Immune Rabbit Serum Research Articles

ACA-1, a new differentiation antigen of activated lymphoid cells

Rabbits were immunized with murine CBA T lymphocytes activated with C57BL/6 antigens (T act). The immune rabbit sera were adsorbed with murine erythrocytes, serum, liver cells, and unstimulated T and B lymphocytes. Upon absorption, the antisera (AT actS) were not cytotoxic for unactivated thymus, lymph node, and spleen cells of different mouse strains in the cytotoxicity assay. AT actS did not inhibit the immune response of lymphocytes from unimmunized animals to SRBC or their proliferation in mixed lymphocyte culture. At the same time AT actS lysed 67% of actively proliferating T-lymphoma EL-4 cells, 31–56% of the T lymphocytes stimulated with allogeneic cells, and 53–56% of Con A- or LPS-stimulated splenocytes. AT actS also inhibited 90–100% of antigen-activated B cells, i.e., plaque-forming cells (PFC) producing 19 S and 7 S antibodies to serologically noncrossreacting antigens, such as sheep, rabbit, and rat RBC. It also decreased the intensity of the secondary immune response to SRBC. The effect of AT actS did not depend on the H-2 or Ala-I phenotype of target cells. Our experiments showed that AT actS activity fell after absorption with activated, but not quiescent lymphoid cells. These results suggest the presence of a special antigenic marker on activated murine T and B cells which differs from the generally recognized cell surface antigens, such as H-2, Ig, Ala-I, idiotype, Thy-I, MBLA, MTLA, Lyt-1,2,3, and others. It has been designated Aca-I (activated cell antigen).

Passive Protection of NMRI Mice Against Serratia marcescens: Comparative Efficacy of Commercial Human IgG Immuno-globulin Preparations and rabbit anti-O, -H, -K, -Life Cell and -Protease Immune Sera

Three commercial, intravenously applicable human IgG preparations passively protected NMRI mice against intraperitoneal challenge with 11 of 19 selected, serologically defined O-antigen reference strains and clinical, nosocomially significant isolates of Serratia marcescens. Conventional anti-O and anti-H, but not anti-K and anti-protease, rabbit immune sera revealed passive protective capacity against homologous challenge strains; however, anti-O rabbit immune sera failed to invariably protect against heterologous test strains that carried identical serogroup O-antigens. The passive protective efficacy of antilive cell immune sera, derived from rabbits following recovery from experimental septicemia, was abolished through dual absorption with homologous O-cells. It was concluded that antibodies directed against O-antigens, i.e., lipopolysaccharide moieties, of the challenge strains of S. marcescens afforded passive protection.

Characterization of antigens on mosquito midgut stages of Plasmodium gallinaceum. I. Zygote surface antigens

We have defined the surface protein antigens on Plasmodium gallinaceum zygotes using radioiodination methods and rabbit anti-zygote serum which blocks transmission of the parasite to Aedes aegypti mosquitoes. Fifteen protein bands (1–15) in the molecular weight range of 40 000–240 000 and one band at the bromophenol blue dye marker were labelled by the lactoperoxidase and IODOGEN (1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril) methods. The localization of these radioiodinated components on the cell surface was confirmed in two ways: (1) They were completely degraded by trypsin or Streptomyces griseus protease treatment of intact viable zygotes. (2) They were immunoprecipitated following incubation of intact zygotes with antibody prior to detergent solubilization. Reactivity with immune rabbit serum demonstrated that the major surface immunogens were components with M r 240 000, 200 000, 180 000, 80 000, 55 000, 50 000 and the band at the dye marker. A band of M r 180 000 was shown immunologically to be a host serum protein selectively adsorbed to the zygote surface. The identity of this protein and the significance of its adsorption to the surface of the zygote are unknown.

Protective effects of antibody against intestinal invasion by Escherichia coli.

