-regulated TNF-alpha-induced MCP-1 mRNA and protein production by ovarian cancer cells .


The effect was mimicked by cobalt chloride and desferrioxamine , consistent with a specific oxygen-sensing mechanism .


Unlike antioxidants , hypoxia did not inhibit nuclear factor KB mobilization .


Monocyte migration in response to MCP-1 was also diminished under hypoxic conditions .


Down-regulation of MCP-1 expression and the inhibition of monocyte migration are independent effects of hypoxia that may contribute to the distribution of macrophages within ovarian tumors .


Activation of nuclear factor kappa B inflammatory bowel disease [ see comments ]


BACKGROUND : Expression of pro-inflammatory cytokines is increased in the intestinal lamina propria of patients with inflammatory bowel disease ( IBD ) .


Nuclear factor kappa B ( NF kappa B ) controls transcription of inflammation genes .


On activation , NF kappa B is rapidly released from its cytoplasmic inhibitor ( I kappa B ) , transmigrates into the nucleus , and binds to DNA response elements in gene promoter regions .


AIMS : To investigate whether increased activation of NF kappa B is important in IBD and may be down-regulated by anti-inflammatory treatment .


METHODS : Activation of NF kappa B was determined by western blot assessment and electrophoretic mobility shift assay in nuclear extracts of colonic biopsy samples as well as lamina propria mononuclear cells .


RESULTS : Nuclear levels of NF kappa B p65 are increased in lamina propria biopsy specimens from patients with Crohn 's disease in comparison with patients with ulcerative colitis and controls .


Increased activation of NF kappa B was detected in lamina propria mononuclear cells from patients with active IBD .


Corticosteroids strongly inhibit intestinal NF kappa B activation in IBD in vivo and in vitro by stabilising the cytosolic inhibitor I kappa B alpha against activation induced degradation .


CONCLUSIONS : In both IBDs , but particularly Crohn 's disease , increased activation of NF kappa B may be involved in the regulation of the inflammatory response .


Inhibition of NF kappa B activation may represent a mechanism by which steroids exert an anti-inflammatory effect in IBD


Insufficient glycemic control increases nuclear factor-kappa B binding activity in peripheral blood mononuclear cells isolated from patients with type 1 diabetes .


OBJECTIVE : The redox-sensitive transcription factor nuclear factor-kappa B ( NF-kappa B ) is believed to contribute to late diabetic complications .


It is unknown whether NF-kappa B is influenced by glycemic control .


RESEARCH DESIGN AND METHODS : To determine whether NF-kappa B is activated in patients with insufficient glycemic control ( HbA1c > 10 % ) , we developed a tissue culture-independent electrophoretic mobility shift assay ( EMSA ) -based semiquantitative detection system that allowed us to determine NF-kappa B activation in ex vivo-isolated peripheral blood mononuclear cells ( PBMCs ) .


We included 43 patients with type 1 diabetes in this cross-sectional study .


10 of those received the antioxidant thioctic acid ( 600 mg/day p.o. ) for 2 weeks .


RESULTS : Monocytes of patients with HbA1c levels > 10 % demonstrated significantly higher NF-kappa B binding activity in an EMSA and a stronger NF-kappa B staining in immunohistochemistry than monocytes of patients with HbA1c levels of 6-8 % .


The increase in NF-kappa B activation correlated with an increase in plasmatic markers of lipid peroxidation .


Treatment with the antioxidant thioctic acid decreased NF-kappa B binding activity .


CONCLUSIONS : Hyperglycemia induces activation of the transcription factor NF-kappa B in ex vivo-isolated PBMCs of patients with type 1 diabetes .


NF-kappa B activation is at least partially dependent on oxidative stress , since the antioxidant thioctic acid significantly lowered the extent of NF-kappa B binding activity .


Synergistic activation of MAP kinase ( ERK1/2 ) by erythropoietin and stem cell factor is essential for expanded erythropoiesis .


Stem cell factor ( SCF ) and erythropoietin ( EPO ) work synergistically to support erythropoiesis , but the mechanism for this synergism is unknown .


By using purified human erythroid colony-forming cells ( ECFC ) , we have found that SCF and EPO synergistically activate MAP kinase ( MAPK , ERK1/2 ) , which correlates with the cell growth and thus may be responsible for the synergistic effects .


Treatment of the cells with PD98059 and wortmannin , inhibitors of MEK and PI-3 kinase , respectively , inhibited the synergistic activation of MAPK and also the cell growth , further supporting this conclusion .


Wortmannin only inhibits MAPK activation induced by EPO but not that by SCF , suggesting that SCF and EPO may activate MAPK through different pathways , which would facilitate synergy .


Furthermore , EPO , but not SCF , led to activation of STAT5 , whereas SCF and wortmannin had no effect on the EPO -induced STAT5 activation , suggesting that STAT5 is not involved in the synergistic action of SCF and EPO .


Together , the data suggest that synergistic activation of MAPK by SCF and EPO is essential for expanded erythropoiesis .


Copyright 1998 by The American Society of Hematology .


The molecular and phenotypic profile of primary central nervous system lymphoma identifies distinct categories of the disease and is consistent with histogenetic derivation from germinal center-related B cells .


Primary central nervous system lymphoma ( PCNSL ) is a major cause of morbidity and mortality among human immunodeficiency virus ( HIV ) -infected individuals .


The precise histogenetic derivation and the molecular pathogenesis of PCNSL is poorly understood .


In an attempt to clarify the histogenesis and pathogenesis of these lymphomas , 49 PCNSL ( 26 acquired immunodeficiency syndrome [ AIDS ] -related and 23 AIDS-unrelated ) were analyzed for multiple biologic markers , which are known to bear histogenetic and pathogenetic significance for mature B-cell neoplasms .


PCNSL associated frequently ( 50.0 % ) with mutations of BCL-6 5 ' noncoding regions , which are regarded as a marker of B-cell transition through the germinal center ( GC ) .


Expression of BCL-6 protein , which is restricted to GC B cells throughout physiologic B-cell maturation , was detected in 100 % AIDS-unrelated PCNSL and in 56.2 % AIDS-related cases .


Notably , among AIDS-related PCNSL , expression of BCL-6 was mutually exclusive with expression of Epstein-Barr virus ( EBV ) -encoded latent membrane protein ( LMP ) -1 and , with few exceptions , also of BCL-2 .


All but one PCNSL expressed hMSH2 , which among mature B cells selectively stains GC B cells .


These data suggest that PCNSL may be frequently related to GC B cells and may be segregated into two major biologic categories based on the expression pattern of BCL-6 , LMP-1 , and BCL-2 .


BCL-6 ( + ) / LMP-1 ( - ) / BCL-2 ( - ) PCNSL occur both in the presence and in the absence of HIV infection and consistently display a large noncleaved cell morphology .


Conversely , BCL-6 ( - ) / LMP-1 ( + ) / BCL-2 ( + ) PCNSL are restricted to HIV-infected hosts and are represented by lymphomas with immunoblastic features .


These data are relevant for the pathogenesis and histogenesis of PCNSL and may be helpful to segregate distinct biologic and prognostic categories of these lymphomas .


Antioxidant regulation of phorbol ester-induced adhesion of human Jurkat T-cells to endothelial cells .


Regulation of adhesion molecule expression and function by reactive oxygen species via specific redox sensitive mechanisms have been reported .


The effects of clinically safe antioxidants in the regulation of adhesion molecule expression in human endothelial cells ( ECV ) , and adherence of human Jurkat T cells to ECV cells were investigated .


The thiol antioxidant , alpha-lipoate , at clinically relevant doses down-regulated phorbol 12-myristate 13-acetate ( PMA ) -induced adhesion molecule expression and cell-cell adhesion .


Inhibition of PMA-induced ICAM-1 and VCAM-1 expression as well as PMA-induced adhesion of Jurkat T-cells to ECV cells by alpha-lipoate was dose dependent ( 50-250 microM ) .


The effect was significant for ICAM-1 ( p < .01 ) and VCAM-1 ( p < .01 ) expression in cells pretreated with 100 microM alpha-lipoate compared to PMA-activated untreated cells .


Inhibition of PMA-induced adhesion molecule expression and cell-cell adhesion was more pronounced when a combination of antioxidants , alpha-lipoate and alpha-tocopherol , were used compared to the use of either of these antioxidant alone .


The regulation of adhesion molecule expression and function by low concentration of antioxidants investigated does not appear to be NF-kappaB regulated or transcription dependent because no change in the mRNA response was observed .


Protein kinase C ( PKC ) has been suggested to regulate PMA-induced adhesion molecule expression by post-transcriptional stabilization of adhesion molecule mRNA .


Alpha-lipoate pretreatment did not influence the response of PKC activity to PMA .


Oxidants are known to be involved in the regulation of cell adhesion processes .


Treatment of ECV cells with PMA induced generation of intracellular oxidants .


Alpha-lipoate ( 100 or 250 microM ) treatment decreased PMA-induced generation of intracellular oxidants .


The inhibitory effect of low concentration of alpha-lipaote alone or in combination with alpha-tocopherol on agonist-induced adhesion processes observed in this study may be of potential therapeutic value .


Binding of human immunodeficiency virus type 1 to CD4 and CXCR4 receptors differentially regulates expression of inflammatory genes and activates the MEK /ERK signaling pathway .


We have previously shown that binding of human immunodeficiency virus type 1 ( HIV-1 ) virions to CD4 receptors stimulates association of Lck with Raf-1 and results in the activation of Raf-1 kinase in a Ras-independent manner .


