Antigen-binding cells in the peripheral blood of sockeye salmon, Oncorhynchus nerka Walbaum, induced by immersion or intraperitoneal injectijon of Vibro anguillarum bacterin

We used an immunocytoadherence assay to monitor the response of antigen-binding cells (ABC) in the peripheral blood of sockeye salmon, Oncorhynchus nerka , after immersion in, or intraperitoneal injection of, Vibrio anguillarum LS 1–74 bacterin. Both methods initiated an elevated ABC response in less than one day; this response persisted one week longer in the injected than in the immersed fish.     

Toxoplasma gondii: a new diagnostic approach based on the specific binding of erythrocytes to lymphoid cells.

Red blood cells coated with toxoplasma antigen were bound to surface receptors, possibly of Ig nature, on lymphoid cells which appeared in the spleen of albino rats a few days after intraperitoneal injection of living T. gondii of the RH strain. Antigen-binding cells appeared before specific antibody, then declined in number, while the antibody response was progressing.Available information stresses the identity of antigen-binding cells with precursors of antibody-forming cells, thus pointing to the early occurrence of the immunocytoadherence phenomenon in the immune response. The applicability of these concepts to the field of human toxoplasmosis was proved by the demonstration of circulating lymphocytes specifically binding toxoplasma antigen early in the course of human toxoplasmic lymphadenitis and only in relation to active disease, thus providing an interesting approach to the early diagnosis and the detection of the active state of the disease.Possible clinical implications are discussed.     

Immuno-cyto-adhesion and sublethal irradiation]

1. In sublethally irradiated CBA mice, the relative and absolute numbers of spontaneous rosetts forming cells against sheep erythrocytes are markedly decreased in bone marrow. 2. The decrease of the absolute number of spontaneous RFC is also important in the spleen in spite of an increase of the RFC relative number above the normal values between the 8th and 12th day after irradiation. 3. The graft of normal bone marrow cells immediately after irradiation of the shielding of a medullary area during irradiation promotes the recovery of the immunocytoadherence capacity of the bone marrow cells but not of the spleen cells.     

The effect of captivity on the immune response of the killifish, Fundulus heteroclitus L.

Fundulus heteroclitus maintained under laboratory for more than four weeks showed a significantly lower immunocytoadherence (rosette) response to thymus-dependent antigens (SRBC and DNP-BSA) than newly captured fish. There was no comparable effect on scale allograft rejection nor in the response to a thymus-independent antigen (DNP-Ficoll) as measured by the rosette response. This suggests that captivity has a differential effect on a sub-population of lymphocytes analogous to the T-helper cells of mammals.     

Immunocyte adherence and leukocyte migration inhibition tests in dogs infested with Leishmania donovani. Importance of lymphocyte conservation in liquid nitrogen]

A method of canine lymphocytes isolation and storage in liquid azote with DMSO is described. It was possible to use these lymphocytes in the immunocytoadherence and leucocytes migration inhibition tests in dogs infected with Leishmania donovani. The sensitisation was made with a soluble antigen from promastigotes of L. donovani braid by ultrasounds. The dogs with Leishmaniasis gave positive results. The controls without Leishmaniasis were negative. The tests with conserved lymphocytes were as satisfactory as the same tests with fresh lymphocytes.     

Specific antigen-binding and antibody-secreting lymphocytes associated with the erythrocyte autoantibody responses of NZB and genetically unrelated mice.

