Number of glucocorticoid receptors in lymphocytes and their sensitivity to hormone action .


The study demonstrated a decreased level of glucocorticoid receptors ( GR ) in peripheral blood lymphocytes from hypercholesterolemic subjects , and an elevated level in patients with acute myocardial infarction .


In the lymphocytes with a high GR number , dexamethasone inhibited [ 3H ] -thymidine and [ 3H ] -acetate incorporation into DNA and cholesterol , respectively , in the same manner as in the control cells .


On the other hand , a decreased GR number resulted in a less efficient dexamethasone inhibition of the incorporation of labeled compounds .


These data showed that the sensitivity of lymphocytes to glucocorticoids changed only with a decrease of GR level .


[ 1 , 25-Dihydroxyvitamin D3 receptors in lymphocytes and T- and B-lymphocyte count in patients with glomerulonephritis ]


Content of receptors to hormonal form of vitamin D3 , 1.25 ( OH ) 2D3 , constituted 27.3 fmole/mg of protein in lymphocytes of peripheric blood of children with glomerulonephritis .


In the patients concentration of total and ionized form of Ca2+ was decreased down to 2.04 mmole/L and 1.09 mmole/L , respectively , while an increase in parathormone ( PTH ) by 36 % and a distinct decrease in 25 ( OH ) D concentration ( lower than 1.25 ng/ml ) was found in blood ; content of cAMP was also decreased in lymphocytes by 33 % .


At the same time , total content of T lymphocytes was decreased 1.5-fold in peripheric blood .


Treatment with I-hydroxyvitamin D3 ( 1-1.5 mg daily , within 4 weeks ) led to normalization of total and ionized form of Ca2+ and of 25 ( OH ) D , but did not affect the PTH content in blood .


Concentration of the receptors to 1.25 ( OH ) 2D3 was elevated up to 39.7 fmole/mg after I week of the treatment , whereas it was decreased to the initial level 24.8 fmole/mg within 4 weeks ; simultaneous alteration in the cAMP content was observed in lymphocytes .


Treatment with 1- ( OH ) D3 normalized also the T lymphocytes content in peripheric blood .


The data obtained suggest that under conditions of glomerulonephritis only high content of receptors to 1.25 ( OH ) 2D3 in lymphocytes enabled to perform the cell response to the hormone effect .


Tumor and serum beta-2-microglobulin expression in women with breast cancer .


To investigate whether the tumor expression of beta-2-microglobulin ( beta 2-M ) could serve as a marker of tumor biologic behavior , the authors studied specimens of breast carcinomas from 60 consecutive female patients .


Presence of beta 2-M was analyzed by immunohistochemistry .


No significant correlations were found between tumor beta 2-M expression and several histologic attributes such as type , histologic and nuclear grades , mitotic index , necrosis , vascular invasion , and lymphocytic infiltration .


Likewise , beta 2-M was not associated with markers of disease extension such as TNM , ( UICC , classification of malignant tumors ) staging and axillary lymph node involvement or with estrogen , progesterone , and glucocorticoid receptor levels .


However , there was a significantly positive association between tumor beta 2-M expression and the degree of lymphocytic infiltration in the tumor tissue .


Beta 2-M serum levels were determined by an enzyme-linked immunosorbent assay in samples from 22 of the above women .


Although some of the highest values had been obtained in women with larger ( T4 ) primary tumors , the authors failed to detect any statistical relationship between beta 2-M expression in the tumor with serum levels or between serum beta 2-M and the above histologic , laboratory , and clinical factors .


[ Preliminary observation of level free-form E receptor levels in serum of normal childbearing-aged and pregnant women ]


In 137 cases of childbearing-aged and pregnant women , free form E receptor levels ( sE ) in serum were measured by ELISA .


The level of sE was significantly decreased during the first trimester , slightly higher in the second trimester , and recovered to normal in the third trimester .


The level remained lower in 29 PIH women but appeared higher in overdue pregnancies as compared with the normal 3rd trimester range .


The results indicate that there is a relationship between a change in T cell function and pregnancy .


Kappa B-specific DNA binding proteins : role in the regulation of human interleukin-2 gene expression .


Transcriptional activation of the human interleukin-2 ( IL-2 ) gene , like induction of the IL-2 receptor alpha ( IL-2R alpha ) gene and the type 1 human immunodeficiency virus ( HIV-1 ) , is shown to be modulated by a kappa B-like enhancer element .


Mutation of a kappa B core sequence identified in the IL-2 promoter ( -206 to -195 ) partially inhibits both mitogen- and HTLV-I Tax-mediated activation of this transcription unit and blocks the specific binding of two inducible cellular factors .


These kappa B-specific proteins ( 80 to 90 and 50 to 55 kilodaltons ) similarly interact with the functional kappa B enhancer present in the IL-2R alpha promoter .


These data suggest that these kappa B-specific proteins have a role in the coordinate regulation of this growth factor-growth factor receptor gene system that controls T cell proliferation .


Novel region within the V kappa gene promoter is responsible for tissue and stage-specific expression of immunoglobulin genes in human lymphoid neoplasms .


Immunoglobulin gene-specific transacting factors have been shown to play a role in lymphoid tissue-specific expression of immunoglobulin genes .


The role of these factors in B-cell differentiation and stage-specific expression of these genes is , however , not fully understood .


We have used a model of human lymphoid neoplasia to address this question .


Different fragments of unrearranged human variable region of immunoglobulin kappa gene ( V kappa ) were used for cell-free in vitro transcription and DNA mobility shift assays .


Previously described enhancement of in vitro transcription that was only seen with nuclear extracts derived from B-cell neoplasms corresponding to the late stages of B-cell differentiation was shown to be dependent on the actions of these factor ( s ) on the DNA region within the V kappa gene promoter .


This region is located within the 920 bp fragment located 210 bp upstream from the coding region and this fragment represents a possible novel DNA region , which plays a role in the stage- and tissue-specific expression of immunoglobulin genes .


[ Determination of the sensitivity to glucocorticoids in vitro ]


A modified method for the determination of glucocorticoid receptors in human lymphocytes is suggested .


The principal distinction of the method is standardization by the lymphocyte count in a sample ( 1 mln ) and the labeled hormone concentration .


The modification saves time and money , limits the range of the data variations , and makes use of a lesser volume of blood .


Examinations of 70 children aged 4 to 15 suffering from the nephrotic form of glomerulonephritis have made it possible to distinguish two groups of patients : with relatively high values of specific binding X = 6820.1 +/- 530.0 ( n = 30 , p = 0.95 , t = 2.04 ) , this corresponding to a clinical form of hormone -sensitive glomerulonephritis , and with relatively low values of specific binding X = 1815.2 +/- 302.8 ( n = 40 , p = 0.95 , t = 1.96 ) , that corresponds to hormone -resistant glomerulonephritis .


Dynamic studies have not shown any statistically significant changes in the specific binding values .


These results permit regarding the specific binding value as a prognostic criterion in the assessment of corticosteroid therapy ; this allows a wide employment of the described method in practical nephrology .


Octamer-binding proteins from B or HeLa cells stimulate transcription of the immunoglobulin heavy-chain promoter in vitro .


The B-cell -type specificity of the immunoglobulin ( Ig ) heavy-chain and light-chain promoters is mediated by an octanucleotide ( OCTA ) element , ATGCAAAT , that is also a functional component of other RNA polymerase II promoters , such as snRNA and histone H2B promoters .


Two nuclear proteins that bind specifically and with high affinity to the OCTA element have been identified .


NF-A1 is present in a variety of cell types , whereas the presence of NF-A2 is essentially confined to B cells , leading to the hypothesis that NF-A2 activates cell-type-specific transcription of the Ig promoter and NF-A1 mediates the other responses of the OCTA element .


Extracts of the B-cell line , BJA-B , contain high levels of NF-A2 and specifically transcribe Ig promoters .


In contrast , extracts from HeLa cells transcribed the Ig promoter poorly .


Surprisingly , addition of either affinity-enriched NF-A2 or NF-A1 to either a HeLa extract or a partially purified reaction system specifically stimulates the Ig promoter .


This suggests that the constitutive OCTA-binding factor NF-A1 can activate transcription of the Ig promoter and that B-cell -specific transcription of this promoter , at least in vitro , is partially due to a quantitative difference in the amount of OCTA-binding protein .


Because NF-A1 can stimulate Ig transcription , the inability of this factor to activate in vivo the Ig promoter to the same degree as the snRNA promoters probably reflects a difference in the context of the OCTA element in these two types of promoters .


Identification of a putative regulator of early T cell activation genes .


Molecules involved in the antigen receptor-dependent regulation of early T cell activation genes were investigated with the use of functional sequences of the T cell activation-specific enhancer of interleukin-2 ( IL-2 ) .


One of these sequences forms a protein complex , NFAT-1 , specifically with nuclear extracts of activated T cells .


This complex appeared 10 to 25 minutes before the activation of the IL-2 gene .


Studies with inhibitors of protein synthesis indicated that the time of synthesis of the activator of the IL-2 gene in Jurkat T cells corresponds to the time of appearance of NFAT-1 .


NFAT-1 , or a very similar protein , bound functional sequences of the long terminal repeat ( LTR ) of the human immunodeficiency virus type 1 ; the LTR of this virus is known to be stimulated during early T cell activation .


The binding site for this complex activated a linked promoter after transfection into antigen receptor-activated T cells but not other cell types .


These characteristics suggest that NFAT-1 transmits signals initiated at the T cell antigen receptor .


Characterization of thyroid hormone receptors in human IM-9 lymphocytes .


Although putatively identified more than 10 years ago , thyroid hormone receptors in human tissues remain poorly characterized .


As a first step towards understanding the mechanism of thyroid hormone action in man we have characterized T3 binding sites in nuclei of the human lymphoblastoid line , IM-9 cells .


In whole cell experiments at 37 degrees C , nuclear binding of [ 125I ] T3 was saturable ( Kd 34 +/- 6 pmol/l ) and of finite capacity ( approximately equal to 350 sites/cell ) .


The binding sites were extracted from a nuclear pellet by treatment with 0.4 mol/l KCl and sonication .


Separation of bound from free [ 125I ] T3 in the extracts was achieved using the calcium phosphate matrix , hydroxyapatite at a concentration of 0.3 ml of a 150 g/l slurry .


Rectilinear Scatchard plots were obtained only when the hydroxyapatite was washed with a buffer containing 0.5 % Triton X-100 .


Under these conditions T3 binding sites in the nuclear extracts were present at a concentration of 22.4 +/- 8.6 fmol/mg protein and showed an affinity of ( Kd , room temperature ) 140 +/- 10 pmol/l .


The same assay system was used to determine the hierarchy of affinities for a range of natural and synthetic analogues .


These results suggest that the T3 binding sites present in human IM-9 lymphocyte nuclei and extracts thereof are thyroid hormone receptors .


These cells may be a useful tool to increase our understanding of human T3 receptors


Definition of T-cell specific DNA-binding factors that interact with a 3'-silencer in the CD4+ T-cell gene Rpt-1 .


