Guizhi-Shaoyao-Zhimu decoction attenuates rheumatoid arthritis by inhibiting mast cell degranulation

Background:In this study,we investigated whether prophylactic treatment with Guizhi-Shaoyao-Zhimu decoction(GSZ)could delay the onset of rheumatoid arthritis by targeting mast cells.Methods:Collagen-induced arthritis was used to evaluate the effect of GSZ in preventing arthritis and joint destruction.Immunohistochemical staining revealed the accumulation of histamine H4 receptor and tryptase alpha/beta-1 in the ankle joint of the model.Then,we explored the effect of GSZ serum on fibroblast-like synoviocytes using standard transwell invasion and migration assays.Real-time quantitative polymerase chain reaction and western blot were used to detect the expression of toll-like receptor 4(TLR4)/myeloid differentiationfactor 88(MyD88)/nuclear factor-κB p65(NF-κB p65).Results:The results showed that pre-rheumatoid arthritis treatment with GSZ could reduce inflammation and maintain cartilage structure in the collagen-induced arthritis model.Moreover,GSZ significantly blocked mast cell degranulation and inhibited the TLR4/MyD88/NF-κB p65 pathway.Since the combined activation of mast cells via TLR4 and immune complexes enhances inflammation in synovial tissue,we concluded that GSZ may block mast cell degranulation by inhibiting the TLR4/MyD88/NF-κB p65 pathway and thus influence rheumatoid arthritis onset.Conclusion:Taken together,our data suggested that GSZ may be a promising therapeutic decoction for the prophylactic treatment of rheumatoid arthritis.

Hemozoin triggers tumor necrosis factor alpha-mediated release of lysozyme by human adherent monocytes:new evidences on leukocyte degranulation in P.falciparum malaria

Objective:Avidly phagocytosed hemozoin(malarial pigment) alters several functions of human monocytes and stimulates generation of several cytokines.Recently,we showed that phagocytosis of hemozoin by human monocytes increases expression and activity of matrix metalloproteinase-9,a proteolytic enzyme available in specific gelatinase granules,which contain several enzymes including lysozyme.Present work investigated active lysozyme release after phagocytosis of hemozoin and its dependence on production of tumor necrosis factor alpha. Methods:After phagocytosis of hemozoin,hemozoin-containing trophozoites or control meals(opsonized nonparasitized red blood cells and latex particles),monocyte supematants were monitored for 2 hours,in presence of blocking anti-human tumor necrosis factor alpha antibodies or recombinant human tumor necrosis factor alpha cytokine in selected experiments.Lysozyme release was evaluated by a specific spectrometric assay measuring lysozyme activity after coincubation of cell supematants with suspensions of Mycrococcus Lysodeikticus,while levels of soluble tumor necrosis factor alpha were analyzed by specific enzyme-linked immunodsorbent assay. Results:Levels of lysozyme activity and soluble tumor necrosis factor alpha protein were increased in hemozoin in-or trophozoites-laden monocytes supematants.Phagocytosis per se(control meals) also increased lysozyme release,but levels were significantly lower than those obtained after phagocytosis of hemozoin or trophozoites. Interestingly,all effects on lysozyme release observed after phagocytosis were abrogated by blocking anti-human tumor necrosis factor alpha antibodies,while they were mimicked by recombinant human tumor necrosis factor alpha cytokine.Conclusions:Present work shows that phagocytosis of hemozoin promotes monocyte degranulation and enhances active lysozyme release.The effect requires tumor necrosis factor alpha mediation.

Studies on the Degranulation of RBL-2H3 Cells Induced by Traditional Chinese Medicine Injections

