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).     

Regulatory role of bone marrow in immunogenesis. II. Analysis of various mechanisms of antibody genesis suppression by the bone marrow B cells in vitro]

The suppressive effect of the cells on the bone marrow of the B-lymphocytic series on the production of antibody-forming cells to sheep red blood cells, observed on their addition into the culture of the spleen cells after Mishell and Dutton, was mediated only by the live cells capable of proliferation, and was independent of the histocompatible differences between the bone marrow and the spleen cells and of the preliminary immunization of the bone marrow donors.     

Effect of antigenic stimulation on acid deoxyribonuclease activity in lymphocytes: I. Antibody-induced response

The effect of antigen-induced stimulation on acid deoxyribonuclease (DNase) activity in BALB/c mouse lymphoid cells was determined. Increase in acid DNase activity was found in intact spleen cell populations of mice from the second or fourth day after immunization with pneumococcal polysaccharide type III and from the fourth day after immunization with SRBC. DNase determinations performed with spleen cell fractions prepared from SRBC-immunized mice, showed that the rise in the enzyme activity was confined to the fraction containing the antibody-forming cells. The DNase activity was also increased in spleen cell cultures, stimulated with SRBC in vitro. Rise in the activity of this enzyme was also observed in peritoneal cell populations taken from SRBC-immunized mice. This change was maximal on the second day after immunization, when no appreciable increase in DNase activity of spleen cells was yet detected. The results obtained suggest, that acid DNase is an enzyme involved in the proliferative/maturation response to antigenic stimulation. It is a consequence of antigenic stimulation rather than being involved in the process of afferent stimulation.     

The immune system response to antigen in cold- and warm-adapted rats

In rat experiments, it was shown that long-term adaptation to cold modified the response pattern of the immune system to antigen stimulus by variously changing the different components of the response. There was a considerable decrease in the number of antigen-binding cells in spleen and peritoneal exudate and also a marked decrease in general antibodies in blood. Conversely, the number of antibody-forming cells in spleen increased twofold. The shift in the thermal conditions after immunization caused changes in the immune response. After the placement of cold-adapted animals to the warm environments the number of antigen binds and antibody-forming cells in spleen, as well as hemagglutinin titers in serum normalized, became indistinguishable from those in the warm-adapted control group. The placement from warm to cold after immunization of warm-adapted rats also produced considerable shifts in parameters of the immune response. However, these shifts were not entirely consistent with those observed for long-term adapted to cold. The obtained data evidence that the thermal factor of the environment is of major importance to the function of immunocompetent cells.     

Plaque-forming cell response in BALB/c mice to two preparations of LPS extracted from Salmonella enteritidis.

The pattern of development of antibody-forming cells in BALB/c mice after immunization with PW-LPS or TCA-LPS was shown to be different. On days 10 and 20, the primary response to PW-LPS was characterized by a low level of IgM synthesis. The plaque-forming cell (PFC) response to TCA-LPS, however, increased from day 10 to day 20. Initially, IgM was the only detectable antibody synthesized but by day 20 a significant number of IgG-producing spleen cells had developed. After a secondary immunization with the appropriate lipopolysaccharide (LPS) preparation, IgG-producing spleen cells were detected in mice immunized with either PW-or TCA-LPS. Partial removal of the LAP or TCA-LPS with phenol or trypsin and pronase significantly reduced the PFC response, suggesting that the protein moiety played an influential role in the immunogenicity of TCA-LPS. The TCA-LPS contained the same antigenic dterminants as PW-LPS, so any difference observed between PFC response was not due to any associated immunogenic moiety.     

