Hemolytic Plaque-forming Cells Research Articles

Effect of JUNCAO-cultivated <i>Ganoderma lucidum</i> spent mushroom substrate-hot water extract on immune function in mice

Purpose: To evaluate the effect of JUNCAO-cultivated Ganoderma lucidum spent mushroom substratehot water extract (SMSG-HWE) on murine immunity. Methods: Five groups of mice (n = 10) received water with 0.00, 0.14, 0.28, 0.84, or 1.68 g/kg of SWSG-HWE, respectively, orally for 30 days. Various biochemical parameters of serum and tissues, including spleen and thymus indices, were determined were determined for the mice Results: The following markers were significantly higher in the 0.84 g/kg SMSG-HWE group than in the control group (all p < 0.05): splenic lymphocyte proliferation, a marker of cell-mediate immunity; dinitrofluorobenzene-induced delayed hypersensitivity; and the number of haemolytic plaque-forming cells, as a marker of humoral immunity. Phagocytic rate, which evaluates mononuclear-macrophage function as a marker of innate immunity, was significantly higher in both the 0.84 g/kg HWE and 1.68 g/kg SWSG-HWE groups, while phagocytic index was significantly higher in the 0.28 g/kg SWSG-HWE group, compared to the control group (all p < 0.05). Natural killer cell activity also was significantly enhanced in the 0.84 g/kg and 1.68 g/kg SWSG-HWE groups (p < 0.05). Conclusion: These findings indicate that SWSG-HWE enhances murine immune function, and may be suitable as a potential additive in animal feed. Keywords: Hot water extract, Cell-mediated immune function, Concanavalin A, Mononuclearmacrophage function, Haemolytic plaque, Humoral immunity, Innate immunity, Animal feed additive

Effect of a seashell protein Haishengsu on the immunological function of mice with Ehrlich ascites tumor

This study was designed to investigate the effect of a seashell protein Haishengsu (HSS) on the immuno logical function in mice with Ehrlich ascites tumor. Ehrlich ascites tumor-bearing mice were divided into three HSS groups (25, 50 and 100 mg/kg, i.v., respectively), cyclophosphamide (10 mg i.p.) and control group. The immunological function was assessed by measuring the phagocytizing capacity of the peritoneal macrophages and neutrophils, as well as the number of spleen hemolytic plaque-forming cells. The percentage of blood T-lymphocytes was also evaluated. The number and the percentage of phagocytizing macrophages and neutrophils in the 50 and 100 mg/kg HSS groups were higher than in the control and the cyclophosphamide groups (P < 0.01). The hemolytic plaque-forming cells in the three HSS groups (10.8 ± 1.2, 16.9 ± 3.9 and 25.3 ± 2.9, respectively), was greater than in the control (7.3 ± 1.4), or the cyclophosphamide group (0.33 ± 0.4) (all P < 0.01). In all HSS groups, the percentage of blood T3, T4 and T8 was higher than in the cyclophosphamide and the control group (all P < 0.01). We conclude that HSS has significant immune-modulating effect in mice with Ehrlich ascites tumor.

Cumulative Effects of Natural and Anthropogenic Stress on Immune Function and Disease Resistance in Juvenile Chinook Salmon

Abstract Previous studies have shown that juvenile chinook salmon Oncorhynchus tshawytscha exposed in the field or the laboratory to polychlorinated biphenyls (PCBs), an anthropogenic stressor, are immunosuppressed. It is not known whether simultaneous exposure to natural stressors can increase this immunosuppression. To examine the effects of natural and anthropogenic stressors on immune function, we infected juvenile chinook salmon with metacercariae of the trematode Nanophyetus salmincola by exposing the fish to infected freshwater snails Juga plicifera. Infected (>300 metacercariae per fish) and noninfected salmon were then injected with either the commercial PCB mixture Aroclor 1254 or an acetone–emulphor carrier. B cell function was examined by in vitro hemolytic plaque-forming cell (PFC) assay. Nanophyetus salmincola infection resulted in significantly lower anterior kidney primary PFCs and lower splenic secondary PFCs. The combination of N. salmincola infection and Aroclor 1254 exposure caused a l...

