不同动物红细胞制备实验教学用溶血素的方法探讨

目的检测绵羊红细胞(SRBC)和猪红细胞(PRBC)分别经2种程序免疫家兔所制得溶血素的效价,并比较几种溶血素在医学免疫学实验中的应用效果,以寻求制备符合免疫学实验及教学需求的高效价溶血素的较好方法。方法 40只雄性家兔分为4组,分别用SRBC和PRBC,间隔2d经不同程序免疫家兔制备溶血素,再用补体溶血试验检测几种溶血素的效价,比较其在免疫学实验教学中的应用,并探讨高效价溶血素的制备方法。结果经补体溶血试验测得几种溶血素效价,A组兔抗-SRBC血清效价为1∶4 800;B组兔抗-PRBC血清效价为1∶1 200;C组兔抗-SRBC血清效价为1∶1 000,D组兔抗-PRBC血清效价为1∶200。结论用SRBC制备的溶血素效价均高于相同方法下用PRBC制备的溶血素;红细胞抗原相同时,采取先皮内全血注射后耳缘静脉接种的免疫方法制备的溶血素效价也要高于单纯采取耳缘静脉注射的免疫方法制备的溶血素;且PRBC可代替SRBC免疫家兔,制备满足实验教学要求的溶血素。     

O型人血清中交叉反应性抗体的初步研究

对Ｏ型人血清中ＩｇＭ和ＩｇＧ交叉反应性抗体的性质作了初步研究。实验证明：Ｏ型人血清经Ａ型细胞和Ｂ型细胞吸收后，血清中除抗Ａ和抗Ｂ被完全吸收外，抗Ｂ和抗Ａ效价也下降１～２个稀释度。Ｏ型人血清中除有抗Ａ和抗Ｂ外，还有一种不可分离的抗Ａ，Ｂ。交叉反应性ＩｇＭ抗Ａ，Ｂ的凝集强度一般效价在２～８。Ｏ型血清经２－ｍｅ处理后，ＩｇＧ抗Ａ，抗Ｂ效价在６４～２５６之间，经Ａ型细胞和Ｂ型细胞吸收后，吸收液中抗Ｂ和抗Ａ效价也分别下降。证明交叉反应性抗Ａ，ＢＩｇＭ及ＩｇＧ二种免疫球蛋白都有，但ＩｇＧ抗Ａ，Ｂ的放散能力比ＩｇＭ抗Ａ，Ｂ明显减低，其机制尚待进一步研究。还证明交叉反应性抗Ａ，Ｂ都能被血型物质中和。     

The significance of erythrocyte antigen site density: II. Hemolysis

The importance of antigen site density has been studied by means of a model passive hemolysis system using red cells coupled with sulfanilic acid groups. Relative site numbers were estimated from the covalent linkage of sulfanilic acid-(35)S to red cell membrane protein, and the effective antigen site number was determined with (125)I-labeled rabbit IgG anti-sulfanilic acid (anti-S).Immune hemolysis was demonstrated for red cells which had greater than a threshold number of antigen sites, the value of which was different for normal human cells (80,000 sites/cell), cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH) (40,000 sites/cell), and sheep red blood cells (RBC) (15,000 sites/cell). Cells with antigen site densities below these values did not hemolyze when tested with 1 mg/ml purified rabbit IgM anti-S. 2-8 times greater antigen site densities were required to obtain hemolysis with IgG anti-S. Above the threshold value, hemolysis titers were proportional to the antigen site number until maximal values were obtained. The greater hemolytic efficiency of IgM antibody was demonstrated in this system, and it was established that the magnitude of the difference was related to the test cell antigen site density.These data, taken with previously reported hemagglutination studies, have been used to develop a general classification of immune hemolysis and hemagglutination based on antigen site density and antibody class. It is suggested that the heterogeneity of blood group systems is caused by differences in the site separation of erythrocyte membrane antigens.     

