Immune Responses In Normal Mice Research Articles

Swine Influenza Virus Infection Decreases the Protective Immune Responses of Subunit Vaccine Against Porcine Circovirus Type 2.

Porcine circovirus type 2 (PCV2) is the primary pathogen of porcine circovirus diseases and porcine circovirus associated diseases. Immunization with a vaccine is considered an effective measure to control these diseases. However, it is still unknown whether PCV2 vaccines have protective immune responses on the animals infected with swine influenza virus (SIV), a pandemic virus in swine herds. In this study, we first compared the effects of 2 different PCV2 vaccines on normal mice and SIV-infected mice, respectively. The results showed that these two vaccines had protective immune responses in normal mice, and the subunit vaccine (vaccine S) had better effects. However, the inactivated vaccine (vaccine I) instead of vaccine S exhibited more immune responses in the SIV-infected mice. SIV infection significantly decreased the immune responses of vaccine S in varying aspects including decreased PCV2 antibody levels and increased PCV2 replication. Mechanistically, further studies showed that SIV infection increased IL-10 expression and M2 macrophage percentage, but decreased TNF-α expression and M1 macrophage percentage in the mice immunized with vaccine S; on the contrary, macrophage depleting by using clodronate-containing liposomes significantly alleviated the SIV infection-induced decrease in the protective immune responses of vaccine S against PCV2. This study indicates that SIV infection decreases the protective immune responses of vaccine S against PCV2. The macrophage polarization induced by SIV infection might facilitate decreased immune responses to vaccine S, which provides new insight into vaccine evaluation and a reference for the analysis of immunization failure.

Alpha-phellandrene-induced apoptosis in mice leukemia WEHI-3 cells in vitro.

Although reports have shown that α-phellandrene (α-PA) is one of the monoterpenes and is often used in the food and perfume industry, our previous studies have indicated that α-PA promoted immune responses in normal mice in vivo. However, there is no available information to show that α-PA induced cell apoptosis in cancer cells, thus, we investigated the effects of α-PA on the cell morphology, viability, cell cycle distribution, and apoptosis in mice leukemia WEHI-3 cells in vitro. Results indicated that α-PA induced cell morphological changes and decreased viability, induced G0/G1 arrest and sub-G1 phase (apoptosis) in WEHI-3 cells. α-PA increased the productions of reactive oxygen species (ROS) and Ca2+ and decreased the levels of mitochondrial membrane potential (ΔΨm ) in dose- and time-dependent manners in WEHI-3 cells that were analyzed by flow cytometer. Results from confocal laser microscopic system examinations show that α-PA promoted the release of cytochrome c, AIF, and Endo G from mitochondria in WEHI-3 cells. These results are the first findings to provide new information for understanding the mechanisms by which α-PA induces cell cycle arrest and apoptosis in WEHI-3 cells in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1640-1651, 2016.

Crude extract of Polygonum cuspidatum stimulates immune responses in normal mice by increasing the percentage of Mac-3-positive cells and enhancing macrophage phagocytic activity and natural killer cell cytotoxicity.

Polygonum cuspidatum is a natural plant that is used in traditional Chinese herbal medicine. The crude extract of Polygonum cuspidatum (CEPC) has numerous biological effects; however, there is a lack of studies on the effects of CEPC on immune responses in normal mice. The aim of the present study was to determine the in vivo effects of CEPC on immune responses in normal mice. CEPC (0, 50, 100, 150 and 200 mg/kg) was orally administered to BALB/c mice for three weeks, following which blood, liver, and spleen samples were collected. CEPC did not significantly affect the total body weight, or tissue weights of the liver or spleen, as compared with the control mice. CEPC increased the percentages of CD3 (T-cell marker), 11b (monocytes) and Mac-3 (macrophages) positive-cells, and reduced the percentage of CD19-positive cells (B-cell marker), as compared with the control mice. CEPC (100 mg/kg) stimulated macrophage phagocytosis of blood samples but did not affect macrophage phagocytosis in the peritoneum. Activity of the splenic natural killer cells was increased in response to CEPC (50 mg/kg) treatment. Furthermore, CEPC inhibited T- and B-cell proliferation when the cells were stimulated with concanavalin A and lipopolysaccharide, respectively.

