Expression of monoclonal antibody-defined cell surface antigens during rat brain development

Using single-cell suspensions of mechanically dissociated, prenatal BDIX-rat brain cells (13th, 15th, and 21st days after fertilization) for immunization, we have established a collection of 37 monoclonal antibodies (Mabs) directed against neural cell surface determinants. The developmental-stage-dependent expression of cell-surface antigens recognized by these Mabs was analyzed both on plasma membranes isolated from whole brains of BDIX rats (prenatal days 13-22 and adults) using an indirect 125I solid-phase radioimmunoassay, and on intact BDIX-rat brain cells (prenatal days 13-22) using a fluorescence-activated cell sorter. Different types of developmental stage-dependent profiles of Mab binding were found, these being indicative of the presence of neural cell surface determinants whose expression increases, decreases, or does not change with brain development. Some of the Mab-binding profiles showed transient changes as a function of developmental stage. These Mabs are currently being used for the characterization, reproducible identification, and isolation of neural cell subpopulations of the developing rat brain, with the aim of investigating the cell type dependence and developmental (differentiation) stage dependence of malignant transformation following pulse exposure to the carcinogen N-ethyl-N-nitrosourea at defined stages of brain development.     

Synthesis of tumor-associated glycopeptide antigens

Carbohydrates and peptides linked together in glycoproteins constitute important components of the molecular communication between cells in multicellular organisms. Cell morphogenesis and tumorigenesis are accompanied by changes in the glycoprotein profiles of the outer cell membranes. Glycopeptide fragments of glycoproteins that have altered structures in tumor cells are of interest as tumor-associated antigens for the distinction between normal cells and tumor cells. In contrast to glycoproteins isolated from biological sources, synthetic glycopeptides are obtained in pure form and exactly specified structures. The methods developed for the synthesis of glycopeptides with tumor-associated antigen structure are outlined in this article by means of a series of typical examples. Beginning with O-glycopeptides of the relatively simple alpha-O-galactosamine-serine/threonine (T(N)-antigen) type, glycopeptide antigens of increasing complexity are described. The review includes syntheses of the saccharide components, the glycosylation reactions to furnish the O-glycosyl amino acid building blocks, their selective C- and N-terminal deprotection and the use of these building blocks for glycopeptide syntheses both in solution and on the solid support. Particular attention is given to glycopeptides containing sialic acid residues, whose syntheses are demanding since reversible protection of the sialic carboxylic group is required. Synthetic methods for the construction of N-glycopeptides carrying the important cell adhesion ligands sialyl Lewis x and sialyl Lewis a antigen are also described. Strategies for the construction of glycopeptides of this type require methods compatible with the presence of the sialic acid residue, as well as with the acid-sensitivity of the fucoside bonds.     

Expression of foreign antigens on the surface ofEscherichia coli by fusion to the outer membrane protein TraT

ThetraT gene is one of the F factor transfer genes and encodes an outer membrane protein which is involved in interactions between anEscherichia coli and its surroundings. This protein was altered so as to permit the expression of foreign proteins on the outer membrane ofE. coli in this study. A 729-bp DNA fragment, including the leader and entire structural gene sequence oftraT, was amplified and obtained by PCR. This sequence was then subcloned downstream of thetac promoter of pDR540, resulting in a TraT expression vector, pT2. Here, we report that the expression of TraT protein, fused either with a partial pre-S antigen of hepatitis B virus (60 and 98 amino acids, respectively) or with the snake venom rhodostomin (72 amino acids), was successfully achieved on the outer membrane ofE. coli, using the pT2 plasmid. This result was demonstrated using dot blot and immunofluorescence analysis. This finding supports the notion that the pT2 plasmid can be used as anE. coli display system. This system can detect a foreign peptide of about 100 amino acid residues in length on the bacterial surface.     

