Incidence of HIV infection in monocyte subpopulations characterized by CD4 and HLA-DR surface density.

OBJECTIVE: The purpose of this study was to determine any correlation between the expression of CD4 antigen on the surface of monocytes, and the frequency with which these cells are infected with HIV. DESIGN: CD4 surface expression on monocytes is significantly less than that expressed on CD4+ lymphocytes. Nevertheless, all monocytes express the HIV CD4 receptor and infected individuals have a significant decrease in the number of monocytes that express a higher density of CD4 surface fluorescence. METHODS: Three-color flow cytometric analysis was used to characterize monocyte-enriched peripheral blood mononuclear cells (PBMC) in terms of surface expression of CD4, CD14 (macrophage antigen), and class II major histocompatibility antigen (HLA-DR). HLA-DR+ monocytes from HIV-positive individuals were sorted into two subpopulations based on either 'bright' or 'dim' CD4 surface expression. A polymerase chain reaction (PCR) assay was used to detect the presence of proviral HIV sequences within the DNA from 10(5) cells from each sorted population. RESULTS: Post-sort analysis revealed that the dim CD4+ monocyte subset expressed dim HLA-DR surface antigen, while the bright CD4+ monocyte subset contained both bright and dim HLA-DR+ cells. PCR results showed that four out of eight dim CD4+ monocyte subsets contained proviral HIV DNA, compared with one out of eight bright CD4+ monocyte subsets.

"Defective" receptors in steroid-resistant conditions may be proteolytic artifacts.

The specific question addressed in this report is whether the resistance to steroid treatment of certain tissues or tumors which appear to contain a normal quantity of steroid-binding sites may be due to structural defects in the receptors. This question may be seen as part of the more general question of whether there are intrinsic variations in the structures of receptors for a given class of steroids in different healthy tissues, in healthy vs. malignant tissues or in different types of tumors. Our experimental approach to these questions has involved the stabilization and precise physicochemical characterization of the receptors. To date, we have studied the estrogen and progestin receptors from human breast cancers and benign and malignant gynecologic specimens and the glucocorticoid receptors from several healthy and malignant rodent tissues and from normal human lymphocytes and various types of leukemic cells. Chromatographic and ultracentrifugal analyses in buffers of low ionic strength, containing 20 mM Na2MoO4 as the stabilizer, have revealed each of these receptors to be a large, oligomeric complex, characterized by remarkably similar values of the Stokes radius, sedimentation coefficient, molecular weight and axial ratio. In the absence of adequate stabilization, however, we found that the receptors for three classes of steroids in extracts of some healthy, steroid-responsive tissues, such as rat kidney and human uterine endometrium, are invariably degraded by endogenous proteinases. The extent of such cleavage is increased considerably by freezing the tissues prior to homogenization. Studies designed to distinguish the intact receptors from the products of proteolysis have included the characterization of receptors in cytosols prepared from mixtures of rat liver and kidney. The results strongly support the interpretation that the smaller size of the receptors detected in kidney cytosol reflects their cleavage by the more active proteinases in that tissue. The sizes and shapes of the receptors in cytosols from various tissues were found to be correlated with the activities of specific endopeptidases, assayed fluorometrically with peptidyl derivatives of 7-amino-4-methylcoumarin (AMC). These studies suggested that the receptors are vulnerable to cleavage by "lysine-specific" endopeptidases, detected with t-butyloxycarbonyl-L-valyl-L-leucyl-L-lysyl-AMC. An enzyme of this specificity was partially purified from rat kidney cytosol and tested for its ability to digest the glucocorticoid receptors from rat liver cytosol.(ABSTRACT TRUNCATED AT 400 WORDS)

Triamcinolone acetonide 21-oic acid methyl ester: a potent local antiinflammatory steroid without detectable systemic effects.