Comparison of the action of carminomycin and rubomycin on the dynamics of the primary and secondary immune responses

The effect of rubomycin and carminomycin on the dynamics of the primary and secondary immune response and formation of the immunologic memory to sheep red cells in mice was studied. Differences in the character of the antibiotics effect indicative of the higher selective action of carminomycin on multiplying cells, precursors of the antibody-forming plasmids, were found. Theoretically interesting discrepancies in the effect of the antibiotics on the content of the antibodies in the serum and the antibody-producing cells in the spleen were shown. It was demonstrated that carminomycin had no effect on formation of the immunologic memory inspite of a noticeable decrease in the total number of the spleen nuclear cells and the number of the antibody-forming cells at the moment of immunization under the effect of the antibiotic.     

Selective release of antibody forming cells into the blood

Cells that are actively producing antibody in the spleen are not randomly released into the blood. At comparable times after immunization, the ratio of IgM/IgG antibody-producing cells in blood differs from that in the spleen and lymph nodes. In contrast to the primary response, a heightened number of antibody-producing cells in the lymphoid tissue during the secondary response is not reflected by an increased number in the blood. The findings suggest that some immunologically active cells are selectively retained by the lymphoid tissues while newly produced antibody-forming cells are released into the blood stream.     

Effect of extremely high frequency electromagnetic radiation of low intensity on parameters of humoral immunity in healthy mice

The modification of indices of the humoral immune response to thymus-dependent antigen (sheep erythrocytes) after a whole-body exposure of healthy mice to low-intensity extremely-high-frequency electromagnetic radiation was studied. Male NMRI mice were exposed in the far-field zone of horn antenna at a frequency of 42.0 GHz and energy flux density of 0.15 mW/cm2 under different regimes: once for 20 min, for 20 min daily during 5 and 20 successive days before immunization, and for 20 min daily during 5 successive days after immunization throughout the development of the humoral immune response. The intensity of the humoral immune response was estimated on day 5 after immunization by the number of antibody-forming cells of the spleen and antibody titers. Changes in cellularity of the spleen, thymus and red bone marrow were also assessed. The indices of humoral immunity and cellularity of lymphoid organs changed insignificantly after acute exposure and series of 5 exposures before and after immunization of the animals. However, after repeated exposures for 20 days before immunization, a statistically significant reduction of thymic cellularity by 17.5% (p < 0.05) and a decrease in cellularity of the spleen by 14.5% (p < 0.05) were revealed. The results show that low-intensity extremely-high-frequency electromagnetic radiation with the frequency and energy flux density used does not influence the humoral immune response intensity in healthy mice but influences immunogenesis under multiple repeated exposures.     

Appendix and γM-antibody formation: VII. Rosette-forming cells in rabbits immunized with sheep erythrocytes

After intravenous immunization with sheep red blood cells the rabbit spleen shows a sharp rise in the number of plaque-forming cells but there is no detectable rise in PFC in the appendix or mesenteric lymph node of the same animals. Repeated immunization via an appendicostomy blind loop results in virtually no local PFC and only a small rise in splenic PFC.In lethally irradiated animals neither thymocytes nor appendix cells alone restore the splenic PFC response. Simultaneous injection of the two cell types restores both direct and indirect plaque formation. The injected cells were labeled with tritiated adenosine and a standard rosette assay for specific antigen-binding cells applied to recipients' spleen cells following immunization. Rosettes appeared by 3 days after immunization whether thymocytes or appendix cells were labeled. Labeled rosettes were observed only in animals receiving labeled appendix cells.This result demonstrates the presence of rosette forming cell precursors in rabbit appendix cell populations and suggests that the cells of gut-associated lymphoid tissues include antibody-forming cell precursors which are normally seeded to the spleen and draining lymph nodes.     

