Anti-Allergic Potential of Cinnamaldehyde via the Inhibitory Effect of Histidine Decarboxylase (HDC) Producing Klebsiella pneumonia.

Allergy is an immunological disorder that develops in response to exposure to an allergen, and histamines mediate these effects via histidine decarboxylase (HDC) activity at the intracellular level. In the present study, we developed a 3D model of Klebsiella pneumoniae histidine decarboxylase (HDC) and analyzed the HDC inhibitory potential of cinnamaldehyde (CA) and subsequent anti-allergic potential using a bacterial and mammalian mast cell model. A computational and in vitro study using K. pneumonia revealed that CA binds to HDC nearby the pyridoxal-5'-phosphate (PLP) binding site and inhibited histamine synthesis in a bacterial model. Further study using a mammalian mast cell model also showed that CA decreased the levels of histamine in the stimulated RBL-2H3 cell line and attenuated the release of ß-hexoseaminidase and cell degranulation. In addition, CA treatment also significantly suppressed the levels of pro-inflammatory cytokines TNF-α and IL-6 and the nitric oxide (NO) level in the stimulated mast cells. A gene expression and Western blotting study revealed that CA significantly downregulated the expressions of MAPKp38/ERK and its downstream pro-allergic mediators that are involved in the signaling pathway in mast cell cytokine synthesis. This study further confirms that CA has the potential to attenuate mast cell activation by inhibiting HDC and modifying the process of allergic disorders.

Small molecules that inhibit the late stage of Munc13-4-dependent secretory granule exocytosis in mast cells.

Ca2+-dependent secretory granule fusion with the plasma membrane is the final step for the exocytic release of inflammatory mediators, neuropeptides, and peptide hormones. Secretory cells use a similar protein machinery at late steps in the regulated secretory pathway, employing protein isoforms from the Rab, Sec1/Munc18, Munc13/CAPS, SNARE, and synaptotagmin protein families. However, no small-molecule inhibitors of secretory granule exocytosis that target these proteins are currently available but could have clinical utility. Here we utilized a high-throughput screen of a 25,000-compound library that identified 129 small-molecule inhibitors of Ca2+-triggered secretory granule exocytosis in RBL-2H3 mast cells. These inhibitors broadly fell into six different chemical classes, and follow-up permeable cell and liposome fusion assays identified the target for one class of these inhibitors. A family of 2-aminobenzothiazoles (termed benzothiazole exocytosis inhibitors or bexins) was found to inhibit mast cell secretory granule fusion by acting on a Ca2+-dependent, C2 domain-containing priming factor, Munc13-4. Our findings further indicated that bexins interfere with Munc13-4-membrane interactions and thereby inhibit Munc13-4-dependent membrane fusion. We conclude that bexins represent a class of specific secretory pathway inhibitors with potential as therapeutic agents.

Inhibitory Activities of Compounds from the Marine Actinomycete Williamsia sp. MCCC 1A11233 Variant on IgE-Mediated Mast Cells and Passive Cutaneous Anaphylaxis.

The compounds of the deep-sea-derived marine Williamsia sp. MCCC 1A11233 (CDMW) were isolated, which are secondary metabolites of the actinomycetes. In this study, seven kinds of CDMW were found to decrease degranulation and histamine release in immunoglobulin E (IgE)-mediated rat basophilic leukemia (RBL)-2H3 cells. The production of cytokines (tumor necrosis factor-α, interleukin-4) was inhibited by these CDMW in RBL-2H3 cells, and their chemical structures were established mainly based on detailed analysis of their NMR spectra. CDMW-3, CDMW-5, and CDMW-15 were further demonstrated to block mast cell-dependent passive cutaneous anaphylaxis in IgE-sensitized mice. Bone marrow mononuclear cells (BMMCs) were established to clarify the effect of CDMW-3, CDMW-5, and CDMW-15 on mast cells. The seven kinds of CDMW decreased the degranulation and histamine release of BMMCs. Furthermore, flow cytometry results indicated that CDMW-3, CDMW-5, and CDMW-15 increased the annexin+ cell population of BMMCs. In conclusion, CDMW-3, CDMW-5, and CDMW-15 have obvious antiallergic activity due to induction of the apoptosis of mast cells.

