Cellular Radioimmunoassay Research Articles

Retention of B-Cell-Specific Monoclonal Antibodies by Human Lymphoma Cells

AbstractThe rates of endocytosis, intracellular degradation, and cell-surface shedding of 125I-labeled monoclonal antibodies (MoAbs) HD-37 (anti-CD19), B1 (anti-CD20), MB-1 (anti-CD37), BC8 (anti-CD45), and DA4-4 (anti-μ) by B-lym-phoma cells were compared by cellular radioimmunoassay, ultrastructural autoradiography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thin layer chromatography using biopsy specimens from 12 patients with non-Hodgkin’s lymphomas. 125I-BC8 was stably retained on the surface of lymphoma cells without appreciable internalization or shedding, whereas 125I-DA4-4 underwent rapid endocytosis and degradation. 12BI-B1 was not internalized or degraded by tumor cells, but rapidly dissociated from the cell surface in intact form. Moderate rates of endocytosis, intracellular metabolism, and cell-surface shedding were shown by 125I-HD37 and 125I-MB-1. The 3 patients with diffuse, small cleaved-cell lymphomas internalized and degraded antibodies more slowly than did patients with other histologic subtypes. These kinetic differences may be important in the selection of MoAbs for immunotoxin and radioimmunoconjugate therapy of B-cell malignancies.

Retention of B-cell-specific monoclonal antibodies by human lymphoma cells

The rates of endocytosis, intracellular degradation, and cell-surface shedding of 125I-labeled monoclonal antibodies (MoAbs) HD-37 (anti- CD19), B1 (anti-CD20), MB-1 (anti-CD37), BC8 (anti-CD45), and DA4–4 (anti-mu) by B-lymphoma cells were compared by cellular radioimmunoassay, ultrastructural autoradiography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thin layer chromatography using biopsy specimens from 12 patients with non- Hodgkin's lymphomas. 125I-BC8 was stably retained on the surface of lymphoma cells without appreciable internalization or shedding, whereas 125I-DA4–4 underwent rapid endocytosis and degradation. 125I-B1 was not internalized or degraded by tumor cells, but rapidly dissociated from the cell surface in intact form. Moderate rates of endocytosis, intracellular metabolism, and cell-surface shedding were shown by 125I- HD37 and 125I-MB-1. The 3 patients with diffuse, small cleaved-cell lymphomas internalized and degraded antibodies more slowly than did patients with other histologic subtypes. These kinetic differences may be important in the selection of MoAbs for immunotoxin and radioimmunoconjugate therapy of B-cell malignancies.

Immunohistochemical characterization of a monoclonal antibody to hyperplastic nodules induced in rat liver by chemical carcinogens.

To investigate the specific phenotype of hyperplastic nodules (HPN) in rat liver, we produced a monoclonal antibody (HAM-6). HAM-6 was a member of the mouse IgG1 class and was specific for hyperplastic nodules, as shown by cellular radioimmunoassay and immunohistochemical studies. The antigen recognized by HAM-6 was located in the rat HPN cell membrane. HAM-6 was also slightly reactive to rat hepatocellular carcinoma, but not to normal or fetal rat liver, other normal rat organs, human hepatocellular carcinoma, or human liver cirrhosis. That is, the antigen recognized by HAM-6 appeared to be differentiated and to occur during chemical carcinogenesis. HAM-6 may be a useful marker for the investigation of premalignant states in chemical carcinogenesis.

Localization of the CD44 Glycoprotein to Fibrous Astrocytes in Normal White Matter and to Reactive Astrocytes in Active Lesions in Multiple Sclerosis

