Reaction In Mice Research Articles

Ubiquitary Expressed Major Histocompatibility (MHC) Class I and Minor Histocompatibility Alloantigens (mHAg) Are No Relevant Targets For Graft-Versus-Tumor (GvT) Reactions In Mice After Allogeneic Bone Marrow Transplantation

IntroductionCell mediated GvT effects after allogeneic stem cell transplantation are heterogeneous with respect to effector cell populations, target antigens and their interrelation with graft-versus-host disease (GvHD). Mutated tumor-associated antigens (TAAs), as well as selectively or aberrantly expressed nonmutated antigens, represent potential targets for T cell-mediated GvT effects that are in principle separable from the generalized graft-versus-host reaction targeting widely expressed MHC alloantigens and mHAg. So far, it remains a matter of debate whether alloantigens ubiquitary expressed on tumor cells and normal host tissue are relevant targets of GvT responses. MethodsTo evaluate the role of MHC class I molecules and mHAg as targets for GvT reactions, we employed three different allogeneic parent-into-F1 murine transplant models (BALB/c [H-2d] or C57BL/6 [H-2b] → F1[BALB/c x C57BL/6] [H-2b/d] and BALB/c → F1[BALB/c x CBA/J] [H-2d/k]). As previously shown, these transplant models allow for separate variation in the histocompatibility between donor cells and normal host tissue on the one hand and between donor cells and tumor tissue on the other hand. Test tumors were the donor derived myeloma / leukemia cell lines MPC11 [H-2d] and C1498 [H-2b], which were stably transfected with MHC class I molecules H-2Kb, H-2Kd, H-2Kk or with the mHAg UTY. ResultsCompared with non-GvHD controls (F1[H-2b/d] mice transplanted with allogeneic bone marrow cells from BALB/c [H-2d] alone), significant GvT effects against the BALB/c derived tumor MPC11 occurred in F1[H-2b/d] recipients with severe GvHD (transplanted with bone marrow and additional splenic lymphocytes from BALB/c). Identical histocompatibility antigens of donor and tumor cells precluded allorecognition of tumor cells, leaving TAAs the only possible target antigens in this particular setting, with GvHD as a driving force for augmentation of tumor specific immune responses. The MPC11 myeloma [H-2d], stably transfected with the C57BL/6 derived MHC class I molecule H-2Kb (MPC11-Kb), showed similar tumor growth compared to the wild type MPC11 in F1[H-2b/d] mice transplanted from BALB/c donors. Similar results could be observed with the C57BL/6 derived C1498 leukemia [H-2b], transfected with the BALB/c MHC class I alloantigen H2Kd, in F1[H-2b/d] mice transplanted from C57BL/6 donors. In both experimental settings, the artificially expressed alloantigens H2Kb and H2Kd were also expressed ubiquitarily on tissues of F1[H-2b/d] recipients. In sharp contrast, MPC11 cells transfected with the CBA/J derived H2Kk (MPC11-Kk) alloantigen showed a significantly reduced tumor growth in F1[H-2b/d] recipients transplanted from BALB/c donors, demonstrating that MHC class I alloantigens are relevant targets, when expressed on tumors but not on healthy recipient tissue. Inoculation of MPC11-Kk in F1[H-2d/k] mice 3 or 7 days after transplantation from BALB/c donors showed a trend towards a reduced tumor growth compared to wild type MPC11 in recipients with severe GvHD. Later application of tumor cells (14 days after transplantation) showed similar growth pattern of both tumors, suggesting that MHC class I antigens might be GvT targets in the early phase after allogeneic transplantation. To evaluate the impact of ubiquitarily expressed mHAg, we established a C1498 cell line, which stably expressed UTY (C1498-UTY), a known immunogenic antigen of the male HY gene. In comparison with the wild type control, C1498-UTY showed similar tumor growth in male or female F1[H-2b/d] recipients, transplanted from naive female C57BL/6 donors. Immunization of female C57BL/6 donors with male splenocytes resulted in a significantly reduced tumor growth of the C1498-UTY in female F1[H-2b/d] recipients. In contrast, tumor growth of the UTY expressing and wild type C1498 was similar in male F1[H-2b/d] mice transplanted from immunized female C57BL/6 donors. ConclusionOur experimental data indicate that a biologically relevant GvT effect targeting MHC class I or mHAgs mainly occurs when these alloantigens are not ubiquitarily expressed, even in the context of severe GvHD. Consequently, TAAs and tissue- /hematopoietic specific antigens are most likely the main targets of GvT effects observed after allogeneic stem cell transplantation. Disclosures:No relevant conflicts of interest to declare.