Structure-activity relationships of vanillic acid ester analogs in inhibitory effect of antigen-mediated degranulation in rat basophilic leukemia RBL-2H3 cells.

Methyl vanillate (1) showed strong degranulation inhibitory activity among vanillin derivatives tested. In order to find structure-activity relationships for developing anti-allergic agents with simple structures and potent activity, we synthesized several vanillic acid (VA) ester derivatives with C1-C4 and C8 alkyl chains and evaluated their degranulation inhibitory activities. The most active compound of VA ester derivatives was derivative 5 with a C4 straight alkyl chain, and derivative 5 exhibited approximately three-fold greater inhibitory activity than that of 1. Moreover, we designed 8 types of analogs based on 5, and we found that the minimum structure for potent degranulation inhibitory activity requires direct connection of the butyl ester moiety on the benzene ring and at least one hydroxyl group on the benzene ring. Butyl meta or para hydroxyl benzoate (10 or 11) has a simpler structure than that of 5 and exhibited more potent degranulation inhibitory activity than that of 5.

Degranulation Inhibitors from Medicinal Plants in Antigen-Stimulated Rat Basophilic Leukemia (RBL-2H3) Cells.

Mast cells and basophils play important roles in both immediate- and late-phase reactions of type 1 allergy. Histamine, which is released from mast cells and basophils stimulated by an antigen or degranulation inducers, is usually determined as a degranulation marker in experiments on immediate allergic reactions in vitro. ß-Hexosaminidase is also stored in secretory granules of the cells and is released concomitantly with histamine when the cells are immunologically activated, and recently this enzyme activity in the medium has been used as a marker of the degranulation. In this paper, we review our studies on the search for degranulation inhibitors, such as flavonoids, stilbenes, and curcuminoids, from medicinal plants using rat basophilic leukemia (RBL-2H3) cells.

Eicosapentaenoic acid and docosahexaenoic acid suppress Th2 cytokine expression in RBL-2H3 basophilic leukemia cells.

It is known that the intake of omega-3 fatty acids, such as eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), is beneficial for preventing and/or treating allergic diseases. The pathogenesis of allergic diseases is associated with overactivation of Th2-skewed immunity. Basophils generate large amounts of Th2 cytokines such as interleukin (IL)-4 and IL-13, which are critically involved in allergic inflammation. We investigated how EPA and DHA affect Th2 cytokine expression in phorbol 12-myristate 13-acetate- and ionomycin (PI)-activated RBL-2H3 basophilic leukemia cells. EPA and DHA induced a dramatic decrease in the production of IL-4 and IL-13 and their transcription in a dose-dependent manner. Luciferase assays of RBL-2H3 cells stably expressing Il4 and Il13 promoter-reporter plasmids demonstrated a significant suppression of PI-induced promoter activation. Analysis of certain transcription factors revealed that nuclear expression of c-Fos and the mRNA expression were suppressed by EPA and DHA. Furthermore, they significantly inhibited the nuclear expression and translocation of nuclear factor of activated T cells (NF-AT)1. In contrast, the expression levels of nuclear factor kappa-B (NF-κB), GATA-binding proteins (GATAs), and CCAAT/enhancer binding protein alpha (C/EBPα) were not significantly affected by EPA and DHA. Phosphorylation of extracellular signal-related kinase was inhibited by EPA and DHA, and phosphorylation of p38 mitogen-activated protein kinase was decreased by DHA, but not by EPA. Taken together, our data suggest that EPA and DHA may suppress Th2-skewed allergic immune responses by inhibiting the expression of basophilic IL-4 and IL-13.