The CD44 antigen is a proteoglycan recently implicated in several adhesion events including that of lymphocytes to endothelium. The CD44 antigen, reactive with monoclonal antibody (MAb) 44D10, has been shown previously to be expressed in normal human white matter homogenates and to be found at higher concentrations in brain homogenates of victims of multiple sclerosis (MS). The cellular localization of CD44 in human brain of normal individuals and in those afflicted with MS has now been determined. Monoclonal antibody 44D10 reacted with astrocyte-like cells in 40 microns thick paraformaldehyde-fixed sections but not in thin (6 microns) fixed sections. A double labeling experiment performed on a frozen brain section with MAb 44D10 and rabbit anti-glial fibrillary acidic protein (GFAP), a cytoplasmic marker of astrocytes, confirmed the co-localization of these two antigens. The reactivity with brain tissue sections of a rabbit antiserum produced against lymphocyte-CD44 could be absorbed by a preparation of the CD44 glycoprotein, purified 2,100-fold from a white matter homogenate. The antiserum was shown by Western blot analysis to be specific for p80 glycoprotein in brain extracts derived from a normal and MS patients. This antibody reacted with fibrous astrocytes predominantly in white matter; staining was also noted in subependymal and subpial regions. Inhibition studies using a cellular radioimmunoassay indicated that the highest concentrations of CD44 in three MS victims were found in plaques, followed by periplaques and non-involved areas of white matter which were higher than normal white matter. Reactive astrocytes, identified in active lesions, expressed high levels of CD44 on their surfaces. Thus, CD44 is associated with astrocytes in human brain and the increased expression observed in MS brain may reflect activation and/or proliferation of astrocytes implicated in the pathogenesis of this disease.

Several epitopes of p85 glycoprotein (CDw44) are dependent on intact disulphide bonds. Isolation of cDNA clones requires a polyclonal antibody raised against the reduced protein

Monoclonal antibodies 50B4 and 50E6 recognize two distinct epitopes of human p85 glycoprotein (CDw44). Both epitopes are destroyed by reduction of the purified glycoprotein as demonstrated by inhibition of cellular radioimmunoassay and Western blot analysis. Endoglycosidase F treated p85 glycoprotein, with an apparent molecular weight of 73,000, is still reactive with both monoclonal antibodies. Thus both epitopes are conformational determinants of the polypeptide chain. A rabbit antibody produced against purified native p85 glycoprotein also reacted only with the non-reduced form of p85. Repeated immunizations with SDS-dissociated and reduced p85 yielded a polyclonal antibody reactive by Western blot analysis with reduced and non-reduced forms of p85 glycoprotein. When a HOON leukemia cell line cDNA expression library was screened with this polyclonal antibody, two cDNA clones were isolated which reacted specifically with the antiserum and not with the control non-immune serum. Preliminary characterization of these clones indicates that they are p85-related.

An antigen specific to hyperplastic liver nodules defined with monoclonal antibody: a new marker for preneoplastic cells in rat chemical hepatocarcinogenesis.

Two monoclonal antibodies (MoAbs) were prepared against the hepatocytes of hyperplastic liver nodules from rats killed in the 13th week during hepatocarcinogenesis with 2-acetylaminofluorene. The specificity of these MoAbs was confirmed by cellular radioimmunoassay. By means of immunoperoxidase staining with 2 MoAbs (HAM 6 and HAM 7), HAM 6-defined antigen was detected on the plasma membrane and/or cytoplasm of the hepatocytes in hyperplastic nodules, while HAM 7 reacted to the hepatocytes surrounding hyperplastic nodules. Therefore, HAM 6 seemed to be specific to hyperplastic nodules, but HAM 7 to be a MoAb developed accidentally to the surrounding hepatocytes by contamination of the cell suspension of hyperplastic nodules.

Identification of an erythrocyte autoantigen using monoclonal autoantibodies induced by immunization of mice with rat erythrocyte.

An erythrocyte autoantigen has been identified by means of monoclonal autoantibodies raised by immunizing mice with rat red blood cells (RBC). The autoantibodies reacted with intact rat and mouse RBC as judged by a cellular radioimmunoassay, and with a 52K band on western blots of rat and mouse RBC. They did not react with intact sheep RBC or blotted sheep erythrocyte membranes. Although anti-rat erythrocyte antibodies did react with bands in the molecular weight region of 50-55 K (on blots of rat erythrocyte membranes), these bands were susceptible to neuraminidase digestion, thus distinguishing them from the 52 K band recognized by monoclonal autoantibodies. The implications of the above results for the known autoantibody specificity of suppression is discussed, and it is suggested that they favour the existence of autoantigen-specific suppressor cells.

The detection of two surface antigens on human lung macrophages using monoclonal antibodies.