Aims: To study RBL-2H3 cell degranulation phenomena induced by some TCMIs through cell morphological and ultra-structural observation, released enzyme activity and establish RBL-2H3 cell degranulation test indicated by β- hexosaminidase activity as a method to evaluate TCMIs at nonclinical stage. Methods: RBL-2H3 cells were used to study the degranulation by co-culture with positive control C48/80 and some TCMIs through morphological and ultra-structure observation, β-hexosaminidase activity detection. RBL-2H3 cell degranulation test was established to detect β-hexosaminidase activity caused by 17 kinds of TCMIs and their ingredients. The cytotoxicity effect of some TCMIs on both RBL 2H3 and BRL cells was measured by CCK-8 assay. Results: Toluidine blue staining and ultra-structure of electronic microscope observation of treated RBL-2H3 cells showed degranulation morphologically. Detection of β-hexosaminidase activity in the supernatant of treated cells showed some TCMIs had elevated enzyme release rates. Further analysis of the ingredients and compound in Tanreqing Injection and Shengmai Injection showed Scutellaria baicalensis Georgi in Tanreqing Injection, Red ginseng and Fructus Schisandrae Chinensis in Shengmai Injection were responsible to the degranulation of RBL-2H3 cells. Osmotic pressures and pH influenced RBL-2H3 degranulation. High Osmotic pressure of Tanreqing Injection and low pH of chlorogenic acid at 2.5 and 5.0 mmol/L congcentration might be responsible to high β-hexosaminidase activity. Most of the TCMIs inducing degranulation had cytotoxicity effect for both RBL-2H3 and BRL cells, but some TCMIs inducing degranulation had no cytotoxicity effect. Conclusion: Some TCMIs can induce degranulation of RBL-2H3 cells;RBL-2H3 cell degranulation test can be used in non-clinical stage to detect the risk causing anaphylactoid reactions. Osmotic pressures and pH influenced RBL-2H3 degranulation, and they should be measured before testing. The mechanism of degranulation caused by some TCMIs is cytotoxic, and some are non-cytotoxic and may be through exicytosis.

Effects of 8-Hydroxyisocapnolactone-2-3-diol and friedelin on mast cell degranulation

Objective: To investigate the effects of friedelin(terpenoid) and 8-hydroxyisocapnolactone-2-3-diol(coumarin) with concentration 10 mM, 30 m M, and 100 mM on inhibiting mast cells(MCs) degranulation.Methods: The investigation was performed in vitro by administering each compound into rat peritoneal MCs and rat basophilic leukemia-2 H3 cells followed by activation with50 mg/m L of compound 48/80 or 1 m M of ionomycin. The concentration of histamine released from each group was measured by a high-performance liquid chromatographyfluorometry system with post-column derivatization using o-phthalaldehyde.Results: 8-Hydroxyisocapnolactone-2-3-diol inhibited degranulation of compound 48/80 activated-rat peritoneal MCs with the histamine release percentages of 74.57%,72.21% and 51.79% when the 10 mM, 30 m M and 100 mM concentrations were used,respectively. Where as about 81% histamine was released by the control group.Degranulation inhibition ability was also observed in ionomycin-activated rat basophilic leukemia-2 H3 cells. In contrast, friedelin failed to inhibit degranulation in either cell type.The inhibition of 8-hydroxyisocapnolactone-2-3-diol was not related to the depletion of histamine synthesis as implied by the total histamine measurement.Conclusions: These results exhibit the promising of 8-hydroxyisocapnolactone-2-3-diol is a potential parent structure for developing a MCs stabilizer.

Studies on the Specific Degranulation of Mast Cell Sensitized by Several Allergens in vitro

Food allergy is a major health issue worldwide. Mast cells play a very important role in the immediate hypersensitivity for which mast cell degranulation needs to be studied extensively. In this study, an approach was taken to study the characteristics of sensitized mast cell degranulation in vitro, which associated with the study of mast cells and animal models. BALB/c mice were immunized respectively by several food allergens, then blood and peritoneal mast cells were collected at different time points. A dynamic determination was carried out between mast cells and serumal IgE. Comparative analysis on sequential time points showed that there was a close coincidence between mast cell degranulation and IgE antibody titers in sensitized BALB/c mice. Furthermore, it is interesting that sensitized mast cells could implement specific degranulation against the challenges in vitro, but the closely tropomyosins induced mast cell degranulation displayed cross reactions. This is very similar to IgE resisting the allergens in vivo. The study disclosed some characteristics on mast cells, coming from sensitized BALB/c mice, degranulation in vitro.

Early CD8 T-cell memory precursors and terminal effectors exhibit equipotent in vivo degranulation

Early after priming, effector CD8 T cells are distinguished into memory precursor and short-lived effector cell subsets （MPECs and SLECs）. Here, we delineated a distinct in vivo heterogeneity in killer cell lectin-like receptor G1 （KLRG-1） expression, which was strongly associated with diverse MPEC and SLEC fates. These in vivo MPECs and SLECs expressed equivalent levels of cytotoxic molecules and effector cytokines. Using a unique in vivodegranulation assay, we found that the MPECs and SLECs similarly encountered infected target cells and elaborated equivalent levels of cytotoxicity in vivo. These data provide direct in vivoevidence that memory-fated ceils pass through a robust effector phase. Additionally, the preferential localization of the MPECs in the lymph nodes, where a lesser degree of cytotoxicity was elaborated, suggests that the MPECs may be protected from excessive stimulation and terminal differentiation by virtue of their differential tissue localization. These data provide novel mechanistic insights into the linear decreasing potential model of memory differentiation.