Butylated hydroxyanisole affects immunomodulation and promotes macrophage phagocytosis in normal BALB/c mice

Butylated hydroxyanisole (BHA), a synthetic antioxidant, has been used in fat and fatty foods to prevent oxidative deterioration. However, the functions of BHA on immune responses in normal mice remain elusive. The aim of the present study was to investigate the effects of oral treatment of BHA on immune responses in normal mice in vivo. BALB/c mice received various treatments. Blood samples were collected and analyzed. Flow cytometry was used to determine the levels of the cell markers. Results showed that BHA did not significantly affect the weight of the animal body and spleen in normal mice. BHA promoted macrophage phagocytosis from peripheral blood mononuclear cells, but did not alter this process in the peritoneal cavity. Furthermore, BHA did not influence natural-killer cell cytotoxicity in normal mice. Notably, BHA promoted the levels of CD3 (T cells) and decreased the level of CD19 (B cells), but did not significantly affect the levels of CD11b (monocytes) and macrophages (Mac-3) in normal mice. Based on these observations it can be concluded that BHA promotes immune responses by increasing T cells and activating phagocytosis by macrophages in normal mice. However, the molecular mechanisms require further investigation.

The effects of B‐cell depletion therapies on humoral immune responses in normal mice

Anti‐CD20 mediated B cell depletion is a therapy for treating rheumatoid arthritis and B‐cell malignancies. To examine how B cell depletion affects an individual's capacity to generate antibody responses we assessed in a mouse model primary and secondary antibody responses after treatment with anti‐mouse CD20 and/or BAFFR‐Ig. BALB/c mice dosed with anti‐CD20 one week prior to primary TNP‐KLH immunization had a 93% decrease in anti‐TNP IgG titers at day 10 compared to controls. Mice treated with anti‐CD20 one week prior to immunization with TNP‐Ficoll had a ~50% increase in TNP‐specific IgG and IgM titers. The secondary antibody response to TNP‐KLH was not significantly affected by anti‐CD20 treatment one week prior to immunization. BAFFR‐Ig treatment reduced the secondary response to TNP‐KLH by 57%. Treatment of mice with the combination of anti‐CD20 and BAFFR‐Ig dramatically reduced secondary antibody responses to either antigen. After B‐cell repopulation all treatment groups mounted a strong response to a tertiary challenge. Therefore, B cell depletion with anti‐CD20 results in a strong reduction in the primary antibody response to a neoantigen, however memory B cells appear resistant to anti‐CD20 treatment. Simultaneous anti‐CD20 and BAFFR‐Ig treatment results in elimination of the secondary response by blocking memory B cell activation but does not deplete memory B cells.

Characterization of flagellin expression and its role in Listeria monocytogenes infection and immunity.

Flagellin is the structural component of flagella produced by many pathogenic bacteria and is a potent proinflammatory molecule that mediates these effects through Toll-like receptor (TLR) 5. In Listeria monocytogenes (LM), flagellin expression is regulated by temperature and has been described as being shut off at 37 degrees C. In this study, we demonstrate that TLR5-mediated cell activation and flagellin expression is maintained at 37 degrees C in some laboratory-adapted strains and in approximately 20% of LM clinical isolates. To determine the role of flagellin in LM infection, a targeted mutation in the structural gene for flagellin (flaA) was generated in a parental LM strain that expressed flagellin under all conditions examined. In vitro studies demonstrated that this deltaflaA mutant was (i). non-motile; (ii). not able to activate TLR5-transfected HeLa cells; and (iii). induced tumour necrosis factor (TNF)-alpha production in approximately 50% fewer CD11b+ cells in splenocytes from normal mice compared with the parental strain. However, there was no significant alteration in virulence of the deltaflaA mutant after either intravenous or oral murine infection. Similarly, there was no difference in the generation of LM-specific CD8 or CD4 T cells after intravenous or oral infection. These data indicate that flagellin is not essential for LM pathogenesis or for the induction of LM-specific adaptive immune responses in normal mice.

