Atrial natriuretic peptide induces rat peritoneal mast cell activation by cGMP-independent and calcium uptake-dependent mechanism

Atrial natriuretic peptide (ANP), a 28 amino acid basic polypeptide, is known to induce histamine release from human and rat mast cells in vitro and cause a wheel formation in rat skin. However, cellular events associated with histamine release are not clearly understood. In this study, we have examined the calcium flux and cGMP formation associated with histamine release in the ANP-treated mast cells. ANP, in vitro, induced mast cell degranulation and histamine release in a dose-dependent manner. ANP also induced an enhanced calcium uptake into cells and increased the cellular level of cGMP in mast cells. A high level of calcium in the media caused an inhibition of ANP-dependent histamine release but enhanced the level of intracellular cGMP of mast cells. ANP inducing a dose-dependent increase in vascular permeability of rat skin was confirmed by the extravasation of the circulating Evans blue. The results indicate ANP induced the histamine release and an increase in vascular permeability through mast cell degranulation in cGMP-independent and calcium uptake-dependent manner.     

Design,Synthesis and Biological Evaluations of a Novel Series of Enediynes Constituted with DNA Cleavage,Alkylating and DNA Binding Agents via Varies Spacers

Among the three series of complicated combinatory enediynes, 3a?d were more active than 2a?c and 4a?b , in which 3a?b showed equally inhibitory activity against the growth of Hepa59T/VGH, KB and Hela with mean IC₅₀ values lower than 10 μg/mL. Compounds 2a and 3b exhibited the most potent inhibitory activity against the growth of Hela cells for 4.39 μg/mL. Derivative 3d displayed the greatest ability against the growth of Hepa59T/VGH cell lines for 6.95 μg/mL, while 3a showed more cytotoxic to KB cells than other analogs. Conjugates 2a‐c presented better responses than 3a‐d for the DNA cleavage assay of supercoiled ΦX174.     

New application of human tumor clonogenic assay to in vitro evaluation of tumor-targeting efficiency of immunoconjugates.

This report proposes an efficient in vitro method for the evaluation of drug targeting with monoclonal antibody as a carrier to tumor cells. Monoclonal antibody (35G; IgG2a) selectively binding to alpha-fetoprotein (AFP) from human hepatoma cells (HuH-7) was conjugated with an anticancer drug, vindesine (VDS). Human tumor clonogenic assay (HTCA) with some modifications was applied to estimate the targeting efficiency of a conjugate (VDS-35G) for the first time. In this assay, VDS-35G was cytotoxically active against HuH-7 cells at a lower concentration (0.5 ng/ml) and for a shorter contact time than VDS (50 ng/ml), while 35G and VDS-normal mouse immunoglobulin conjugate (VDS-n-IgG) were not active against the cells. Both VDS-35G and VDS-n-IgG were inactive against HuH-13 cells established from a human hepatocellular carcinoma producing no AFP. In the conventional monolayer culture assay (MCA), VDS-35G showed little effect on HuH-7 cells at the concentration effective in HTCA. The cytotoxic activity of VDS in MCA was similar to that in HTCA but the cytotoxic activity of VDS-35G in MCA was considerably different from that in HTCA. This discrepancy could be explained by the hypothesis that VDS-35G was directed at stem cells of the HuH-7 cell population sensitively and selectively. HTCA was shown to be a useful in vitro evaluation method for drug targeting.     

Effects of low power laser irradiation on intracellular calcium and histamine release in RBL-2H3 mast cells

Although laser irradiation has been reported to promote skin wound healing, the mechanism is still unclear. As mast cells are found to accumulate at the site of skin wounds we hypothesized that mast cells might be involved in the biological effects of laser irradiation. In this work the mast cells, RBL-2H3, were used in vitro to investigate the effects of laser irradiation on cellular responses. After laser irradiation, the amount of intracellular calcium ([Ca2+]i) was increased, followed by histamine release, as measured by confocal fluorescence microscopy with Fluo-3/AM staining and a fluorescence spectrometer with o-phthalaldehyde staining, respectively. The histamine release was mediated by the increment of [Ca2+]i from the influx of the extracellular buffer solution through the cation channel protein, transient receptor potential vanilloid 4 (TRPV4). The TRPV4 inhibitor, Ruthenium Red (RR) can effectively block such histamine release, indicating that TRPV4 was the key factor responding to laser irradiation. These induced responses of mast cells may provide an explanation for the biological effects of laser irradiation on promoting wound healing, as histamine is known to have multi-functions on accelerating wound healing.     