Analysis of the region 3 ' to the CD4+ T-cell gene Rpt-1 ( encoding regulatory protein T-lymphocyte 1 ) led to the definition of a silencer element that inhibits heterologous gene expression in certain CD4+ T-cell lines but not in B-cell or non-lymphoid cell lines .


Functional silencer activity in vivo was associated with the presence of a specific silencer-DNA-protein complex in electrophoretic mobility shift assays with T-cell extracts .


Formation of this complex was selectively inhibited by the region in HIV-1 containing a silencer element .


We discuss the possibility that DNA-binding factors may coregulate HIV-1 and Rpt-1 gene expression through a common transcriptional silencer element .


Congenital immunodeficiencies associated with absence of HLA class II antigens on lymphocytes result from distinct mutations in trans-acting factors .


Coordinate regulation of HLA class II gene expression during development and coinduction of class II genes by soluble factors suggests that common trans-acting factor ( s ) control expression of these genes .


In B-lymphoblastoid cell lines derived from two independent class II-deficient bare lymphocyte syndrome patients , we observed a drastic decrease in transcription rates of the class II genes .


When these cell lines are fused , class II genes are reexpressed , indicating that immunodeficiencies in bare lymphocyte syndrome patients are the result of two distinct mutations .


Further studies show that genes governing the expression of class II antigens fall into at least three complementation groups ; two of these were previously unidentified in mutant cell lines generated in vitro .


In addition , we report the identification of two discrete complexes , NFX1.1 and NFX1.2 , that bind to the DRA X consensus element .


Though the mutation in at least one mutant line generated in vitro ( RJ2.2.5 ) affects products functioning via interaction with the X box , clear alterations in either NFX1.1 or NFX1.2 are not found in any of the mutant cell lines .


In vivo responsiveness to glucocorticoid correlated with glucocorticoid receptor content in peripheral blood leukocytes in normal humans .


Dexamethasone loading tests ( 0.1 mg dexamethasone/kg , iv ) were performed in 18 normal males to evaluate the individual responsiveness to glucocorticoid .


There were inter-individual differences in increase in peripheral blood polymorphonuclear leukocyte count , decrease in peripheral blood lymphocyte count , and increase in plasma free fatty acids levels after dexamethasone injection .


In addition , there was a significant correlation between the maximum increase in polymorphonuclear leukocytes and the maximum decrease in lymphocytes ( r = 0.7514 , p less than 0.0003 ) .


Simultaneous measurements of glucocorticoid receptor content by whole-cell assay revealed that glucocorticoid receptor content in polymorphonuclear leukocytes linearly correlated with that in the corresponding lymphocytes ( r = 0.9482 , p less than 0.0001 ) .


There were also significant correlations between the maximum increase in polymorphonuclear leukocytes and glucocorticoid receptor content in polymorphonuclear leukocytes ( r = 0.7239 , p less than 0.0007 ) , and between the maximum decrease in lymphocytes and glucocorticoid receptor content in lymphocytes ( r = 0.7703 , p less than 0.0002 ) .


These results suggest that individual differences are preserved both in glucocorticoid responsiveness and in glucocorticoid receptor content in peripheral blood leukocytes in normal humans .


Estradiol receptors in the cytosol of peripheral blood mononuclear cells in hepatitis B virus carriers treated with interferon-alpha .


Estradiol receptors in the cytosol of peripheral blood mononuclear cells and the effects of interferon-alpha ( IFN-alpha ) on estradiol receptors were studied in asymptomatic hepatitis B virus ( HBV ) carriers , patients with chronic hepatitis B and normal controls .


The level of estradiol receptors in the cytosol of mononuclear cells was significantly lower in asymptomatic HBV carriers and patients with chronic hepatitis B , compared to normal controls .


This low level of cytosol estradiol receptors in patients with chronic hepatitis B was increased by the administration of IFN-alpha .


In addition , when peripheral blood mononuclear cells from patients with chronic hepatitis B were incubated with IFN-alpha in vitro , the level of cytosol estradiol receptors also increased by increasing the concentration of IFN-alpha .


We previously reported that the response of mononuclear cells to estrogen is impaired in HBV carriers , and our present results suggested that this may be due to the low level of estradiol receptors in the cytosol of mononuclear cells .


Association of increased lytic effector cell function with high estrogen receptor levels in tumor-bearing patients with breast cancer .


Tumor-bearing patients with breast cancer were assayed for their natural killer ( NK ) cell activity and for the function of activated cytotoxic T-cells , as assessed by lectin-dependent cellular cytotoxicity ( LDCC ) .


Tumor-bearing patients with breast cancer had a significant increase in NK activity and in LDCC , as compared with healthy control individuals .


Although the enhanced NK cell activity and LDCC were closely associated with high levels ( greater than 31 fmol/mg ) of estrogen receptor ( ER ) content in the primary tumor , no other clinical or histologic correlation between the increase in either parameter of cytotoxic effector cell function could be found .


Thus , ER levels greater than 31 fmol/mg might be associated with increased cytotoxic effector cell function in tumor-bearing patients with breast cancer .


Properties of glucocorticoid receptors in Epstein-Barr virus-transformed lymphocytes from patients with familial cortisol resistance .


In a previous report of two patients with familial glucocorticoid resistance due to reduced numbers of glucocorticoid receptors ( GR ) , we have shown decreased numbers of GR in peripheral mononuclear cells and cultured fibroblasts but normal affinity of GR in both patients .


In this study , peripheral lymphocytes from these patients , one patient 's son and daughter , and normal subjects were transformed with Epstein-Barr virus .


Reduced numbers and normal affinity of GR were found in the Epstein-Barr virus-transformed lymphocytes from both patients while the son and daughter had normal numbers and affinity of GR .


The thermal stability of GR and thermal activation of cytosolic receptors in both patients were found to be normal .


Although the percentages of nuclear bound GR were similar in both patients and normal controls , the absolute amounts of nuclear bound GR of the patients were about one-half that of normal controls .


These abnormal properties of GR ( reduced numbers of GR ) were preserved in the transformed cells from the patients .


Octamer transcription factors 1 and 2 each bind to two different functional elements in the immunoglobulin heavy-chain promoter .


Immunoglobulin heavy-chain genes contain two conserved sequence elements 5 ' to the site of transcription initiation : the octamer ATGCAAAT and the heptamer CTCATGA .


Both of these elements are required for normal cell-specific promoter function .


The present study demonstrates that both the ubiquitous and lymphoid-cell-specific octamer transcription factors ( OTF-1 and OTF-2 , respectively ) interact specifically with each of the two conserved sequence elements , forming either homo- or heterodimeric complexes .


This was surprising , since the heptamer and octamer sequence motifs bear no obvious similarity to each other .


Binding of either factor to the octamer element occurred independently .


However , OTF interaction with the heptamer sequence appeared to require the presence of an intact octamer motif and occurred with a spacing of either 2 or 14 base pairs between the two elements , suggesting coordinate binding resulting from protein-protein interactions .


The degeneracy in sequences recognized by the OTFs may be important in widening the range over which gene expression can be modulated and in establishing cell type specificity .


Identification of a novel lymphoid specific octamer binding protein ( OTF-2B ) by proteolytic clipping bandshift assay ( PCBA ) .


The octamer sequence ATGCAAAT is found in the promoters of immunoglobulin ( Ig ) heavy and light chain genes and in the heavy chain enhancer and is a major determinant of the cell type specific expression of Ig genes in B cells .


An apparent paradox is that the same sequence serves as an upstream promoter or enhancer element in a variety of housekeeping genes such as the histone H2B and U snRNA genes .


The differential usage of this regulatory sequence motif is thought to be mediated by different species of octamer binding proteins .


One species of 100 kd , designated OTF-1 , is present in all cell types and may exert its activating function only when it can interact with additional adjacent transcription factors .


The lymphoid cell specific octamer binding protein of 60 kd ( OTF-2A ) specifically stimulates Ig promoters which consist essentially of a TATA-box and an octamer sequence upstream of it .


Here we present evidence for yet another B cell specific octamer binding protein of 75 kd ( OTF-2B ) .


From several findings , including the absence of OTF-2B ( but not OTF-2A ) from a lymphocyte line that can not respond to the IgH enhancer , we propose a role of the novel octamer factor in the long range activation by the IgH enhancer .


We have used the proteolytic clipping bandshift assay ( PCBA ) technique to distinguish the three different forms found in B cells .


This analysis indicates that the 75 kd-species OTF-2B is closely related to the 60 kd species OTF-2A .


Inhibition of interleukin 2 -induced proliferation of cloned murine T cells by glucocorticoids .


Possible involvement of an inhibitory protein .


The ability of glucocorticoids to inhibit interleukin 2 ( IL 2 ) -induced T cell proliferation in two cytotoxic T cell ( CTL ) clones has been studied .


A complete inhibition of DNA synthesis by dexamethasone ( Dx ) could be observed when IL 2-depleted cultures of CTL were either incubated for 6 h with the hormone prior to the addition of IL 2 or treated simultaneously with Dx and a low concentration of IL 2 .


No significant reduction in the number and affinity of IL 2 receptors was seen after 6 h incubation with Dx .


The order of potency observed with the different steroids indicated that this inhibitory effect was mediated through binding to a specific glucocorticoid receptor .


The action of these hormones possibly involves the synthesis of an inhibitory protein ( s ) , since the presence of cycloheximide during the incubation with Dx prevented the inhibition of DNA synthesis .


Furthermore , supernatant from Dx-treated CTL contained a nondialyzable factor which inhibited DNA synthesis and cell growth of CTL clones induced by IL 2 .


Blocking of IL 2 synthesis and IL 2 receptor formation have been proposed as one of the major mechanisms of glucocorticoid-induced immunosuppression .


Our results indicate that these hormones may also affect T cell proliferation by inhibiting IL 2 activity .


Identification and purification of a human immunoglobulin-enhancer-binding protein ( NF-kappa B ) that activates transcription from a human immunodeficiency virus type 1 promoter in vitro .


The enhancer-binding factor NF-kappa B , which is found only in cells that transcribe immunoglobulin light chain genes , has been purified from nuclear extracts of Namalwa cells ( human Burkitt lymphoma cells ) by sequence-specific DNA affinity chromatography .


The purified NF-kappa B has been identified as a 51-kDa polypeptide by UV-crosslinking analysis .


`` Footprint '' and methylation-interference analyses have shown that purified NF-kappa B has a binding activity specific for the kappa light chain enhancer sequence .


The purified factor activated in vitro transcription of the human immunodeficiency virus type I promoter by binding to an upstream NF-kappa B-binding site


Lymphocyte glucocorticoid receptor binding in depression : normal values following recovery .


The number of glucocorticoid receptor sites in lymphocytes and plasma cortisol concentrations were measured in 20 patients who had recovered from major depressive disorder and 20 healthy control subjects .


The number of glucocorticoid receptor sites in lymphocytes from the recovered depressed group was not significantly different from that of the control group .


Although the mean plasma cortisol concentration in recovered depressives was higher than in control subjects , the difference only just reached significance .


This study shows that the reduction in glucocorticoid receptor numbers which occurs during acute depressive illness does not persist on recovery and is , therefore , state-dependent .