The receptor characteristics as well as incidence of antigen-binding lymphocytes (ABL) or B and T cell classes with membrane receptors specific for the exposed (X) and cryptic (HB) murine erythrocyte autoantigens were examined in NZB and nine control strains of mice. Whereas only NZB and NZB hybrid mice synthesize anti-X autoantibody pathogenetically implicated in the genetically determined autoimmune hemolytic anemia, the NZB as well as control strains synthesize the ubiquitous anti-HB anti-erythrocyte autoantibody. By utilizing immunocytoadherence assays, maximum numbers of specific ABL of both B and T lymphocyte classes were optimally demonstrated at erythrocyte:lymphocyte ratios of 20:1 and after lymphocyte fixation at 56 degrees C for 20 min. Surface membrane receptor specificity was established by inhibition with semi-purified soluble X or HB autoantigen. Inhibition of immunocyto-adherence with class specific antisera to mouse immuno-globulins demonstrated that the receptors on both B and T cells were of IgM class. Specific receptors regenerated in vitro after trypsinization which excluded the role of cytophilic antibody in the immunocytoadherence reactions. B lymphocyte ABL reactive with the X autoantigen were demonstrable in NZB, NZB hybrid, and control mice. Only in NZB and NZB hybrid mice, strains that uniformly synthesize anti-X autoantibody, were X ABL of T lymphocyte class demonstrated. The presence and incidence of T lymphocyte X ABL is compatible with the expression of a single dominant gene carried by the NAB strain. The incidence of B lymphocyte X ABL increased with age, suggesting proliferation of this cell population. HB ABL of both B and T lymphocyte classes were observed in all strains, concordant with the ubiquitous presence of humoral anti-HB autoantibodies. Differentiation of precursor B cells are evaluated by PFC assay of cells secreting specific autoantibodies. Anti-X PFC were observed only in NZB and NZB hybrid mice; and the observed frequency suggested that less than 3.5% of the specific ABL were differentiated for the secretion of anti-X autoantibody. Anti-HB PFC were observed in all strains and represented as high as 11.8% of specific ABL. Genetic determination of the anti-X anti-erythrocyte autoantibody response does not prescribe the presence of precursors of the antibody-forming cell, but rather appears to influence regulation of the differentiation of these cells. These data suggest that circumvention of immunologic tolerance to this specific erythrocyte autoantigen may occur at the level of the T lymphocyte; or alternatively, that T lymphocytes as well as B lymphocytes, are induced to proliferate and differentiate in the NZB strain.     

蟾蜍红细胞融合条件的正交设计优化

目的：用聚乙二醇法探讨蟾蜍红细胞融合的最佳条件。方法：采用3因素3水平的正交实验法,以蟾蜍红细胞为材料,从聚乙二醇浓度、反应时间、反应温度三方面计算细胞融合率,探讨和比较蟾蜍红细胞融合的最适条件及影响因素。结果：蟾蜍红细胞融合的最佳条件是：选用50%的聚乙二醇,反应温度为37℃,反应时间为15min。     

A fast and highly sensitive method for detecting freemartinism in bovine twins using immunomagnetic beads and Y-specific PCR primers

A method for detecting male cells in the blood of the female calf in bovine heterosexual twin pregnancies has been established. Nucleated cells were isolated from full blood by immunomagnetic separation, lysed by boiling and then subjected to polymerase chain reaction (PCR) amplification with Y chromosome specific primers. Diagnosis was achieved within one day. The method was successfully used on blood samples that had been stored at +4°C for more than one month. Dilution of male blood in female blood showed that XY cells were detectable down to a concentration of 0.1%. This method should be amenable to automatization and can be adapted to any PCR-based genetic test.     

Simplified method for isolation of white cells from whole blood suitable for direct polymerase chain reaction

A simple method for isolating mononuclear cells from whole blood is described. The procedure utilizes phytohemagglutinin to agglutinate the erythrocytes, separating white cells from whole blood in a very brief handling time. The isolated cells are readily subjected to DNA isolation simply by boiling, and the released DNA can be directly employed for the polymerase chain reaction analysis. The efficiency of this method is similar to other conventional methods, but less costly and less time-consuming. This method is particularly useful in analyzing DNA samples from the peripheral blood cells when the simplicity and low cost of the assay are preferable.     

Immunoglobulin and enzyme-conjugated dextran polymers enhance u-PAR staining intensity of carcinoma cells in peripheral blood smears.

