CD96 Blockade Ameliorates Imiquimod-Induced Psoriasis-like Dermatitis via Suppression of IL-17A Production by Dermal γδ T Cells.

Psoriasis is a chronic inflammatory skin disease. IL-23 plays a critical role in its pathogenesis by inducing production of IL-17A from pathological Th17 cells and IL-17A-producing γδ T cells. However, the mechanisms regulating the IL-23/IL-17 axis in psoriasis are incompletely understood. In this study, we show that, in comparison with wild-type mice, those deficient in the CD96 immunoreceptor had lower production of IL-17A in their dermal γδ T cells and milder psoriasis-like dermatitis after topical application of imiquimod (IMQ). Moreover, transfer of CD96-deficient dermal γδ T cells into the skin of Rag1-deficient mice resulted in them developing milder IMQ-induced dermatitis compared with Rag1-deficient mice transferred with wild-type dermal γδ T cells. In γδ T cells in vitro, CD96 provides a costimulatory signal for the production of IL-23-induced IL-17A. In mice given an anti-CD96 neutralizing Ab, IL-17A production from dermal γδ T cells decreased and IMQ-induced dermatitis was milder compared with mice given a control Ab. These results suggest that CD96 is a potential molecular target for the treatment of psoriasis.

CD96 Blockade Ameliorates Imiquimod-Induced Psoriasis-like Dermatitis via Suppression of IL-17A Production by Dermal γδ T Cells.

Psoriasis is a chronic inflammatory skin disease. IL-23 plays a critical role in its pathogenesis by inducing production of IL-17A from pathological Th17 cells and IL-17A-producing γδ T cells. However, the mechanisms regulating the IL-23/IL-17 axis in psoriasis are incompletely understood. In this study, we show that, in comparison with wild-type mice, those deficient in the CD96 immunoreceptor had lower production of IL-17A in their dermal γδ T cells and milder psoriasis-like dermatitis after topical application of imiquimod (IMQ). Moreover, transfer of CD96-deficient dermal γδ T cells into the skin of Rag1-deficient mice resulted in them developing milder IMQ-induced dermatitis compared with Rag1-deficient mice transferred with wild-type dermal γδ T cells. In γδ T cells in vitro, CD96 provides a costimulatory signal for the production of IL-23-induced IL-17A. In mice given an anti-CD96 neutralizing Ab, IL-17A production from dermal γδ T cells decreased and IMQ-induced dermatitis was milder compared with mice given a control Ab. These results suggest that CD96 is a potential molecular target for the treatment of psoriasis.

Pre-implantation evaluation of a small-diameter, long vascular graft (Biotube®) for below-knee bypass surgery in goats.

There are no small-diameter, long artificial vascular grafts for below-knee bypass surgery in chronic limb-threatening ischemia. We have developed tissue-engineered vascular grafts called "Biotubes®" using a completely autologous approach called in-body tissue architecture (iBTA). This study aimed at pre-implantation evaluation of Biotube and its in vivo preparation device, Biotube Maker, for use in below-knee bypass surgery. Forty nine makers were subcutaneously embedded into 17 goats for predetermined periods (1, 2, or 3 months). All makers produced Biotubes as designed without inflammation over all periods, with the exception of a few cases with minor defects (success rate: 94%). Small hole formation occurred in only a few cases. All Biotubes obtained had an inner diameter of 4 mm and a length of 51 to 52 cm with a wall thickness of 594 ± 97 µm. All Biotubes did not kink when completely bent under an internal pressure of 100 mmHg and did not leak without any deformation under a water pressure of 200 mmHg. Their burst strength was 2409 ± 473 mmHg, and suture retention strength was 1.75 ± 0.27 N, regardless of the embedding period, whereas tensile strength increased from 7.5 ± 1.3 N at 1 month to 9.7 ± 2.0 N at 3 months with the embedding period. The amount of water leakage from the needle holes prepared in the Biotube wall was approximately 1/7th of that in expanded polytetrafluoroethylene vascular grafts. The Biotubes could be easily connected to each other without cutting or anastomosis leaks. They could be stored for at least 1 year at room temperature. This study confirmed that even Biotubes formed 1 month after embedding of Biotube Makers had properties comparable to arteries.