Monoclonal antibodies (McAb), designated AMH1 (IgM, lambda) and AMH2 (IgG1, Kappa), against specific surface antigens of human lung macrophages were produced by the fusion of the NS-1 plasmacytoma cell line with spleen cells from BALB/c mice immunized with bronchoalveolar lavaged (BAL) cells obtained from selected smoking subjects. The screening and characterization of these McAb were carried out employing cellular radioimmunoassay, flow cytofluorography, and immunohistochemical methods. These two antibodies specifically reacted with macrophages in the alveolar spaces and BAL fluids. AMH1 did not react with peripheral blood cells including freshly separated monocytes, cultured monocytes, lymphocytes, granulocytes, and platelets. In addition, AMH1 did not react with peritoneal exudate cells or pleural exudate cells. On the other hand AMH2 showed the dull-positive reaction with some monocytes and pleural exudate cells among above-mentioned cells. These two McAb seemed to detect cell surface antigens that are expressed by highly differentiated or mature macrophages compared to OKM1. These antibodies will allow not only better characterization of immune cells but also assessment of maturity of lung macrophages.

STRAIN DIFFERENCES IN THE EXPRESSION OF THE EPA‐1‐RESTRICTING ELEMENT

Epa-1-specific cytotoxic T lymphocytes (CTL) lyse epidermal cells (EC) of different Epa-1+ H-2k strains, such a AKR, CBA, C58, and RF, at different levels. We used an H-2Kk-specific monoclonal antibody (mAb) to test the hypothesis that this phenomenon is due to differences in the H-2-restricting element. Initially, we established the specificity of this mAb for the Epa-1-restricting element by demonstrating its capacity to inhibit the lysis of CBA EC by Epa-1-specific CTL. We then used it as the probe in a cellular radioimmunoassay to quantify the expression of the restricting element by EC of different H-2k strains. We found that C58 and RF EC bound significantly less of the mAb than did CBA EC. Although AKR also bound less of the mAb than did CBA EC, the difference was not statistically significant. To examine the generality of this phenomenon, we quantified the expression of Kk antigens on spleen cells (SC) of the same four strains. We found that RF SC, but not AKR or C58 SC, bound significantly less of the Kk mAb than did CBA SC. Thus, the differential CTL lysis of Epa-1+ EC of different strains probably reflects differences in expression of the H-2-restricting element rather than of the nominal antigen.

Distinct human leukemia-associated cell surface glycoprotein GP160 defined by monoclonal antibody SN6.

In this study, a monoclonal antibody (mAb) termed SN6 was generated by immunizing a mouse with a non-T-cell leukemia antigen preparation isolated from cell membranes of leukemia cells derived from a patient (FJ) with non-T/non-B-cell-type acute lymphoblastic leukemia (ALL). SN6 was tested against a variety of cultured and uncultured human cell specimens by using a sensitive cellular radioimmunoassay. Among the 26 cultured malignant and nonmalignant cell lines tested, SN6 reacted with all of the 6 leukemic non-T/non-B (including pre-B)-cell lines tested--i.e., KM-3, NALM-16, REH, NALL-1, NALM-1, and NALM-6. Of these cell lines, 5 were derived from individual patients with ALL; the remaining 1 was from a patient with chronic myelocytic leukemia in blast crisis. In addition, SN6 reacted with 3 of 3 leukemic myelo-monocytic cell lines tested--i.e., ML-2, HL-60, and U937. SN6 did not react with any other cell lines. A consistent result was obtained with 42 fresh (uncultured) cell specimens derived from individual patients with several different types of leukemias. SN6 reacted with 11 of 16 non-T/non-B (including pre-B)-cell ALL specimens. In addition, it reacted with various myelo-monocytic leukemia cell specimens to various degrees. SN6 did not show a significant reaction with normal peripheral blood cells tested, which included B cells, T cells, granulocytes, monocytes, and erythrocytes. However, it reacted with a small population (approximately 1% as determined by immunofluorescence staining) of normal bone marrow cells. The approximate molecular mass of the glycoprotein antigen defined by SN6 was determined to be 160,000 by radioimmunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Only one component of 80,000 daltons was formed upon reduction of the 160,000 molecular mass antigen. Therefore, this antigen is apparently a homodimer of a 80,000-dalton subunit. This conclusion was further corroborated by two-dimensional gel analysis, which showed a single well-defined spot for the reduced antigen. We designate this distinct human leukemia-associated cell surface antigen "GP160."

