Antigenic and blood group activity of lamb gastric mucoproteins]

The antigenic properties of a lamb mucin and of a glycoprotein isolated from it are investigated. The proteins are studied by immunoelectrophoresis of the mucin and of the glycoprotein against either a sheep serum antiserum or an exclusively glycoprotein antiserum. No serum protein could be shown in the mucin. In addition, the glycoprotein gives a precipitin line only with the specific antiserum; its antigenic power seems to be concealed in the original mucin. The carbohydrates are studied through the investigation of the blood group activities by the hemagglutination inhibition technique. The mucin and the isolated glycoprotein show the same types of activities: they both prevent the agglutination of A1 and 0 red cells. The presence of two blood group activities in that glycoportein and its non-reactivity against A2 red cells corroborate the hypothesis that carbohydrate chains must be branched for the elicitation of these activities in a single molecule.     

Colony formation in agar by murine plasmacytoma cells: potentiation by hemopoietic cells and serum

Clonal growth in semisolid agar medium was obtained using cells from 19 of 25 transplanted murine plasmacytomas when the medium was supplemented by whole mouse blood or washed red cells. With different tumors cloning efficiency ranged from 0.01% to 21.6%. With two exceptions, mouse blood did not potentiate colony formation in agar by cells from transplantable myelomonocytic, myeloid, and lymphoid leukemias, reticulum cell sarcomas and fibrosarcomas. The clonal growth of some plasmacytomas was also potentiated by syngeneic thymic, spleen or bone marrow cells. Plasmacytoma colony growth was not stimulated by normal mouse serum but serum from mice injected with endotoxin or polymerised flagellin stimulated colony growth by some plasmacytomas. The active serum factor was not the colony stimulating factor (CSF) and its appearance after antigenic stimulation was not T cell-dependent. Preimmunised mice failed tq respond to antigenic stimulation. Whole body irradiation did not induce a rise in the capacity of serum to stimulate colony formation by plasmacytoma cells.     

Red blood cell mechanics and capillary blood rheology.

Blood contains a high vol fraction of erythrocytes (red blood cells), which strongly influence its flow properties. Much is known about the mechanical properties of red cells, providing a basis for understanding and predicting the rheological behavior of blood in terms of the behavior of individual red cells. This review describes quantitative theoretical models that relate red cell mechanics to flow properties of blood in capillaries. Red cells often flow in single file in capillaries, and rheological parameters can then be estimated by analyzing the motion and deformation of an individual red cell and the surrounding plasma in a capillary. The analysis may be simplified by using lubrication theory to approximate the plasma flow in the narrow gaps between the cells and the vessels walls. If red cell shapes are assumed to be axisymmetric, apparent viscosities are predicted that agree with determinations in glass capillaries. Red cells flowing in microvessels typically assume nonaxisymmetric shapes, with cyclic "tank-treading" motion of the membrane around the interior. Several analyses have been carried out that take these effects into account. These analyses indicate that nonaxisymmetry and tank-treading do not significantly influence the flow resistance in single-file or two-file flow.     

Red blood cell mechanics and capillary blood rheology

Blood contains a high vol fraction of erythrocytes (red blood cells), which strongly influence its flow properties. Much is known about the mechanical properties of red cells, providing a basis for understanding and predicting the rheological behavior of blood in terms of the behavior of individual red cells. This review describes quantitative theoretical models that relate red cell mechanics to flow properties of blood in capillaries. Red cells often flow in single file in capillaries, and rheological parameters can then be estimated by analyzing the motion and deformation of an individual red cell and the surrounding plasma in a capillary. The analysis may be simplified by using lubrication theory to approximate the plasma flow in the narrow gaps between the cells and the vessel walls. If red cell shapes are assumed to be axisymmetric, apparent viscosities are predicted that agree with determinations in glass capillaries. Red cells flowing in microvessels typically assume nonaxisymmetric shapes, with cyclic “tank-treading” motion of the membrane around the interior. Several analyses have been carried out that take these effects into account. These analyses indicate that nonaxisymmetry and tank-treading do not significantly influence the flow resistance in single-file or two-file flow.     