CD300a blockade enhances efferocytosis by infiltrating myeloid cells and ameliorates neuronal deficit after ischemic stroke.

Immune responses contribute to tissue injury and repair during and after ischemic stroke. However, the spatiotemporal and initiating molecular events remain incompletely understood. Here, we show that mice deficient in the phosphatidylserine receptor CD300a, which is highly expressed on brain myeloid cells including Ly6Chi monocytes, exhibited ameliorated neurological deficit after middle cerebral artery occlusion (MCAO). CD300a inhibited signaling through the CD300b-DNAX-activation protein 12 (DAP12) signaling pathway to prevent efferocytosis of apoptotic cells. Deficiency of CD300a enhanced efferocytosis by myeloid cells infiltrating the brain as early as 1 hour after MCAO and reduced release of damage-associated molecular patterns from dead cells, resulting in milder inflammation in the penumbral region. Treatment with an anti-CD300a neutralizing antibody ameliorated the neurological deficit after MCAO. These findings reveal an important role of efferocytosis in the super-acute phase of ischemic stroke pathology and identified CD300a as a target for immunotherapy in treating ischemic stroke.

DNAM-1 regulates Foxp3 expression in regulatory T cells by interfering with TIGIT under inflammatory conditions.

Regulatory T (Treg) cells that express forkhead box P3 (Foxp3) are pivotal for immune tolerance. Although inflammatory mediators cause Foxp3 instability and Treg cell dysfunction, their regulatory mechanisms remain incompletely understood. Here, we show that the transfer of Treg cells deficient in the activating immunoreceptor DNAM-1 ameliorated the development of graft-versus-host disease better than did wild-type Treg cells. We found that DNAM-1 competes with T cell immunoreceptor with Ig and ITIM domains (TIGIT) in binding to their common ligand CD155 and therefore regulates TIGIT signaling to down-regulate Treg cell function without DNAM-1-mediated intracellular signaling. DNAM-1 deficiency augments TIGIT signaling; this subsequently inhibits activation of the protein kinase B-mammalian target of rapamycin complex 1 pathway, resulting in the maintenance of Foxp3 expression and Treg cell function under inflammatory conditions. These findings demonstrate that DNAM-1 regulates Treg cell function via TIGIT signaling and thus, it is a potential molecular target for augmenting Treg function in inflammatory diseases.

Suppression of Th1 and Th17 Proinflammatory Cytokines and Upregulation of FOXP3 Expression by a Humanized Anti-DNAM-1 Monoclonal Antibody.

DNAM-1 is an activating immunoreceptor expressed on hematopoietic cells, including both CD4+ and CD8+ T cells, natural killer cells, and platelets. Since DNAM-1 is involved in the pathogenesis of various inflammatory diseases and cancers in humans as well as mouse models, it is a potential target for immunotherapy for these diseases. In this study, we generated a humanized neutralizing antihuman DNAM-1 monoclonal antibody (mAb), named TNAX101A, which contains an engineered Fc portion of human IgG1 to reduce Fc-mediated effector functions. We show that TNAX101A efficiently interfered the binding of DNAM-1 to its ligand CD155 and showed unique functions; it decreased production of the inflammatory cytokines such as interferon-gamma, tumor necrosis factor alpha, interleukin (IL)-6, IL-17A, and IL-17F by anti-CD3 antibody-stimulated or alloantigen-stimulated T cells and increased FOXP3 expression in anti-CD3-stimulated regulatory T (Treg) cells. These dual functions of TNAX101A may be advantageous for the treatment of T cell-mediated inflammatory diseases through both downregulation of effector T cell function and upregulation of Treg cell function.

Identification and isolation of splenic tissue-resident macrophage sub-populations by flow cytometry.