Monoclonal antibodies to human choriocarcinoma.

We have established two monoclonal antibodies (TM7-3 and TM3-8) that react to choriocarcinoma cells. Both of these monoclonal antibodies have shown a similar reactive pattern to human cell lines, normal and neoplastic trophoblast tissues, and other fetal and adult tissues. They have reacted to nine of the ten choriocarcinoma cell lines, as well as to Hela cells (a cervical carcinoma cell line). During a cellular radioimmunoassay, neither TM7-3 nor TM3-8 reacted to two T lymphoblastoid cell lines or three B lymphoblastoid cell lines. Immunofluorescence and immunoperoxidase staining showed that both monoclonal antibodies reacted selectively to the cytotrophoblast-like tumor cells of a choriocarcinoma and a hydatidiform mole but not to syncytiotrophoblast-like tumor cells. TM7-3 and TM3-8 also reacted slightly to the normal cytotrophoblast of early human chorionic villi under the same conditions as they did to choriocarcinoma tissues, TM7-3 and TM3-8 bind only to a part of the urinary tubles of the kidney and to the ducts of the pancreas of both adult and fetus.

New micro-glass-tube leukocyte adherence inhibition assay assessing cell adherence of mononuclear cell subpopulations defined by monoclonal antibodies

A new micro-glass-tube leukocyte adherence inhibition (LAI) assay which is appropriate for detecting delayed type hypersensitivity in vitro has been developed for human leukocytes. Enumeration of adherent cells is replaced by a cellular radioimmunoassay determining antibody binding of the monoclonal reagents, OKT4, OKT8, and OKM1, to glass-adherent cells, fixed by glutaraldehyde or formaldehyde. An LAI reactivity to purified protein derivative of tuberculin (PPD) was detectable in donors giving a positive PPD skin test with OKT4 reagent, but not with the other two reagents.

Human early pregnancy factor: application of the monoclonal antibody anti-HuLyt 3 in the rosette inhibition test and in a cellular radioimmunoassay

Using the monoclonal antibody anti-HuLyt 3, raised rosette inhibition titres were observed when lymphocytes were preincubated with early human pregnancy sera, although this was not a consistent finding. Results obtained from a cellular radioimmunoassay suggest that this enhancement of rosette inhibition is not due to interference with antibody binding.

Human Ia molecules carrying MT1 determinants: Purification with a monoclonal antibody

A monoclonal antibody 77.34, reactive with polymorphic HLA class II molecules, was produced. The allotype specificity of this IgG2a antibody was analyzed by cytotoxicity, flow cytometry, and cellular radioimmunoassay. Cytotoxic reactivity on a panel of B cells from 88 unrelated individuals was concordant with the MT1 (DC1) allospecificity (r = 0.83). Immunoanalysis by flow cytometry showed that cells from MT1 + homozygous cell lines were reactive, whereas MT2 + and MT3 + homozygous cells were not. A cellular radioimmunoassay performed under saturating conditions indicated that three MT1 + cell lines bound 14–45 × 10 5 molecules of antibody per cell representing 30–40% of the amount detected with monomorphic anti-DR monoclonal antibody 21w4. The subset of molecules bearing the MT1 allospecificity was purified with a 77.34 IgG immunoadsorbent. The purified molecules were antigenically reactive with several antibodies directed at DQw1 molecules but were devoid of reactivity to monomorphic anti-DR antibodies. Two-dimensional gel electrophoresis showed that the α subunit is composed of several acidic spots of Mr 32,000 whereas the β subunit was seen as a single spot of Mr 25,000, corresponding to DQw1 molecules. DR molecules purified by monoclonal antibody affinity were unreactive with 77.34 antibody. All of the 77.34 reactivity was observed with the fractions depleted of DR molecules. Two-dimensional gel analysis showed marked differences between the purified DR and DQw1 molecules. Two-dimensional gel analysis showed marked differences between the purified DR and DQw1 molecules. The presence of the MT1 determinant on Ia molecules referred to as the DQw1 molecules and distinct from those bearing the DR epitopes was confirmed on two DR1, MT1 homozygous cell lines. Thus, DQw1 molecules can be purified away from DR1 molecules by affinity chromatography to 77.34 IgG, specifically reactive with the MT1 (DQw1) allospecificity. The binding of 77.34 IgG to MT1 + cells was not inhibited by all monoclonal antibodies reported to be correlated with the MT1 allospecificity suggesting that the latter might be comprised of more than a single epitope.