In the present study , we demonstrate that HIV-1 envelope glycoproteins of both T-cell-tropic and macrophagetropic strains rapidly activate the ERK/mitogen-activated protein ( MAP ) kinase pathway and the binding of nuclear transcription factors ( AP-1 , NF-kappaB , and C/EBP ) and stimulate expression of cytokine and chemokine genes .


The activation of this signaling pathway requires functional CD4 receptors and is independent of binding to CXCR4 .


Binding of the natural ligand stromal cell-derived factor 1 ( SDF-1 ) to CXCR4 , which inhibits entry of T-cell-tropic HIV-1 , activates also the ERK/ MAP kinase pathway .


However , SDF-1 did not affect the CD4-mediated expression of cytokine and chemokine genes .


These results provide firm molecular evidence that binding of HIV-1 envelope glycoproteins to CD4 receptor initiates a signaling pathway ( s ) independent of the binding to the chemokine receptor that leads to the aberrant expression of inflammatory genes and may contribute significantly to HIV-1 replication as well as to deregulation of the immune system .


Antigen receptor signaling induces MAP kinase-mediated phosphorylation and degradation of the BCL-6 transcription factor .


The bcl-6 proto-oncogene encodes a POZ/zinc finger transcriptional repressor expressed in germinal center ( GC ) B and T cells and required for GC formation and antibody affinity maturation .


Deregulation of bcl-6 expression by chromosomal rearrangements and point mutations of the bcl-6 promoter region are implicated in the pathogenesis of B-cell lymphoma .


The signals regulating bcl-6 expression are not known .


Here we show that antigen receptor activation leads to BCL-6 phosphorylation by mitogen-activated protein kinase ( MAPK ) .


Phosphorylation , in turn , targets BCL-6 for rapid degradation by the ubiquitin / proteasome pathway .


These findings indicate that BCL-6 expression is directly controlled by the antigen receptor via MAPK activation .


This signaling pathway may be crucial for the control of B-cell differentiation and antibody response and has implications for the regulation of other POZ/zinc finger transcription factors in other tissues .


Glucocorticoid receptors are differentially expressed in the cells and tissues of the immune system .


Cytosolic glucocorticoid receptor ( GR ) binding studies on immune tissues demonstrate that the thymus exhibits three to four times higher levels of GR protein than the spleen .


High levels of GR are consistent with the exquisite sensitivity of the thymus to glucocorticoid exposure .


Nevertheless , whole cell binding studies reveal similar levels of GR in immature thymic T lymphocytes and more mature , splenic T lymphocytes .


Moreover , whole cell binding techniques indicate that neutrophils ( which represent roughly 30 % of splenic leukocytes ) exhibit higher GR than both T and B lymphocytes , further contradicting results from cytosolic binding studies .


To address these inconsistencies , GR protein was assessed in immune cells and tissues using cytosolic radioligand binding .


Western blot analysis , and immunocytochemistry .


Consistent with previous cytosolic receptor binding studies on immune tissue homogenates , thymic T cells were found to have higher levels of GR than T cells isolated from the spleen .


In addition , neutrophils were found to have fewer GR than lymphocytes and monocytes .


These results indicate a meaningful relationship between receptor expression and known sensitivity to glucocorticoids .


Functional replacement of the mouse E2A gene with a human HEB cDNA .


The mammalian E2A , HEB , and E2-2 genes encode a unique class of basic helix-loop-helix ( bHLH ) transcription factors that are evolutionarily conserved and essential for embryonic and postnatal development .


While the structural and functional similarities among the gene products are well demonstrated , it is not clear why deletion of E2A , but not HEB or E2-2 , leads to a complete arrest in B-lymphocyte development .


To understand the molecular basis of the functional specificity between E2A and HEB / E2-2 in mammalian development , we generated and tested a panel of E2A knockin mutations including subtle mutations in the E12 and E47 exons and substitution of both E12 and E47 exons with a human HEB cDNA .


We find that the alternatively spliced E12 and E47 bHLH proteins of the E2A gene play similar and additive roles in supporting B lymphopoiesis .


Further , we find that HEB driven by the endogenous E2A promoter can functionally replace E2A in supporting B-cell commitment and differentiation toward completion .


Finally , the postnatal lethality associated with E2A disruption is fully rescued by the addition of HEB .


This study suggests that the functional divergence among E12 , E47 , and HEB in different cell types is partially defined by the context of gene expression .


Biased dependency of CD80 versus CD86 in the induction of transcription factors regulating the human IL-2 promoter .


In addition to the signals obtained by ligation of the TCR , T cells need additional , co-stimulatory signals to be activated .


One such co-stimulatory signal is delivered when CD28 on T cells binds to CD80 or CD86 on antigen-presenting cells ( APC ) .


In the present study , we analyzed the ability of CD80 and CD86 to co-stimulate human T cells activated by superantigen .


Using the Raji B cell lymphoma , which express similar levels of CD80 and CD86 , it was found that T cell proliferation was mainly co-stimulated by CD80 .


To further characterize the consequences of this biased co-stimulatory dependency , we employed a well-defined system of transfected CHO cells expressing human MHC class II together with CD80 , CD86 or CD80 and CD86 .


Proliferation of freshly prepared CD4+ T cells required the presence of either CD80 or CD86 .


However , IL-2 production reached only suboptimal levels in the presence of CD86 but optimal levels with CD80 .


To analyze IL-2 transcriptional activity in CD80 and CD86 co-stimulated T cells we used Jurkat T cells transfected with luciferase reporter gene constructs .


CD80 induced higher levels of IL-2 promoter -enhancer activity compared to CD86 .


Furthermore , the activity of transcription factors regulating the IL-2 promoter-enhancer region including activation protein-1 , CD28 response element and nuclear factor kappaB were 4-8 times higher after CD80 compared to CD86 ligation .


Our results suggest that the eventual appearance of CD80 on recently activated CD86+ APC is important for the superinduction of IL-2 production and to support vigorous T cell proliferation .


Duplication of the DR3 gene on human chromosome 1p36 and its deletion in human neuroblastoma .


The human DR3 gene , whose product is also known as Wsl-1/APO-3/TRAMP/LARD , encodes a tumor necrosis factor-related receptor that is expressed primarily on the surface of thymocytes and lymphocytes .


DR3 is capable of inducing both NF-kappa B activation and apoptosis when overexpressed in mammalian cells , although its ligand has not yet been identified .


We report here that the DR3 gene locus is tandemly duplicated on human chromosome band 1p36.2-p36.3 and that these genes are hemizygously deleted and/or translocated to another chromosome in neuroblastoma ( NB ) cell lines with amplified MYCN .


Duplication of at least a portion of the DR3 gene , including the extracellular and transmembrane regions but not the cytoplasmic domain , was demonstrated by both fluorescence in situ hybridization and genomic Southern blotting .


In most NB cell lines , both the DR3 and the DR3L sequences are simultaneously deleted and/or translocated to another chromosome .


Finally , DR3/ Wsl-1 protein expression is quite variable among these NB cell lines , with very low or undetectable levels in 7 of 17 NB cell lines


Differential protection of normal and malignant human myeloid progenitors ( CFU-GM ) from Ara-C toxicity using cycloheximide .


Cycloheximide , a reversible protein synthesis inhibitor , is thought to block DNA replication in normal cells by preventing synthesis of a labile protein .


In animal systems , cycloheximide protects normal cells from cytotoxic S-phase specific agents , such as cytosine arabinoside ( Ara-C ) .


Malignant cells appear not to be susceptible to cycloheximide-induced cycle arrest and , subsequently , may not be protected from Ara-C cytotoxicity .


The effect of cycloheximide on granulocyte/macrophage progenitors ( CFU-GM ) after in vitro Ara-C exposure was examined using normal human bone marrow , malignant progenitors from patients with chronic myelogenous leukemia ( CML ) , and clonogenic cells from the human acute nonlymphocytic leukemia cell lines HL-60 and KG-1 .


Mononuclear or clonogenic cells were incubated for one hour with cycloheximide , followed by the addition , for three or 17 hours , of Ara-C before being plated in a methylcellulose culture system .


CFU-GM survival was significantly increase if normal cells were treated with cycloheximide before Ara-C exposure .


Similar cycloheximide pretreatment of CML progenitors and clonogenic HL-60 and KG-1 cells failed to protect CFU-GM from Ara-C-induced cytotoxicity .


Primary cortisol resistance accompanied by a reduction in glucocorticoid receptors in two members of the same family .


This report describes studies of a man suspected of having primary cortisol resistance .


This conclusion is based on his high plasma cortisol levels and high 24-h urinary 17-hydroxycorticosteroid and cortisol excretion , plus the fact that he had no manifestations of Cushing 's syndrome .


Among family members tested , his mother also had hypercortisolemia .


Both mother and son had high levels of unbound plasma cortisol , but their plasma ACTH concentrations were within the normal range .


Both were partially resistant to dexamethasone adrenal suppression , and both had mild hypertension without hypokalemia .


To study this apparent end-organ resistance to cortisol , we examined the glucocorticoid receptors in peripheral mononuclear cells .


Using whole cell assays , glucocorticoid receptors in both patients were found to have reduced total binding capacity .


We conclude that these two patients , members of the same family , have primary cortisol resistance accompanied by a reduced number of glucocorticoid receptors .


[ Glucocorticoid receptors and response to polychemotherapy in acute lymphatic leukemia ]


Glucocorticoid receptor ( GR ) levels were quantified in leukemic blasts from peripheral blood of 86 patients with acute lymphoblastic leukemia .


The subsequent achievement of complete remission after combination chemotherapy was correlated with high receptor levels .


Forty-seven of 50 patients with leukemic cells containing more than 6 , 000 receptor sites and 22 of 36 patients with cells containing less than 6 , 000 receptor sites achieved remission .


