Number Of Antibody-forming Cells Research Articles

Immunopharmacological aspects of studying immunotropic properties of a novel biocompound

The article presents an experimental study of immunotropic activity of a new compound, a metabiotic, based on metabolites (biologically active substances) produced by a of saprophytic Bacillus subtilis B-9909 strain, being safe for health and standardized at Russian National Collection of Industrial Microorganisms (RNCIM). The aim of our study was to experimentally evaluate the immunotropic effects of metabolites produced by the probiotic Bacillus subtilis B-9909 upon the parameters of cellular immunity tested in the animal model of induced toxic liver damage. The metabolites were isolated from the cultural liquid of Bacillus subtilis culture (RNCIM strain B-9909) during its deep cultivation in a medium containing hydrochloric acid hydrolyzate of soy flour, or pancreatic hydrolyzate of casein. At 16-18 hours of cultivation, the cells were in the exponential growth phase. The indices of cellular status were studied in experimental groups of animals, when assessing therapeutic efficacy of an experimental metabiotic sample, as compared to a group of laboratory animals treated with the reference drug (ursosan). We assessed the quantitative indicators of blood as follows: determination of phagocytic activity (FA) of peripheral blood neutrophils; measurement of metabolic activity in peripheral blood neutrophils by means of NBT-test; quantitative determination of T and B lymphocytes; the number of antibody-forming cells (AFC). Liver damage was studied in a model of acute toxic hepatitis in albino rats. Experimental toxic hepatitis was induced by 40% solution of CCl4 in vaseline oil injected intragastrically for 2 weeks at the dose of 0.2 g/kg. The results of experimental studies showed activation of phagocytic activity by peripheral blood neutrophils at the early terms of experiment, being also confirmed by the results of NBT-testing. Moreover, at the early observation terms, a significantly increased representation of T and B lymphocyte populations as well as AFC numbers, were revealed thus suggesting activation of all components of cellular immunity in response to the carbon tetrachloride toxicity. Thus, the reported studies on the cellular status of laboratory animals treated with metabolites produced by probiotic microorganisms of the genus Bacillus subtilis (B-9909) on laboratory animals with toxic liver damage allow us to conclude that the test sample of this metabiotic preparation exerts a significant immunomodulatory effect, when compared with ursosan. This evidence allows us to consider this compound a promising candidate drug for a new hepatoprotector with immunotropic effect.

Effect of endomorphins on humoral immune response, Th1/Th2/Th17 cytokine production and CD4<sup>+</sup>, CD8<sup>+</sup> lymphocyte apoptosis <i>in vivo</i>

Endogenous opioid peptides are a large group of physiologically active compounds with a pronounced affinity for opioid-type receptors, capable of showing pronounced analgesic activity, as well as having additional effects on the periphery, due to their wide distribution on the cells of many organs and tissues. Little studied representatives of this group are endomorphins, which due to their structure and properties, are capable of producing a strong antinociceptive effect after central administration, which means that, in the future, they can be considered as potential substitutes for low molecular weight opiates. The aim of this study is to evaluate the effect of endomorphins on the humoral immune response, the production of Th1/Th2/Th17 cytokines and apoptosis of CD4+, CD8+ lymphocytes in vivo. The splenocytes of Swiss white mice were used as the object of the study. The number of antibody-forming cells in the spleen was assessed using the method of local hemolysis in agarose gel according to Jerne. Quantitative determination of cytokines was carried out by enzyme-linked immunosorbent assay using kits (R&D, USA) according to the method proposed by the manufacturer. Apoptosis was assessed using Annexin V-FITC/7-AAD kit reagents (Beckman Coulter, USA) according to the manufacturer’s instructions by flow cytometry on a CytoFLEX S flow cytometer (Beckman Coulter, USA). In the course of the study, it was found that endomorphins enhance the antibody genesis of the spleen, and the preliminary blockade of opiate receptors with naloxone led to the cancellation of the stimulating effect of peptides. Endomorphins didn’t affect splenocyte production of IL-2, IL-4, and IFNg, however, the introduction of endomorphin-2 naloxone-independent enhanced the induced production of IL-17. Evaluation of the effect of endomorphins on apoptosis of splenocytes in 24-h cultures showed that endomorphin-2 in unstimulated cultures of naloxone-dependently increased the percentage of late apoptosis of CD8+ lymphocytes, however, in stimulated cultures, both endomorphins increased the apoptotic activity of CD8+ lymphocytes, regardless of the preliminary blockade of opioid receptors. In summary, we can say that in the in vivo system, endomorphins have a wide range of multidirectional immunomodulatory effects, which may be of interest for practical use in the future.