There have been many previous attempts to prepare glucocorticoid analogs that would have high antiinflammatory activity at the site of application but minimal systemic side effects. In principle, esters of cortoic acids could fulfill these criteria, if they had sufficient affinity for the glucocorticoid receptors but were rapidly hydrolyzed to the inactive acids in the circulation. With this rationale, we have synthesized esters of the 21-oic acid of triamcinolone acetonide (TA, 9 alpha-fluoro-11 beta, 16 alpha, 17 alpha, 21-tetrahydroxy-pregna-1, 4-diene-3,20-dione 16,17-acetonide), a potent synthetic glucocorticoid, in both tritiated and unlabeled forms. The synthesis involves 1) oxidation to the 21-dehydro compound with methanolic cupric acetate, 2) further oxidation to the acid with methylene blue in the presence of KCN at pH 6.5, 3) esterification with diazomethane in the presence of methanol or ethanol, to produce the methyl ester of TA (TAme) or ethyl ester, respectively, and 4) purification of the products by TLC and HPLC. The molecular weights and structures of the esters were established by mass spectrometry and nuclear magnetic resonance. The binding of [3H]TAme to steroid receptors or serum steroid-binding proteins and the in vitro hydrolysis of the ester were evaluated simultaneously, by chromatography on Sephadex LH-20 columns in aqueous buffer. [3H]TAme is bound with high affinity by receptors from human leukemic cells and rat liver. The pattern of competition for this binding is characteristic of glucocorticoid receptors: TA approximately equal to TAme greater than R5020 (a synthetic progestin) approximately equal to aldosterone greater than 5 alpha-dihydrotestosterone. [3H]TAme is not bound detectably by serum steroid-binding proteins and is rapidly hydrolyzed during incubation with serum at 37 C. The acidic product has a very low affinity for the glucocorticoid receptor. Complexes of [3H]TAme with human and rat receptors have sedimentation coefficients of 9-10S in hypotonic buffer containing 20 mM Na2MoO4 and approximately 4S in hypertonic, molybdate-free buffer. These values of s20,w are similar to those of the oligomeric and monomeric forms, respectively, of the same receptors labeled with [3H]TA, and of mammalian steroid receptors, in general. The antiinflammatory activity of TAme in rats is comparable to that of prednisolone, but the ester is devoid of the side effects associated with prednisolone treatment (suppression of thymic weight and of serum corticosterone concentration). These bioassay data and the high affinity of the ester for human glucocorticoid receptors suggest that TAme may eventually be useful clinically, as a loc

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

Therapy with glucocorticoids is generally more effective in acute lymphoblastic leukemia than in other types of human leukemia. Previous studies, however, have not revealed any consistent relationship between clinical responsiveness and the cellular or cytosolic concentration of glucocorticoid-binding sites. The objectives of this study were to determine whether there are intrinsic structural differences among the glucocorticoid receptors in various types of leukemic cells and normal lymphocytes and to investigate the role of endogenous peptidases in receptor degradation. Cytosols were prepared from fresh or rapidly frozen leukocytes from 6 healthy adults and 35 high-risk leukemia patients (median white blood cell count, 150,000 cells/microliter; median age, 13 years). Receptors were labeled with [3H]triamcinolone acetonide and quantitated by charcoal-dextran treatment or Sephadex LH-20 chromatography. Mean and median cytosolic receptor concentrations in 12 acute lymphoblastic leukemia specimens lacking the standard B-cell or T-cell markers ("null cells") were approximately 4-fold higher than in 23 other leukemic cell specimens. No other consistent differences in receptor content were observed. Agarose filtration and ultracentrifugation in hypotonic buffers containing 20 mM Na2MoO4 revealed complexes of similar size and shape in all clinical specimens tested and two established leukemic cell lines. They had Stokes radii (Rs) of 8.1 +/- 0.5 (S.D.) nm (n = 50), sedimentation coefficients of 9.5 +/- 0.3S (n = 40), molecular weights of approximately 330,000, and axial ratios (a/b) of approximately 12. In hypertonic, molybdate-free buffer, these oligomeric complexes were dissociated into subunits with Rs of 5.9 +/- 0.3 nm (n = 12) and a/b of 11 to 12, as observed previously for other receptors. Fragmentation of the oligomer and the subunit was evident in some cytosols. High activities of peptidases of various specificities were detected in leukemic cell cytosols, as in other cytosols, by fluorometric assays with derivatives of 7-amino-4-methylcoumarin. Receptor cleavage by these and other endogenous enzymes may account for previous observations of "abnormal" receptors in cytosols from some leukemic specimens. We conclude that intrinsic structural defects in the receptors are unlikely explanations for the unresponsiveness of some types of leukemia to steroid therapy.

Human breast tumor estrogen receptor: effects of molybdate and electrophoretic analyses.

The largest and smallest discrete forms of the estrogen receptor in human breast tumor cytosol were characterized by competitive steroid binding, ultracentrifugation, gel filtration, and electrophoresis in polyacrylamide gels of several concentrations. Incubation of cytosol with [3H]estradiol and centrifugation in glycerol gradients containing 20 mM Na2MoO4 and 0 or 150 mM KCl revealed a 9-10S form of the receptor. It resembles the molybdate-stabilized complexes in cytosols of other human and rodent, malignant and healthy tissues, and the complex detected in breast tumor cytosol containing leupeptin, a bacterial protease inhibitor. Preservation of receptor integrity during purification and discrimination from serum steroid-binding components are facilitated by inclusion of molybdate in all buffers. Possible mechanisms of action of molybdate include the inhibition of ribonuclease action on RNA-associated receptor forms and protection against specific proteolytic cleavage by stabilization of a phosphate group on the vulnerable residue or a neighboring one. During fractionation of tumor cytosol in the absence of molybdate, the receptor is converted to a mixture of fragments. The smallest that retains the bound steroid, the mero-receptor, resembles the products of endogenous and exogenous protease action on receptors for all classes of steroids in a wide range of tissues. The similarities between both the largest and the smallest known forms of the breast tumor estrogen receptor and corresponding forms of other receptors support the notion of the common architecture of steroid receptors in normal and malignant tissues of diverse origins.

