A comparison of a solid phase IRC assay and the PSIFT for detection of antibodies to platelets.

A rapid solid phase indicator red cells assay (IRCA) for detection of platelet antibodies was developed and its sensitivity compared with PSIFT. Platelets were attached to the surface of polystyrene microtitre plate wells by means of a sodium carbonate buffer and centrifugation. Uncovered areas were blocked by a gelatin blocking buffer. After serum incubation bound platelet-specific antibodies were made visible by anti-IgG-coated indicator red cells and a brief centrifugation. A positive result, meaning the presence of an anti-platelet antibody was indicated by red cell adherence over the reaction surface. In the absence of serum antibodies to platelets the indicator red cells formed a pellet. The IRCA showed a high sensitivity; the anti-platelet antibody Thrombocyte was detectable until a dilution of 1:1,600 whereas the same antibody in the PSIFT could only be detected until a dilution of 1:400.     

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.     

Morphological properties and adhesiveness of bacteria of the genus Proteus

Out of 100 Proteus strains isolated from patients with purulent inflammatory, urological and enteric diseases, from healthy persons and from the environment, 29 stains showed the positive D-mannose-resistant reaction of hemagglutination with chick red blood cells and 18 strains showed such reaction with goose and duck red blood cells. The results of these studies permit the use of chick red blood cells as target cells for the detection of Proteus adhesin. Human red blood cells of groups O, A, B and AB, sheep, bovine, dog, rat and rabbit red blood cells gave no positive D-mannose-sensitive reaction and D-mannose-resistant reaction of hemagglutination. In bacterial cells pili function as organelles which determine Proteus adhesiveness, while flagellae play no positive role.     

Detection of human B cells and chronic myelocytic leukemic cells by rosette formation with sheep erythrocytes and fresh human sera.

A new method of detecting the C3b receptor is reported. A particular merit of this method is that anti-RBC rabbit antiserum is not required. Rosettes were formed with human B lymphocytes, B lymphoblasts and granulocytes, using sheep erythrocytes (SRBC) sensitized with fresh human serum (FHS). T lymphocytes and T lymphoblasts did not form rosettes. The percentage of cells forming rosettes with this method approximated the percentage of rosettes formed with EACm. However, FHS coated SRBC did not react with most cells of B cell type chronic lymphocytic leukemia (CLL), whereas EACm rosette formations showed a definite reaction. On the other hand, 34--58% of cells of chronic myelocytic leukemia (CML) bound with the indicator red cells. SRBC sensitized with fresh rabbit or guinea pig serum formed rosettes with PBL, tonsil cells, B lymphoblasts and granulocytes. Complement and IgM antibody were required for this reaction, as in EAC rosette formation.     

THE AGGLUTINATION OF RED BLOOD CELLS IN THE PRESENCE OF BLOOD SERA

1. The addition of blood serum displaces the optimum for agglutination of red blood cells in a salt-free medium to the reaction characteristic of flocculation of the serum euglobulin. 2. This effect is not due merely to a mechanical entanglement of the cells by the precipitating euglobulin, since at reactions at which the latter is soluble it protects the cells from the agglutination which occurs in its absence. 3. A combination of some sort appears therefore to take place between sheep cells and sheep, rabbit, and guinea pig serum euglobulin, and involves a condensation of the serum protein upon the surface of the red cell. 4. At the optimal point for agglutination of persensitized cells both mid- and end-piece of complement combine with the cells. 5. Agglutination is closely related to an optimal H ion concentration in the suspending fluid, and probably of the cell membrane, and not to a definite reaction in the interior of the cell.     