Role of MP-4 myelopeptide in regulation of antibody production and functional activity of macrophages under stress conditions

Myelopeptides are endogenous low-molecular weight peptides with immunoregulatory activity. Each of these myelopeptides has an individual sequence and plays a role in correction of immune system disorders. In our earlier studies, we have shown immunomodulatory effect of MP-3, MP-5, MP-6 myelopeptides upon functioning of peritoneal macrophages under the conditions of immobilization and cold stresses. The aim of this work was to investigate the effect of MP-4 myelopeptide upon antibody formation, production of reactive oxygen species, and absorptive activity of peritoneal macrophages in mice under the immobilization stress. Materials and methods. The in vivo experiments were carried out in 24 male Swiss mice weighing 17-22 g. Two-or six-hour immobilization stress was produced in the animals. MP-4 was injected intraperitoneally 30 min before the onset of stress, at a dose of 40 μg/kg. The animals were divided into 4 groups: 1, control; 2, stress; 3, stress + MP-4; 4, MP-4 injection. On day +5, cellularity and the number of antibody-forming cells (AOC) in spleen were assessed by the method of local hemolysis in an agarose gel according to Jerne. Production of reactive oxygen species by peritoneal cells was assessed using the luminol-dependent chemiluminescence (LZHL) reaction. The absorption capacity of peritoneal macrophages was assessed using a standard method using a BDFACSCalibur laser flow cytometer. Labeled St. aureus at a final concentration of 108 cells/ml were used as phagocytosis targets. Statistical analysis was performed using the unpaired Student's t-test. Results. It was found that two-hour immobilization stress did not affect the absorption activity of peritoneal leukocytes, whereas six-hour stress suppressed phagocytosis. Injection of MP-4 in stress-exposed animals did not lead to changes in phagocytic activity of peritoneal macrophages. MP-4 did not correct the stress-induced decrease in the number of antibody-forming cells from the spleen during six hours of immobilization, nor stress-induced increase in absolute amounts of AOC under the two-hour immobilization stress. In the group of animals receiving MP-4 and immobilized for two-hours, an increase in the number of nucleated cells was increased, as compared with the control group. Thus, in contrast to the previously studied myelopeptides, MP-4 did not show pronounced immunomodulatory effects upon these parameters.

The composition of peripheral immunocompetent cell subpopulations and cytokine content in the brain structures of mutant Disc1-Q31L mice.

The DISC1 (disrupted in sсhizophrenia 1) gene is associated with brain dysfunctions, which are involved in a variety of mental disorders, such as schizophrenia, depression and bipolar disorder. This is the first study to examine the immune parameters in Disc1-Q31L mice with a point mutation in the second exon of the DISC1 gene compared to mice of the C57BL/6NCrl strain (WT, wild type). A flow cytometry assay has shown that intact Disc1- Q31L mice differ from the WT strain by an increase in the percentage of CD3+ T cells, CD3+CD4+ Т helper cells and CD3+CD4+CD25+ T regulatory cells and a decrease in CD3+CD8+ T cytotoxic/suppressor cells in the peripheral blood. A multiplex analysis revealed differences in the content of cytokines in the brain structures of Disc1-Q31L mice compared to WT mice. The content of pro-inflammatory cytokines was increased in the frontal cortex (IL-6, IL- 17 and IFNγ) and striatum (IFNγ), and decreased in the hippocampus and hypothalamus. At the same time, the levels of IL-1β were decreased in all structures being examined. In addition, the content of anti-inflammatory cytokines IL-4 was increased in the frontal cortex, while IL-10 amount was decreased in the hippocampus. Immune response to sheep red blood cells analyzed by the number of antibody-forming cells in the spleen was higher in Disc1-Q31L mice at the peak of the reaction than in WT mice. Thus, Disc1-Q31L mice are characterized by changes in the pattern of cytokines in the brain structures, an amplification of the peripheral T-cell link with an increase in the content of the subpopulations of CD3+CD4+ T helpers and CD3+CD4+CD25+ T regulatory cells, as well as elevated immune reactivity to antigen in the spleen.