Estrogen receptor cleavage and plasminogen activation by enzymes in human breast tumor cytosol.

Estrogen receptors in cytoplasmic extracts of breast tumors from more than 40 patients were separately analyzed by gel filtration and/or ultracentrifugation under diverse conditions. Resultant patterns are presented for specimens from 11 women with infiltrating duct carcinoma and are representative of results obtained in all samples of sufficient size and receptor content (approximately 40 fmol/mg cytosol protein) for accurate determination of hydrodynamic parameters. Estradiol-binding components of intracellular origin were distinguished from the serum contaminant, sex hormone-binding globulin by their high affinity for diethylstilbestrol and negligible affinity for 5 alpha-dihydrotestosterone. The predominant molecular forms of the receptors, but not the steroid specificity, varied dramatically with experimental factors, including the duration of the fractionation procedure, ionic strength, and the presence of protease inhibitors, particularly the bacterial tripeptides N-acetyl- and N-propionyl-L-leucyl-L-leucyl-DL-arginine aldehydes (leupeptin). At least three discrete forms of the intracellular receptors were detected. The smallest labeled complex, the mero-receptor, with a sedimentation coefficient of about 3S and a Stokes radius of about 24 A, was formed during prolonged analysis of control cytosol in hypotonic or hypertonic buffers. Complexes with an intermediate sedimentation coefficient (approximately 5S) and Stokes radius (approximately 34A) were detected when control cytosol was analyzed rapidly in hypotonic buffer or when cytosol containing 50 nM leupeptin was analyzed in hypertonic buffer. The largest receptor form (10.5S, 71A) was predominant in cytosol prepared with 50 mM leupeptin and analyzed in hypotonic buffer. In this small series of patients, there was no obvious correlation between the molecular form of the receptors and the clinical status or eventual responsiveness to endocrine therapy. Preliminary studies of endogenous proteolytic enzymes in breast tumor cytosol that may be involved in mero-receptor formation included assays of plasminogen activators (EC 3.4.21.-) by fibrinolytic and spectrofluorometric techniques. The detection of high concentrations of plasminogen activators in several tumor cytosols and the inhibition of this activity by leupeptin, which stabilizes the large receptor forms in this and other systems, are consistent with a possible role of these enzymes in receptor cleavage.

PIP: The multiple molecular forms of steroid hormone receptors in human breast tissue were characterized and are reported. 40 patients provided cytoplasmic extracts for assay by either gel filtration or ultracentrifugation under diverse conditions. In general, the predominant molecular forms of the receptors, but not the steroid specificity, varied dramatically with experimental factors, including the duration of the fractionation procedure, ionic strength, and the presence of protease inhibitors, particularly the bacterial tripeptides N-acetyl- and N-propionyl-L-leucyl-Dl-arginine aldehydes (leupeptin). 3 discrete forms of intracellular receptors were detected. The mero-receptor, the smallest labeled complex, had a sedimentation coefficient of 3S and a Stokes radius of 24 angstroms; it was formed during prolonged analysis of control cytosol in hypotonic or hypertonic buffers. Complexes with an intermediate sedimentation coefficient (5S) and Stokes radius (34 angstroms), were detected when control cytosol was analyzed rapidly in hypotonic buffer or when cytosol containing 50 mM leupeptin was analyzed in hypertonic buffer. There was no obvious correlation between the molecular form and clinical status or eventual responsiveness to endocrine therapy.

Multiple forms of oviduct progesterone receptors analyzed by ion exchange filtration and gel electrophoresis.

