Characterization and biological relevance of a 29-kDa, oestrogen receptor-related protein

The properties of a monoclonal antibody (D5) that can immunoprecipitate human oestradiol receptor (ER) under some but not all conditions are described. The antibody recognises a 29-kDa serine phosphoprotein that is qualitatively and quantitatively related to ER but not other steroid receptors or binding proteins. p29 will not complex with untreated cytosol ER but, after ammonium sulphate, KCl, heat or phosphatase treatments, interaction occurs that can be detected by immunoprecipitation with D5; molybdate and GTP inhibit complex formation. In human endometrium, p29 is increased by oestrogen and decreased by progestins. IRMA and histochemical assays for p29 have been developed and applied to a large series of human breast tumours. Most, but not all ER+ tumours are p29+, whilst ER-tumours are rarely p29+ unless they are also PR+. p29 predicts for clinical response to hormone therapy. ER+ p29+ tumours have a higher response rate than the ER+ p29-tumours. We do not know if p29 is a previously undetected component of the oestradiol receptor machinery or whether it is a product of oestrogen action.     

Oestradiol-BSA conjugates for receptor histochemistry: problems of stability and interactions with cytosol

Summary The validity of histochemical methods for the localization of hormone receptors based on the binding of fluorescent bovine serum albumin conjugates of oestradiol was examined with respect to their stability and their interactions with the oestrogen receptor type I. Stability was assessed by measuring free oestrogen in conjugates by radioimmunoassay and/or receptor protein binding assay. Sufficient free oestrogen-in order to saturate type I and type II binding sites (ER I, ER II)-was detected in freshly prepared conjugates. This free oestrogen originates in inadequate removal of adsorptively bound original ligand after synthesis. Apart from this fact, conjugates appeared to be unstable in aqueous solutions, especially under the conditions used for histochemical methods. Free oestrogen extracted from the conjugates was subjected to high performance liquid chromatography. Amongst the eluted peaks, oestradiol and/or the original ligand used for synthesis were identified. Thein vitro interaction of conjugates with oestrogen receptors was studied by competitive binding analysis and by incubation of cytosol with a Sepharose-bound conjugate. The results, especially those concerning the amount of free oestrogen, suggest that neither ER I nor ER II is involved in the staining mechanism of conjugates.This paper is dedicated to Professor Dr E. Schauenstein on the occasion of his 65th birthday.     

Measurement of dopamine D2-like receptors in postmortem CNS and pituitary: differential regional changes in schizophrenia

In situ radioligand binding with autoradiography and anti-human dopamine D(2) receptor antibodies with Western blots have been used to measure the density of dopamine D(2)-like receptors in the caudate-putamen and pituitary from schizophrenic subjects who did or did not have residual antipsychotic drugs in their tissue at death. There was a significant decrease in the Ki for haloperidol displaceable [(125)I]iodosulpride binding in the pituitary (p < 0.01) and caudate-putamen (p < 0.05) from subjects with schizophrenia with residual drugs in their tissue. There was a significant decrease in the density of [(125)I]iodosulpride in the pituitary (p < 0.001) and a strong trend to a decrease in binding in the caudate-putamen (p = 0.055) from subjects with schizophrenia. By contrast, [(3)H]spiperone binding was decreased in the caudate-putamen (p < 0.05) with a trend to decreased binding in the pituitary (p = 0.07) from subjects with schizophrenia. There was no difference in the density of dopamine D(2) receptors in the caudate-putamen from subjects with schizophrenia (p = 0.31). All the findings on receptor densities were independent of drug status. [(125)I]iodosulpride binds to the dopamine D(2&3) receptors. We have shown that there is no change in the dopamine D(2) receptor in the caudate-putamen from subjects with schizophrenia and therefore, these data would be consistent with there being a decrease in the dopamine D(3) in the caudate-putamen from subjects with schizophrenia. Since dopamine D(3) receptors are absent or present at low concentrations in the pituitary, our data would suggest the dopamine D(2) receptor is decreased in that tissue from schizophrenic subjects.     