Identification of several cell surface proteins of non-T, non-B acute lymphoblastic leukemia by using monoclonal antibodies.

Monoclonal antibodies (MAb) were produced by immunization of BALB/c mice with cells from a non-T, non-B acute lymphoblastic leukemia (ALL) cell line. Nine distinct antigens (groups I to IX) were defined by these monoclonal antibodies, some of which appear to be associated with specific stages of cellular differentiation. The number of molecules of each MAb reactive with the ALL cell line, measured in a quantitative cellular radioimmunoassay, varied from 0.6 X 10(5) to 11 X 10(5) molecules/cell, indicating that the antigens identified represent major constituents of the cell surface. The biochemical nature of the antigens was examined on the ALL cell line by antibody affinity chromatography and/or immunoprecipitation and SDS-polyacrylamide gel electrophoresis. Groups I through III are composed of previously described antigens: HLA class I, HLA class II molecules, and CALLA, the common ALL antigen. The other MAb define antigens previously undescribed on non-T, non-B ALL cells. Group IV antigen is a polypeptide of apparent m.w. 95,000 distinct from CALLA. It is expressed on some ALL samples and on the vascular endothelial cells of several tissues. Group V antigen is a single polypeptide chain of m.w. 94,000, also distinct from CALLA and expressed by lymphocytes, thymocytes, acute myelogenous leukemia (AML) cells, and ALL cells. Group VI is a molecular complex composed of two noncovalently associated polypeptides of apparent m.w. 125,000 and 87,000 and appears to be restricted to ALL, AML, macrophages, and hematopoietic precursor cells. Group VII is a glycoprotein of apparent m.w. 85,000, which, within the thymus, is primarily restricted to the medullary area. It is also present on AML, bone marrow cells, and mature T and B lymphocytes. Group VIII is a disulfide-linked complex of apparent m.w. greater than 120,000 under nonreducing conditions. It is resolved into three major polypeptides of apparent m.w. 57,000, 47,000, and 41,000 under reducing conditions. This complex is found in greatest amounts on the non-T, non-B ALL cell line but is also present on AML, ALL, and on subpopulations of normal bone marrow and tonsil cells. Group IX antigen is a single polypeptide chain of apparent m.w. 51,000 on the ALL cell line. This antigen is expressed strongly on ALL and AML samples and on normal bone marrow; much lower antigenic density is found on thymus and tonsil cells. The antigens described here with a series of MAb produced in a single fusion represent a unique array of cell surface molecules of non-T, non-B ALL cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative expression of epitopes defined by murine monoclonal antibodies on DR molecules from different HLA haplotypes

Monoclonal antibodies 50D6 and 21r5, reactive with human class II molecules, were analyzed quantitatively by flow cytometry and cellular radioimmunoassay for their binding to cells of different HLA-DR types. Monoclonal antibody 50D6 bound equally to cells of all DR types tested except DR7, where no reactivity was observed. Monoclonal antibody 21r5 was reactive with all cells. However, the percentage of DR molecules at the cell surface expressing 21r5 epitope varied with the DR type and increased as follows: DR3 = DR7 less than DR2 less than DR5 less than DR4 less than DR1. MAb 50D6 reacted with an epitope spatially related to but distinct from the 21w4 epitope present on all DR molecules. The 50D6 epitope was shown to be present on isolated DR1 molecules.

Use of an automatic cell harvester in a cellular radioimmunoassay

An automatic cell harvester was used in the final step of a cellular radioimmunoassay to collect cell bound anti-rat IgG 125I-F(ab′) 2. Studies on the reliability of this collection method were performed with antibodies directed against cell surface antigens induced by the Gross murine leukemia virus and produced by immunization of W/Fu rats with the syngeneic (C58NT)D lymphoma. Glutaraldehyde-fixed as well as untreated Gross virus induced lymphoma cells could be used. Similar and specific antibody binding curves were obtained when the cells were incubated with the anti-(C58NT)D serum and anti-rat IgG 125I-F(ab′) 2 in the presence of 0.1% NaN 3. Background levels of non-specific binding of anti-rat 125I-F(ab′) 2 to mouse lymphoma cells or rat thymocytes were only a few cpm above the background of the gamma-counter. This allowed detection of surface immunoglobulin positive lymphocytes among as few as 30,000 rat splenocytes. In addition, this cellular radioimmunoassay was found to be suitable for the measurement of solubilized cell surface antigen by its capacity to inhibit binding of the specific antibodies to the target cells.