Effect of hot water extract from Agaricus blazei Murill on antibody-producing cells in mice

We investigated the immunopotentiating activities of boiled water-soluble extracts from desiccated Agaricus blazei Murill (ABM). Effect of ABM extract on antibody production was investigated by method of hemolytic plaque-forming cells (PFC) against sheep red blood cells (SRBC) antigen. ABM extracts significantly ( p<0.01) increased the number of PFC in spleen with intraperitoneal administration at doses of 25 mg/kg as compared with control group. The populations of Mac-1- or CD25-positive cells significantly ( p<0.01, p<0.001) increased, but in CD19-positive cells, there were no differences in ABM-treated mice as compared with control mice. The expressions of IL-6 and IL-1β mRNA were augmented by ABM extract in both peritoneal macrophages and spleen cells. These results suggested that ABM extract might be an effective stimulator for T cell and macrophage to IL-1β and IL-6 release, resulting in augmentation of antibody production against SRBC antigen.

Immunomodulatory Effects of Aged Garlic Extract

Using various kinds of models, we examined the effects of aged garlic extract (AGE) on immune functions. In the immunoglobulin (Ig)E-mediated allergic mouse model, AGE significantly decreased the antigen-specific ear swelling induced by picryl chloride ointment to the ear and intravenous administration of antitrinitrophenyl antibody. In the transplanted carcinoma cell model, AGE significantly inhibited the growth of Sarcoma-180 (allogenic) and LL/2 lung carcinoma (syngenic) cells transplanted into mice. Concomitantly, increases in natural killer (NK) and killer activities of spleen cells were observed in Sarcoma-180--bearing mice administered AGE. In the psychological stress model, AGE significantly prevented the decrease in spleen weight and restored the reduction of anti-SRBC hemolytic plaque-forming cells caused by the electrical stress. These studies strongly suggest that AGE could be a promising candidate as an immune modifier, which maintains the homeostasis of immune functions; further studies are warranted to determine when it is most beneficial.

Study of the immunostimulating effect of IM-104 in mice.

The results of this study show that the product IM-104 has a marked immunostimulant effect, when administered intraperitoneally in mice, as seen by the increase in the number of haemolytic plaque-forming cells producing antibodies against sheep erythrocytes, as compared with saline-treated controls.

An improved haemolytic plaque assay for the detection of cells secreting antibody to bacterial antigens

Recent advances in the development of conjugate polysaccharide vaccines for human use have stimulated interest in the use of assays detecting antibody-secreting cells (AbSC) with specificity for bacterial antigens. Here we present improved haemolytic plaque-forming cell (PFC) assays detecting AbSC with specificity for tetanus and diphtheria toxoid as well as for Haemophilus influenzae type b and pneumococcal capsular polysaccharides. These assays were found to be less time consuming, more economical and yielded 1.9–3.4-fold higher plaque numbers than traditional Jerne-type PFC assays. In the case of anti-polysaccharide AbSC of the IgG isotype, the increase was as high as 7.4–11.8 times. Evidence is presented that the pronounced improvement in the detection of the latter is due to the presence of aggregating anti-IgG antibody from the beginning of the assay. It is proposed that in the case of low affinity of anti-polysaccharide antibodies aggregation of secreted monomeric antibody (IgG) is critical for plaque formation and increases the avidity of binding to target cells.

