Mechanism of the protective effect of antiserum against isologous aggregated mouse immunoglobulins in transplantation immunity]

The injection of antiserum against isogeneic aggregated mouse immunoglobulins prolonged the survival time of skin allografts in mice by 5-6 days. The study of the possible mechanisms of this phenomenon revealed that the injection of aggregated immunoglobulin antiserum decreased the ability of the lymphocytes of the animals receiving the antiserum to induce local "graft versus host" reaction and to proliferate in the blast transformation test in response to phytohemagglutinin treatment and in the mixed lymphocyte culture test. When added in vitro, the antiserum did not influence either the mixed lymphocyte, or the specific cytotoxic effect of immune lymphocytes, but stimulated 3H-thymidine incorporation into the suspension of lymphocytes incubated with syngeneic irradiated cells. The latter fact may be indicative of the mitogenic action of the antiserum. The analogy between the mitogenic action of the antiserum and that of nonspecific mitogens, such as concanavalin A and phytohemagglutinin which also show opposite effects in vivo and in vitro, is discussed. The conclusion has been made that the increase of the survival time of the skin allograft after the multiple injection of aggregated immunoglobulin antiserum was primarily due to its action on the recognizing and proliferative functions of T lymphocytes, and later due to an increase in IgG synthesis.     

Effect of mouse antiserum against isologous aggregated immunoglobulins on the transplantation and antitumor immunity]

It was shown that mouse antiserum against isologous thermically aggregated immunoglobulins MAAS injected repeatedly to the animals prolonged the survival of the skin allotransplant, induced Moloney's sarcoma diminished the latent period of the tumour development sharply increased the formation of the tumours which in the majority of cases, led to the death of mice, deranged the normal age barrier in fibrosarcoma induction, and sharply inhibited the intensity of Rauscher's leukemia development. This action of MAAS on the transplantation and antitumour immunity was of immunological nature.     

Effect of mouse antiserum against isologous aggregated immunoglobulins on the accumulation of rosette- and antibody-forming cells in mice immunized with ram erythrocytes

The paper describes the effect of mouse antiserum against isologous aggregated immunoglobulins (termed MAAS) on the kinetics of rosette-forming and antibody-forming cells (RFC and AFC, respectively) in mice immunized with SRBC. MAAS effect was assessed in vivo by injecting this serum for 5 days to mice CBA, combining the first injection with the injection of 5.10(7) SRBC. MAAS administration to mice immunized with SRBC induced a marked reduction of RFC in the spleen on the 5th and 9th days after the immunization. At the same periods MAAS produced no significant effect on the proliferation of AFC producing IgM-hemagglutinins. At the same time MAAS intensified the IgG-AFC proliferation in the period of the maximal content of these cells in the spleen of the immunized mice. After the MAAS absorption with the immune complexes formed by the mouse IgG-antibodies this serum largely lost its capacity to block RFC in vivo. On the basis of the data obtained it is suggested that the property of MAAS to influence the accumulation of RFC and AFC producing IgG-hemagglutinins is caused by the factor reacting with the immune complex formed by mouse IgG-antibodies. Possibly this factor represented antibodies against the aggregated immunoglobulins of this class.     

Effect of antiserum against isologous aggregated immunoglobulins on the delayed-type hypersensitivity in mice immunized with sheep red blood cells

The mouse antiserum against isologous aggregated immunoglobulins (MAAS) injected to mice sensitized with 10(5) sheep red blood cells (SRBC) did not influence the delayed-type hypersensitivity (DTH) tested on the peak of sensitization (the 4th day) but enhanced significantly DTH tested on the 6th day. MAAS completely abolished the DTH suppression observed after sensitization with 5 x 10(7) SRBC. In transfer experiments the number of the DTH suppressor cells decreased in the spleen of sensitized mice under the MAAS action. MAAS did not affect the proliferation of antibody-forming cells (AFC) and hemagglutinin production but reduced by 70% the number of rosette-forming cells (RFC) in the spleen on the peak of the initial immune response. The data obtained may indicate that RFC participate in DTH suppression.     

