Immunogenic properties of modified antigen E. III. Effect of repeated injections of modified antigen on immunocompetent cells specific for native antigen.

It has been shown that ragweed antigen E loses its major antigenic determinants after denaturation in 8 M urea, but urea-denatured (UD) antigen and an alpha-polypeptide chain isolated from the denatured molecules are capable of priming mouse T cells specific for native antigen. Weekly injections of 10mug UD antigen or alpha-chain into antigen E-primed animals depressed the ongoing IgE antibody response, whereas injections of the same dose of antigen E failed to depress the antibody response. It was found by adoptive transfer experiments that helper activity of antigen E-primed splenic T cells was depressed by the treatment of the donors with either modified antigen or native antigen E. The same treatment of antigen E-primed animals depressed the DNA synthetic response of their splenic T cells to antigen E. The treatment of antigen E-primed animals with UD antigen resulted in a decrease of antigen E-specific IgE-B cells and IgG-B cells in their spleen, whereas the treatment with native antigen expanded the B cell populations. In view of the results obtained in the mouse, cellular basis for the immunologic effects of hyposensitization treatment is discussed.     

Transformation of type polysaccharide antigen synthesis and hemolysin synthesis in streptococci

Transformation of the ability to synthesize type polysaccharide antigen and beta-hemolysin has been obtained in group F streptococci. Colonies possessing cells transformed to antigen synthesis were detected on the agar surface with fluorescein-labeled anti-type serum. This selection method, in contrast to those with antibiotics, allowed both transformed and nontransformed cells to grow, resulting in sectored colonies. These colonies could be subcultured to further establish the synthesis of antigen. Group F, group A, and group-like z deoxyribonucleic acid (DNA) labeled with type II antigen and hemolysin, and streptomycin resistance transferred each marker to a group F strain lacking a type antigen. DNA from group F and z3 strains labeled with type III antigen, and streptomycin resistance transferred both markers to group F and z3 strains lacking type antigen. A second F strain without type antigen was not transformed with any of these markers. A group H strain was transformed to streptomycin resistance only by the same types of DNA. Transformation to type II antigen synthesis always resulted in the formation of beta-hemolysin. All strains isolated from natural sources contained both markers. A mutant, obtained by nitrosoguanidine treatment of an FII(sr) strain, did not synthesize either the hemolysin or the antigen. This mutant still possessed the group antigen and streptomycin resistance. A close linkage of type II antigen and beta-hemolysin is indicated. The fluorescent-antibody staining of cells containing both group and type antigens showed a more intense ultraviolet adsorption for type than group antigen. A surface location (microcapsular) for the type antigen appeared likely. These results are of interest for studies on antigen biosynthesis, genetics, and classification of the streptococci.     

B cells extract and present immobilized antigen: implications for affinity discrimination

Binding of antigen to B-cell antigen receptor (BCR) leads to antigen internalization and presentation to T cells, a critical process in the initiation of the humoral immune response. However, antigen internalization has been demonstrated for soluble antigen, in vivo antigen is often encountered in insoluble form or tethered to a cell surface. Here, we show that not only can B cells internalize and present large particulate antigen (requiring a signalling-competent BCR to drive antigen uptake), but they can also extract antigen that is tethered tightly to a non-internalizable surface. The form in which the antigen is displayed affects the B cell's ability to discriminate antigen-BCR affinity. Thus, arraying an antigen on a particle or surface allows efficient presentation of low affinity antigens. However, the presentation efficiency of antigen arrayed on an internalizable particle plateaus at low affinity values. In contrast, extraction and presentation of antigen from a non-internalizable surface depends on antigen-BCR affinity over a wide affinity range. The results have implications for understanding both the initiation and affinity maturation of the immune response.     

Endocytosis and recycling of specific antigen by human B cell lines.