Modulation of two forms of tumor necrosis factor receptors and their cellular response by soluble receptors and their monoclonal antibodies.

Recently, two different receptors for human tumor necrosis factor (TNF) with molecular masses of 60 kDa (p60) and 80 kDa (p80) have been identified. In this report, we investigated the effect of the soluble forms of these receptors and monoclonal antibodies against them on ligand interaction, receptor down-regulation, and mediation of cellular response in U-937 cells. Our results indicate that p60 and p80 constitute 20-30 and 60-80% of the total TNF-binding sites on U-937 cells, respectively. However, by cross-linking, only the p80 form of the receptor could be detected. In contrast to unlabeled TNF, the anti-p60 and anti-p80 antibodies together only partially inhibited ligand binding, and this inhibition was not additive. Lack of additive inhibition of binding was found to be not due to stereo-chemical hindrance. TNF binding to cells can be completely displaced by soluble forms of either the p60 or p80 receptor. However, 100-fold more of the p80 than the p60 form of the soluble receptor is needed for equivalent displacement. Under optimum conditions, TNF and the anti-p80 and anti-p60 antibodies down-regulated 30, 80, and 20% of the TNF receptors, respectively. The anti-p60 and anti-p80 antibodies down-regulated not only their own receptors, but also reciprocal receptors, suggesting a cross-communication between the p60 and p80 forms of the TNF receptor. In spite of inhibiting as much as 80% of TNF binding, none of the receptor antibodies significantly inhibited the cytotoxic response to TNF in U-937 cells. Soluble forms of both receptors, however, completely abrogated the cellular response to TNF. Thus, overall, our results indicate that the antibodies against both receptors together inhibit the majority of the receptor-ligand interaction without any significant effect on the biological response to TNF.     

The role and proposed mechanism by which oestradiol 17β-hydroxysteroid dehydrogenase regulates breast tumour oestrogen concentrations

Synthesis of the biologically active oestrogen, oestradiol, within breast tumours makes an important contribution to the high concentrations of oestrogens which are present in malignant breast tissues. In breast tumours, oestrone is preferentially converted to oestradiol by the Type I oestradiol 17β-hydroxysteroid dehydrogenase (E2DH). Several growth factors, such as insulin-like growth factor Type I, and cytokines, such as Tumour Necrosis Factor (TNF), have been shown to stimulate E2DH activity in MCF-7 breast cancer cells. As little is known about the regulation of Type I E2DH expression and activity in other breast cancer cell lines, the expression and activity of this enzyme was examined in other oestrogen receptor positive and also oestrogen receptor negative breast cancer cell lines. As it is possible that E2DH activity may be limited by co-factor availability, the effects of exogenous co-factors on enzyme activity in these cell lines was also investigated. For T47D and BT20 breast cancer cells, the addition of exogenous co-factors was found to enhance enzyme activity. TNF, in addition to stimulating E2DH activity in MCF-7 cells, also increased activity in T47D and MDA-MB-231 cells, although to a lesser extent than in MCF-7 cells. An investigation of signalling pathways involved in the regulation of E2DH activity revealed that stimulation of both the protein kinase C (PKC) and PKA pathways may be involved in regulation of E2DH activity. As several growth factors and cytokines have now been found to be involved in regulating E2DH activity, the role that macrophages and lymphocytes have in supplying these factors and the mechanism by which these factors may stimulate tumour growth, is also reviewed.     