Investigation of hemolytic activity of leptospirae on solid culture media

Results of investigation of hemolytic activity of leptospirae against red blood cells of various animal species on solid culture media using the technique of agar layers are presented. It has been shown that hemolytic properties of pathogenic and saprophytic leptospirae differ with respect to red blood cells of the sheep, the rabbit, the hamster, the albino rat and the albino mouse. Hemolytic activity of leptospirae regarding the erythrocytes of the cattle, the horse and the fowl depends on the strain of leptospirae. The advantage of using solid nutrient media for the determination of hemolytic properties of leptospirae in comparison with liquid media has been demonstrated.     

In vitro attachment of mono- and oligosaccharides to surface glycoconjugates of intact cells

We have synthesized glycosylhydrazines of various mono- and oligosaccharides and coupled these to periodate- or galactose oxidase-treated human red cells and K562 erythroleukemia cells. The optimal conditions for this carbohydrate modification of cells have been established. This method makes it possible to specifically elongate oligosaccharide chains of cell surface glycoconjugates with desired carbohydrates. In this way, new antigenic and receptor properties can be conferred to cells, and the functional roles of carbohydrates in cell surface glycoconjugates can be studied. The method has been used to make red cells of blood group O reactive with anti-A and anti-B sera, and in rendering K562 cells or red cells of blood group O agglutinable with the alpha-N-acetylgalactosamine-specific Helix pomatia lectin.     

Human red blood cell insulin-degrading enzyme and rat skeletal muscle insulin protease share antigenic sites and generate identical products from insulin.

The mechanisms of cellular insulin degradation remain uncertain. Considerable evidence now exists that the primary cellular insulin-degrading activity is a metallothiol proteinase. Two similar degrading activities have been purified and characterized. Insulin protease has been purified from rat skeletal muscle and insulin-degrading enzyme from human red blood cells. Whereas the two degrading activities share a number of similar properties, significant differences have also been reported; and it is not at all established that they are the same enzyme. To examine this, we have compared antigenic and catalytic properties of the two enzymatic activities. Monoclonal antibodies against the red blood cell enzyme adsorb the skeletal muscle enzyme; and on Western blots, the antibodies react with an identical 110-kDa protein. Immunoaffinity-purified enzymes from both red blood cells and skeletal muscle degrade [125I]iodo(B26)insulin to the same products as seen with purified insulin protease and with intact liver and kidney. Chelator-treated muscle and red blood cell enzymes can be reactivated with either Mn2+ or Ca2+. Thus, insulin-degrading enzyme and insulin protease have similar properties. These results support the hypothesis that these activities reside in the same enzyme.     

Hybridomas specific for carbohydrates; synthetic human blood group antigens for the production, selection, and characterization of monoclonal typing reagents

A general method for the production of carbohydrate-specific hybridoma antibodies is illustrated by generation of monoclonal antibody to the antigenic determinant of human blood group B. This trisaccharide determinant was chemically synthesized and covalently coupled to bovine serum albumin and human blood group O red cells. Soluble protein antigen and the 'artificial' B red cells were used to immunize BALB/c mice before fusion of spleen cells with the Sp2/0 plasmacytoma cell line. ELISA screening of putative hybrids for B-specific binding activity was facilitated by the availability of a second synthetic conjugate, B-horse hemoglobin. IgM-producing clones were identified by class-specific ELISA reagents and by hemagglutination assay. In this way, clones suitable for blood typing were rapidly identified. The precise antigenic specificity and Ig class of such monoclonal antibodies were defined by inhibition of precipitation and by gel filtration. Hybridoma antibodies were obtained from two separate fusion experiments. One of these, clone 3E-4, was of the IgM class and possessed a binding site that was completely satisfied (100% inhibition) by the trisaccharide determinant of the B blood group. This antibody is shown to be suitable for use in blood typing.     

Red blood cell orientation in pulmonary capillaries and its effect on gas diffusion.