The presence of disseminated carcinoma cells in bone marrow and peripheral blood has prognostic importance in patients with carcinomas. Much evidence indicates that dissemination of tumor cells may depend on activation of a variety of degradative enzymes. A strong positive correlation has been shown between the expression of tumor cell proteases and tumor invasion. Therefore, phenotypic characterization of disseminated carcinoma cells for expression of protease activators might define the invasive potential of the cells. We present an immunocytochemically enhanced staining method that allows phenotyping of disseminated carcinoma cells in bone marrow and peripheral blood smears. In the first step, the cells were incubated with antibodies against urokinase plasminogen activator receptor (u-PAR) and subsequently with secondary antibodies conjugated to peroxidase-labeled dextran polymers. A brown color reaction was developed with diaminobenzidine as chromogen. In the second step, the cells were incubated with alkaline phosphatase-conjugated murine monoclonal antibodies against a common cytokeratin epitope and a red color reaction was developed with new fuchsin as substrate. This method allows simultaneous and unambiguous immunolabeling of intracellular cytokeratin and of u-PAR intracellularly and on the surface of carcinoma cells. This novel approach can be used for detection and phenotyping of carcinoma cells in blood smears for u-PAR or, presumably, for any other heterogeneously expressed antigen on the surface of the detected cells.     

Serotonin and neurotensin-containing cells in the mucosa of the alimentary tract

Localization of serotonin (5-OT)- and neurotensin (NT)-containing cells in the tunica mucosa of the gastric antral part and the duodenum of the rat and chick has been revealed by means of the immunofluorescent method and the method with application of the peroxidase-antiperoxidase complex. For immune-histochemical revealing of biologically active substances in endocrine cells Sovient antisera are used for the first time. Experiments are performed on injection of antiserotonin serum of various dosage into the rat blood bed. The reaction of serotonin-containing cells of the gastric antral part tunical mucosa and those of the duodenum to changes in serotonin concentration in blood serum is demonstrated.     

A modified immunohistochemical procedure for the detection of estrogen receptor in mouse tissues

A horseradish peroxidase (HRP) labeled antibody method was developed for use with a monoclonal antibody to detect estrogen receptor (ER) in mouse tissue. Combined use of HRP labeled F(ab')2 fragment absorbed with mouse liver protein to minimize background staining and imidazol-DAB reaction gave the most reliable and sensitive immunostaining. The method was applied to uterine, vaginal, pituitary and liver tissues in ovariectomized adult mice. In uterus and vagina, ER was recognized in nuclei of epithelial cells, stromal cells and smooth muscle cells of the muscle layer and blood vessels. Liver tissue showed positive nuclear immunostaining in parenchymal cells; however, no reaction was present in endothelial cells, Kupffer cells, bile ductal cells, and smooth muscle cells of blood vessels. ER was localized in the nuclei of anterior pituitary cells while weak reaction was also recognized in cells of the intermediate lobe. No staining was detected in the posterior pituitary. Results demonstrate that both occupied and unoccupied ER are localized in the cell nucleus from several target tissues. Weak immunostaining in samples could not be enhanced by multiple procedures. It is suggested that nuclear ER is partially hidden by nuclear components such as nucleic acid and chromatin proteins.     

Application on Timm sulphide silver method for electron microscope localization of lead ions in blood cells.

The application of the Timm sulphide silver method for the demonstration of lead ions in peripheral blood cells is investigated, using male white adult Wistar rats treated with a single dose of lead acetate, range 150 mg/kg b.w. To improve the Timm reaction for electron microscopy, fixation of whole cells with glutaraldehyde fixative solution saturated with H2S, an agarose embedding and physical development of thick sections without prior cryostat sectioning are presented. At 6.0 hours after injection both erythrocytes as well as white cells reveal the positive Timm reaction. All types of blood cells contain numerous cytoplasmic precipitates illustrating the intracellular lead accumulation. It is shown that the invaginations of white cell nuclear membrane possess a storing function as areas of lead depots. As a rule, the neutrophils display a highest amount of cytoplasmic precipitates and exclusively a low amount of reaction products in basophils is observed. At 14 days after injection, precipitates are present only in erythrocytes and monocytes. A suggestion of a possible functional significance of changes in the Timm staining pattern in blood cell types is discussed in this paper.     

An immunoinhibitory substance in the serum of laboratory held killifish, Fundulus heteroclitus L.