Immune Activity of Polysaccharide Fractions Isolated from Korean Red Ginseng

Korean red ginseng (KRG)’s pharmacological efficacy and popular immunomodulatory effects have already been proven in many studies; however, the component of KRG that is effective in immune activity has not been studied before. Therefore, this study extracted and separated KRG for an immune activity comparison. In the water fraction obtained by extracting KRG powder with water, a red ginseng neutral polysaccharide (RGNP) fraction and a red ginseng acidic polysaccharide (RGAP) fraction were obtained. Each fraction was orally administered for 10 days to mice with reduced immunity, and the number of IgM antibody-forming cells (AFCs) in splenocytes was measured to compare the immune activity of the water fractions. The results showed that the RGAP fraction has the greatest number of AFCs. To set the optimal dose of the RGAP fraction, which had the highest immune activity, the AFCs, macrophage activity, and splenocyte subtype in the mice were analyzed. As a result, the number of AFCs was significantly increased in the RGAP fraction compared to RGNP. The intraperitoneal macrophage phagocytosis activity and the number of T cells, B cells, and macrophages in the spleen increased significantly. It can, therefore, be confirmed that immune activity increases by a fraction containing higher RGAP content, and we hypothesize that RGAP activates immune activity.

Effect of carpesium abrotanoides petroleum ether fraction on phagocytic function of macrophages and antibody forming cell in mice

Objective To study the effects of carpesium abrotanoides petroleum ether fraction on phagocytic function of macrophages and antibody forming cell in mice. Methods The extract carpesium abrotanoides petroleum ether fraction, forty five mice were divided into the control group and low and high dose groups of carpesium abrotanoides petroleum ether fraction by random number table method, 15 mices in each group. The low-dose and high-dose groups of carpesium abrotanoides petroleum ether fraction were given 50 mg/kg and 100 mg/kg respectively by gavage for 10 days. The spleen weight, growth index, the phagocytosis of macrophages and the number of antibody-forming cells in peritoneal macrophages of mice were detected by chicken erythrocyte phagocytosis test and hemolytic plaque test. Results Compared with the control group, the spleen weight (192.4 ± 11.49 mg, 204.6 ± 10.59 mg vs. 117.6 ± 10.89 mg), the growth index (6.04 ± 0.54, 6.06 ± 0.40 vs. 3.89 ± 0.14), antibody forming (1 216.4 ± 94.1, 1 548.8 ± 86.4 vs.361.0 ± 11.7), phagocytosis percentage of macrophages (58.60% ± 2.60%, 72.0% ± 3.08% vs. 35.49% ± 1.64%), and Phagocytosis index (2.01 ± 0.10, 2.69 ± 0.15 vs. 0.37 ± 0.06) of the groups of low doses and high doses of Carpesium abrotanoides Petroleum ether fraction significantly increased (P<0.05). Conclusions The low doses and high doses of Carpesium abrotanoides Petroleum ether fraction can enhance the mice's specific and nonspecific immune function,and protect the immune system of mice. Key words: Carpesium abrotanoides; Macrophages; Immune function; Mice

Иммуномодулирующая активность форситозида В при экспериментальной иммуносупрессии

