Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients.Allergy and asthma to cat ( Felis domesticus) is one of the most frequent pets and approximately 10% of the general population in industrialized countries is sensitized to cat allergens The domestic cat  The T cell receptor on primed CD4+ T cells recognize antigen presenting cells carrying MHC II molecules with tightly bound enzyme-digested linear polypeptides of 12 to 25 amino acids derived from the primary structure of the antigen + T cells found in bronchial mucosa, BAL fluid, and sputum in these patients.Allergen-activated T cells are responsible for late-phase reactions (LPR), occurring 6–8 hours after allergen challenge eliciting symptoms ranging from oedema and itching to eczema, and also play a prominent and pivotal role in the pathogenesis of allergic asthma + T cells The most common treatment of allergic diseases is either alleviation of symptoms using drugs or advice the patient to avoid the allergen source T cell epitope (TCE) mappings of Fel d 1 have shown a scattered distribution of TCE located on both chains of Fel d 1 Due to the close interactions between allergen, T, B and basophil or mast cell effector cells, the optimal allergy vaccine should be depleted of IgE-binding epitopes to evade cross-linking of high affinity receptors on effector cells and IgE-facilitated allergen presentation, and show a moderate T cell activation profile to avoid LPR and bronchial hypersensitivity. Modern biotechnology allows fast, easy cloning and production of allergens, allergen isoforms and hypoallergen variants with reduced IgE binding but conserved T cell reactivity in vivo was shown by mouse immunization experiments.In this study we used a novel strategy to engineer full sized and folded allergy vaccine candidates with reduced number of T cell epitopes and reduced risk of inducing anaphylaxis, while maintaining the immunogenic properties. The design has several advantages over previously established methods for construction of hypoallergenic derivatives, which mainly involve disruption of structural B-cell epitopes, while essentially preserving T-cell reactivity. Although treatment with hypoallergens may be efficacious, it also carry the risk of inducing LPR 5 individual clones.Following epPCR, ligation and transformation, the generated mutated Fel d 1 phage libraries typically contained 10vice versa occurring at position 13, 17, 50, 63, 77, 78, 82, 102, 117, 118, 122 and 137  were identified and used as templates for further rounds of epPCR and panning. The iterative mutational procedure for clone 3 was stopped after 4, for clone 11 after 5, and for clone 6 after 7 rounds of panning, when no or few additional mutations could be detected . The fin and 137 . Interes and 137 .E. coli cytoplasm. Final purity of the recombinant proteins was estimated to be >95% by Coomassie-stained SDS PAGE . Proliferative responses were comparable between the three antigen concentrations, with the concentration 10 µg/ml showing the lowest coefficient of variation. PBMCs stimulated with mutants 3.4.7 and 6.7.3 exhibited significantly lower mean proliferation indices in comparison to PBMCs with rFel d 1, P<0.05, . The mutBlocking antibodies of the IgG isotype have been suggested as an important mechanism behind successful SIT. Immunogenicity of rFel d 1 and each of the mutants was investigated by ELISA. Interestingly, all mutants did induce higher titers of antigen specific IgG than the wild-type protein , and imp−5, 9.1E−5 and 7.1E−5 1/s respectively), whereas 6.7.3 and 11.5.2 showed a higher off-rate (kd = 2.6E−4 and 2.7E−4 1/s respectively), , .The ability of the mutants 3.4.7 and 6.7.1 to induce an IgE blocking IgG response upon vaccination were investigated in a mouse model for cat allergy Treatments utilizing high dose hypoallergens, such as attenuated derivatives of wild type proteins or short peptides, covering the T cell repertoires of allergens have been tested in clinical trials. However, such strategies have induced LPRs, where symptoms evolve several hours after administration.in vitro evolution of allergens with a native-like structure for a maintained immunogenicity and treatment efficacy, but with reduced IgE-binding and T cell activation for reduction of immediate and late phase side effects during allergen-specific immunotherapy. Numerous studies have demonstrated sensitization to allergens as a risk factor for the development of allergy and asthma. In more severe cases, asthma is a chronic condition without curative therapeutic options. An alternative rarely considered for this group of patients is alleviation of allergic and asthmatic symptoms by SIT. One of the reasons for the restricted use of SIT in the treatment of extrinsic allergic asthma is the risk of adverse reactions which even may have a fatal outcome The aim of this study was to develop a technology for the Novel candidate vaccines would be expected to have a high safety profile including reduced immediate side effects due to a decrease in IgE-mediated degranulation of effector cells and a reduced capability of the TCE to activate T cells, which could result in mild or absent LPR. We hypothesized that