Antigenic differences between tumor-associated fetal antigens and rat histocompatibility antigens

Certain molecular properties of purified tumor-associated fetal antigens (TAFA) were analyzed by sequential immune precipitation (SIP), isoelectric focusing, and high-pressure liquid chromatography (HPLC). The antigenic relatedness of rat histocompatibility antigens and the various TAFA were determined by SIP. SIP of chloramine-T-labeled purified TAFA or lactoperoxidase-iodinated tumor cell membranes, in the presence of rat alloantisera and monospecific rabbit anti-TAFA sera demonstrated no antigenic cross-reactivities or similarities between H-antigens and TAFA. TAFA were also compared with histocompatibility antigens for isoelectric point optima and molecular weight. Rat H-antigens had isoelectric points in the 7.0–8.5 pH range, whereas all TAFA focused at pH 5.0–6.5 or above pH 8.0. Molecular weights were determined by HPLC. TAFA-I and TAFA-III had molecular weights of 16,000–17,500 daltons, whereas TAFA-II had a molecular weight of 12,000. The antigens were not coprecipitated by the rat alloantisera. Each TAFA was also isolated (via immune precipitation) from NP-40-solubilized tumor cell membranes. These TAFA were identical to the chloramine-T-labeled TAFA which had been previously extracted and purified from rat fibrosarcomas and osteosarcomas. These studies demonstrated that although TAFA and H-antigens cocap on embryonic and transformed cell membranes, these determinants are different molecules; they are not covalently linked on cell membranes; and TAFA are not cleavage products of normal NBR H-antigens.     

HLA-A,B antigens Ia-like antigens, and tumor-associated antigens on prostate carcinoma cell lines: serologic and immunochemical analysis with monoclonal antibodies

Serologic and immunochemical analysis of the antigenic profile of the 2 human prostate carcinoma cell lines DU-145 and H494 with a battery of monoclonal antibodies has shown that both cell lines express HLA-A,B alloantigens and the 94,000 m.w. tumor-associated glycoprotein recognized by the monoclonal antibody 376.96S. In addition, the cell line H494 unexpectedly expresses Ia-like antigens, which are similar in their antigenic profile and structure to B lymphoid cell derived Ia-like antigens. Both Ia-like antigens and tumor-associated antigens can function as targets of cell-dependent lysis mediated by the corresponding monoclonal antibodies.     

The Golgi protein RCAS1 controls cell surface expression of tumor-associated O-linked glycan antigens

Tumor immunology has received a large impetus from the identification of tumor-associated antigens. Among them, a monoclonal antibody, 22.1.1, was instrumental in defining a novel tumor-associated antigen that was termed "receptor binding cancer antigen expressed on SiSo cells" (RCAS1). RCAS1 was proposed to induce growth arrest and apoptosis on activated immune cells, mediated by a putative death receptor. Structurally, RCAS1 was predicted to exist as a type II transmembrane protein and in a soluble form. Here, we analyzed occurrence, membrane topology, and subcellular localization of the RCAS1-encoded gene product. RCAS1 was shown to be a ubiquitously expressed type III transmembrane protein with a Golgi-predominant localization. Monoclonal antibody 22.1.1 failed to recognize RCAS1, as demonstrated by confocal microscopy. Instead, we showed that the cognate 22.1.1 epitope is identical with the tumor-associated O-linked glycan Tn (N-acetyl-d-galactosamine, GalNAc). Overexpression of RCAS1 in cell lines that are negative for 22.1.1 surface staining led to the generation of Tn and the closely related TF (Thomsen-Friedenreich, Galbeta1-3GalNAc) antigen, thus providing a functional link to the generation of the 22.1.1 epitope. We suggest that RCAS1 modulates surface expression of tumor-associated, normally cryptic O-linked glycan structures and contributes indirectly to the antigenicity of tumor cells.     

Fixation, processing, and immunochemical reagent effects on preservation of T-lymphocyte surface membrane antigens in paraffin-embedded tissue

Fixatives, fixation additives, paraffin processing reagents, and immunochemical reagents were investigated for effects on preservation of T-lymphocyte surface membrane antigens CD3, CD4, and CD8 in human tonsil. Individual reagent effects were assessed in frozen sections by use of monoclonal antibodies and this information was used to optimize T-cell immunostaining in paraffin sections. Harmful factors were fixation delay, fixation at acid pH, fixation and processing at temperatures above 4 degrees C, hot paraffin wax, proteolytic enzymes, methanolic hydrogen peroxide, Triton X-100, and prolonged iodine treatment. Optimal T-cell demonstration in paraffin sections followed tissue fixation in periodate-lysine-paraformaldehyde dichromate at 4 degrees C, pH 7.5; processing through isopropanol, then xylene or chloroform, at 4 degrees C; and embedding in low melting point wax at 45-50 degrees C. Graded antigen stability occurred: CD3 most stable, CD8 least, and CD4 intermediate. CD4 and CD8 antigen preservation in paraffin sections required critical optimal tissue handling. CD3 was more stable and was also demonstrated in tissue fixed in commercial formalin, glutaraldehyde, and Bouin's fluid when fixation and processing conditions were optimized for pH and temperature. Of the fixation additives studied, polyethylene glycol and several potassium and magnesium salts enhanced immunostaining, whereas calcium chloride and lidocaine were deleterious.     