Heterophile infectious mononucleosis antigen used in the latex diagnostic test. I. A method of antigen isolation and its serologic activity

The aim of this study was to elaborate a method of heterophile mononucleosis antigen preparation useful for latex coating. This antigen was isolated from bovine red blood cells stroma by the technique of Schwarzweiss and Tomcsik with author's own modification, in which introductory extraction of erythrocytes stroma ++ was performed by means of trichloracetic acid, aqueous extraction and elution of active substance with 80% ethanol. Besides of heterophile antigen preparation obtained by the method of Schwerzweiss and Tomcsik (preparation S-T) two serologically++ active preparations were obtained (fraction I and IV), which ability to inhibit PBD agglutinating reaction and bovine red blood cells haemolysis was 16 and 8 times lower, respectively, than S-T preparation. The preparation of heterophile mononucleosis antigen obtained differed in latex coating efficacy. In order to prepare latex reagent MZ-I (from fraction I) a solution of preparation of 125 micrograms/ml concentration was used, for MZ-II (from fraction IV)--50 micrograms and for MZ-III (from preparation S-T)--15 micrograms/ml. The reagent MZ-I showed, the highest activity in agglutinating test with human serum containing heterophile mononucleosis antibodies while two others reacted with 2-4 times lover serum dilutions. Similar differentiated reactivity with these reagents was found in latex test with 15 sera from patients suspected of having infectious mononucleosis.     

Rosette formation by human thymocytes

A proportion of lymphocytes in human fetal and post-natal thymus, and in blood, formed rosettes with red blood cells from sheep and pig. The count of rosette-forming cells (RFC) among human thymocytes varied widely, from 2–216 per thousand cells, and was higher in fetal than in post-natal life. The count of RFC among human thymocytes was not reduced by specific rabbit anti-human immunoglobulin sera, indicating that the receptor was not of immunoglobulin character; the reaction was inhibited by antithymocyte serum and metabolic poisons and certain enzymes. The receptor may be equivalent to other “non-specific” glycoprotein hemagglutinins in plants and viruses.The importance of species differences in immunological assays is emphasized. Thus human thymocytes gave high counts of RFC only with red blood cells of sheep and pig; moreover thymus lymphocytes from only man and pig, but not several other species including rodents, were highly reactive with sheep red blood cells. The capacity for rosette formation could be a marker for T cells in human blood.     

Abnormal blood group galactosyltransferase in blood type A1B-subjects whose sera contain anti-B agglutinin

Summary A blood type A₁B female, whose plasma agglutinates B red blood cells from other subjects but not her own, was found. Her sister with blood type A₁B (sister-1) exhibited the same peculiarity. The agglutination titer of red cells from these two subjects is lower than that of normal B. Her father is blood type B, and his plasma did not agglutinate B red cells from other subjects and his own. Her mother and another sister (sister-2) were usual blood type A₁. Blood group galactosyltransferase (B-enzyme) activity of plasma from the propositus, sister-1, and their father was very low. Km for 2-fucosyllactose of B-enzyme of these subjects was 1.3 mM, which was more than two times higher than that of the normal value. Km for UDP-Gal was similar, but the pH-activity profile differed for the two enzymes.Red cell membranes from her father contained about 70% ungalactosylated H-sites, whereas, virtually all H-sites are galactosylated in the usual B red cell membranes. The blood group ABH components are known to be heterogeneous. Because of the abnormal B-enzyme with low activity and low substrate affinity, some H components might not be galactosylated, particularly in A₁B cases (i.e., the propositus and her sister-1), due to competition by A₁ enzyme. The lack of certain B components is likely to be the cause of the existence of the anti-B agglutinin in their sera.     

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.     

Transbilayer redistribution of phosphatidylserine in erythrocytes of a patient with autoerythrocyte sensitization syndrome (psychogenic purpura).

We have investigated a female patient with autoerythrocyte sensitization syndrome (AES syndrome), having a positive skin response to her own red blood cells (RBC) and to phosphatidylserine (PS). Using 2,4,6-trinitrobenzenesulfonic acid (TNBS), bee venom phospholipase A2 and merocyanine 540 binding, we have demonstrated that in RBC of patient more than 50% of PS is redistributed into the outer leaflet of the plasma membrane. Using homologous RBC from a healthy donor we were able to induce transbilayer PS redistribution by incubation with the patient plasma. The presence of immunoglobulin E against cardiolipin and PS was proved in patient's plasma. We elaborated a method for cytoskeleton visualization using indirect immunofluorescence technique. We found disorders in cytoskeleton organization in RBC of the patient. We recommend in vitro testing for AES syndrome diagnosis. The positive effect of chlorpromazine treatment is described.     