Human B cell lines expressing membrane immunoglobulin specific for tetanus toxoid/toxin were used to study the receptor-mediated endocytosis of antigen. Monovalent antigen, initially bound to cell surface immunoglobulin at 0 degree C, was rapidly endocytosed upon warming the cells to 37 degrees C. The kinetics of endocytosis of antigen were independent of the number of occupied binding sites and indicated a half-life for antigen on the cell surface of 8.5 min. Endocytosis of antigen apparently ceased after approximately 15 min at 37 degrees C, although some 40-50% remained on the cell surface at this time. We show, using biotinylated antigen and an avidin detection assay, that this is due to recycling of antigen to the cell surface. By labelling the antigen on the cell surface with Fabs against different epitopes we show that antigen continues to be endocytosed for at least 1 h after the initial rapid phase of endocytosis, again indicating that there must be recycling of immunoglobulin/antigen complexes. As a consequence of the stable interaction between antigen and membrane immunoglobulin, the capacity of the cells to accumulate antigen was limited when the synthesis of membrane immunoglobulin was blocked; under these conditions only 2-3 times as much antigen was endocytosed and degraded when antigen was supplied continuously over a 4-h period at 37 degrees C as could be bound to the cells at 0 degree C. These results reveal a rapid and efficient pathway for the endocytosis and recycling of monovalent antigen in B cells.     

Antigenic relatedness of small ribonucleoprotein particles

We have examined the relationships among small ribonucleoprotein particles found in eucaryotic cells by an antigen depletion technique using autoimmune antibodies. We have confirmed that the (U1) ribonucleoprotein particle antigen is found on the same complex as the Sm antigen. We have also shown that the Ro antigen is found on the same complexes as the La antigen. However, both Sm and La antigens are also found on complexes that are never associated with (U1) ribonucleoprotein particle and Ro, respectively. Further, U1 containing complexes can exist that contain the Sm antigen but not the (U1) ribonucleoprotein particle antigen. In a similar manner, we find several La-Ro RNA containing complexes that carry the La antigen but do not always carry the Ro antigen. Sm and La antigen are quantitatively associated with their specific ribonucleoprotein complexes.     

The actin cytoskeleton coordinates the signal transduction and antigen processing functions of the B cell antigen receptor

The B cell antigen receptor （BCR） is the sensor on the B cell surface that surveys foreign molecules （antigen） in our bodies and activates B cells to generate antibody responses upon encountering cognate antigen. The binding of antigen to the BCR induces signaling cascades in the cytoplasm, which provides the first signal for B cell activation. Subsequently, BCRs internalize and target bound antigen to endosomes, where antigen is processed into T cell recognizable forms. T helper cells generate the second activation signal upon binding to antigen presented by B cells. The optimal activation of B cells requires both signals, thereby depending on the coordination of BCR signaling and antigen transport functions. Antigen binding to the BCR also induces rapid remodeling of the cortical actin network of B cells. While being initiated and controlled by BCR signaling, recent studies reveal that this actin remodeling is critical for both the signaling and antigen processing functions of the BCR, indicating a role for actin in coordinating these two pathways. Here we will review previous and recent studies on actin reorganization during BCR activation and BCR- mediated antigen processing, and discuss how actin remodeling translates BCR signaling into rapid antigen uptake and processing while providing positive and negative feedback to BCR signaling.     

Peptide map analysis of normal plasma and platelet factor VIII antigen

Factor VIII antigen from platelet intracellular granules was immunoprecipitated using a monospecific rabbit antibody to normal plasma factor VIII antigen. The factor VIII antigen in the immunoprecipitate was isolated on sodium dodecyl sulfate polyacrylamide gels, radiolabeled with ¹²⁵I, trypsinized, and subjected to peptide mapping using two dimensional high voltage electrophoresis and thin layer chromatography. The platelet protein was compared to purified plasma factor VIII antigen. The two dimensional tryptic ¹²⁵I peptide map of platelet granule factor VIII antigen was similar but not identical to the plasma factor VIII antigen peptide map. The platelet and plasma proteins shared approximately 34 radioactive peptide spots. Seven plasma factor VIII antigen peptides were not detected in platelet factor VIII antigen. The reason for the structural differences of plasma and platelet granule factor VIII antigen are unknown. The possibility is raised that proteolysis has altered the platelet protein in vitro. It is also possible that factor VIII antigen synthesized by megakaryocytes differs from the plasma protein.     

