Number Of Antibody-producing Cells Research Articles

Immunomodulatory properties of Cumaside

The medical lead, so-called Cumaside, was created on the basis of triterpene oligoglycosides from the Far-Eastern edible sea cucumber (holothurian) Cucumaria japonica and its immunomodulatory properties were studied. The haemolytic activity of Cumaside was significantly reduced in comparison with original glycosides due to the glycoside-cholesterol complex formation. The influence of Cumaside on mouse macrophages in low doses was accompanied by more then two-fold stimulation of lysosomal activity. This preparation was found to increase significantly the animal resistance against bacterial infections elicited by various pathogens. It stimulated phagocytosis, ROS formation, IL6 and TNF-α production in lymphocytes, increased the number of antibody producing cells and amplified the expression of several cell surface molecules (CD3, CD4, CD8) preliminary cultured with hydrocortisone. At the same time the preparation did not affect the delayed-type hypersensitivity, proliferative activity of lymphocytes, cytotoxic activity of NK-cells and cytokine IFNγ and IL12p70 release. The mechanism of Cumaside action is discussed.

Effect of sex ratio on the immune system of Oreochromis mossambicus (Peters)

The social environment of fish has a crucial role to play on the immune system and hence on the overall health status. Stressors of social origin such as dominance, subordination, and fight for mate have a major impact on the immune system of fish. The present study was designed with the objective of finding the effect of sex ratio of the population on the immune system of Oreochromis mossambicus. Groups of fish were maintained for 28 days in three different sex ratios i.e., (i) all-male (ii) all-female (iii) equal male and female (mixed). The specific immune response of fish was assessed by antibody response to Aeromonas hydrophila by ELISA and bacterial agglutination assay, and to SRBC by plaque forming cell assay. Nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, production of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) by peripheral blood leukocytes. Disease resistance against live, virulent A. hydrophila was performed to assess the overall functional immunity. The results showed that antibody responses and numbers of antibody producing cells were increased in fish in the equal male and female sex ratio group compared to fish in monosex ratio groups. Similar enhancement was also observed in nonspecific serum lysozyme level and the ROS and RNS production. The host resistance test revealed that enhanced immunity in equal male and female sex ratio group was protective against A. hydrophila infection. The study clearly reveals positive and negative effect of sex ratio on the immune system of O. mossambicus.

Effect of chronic confinement stress on the immune responses in different sex ratio groups of Oreochromis mossambicus (Peters)

Fish live in a complex and diverse aquatic environment. Various environmental stressors continuously challenge the physiology of fish. Social stress has a crucial role to play in immunity of fish. Hence, an attempt was made to investigate whether a stressor would have differential effect on the immune system of Oreochromis mossambicus when maintained in different sex ratios. Fish were crowded by confining at three different densities, i.e., 4 g l − 1 (control), 8 g l − 1 or 16 g l − 1 and in three different sex ratios, i.e., (i) all-male, (ii) all-female and (iii) equal male and female (mixed). Confinement stress was maintained for a period of 28 days and the fish were assessed for their specific immune response in terms of antibody response to Aeromonas hydrophila assayed by ELISA and plaque forming cell assay, nonspecific immune response in terms of intracellular ROS production by peripheral blood leukocytes, serum lysozyme level and integrated immunity by host resistance. The results showed that chronic confinement enhanced specific immunity in monosex ratio groups in terms of both antibody response, in the number of antibody producing cells. The nonspecific immune parameter and serum lysozyme level showed enhancement on confinement in all-male sex ratio group, whereas the ROS production showed suppression on confinement irrespective of sex ratio. The host resistance test revealed that enhanced specific immunity and lysozyme activity were not protective by showing 56% mortality in all-male confined group. Fish maintained as equal male and female group showed marginal level of protection in control group. Hence, the social environment in terms of sex ratio does have both positive and/or negative impact on the immune system depending on the degree of confinement.