the conservation of a native like tertiary structure with concomitant reduction of the IgE-binding capacity would keep the ability to induce strong protective allergen-specific IgG immune responses, which in turn would be able to compete with IgE for epitope binding on the native allergen. The use of freshly prepared serum pools were used to reduce the risk of epitope biased selection.We used Fel d 1, the major and most dominant cat allergen, as a prototype allergen to test the feasibility of our approach. In a first step the sequence coding for rFel d 1 E. coli either as inclusion body (clones 6.7.1 and 6.7.3) or as soluble cytoplasmic proteins (clone 3.4.7 and 11.5.2), folded well using established methods Four candidate sequences derived from three different panning experiments showing the highest numbers of mutations compared to the rFel d 1 sequence  were choin vitro data from the basophil activation assay. The mutant 6.7.1 generally displayed the highest IgE-binding and degranulating capacity, while the other mutants showed a weaker binding to IgE, with 3.4.7 generally showing the lowest degranulating capacity. We assume that the high number of mutations introduced has reduced the number or conformation of IgE epitopes of Fel d 1 by substitution of amino acids essential for interaction with IgE. We are, however, not able to confirm this hypothesis because no convincing information about conformational IgE epitopes on Fel d 1 is available. Certainly, due to the selection procedure based on serum IgE from cat allergic patients, at least one IgE-binding epitope has to be retained by all mutants. A reduction of the number of IgE-binding epitopes can explain the reduced IgE-binding activity of the mutants and in agreement, all mutants showed a reduction in the capacity to stimulate activation of basophil from cat allergic patients demonstrating the hypoallergenic potential of these mutants , were used to analyze IgE-binding to the Fel d 1 mutants and to rFel d 1 used as positive control. A pool from these sera was prepared for use in allergen competition ELISA.New serum pool from 3–4 cat-sensitized subjects were mixed for each round of biopanning. The inclusion criterion was a cat dander specific IgE content exceeding 10% of the total IgE as measured by ImmunoCAP (Phadia). Serum pools from healthy individuals without serum IgE to cat dander were used as negative controls. Sera mentioned above were collected at the routine allergy diagnostic lab at Karolinska University hospital. Ten consecutively selected well-characterized cat allergic and asthmatic patients were recruited at the Karolinska University Hospital, Lung Allergy clinic, and PBMC were isolated The study was approved by the local ethics committee and all patients gave an informed written consent.Solid-phase ELISA measurements were carried out essentially as described Allergen inhibition ELISA was performed as described above, but with a prior over night incubation at +4°C of serum from a pool of cat sensitized patients (n = 20) mixed with 3-fold serial dilutions (range 170 pg/ml–270 µg/ml) of mutants or rFel d 1 as competing antigen.5′ caccggaatt cgttaaaatg gctgaaacct gc and 5′ cgcgccgctc gaggcacagc gggg, with overhangs designed for the Eco RI/Xho I cloning sites of phagemid pJuFo Libraries containing random mutations were created by epPCR using dNTP-Mutagenesis Kit . The rFel d 1 Phagemids carrying the mutated Fel d 1 coding sequences were transformed into XL1-Blue MRF' Electroporation-Competent Cells  by electroporation , immediately added to 3 ml SOC medium for 1 h at 37°C followed by titration. Transformed cells were added to 12 ml 2×YT medium containing ampicillin (100 mg/L) and tetracycline (10 mg/L) and incubated for 2 h  before addition of helper phage  at a multiplicity of infection (moi) of 10. The medium was supplemented with kanamycin (25 mg/L) after an additional 2 h and the culture incubated over night. The steps described above were repeated 4–7 times depending on the clone.Phage-infected XL1-Blue cell were centrifuged  at 5000× g, 15 min, 4°C. The supernatant was collected and phagemids precipitated with 5% PEG-8000, 4% NaCl final concentration, incubated on ice for 2 h followed by centrifugation . Phages were resuspended in ice cold Tris-buffered saline (TBS) centrifuged  to eliminate cell debris and supernatants stored at 4°C.5 cfu) was added and incubated with shaking at 100 rpm for 2 h at 37°C. The supernatant containing unbound phages was discarded and the wells washed using TBS with 0,1% Tween with increasing stringency , to isolate clones with the highest IgE- binding capacity. Bound phages were eluted with 0.1 M glycin buffer, pH 2.2, neutralized with 0.5 M Tris-HCl pH 10.9, and added to 5 ml of exponentially growing XL1-Blue, optical density (OD) 1.0 at 600 nm. Number of phages before (input) and after biopanning (output) were calculated by titrations counting single colony forming units on agar plates. To prepare phage for the next round of panning ampicillin (100 mg/L) and tetracycline (10 mg/L) were added to the infected XL1-Blue cells. Helper phage (moi of 10) and kanamycin (25 mg/L) were added after 2 h and the cultures incubated over night at 37°C with shaking at 250 rpm.100 µl, 2 µg/ml of monoclonal anti-human IgE  was coated to 96-well microtiter plates (Nunc). 