Different time course patterns of local expression of delayed-type hypersensitivity to sheep red blood cells in mice.

The delayed-type hypersensitivity (DTH) reaction, a peripheral expression of cell-mediated immunity is still a crucial in vivo immunological test. Nevertheless, the biological significance of its time course remains unclear. Thus, an exhaustive study of DTH was undertaken in mice immunized with increasing doses of sheep red blood cells (SRBC) inoculated intravenously (iv) or subcutaneously. The results showed that overall DTH reactions peaked at 18 hr except in mice iv immunized with the lowest doses (10(5) and 10(6)) and elicited at Day 4. The protracted DTH reaction was shown to be associated with an histological picture of tuberculin-type reaction. A part of the 18-hr DTH reaction is mediated by serum in mice inoculated with large doses of SRBC; nevertheless, numeration by limiting dilution analysis of circulating DTH cells showed that the frequency of these cells correlates with the 18-hr DTH level. The protracted DTH shown at 42 and 48 hr, 4 days after immunization with 10(5) and 10(6) SRBC, could not be transferred in naive recipients with immune spleen cells; it was independent of the antigen life span and did not result from immunization modulation at the bone marrow level on recruitable cells.     

Role of mediators of cellular hypersensitivity in the stimulation of the antibody formation to unrelated antigen

The effect of a concurrent delayed hypersensitivity reaction on the antibody response to sheep red cells was assessed by a plaque assay. Guinea pigs with delayed hypersensitivity to tuberculin purified protein derivative (PPD) or egg albumin showed an increased antibody response to sheep red cells when the cells were injected intravenously at the same time as PPD or egg albumin. This effect was transferred to normal guinea pigs by serum from guinea pigs with delayed hypersensitivity to PPD or egg albumin taken 24 hr after injecting the corresponding antigen. Supernatants containing migratory inhibitory factor were prepared by incubating lymphocytes from sensitized rabbits with antigen. These supernatants were injected with sheep red cells and gave rise to an enhanced plaque response. Similar results were obtained with supernatants from normal rabbit thymus cells. The role of mediators of delayed hypersensitivity in enhancing antibody formation and in T cell/B cell cooperation is discussed.     

ELECTROKINETIC PHENOMENA. III : THE "ISOELECTRIC POINT" OF NORMAL AND SENSITIZED MAMMALIAN ERYTHROCYTES

A survey of the published electrophoretic mobilities of certain mammalian red cells reveals that the isoelectric points accorded to these cells are the result of equilibria incidental to red cell destruction. The electrophoretic mobilities of normal washed sheep and human cells have now been studied in 0.85 per cent NaCl solutions from about pH 3.6 to 7.4. All measurements were made within 2 minutes of the preparation of the suspension of red cells. In no case was reversal of sign of charge observed under these conditions. Reversal of sign of charge occurred only after sufficient time had elapsed to permit sufficient adsorption of the products of red cell destruction. There is little change in mobility as the pH of the medium is decreased. Reversal of sign of charge does occur in the presence of normal and immune (anti-sheep) rabbit sera. The isoelectric point determined under these conditions does not appear to be connected specifically with the immune body but is perhaps associated with phenomena incidental to red cell destruction and the presence of serum. The characteristic lowering of mobility by amboceptor occurs, however, from pH 4.0 to pH 7.4. The curves of mobility plotted against pH for normal and for immune sera support the viewpoint that the identity of the isoelectric points for normal and sensitized sheep cells is not primarily concerned with the immune reaction. It is most unlikely that an "albumin" or a "globulin" surface covers red cells with a complete protein film. Although serum protein reacts with red cells in acid solutions, this is not demonstrable for gelatin. The lowering of mobility usually ascribed to anti-sheep rabbit serum may also occur, but to a lesser degree, in normal rabbit serum. This diminution of mobility is not, in the first place, associated with sensitization to hemolysis induced by complement. This supports the view that only a very small part of the red cell surface need be changed in order to obtain complete hemolysis in the presence of complement.     