Assays for B Lymphocyte Function

This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first basic protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. Alternatively, the number of antibody-producing cells can be quantified by plaque-forming cell (PFC) assays presented in this unit: the Cunningham-Szenberg and the Jerne-Nordin techniques. Both methods employ specially prepared slide chambers, described here, in which the antibody-producing B cells are mixed with complement and indicator sheep red blood cells (SRBC), or with trinitrophenol-modified SRBC (TNP-SRBC), with subsequent lysis and counting of plaques. Because IgM antibodies fix complement efficiently, whereas IgG and IgA antibodies do not, unmodified PFC assays measure only IgM antibodies. The assay can be modified, however, to measure all classes of antibodies or to enumerate total immunoglobulin-secreting B cells, as described in alternate protocols. Yet another method of measuring the number of antibody-producing B cells (in a class-specific fashion) is to use the ELISPOT technique described in UNIT 7.14. The resting B cells used in these procedures are prepared as described in the final support protocols for Percoll gradient centrifugation.

DSLET and ACTH(4-10) increase mitotic activity of hepatocytes and suppress antibody production.

The mitotic index of hepatocytes remained unchanged after 10 intraperitoneal injections of DSLET and ACTH(4-10) in doses of 0.5, 1.5, and 5 microg/kg, but increased after injection of these substances in doses of 50 and 150 microg/kg. DSLET in doses of 5, 50, and 150 microg/kg decreased the number of antibody-producing cells in the spleen. ACTH(4-10) possessed immunosuppressive activity not only in these doses, but also in a dose of 1.5 microg/kg. As differentiated from mitotic activity of hepatocytes, the degree of immunosuppression increased with increasing the dose of test peptides.

Immunotropic effects of gonadotropin-releasing hormone analog under conditions of emotional painful stress.

Surfagon, a synthetic analog of gonadotropin-releasing hormone injected before emotional painful stress dose-dependently changed the number of antibody-producing cells in rats and phagocytic and functional activities of neutrophils in mice. In castrated animals this peptide increased all studied parameters. This suggests that sex steroids are not involved in the realization of these effects.

Antibody-producing cells correlated to body weight in juvenile chinook salmon (Oncorhynchus tshawytscha) acclimated to optimal and elevated temperatures

The immune response of juvenile chinook salmon (Oncorhynchus tshawytscha) ranging in weight from approximately 10 to 55g was compared when the fish were acclimated to either 13 or 21°C. A haemolytic plaque assay was conducted to determine differences in the number of antibody-producing cells (APC) among fish of a similar age but different body weights. Regression analyses revealed significant increases in the number of APC with increasing body weight when fish were acclimated to either water temperature. These results emphasise the importance of standardising fish weight in immunological studies of salmonids before exploring the possible effects of acclimation temperatures.

Changes in Immunoglobulin Producing Cells in Response to Gonadal Maturation in Rainbow Trout

Changes in the number of immunoglobulin producing cells (IPC), immunoglobulin M (IgM) levels and leukocyte counts were investigated in rainbow trout reared under constant water temperature with special reference to gonadal maturation. During the spawning season, leucocytopenia, decreased number of antibody producing cells and low plasma IgM levels were observed. These reductions were apparently correlated with elevations in plasma cortisol (F), testosterone (T) and estradiol-17β in female and F, T and 11-ketotestosterone in males. In addition, decreased number of antibody producing cells in the skin lymphocytes and low mucus IgM levels were observed during the spawning season. These results suggest that endocrine changes related to gonadal maturation cause overall suppression of immune functions.

A 24,000 MW Trypanosoma cruzi antigen is a B-cell activator.

Trypanosoma cruzi, the causative agent of Chagas' disease, is a protozoan parasite that infects humans and other mammals in Central and Latin America. Several alterations of the immune response after infection have been described, such as severe immunosuppression of both cellular and humoral responses and massive polyclonal B- and T-cell activation, including the expansion of self-reactive clones. We have investigated the effects of the intraperitoneal injection of a recombinant 24,000 MW T. cruzi-specific antigen (rTc24) on the immune response of normal and deficient strains of mice. We analysed the in vivo and ex vivo levels of lymphocyte activation and the proliferative responses to rTc24 by determining the expression of CD69 activation marker and the levels of thymidine incorporation by spleen cells. The numbers of antibody-producing cells were determined by ELISPOT and the levels of immunoglobulin in the sera by isotype-specific enzyme-linked immunosorbent assay. We observed an increased [3H]thymidine ([3H]TdR) incorporation by spleen cells after rTc24 stimulation in vivo and in vitro. This proliferative activity induced by rTc24 was independent of the mouse strain used in the experiments (including C3H/HeJ mice) and ruled out the possibility that rTc24 preparations were contaminated by lipopolysaccharide. The injection of rTc24 protein induced preferentially the activation of B cells, as determined by the increased expression of CD69 molecules on IgM+ spleen cells. Considerable increases of IgM-secreting B cells were determined in both athymic and euthymic BALB/c mice. Mice that are deficient in B cells (BALB.Xid) responded to rTc24 but to a lesser extent. These increases in IgM B-cell numbers were accompanied by elevated levels of IgM immunoglobulins in the sera of injected animals. Our results suggest a role for rTc24 in B-cell activation.