IgE antibody-forming cells in rats infected with Nippostrongylus brasiliensis and immunized with antigens

The distribution of IgE antibody-forming cells was examined in rats infected with Nippostrongylus brasiliensis (Nb) or immunized with Nb antigen or with OA. The frequency of antigen-specific IgE antibody-forming cells was detected by a passive cutaneous anaphylactic (PCA) reaction using cell extract from lymphoid organs. In Nb-infected rats, anti-Nb and anti-4th stage larvae (L4) IgE-forming cells distributed mainly in the mesenteric and the bronchial lymph nodes (LN) near the parasite-harboring sites. After intraperitoneal (ip) immunization with Nb antigen mixed with Al(OH)3 and Bordetella pertussis (Bp) as adjuvants, anti-Nb IgE antibody-forming cells were detected in the mesenteric and the bronchial LN. Anti-Nb or OA IgE antibody-forming cells after subcutaneous (sc) immunization were found in the inguinal and the axillary LN. An effect of Bp on the distribution of IgE antibody-forming cells seems to be ruled out. The distribution of IgG2a antibody-forming cells was similar to that of IgE antibody-forming cells, indicating that the distribution of the IgE antibody-forming cells is not preferential. IgE antibody-forming cells were stimulated in the regional LN near the site of antigen administration. IgE antibody-forming cells induced by potentiated IgE antibody production were also examined. Rats were immunized ip or sc with OA and infected with Nb. Anti-OA IgE antibody-forming cells were found in all of the lymphoid organs and especially in the regional LN near the Nb parasite-harboring and antigen administration sites.     

Suppression of humoral antibody synthesis on exposure to hyperbaric oxygenation

The influence of hyperbaric oxygenation (HBO) on the immune response in mice, immunized intraperitoneally with sheep red blood cells, was studied. HBO was shown to reduce hemagglutinin and hemolysin titres in peripheral blood as well as to decrease the amount of antibody-forming cells in the spleen. The most pronounced immunodepressant HBO effect is seen when hyperbaric oxygenation is carried out under toxic conditions before immunization of the animals with low antigen doses. Relationship is shown between the immunodepressant HBO effect and reduced leucocyte and lymphocyte counts in peripheral blood of the animals.     

Primary anti-trinitrophenyl memory cells investigated by plaque-forming cells and ELISA.

Primary anti-trinitrophenyl antibody production was investigated from spleen cells of mice immunized with trinitrophenylated-keyhole limpet hemocyanin, using the plaque-forming cell method and ELISA. Cells taken 5 days after antigen injection do not produce IgE, but do produce IgM and IgG1 anti-trinitrophenyl antibodies as demonstrated by plaque-forming cells. Substantial increase of IgM, IgG1, and IgE antibody production was seen from cells taken 7 days after immunization, followed by a rapid decline. By ELISA it was seen that cells taken 3 days after immunization already produce small amounts of anti-trinitrophenyl antibodies. Presence of antigen from the start of the cultures did not increase antibody production from cells taken 3 days after immunization, but potentiated antibody secretions from cells taken 5 days or later after immunization. This potentiation was interpreted as recruitment of antibody-forming cells from early memory B cells. The presence of IL-4 from the start of the cultures had no appreciable effect. Cell sorting with specific antibody-coated magnetic beads showed that plaque-forming cells from nonsorted cells, membrane IgE+ or membrane IgE- cells secreted similar amounts of anti-trinitrophenyl IgG1 and IgE antibodies. No difference in anti-trinitrophenyl IgM, IgG1, or IgE production was found in controls; cells sorted negatively or positively for CD23. The data show that memory B cells can be demonstrated already on Day 5 after immunization, and their antigen-induced antibody secretion is IL-4 dependent.     

Migration of specific antibody-forming cells during the secondary immune response against horseradish peroxidase

Mice were primed subcutaneously in the hind footpads with horseradish peroxidase (HRP) and boosted intravenously 10 weeks later. Removal of the popliteal lymph nodes (draining the site of primary immunization) before the booster injection markedly depressed the secondary immune response in the spleen and bone marrow. This was taken as evidence that the secondary humoral immune response against HRP in the spleen and bone marrow is largely dependent upon immigration of cells from the popliteal lymph nodes after the booster injection. In rats primed subcutaneously in the hind footpads with HRP, antibody-forming cells were demonstrated in the blood, but not in the thoracic duct lymph 3 days after an intravenous booster injection with HRP.     

