Species Specificity in Thermostable and Ethanol Insoluble Tissue Antigens

In a previous experiment, rabbits and goats were immunized with boiled and ethanol precipitated (BE) bovine kidney antigen, and the specificity of the antibodies produced was compared (Andersen 1975). The caprine sera were species specific while the rabbit sera, however, cross-reacted with BE antigens from other ruminant species. Sera from 2 rabbit littermates differed somewhat in that 1 serum seemed to be mainly species specific giving only weak reactions against BE antigens from kidney and spleen from other ruminants, whilst the other serum was more organ specific and reacted equally with homologous and heterologous kidney antigens.     

Surface K antigen in Salmonella paratyphi B.

It was established by agar immunoelectrophoresis that Salmonella paratyphi B lysate contains a large number of soluble antigens which display a varying degree of serological specifity as well as different diffusion and electrophoretic mobility. Salmonella paratyphi B was found to possess, apart from specific O and H antigens, a surface K antigen. This is a distinct antigen having strict serological specificity. Purified K antigen displayed anodic mobility in immunoelectrophoresis. A detailed study of K antigen properties in cultures treated by different methods as well as immunochemical investigations of purified K antigen showed that the surface K antigen of S. paratyphi B differs from its O, M, Vi, H and other known antigens in terms of basic characteristics.     

Expression of ABH and X (Lex) antigens in various cells

Using a panel of reagents specific to the various subtypes of ABH antigens, it could be demonstrated that platelets carry ABH type 2 monofucosylated determinants on intrinsic glycoproteins. The presence of these antigens is controlled by the H gene and correlates with the presence of alpha-2-L-fucosyltransferase and the absence of alpha-3-L-fucosyltransferase. In contrast, intrinsic ABH antigens were not found on mononuclear cells, correlating with the absence of alpha-2-L-fucosyltransferase on these cells. However, after transformation with the Epstein-Barr virus and stimulation with 12-O-tetradecanoylphorbol-13-O-acetate (TPA), B lymphocytes were found to express the H antigen under control of the H gene and not the Se gene. The lymphoblastoid cell lines also expressed the X and sialylated X antigens which are normally markers of the myeloid lineage. These antigens are also normally found in epithelial cells of the digestive tract, kidney proximal convoluted tubules and hepatocytes. The alpha-3-L-fucosyltransferase responsible for the synthesis of this antigen is present in the serum but we report the existence of two individuals, a mother and her daughter, who lack more than 90% of this serum enzyme. The young girl suffers from a congenital kidney anomaly: oligomeganephronic hypoplasia. Her kidney tubules are devoid of X antigen. However, she and her mother have the X antigen on their granulocytes and its sialylated form on their monocytes. It therefore appears that there are distinct genetic controls for the expression of antigen X in different body compartments. This would be quite similar to the H and Se gene controls in tissues of distinct embryological origins.     

Changes in cell surface antigens during in vitro lizard myogenesis

Indirect immunofluorescence has been used to examine surface antigens of lizard myogenic cells during in vitro differentiation. At least two developmental stage-specific surface alterations have been identified. One of these is a compositional change and involves the appearance of a cell-surface antigen(s) as the cells differentiate. This antigen(s) (Ag1422) is muscle specific and is characteristic of some rounded-up G0 myosin-positive myocytes, all stretched-back, G0 myosin-positive myocytes, and all identifiable myotubes. The antigen is not found on proliferating myoblasts, extended G1 (myosin-negative) cell-cycle-competent myoblasts or newly differentiated rounded-up, G0 myosin-positive myocytes. Pretreatment of cells with neuraminidase, trypsin, or proteinase K indicates the antigen is not present in "masked" form on normally nonreactive cells. Proteinase K is effective in the removal or destruction of the antigen, indicating it is at least partially protein in nature. The antigen is expressed in a similar developmental stage-specific fashion on early-passage myogenic cells taken from both adult lizard tail regenerates and embryonic muscle. The antibodies identifying Ag1422 can be removed by adsorption with homogenates of mature skeletal muscle. Therefore, Ag1422 is not an artifact due to in vitro conditions or the expression of a transformation antigen unique to the continuous culture line. The second alteration is an apparent restriction in the mobility of surface components (antigens and lectin receptors). Upon treatment with multivalent ligands, undifferentiated myosin-negative myoblasts exhibit rapid patching and capping of cell surface components while well-differentiated myocytes and myotubes do not. This mobility restriction is evident after the appearance of Ag1422. Treatment with cytochalasin B (15 micrograms/ml) and/or colchicine (100 microM) does not alter the restricted mobility of surface components seen on differentiated cells. Therefore, neither microfilaments nor microtubules seem to be involved in the mobility restriction. These observations are discussed in relation to current views of myogenesis.     