The effect of calcineurin activator, extracted from Chinese herbal medicine, on memory and immunity in mice

Calcineurin (CN) is a highly abundant phosphatase in the brain and it is the only Ca 2+- and calmodulin-dependent protein serine/threonine phosphatase. There is considerable evidence to suggest that CN plays an essential role in activity-dependent modulation of synaptic efficacy. It has been shown recently that inhibitors of CN, such as CsA or FK506, impair memory formation in day-old chicks. In our present study, extract of Fructus cannabis (EFC) with activation of CN, extracted from Chinese traditional medicine, was used to determine the effects on memory and immunity. In the step-down-type passive avoidance test, the plant extract (0.2 g/kg) significantly improved amnesia induced by chemical drugs in mice, and greatly enhanced the ability of cell-mediated type hypersensitivity and nonspecific immune responses in normal mice. The present study provided pharmacological evidence for Chinese herbal medicine screening from molecular model.

In vivo priming of CD4 T cells that produce interleukin (IL)-2 but not IL-4 or interferon (IFN)-gamma, and can subsequently differentiate into IL-4- or IFN-gamma-secreting cells.

The differentiation of antigen-stimulated naive CD4 T cells into T helper (Th)1 or Th2 effector cells can be prevented in vitro by transforming growth factor (TGF)-beta and anti-interferon (IFN)-gamma. These cells proliferate and synthesize interleukin (IL)-2 but not IFN-gamma or IL-4, and can differentiate into either Th1 or Th2 cells. We have now used two-color Elispots to reveal substantial numbers of primed cells producing IL-2 but not IL-4 or IFN-gamma during the Th1- or Th2-biased immune responses induced by soluble proteins or with adjuvants. These cells were CD4(+)CD44(high) and were present during immediate and long-term immune responses of normal mice. Naive T cell receptor for antigen (TCR) transgenic (DO11.10) T cells were primed in vivo after adoptive transfer into normal hosts and FACS((R)) cloned under conditions that did not allow further differentiation. After clonal proliferation, aliquots of each clone were cultured in Th1- or Th2-inducing conditions. Many in vivo-primed cells were uncommitted, secreting IL-2 but not IL-4 or IFN-gamma at the first cloning step, but secreting either IL-4 or IFN-gamma after differentiation in the appropriate conditions. These in vivo-primed, uncommitted, IL-2-producing cells may constitute an expanded pool of antigen-specific cells that provide extra flexibility for immune responses by differentiating into Th1 or Th2 phenotypes later during the same or subsequent immune responses.

Artemisinin and Its Derivatives Enhance T Lymphocyte-Mediated Immune Responses in Normal Mice and Accelerate Immunoreconstitution of Mice with Syngeneic Bone Marrow Transplantation

Artemisinin and its derivatives, dihydroartemisinin and sodium artesunate, enhanced DNA synthesis of mouse spleen cells that had been activated with alloantigens or T cell mitogen Con A, but not B cell mitogen LPS, both in vitro and ex vivo. In vivo experiments showed that DTH response and antibody response against sheep erythrocytes were augmented in sodium artesunate-treated animals. In addition, sodium artesunate, a representative of these compounds, increased IL-2 production from mouse splenocytes stimulated with Con A. Because of the long-term T cell function deficiency in mice after bone marrow transplantation, the effect of sodium artesunate on the immune reconstitution was assessed. The results demonstrated that 10 mg/kg sodium artesunate significantly accelerated the immune reconstitution in mice after syngeneic bone marrow transplantation. These data suggested that artemisinin and its derivatives appeared to promote T cell function selectively, and these compounds had a potential application for the recovery of immune function.