Tissue-resident macrophages in the spleen, including red pulp and white pulp macrophages, marginal zone macrophages (MZMs) and marginal zone metallophilic macrophages (MMMs), are highly heterogeneous as a consequence of adaptation to tissue-specific environments. Each macrophage sub-population in the spleen is usually identified based on the localization, morphology and membrane antigen expression by immunohistochemistry. However, their phenotypical and functional characteristics remain incompletely understood due to the difficulty of identification and isolation by flow cytometry. We used a cocktail of three enzymes (Collagenase D, Dispase I and DNase I), rather than traditional mechanical grinding, for isolation of each sub-population, which resulted in significant improvement of isolation of these macrophage sub-populations, particularly MZMs and MMMs, as determined by CD11bhiF4/80medTim4hi and CD11bhiF4/80medTim4med, respectively. This method should be helpful for molecular and functional characterization of each splenic resident macrophage sub-population.

Glycoprotein nmb Is Exposed on the Surface of Dormant Breast Cancer Cells and Induces Stem Cell-like Properties.

Glycoprotein nmb (GPNMB) is a type I transmembrane protein that contributes to the initiation and malignant progression of breast cancer through induction of epithelial-mesenchymal transition (EMT). Although it is known that EMT is associated with not only cancer invasion but also acquisition of cancer stem cell (CSC) properties, the function of GPNMB in this acquisition of CSC properties has yet to be elucidated. To address this issue, we utilized a three-dimensional (3D) sphere culture method to examine the correlation between GPNMB and CSC properties in breast cancer cells. Three-dimensional sphere cultures induced higher expression of CSC genes and EMT-inducing transcription factor (EMT-TF) genes than the 2D monolayer cultures. Three-dimensional culture also induced cell surface expression of GPNMB on limited numbers of cells in the spheres, whereas the 2D cultures did not. Therefore, we isolated cell surface-GPNMBhigh and -GPNMBlow cells from the spheres. Cell surface-GPNMBhigh cells expressed high levels of CSC genes and EMT-TF genes, had significantly higher sphere-forming frequencies than the cell surface-GPNMBlow cells, and showed no detectable levels of proliferation marker genes. Similar results were obtained from transplanted breast tumors. Furthermore, wild-type GPNMB, but not mutant GPNMB (YF), which lacks tumorigenic activity, induced CSC-like properties in breast epithelial cells. These findings suggest that GPNMB is exposed on the surface of dormant breast cancer cells and its activity contributes to the acquisition of stem cell-like properties.Significance: These findings suggest that cell surface expression of GPNMB could serve as a marker and promising therapeutic target of breast cancer cells with stem cell-like properties. Cancer Res; 78(22); 6424-35. ©2018 AACR.

Development and Characterization of Novel Monoclonal Antibodies Against Human DNAM-1.

DNAM-1 (CD226) is an activating immunoreceptor expressed on lymphocytes and myeloid cells. CD155 and CD112 are the ligands for DNAM-1. DNAM-1 plays an important role in tumor immunity mediated by CD8+ T cells and NK cells. Moreover, the interaction of DNAM-1 with the ligands contributed to the development of acute graft versus host disease (GVHD) and treatment with anti-DNAM-1 monoclonal antibodies (mAb) dramatically improved acute GVHD in a mouse model, suggesting that DNAM-1 may be a good molecular target for therapy to acute GVHD in human. In this study, we generated and characterized five novel clones of anti-human DNAM-1 mAbs, named TX94, TX95, TX96, TX107, and TX108. Among these mAbs, TX94 is a unique neutralizing mAb that most efficiently blocked the interaction between DNAM-1 and CD155. Furthermore, TX94 inhibited NK cell-mediated cytotoxicity against a tumor cell line and suppressed CD8+ T cell proliferation mediated by allogeneic mixed lymphocyte reaction. Thus, TX94 may be useful for molecular therapy targeting DNAM-1.

Increased CD112 expression in methylcholanthrene-induced tumors in CD155-deficient mice.