Synthesis of benzimidazole derivatives as antiallergic agents with 5-lipoxygenase inhibiting action

Syntheses were conducted of novel benzimidazole derivatives that suppress histamine release from mast cells, inhibit 5-lipoxygenase, and possess antioxidative action. Among the compounds synthesized, 1-[2-[2-(4-hydroxy- 2,3,5-trimethylphenoxy)ethoxy]ethyl]-2-(4-methyl-1- homopiperazino)benzimidazole (22) potently suppressed histamine release from rat peritoneal mast cells triggered by the antigen-antibody reaction, inhibited 5-lipoxygenase in rat basophilic leukemia-1 (RBL-1) cells, and prevented the NADPH-dependent lipid peroxidation induced by Fe(3+)-ADP in rat liver microsomes, in addition to an antagonizing the contraction of guinea pig ileum caused by histamine.     

Assay of Histamine in Single Mast Cells by Capillary Zone Electrophoresis with Electrochemical Detection

Histamine is one of the chemical mediators in connection with allergies. Only Pihel et al.1 reported a method for detection of histamine in isolated mast cells by high- performance liquid chromatography with electrochemical detection. In this method, single cells were removed and transferred to 300-nL microvials. A part of the supernatant from individual vials was injected into the chromatography column. The analysis of histamine in individual rat peritoneal mast cells using CZE with amp…     

The human mast cell line‐1 cell membrane chromatography coupled with HPLC‐ESI‐MS/MS method for screening potentical anaphylactic components from chuanxinlian injection

Chuanxinlian injection is a traditional Chinese medicine injection widely used in China to treat sore throat, cough and dysentery, although a high occurrence of severe adverse reactions has been reported in clinical practice in recent years. In the present study, a human mast cell line‐1 cell membrane chromatography coupled with HPLC‐ESI‐MS/MS method was established to screen and identify potentical anaphylactic components in chuanxinlian injection, and the dehydroandrographolide was identified as a potential anaphylactic component. In vitro anaphylactic assay showed that intracellular Ca²⁺ concentration clearly increased under dehydroandrographolide (100 μm ) treatment. β ‐Hexosaminidase and histamine release in human mast cell line‐1 cells were both markedly enhanced with increased concentrations of dehydroandrographolide, confirming the anaphylactic activity of dehydroandrographolide. The application for chuanxinlian injection in this study suggested that the developed human mast cell line‐1 cell membrane chromatography coupled with HPLC‐ESI‐MS/MS system may be effective and rapid for screening the potentical anaphylactic components from complex samples.     

Induction of Effective Antitumor Immune Response by Combined Administration of hIL-18 and NDV HN

To analyze the antitumor potential and mechanism of action of simultaneous Newcastle disease virus (NDV) hemagglutinin-neuraminidase(HN) and human interleukin 18(hIL-18) gene transfer in C57BL/6 mice with H22 hepatoma,the mouse model with H22 hepatoma was established in C57BL/6 mice, and the antitumor effects of the combined application of NDV HN and hIL-18 were evaluated in vivo. The results show that the growth of established tumors in mice immunized with adenovirus(Ad)-HN in conjunction with Ad-hIL-18 was significantly inhibited compared with that in mice immunized with Ad-HN, Ad-hIL-18 alone, or the empty vector(Ad-mock). Furthermore, the immunization of mice with Ad-HN in conjunction with Ad-hIL-18 elicited strong natural killer activity and H22 tumor-specific cytotoxic T lymphocyte(CTL) responses in vivo. In addition, T cells from the lymph nodes of mice immunized with Ad-hIL-18 or Ad-HN+Ad-hIL-18 secreted high levels of the Th1 cytokine IL-2 and interferon-γ (IFN-γ), indicating that the regression of tumor cells is related to a Th1-type dominant immune response. These results demonstrate that vaccination with NDV HN together with hIL-18 may be a novel and powerful strategy for cancer immunotherapy.     

Inhibitory effects of epigallocatechin gallate on compound 48/80-induced mast cell activation and passive cutaneous anaphylaxis

Epigallocatechin gallate (EGCG) is a principle phenolic antioxidant found in a variety of plants, including green and black tea. The anti-allergic effect of EGCG is unknown. The purpose of this study is to investigate the effects of EGCG on compound 48/80-induced mast cell activation and passive cutaneous anaphylaxis. For this, the influences of EGCG on the compound 48/80-induced cutaneous reaction were measured in vivo and the effects of EGCG on the compound 48/80-induced mast cell activations were examined in vitro. Results are below: as 1) EGCG significantly inhibited compound 48/80-induced passive cutaneous anaphylaxis, 2) the compound 48/80-induced degranulation, calcium influx and histamine release of rat peritoneal mast cells (RPMCs) were significantly inhibited by the pretreatment with EGCG, and 3) the compound 48/80-mediated inhibition of cAMP level in RPMCs was significantly increased by the pretreatment with EGCG. These results suggested that EGCG, the most abundant polyphenol in green tea, inhibits the compound 48/80-induced mast cell activation and the increase of vascular permeability, and potentially serve as effective therapeutic tools for allergic diseases.     