Immunomodulating activities of water extract fromXanthium strumarium (II): Immunostimulating effects of the water layer after treated with chloroform

One of water and/or methanol extracts from 14 herbal drugs which were screened using murine splenocytes showed immunosuppressive activities previously. After water extract fromXanthium strumarium was treated with chloroform, 100μg/ml of water layer (XS-WC1) has very strong immunostimulating activities tested by3H-thymidine incorporation (control vs 100μg/ml, 4962 cpm vs 69515 cpm). MLR also appears to be stimulated strongly (control vs 100μg/ml, 26345 cpm vs 78688 cpm). When 100μg/ml of XS-WC1 and 0.8μg/ml of concanavalin A (ConA) were added, more3H-thymidine were incorporated significantly, compared with 0.8μg/ml of ConA only. In contrast with ConA, results from 5μg/ml of lipopolysaccharide (LPS) and 100μg/ml of XS-WC1 were not different, compared with 5μg/ml of LPS only. These results indicated that responses of XS-WC1 to B cell and T cell may be different. XS-WC1 was injected intraperitoneally (10mg/kg, 50mg/kg, 100mg/kg) for 4 days or 10 days and tested secretion of IgM or IgG by direct and indirect hemolytic plaque-forming cell assays, respectively. Numbers of hemolytic plaques for both IgM and IgG were increased significantly. Especially, secretion of IgGs was increased more than 10 times. After administration of XS-WC1 for 7 days (50mg/kg, 100mg/kg) splenomegaly due to graft vs host reaction was observed. Human lymphocytes separated from whole blood by Ficoll-Hypaque method were also proliferated after treatment of 10μg/ml and 50μg/ml of XS-WC1. As seen in murine lymphocytes, human lymphocyte proliferation was increased synergistically after treatment with both of XS-WC1 and phytohemagglutinin (PHA). It appears that XS-WC1 may have potential immunostimulating activities and that it remains to be purified further for isolation of active components.

Effects of Dietary Restriction on Age-Related Immune Dysfunction in the Senescence Accelerated Mouse (SAM)

The effects of age and dietary restriction on immune response were investigated using an animal model of accelerated senescence (senescence accelerated mouse, SAM). The experimental groups consisted of control (ad libitum fed) and restricted groups (fed 60% of energy intake of the controls). Spleen weight and total number of splenic cells were significantly lower in the food-restricted group at 8 mo of age. Percentages of T (Thy-1.1+) and B (surface Ig+) cells in the splenic cells were not significantly different between the two groups. The number of direct hemolytic plaque-forming cells per 10(6) spleen cells 4 d following immunization with sheep red blood cells and dinitrophenyl-Ficoll was significantly greater in the 8-mo-old mice in the food-restricted group than in the control group. In the latter group, antibody responses Progressively decreased with age. Mitogen responses to concanavalin A and lipopolysaccharide were maintained in the food-restricted group but were depressed in the control group at 8 mo. In addition, though autoantibody to single-stranded DNA increased in the control group with advancing age, there was a steady decrease in the food-restricted group until 8 mo. Serum immunoglobulin (IgA and IgM) concentrations were significantly lower in the food-restricted group than in controls at 8 mo of age. Therefore, our results suggest that when senescence accelerated mice are subjected to food restriction, there may be a modulatory effect on the immune dysfunction associated with advancing age.

Quantitation of Antibody‐Secreting Cells in the Blood after Vaccination with aHaemophilus influenzaeType b Conjugate Vaccine

The human B-lymphocyte response to protein-conjugated polysaccharide antigens has not previously been studied at the cellular level. In order to do so, we developed and evaluated haemolytic plaque-forming cell assays detecting Haemophilus influenzae type b (Hib) capsular polysaccharide-specific antibody-secreting cells (AbSC) of the isotypes IgM, IgG, and IgA. The appearance of AbSC in the blood after vaccination of adults with diphtheria toxoid-conjugated Hib polysaccharide was investigated. AbSC were detected from post-vaccination day 5 to day 14. IgA was the predominant isotype among these cells. IgM AbSC peaked slightly earlier (median day 7) than IgG and IgA AbSC (both day 8). On post-vaccination day 8 the numbers of AbSC were: IgA, 1217/10(6) mononuclear cells (median); IgG, 211; and IgM, 30 (n = 11). Similar isotype distribution has earlier been found after vaccination with pure capsular polysaccharides from Hib and pneumococci. The predominance of IgA AbSC in response to both conjugate and pure polysaccharide vaccines is probably due to reactivation of the same clones of IgA-committed memory B cells originally primed at the mucosa by natural exposure to the polysaccharide or cross-reacting antigens.