Effect of sera against aggregated mouse immunoglobulins on a population of lymphoid and hematopoietic cells]

The in vitro treatment of the mouse spleen cells immunized by the ram erythrocytes with the rabbit and mouse sera against the thermoaggregated mouse immunoglobulins resulted in the inhibition of antigen binding receptors of rosette forming cells. The mouse serum, unlike the rabbit one, induced the inactivation of receptors in rosette forming lymphocytes both in the non-immune and immune mice on the 8th day after the antigenic stimulation. The treatment of bone marrow cells from the intact mice with these sera increased insignificantly the number of hemopoietic colonies in the spleens of lethally irradiated syngenic recipients and stimulated markedly the migration of spleen cells. This may be due both to the direct effect of these sera and to their mediated (through the humoral factor) influence. The inactivation of antigen binding receptors in the spleen rosette forming cells suggests the presence of immunoglobulins on the membrane of B-lymphocytes in the aggregated state or in the form of antigen--antibody complexes.     

Description of the effect of polyclonal mitogens on a population of splenic lymphocytes in the presence of serum against isologous aggregated mouse immunoglobulins

Antiserum against isologous aggregated mouse immunoglobulins was shown to be mitogenic for spleen cells during the first 48 hours of culture. The mitogenic effect of the serum factor is mediated through T lymphocytes and may be macrophage-dependent. Whe incubated with spleen cells the serum was demonstrated to inhibit the DNA synthesis in B cells in response to sulfate dextran and lipopolysaccharide. Experiments with B cells and the thymidine "suicide" test suggest that the target cells for the serum factor may reside in the population of radiosensitive and highly proliferating B lymphocytes. The degree of suppressing by the antiserum factor depends on the differentiation stage and the presence of antigen-binding receptors on the membrane of B cells.     

Thymic hormones and prostaglandins II. Synergistic effect on mouse spontaneous rosette forming cells

Prostaglandins (PGs) have been assummed to play a role in the biological activity of thymic hormones (TH). Indeed, it has been shown that type E-PGs are able to mimic the action of several TH. Moreover, indomethacin interferes in the rosette assay, which still represents the most commonly used bioassay for the evaluation of TH and, in particular thymulin levels, in biological fluids. Previously, our attempt to modulate PG production by different TH showed that none of the TH tested affect PGE2, 6-keto-PGF1α, PGF2α and TXB2 production by spleen cells from control and thymectomized (Tx) mice, while indomethacin was able to inhibit the spontaneous PG production. Here, we investigated a possible role for each endogenously produced PG in the experimental conditions of the rosette assay, in order to define : 1) whether or not there was a specificity of action of a given PG ; and 2) to analyze the pattern of action between thymulin and the endogenously produced PGs. We demostrated that PE2 and 6-keto-PGF1α are the PGs which are physiologically involved in the rosette assay, according to their levels of endogeneous production, and that they are able to synergize with thymulin. This synergy was demonstrated in two ways: 1) by adding anti-PGE2 and anti-6-keto PGF1α-antibodies, which prevent part of the thymulin effect, or 2) by simultaneous addition of PG and thymulin, at concentrations far lower than those which correspond to their thymulin-like effect. Moreover, PGE2 addition, at concentration close to that found to be endogenously produced, partially reversed the indomethacin-induced effect in the rosette assay. In conclusion, if PGs do not act as mediators of thymulin, they are able to synergize in one of its biological action.     

Thymic hormones and prostaglandins. II. Synergistic effect on mouse spontaneous rosette forming cells

Prostaglandins (PGs) have been assumed to play a role in the biological activity of thymic hormones (TH). Indeed, it has been shown that type E-PGs are able to mimic the action of several TH. Moreover, indomethacin interferes in the rosette assay, which still represents the most commonly used bioassay for the evaluation of TH and, in particular thymulin levels, in biological fluids. Previously, our attempt to modulate PG production by different TH showed that none of the TH tested affect PGE2, 6-keto-PGF1 alpha, PGF2 alpha and TXB2 production by spleen cells from control and thymectomized (Tx) mice, while indomethacin was able to inhibit the spontaneous PG production. Here, we investigated a possible role for each endogenously produced PG in the experimental conditions of the rosette assay, in order to define: 1) whether or not there was a specificity of action of a given PG; and 2) to analyze the pattern of action between thymulin and the endogenously produced PGs. We demonstrated that PGE2 and 6-keto-PGF1 alpha are the PGs which are physiologically involved in the rosette assay, according to their levels of endogenous production, and that they are able to synergize with thymulin. This synergy was demonstrated in two ways: 1) by adding anti-PGE2 and anti-6-keto PGF1 alpha-antibodies, which prevent part of the thymulin effect, or 2) by simultaneous addition of PG and thymulin, at concentrations far lower than those which correspond to their thymulin-like effect. Moreover, PGE2 addition, at concentration close to that found to be endogenously produced, partially reversed the indomethacin-induced effect in the rosette assay. In conclusion, if PGs do not act as mediators of thymulin, they are able to synergize in one of its biological action.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.     