The amino acids differences in epitopes may promote the different allergenicity of ovomucoid derived from hen eggs and quail eggs

Quail egg ovomucoid can inhibit activation of basophils and eosinophils,while hen egg ovomucoid has been shown to be a major allergen,named Gal d 1.At present,the differences in structure and function between two ovomucoid are unclear.We found the homology of ovomucoid in quail eggs and hen eggs reached77%.Compared with hen egg ovomucoid,the distribution of secondary structure was different in AA52-53,AA57-58,AA66-68,AA71-72,AA131-133,AA139-140,AA157-159 and AA184-185.Among 9 epitopes of egg ovomucoid,there were different amino acids from quail egg ovomucoid in 8 epitopes.Recombination quail egg ovomucoid had trypsin inhibition activity and quail egg ovomucoid didn't specifically bind to serum of eggs allergic patients.Quail egg ovomucoid can significantly inhibit RBL-2 H3 cells degranulation and protect cells morphology to a certain extent,indicating quail egg ovomucoid can inhibit cells activation and have potential anti-allergic effects,which is related to trypsin inhibitory activity.The difference in sensitization compare to hen egg ovomucoid may be due to amino acids differences affecting protein structure by changing antigenic epitopes.

豚鼠变应性鼻炎模型鼻黏膜嗜酸性粒细胞的脱颗粒

鼻黏膜局部嗜酸性粒细胞（eosinophils,EOS）参与变应性鼻炎（allergic rhinitis,AR）发病,EOS脱颗粒与鼻黏膜组织病理学改变有关,广泛的脱颗粒是EOS发挥病理学作用的先决条件。呼吸道黏膜EOS聚集的本身不引起高反应性,只有聚集的EOS激活后才引起相关症状。活化的标志就是EOS的形态和功能发生变化。本研究通过电镜观察豚鼠AR模型鼻黏膜EOS脱颗粒的状态。

Oral polymorphonuclear neutrophil characteristics in relation to oral health： a cross-sectional, observational clinical study

Polymorphonuclear neutmphils （PMNs） have a major role in the innate immune system. However, little is known about PMN contribution in relation to oral health. The objective of this study was to investigate the numbers and functional characteristics of oral PMNs （oPMNs） compared with circulatory PMNs （cPMNs）. Oral rinse and venous blood samples were obtained from 268 systemically and orally healthy volunteers in a cross-sectional observational study. PMN counts, cell cycle analysis and cellular activation state were investigated. Also, reactive oxygen species （ROS） production was analyzed, with and without bacterial stimulation （Fusobacterium nucleatum）. In males, 1.2 × 10^6- 1.0 ×10^6 oPMNs were collected, and showed a tendency to correlate with the levels of gingival bleeding （r= 0.215, P= 0.008）. Comparable oPMNs counts were found among females （1.0 ×10^6＋ 0.7 × 10^6）. More late-stage apoptotic/necrotic cells were found among the oPMNs （53.1%） compared with the cPMNs （8.5%; P〈 0.001）. Without additional stimulation, oPMNs were more activated than cPMNs, as indicated by higher expression of CD11b, CD63 and CD66b, and higher constitutive ROS levels （P〈 0.001）. Notably, in response to bacterial stimulation, oPMNs released comparable ROS levels as cPMNs （P= 0.042）. In conclusion, this study provides data on viable oPMNs showing high levels of activation in orally and systemically healthy individuals, free of apparent caries lesions and periodontal disease. These data suggests that although the oPMNs are in a more mature stage of their life cycle compared with the cPMNs, oPMNs are still responsive to stimulation, which indicates their functional potential and possible contribution to a healthy oral ecosystem.

Anaphylactic cardiovascular collapse during hemodialysis: Kounis syndrome in the dialysis room

Kounis syndrome seems to be not a rare disease but a rarely diagnosed disorder. Multiple causes can join forc-es and trigger the development of this syndrome. We report the first case of Kounis syndrome manifesting as myocardial infarction with cardiovascular collapse that occurred in the dialysis room following an allergic reac-tion. The dialysis apparatus material of polyurethane, polyamide, polycarbonate, silicon rubber and polypro-pylene were incriminated causes. Physicians should be aware of the causality and existence of this disorder in order to achieve early and correct diagnosis and apply the appropriate therapeutic measures.