Цель исследования - изучение иммуномодулирующего дейтсивя фенилпропаноида (форситозида В, ForВ), выделенного из клубней зопника клубненосного ( Phlomoides tuberosa (L.) Moench) на показатели клеточного и гуморального звеньев иммунного ответа при азатиоприновой иммуносупрессии. Методика. Опыты проведены на 100 мышах-самцах линии F1 (СВАхС57Вl/6) массой 18-20 г. Иммунодефицит моделировали внутрижелудочным введением азатиоприна в дозе 50 мг/кг в течение 5 сут. (контрольная группа). Опытная группа животных получала ForВ 1 раз в сутки внутрижелудочно в дозе 1 мг/кг в течение 14 сут. на фоне азатиоприновой иммуносупрессии. Интактная группа мышей получала очищенную воду по аналогичной схеме. Показатели клеточного звена иммунного ответа оценивали в реакции гиперчувствительности замедленного типа (ГЗТ) согласно стандартной методике локальной ГЗТ, гуморального звена - по количеству антителообразующих клеток (АОК), определяемых методом локального гемолиза по A.J. Cunningham (1965). Статистическую обработку результатов проводили стандартными методами вариационной статистики с использованием параметрического t -критерия Cтьюдента. Результаты. Установлено, что курсовое применение форситозида В способно ослаблять иммуносупрессивное действие цитостатика азатиоприна - индекс реакции гиперчувствительности замедленного типа возрастал в 1,5 раза, абсолютное и относительное число антителообразующих клеток в 1,3 и 1,9 раза соответственно. Заключение. Форситозид В, выделенный из клубней P.tuberosa обладает иммуномодулирующим действием в отношении клеточного и гуморального звеньев иммунного ответа при азатиоприновой иммуносупрессии, что обосновывает целесообразность его дальнейшего исследования для создания новых эффективных иммуномодуляторов. The purpose of the study is to determine the immune modulating activity of phenylpropanoid (forsythoside B, ForB) isolated from the tubers of Phlomoides tuberosa (L.) Moench (Lamiaceae) on the cellular and humoral immune response at the azathioprine immune suppression. Methods. Experiments were carried out on CBA male mice (n = 100, 18-20 g). Immune deficiency was modeled by the intragastrical introduction of azathioprine in the dose 50 mg/kg once a day for 5 days. The experimental group of animals administered ForB once a day intragastrically in dose 1 mg/kg for 14 days against the background of azathioprine immune suppression. The intact group administered the purified water according to the analogous scheme. The activity of the tested compound on the cellular immunity was evaluated in the reaction of delayed-type hypersensitivity (DTH) according to the standard method of local DTH. Condition of the humoral immunity was estimated by the number of antibody-forming cells (AFC) determined by the method of local hemolysis by A.J. Cunningham (1965). Statistical analysis was performed using standard methods of variation statistics using parametric Student’s t-test. Results. It was established that the administration of the ForB to animals significantly reduced suppressive effect of cytostatic azathioprine on the DTH reaction and antibody response. The use of the tested compound was resulted to the increase of the DTH-index (1.5 times) and the value of absolute and relative numbers of AFC in 1.3 and 1.9 times comparing with the control group. Conclusion. The results obtained allow to conclude that the ForB showed the marked immune modulating activity at the azathioprine-induced immune suppression demonstrating its perceptiveness as a new and effective immune modulators.

Phenolic compounds of ethanol extracts of Lemna minor L., Lemna trisulca L. and Lemna polyrrhiza L. Schleid and their immunomodulating activity