猕猴血清B病毒抗体三种检测方法的比较

目的对B病毒（B virus）抗体检测的3种方法进行比较,寻求准确、可靠、经济的检疫方法。方法对以HSV-1为抗原的玻片酶免疫法、B病毒为抗原的玻片酶法（EIA）和酶联免疫吸附法（ELISA）的猕猴血清B病毒抗体检测结果进行比较。结果HSV-1为抗原的EIA与B病毒为抗原的EIA、ELISA检测结果符合率分别为97.7%和95.5%。结论HSV-1为抗原EIA的检测结果与B病毒抗原EIA和ELISA的检测结果一致性较好,可以做为初筛手段,且检测效果较好,投入资金相对最低,达到节约成本的目的。     

Purification, characterization, and quantitation of the antigen employed in the immunodiffusion test for diagnosis of equine infectious anemia.

Equine infectious anemia (EIA) antigen extracted from the spleen of horses infected with EIA virus was purified by pH treatment, (NH4)2SO4 fractionation and affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A S20,w of 0.51 was determined and a molecular weight of 7600 was calculated from sedimentation equilibrium analysis. The amino acid composition of the pure antigen indicated the antigen is an acidic protein. Employing radical immunodiffusion (RID) and pure antigen a method for quantitating antigen content of antigen containing preparations was developed.     

Removal of glomerular deposits induced by either preformed immune complexes or by a chronic immune complex model in NZB/W mice

The solubilization and removal of defined glomerular immune complex deposits by excess antigen was examined in NZB/W female mice. Glomerular deposits were induced by administering preformed immune complexes to young (2 to 4 mo) mice before they naturally acquired deposits from endogenous disease and to old (7 mo) mice with deposits from naturally acquired disease. The administration of excess antigen specifically removed deposits of preformed immune complexes in both groups. This was associated with a reduction in circulating large latticed complexes containing more than two antigen and two antibody molecules (greater than Ag2Ab2). Established deposits in old mice therefore did not interfere with removal of newly induced deposits of preformed immune complexes. Glomerular deposits were also induced in young mice by a chronic human serum albumin (HSA) immune complex model. The antigen in immune deposits induced by 2 wk of chronic antigen administration was solubilized and was removed within 48 hr of administering excess antigen. Circulating antibodies to the antigen were also reduced by excess antigen. Glomerular deposits of mouse immunoglobulin and complement were not significantly reduced by excess antigen but remained more intense than in mice of comparable age given preformed complexes. Thus deposits of other antigen antibody systems and possibly endogenous disease were induced by the chronic HSA immune complex model in NZB/W mice. However, defined antigen deposits within deposits containing multiple antigen antibody systems can clearly be removed by administering excess antigen.     

Chemotherapy Enhances Cross-Presentation of Nuclear Tumor Antigens

Cross-presentation of tumor antigen is essential for efficient priming of naïve CD8⁺ T lymphocytes and induction of effective anti-tumor immunity. We hypothesized that the subcellular location of a tumor antigen could affect the efficiency of cross-presentation, and hence the outcome of anti-tumor responses to that antigen. We compared cross-presentation of a nominal antigen expressed in the nuclear, secretory, or cytoplasmic compartments of B16 melanoma tumors. All tumors expressed similar levels of the antigen. The antigen was cross-presented from all compartments but when the concentration was low, nuclear antigen was less efficiently cross-presented than antigen from other cellular locations. The efficiency of cross-presentation of the nuclear antigen was improved following chemotherapy-induced tumor cell apoptosis and this correlated with an increase in the proportion of effector CTL. These data demonstrate that chemotherapy improves nuclear tumor antigen cross-presentation and could be important for anti-cancer immunotherapies that target nuclear antigens.     