Lignified natural products as potential medicinal resources. I. Potentiation of hemolytic plaque-forming cell production in mice.

We have found that water-soluble extracts of lignified natural products are strong potentiaters of hemolytic plaque-forming cell production in mice. Apparently, the active components involved belong to a lignin family. Consequently, study of medicinal potential, especially immunopotentiating capacity, of lignified materials is important.

Immunomodulatory Effect of Methimazole on Inbred Mice

The effects of antithyroid drug, methimazole (MMI, 1-methyl-2-mercaptoimidazole), on the immune system of inbred mice, C57BL/6, were studied. The proliferative response of splenic lymphocytes to concanavalin A (Con A) and allogeneic stimulator cells was reduced when the mice were provided with 0.1 % MMI in tap water ad libitum for two to four weeks. The reduction of proliferative response was correlated with a lower frequency of proliferative spleen cells in the MMI-treated mice. The ability to produce macrophage activating factor of these spleen cells and the levels of hemolytic plaque-forming cells were also reduced. However, the mitogenic response of the splenic lymphocytes to lipopolysaccharide (LPS) was enhanced. In order to investigate whether the impaired cell-mediated and humoral immune systems would increase the susceptibility of the MMI-treated animal to the growth of tumor cells, mice were challenged with a lethal dose of Moloney virus-induced T cell lymphoma of C57BL/6 mice (MBL-2). The time required for 50 % of the animals to die was reduced from 15 days for the normal mice to 10 days for the MMI-treated mice.

Immunosuppressive Principles ofRehmannia glutinosavar.hueichingensis1

Separation of the immunosuppressive principles from Rehmanniae radix was done by monitoring hemolytic plaque-forming cells (HPFC) inhibitory activity to give two new phenethyl alcohol glycosides: jionosides A (1) ( 4) and B (1) ( 5), along with six known compounds: acetoside, isoacteoside, purpureaside C, echinacoside, and cistanosides A and F.

Suppression of B cell responses by natural killer cells is mediated through direct effects on T cells

We examined the ability of human natural killer (NK) cells to modulate T cell-dependent mitogen-induced B cell responses. Highly purified NK cells inhibited the polyclonal antibody responses of autologous pokeweed mitogen (PWM)-stimulated unfractionated mononuclear cells in a reverse hemolytic plaque-forming cell (PFC) assay. Investigation of the possible mechanism(s) of the suppressor activity of NK cells revealed that lysis of mitogen-stimulated cells was unlikely. Chromium-51 release cytotoxicity assays of PWM-stimulated mononuclear cells did not demonstrate lysis by NK cells. Additionally, the monoclonal antibody 13.3, which abrogates NK cell cytolysis, did not reverse NK cell-dependent suppression of PFC formation. The putative lytic molecule elaborated by NK cells, NK cytotoxic factor, did not suppress B cell responses, further supporting a nonlytic inhibitory mechanism. That NK cell-derived lymphokines such as IFN-α, IFN-γ, or IL-2 were uninvolved in the down-regulation of B cells was corroborated by the failure of antibodies to these mediators to reverse the suppression. NK cells did not suppress PFC formation when T cells were replaced by supernatants from PWM-stimulated T cells; additionally, NK cells had no effect on the generation of these necessary T cell factors. However, the coculture of T cells with NK cells resulted in the induction of suppressor activity within the T cell population suggesting that this was the mechanism of NK cellmediated Suppression of B cell responses.