The study of glucocorticoid receptors in leukemic cells may predict response to combination chemotherapy in patients with acute lymphoblastic leukemia .


Specific uptake of 1 , 25-dihydroxycholecalciferol by human chronic myeloid leukemia cells .


We have examined mononuclear cell preparations from patients with chronic myeloid leukemia [ CML ] for binding of and response to 1 , 25-dihydroxycholecalciferol [ 1 , 25- ( OH ) 2D3 ] .


Whole cells specifically took up [ 3H ] -1 , 25- ( OH ) 2D3 with high affinity ( Kd 3.6 X 10 ( -11 ) M ) and low capacity .


Subcellular fractionation of labeled cells showed that binding was restricted to cytosols and nuclei .


Sucrose gradient centrifugation of cells preincubated with [ 3H ] -1 , 25- ( OH ) 2D3 revealed a single 3.6S peak which was totally displaced with 100-fold excess nonradioactive hormone .


However , we were unable to demonstrate specific binding of 1 , 25- ( OH ) 2D3 by postlabeling standard cytosol preparations .


In addition , cytosols prepared from a mixture of CML cells and 1 , 25- ( OH ) 2D3 receptor-positive T47D ( human breast cancer ) cells had less than 10 % of the binding measured in T47D cytosol alone .


However , the levels of binding in T47D cytosols were not reduced if the receptors were occupied with [ 3H ] -1 , 25- ( OH ) 2D3 prior to the addition of the CML cytosols .


Thus , CML cells appear to contain both the receptor for 1 , 25- ( OH ) 2D3 and an unknown substance which prevents its detection following the preparation of cytosol .


Cells from patients with CML in the chronic phase specifically bound more 1 , 25- ( OH ) 2D3 [ 18.0 +/- 3.2 ( S.E. ) fmol/10 ( 7 ) cells ] than did those in acute myeloid transformation [ 7.2 +/- 1.5 ] or than did cells from patients with acute myeloid leukemia [ 2.6 +/- 0.8 ] .


Only cells from the first group of patients responded to the addition of 1 , 25- ( OH ) 2D3 by differentiating along the monocyte-macrophage pathway .


We conclude that the differentiation-induction effect of 1 , 25- ( OH ) 2D3 is likely to depend on adequate levels of receptor and that intact cells rather than cytosol preparations should be studied before cells of a particular tissue are designated as receptor negative .


Effect of cell cycle position on dexamethasone binding by mouse and human lymphoid cell lines : correlation between an increase in dexamethasone binding during S phase and dexamethasone sensitivity .


We determined the effect of cell cycle position on the amount of dexamethasone that was specifically bound by mouse and human lymphoid cell lines .


Cell lines that were either sensitive or resistant to growth inhibition by dexamethasone were compared .


Exponentially growing cells were separated by centrifugal elutriation into fractions that corresponded to different positions in the cell cycle .


The cell cycle phase distribution of each fraction was estimated by flow cytometry and autoradiography .


The amount of dexamethasone bound per cell in each fraction was measured by a whole cell binding assay .


In three dexamethasone-sensitive cell lines ( two mouse and one human ) , we found that the amount of dexamethasone bound per cell increased 2-4-fold between G1 phase and S phase , and then decreased during G2/M phase .


Results were the same when the amount of dexamethasone bound per milligram of cell protein was measured .


Binding affinity was the same during G1 phase and S phase , but the proportion of bound dexamethasone that translocated to the nucleus was greater during S phase .


In contrast , we found that the amount of dexamethasone bound per cell by three dexamethasone-resistant cell lines ( two mouse and one human ) did not increase during S phase .


Our results indicate that cell cycle changes in dexamethasone binding are not simply related to changes in cell protein or cell volume during the cell cycle .


An increase in dexamethasone binding during S phase may be required for dexamethasone to inhibit cell growth , and a failure of dexamethasone binding to increase during S phase might represent a new mechanism of dexamethasone resistance in lymphoid cells .


Presence and steroid inducibility of glutamine synthetase in human leukemic cells .


Glutamine synthetase ( EC 6.3.1.2 ; GS ) is present in lymphoblasts from patients with acute lymphoblastic leukemia ( ALL ) as well as in normal peripheral blood lymphocytes .


In 16 out of 20 ALL patients studied exposure of the cells to physiological concentrations of dexamethasone in vitro increased enzyme activity above the control levels .


The increase was specific for glucocorticoid receptor ligands .


A direct correlation was found between the magnitude of glucocorticoid-mediated increase of GS activity and the cellular levels of specific glucocorticoid receptors assayed in the same cell specimen .


Moreover , the basal levels of the enzyme measured in cells prior to exposure to dexamethasone correlated negatively with receptor density .


It is suggested that the presence of steroid-inducible GS in ALL cells may prove to be a marker for functional receptor sites .


Binding of progestins to the glucocorticoid receptor .


Correlation to their glucocorticoid-like effects on in vitro functions of human mononuclear leukocytes .


A number of physiological and synthetic progestins were tested for their ability to compete with [ 3H ] dexamethasone for the binding to the glucocorticoid receptor of human mononuclear leukocytes and their ability to elicit glucocorticoid-like effects on the same cells .


As compared to the reference compound dexamethasone ( relative receptor binding affinity defined as 100 % ) , two potent synthetic progestins with a pregnane-type structure , megestrol acetate and medroxyprogesterone acetate , were found to display a considerable binding affinity towards the receptor ( 46 and 42 % , respectively ) .


The relative binding affinity of the naturally occurring ligand , cortisol , to the receptor was clearly lower ( 25 % ) .


The effective binding of medroxyprogesterone acetate to the glucocorticoid receptor was confirmed by direct binding studies utilizing a tritiated derivative of this steroid .


No evidence for the existence of a specific progesterone receptor in human mononuclear leukocytes was obtained as judged by the results of competition experiments where a progesterone receptor-specific ligand [ 3H ] Org 2058 was used .


Medroxyprogesterone acetate and megestrol acetate also induced glucocorticoid-like effects on the lymphocyte functions .


These included inhibition of the proliferative responses to the T-cell mitogens concanavalin A and phytohaemagglutinin and an enhanced accumulation of immunoglobulin secreting cells in pokeweed mitogen-stimulated cultures .


The progestin effect appears to be mediated through a radiosensitive ( suppressor ) subpopulation of T lymphocytes .


In contrast , the synthetic progestins related structurally to 19-nortestosterone , norethisterone and d-norgestrel , were virtually devoid of binding affinity towards the glucocorticoid receptor nor did they measurably influence the in vitro lymphocyte functions .


These studies demonstrate that certain progestins in common clinical use probably possess inherent glucocorticoid activity and suggest that side effects attributable to this character ( e.g. suppression of the pituitary-adrenal axis ) might be expected when these compounds are used in pharmacological doses .


Metabolic and ultrastructural aspects of the in vitro lysis of chronic lymphocytic leukemia cells by glucocorticoids .


Human chronic lymphocytic leukemia ( CLL ) cells like prothymocytes and immunoactivated T-lymphocytes are readily lysed in vitro by pharmacological concentrations of glucocorticoids such as cortisol , whereas peripheral blood lymphocytes and thymocytes are unaffected by the hormone .


In this study , metabolic and ultrastructural aspects of the cortisol-induced killing process of CLL cells are recorded .


In vitro lysis was found to be temperature dependent and was detected only after 6 to 8 hr incubation with cortisol by means of the trypan blue exclusion test .


However , 30 min of incubation with cortisol at either 37 degrees or 4 degrees followed by the removal of the hormone was still sufficient to induce the lytic process .


Ultrastructural studies demonstrated sequential changes in the cytoplasm , including swelling of mitochondria and cytoplasmic decompartmentalization , followed by loss of surface microvilli with the appearance of `` holes '' in the cell membrane , and subsequent condensation of nuclear chromatin .


The large holes in the membrane appearing after 6 hr of incubation with the hormone may be the cause for the penetration of the viable stain into the dead cells , as seen by light microscopy .


Addition of metabolic inhibitors including actinomycin D , puromycin , and cycloheximide following administration of cortisol resulted in inhibition of the cell lysis .


An excess of an antagonist such as cortexolone was found to inhibit the cortisol-induced cytolysis of the CLL cells .


It is suggested that the glucocorticoid-induced lysis of human CLL cells is similar to the phenomenon observed in rat or murine lymphocytes and is mediated by interaction of the steroid molecule with the cytoplasmic receptor .


The resulting complex appears to activate specific gene ( s ) the products of which eventually cause cytolysis .


Glucocorticoid receptors in lymphoid tumors .


There is a range of levels of glucocorticoid receptor numbers seen in the various subclasses of acute lymphatic leukemia ( ALL ) .


This variability can not be explained by the known correlation between active cell proliferation and an increase in the number of receptors , since the tumors with the highest growth fraction ( i.e. , Burkitt 's lymphoma and T-cell leukemia ) tend to have lower average receptor numbers than do tumors with lower growth fractions such as common ALL .


All clinical specimens from patients with lymphatic leukemia have some measurable level of glucocorticoid receptors ; therefore , the resistance seen in vivo can not be explained by the lack of receptors .


However , there has been a positive correlation , in our hands , with receptor level and prognosis .


On the basis of in vitro models , it is proposed that perhaps the high receptor cell lines ( i.e. , common ALL of childhood ) have relative stability of their genetic material making glucocorticoid-resistant mutations less likely to occur in patients with these cells than in low-receptor cell lines ( i.e. , T-cell leukemia ) .


This greater genetic variability in the low-receptor lines could account for the earlier emergence of clinical glucocorticoid resistance in these patients .