The purpose of the study is to determine the composition of the phenolic compounds of ethanol extracts isolated from three species of duckweed: Lemna minor L., Lemna trisulca L. and Lemna polyrrhiza L. (the synonym is Spirodella polyrrhiza Schleid.) and to study its effect on immune system activation. Materials and methods. The objects of the study are: air-dried grass samples of Lemna minor L., Lemna trisuica L. and Lemna polyrrhiza L. collected during their growing season in 2010–2011 in low-flow and stagnant ponds of Kozhevnikovsky and Tomsk districts of Tomsk region. Isolation of polyphenolic complexes (PFC) was carried out by extraction of air-dried raw material with ethyl alcohol. In qualitative and quantitative analysis of the samples studied the method of reversed-phase high-performance liquid chromatography on an Agilent 1100 Series instrument (USA) was used in isocratic mode. In the experiments, 200 male C57BL / 6 and BALB / C mice aged 8–12 weeks were used to determine immunomodulatory activity. Cell proliferation was assessed by a colorimetric method. The absorption of the obtained solutions was measured with a multi-channel spectrophotometer at the wavelength of 540 nm. The determination of antibody-forming cells in the spleen was performed by local hemolysis. The titer of antibodies in serum was evaluated in the hemagglutination reaction. The local hypersensitivity reaction of immediate type was assessed by the author’s modification. Results. For the first time the study of the qualitative composition and quantitative content of PFC of Lemna minor L. (LM) , Lemna trisulca L. (LT) trisulkas, and Lemna polyrrhiza L. (LP) : (4,7 ± 0,4)%, (3,3 ± 0,3)%, (12,8 ± 0,7)% was carried out. The highest content of phenolic acids (10,76%) and the sum of flavonoids, isoflavonoids and coumarins (14,7%) were found in the LP sample. The content of chlorogenic and 3,5-dihydroxybenzoic acids was 2–9 times higher in LP than in other species of duckweed. In LM and LT with concentrations of 5 μg/ml and LP in the range of 0,5–160 μg/ml did not have a toxic effect on antigen presenting cells. When incubated with LT (20 μg/ml), the proliferation of macrophages was reduced by 1,2 times, and when incubated with LM and LT (80 μg/ml), by 1,2 and 1,8 times, respectively. PFC duckweed (LP) did not have a similar effect. LM and LP had a stimulating effect on the production of nitrites in the concentrations of 5 and 20 μg/ml, increasing it by 1,3–1,6 times. Course introduction of LT and LP led to a significant decrease in the number of antibody-forming cells by 1,5 and 2,3 times and a decrease in the local hypersensitivity reaction by 1,9 and 1,5 times, respectively.

Results of Experimental Studies of the Immunotropic Action of Polyprenols Extracted from Alcea Nudiflora

Methods of extracting polyprenols from Alcea nudiflora and their immunotropic actions were studied. Administration of Alcea nudiflora polyprenols to mice simultaneously with immunization with sheep erythrocytes produced marked stimulation of primary antibody formation, apparent as an increase in the number of antibody-forming cells in the spleen. In addition, there were increases in the cellularity of the central and peripheral organs of immunity, along with increases in blood erythrocyte and leukocyte levels. The immunomodulatory action of Alcea nudiflora polyprenols was seen not only in normal animals, but also in animals with secondary immunodeficiency states developing in acute toxic hepatitis and sublethal irradiation. In terms of activity in these experiments, Alcea nudiflora polyprenols were as active as the known immunostimulatory substance Immunal.

Effect of Diprotin A, an Inhibitor of Dipeptidyl Peptidase IV, on Immunological Parameters of Lymphocytes in Intact Animals and Animals with Experimental Autoimmune Process.

We found the peculiarities of the effects of dipeptidyl peptidase IV inhibitor diprotin A on immunological parameters of lymphocytes in lymphoid organs in intact animals and in animals with experimental autoimmune process. In intact animals, diprotin A increased cellularity of the thymus and spleen, number of CD4+, CD8+, and CD4+8+ thymocytes and CD3+ splenocytes and reduced cellularity of lymph nodes and the number of antibody-forming cells in the spleen. Diprotin A administered against the background of autoimmune process, reduced thymus cellularity, number of antibody-forming cells in the spleen, and number of CD4 and CD4+8+ thymocytes in comparison with the corresponding parameters of autoimmune process.