A comparison of the effects of dexamethasone-pharmacopuncture and dexamethasone-oral administration based on traditional Korean medicine theory on anaphylactic reaction in mice

Pharmacopuncture, or herbal acupuncture, is a new form of therapy derived from combinations of two traditional therapeutic methods, herbal medicine and acupuncture therapy. To compare the efficacy between dexamethasone-pharmacopuncture (DP) and dexamethasone-oral administration (DO), the effect of DP was investigated in murine models. Anti-anaphylactic effects of dexamethasone treatments were investigated in compound 48/80-induced systemic anaphylactic reaction, ear swelling response, and passive cutaneous anaphylaxis (PCA). DP treatment significantly inhibited the compound 48/80-induced systemic anaphylactic reaction, ear swelling response, and PCA. The effects between DP and DO were on a similar level. These results indicate that DP can be used as an alternative method for DO in case of emergency.

Cholinergic Receptors Modulate Immune Complex–Induced Inflammation In Vitro and In Vivo

Cholinergic neural output has been shown to modulate innate immune responses to infection, injury and ischemia through stimulation of α7 nicotinic acetylcholine receptors (α7nAChR) on mononuclear phagocytes. We tested the hypothesis that cholinergic neurotransmitters, similar to those released through activation of a neural reflex, regulate responses to products of the adaptive immune system, specifically immune complex (IC)-mediated activation of effector cells. In this study, we show that stimulation of α7nAChR on human polymorphonuclear neutrophils (PMNs) and blood mononuclear phagocytes in vitro attenuates C5aR- and FcγR-triggered generation of reactive oxygen species, expression of leukocyte markers involved in cell recruitment and adhesion, and release of TNF-α and other proinflammatory cytokines. We show that this pathway is operative in vivo. Ligation of cholinergic receptors blunts IC-triggered responses in the reverse peritoneal Arthus reaction in mice. The selective 7nAChR agonist GTS21 decreased PMN accumulation and release of cytokines and chemokines at sites of IC deposition. In addition, mice lacking α7nAChR had exaggerated responses to reverse peritoneal Arthus reaction characterized by increased infiltration of PMNs and elevated of levels of TNF-α and CXCL1 in peritoneal fluid compared with wild-type mice. Taken together, these findings suggest that cholinergic output has the potential to exert tonic inhibitory activity that dampens responses to ICs and C5a and thus may be a target to minimize tissue damage in autoimmune diseases.

The inhibition of 2,3-dichloro-1-propanol on T cell in vitro and in vivo

2,3-Dichloro-1-propanol (2,3-DCP) is a member of a group of chemicals known as chloropropanols. Currently, immunotoxicity of 2,3-DCP has not been reported. In the present study, we studied its inhibitory effects on T cell both in vivo and in vitro. The results showed that 2,3-DCP markedly inhibited ConA-induced splenocyte proliferation, Th1 and Th2 cytokine production, CD4(+) T cell populations, and the ratio of CD4(+)/CD8(+) T cells and cell cycle arrest in vitro. In addition, 2,3-DCP markedly suppressed DNFB-induced T-cell-mediated delayed-type hypersensitivity (DTH) reaction in mice. Furthermore, Western blot was used to study how 2,3-DCP affects signal transduction mechanisms. The data revealed that 2,3-DCP could down regulate activation of ConA-induced NF-κB and NFAT signal transduction pathways. These observations indicated that 2,3-DCP exhibited negative regulatory effects by directly suppressing T-cell-mediated immune responses in vitro and in vivo.