Antibody formation and B-cell migration from bone marrow to spleen in mice under conditions of stimulation and suppression of erythropoiesis]

The effect of erythropoietic stimulation and suppression on the production of antibody-forming cells (AFC) in the spleen and on the B-cell migration from the bone marrow to the spleen was investigated in the CBA mice. Erythropoiesis stimulation proved to sharply increase the AFC count in the spleen and the B-cell migration from the bone marrow to the spleen 1 and 4 days after the bleeding. Erythropoiesis suppression resulted in a slight increase of the AFC count in the spleen 4 and 7 days after the transfusion of syngeneic red blood cells. However, the erythropoietic suppression inhibited the B-cell migration from the bone marrow to the spleen. Possible mechanisms of the effect of the erythropoietic stimulation and suppression on the antibody production are discussed.     

Transfer Agent of Immunity

Antibody formation in vitro was studied using erythrocytes (RBC) as antigen and immunocytoadhesion as the technique for detection of antibody-forming cells. Spleen cells (SPC) of nonimmune mice gained the ability to produce antibody after treatment with ribonucleic acid (RNA) preparation extracted from allogeneic mice immunized with xenogeneic or allogeneic RBC. It was also found that a small proportion of SPC from individual mice of certain strains formed antibody against autologous RBC when the cells were treated in vitro with RNA preparation obtained from the spleen of an allogeneic mouse immunized with RBC of that individual. No converting ability was observed in the RNA preparation from spleen of nonimmune autologous or allogeneic mice. The converting activity of immune RNA preparation was shown to be sensitive to ribonuclease treatment. These evidences exclude the possible contribution of antigen or fragments thereof in the RNA preparation to the induction of antibody formation in RNA recipient cells.     

Studies on the adjuvant effect of Bordetella pertussis vaccine to sheep erythrocytes in the mouse. I. In vitro enhancement of antibody formation with normal spleen cells.

The adjuvant effect of Bordetella pertussis vaccine (PV) on the antibody response to sheep erythrocytes (SRBC) has been studied in vitro with the Mishell-Dutton immunization technique. The addition of PV to cultures of spleen cells obtained from normal non-immunized mice markedly enhanced the plaque-forming cell response to SRBC. The greatest enhancement was evident at 24 hr of culture. PV was also shown to enhance the antibody response of spleen cells that had been depleted of either T lymphocytes or adherent cells, presumably macrophages. In addition, it was found that PV, per se, released into the culture medium a soluble cell-free component(s) that contributed significantly to adjuvanticity. The results suggest that at least one of the ways that PV enhances the in vitro immune response to SRBC is by direct stimulation of precursors of antibody-forming cells.     

In Vitro Reconstitution of Anti-Sheep Erythrocyte Antibody Response of T Cell-Depleted Spleen Cells by Allogeneic T Cells or by Factors Derived from Them

Restoration of the impaired antibody response to sheep erythrocytes (SRBC) in cultures of mouse spleen cells, which were deprived of thymus-derived lymphocytes (T cells) by treatment with anti-mouse brain-associated θ (BAθ) antiserum and complement, was studied by adding a small portion of syngeneic or allogeneic normal spleen cells in vitro. Allogeneic spleen cells had a far greater effect than syngeneic spleen cells on the restoration, as far as the normal spleen cells added were able to recognize the alloantigens on the anti-BAθ serum-treated spleen cells (bone marrow-derived lymphocytes). Treatment of the allogeneic spleen cells with mitomycin C did not affect their activity in the restoration of the impaired antibody response. The possibility that the role of T cells in the antibody response to SRBC may be replaced by a nonspecific mediator derived from T cells reacting with allogeneic cells was proven by the finding that supernatant of the mixed allogeneic spleen cell cultures restored the impaired anti-SRBC antibody response of the T cell-depleted spleen cells. The effect of such culture supernatant on the restoration of the antibody response was greatest when it was added to the T cell-depleted spleen cell cultures one day after cultivation with SRBC, suggesting that the effectiveness may result from triggering of the proliferation and differentiation of antibody-forming cell precursors, which have already reacted with the antigen, to antibody-forming cells.     