Identification and purification of a human lymphoid-specific octamer-binding protein ( OTF-2 ) that activates transcription of an immunoglobulin promoter in vitro .


The octamer sequence 5'-ATGCAAAT , in either orientation , serves as an upstream element in a variety of promoters and also occurs as a modular enhancer element .


It is of particular interest in immunoglobulin genes since it is found in the upstream regions of all heavy and light chain promoters and in the heavy chain enhancer , both of which are known to be necessary for cell-specific expression .


We report here the chromatographic separation of ubiquitous and B cell-specific octamer-binding proteins .


The B cell factor was purified to homogeneity using affinity chromatography and consists of three peptides of 62 , 61 , and 58.5 +/- 1.5 kd .


Each of the polypeptides was renatured after SDS-PAGE and shown to bind to the octamer sequence .


The specific DNA binding activity of the pure B cell-specific factor was indistinguishable from that of the affinity-purified ubiquitous factor .


This B cell-specific octamer-binding factor , in pure form , activated transcription from a kappa light chain promoter in vitro , thus demonstrating that it is indeed a B cell-specific transcription factor for this gene .


In addition to the ubiquitous and B cell-specific octamer-binding factors , we identified several additional proteins , one of which is B cell -specific , that interact with the kappa promoter .


Decreased deoxyribonucleic acid binding of glucocorticoid-receptor complex in cultured skin fibroblasts from a patient with the glucocorticoid resistance syndrome .


A patient with the syndrome of glucocorticoid resistance was studied .


A 27-yr-old woman initially was diagnosed as having Cushing 's disease , based on the findings of high plasma ACTH and serum cortisol levels , increased urinary cortisol secretion , resistance to adrenal suppression with dexamethasone , and bilateral adrenal hyperplasia by computed tomography and scintigraphy of the adrenal glands .


However , she had no signs or symptoms of Cushing 's syndrome .


During a 5-yr follow-up , no clinical abnormalities developed , although hypercortisolism persisted .


End-organ resistance to cortisol was suspected .


To explain the end-organ resistance to cortisol , the glucocorticoid receptors ( GR ) in peripheral mononuclear leukocytes and cultured skin fibroblasts from a forearm skin biopsy were characterized and compared with the results of similar studies in normal subjects .


The patient 's GR in whole cell assays had an increased dissociation constant ( Kd ) .


In the cytosol of cultured skin fibroblasts from the patient , there was also decreased binding capacity .


The thermal stability and the sedimentation coefficient in a sucrose density gradient of the receptors in the cytosol of cultured skin fibroblasts from the patient and normal subjects were similar .


GR complex activation , analyzed by DEAE-cellulose chromatography , was decreased in the patient .


DNA binding of the GR complex after temperature-induced activation was lower in the patient than in normal subjects .


Nuclear translocation of GR complexes from the patient was also slightly decreased .


These results suggest that the patient 's glucocorticoid resistance was due to a decrease in the affinity of the receptor for glucocorticoids and a decrease in the binding of the GR complex to DNA .


Granulocyte-macrophage colony-stimulating factor .


Sensitive and receptor-mediated regulation by 1 , 25-dihydroxyvitamin D3 in normal human peripheral blood lymphocytes .


We show that 1 , 25-dihydroxyvitamin D3 ( 1 , 25 [ OH ] 2D3 ) , the most hormonally active metabolite of vitamin D3 , modulates sensitively and specifically both the protein and messenger RNA accumulation of the multilineage growth factor granulocyte-macrophage colony-stimulating factor ( GM-CSF ) .


The regulation of GM-CSF expression is seen in both normal human mitogen-activated T lymphocytes and T lymphocytes from a line ( S-LB1 ) transformed with human T cell lymphotropic virus 1 ( HTLV-1 ) .


In contrast , cells from a HTLV-1 transformed T lymphocyte line ( Ab-VDR ) established from a patient with vitamin D-resistant rickets type II with undetectable 1 , 25 ( OH ) 2D3 cellular receptors are resistant to the action of 1 , 25 ( OH ) 2D3 .


Inhibition of GM-CSF expression by 1 , 25 ( OH ) 2D3 can occur independently of interleukin 2 regulation and is probably mediated through cellular 1 , 25 ( OH ) 2D3 receptors .


We conclude that 1 , 25 ( OH ) 2D3 may be important in the physiology of hematopoiesis .


Altered interaction between triiodothyronine and its nuclear receptors in absence of cortisol : a proposed mechanism for increased thyrotropin secretion in corticosteroid deficiency states .


Thyroid hormones occasionally appear less effective when administered alone to patients with panhypopituitarism , and manifestations suggestive of hypothyroidism have been reported in patients suffering from untreated Addison 's disease .


In the latter condition , thyrotropin secretion is increased : this occurs already after as little as 2 days of temporary withdrawal of therapy with substitution doses of corticosteroids while circulating levels of thyroid hormones remain within normal limits .


Therefore , a possible role of cortisol in interaction between triiodothyronine and its nuclear receptors was examined at the level of circulating lymphocytes obtained from patients with primary or secondary adrenocortical failure .


The affinity of these receptors was found to be decreased , by more than 50 % on average , in the absence of cortisol treatments .


This change was promptly corrected upon resumption of therapy .


The number of binding sites was not significantly modified .


The influence of cortisol on thyroid hormone receptors discussed here might account for the clinical observations mentioned above .


Inhibition by cortisol of human natural killer ( NK ) cell activity .


The effects of cortisol on the natural killer ( NK ) activity of human peripheral blood mononuclear ( PBM ) cells were studied in vitro using a direct 4-h 51Cr-release assay and K 562 cell line as a target .


Preincubation for 20 h of PBM cells drawn from healthy donors with 1 X 10 ( -8 ) to 1 X 10 ( -5 ) M cortisol resulted in a significant decrease of NK cell activity .


The magnitude of the suppression was directly related to the steroid concentration and inversely related to the number of effector cells .


Cortisol was able to minimize the enhancement of NK cytotoxicity obtainable in the presence of immune interferon ( IFN-gamma ) .


A significantly higher suppression was achieved after sequential exposure of PBM cells to cortisol and equimolar levels of prostaglandin E2 ( PgE2 ) .


The concomitant incubation with theophylline and isobutyl-methylxanthine failed to enhance the cortisol-induced suppression , whereas PgE2-dependent inhibition significantly increased after exposure of PBM cells to methyl-xanthines .


The inhibitory effect of cortisol was partially or totally prevented by the concomitant incubation with equimolar amounts of 11-deoxycortisol and RU 486 but not of progesterone .


Treatment of NK effectors with a monoclonal anti-human corticosteroid-binding globulin ( CBG ) antibody produced an enhancement of the spontaneous NK activity and a partial suppression of cortisol-mediated effects .


Our results suggest that endogenous glucocorticoids play a role in the regulation of NK cell-mediated cytotoxicity .


Since the effect of cortisol was additive to that of PgE2 and was not changed by phosphodiesterase inhibitors , it is conceivable that the hormone acts at a level different from the adenylate cyclase - phosphodiesterase system .


Data obtained with the use of antiglucocorticoids and the anti-CBG antibody are compatible with a role both of high-affinity glucocorticoid receptors and of CBG in mediating cortisol action on the human NK cell activity .


Interaction of cell-type-specific nuclear proteins with immunoglobulin VH promoter region sequences .


All human and murine immunoglobulin heavy chain variable region ( VH ) genes contain the sequence ATGCAAAT approximately 70 nucleotides 5 ' from the site of transcription initiation .


This octanucleotide , in reverse orientation , is also found in all light chain variable region ( VL ) genes , and in the immunoglobulin heavy chain transcriptional enhancer .


Transfection studies have established that this octamer is involved in the lymphoid-specific transcription of immunoglobulin genes .


Octamer-containing fragments have been reported to bind a factor present in nuclear extracts of human cell lines ; however , identical binding activity was detected in both B lymphoid and non-lymphoid cells .


Here we establish that nuclear extracts from distinct cell types differ in their ability to interact with octamer-containing fragments .


We have also detected a DNA-protein interaction that may be involved in the cell-type specificity of immunoglobulin expression , and we have determined that a sequence upstream of the octamer participates in an interaction with a nuclear protein ( s ) .


Preferential transcription of HTLV-I LTR in cell-free extracts of human T cells producing HTLV-I viral proteins .


The promoters of the adenovirus 2 major late gene , the mouse beta-globin gene , the mouse immunoglobulin VH gene and the LTR of the human T-lymphotropic retrovirus type I were tested for their transcription activities in cell-free extracts of four cell lines ; HeLa , CESS ( Epstein-Barr virus-transformed human B cell line ) , MT-1 ( HTLV-I-infected human T cell line without viral protein synthesis ) , and MT-2 ( HTLV-I-infected human T cell line producing viral proteins ) .


LTR was preferentially transcribed in the extracts of MT-2 although the other three genes were transcribed with relatively constant efficiencies in different extracts .


The results agree well with the previous in vivo studies on the promoter activity of HTLV-I LTR .


Mixing of HeLa and MT-2 extracts revealed the presence of a LTR-specific stimulating activity in MT-2 extracts .


A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes .


Trans-acting factors that mediate B-cell specific transcription of immunoglobulin genes have been postulated based on an analysis of the expression of exogenously introduced immunoglobulin gene recombinants in lymphoid and non-lymphoid cells .


Two B-cell-specific , cis-acting transcriptional regulatory elements have been identified .


One element is located in the intron between the variable ( V ) and constant ( C ) regions of both heavy and kappa light-chain genes and acts as a transcriptional enhancer .


The second element is found upstream of both heavy and kappa light-chain gene promoters .


This element directs lymphoid-specific transcription even in the presence of viral enhancers .


We have sought nuclear factors that might bind specifically to these two regulatory elements by application of a modified gel electrophoresis DNA binding assay .


We report here the identification of a human B-cell nuclear factor ( IgNF-A ) that binds to DNA sequences in the upstream regions of both the mouse heavy and kappa light-chain gene promoters and also to the mouse heavy-chain gene enhancer .


This sequence-specific binding is probably mediated by a highly conserved sequence motif , ATTTGCAT , present in all three transcriptional elements .


Interestingly , a factor showing similar binding specificity to IgNF-A is also present in human HeLa cells .


The new world primates as animal models of glucocorticoid resistance .


Many New World primate species have greatly increased plasma cortisol concentrations , decreased plasma cortisol binding globulin capacity and affinity , marked resistance of the hypothalamic-pituitary-adrenal axis to suppression by dexamethasone , and no biological evidence of glucocorticoid excess .


These primates also have high levels of circulating progesterone , estrogen , mineralocorticoid , androgen and vitamin D .


The glucocorticoid target tissues that have been examined ( circulating mononuclear lymphocytes and cultured skin fibroblasts ) have normal concentrations of glucocorticoid receptors with decreased affinity for dexamethasone .


Transformation of B-lymphocytes with the Epstein-Barr virus leads to glucocorticoid receptor induction that is less than that observed with cells from Old World primates .


The receptor in these cells has a low affinity for dexamethasone .


The low affinity leads to an increased loss of specific bound ligand during thermal activation .