Solid phase enzyme-linked immunosorbent assay (ELISA) for anti-sheep erythrocyte antibody in mouse serum

The solid phase enzyme-linked immunosorbent assay (ELISA) was developed to measure IgM and IgG anti-SRBC antibody titers in mouse serum. Sheep erythrocytes, which have a surface negative charge, were attached directly to the bottom of an aminoplate well (96-well flat bottom, Sumitomo Bakelite Co.) which is charged positively to provide a solid phase for the ELISA antigen. Serum samples titrated were obtained from mice 5 and 10 days after SRBC immunization. Alkaline phosphatase-labeled goat anti-mouse IgM and/or IgG preparations were used as second antibody. Alkaline phosphatase activity in a well was measured by the Kind and King method [Kind, P.R.N. & King, E.J. (1954). J. clin. Path., 7, 322–326]; the optical density (OD) value of quinone as a final product was measured at 492 nm. (1) A linear relationship was observed between the antiserum concentration and OD value over a wide enough dilution range to assay antibody titers of serum samples; (2) IgM and IgG fractions from antiserum showed almost the same antibody titer as did the reconstituted samples; (3) a good correlation was observed between the serum IgM titer and the number of IgM hemolytic plaque-forming cells (PFCs) in spleen 5 days after the immunization, and was also observed between serum IgG antibody titer and the number of IgG PFCs 5 and 10 days after. Therefore, the ELISA described here requires no fixative for preparation of cell-coated plates, and serum IgM and IgG anti-SRBC antibody titers can be measured without any fractionation technique.

Effect of cysteine ethylester hydrochloride (Cystanin) on host defense mechanism (IV): Potentiating effects on the function of peritoneal or spleen macrophages.

L-Cysteine ethylester hydrochloride (ethylcysteine; 30 mg/kg, p.o.) increased the number of la-positive cells (antigen presenting cells) in spleen adherent cells (SAC) and that of Lyt 1.2-positive cells (helper T cells), but not that of Lyt 2.2-positive cells (suppressor T cells) of C57BL/6 mice immunized with sheep red blood cells. The production of hemolytic plaque forming cells (HPFC) in spleens of syngeneic recipient mice was enhanced by the transfer of SAC or spleen lymphocytes of the donor mice pretreated with ethylcysteine. This drug augmented phagocytosis of yeast particles by peritoneal macrophages of ICR mice at concentrations of 1-100 microM. In ex vivo experiments, this drug (30 mg/kg, p.o.) augmented the phagocytosis of yeast particles by mouse macrophages and showed a tendency to increase the macrophage number in the peritoneal cavity. Ethylcysteine (30 mg/kg, p.o.) significantly accelerated the decrease of viable E. coli number in the liver of normal mice 2 and 48 hr after challenge. Furthermore, this drug at the same dose restored the suppression of the decrease of E. coli number in the blood and liver of mice treated with cyclophosphamide (200 mg/kg, i.p.). These results suggest that ethylcysteine augments the functions of macrophages in vitro and ex vivo, and these enhancing effects may lead to the enhancement of host resistance to infections in compromised hosts.

Effect of Cysteine Ethylester Hydrochloride (Cystanin®) on Host Defense Mechanisms (IV): Potentiating Effects on the Function of Peritoneal or Spleen Macrophages

L-Cysteine ethylester hydrochloride (ethylcysteine; 30 mg/kg, p.o.) increased the number of la-positive cells (antigen presenting cells) in spleen adherent cells (SAC) and that of Lyt 1.2-positive cells (helper Τ cells), but not that of Lyt 2.2-positive cells (suppressor Τ cells) of C57BL/6 mice immunized with sheep red blood cells. The production of hemolytic plaque forming cells (HPFC) in spleens of syngeneic recipient mice was enhanced by the transfer of SAC or spleen lymphocytes of the donor mice pretreated with ethylcysteine. This drug augmented phagocytosis of yeast particles by peritoneal macrophages of ICR mice at concentrations of 1-100 μM. In ex vivo experiments, this drug (30 mg/kg, p.o.) augmented the phagocytosis of yeast particles by mouse macrophages and showed a tendency to increase the macrophage number in the peritoneal cavity. Ethylcysteine (30 mg/kg, p.o.) significantly accelerated the decrease of viable E. coli number in the liver of normal mice 2 and 48 hr after challenge. Furthermore, this drug at the same dose restored the suppression of the decrease of E. coli number in the blood and liver of mice treated with cyclophosphamide (200 mg/kg, i.p.). These results suggest that ethylcysteine augments the functions of macrophages in vitro and ex vivo, and these enhancing effects may lead to the enhancement of host resistance to infections in compromised hosts.