Suppressive effects of fisetin on mice T lymphocytes in vitro and in vivo

BackgroundMost of the immunosuppressive drugs have satisfactory therapeutic effects on organ transplantation and autoimmune disease. However, their clinical application is limited by side effects. Therefore, new and safe immunosuppressive drugs against acute and chronic rejections are eagerly awaited. Fisetin, a flavonoid present in various types of vegetables and fruits, has few side effects and low level of toxicity, which would be a desirable clinical feature. In the present study, we investigated the immunosuppressive effects and underlying mechanisms of fisetin against T-cell activation in vitro and in vivo. MethodsWe measured the effect of fisetin on T-lymphocyte proliferation, T-cell subsets, cell cycle progression, cytokine production, and nuclear factor activation in vitro, as well as its influence on T cell–mediated delayed-type hypersensitivity reaction in vivo. ResultsIn vitro, the results showed that fisetin significantly suppressed mouse splenocytes proliferation, Th1 and Th2 cytokine production, cell cycle and the ratio of CD4+/CD8+ T cells. Furthermore, fisetin exerts an immunosuppressive effect in mouse T lymphocytes through the suppression of nuclear factor kappa B activation and nuclear factor of activated T cells signaling in a dose-dependent manner. In vivo, fisetin treatment also significantly inhibited the dinitrofluorobenzene-induced delayed-type hypersensitivity reactions in mice. ConclusionsFisetin had strong immunosuppressive activity in vitro and in vivo, suggesting a potential role for fisetin as an immunosuppressive agent.

The drug efficacy and adverse reactions in a mouse model of oral squamous cell carcinoma treated with oxaliplatin at different time points during a day

BackgroundRecent studies have shown that the growth and proliferation of cancer cells in vivo exhibit circadian rhythm, and the efficacy and adverse reactions of platinum-based anticancer drugs administered at different times of the day vary significantly on colon cancer. However, since the circadian rhythms of growth and proliferation of various cancer cells often differ, the question of whether the administration of platinum anticancer drugs at different times of the day exerts significantly different efficacy and adverse effects on oral cancers remains to be elucidated. This study has compared the efficacy and adverse effects of oxaliplatin (L-OHP) administration at different times during a day on oral squamous cell carcinoma in mice and has analyzed cellular circadian rhythms.MethodsThe mouse model for oral squamous cell carcinoma was established in 75 nude mice, housed in a 12 hour light/12 hour dark cycle environment. The mice were randomly divided into five groups; four experimental groups were intravenously injected with L-OHP at four time points within a 24-hour period (4, 10, 16, and 22 hours after lights on [HALO]). The control group was intravenously injected with the same volume of saline. Treatment efficacy and adverse reactions were compared on the seventh day after the injection, at 22 HALO. The existence of circadian rhythms was determined by cosine analysis.ResultsOnly injections of L-OHP at 16 and 22 HALO significantly prolonged animal survival time. The adverse reactions in mice injected with L-OHP at 16 and 22 HALO were significantly less than those observed in mice administered L-OHP at 4 and 10 HALO. The cosine fitting curve showed that the survival time and adverse reactions exhibited circadian rhythm.ConclusionThe time factor should be considered when treating patients with oral squamous cell carcinoma with L-OHP in order to achieve better efficacy, reduce the adverse reactions, and improve the patients’ survival time and quality of life.

Conformational and Functional Effects Induced by D- and L-Amino Acid Epimerization on a Single Gene Encoded Peptide from the Skin Secretion of Hypsiboas punctatus

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH2) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy 1H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways.

Type BCpG oligodeoxynucleotides induce Th1 responses to peanut antigens: Modulation of sensitization and utility in a truncated immunotherapy regimen in mice

Peanut allergy stems from a Th2-biased immune response to peanut allergens leading to IgE production and allergic reactions upon ingestion. A model of peanut allergy in C3H/HeJ mice was used to assess whether type A, B, or C CpG oligodeoxynucleotide (ODN) molecules would be effective in: (i) a prophylactic approach to prevent peanut allergy when administered simultaneously with a Th2-skewing adjuvant, and (ii) a therapeutic model to allow for shortened immunotherapy. Type B ODNs were extremely effective in inhibiting anaphylaxis in the sensitization protocol as evidenced by differences in symptom scores, body temperature, and mouse mast cell protease 1 release compared to sham treatment. In the therapeutic model, co-administration of type B ODN plus peanut proteins was highly effective in reducing anaphylactic reactions in mice with established peanut allergy. The therapeutic effect was accompanied by an increase in IFN-γ and peanut-IgG2a, without a significant decrease in peanut IgE or IL-4 responses. CpG ODNs, especially type B, were highly effective in inducing Th1 responses in mice undergoing induction of peanut allergy, as well as in mice undergoing therapy for established peanut allergy. Interestingly, the IgE response was not significantly altered, suggesting that IgG antibodies may be enough to prevent peanut-induced anaphylaxis.