Inhibitory effects of panduratin A on allergy-related mediator production in rat basophilic leukemia mast cells.

Immediate-type hypersensitivity is characterized by elevated levels of immunoglobulin E (IgE) and activated mast cell plays a crucial role by releasing granule contents, lipid-derived mediators, cytokines, and chemokines. To evaluate the antiallergic effects of panduratin A isolated from Boesenbergia pandurata Roxb., we determined its effects on calcium (Ca(2+)) influx, degranulation, and inflammatory mediators in calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA)-stimulated rat basophilic leukemia (RBL-2H3) cells. Panduratin A (20 µM) inhibited secretion of ß-hexosaminidase (46.69 ± 9.6 %), histamine (34.32 ± 2.1 %), and Ca(2+) influx (43.84 %). Panduratin A reduced the production of prostaglandin E(2) (PGE(2), 47.58 ± 3.4 %), leukotriene B(4) (LTB(4), 98.15 ± 1.6 %), and the mRNA expression of cyclooxygenase-2, 5-lipoxygenase, interleukin (IL)-4, IL-13, and tumor necrosis factor-α. Furthermore, panduarin A attenuated phosphorylation of Akt, the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) expression. These results indicate that panduratin A might be useful as an agent against immediate-type hypersensitivity.

Russula cutefracta inhibits antigen-induced degranulation and Syk and MAPK phosphorylation in rat basophilic leukaemia cells

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Conjugation of sulfonated aluminum phthalocyanine to doxorubicin can improve the efficacy of photodynamic cancer therapy.

Sulfonated aluminum phthalocyanine (AlPcS), a widely used photosensitizer for photodynamic therapy of cancer, was conjugated to doxorubicin (Dox), a chemotherapy drug, through electrostatic binding. The fluorescence resonance energy transfer from Dox to AlPcS showed the formation of AlPcS-Dox conjugates, as the fluorescence intensity of conjugated Dox was decreased and that of the AlPcS moiety was enhanced. This AlPcS-Dox conjugation was further confirmed by electrophoresis. The AlPcS-Dox conjugates enhanced the cellular uptake of AlPcS three times more than unconjugated AlPcS in both human hepatocellular carcinoma cell line 7701 and rat basophilic leukemia cell line. Moreover, the photodynamic killing effect of the conjugates was markedly increased as compared with that of AlPcS alone or the cytotoxicity of Dox alone, showing an enhanced effect of the AlPcS-Dox conjugates. These results indicate that the conjugation of a photosensitizer with a chemotherapy drug may improve photodynamic cancer therapy.

Anti-inflammatory effects of low-molecular weight chitosan oligosaccharides in IgE-antigen complex-stimulated RBL-2H3 cells and asthma model mice.

The anti-inflammatory effects of low-molecular weight chitosan oligosaccharides (LM-COS) prepared from high-molecular weight chitosan by enzymatic digestion were investigated against allergic reaction and allergic asthma in vivo and in vitro. Allergic asthma is an inflammatory disease of the airways associated with enhanced degranulation and cytokine generation. The LM-COS (<1 kDa), consisting of glucosamine (GlcN)(n), n=3-5, were capable of inhibiting both antigen-stimulated degranulation and cytokine generation in rat basophilic leukemia RBL-2H3 cells. The protective effect of LM-COS against ovalbumin (OVA)-induced lung inflammation in asthma model mice was also examined. Oral administration of LM-COS (16 mg/kg body weight/day) resulted in a significant reduction in both mRNA and protein levels of interleukin (IL)-4, IL-5, IL-13, tumor necrosis factor (TNF)-α in the lung tissue and bronchoalveolar lavage fluid (BALF); The protein levels of IL-4, IL-13 and TNF-α in BALF were decreased by 5.8-fold, 3.0-fold and 9.9-fold, respectively, compared to those in the OVA-sensitized/challenged asthma control group. These results suggest that the oral administration of LM-COS is effective in alleviating the allergic inflammation in vivo and thus can be a good source material for the development of a potent therapeutic agent against mast cell-mediated allergic inflammatory responses and airway inflammation in allergic inflammatory diseases, including asthma.