Anti-cell surface monoclonal antibodies which antagonize the action of VIP in a human adenocarcinoma cell line (HT 29 cells)

Hybridoma cells have been obtained by fusing RCY 3 Ag 1-2-3 rat myeloma cells with spleen cells from a rat hyperimmunized with human adenocarcinoma cells (HT 29 cell line) grown in serum-free medium. Immunoglobulins secreted by hybridomas were screened for: (i) specific binding to HT 29 cells; (ii) their ability to inhibit the binding of [125I]-vasoactive intestinal peptide (VIP) to HT 29 cells; (iii) their capacity to modulate the cAMP production induced by VIP. The monoclonal antibodies we have obtained from clones 109-10-16 and 109-10-19 compete for the binding of radiolabelled VIP to HT 29 cells and partially inhibit the production of cAMP induced by VIP while they are ineffective in reducing the intracellular level of cAMP attained after stimulation of HT 29 cells by isoproterenol. We never found antibodies which increase the cAMP level in HT 29 cells. The binding of the purified monoclonal antibody 109-10-16 Ig gamma 2c to HT 29 cells was visualized by indirect immunofluorescence and was not present at the surface of all cells. These observations strongly support the hypothesis that the monoclonal antibodies we have characterized interact with an antigenic determinant which belongs to the VIP receptor or at least to a cell surface component closely associated with the receptor.     

Oestrogen receptors in chick oviduct. Characterization and subcellular distribution.

Macromolecular components with properties of oestrogen receptors have been identified in the 0.5 M KCl nuclear soluble, the nuclear insoluble and the cytosol fractions of laying hen and immature (2--4 weeks, untreated by hormone) chicken oviduct. 7n the 0.5 M KCl extract of laying hen oviduct nuclei, a receptor, of protein nature according to the effects of enzymic treatments, has been identified. It exhibits high affinity for oestradiol with an apparent equilibrium association constant KA = 4 - 109 M-1 at 4 degrees C. The binding of [3H] oestradiol is abolished by 1 muM oestriol, oestrone and diethylstilboestrol, but not by the same concentration of progesterone, testosterone, and cortisol. Sucrose gradient ultracentrifugation studies in the presence of 0.5 M KCl indicate a sedimentation coefficient of 4.3 S, and there is partial aggregation in low-ionic-strength medium. The estimated number of binding sites per nucleus is about 5000, as calculated from DNA content of chick diploid genome. Most of the binding sites were found to be occupied by endogenous oestrogen(s). Oestradiol dissociates from the receptor according to an apparent two-step mechanism. The half-life time for the faster dissociation step is 18 h at 0 degrees C, 25 min at 20 degrees C and 10 min at 30 degrees C, and for the slower one is 180 h, 115 min and 60 min, respectively. In the 0.5 M KCl extract of immature chicken oviduct nuclei, there are approximately 500 receptor sites per nucleus; their affinity for oestradiol is the same as in the case of laying hen soluble nuclear receptor. After repeated extractions of nuclei with 0.5 M KCl medium, a substantial quantity of oestrogen binding sites remains in the residual fraction. Binding characteristics of this insoluble nuclear receptor resemble those of the soluble nuclear receptor: high affinity for oestradiol (KA = 7 - 10(8) M-1 at 37 degrees C) and specificity for oestrogens. The estimated number of binding sites are approximately 2000/cell for laying hen, and approximately 1000/cell for immature chicken. In the high-speed supernatant fraction of laying hen oviduct homogenates, an oestrogen receptor is also present, but its concentration is low (less than or equal to 100 sites/cell) and at the limits of sensitivity of the methods used. In the cytosol of immature chicken oviduct, there are approximately 2500 oestradiol receptor sites per cell.     

Evidence for structurally conserved recognition of the major carbohydrate xenoantigen by natural antibodies.

Natural or preformed antibodies that react with oligosaccharides bearing terminal galactose-alpha(1,3)-galactose [Gal alpha(1,3)Gal] stuctures are present in the sera of all humans. Antibodies against Gal alpha(1,3)Gal epitopes initiate hyperacute rejection of xenografts of porcine organs in human recipients. Despite the enormous clinical potential for xenotransplantation, very little is known about the 3D structural basis for natural antibody recognition of the major xenoantigen (i.e. Gal alpha(1,3)Gal). In this review, we discuss general binding patterns that have been repeatedly identified in antibody complexes with small molecules (haptens), carbohydrate and peptide ligands because similar mechanisms will almost certainly mediate recognition of the major xenoantigen by natural antibodies.     