The molecular weight of the receptor , determined by SDS-PAGE , is similar to that of Old World primates ( approximately 92 , 000 ) and the activation pattern per se , examined in vitro by heating cytosol and performing phosphocellulose chromatography , appears similar to that of human controls .


The ratios of nuclear to cytosolic hormone-receptor-complexes and of cytosolic activated to unactivated receptor complexes in intact cells are similar to Old World primates .


Results from mixing studies do not support the hypothesis that a binding inhibitor ( s ) or a deficient cytosolic positive modifier ( s ) of binding underlies the findings in these primates .


The New World primates , unlike men with the syndrome of primary cortisol resistance , have compensated for their condition with intra-adrenal and mineralocorticoid receptor adaptations .


Thus , unlike Old World primates , cortisol in New World primates has only weak sodium-retaining potency because the aldosterone receptor has a low affinity for cortisol .


The common element that would explain the apparent resistance to six steroid hormones in New World primates


Acetylation and modulation of erythroid Kruppel-like factor ( EKLF ) activity by interaction with histone acetyltransferases .


Erythroid Kruppel-like factor ( EKLF ) is a red cell-specific transcriptional activator that is crucial for consolidating the switch to high levels of adult beta-globin expression during erythroid ontogeny .


EKLF is required for integrity of the chromatin structure at the beta-like globin locus , and it interacts with a positive-acting factor in vivo .


We find that EKLF is an acetylated transcription factor , and that it interacts in vivo with CBP , p300 , and P/CAF .


However , its interactions with these histone acetyltransferases are not equivalent , as CBP and p300 , but not P/CAF , utilize EKLF as a substrate for in vitro acetylation within its trans-activation region .


The functional effects of these interactions are that CBP and p300 , but not P/CAF , enhance EKLF 's transcriptional activation of the beta-globin promoter in erythroid cells .


These results establish EKLF as a tissue-specific transcription factor that undergoes post-translational acetylation and suggest a mechanism by which EKLF is able to alter chromatin structure and induce beta-globin expression within the beta-like globin cluster .


Recognition of herpes simplex virus type 2 tegument proteins by CD4 T cells infiltrating human genital herpes lesions .


The local cellular immune response to herpes simplex virus ( HSV ) is important in the control of recurrent HSV infection .


The antiviral functions of infiltrating CD4-bearing T cells may include cytotoxicity , inhibition of viral growth , lymphokine secretion , and support of humoral and CD8 responses .


The antigens recognized by many HSV-specific CD4 T cells localizing to genital HSV-2 lesions are unknown .


T cells recognizing antigens encoded within map units 0.67 to 0.73 of HSV DNA are frequently recovered from herpetic lesions .


Expression cloning with this region of DNA now shows that tegument protein VP22 and the viral dUTPase , encoded by genes UL49 and UL50 , respectively , are T-cell antigens .


Separate epitopes in VP22 were defined for T-cell clones from each of three patients .


Reactivity with the tegument protein encoded by UL21 was identified for an additional patient .


Three new epitopes were identified in VP16 , a tegument protein associated with VP22 .


Some tegument-specific CD4 T-cell clones exhibited cytotoxic activity against HSV-infected cells .


These results suggest that herpes simplex tegument proteins are processed for antigen presentation in vivo and are possible candidate compounds for herpes simplex vaccines .


Fibrinogen activates NF-kappa B transcription factors in mononuclear phagocytes .


Adhesion to extracellular matrices is known to modulate leukocyte activation , although the mechanisms are not fully understood .


Mononuclear phagocytes are exposed to fibrinous provisional matrix throughout migration into inflammatory foci , so this study was undertaken to determine whether fibrinogen triggers activation of NF-kappa B transcription factors .


U937 cells differentiated with PMA in nonadherent culture were shown to express two fibrinogen-binding integrins , predominately CD11b/CD18 , and to a lesser extent , CD11c/CD18 .


Cells stimulated with fibrinogen ( 10-100 microg/ml ) /Mn2+ ( 50 microM ) for 2 h were examined by electrophoretic mobility shift assay .


NF-kappa B activation , minimal in unstimulated cells , was substantially up-regulated by fibrinogen .


Fibrinogen also caused activation of AP-1 , but not SP1 or cAMP response element-binding protein ( CREB ) factors .


Blocking mAbs against CD18 and CD11b abrogated fibrinogen -induced NF-kappa B activation .


To determine the effects on transcriptional regulation , U937 cells were transfected with a plasmid containing the HIV-1 enhancer ( bearing two NF-kappa B sites ) coupled to a chloramphenicol acetyltransferase ( CAT ) reporter .


Cells were subsequently stimulated with 1 ) PMA for 24 h , inducing CAT activity by 2.6-fold , 2 ) fibrinogen /Mn2+ for 2 h , inducing CAT activity by 3.2-fold , or 3 ) costimulation with fibrinogen and PMA , inducing 5.7-fold the CAT activity induced by PMA alone .


We conclude that contact with fibrinogen-derived proteins may contribute to mononuclear phagocyte activation by signaling through CD11b/CD18 , resulting in selective activation of transcriptional regulatory factors , including NF-kappa B .


Peripheral blood T cells and monocytes and B cell lines derived from patients with lupus express estrogen receptor transcripts similar to those of normal cells .


OBJECTIVE : To identify and characterize estrogen receptor ( ER ) transcripts expressed in immune cells of patients with systemic lupus erythematosus ( SLE ) and healthy donors .


METHODS : Peripheral blood monocytes and T cells were prepared from patients with SLE ( n = 6 ) and healthy donors ( n = 8 ) .


T cells were separated into CD4 and CD8 .


Some monocytes and T cells were stimulated with estradiol , PMA , and ionomycin .


Epstein-Barr virus-transformed B cell lines ( n = 7 ) and B cell hybridomas ( n = 2 ) established from patients with SLE and a healthy individual were used as a B cell source .


These cells were examined for ER mRNA by reverse transcription nested polymerase chain reaction .


Amplified cDNA were sequenced by standard methods .


RESULTS : In all cells tested , ER mRNA was expressed without prior in vitro stimulation .


Partial sequences from exons 1-8 were nearly identical to the published sequence of the human ER mRNA .


There were no notable differences in the ER transcripts between patients and healthy controls .


Variant receptor transcripts lacking exon 5 or exon 7 , which encodes the hormone binding domain , were identified in the majority of the cells .


Precise deletion of the exons suggests that they are alternatively spliced transcripts .


Whether the detected transcripts are translated into functional receptor proteins remains to be determined .


In vitro stimulation did not affect ER mRNA expression .


The presence of variants did not correlate with disease activity or medication .


CONCLUSION : Monocytes , T cells , and B cells in patients express transcripts of the normal wild type ER and the hormone binding domain variants in vivo .


DNA damaging agents induce expression of Fas ligand and subsequent apoptosis in T lymphocytes via the activation of NF-kappa B and AP-1 .


Apoptosis induced by DNA damage and other stresses can proceed via expression of Fas ligand ( FasL ) and ligation of its receptor , Fas ( CD95 ) .


We report that activation of the two transcription factors NF-kappa B and AP-1 is crucially involved in FasL expression induced by etoposide , teniposide , and UV irradiation .


A nondegradable mutant of I kappa B blocked both FasL expression and apoptosis induced by DNA damage but not Fas ligation .


These stimuli also induced the stress-activated kinase pathway ( SAPK/JNK ) , which was required for the maximal induction of apoptosis .


A 1.2 kb FasL promoter responded to DNA damage , as well as coexpression with p65 Rel or Fos/Jun .


Mutations in the relevant NF-kappa B and AP-1 binding sites eliminated these responses .


Thus , activation of NF-kappa B and AP-1 contributes to stress-induced apoptosis via the expression of FasL .


A small , nonpeptidyl mimic of granulocyte-colony-stimulating factor [ see commetns ]


A nonpeptidyl small molecule SB 247464 , capable of activating granulocyte-colony-stimulating factor ( G-CSF ) signal transduction pathways , was identified in a high-throughput assay in cultured cells .


Like G-CSF , SB 247464 induced tyrosine phosphorylation of multiple signaling proteins and stimulated primary murine bone marrow cells to form granulocytic colonies in vitro .


It also elevated peripheral blood neutrophil counts in mice .


The extracellular domain of the murine G-CSF receptor was required for the activity of SB 247464 , suggesting that the compound acts by oligomerizing receptor chains .


The results indicate that a small molecule can activate a receptor that normally binds a relatively large protein ligand .


Minimal residual disease in acute myelogenous leukemia with PML/RAR alpha or AML1/ETO mRNA and phenotypic analysis of possible T and natural killer cells in bone marrow .


Here we studied minimal residual disease ( MRD ) of patients with acute myeloid leukemia ( AML ) who have PML/RAR alpha or AML1/ETO as well as the phenotypic analysis of lymphocyte subsets involved in antitumor immunity .


Eight patients in long-term ( LT ; 3 to 15 years ) and 15 patients in short-term ( ST ; up to 3 years ) remission were studied .


Using the reverse transcription-polymerase chain reaction ( RT ) assay , the limit of detection was 10 ( -5 ) to 10 ( -6 ) for PML/RAR alpha transcript and 10 ( -4 ) to 10 ( -5 ) for the AML1/ETO transcript .


Simultaneously , T lymphocyte subsets and NK cells from the peripheral blood ( PB ) and bone marrow ( BM ) were investigated by flow cytometric analysis .


Four of the eight patients in LT and 7 of the 15 patients in ST remission were MRD-positive .


Although all MRD-positive patients in LT remission are still until now event-free , 3 of the 7 MRD-positive ( MRD+ ) patients in ST remission soon relapsed .


The total populations of CD4 + , CD8 + and CD56 + [ possible T-cell and natural killer ( T/NK ) populations ] in the BM of ST patients and MRD+/LT patients were significantly ( p < .01 ) low .


The CD8+ CD28+ population showed the same tendency ( p < .01-.02 ) .


The T/NK subsets in the BM of MRD-negative ( MRD- ) LT ( MRD-/LT ) patients showed similar numbers of cells as normal volunteers .


Basically , the total percentage of the CD4+ , CD8+ and CD56+ cell populations in the BM was increased and in the following order : MRD-/LT patients , normal volunteers , MRD+/LT patients and MRD+ or -/ST patients .


The percentages of the T/NK-cell subsets in the PB were not significantly different among these groups .


Thus , the difference of the possible T/NK-cell phenotype in the BM may strongly influence clinical and molecular remission .


These results still remain to be confirmed by further studies of the functional anti-tumor immunity of T/NK cells of AML in remission .


Mycobacterium tuberculosis mannose-capped lipoarabinomannan can induce NF-kappaB -dependent activation of human immunodeficiency virus type 1 long terminal repeat in T cells .


Tuberculosis has emerged as an epidemic , extended by the large number of individuals infected with human immunodeficiency virus type 1 ( HIV-1 ) .


The major goal of this study was to determine whether the mycobacterial cell wall component mannose-capped lipoarabinomannan ( ManLAM ) of Mycobacterium tuberculosis ( M. tuberculosis ) could activate transcription of HIV-1 in T cells with the use of an in vitro cell culture system .