Radiant heat-induced hyperthermia in mice: in vivo effects on the immune system

Whole-body hyperthermia (WBH) in mice was induced by 2-4-h exposure to radiant heat resulting in core body temperatures of 38.5-40.4 degrees C, and correlated directly with the magnitude and duration of heat treatment. Two-hour heat treatments in this temperature range did not consistently affect generation of antibody-forming cells in vivo, while 4-h treatments at temperatures greater than or equal to 40 degrees C significantly suppressed the antibody-forming cell response. The capacity of lymphocytes from similarly heated mice to generate antibody-forming cells in vitro was not affected, suggesting that the observed in vivo suppression may be mediated by circulating factors rather than by some heat-induced alteration in the cells themselves. In vivo treatment did not alter T-cell responsiveness to the mitogen Con-A or delayed-type hypersensitivity responses to sheep red blood cells. Quantitative and flow cytometric assessment of splenic and thymic lymphocyte numbers showed that WBH did not alter absolute numbers of lymphocytes but did temporarily change the proportions of lymphocyte subsets. An immediate increase in splenic L3T4+ cells was observed, followed within 18 h by an overall decrease in Lyt 2+ and Thy 1.2+ T-cells. In the thymus the percentages of mature T cells increased. In general, only minimal effects of heat on the immune responses of normal mice could be demonstrated.

Effects of artemisinin, dihydroartemisinin and arteether on immune responses of normal mice

Artemisinin (Qinghaosu) is a potent antimalarial sesquiterpene lactone isolated from the Chinese herb Artemisia annua. Arteether, a potent semisynthetic analogue of dihydroartemisinin is being developed by the World Health Organization as the artemisinin derivative of choice for the treatment of malaria. All three agents in doses of 400 and 600 mg/kg body weight were found to exhibit marked suppression of humoral responses, as measured by the hemolytic plaque assay, with arteether being the most potent. These agents did not alter the delayed-type hypersensitivity response to sheep erythrocytes at the same dose levels. In addition, all three agents were found not to possess any anti-inflammatory activity when tested on carrageenan-induced oedema. These results indicated that these agents have a selective immunosuppressive activity. They did not exhibit immunostimulating activity in contrast to what has been reported for sodium artesunate.

In vivo stimulation and restoration of the immune response by the noninflammatory fragment 163-171 of human interleukin 1 beta.

The synthetic nonapeptide VQGEESNDK, corresponding to the fragment 163-171 of human IL-1 beta, showed in vivo immunomodulatory capacities qualitatively and quantitatively comparable to those of the mature human IL-1 beta protein. In fact, both IL-1 beta and the 163-171 fragment stimulated the immune response of normal mice and restored immune reactivities of immunocompromised animals. In addition, the synthetic IL-1 peptide was as efficient as the entire protein in inducing tumor rejection and radioprotection. On the other hand, the 163-171 fragment did not cause any of several inflammation-associated metabolic changes inducible by the whole IL-1 beta molecule in vivo: hypoferremia, hypoglycemia, hyperinsulinemia, increase in circulating corticosterone, SAA and fibrinogen, decrease in hepatic drug-metabolizing enzymes. Furthermore, at variance with IL-1 beta, the 163-171 peptide did not show the toxic effects causing shock and death in adrenalectomized mice. Thus, these results confirm our previous in vitro observations that functional domains are identifiable within the multipotent cytokine IL-1 beta, and demonstrate the biological relevance of this finding in a variety of in vivo systems. The identification of a selectively active fragment of a cytokine may thus represent a significant step towards a better directed and more rational immunotherapeutic approach.

Tolerance induction in mice by conjugates of monoclonal immunoglobulins and monomethoxypolyethylene glycol. Transfer of tolerance by T cells and by T cell extracts.