Immunotropic and immunogenic properties of Burkholderia pseudomallei surface and membrane antigens

The immunotropic and immunogenic properties of some chromatographic fractions of B. pseudomallei surface antigenic complex, as well as the preparations of B. pseudomallei outer and cytoplasmic membranes, were studied. The difference between the biopolymers under study in cytotoxicity, humoral and cell-mediated immunity characteristics, phagocytic activity were established. Some antigenic fractions (B, C, C1, H) showed perceptible protective activity (25-60%) in experiments on mice infected with B. pseudomallei virulent strain. One of the preparations of cytoplasmic membrane (CM-1) was also found to have protective properties (30%). Complex immunization with the antigenic complexes under study, introduced in combination with the immunomodulating agent Bromantan, was shown to enhance the protective effect.     

Immunochemical characterization of synaptosomal membrane antigens from chicken brain

A set of synaptic membrane antigens has been investigated in a number of tissues by indirect immunofluorescence histochemistry, using an antiserum (SPM-I) raised against a purified synaptosomal plasma membrane fraction prepared from day-old chick forebrain. The antigens were found to be present in both the central and peripheral nervous systems and none of them were restricted to the forebrain. The antigens were not detectable in nonneural tissues except for the adrenal medulla. Since the antigens could not be detected in a number of clearly defined glial cell populations or a surgically induced gliosis of the optic nerve, the antigens appear to be nerve specific. The antigens were not present in all types of neurons, thus indicating that surface membrane differences exist between different classes of neurons. Within the plasma membrane of the nerve cell the antigens were not uniformly distributed: they were present in the synaptic region and, in some nerve cells, also in axonal region but were absent from perikaryal membranes and extended regions of dendritic membranes. In the sciatic nerve the antigens were transported at the fast rate of anterograde axonal transport as well as in the retrograde direction. These results have been compared with previous attempts to detect nerve-specific membrane components by immunological means.Part of this work has been presented at American Society for Neurochemistry Meeting, New Orleans, March 1974.     

Immunochemical characterization of synaptosomal membrane antigens from chicken brain

The developmental changes in the distribution of a number of nerve specific synaptic membrane antigens have been investigated in a number of tissues using indirect immunofluorescence histochemistry. In all tissues examined, by the time synaptic contacts were made, all regions of tissue that were going to display these antigens had already done so. Areas of tissue rich in actively dividing neuroblasts or postmitotic undifferentiated neurons showed little fluorescence. Characteristic strong fluorescence was only visible where regions of differentiated synapses or axons were present. In the optic nerve, and perhaps also in the white matter of the cerebellum and the outer plexiform layer of the retina, the antigens were already present before synapse formation. After synapse formation the antigens disappeared from the outer plexiform layer of the retina and the white matter of the cerebellum and spinal cord. The amount of fluorescence was drastically reduced in the deep cerebellar nuclei but did not change appreciably in the other areas examined. The loss of these antigens occurred at a different time in each of the tissue areas, indicating that it was related to the maturation of a particular network of neurons rather than the animal as a whole. By considering the pattern of developmental change of this set of antigens, the possibility that some of them may be involved in the processes of intercellular recognition or synaptogenesis has been examined.     

Immunochemical characterization of synaptosomal membrane antigens from chicken brain

Quantitative microcomplement fixation assays have been used to partially characterize a set of nerve-specific membrane antigens. Their sensitivity to proteolytic enzymes suggested that most, or all, of the antigens contain protein, and the stimulatory effect of neuraminidase treatment suggested that some were glycoproteins. The antigens were found to be approximately 100 times more concentrated in neuronal tissues than in nonneural tissues, except for the adrenal, and they were found to be absent from the forebrains of nonavian species. The changes in total and specific activity have been measured during development in a number of neuronal tissues and compared with previously obtained immunohistochemical data.     