Characterization of a membrane-associated 3,3',5-triiodo-L-thyronine binding protein by use of monoclonal antibodies

Four mouse hybridoma cell lines have been isolated which secrete antibodies to the membrane-associated thyroid hormone binding protein (Mr 55,000) from human epidermoid carcinoma A431 cells. J6 is rat specific; J2 is human and monkey specific; J8 and J9 have a wider specificity and react with similar thyroid hormone binding proteins (p55) from human, monkey, rat, and hamster. None of these antibodies reacts with mouse cells. J2, J6, and J9 are of the IgG1k class, and J8 is an IgAk antibody. p55 was characterized by using these monoclonal antibodies. It is not posttranslationally processed by glycosylation, phosphorylation, or sulfation. It has a cellular degradation rate t1/2 approximately equal to 3.2 h. Using immunofluorescence and electron microscopic immunocytochemistry, p55 was found to be associated with the lumenal face of the endoplasmic reticulum and nuclear envelope. When cell homogenates were prepared, significant amounts of p55 were released into the 110000g supernatant, indicating that p55 is loosely associated with the endoplasmic reticulum and nuclear envelope.     

Isolation of a major human placental substrate for the epidermal growth factor (urogastrone) receptor kinase: immunological cross-reactivity with transducin and sequence homology with lipocortin

Using as a starting material either a detergent extract or a protein fraction eluted from membranes with ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, we have isolated from human placental membranes a major substrate for the epidermal growth factor (urogastrone) receptor kinase (EGF kinase). The substrate was isolated both in an intact form, having a molecular mass of approximately 38-kDa (p38), and in a 35-kDa form (p35) representing a proteolytic cleavage product of p38. Both p38 and p35 cross-reacted with antibodies directed against bovine retinal transducin, but did not cross-react with antibodies directed against the 35-kDa beta subunit of human placental G-protein. Antisera directed against the placental EGF kinase substrate failed to react with either bovine or human placental src kinase substrate, p36. Conversely, antisera directed against p36 reacted only poorly with placental p38 or p35. Although p38 had a blocked amino terminus that precluded sequence analysis, p35 yielded an N-terminal sequence that was identical with residues 13-36 of human lipocortin. Our data clearly distinguish p38 from the previously described intestinal calcium binding protein calpactin I or p36 that is also a tyrosine kinase substrate, and our work points to a close relationship (if not identity) between p35 and a 35-kDa EGF receptor kinase substrate previously characterized in A431 cells. We conclude that p38 and p35, which very likely represent human placental lipocortin, may share only limited epitope homology with transducin alpha subunit; however, the possibility that p38, along with intestinal p36 and with a family of related calcium binding proteins, may, like transducin, play a role in receptor-mediated transmembrane signaling is discussed.     

Antibody cross-reactivity with CD46 and lack of cell surface expression suggest that moesin might not mediate measles virus binding.

The binding of antimoesin antibodies from ascites fluids to the surfaces of human and rodent cells was found to parallel the level of CD46 expression. No such reactivity was detected with a purified antimoesin antibody which recognized intracellular moesin. In Western blots, antimoesin antibodies were found to react with solubilized CD46 and a recombinant soluble form of CD46. Antimoesin antibodies also reacted with CD46/CD4 molecules containing only the SCR I and II domains required for measles virus (MV) hemagglutinin binding onto CD46. We suggest that the weak cross-reactivity of antimoesin antibodies with CD46 explains the inhibitory effect of these antibodies on MV entry and that moesin is not directly involved in MV binding.     

Effects of a single cleavage in insulin-like growth factors I and II on binding to receptors, carrier proteins and antibodies.

