Effect of rubomycin and carminomycin on suppressor cells participating in the regulation of the immune response

The effect of rubomycin and carminomycin on induction of suppressor cells in immunization of mice with high doses (2 . 10(9) cells) of sheep red blood cells (SRBC) or with the allogeneic spleen cells was studied. When the spleen cells of syngeneic animals hyperimmunized with SRBC were administered to intact cells, a marked specific suppression of both the antibody formation and the reaction of the delayed type hypersensitivity were observed. A decrease in the suppressor activity or its complete elimination as a result of exposure of the spleen cell donors to the antibiotics was indicative of a relatively selective effect of these drugs on the suppressor cells. This effect of the antibiotics increased after treatment of the spleen immune cells with the anti-T-serum and complement. However, the blocking effect of the antibiotics on the suppressor cells was not strictly specific, since they were detected in the syngeneic and allogenic systems with the cell transfer.     

Comparative characteristics of the antitumor and immunodepressive activity of carminomycin on the L-1210 experimental model]

The antitumor activity of carminomycin was estimated by the number of lymphoma colonies formed in the spleen of DBA/2 mice on their inoculation with the bone marrow cells from mice with transplantable leukemia L-1210. The immunodepressive properties of carminomycin were determined by the number of the antibody forming cells in the spleen of CBA and DBA/2 mice with leukemia L-1210 after immunization with sheep red blood cells. It was found that in a single dose of 1.5 mg/kg carminomycin inhibited the lymphoma colonies by 50 per cent. The maximum immunodepressive effect was observed when carminomycin was used in a single dose of 1.5 mg/kg 48 hours after the antigen stimulation. In this case the number of the antibody forming cells in DBA/2 mice with leukemia L-1210 was lower than that in CBA mice without leukemia.     

Cyclic kinetics and mathematical expression of the primary immune response

The kinetics of antibody-forming cells (AFC) in the spleen of rats immunized with Salmonella typhi O-antigen was investigated. The number of nucleated cells of spleen and blood serum antibody titres in passive haemagglutination were determined in parallel. Cyclic changes in the number of antibody-forming cells were detected as three peaks on the 4th, 9th, and 13th days following immunization. The fluctuations of their number were not related to the total number of nucleated cells of spleen. The antibody titres reached their peak on the 10th day following immunization, decreased by the 14th day and rose again on the 16th day after immunization. Repeated increases of the number of AFC were probably due to the regular, not accidental, recruitment of committed precursors cells (memory cells).     

The generation of memory B-cell subpopulations capable of proliferation and expansion of the pool: effect of time and antigen

The ability of antigen to induce proliferation of memory B lymphocytes, thus perpetuating and expanding the memory cell pool, has been examined using an antibody-forming cell (AFC) assay. This method provided confirmation of previous studies in which serum antibody titer was used as a relative measure of pool size and demonstrated directly that the number of antibody-forming cells is increased. Memory cell subpopulations were prepared by lg velocity sedimentation from recently immunized donors (2 weeks) and tested for their ability to proliferate, thus expanding the memory cell pool. Both large and small immature DNP-specific memory cells displayed antigen-dependent and antigen-independent proliferation while mature cells (8 weeks postpriming) were capable only of antigen-dependent proliferation. Chicken γ-globulin (CGG) specific memory cells were also evaluated in this system and were found to differ from DNP-specific cells in several ways. (A) DNP-specific AFC were found to be concentrated in the spleen while CGG-specific AFC were found predominantly in the bone marrow early after transfer and in the spleen upon delayed challenge. (B) The rate of maturation of CGG-specific memory cells capable of antigen-driven proliferation and pool expansion was delayed in comparison to DNP-specific memory cells. The relationship of these functionally defined subsets to previously described memory cell subpopulations is discussed.     

Suppression of DNA synthesis in mice by the anthracycline antibiotics daunorubicin, carminomycin and doxorubicin

Inhibition of DNA synthesis by rubomycin (daunorubicin), carminomycin and doxorubicin in the spleen, liver, kidneys and heart was studied on mice. The antibiotics were administered intravenously in a dose of 0.3 LD50. The inhibition level was estimated by incorporation of 3H-thymidine. The time courses of DNA synthesis inhibition by daunorubicin, carminomycin and doxorubicin markedly differed, whereas the patterns of their inhibition curves for all the organs were close. The maximum inhibition of DNA synthesis by carminomycin was observed in 6 hours. After that period it gradually restored. Doxorubicin induced the maximum inhibition of DNA synthesis in 24-48 hours after its administration. Daunorubicin induced two maxima in inhibition of DNA synthesis i. e. in 6 and 48 hours. Definite correlation between the levels of DNA synthesis inhibition by the antibiotics and their toxic action was shown.     