Studies on hemagglutination and hemolysis by escherichia coli antisera

A study on hemagglutination and hemolysis by Escherichia coli O111 and O55 (rabbit) antisera and on hemagglutination and hemolysis inhibition by E. coli O111 and O55 antigens revealed the following facts. 1. Red blood cells of man, dog, rabbit, guinea pig, sheep, rat, and chicken adsorb E. coli O111 and O55 antigens and thus become specifically agglutinable by the homologous E. coli antisera. 2. The adsorption of these E. coli antigens is a function of the concentration of the antigen, the time (from 5 minutes to 2 hours) of treatment of the red blood cells with the antigen, and the concentration of the red blood cells used. 3. Red blood cells of man and sheep adsorb simultaneously both antigens, as indicated by the fact that both antisera give agglutination of all red blood cells. Complete agglutination does not occur when a mixture of red blood cells treated separately with the two antigens is added to one or the other of the two antisera. 4. Treatment of red blood cells of man with one of the antigens does not block the adsorption of the second antigen. Human cells treated with either or both antigens are still agglutinated by the homologous blood group (A, B, and Rh)-specific antibodies. 5. In the presence of guinea pig complement, E. coli O111 and O55 antisera produce hemolysis of modified human red blood cells in titers of the same order of magnitude as those giving hemagglutination and bacterial agglutination. The same antisera produce hemolysis of sheep cells treated with the identical antigens in titers exceeding by far those giving agglutination of modified human or sheep red blood cells. 6. Both sediment and supernate of a boiled E. coli suspension are capable of modifying red blood cells for E. coli hemagglutination; in contrast, the supernate obtained from an unboiled suspension and then heated does not modify red blood cells for hemagglutination, although it contains the antigen which can specifically adsorb E. coli antibodies, as shown by means of the hemagglutination and hemolysis inhibition tests. 7. Both the unheated and the boiled suspensions of E. coli O111 and O55 inhibit hemagglutination and hemolysis specifically. 8. Rabbit red blood cells modified by either E. coli O111 or 055 antigens, upon intravenous injection into rabbits, engender specific E. coli antibodies. The significance of the results is discussed.     

Presence of Forssman-like and Hanganutziu Deicher-like antibodies in Japanese sera

Absorption studies were performed to analyse the properties of heterophile antibodies in Japanese sera. The conventional absorption test using ox erythrocytes and guinea pig kidney tissues classified 28 of 3570 pathologic sera and 12 of 19 IM-like syndrome sera into 2 categories of Forssman type and Hanganutziu Deicher type. No sera contained the antibody of Paul-Bunnell type. Absorptions of the representative sera with cells of various species disclosed the presence of Hanganutziu Deicher-like and Forssman-like antibodies in Japanese IM-like syndrome sera. The activity of both antibodies were removed partly or non-significantly with rat cells. The antibody properties in serum sickness serum between Japanese and a Caucasian were compared. Cells of ox, rabbit, guinea peg and mouse removed the antibody in Japanese sera, however, rabbit cells did not absorb it in a Caucasian case. It could be proposed that various cells other than ox and guinea pig should be employed in the absorption study of the heterophile antibodies.     

The effect of immunotherapy on humoral and cellular responses in ragweed hayfever.

The effect of specific immunotherapy on several in vitro responses to ragweed antigen E has been evaluated in 17 atopic patients with ragweed hayfever. The methods employed were leukocyte histamine release, measurement of specific IgE anti-ragweed antibody and specific IgG anti-ragweed antibody, lymphocyte proliferation, and the production of two lymphocyte mediators (migration inhibitory factor and mitogenic factor). The duration of treatment and symptom improvement were also recorded for comparison. Immunotherapy was associated with a decrease in leukocyte sensitivity for histamine release to ragweed antigen E in a majority of the patients. In addition, there was a significant decrease in IgE anti-ragweed antibody and a significant increase in IgG anti-ragweed antibody. Immunotherapy also resulted in a significant decrease in lymphocyte responsiveness to ragweed antigen E as measured by proliferation and the production of mediators. Symptomatic improvement was best correlated with the presence of IgG anti-ragweed antibody responses. The production of this antibody was also associated with a decrease in lymphocyte responsiveness. The results of this study indicate that specific immunotherapy in ragweed-sensitive patients induces alterations in immunologic reactivity to ragweed antigen in vitro. This response is antigen specific, includes elements of both humoral and cellular immunity, and may account for the clinical improvement that is often observed in patients who undergo this form of therapy.     

