Hemolysis In Agar Research Articles

MECHANISMS OF REDUCTION OF ANTITUMOR DRUG TOXICITY BY LIPOSOME ENCAPSULATION *

Actinomycin D, when encapsulated within liposomes, has been previously shown to be less toxic to mice than nonencapsulated actinomycin D, but to retain its tumoricidal activity. We have compared the toxic effects of Act D encapsulated either in the aqueous phase or in the lipid phase of liposomes (APL and LPL, respectively), and the nonencapsulated Act D on the blood forming system, on cell proliferation in the intestine, and on antibody production by spleen lymphocytes. At a single dose of 0.4 mg/kg, APL-encapsulated Act D wass less toxic to white blood cells and to the nucleated cells and colony-forming stem cells of the bone marrow. During toxicity in the proliferating intestinal cells, measured by 3H-thymidine incorporation, was reduced by about a factor of 4 with encapsulation in APL, particularly 24 hours after Act D administration. The toxiciaty of LPL-encapsulated Act D to both the blood-forming system and the intestinal proliferating cells was, however, not significantly different from that of the nonencapsulated Act D. Effects of Act D on the antibody production by spleen cells, determined by the "limited hemolysis in agar" assay, showed that immunosuppression was most markedly reduced by liposome encapsulation either in APL or in LPL, when the drug was given one day before the antigen. These findings are important for considerations of liposome application in cancer chemotherapy.

Origin of cells reducing normal hemolysins in guinea pigs

Cells producing normal antibodies against sheep's erythrocytes were revealed by local hemolysis in agar in the spleen of embryos in the late stage of development and of newborn guinea pigs. Preliminary immunization of the pregnant animals with sheep's red cells did not affect the number of hemolysin-producing cells in the spleen of the embryos or newborn guinea pigs. The number of normal antibody-forming cells in the spleen of sterile adult guinea pigs was the same as in animals of the same species kept under ordinary conditions. The results suggest that normal hemolysin-producing cells arise in guinea pigs not by antigenic stimulation through cross-reacting antigens of the external environment, but by physiological differentiation of predetermined precursor cells. This hypothesis corresponds to one of the postulates of the clonal selection theory of immunogenesis.

Cellular mechanisms of adjuvant action of bacterial lipopolysaccharide in anti-sheep red blood cell antibody response.

Adjuvant action of lipopolysaccharide (LPS) extracted from Salmonella typhimurium LT2 on anti-sheep red blood cell (sRBC) antibody response in mouse spleen was studied at the cellular level by the technique of localized hemolysis in agar. Injection of LPS caused significant increase in direct 19S and indirect 7S plaque-forming cells (PFC) in mouse spleen after the primary antigenic stimulation, while the adjuvant effect of LPS could hardly be assessed in the secondary PFC response. The adjuvant effect on the primary PFC response was most conspicuous when LPS was injected at the same time as the sRBC, and no effect could be observed when LPS was injected 1 or 5 hr before PFC determination. The mice who had received LPS with the primary sRBC stimulation showed much higher PFC level after the secondary sRBC stimulation, indicating the increase in the memory cells. Cell population consisting of lymphoid cells and macrophages was separated into ‘adherent’ (macrophage-rich) and ‘non-adherent’ (lymphoid cell-rich) fractions by incubating in petri dishes, and effect of LPS on each fraction was examined. Transfer of non-adherent lymphoid cells treated with LPS in vitro and simultaneous sRBC injection resulted in a significant increase of PFC in the spleen of the syngeneic recipient mice, but transfer of LPS-treated macrophages did not show any effect, suggesting that LPS acts on lymphocytes but not on macrophages. This idea appeared also to be supported by the results that LPS exerted antagonistic effect on the immune suppression caused by heterologous anti-lymphocyte serum, anti-sRBC serum and cortisone which are currently supposed to suppress the antigen reactive cells.

Cells Involved in the Immune Response. XX. The “All or None” Nature of the Primary Immune Response (19S Antibody) by Individual Antibody-Forming Cells in the Rabbit

Abstract The cells that synthesize 19S antibodies in the rabbit following primary intravenous immunization have been shown to be localized in the spleen, using the hemolysis in agar (plaque) technique (1). The temporal relationships are such that the 19S antibody-forming cells (AFC) cannot be detected in appreciable numbers before 4 to 5 days after immunization, but they then increase in number markedly within 24 hr. However, what has not been ascertained is whether this explosive increase in the AFC population is due to a) rapid division of these cells, each progeny being immediately capable of synthesizing large quantities of antibody, b) a synchronous phase of rapid antibody formation by non-dividing cells, or c) increasing amounts of antibody being synthesized by the AFC, thus permitting the detection of increasing numbers of non-dividing AFC. The results presented below strongly support but do not discriminate between the first two hypotheses.