[ Tumor histology and steroid receptors in breast carcinoma ]


In Specimens of 115 patients with breast cancer 4 tumorparameters ( tumorsize , tumorboder , nucleargrade , lymphocytic stromal reaction ) 3 features of regional lymphnodes ( sinushistiocytosis , T-cellreaction , lymphnode metastases ) and estrogen and progesteron receptors were determined .


A strong sinushistiocytosis and T-cellreaction could be verified mainly in metastases in free lymphnodes .


The steroid receptor content does not correlate with histological parameters


Aldosterone-receptor deficiency in pseudohypoaldosteronism .


Pseudohypoaldosteronism , a syndrome characterized by salt wasting and failure to thrive , usually presents in infancy as high urinary levels of sodium despite hyponatremia , hyperkalemia , hyperreninemia , and elevated aldosterone levels .


We have investigated this syndrome for the possibility of abnormal Type I or `` mineralocorticoid-like '' receptors , which have intrinsic steroid specificity indistinguishable from that of renal mineralocorticoid receptors and are found in many tissues and cells , including mononuclear leukocytes .


We have studied three patients with pseudohypoaldosteronism : the 28-year-old index case in Melbourne ( Patient 1 ) and two siblings in Munich , eight and two years of age ( Patients 2 and 3 ) ; clinically , Patient 3 had a less severe case than his sister .


Percoll-separated control monocytes bound [ 3H ] aldosterone with high affinity ( Kd approximately 3 nM ) and limited capacity ( 150 to 600 sites per cell ) .


On repeated examination , no [ 3H ] aldosterone binding was found in monocytes from Patients 1 and 2 ; in Patient 3 , the levels were 62 sites per cell , more than 2 S.D. below those of the control .


Levels in the parents of the Munich patients ( first cousins ) were normal .


It appears that pseudohypoaldosteronism is caused by a Type I receptor defect , that the defect may be complete or partial , that transmission may be autosomal recessive , and that the study of patients with pseudohypoaldosteronism may indicate physiologic roles for Type I receptors in nonepithelial tissues .


Glucocorticoid inhibition of urokinase-like plasminogen activators in cultured human lymphoblasts .


Two human lymphoblast cell lines , LICR-LON-HMy2 ( HMy2 cells ) and GM4672A cells , are moderately growth inhibited by dexamethasone ( 1 , 4-pregnadien-9-fluoro-16 alpha-methyl-11 beta , 17 alpha , 21-triol-3 , 20-dione ) ( Dex ) .


Both cell types secrete a urokinase ( UK ) -like plasminogen activator ( PA ) .


Treatment of both HMy2 and GM4672A cells with Dex for 1-4 days inhibits extracellular PA activity in a concentration-dependent manner , being half-maximal at approximately 1 X 10 ( -9 ) M .


Inhibition of PA in both cell types is specific for active glucocorticoids , and this specificity parallels the ability of various steroids to bind to glucocorticoid receptors .


HMy2 cell PA is fully suppressible by Dex , whereas up to one third of the activator expressed by GM4672A cells is resistant to glucocorticoid inhibition .


Mixing experiments using a UK standard and conditioned media from Dex-treated cells suggest an absence of glucocorticoid-inducible inhibitors to UK or plasmin in both cell types .


However , conditioned media from Dex-treated GM4672A cells inhibits a portion of the homologous cellular activator in conditioned media from control GM4672A cells .


Thus , low levels of glucocorticoid-inducible inhibitors may contribute to , but can not fully account for , Dex inhibition of GM4672A PA activity .


Glucocorticoid-inducible inhibitors in HMy2 cells are either totally absent or are present at undetectable levels .


Thus , regulation of UK-like PAs in HMy2 and GM4672A cells differs with respect to the extent to which glucocorticoids inhibit constitutively expressed activator levels , as well as the possible contribution of glucocorticoid-inducible inhibitors to the regulatory process in GM4672A cells .


Characterization of aldosterone binding sites in circulating human mononuclear leukocytes .


Aldosterone binding sites in human mononuclear leukocytes were characterized after separation of cells from blood by a Percoll gradient .


After washing and resuspension in RPMI-1640 medium , cells were incubated at 37 degrees C for 1 h with different concentrations of [ 3H ] aldosterone plus a 100-fold concentration of RU-26988 ( 11 alpha , 17 alpha-dihydroxy-17 beta-propynylandrost-1 , 4 , 6-trien-3-one ) , with or without an excess of unlabeled aldosterone .


Aldosterone binds to a single class of receptors with an affinity of 2.7 +/- 0.5 nM ( means +/- SD , n = 14 ) and a capacity of 290 +/- 108 sites/cell ( n = 14 ) .


The specificity data show a hierarchy of affinity of desoxycorticosterone = corticosterone = aldosterone greater than hydrocortisone greater than dexamethasone .


The results indicate that mononuclear leukocytes could be useful for studying the physiological significance of these mineralocorticoid receptors and their regulation in humans .


Mineralocorticoid and glucocorticoid receptors in circulating mononuclear leukocytes of patients with primary hyperaldosteronism .


Mineralocorticoid and glucocorticoid receptors were measured in circulating mononuclear leukocytes in 5 patients affected by Conn 's syndrome ( 3 cases of bilateral adrenal hyperplasia and 2 cases of adenoma plus unilateral hyperplasia ) .


The number of the binding sites per cell resulted significantly lower ( 189 +/- 114 , mean +/- SD ) , as compared with the normal controls ( 298 +/- 105 ) .


The affinity of aldosterone for the receptor was found to be not different than that of healthy control subjects .


The capacity and the affinity of dexamethasone for glucocorticoid receptors ranged in the normal values .


These data suggest a possible down-regulation of mineralocorticoid receptors in humans .


Short-term and long-term effects of estrogen on lymphoid tissues and lymphoid cells with some remarks on the significance for carcinogenesis .


Estrogens have long been thought to play a role in regulating the immune system .


The difference in some types of immune responses between males and females is well-known , as is the pronounced thymic involution induced by exogenous estrogens .


Estrogens stimulate some aspects of macrophage activity and , depending on dose and mitogen , inhibit or stimulate lymphocyte proliferative response in vitro .


Another example is the estrogen effect on the delayed type hypersensitivity response .


A broad review is given of such estrogen effects on lymphoid tissue and immune response .


Most of the studies published so far are phenomenological .


However , the recent description of estrogen receptors in the thymus and in some lymphocyte subpopulations , as well as a deeper understanding of regulating factors in the immune system , open the possibility of a more detailed understanding of the estrogen mechanism of interference .


Estrogen effects in adults are reversible .


After treating neonatal mice with the synthetic estrogen diethylstilbestrol ( DES ) , disturbances are induced in lymphocyte populations and lymphocyte functions which are permanent and irreversible .


Lymphocytes from adult , neonatally DES-treated female mice have a reduced mitogen response to ConA and LPS ( T and B cell mitogen ) and the delayed type hypersensitivity response is depressed .


A detailed analysis demonstrated a decreased T helper cell population .


The activity of Natural Killer cells is permanently reduced and this functional impairment is related to a decreased number of these cells , in turn determined at the bone marrow level .


The same animals have an increased sensitivity to chemical carcinogens ( methylcholanthrene ) and they spontaneously develop epithelial changes in the uterine cervix which morphologically are similar to adenocarcinoma .


The association between estrogen-associated malignancy and estrogen effects in lymphocyte functions deserves further study .


Drugs affecting the hormonal receptors of normal and leukaemic peripheral leucocytes .


The authors investigated the behaviour of steroid hormone uptake in leukaemic cells ( CML , CLL , AML , ALL ) , in basal conditions and after incubation with drugs which modify the cellular concentration of cAMP , PGE and PGF .


The results demonstrated the presence in leukaemic cells of an alteration in the incorporation of steroid hormones .


This alteration was scarcely modified by incubation with theophylline , which increases cellular concentration of cAMP .


On the other hand , it was moderately counteracted by thioproline and was evidently inhibited by flurbiprofen , which also reduced cellular concentrations of prostaglandins , particularly PGE2 , with the exception of PGF2 which showed a poor response .


Differences were observed in the behavior of hormonal uptake of CML , in contrast to that of AML , CLL and ALL peripheral leucocytes .


Human breast cancer and impaired NK cell function .


Recent advances in tumor immunology have led to the discovery of a new lymphoid cell with unique antitumor activity .


Natural killer ( NK ) cells form an antitumor surveillance system and appear to be vital in preventing tumor growth and metastasis in animal models .


We studied NK activity in patients with benign and malignant breast disease , using a chromium-51 release microtiter cytotoxicity assay with K562 cells as targets .


Compared with benign controls , patients with malignancies had significantly depressed NK -mediated lysis ( P less than 0.01 ) .


Furthermore , lysis in those with advanced disease ( stages II , III , and IV ) was significantly less than in those with limited disease ( stage I ) ( P less than 0.01 ) .


NK activity was not correlated to estrogen or progesterone receptor states .


Positive correlation of a depressed natural killer activity with the extent of tumor spread supports the concept of an NK cell immune surveillance system in breast cancer and emphasizes its importance in this malignancy .


Immunosuppressive effect of serum progesterone during pregnancy depends on the progesterone binding capacity of the lymphocytes .


Cytotoxic activity and progesterone binding capacity of the lymphocytes , together with serum progesterone concentrations , were determined in women with normal pregnancy or with a clinical diagnosis of threatened abortion or threatened premature labour .


The lymphocytes of women with threatened abortion or threatened premature labour showed significantly higher cytotoxic activity ( P less than 0.001 ) and significantly lower progesterone binding capacity ( P less than 0.001 ) than did lymphocytes obtained from the healthy pregnant women .