The effect of in utero exposure to hexachlorobenzene on the developing immune response of BALB/C mice

BALB/c mice were exposed to 0.0, 0.5 and 5.0 mg/kg maternal body weight hexachlorobenzene (HCB) throughout gestation by daily per os dosing of the females. At 45 days of age selected immune functions of the offspring were assessed. The delayed-type hypersensitivity (DTH) response to oxazolone was severely depressed in animals exposed to either 0.5 or 5.0 mg/kg HCB, however, only those animals exposed to 5.0 mg/kg HCB showed a significant decrease in their mixed lymphocyte response (MLR) levels. The ability of isolated spleen cells to undergo a blastogenic response to concanavalin A (ConA), phytohemagglutinin (PHA) and lipopolysaccharide (LPS) showed no significant changes due to HCB exposure. Similarly, no significant difference in the induction of direct hemolytic plaque-forming cells was seen. A significant increase in the relative distribution of splenic T cells and a significant decrease in splenic B cells was measured in the offspring of HCB-treated females. These results suggest that HCB is capable of affecting the development or maturation of the immune response in mice, perhaps at the T cell level.

新規抗炎症薬Ｐｉｒａｚｏｌａｃ，４‐（４‐ｃｈｌｏｒｏｐｈｅｎｙｌ）‐１‐（４‐ｆｌｕｏｒｏｐｈｅｎｙｌ）ｐｉｒａｚｏｌｅ‐３‐ａｃｅｔｉｃ ａｃｉｄの免疫薬理学的研究

Pirazolac is known as a compound having both anti-inflammatory and immunomodulating activities. We studied the effects of Pirazolac on anti-SRBC response in BALB/c mice (female, 7w) and in MRL/Mp-lpr/lpr (MRL/1) mice (male, 20w) with spontaneously occurring autoimmune diseases. In BALB/c mice, Pirazolac enhanced spleen hemolytic plaque forming cell (HPFC) and rosette forming cell (RFC) productions at 3 or 30 mg/kg p.o. On the other hand it suppressed spleen HPFC productions in MRL/l mice at 3 or 30 mg/kg p.o. T cell subsets analysis showed that the effect of Pirazolac in MRL/l mice was based on the increase in suppressor/cytotoxic T cells (Lyt-1-2+) in the spleen cells. To clarify the mechanisms of immunomodulating activities of Pirazolac, the effect of Pirazolac on phagocytosis of peritoneal exudate cells of BALB/c mice was studied using Candida parapsilosis as a target cell. Pirazolac enhanced candidacidal activities at 10-7-10-6M. This result suggests that some part of immunomodulating activities of Pirazolac was due to activation of macrophages.

Recovery from immunesuppression in mice infected with Schistosoma japonicum by treatment of praziquantel.

To learn the possible alteration of immune response, hemolytic plaque-forming cells (PFC) produced in the spleen of Schistosoma japonicum infected mice treated with Praziquantel and untreated group were counted. There was no significant difference in the immunesuppression percentage between the treated and untreated groups 1 and 3 weeks after treatment. In 5 weeks after treatment, however, the immunesuppression percentage in the treated mice was markedly reduced in comparison with that of the untreated group. Recovery from immunesuppression appears to be associated with elimination or impairment of adult worms.