Antibody formation in experimental kidney failure and in the early stages of restorative kidney growth in mice

The ability of spleen cells to respond with antibody formation to a foreign antigen (sheep erythrocytes) was studied in mice at different kidney lesions (uni- and bilateral nephrectomy, ureter ligation, pseudo-operation, wound of one kidney) during the early postoperation period (1-72 h). In the case of bilateral nephrectomy, the reliable increase in the number of antibody-forming cells (AFC) was noted already within 1 h after the operation, in the case of unilateral nephrectomy within 12 h. In the case of bi- and unilateral ligations of ureter, the response was delayed by 3 and 5 h, respectively. Sham operation and kidney wound did not stimulate antibody formation. It is suggested that the antibody-forming ability of the spleen cells does not depend on stress, renal deficiency or destructive changes and that the antibody formation is activated by disturbances in the ratio of immunoregulatory cells.     

Transfer Agent of Immunity

Using erythrocytes as antigen particles, number of antibody-forming cells was enumerated by immunocytoadehesion technique, in which formation of rosette was shown to be inhibited by anti-mouse immunoglobulin sera. This number increased in vitro after treatment of spleen cells of mice for 60 min with RNA fraction extracted from spleen of mice immunized with erythrocytes used in the enumeration, and incubation of cells for 12 hr at 37 C. Response of cells treated with immune RNA fraction was immunologically specific and was inhibited by puromycin or cycloheximide. The activity of immune RNA capable of converting nonimmune cells to antibody-forming cells was shown to be sensitive to ribonucleases but resistant to deoxyribonuclease and proteolytic enzyme.     

Selective concurrent suppression of the process of rosette-forming cell accumulation during the immune response]

Repeated injections to mice of normal rabbit immunoglobulins preceding immunization with sheep erythrocytes inhibited the accumulation of rosette-forming cells (RFC) in the spleen, without influencing the proliferation of the antibody-forming cells and hemaggutinin production. Reduction of the RFC under these conditions occurred on account of B-cells whose antigen-binding receptors could be blocked by antibodies against the aggregated mouse immunoglobulins and a complex of polyadenylic-polyuridylic acids. Repeated injections of the competitive antigen enhanced the formation of the immunological memory to the second antigen. The problem of the origin of the immune rosette-forming B-cells and their influence on the formation on the immunological memory is discussed.     

Transfer of agammaglobulinemia in the chicken. II. Characterization of the target of suppression

The T-independent adoptive primary and secondary responses of FP chicken spleen cells to B. abortus (BA) were suppressed by simultaneously transferred histocompatible spleen cells from agammaglobulinemic (Aγ) chickens previously injected with bursa cells. The response of spleen cells transferred with BA 2 days prior to suppressor cells was partially inhibited. Highly significant suppression of both 19 S and 7 S antibody formation was seen during the second week after memory cell transfer. When suppressor cells and primed spleen cells were transferred together and challenged with BA 2 weeks later the secondary responses were more inhibited in recipients that showed persistently decreased serum IgM than in those showing recovery toward normal IgM levels. Transfer of histocompatible suppressor cells into surgically bursectomized (SBX). irradiated, 4-week-old SC and FP strain recipients caused reduction or complete disappearance of serum IgM and lowered IgG levels, which correlated well with a decrease in plasma cell numbers in spleen and trident. Many recipients of both strains, examined 1 to 2 weeks after transfer, completely lacked plasma cells from gut-associated lymphoid tissues and from spleen. When such animals had been sensitized to BA prior to transfer and were challenged after recovery from suppression, they showed good secondary responses. Experiments with serially transferred memory cells using suppressed intermediate hosts also indicated survival of such cells in the face of marked suppression of antibody synthesis. Bursa follicles that were not destroyed by irradiation did not show any effect of the suppressor cells, although plasma cells often disappeared from the interfollicular areas. Intact irradiated recipients showed a greater tendency toward recovery from suppression than did SBX recipients. Although Ig-bearing cells in spleen and peripheral blood of suppressed animals were significantly lower than in irradiated SBX controls, they never disappeared as completely as did plasma cells and serum IgM. The results suggest a direct effect of suppressor cells on antibody-forming cells with a less marked effect on their precursors.     