DX5+CD4+T cells modulate CD4+T‐cell response via inhibition of IL‐12 production by DCs

DX5(+) CD4(+) T cells have been shown to dampen collagen-induced arthritis and delayed-type hypersensitivity reactions in mice. These cells are also potent modulators of T-helper cell responses through direct effects on CD4(+) T cells in an IL-4 dependent manner. To further characterize this T-cell population, we studied their effect on DCs and the potential consequences on T-cell activation. Here, we show that mouse DX5(+) CD4(+) T cells modulate DCs by robustly inhibiting IL-12 production. This modulation is IL-10 dependent and does not require cell contact. Furthermore, DX5(+) CD4(+) T cells modulate the surface phenotype of LPS-matured DCs. DCs modulated by DX5(+) CD4(+) T-cell supernatant express high levels of the co-inhibitor molecules PDL-1 and PDL-2. OVA-specific CD4(+) T cells primed with DCs exposed to DX5(+) CD4(+) T-cell supernatant produce less IFN-γ than CD4(+) T cells primed by DCs exposed to either medium or DX5(-) CD4(+) T-cell supernatant. The addition of IL-12 to the co-culture with DX5(+) DCs restores IFN-γ production. When IL-10 present in the DX5(+) CD4(+) T-cell supernatant is blocked, DCs re-establish their ability to produce IL-12 and to efficiently prime CD4(+) T cells. These data show that DX5(+) CD4(+) T cells can indirectly affect the outcome of the T-cell response by inducing DCs that have poor Th1 stimulatory function.

Morus bombycis extract suppresses mast cell activation and IgE-mediated allergic reaction in mice

Ethnopharmacological relevanceMorus bombycis Koidzumi (MB) is widely distributed throughout Korea, where it is used as a traditional folk remedy for the treatment of allergic diseases including asthma. However, the pharmacological effect and the mechanistic study of MB have not been investigated. We aimed to investigate the anti-allergic activity of MB in vitro and in vivo and the mechanism of its action on mast cells. Materials and methodsThe anti-allergic activity of MB extract (MBE) was assessed using passive cutaneous anaphylaxis (PCA) in mice and mouse bone marrow-derived mast cells (BMMCs) in vitro. The effects of MBE on mast cell activation were evaluated by using the β-hexosaminidase release assay, reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting analysis. ResultsMBE reversibly inhibited degranulation and generation of cytokines (TNF-α and IL-4) in antigen-stimulated mast cells. With regard to its mechanism of action, MBE inhibited the activation of Lyn and Syk, which have essential roles in degranulation and the production of various inflammatory cytokines. MBE also inhibited the activating phosphorylation of mitogen-activated protein (MAP) kinases, Erk1/2, p38, JNK, and Akt. In agreement with its in vitro effect, MBE significantly inhibited mast cell-mediated PCA reactions in IgE-sensitized mice. ConclusionsThe present results strongly suggest that MBE exerts an anti-allergic effect, both in vitro and in vivo by inhibiting the Lyn and Syk pathways in mast cells. Therefore, MBE may be useful for the treatment of allergic diseases, including atopic dermatitis and allergic asthma.

Особенности и возможность химической модификации поведенческих реакций в приподнятом крестообразном лабиринте хронически облученных мышей с различной генетически детерминированной радиочувствительностью

Structural elements of the central nervous system--neurons, along with the higher neuroendocrine structures and the hypothalamus centres, show high sensitivity to a chronic action of low doses of ionizing radiation (IR) in view of their extreme enrichment by phospholipids and intensive supply by oxygen, creating favorable conditions for the development of oxidizing stress. Stressful influences cause negative emotions in the behaviour of animals manifested as fear or uneasiness. The study represents the results of comparative research into the behavioral reactions characterized by uneasiness in the Balb/c and C57bl/6 mice exposed to a chronic irradiation at low doses. The chitin-melanin-glucan complex from fungi Fomes fomentarius (ChMG) was approved as an adaptive agent. It has been shown that under identical conditions, deposition levels of radionuclides 137Cs and 90Sr are raised in mice with IR hypersensitivity--line Balb/c, in comparison with less radio sensitive mice--line C57bl/6. Simultaneously, Balb/c mice were observed to exhibit the signs of a more anxious behaviour in the new environment. Chronic external and internal radiation exposure to rare ionizing radiation at low doses promotes strengthening of anxiety and phobic reactions in mice with IR hypersensitivity. The use of ChMG in animals neutralized the increase in anxiety and phobic reactions after a prolonged irradiation, thus indicating the presence in ChMG of the anxiolitic activity along with the above mentioned powerful radiosorbent, antioxidant, gene protective and immunomodulatory properties.

Novel birch pollen specific immunotherapy formulation based on contiguous overlapping peptides

BackgroundSynthetic contiguous overlapping peptides (COPs) may represent an alternative to allergen extracts or recombinant allergens for allergen specific immunotherapy. In combination, COPs encompass the entire allergen sequence, providing all potential T cell epitopes, while preventing IgE conformational epitopes of the native allergen.MethodsIndividual COPs were derived from the sequence of Bet v 1, the major allergen of birch pollen, and its known crystal structure, and designed to avoid IgE binding. Three sets of COPs were tested in vitro in competition ELISA and basophil degranulation assays. Their in vivo reactivity was determined by intraperitoneal challenge in rBet v 1 sensitized mice as well as by skin prick tests in volunteers with allergic rhinoconjunctivitis to birch pollen.ResultsThe combination, named AllerT, of three COPs selected for undetectable IgE binding in competition assays and for the absence of basophil activation in vitro was unable to induce anaphylaxis in sensitized mice in contrast to rBet v 1. In addition no positive reactivity to AllerT was observed in skin prick tests in human volunteers allergic to birch pollen. In contrast, a second set of COPs, AllerT4-T5 displayed some residual IgE binding in competition ELISA and a weak subliminal reactivity to skin prick testing.ConclusionsThe hypoallergenicity of contiguous overlapping peptides was confirmed by low, if any, IgE binding activity in vitro, by the absence of basophil activation and the absence of in vivo induction of allergic reactions in mouse and human. Trial registrationClinicalTrials.gov NCT01719133

Macrophage Activation - Mediated Hydrogen Peroxide Generation by the Royal Sun Medicinal Mushroom Agaricus brasiliensis (Higher Basidiomycetes)

Agaricus brasiliensis has been demonstrated to have potent antitumor activity. The activity is postulated to act through mediation of the host immune system. We have reported that A. brasiliensis extract (ABE) inhibited compound 48/80 induced a systemic anaphylaxis-like reaction, ear swelling response, and passive cutaneous anaphylaxis-like reaction in mice. There is some recent information available on the mechanism of antiallergic effects resulting from oral administration of ABE. However, information regarding how ABE may activate macrophages through intestinal epithelial cells is still limited. To clarify the mechanism of macrophages activation by ABE, a gut in vitro model constructed of Caco-2 and RAW264.7 cells was applied. Treatment of ABE to the apical compartment resulted in significant increases in tumor necrosis factor (TNF)-α production in the basolateral compartment. Moreover, addition of catalase to the basolateral compartment before ABE treatment suppressed TNF-α production completely, but the addition of superoxide dismutase did not suppress this at all. These data suggest that ABE could potentiate hydrogen peroxide emissions from Caco-2 cells into the basolateral side and activate macrophages, which is important in the immune system.

진피(陳皮)의 저장기간에 따른 항알러지효과 비교

Objectives : The present study was undertaken to determine whether the ethanol extact of Citri Pericarpium (CP, Pericarp of Citrus unshiu Markovich, Rutaceae) is effective against atopic dermatitis according to it's storage period. Methods : To evaluate antiallergic effect of CP and OCP (Old Citri Pericarpium) evaluated in vivo their inhibitory effects against passive cutaenous anaphylaxis (PCA) reaction induced by IgE-antigen complex and scratching behaviors by compound 48/80. The Anti-atopic effects were measured by contact dermatitis, prurient animal model and PCA reaction. Contact dermatitis in mice as a model of the Type IV reaction caused by Oxazolone. Results : The results showed that anti-pruritus effects, analgesic effects of CP was depends on its hesperetin contents. And It also showed that keep longer in storage appeared to be higher in hesperetin contents. Both CP and OCP(Old CP) have a dose-dependent analgesic action in acetic acid induced writhing syndrome. OCP Potently inhibited PCA reaction in mice, although OCP weakly inhibited in long term contact dermatitis model in mice. Conclusions : These results suggest that the Proportional to the storage period, Citri Pericarpium possesses analgesic effects and anti-allergic effects.

The Rhizomes of Acorus gramineus and the Constituents Inhibit Allergic Response In vitro and In vivo

The rhizomes of Acorus gramineus have frequently been used in traditional medicine mainly for sedation as well as enhancing brain function. In this study, the anti-allergic activity of A. gramineus was investigated. The 70% ethanol extract of the rhizomes of A. gramineus was found to inhibit the allergic response against 5-lipoxygenase (5-LOX)-catalyzed leukotriene (LT) production from rat basophilic leukemia (RBL)-1 cells and β-hexosaminidase release from RBL-2H3 cells with IC50’s of 48.9 and >200 μg/ml, respectively. Among the 9 major constituents isolated, β-asarone, (2R,3R,4S,5S)-2,4-dimethyl-1,3-bis (2',4',5'-trimethoxyphenyl)tetrahydrofuran (AF) and 2,3-dihydro-4,5,7-trimethoxy-1-ethyl-2-methyl-3-(2,4,5-trimethoxyphenyl)indene (AI) strongly inhibited 5-LOX-catalyzed LT production in A23187-treated RBL-1 cells, AI being the most potent (IC50=6.7 μM). Against β-hexosaminidase release by antigen-stimulated RBL-2H3 cells, only AI exhibited strong inhibition (IC50=7.3 μM) while β-asarone and AF showed 26.0% and 39.9% inhibition at 50 μM, respectively. In addition, the ethanol extract of A. gramineus showed significant inhibitory action against the hapten-induced delayed hypersensitivity reaction in mice by oral administration at 200 mg/kg. Therefore, it is suggested that A. gramineus possesses anti-allergic activity and the constituents including β-asarone and AI certainly contribute to the anti-allergic activity of the rhizomes of A. gramineus.

Straub tail reaction in mice treated with σ1 receptor antagonist in combination with methamphetamine

Straub tail reaction (STR) was observed in male ddY mice after simultaneous administration with BMY 14802 (a non-specific σ receptor antagonist) and methamphetamine (METH). The intensity and duration of STR depended on the dose of BMY 14802. The tail reaction was inhibited completely by (+)-SKF 10,047 (a putative σ(1) receptor agonist) and partially by PB 28 (a putative σ(2) receptor agonist). The STR was mimicked in mice treated with BD 1047 (a putative σ(1) receptor antagonist), but not SM-21, a putative σ(2) receptor antagonist, in combination with METH. STR evoked with BD 1047 plus METH was inhibited by (+)-SKF 10,047. STR induced by BMY 14802 and METH was abolished by naloxone (a relatively non-selective opioid receptor antagonist) or U-50,488H (a selective κ-agonist), suggesting that the STR may be mediated by activation of opioid receptor system.

Inhibitory Effect of Eriodictyol on IgE/Ag-Induced Type I Hypersensitivity

Mast cells are the principal effector cells involved in the allergic response, through the release of histamine. We investigated the effect of eriodictyol, derived from the painted maple and yerba santa, on mast cell degranulation and on an allergic response in an animal model. We also investigated its effect on the expression of the ceramide kinase (CERK) involved in calcium-dependent degranulation, and on ceramide activation by multiple cytokines. Eriodictyol suppressed the release of beta-hexosaminidase, a marker of degranulation, and the expression of interleukin (IL)-4 mRNA. It inhibited the expression of CERK mRNA, reduced the ceramide concentration in antigen-stimulated mast cells, and suppressed the passive cutaneous anaphylaxis (PCA) reaction in mice in a dose-dependent manner. These results suggest that eriodictyol can inhibit mast cell degranulation through inhibition of ceramide kinase, and that it might potentially serve as an anti-allergic agent.