Tumor recognition by immune effector cells is mediated by antigen receptors and a variety of adhesion and costimulatory molecules. The evidence accumulated since the identification of CD155 and CD112 as ligands for DNAM-1 in humans and mice has suggested that the interactions between DNAM-1 and its ligands play an important role in T cell- and natural killer (NK) cell-mediated recognition and lysis of tumor cells. We have previously demonstrated that methylcholanthrane (MCA) accelerates tumor development in DNAM-1-deficient mice, and the Cd155 level on MCA-induced tumors is significantly higher in DNAM-1-deficient mice than in wild-type (WT) mice. By contrast, Cd112 expression on the tumors is similar in WT and DNAM-1-deficient mice, suggesting that CD155 plays a major role as a DNAM-1 ligand in activation of T cells and NK cells for tumor immune surveillance. To address this hypothesis, we examined MCA-induced tumor development in CD155-deficient mice. Unexpectedly, we observed no significant difference in tumor development between WT and CD155-deficient mice. Instead, we found that Cd112 expression was significantly higher in the MCA-induced tumors of CD155-deficient mice than in those of WT mice. We also observed higher expression of DNAM-1 and lower expression of an inhibitory receptor, TIGIT, on CD8+ T cells in CD155-deficient mice. These results suggest that modulation of the expression of receptors and CD112 compensates for CD155 deficiency in immune surveillance against MCA-induced tumors.

Drug interaction between sunitinib and cimetidine and contribution of the efflux transporter ATP-binding cassette C2 to biliary excretion of sunitinib in rats.

The present study investigated the effect of the H2 antagonist cimetidine on the pharmacokinetics of a multi-targeted receptor tyrosine kinase (RTK) inhibitor, sunitinib, in Sprague-Dawley (SD) rats and Eisai hyperbilirubinemic mutant rats (EHBR) lacking the efflux transporter, ATP-binding cassette C2 protein (ABCC2). Rats received an intraperitoneal injection of cimetidine (10 mg/kg) once a day for three days. On day 4, sunitinib (3 mg/kg) was administered intravenously 30 min after the final injection of cimetidine or saline to SD rats. Disappearance of sunitinib from plasma was significantly delayed by cimetidine. The pharmacokinetic parameter of sunitinib, systemic clearance (CLSYS), was significantly reduced and the half-life was significantly prolonged, with no change in the volume of distribution at steady-state (VSS). When the effect of cimetidine on the biliary excretion of sunitinib at steady-state condition was investigated in SD rats, cimetidine had no effect on some transporter-mediated biliary excretion of sunitinib. Furthermore, the contribution of ABCC2 to the biliary excretion of sunitinib was also examined in SD rats and EHBR. The biliary clearance of sunitinib was significantly lower in EHBR, but the biliary excretion rate of EHBR was not different from that of SD rats, and the contribution of biliary excretion to systemic elimination was small, suggesting that sunitinib is mainly eliminated by cytochrome P450 3A4 (CYP3A4)-mediated metabolism and is not excreted into the bile via ABCC2. These findings indicate that co-administration of cimetidine alters the pharmacokinetics of sunitinib probably due to inhibition of CYP3A4, suggesting the possibility that cimetidine should be used carefully for patients with cancer being treated with sunitinib therapy.

[Preparation of dealcholized onetaxotere®, docetaxel injection formulation and its pharmacokinetics in rats].

Onetaxotere®(OTAX)injection, which is a docetaxel(DOC)injection formulation, cannot be administered to those patients with alcohol intolerance or hypersensitivity, because it contains ethanol as a dissolving agent. To broaden treatment options for those patients, we tried to eliminate ethanol from OTAX injection. Under sterile conditions, dealcoholization was carried out using nitrogen gas in a hot water bath at 50°C. By this method, the ethanol included in OTAX injection was almost completely removed and DOC in the formulation was stable for 28 days. When the dealcoholized or untreated OTAX injection was intravenously injected in rats, no significant differences in the pharmacokinetic parameters of DOC were observed between those with dealcoholized and untreated OTAX injections. It is expected that dealcoholized OTAX may be useful in patients with alcohol-related difficulties.

Establishment and characterization of a novel anti-DNAM-1 monoclonal antibody.

DNAM-1 (CD226) is expressed on the majority of NK cells, CD8(+) T cells, and CD4(+) T cells and mediates an activating signal in these cells upon binding to the ligands CD155 or CD112 expressed on target cells or antigen-presenting cells. DNAM-1 plays an important role in tumor immunity mediated by NK cells and CD8(+) T cells and the development of graft-versus-host disease (GVHD) in mice. We previously generated a monoclonal antibody against mouse DNAM-1, TX42, which inhibited DNAM-1 binding to its ligands CD155 and CD112 and inhibited activation of NK cells and CD8(+) T cells in vitro. Injection of mice with TX42 ameliorated the development of GVHD in mice. Here, we generated a new clone of anti-DNAM-1 MAb, TX92. TX92 similarly stained primary spleen cells, including CD8(+) and CD4(+) T cells and NK cells. TX92 as well as TX42 interfered with the interaction between DNAM-1 and ligands CD155 and CD112. However, TX92 recognizes a different epitope and, unlike TX42 partially, but not completely, depleted peripheral blood (PB) CD8(+) T cells in vivo. Thus, TX92 is a unique MAb that is characterized not only by inhibitory function of DNAM-1 binding to the ligands but also by function of partial depletion of PB CD8(+) T cells.

De-alcoholization of paclitaxel injection for clinical application.

A generic drug of Taxol® Injection, Paclitaxel Injection NK (PTX injection), cannot be used for patients with severe hypersensitivity or overwhelming intolerance to alcohol because it contains ethanol as a dissolving agent. Therefore, we evaluated the suitability of de-alcoholized PTX injection for clinical application. De-alcoholization was carried out using inactive N2 gas under sterile conditions. The pharmacokinetic properties of the de-alcoholized PTX injection were evaluated in rats after intravenous injection. Finally, the de-alcoholized PTX injection was administered to a patient with alcohol intolerance to evaluate its suitability for clinical application. The ethanol included in the supplied PTX injection was almost completely removed (>99.9%). PTX, the major component of the de-alcoholized PTX injection, was stable with no decomposed compounds or bacterial contamination, although its viscosity was increased by 29-fold compared with untreated PTX injection. No significant differences in the pharmacokinetic parameters of PTX were observed between the de-alcoholized and untreated PTX injections. No drunkenness was observed in the patient with severe alcohol intolerance after injection of de-alcoholized PTX injection. Adverse events such as nausea, muscle pain, joint pain, neuropathy and myelosuppression were observed at similar degrees to those after injection of untreated PTX injection. The plasma concentrations of PTX after injection of the de-alcoholized PTX injection were similar to those after injection of untreated PTX injection. The present findings suggest that almost complete de-alcoholization of PTX can be achieved easily under sterile conditions and that the resulting product can be used safely for patients with severe alcohol intolerance.

Panipenem does not alter the pharmacokinetics of the active metabolite of irinotecan SN-38 and inactive metabolite SN-38 glucuronide (SN-38G) in rats.

The present study has investigated the effect of panipenem, a widely used antibiotic, on the pharmacokinetics of an active metabolite of irinotecan (CPT-11), 7-ethyl-10-hydroxy-camptothecin (SN-38) and SN-38 glucuronide (SN-38G) produced by uridine-diphosphate glucuronosyltransferase (UGT) 1A isoform-mediated glucuronidation in rats. Rats received a 1 h infusion with panipenem at a loading dose of 10 mg/kg and a maintenance dose of 15 mg/min/kg once a day for 5 days. When the effect of pretreatment with panipenem on glucuronidation activities of substrates for hepatic UGT1A isoforms was investigated using substrates 4-methylumbelliferone (4MU), estradiol and SN-38, the rate of 4MU glucuronide formation was significantly increased, but that of estradiol glucuronide formation was unchanged. However, the rate of SN-38G formation showed a tendency to increase. One hour after the final infusion of panipenem or saline, SN-38 (2 mg/kg) was administered intravenously in rats with or without bile duct cannulation. Pretreatment with panipenem had no effect on the plasma concentration-time curves and biliary excretion of SN-38 and SN-38G in rats with and without bile duct cannulation. There were also no significant differences in the relative extent of glucuronidation of SN-38 to SN-38G (AUC(2 h, SN-38G)/AUC(2 h, SN-38)) between panipenem-treated and untreated rats. These findings suggest that pretreatment with panipenem does not alter the pharmacokinetics of SN-38 and SN-38G, suggesting the possibility that panipenem can be used safely for cancer patients undergoing irinotecan chemotherapy.