Two somatomedin-like peptides were extracted from Cohn fraction IV of human plasma and brought to homogeneity: one focused at pH 7.8 and the other at pH less than 5.6. Each consisted of two peptide chains interlinked by disulphide bonds. The basic peptide was identical to insulin-like growth factor I (IGF-I) and had a single cleavage in the C-domain before Arg37 [IGF-I(Arg36cl)]. The acid peptide showed identity with IGF-II, with a cleavage in the B-domain before Arg30 [IGF-II(Ser29cl)]. The effects of these cleavages on the characteristics of binding to type I and type II receptor sites, to binding proteins and to antibodies was studied. Binding of IGF-I(Arg36cl) to antibodies directed against the B-domain or against the AD-domain of IGF-I was the same as IGF-I binding. Thus the cleavage does not influence these antigenic sites. In contrast, binding of IGF-I(Arg36cl) to the type I receptor on human and bovine placental cell membranes was markedly decreased compared with IGF-I binding. Binding to the insulin receptor on human placental cell membranes was slightly diminished, whereas the interaction with specific type II receptors on bovine placental cell membranes was unaffected. There was only a minor influence of the cleavage on the region involved in binding to binding proteins. The cleavage in IGF-II(Ser29cl) diminished binding to antibodies directed against the C-domain of IGF-II, compared with binding of IGF-II itself. Binding to receptors (type I and type II) was changed less profoundly. With 125I-labelled IGF-II(Ser29cl), less insulin was needed in order to obtain 50% displacement of the tracer compared with displacement of 125I-labelled IGF-II. The cleaved form of IGF-II probably has a greater affinity towards the common receptor population than does native IGF-II. Binding to binding proteins was not affected by the cleavage in IGF-II.     

Monoclonal antibodies to different sites on human transcobalamin II

Two IgG1K monoclonal antibodies to human transcobalamin II (TC II) were generated. These antibodies, 16.1 and 16.6, did not cross-react with the other two types of human cobalamin-binding proteins, intrinsic factor and R binder (TC I). Both antibodies cross-reacted with orangutan and simiang TC II but not with TC II from cynomolgus and howler monkeys, who are less closely related to humans. This finding suggests close structural similarity of human to ape TC II. The antibodies also did not react with TC II of lower mammals which included the horse, dog, guinea pig, and mouse; in particular, reaction did not occur with rabbit TC II, which has been considered structurally close to human TC II. Neither of the two antibodies was directed at the cobalamin-binding site of TC II. However, antibody 16.6 hindered TC II binding to cell receptor. This reactivity with the receptor-binding site should prove particularly useful in studies of that region of the TC II molecule.     

Identification of an interleukin-15alpha receptor-binding site on human interleukin-15

To identify the epitopes in human interleukin-15 (IL-15) that are responsible for binding to the interleukin-15 receptor alpha chain, antibody and receptor mapping by peptide scanning and site-directed mutagenesis was used. By using peptide scanning, we identified four regions in IL-15. The first region ((85)CKECEELEEKN(95)) is located in the C-D loop and is recognized by a set of non-inhibitory antibodies. The second region ((102)SFVHIVQMFIN(112)) is located in helix D and is recognized by two antibodies that are inhibitory of IL-15 bio-activity but not of IL-15 binding to IL-15Ralpha. The two remaining regions react with a recombinant soluble form of the IL-15Ralpha; the first ((44)LLELQVISL(52), peptide 1) corresponds to a sequence located in the B-helix and the second ((64)ENLII(68), peptide 2) to a sequence located in helix C. The latter is also contained in the epitope recognized by an antibody (monoclonal antibody B-E29) that prevents IL-15 binding to IL-15Ralpha. By site-directed mutagenesis, we confirmed that residues present in peptide 1 (Leu-45, Glu-46, Val-49, Ser-51, and Leu-52) and peptide 2 (Leu-66 and Ile-67) are involved in the binding of IL-15 to IL-15Ralpha. Furthermore, the results presented indicate that residues in the second peptide (Glu-64, Asn-65, and Ile-68) participate in IL-2Rbeta recruitment. This finding could have implications for the dynamics of receptor assembly. These results also indicate that the modes of interaction of IL-15 and IL-2 with their respective alpha chains are not completely analogous. Finally, some of the IL-15 mutants generated in this study displayed agonist or antagonist properties and may be useful as therapeutic agents.     