These experiments are of prime importance considering that CD4 -expressing T lymphocytes represent the major virus reservoir in the peripheral blood of infected individuals .


Using the 1G5 cell line harbouring the luciferase reporter gene under the control of the HIV-1 LTR , it was first found that culture protein filtrates ( CFP ) from M. tuberculosis or purified ManLAM could activate HIV-1 LTR-dependent gene expression unlike similarly prepared CFP extracts devoid of ManLAM .


The implication of protein tyrosine kinase ( s ) , protein kinase A and/or protein kinase C was highlighted by the abrogation of the ManLAM-mediated activation of HIV-1 LTR-driven gene expression using herbimycin A and H7 .


It was also determined , using electrophoresis mobility shift assays , that M. tuberculosis ManLAM led to the nuclear translocation of the transcription factor NF-kappaB .


M. tuberculosis ManLAM resulted in clear induction of the luciferase gene placed under the control of the wild-type , but not the kappaB-mutated , HIV-1 LTR region .


Finally , the ManLAM-mediated activation of HIV-1 LTR transcription was found to be independent of the autocrine or paracrine action of endogenous TNF-alpha .


The results suggest that M. tuberculosis can upregulate HIV-1 expression in T cells and could thus have the potential to influence the pathogenesis of HIV-1 infection .


Human immunodeficiency virus type 1 long terminal repeat quasispecies differ in basal transcription and nuclear factor recruitment in human glial cells and lymphocytes .


The generation of genomic diversity during the course of infection has the potential to affect all aspects of HIV-1 replication , including expression of the proviral genome .


To gain a better understanding of the impact of long terminal repeat ( LTR ) sequence diversity on LTR-directed gene expression in cells of the central nervous system ( CNS ) and immune system , we amplified and cloned LTRs from proviral DNA in HIV-1-infected peripheral blood .


Sequence analysis of nineteen LTRs cloned from 2 adult and 3 pediatric patients revealed an average of 33 nucleotide changes ( with respect to the sequence of the LAI LTR ) within the 455-bp U3 region .


Transient expression analyses in cells of neuroglial and lymphocytic origin demonstrated that some of these LTRs had activities which varied significantly from the LAI LTR in U-373 MG cells ( an astrocytoma cell line ) as well as in Jurkat cells ( a CD4-positive lymphocyte cell line ) .


While LTRs which demonstrated the highest activities in U-373 MG cells also yielded high activities in Jurkat cells , the LTRs were generally more active in Jurkat cells when compared to the LAI LTR .


Differences in LTR sequence also resulted in differences in transcription factor recruitment to cis-acting sites within the U3 region of the LTR , as demonstrated by electrophoretic mobility shift assays .


In particular , naturally occurring sequence variation impacted transcription factor binding to an activating transcription factor / cAMP response element binding ( ATF / CREB ) binding site ( located between the LEF-1 and distal NF-kappaB transcription factor binding sites ) that we identified in previous studies of the HIV-1 LTR .


These findings suggest that LTR sequence changes can significantly affect basal LTR function and transcription factor recruitment , which may , in turn , alter the course of viral replication in cells of CNS and immune system origin .


HMG box containing transcription factors in lymphocyte differentiation .


The identification of the mammalian sex-determining gene Sry has led to the discovery of a large family of related ( ' HMG box ' ) transcription factors that control developmental events in yeast , C. elegans , Drosophila and vertebrates .


In lymphocyte differentiation , several HMG box proteins play a decisive role .


Sox-4 is important for very early B-cell differentiation , while TCF-1 / LEF-1 play a crucial role in early thymocyte development .


TCF/LEF proteins have recently been found to constitute a downstream component of the Wingless/Wnt signal transduction pathway .


In flies , this pathway controls segment polarity ; in Xenopus it controls the definition of the body axis .


Deregulation of the pathway occurs in several human tumors .


These insights in the molecular events that are involved in TCF/LEF function in these organisms may eventually lead to the understanding of the function of these HMG box proteins in lymphoid development


Transcriptional regulation by C/EBP alpha and -beta in the expression of the gene for the MRP14 myeloid calcium binding protein .


Transcriptional regulation of the gene for the myeloid calcium binding protein , MRP14 , was investigated in human monocytic leukemia cell lines .


The MRP14 gene was not expressed in monoblastic ML-1 cells , promonocytic U-937 cells , or promyelocytic HL-60 cells .


On the other hand , the gene was expressed in monocytic THP-1 cells and in the HL-60 cells treated with 1 , 25-dihydroxyvitamin D3 ( VD3 ) .


The level of MRP14 in VD3-treated HL-60 cells was two-fold higher than that in THP-1 cells .


Among several known transcription factor binding motifs , nuclear protein ( s ) of VD3-treated HL-60 cells and THP-1 cells bound to the CCAAT/enhancer binding protein ( C/EBP ) -binding motif that was located in the upstream region of the MRP14 gene ( -81 ) , as evidenced by the competitive gel mobility-shift assay .


An antibody for C/EBP alpha super-shifted the nucleoprotein complex in THP-1 cells but not in the VD3-treated HL-60 cells , whereas an antibody for C/EBP beta blocked the formation of the complex with the nuclear factor of the HL-60 cells but not with that of THP-1 cells .


An anti-C/EBP delta antibody had no effect on the complex in either cell .


Thus , it was concluded that C/EBP alpha and -beta were able to bind to the C/EBP motif , and that C/EBP alpha bound to the motif in THP-1 cells and C/EBP beta bound to that in the VD3-treated HL-60 cells .


Furthermore , to examine the transcriptional activity of the C/EBP motif , we transfected several constructed luciferase reporter DNAs into HL-60 cells and THP-1 cells .


The luciferase activity of the C/EBP motif in HL-60 cells was increased by VD3 treatment .


The C/EBP motif in the MRP14 gene was confirmed to function as a regulatory region in VD3-treated HL-60 cells and THP-1 cells by the assay .


Since C/EBP beta was also detected in VD3-untreated HL-60 cells by immunoblotting , VD3 activated C/EBP beta to bind to the motif , probably through post-translational modification .


Patients with high-risk myelodysplastic syndrome can have polyclonal or clonal haemopoiesis in complete haematological remission .


The clonality of mature peripheral blood-derived myeloid and lymphoid cells and bone marrow haemopoietic progenitors from 18 females with myelodysplasia ( MDS ) ( five refractory anaemia , RA ; one RA with ringed sideroblasts , RARS ; three chronic myelomonocytic leukaemia , CMML ; four RA with excess of blasts , RAEB ; five RAEB in transformation , RAEB-t ) was studied by X-chromosome inactivation analysis .


Using the human androgen-receptor ( HUMARA ) assay , we analysed the clonal patterns of highly purified immature CD34+ 38- and committed CD34+ 38+ marrow-derived progenitors , and CD16+ 14- granulocytes , CD14+ monocytes , CD3+ T and CD19+ B lymphocytes from peripheral blood .


In high-risk patients ( RAEB , RAEB-t ) , clonality analysis was performed before and after intensive remission-induction treatment .


All patients , except one with RA , had predominance of a single clone in their granulocytes and monocytes .


The same clonal pattern was found in CD34+ progenitor cells .


In contrast , CD3+ T lymphocytes were polyclonal or oligoclonal in 14/18 patients .


X-chromosome inactivation patterns of CD19+ B cells were highly concordant with CD3+ T cells except for two patients ( one RA , one CMML ) with monoclonal B and polyclonal T lymphocytes , therefore suggesting a clonal mutation in a progenitor common to the myeloid and B-lymphoid lineages or the coexistence of MDS and a B-cell disorder in these particular patients .


After high-dose non-myeloablative chemotherapy , polyclonal haemopoiesis was reinstalled in the mature myeloid cells and immature and committed marrow progenitors in three of four patients achieving complete haematological remission .


Therefore we conclude that most haematological remissions in MDS are associated with restoration of polyclonal haemopoiesis .


Prominent sex steroid metabolism in human lymphocytes .


Steroid metabolism was investigated in cultured human B-lymphoblastoid cells ( B-LCL ) , and peripheral blood T and B cells .


Gene expression was examined by reverse-transcription polymerase chain reaction amplification ( RT-PCR ) .


Appropriate sized transcripts were detected in both cultured and fresh peripheral lymphocytes for CYP11A , CYP17 , HSD11L ( 11beta-hydroxysteroid dehydrogenase I ) , HSD17B1 ( 17beta-hydroxysteroid dehydrogenase type I ) and SRD5A1 ( 5alpha-reductase I ) .


B-LCL , but not T and B cells , expressed CYP11B .


There was minimal expression of HSD3B1 and HSD3B2 ( 3beta-hydroxysteroid dehydrogenase I and II ) in B-LCL and T cells .


Transcripts for CYP19 and HSD11K were not detected .


Corresponding enzymatic activity was detectable only for 17-hydroxysteroid dehydrogenase and 5alpha-reductase , respectively producing testosterone and 5alpha-dihydrotestosterone .


Steroid identities were confirmed by gas chromatography/mass spectrometry ( GC/MS ) .


One metabolite thought to be deoxycorticosterone was identified by GC/MS as 6alpha-hydroxypregnanolone .


It was concluded that sex hormone metabolism , including androgen synthesis , occurs in lymphocytes , and may modulate immune response .


Human white blood cells and hair follicles are good sources of mRNA for the pterin carbinolamine dehydratase/dimerization cofactor of HNF1 for mutation detection .


Pterin carbinolamine dehydratase/dimerization cofactor of HNF1 ( PCD/DCoH ) is a protein that has a dual function .


It is a pterin 4alpha-carbinolamine dehydratase that is involved in the regeneration of the cofactor tetrahydrobiopterin during the phenylalanine hydroxylase- catalyzed hydroxylation of phenylalanine .


In addition , it is the dimerization cofactor of HNF1 that is able to activate the transcriptional activity of HNF1 .


Deficiencies in the gene for this dual functional protein result in hyperphenylalaninemia .


Here we report for the first time that the PCD/DCoH mRNA is present in human white blood cells and hair follicles .


Taking advantage of this finding , a sensitive , rapid and convenient method for screening mutations occurring in the coding region of this gene has been described .


Biochemical characterization of the NF-Y transcription factor complex during B lymphocyte development .


The transcription factor , NF-Y , plays a critical role in tissue-specific major histocompatibility complex class II gene transcription .


In this report the biochemical properties of the heterotrimeric NF-Y complex have been characterized during stage-specific B-cell development , and in several class II- mutant B-cell lines , which represent distinct bare lymphocyte syndrome class II genetic complementation groups .


The NF-Y complex derived from class II+ mature B-cells bound with high affinity to anion exchangers , and eluted as an intact trimeric complex , whereas , NF-Y derived from class II- plasma B-cells , and from bare lymphocyte syndrome group II cell lines , RJ2.2.5 and RM3 , dissociated into discrete NF-YA and NF-YB : C subunit fractions .


Recombination of the MPC11 plasma B-cell derived NF-Y A : B : C complex with the low molecular mass protein fraction , NF-Y-associated factors ( YAFs ) , derived from mature A20 B-cell nuclei , conferred high affinity anion exchange binding to NF-Y as an intact trimeric complex .