Significant inverse correlation was found between progesterone binding capacity and cytotoxic activity of the lymphocytes ( P less than 0.001 ) , but the progesterone concentration of the pregnancy serum appeared to have no influence on the other two parameters .


The findings indicate that intact progesterone binding capacity of the lymphocytes is an essential factor for the manifestation of the blocking effect exerted by pregnancy serum on lymphocyte cytotoxicity in vitro .


Serum sex steroid and peptide hormone concentrations , and endometrial estrogen and progestin receptor levels during administration of human leukocyte interferon .


Five normally cycling healthy women were given daily subcutaneous injections of human leukocyte interferon ( 3 X 10 ( 6 ) units/day ) from the 3rd through 23rd day of the menstrual cycle , and serum steroid and peptide hormone concentrations monitored at 3-day intervals during the treatment and the preceding control cycle .


Concentrations of cytosol and nuclear estrogen receptors ( ERC and ERN , respectively ) and progestin receptors ( PRC and PRN ) were also measured from endometrial biopsies taken on the 24th day of the control and treatment cycle .


In addition , an extensive monitoring of clinical chemical and hematological tests from the blood samples were performed .


Serum estradiol and progesterone concentrations were significantly decreased during the treatment cycle , suggesting that interferon interacts in vivo with the function of both FSH and LH .


No significant changes were observed in the serum peptide hormone concentrations measured ( FSH , LH , prolactin , insulin , growth hormone and TSH ) ; neither were the levels of endometrial ERC , ERN , PRC and PRN affected by interferon administration .


As expected , interferon administration resulted in decreased leukocyte counts .


Moreover , an increasing tendency in the activities of serum alkaline phosphatase and gamma-glutamyltransferase during the interferon therapy shows that interferon may slightly interfere with the liver function .


These results suggest that one of the mechanisms by which interferon treatment may affect the growth of hormone-dependent neoplasms could be the interaction with production and/or function of circulating hormonal compounds .


[ Glucocorticoid receptor level in the blood leukocytes in different acute diseases ]


Content of glucocorticoid receptors in cytosol of blood leukocytes , concentration of cortisol and amount of leukocytes in blood were studied in 20 patients with acute impairments within the second day of the disease .


Content of receptors in cytosol of blood leukocytes was studied using 3H-triamcinolone acetonide .


Distinct increase in amount of the leukocyte glucocorticoid receptors was found in patients with poisoning by dichlorethane and hypnotic drugs under conditions of acute myocardial infarction .


In acute pancreatitis content of the leukocyte receptors was not altered as compared with controls .


Concentration of endogenous cortisol was increased in blood of all the patients , except of the cases of acetate intoxication .


Reverse correlation was observed between concentration of cortisol in blood and content of glucocorticoid receptors in leukocytes .


But in the patients with acute pancreatitis the decrease in content of leukocyte glucocorticoid receptors was not observed although there was an increase in cortisol concentration in blood .


The role of glucocorticoid receptors in immunological processes under conditions of purulent complications and possibility to regulate the metabolism in leukocytes


Therapeutic concentrations of glucocorticoids suppress the antimicrobial activity of human macrophages without impairing their responsiveness to gamma interferon .


By exposing human blood-derived macrophages and alveolar macrophages in vitro to dexamethasone , we showed in these studies that glucocorticoids markedly suppress the antimicrobial activity of macrophages but not macrophage activation by lymphokines .


As little as 2.5 X 10 ( -8 ) mol/liter of dexamethasone prevented macrophages from inhibiting germination of Aspergillus spores or from eliminating ingested bacteria such as Listeria , Nocardia , or Salmonella .


Damage to macrophage function was inhibited by progesterone and appeared to be receptor-mediated .


In accordance with in vivo observations , dexamethasone required 24-36 h to suppress antimicrobial activity .


While glucocorticoids interfered with base-line activity of macrophages , dexamethasone concentrations comparable to drug levels in patients had no effect on macrophage activation .


Proliferating lymphocytes and gamma-interferon thus increased the antimicrobial activity of phagocytes exposed to glucocorticoids over that of control cells .


Macrophage activation and correction of the dexamethasone effect by gamma-interferon , however , was dependent on the pathogen .


The lymphokine enhanced the antimicrobial activity of dexamethasone-treated macrophages against Listeria and Salmonella but not against Aspergillus or Nocardia .


Dexamethasone-induced damage to the antimicrobial activity of human macrophages in vitro parallels observations that glucocorticoids render laboratory animals susceptible to listeriosis and aspergillosis by damaging resident macrophages .


Suppression of macrophage antimicrobial activity should thus be considered when treating patients with glucocorticoids ; its prevention by gamma-interferon might be beneficial for some but not all pathogens .


Interleukin 2 receptor ( Tac antigen ) expression in HTLV-I-associated adult T-cell leukemia .


Interleukin-2 ( IL-2 ) is a lymphokine synthesized by some T-cells following activation .


Resting T-cells do not express IL-2 receptors , but receptors are rapidly expressed on T-cells following interaction of antigens , mitogens , or monoclonal antibodies with the antigen-specific T-cell receptor complex .


Using anti-Tac , a monoclonal antibody that recognizes the IL-2 receptor , the receptor has been purified and shown to be a Mr 33 , 000 peptide that is posttranslationally glycosylated to a Mr 55 , 000 mature form .


Normal resting T-cells and most leukemic T-cell populations do not express IL-2 receptors ; however , the leukemic cells of the 11 patients examined who had human T-cell lymphotropic virus-associated adult T-cell leukemia expressed the Tac antigen .


In human T-cell lymphotropic virus-I infected cells , the Mr 42 , 000 long open reading frame protein encoded in part by the pX region of this virus may act as a transacting transcriptional activator that induces IL-2 receptor gene transcription , thus providing an explanation for the constant association of IL-2 receptor expression with adult T-cell lymphotropic virus-I infection of lymphoid cells .


The constant expression of large numbers of IL-2 receptors which may be aberrant may play a role in the uncontrolled growth of adult T-cell leukemia cells .


Two patients with Tac-positive adult T-cell leukemia have been treated with the anti-Tac .


One of the patients had 6- and 3-mo remissions of his leukemia following two courses of therapy with this monoclonal antibody directed toward this growth factor receptor .


Lymphocyte glucocorticoid receptor binding in depressed patients with hypercortisolemia .


Despite elevated levels of serum and urinary cortisol , patients with depressive illness manifest none of the clinical stigmata of glucocorticoid excess .


This hypercortisolemia in the absence of clinical effects suggests a state of hormone resistance and could be mediated by alterations in the glucocorticoid receptor .


Earlier studies have shown that small doses of glucocorticoids cause a decrease in glucocorticoid receptor binding in normal human lymphocytes .


White cells from depressed patients with significant hypercortisolemia would be expected to show a similar change in receptor concentration if peripheral tissues are adequately exposed to and sensitive to the hormone .


In this study we compared the binding of [ 3H ] dexamethasone to lymphocytes from normal subjects and depressed patients with hypercortisolemia .


Lymphocytes from normal subjects had a mean receptor concentration of 10.2 +/- 0.66 fm/10 ( 6 ) cells ( S.E.M. ) and a dissociation constant of 4.8 +/- 0.47 nM .


Lymphocytes from depressed patients with abnormal 0800 h serum cortisol after dexamethasone had a mean receptor concentration of 8.8 +/- 0.75 fm/10 ( 6 ) cells , which was not significantly different from that in lymphocytes from normal subjects or from depressed subjects with normal post-dexamethasone cortisol levels ( 9.4 +/- 0.95 fm/10 ( 6 ) cells ) .


Lymphocytes from depressed patients with elevated urinary free cortisol excretion ( UFC ) also had normal receptor concentration and binding affinity for dexamethasone .


The lack of a change in lymphocyte glucocorticoid receptor concentration in the presence of cortisol excess suggests the possibility that hypercortisolemia in depressive illness represents a state of peripheral glucocorticoid resistance .


Structure and regulation of the glucocorticoid hormone receptor .


The glucocorticoid receptor is an intracellular protein which possesses three distinct domains , one that binds agonist and antagonist steroids , one that binds DNA , and one that binds anti-receptor antibodies and is required for glucocorticoid modulation of gene expression .


In intact cells , receptor number , affinity and activity can change in response to factors that bind to the receptor , or that act indirectly through ill-defined mechanisms which may include resumption or arrest of cell cycling and variations in intracellular calcium ion concentrations .


Some of these factors appear to exert their effect by controlling critical receptor properties such as ATP-dependent phosphorylation , integrity of thiol groups , and exposure of key amino acid residues .


Glucocorticoid agonists promote the 'transformation ' of the receptor into the DNA-binding state , which is competent for modulating gene expression .


Glucocorticoid antagonists are steroids that interact with the receptor but either fail to produce a stable complex or produce a stable but inefficient complex .


Although substituent groups that confer agonist or antagonist activity to the steroid have been identified , the molecular determinants of this difference at the receptor level remain unknown .


Most in vitro and in vivo data on receptor regulation can be accommodated by postulating the existence of an intracellular cycle that involves five states of the receptor .


The active free receptor is phosphorylated , reduced , and presumably oligomeric ( state A ) .


Following binding of an agonist ( state B ) , it can become transformed by dissociation into its subunits and dephosphorylation ( state C ) .


The transformed receptor then interacts with chromatin ( state D ) .


Dissociation of the steroid and oxidation of receptor thiol group ( s ) lead to the inactive receptor form ( state E ) .


Reduction and rephosphorylation of the receptor enable it to bind steroids again so that the cycle is closed .


Thermodynamics of steroid binding to the human glucocorticoid receptor .


The thermodynamics of the interaction of glucocorticoids with their receptor were studied in cytosol from human lymphoblastoid cells .