Evolutionary pattern of intra-host pathogen antigenic drift: effect of cross-reactivity in immune response.

Several viruses are known to change their surface antigen types after infecting a host, thereby escaping the immune defence and ensuring persistent infection. In this paper, we theoretically study the pattern of intra-host micro-evolution of pathogen antigen variants under the antigen specific immune response. We assume that the antigen types of the pathogen can be indexed in one-dimensional space, and that a mutation can produce a new antigen variant that is one step distant from the parental type. We also assume that antibodies directed to a specific antigen can also neutralize similar antigen types with a decreased efficiency (cross-reactivity). The model reveals that the pattern of intra-host antigen evolution critically depends on the width of cross-reactivity. If the width of cross-reactivity is narrower than a certain threshold, antigen variants gradually evolve in antigen space as a travelling wave with a constant wave speed, and the total pathogen density approaches a constant. In contrast, if the width of cross-reactivity exceeds the threshold, the travelling wave loses stability and the distribution of antigen variants fluctuates both in time and in genotype space. In the latter case, the expected episodes after infection are a series of intermittent outbreaks of pathogen density, caused by distantly separated antigen types. The implication of the model to intra-host evolution of equine infectious anaemia virus and human immunodeficiency virus is discussed.     

Mutational analysis of polyomavirus small-T-antigen functions in productive infection and in transformation.

The function of polyomavirus small T antigen in productive infection and in transformation was studied. Transfection of permissive mouse cells with mixtures of mutants that express only one type of T antigen showed that small T antigen increased large-T-antigen-dependent viral DNA synthesis approximately 10-fold. Under the same conditions, small T antigen was also essential for the formation of infectious virus particles. To analyze these activities of small T antigen, mutants producing protein with single amino acid replacements were constructed. Two mutants, bc1073 and bc1075, were characterized. Although both mutations led to the substitution of amino acid residues of more than one T antigen, the phenotype of both mutants was associated with alterations of the small T antigen. Both mutant proteins had lost their activity in the maturation of infectious virus particles. The bc1075 but not the bc1073 small T antigen had also lost its ability to stimulate viral DNA synthesis in mouse 3T6 cells. Finally, both mutants retained a third activity of small T antigen: to confer on rat cells also expressing middle T antigen the ability to grow efficiently in semisolid medium. The phenotypes of the mutants in these three assays suggest that small T antigen has at least three separate functions.     

Histochemical reactivity of soybean agglutinin with blood group antigens and their precursor substances in acinar cells of human pancreas

In human pancreas, soybean agglutinin (SBA) conjugated to horseradish peroxidase reacted with the acinar cells secreting blood group A and/or H antigen, but not with those secreting only B antigen. For detailed histochemical characterization of SBA staining, the effects of treatment with unlabeled lectins and of digestion of certain enzymes on SBA staining were investigated in formalin-fixed, paraffin-embedded pancreatic tissue from individuals of different blood groups. Pre-incubation of sections with unlabeled Dolichos biflorus agglutinin to block A antigen eliminated subsequent SBA staining in the cells secreting A antigen, although failing to induce any effects in those secreting H antigen. In contrast, pre-incubation with unlabeled Ulex europaeus agglutinin-I (UEA-I) to block H antigen abolished SBA staining in cells secreting H antigen but not in those secreting A antigen. Treatment with galactose oxidase yielded the same results as those with unlabeled UEA-I, i.e., SBA reactivity was significantly diminished in cells secreting H antigen but not in those secreting A antigen. Digestion with beta-galactosidase resulted in a slight decrease of SBA staining in the cells secreting H antigen. Accompanying the decrease of SBA staining, reactivity with Griffonia simplicifolia agglutinin-II (GSA-II) appeared for the first time in the enzyme-susceptible, SBA-reactive cells secreting H antigen. Pre-treatment with galactose oxidase abolished this effect of beta-galactosidase. The GSA-II reactivity disclosed by treatment with galactosidase was completely eliminated by digestion with beta-N-acetylhexosaminidase, indicating that GSA-II staining after digestion with galactosidase is due to exposed penultimate beta-N-acetyl-D-glucosamine residues. These results demonstrate that at least two substances react with SBA in acinar cells of human pancreas, one being terminal beta-N-acetyl-D-galactosamine residues of A antigen, and the other being terminal beta-D-galactose-(1----3 or 1----4)-beta-N-acetyl-D-glucosamine dimers in the precursor of blood group H antigen. Such dimers may exist in close proximity to L-fucose residues of H antigen, since unlabeled UEA-I blocked SBA staining.     