Antibody Elicited by Brucella Abortus Antigen in Vitro: Prolonged Production by Cell Suspensions

Summary It has been found that Brucella antibody can be elicited in vitro. Spleen cell suspensions from rabbits immunized 6 to 13 months earlier to Brucella abortus antigen were antigenically stimulated in vitro and cultured up to 30 days. Four to five days after initiation of the response antibody-synthesizing cells could be detected by passive localized hemolysis in agar. For detecting the cells, the ratio of Brucella erythrocyte-sensitizing substance to erythrocytes proved critical. After the exponential phase of their appearance, at 4 to 6 days, plaque-forming cells could no longer be demonstrated. The Brucella-stimulated cells synthesized high concentrations of specific bacterial agglutinins. Extracellular antibody reached a maximum by 8 to 11 days and remained at this level for 1 to 2 weeks. Titers of 1:160 and 1:320 were attained regularly. The prolonged “steady-state” of antibody production permitted an examination of the late phase of the immune response in vitro and the effect of various factors on it. Within limits, the antibody response of the cultured spleen cells was a function of antigen concentration. Although 106 brucellae elicited a high level of antibody from 107 spleen cells, antibody synthesis was sustained for 1 to 2 weeks only when higher numbers of brucellae were present. The amount of antibody produced depended markedly on the normal rabbit serum used in the medium, the atmosphere, and the physical conditions of culture. It was found that actinomycin D at 0.1 to 1.0 µg/ml completely inhibited the secondary immune response of cultured cells to Brucella abortus antigen when added at 0 hr or up to day 3. Aflatoxin at 1 µg/ml reduced the response significantly when the agent was added up to day 3. On disc electrophoresis, the antibody elicited in vitro migrated as a single band with the slow γ globulins, at the same rate as Brucella antibody produced in the rabbit. On the basis of sensitivity to 0.1 M 2-mercaptoethanol, over 90% of the antibody was IgM. It was concluded that the prolonged production of antibody provided a sensitive means of studying the effect of various factors on the secondary immune response in vitro. With cultures of dispersed cells initiated at 107 spleen cells/ml, antigenically stimulated with 108 killed Brucella abortus and grown under rather rigid conditions, the results were reproducible.

Critical Variables of the Jerne Plaque Technique As Applied to Rodent Antibody-Forming Systems Responding to Heterologous Red Cell Antigens

Summary Improved conditions were developed for the Jerne plaque technique; a simple test (“hemolytic spot-testing”) allows a study of interspecies antigen-antibody systems and interspecies complement effects as well as ionic requirements for hemolysis in agar. These optimal conditions were applied to the Jerne plaque test with parallel results. The Jerne technique can easily be applied to study the hemolytic antibody response of C3BF1 mice, Golden Syrian hamsters, and Wistar rats immunized with heterologous RBC. Optimal results depend on the type and dilution of complement used for a certain heterologous combination: guinea pig complement gives best results in the mouse anti-sheep system; rabbit complement is superior in the mouse anti-rat system; and human complement is hemolytically most active in the mouse anti-hamster, hamster anti-mouse, and rat anti-mouse combination. Each C′ requires different ionic conditions of the reaction medium. It is demonstrated that optimal results of the Jerne plaque test depend on adequate concentrations of Ca++ and Mg++ in the agar. The data suggest also that the anticomplementary effect of agar is ionic. This hemolysis-reducing or inhibiting factor is overcome by DEAE dextran and higher cation concentrations of the agar. The mechanism of action of the polycation and Ca++ and Mg++ cations is discussed. The hemolytic activity of complement of different species is not only reflected in variation of the number of plaques but also in average plaque size. A quantitative complement-antibody correlation is considered as a possible explanation for this observation, and suggests that this method may provide a model for the study of the dynamics of cell lysis.

Influence of chloramphenicol and cetophenicol on antibody formation in mice.

SummaryThe immunosuppressive effect of chloramphenicol in vivo has been demonstrated in mice at low dose in short-term experiments by application of the technique of localized hemolysis in agar. The antibiotic suppressed the increase in numbers of hemolysin-forming spleen cells found 42 hr after immunization with sheep red blood cells or treatment with endotoxin. Cetophenicol, an analog of chloramphenicol having the same antibacterial spectrum and ability to inhibit de novo protein synthesis, also suppressed the response to the specific antigen, sheep erythrocytes. In contrast, however, cetophenicol enhanced the proliferation of hemolysin-forming cells induced by endotoxin. These opposite influences disappeared when the two antibiotics were injected into the same endotoxin-stimulated mice. Neither analog by itself modified the normal background of antibody-forming cells found in unstimulated mice. The implications of these findings for the modes of action of the analogs and for the influence of endotoxin ...

Local hemolysis in agar as a test of inactivation of nonsyngenetic stem cells

Local hemolysis in agar as a test of inactivation of nonsyngenetic stem cells

Detection of cells producing antibodies to O-antigen of S. typhi by the method of local passive hemolysis in agar

The reaction of passive agglutination with O-antigenS.typhi can be transformed by adding complement into a highly sensitive passive hemolysis test. Modification of Jerne's test with the use of erythrocytes sensitized by O-antigen enables one to detect cells producing O-antibodies in the lymphoid tissue of immunized animals. This makes it possible to extend the sphere of use of Jerne's test to a number of experimental models not connected with the production of hemolysins.