A hemolysin associated with the mumps virus

1. A factor capable of causing the hemolysis of the erythrocytes of man, chicken, and sheep occurs in the amniotic and allantoic fluids of chick embryos infected with the virus of mumps. 2. The hemolysin has not been found in normal fluids or in those infected with PR8 or Lee B strains of influenza virus. 3. The hemolysin is definitely inhibited by the serum of man and monkey convalescent from mumps, but only slightly by the serum of the acute phase. 4. The hemolysin is almost completely inactivated at 50 degrees C. after 10 minutes. It exhibits maximal activity at 37 degrees C. and is completely inactive at 4 degrees C. A pH range from about 7.0 to 8.0 allows for maximum activity. 5. Adsorption and elution of the hemolysin with red blood cells has been demonstrated. After elution of the hemolysin, the red blood cells exhibit an increased osmotic fragility. Similar treatment of red cells with influenza virus did not alter this property. 6. The relationship of the hemolysin to the hemagglutinin and the enzyme-like behavior of the former have been discussed.     

Nonspecific Warm Hemolysins of Papain-Treated Cells: Serologic Characterization and Transfusion Risk

Over a 13-year period, 130,000 sera were screened for atypical antibodies with untreated and papain-treated cells. A warm hemolysin of the papain treated screening cells was found in 130 (0.1%) sera. The hemolysin was characterized serologically and its clinical significance evaluated. The hemolysin could be misinterpreted as anti-H or anti-HI if preferential lysis of stored red cells was not noted. The hemolysins reacted most strongly with papainized cells, variably with trypsinized cells, and did not lyse red blood cells treated with bromelin or neuraminidase. The hemolysin appeared in a wide variety of clinical conditions and included normal individuals with no correlation to any specific disease process. Transfusions of stored rather than fresh blood were given to 23 patients with no untoward reaction. The phenomenon is concluded to be an in vitro effect creating technical difficulties, but of no clinical significance.     

Cell-Mediated Immune Response of Ragweed-Sensitive Patients to Ragweed Antigen E: IN VITRO LYMPHOCYTE TRANSFORMATION AND ELABORATION OF LYMPHOCYTE MEDIATORS

The in vivo and in vitro responses to ragweed antigen E were evaluated in 28 untreated atopic patients with ragweed hayfever. The methods employed included direct skin testing, measurement of total serum IgE, measurement of specific IgE anti-ragweed antibodies, leukocyte histamine release, lymphocyte transformation, and release of lymphocyte mediators (migration inhibitory factor and mitogenic factor). The patients could be divided into sensitive and insensitive groups on the basis of their in vitro reactivity to antigen E. 20 patients in the sensitive group had statistically higher levels of total serum IgE, higher levels of specific IgE anti-ragweed antibodies, and greater leukocyte sensitivity as measured by antigen-induced histamine release than did eight patients in the insensitive group. Lymphocytes from sensitive patients produced greater amounts of migration inhibitory factor and mitogenic factor when challenged by antigen E than did lymphocytes from insensitive patients. A possible role for the lymphocyte in this allergic disease is discussed. The results of this study indicate that the immune response to ragweed antigen is complex and involves components of both immediate and delayed hypersensitivity.     

OXYGEN-STABLE HEMOLYSINS OF GROUP A STREPTOCOCCI : IV. STUDIES ON THE MECHANISM OF LYSIS BY CELL-BOUND HEMOLYSIN OF RED BLOOD CELLS AND EHRLICH ASCITES TUMOR CELLS

In studies of the mechanism of lysis of red blood cells by washed streptococci with hemolytic activity (cell-bound hemolysin, CBH) no components released spontaneously by RBC or streptococci, or by interaction between these cells, could be found to induce the formation of soluble hemolysin by the streptococci. It was also found that separation of RBC from streptococci even by Millipore filter or a very thin layer of agar could prevent their hemolysis. By means of cellulose columns it was possible to separate RBC from streptococci after a short incubation. RBC thus separated from streptococci with which they had been incubated underwent hemolysis on subsequent incubation at 37°C. By varying the period of incubation prior to separation it was possible to demonstrate the transfer of increasing amounts of hemolysin from streptococci to RBC with increasing periods of incubation. A considerable part of this appeared to be at a constant rate. A theory is presented on the relationship between the streptococcal cell-bound hemolysin and the group of oxygen-stable streptococcal hemolysins, in terms of a transferable hemolytic moiety and binding sites for this moiety on the streptococcal cell, on various molecular species which can act as inducers of the oxygen-stable hemolysins, and on the RBC, with the affinity of the respective binding sites for the hemolytic moiety increasing in that order.     