When alveoli are inflated, the stretched alveolar walls draw their capillaries into oval cross sections. This causes the disk-shaped red blood cells to be oriented near alveolar gas, thereby minimizing diffusion distance. We tested these ideas by measuring red blood cell orientation in histological slides from rapidly frozen rat lungs. High lung inflation did cause the capillaries to have oval cross sections, which constrained the red blood cells within them to flow with their broad sides facing alveolar gas. Low lung inflation stretched alveolar walls less and allowed the capillaries to assume a circular cross section. The circular luminal profile permitted the red blood cells to have their edges facing alveolar gas, which increased the diffusion distance. Using a finite-element method to calculate the diffusing capacity of red blood cells in the broad-side and edge-on orientations, we found that edge-on red blood cells had a 40% lower diffusing capacity. This suggests that, when capillary cross sections become circular, whether through low-alveolar volume or through increased microvascular pressure, the red blood cells are likely to be less favorably oriented for gas exchange.     

Studies on the osmotic resistance and the viability of amidinated erythrocytes]

The present studies are concerned with properties of amidinated erythrocytes. The reactions of dimethyladipimidate with proteins in solution and red blood cells, respectively, result in an intermolecular cross-linking. Following an amidination of human serum albumin or human gamma-globulin cross-linked products of increased molecular weight have been demonstrated by polyacrylamide gel and immune electrophoresis. Human erythrocytes previously amidinated intensely, exhibit a restricted motility of membrane particles and cross-linked hemoglobin. Intensely amidinated erythrocytes are resistant against distilled water, and they do no longer agglutinate. The findings presumably indicate an increased permeability of the amidinated red cell membrane. The glycolytic activity was found to be normal in moderately amidinated erythrocytes. In comparison with normal red blood cells, previously moderately amidinated erythrocytes of the rat become sequestered more quickly after re-injection into the vascular system.     

Influence of the amount of antigen and the interval on the secondary reaction

The course of the revaccination reaction in mice immunized with different doses of sheep red blood cells was determined at different intervals after the primary stimulus. The maximum level of haemagglutinating antibodies in the secondary reaction was found after a high primary and secondary antigenic stimulus. On the contrary, if the level of haemolytic antibodies was determined, the higher was the primary antigenic stimulus, the lower was the secondary antibody response. Differences between haemagglutinins and haemolytic antibodies were also manifested in the earlier onset of the maximum haemolytic secondary reaction (five months after the first dose of antigen); the maximum haemagglutination response was not attained until eight months after the primary dose of antigen. The results comfirm that the basis of preparation for the secondary reaction is proliferation of immunologically activated Y cells; differences in the haemolytic and haemagglutination response are related to differences in the character of the antigenic determinants of sheep red cells.     

Differences Between Brucella Antigens Involved in Indirect Hemagglutination Tests with Normal and Tanned Red Blood Cells

Brucella antigens capable of sensitizing normal and tanned sheep red blood cells for indirect hemagglutination were compared with antigens involved in agglutination, gel diffusion, and immunoelectrophoresis. Hyperimmune rabbit sera, before and after absorption with various antigenic preparations from smooth and rough B. abortus, were used in the tests. Normal erythrocytes could be sensitized with an NaOH-treated ether-water extract (EW-T) of smooth Brucella. Tanned erythrocytes could be sensitized with a water-soluble extract from ultrasonically disrupted smooth or rough Brucella. The EW-T produced a single precipitation band and the water-soluble antigens produce 6 to 23 bands in immunoelectrophoresis with unabsorbed sera. After absorption of antisera with water-soluble extracts from smooth or rough Brucella cells or from smooth or rough cell walls, the hemagglutinins for sensitized tanned erythrocytes and the precipitins for water-soluble antigens were removed. Absorption with living smooth or rough Brucella cells or with EW-T did not remove these antibodies. The precipitins and hemagglutinins for the antigen EW-T, and agglutinins for smooth cells, were absorbed by smooth antigens but not by rough antigens. It appears that the antigens which sensitize tanned erythrocytes and diffuse through agar gels are present in both smooth and rough forms and may be situated in the cytoplasm or in the internal part of the cell wall, whereas the agglutinogen and the antigen which attaches to normal erythrocytes are surface antigens found only on the smooth Brucella cell.     

Studies on the role of vitamin E in the oxidation of blood components by fatty hydroperoxides.