Recombination of the native NF-YA : B : C complex with the transcriptional cofactor , PC4 , likewise conferred high affinity NF-Y binding to anion exchangers , and stabilized NF-Y interaction with CCAAT-box DNA motifs in vitro .


Interaction between PC4 and NF-Y was mapped to the C-terminal region of PC4 , and the subunit interaction subdomain of the highly conserved DNA binding-subunit interaction domain ( DBD ) of NF-YA .


These results suggest that in class II+ mature B-cells NF-Y is associated with the protein cofactor , PC4 , which may play an important role in NF-Y-mediated transcriptional control of class II genes .


Arrest of B lymphocyte terminal differentiation by CD40 signaling : mechanism for lack of antibody-secreting cells in germinal centers .


Despite extensive research , the role of CD40 signaling in B cell terminal differentiation remains controversial .


Here we show that CD40 engagement arrests B cell differentiation prior to plasma cell formation .


This arrest is manifested at a molecular level as a reduction in mRNA levels of secretory immunoglobulin gene products such as mu ( s ) and J chain as well as the loss of the transcriptional regulator BLIMP-1 .


Furthermore , the inhibition of B cell differentiation by CD40 engagement could not be overcome by either mitogens or cytokines , but could be reversed by antibodies that interfere with the CD40 / gp39 interaction .


These data suggest that secretory immunoglobulin is not produced by B cells that are actively engaged by gp39-expressing T cells .


A positively charged alpha-lipoic acid analogue with increased cellular uptake and more potent immunomodulatory activity .


alpha-Lipoic acid ( LA ) is taken up by cells and reduced to its potent dithiol form , dihydrolipoate ( DHLA ) , much of which is rapidly effluxed out from cells .


To improve retention in cells , the LA molecule was modified to confer a positive charge at physiological pH .


N , N-dimethyl , N'-2-amidoethyl-lipoate was synthesized .


The protonated form of the new molecule is referred to as LA-Plus .


The uptake of LA-Plus by human Wurzburg T cells was higher compared to that of LA .


Several-fold higher amounts of DHLA-Plus , the corresponding reduced form of LA-Plus , were detected in LA-Plus treated cells compared to the amount of DHLA found in cells treated with LA .


At 100 microM , LA did not but LA-Plus inhibited H2O2 induced NF-kappaB activation and NF-kappaB directed IL-2 receptor expression .


Both LA and LA-Plus synergised with selenium in inhibiting H2O2 induced NF-kappaB activation .


At 150 microM LA-Plus , but not LA , inhibited TNFalpha induced NF-kappaB activation .


At 5 microM LA-Plus , but not LA , protected against both spontaneous and etoposide induced apoptosis in rat thymocytes .


LA-Plus is thus an improved form of LA with increased therapeutic potential .


Carrier identification in X-linked immunodeficiency diseases .


OBJECTIVE : Carrier identification in X-linked immunodeficiency disorders can be based on the demonstration of non-random X inactivation ( NRXI ) in affected blood cell lineages when growth is impaired in cells expressing the abnormal gene .


We examined the utility of seeking evidence of NRXI to test the carrier status of women in families affected by X-linked severe combined immunodeficiency ( XSCID ) and X-linked hypogammaglobulinaemia ( XLH ) , to identify as carriers the mothers of boys with SCID or hypogammaglobulinaemia whose phenotype suggested X-linkage and to infer X-linkage in boys with SCID or hypogammaglobulinaemia whose disease was not clearly X-linked on the basis either of family history or clinical and immunological characteristics .


METHODOLOGY : A polymerase chain reaction-based method was used to amplify a polymorphic CAG repeat in the first exon of the androgen receptor gene after selective digestion of the active X chromosome with a methylation-sensitive enzyme , HpaII to distinguish between the paternal and maternal alleles and to identify their methylation status .


RESULTS : Heterozygosity was found in 24 of 31 female subjects ( 77 % ) .


As anticipated , NRXI could be demonstrated in all lymphoid cells studied from obligate carriers of XSCID and an obligate carrier of XLH but not on a carrier of X-linked immunodeficiency with hyper-IgM .


The finding of NRXI in the mother of a boy with a SCID variant showed her to be a carrier of XSCID and establishes that her son has XSCID , not otherwise evident from available data .


CONCLUSIONS : This PCR assay provides a rapid method for carrier detection of X-linked immunodeficiencies , and has allowed us to expand the phenotype of XSCID


Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycemia in a NF-kB-dependent fashion .


We addressed the role of hyperglycemia in leukocyte-endothelium interaction under flow conditions by exposing human umbilical vein endothelial cells for 24 h to normal ( 5 mM ) , high concentration of glucose ( 30 mM ) , advanced glycosylation end product-albumin ( 100 microg/ml ) , or hyperglycemic ( 174-316 mg/dl ) sera from patients with diabetes and abnormal hemoglobin A1c ( 8.1+/-1.4 % ) .


At the end of incubation endothelial cells were perfused with total leukocyte suspension in a parallel plate flow chamber under laminar flow ( 1.5 dyn/cm2 ) .


Rolling and adherent cells were evaluated by digital image processing .


Results showed that 30 mM glucose significantly ( P < 0.01 ) increased the number of adherent leukocytes to endothelial cells in respect to control ( 5 mM glucose ; 151+/-19 versus 33+/-8 cells/mm2 ) .


A similar response was induced by endothelial stimulation with IL-1beta , here used as positive control ( 195+/-20 cells/mm2 ) .


The number of rolling cells on endothelial surface was not affected by high glucose level .


Stable adhesion of leukocytes to glucose-treated as well as to IL-1beta-stimulated endothelial cells was preceded by short interaction of leukocytes with the endothelial surface .


The distance travelled by leukocytes before arrest on 30 mM glucose , or on IL-1beta-treated endothelial cells , was significantly ( P < 0.01 ) higher than that observed for leukocytes adhering on control endothelium ( 30 mM glucose : 76.7+/-3.5 ; IL1beta : 69.7+/-4 versus 5 mM glucose : 21.5+/-5 microm ) .


Functional blocking of E-selectin , intercellular cell adhesion molecule-1 , and vascular cell adhesion molecule-1 on endothelial cells with the corresponding mouse mAb significantly inhibited glucose-induced increase in leukocyte adhesion ( 67+/-16 , 83+/-12 , 62+/-8 versus 144+/-21 cells/ mm2 ) .


Confocal fluorescence microscopy studies showed that 30 mM glucose induced an increase in endothelial surface expression of E-selectin , intercellular cell adhesion molecule-1 , and vascular cell adhesion molecule-1 .


Electrophoretic mobility shift assay of nuclear extracts of human umbilical vein endothelial cells ( HUVEC ) exposed for 1 h to 30 mM glucose revealed an intense NF-kB activation .


Treatment of HUVEC exposed to high glucose with the NF-kB inhibitors pyrrolidinedithiocarbamate ( 100 microM ) and tosyl-phe-chloromethylketone ( 25 microM ) significantly reduced ( P < 0.05 ) leukocyte adhesion in respect to HUVEC treated with glucose alone .


A significant ( P < 0.01 ) inhibitory effect on glucose-induced leukocyte adhesion was observed after blocking protein kinase C activity with staurosporine ( 5 nM ) .


When HUVEC were treated with specific antisense oligodesoxynucleotides against PKCalpha and PKCepsilon isoforms before the addition of 30 mM glucose , a significant ( P < 0.05 ) reduction in the adhesion was also seen .


Advanced glycosylation end product-albumin significantly increased the number of adhering leukocytes in respect to native albumin used as control ( 110+/-16 versus 66+/-7 , P < 0.01 ) .


Sera from diabetic patients significantly ( P < 0.01 ) enhanced leukocyte adhesion as compared with controls , despite normal levels of IL-1beta and TNFalpha in these sera .


These data indicate that high glucose concentration and hyperglycemia promote leukocyte adhesion to the endothelium through upregulation of cell surface expression of adhesive proteins , possibly depending on NF-kB activation .


Ikaros in hemopoietic lineage determination and homeostasis .


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


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


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


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


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


Induction of early B cell factor ( EBF ) and multiple B lineage genes by the basic helix-loop-helix transcription factor E12 .


The transcription factors encoded by the E2A and early B cell factor ( EBF ) genes are required for the proper development of B lymphocytes .


However , the absence of B lineage cells in E2A- and EBF-deficient mice has made it difficult to determine the function or relationship between these proteins .


We report the identification of a novel model system in which the role of E2A and EBF in the regulation of multiple B lineage traits can be studied .


We found that the conversion of 70Z/3 pre-B lymphocytes to cells with a macrophage-like phenotype is associated with the loss of E2A and EBF .


Moreover , we show that ectopic expression of the E2A protein E12 in this macrophage line results in the induction of many B lineage genes , including EBF , IL7Ralpha , lambda5 , and Rag-1 , and the ability to induce kappa light chain in response to mitogen .


Activation of EBF may be one of the critical functions of E12 in regulating the B lineage phenotype since expression of EBF alone leads to the activation of a subset of E12 -inducible traits .


Our data demonstrate that , in the context of this macrophage line , E12 induces expression of EBF and together these transcription factors coordinately regulate numerous B lineage-associated genes .


Activation-induced down-regulation of retinoid receptor RXRalpha expression in human T lymphocytes .


A 5.4-kilobase mRNA , the expression of which is down-regulated after treatment of human peripheral blood mononuclear cells ( PBMCs ) with various T cell-activating agents , was isolated using an mRNA differential display method .


Nucleotide sequence analysis identified the 5 ' end of this RNA as human retinoid receptor RXRalpha mRNA .


Here , we report the nucleotide sequence of 3.6 kilobases of this RNA , which represents the 3 ' end of RXRalpha mRNA , the sequence of which has not been previously described .


Activated PBMCs also expressed lower levels of RXRalpha protein , and a DNA binding assay showed that the activation-induced loss of RXRalpha mRNA and protein expression correlated with the loss of DNA binding activity of this protein .


We present evidence that the transition from G0/G1 to S phase of the cell cycle results in the down-regulation of RXRalpha expression and that cell cycle inhibitors , which block the cells in G1 phase , prevent this down-regulation .


The decrease in the levels of RXRalpha mRNA was found to be regulated at the post-transcriptional level and involved new protein synthesis .


These observations indicate that the levels of RXRalpha expression in T lymphocytes are coupled to cell cycle progression , and there is tight regulatory control of RXRalpha expression during the transition from G0/G1 to S phase of the cell cycle .


Epidemiology and pathogenesis of AIDS-related lymphomas .


Among patients with congenital and acquired immunodeficiencies , non-Hodgkin 's lymphoma ( NHLs ) are the most common tumors of the immune system .


In the setting of human immunodeficiency virus ( HIV ) infection , as many as 10 % to 20 % of people ultimately developed NHLs .


These tumors are clinically aggressive , frequently involve extranodal sites , and often exhibit unique features that distinguish them from NHL arising in individuals with other forms of immunosuppression .


Important in the development of HIV-associated NHL are cytokines and other factors that induce B-cell proliferation and increase the likelihood of mutations of c-myc , bcl-6 , and other tumor-suppressor genes with carcinogenic potential .