The rate and affinity constants of dexamethasone and cortisol between 0 degree and 25 degrees C were calculated by curve-fitting from time-course and equilibrium kinetics .


The data were consistent with a simple reversible bimolecular interaction .


Arrhenius and Va n't Hoff plots were curvilinear for both steroids .


At equilibrium , the solution for the equation delta G = delta H - T X delta S ( eqn. 1 ) was ( in kJ X mol-1 ) -47 = 36 - 83 ( dexamethasone ) and -42 = -9 - 33 ( cortisol ) at 0 degree C .


Enthalpy and entropy changes decreased quasi-linearly with temperature such that , at 25 degrees C , the respective values were -50 = -75 + 25 and -43 = -48 + 5 .


Thus , for both steroids , the interaction was entropy-driven at low temperature and became entirely enthalpy-driven at 20 degrees C .


Thermodynamic values for the transition state were calculated from the rate constants .


For the forward reaction , eqn. ( 1 ) gave 45 = 84 - 39 ( dexamethasone ) and 46 = 60 - 14 ( cortisol ) at 0 degree C , and 44 = 24 + 20 ( dexamethasone ) and 46 = 28 + 18 ( cortisol ) at 25 degrees C .


These data fit quite well with a two-step model [ Ross & Subramanian ( 1981 ) Biochemistry 20 , 3096-3102 ] proposed for ligand-protein interactions , which involves a partial immobilization of the reacting species governed by hydrophobic forces , followed by stabilization of the complex by short-range interactions .


On the basis of this model , an analysis of the transition-state thermodynamics led to the conclusion that no more than half of the steroid molecular area is engaged in the binding process .


Cell cycle-related changes in number of T-lymphocyte receptors for glucocorticoids and insulin .


Enriched human peripheral T-lymphocytes were stimulated with PHA and examined for variations in insulin and glucocorticoid ( dexamethasone ) receptor numbers during the early phases of the cell cycle .


Cells in G0 , G1a and G1b phases , where the G1a - G1b transition is an Interleukin 2 dependent event , were quantitated by flow cytometry .


Few but significant numbers of glucocorticoid receptors ( 2700/cell ) and no insulin receptors ( -1/cell ) were found in the resting ( G0 ) phase .


As cells entered the G1a phase the specific binding of dexamethasone increased and of insulin took place .


Although the specific binding further increased as T-cells entered the G1b phase ( as measured at 44 h of incubation and using hydroxyurea-treated cells ) , the major changes in the specific binding of dexamethasone took place during the period 16 - 20 h after stimulation .


Based on these findings , it is concluded that both receptor types ( cell membrane and cytoplasmic receptors ) are being formed and increased at G1 phase prior to cell proliferation , indicating the importance of G1 phase in immunoregulation .


Glucocorticoid receptors and cortico-sensitivity in a human clonal monocytic cell line , CM-SM .


CM-SM is a clonal line of human precursor mononuclear phagocytes inducible to macrophage differentiation in response to the tumor promoter phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate ( TPA ) .


Untreated CM-SM cells contain single class , high-affinity ( KD = 4.0 X 10 ( -9 ) M ) glucocorticoid-specific receptor sites ( approximately 60 , 000 per cell ) , as measured by a whole cell assay , at 37 degrees C , using [ 3H ] triamcinolone acetonide ( TA ) .


Exposure of CM-SM to dexamethasone ( DEX ) produced a progressive , dose- and time-related series of changes in CM-SM cell growth , saturation density , morphology , and functional properties , with half-maximal effects at about 10 ( -9 ) M for DEX .


TA-receptor sites rapidly decreased ( about 70 % ) after DEX treatment , without any apparent change in steroid specificity and affinity .


After 5 days in culture with a saturating concentration ( 3.6 X 10 ( -8 ) M ) of hormone , the cells reached a saturation density of about 9.0 X 10 ( 6 ) viable cells/ml ( about 4.0 X 10 ( 6 ) viable cells/ml in the controls ) , while the modal volume of the resulting cell population was approximately 60 % , as compared to the volume of untreated cells .


DEX-treated cells appeared less differentiated than controls , as assessed by combined morphologic , antigenic , and cytoenzymatic analyses .


DEX almost completely inhibited TPA activation of the following macrophage functions : adherency to the culture plate , expression of lysosomal enzymes , Fc and C3 receptors , and stimulation of phagocytosis .


After removal of DEX , the cells , within a few passages , returned to a state apparently identical to the untreated controls and could be induced to macrophage differentiation in response to TPA .


Acute lymphoblastic leukemia in children : current status , controversies , and future perspective .


Disease-free survival ( DFS ) in childhood ALL is 60 % , and survival in good , average , and poor prognostic groups defined by initial WBC and age is 90 , 60 , and 45 % , respectively .


Additional immunological , morphological , biochemical , cytokinetic , and cytogenetic factors have been identified , illustrating the heterogeneity of ALL and its derivation from malignant clones at various stages of differentiation and with varying rates of proliferation .


Of biologic importance , these factors may refine further the characteristic features of clinically-determined prognostic groups .


Multivariate analysis of large prospective trials with homogeneous therapy will be required to determine the independent prognostic importance of these factors .


Current treatment strategies in ALL include ( 1 ) tailoring therapy and its intensity to prognostic groups ; ( 2 ) multiple-drug combinations in induction ; ( 3 ) early use of intrathecal ( IT ) methotrexate ( MTX ) ; ( 4 ) CNS prophylaxis with IT MTX alone in good prognosis patients and combined cranial radiation ( CXRT ) , 1800 rads plus IT MTX , in average and poor prognosis patients .


Current studies show a CNS relapse rate of 5 % in all prognostic groups .


Late neuropsychological defects caused by cranial XRT and IT MTX have prompted programs designed to reduce the potential late toxicity of CNS prophylaxis .


More pronounced in younger children , these abnormalities include decreased IQ , visual-motor incoordination , poor performance in mathematics , and memory dysfunction .


Until 1980 , more intensive induction , consolidation , and maintenance therapy had failed to prolong DFS in children with a poor prognosis .


In West Germany ( Berlin-Frankfurt-Muenster protocol ) a 70 to 75 % DFS is seen in all patients regardless of initial WBC , suggesting that effective therapy will override prognostic factors .


Ultra-high-dose MTX , without cranial radiation , is also showing promise in poor prognosis patients .


Other issues include the optimal duration of therapy , the role of testicular biopsies , and prophylactic testicular radiation .


Recent studies suggest that prognostic factors lose their significance after 2 years of continuous complete remission and that 2 years of maintenance therapy is adequate .


Bilateral open-wedge testicular biopsies have identified occult testicular disease in 8 to 10 % of males .


A unified approach to children with leukemia/lymphoma , a group with a particularly poor prognosis , utilizing NHL-type therapy may be more effective than conventional ALL therapy .


Glucocorticoid receptor and in vitro sensitivity to steroid hormones in human lymphoproliferative diseases and myeloid leukemia .


The glucocorticoid receptor ( GR ) quantitation by a whole-cell assay and/or cytosol technique and the in vitro sensitivity to steroids have been assessed in peripheral blood cells from normal donors and patients with chronic lymphatic leukemia ( CLL ) , acute lymphoblastic leukemia ( ALL ) , lymphosarcoma cell leukemia ( LSCL ) , acute nonlymphatic leukemia ( ANLL ) , and chronic myeloid leukemia ( CML ) .


Within the lymphoproliferative diseases , ALL cells exhibited the highest GR concentration ( regardless of the method used ) and the highest in vitro inhibition of spontaneous [ 3H ] thymidine ( [ 3H ] TdR ) uptake by glucocorticoids .


A significant relationship between GR concentration ( whole-cell assay ) and in vitro sensitivity to dexamethasone was also found .


On the contrary , CLL cells presented the highest sensitivity to glucocorticoids in PHA-stimulated cell cultures .


Cells from the only two ALL patients who did not undergo a remission after glucocorticoid-inclusive chemotherapy had both the lowest in vitro sensitivity to dexamethasone and the lowest GR concentration with whole-cell assay .


Concerning myeloid leukemia , ANLL patients had GR concentrations slightly higher than those found in the ALL group but exhibited the lowest degree of inhibition of spontaneous [ 3H ] TdR uptake by dexamethasone ( stimulatory effects occurred in some cases ) .


CML cells exhibited an inhibition degree by in vitro glucocorticoids significantly higher than that of ANLL cells but not different from that of lymphoproliferative diseases .


No clear relationship among GR pattern , in vitro cell sensitivity to glucocorticoids , and clinicohematologic parameters was observed in myeloid leukemia-bearing patients .


Glucocorticoid receptors and in vitro corticosensitivity of peanut-positive and peanut-negative human thymocyte subpopulations .


In 6 human thymus glands , the immature subset of thymocytes was separated from the more mature one , by differential peanut lectin agglutination .


These 2 cell subpopulations were analyzed for glucocorticoid receptor content by using a whole cell assay , with ( 3H ) -triamcinolone acetonide as tracer .


The unagglutinated thymocytes ( peanut negative ) contained about 2 times more receptor sites per cell than agglutinated ( peanut positive ) ones ( 7650 +/- 1550 S.D. verus 3195 +/- 896 S.D. ) .


The affinity for steroid was similar in both cell subsets , as was the stereospecificity for glucocorticoids , the time-course of steroid-receptor association , and cytoplasmic to nuclear translocation .


Despite the greater number of glucocorticoid receptor sites , the peanut-negative thymocyte subpopulation did not differ from the peanut-positive one in its sensitivity to the inhibitory effects of triamcinolone acetonide , as determined by measurements of the incorporation of radiolabeled precursors of protein and DNA .