The specific tolerance induced in mice by conjugates of human monoclonal IgG (HIgG) with monomethoxypolyethylene glycol (mPEG) was transferred to normal mice by spleen cells or a surface immunoglobulin negative (sIg-) Lyt-2+ subpopulation of these cells. Although transferable tolerance was demonstrable 6 to 14 days after treatment of the cell donors with tolerogen, the state of tolerance persisted in the treated mice for at least 43 days. Moreover, an extract prepared by freezing and thawing of the sIg- spleen cells obtained from mice 6 days after treatment with HIgG(mPEG)20 was capable of reducing (greater than 85%) the immune response of normal mice to heat aggregated HIgG. On the basis of these results, it is suggested that similar tolerogenic mPEG derivatives of xenogeneic monoclonal immunoglobulins (XIg) may prove to be useful therapeutic agents in man when administered before treatment with the unmodified XIg.

Chitin: an immunological adjuvant and a polyclonal B-lymphocyte activator.

Chitin, a linear beta 1-4 linked poly-N-acetylglucosamine, enhanced the in vivo immune response of normal mice to sheep red blood cells, but not that of mice with thymic aplasia (nude mice). The compound is mitogenic for murine B lymphocytes but not for thymocytes or splenic T lymphocytes. Similarly to well-known polyclonal B-lymphocyte activators; e.g. lipopolysaccharide, chitin induces differentiation of B lymphocytes into antibody-forming cells in vitro.

Suppressive effects of thiopental and halothane on specific arms of the immune response.

The effect of thiopental, halothane and surgery on specific arms of the immune response of normal mice was studied. These experiments represent the first step in localizing a potentially correctable anesthesia/surgery-induced defect in immune reactivity which may be involved with postoperative increases in tumor growth. The delayed-type hypersensitivity (DTH) response of mice to 2,4-dinitrochlorobenzene (DNCB) was studied. Combinations of induction and inhalation anesthesia and surgery were administered at various phases of the immune response to DNCB. Thiopental impaired the afferent response while halothane impaired the efferent response. When the agents were combined, both arms of the immune response were suppressed. A surgical procedure, in most experiments, did not produce a greater immunosuppression than thiopental and halothane. The administration of an immunorestorative agent, thiabendazole, returned reactivity to normal levels. Thiopental and halothane either affect different immune cell populations or they affect different functions of a cell population active in both arms of the DTH response. In relation to tumor growth, the degree of suppression may not be as significant as the cell population impaired.

The effect of thymectomy and anti-thymocyte serum on the immunological competence of adult mice.

AbstractThe immune response of normal mice, adult thymectomized mice, micetreated with anti‐thymocyte serum and ice which were treated with antithymocyte serum (ATS) after hymectomy were studied for their response to tobacco mosaic virus (TMV) as antigen, 52, 84 and 316 days after the beginning of treatment. Adult mice, treated after thymectomy with ATS, failed to produce detectable antibodies to TMV. The role of the thymus in the adult mouse in the immune response to a thymus‐dependent antigen is discussed.

Quantity and quality of anti-hapten antibodies in normal and in T cell-deprived mice studied at the cellular level.

The Immune response of normal mice as well as of thymus‐deprived (TxB) mice against 2 different hapten‐protein conjugates (NNP‐BSA and NNP‐CG) was tested at various times after immunization at the cellular level. Normal mice developed both direct and indirect plaque‐forming cells against the hapten, as determined by a modified local haemolysis in gel assay for detection of anti‐hapten antibody‐secreting cells. However, TxB mice produced direct plaque‐forming cells to the same extent as normal mice, whereas the indirect plaque‐forming cell response was greatly impaired. In fact, significantly elevated levels of indirect plaque‐forming cells were not detected in such mice. Studies on the affinity of secreted antibodies by means of hapten inhibition revealed that the indirect plaque‐forming cells derived from normal immunized mice could be inhibited by decreasing concentrations of free hapten with time after immunization, indicating a gradual increase in the number of cells secreting antibodies of high affinity. No such change was detected in normal or TxB mice with regard to antibodies giving rise to direct lysis, presumably IgM antibodies.