Specific forms of HIV-associated NHL are linked to expression of Epstein-Barr virus ( EBV ) -latent proteins ; the newly described DNA virus , Karposi 's sarcoma-associated herpesvirus/human herpesvirus-8 ( KSHV/HHV-8 ) ; and perhaps HIV .


Elucidation of the factors that contribute to the high incidence of NHL among patients infected with HIV provides insights into important elements of lymphomagenesis .


Matrix metalloproteinase expression in human breast cancer : an immunohistochemical study including correlation with cathepsin D , type IV collagen , laminin , fibronectin , EGFR , c-erbB-2 oncoprotein , p53 , steroid receptors status and proliferative indices .


Matrix metalloproteinase s ( MMPs ) are a group of enzymes thought to be responsible for both normal connective tissue matrix remodelling and accelerated breakdown associated with tumour development .


The current study aimed to investigate the immunohistochemical expression of matrix metalloproteinase 3 ( MMP-3 , stromelysin-1 ) in correlation with the expression of Basement Membrane ( BM ) antigen ( type IV collagen , laminin ) , fibronectin , cathepsin D , p53 , c-erbB-2 , proliferative activity ( Ki-67 , PCNA ) , steroid receptor content as well as to the other conventional clinicopathological parameters in breast cancer .


This study was performed on a series of frozen and paraffin sections from 84 breast cancer specimens by immunohistochemistry using the monoclonal antibody MMP-3 ( Ab-1 ) .


Stromelysin-1 ( ST1 ) was observed in about 10 % of epithelial cells in the control groups ( cases of fibrocystic and benign proliferative breast disease ) , while expression ( > 10 % of expression ) was detected in 89.7 % of tumours .


The expression of ST1 in carcinoma cells was strongly associated with its presence in the stroma ( p < 0.001 ) .


A significantly positive correlation was found between ST1 expression , and p53 tumour suppressor gene product ( p = 0.004 ) , and a relationship with c-erbB-2 protein and progesterone receptor status was also indicated .


These findings suggest that ST1 expression in breast cancer tissue is irrespective of the expression of the extracellular matrix component , the proteolytic enzyme cathepsin D and the growth fraction of the tumour , and that it could be a potential new prognostic marker in breast cancer .


Use of transfected liver cells to evaluate potential mechanisms of alcohol-induced liver injury [ see comments ]


There is increased activity of the proinflammatory cytokine , tumor necrosis factor ( TNF ) in alcoholic liver disease ( ALD ) .


Hepatic neutrophil infiltration is a principal injurious manifestation of ALD .


TNF can induce cellular oxidative injury directly , and indirectly by inducing neutrophil chemotactic factor ( IL-8 ) production by hepatocytes .


IL-8 activates and chemotactically attracts neutrophils to the liver where they release oxidizing substances .


Patients with ALD also have decreased protective factors for cellular oxidative injury .


Manganous superoxide dismutase ( MnSOD ) is an antioxidant protective factor .


The objectives of these studies were to investigate mechanisms for induction of an injurious factor ( IL-8 ) and a protective factor ( MnSOD ) in the HepG2 human hepatoma cell line .


In the first set of experiments , IL-8 gene reporter constructs were used to transiently transfect a derivative ( MVh2E1-9 ) of the HepG2 cell line which expresses P-4502E1 and metabolizes ethanol .


Inactivation of the NF-kappaB and 3'NF-IL-6 DNA binding sites decreased IL-8 gene transcriptional activation in response to TNF while inactivation of the 5'NF-IL-6 binding site increased IL-8 gene transcriptional activity in response to TNF .


This system may be useful to assess the effects of ethanol on TNF -induced hepatocyte IL-8 production .


In the second set of experiments , HepG2 cells were cultured in 25 to 100 mmol concentrations of ethanol .


Both TNF and ethanol increased HepG2 cell MnSOD activity in short-term ( 72 hr ) cultures with ethanol .


However , after long-term ( 10 weeks ) culture with ethanol , there was no induction of MnSOD by ethanol and there was a diminished induction of MnSOD in response to TNF .


Further studies are needed to assess the effect of this diminished induction of MnSOD with chronic ethanol culture on HepG2 cell susceptibility to TNF cytotoxicity .


We conclude that transfected liver cell lines can be used to evaluate mechanisms for increased injurious factors and decreased protective factors in alcoholic liver injury .


Tissue factor transcription driven by Egr-1 is a critical mechanism of murine pulmonary fibrin deposition in hypoxia .


Local hypoxemia and stasis trigger thrombosis .


We have demonstrated previously that in a murine model of normobaric hypoxia pulmonary fibrin deposition is a result of expression of tissue factor , especially in oxygen-deprived mononuclear phagocytes ( MPs ) .


We now show that transcription factor early-growth-response gene product ( Egr-1 ) is rapidly activated in hypoxia , both in vitro and in vivo , and is responsible for transcription and expression of tissue factor in hypoxic lung .


MPs and HeLa cells subjected to hypoxia ( pO2 approximately 13 torr ) had increased levels of tissue factor transcripts ( approximately 18-fold ) and an increased rate of transcription ( approximately 15-fold ) , based on nuclear run-on analysis .


Gel-shift analysis of nuclear extracts from hypoxic MPs and HeLa cells demonstrated increased DNA-binding activity at the serum response region ( SRR ; -111/+14 bp ) of the tissue factor promoter at Egr-1 motifs .


Using 32P-labeled Egr consensus oligonucleotide , we observed induction of DNA-binding activity in nuclear extracts from hypoxic lung and HeLa cells because of activation of Egr-1 , by means of supershift analysis .


Transient transfection of HeLa cells with chimeric plasmids containing wild-type or mutant SRR from the tissue factor promoter showed that intact Sp1 sites are necessary for basal promoter activity , whereas the integrity of Egr-1 sites was required for hypoxia-enhanced expression .


A central role for Egr-1 in hypoxia-mediated tissue factor expression was confirmed by experiments with homozygous Egr-1 null mice ; wild-type mice subjected to oxygen deprivation expressed tissue factor and showed fibrin deposition , but hypoxic homozygous Egr-1 null mice displayed neither tissue factor nor fibrin .


These data delineate a novel biology for hypoxia-induced fibrin deposition , in which oxygen deprivation-induced activation of Egr-1 , resulting in expression of tissue factor , has an unexpected and central role .


Kinetics of cytokine and NFAT gene expression in human interleukin-2-dependent T lymphoblasts stimulated via T-cell receptor .


T cells respond to mitogenic or antigenic stimulation by proliferation and by turning on cytokine gene expression .


Here we have analysed the kinetics and nature of cytokine production in human peripheral blood-derived T lymphoblasts stimulated with anti-CD3 antibodies or Lens culinaris lectin ( LCL ) .


T cells were purified from peripheral blood mononuclear cells ( PBMC ) and primarily activated with anti-CD3 antibodies and cultured in the presence of interleukin-2 ( IL-2 ) .


Anti-CD3-restimulated T cells ( mainly CD8+ ) produced IL-2 , interferon-gamma ( IFN-gamma ) and tumour necrosis factor-alpha ( TNF-alpha ) and low levels of IL-4 and IL-10 transcripts and proteins .


In LCL-stimulated cells the cytokine production pattern was very similar .


Steady-state mRNA levels of IL-2 , IL-10 and IFN-gamma peaked at 3 hr after anti-CD3 stimulation and declined rapidly thereafter .


The kinetics of TNF-alpha mRNA expression was faster , being at its peak level 1 hr after stimulation .


Anti-CD3-stimulated IL-2 gene expression was down-regulated by protein synthesis inhibitor , whereas IL-10 , IFN-gamma and TNF-alpha genes were readily induced independent of ongoing protein synthesis .


T-cell receptor stimulation also induced a very rapid expression of c-jun , c-fos and NFATc1 ( NFATc ) genes , the gene products of which are involved in cytokine gene expression .


In conclusion , the cytokines synthesized by IL-2-dependent T cells were predominantly IL-2 , IFN-gamma and TNF-alpha .


An animal model to study local oxidation of LDL and its biological effects in the arterial wall .


Oxidized LDL ( oxLDL ) is present in atherosclerotic lesions and is believed to play a key role in atherogenesis .


Mainly on the basis of cell culture studies , oxLDL has been shown to produce many biological effects that influence the atherosclerotic process .


To study LDL oxidation in vivo , we have established a model in which Sprague-Dawley rats are given a single injection of unmodified human LDL ( > or = 4 mg/kg body weight ) .


Within 6 hours , an accumulation of apolipoprotein B and epitopes present on oxLDL are detected in the arterial endothelium and media .


The presence of oxLDL is associated with activation of the transcription factor nuclear factor-kappaB in the endothelium as well as endothelial expression of intercellular adhesion molecule-1 .


Injection of LDL enriched with the antioxidant probucol resulted in arterial accumulation of apolipoprotein B , but the expression of oxLDL-specific epitopes was reduced at 24 hours .


Thus , this simple model has the potential to analyze the mechanisms behind and biological effects of LDL oxidation in vivo .


Molecular mechanisms of promoter regulation of the gp34 gene that is trans-activated by an oncoprotein Tax of human T cell leukemia virus type I .


We investigated the molecular mechanism of transcriptional activation of the gp34 gene by the Tax oncoprotein of human T cell leukemia virus type I ( HTLV-I ) .


gp34 is a type II transmembrane molecule belonging to the tumor necrosis factor family and is constitutively expressed on HTLV-I-producing cells but not normal resting T cells .


The transcriptional regulatory region of the gp34 gene was activated by HTLV-I Tax in the human T cell line Jurkat , in which endogenous gp34 is induced by Tax .


Sequence analysis demonstrated that two NF-kappaB-like elements ( 1 and 2 ) were present in the regulatory region .


Both NF-kappaB-like elements were able to bind to NF-kappaB or its related factor ( s ) in a Tax -dependent manner .


Chloramphenicol acetyltransferase assays indicated that NF-kappaB-like element 1 was Tax -responsive , although the activity was lower than that the native promoter .


NF-kappaB -like element 2 elevated promoter activity when combined with NF-kappaB-like element 1 , indicating cooperative function of the elements for maximum promoter function .


Unlike typical NF-kappaB elements , the NF-kappaB-like elements in gp34 were not activated by treatment of Jurkat cells with phorbol ester despite induction of the NF-kappaB -like binding activity .


Chloramphenicol acetyltransferase reporter assays using the region upstream of the NF-kappaB-like elements identified an upstream region that reduced transcription from cognate and noncognate core promoters in a Tax-independent manner .


Our results imply complex regulation of expression of the gp34 gene and suggest implication of gp34 in proliferation of HTLV-I infected T cells .


beta-Amyloid fibrils activate parallel mitogen-activated protein kinase pathways in microglia and THP1 monocytes .


The senile plaques of Alzheimer 's disease are foci of local inflammatory responses , as evidenced by the presence of acute phase proteins and oxidative damage .


Fibrillar forms of beta-amyloid ( Abeta ) , which are the primary constituents of senile plaques , have been shown to activate tyrosine kinase-dependent signal transduction cascades , resulting in inflammatory responses in microglia .