Studies are reported on the oxidation of vitamin E and changes in lipid and fatty acid composition of rat blood components incubated in vitro with hydroperoxides prepared from autoxidized methyl linoleate. Red blood cells, plasma, serum, and hemoglobin free stroma were incubated at 37 °C with suspensions of linoleate hydroperoxide in Tris buffer at pH 7.4. The RBC were destroyed and substances with excitation-fluorescent properties were produced. Phosphatidylethanolamine, vitamin E and unsaturated fatty acids were oxidized in the reaction. Among the reaction products were substances that gave a positive thiobarbituric acid value, tocoquinone, and an unidentified substance isolated in the nonsaponifiable fraction of the lipid extract of the hemolyzed red cells. The reaction of linoleate hydroperoxide with stroma was similar to that with red blood cells and the same products were observed. In contrast there was little reaction of linoleate hydroperoxide with vitamin E or lipids of the serum or plasma in the absence of red blood cells. The destruction of the red blood cells appeared to be closely related to the oxidation of vitamin E indicating that the strong antioxygenic action of vitamin E in vivo was due to its particular form or structural orientation in the red cell membrane.     

Immunosuppressive Effect of Bacterial Lipopolysaccharide on Antibody Response

Injection of endotoxins (bacterial lipopolysaccharide: LPS) several days prior to immunization causes the suppression of antibody response. The suppressive effects of several kinds of LPS preparations on the plaque-forming cell (PFC) antibody response in the spleen of mice were examined after immunization with sheep red blood cells (SRBC). Glycolipids obtained from heptoseless mutants (Re form) of salmonella or its lipid A preparation coupled artificially with bovine serum albumin (BSA) are capable, like LPS obtained from a wild type (S form) strain, of inducing suppression of the PFC response, while alkaline-detoxified LPS can not. The refractory periods of the PFC response induced by LPS injection last only a few days. However, the use of cyclophosphamide (CY) together with LPS can extend the refractory periods of antigenic stimulation for several weeks. Injections of LPS and CY can also induce unresponsive states of OH agglutinin antibody response to antigenic stimulation with formalin-killed organisms of Escherichia coli or Salmonella enteritidis (presumably both thymus-independent antigens). These unresponsive states induced by LPS and CY are easily terminated by a transfer of syngeneic bone marrow cells but not by thymocyte transfer.     

Expression of an onco-developmental antegen among avian species.

Rabbit antisera directed against an onco-developmental antigen on chicken red blood cells have been serologically dissected through specific adsorptions. It is now possible to detect 13 antigenic determinants with the fractionated antisera. The onco-developmental antigen referred to as chicken fetal-leukemic antigen (CFA) is fetal-specific in the white Leghorn chicken, being present on the embryonic but not adult peripheral red blood cells of non-being present on the embryonic but not adult peripheral red blood cells of non-leukemic birds. However, one or more of the onco-developmental antigenic determinants have been detected on adult peripheral red blood cells of non-Gallus avian species, as well as on red blood cells from two adult chicken varieties. For phylogenetic purposes, red blood cells from avian species were characterized for their combinations of CFA determinants. Comparisons among species revealed specific patterns of antigenic expression within phylogenetic groups. Several CFA determinants were restricted in their occurrence to species within a single family, and one determinant was found in all cases where CFA was expressed. The distribution of CFA determinants was used to determine immunological distances among four Galliform species. These distances agreed with the immunological relationships established using different serological markers.     

Specificity of antierythrocyte autoantibodies secreted by a NZB-derived hybridoma and NZB peritoneal cells

The specificity of monoclonal IgM antierythrocyte autoantibody produced by a NZB-derived hybridoma and the specificity of autoantibodies produced by uninduced NZB peritoneal cells in culture were determined. Supernatant fluids from cultures of hybridoma and peritoneal cells reacted in direct hemagglutination assays with bromelin-treated mouse erythrocytes, and, to a lesser extent, with sheep red blood cells; no agglutination was observed with intact mouse red blood cells or human O⁺ erythrocytes. These results suggest the presence of previously characterized anti-HB, but not anti-X or cold reactive autoantibodies, with a cross-reaction between antigenic constituents on sheep and bromelin-treated mouse erythrocytes. Specificity was affirmed by neutralization of agglutination or of direct hemolysis of bromelin-treated mouse erythrocytes with partially purified SEA-HB, the soluble plasma analog of the erythrocyte-bound HB autoantigen. Plaque formation in direct plaque-forming cell assays by both hybridoma and peritoneal cells was specifically inhibited by SEA-HB. These results demonstrate that NZB-derived hybridoma as well as NZB peritoneal cells secrete anti-HB autoantibody, an autoantibody that spontaneously appears in the serum of NZB as well as other strains of mice.     