Effect of tylosin tartrate on humoral immune responses in chickens.

While many antimicrobial agents have been reported to cause immunosuppression in animals, macrolide antibiotics enhance the immune function. Tylosin tartrate is a macrolide antibiotic approved for the control of mycoplasmosis in poultry. The purpose of this investigation was to determine the effect of tylosin on the humoral immune functions in chickens. Three days after oral tylosin tartrate administration, 4- or 8-week-old chickens were immunized intravenously with carbolic acid-killed Brucella abortus bacterin or sheep red blood cells. Seven days (plasma antibodies titre) or 4 days (antibody forming cells) post-immunization, there was a significant increase in antibody production as well as in the numbers of antibody-producing cells in tylosin tartrate-administered chickens compared with the untreated controls. However, 3 days after tylosin tartrate administration, there was no difference in the distribution of T-lymphocyte subpopulations (CD4 or CD8 positive cells) or B lymphocytes (surface immunoglobulin positive cells) in either the peripheral blood or spleens or untreated control chickens.

Effect of vitamin D on humoral and cell-mediated immune response and functional activity of peritoneal macrophages

1,25-Dihydroxyvitamin D3, an active metabolite of vitamin D, decreases the number of antibody-producing cells in C57B1/6 mice immunized with sheep erythrocytes, suppresses lymphocytes proliferation in response to stimulation with pokeweed mitogen and concanavalin A, and stimulates functional activity of macrophages.

Androgens and immunocompetence in salmonids: Specific binding in and reduced immunocompetence of salmonid lymphocytes exposed to natural and synthetic androgens

Leukocytes from spleen and anterior kidney of both juvenile chinook salmon ( Oncorhynchus tshawytscha) and rainbow trout ( O. mykiss) demonstrated specific binding of 3H-testosterone and the synthetic androgen 3H-mibolerone. Saturation analysis of leukocytes from anterior kidney of rainbow trout using 3H-mibolerone indicated a single class of high affinity binding sites with K d = 1.04 nM ± 0.31 nM and B max = 0.11 nM ± 0.01 nM. Specific binding of 3H-mibolerone could be displaced by the natural androgens testosterone and 11-ketotestosterone and by the synthetic androgens mibolerone and 17 α-methyltestosterone, while even 500-fold excess of the known immunosuppressive steroid cortisol did not displace specifically bound 3H-mibolerone. A link between these steroid binding sites and androgen induced immunosuppression is indicated by the fact that the androgens which demonstrated specificity for the binding sites (testosterone, mibolerone and 17α-methyltestosterone) all significantly reduced the number of antibody producing cells in in vitro cultures of chinook salmon anterior kidney leukocytes.

Correlation of pain sensitivity and the humoral immune response in mice during thermal stimulation

A hypothesis about a correlation between threshold pain sensitivity and antibody production is proposed and experimentally validated. Immunodeficient mouse strains are characterized by a higher threshold sensitivity to pain than animals with a normal immune response. A highly reliable negative correlation between threshold sensitivity to pain assessed by the hot plate test and the number of antibody-producing cells in the spleen after immunization with sheep red cells is observed in 77% of (CBA×C57B1/6) F1 mice examined. The negative correlation is observed both in spontaneous variations of threshold pain sensitivity and during an elevation of this threshold under the effect of preceding nociceptive stimulation.