High affinity human IgE receptor (Fc epsilon RI). Analysis of functional domains of the alpha-subunit with monoclonal antibodies.

The binding of IgE to the high affinity Fc epsilon receptor (Fc epsilon RI) on mast cells and basophils is mediated by the alpha-subunit of the tetrameric receptor complex. Based on sequence homologies, the 50-kDa alpha-subunit is a member of the immunoglobulin superfamily of proteins and has two predicted disulfide-bonded loops. Monoclonal antibodies specific for the human alpha-subunit have been identified and separated into two major classes: inhibitory and noninhibitory antibodies. Inhibitory antibodies (i.e. 15A5) block 125I-IgE binding to a recombinant chimeric alpha-subunit (ch-alpha-protein) expressed on Chinese hamster ovary cells and immunoprecipitate 125I-labeled purified ch-alpha-protein. Noninhibitory antibodies (i.e. 22E7) immunoprecipitate both 125I-labeled ch-alpha-protein and the soluble complex of 125I-IgE cross-linked to ch-alpha-protein but do not block 125I-IgE binding to the ch-alpha-protein expressed on Chinese hamster ovary cells. Both classes of antibodies bind to natural Fc epsilon RI present on human basophils and induce histamine release from these cells. Inhibitory antibody 15A5 specifically binds to a peptide corresponding to amino acids 125-140 of the putative second domain of the alpha-subunit sequence. All the inhibitory antibodies compete with 125I-15A5 for binding to the ch-alpha-protein, indicating that these antibodies recognize inhibitory epitopes that are either identical or sterically overlapping. Noninhibitory antibodies (i.e. 22E7) do not block 125I-15A5 binding to the ch-alpha-protein. These data suggest that antibodies binding to the predicted second domain of the alpha-subunit can inhibit IgE binding to the alpha-subunit, while antibodies binding at a distance from this site do not inhibit IgE binding. These inhibitory antibodies may block IgE binding to the ch-alpha-protein by direct overlap, steric inhibition, or induced conformational changes of the receptor contact points for IgE.     

Characterization of p29, an estrogen-receptor associated tumor marker

Monoclonal antibody D5, raised against cytosolic human estrogen receptor (ER) reacts with p29, a receptor-associated cytoplasmic serine phosphoprotein which does not bind steroid, While p29 selectively binds GTP and to a lesser extent ATP, in vitro GTP binding does not result in p29 phosphorylation. Under ER activating conditions, p29 associates with cytosolic ER; GTP, ATP and sodium molybdate block formation of immunoprecipitable p29-ER complexes. Nucleotide binding data suggest a role for p29 in the estrogen response machinery, possibly at the level of phosphate or nucleotide metabolism.     

Human interferon-gamma receptor. Mapping of epitopes recognized by neutralizing antibodies using native and recombinant receptor proteins.

Monoclonal antibodies produced against native interferon-gamma receptor (IFN gamma-R) have been characterized for their capacity to react with purified receptor and receptor-positive cells, to inhibit the binding of IFN gamma to cellular receptor, to precipitate the receptor protein when cross-linked to IFN-gamma, and to recognize the recombinant interferon-gamma receptor and 19 overlapping fragments of this protein expressed in Escherichia coli. The results of this analysis showed that: (i) the extracellular portion of human IFN gamma-R is located between the N terminus and the transmembrane region (amino acids 18-246). (ii) The intracellular domain is between the transmembrane region and the C terminus (amino acids 269-489). (iii) The monoclonal antibodies that react with the IFN gamma-R intracellular domain recognize small linear epitopes. (iv) The human IFN gamma-R binding site is located between the N terminus and the transmembrane region. (v) The monoclonal antibodies that react with IFN gamma-R extracellular domain and inhibit the binding of IFN gamma recognize two different epitopes. One of these epitopes (included between amino acids 26 and 133) is very close to the binding site for IFN gamma. The second (included between amino acids 70 and 210) is related to the binding site for IFN gamma without including it. (vi) These two functional epitopes are conformational and need S-S bridges to maintain their architecture. (vii) These conformational epitopes are formed in receptor fragments expressed in E. coli.     