L-histidine decarboxylase as a probe in studies on histamine

Because the Falck-Hillarp formaldehyde fluorescence method, which was superbly applied to identify catecholaminergic and serotonergic neurons, is not applicable to histamine, the first author (T.W.) developed an antibody to L-histidine decarboxylase (HDC) for identification of the histaminergic neuron system in the brain. The anti-HDC antibody was of great use for mapping the location and distribution of this histaminergic neuron system. (S)-alpha-fluoromethylhistidine, a specific and potent irreversible inhibitor of HDC, was also very useful in studies on functions of the neuron system. The activity of HDC is increased by various agents, treatments, and physiological conditions. We found new compounds that increased HDC activity (i.e., tetradecanoylphobol acetate (TPA), other tumor promoters, and staphylococcal enterotoxin A); and using mast cell-deficient mutant (W/W(v)) mice, we obtained evidence that this increase occurred in macrophages. To further characterize the mechanism of increases in HDC activity, the second author (H.O.) cloned human HDC cDNA and a human HDC gene. In studies on the regulation mechanism of the HDC gene, which is expressed only in limited types of cells such as mast cells, enterochromaffin-like cells in the stomach, cells in the tuberomammillary nucleus of the brain, and macrophages, CpG islands in the promoter region of the HDC gene were found to be demethylated in cells expressing the gene, whereas they are methylated in other cells that do not express the HDC gene. In collaboration with many other researchers, we developed HDC knockout mice. The resulting research is producing a lot of interesting findings in our laboratory as well as in others. In summary, HDC has been and will be useful in studies on functions of histamine.     

A proteome-wide CDK/CRK-specific kinase inhibitor promotes tumor cell death in the absence of cell cycle progression

The identification of molecular determinants of tumor cell survival is an important objective in cancer research. Here, we describe a small-molecule kinase inhibitor (RGB-286147), which, besides inhibiting tumor cell cycle progression, exhibits potent cytotoxic activity toward noncycling tumor cells, but not nontransformed quiescent fibroblasts. Extensive yeast three-hybrid (Y3H)-based proteome/kinome scanning with chemical dimerizers revealed CDK1/2/3/5/7/9 and the less well-characterized CDK-related kinases (CRKs) p42/CCRK, PCTK1/3, and PFTK1 as its predominant targets. Thus, RGB-286147 is a proteome-wide CDK/CRK-specific kinase inhibitor whose further study could yield new insight into molecular determinants of tumor cell survival. Our results also suggest that the [1, 3, 6]-tri-substituted-pyrazolo[3,4-d]-pyrimidine-4-one kinase inhibitor scaffold is a promising template for the rational design of kinase inhibitors with potential applications to disease indications other than cancer, such as neurodegeneration, cardiac hypertrophic growth, and AIDS.     

Influence of UVB,UVA and UVA1 irradiation on histamine release from human basophils and mast cells in vitro in the presence and absence of antioxidants

UV irradiation is widely used for the treatment of atopic eczema. In recent years, UVA1 phototherapy has gained increasing attention. This study analyzed the influence of different UV wavelengths--especially UVA1--on histamine release from human basophils and mast cells. The modulation of this parameter might be responsible for some of the therapeutic effects of UV irradiation. Enriched human basophils and human mast cells (HMC1 cell line) were irradiated with increasing doses of UVB, UVA and UVA1 in vitro. After irradiation, different stimulants were added to induce histamine release. In additional experiments, basophils were preincubated with superoxide dismutase, ascorbate or trolox to study the role of antioxidants in the modulation of histamine release after UV irradiation. UVA and UVA1 significantly inhibited histamine release from basophils and mast cells. UVB only had an inhibitory effect on mast cells. Preincubation with superoxide dismutase and ascorbate did not influence the inhibitory effect of UVA1 on basophil histamine release, whereas trolox decreased significantly the histamine release from nonirradiated basophils.     