Changes of cell-mediated and humoral immune response, absolute cell number and their phagocytic activity in peritoneal cavity at experimental thyrotoxicosis in rats

Changes of cell-mediated and humoral immune response, absolute cell number and their phagocytic activity in peritoneal cavity at experimental thyrotoxicosis in rats

EFFECT OF POLYPHENOLIC COMPOUNDS ISOLATED FROM CARTHAMUS TINCTORIUS AND CALENDULA OFFICINALIS L., ON FUNCTIONAL ACTIVITY OF IMMUNE CELLS UNDER CONDITIONS OF CYTOSTATIC IMMUNOSUPPRESSION

The purpose of the study – to study the effect of polyphenolic compounds extracted from the flowers of safflower oil and calendula, the functional activity of immune cells in cytotoxic immune suppression.Conventional methods determined the total number of splenocytes, relative (%) and absolute (106), the number of antibody-forming cells (AFC) in the spleen of mice by local hemolysis by Cunningham.Evaluated the effect of compounds of natural origin on the cellular immune response in the delayed-type hypersensitivity (DTH). Phagocytic activity of peritoneal macrophages was determined by the method based on the intensity of their capture ink particles. We studied the functional activity of peritoneal macrophages by NBT test (spontaneous and stimulated). Studies were conducted on male mice Category 1 (conventional linear mouse) line CBA/CaLac aged 2–2.5 months, weighing 20–22 g. After the introduction mice line CBA/CaLac of polyphenolic compounds derived from flowers of Carthamus tinctorius and flowers of Calendula officinalis L. during the 5-day course in dose 50 mg/kg was observed stimulation of the humoral immune response (total number of splenocytes, the number of antibodies in the spleen cells) and the functional activity of macrophages and Immunomodulating effect on the humoral immunity and the functional activity of macrophages after a single injection of cyclophosphamide in dose 250 mg/kg. Immunotropic activity of polyphenolic compounds is higher than that those of the reference product of tincture of Echinacea purpurea.

Popliteal lymph node (PLN) assay to study adjuvant effects on respiratory allergy

Different variants of the popliteal lymph node (PLN) assay have been published. Here we describe the adjuvant popliteal lymph node assay, an immune response assay to study the adjuvant activity of soluble substances as well as particulate matter. The substance to be studied for adjuvant activity is injected into the hind footpad of mice or rats together with an antigen. Adjuvant activity is determined as the increase in PLN weight and cell numbers in animals receiving antigen together with the substance under study, compared with PLN weight and cell numbers in animals given the antigen without the substance in question, and animals given the putative adjuvant alone. Because lymph node weight and cell numbers are immunologically non-specific parameters, specific immune response assays like serum antibody responses or antibody-forming cell numbers should additionally be performed. Different antigens and immune response assays may be used, depending on the research question asked. In relation to respiratory (or food) allergy, the assays should as a minimum include determination of specific IgE in serum, and preferably also IgG1 (mouse). Serum specific IgG2a antibody determination may be added to get an indication of the Th1–Th2-balance of the response. The adjuvant PLN assay, with cellular response assays performed in the draining popliteal lymph node and antibody determinations in serum, requires small amounts of test material. The assay offers a practical, sensitive and reproducible method to determine the adjuvant activity of soluble substances as well as particulate material, with the possibility to also perform mechanistic studies.

In Vivo and in Vitro Immunosuppressive Effects of Benzo[k]fluoranthene in Female Balb/c Mice

Although polycyclic aromatic hydrocarbons (PAHs) have been known to suppress immune responses, few studies have addressed the immunotoxicity of benzo[k]fluoranthene (B[k]F). In this study, we investigated the immunosuppression by B[k]F, both in vivo and in vitro, in female BALB/c mice. To assess the effects of B[k]F on humoral immunity as splenic antibody response to sheep red blood cells (SRBCs), B[k]F was given a single dose or once daily for 7 consecutive days po with 30, 60, and 120 μmol/kg. B[k]F reduced the number of antibody-forming cells (AFCs) in a dose-dependent manner. Subacute treatment with B[k]F caused weight increases in liver and decreases in spleen and thymus. The number of AFCs was dramatically decreased by B[k]F in a dose-dependent manner. In a subsequent study, mice were subacutely exposed to the same doses of B[k]F without an immunization with SRBCs, followed by splenic and thymic lymphocyte phenotypings using a flow cytometry and ex vivo mitogen-stimulated proliferation. B[k]F-exposed mice exhibited reduced splenic and thymic cellularity, decreased numbers of total T cells, CD4+ cells, and CD8+ cells in spleen, and immature CD4+CD8+ cells, CD4+CD8− cells, and CD8+CD4− cells in thymus. The number of CD4+ IL-2+ cells was reduced by about 11%, 31%, and 53% following exposure of mice to 30, 60, and 120 μmol/kg of B[k]F, respectively. In the ex vivo lymphocyte proliferation assay, B[k]F inhibited splenocyte proliferation by LPS and Con A. In the in vitro mitogen-stimulated proliferation by untreated splenic suspensions, B[k]F only suppressed splenocyte proliferation to LPS. These results suggested that B[k]F-induced immunosuppression might be mediated, at least in part, through the IL-2 production, and caused by mechanisms associated with metabolic processes.