Seven Escherichia coli isolates from newborn calves with diarrhea were examined for enteropathogenic properties. One isolate penetrated into HeLa cells, four produced enterotoxin(s) and the remaining two possessed neither of these properties. Penetration of E. coli into HeLa cells was inhibited by antibody in bovine colostrum and in bovine and rabbit immune sera. The effective antibodies appeared to be mostly of the IgM class. The invasion by E. coli isolates was also examined by inoculation of the bacteria into the small intestine of E. coli-immunized and non-immunized guinea pigs. The isolate which penetrated into HeLa cells could penetrate the intestinal mucosa to be disseminated into various organs of non-immunized guinea pigs, but not of immunized guinea pigs, whereas no other isolates showed such pathogenicity in vivo. The inhibition of the invasion was observed when non-immunized guinea pigs were inoculated with the bacteria together with colostral or serum antibody. The results show the importance of antibody in the local defense mechanism against E. coli invasion.

Antigen analysis of several pathogenic strains of Trichomonas vaginalis

Analysis of several human strains of Trichomonas vaginalis and one bovine strain of Tritrichomonas foetus was accomplished with standard sodium dodecyl sulfate-gel electrophoresis and fluorography technology. Highly motile, live trichomonads were radiolabeled, and total trichloroacetic acid-precipitated proteins were electrophoresed. Complex protein profiles of the various human strains of T. vaginalis were obtained with proteins ranging in molecular weight from 20,000 to greater than 200,000. The parasite biosynthesis of the Coomassie brilliant blue-stained protein bands was demonstrated by efficient radiolabeling of trichomonads with [35S]methionine or a 3H-amino acid digest before electrophoresis and fluorography. Immunogenic trichomonal proteins were then identified by a radioimmunoprecipitation method. A detergent extract of [35S] methionine-labeled T. vaginalis proteins was mixed with serum from an immunized rabbit or pooled sera from subcutaneously infected mice and soluble antibody-antigen complexes isolated by adsorption to protein A-bearing Staphylococcus aureus. The radiolabeled protein antigens were then identified by gel electrophoresis and fluorography. Immunized rabbit serum and pooled sera from challenged mice contained high-titered antibody which reacted with numerous high- and low-molecular-weight proteins. Individual subcutaneously infected mice were found to possess identical antibody responses to these immunogenic trichomonal proteins. A high degree of serological cross-reactivity among the various trichomonads was demonstrated. No differences in the composition of immunogenic proteins were observed among cultures grown in vitro for various lengths of time under the experimental conditions employed. Finally, electrophoretic analysis of cloned colonies of T. vaginalis organisms revealed no differences in their protein composition. The biological relevance of these observations is discussed.

Cross-reactivity of Haemophilus somnus antibody in agglutination and complement fixation tests and in the enzyme-linked immunosorbent assay.

The specificity and sensitivity of agglutination, complement fixation, and enzyme-linked immunosorbent assay (ELISA) procedures in the detection of antibodies to Haemophilus somnus was investigated. H. somnus rabbit immune sera were found to agglutinate Pasteurella multocida, Staphylococcus aureus, and Haemophilus agni and, in some instances, also Pasteurella haemolytica, Salmonella dublin, Streptococcus agalactiae, and Corynebacterium pyogenes. In complement fixation tests with saline extracts as antigens, only H. agni reacted with H. somnus antisera to any significant degree. In ELISA tests with sonicated or heat-extracted antigens, cross-reactions were seen with the two Pasteurella spp. and with H. agni. When whole cells and saline extracts were used as antigens in ELISAs, only H. agni showed any cross-reactivity. The greatest specificity in distinguishing homologous from heterologous reactions was achieved by ELISA with saline extracts as antigens. Escherichia coli and Brucella abortus antigens failed to react with H. somnus antibody in any of the tests. A rabbit serum containing antibody to bovine type isolates of P. multocida, P. haemolytica, S. aureus, S. agalactiae, S. dublin, C. pyogenes, and E. coli gave no positive reaction in ELISA tests with saline extract of H. somnus as antigen. It is concluded that such saline extract, which appears to consist largely of H. somnus common antigen, has the potential of being a useful diagnostic reagent in the study by ELISA of antibody response to H. somnus.

Detection of Sarcocystis antigens in the sera of experimentally-infected pigs and mice by an immunoenzymatic assay.

Immunoglobulins raised against Sarcocystis miescheriana and Sarcocystis muris cystozoite antigens were isolated from rabbit immune sera by affinity chromatography (using CNBr-activated Sepharose 4B for antigen immobilization). The specific immunoglobulins were incorporated into double-antibody sandwich immuno-enzymatic assays which were firstly quantitated and then used to detect soluble Sarcocystis antigens in the sera of experimentally-infected pigs and mice. Assays employing immunoglobulins attached to the solid-phase at concentrations of 80 micrograms/ml were capable of detecting homologous soluble cystozoite and sporozoite reference antigens at concentrations as low as 8 micrograms/ml. Circulating antigens were detected both in the presence and absence of acute clinical disease in pigs experimentally-infected with high and low doses of S. miescheriana sporocysts. The antigenemia detected was transitory (occurring from 3-20 days post-inoculation: dpi) and coincided well with the sporozoite and merozoite phases of parasite development. Circulating antigens were also detected during subclinical infections in mice (from 4-49 dpi) following their experimental infection with low doses of S. muris sporocysts. Specific immuno-enzymatic assays for circulating Sarcocystis antigens may therefore prove useful in the clinical diagnosis of acute sarcocystosis.

Interaction of Treponema pallidum with isolated rabbit capillary tissues.

Within infected tissue Treponema pallidum shows a characteristic predilection for perivascular areas. After intact capillaries had been prepared from rabbit brain tissue treponemes were incubated with isolated capillaries and visualised by darkfield, phase contrast, and scanning electron microscopy. The organisms rapidly attached to the surface of the capillaries at the tip of the treponeme; attached organisms retained motility for longer periods than unattached organisms. Treponema pertenue also attached to capillaries. Heat-inactivated T pallidum and three non-pathogenic treponemes did not, however, attach to the capillaries. Immune rabbit serum contains a factor that blocks the attachment of T pallidum to capillaries. Compared with cultured mammalian cells capillaries should provide a better tool for investigating host-parasite relationships in syphilis.

Identification of the virus-specific proteins of respiratory syncytial virus temperature-sensitive mutants by immunoprecipitation.

The proteins of Long strain RSV and three temperature-sensitive (ts) mutants of the A2 strain were compared by pulse labeling virus-infected cells with [35S]methionine and [3H]glucosamine followed by analysis of the cell lysates by polyacrylamide gel electrophoresis. At the permissive temperature (30 degrees) proteins ranging in molecular weight from 24,000 to 50,000 (VP24, VP27, VP33, VP44) could be identified. Immunoprecipitation of viral lysates by immune rabbit serum demonstrated antigenic similarity with VP27, VP44, VP50, and VP67 in all ts mutants and Long strain RSV. [3H]Glucosamine labeling demonstrated glycoproteins of 90,000 (GP90) and 50,000 (GP50) in Long strain and GP90 in the ts mutants.

Immunodiffusion and complement fixation assays with sera from mucormycotic-infected mice.

Results using homogenate antigens of Rhizomucor pusillus against immunized rabbit sera, mouse ascites fluid and sera from mucormycotic-infected mice, detected by immunodiffusion and complement fixation assays are presented. Using immunodiffusion techniques, common antigens and unique antigens from Rh. pusillus, Rhizopus oryzae and Absidia corymbifera were observed. Complement fixation titers of greater than 256 and 128 were obtained in immunized rabbit sera and mouse ascites fluid, respectively. However, no appreciable titers of antibody were measured from mucormycotic-infected animals by either immunodiffusion or complement fixation.

Monoclonal antibody against the carboxy terminal peptide of pp60src of Rous sarcoma virus reacts with native pp60src.

A monoclonal mouse antibody has been prepared against a synthetic peptide corresponding to the six carboxy-terminal amino acids (C' peptide) of the src gene product pp60v -src of Rous sarcoma virus (RSV). The antibody was able to precipitate pp60v -src and to bind pp60v -src kinase activity in a competition test, indicating that this peptide can serve as an antibody-binding site (epitope). Furthermore, the finding that three out of 28 pp60src-specific tumor-bearing rabbit (TBR) sera contained antibody against the C' peptide argues for an in vivo role for the carboxy terminus of pp60src. C' peptide-specific IgG was purified from one TBR serum using affinity chromatography, and was shown to precipitate significant amounts of pp60src, and bind most of the pp60src kinase activity from SRA, PrA, and B77-C strains of avian sarcoma virus (ASV), but not endogenous pp60c -src, a cellular homologue to the viral pp60v -src. Similar results were obtained with IgG isolated from a C' peptide immune rabbit serum. None of the three C' peptide-specific IgGs could serve as a phosphate acceptor in an immune complex protein kinase reaction.

Opsonization requirements of serratia marcescens

Dextran-separated human peripheral blood leukocytes (55 vol%) from healthy adults served to delineate the opsonic requirements of various serologically defined assay strains of Serratia marcescens. 'Natural' antibodies of three commercial IgG immunoglobulin preparations (25 vol%), in the absence of complement (C), did not opsonize all 19 test strains, in particular certain strains carrying O-antigens O6 and/or O14, despite documented (ELISA technique) contents of anti-O6 and -O14 IgG antibodies. The 'natural' IgG opsonins proved O-antigen specific. Several of 13 selected assay strains of S. marcescens differed in opsonic requirements for 'classically' or 'alternatively' activated human C; opsonization was optimal with intact fresh human serum (25 vol%). All 13 tested strains activated both pathways of C. Anti-live cell rabbit immune sera (RIS) surpassed conventional anti-O and anti-H RIS (25 vol%) in terms of opsonizing activity, which proved partially heat-labile and O-antigen specific for two, but not for two other test strains. Isolated IgM immunoglobulin fractions of anti-live cell RIS either totally lacked or displayed inferior opsonizing activity in the absence of C, although O-agglutinin activity was only from 4- to 8-fold lower than that of unfractionated anti-live cell RIS. Human and rabbit 'natural' IgM antibodies opsonized moderately. Added guinea pig C (10 vol%) failed to enhance opsonizing activity of 'natural' and 'immune' rabbit IgM and 'natural' human IgG and IgM antibodies.

Use of Monoclonal Antibody in an Enzyme-Linked Immunosorbent Assay to Detect the Presence of Trichoplusia ni (Lepidoptera: Noctuidae) S Nuclear Polyhedrosis Virus Polyhedrin in T. ni Larvae

A sandwich type of enzyme-linked immunosorbent assay (ELISA) incorporating monoclonal antibody and rabbit immune serum was used to detect polyhedrin in Trichoplusia ni S nuclear polyhedrosis virus (TSN)-infected T. ni (Hubner) larvae. Polyhedrin, the major component of nuclear polyhedrosis virus (NPV) polyhedra, is coded for by the virus, and its presence in larvae indicates the presence of NPV or an NPV infection. The ELISA and the judgment of a trained observer were compared with regard to detecting NPV or NPV disease in larvae exposed to high, medium, and low doses of TSN. The ELISA was the more sensitive diagnostic technique of the two, and detected the presence of NPV 2 to 3 days in advance of the observer. At the medium and high dosages, the ELISA detected polyhedrin in or on larvae 2 h after exposure to the virus, before any protein amplification could have taken place, indicating a high degree of assay sensitivity.

Role of surface components in serum resistance of virulent Aeromonas salmonicida.

The ability of virulent strains of Aeromonas salmonicida to resist the bactericidal activity of serum was quantitated. The A. salmonicida strains tested included virulent strains, mutants lacking the major surface A-protein, and mutants lacking A-protein and having a modified lipopolysaccharide structure. The sera evaluated included normal human, rabbit, and trout sera, immune trout serum, and immune rabbit serum containing antibodies to A-protein and lipopolysaccharide. Virulent strains of A. salmonicida displayed high or intermediate resistance to the bactericidal activity of complement both in the presence and absence of specific antibody. In normal sera, both A-protein and lipopolysaccharide contributed to serum resistance. In immune trout serum, the protection was conferred by A-protein.

Immunofluorescent detection of erythrocyte sialoglycoprotein antigens on murine erythroid cells.

A sialoglycoprotein fraction isolated from murine (DBA/2) erythrocytic ghosts (see companion article, Sarris and Palade, 1982, J. Cell. Biol. 93:583-590) was used to raise antibodies in rabbits. By immune-IgG (serum)-[125I] protein A overlays, the antibodies were found to react positively with the four sialoglycoprotein monomers (gp-2.1, gp-2.2, gp-3.1, and gp-3.2) of the original fraction, with the sialoglycoproteins detected in erythrocytic ghosts (gp-2.1 and gp-3.1), with a diffuse component (probably a macroglycolipid) trailing around gp-3.1 in SDS polyacrylamide gel electrophoretograms of solubilized ghosts, and with a minor sialoglycoprotein hidden under this trail. IgG's isolated from immune and nonimmune rabbit sera were conjugated to tetramethylrhodamine isothiocyanate and used to survey, by fluorescence microscopy, the distribution of the cognate antigens on the three different erythroid lines known to succeed each other during the life span of the mouse. In the peripheral blood of the adult, the antibodies recognized only mature erythrocytes; they did not crossreact with either platelets, monocytes, or different types of granulocytes. In the spleen of adult anemic mice, the antibodies reacted weakly with proerythroblasts and strongly with all types of erythroblasts. In enucleating erythroblasts, antigens were preferentially segregated on the cell membrane of the nascent reticulocyte. In the 10-day-old embryo, antigens were already present on the primitive nucleated erythrocytes (produced by the blood islets of the yolk sack), and in the 14-d fetus they were found on all hepatic erythroblasts and derived non-nucleated erythrocytes. A positive immunoreaction was also obtained on Friend erythroleukemic cells, before or after induction by dimethyl sulfoxide. Nonimmune serum, or nonimmune IgGs gave negative reactions in all cases. The antibodies were species-specific: they did not crossreact with either human or rat erythrocytes.

Préparation d'anticorps monospécificiques anti-LPS de Salmonella sur bactéries immobilisées dans un gel de polyacrylamide

The use of an immunoabsorbent obtained by entrapping Salmonella cells into a poylacrylamide gel lattice enabled us to obtain anti-O monospecific immune sera which can be used for a quick serological identification of some species of Salmonella in the course of a diagnostic. In this paper we describe a method for immbilization of S. typhimurium as well as the preparation of monospecific anti-O5 antibodies from plurispecific anti- S. haifa rabbit immune sera. This separation of anti-O monospecific antibodies avoids the repeated and often tedious adsorption of anti- Salmonella immune sera by the corresponding whole bacteria. Such Salmonella, entrapped into the polyacrylamide gel lattice, can be used several times without appreciable loss of their affinity properties.

Xenogeneic antisera against T-lymphocyte receptors recognizing allogeneic transplantation antigens: Preparation and properties

Antireceptor sera ARS-1 (anti-CBA-anti-C57BL/6) and ARS-2 (anti-C57BL/6-anti-BALB/c) were prepared in a xenogeneic system by immunizing rabbits with alloimmune CBA anti-C57BL/6 and C57BL/6 anti-BALB/c lymphocytes and subsequent exhaustive absorption of rabbit immune sera with red blood cells, lymphocytes, liver cells, and serum from intact mice. In the cytotoxicity test, both ARS sera lysed only activated T lymphocytes of corresponding, but no other specificities, and did not affect intact lymphocytes. They did not inactivate in vitro the PFC-producing antibodies to SRBC or inhibit the immune response to SRBC and Viantigen of Salmonella typhi in adoptive transfer experiments. Pretreatment of intact CBA lymphocytes in vitro with ARS-1 plus complement suppressed their ability to induce lethal GvH disease in irradiated (CBA × C57BL/6)F 1, but not (CBA × BALB/c)F 1 recipients; similar pretreatment of C57BL/6 lymphocytes did not lower lethality among irradiated (CBA × C57BL/6)F 1 recipients. This method of preparing ARS may help to obtain highly specific antireceptor sera for regulation of the immune response to different antigens in outbred animals and in humans.

Opsonic requirements for phagocytosis of Borrelia hermsii by human polymorphonuclear leukocytes.

Opsonic requirements for phagocytosis by human polymorphonuclear leukocytes (PMNLs) of a laboratory strain of Borrelia hermsii were examined. Intracellular localization of spirochetes was confirmed by electron microscopy and cinephotomicroscopy. Phagocytosis of 3H-labeled spirochetes was increased with higher concentrations of pooled human serum or greater ratios of spirochetes to PMNLs and was unaffected by diminished classical complement pathway activity. Immune rabbit serum markedly increased phagocytosis of spirochetes beyond levels observed with preimmune rabbit serum. Immune rabbit serum also demonstrated direct activity against B. hermsii, which decreased the motility and viability and increased agglutination of the spirochetes independently of complement. Both the direct activity against B. hermsii and the opsonic activity of immune rabbit serum were eliminated by immunoabsorption of immunoglobulins. These observations suggest a role for the alternative complement pathway and for immunoglobulins in the opsonization of spirochetes for phagocytosis by PMNLs. Immunoglobulins may also play a role in the direct clearance of spirochetes.

Towards serodiagnosis of Serratia marcescens infections: Examination of sera from noninfected patients and from experimentally infected rabbits for anti-H and anti-O antibodies; S. marcescens O-antigen cross-reactions with those of other enterobacteriaceae

Sera from 100 patients not infected with Serratia marcescens at the time of hospital admission lacked detectable H-immobilizing antibodies against all 20 currently recognized H-antigens of this microorganism. However, various patient sera revealed elevated titers of O-agglutinins against several of the 20 O-antigens of S. marcescens, in particular O-antigens O1, O3, O4, O5, O7, O8, O10, O11, O16, O17, O18, O19, and O20. Rabbit anti-Shigella serogroup B immune serum cross-reacted with S. marcescens O-antigens O1 and O10; anti-Shigella serogroup C serum cross-reacted weakly with S. marcescens O-antigen O8. Conversely, rabbit anti-S. marcescens O1 and O10 immune sera cross-reacted with a clinical isolate of Shigella flexneri. None of the anti-S. marcescens O1-O20 rabbit immune sera reacted with commercial febrile antigens of Salmonella serogroups A, B, C1,2, D, E1,2,3,4, Brucella abortus, Francisella tularensis, and Proteus OX19. However, a reference strain of Salmonella typhi (9;-,-) was agglutinated by anti S. marcescens anti-O8 and O10 sera, a reference strain of S. paratyphi B (4,5;-,-) weakly by anti-S. marcescens O8 serum, and a reference strain of S. paratyphi C (6,8;-,-) by anti-S. marcescens anti-O10 and O16 rabbit immune sera. None of the anti-S. marcescens H-antisera cross-reacted with H-antigens of S. typhi (9;d,-), S. paratyphi A (1,2;a,-), S. paratyphi B (4,5;b,-), S. choleraesuis (6,7;-,1,5), S. typhimurium (4,5;,i,-), and S. enteritidis (9;gm,-). Yersinia enterocolitica reference strain Ye 75 (O1 = O3) was agglutinated weakly by anti-S. marcescens O2 serum, whereas Y. enterocolitica reference strain Ye 373 (OV = O9) cross-reacted with S. marcescens anti-O5 rabbit immune serum. Intravenously and tissue cage-infected rabbits developed anti-Hand anti-O antibodies within 5 to 12 days after infection with representative test strains of S. marcescens. Therefore, it is suggested that the serodiagnosis of human S. marcescens infections consist of serial monitoring of both anti-H and anti-O agglutinins, because determinations of the latter alone might yield false-positive, i.e., potentially misleading results.

Ether derivatives of alpha-amanitin. Introduction of spacer moieties, lipophilic residues, and radioactive labels.

Etherification of alpha-amanitin with tritiated methyl iodide yielded a radioactively labeled amatoxin of high specific activity (similar to or approximately 4 Ci/mmol) which, in its inhibition capacity for RNA polymerase II, was very similar to alpha-amanitin. The labeled toxin was used successfully in binding assays with RNA polymerases II and in radioimmunological determinations of amatoxins. If long-chained alkyl bromides were reacted with alpha-amanitin, lipophilic ether derivatives were obtained with a facilitated penetration capacity into cells. As a consequence of the improved permeability, two derivatives, O-hexyl- and O-decyl-alpha-amanitin, were more toxic in vivo than alpha-amanitin, although their affinity to RNA polymerases II was much reduce. By reaction of N-tert-butyloxy-carbonyl-N'-(6-bromocaproyl)ethylenediamine with alpha-amanitin, a ten-atom spacer with a terminal amino group could be introduced into the toxin, which allowed the attachment of alpha-amanitin to proteins, solid-phase supports, or reporter groups. For example, by reaction with fluoresceinyl isothiocyanate, a fluorescent amatoxin was prepared for visualizing amatoxin-binding structures in cells. After succinylation of the spacer moiety, alpha-amanitin could be attached to proteins, e.g., fetuin, yielding a derivative with good antigenic properties. When an alpha-amanitin derivative was coupled to Sepharose 6B, an adsorbent for affinity chromatography was obtained suitable for a one-step purification of amatoxin-binding immunoglobulins from the sera of immunized rabbits.