SV40 T antigen and the exocytotic pathway.

A chimeric gene consisting of DNA coding for the 15-amino acid signal peptide of influenza virus hemagglutinin and the C-terminal 694 amino acids of SV40 large T antigen was inserted into a bovine papilloma virus (BPV) expression vector and introduced into NIH-3T3 cells. Cell lines were obtained that express high levels (approximately 5 X 10(6) molecules/cell) of the chimeric protein (HA-T antigen). The biochemical properties and intracellular localization of HA-T antigens were compared with those of wild-type T antigen. Wild-type T antigen. Wild-type T antigen is located chiefly in the cell nucleus, although a small fraction is detected on the cell surface. By contrast, HA-T antigen is found exclusively in the endoplasmic reticulum (ER). During biosynthesis, HA-T antigen is co-translationally translocated across the membrane of the ER, the signal peptide is cleaved and a mannose-rich oligosaccharide is attached to the polypeptide (T antigen contains one potential N-linked glycosylation site at Asn154). HA-T antigen does not become terminally glycosylated or acylated and little or none reaches the cell surface. These results suggest that T antigen is incapable of being transported along the exocytotic pathway. To explain the presence of wild-type T antigen on the surface of SV40-transformed cells, an alternative route is proposed involving transport of T antigen from the nucleus to the cell surface.     

Species-dependent regulation of monocyte/macrophage Ia antigen expression and antigen presentation by prostaglandin E

The expression of Ia antigen by various murine and human macrophage populations and the ability of prostaglandins of the E series to regulate Ia antigen expression were explored. Monocytes and macrophages from human and murine populations demonstrated a dichotomy in the expression of Ia antigen. Both human monocytes and macrophages expressed elevated levels of Ia antigen compared to their murine counterpart. Murine macrophages appear to express elevated levels of Ia antigen only when actively interacting with T lymphocytes in vivo or with lymphokines in vitro. Prostaglandins of the E series can suppress murine macrophage Ia antigen expression, but have little effect on the expression of Ia antigen by human monocytes and macrophages. Also, prostaglandins of the E series do not modulate the ability of human monocytes to present antigen to autologous lymphocytes when studied over a broad concentration range. These data suggest that prostaglandin E compounds do not profoundly affect human monocyte/macrophage Ia antigen expression or human monocyte antigen presenting activity.     

The cell cycle associated change of the Ki-67 reactive nuclear antigen expression

The change of human nuclear antigen expression in proliferating cells recognized by a monoclonal antibody, Ki-67, during the cell cycle was investigated in HeLa S3 cells using a bivariate-flow-cytometric analysis. The antigen was immunocytochemically stained with FITC, and DNA was stained with propidium iodide (Pl). The expression of the antigen increased with cell-cycle progression, especially in the latter half of S-phase and reached a maximum at G2M-phase, although its content varied greatly from cell to cell. The cells in which DNA synthesis was inhibited by treatment with hydroxyurea increased markedly in the antigen expression (as compared to untreated cells). Treatment with adriamycin also elevated the antigen content. After digestion with DNase I, but not after RNase treatment, FITC fluorescence from the antigen disappeared. These results suggest that the Ki-67 antigen is bound to DNA and its expression does not depend on DNA replication. Although the biological implications of the antigen remain unresolved, the antigen may be considered to be essential for maintaining the proliferating state of cells.     