Loss in transformed cells of cell cycle regulation of expression of a nuclear protein recognized by SLE patient antisera

The identification of cellular proteins involved in the control of cell proliferation in normal cells is essential for understanding the mechanism underlying growth regulation and cellular transformation. A nuclear protein termed Ki antigen with a relative mobility of 32,000 (Mr 32K) and which is recognized by SLE patient antisera has been identified in cells of human, bovine, and murine origin. Recently, cDNA clones for the bovine and human Ki antigens have been isolated using SLE patient antisera (T. Nikaido, et al., in preparation). The nucleotide sequence predicted a protein of 239 amino acids with a possible nuclear localization signal resembling that identified in SV40 T antigen and other nuclear proteins. Here we show that the expression of Ki antigen is regulated in the normal cell, but not in the transformed cell. Furthermore, in the K-ras temperature-sensitive mutant cell line, ts 371 normal rat kidney (NRK), Ki antigen expression increases several-fold at the permissive temperature relative to the nonpermissive temperature. These results suggest that expression of Ki antigen might be correlated with cellular transformation as well as with cell growth regulation.     

Cytotoxic immune cells with specificity for defined soluble antigens. I. Assay with antigen-coated target cells

An in vitro cytotoxic system is described, in which immune cells specific for a given soluble antigen exert a specific cytotoxic effect on target cells to which this antigen has been covalently linked. The nature of the target cell is important in this system. When antigen-coated P 815-X2 mastocytoma cells and antigen-coated chicken red blood cells were incubated for several hours in culture medium at 37 °C, the presence of membrane-bound antigen could still be demonstrated on the latter, but not on the former target cells. This might be the reason why antigen-specific target cell destruction by specific immune cells was observed only with antigen-coated chicken red blood cells as target cells. The specificity of the cytotoxic effect was controlled in each experiment in a criss-cross way by using two non cross-reacting antigens both as immunogens and for coating the target cells. Specific cytotoxicity was demonstrable with both guinea pig and mouse immune cells and with different kinds of antigens: foreign proteins, hapten-heterologous protein conjugates and hapten-autologous protein conjugates.     

Identification, isolation and physico-chemical properties of neurospecific alpha2-globulin]

In the immunization process of rabbits with the protein fraction of the water-salt extract from human brain partially and concentrated at 500-600 times was received antiserum revealed brain specific alpha 2-globulin that is not identical to the known cytoplasmatic brain specific antigens. This antigen has got electrophoretic mobility of alpha 2-globulins, molecular weight 90 +/- 10 kD and isoelectric point 4.1-5.4. Develop the procedure for purification of this antigen on the basis of the combination ion change, affinity, hydrophobic chromatography gel-filtration and isochromatofocusing.     

Antigens common to Mycobacterium bovis (BTsZh) and several malignant and normal tissues of laboratory animals]

Agglutination, gel precipitation and immunoelectrophoresis tests demonstrated the presence of antigens common for the Mycobacterium bovis (BCG) and some malignant and normal rat tissue cells. The method of absorption of immune sera in the agglutination test showed that this antigen was specific for the BCG microorganisms and cells of primary induced tumours of rat muscle tissue. This antigen had the electrophoretic mobility in the beta-globulin zone.     

Inhibition of modulation of thymus-leukemia antigens by concanavalin A.