The Effects of Rotation Stress on Measures of Immunity. The Role of Opiate Receptors

Experiments were performed on mongrel male rats to study the effects of blockade of delta, mu, and kappa opiate receptors on antibody formation, delayed-type hypersensitivity (DTH), and changes in the numbers of antibody-forming cells (AFC) and nucleated cells in the lymph nodes and spleen in a local form of immune response in a model of rotation stress. These studies showed that rotation stress led to mild suppression of immune inflammation in DTH, significant increases in the numbers of AFC and nucleated cells in regional lymph nodes, but no change in the peripheral blood antibody titer. Blockade of delta, mu, and kappa opiate receptors with naloxone altered these stress-induced effects. It is suggested that abolition of the stimulating effect of rotation stress on the number of AFC and depression of DTH may be associated with blockade of the effects of beta-endorphin and met-enkephalin, which act predominantly via stimulation of delta receptors.

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.

Benzo[ a]pyrene-induced immunotoxicity in Japanese medaka ( Oryzias latipes): relationship between lymphoid CYP1A activity and humoral immune suppression

Exposure to the environmental contaminant benzo[ a]pyrene (BaP) results in suppression of immune function in both mammalian and fish species. This laboratory has previously demonstrated that a single intraperitoneal (IP) injection of BaP reduced lymphocyte proliferation, phagocyte-mediated superoxide generation, and antibody-forming cell (AFC) numbers in Japanese medaka ( Oryzias latipes). The objective of the current study was to determine the role of BaP metabolism in the observed immunosuppression. Results from rodent studies have suggested that BaP elicits its immunotoxic effects via upregulation of cytochrome P4501A1 (CYP1A1) and the subsequent production of immunosuppressive BaP metabolites. In this study, exposure of medaka to 200 μg BaP/g BW significantly induced CYP1A expression or activity within lymphoid tissue 48 h post-IP injection; induction was observed specifically within distinct subpopulations of kidney mononuclear cells. Concurrent injection of fish with BaP and the CYP1A1 inhibitors α-naphthoflavone (ANF) or dehydroepiandrosterone (DHEA) resulted in inhibition of renal EROD activity and amelioration of BaP-induced suppression of medaka AFC numbers. Results of this study suggest that (1) BaP-induced suppression of medaka humoral immunity relies upon the CYP1A-catalyzed production of immunotoxic BaP metabolites and (2) BaP metabolites may be created in situ, directly by specific cells within kidney lymphoid tissue. Thus, apparently, mechanisms involved in BaP-induced immunosuppression have been phylogenetically conserved from fish to mammals.

Local and systemic antibody responses in mice immunized intranasally with native and detergent-extracted outer membrane vesicles from Neisseria meningitidis.