RESPONSES TO IMMUNIZATION IN THE THYMUS OF THE ADULT MOUSE

The rate of synthesis of RNA in the thymus glands of adult mice increased after immunization with sheep red blood cells (SRBC). The specific activity of some fractions of RNA, separated first by density gradient centrifugation and then by polyacrylamide gel electrophoresis, was 16-fold higher on day 3 after immunization than control mice not injected. RNA synthesis in the thymus was inhibited by rabbit anti-mouse thymus serum, injected along with antigen. A material was found in RNA extracts from the thymus glands of mice immunized with SRBC which converted a small proportion of either spleen cells or peritoneal cells from nonimmunized mice to form sheep cell hemolysins. Neither extracts from the glands of nonimmunized mice nor the livers of immunized mice were active. Extracts from the thymus glands of mice immunized with rabbit red blood cells (RRBC) were inactive and activity was destroyed by ribonudease. The residual antigen content was not determined. Biologically active extracts from the thymus had a different electrophoretic mobility from active extracts from the spleen.     

PRIMARY SERUM TOXICITY AS DEMONSTRATED BY THE CHICKEN EMBRYO

1. The 3 day old chicken embryo removed from its shell is a suitable test object for the demonstration of primary serum toxicity. Addition of normal rabbit type sera as well as Forssman antiserum causes the vascular network to contract and the embryo sinks in the yolk and dies. 2. Only sera of animals of the so called rabbit type produce this phenomenon. Sera of the guinea pig type are ineffective. 3. Heating to 51°C. destroys the complement content of normal human serum as also its effectiveness to produce the vascular phenomenon. 4. Up to the present it has not been possible to reactivate heat-inactivated normal serum by the addition of complement, while inactivated Forssman antiserum can be easily reactivated. 5. The vascular phenomenon of the chicken embryo is produced not only by the addition of a mixture of Forssman antiserum and complement but also by separate addition of both components. 6. Guinea pig type sera, containing dissolved Forssman antigen, are not only ineffective but actually exert an inhibitory influence on effective rabbit type sera as well as on Forssman antiserum.     

The characteristics of ABO antibodies in group O Thai blood donors

This study aimed to characterize anti-A and anti-B hemolysins, IgM, and IgG titers in Thai blood donors. Altogether, 300 serum samples from group O donors at the National Blood Centre, Thai Red Cross Society, were screened for anti-A and anti-B hemolysins and treated with 0.01 M dithiothreitol to characterize IgM and IgG titers by standard tube technique. Antibody titers were compared with hemolysis grade. Male and female ratio = 1:1.3 and ages ranged from 17 to 60 years. The overall prevalence of anti-A and anti-B hemolysins was 69%. Anti-A and anti-B hemolysins comprised 18.3% and 16.7%, respectively and 34% had both antibodies. High titers of anti-A hemolysins were associated with females (P< 0.05), and only anti-B IgM titers were associated with age (P< 0.05). Interestingly, the association of anti-A IgM titers, anti-A IgG titers, and hemolysin grade was demonstrated (P< 0.05). A significant association between hemolysin grade and anti-B IgM titers was found (P< 0.05). The prevalence of anti-A and anti-B hemolysins and high titers of IgM and IgG in Thais are high. Hemolysin grade showed significant associations with IgM titers; therefore, when providing ABO-incompatible platelet transfusion, especially for female plateletpheresis donors, IgM high titers of anti-A and anti-B screening is suggested.     

STUDIES ON HETEROGENEOUS HEMOLYTIC SERA

Red corpuscles with nuclei contain heterogeneous sheep antigen when the organs of the same animal possess it. The sera in question contain not only heterogeneous hemolysins but also heterogeneous agglutinins. The hemolytic sera of rabbits immunized with hen red corpuscles are toxic to guinea pigs and kill them, the clinical picture being that of anaphylactic shock. Heterogeneous hemolysins are obtained by means of non-toxic antigen (hen red corpuscles injected into veins of rabbits). This fact refutes the theory of Friedberger, who attributed the antigenic power of a suspension of organs to its toxic character.     