血型检测用反向定型试剂的研制

将正常的红细胞在特定条件下用甲醛处理,使红细胞膜固定但不影响膜表面糖蛋白血型抗原的活性。采用与正向定型相同的平板凝集试验方法,４０６０份血样正向和反向定型结果完全一致。经稳定性观察９０天,处理后的红细胞与相应抗体的凝集性能未见明显改变。实验结果表明本文介绍的红细胞试剂可用于ＡＢＯ血型鉴定的反向定型试验。     

Reaction of antibody to normal human hypoxanthine phosphoribosyltransferase with products of mutant genes.

Immunoglobulin produced in rabbits against normal human red cell hypoxanthine phosphoribosyl transferase (HPRT, EC 2.4.2.8) was used to study cell lysates of individuals with deficient enzyme activity. The reaction of immunoglobulin with HPRT formed partially active insoluble and fully active soluble complexes. The insoluble complexes were separated from soluble complexes and the free enzyme by centrifugation. The soluble complexes and free enzyme were separated by electrophoresis. Hemolysates from 13 patients with the Lesch-Nyhan syndrome who have virtually total deficiency of HPRT activity and 2 patients with hyperuricemia and 2–5% of normal activity were unable to neutralize immunoglobulin and showed no evidence of cross-reacting material (CRM). In contrast, 2 other partially deficient males with 4.5 and 50% of normal actvity, and a partially deficient heterozygous female with 34% of normal activity, were CRM⁺ in this assay. The amount of CRM present in the cells of these 2 males appeared to be disproportionate to their HPRT activity. The heterozygous female contained about 30% of normal CRM which was consistent with the estimated activity provided by her normal cell population. This indicated that her abnormal cells were CRM^?. Absence of CRM in her abnormal cells was consistent with the observed lack of CRM in hemolysates of her hyperuricemic half-brother. These data indicate the presence of considerable heterogeneity in human mutation at the HPRT locus.     

南方鲇血型的初步鉴定

本研究旨在通过观察南方鲇血清与其红细胞的交叉反应以鉴定南方鲇的血型.实验结果表明:南方鲇的血清与同种其他个体的红细胞进行交叉反应时均未出现凝集现象,这表明南方鲇可能不存在血型或南方鲇具备血型但血清中相应的凝集素含量不足.以南方鲇的红细胞为抗原免疫日本种大耳白兔制备的抗血清与南方鲇的红细胞进行交叉反应,出现了不同程度的凝集反应,这表明南方鲇存在血型.据上述两个实验结果可以推断,南方鲇可能存在4种血型,分别命名为NA、NB、NAB和NO型;同时也证实,在鉴定南方鲇血型的研究中,通过制备抗血清与红细胞进行交叉反应的方法更为可靠.     

Autologous immune complex nephritis associated with sickle cell trait: diagnosis of the haemoglobinopathy after renal structural and immunological studies.

A renal tubular epithelial antigen (RTE)--anti-RTE autologous immune complex nephritis associated with sickle cell anaemia (SS) has been reported, but immune complex nephritis has never been described in patients with sickle cell trait (SA). During investigation of a child with "asymptomatic proteinuria" cryoprecipitable complexes of RTE-anti-RTE were detected in the serum and granular deposits of RTE, immunoglobulins, and complement localised on the glomerular basement membranes. Morphological and ultrastructural studies showed increased mesangial matrix, sickled red blood cells in the glomeruli and vessels, and tubular and interstitial abnormalities. These findings prompted haemoglobin electrophoretic studies, which showed previously undiagnosed haemoglobin SA in this patient and her family. These observations suggest that nephritis mediated by similar immunopathogenic mechanisms may be associated with SS and SA haemoglobinopathy. Under some conditions patients with sickle cell trait may experience haemodynamic and oxygenation abnormalities, which may be aetiological factors in the immune complex nephritis associated with SS disease.     