However , the downstream signaling pathways mediating Abeta -induced inflammatory events are not well characterized .


We report that exposure of primary rat microglia and human THP1 monocytes to fibrillar Abeta results in the tyrosine kinase -dependent activation of two parallel signal transduction cascades involving members of the mitogen-activated protein kinase ( MAPK ) superfamily .


Abeta stimulated the rapid , transient activation of extracellular signal-regulated kinase 1 ( ERK1 ) and ERK2 in microglia and ERK2 in THP1 monocytes .


A second superfamily member , p38 MAPK , was also activated with similar kinetics .


Scavenger receptor and receptor for advanced glycated end products ( RAGE ) ligands failed to activate ERK and p38 MAPK in the absence of significant increases in protein tyrosine phosphorylation , demonstrating that scavenger receptors and RAGE are not linked to these pathways .


Importantly , the stress-activated protein kinases ( SAPKs ) were not significantly activated in response to Abeta .


Downstream effectors of the MAPK signal transduction cascades include MAPKAP kinases , such as RSK1 and RSK2 , as well as transcription factors .


Exposure of microglia and THP1 monocytes to Abeta resulted in the activation of RSK1 and RSK2 and phosphorylation of cAMP response element-binding protein at Ser133 , providing a mechanism for Abeta -induced changes in gene expression


Regulation of cellular retinoic acid binding protein ( CRABP II ) during human monocyte differentiation in vitro .


Cellular retinoic acid binding proteins ( CRABP ) are low molecular weight proteins whose precise function remains unknown .


They bind retinoids and may thereby modulate the intracellular steady-state concentration of retinoids .


Whereas CRABP I is ubiquitously expressed , CRABP II is mainly detected in various cell types of the skin .


By representative difference analysis we found that CRABP II is also strongly expressed in human monocyte-derived macrophages ( MAC ) but not in freshly isolated monocytes ( MO ) .


The CRABP II mRNA was gradually upregulated during differentiation from MO to MAC in the presence of 2 % serum .


Adherence , which is important for MO differentiation , induced CRABP II expression , but the addition of 10 ( -7 ) M retinoic acid inhibited the upregulation of CRABP II expression during MO/MAC differentiation .


As MO can differentiate along the classical pathway not only to MAC but also to dendritic cells we analyzed the expression of CRABP II in MO-derived dendritic cells cultured with 10 % FCS , IL-4 , and GM-CSF .


In contrast to MAC , MO-derived dendritic cells showed an extremely low expression of CRABP II .


From these results we conclude ( 1 ) that the availability and the metabolism of retinoids may be different in MAC compared to MO and dendritic cells and ( 2 ) that this may influence differentiation and activation of those cells .


Transcription factor B-cell-specific activator protein ( BSAP ) is differentially expressed in B cells and in subsets of B-cell lymphomas .


The paired box containing gene PAX-5 encodes the transcription factor BSAP ( B-cell-specific activator protein ) , which plays a key role in B-lymphocyte development .


Despite its known involvement in a rare subtype of non-Hodgkin 's lymphoma ( NHL ) , a detailed examination of BSAP expression in NHL has not been previously reported .


In this study , we analyzed normal and malignant lymphoid tissues and cell lines , including 102 cases of B-cell NHL , 23 cases of T- and null-cell NHL , and 18 cases of Hodgkin 's disease .


Normal lymphoid tissues showed strong nuclear BSAP expression in mantle zone B cells , less intense reactivity in follicular center B cells , and no expression in cells of the T-cell-rich zones .


Monocytoid B cells showed weak expression , whereas plasma cells and extrafollicular large transformed B cells were negative .


Of the 102 B-cell NHLs , 83 ( 81 % ) demonstrated BSAP expression .


All of the 13 ( 100 % ) B-cell chronic lymphocytic leukemias ( B-CLLs ) , 21 of ( 100 % ) mantle cells ( MCLs ) , and 20 of 21 ( 95 % ) follicular lymphomas ( FLs ) were positive .


Moderate staining intensities were found in most B-CLL and FL cases , whereas most MCLs showed strong reactions , paralleling the strong reactivity of nonmalignant mantle cells .


Eight of 12 ( 67 % ) marginal zone lymphoma cases showed negative or low BSAP levels , and 17 of 24 ( 71 % ) large B-cell lymphomas displayed moderate to strong expression .


of the 23 T- and null-cell lymphomas reacted with the BSAP antisera , whereas in Hodgkin 's disease , 2 of 4 ( 50 % ) nodular lymphocytic predominance and 5 of 14 ( 36 % ) classical cases showed weak nuclear or nucleolar BSAP reactions in a fraction of the tumor cells .


Western blot analysis showed a 52-kD BSAP band in B-cell lines , but not in non-B-cell or plasma cell lines .


We conclude that BSAP expression is largely restricted to lymphomas of B-cell lineage and that BSAP expression varies in B-cell subsets and subtypes of B-cell NHL .


The high levels of BSAP , especially those found in large-cell lymphomas and in some follicular lymphomas , may be a consequence of deregulated gene expression and suggest a possible involvement of PAX-5 in certain B-cell malignancies .


Mitogen and growth factor-induced activation of a STAT-like molecule in channel catfish lymphoid cells .


This article describes the identification of a putative STAT molecule in the channel catfish ( Ictalurus punctatus ) , the first report of such a molecule in a 'lower ' vertebrate .


A monoclonal antibody against human STAT6 recognizes an approximately 100 kDa molecule that becomes activated and translocates to the nucleus upon both growth factor and mitogen stimulation of catfish leukocytes .


This presumed catfish STAT binds the mammalian interferon-gamma activation site , a known motif of mammalian STAT binding , as shown by electromobility shift assays .


Purification of the proteins present in these DNA complexes confirms that the catfish reactive molecule binds to the interferon-gamma activation site sequence .


These results suggest that STAT molecules have been highly conserved in vertebrate evolution .


Isolation and analysis of a T cell clone variant exhibiting constitutively phosphorylated Ser133 cAMP response element-binding protein .


In driving T cell proliferation , IL-2 stimulates a new program of gene expression that includes proliferating cell nuclear antigen ( PCNA ) , a requisite processivity factor for DNA polymerase delta .


PCNA transcription is regulated in part through tandem CRE sequences in the promoter and CRE binding proteins ; IL-2 stimulates CREB phosphorylation in the resting cloned T lymphocyte , L2 .


After culturing L2 cells for greater than 91 days , we consistently isolate a stable variant that exhibits constitutive CREB phosphorylation .


L2 and L2 variant cells were tested for IL-2 responsiveness and rapamycin sensitivity with respect to specific kinase activity , PCNA expression and proliferation .


In L2 cells , IL-2 stimulated and rapamycin inhibited the following : cAMP-independent CREB kinase activity , PCNA expression and proliferation .


In L2 variant cells , CREB kinase activity was constitutively high ; IL-2 stimulated and rapamycin blocked PCNA expression and proliferation .


These results indicate that IL-2 induces a rapamycin-sensitive , cAMP-independent CREB kinase activity in L2 cells .


However , phosphorylation of CREB alone is not sufficient to drive PCNA expression and L2 cell proliferation in the absence of IL-2 .


Serotonin derivative , N- ( p-coumaroyl ) serotonin , inhibits the production of TNF-alpha , IL-1alpha , IL-1beta , and IL-6 by endotoxin-stimulated human blood monocytes .


We have reported that N- ( p-coumaroyl ) serotonin ( CS ) and its derivatives with antioxidative activity are present in safflower seeds .


As reactive oxygen species ( ROS ) are implicated in the signaling of lipopolysaccharide ( LPS ) , we examined whether CS has a suppressive effect on inflammatory cytokine generation from human monocyte s in vitro .


CS at 50-200 microM reduced tumor necrosis factor ( TNF ) , interleukin-1 ( IL-1 ) , and IL-6 activities in the culture supernatants from LPS-stimulated human blood monocytes without cytotoxicity .


ELISA assay revealed that the production of TNF-alpha , IL-1alpha , IL-1beta , and IL-6 was inhibited by CS .


Northern blot analysis showed that LPS-induced expression of these cytokine mRNA in monocytes was suppressed by CS .


NF-kappaB activation was also inhibited by CS .


These findings indicate that CS has a suppressive effect on proinflammatory cytokine production from monocytes , and this effect is based in part on the suppression of cytokine mRNA expression through inhibition of NF-kappaB activation .


Elevated expression of differentiation inhibitory factor nm23 mRNA in monoblastic crisis of a patient with chronic myelogenous leukemia .


Differentiation inhibitory factor nm23 gene has been found to be expressed in high quantities in acute myelogenous leukemia ( AML ) , especially in acute monocytic leukemia ( AML-M5 ) and is suggested as a new prognostic factor in AML-M5 .


We report an example of elevated expression of nm23 mRNA in a patient with chronic myelogenous leukemia ( CML ) who developed monoblastic crisis .


Relative levels of nm23-H1 and -H2 mRNA extracted from the patient 's peripheral blood mononuclear cells and bone marrow mononuclear cells were measured by quantitative reverse transcriptase polymerase chain reaction .


The level of nm23-H1 mRNA in CML cells at the chronic phase was as high as that in bone marrow cells from healthy volunteers .


The mRNA level of nm23-H2 was slightly below the normal level .


At blastic crisis , however , expression of both nm23-H1 and -H2 mRNA was elevated to about three to nine times of that at the chronic phase .


Proliferated blastic cells were positive for non-specific esterase , and the serum lysozyme level was elevated and diagnosed as monoblastic crisis .


The patient received combined chemotherapy but response was partial .


These findings are compatible with our previous report that nm23 gene is overexpressed in monocytic leukemia .


Increased transcription decreases the spontaneous mutation rate at the thymidine kinase locus in human cells .


Transcription increases DNA repair efficiency and modulates the distribution of certain types of DNA damage .


Furthermore , increased transcription level stimulates spontaneous mutation rate in yeast .


We explored whether transcription level affects spontaneous mutation rate in human cells .


We first developed two thymidine kinase ( tk ) inducible human cell lines using the Gal4-Estrogen receptor system .


In our TK6i-G3 and G9 tk heterozygous cell lines , the active tk allele is linked to an inducible promoter element .


Tk mRNA is induced following treatment with estrogen .


Spontaneous mutation rate was significantly decreased in human cell lines after induction in contrast to the report in yeast .


Thus , humans may have evolved different or additional mechanisms to deal with transcription related spontaneous mutagenesis .


Mutation of BCL-6 gene in normal B cells by the process of somatic hypermutation of Ig genes .


Immunoglobulin ( Ig ) genes are hypermutated in B lymphocytes that are the precursors to memory B cells .


The mutations are linked to transcription initiation , but non-Ig promoters are permissible for the mutation process ; thus , other genes expressed in mutating B cells may also be subject to somatic hypermutation .


Significant mutations were not observed in c-MYC , S14 , or alpha-fetoprotein ( AFP ) genes , but BCL-6 was highly mutated in a large proportion of memory B cells of normal individuals .


The mutation pattern was similar to that of Ig genes .


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


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


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