Resolution of the multiple forms of steroid receptors in small samples has been improved by two new techniques: preparative ion exchange filtration and electrophoresis in highly cross-linked polyacrylamide gels of varied concentration. These techniques were used in conjunction with protamine precipitation, gel filtration, and density gradient centrifugation to separate five forms of the progesterone receptor of chick oviduct cytosol. These complexes, numbered I to V in order of elution from agarose gel columns, have been characterized with respect to apparent molecular weight, shape, and relative net charge. Form I, which is eluted in the void volume after gel filtration of cytosol in hypotonic media, is heterodisperse with respect to sedimentation coefficient and electrophoretic mobility (Rf). Form I is converted to form III by KC1. Form II has the highest axial ratio and the highest Rf at pH 10.2. This 4.2S complex can be extracted from DEAE filters, but not from protamine-precipitated cytosol, by 0.3 to 0.5 M KC1. Form III is slightly smaller (3.9S) and less asymmetric than form II. It is relased from DEAE filters and protamine-precipitated cytosol by 0.15 M KC1 and displays increased Rf upon purification. Forms II and III correspond to the B and A components described by W. T. Schrader and B. W. O'Malley ((1972), J. Biol. Chem 247, 51). Form IV may result from the proteolytic cleavage of forms II and/or III. Form V is a globular polypeptide obtained in the presence of certain divalent cations. This complex has been named the "mero-receptor" since it is the smallest part or fragment of the receptor that contains the steroid-binding site.

Steroid-receptor quantitation and characterization by electrophoresis in highly cross-linked polyacrylamide gels.

Conditions for discontinuous polyacrylamide gel electrophoresis have been defined in which progesterone receptors of chick oviduct cytosol and a variety of steroid-binding proteins from other sources are stable and amenable to quantitative analysis. The essential modifications from standard procedures include the use of (1) separation gels in which the cross-linking agent/acrylamide monomer = 15:85, (2) glycerol (10% v/v) in all phases of the Trisglycine-HCl buffer system (pH 10.2 in the separation phase during electrophoresis at 0 degrees), and (3) a layer of a charged reducing agent, thioglycolate, beneath the sample layer. Electrophoresis of untreated oviduct cytosol labeled with [3H]progesterone +/- competing steroids revealed a heterodisperse slow peak and a sharp fast peak. Both peaks displayed the steroid-binding specificity and saturability that are characteristic of intracellular receptors. Recovery of steroid from both the slow and fast components increased linearly with sample load up to 60 mul of cytosol (1.2 mg of protein)/gel (6 mm diameter). The specific progesterone binding detected by this technique was comparable to that detected by charcoal-dextran treatment or ion exchange filtration. Relative electrophoretic mobilities (Rf) of globular protein standards and steroid-protein complexes in cytosol and chick serum were measured in separation gels with total gel concentrations (T) systematically varied from 5 to 15% (w/v). Data were processed by computer programs to obtain weighted linear regressions of log Rf on T (Ferguson plots) and the joint 95% confidence limits of the slopes (-KR) and intercepts of these plots. Molecular radii (R) of the binding components and apparent molecular weights (M) were calculated from the linear correlation of R with KR 1/2 for the standards. The value of M is approximately 158,000 obtained for the cytosol fast component was independent of the length of the separation gel, the presence of a stacking gel or prior exposure of the cytosol to KCl. It was higher than expected from the sedimentation coefficient of 4.2 S in the same pH 10.2 buffer. Electrophoresis in 170-mm separation gels without stacking gels revealed that KCl extracts of protamine-precipitated cytosol contain a different receptor form, of lower net negative charge than the cytosol fast form. The results demonstrate the utility of electrophoresis in highly cross-linked gels of several concentrations to discriminate between various receptor forms and steroid-binding components of serum. This method may lead to overestimates of M for highly asymmetric receptor forms.

Progesterone "receptors" in the cytoplasm and nucleus of chick oviduct target tissue.

This report demonstrates that the chick oviduct, a specific target organ for progesterone, contains both cytoplasmic and nuclear macromolecules which bind progestins. These binding molecules can be clearly distinguished from transcortin by centrifugation through sucrose gradients of low ionic strength and by agarose gel filtration. The cytoplasmic progesterone-binding molecules also bind 5-alpha-pregnane-3,20-dione, but have significantly lower affinity for cortisol, estrone, or aldosterone. They are absent from blood and nontarget organs such as lung and spleen. The tissue-specific binding components appear to be heat-labile proteins with an average dissociation constant for progesterone of about 8 x 10(-10) M at 2 degrees C. These results are consistent with the identification of the progesterone-binding molecules as the functional hormone receptors. In further support of this concept is the finding that treatment of the chicks with estrogen coordinately induces a 20-fold increase in the number of progesterone-binding molecules and enhances the capacity of progesterone to induce avidin synthesis.A progesterone-"receptor" complex can be detected in both the cytoplasm and nuclei of oviduct tissue after an injection of [(3)H]progesterone to estrogen-treated chicks. By contrast, incubation of oviduct tissue with [(3)H]progesterone in vitro at 2 degrees C for 5 min leads to labeling of the cytoplasmic "receptor" only. Transfer of the "receptor"-steroid complex into the nucleus then appears to occur upon subsequent incubation in vitro at 37 degrees C. This observation suggests that the transfer of bound progesterone across the nuclear membrane may be an energy-requiring enzymatic process.