Suppression of the formation of cells secreting antibodies and antigen-dependent nonspecific immunoglobulins in mice receiving isologous antierythrocyte immunoglobulins

Mice injected with syngeneic cellulose-conjugated immunoglobulins (Ig) containing antibodies to sheep red blood cells (SRBC) develop a specific non-responsiveness to SRBC. Such animals demonstrate a sharp decrease not only in the formation of anti-SRBC antibody producers but also of the cells secreting antigen-dependent nonspecific Ig. The inhibition of both these processes is antigen-specific. It is suggested that inhibition of the cells forming antigen-dependent nonspecific Ig is due to suppression of either hypothetic inductors or precursors of these cells expressing an idiotype spectrum similar to that of anti-SRBC antibody producers.     

The effect of cholesterol oleate treatment of mice on the rosette forming cell response against sheep erythrocytes.

Mice injected with a single dose of 60 mg cholesterol oleate emulsion showed substantial blockade of the monoclear phagocyte system measured by the rate of vascular clearance of radio-labelled sheep erythrocytes. The labelled rythrocytes, in lipid treated mice, localized mainly in the spleen, contrasting with control mice in which localization was mainly in the liver. Treatment with this lipid, 24 hr before the intravenous of two different doses of sheep erythrocytes, resulted in significant depression of the rosette forming cell response in the spleen, whereas the responses in the lymph nodes of both control and lipid treated mice were at a low level and not significantly different. Intravenously administered cholesterol oleate emulsion is known to localize mainly in the Kupffer cells and in splenic red pulp macrophages. Cultured macrophages treated with this lipid show inhibition of antigen-binding and depressed phagocytosis of heterologous erythrocytes. The lipid does not affect lymphocytes. These findings are in keeping with the hypothesis that macrophages play a direct role in the induction of an immune response against a particulate antigen.     

抗血管紧张素原多克隆抗血清的制备及鉴定

为深入研究血管紧张素原（angiotensinogen,AGT)的表达和组织分布特征,我们以原核表达的AGT蛋白C末端片段为免疫原免疫家兔,制备了针对AGT的多克隆抗血清,并对其进行了ELISA、Western印迹分析及免疫组化鉴定。结果发现,经多次免疫后获得的多克隆抗血清不仅效价高（1：25600）,而且能特异性地针对组织内源性及原核表达的AGT蛋白,同时在人和大鼠组织AGT之间会发生交叉反应。以此抗血清进行免疫组化染色,观察到人胰腺的胰岛细胞及杆内胆管上皮细胞胞浆均有AGT蛋白表达。以上结果提示,利用大鼠杆菌融合表达的AGT蛋白可有效刺激家兔产生AGT抗体。本工作为进一步研究AGT乃至局部RAS的生理、病理意义提供了重要的实验工具。     

镉诱导黄瓜金属硫蛋白抗体的制备及纯化

用镉诱导黄瓜叶片组织产生金属硫蛋白（MT）,经分离纯化,血清白蛋白偶联后作为抗原。采用背部皮内、皮下和耳缘静脉注射免疫家兔,制备出效价为1：16的免疫血清。用饱和硫酸铵盐析法和Sephadex G-200柱层析分离纯化IgG。经测定纯化后的IgG蛋白含量为0．9364mg·ml^-1。用纯化的IgG建立黄瓜Cd-MT的斑点免疫试验检测方法可检测黄瓜Cd-MT的最低含量为200Pg·ml^-1。     

兔抗人NESTIN抗血清的制备与鉴定（简报）

Nestin belongs to the class VI intermediate filament family and it is a marker for neural progenitor cells. In this work, the 3'-terminal coding sequence(396 bp) of human nestin gene was cloned into pGEX-3X plasmid and introduced into BL21 E. coli cells. The GST-nestin protein was purified with an affinity column. Anti-human nestin antiserum was raised by immunizing a rabbit with the fusion protein. The high specificity of the antibody against human nestin was confirmed by western-blot and immunocytochemistry analysis.