The mouse humoral immune response toward native or detergent-extracted outer membrane vesicles (NOMVs and DOMVs, respectively) from Neisseria meningitidis was determined after intranasal immunization. Both preparations elicited high frequencies of NOMV-specific antibody-forming cells (AFCs) locally in the nasal associated lymphoid tissue (NALT) after three or four weekly doses. The diffuse NALT (D-NALT) contained ca. 10-fold more NOMV-specific AFCs than those observed in the mediastinal lymph node, spleen, and bone marrow. AFCs observed in the D-NALT were primarily immunoglobulin A positive (IgA(+)) and were maintained for at least 1 month. In contrast, the organized NALT (O-NALT) contained low numbers of AFCs, and the response was relatively short-lived. In other lymphoid tissues, AFCs producing various IgG subclasses and IgM were present with IgG2b-producing AFCs being dominant or codominant with IgA or IgG2a. In serum and in all of the tissues examined, with the exception of the NALT, NOMVs clearly induced a stronger antibody response and a broader range of antibody isotypes than DOMVs. The development of NOMV-specific AFCs in spleen and bone marrow after intranasal immunization was slow compared to intravenous immunization but, once established, the intranasally elicited responses increased steadily for at least 75 days. NOMV-specific antibodies induced via several routes of immunization had high bactericidal activities in serum. Our results indicated that intranasally administered OMVs induced strong local and systemic antibody responses in mice that were relatively long-lived.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters the regulation and posttranslational modification of p27kip1 in lipopolysaccharide-activated B cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters B-cell differentiation, as evidenced by a marked decrease in immunoglobulin M (IgM) secretion and in the number of antibody-forming cells (AFC) induced by antigenic stimulation. The objective of the present studies was to evaluate the effect of TCDD on the level of p27kip1, a cyclin-dependent kinase inhibitor that is a critical regulator of cellular differentiation. In the well-characterized B-cell line, CH12.LX, a modest decrease in p27kip1 was observed during the initial 24-h post-LPS (lipopolysaccharide) activation, which then gradually increased above background at 48 and 72 h. Conversely, in the presence of TCDD, p27kip1 was not induced and remained unchanged from LPS unstimulated cells throughout the entire 72-h period post-LPS activation. In addition, Western blotting revealed that TCDD treatment altered the profile of p27kip1 migration as compared to the LPS-activated control. Time-of-addition studies demonstrated that the greatest sensitivity of p27kip1 to TCDD treatment occurred within the initial 24-h post-LPS activation. Interestingly, LPS-induced Ig kappa light chain and IgM secretion also exhibited the greatest period of sensitivity (i.e., inhibition) to TCDD during the first 24-h post-LPS activation. In addition, TCDD markedly suppressed the LPS-induced differentiation of CH12.LX cells into IgM secreting AFC, with a modest but cumulative effect on cell proliferation over a 72-h period. Collectively, these findings show that TCDD altered the cellular concentration and posttranslational modification of p27kip1 in this activated B-cell line model, which occurred concomitantly with altered B-cell differentiation and suggests that cyclin-dependent kinase inhibitors may be an important intracellular target in TCDD-mediated inhibition of B-cell differentiation.

The Japanese medaka (Oryzias latipes) model: applicability for investigating the immunosuppressive effects of the aquatic pollutant benzo[a]pyrene (BaP)

Despite the fact that BaP is a carcinogen, mammalian immunosuppressant, and ubiquitous aquatic pollutant, knowledge regarding the effects of BaP on the immune system of fish is still lacking. To begin to fill this gap, studies were conducted in medaka to examine the effects and mechanisms by which BaP exposure might alter host immunocompetence. Fish, exposed by IP injection of BaP (2–600 μg/g BW), were examined after 48 h for effects upon immune function and CYP1A expression/activity. Benzo[a]pyrene, at a concentration below that which increased levels of CYP1A expression/activity (2 μg BaP/g BW) suppressed lymphocyte proliferation. Concentrations of BaP at 20 and 200 μg/g BW, suppressed antibody-forming cell (AFC) numbers, superoxide production, and host resistance against bacteria. In contrast, exposure to the low affinity aryl hydrocarbon receptor (AhR) agonist, benzo[e]pyrene (BeP), neither induced CYP1A expression nor altered immune function. Given the lack of immunosuppressive effects produced by BeP, and the fact that exposure to the AhR antagonist (and CYP1A inhibitor) α-naphthoflavone (ANF) ameliorated the suppressive effects of BaP upon AFC numbers, the AhR pathway (including CYP1A-mediated production of reactive BaP metabolites) appears important in mediating BaP-induced immunotoxicity in fish, as in mammals. In the past, the medaka has proven a successful model for assessing carcinogenic agents. These studies have demonstrated its utility for also determining the immunosuppressive effects of an important aquatic contaminant.