When incubated at 37 °C in medium containing antibodies specific for thymus-leukemia (TL) antigens, viable cells bearing these antigens become resistant to the cytolytic effects of guinea pig complement, a process termed antigenic modulation. Antibody-induced membrane redistribution of the TL antigens, detected by indirect immunofluorescence, occurs with a similar pace. When high concentrations of concanavalin A (Con A) were included with antibodies in the incubation medium, TL antigenic modulation as well as antigen patching and capping were markedly inhibited, similar to effects of Con A on membrane immunoglobulin redistribution with murine spleen cells. Colchicine antagonized the inhibition by Con A suggesting the involvement of microtubules. In parallel experiments high concentrations of Con A failed to alter the quantity of TL antigen expression or its rate of change with time during incubation in cognate antisera. These results support the hypotheses that (a) generalized alterations in membrane receptor mobility may be induced by ligand binding to the cell membrane, and (b) under certain conditions stable interactions occur between normally independent cell surface antigens.     

Ultrastructural localization of human kidney antigens using monoclonal antibodies.

A highly sensitive method of ultrastructural-immunoperoxidase staining was developed for use with monoclonal antibodies which have been raised in this laboratory to a variety of antigens of the human kidney. Because of the susceptibility of the antigens to fixation and processing, a four layer, pre-embedding method of staining was used. Results confirmed and clarified previously reported light microscopy results, indicating that an antigen recognized by the PHM5 antibody was found on the podocyte cell membrane within the glomerulus and was not present within the glomerular basement membrane. The antigen was also present on the extraglomerular endothelial cell membrane. The study also demonstrated the presence of an antigen specific to endothelial cells throughout the renal cortex, and gave further insight into the precise localization of glomerular basement membrane components including fibronectin. The method of staining is now being used together with detailed ultrastructural studies to identify the cells produced from isolated glomeruli in tissue culture.     

An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates.

Polyoma T antigen immunoprecipitates contain a protein kinase-like activity which preferentially phosphorylates material of 50-60,000 daltons molecular weight. Phosphorylation is not diminished in extracts of polyoma tsA mutant-infected cells shifted to the nonpermissive temperature late in infection, conditions which inactivate the large T antigen. Phosphorylation is reduced or absent in cells infected with polyoma host range nontransforming (hr-t) mutants, which have defective small and medium T antigens. The major acceptor of phosphate is not the heavy chain of immunoglobulin, but appears to be the polyoma medium T antigen. The large T antigen is also phosphorylated, but usually to a lower specific activity. In terms of acid and alkali sensitivity and electrophoretic and chromatographic mobility in one and two dimensions, the phosphorylated residue behaves identically to phosphotyrosine and differently than phosphorylated serine, threonine, lysine and histidine.     

Schistosoma mansoni: Characterization of two circulating polysaccharide antigens and the immunological response to these antigens in mouse,hamster, and human infections

In this study the nature and occurrence of two circulating polysaccharide antigens of Schistosoma mansoni, circulating anodic antigen (CAA) and circulating cathodic antigen (CCA), and the immunological response to these antigens in mouse, hamster, and human infections were investigated. Both CAA and CCA showed a large molecular weight range, less than 50,000 to over 300,000 for CAA and 50,000 to over 300,000 for CCA, possibly representing monomers and polymers. CAA and CCA could be purified from the trichloroacetic acid-soluble fraction of adult worm antigen (AWA-TCA) by means of DEAE ion exchange chromatography. The presence of at least two other components in AWA-TCA was shown. Both CAA and CCA were found to be gut associated, and could be demonstrated in the vomitus and in the excretory and secretory antigens of adult worms. Both antigens were present in the kidney eluates of infected hamsters, while CCA could normally be detected in the urine of these hamsters and CAA only occasionally. CAA was demonstrated in the Kupffer cells of the livers of infected mice and hamsters. Antibodies against CAA and CCA were shown in mouse, hamster, and human infections. In human infections specific IgM titers against these antigens were especially elevated in children and in recent infections of adults.     

A specific IgG in Graves' ophthalmopathy and its relation to retro-orbital and thyroid autoimmunity.