Polypyrimidine tract-binding protein and heterogeneous nuclear ribonucleoprotein A1 bind to human T-cell leukemia virus type 2 RNA regulatory elements.

Efficient expression of human T-cell leukemia virus (HTLV) and human immunodeficiency virus structural proteins requires Rx and Rev proteins, respectively. Decreased expression of Gag and Env appears to be due, in part, to intragenic RNA sequences, termed cis-acting repressive sequences (CRS), and may be mediated by binding of specific cellular factors. We demonstrated previously that two cellular proteins, p60CRS and p40CRS, interact with HTLV type 2.5' long terminal repeat CRS RNA and that the interaction of both proteins with CRS RNA correlates with function (A. C. Black, C. T. Ruland, J. Luo, A. Bakker, J. K. Fraser, and J. D. Rosenblatt, Virology 200:29-41, 1994). By radioimmunoprecipitation of HeLa nuclear proteins UV cross-linked to CRS RNAs with murine monoclonal antibodies, we now show that p40CRS is heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and p60CRS is polypyrimidine tract-binding protein or hnRNP I. These immunoprecipitation results were confirmed by an immunobinding assay with hnRNP I and hnRNP AI antibodies and by cross-competition electrophoretic mobility shift experiments. In addition, we mapped a putative hnRNP A1 binding site in U5 RNA and demonstrated that p40CRS (hnRNP A1) binding to that site correlates with CRS function. Since both hnRNP I and hnRNP A1 have been shown to influence splicing and potentially other steps in RNA processing, the binding of both hnRNP I and hnRNP A1 to HTLV RNA regulatory elements may alter retrovirus RNA processing and may be involved in regulation by Rex.     

Mannose receptor (MR) engagement by mesothelin GPI anchor polarizes tumor-associated macrophages and is blocked by anti-MR human recombinant antibody

Tumor-infiltrating macrophages respond to microenvironmental signals by developing a tumor-associated phenotype characterized by high expression of mannose receptor (MR, CD206). Antibody cross-linking of CD206 triggers anergy in dendritic cells and CD206 engagement by tumoral mucins activates an immune suppressive phenotype in tumor-associated macrophages (TAMs). Many tumor antigens are heavily glycosylated, such as tumoral mucins, and/or attached to tumor cells by mannose residue-containing glycolipids (GPI anchors), as for example mesothelin and the family of carcinoembryonic antigen (CEA). However, the binding to mannose receptor of soluble tumor antigen GPI anchors via mannose residues has not been systematically studied. To address this question, we analyzed the binding of tumor-released mesothelin to ascites-infiltrating macrophages from ovarian cancer patients. We also modeled functional interactions between macrophages and soluble mesothelin using an in vitro system of co-culture in transwells of healthy donor macrophages with human ovarian cancer cell lines. We found that soluble mesothelin bound to human macrophages and that the binding depended on the presence of GPI anchor and of mannose receptor. We next challenged the system with antibodies directed against the mannose receptor domain 4 (CDR4-MR). We isolated three novel anti-CDR4-MR human recombinant antibodies (scFv) using a yeast-display library of human scFv. Anti-CDR4-MR scFv #G11 could block mesothelin binding to macrophages and prevent tumor-induced phenotype polarization of CD206(low) macrophages towards TAMs. Our findings indicate that tumor-released mesothelin is linked to GPI anchor, engages macrophage mannose receptor, and contributes to macrophage polarization towards TAMs. We propose that compounds able to block tumor antigen GPI anchor/CD206 interactions, such as our novel anti-CRD4-MR scFv, could prevent tumor-induced TAM polarization and have therapeutic potential against ovarian cancer, through polarization control of tumor-infiltrating innate immune cells.     

Recognition of two Dermatophagoides pteronyssinus-specific epitopes on antigen P1 by using monoclonal antibodies: binding to each epitope can be inhibited by serum from dust mite-allergic patients

Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen.