Antigenic proteins of Eimeria maxima gametocytes: cell-free translation and detection with recovered chicken serum

RNA was extracted from isolated Eimeria maxima gametocytes and translated in a rabbit reticulocyte cell-free protein synthesis system. The major cell-free translation products from E. maxima gametocyte RNA ranged from 225 to 50 kDa, distinct and different from uninfected chicken intestine cell-free translation products. Rabbit antiserum to E. maxima gametocytes as well as recovered chicken sera specifically precipitated some of the major gametocyte cell-free products. A time course of infected intestine RNA indicated that these cell-free synthesized gametocyte antigens appear at 130 to 138 hr postinfection.     

Conservation of proteins involved in oocyst wall formation in Eimeria maxima, Eimeria tenella and Eimeria acervulina

Vaccination with proteins from gametocytes of Eimeria maxima protects chickens, via transfer of maternal antibodies, against infection with several species of Eimeria. Antibodies to E. maxima gametocyte proteins recognise proteins in the wall forming bodies of macrogametocytes and oocyst walls of E. maxima, Eimeria tenella and Eimeria acervulina. Homologous genes for two major gametocyte proteins - GAM56 and GAM82 - were found in E. maxima, E. tenella and E. acervulina. Alignment of the predicted protein sequences of these genes reveals that, as well as sharing regions of tyrosine richness, strong homology exists in their amino-terminal regions, where protective antibodies bind. This study confirms the conservation of the roles of GAM56 and GAM82 in oocyst wall formation and shows that antibodies to gametocyte antigens of E. maxima cross-react with homologous proteins in other species, helping to explain cross-species maternal immunity.     

Biochemical characterisation of the 56 and 82 kDa immunodominant gametocyte antigens from Eimeria maxima

Two immunodominant gametocyte antigens from Eimeria maxima with M(r) 56 kDa and M(r) 82 kDa have been identified previously as potential candidates for inclusion in a recombinant subunit vaccine against coccidiosis in poultry. Here, these proteins have been biochemically characterised, immunolocalised within the parasite, and sequences for their amino termini determined. These antigens co-purify by affinity chromatography suggesting an interaction with each other. However, separation of the proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of beta-mercaptoethanol did not reveal the presence of inter-chain disulphide bonds. The true masses of the 56 and 82 kDa antigens are 52450 and 62450 Da, respectively, as determined by mass spectrometry. TX-114 separations suggested that they exist, in part, as soluble proteins within the parasite, and immunolocalisation studies indicated that they were found in the wall forming bodies of macrogametocytes. Separation of the proteins by 2D SDS-PAGE revealed that they are acidic in nature and heterogeneous in charge. Cleavage by neuraminidase and O-glycosidase indicated that the presence of O-linked glycans contributed to some of the charge microheterogeneity of both proteins. The absence of these O-glycans however, did not abolish antibody recognition, suggesting that the development of a recombinant subunit vaccine is possible. A more extensive investigation of the carbohydrate moieties of these proteins revealed that they also possess glucose, fucose, mannose and galactose. There was no evidence for the presence of N-linked glycans. The 56 and 82 kDa antigens were separated from a mixture of proteins in a crude gametocyte lysate by 2D SDS-PAGE, the proteins isolated, and the N-terminus amino acid sequence determined. They showed no homology to each other at the N-terminus, or to any other previously characterised protein. Characterisation of these novel proteins has provided further insights into the molecular mechanisms of gametocyte differentiation in E. maxima.     

Plasmodium falciparum and P. vivax gametocyte-specific exoantigens stimulate proliferation of TCR gammadelta+ lymphocytes

Immune modulation of Plasmodium vivax and P. falciparum gametocytes occurs over the course of erythrocytic infection. The response is linked to proliferative and inflammatory responses, which may be stimulated by stage-specific gametocyte proteins. Stage-specific exoantigens were purified from supernatants of P. falciparum and P. vivax gametocyte cultures, and either primary or secondary postinfection lymphocytes were stimulated for proliferation. Five of 25 exoantigens purified from P. falciparum gametocyte cultures and 6 of 28 exoantigens isolated from P. vivax were gametocyte stage specific. Metabolic labeling of soluble P. falciparum gametocyte proteins confirmed synthesis and secretion of 5 stage-specific exoantigens, with molecular masses of 118, 62, 52, 37, and 33 kDa. Purified gametocyte exoantigens within the range of 50 to 100 kDa stage-specifically stimulated proliferation of lymphocytes from postprimary P. falciparum infections, and from postprimary and secondary P. vivax infection patients with homologous purified exoantigens. T-cell receptor (TCR)gammadelta+, and CD3+ CD8+ and CD3+ CD4- CD8- T cells were specifically upregulated from P. falciparum primary- and P. vivax secondary-infection lymphocytes, respectively, using gametocyte stage-specific exoantigens. CD25+ was the major activation marker expressed by CD3+ and gammadelta T cells when stimulated with gametocyte exoantigens. None of the T cell markers was significantly upregulated using gametocyte stage-specific exoantigens with primary-infection P. vivax lymphocytes.     

Identification and partial characterization of new antigens from simian virus 40-transformed mouse cells.

Two new species of antigens were detected in simian virus 40-transformed mouse cells, in addition to the large (94,000 daltons) and small (20,000 daltons) tumor antigens. These antigens were immunoprecipitated from cell extracts by using anti-T serum and not normal, nonimmune serum. One of these was a protein with a molecular weight of approximately 130,000 and was present in some but not all SV40-transformed mouse cells. The other, which we have named Tau antigen, has a molecular weight of 56,000 as estimated by electrophoresis through acrylamide gels and was found in all virus-transformed cells examined. The 13,000-daltons antigen contained about 15 methionine-tryptic peptides which were also present in the large SV40 tumor antigen as determined by ion-exchange chromatography. This strongly suggested that the protein was virus coded. The 56,000-dalton Tau antigen appeared to share only two methionine-tryptic peptides with the large species of SV40 tumor antigen, as determined by ion-exchange and paper chromatographies. Our results are compatible with a cellular origin for Tau antigen. However, our data do not exclude the possibility that this protein contains sequences specified by the virus DNA.     

Chemical and immunological characterization of developmentally expressed chicken erythroid surface membrane antigens

The expression of two hematopoietic-lymphoid membrane antigens, referred to as chicken fetal antigen (CFA) and chicken adult antigen (CAA) were investigated on primitive and definitive peripheral red blood cells (RBC) from different-aged chickens using chemical and immunological techniques. Differential adsorptions of antisera specific for adult RBC membrane antigens permitted the serological dissection of CAA into eight antigenic determinants. CFA and CAA were assayed by hemagglutination, hemolysis, and immune precipitation of radioiodinated surface antigens of RBC from different-aged chickens. Primitive RBC express CFA, while definitive RBC express three distinct phenotypes: CFA, both CFA and CAA, or CAA, depending on the developmental age of the chicken from which the RBC were obtained. When solubilized membrane extracts of radioiodinated peripheral RBC from chickens at 5 and 18 days embryonic development (E5 and E18, respectively), 13 days posthatch development (H13), and adult chickens were immunoprecipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the major antigen detected by anti-CFA sera was associated with proteins having apparent molecular weights (M_r) of 50,000 daltons (50 kd). The antigens detected by anti-CAA sera were associated with proteins having apparent M_r of 102, 81, 48, and 43 kd.     

Immunogenicity of a single sporocyst of Eimeria maxima

Eight out of a total 40 chickens infected with single sporocysts of Eimeria maxima produced infective oocysts. These 8 chickens, when challenged with 4 X 10(5) oocysts per bird at 28 days postinfection, showed at least twice the percent average weight gain of the previously unexposed chickens, indicating that partial immunity was probably conferred by these infections. Also, these successful single sporocyst infections suggest that like E. tenella, sporozolles of E. maxima are probably sexually undifferentiated.     

Membrane vesicles released by Avibacterium paragallinarum contain putative virulence factors

Avibacterium paragallinarum, the causative agent of infectious coryza, releases extracellular membrane vesicles (MVs), containing immunogenic proteins, proteases, putative RTX proteins, haemagglutinin, and nucleic acids, into the medium. MVs ranging 50-300 nm in diameter were observed by electron microscopy. They contained immunogenic proteins in the range of 20-160 kDa, detected using vaccinated or experimentally infected chicken sera raised against Av. paragallinarum, but not in pooled sera from specific pathogen-free chickens. Proteolytic activity was not detected in MVs through zymograms; however, immune recognition of high molecular mass bands was observed by Western blotting using an antiprotease serum against Actinobacillus pleuropneumoniae serotype 1 purified protease, suggesting its presence. MVs agglutinated glutaraldehyde-fixed chicken red blood cells indicating the presence of haemagglutinating antigens. Nucleic acids were also detected inside MVs. Avibacterium paragallinarum releases MVs containing putative virulence factors, which could be important in the pathogenesis of infectious coryza.     

Immunization against Taenia taeniaeformis in mice: studies on the characterization of antigens from oncospheres

Mature eggs of Taenia taeniaeformis hatched readily in the presence of sodium hypochlorite and no loss in infectivity of oncospheres for mice was observed after hatching. Crude and sodium deoxycholate-solubilized antigens (termed TtO-DOC) prepared from such oncospheres stimulated high levels of protection against T. taeniaeformis infection in immunized mice similar to those described previously for oncospheres prepared by other methods. Mice immunized with TtO-DOC antigens that had been exposed to potassium metaperiodate remained significantly protected against infection. Exposure of TtO-DOC antigens to pronase and thermolysin, or to trypsin, significantly reduced the ability of these antigens to protect mice against infection. These data suggest that the antigens which immunize mice against infection include protein components. ¹²⁵I-labelled TtO-DOC antigens were immunoprecipitated with sera from mice infected with T. taeniaeformis and the immunoprecipitates analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoprecipitation with sera from C3H/He mice infected for 28 days revealed a single major labelled protein antigen having a relative molecular mass (M_r) of 31,000. Sera from 5-month infected C3H/He mice immunoprecipitated at least thirteen labelled antigens, including one at M_r 31,000. Attempts to use SDS-PAGE separated proteins to immunize mice showed that oncosphere antigens exposed to the reducing conditions prior to SDS-PAGE lost their ability to protect mice against infection. It was concluded that SDS-PAGE was an unsatisfactory technique for the isolation of a host protective fraction of TtO-DOC antigens. TtO-DOC proteins were resolved by PAGE performed in the presence of sodium deoxycholate (DOC-PAGE) and mice were vaccinated with cut-outs from the gel. A fraction of the DOC-polyacrylamide gel was found to be effective in immunizing mice against infection. Thus, although the characteristics of the protein antigens in this DOC-PAGE fraction have yet to be determined, an important fractionation technique has been identified. It was shown that partial removal of DOC from oncosphere antigen preparations solubilized in 1% DOC was required for the antigen to stimulate protective immunity. These findings will facilitate further antigen characterization studies towards the development of a defined-antigen vaccine in murine cysticercosis. This is particularly so as attempts to raise anti-oncospheral monoclonal antibodies capable of passively transferring protection to mice by using crude antigen preparations to immunize donor mice have not been successful.     

Proteins and antigens of merozoites and sporozoites of Eimeria bovis (Apicomplexa)

Proteins and antigens of first-generation merozoites and sporozoites of Eimeria bovis were examined using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and lactoperoxidase iodination procedures. SDS-PAGE gels revealed both common and unique protein bands in merozoite and sporozoite extracts, ranging in molecular weight (Mr) from 15,000 to 215,000. Nitrocellulose immunoblots of separated proteins, when probed with sera obtained from immunized calves, revealed numerous IgG-binding antigens of Mr 18,000 to 180,000 in merozoites and Mr 28,000 to approximately 118,000 in sporozoites. Although merozoite and sporozoite preparations each contained antigens of different molecular weights, 4 antigens had the same migratory distance in both preparations (Mr 58,000, 70,000, 83,000, 98,000). Of 3 types of immune sera used to probe immunoblots, serum taken from a calf that had been inoculated with oocysts of E. bovis and boosted 10 wk later by subcutaneous injection with 2 X 10(7) live merozoites emulsified in Freund's complete adjuvant consistently identified and reacted more intensely with more antigens of merozoites and sporozoites than the other immune sera tested. Autoradiographic analysis of radioiodinated parasites revealed major surface proteins on merozoites of between 15,000 and 18,000 Mr and 3 surface proteins on sporozoites of Mr 28,000, 77,000, and 183,000. All but the 183,000 protein elicited an IgG antibody response in the host.     

A study of adipocyte precursor cells derived from brown adipose tissue: the expression of specific cell surface antigens during their differentiation in culture

Using cell specific anti-adipocyte sera and an immuno-precipitation procedure, the nature of the cell surface antigens characterizing adipocytes from rat brown adipose tissue was investigated. Initially the ability of anti-sera, raised against adipose plasma membrane preparations of white or brown adipose tissue, to distinguish between membrane preparations derived from either tissue was confirmed. Analysis of the plasma membranes derived from brown adipose and similar preparations labelled with 125I revealed the presence of specific externally disposed mature brown adipocyte-specific antigens. The specifically immunoprecipated antigens had molecular weights of 70,000, 56,000 and 23,000. None of these antigens were cross immunoprecipated by antisera to mature white adipocyte membranes. The presence of the brown adipose specific antigens on the surface of differentiating adipocyte precursor cells derived from rat brown adipose tissue was demonstrated using a labelled-secon antibody cellular immunoassay. The expression of the immunoreactivity associated with these antigens was shown to be an early event in the differentiation programme of the cells in vitro. The functional identity and possible roles of these antigens in the control of brown adipocyte differentiation now becomes accessible to further experimental investigation.     

Demonstration of immunoreactive retinol-binding protein and prealbumin in developing chicken embryo.

The immunoreactive retinol-binding protein (RBP) and prealbumin (PA) were identified in chicken embryo by the method of double immunodiffusion using antisera against purified chicken serum RBP and PA, respectively. The embryonic RBP studied by a fluorospectrophotometric analysis showed presence of vitamin A (retinol) within the molecule. The RBP and PA fractionated on a column of Sephadex G-200 had molecular weight of approximately 20,000 and 56,000, respectively. RBP and PA formed a complex with vitamin A which had a molecular weight of approximately 76,000. The developmental changes of RBP and PA in the chicken embryo were determined in the eye, brain, serum and liver by the single radial immunodiffusion. In the brain and eye, the maxima for the concentration of RBP and PA were detected at day 6 for RBP, and day 6 and day 13 for PA during development. However, these proteins were not detected in the tissues of young chicken. The concentration of the serum embryonic RBP and PA showed a maximum at day 6. With regard to the liver, the PA was observed in the embryo only at day 13, but the RBP only after hatching.     

Characterization of tau antigens isolated from uninfected and simian virus 40-infected monkey cells and papovavirus-transformed cells.

Tau antigens (also known as cellular or nonviral tumor antigens) were detected in uninfected and simian virus 40-infected monkey cells after immunoprecipitation with serum from hamsters bearing simian virus 40-induced tumours (anti-T serum). These two proteins (56,000 daltons) were digested to similarly sized peptides with various amounts of Staphylococcus aureus V8 protease. The Tau antigen isolated from infected monkey cells was closely related but was not identical to the corresponding protein from human cells transformed by simian virus 40, as determined by two-dimensional mapping of their methionine-labeled tryptic peptides. Hamster cells transformed by various primate papovaviruses (simian virus 40, BK virus, and JC virus) synthesized indistinguishable Tau antigens, as determined by two-dimensional peptide mapping. When tested by the same procedure, these proteins and the ones made in monkey and human cells were found to be related to the Tau antigens isolated from simian virus 40-transformed mouse and rat cells. Based on these results, an "evolutionary tree" was constructed to show the relationship among the methionine-containing tryptic peptides of all of these proteins.     

Development of enzyme-linked immunosorbent assay for detection of Mycoplasma pneumoniae antigens

The variant of enzyme-linked immunosorbent assay (ELISA) for detection of Mycoplasma pneumoniae (Mp) antigens in sera of patients with respiratory infections was developed. Sensitivity of detection of soluble antigens of Mp in modeling experiment varied from 1.5 to 1.0 ng/ml (on protein). Approbation of the assay was performed using 50 serum samples obtained from patients with confirmed diagnosis of respiratory mycoplasmosis. In the ELISA test Mp antigens were detected in 96% of samples. Obtained results were confirmed by testing of these serum samples and isolated from them circulating immune complexes (CICs) in immunoblotting using polyclonal antibodies labeled by horse-radish peroxidase. Mp antigenswere detected both in free state and as components of CICs. Specific reaction was observed with proteins, which molecular mass varied from 30 to 170 kDa (30, 37, 45, 56, 58, 72, 90, 130 and 170 (160) kDa). Obtained results point to appropriateness of use of developed assay for detection Mp antigens in sera of patients with respiratory infections.     

Relationship Among Tau Antigens Isolated from Various Lines of Simian Virus 40-Transformed Cells

In addition to the virus-specified tumor antigens, simian virus 40-transformed cells contain at least one other protein which can be immunoprecipitated with serum from animals bearing simian virus 40-induced tumors. This protein, which is designated Tau antigen, has an apparent molecular weight of 56,000 as determined by electrophoresis on acrylamide gels. The relationship among Tau antigens isolated from different lines of simian virus 40-transformed cells was examined by comparing the methionine-labeled tryptic peptides of these proteins by two-dimensional fingerprinting on thin-layer cellulose plates. In this fashion, we initially determined that the Tau antigens isolated from three different lines of transformed mouse cells were very similar. Second, we found that Tau antigen isolated from a line of rat transformants was closely related, but not identical, to the mouse cell Tau antigens. Approximately 70% of their methionine peptides comigrated in two dimensions. Finally, we showed that Tau antigen isolated from a line of transformed human cells was only partially related to the mouse and rat proteins. About 40% of the methionine peptides of the human protein were also contained in the Tau antigens from the other two species. These results strongly indicate that the Tau antigens isolated from these various simian virus 40-transformed cell lines contain common amino acid sequences.     

Antigens of Cryptosporidium sporozoites recognized by immune sera of infected animals and humans

The humoral response of humans, calves, and horses to Cryptosporidium sporozoite antigens was evaluated using a western blot technique. Sera from calves, humans, and horses were obtained at various times following the detection of infection. Sera were reacted with detergent-solubilized, sodium dodecyl sulfate polyacrylamide gel-electrophoresed (SDS-PAGE) sporozoite antigens. The number of antigens recognized by immune sera from humans and animals increased with time postinfection. A 20-kDa antigen appears to be a major sporozoite surface determinant labeled via membrane protein biotinylation and recognized by mouse monoclonal antibodies using indirect immunofluorescence and western blotting. Detectable recognition of the 20-kDa band occurred in 3-wk postinfection (PI) sera from all species tested. Reactivity to the 20-kDa band diminished significantly in sera 5 mo PI or longer from infected humans with no known recurrence of cryptosporidial diarrhea. In contrast, 12-mo PI sera from an individual constantly exposed to oocysts under working conditions was as strongly reactive as the 3-wk convalescent sera. Serum reactivity to the 20-kDa antigen appears to be a good indicator of exposure to Cryptosporidium.     

Eimeria acervulina: DNA cloning and characterization of recombinant sporozoite and merozoite antigens

Genes encoding antigens of Eimeria acervulina were cloned from cDNA expression libraries prepared from the sporozoite and merozoite stages in order to examine humoral and cellular immune responses to this protozoan parasite. Two clones expressing surface antigens were characterized by DNA hybridization studies to identify homologous genomic DNA fragments. The proteins they encode were identified by 125I-labeling, immunoblotting, immunofluorescence, and T-cell activation experiments. One, designated cSZ-1, encodes a 130-kDa beta-galactosidase fusion protein which represents a portion of a p240/p160 immunodominant sporozoite surface antigen. Immunofluorescence studies using anti-cSZ-1 sera and live or 1% paraformaldehyde-fixed E. acervulina sporozoites have confirmed this surface locale. Purified cSZ-1 fusion protein, which is not recognized by sera from E. acervulina-infected chickens, induced the activation of immune T lymphocytes in vitro. Another cDNA clone, designated cMZ-8, gives rise to a 150-kDa fusion protein and encodes a portion of a p250 immunodominant merozoite surface antigen. This was established by immunoblotting of 125I-labeled merozoite proteins with anti-cMZ-8 sera and immunofluorescence staining of live and 1% paraformaldehyde-fixed E. acervulina merozoites. Purified cMZ-8 is recognized by sera from E. acervulina-infected chickens and induces a significant activation of immune T lymphocytes in vitro.     

Antibodies to meningococcal antibodies in the sera of patients and donors

The comparative study of sera taken from healthy persons (pooled sera of 100 donors, 6 individual serum specimens) and sera taken from patients with meningococcal meningitis (pooled sera of 10 patients with meningococcal infection, group A, and 6 individual serum specimens from patients with meningococcal infection, groups A, B, C) was carried out by the method of immunoblotting. All proteins from healthy donors were found to contain antibodies to meningococcal iron-regulated protein (IRP) of 85 kD, designated as TbpB. In 30% of donor sera the presence of antibodies to meningococcal IRP of 34 kD (FbpA) was registered. Moreover, donor sera were found to contain antibodies to meningococcal IRP of 45 kD. The sera taken from convalescents were found to have the increased content of antibodies to IRP of 70 and 85 kD and somewhat lesser content of antibodies to proteins of 98, 44 and 34 kD. As regards other (non iron-regulated) proteins, in the process of convalescence the most intensive antibody production was observed with respect to minor protein with a molecular weight of 50 kD, as well as proteins of class 5, characterized by molecular weights of 30 kD and less.     

Studies on the expression of adipocyte-specific cell surface antigens during the differentiation of adipocyte precursor cells in vitro

Using species and cell specific antiadipocyte sera an immunoprecipitation procedure was developed which allowed the nature of adipocyte cell surface antigens to be investigated. Analysis of immunoprecipitates from mature adipocyte plasma membranes of rat, ox and chicken and similar 125I-labelled membranes revealed the presence of specific externally disposed adipocyte specific antigens which were also species specific. For mature cells the specific antigens had molecular weights of 124,000, 92,000 and 59,000 in the case of the rat, 87,000 in the case of the ox and 56,000, 47,000 and 37,000 in the case of the chicken. None of these antigens were cross immunoprecipated by antisera to non-homologous adipocytes. The presence of the antigens at the surface of differentiating rat while adipocyte precursor cells was demonstrated using a labelled-second antibody cellular immunoassay and the expression of this reactivity revealed to be an early event in the differentiation programme of the cells. The increase in cell surface immunoreactivity during the differentiation of the cells was shown to be dependent upon the expression of two of the antigens previously shown to be markers of the mature adipocyte phenotype. The functional identity and possible role of these antigens in the control of adipocyte differentiation in vitro and in vivo now becomes accessible to investigation experimentally.     

人肺鳞癌组织的血清蛋白质组学的比较分析

采用以肿瘤免疫学与蛋白质组学(proteomics)研究技术有机地结合为基础的血清蛋白质组学研究体系(serologicproteomeanalysis ,SERPA)筛选肺癌分子标志物.对10例人肺鳞癌组织,应用双向凝胶电泳(two dimensionalelectrophoresis ,2 DE)技术对同一肺鳞癌组织的细胞总蛋白同时进行电泳后获得3张相同的凝胶,其中一块2 DE凝胶经银染显色作为平行胶,其余两块2 DE凝胶经电转膜将凝胶中的蛋白质转至硝酸纤维素(NC)膜上,然后分别与肺癌患者的自身血清以及正常对照血清进行Western印迹分析,获取Western印迹反应图谱.经计算机图像分析识别差异反应的蛋白质,然后与平行胶比较找出相应的差异反应蛋白质点.获得了分辨率较高的人肺鳞癌组织与患者的自身血清以及正常对照血清的Western印迹反应图谱;图像分析共识别36±8个差异反应的蛋白质;在平行胶上找到了匹配的差异反应蛋白质点.对2 0个差异蛋白质点进行了肽质指纹图分析,鉴定出14个与细胞生长增殖、细胞代谢、细胞周期调控、信号转导等有关的肺鳞癌相关抗原.通过血清蛋白质组技术对肺鳞癌组织进行的研究,建立了分辨率较高的人肺鳞癌组织与患者的自身血清以及正常血清的Western印迹反应图谱,成功鉴定14个肺鳞癌相关抗原,为进一步筛选用于肺鳞癌诊断、治疗和预后评估