Morphofunctional Properties of Human Platelets, Treated with Low Doses of Hydrogen Peroxide

We studied morphofunctional properties of human platelets, treated with low doses of hydrogen peroxide (H2O2). It was shown that platelet incubation with 20 - 75 μM H2O2 during 24 hours at 20°C - 22°C did not cause visible structural deficiencies in cells. Platelets maintained their adhesion activity and velocity of degranulation at 20 μM H2O2. 30 - 60 μM H2O2 dose-dependently reduced lamella’s growth and allowed retaining granule bulk inside cytoplasm of spreading platelets. After 1 - 2 hours, the most effect of platelet granules’ stabilization was registered at samples, previously incubated with 60 μM H2O2. Adrenalin activation led to rapid degranulation of stabilized platelets, whereas lamella’s growth in activated platelets was low or medium.

Intracellular localisation of Mycobacterium marinum in mast cells

AIM:To study the bacteriocidal or bacteriostatic role of mast cells during infection with Mycobacterium.METHODS:Mycobacterium marinum(M.marinum)(BAA-535/M strain)was investigated for its ability to grow at a temperature relevant to the mammalian host.Primary mast cells were differentiated from bone marrows of mice,a human mast cell line(HMC-1)and a human monocytic cell line(Mono Mac6)were maintained in culture.Mice were stimulated by intraperitoneal injection of heat-killed M.marinum to study cytochemically the degranulation of peritoneal mast cells.HMC-1 cells were stimulated with M.marinum to analyse m RNA expression for inflammatory reactant genes,while HMC-1 and primary mouse mast cells were infected with M.marinum to establish in parallel cell viability(lactate dehydrogenase release and cell counts)and viable mycobacterial counts.Flow cytometry was used to assess intracellular presence of fluorescein isothiocyanate labelled M.marinum after trypan blue quenching and to measure the extent of infection-induced apoptosis or necrosis in HMC-1.A GFP expressing recombinant M.marinum strain was used to assess intracellular location by fluorescence microscopy.Light microscopy of osmium tetroxide and Gram Twortstained sections of 0.5 μm and transmission electron microscopy were undertaken as sensitive methods.RESULTS:Since its isolation,M.marinum has adapted to grow at 37 ℃.This study found that M.marinum infects HMC-1 cells and primary murine mast cells,where they survive,replicate,and cause dose dependent cell damage over the analysis period of up to 120 h.Amikacin was an effective aminoglycoside antibiotic to eliminate extracellular or membrane attached M.marinum in order to adequately quantify the intracellular bacterial loads.In vivo,intraperitoneal injection of heat-killed M.marinum led to the release of mast cell granules in mice.HMC-1 cells stimulated with M.marinum showed a biphasic pattern of increased mR NA expression for LL-37 and COX-2/TNF-a during 24 h of stimulation.In HMC-1,M.marinum localised to the cytoplasm whereas in primary mast cells,M.marinum were found in vacuoles.CONCLUSION:The effector role of mast cells in infection with M.marinum can be studied in vitro and in vivo.

Comparison of the Biological Activity and Constituents in Japanese Ambers

Background/Aim: Kuji amber is an interesting natural source for drug discovery because a new anti-allergic compound, named kujigamberol and several new compounds have been isolatated from it. It was important to evaluate the yield, biological activities and constituents of each methanol extract of Kuji, Iwaki, Choshi, Mizunami and Ube ambers in Japan in order to establish if additional new compounds could be identified in these ambers. Materials and Method: Biological activities of each extract were evaluated using growth-restoring activity of the mutant yeast strain involving Ca2+-signal transduction and inhibition activity of degranulation in rat basophilic leukemia (RBL)-2H3 cells. Constituents of each extract were analyzed by high performance liquid chromatography (HPLC). Results: All ambers except Ube amber have growth-restoring activity against the mutant yeast. Both Kuji and Iwaki ambers inhibited the degranulation of RBL-2H3 cells induced by the calcium ionophore A23187 in a dose dependent manner. The main biologically active compound in Kuji amber, kujigamberol, was also isolated from Iwaki amber and analyzed by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Conclusion: Kuji and Iwaki ambers appeared to have the same origin. Choshi, Mizunami, and Ube ambers are valuable sources for biologically active compounds which are different from those of Kuji amber.