Transformation of anthracycline antibiotics and their semisynthetic derivatives as affected by the liver aldo-keto reductase of rats

Formation of 13-dihydro derivatives of rubomycin (daunorubicin), carminomycin, doxorubicin and some of their semisynthetic derivatives under the effect of pure aldo-keto reductase from the rat liver was studied. Attachment of an oxy group to C-14 markedly retarded formation of the 13-dihydro derivatives while attachment of the bulky radicals to the same position prevented their formation. Binding of the anthracycline antibiotics to human serum albumin had no impact on the fermentative reaction rate. Rubomycin, carminomycin and doxorubicin significantly differed in their lipophilic properties and capacity for binding to serum albumin.     

Effect of the peroral use of the antitumor antibiotic, carminomycin, on the immunological reactivity of the body of animals]

Carminomycin administered orally to mice for many times in doses of 2.5 and 1.25 mg/kg induced suppression of hemagglutinine production to sheep erythrocytes and formation of immunologically competent cells in the spleen of test animals. The content of DNA and RNA in the spleen of the test animals treated with carminomycin and sheep erythrocytes was somewhat lower than that in the control mice immunized but not treated with the antibiotic. Carminomycin prolongated the life time of the skin graft by 6.5 days as compared to that of the skin homotransplant in the control animals. The oral use of carminomycin in a dose of 2.5 mg/kg induced a statistically significant decrease in the absorption capacity of the cells of the reticuloendothelial system of the animals.     

Growth and senescence of antibody-forming cells

Studies were performed on the capacity of mice for hemagglutinating antibody production throughout their life-span. An in vivo culture method was used for assessment of primary and secondary antibody-forming potentials of spleen cells of mice ranging in age from 1 to 130 weeks. There was a marked growth of potential for antibody formation during neonatal and juvenile life followed by a gradual decline in potential with advancing age. It was possible to show that the changes in potential were principally due to changes in the number of competent progenitor cells and not to changes in their performance. Death of very old animals was correlated with decline in number of immunologically competent progenitor cells. The decay in number of progenitor cells during aging of mice was random. Loss of progenitor cells was not entirely attributable to either generative failure of the pool of progenitor cells or the capacity of the milieu of the animal to support such cells. Thus, spleen cells from aged animals displayed increasing capacity for primary antibody formation during a 3-week period of culture in young, irradiated mice; identical cultures in old, irradiated recipients failed in respect to growth of primary antibody-forming potential. Progressive imparirment of the milieu of aging animals was suggested by the fact that spleen cells from very old animals were “toxic” when infused into lightly irradiated recipients which were themselves of advanced age but far short of the senescent phase of their life-span. These results lead to the argument that senescence may be, to a major degree, the result of progressive loss of progenitor, or “stem,” cells which are normally utilized to replace terminally differentiated, dying cells.     

Inhibition of synthesis of nucleic acids, proteins and respiration in isolated thymocytes by anthracyclines

The effect of anthracycline antibiotics such as carminomycin, daunomycin (rubomycin) and adriamycin on respiration and synthesis of nucleic acids and protein was studied comparatively. The anthracyclines inhibited the processes. By their efficacy in that respect they could be arranged in the following order: carminomycin greater than rubomycin greater than adriamycin. Thus, 50 per cent inhibition of nucleic acid synthesis in the thymocytes required 0.027, 0.044 and 0,173 mM of carminomycin, rubomycin and adriamycin respectively. Protein synthesis and respiration in the thymocytes were less sensitive to the effect of the anthracyclines than synthesis of nucleic acids. The study results were compared with the literature data on the effect of the compounds on respiration and synthesis of nucleic acids and protein in tumour and bacterial cells.     

Hypothalamo-pituitary system controls the development of humoral immune response during prenatal ontogenesis in rats

We studied the influence of the neuroendocrine system on the development of humoral immune response to sheep erythrocytes in rat fetuses. The removal of brain in utero by decapitation of 18-day fetuses induced a fourfold increase in the number of antibody-forming cells in the liver, as compared to the unoperated fetuses. After the removal of the forebrain, including hypothalamus (encephalectomy), the number of antibody-forming cells was comparable to that in unoperated fetuses. The observed increase in the number of antibody-forming cells in the liver was not due to a disturbed migration of precursors of B-lymphocytes in the spleen, since their content in the spleen was also four times that in the encephalectomized and unoperated fetuses. The increased number of antibody-forming cells in decapitated fetuses could be due to an enhanced proliferative activity of the lymphocytes in the liver of these fetuses. It has been proposed that humoral immunity is controlled by the hypothalamo-pituitary-adrenal system already during prenatal development; the adrenocorticotropic hormone and glucocorticoids appear to be involved in this regulation.     

Antibody-producing ability of splenic cells of the progeny of female rats with chronic lesions of the liver

Spleen antibody-forming function in the offspring of female rats with experimental chronic autoimmune liver affection has been studied for its specificities. The data obtained testify to that these animals are characterized by the increased immunoreactivity at all the stages of investigation, that is confirmed by an increase in the number of antibody-forming cells in their spleen. Besides, the splenocytes of immunized animals have an immunostimulating effect on the productive phase of antibodygenesis and has no such effect on the inductive phase.     