Simultaneous analysis of DNA content and surface antigens in human bone marrow

In order to identify when cellular expansion occurs during hematopoietic maturation, a method was developed for the simultaneous analysis of one or two cell-surface antigens and DNA content on bone marrow cells while preserving their light-scatter properties. Proliferation in a population defined by light-scatter and surface-antigenic characteristics was assessed by measuring the percentage of cells in this population having more than 2C amount of DNA ("proliferation index"). Viable, low-density (1.077 g/cm3), bone marrow cells, stained with monoclonal antibodies conjugated with fluorescein or phycoerythrin, were fixed with paraformaldehyde and subsequently treated with the detergent, Tween 20. The UV-excitable DNA stain Hoechst 33342 was used to quantify DNA content in the cells without interference with immunofluorescence. A FACS IV flow cytometer was used, equipped with the first laser at 488 nm emitting for light scattering and immunofluorescence measurements and the second laser emitting at 360 nm for the Hoechst excitation. The Hoechst uptake was the same for all bone marrow populations, yielding a tight coefficient of variation (CV) (average 5.0%) for the G0/G1 DNA peak. This permitted high sensitivity of cell detection in S, G2, and M phases of the cell cycle, while preserving light-scattering properties of the cells and maintaining cell surface immunofluorescence. The lowest "proliferation index" detected using this technique was 0.08% in a sample obtained from a patient with chronic lymphocytic leukemia. Normal helper T lymphocytes in marrow had approximately 0.5% of the cells in S, G2, or M phase. We show that the erythroid lineage, in the adult normal bone marrow, is the most active in proliferation among all hematopoietic lineages.     

Iodination and identification of surface membrane antigens in procyclic Trypanosoma rhodesiense

Surface membrane proteins and glycoproteins of procyclic Trypanosoma rhodesiense were labeled with 125I by the use of the insoluble catalyst Iodo-Gen. Autoradiography of whole solubilized procyclic trypanosomes after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a minimum of 25 surface components to have incorporated radioactivity. These components ranged in m.w. from approximately 10,000 to approximately 285,000. Immunoprecipitation with rabbit antisera of Triton X-100 extracts of radiolabeled trypanosomes revealed a subset of at least 14 surface antigens. Two of these antigens (m.w. of 63,000 and 96,000) showed heavy incorporation of label and may be major proteins of the procyclic membrane. Sera from trypanosome-infected mice recognized an overlapping but different subset of surface antigens, including a doublet of very high m.w. Lectin precipitation using antilectin conjugates or bead-bound lectins indicated that many of the labeled surface components are glycoproteins including the two major proteins precipitated by rabbit antisera. Radiolabeled glycoproteins identified by these methods bear alpha-methyl-mannopyranoside and/or galactose residues but not N-acetyl glucosamine or fucose residues in quantity. The use of these methods in identifying potentially pathogenic trypanosomal antigens is suggested.     

Expression of membrane activation antigens on murine B lymphocytes stimulated with lipopolysaccharide

The expression of two membrane glycoproteins, RL388 antigen and transferrin receptor (TfR), was examined on murine B cells stimulated with lipopolysaccharide (LPS) in vitro. Immunofluorescent staining with monoclonal antibodies and flow cytofluorometric analysis were used to monitor the expression of these markers as a function of the time in culture, the state of membrane Ia antigen expression, the position in cell cycle, and the degree of B-cell differentiation. Freshly explanted splenic B cells expressed low levels of RL388 antigen and TfR. Following LPS stimulation, increased expression of RL388 antigen was detectable by 8 to 12 hr of culture, a time span characterized by increased Ia antigen expression, blast transformation, and G0 to G1 phase transition. The increased expression of TfR was apparent later and correlated with entry into late G1 phase and the onset of S phase. LPS-stimulated cell cultures treated with actinomycin D (G0/G1 block) exhibited increased expression of Ia antigen, but neither RL388 antigen nor TfR, whereas hydroxyurea treatment (G1/S block) allowed expression of all three markers. These results indicate that hyperexpression of RL388 antigen and TfR occurs during G1 phase and that these events are subsequent to Ia antigen hyperexpression. Finally, B cells in late G1 through M phase of the cell cycle simultaneously express high levels of RL388 antigen and TfR. These findings suggest that the expression patterns of RL388 antigen and TfR might be useful parameters for defining compartments of the murine B-cell cycle.     