Fine-tuning Mast Cells is Essential for the Maintenance and Regulation of the Systemic and Immune Homeostasis

During the past decades,populous expansion in mast cell scientific literature came forth with more,than forty-four thousand PubMed publications available to date.Such surge is due to the appreciation of the momentous role of mast cells in the evolution of species,in the development and maintenance of vital physiological functions,such as reproduction,homeostasis,and fluids,diverse immunological roles,and the potential of far-reaching effects despite minute numbers.While the emerging knowledge of the importance of mast cells in equilibrium comes of age when looking at the matter from an evolutionary perspective,the recognition of mast cells beyond detrimental performance in allergies and asthma,during protection against parasites,falters.Beyond wellknown classical functions,mast cells can process and present antigens,can serve as a viral reservoir,can respond to hormones and xenobiotics,initiate antiviral and antibacterial responses,phagocytosis,apoptosis,and participate in important developmental cornerstones.During evolution,upon the development of a sophisticated niche of innate and adaptive cell populations,certain mast cell functions became partially transmutable,yet the potency of mast cells remained considerable.Reviewing mast cells enables us to reflect on the certitude,that our sophisticated,complex physiology is rooted deeply in evolution,which we carry ancient remnants of,ones that may have decisive roles in our functioning.This communication sets out the goal of characterizing mast cells,particularly the aspects less in limelight yet of immense significance,without the aspiration exhaust it all.

A Fatal Case of Chronic Eosinophilic Leukemia Not Otherwise Specified (CEL-NOS) in a 19-Year-Old Male with Acute Transformation into Blast Crisis

Chronic eosinophilic leukemia (CEL) is a rare disorder that is characterized by hypereosinophilia with increased number of blood or marrow blasts (>5% and <20%). CEL is distinguished from hypereosinophilic syndrome (HES) by the presence of eosinophilic clonality. Chronic eosinophilic leukemia not otherwise specified (CEL-NOS) diagnosis is made when no fusion genes are detected by most modern molecular testing, particularly the most common fusion gene FIP1L-1/PDGFRA (Factor Interacting with PAP like-1/Platelet-Derived Growth Factor Receptor Alpha). This disease is very rare, and its description in the literature is not well characterized. We report a fetal case of severe CEL-NOS in a 19-year-old male who presented with a plethora of clinical features consists of constitutional symptoms, pancytopenia, intravascular thrombosis, acute stroke and endomyocardial infiltrates. The course of his disease was aggressive and resistant to conventional treatment. After a short period of improvement, an acute transformation into blast crisis (BC) had occurred. The diagnosis was confirmed by morphology and immunophenotyping of bone marrow biopsy. The patient eventually died of heart failure and sepsis. To our knowledge this is the first case report of fatal CEL-NOS transforming into severe blast crisis.

Mast Cell Stabilizing,Antianaphylactic and Antihistaminic Activity of Coccinia grandis Fruits in Asthma

Coccinia grandis Linn(Curcubitaceae) is a climber herb cultivated throughout India.In traditional medicine fruits have been used to treat leprosy,fever,asthma,bronchitis and jaundice.In present study,ethanol extract of C.grandis fruit(ECGF) at 100,125 and 150 mg·kg-1,i.p.,was evaluated for mast cell stabilizing,antianaphylactic and antihistaminic activity using egg albumin induced mast cell degranulation in mice;passive cutaneous anaphylaxis in rats and clonidine induced catalepsy in mice respectively.ECGF at(100-150 mg·kg-1,i.p.) significantly protected egg albumin induced degranulations of mast cells and caused reduction of blue dye leakage in passive cutaneous anaphylaxis in dose dependently.The treatment ECGF also inhibited clonidine induced catalepsy in dose dependent manner.Phytochemical studies observed presence of saponin,steroids,alkaloids,flavonoids and glycosides.In conclusion ECGF possesses mast cell stabilizing;anti anaphylactic and antihistaminic potential which might be used in treatment of asthma.

Panax Notoginseng Saponins Restrains Ischemia-reperfusion-induced Rat Mesenteric Microcirculatory Disturbance

Objective: To investigate the effect of Panax notoginseng saponins(PNS) on ischemia reperfusion(I/R) induced rat mesenteric microcirculatory dysfunctions.Methods: Male Wistar rats weighting 200–250 g were subjected to 10 min ligation of the superior mesenteric artery and vein, followed by60 min reperfusion. PNS(5 mg/kg/hr) was continuously administrated starting from 10 min before ischemia or 10 min after reperfusion until60 min after reperfusion via left jugular vein. Leukocytes adhesion, mast cell degranulation, endothelial peroxidation, and albumin leakage of rat mesenteric venules were observed. Serum myeloperoxidase(MPO) level, intercellular adhesion molecule-1(ICAM-1) expression and Src phosphorylation were examined.Results: PNS ameliorated leukocyte adhesion and mast cell degranulation, while with no obvious effects on endothelial peroxidation and albumin leakage. In addition, PNS inhibited serum MPO increase, intestinal ICAM-1 expression and Src phosphorylation induced by I/R.Conclusions: PNS ameliorated I/R-induced leukocyte adhesion and mast cell degranulation, the former is related to its inhibition of Src phosphorylation and ICAM-1 expression.