Serum of the killifish, Fundulus heteroclitus , maintained under laboratory conditions for more than four weeks, contained a factor that depressed the immunocytoadherence response to SRBC. When serum was injected into immunocompetent fish 48 h prior to antigen stimulation it was effective; when injected simultaneously with the antigen there was no effect. Fractionation of the serum by ultracentrifugation suggests that the inhibitor is either low or very low density lipoprotein. Hypophysectomy decreased immune inhibition suggesting that the production of the inhibitor may be under the control of the pituitary.     

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.     

A Simple and Efficient Method for DNA Purification from Samples of Highly Clotted Blood

Rapid purification of DNA from samples of highly clotted blood is a challenging problem due to the difficulty in recovering and dispersing blood clots. We developed a new method for discarding the serum-separator gel and rapidly dispersing the blood clots. A special disposable tip was inserted into the serum-separator gel so that the serum-separator gel could be discarded. The blood clot obtained was dispersed into small pieces through a copper mesh (pore size, 250 μm) in a special dispersing instrument by centrifugation. After lysis of red blood cells and white blood cells, genomic DNA was concentrated and desalted by isopropanol precipitation. The mean yield of DNA purified from a 0.3-ml blood clot was 22.70 μg in 173 samples of clotted blood cryopreserved for 1 month, and 19.02 μg in 1,372 samples of clotted blood cryopreserved for >6 months. DNA samples were successfully performed through polymerase chain reaction, real time polymerase chain reaction, and melt curve analysis. Their quality was comparable with that purified directly from EDTA-anticoagulated blood. The new method overcomes the difficulties in recovering and dispersing blood clots, allowing efficient purification of DNA from samples of highly clotted blood.     

Absorption of elastase through the jejunal mucosa of the rat. An immunocytochemical study

In order to reveal the absorption process of elastase from the intestine, hog pancreatic elastase was injected into the ligated jejunum lumen of the rat, and the tissues were cytochemically observed at various times after injection. The peroxidase anti-peroxidase (PAP) method using anti-hog-elastase rabbit antibody was used for light microscopy, and the anti-elastase Fab'-peroxidase conjugate was used for electron microscopy. The tissues stained by the PAP method exhibited a dense deposition of reaction products on the luminal surface of epithelial cells and a moderate deposition in the blood and lymph capillaries of the intestinal villi. Immunoelectron microscopy revealed that the reaction product was deposited on the surface of the microvilli and in their pocketing; some was found in the pinocytotic vesicles in the terminal-web area and on the inner surface of the enlarged smooth endoplasmic reticulum. Round droplets which gave a positive reaction were found in the widened intercellular cleft and the thick basement membrane lining the blood capillaries and lymphatics. The jejunum retained its normal ultrastructure. The results indicate that the elastase molecules, which were introduced into the rat jejunum lumen, were absorbed without being decomposed through healthy intestinal epithelial cells by pinocytosis and translocated into blood and lymph capillaries.     

A modified immunohistochemical procedure for the detection of estrogen receptor in mouse tissues

Summary A horseradish peroxidase (HRP) labeled antibody method was developed for use with a monoclonal antibody to detect estrogen receptor (ER) in mouse tissue. Combined use of HRP labeled F(ab)₂ fragment absorbed with mouse liver protein to minimize background staining and imidazol-DAB reaction gave the most reliable and sensitive immunostaining.The method was applied to uterine, vaginal, pituitary and liver tissues in ovariectomized adult mice. In uterus and vagina, ER was recognized in nuclei of epithelial cells, stromal cells and smooth muscle cells of the muscle layer and blood vessels. Liver tissue showed positive nuclear immunostaining in parenchymal cells; however, no reaction was present in endothelial cells, Kupffer cells, bile ductal cells, and smooth muscle cells of blood vessels. ER was localized in the nuclei of anterior pituitary cells while weak reaction was also recognized in cells of the intermediate lobe. No staining was detected in the posterior pituitary.Results demonstrate that both occupied and unoccupied ER are localized in the cell nucleus from several target tissues. Weak immunostaining in samples could not be enhanced by multiple procedures. It is suggested that nuclear ER is partially hidden by nuclear components such as nuclei acid and chromatin proteins.