The effect of carbamylcholine on CFU-s differentiation

The proportion of spleen colony-forming units (CFU-s) killed by hydroxyurea was greatly increased after bone marrow cells (BMCs) from LACA mice were exposed to carbamylcholine (Cach; 1 X 10(-13) to 1 X 10(-9) in vitro and there was a marked change in the proportion of spleen colony types. Following treatment with Cach, granulocytic and mixed erythroid-type colonies increased from 20 to 26.3% and 16.1 to 29.6% in 9-day colonies and from 8.3 to 28.2% and 21.7 to 39.4% in 13-day colonies, respectively. Single cell suspensions of spleen colonies were made for granulocyte-macrophage progenitor (CFU-gm) and late erythroid progenitor (CFU-e) assays. The number of CFU-gm from Cach-treated BMC was about twice that from control BMC for both day 9 and day 13 groups; the number of CFU-e decreased relatively. The results suggest that cholinergic receptors on CFU-s may increase the tendency to differentiate into the granulocytic/monocytic line.     

Trypanosoma cruzi: shedding of surface antigens as membrane vesicles

Tissue culture-derived trypomastigotes from Trypanosoma cruzi spontaneously shed surface antigens into the culture medium. The shedding is a temperature- and time-dependent phenomenon and is independent of the presence of proteins or immune serum in the medium. The analysis of this process in four strains (Y, YuYu, CA1, and RA) showed differences in the amounts of polypeptides released. However, for all strains the liberation of the entire set of surface polypeptides ranging in molecular mass from 70 to 150 kDa was observed. Biochemical and electron microscopic data strongly suggest that most of the surface antigens are released as plasma membrane vesicles, ranging from 20 to 80 nm in diameter.     

Trypanosoma cruzi: expression of antigens on the membrane surface of parasitized cells

A direct immunofluorescent antibody test with an anti-Trypanosoma cruzi F(ab')2 conjugate was used to demonstrate antigens of T. cruzi on the membrane surface of intact live or fixed macrophages and L929 mouse fibroblasts infected with the organism. Antigens were demonstrated in 5 to 50% of infected cells, and their presence was not directly related to the number of intracellular organisms. Cells with as few as four intracellular amastigotes had demonstrable surface antigens, whereas some cells with as many as twelve or more organisms did not. Capping of antigen-antibody complexes was noted to begin a few minutes after the addition of the anti-T cruzi F(ab')2 conjugate; by 30 min, most of the parasitized cells had eliminated the complexes, and no surface antigen of parasitic nature could be demonstrated. Although capping may have caused a negative result in a previously positive cell, other mechanisms may be involved, because antigens were not demonstrated in some heavily parasitized cells examined immediately after completion of the test. Treatment of the infected cells with trypsin or chymotrypsin resulted in the absence of demonstrable parasite antigens on the cell membrane surface. However, the antigens were again demonstrated 12 hr after the enzymes were removed. The reappearance of parasite antigens on the surface of infected cells was prevented by treatment of the monolayers with puromycin or tunicamycin. A T cell-enriched population of spleen lymphocytes from mice chronically infected with T. cruzi recognized the membrane-bound antigens and proceeded to destroy the host cell and the intracellular organisms. In this process, noninfected cells were also destroyed, possibly because they were coated with antigens released from intact infected cells or from infected cells that had been lysed by the action of the sensitized lymphocytes or their products.     

Cancer vaccines based on dendritic cells loaded with tumor-associated antigens

Dendritic cells (DCs) can be used as an antigen presentation platform for vaccination against cancer. In this approach, DCs are expanded in vitro from monocyte-derived progenitors, and subsequently loaded with well-characterized tumor-associated antigens (TAAs). TAAs can be incubated with DCs in various forms, including peptides, recombinant proteins, plasmid DNA, formulated RNA, or recombinant viruses. Advantages and limitations of DC-based cellular vaccines against cancers, as well as preliminary results of clinical studies already performed in humans, are discussed. Importantly, significant advances in our understanding of the biology of DCs can be used to support the design of new vaccines or adjuvants in order to elicit T_H1 cellular immune responses. This revised version was published online in July 2006 with corrections to the Cover Date.