Selective binding of biotinylated albumin to the lymphoid microvasculature

Chemically modified albumin binds to the surface of microvascular endothelia lining the vessel wall in several tissues. In this paper, we report that following their biotinylation, ovalbumin (bioOVA) and bovine serum albumin (BSA) [biotinyated albumin (bioAlb)] showed heterogeneous binding to distinct vascular subsets in different lymphoid tissues. The binding of bioAlb could be demonstrated both by fluorescent and enzymohistochemical techniques. In the spleen, the reaction was restricted to the red pulp sinuses whereas the white pulp vessels (including the central arteriole) and the marginal sinus were negative for bioAlb binding. In lymph nodes, the strongest labeling was observed in the medullary sinuses. In the thymus, the most prominent labeling of capillaries was restricted to the corticomedullary area where it was found to be less intense compared with the splenic reaction. The splenic reactivity of bioAlb in the mouse was defined using antibodies against endothelial cell subsets in distinct vascular beds in the red pulp and marginal zone, respectively. The bioAlb-binding elements of the splenic red pulp sinus architecture corresponded to the display of hyaluronan receptor stabilin-2 and subset-specific marker IBL-9/2 while they differed from the expression pattern of both the complementary red pulp sinus subset and the marginal sinus-lining cells expressing MAdCAM-1 antigen, respectively. Similar red pulp sinus-restricted reactivity could be demonstrated in the human, rat, and guinea pig. The use of bioAlb may thus offer a reliable probe for the histological identification of select microvascular endothelia in lymphoid tissues.     

Adoption of an additional infant by a Western lowland gorilla (Gorilla gorilla gorilla).

A 10-year-old Western lowland gorilla, already caring for her own 14-month-old son, adopted a female neonate. The infant's mother (aged 7 years, 4 months) showed no interest in the infant, and it is unclear whether she abandoned the infant or whether it was seized by the dominant foster-mother. The foster-mother gave more maternal attention to the adoptee than to her own son but gave both infants the same protection. She adjusted her forms of transport to the age of each infant. The subadult mother of the neonate did not seek contact with her offspring during the first 4 weeks and in fact showed more interest in the 14-month-old male infant. Interactions between the two mothers were rare. The foster-mother's own male infant died 2 months after she had adopted the female infant. She looked after the adopted infant for 1 year, but then lost interest so that the adoptee had to be separated.     

Pinocytotic response of circulating erythrocytes to specific blood grouping antibodies

Human blood samples from adults and newborns of blood groups O, A, and B were treated with either anti-A blood grouping serum, ferritin-conjugated anti-A serum, free ferritin, or saline and then prepared for electron microscopy. Morphological differences were observed between the untreated erythrocytes of infants and adults. Circulating red cells of newborns were frequently vesiculated (25.5%), whereas those of adults only occasionally showed vesicles (5.5%). On the basis of morphology and incidence, the majority of these vesiculated cells seemed to be mature erythrocytes. The introduction of anti-A serum to group A erythrocytes of infants appeared to stimulate vesicle formation, but anti-A serum did not have a similar effect on group O or B cells of infants or on group A cells of adults. Vesicles which formed in response to antiserum treatment appeared to be the result of pinocytosis. In contrast to the well dispersed ferritin along the membrane of agglutinated adult cells, the ferritin particles on the infants' cells were frequently clustered at irregular intervals. These accumulations seemed to lead to invaginations of the cell membrane, resulting in ferritin-lined intracytoplasmic vesicles. The addition of free ferritin or ferritin-conjugated antibodies of the wrong specificity to red cells did not increase vesicle formation.