Immunological responses during the organism's adaptation to a dosed thermal factor

Overheating of mice over the course of 10 and 20 days suppresses the proliferative activity of splenic cells in response to stimulation with phytohemagglutinin, concanavalin A, lipopolysaccharide, pokeweed mitogen, and alloantigens. The number of antibody-producing cells in the spleen drops on day 5 of overheating and is still low on days 10–20. Forty days after the start of overheating the functional activity of lymphocytes is restored. Overheating of animals does not change the colony-forming activity of hemopoietic stem cells.

Low number of antibody producing cells in patients with sickle cell anemia

B cell function is impaired in patients with sickle cell anemia. Although the number of surface IgM positive cells was similar in sickle cell patients and controls, in vitro spontaneous IgM, and PWM stimulated IgA, IgM, and IgG synthesis was significantly lower in the patients than in controls. The number of PWM induced and antigen specific immunoglobulin producing cells after immunization with Pneumovax, containing 21 serotypes of Streptococcus pneumoniae, was about 100-fold lower in the patients as compared with controls. Finally, the ability of the patients' peripheral blood mononuclear cells to proliferate in response to mitogens (PWM, SAC, PHA) was diminished. Because of the observed impairments in both nonspecific and antigen specific immunoglobulin synthesis and cell proliferation assays in the patients, we determined serum concentrations of IL-4 and IL-6, two cytokines associated with antibody production. IL-4 concentrations appeared low in sickle cell patients, and correlated with that of serum IgM. We hypothesize that B cell maturation in sickle cell patients is arrested at an IL-4 dependent stage.

Mechanisms of augmented resistance of cyclosporin A-treated mice to influenza virus infection by trehalose-6,6'-dimycolate.

Cyclosporin A (CsA), which is an immunosuppressive drug of helper T lymphocytes, diminished a resistance of mice to influenza virus infection. Mice inoculated intravenously with trehalose-6,6'-dimycolate (TDM, a glycolipid component of the cell wall of Mycobacterium) in an oil-in-water emulsion (TDM emulsion) recovered the resistance to influenza virus infection impaired by CsA. Number of antibody-producing cells was markedly reduced in CsA- and/or TDM-treated mice. Interferon production in lung of TDM-treated mice was augmented; however, it was extremely reduced not only in CsA-treated mice, but also in CsA- and TDM-treated mice. Activities of natural killer cells of CsA- and/or TDM-treated mice were not different from that of control mice. Numbers of lymphocytes in lung of TDM-treated mice and CsA- and TDM-treated mice were more predominantly increased than that of control mice. Analysis of lung lymphocytes by flow cytometry revealed no difference between the populations of L3T4+ T lymphocytes and Lyt-2.2+ T lymphocytes in CsA- and/or TDM-treated mice and the populations in control mice. However, the population of gamma delta T cell receptor positive (gamma delta TCR+) lymphocytes increased markedly in lung of TDM-treated mice and also CsA- and TDM-treated mice. In vitro experiments showed that macrophage cultures treated with TDM emulsion released a mediator(s) which activates T lymphocytes, but not B lymphocytes. These and our earlier results suggest that the recovered anti-influenza virus resistance of CsA-treated mice by treatment with TDM emulsion was caused by elicitation of macrophages with TDM, then activation of T lymphocytes, especially gamma delta TCR+ lymphocytes.

Preferential Proliferation of Anti-Dna Producing Cells of Nzb Mice in Nzbxzd Recipients

B cells from autoimmune NZB mice were transferred into unmanipulated non-autoimmune NZB.xid mice. The number of antibody-producing cells against various antigens in recipient mice was monitored at varying time after cell transfer using ELISPOT assay. NZB B cells producing antibody against all antigens we examined were able to proliferate in NZB.xid mice, which supports the idea of polyclonal B cell activation. However, anti-DNA producing cells proliferated most rapidly, and anti-BrMRBC producing cells proliferated more rapidly than B cells of other antigenic specificities. The percentage of anti-DNA producing cells in total immunoglobulin-producing cells increased over time whereas the percentage of anti-ovalbumin producing cells kept the same level. This indicates directly the preferential proliferation of NZB anti-DNA producing cells in NZB.xid mice. The result shows the responsibility of antigen-specific stimulation or activation on autoimmunity in the context of polyclonal B cell activation.