Endotoxin does not alter the pharmacokinetics of micafungin, but it impairs biliary excretion of micafungin via multidrug resistance-associated protein 2 (ABCC2/Mrp2) in rats.

Micafungin, a newly developed echinocandin-type antifungal agent, is widely used for the treatment of deep-seated fungal infections including those of Candida species and Aspergillus species. In the present study, the possible alterations in the pharmacokinetics and biliary excretion of micafungin were investigated in endotoxemic rats induced by Klebsiella pneumoniae endotoxin. Endotoxin (2 mg/kg) was injected intraperitoneally 24 h before an intravenous injection of micafungin (1 mg/kg). No significant differences in the plasma concentration-time curves and pharmacokinetic parameters of micafungin were observed between endotoxin-treated and endotoxin-untreated rats. When endotoxin-treated rats received a constant-rate infusion of micafungin, the biliary clearance of micafungin was significantly decreased, whereas the steady-state plasma concentration did not change. By protein immunoblot analysis, a significant decrease in the expression of hepatic multidrug resistance-associated protein 2 (ABCC2/Mrp2), which is an efflux protein for micafungin, was observed in endotoxin-treated rats. These results suggest that endotoxin-induced decrease in the hepatobiliary excretion of micafungin is caused, at least in part, by the reduction of Mrp2-mediated hepatobiliary transport ability. The present study may provide information suggesting that micafungin can be used for patients with endotoxemia without the need for dosage adjustment.

Involvement of sulfate conjugation and multidrug resistance-associated protein 2 (Mrp2) in sex-related differences in the pharmacokinetics of garenoxacin in rats.

In this study, the involvement of sulfate conjugation and drug efflux transporter multidrug resistance-associated protein 2 (Mrp2) in sex-related differences in the pharmacokinetics of a new quinolone antimicrobial agent, garenoxacin, was investigated in Sprague-Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBRs) lacking Mrp2. The disappearance of garenoxacin from plasma in female SD rats was significantly faster than that in male SD rats after a single intravenous injection of garenoxacin (5 mg/kg). The systemic clearance of garenoxacin in female rats was approximately threefold larger than that of male rats (2.43 ± 0.31 and 0.87 ± 0.06 l/h/kg, respectively), suggesting the existence of sex-related differences in the pharmacokinetics of garenoxacin. When rats received a constant-rate infusion of garenoxacin, the contribution of biliary and renal excretion of garenoxacin was small, and no significant difference in the biliary (CL(BILE)) clearance of garenoxacin was observed between male and female SD rats. The metabolic clearance [CL(M (SULF))] of garenoxacin to garenoxacin sulfate conjugate (which is mainly excreted into the bile) in female SD rats was 8.5-fold larger than that in male SD rats (27.9 ± 2.94 and 3.28 ± 0.07 ml/h/kg, respectively). The CL(BILE) of garenoxacin was decreased in male and female EHBRs by approximately 50% compared with that in male and female SD rats. These results suggest that sulfate conjugation, but not Mrp2, is mainly involved in the sex-related differences in the pharmacokinetics of garenoxacin.

Effect of a Kampo preparation, byakkokaninjinto, on pharmacokinetics of ciprofloxacin and tetracycline.

The effect on the bioavailability of the antimicrobial agents (ciprofloxacin and tetracycline), which are well known to form chelates with cationic metals such as calcium, was evaluated in 20 healthy male volunteers according to an open, random crossover fashion using a Kampo preparation, byakkokaninjinto (TJ-34) which contains various cationic metals including calcium. Each subject received a single oral dose of tetracycline (250 mg) alone or ciprofloxacin (200 mg) alone along with a single coadministration of one pack (3 g) of the Kampo preparation, at one-week intervals. Concentrations of the drugs in plasma and urine were analyzed by HPLC. Concomitant administration of the Kampo preparation significantly decreased the peak plasma concentration (C(max)) and area under the plasma concentration-time curves (AUC), but not time to reach C(max) (T(max)), of ciprofloxacin and tetracycline. However, the decrease in bioavailability of ciprofloxacin was slight (15%) compared with that of tetracycline (30%). The Kampo preparation significantly decreased the urinary recovery of tetracycline, but not ciprofloxacin, and it had no effect on the renal clearance of either ciprofloxacin or tetracycline. These results indicate that the Kampo preparation tested in this study reduces the extent of bioavailability of ciprofloxacin and tetracycline, but not renal excretion, by decreasing the gastrointestinal absorption due to the formation of insoluble chelates with calcium. We recommend that the dose timing of the Kampo preparation should be carefully controlled to avoid therapeutic failure especially for patients receiving the treatment with tetracycline.

Heme oxygenase-1 mediates the anti-allergic actions of quercetin in rodent mast cells.

OBJECTIVE AND DESIGN: We investigated the involvement of heme oxygenase (HO)-1 in the anti-allergic action of quercetin against degranulation of rat basophilic leukemia (RBL-2H3) cells, rat peritoneal mast cells, and mouse bone marrow-derived mast cells. METHODS: The strength of allergic reaction was evaluated by the extent of degranulation in mast cells sensitized with various stimulants. The levels of HO-1, HO-2, and nuclear factor erythroid 2-related factor 2 (Nrf2) expressions were determined by quantitative RT-PCR, western blotting, or immunocytochemistry. RESULTS: Heme oxygenase activity was upregulated after short exposure to quercetin, followed by the induction of HO-1 expression after long exposure to quercetin. The inhibition of degranulation by quercetin was reversed using tin protoporphyrin IX (SnPP), an HO-1 inhibitor. HO-1 metabolites, bilirubin and CO, led to inhibit degranulation, and quercetin translocated Nrf2 from cytoplasm into nucleus in RBL-2H3 cells. CONCLUSION: These results strongly suggest that quercetin exerted anti-allergic actions via activation of Nrf2-HO-1 pathway.

Effect of thalidomide on endotoxin-induced decreases in activity and expression of hepatic cytochrome P450 3A2.

Thalidomide has been reported to inhibit the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) that are involved in the down-regulation of hepatic cytochrome P450 (CYP) induced by endotoxin. In the present study, we investigated the effects of thalidomide on endotoxin-induced decreases in the activity and expression of hepatic CYP3A2 in rats. Thalidomide (50 mg/kg) was administered orally 22 h and 2 h before intraperitoneal injection of endotoxin (1 mg/kg). Twenty-four hours after the injection of endotoxin, antipyrine clearance experiments were conducted, in which the rats were sacrificed and protein levels of hepatic CYP3A2 were measured. There were no significant differences in the histopathological changes in the liver between the endotoxin-treated and endotoxin plus thalidomide-treated rats. Thalidomide had no effect on the systemic clearance of antipyrine, which is a proper indicator for hepatic CYP3A2 activity, whereas it enhanced endotoxin-induced decrease in the systemic clearance of antipyrine. Western blot analysis revealed that thalidomide had no effect on the protein levels of hepatic CYP3A2, whereas it enhanced the down-regulation of hepatic CYP3A2 by endotoxin. However, there were no significant differences in the concentrations of TNF-alpha and NO in plasma between the endotoxin-treated and endotoxin plus thalidomide-treated rats. The present findings suggest that thalidomide enhances endotoxin-induced decreases in the activity and expression of hepatic CYP3A2.