Tolerance-like condition developing in mice under the effect of antigen of erythrocyte lysate]

Lysate of sheep red blood cells obtained by the treatment of these cells with distilled water and purified by ultracentrifugation in cold possessed a weak immunogenicity. Its administration to mice caused the state of hyporeactivity to sheep red blood cells (a reduction of the immune response level to 10-25% of control. The capacity of the mise spleen cells to respond by immune reaction to the red blood cells following adoptive transfer was not disturbed. At the early periods after the lysate administrations the mouse spleen cells possessed a weak supressive activity in case of their transfer to the intact animals. The blood serum of mice treated with the lysate possessed a blocking activity which disappeared after the serum absorption with sheep red blood cells. A conclusion was drawn that hyporeactivity originating in mice after the lysate administration was caused by the presence in the serum of antibodies inhibiting the immune response.     

Influence of storage and buffer composition on the mechanical behavior of flowing red blood cells

On-chip study of blood flow has emerged as a powerful tool to assess the contribution of each component of blood to its overall function. Blood has indeed many functions, from gas and nutrient transport to immune response and thermal regulation. Red blood cells play a central role therein, in particular through their specific mechanical properties, which directly influence pressure regulation, oxygen perfusion, or platelet and white cell segregation toward endothelial walls. As the bloom of in-vitro studies has led to the apparition of various storage and sample preparation protocols, we address the question of the robustness of the results involving cell mechanical behavior against this diversity. The effects of three conservation media (EDTA, citrate, and glucose-albumin-sodium-phosphate) and storage time on the red blood cell mechanical behavior are assessed under different flow conditions: cell deformability by ektacytometry, shape recovery of cells flowing out of a microfluidic constriction, and cell-flipping dynamics under shear flow. The impact of buffer solutions (phosphate-buffered saline and density-matched suspension using iodixanol/Optiprep) are also studied by investigating individual cell-flipping dynamics, relative viscosity of cell suspensions, and cell structuration under Poiseuille flow. Our results reveal that storing blood samples up to 7 days after withdrawal and suspending them in adequate density-matched buffer solutions has, in most experiments, a moderate effect on the overall mechanical response, with a possible rapid evolution in the first 3 days after sample collection.     

The Antigens Associated with the Cell Walls of Members of the Genus Pseudomonas

Three antigens were associated with the cell walls of pseudomonads. A highly antigenic, strain-specific antigen of high molecular weight and protein or lipoprotein in nature, occurred as an envelope around the cells. It could be washed off the cells closely associated with carbohydrate material but its antigenicity was not dependent on the carbohydrate present. Another antigen, common to all strains tested, was situated below the first antigen. This was less antigenic than the strain-specific antigen and was polysaccharide or lipopolysaccharide in nature. A second common antigen was the mucopeptide of the cell walls. This had an antigenicity similar to that of the second antigen and was dependent on both the carbohydrate and polypeptide components of the macromolecule. There appears to be some correlation between these findings and the structure of cell walls of pseudomonads are shown by electron microscopy.     

The cell wall of Chlamydomonas eugametos. Immunological aspects

An antiserum was raised against the major cell wall glycoprotein of Chlamydomonas eugametos which after purification reacted specifically with all individual wall components but not with intact cell walls. The antigenic sites in intact walls appear to be cryptic but become exposed on partial enzymatic degradation or in situ during daughter-cell release when the insoluble component is digested. Using the antiserum as a specific label for cell walls in various stages of disintegration, cell wall digestion during asexual and sexual reproduction was studied. It is also shown that while cell wall material is associated with isolated flagella, it is not normally associated with the flagella of intact cells.