CELLS INVOLVED IN THE IMMUNE RESPONSE : VI. THE IMMUNE RESPONSE TO RED BLOOD CELLS IN IRRADIATED RABBITS AFTER ADMINISTRATION OF NORMAL, PRIMED, OR IMMUNE ALLOGENEIC RABBIT BONE MARROW CELLS

Irradiated rabbits given allogeneic bone marrow cells from normal adult donors responded to an injection of sheep red blood cells by forming circulating antibodies. Their spleen cells were also capable of forming many plaques using the hemolysis in gel technique, and were also capable of undergoing blastogenesis and mitosis and of incorporating tritiated thymidine upon exposure to the specific antigen in vitro. However, irradiated rabbits injected with allogeneic bone marrow obtained from rabbits injected with sheep red blood cells 24 hr prior to sacrifice (primed donors) were incapable of mounting an immune response after stimulation with sheep red cells. This loss of reactivity by the bone marrow from primed donors is specific for the antigen injected, since the immune response of the irradiated recipients to a non-cross-reacting antigen, the horse red blood cell, is unimpaired. Treatment of the bone marrow donors with high-titered specific antiserum to sheep red cells for 24 hr prior to sacrifice did not result in any diminished ability of their bone marrow cells to transfer antibody-forming capacity to sheep red blood cells. The significance of these results, with respect to the origin of the antigen-reactive and antibody-forming cells in the rabbit, is discussed.     

Regulation of immunoglobulin E antibody synthesis in man by antiidiotypic antibodies

The effect of rabbit antiidiotypic antibody raised against human IgG F(ab')2 anti-tetanus toxoid (TT) antibodies on the in vitro synthesis of TT-specific IgE antibody by peripheral blood lymphocytes (PBL) was examined in three subjects. Two of these subjects were allergic twins whose sera persistently contained IgE anti-TT antibodies. The third subject was a nonallergic individual who had a slightly elevated serum IgE (250 IU/ml) and who exhibited a transient serum IgE anti-TT response after booster immunization with TT. After appropriate absorptions rabbit anti-idiotype (Id) IgG reacted with anti-TT antibodies of both IgG and IgE isotypes in an idiotype- and antigen-specific fashion. PBL and B cells from the three subjects studied spontaneously synthesized TT-specific IgE in culture. In all three cases, adsorption of B cells over plastic plates coated with anti-Id before culture specifically decreased the synthesis of IgE antibodies to TT but did not affect the synthesis of IgE antibodies to ragweed antigen E by PBL from the twin allergic subjects. Addition of anti-Id to cultures of PBL from all three subjects specifically inhibited the synthesis of TT-specific IgE. This inhibition was shown to be exerted both at the level of the B cells and via the generation of antigen-specific suppressor T cells from radiosensitive precursors. The present results indicate that the synthesis of antigen-specific IgE in man is subject to regulation by idiotypic anti-idiotypic interactions that can involve both B and T lymphocytes.     

HEMOLYSINS OF VEGETABLE ORIGIN

1. The sap of Cotyledon scheideckeri possesses hemolysins for the red corpuscles of different animals. 2. The hemolysins of vegetable sap can be bound by erythrocytes and cannot be separated. 3. A definite quantity of erythrocytes is able to extract from the sap only a part of the hemolysins it contains. 4. The quantity of hemolysins in the sap of different plants is subjected to the same fluctuations as that of bacterial agglutinins, precipitins, and hemagglutinins. 5. As the hemolysins are bound by erythrocytes, the hemolysis can take place not only at 37° but also at 15–16°. 6. The thermostability of the hemolysins varies from one individual plant to another. 7. In many cases the vegetable sap loses its hemolytic properties at a certain temperature and recovers them at a higher one. 8. The vegetable sap is unable to produce complete hemolysis of erythrocytes. 9. But the sap of many plants acquires the power to dissolve red corpuscles completely after I hour of heating at 134° and 144°. 10. Erythrocytes modified by Cotyledon sap cannot be dissolved completely even by distilled water. 11. The agglutination of the erythrocytes and their hemolysis are conditioned, probably, by different substances. 12. The hemolytic amboceptor and the hemolysin of Cotyledon scheideckeri can be bound with the same receptor of the erythrocytes.     