透明质酸-量子点的制备及应用

基于量子点(QD)独特的光学成像特性, 采用化学合成法制备了透明质酸(HA)修饰的水溶性纳米量子点(HA-QD), 并将其应用于特异性受体CD44的识别研究中. 体外细胞实验结果证实, 在透明质酸受体的介导下, 该纳米复合物可使小鼠肺腺癌细胞LA795显示特异性的荧光成像. 本研究为建立针对透明质酸受体的肿瘤活体检测及研究肿瘤的发生发展提供了重要的纳米靶向荧光探针.     

Resin-packed nanoelectrospray in combination with video and mass spectrometry for the direct and real-time molecular analysis of mast cells

A nano-electrospray ionization (nanoESI) emitter for analysis of a biological solution was developed by packing a nanoESI needle with two types of resins for desalting and preconcentration of target molecules. Determination of secreted histamine and serotonin molecules in cell culture buffers was demonstrated using 5-methyltryptamine as internal standard. The results showed good linearity of target signals in the concentration range from 0.25 to 50.0 ng/mL of histamine or serotonin. These molecules were monitored to be secreted by A23187 (calcium ionophore) stimulant in rat peritoneal mast cells. Using a combination of a video-microscope and a mass spectrometer, we could visualize exocytotic moments and analyze secreted molecules by mass spectrometry simultaneously. Time-dependent release of histamine and serotonin from activated mast cells showed that significant production of these molecules occurred and reached a maximal level at 15 min for serotonin and at 30 min for histamine, respectively. These results showed that this method allows the direct and timely analysis of secreted molecules in biological responses.     

Characterization of a brain tumor cell line established from transgenic mice expressing the vasopressin SV-40 T antigen

We previously reported that transgenic mice produced with a transgene consisting of the SV40 T antigen and vasopressin without the 3'-flanking region exhibit brain tumors and lymphoma. In this study, transgenic mice were produced with the fusion gene containing the SV40 T antigen and the whole vasopressin gene with the 3'-flanking region. Six transgenic mice were generated, five which died after 2-6 weeks. The remaining founder mouse was investigated for fusion gene expression and tumor progression at the age of 6 weeks. Brain tumor cells were characterized for phenotypes and transgene expression. During in vitro cell cultures, the phenotypic appearances at 10, 20, and 30 passages were as a uniform monolayer with similar growth rates. The site of SV40 T antigen integration was in the A2 region of chromosome 11, and SV40 T antigen was expressed at the same level in cells of both earlier and later passages. Thirty passages were probably insufficient to reach crisis and immortalization. These cells enriched brain tumor cell compositions with astrocytes and neuronal cells.     

Real-time dynamics of label-free single mast cell granules revealed by differential interference contrast microscopy

We demonstrate the capability of differential interference contrast (DIC) microscopy as a simple and useful tool for studying cellular events without fluorescence labeling. By coupling an advanced DIC microscope to a computer-controlled motorized vertical stage and a high-speed, high-resolution CCD camera, real-time three-dimensional monitoring is possible in a high-throughput manner. The performance among three modes of microscopy, bright-field, dark-field and DIC, in terms of horizontal resolving power and vertical sectioning was investigated. As a model, exocytosis of rat peritoneal mast cells was recorded on the subsecond time scale. Three-dimensional tracking of granules during degranulation was achieved and granule–granule fusion before plasma membrane fusion was recorded. Electronic Supplementary Material Supplementary material is available for this article at     

Proteomic analysis of conditioned media from glucose responsive and glucose non-responsive phenotypes reveals a panel of secreted proteins associated with beta cell dysfunction

Media conditioned by dysfunctioning pancreatic beta cells offer an excellent source of potential protein markers associated with this phenotype. Proteins identified from cell culture model systems are often found to be of importance clinically. Previous work by us and others have shown that low-passage MIN-6 cells (MIN-6(L)) respond to changes in glucose concentrations, producing an approximately 5.5-fold glucose-stimulated insulin secretion (GSIS) in response to 26.7 mmol/L, compared with 3.3 mmol/L, glucose. After continuous culture or high-passage (MIN-(H)), this GSIS was no longer present and thus represents an excellent model system for investigating beta cell dysfunction. Employing 2-D difference gel electrophoresis and mass spectrometry a panel of protein markers were identified in conditioned media (CM) from MIN-6(L) and MIN-6(H) beta cells. These proteins, including secretogranin II, secretogranin III and transthyretin, are associated with secretory granule biogenesis and were found to have substantially increased levels in the CM from the non-responsive high-passage MIN-6 beta cells. A panel of protein markers found to have increased abundance levels in CM from MIN-6(H) compared with MIN-6(L) beta cells may have the potential to be used clinically for assessing beta cell function and to monitor the effects of specific therapeutics.     