Episome-carried surface antigen K88 of Escherichia coli. I. Transmission of the determinant of the K88 antigen and influence on the transfer of chromosomal markers

?rskov, Ida (Statens Seruminstitut, Copenhagen, Denmark), and Frits ?rskov. Episome-carried surface antigen K88 of Escherichia coli. I. Transmission of the determinant of the K88 antigen and influence on the transfer of chromosomal markers. J. Bacteriol. 91:69-75. 1966.-The transmission of the determinant of the Escherichia coli K88 antigen in mixed cultures of E. coli strains is described. The K88 factor could not be transferred by filtrates, nor could responsible phages or colicines be detected. Acriflavine was shown to "cure" the bacteria for the K88 antigen. Generally, the strains having acquired the K88 antigen also acquired the ability to transfer chromosomal markers, but this ability was in some cases retained by segregants which had lost the K88 antigen. Introduction into an F(+) strain caused reduction of the recombination frequency and disappearance of the f(+) antigen. Not all wild-type strains with the K88 antigen are genetic donors of this antigen, at least not to a discernible degree. It was concluded that the K88 antigen determinant is carried by an episome.     

The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion

The E48 antigen, a putative human homologue of the 20-kD protein present in desmosomal preparations of bovine muzzle, and formerly called desmoglein III (dg4), is a promising target antigen for antibody- based therapy of squamous cell carcinoma in man. To anticipate the effect of high antibody dose treatment, and to evaluate the possible biological involvement of the antigen in carcinogenesis, we set out to molecularly characterize the antigen. A cDNA clone encoding the E48 antigen was isolated by expression cloning in COS cells. Sequence analysis revealed that the clone contained an open reading frame of 128 amino acids, encoding a core protein of 13,286 kD. Database searching showed that the E48 antigen has a high level of sequence similarity with the mouse ThB antigen, a member of the Ly-6 antigen family. Phosphatidylinositol-specific (PI-specific) phospholipase-C treatment indicated that the E48 antigen is glycosylphosphatidylinositol-anchored (GPI-anchored) to the plasma membrane. The gene encoding the E48 antigen is a single copy gene, located on human chromosome 8 in the 8q24-qter region. The expression of the gene is confined to keratinocytes and squamous tumor cells. The putative mouse homologue, the ThB antigen, originally identified as an antigen on cells of the lymphocyte lineage, was shown to be highly expressed in squamous mouse epithelia. Moreover, the ThB expression level is in keratinocytes, in contrast to that in lymphocytes, not mouse strain related. Transfection of mouse SV40-polyoma transformed mouse NIH/3T3 cells with the E48 cDNA confirmed that the antigen is likely to be involved in cell-cell adhesion.     

Cytolysin A-mediated protein exportation efficiency and its role in enhancing the fitness of live recombinant Salmonella Typhi vaccine strain

The genetic fusion of cytolysin A (clyA) to heterologous antigen expressed in live Salmonella vector demonstrated efficient translocation into periplasmic space and extracellular medium. Accumulating evidence has shown that clyA-mediated antigen delivery improved growth fitness and enhanced immunogenicity of live vector vaccine, but the factors influencing this protein exportation has not been investigated. In this study, Toxoplasma gondii antigen fused at C-terminal of clyA protein was expressed in live S. Typhi vector via both plasmid and chromosomal-based expressions. The bivalent strains showed comparable growth rates as monovalent strains, but in varies antigen exportation efficiency. ClyA-fusion antigen with positive charges was translocated to the extracellular spaces, whereas those with negative charges were retained in the cytoplasm. Furthermore, excessive cellular resources expenditure on antigen expression, especially antigen with larger size, could limit the clyA-fusion antigen exportation, resulting in undesirable metabolic burden that eventually affects the growth fitness. Altogether, the present work indicates potential linkage of factors mainly on antigen properties and expression platforms that may affect clyA-mediated antigen delivery to enhance the growth fitness of live vector strain.