The green tea polyphenol (-)-epigallocatechin-3-gallate inhibits leukocyte activation by bacterial formylpeptide through the receptor FPR.

Although green tea polyphenol catechin is considered as a potential anti-inflammatory agent, its effect on bacterial component-induced inflammation has been poorly investigated. We examined the capacity of epigallocatechin gallate (EGCG) to regulate leukocyte responses to bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLF), which is recognized by a human G protein-coupled receptor FPR on phagocytic leukocytes. Pretreatment of human monocytic cells or FPR-transfected rat basophilic leukemia cells (ETFR cells) with EGCG significantly inhibited fMLF-induced chemotaxis. Intraperitoneal administration of EGCG in mice suppressed fMLF-induced leukocyte infiltration into the air pouch created in the skin. Mechanistic studies revealed that EGCG dose-dependently suppressed fMLF-induced calcium flux in monocytic cells and ETFR cells. fMLF-induced ETFR cell migration was significantly inhibited by a specific MEK1/2 inhibitor, PD98059, which was associated with reduction in fMLF-induced ERK1/2 phosphorylation. These results suggest that EGCG inhibits FPR-mediated leukocyte activation thus is a promising anti-inflammatory compound.

Gab2 antisense oligonucleotide blocks rat basophilic leukemic cell functions.

Adapter molecule Grb2-associated binder-like protein 2 (Gab2) plays a critical role in FcepsilonRI-induced mast cell degranulation and activation. The present study aimed to investigate the pharmacological effects of an antisense oligonucleotide (ASO) targeted at Gab2 on the immune responses of rat basophilic leukemic (RBL)-2H3 cells. Gab2 ASOs were rationally designed and transfected into RBL-2H3 cells. Gab2 mRNA and protein knockdown was confirmed by real-time RT-PCR and immunoblotting, respectively. Effects of Gab2 ASO on FcepsilonRI-induced release of histamine and beta-hexosaminidase was measured by EIA and an enzymatic assay, respectively; signaling events by immunoblotting; and cytokine mRNA expression by RT-PCR. Effects of Gab2 ASO on cell adhesion and migration were performed on fibronectin-coated 96-well plate and transwells cell culture chambers, respectively. We have characterized a phosphorothioate-modified ASO targeted at Gab2 mRNA that was able to knockdown Gab2 mRNA and protein in RBL-2H3 cells. Gab2 ASO significantly blocked IgE-mediated mast cell release of beta-hexosaminidase and histamine; phosphorylation of Akt, p38 mitogen-activated protein kinase and PKCdelta; and up-regulation of cytokine mRNA levels (e.g. IL-4, -6, -9 and -13, and TNF-alpha). In addition, Gab2 ASO markedly prevented mast cell adhesion to fibronectin-coated plates and restrained random migration of RBL-2H3 cells in cell culture chambers. Our findings show that Gab2 knockdown in RBL-2H3 cells by ASO strategy can suppress many aspects of the mast cell functions and, therefore, a selective Gab2 ASO may have therapeutic potential for mast cell-dependent allergic disorders.

Monosomy 7 as the sole abnormality of an acute basophilic leukemia.

We report the case of a 72-year-old man who had the very rare disease acute basophilic leukemia with the sole chromosomal finding of a monosomy 7. Most nuclear cells in the peripheral blood and bone marrow samples were either basophils or blasts. The blasts showed negative reaction with myeloperoxidase, periodic acid Schiff, chloroacetate esterase, alpha-naphthyl butyrate esterase, acid phosphatase, and Sudan black B. Metachromatic features of the blasts, however, were observed with toluidine blue stain. Electron microscopic evaluation showed the typical ultrastructure, with basophil and immature mast cell granules. Cytogenetic study revealed monosomy 7 in all metaphase cells, and this finding was confirmed by fluorescence in situ hybridization. The Philadelphia chromosome was absent. Review of the literature revealed abnormalities in cases of ABL. To our knowledge, the case reported here is the first to have basophilic leukemia with monosomy 7 as the only chromosome abnormality.

A fibronectin fragment induces tumor necrosis factor production of rat basophilic leukemia cells.

Proteolytic digest of fibronectin (FN), but not intact FN, induced TNF-alpha secretion of rat basophilic leukemia (RBL-2H3) cells. As a result of the identification of FN fragment responsible for TNF-alpha secretion, a 30-kDa fragment derived from the carboxyl-terminal heparin-binding (Hep 2) domain of FN was isolated from the FN digest. The TNF-alpha secretion was abrogated by treatment of RBL-2H3 cells with cycloheximide, indicating the de novo synthesis of TNF-alpha, but not with polymyxin B, excluding the possible TNF-alpha induction by some contaminated lipopolysaccharides. A 22-mer synthetic peptide originated from the Hep 2 domain, termed FNIII14, which has been found to negatively modulate the beta1 integrin activation, had the ability to induce TNF-alpha production, whereas this activity of FNIII14 disappeared by shuffling a YTIYVIAL sequence essential for the integrin-inactivating activity. FNIII14 suppressed the spreading of RBL-2H3 cells on FN substrate, wherein RBL-2H3 cell proliferation was inhibited with FNIII14 in a dose-dependent manner. Thus, it appears that FN fragments containing the YTIYVIAL anti-adhesive site affect the activation status of RBL-2H3 mast cells, characterized by the stimulation of TNF-alpha production and growth suppression, probably due to negative regulation of beta1 integrin activity.

Ephedrae herba in Mao-Bushi-Saishin-To inhibits IgE-mediated histamine release and increases cAMP content in RBL-2H3 cells.

Acute effect of Mao-Bushi-Saishin-To (Ma-Huang-Fu-Zi-Xi-Xin-Tang in Chinese: MBS) on histamine release was investigated. The IgE-mediated anaphylaxic response in Wistar rats was significantly suppressed by MBS and Mao. However, Saishin and Bushi had no or little effect on the antigen-mediated anaphylaxic reaction. Mao as well as MBS but not Saishin nor Bushi inhibited IgE-mediated histamine release from rat basophilic leukemia (RBL-2H3) cells. Consistently, MBS and Mao but not Bushi nor Saishin increased cAMP levels in RBL-2H3 cells. However, ephedrine, methylephedrine, and pseudoephedrine, the main components in Mao, did not affect histamine release. From these results, increase of cAMP levels may account for the inhibitory effect of Mao on histamine release. Furthermore, these inhibitory actions of MBS were mainly due to Mao with an ingredient(s) different from ephedrines.

Function oriented synthesis: the design, synthesis, PKC binding and translocation activity of a new bryostatin analog.

Bryostatin 1 represents a novel and potent therapeutic lead with a unique activity profile. Its natural and synthetic availability is severely limited. Function oriented synthesis provides a means to address this supply problem through the design of synthetically more accessible simplified structures that at the same time incorporate improved functional activity. Pharmacophore searching and a new computer aided visualization of a possible binding mode are combined with an understanding of function and knowledge of synthesis to design and prepare a new and simplified compound with bryostatin-like function in biological systems. This new compound is a potent ligand for protein kinase C in vitro (K(i) = 8.0 nM). More significantly, the described molecule retains the functional ability to translocate a PKCdelta-GFP fusion protein in RBL cells. The extent of protein translocation and the sub-cellular localization induced by this new compound is similar to that seen in response to bryostatin 1, indicating that the new molecule retains the functional activity of the natural product but is simpler and can be synthesized in a practical fashion.