THE EFFECT OF PROTOPORPHYRIN ON HISTAMINE SECRETION BY RAT PERITONEAL MAST CELLS: A DUAL PHOTOTOXIC REACTION

Abstract— Protoporphyrin-induced phototoxicity in rat peritoneal mast cells was manifested either by inhibition of 48/80-stimulated histamine secretion or by cell lysis. At a protoporphyrin concentration of 100ng/m/ (0.17 μM), histamine secretion was completely inhibited after 30min illumination. After initiation, the inhibited state progressed in the dark, and was irreversible, however, it did not develop into cell lysis. More severe phototoxic reactions in mast cells could not be produced by increasing the PP concentration or the incubation time; however, cell lysis was evoked by increasing the light intensity between 180–950W/m², using a light source with emission maxima in the 350–470nm region. Dual phototoxic effects could also be demonstrated in erythrocytes by manipulating the illumination conditions. Increased resistance to osmotic lysis was seen under moderate conditions, and decreased resistance and cell lysis were seen under severe conditions. In the absence of protoporphyrin, the effect of light alone on mast cells was similar to protoporphyrin-phototoxicity, although the light intensities required were higher both for inhibition (60–130W/m²) and lysis (280–950W/m²). The data therefore indicate that certain cell functions can be specifically disrupted by phototoxic reactions that are not cytotoxic; however, phototoxic reactions that lead to severe membrane protein denaturation and cell lysis also occur. The manifestation of these dual effects depends on the intensity of illumination in the 350–470nm region.     

Cell membrane chromatography coupled online with LC‐MS to screen anti‐anaphylactoid components from Magnolia biondii Pamp. targeting on Mas‐related G protein‐coupled receptor X2

Mas‐related G protein‐coupled receptor X2 was a mast cell–specific receptor mediating anaphylactoid reactions by activating mast cells degranulation, and it was also identified as a target for modulating mast cell–mediated anaphylactoid and inflammatory diseases. The anti‐anaphylactoid drugs used clinically disturb the partial effect of partial mediators released by mast cells. The small molecule of Mas‐related G protein‐coupled receptor X2 specific antagonists may provide therapeutic action for the anaphylactoid and inflammatory diseases in the early stage. In this study, the Mas‐related G protein‐coupled receptor X2 high expression cell membrane chromatography was coupled online with liquid chromatography and mass spectrometry and successfully used to screen anti‐anaphylactoid components from Magnolia biondii Pamp. Fargesin and pinoresinol dimethyl ether were identified as potential anti‐anaphylactoid components. Bioactivity of these two components were investigated by β hexosaminidase and histamine release assays on mast cells, and it was found that these two components could inhibit β hexosaminidase and histamine release in a concentration‐dependent manner. This Mas‐related G protein‐coupled receptor X2 high expression cell membrane chromatography coupled online with liquid chromatography and mass spectrometry system could be applied for screening potential anti‐anaphylactoid components from natural medicinal herbs. This study also provided a powerful system for drug discovery in natural medicinal herbs.     

A dendritic cell-like biomimetic nanoparticle enhances T cell activation for breast cancer immunotherapy

Cancer immunotherapy has remarkably improved the therapeutic effect of melanoma and non-small cell lung cancer in the clinic. Nevertheless, it showed disappointing clinical outcomes for treating immunosuppressive tumors, wherein aggressive T cells are rather limited in tumor sites. Therefore, regulating the behavior of T cells in tumor sites to increase their attack ability for suppressing the immunosuppressive tumor is highly desirable. Inspiringly, we designed a dendritic cell-like biomimetic nanoparticle (DMSNs³@HA) to regulate the behavior of T cells for improving the immunotherapy effect against immunosuppressive tumors. In this work, anti-CD3 and anti-CD28 were responsible for mimicking dendritic cells to activate T cells, and anti-PD-1 for blocking the pathway of PD-1/PD-L1 to break the immune “brake”, which synergistically regulated the behavior of T cells to attack cancer cells. Experimental results indicated that DMSNs³@HA can effectively activate T cells and improve their immune response to significantly inhibit the growth of breast cancer. Moreover, it also proved that T cell activation combining immune checkpoint blocking induced the “1 + 1 >2” immunotherapy effect against immunosuppressive tumors. We expect that this strategy will provide new insights into tumor immunotherapy by modulating T cell behavior.

A dendritic cell-like biomimetic nanoparticle has been designed to regulate the behavior of T cells for improving the immunotherapy effect against immunosuppressive tumors.