Expression of Forssman glycolipid and blood group-related antigens A, Le(x), and Le(y) in human gastric cancer and in fetal tissues

The expression of Forssman glycolipid antigen in human gastric cancers was investigated by thin-layer chromatogram immunostaining of glycolipid fractions. Incompatible blood group A antigen, Le(x) and Le(y) antigen were also studied in comparison with Forssman antigen. Forssman glycolipid as the pentaglycosylceramide was demonstrated in nine out of 12 gastric cancers, and in three out of 10 adjacent uninvolved tissues. In most cases the content of Forssman glycolipid was increased in the cancers compared with that in the uninvolved counterparts. Forssman pentaglycosylceramide was also detected in some normal gastric mucosae (two out of four), and in a fetal gastrointestinal tract tissue. In immunohistochemical examination of gastric cancer tissues, sialidase treatment revealed a positive staining with anti-Forssman antibody. Some cancer tissues from patients with blood group O were found to contain blood group A-active glycolipids, which could be distinguished from Forssman glycolipid by thin-layer chromatogram immunostaining. The incidence of incompatible A-active glycolipids was two out of 10 cancers from patients with blood group O or B. Le(x)- and Le(y)-active glycolipids were detected in most of the preparations and were not accumulated consistently in the cancers.     

Immunological studies in ulcerative colitis. II. "Colon" antigen and human blood group A- and H-like antigens in germfree rats

Sera from patients with ulcerative colitis contain antibodies which hemagglutinate sheep red cells, sensitized with phenol-water extracts from. colon, cecum, or feces of germfree rats. Minor concentrations of such antibodies are also present in a certain fraction of normal human sera. Hemagglutination and hemagglutination inhibition experiments with human erythrocytes and with the rat extracts showed that the latter contained an antigen similar to human blood group A antigen. In contrast, a blood group B-like antigen could not be detected in these extracts. However, experiments with eel serum indicated that these extracts also contained an antigen similar to the H antigen of the human ABO system. Absorption of ulcerative colitis sera with human A₁ erythrocytes but not that with B or O erythrocytes gave, in a few cases, a slight reduction of the hemagglutinating titers against rat cecum-sensitized sheep erythrocytes. In contrast, this treatment considerably reduced such titers when found in sera from healthy persons or from patients with unrelated diseases. It could be concluded that the rat extracts also contained a "colon" antigen, detected with antibodies, present at elevated titers, in the sera of ulcerative colitis patients, but not in those of the controls. This colon antigen is immunologically distinct from the blood group antigens studied. Hemagglutination inhibition experiments indicated that A, H and colon antigen were widely distributed throughout the gastrointestinal tract of the germfree rats. The colon antigen was found to be enriched in the extracts from colon, cecum, and feces. Fluorescent antibody staining provided evidence that both the colon antigen and the A antigen were present in similar sites of the colon and cecum mucosa, particularly in goblet cells of the crypts, and in the mucus.     

Modulation of Fc receptors of thymus-derived lymphocytes by antigen E in ragweed-sensitive and ragweed non-sensitive subjects

In ragweed-sensitive and ragweed non-sensitive subjects the proportions of T lymphocytes bearing receptors for the Fc portion of IgG (T gamma) and IgM (T mu) were examined as obtained from the blood, and after treatment in vitro with ragweed antigen E or concanavalin-A. The proportion of T gamma and T mu cells, from the peripheral blood of ragweed-sensitive and ragweed non-sensitive persons, untreated in vitro, were not statistically different. However, when T cells from ragweed-sensitive subjects were exposed to ragweed antigen E, the T mu subpopulation was significantly increased (P less than 0.001) without change in the T gamma population. The reverse change occurred when cells of ragweed non-sensitive subjects were treated with antigen E; there was an increase in the T gamma subpopulation (P = 0.01) but no change in number of the T mu cells. Cells from both the sensitive and non-sensitive groups showed increase in number of T gamma and reduced numbers of T mu cells when incubated with concanavalin A. Since T gamma and T mu cells appear to have a regulatory function on B lymphocyte differentiation and antibody production, the pattern of responses of T gamma and T mu subpopulations in vitro to antigen E in ragweed-sensitive and ragweed non-sensitive subjects may reflect a difference in the cellular control of the immune response to ragweed antigen E.