Ｖｉｂｒｉｏ ａｎｇｕｉｌｌａｒｕｍ Ｏ抗原で免疫した数種魚類における抗体産生細胞の出現

(Changes in the number of antibody producing cells were studied by measuring plaque forming cells (PFC) of the spleen and kidney in carp (Cyprinus carpio), rainbow trout(On corhynchus mykiss), ayu (Plecoglossus altivelis), Japanese eel(Anguilla japonica),Japanese flounder (Paralichthys olivaceus), and red seabream (Pagrus major) after an intraperitoneal injection with Vibrio anguillarum 0-antigen. It was confirmed in carp and ayu that the injected antigen was distributed at the highest levels in the spleen and kidney 3 to 6 h after the injection. PFCs appeared in the spleen and kidney of all the fish species tested. The number of PFC reached maximum value after 8-17 days. The number of PFC in the spleen was always higher than that in the kidney in carp, rainbow trout, ayu and eel, whereas in red seabream the former was lower than the latter. There was no difference in PFC number between the two organs in the Japanese flounder. In an additional experiment, ayu was immunized by immersing in the 0-antigen suspension (3g/l) for 30 min. As a result, no PFCs were detected in the spleen or kidney and no ag gulutinating antibody was measured in the serum.

Biochemical and immunological changes in mice following postweaning copper deficiency

Weanling albino male mice rapidly develop biochemical signs of copper deficiency when fed a purified diet containing 0.5 mg Cu/kg. Plasma ceruloplasmin activity of copper-deficient (-Cu) mice was 5% of that of copper-adequate (+Cu) control mice after only 3 d on the diet. More gradual loss of organ (liver, spleen, and thymus) cytochrome c oxidase activity was observed during the next 4 wk. Body weight was equivalent between +Cu and -Cu mice, but thymus weight dropped faster in -Cu mice than +Cu mice. The number of antibody producing cells to sheep erythrocytes was lower in -Cu mice compared to +Cu mice after 17 d on the diet. Spleen cytochrome oxidase activity of -Cu mice was 50% of that of +Cu mice by 10 d on the diet. Mitogenic response of splenic and thymic lymphocytes to concanavalin A (con A) was not greatly different between +Cu and -Cu mice. Splenocytes from -Cu mice had a 3-fold higher thymidine incorporation rate in the absence of mitogen compared to +Cu mice. The depressed antibody and high mitogenic background responses of -Cu mice were similar to previous work with another strain (C58) of mice that had been started on copper-deficient treatment from birth. However, the normal proliferative response to con A stimulation in postweaning copper deficiency differs from the previous model. Mice of both studies were very copper-deficient as judged by liver copper levels. Timing of the copper-deficient treatment influences the manner in which copper deficiency alters the immune response.

Establishment of a memory in vitro murine IgE response to benzylpenicillin and its resistance to suppression by anti-IL-4 antibody.

Regulation of a memory IgE antibody response may be different from the induction of a primary response and may, therefore, be more relevant to the study of allergic diseases and the therapeutic manipulation of IgE antibody formation. In this paper a murine hapten-specific in vitro memory IgE antibody response to benzylpenicilloyl(BPO)-KLH is described. The response was analyzed by determining the number of antibody-producing cells (APC) in an ELISA spot assay. Of the total number of BPO-specific APC (10,000 APC/10(6) cultured spleen cells), about 1% were IgE-producing cells (100/10(6) cultured cells), as detected on day 6 of culture. The level of the antibody response is antigen dose-dependent, and the detected APC are BPO specific. The memory IgE response is not inhibited by the addition of anti-IL-4 antibody (11B11), even at a high excess. In the presence of the mitogen lipopolysaccharide, it has been shown that switch of B cells to IgE is induced by IL-4, a process which can be inhibited by anti-IL-4 antibody. Because the antigen-induced IgE response cannot be inhibited by anti-IL-4 antibody, in vitro responding cells derived from BPO-KLH-preimmunized mice may, therefore, have already switched in vivo to IgE. On the other hand, B cells switching to IgE in a situation of cognate T-B cell interaction might receive IL-4 in a transsynaptical way from T cells which might not be accessible to inhibition by anti-IL-4 antibody. The identification of the two possibilities in situations of established allergic disorders will be decisive for determining whether pharmacological inhibition of IL-4 (or IL-4-induced switch)--e.g., by putative low molecular weight compounds--will ever be a meaningful approach to suppress allergic diseases.