A Novel Role of the L-Type Calcium Channel α1D Subunit as a Gatekeeper for Intracellular Zinc Signaling: Zinc Wave

Recent studies have shown that zinc ion (Zn) can behave as an intracellular signaling molecule. We previously demonstrated that mast cells stimulated through the high-affinity IgE receptor (FcεRI) rapidly release intracellular Zn from the endoplasmic reticulum (ER), and we named this phenomenon the “Zn wave”. However, the molecules responsible for releasing Zn and the roles of the Zn wave were elusive. Here we identified the pore-forming α1 subunit of the Cav1.3 (α1D) L-type calcium channel (LTCC) as the gatekeeper for the Zn wave. LTCC antagonists inhibited the Zn wave, and an agonist was sufficient to induce it. Notably, α1D was mainly localized to the ER rather than the plasma membrane in mast cells, and the Zn wave was impaired by α1D knockdown. We further found that the LTCC-mediated Zn wave positively controlled cytokine gene induction by enhancing the DNA-binding activity of NF- κB. Consistent with this finding, LTCC antagonists inhibited the cytokine-mediated delayed-type allergic reaction in mice without affecting the immediate-type allergic reaction. These findings indicated that the LTCC α1D subunit located on the ER membrane has a novel function as a gatekeeper for the Zn wave, which is involved in regulating NF-κB signaling and the delayed-type allergic reaction.

Using the reactome database.

There is considerable interest in the bioinformatics community in creating pathway databases. The Reactome project (a collaboration between the Ontario Institute for Cancer Research, Cold Spring Harbor Laboratory, New York University Medical Center, and the European Bioinformatics Institute) is one such pathway database and collects structured information on all the biological pathways and processes in the human. It is an expert-authored and peer-reviewed, curated collection of well-documented molecular reactions that span the gamut from simple intermediate metabolism to signaling pathways and complex cellular events. This information is supplemented with likely orthologous molecular reactions in mouse, rat, zebrafish, worm, and other model organisms. This unit describes how to use the Reactome database to learn the steps of a biological pathway; navigate and browse through the Reactome database; identify the pathways in which a molecule of interest is involved; use the Pathway and Expression analysis tools to search the database for and visualize possible connections within user-supplied experimental data set and Reactome pathways; and the Species Comparison tool to compare human and model organism pathways.

Mechanism of walnut induced anaphylaxis-like shock reaction in mice (175.1)

Abstract Walnut is a leading cause of life-threatening anaphylaxis in humans. Such reactions are largely thought to be mediated by the Type-I hypersensitivity mechanism. We have found that English walnut extract (EWE) upon oral or i.p., administration in naive mice without pre-sensitization results in anaphylaxis-like shock response as quantified by hypothermia. We have been studying the mechanism underlying this unusual reaction. The Fcg receptor knockout (KO) mice, IgE non-responder SJL mice and TLR4 mutant C3H/HeJ mice, all exhibited hypothermia response indicating that the reaction was antibody and LPS independent respectively. We then examined the role of complement system. The C3a receptor (C3aR) KO mice were protected from shock responses. Among the C5 deficient (DBA/1J) and C5 sufficient (DBA/2J) mice, both exhibited hypothermia responses with DBA1 being a slightly lower responder. Since the shock reaction was complement dependent, we reasoned and tested whether EWE causes hemagglutination (HA) of RBC. Using a standard HA assay protocol we evaluated the HA activity of EWE. We found that the EWE had a significant HA activity for RBC obtained from BALB/c, DBA/1J and DBA/2J mice (HA titre: 1/32; translates to ~35,555 HA units per gram of EWE protein). These data are consistent with the hypothesis that EWE causes HA in vivo leading to complement system activation with consequent C3aR dependent anaphylaxis-like shock reaction.