Moreover , the peanut-negative subset appeared more resistant in vitro to the steroid-induced cell lysis as compared to the peanut-positive one .


Thus , our data suggest that glucocorticoid receptor density and corticosensitivity are not directly correlated and that the number of glucocorticoid receptor sites may be dependent on the degree of immunologic maturation


Defective binding and function of 1 , 25-dihydroxyvitamin D3 receptors in peripheral mononuclear cells of patients with end-organ resistance to 1 , 25-dihydroxyvitamin D .


Lectin-induced DNA synthesis by peripheral mononuclear cells from 17 normal donors was inhibited ( 40-60 % ) by 1 , 25-dihydroxyvitamin D3 ( 1 , 25 [ OH ] 2D3 ) at physiological concentrations ( 10 ( -10 ) -10 ( -9 ) M ) .


The lymphocytes acquire specific receptors for 1 , 25 ( OH ) 2D3 upon activation by the lectins .


This process precedes the inhibitory effect of 1 , 25 ( OH ) 2D3 .


We studied lymphocytes from six patients from four different kindreds with the syndrome of hereditary end-organ resistance to 1 , 25 ( OH ) 2D ( the so-called vitamin D-dependent rickets type II ) .


In five patients ( three kindreds ) peripheral blood mononuclear cells did not acquire receptors for 1 , 25 ( OH ) 2D3 upon phytohemagglutinin-induced activation .


Moreover , in contrast to normal lymphocytes , the mitogenic stimulation of these patients ' lymphocytes by phytohemagglutinin and concanavalin A was not inhibited by 1 , 25 ( OH ) 2D3 .


Activated lymphocytes of the sixth patient from a fourth kindred exhibited normal binding of [ 3H ] 1 , 25 ( OH ) 2D3 but the hormone failed to inhibit the mitogenic stimulation .


A similar pattern of the vitamin D effector system was previously observed in fibroblasts cultured from skin biopsies of the same group of patients .


The conclusions from these findings are : ( a ) the inhibition of mitogenic stimulation by 1 , 25 ( OH ) 2D3 is mediated by specific functional receptors to the hormone ; and ( b ) the receptors for 1 , 25 ( OH ) 2D3 in mononuclear cells are probably controlled genetically by the same mechanisms as the effector system in well-characterized target organs of the hormone , such as intestine and kidney .


Glucocorticoid receptors of mononuclear leukocytes from myasthenia gravis patients .


The present study was performed to analyse glucocorticoid receptor ( GR ) binding in peripheral blood mononuclear leukocytes ( MNL ) from 39 myasthenia gravis ( MG ) patients ( unoperated patients ( n = 13 ) , thymectomized patients ( n = 14 ) and patients receiving glucocorticoids : thymectomized ( n = 11 ) and unoperated ( n = 6 ] .


A whole cell binding assay with 3 ( H ) dexamethasone was used .


GR mean values were significantly higher in the MNL of MG patients ( thymectomized or not ) not receiving glucocorticoid than in the MNL of healthy donors .


Sex , age or clinical forms of illness did not influence the results .


In patients receiving prednisone ( Pd ) the GR values were significantly lower than in MG patients without Pd therapy , independent of Pd dose or time of administration .


No differences in receptor binding between normal subjects and MG patients receiving Pd have been found .


Immunological interference of high dose corticosteroids .


High-dose corticosteroids ( HDC ) will influence cellular as well as humoral participants of the immune response .


The lymphoid tissue will decrease in size and weight after prolonged treatment with HDC .


Reduced synthesis of B- as well as T-lymphocytes will be seen .


The inhibitory effect on B-cell function can be observed both as decreased serum levels of immunoglobulins and as impaired binding of antibodies and complement to the cellular surface .


Reduced T-cell function indicated by impaired stimulation by PHA and porkweed as well as by impaired lymphokinin effects on leukocyte migration inhibition has been reported .


Reduced lymphocyte adherence to antigen and suppressed lymphocyte reaction have also been observed .


Humoral factors involved in chemotaxis , opsonisation , phagocytosis , vascular permeability leading to leakage of fluid and cells and factors involved in lysis of antigens are impaired .


This can be explained partly by the observed reduced complement activation via the alternative as well as the classical pathway in association with HDC therapy .


Acute processes with increased vascular permeability and accumulation of leukocytes as impairing factors could be influenced beneficially by HDC therapy .


This positive effect can be seen in treatment of septic shock or rejection of a transplant .


However , if sepsis or rejection is not rapidly reversed , complications such as multisystem organ failure and bacteremia are prone to appear .


Identification of human leukemic glucocorticoid receptors using affinity labeling and anti-human glucocorticoid receptor antibodies .


Antisera raised against human lymphoid glucocorticoid receptors were used in combination with the glucocorticoid receptor affinity label [ 3H ] dexamethasone 21-mesylate [ ( 3H ] DM ) to identify the glucocorticoid receptors of the human B-lymphoblastoid cell line IM-9 and the human T-cell leukemic cell line CEM-C7 .


Antisera were obtained following immunization of New Zealand White rabbits with [ 3H ] triamcinolone acetonide [ ( 3H ] TA ) -glucocorticoid receptor complexes partially purified by two-stage DNA-cellulose chromatography .


The presence of anti-human glucocorticoid receptor antibodies was verified by : ( a ) adsorption of [ 3H ] TA-receptor-antibody complexes to Protein A ; ( b ) a shift to higher apparent molecular weight in the elution position from Sephacryl S300 of [ 3H ] TA-receptor complexes incubated with immune serum ; and ( c ) the ability of immune serum to displace [ 3H ] TA-receptor complexes on sucrose gradients .


These antibodies also recognized rat liver and murine S49 cell glucocorticoid receptors .


Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [ 3H ] DM-labeled IM-9 cytosol identified a major competable band with a molecular weight of approximately 90 , 000 , three minor competable components with molecular weights of approximately 78 , 000 , approximately 51 , 000 , and approximately 38 , 500 , and at least 21 other noncompetable components .


Following immunoprecipitation of [ 3H ] DM-labeled cytosol with immune serum , only the Mr 90 , 000 and 78 , 000 components were seen .


Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [ 3H ] DM-labeled CEM-C7 cytosol revealed a larger number of [ 3H ] DM-labeled components .


However , after immunoprecipitation of [ 3H ] DM-labeled CEM-C7 cytosol , a predominant competable component with a molecular weight of 90 , 000 was easily identified .


This component was markedly diminished when cytosols from the glucocorticoid receptor-deficient cell line ICR-27 were used .


Thus , the combination of affinity labeling and anti-human glucocorticoid receptor antibodies is capable of providing direct physical identification of human lymphoid glucocorticoid receptors .


Effect of thymosin on glucocorticoid receptor activity and glucocorticoid sensitivity of human thymocytes .


Incubation with thymosin fraction 5 , ( TMS F5 at 300 micrograms/ml ) a partially purified thymic factor , reduced the steroid binding activity of human infant thymocytes from 9.6 +/- 2.1 fmole/ml to 5.0 +/- 2.0 fmole/ml .


The glucocorticoid receptor activity in normal infant thymocytes was found to be 2 , 146 +/- 726 ( s.d. ) sites per cell with dissociation constant of 1.4 +/- 0.6 X 10 ( -8 ) M .


TMS F5 also increased the resistance of human thymocytes to the cytolytic effect of dexamethasone ( 2.5 X 10 ( -8 ) M ) to 168.6 +/- 30.2 % of control ( P less than 0.01 ) .


In animals , medullary and peripheral blood T cells are more resistant to glucocorticoids than immature thymic T cells .


The results show that thymosin can induce changes consistent with differentiation in human thymocytes .


These in vitro results are consistent with a physiological role of thymosin in intrathymic T cell maturation in man .


Incubation of a human malignant thymus derived T cell line ( MOLT 3 ) with TMS F5 also resulted in a significant reduction of the number of steroid binding sites to 44.2 +/- 15.3 % of control ( P less than 0.05 ) , but TMS F5 did not significantly reduce the glucocorticoid sensitivity of MOLT 3 cells .


Specific high-affinity receptors for 1 , 25-dihydroxyvitamin D3 in human peripheral blood mononuclear cells : presence in monocytes and induction in T lymphocytes following activation .


Human peripheral blood monocytes have high affinity binding sites for 1 , 25- ( OH ) 2D3 ( Kd 0.14 nM , sedimentation coefficient 3.7S ) .


Resting human peripheral blood T lymphocytes , however , do not have a demonstrable 1 , 25- ( OH ) 2D3 receptor .


After activation with phytohemagglutinin the T cells exhibit the receptor within 24 h , and this expression is blocked by cycloheximide .


The receptor in activated T lymphocytes has a sedimentation coefficient of 3.7S and a high affinity ( Kd 0.10 nM ) for the ligand .


Effects of chronic glucocorticoid excess in man on insulin binding to circulating cells : differences between endogenous and exogenous hypercorticism .


We measured [ 125I ] insulin binding to circulating monocytes or erythrocytes from 16 patients with chronic glucocorticoid excess , 9 chronically treated with prednisone and 7 with adrenocortical hyperfunction .


With monocytes , [ 125I ] insulin binding was iincreased in all patients .


Analysis of binding data indicated that increased binding in patients treated with prednisone was due to an ncrease in receptor concentration , whereas in patients with adrenocortical hyperfunction , it was due to an increase in receptor affinity .


With erythrocytes from patients with adrenocortical hyperfunction there was an increase in receptor affinity and a decrease in receptor concentration , so that the binding of [ 125I ] insulin was normal .


The disparity of results between endogenous and exogenous hypercorticism , between the two cell types , and between the present studies and previous studies suggest that the effects of glucocorticoid excess on the insulin receptor are extremely complex and wide-ranging and that in this condition , extrapolations in humans from data with circulating cells to liver and muscle may not be appropriate .