An IgG (ophthalmopathic immunoglobulin) that binds to retro-orbital antigen was identified in serum from patients with active Graves'' ophthalmopathy, and its nature and specificity were investigated. Dose related binding of this immunoglobulin to retro-orbital antigens prepared from guinea pig harderian gland or porcine eye muscle was found, which could be abolished by prior incubation with antigen. The immunoglobulin did not bind to thyroid membranes, thyroid microsomes, or thyroglobulin or interact with liver, skeletal muscle, or fat membranes. Serum with high activity of thyrotrophin binding inhibiting immunoglobulin did not react with retro-orbital antigen, and this activity was not affected by preincubation of the serum with retro-orbital antigen. Thyroid stimulating hormone was also without effect on retro-orbital antigen. It is concluded that Graves'' ophthalmopathy is associated with a specific ophthalmopathic immunoglobulin that reacts with retro-orbital antigen as distinct from thyroid antigens, and that the autoimmune response is directed towards retro-orbital antigens. This suggests that the ophthalmopathy is an entity distinct from autoimmune thyroid disease.     

Immunological characterization of cytochrome H-450 in rat tissues

Rabbit antiserum produced against rat liver cytochrome H-450 was specific for cytochrome H-450. The antiserum did not react with hemolysate, microsomal and mitochondrial fractions of liver, and tissue extracts from heart, lung skeletal muscle, and testis of rat. With the monospecific antiserum, a rocket immunoelectrophoretic assay method was developed for the quantitation of the antigen with a sensitivity of 25 ng. By using rocket immunoelectrophoresis, the total amounts of the antigen found in liver, kidney, and brain of 20 rats were 33.6, 3.6, and 1.3 mg, respectively. It appears that the antigens in liver, kidney, and brain are immunologically identical. From immunological studies with subcellular fractions of rat liver, the antigen was found only in the postmicrosomal fraction. This indicates that the antigen is not a precursor or a proteolytic product of known cytochromes in mitochondria or microsomes. Therefore, cytochrome H-450 is a unique cytosolic protein found in brain, kidney, and liver.     

Comparative study of appearance of water-soluble antigens and brain esterase fractions in ontogenesis of two strains of rats

By means of immunoelectrophoresis of rat brain, 12 water-soluble antigens were detected, five of which were found to be specific to the brain. Histochemical reactions have identified two antigens that are not specific to the brain, lactate dehydrogenase and esterase. By means of enzymoelectrophoresis, 14 esterase fractions were determined. An immunoautoradiographic study of the synthesis of some antigens specific to the brain was carried out. It was found that rats responding to sound by epileptic seizures develop more slowly than normal rats. In particular, in rats resistant to auditory stimulus the antigenic spectrum typical of the brain of the adult rat (12 antigens) is formed by day 14 of postnatal life, while in rats of the sensitive strain this pattern takes 17 days to completely form. The last brain-specific antigen for rats susceptible to audiogenic epilepsy appears on day 17, while this antigen is present in normal rats on day 14. Fast-moving esterase fractions are detected earlier in ontogenesis in rats resistant to sound than in sensitive rats.     

On the control between cell-mediated, IgM and IgG immunity

An hypothesis is proposed here describing some of the conditions that determine the type of response an antigen will induce, and explaining how the induction of one type of immunity affects the induction of other types of immunity. In more detail, the hypothesis attempts to account for the following observations: Some antigens induce only cell-mediated immunity, whereas others can, under different conditions, induce either cell-mediated or humoral immunity. The humoral response to most antigens consists of an initial period of IgM antibody synthesis, followed by a period of IgG synthesis. Some polymeric antigens induce the synthesis of only IgM antibody. There is a tendency for the immune response to an antigen, at a particular time, to be exclusively of the cell-mediated, IgM or IgG type.The hypothesis may also be relevant to some observations that, I believe, have been incorrectly interpreted to mean that “tolerance” to some antigens requires the presence of T (thymus-derived) cells specific for these antigens. The hypothesis suggests teleological reasons for the existence of the different types of immunity. It also suggests ways of controlling the type of response an antigen induces.     

Leukocyte-migration inhibition in guinea pigs. I. Correlation with skin test reactivity and macrophage-migration inhibition.