Elicitation of the maternal antibody response to the fetus by a broadly shared MHC class I antigenic determinant.

The antibody response to the semiallogeneic fetus in the rat was examined in eight mating combinations that were chosen specifically to cross different major histocompatibility complex (MHC) and nonMHC loci. The BN X DA and BN X BN.1A(DA) matings (n anti-a) were the only ones to show an antibody response to the fetus, and the response occurred in 25-64% of the matings. All the other six mating combinations studied were negative by hemagglutination, cytotoxicity, and cellular radioimmunoassay. The response appeared in the postpartum period after the first litter and reached its maximal frequency during the postpartum periods of the second and third matings. There was no correlation between the litter size and the presence or absence of an antibody response to the fetal antigens. The antibodies elicited by the fetus were hemagglutinating but not cytotoxic, and consisted of only one isotype, IgG2a, whereas in the alloantiserum raised by skin grafting and the injection of lymphocytes, they were distributed among the IgG2a greater than IgG1 greater than IgM isotypes and were both hemagglutinating and cytotoxic. Using reciprocal congenic strains, the pregnancy-induced antibody response was shown to be against MHC antigens only. This finding was confirmed by testing the sera against rats from a population that was segregating for the haplotypes a/a, a/n, and n/n. Comparison of the pregnancy-induced antibody with the BN anti-DA alloantibodies by absorption studies showed that the former was less crossreactive, did not react with the RT1.Aa haplotype-specific antigenic determinant, and did react with an antigenic determinant shared among the a, d, o, i, e, f, r10, b, and m haplotypes--but not with the n, c, g, k, l, h, or u haplotypes. Using the DA.1I and r10 strains in cellular radioimmunoassay, absorption, and immunoprecipitation experiments, it was shown that the pregnancy-induced antibody was elicited by a class I antigenic determinant, provisionally designated Pa, which is encoded by a locus in the vicinity of RT1.A.

Characterization of new non-T, non-B acute lymphoblastic leukemia cell lines: Analysis of surface antigens by quantitative cellular radioimmunoassay and flow cytometry

Two novel acute lymphoblastic leukemia (ALL) cell lines, HOON and HYON, have been established from patients with non-T, non-B ALL. The cell lines have been characterized and shown to express phenotypic markers of non-T, non-B ALL. By indirect immunofluorescence and flow cytometry they express Ia and common ALL (CALLA) antigens and are reactive with monoclonal antibodies BA-1, BA-2 and OKT-9. However, the cells do not express detectable amounts of B1 antigen or of cytoplasmic μ chain, which are markers of pre-B cells. Quantitation of Ia and CALLA antigens on HOON and HYON cell lines using a cellular radioimmunoassay revealed that both cells bind high levels of anti-Ia antibodies, 110 × 10 4 molecules per cell, and low levels of anti-CALLA antibodies 7 × 10 4 molecules per cell. Although both HOON and HYON carry equivalent amounts of Ia on their surface, only the former is a good stimulator of the one-way mixed lymphocyte reaction.

A private strain-specific idiotype-like structure discovered on CBA-anti-C57BL/6 T lymphocytes

Using rabbit anti-CBA-anti-C57BL/6 sera, a private strain-specific idiotype-like determinant(s) was detected by cellular radioimmunoassay on activated CBA—anti-C57BL/6 T cells. This structure was not expressed on other activated T lymphocytes: CBA—anti-BALB/c; C3H—anti-C57BL/6; AKR—anti-C57BL/6; A/Sn—anti-C57BL/6; BALB/c-anti-C57BL/6; DBA/2-anti-C57BL/6. It is suggested that the gene(s) coding for the private strain-specific idiotype-like determinant(s) of T-cell antigen-recognizing receptors is not located within the murine H-2 complex.