Modulation of mitogen-induced spleen cell proliferation and the antibody-forming cell response by beta-endorphin in vivo

Experiments were conducted which compared the in vivo effects of beta-endorphin (BEP), gamma-endorphin (gamma EP), methionine-enkephalin (Met-ENK), and acetylated BEP(1-27) on the in vitro proliferative response of rat spleen cells to concanavalin A (ConA). In addition, the influence of BEP administration on the primary and secondary antibody-forming cell (AFC) response to the soluble antigen keyhole-limpet hemocyanin (KLH) was examined. Intravenous administration of BEP enhanced the spleen cell proliferative response to ConA assessed 3 hr after a single bolus infusion. Conversely, infusion with AcBEP(1-27) suppressed the proliferative response, whereas no effects of intravenous gamma EP or Met-ENK treatment were observed. The enhancing effect of BEP administration was not detectable 24 hr after a single infusion, but could be maintained over a 44 hr period by multiple infusions. The primary AFC response to KLH was suppressed by a dose of 1 nmole BEP only. On the other hand, the secondary IgG AFC response to KLH was enhanced by 10 pmoles BEP, while the IgM and IgA AFC responses remained unaltered by BEP treatment. The anamnestic in vitro proliferative response of spleen cells cultured with KLH was not altered if BEP was administered at the time of secondary KLH immunization. These results extend previous observations of BEP-induced modulation of in vitro immune function by demonstrating that opioid and nonopioid forms of BEP administered in vivo alter the capacity of spleen cells to proliferate and develop antibody responses to antigen.     

Protective effect of the aerosol immunization of mice with the polycomponent vaccine Immunovac VP-4 after the challenge of the animals with Klebsiella pneumoniae virulent strain

Used four schemes of the administration of the preparation with different time of the exposition of the animals in an aerosol chamber were tested with their subsequent intraperitoneal challenge with K. pneumoniae virulent strain K16. Irrespective of the number of immunization courses, the administration of the preparation made at intervals of 1 day, or daily, did not ensure any protective effect, but only led to an insignificant increase in their survival time in comparison with nonimmunized animals. After intervals between immunizations were increased to 3 days the protective effect of aerosol immumization was obtained (the survival rate was 65-80 % and considerably differed from that of the controls). The protective effect of aerosol immunization thus obtained was comparable with the effectiveness immunization made in a single subcutaneous injection. Aerosol immunization resulted in low antibody titers to the antigens contained in the vaccine, while after a single subcutaneous injection high antibody titers to Klebsiella and Proteus antigens were detected. The antigen-stimulated blast transformation of spleen lymphocytes in mice subjected to aerosol immunizations in 5 exposures was high. After subcutaneous immunization significant changes in such characteristics were detected on day 15. The data thus obtained were indicative of good prospects in the development Immunovac VP-4 as the medicinal form intended for use in aerosols.     

Changes in the histostructure of functional activity of the immunocompetent organs in damaged portal blood supply of the liver

In CBA mice calibrated stenosis of the portal vein was produced. Liver and immunocompetent organs were morphologically analyzed. The total number of hemopoietic stem cells in the bone marrow was estimated by the colony-forming cells and in the spleen after immunization with sheep red cells by the plaque forming method. It is established that stenosis of the portal vein (on the average by 45% and 58%) produced the histostructural changes in the liver and in the immunocompetent organs. Expression of morphological changes depended on the time elapsed after operation and the degree of the portal vein stenosis. These changes were the most pronounced on the 16-17th day when stenosis of the portal vein was 58%. The character of the changes in the number of the hemopoietic stem cells in the bone marrow and in that of antibody-forming cells in the spleen depended on the degree of the liver damage. These changes increased with the degree of the liver histostructure damage. The maximal liver damage was accompanied by a decrease of these indices.