Reduced level of cellular glucocorticoid receptors in patients with anorexia nervosa .


Specific glucocorticoid receptors were measured in circulating mononuclear leukocytes from 12 patients with anorexia nervosa and 21 healthy control subjects .


Cells from patients were found to contain a significantly ( p less than 0.01 ) lower level of glucocorticoid receptor ( 3830 +/- 210 sites/cell , mean +/- SE ) than those from controls ( 4930 +/- 250 sites/cell ) .


A partial glucocorticoid receptor defect may well explain the abnormal cortisol metabolism and glucocorticoid resistance commonly found in patients with anorexia nervosa .


Regulation of the glucocorticoid receptor in human lymphocytes .


The presence of a glucocorticoid receptor in human lymphocytes is well established , but factors affecting its regulation have not been described .


Using a competitive binding whole cell assay , we have examined the binding of [ 3H ] -dexamethasone at 24 and 37 degrees C in untreated normal subjects and in healthy subjects taking various glucocorticoid preparations .


At 24 degrees C normal human lymphocytes had 6000 binding sites/cell and a dissociation constant of 4 x 10 ( -9 ) M .


The administration of 1 mg of dexamethasone , 5 mg of prednisone , and 37.5 mg of cortisone acetate resulted in a 30 % decrease in binding sites after 1 week with no change in binding affinity .


No changes in the number of binding sites was noted before 1 week and the diminished number persisted for 1 week after discontinuation of glucocorticoid treatment .


Lymphocytes from hospitalized patients taking 40-60 mg of dexamethasone daily demonstrated the same change in number of binding sites that was seen in normal subjects taking 1 mg of dexamethasone .


When binding assays were carried out at physiologic temperature there was the same decrease in number of binding sites after dexamethasone administration , and in addition , there was a two-fold increase in binding affinity .


Glucocorticoid administration results in a time-dependent decrease in the number of lymphocyte glucocorticoid binding sites that is independent of the type of glucocorticoid administered .


This is the first in vivo demonstration that glucocorticoids modulate their own receptors in man .


Immunoglobulin localization in benign and malignant lesions of the human mammary gland .


Using direct imunofluorescence , lesions from 266 human breast specimens were studied for the presence of IgA , IgM , or IgG localization .


The lesions included benign elements from 66 subcutaneous mastectomy specimens in which the absence of simultaneous breast malignancy was documented , primary breast carcinomas from 153 mastectomy specimens , and 47 biopsies containing metastatic breast cancer .


A statistically significant association of IgA and IgM with benign lesions was contrasted to the association of IgG with malignant lesions .


In both primary and metastatic lesions , IgG localization was associated with estrogen-receptor-poor primary cancers as compared with estrogen-receptor-rich primary cancers .


Among primary breast cancer patients , IgG localization in the tumor correlated with relative lymphopenia .


A shorter disease-free interval was noted in association with IgG localization among the metastatic breast lesions .


No statistically significant association between stage of disease and immunoglobulin presence was demonstrable .


Moderate-to-severe intraductal epithelial hyperplasias were more often associated with immunoglobulin G localization that were other benign lesions


Correlation of steroid receptors with histologic differentiation in mammary carcinoma .


Cancer of the breast is the most common tumor in females in Singapore , with the rate of 20.7 per 100 , 000 per year ( 1977 estimate ) , which is predicted to increase to 29.8 per 100 , 000 women per year by 1995 .


A detailed histopathologic review of 50 primary breast cancer tumors analyzed for estrogen receptor ( ER ) level was carried out and a variety of morphologic features correlated with ER results to identify any factors that will improve the management and prognosis for breast cancer .


Cytosol was incubated with 3H-estradiol in the presence and absence of cold diethylstilbestrol , and bound and free hormone were separated by Dextran-coated charcoal method .


Tumors binding more than 5 fmol/mg cytosol protein were classified as ER -positive .


Progesterone receptor ( PR ) level was analyzed in some specimens with the use of a similar method .


Most of the patients were Chinese ( 90 % ) .


Three patients were Malays , one was Indian , and one was European in this series .


Results indicated that there was strong correlation between ER level , age , and histologic grade of the tumors .


No correlation existed between absence or presence of lymph node metastases and ER .


Although there was a trend for ER -positive tumors to have a low-grade lymphocytic infiltration , the difference was not statistically significant .


Mononuclear cells infiltrating human mammary carcinomas : immunohistochemical analysis with monoclonal antibodies .


Breast carcinomas were examined by the immunoperoxidase technique using antisera specific for lymphocyte subsets , monocytes , NK cells and major histocompatibility antigens ( HLA-A , -B , -C ; Ia-like ) .


Sixty-four per cent of the patients had a moderate or strong mononuclear cell infiltration , 77 % of the patients without mononuclear cell infiltration had receptors for estrogens as compared to 51 % of the patients with infiltration .


The majority of the infiltrating mononuclear cells were T cells ; generally the OKT8 cells were predominant .


The Leu 3A/OKT8 cell ratio was not related to histological type , tumor size , age of the patient or presence of metastases .


Some of the T cells had the Ia antigen and were thus probably activated .


The B cells were either absent or less numerous than the T cells .


There was no relation between their distribution and the various parameters studied .


A few monocytes were heterogeneous according to their markers ( OKM I and acid phosphatase ) .


In 6 cases only there was a strong infiltration of mononuclear cells positive for acid phosphatase .


The number of the natural killer cells was also low .


Only a few mononuclear infiltrating cells had receptors for transferrin .


There was a positive correlation between the inflammatory infiltration and the presence of HLA class-I antigens on tumor cell s .


Some of the antisera specific for lymphocyte subsets also stained the breast carcinoma cells .


The great variations in the subsets of mononuclear cells in breast carcinomas may correspond to various systems of defense against neoplasm .


A case of male pseudohermaphroditism with normal androgen receptor binding and 47 , XYY karyotype .


A case of male pseudohermaphroditism with 47 , XYY karyotype in blood and cutaneous fibroblasts is described .


The plasma testosterone response to HCG stimulation was slightly below the normal range on two occasions suggesting a deficit of gonadal function .


A study of the receptors for dihydrotestosterone in fibroblasts of genital and nongenital skin showed a normal concentration of receptors in genital skin ; 5-alpha-reductase activity in fibroblasts of the genital skin was low , but the plasma relationship testosterone/dihydrotestosterone under HCG stimulation was normal .


The diagnostic possibility of a complete testicular feminization syndrome with normal receptors for dihydrotestosterone is commented on .


1 , 25-Dihydroxyvitamin D3 inhibits antigen-induced T cell activation .


The proliferative response of murine spleen and thymus cells to antigen but not to lectin was inhibited by the active metabolite of vitamin D3 , 1 , 25- ( OH ) 2D3 .


To directly examine the effect of 1 , 25- ( OH ) 2D3 on T cell activation in the absence of other complicating interactions , we utilized a panel of cloned Ia-restricted T cell hybridomas that secrete IL 2 on activation by cloned Ia-bearing stimulator cells ( TA3 ) or when stimulated by mitogen .


Physiologic concentrations of 1 , 25- ( OH ) 2D3 ( 0.01 to 0.1 nm ) inhibited the antigen-induced secretion of IL 2 by several of these T cell hybridomas .


This inhibition was dependent on the concentration of the free hormone and could be overcome by increasing the number of Ia-bearing stimulator cells used .


Pretreatment of the T hybridoma but not the TA3 stimulator cell with 1 , 25- ( OH ) 2D3 resulted in inhibition of activation .


These results are consistent with the finding that specific 1 , 25- ( OH ) 2D3 receptors are present on the T cell hybridomas but are lacking in TA3 cells .


1 , 25- ( OH ) 2D3 failed , however , to inhibit the activation of the T cell hybridomas by lectin or by an anti-Thy-1 antibody .


These findings suggest that 1 , 25- ( OH ) 2D3 may be interfering with early events of antigen-induced T cell activation , perhaps by hindering T cell recognition of the relevant antigen on stimulator cell surfaces .


This system should prove useful in studying the molecular mechanisms by which 1 , 25- ( OH ) 2D3 acts to inhibit T cell activation and subsequent IL 2 production .


Glucocorticoid receptors and steroid sensitivity in normal and neoplastic human lymphoid tissues : a review .


The determination of estrogen and progesterone receptors in breast cancer has been shown to be useful in predicting the response to endocrine therapy .


Given their well-known inhibitory effects on lymphoid tissue , glucocorticoids have been used widely in the treatment of leukemia .


Given these facts , over the last 10 years , several investigators have measured the number of glucocorticoid receptors in normal and neoplastic lymphoid tissue to see whether their number correlated with glucocorticoid responsiveness in vitro or in vivo .


No clear correlation could be established between the level of glucocorticoid receptor and the in vitro action of steroids in normal and neoplastic lymphoid tissue .


In contrast , attempts to correlate glucocorticoid receptor levels in acute lymphocytic leukemia to in vivo steroid responsiveness and immunological type using the whole-cell-binding assay for receptor determination and selecting the patients according to age and immunological criteria have been more successful .


[ Glucocorticoid receptors in normal human lymphocytes ]


Glucocorticoid ( GC ) receptors were studied in intact lymphocytes from 11 donors .


GC binding parameters were found to be highly reproducible in repeated experiments with lymphocytes .


It was shown that GC receptors in donors ' lymphocytes could be distributed into two different classes similarly to the pattern seen in skin fibroblasts .


Human lymphocytes are an adequate object for studying genetically determined variability of GC receptors and its clinical importance .


Specific estrogen binding sites in human lymphoid cells and thymic cells .