Fifteen guinea pigs, immunized with one of three soluble antigens, repeatedly demonstrated inhibition of leukocyte migration and positive skin tests to the immunizing antigens. An additional five animals immunized with ovalbumin demonstrated inhibition of macrophage migration as well as direct and indirect inhibiton of leukocyte migration. Only one of fifteen animals demonstrated inhibition of leukocyte migration and none had a positive skin test with an antigen to which it had not been sensitized, indicating that the assay is antigen specific.     

Cytotoxic immunotherapy strategies for cancer: mechanisms and clinical development

Traditional therapies for cancer include surgery, chemotherapy, and radiation. Chemotherapy has widespread systemic cytotoxic effects against tumor cells but also affects normal cells. Radiation has more targeted local cytotoxicity but is limited to killing cells in the radiation field. Immunotherapy has the potential for systemic, specific killing of tumor cells. However, if the immune response is specific to a single antigen, tumor evasion can occur by down-regulation of that antigen. An immunotherapy approach that induces polyvalent immunity to autologous tumor antigens can provide a personalized vaccine with less potential for immunologic escape. A cytotoxic immunotherapy strategy creates such a tumor vaccine in situ. Immunogenic tumor cell death provides tumor antigen targets for the adaptive immune response and stimulates innate immunity. Attraction and activation of antigen presenting cells such as dendritic cells is important to process and present tumor antigens to T cells. These include cytotoxic T cells that kill tumor cells and T cells which positively and negatively regulate immunity. Tipping the balance in favor of anti-tumor immunity is an important aspect of an effective strategy. Clinically, immunotherapies may be most effective when combined with standard therapies in a complimentary way. An example is gene-mediated cytotoxic immunotherapy (GMCI) which uses an adenoviral vector, AdV-tk, to deliver a cytotoxic and immunostimulatory gene to tumor cells in vivo in combination with standard therapies creating an immunostimulatory milieu. This approach, studied extensively in animal models and early stage clinical trials, is now entering a definitive Phase 3 trial for prostate cancer.     

Heterologous antigenic stimulation in induction of delayed hypersensitivity.

An influence of a delayed hypersensitive reaction to a primary antigen on the induction of delayed hypersensitivity to a second unrelated antigen was observed in guinea pigs immunized with azobenzenearsonate-N-acetyl-L-tyrosine (ABAT), and injected intradermally 3 weeks later with a mixture of ABAT and secondary antigen. Animals so treated developed delayed hypersensitivity to sheep red blood cells (SRBC) or Type II pneumococcal polysaccharide as secondary antigens, as measured by skin test reactivity and inhibition of macrophage migration, whereas ABAT unsensitized control groups did not. However, attempts to induce delayed reactivity to proteins as secondary antigens were unsuccessful. The injection of secondary antigen into a mineral oil-induced inflammatory lesion did not induce delayed hypersensitivity, suggesting that specific reactivity to ABAT is a prerequisite for heterologous induction. Possible mechanisms for the observed phenomenon, including a role for macrophages, are discussed.     

Entactin, a novel basal lamina-associated sulfated glycoprotein

A sulfated glycoprotein, entactin, of apparent molecular weight 158,000 has been isolated from an extracellular basement membrane-like matrix. This matrix is elaborated in cell culture by a mouse endodermal cell line. Antibodies prepared in rabbits against this sulfated glycoprotein react with mouse and rat basement membranes from a variety of tissues. These antibodies also react in a specific manner with a discrete component of mouse and rat kidney glomeruli. The electrophoretic mobility of this component is identical to that of entactin. The mouse kidney antigen, as shown by immunoelectron microscopic studies, is predominantly localized at the surface of epithelial cells of tubules and glomeruli adjacent to the basement membrane. Some antigen is also present in the basal lamina adjacent to the epithelial cells. Entactin is distinct from the basement membrane-associated protein GP-2, a protein similar to laminin. Entactin differs from GP-2 in electrophoretic mobility, cyanogen bromide peptide fragmentation pattern, immunological cross-reactivity, and incorporation of H235SO4. Entactin is insensitive to treatment with chrondroitinase ABC. It is suggested that this molecule plays a role in the interaction of the extracellular matrix and the cell surface.