Interaction with DNA of semisynthetic carminomycin and rubomycin derivatives

The apparent binding constants and the effect of semisynthetic derivatives of carminomycin and rubomycin (anthracycline antibiotics) on DNA fusion were studied. The following semisynthetic derivatives were used. 13-dihydrocarminomycin, 14-hydroxycarminomycin, 13-(4-methylpiperazinyl) imine carminomycin, 13-benzoylhydrazone carminomycin (carminazone), 13-tret-butoxycarbonyl hydrazone rubomycin, 13-(4-methylpiperazinyl) imine rubomycin, 14-(1-hydroxyl-2,2,6,6-tetramethylpiperidyl-4)-acetoxyrubomycin (spin-labeled rubomycin). The above derivatives slightly differed from the initial antibiotics by their affinity to DNA. The binding constants of methylpiperazinyl imines was 2-3 times higher than those of the respective antibiotics.     

The effect of myelopid on antibody formation in gamma-irradiated animals

The effect of a new immunocorrecting preparation, Myelopid, on the antibody-forming cell content of mouse spleen after gamma-irradiation (1-3 Gy) has been investigated. The preparation administered after immunization of mice with sheep erythrocytes increases the number of antibody-forming cells in the spleen, the effect being a function of radiation dose and time interval between the exposure and immunization. The preparation is ineffective when delivered after irradiation, but prior to immunization.     

Structural and serological studies of lipopolysaccharides of Citrobacter O35 and O38 antigenically related to Salmonella

Abstract Lipopolysaccharide (LPS) was administered into sheep red blood cells (SRBC)-primed mice, and the effect of LPS on SRBC-specific memory cells was investigated. Spleen cells from SRBC-primed mice which were injected with LPS exhibited much lower in vitro secondary plaque-forming cells (PFC) responses to SRBC than those from untreated SRBC-primed mice. The in vitro anti-SRBC response of the spleen cells to LPS was also reduced. The combination experiments of B cells and T cells from SRBC-primed mice which were injected with or without LPS demonstrated that the reduction of immune responses to SRBC after administration of LPS was caused by the defect of SRBC-specific B memory cells, but not T memory cells. B cell type rosette-forming cells (RFC) for SRBC markedly decreased after injection of LPS, while PFC as antibody-forming cells did not increase subsequently. Therefore, the reduction of RFC was not due to their differentiation into PFC. The lymphoid follicles in the spleens from mice injected with LPS were stained positively by in situ nick end labeling specific for fragmented DNA. A large percentage of Ig⁺ spleen cells from SRBC-primed mice which were injected with LPS was also stained positively. The injection of glucocorticoids into SRBC-primed mice induced similar reduction of B memory cells. It was suggested that LPS might induce apoptosis of B memory cells and regulate B cell memory in antigen-nonspecific manner.     

Semisynthetic derivatives of daunorubicin and carminomycin: inhibition of DNA synthesis after intravenous administration to mice

Inhibition of DNA synthesis in the liver, kidneys, spleen and heart of mice after intravenous administration of 0.1 and 0.3 LD50 of semisynthetic derivatives of rubomycin (daunorubicin) and carminomycin was studied. The level of DNA synthesis inhibition was estimated by a decrease in incorporation of (methyl-3H) thymidine. Under the action of 13-trebutoxycarbonyl hydrazone and 14-salicyloiloxy derivatives of rubomycin and carminomycin maximum inhibition of DNA synthesis was reached later while its recovery started earlier as compared to the initial antibiotics.     

New derivatives of carminomycin and rubomycin

For preparing new semisynthetic analogs of anthracycline antibiotics, hydrolysis of 13-dimethylketals of 14-bromrubomycin and 14-brom-arminomycin in solution of diluted hydrochloric acid was studied. It was shown that such hydrolysis yielded 14-chlorrubomycin and 14-chlorcarminomycin. Conditions for separating the mixture of 14-chlor- and 14-bromrubomycins and the mixture of 14-chlor- and 14-bromcarminomycins by HPLC were developed. Interaction of 14-chlorine derivatives of rubomycin and carminomycin with potassium formate in the presence of the crown ether yielded 14-formyloxy derivatives of rubomycin and carminomycin. Interaction of rubomycin and carminomycin with formic acid in the presence of N-oxysuccinimide and dicyclohexylcarbodiimide resulted in formation of N-formyl derivatives of rubomycin and carminomycin.