100 µl of a patient serum pool (n = 3) containing Fel d 1-specific IgE, Fel d 1 negative serum, or TBS as negative control was added with shaking at 100 rpm for 2 h at 37°C. After washing, phage library  and used as templates for sequencing . The repetitive biopanning process was terminated when no new dominant clones could be amplified, as confirmed by sequencing of 12 randomly picked clones.The inserts of twelve randomly picked colonies from the input and output of each panning round were amplified by colony PCR with pJuFo sequencing primers Finally selected clones were PCR-amplified using rFel d 1 primers, cleaved by XhoI and NdeI restriction endonucleases and ligated into the expression vector pET20b (Novagen). The constructs were transformed into BL21(DE3)pLysS (Novagen), plated on LB plates containing 100 mg/L ampicillin and 35 mg/L chloramphenicol and incubated overnight at 37°C. Single colonies were picked, cultured in 15 ml LB medium containing ampicillin (100 mg/L) and chloramphenicol (35 mg/L) overnight at 37°C and 250 rpm and used to inoculate two liters of LB medium containing ampicillin (100 mg/L) and chloramphenicol (35 mg/L) for protein production. Cells at log phase were induced using isopropyl thiogalactoside at a final concentration of 0.4 mM, with simultaneous addition of ampicillin to 100 mg/l final concentration. After 3 h incubation at 37°C, the cells were collected by centrifugation , resuspended in 30 ml PBS and frozen at −20°C.2+-HiTrap IMAC column  followed by elution with 5 mM Tris-HCl containing 0.5 M NaCl, 0.5 M imidazol and 6 M guanidine-HCl. The guanidine was removed by dialysis in 20 mM Tris-HCl, pH 7.4. Proteins were concentrated to 3 ml in a 10 ml Amicon cell  prior to size exclusion chromatography on a 16/60 Superdex 200 pg column equilibrated in PBS pH 7.4 using FPLC system . The peaks corresponding to monomeric proteins (30 kDa) were diluted 1∶1 in 20 mM Tris-HCl pH 8.7, and loaded on 10 ml anion exchange Q-Sepharose HP columns  equilibrated in 20 mM Tris-HCl pH 8.7. Proteins were fractioned with a 15 column volumes linear gradient using 20 mM Tris-HCl, 1 M NaCl, pH 7.4, aliquoted and stored at −80°C. Protein concentration was determined using the BCA protein assay  using bovine serum albumin as standard. The purity was estimated by SDS-PAGE under reducing and non-reducing conditions, and by analytical SEC using a Superdex 200 32/300 column  equilibrated in PBS at a flow rate of 40 µl/min using ÄKTA Purifier .Thawed pellets were adjusted to 6 M guanidine-HCl, sonicated  by 5 bursts×10 sec (10 ma) on ice and centrifuged at 18600× g for 10 min. Buffer was exchanged on Sephadex G-25 equilibrated in 5 mM Tris-HCl containing 0.5 M NaCl, 20 mM imidazol and 6 M guanidine-HCl, loaded on a 5-ml NiFor control of allergen-specific T cell proliferation, two samples of purified rFel d 1 containing 20 and 40 ng lipopolysaccharide (LPS)/mg protein, respectively, were used. The LPS content was determined with the limulus amebocyte lysate endochrome assay according to the instructions .SDS-PAGE was performed under reducing conditions as described + cells using FACS Calibur . The magnitude of reactivity  obtained from the cell cultures were divided by cpm values from unstimulated wells and expressed as a stimulation index (SI). SI values ≥2.0 were considered to be positive. The proliferation varied between patients  and was normalized for purposes of comparison. For each patient, a mean proliferation value  was calculated. This value was set to 100%. The stimulation for single antigens is presented as percentages in relation to the MPV.Allergen-specific proliferation of cultured PBMC from 10 cat allergic patients was analyzed in triplicates with 2×10ad libitum. Animal experiments were approved by the Swedish local ethics committee for animal welfare.Six to eight week old female BALB/c mice were obtained from Charles River  and housed with food and water The ability of the four mutants to generate IgE-blocking IgG antibodies was tested. Groups of mice (n = 4) were immunized subcutaneously in the neck with 10 µg of the mutant proteins and as positive control rFel d 1, each protein adsorbed to 1 mg aluminium hydroxide . Injections were given on day 0, 14, 28 and 42. On day 47, mice were sacrificed and blood collected. Sera from mice immunized with a distinct protein were pooled and a serum pool from naïve mice was used as a negative control.In a first set of experiments titers were normalized between the five different mouse immune serum pools by 5-fold serial dilutions from 1/50 to 1/781250 using the respective antigen by ELISA as described  [7].A/L, and serum from an IgE-myeloma of 2000 kU/L as negative control was added, followed by IgE detection as described.The ability of mouse IgG-antibodies to block binding of patient's serum IgE to allergen was determined by ELISA. Plates were coated with rFel d 1 and blocked with BSA. The normalized mouse serum pools were added and incubated over night at 4°C. The following day, patient serum containing IgE to rFel d 1 diluted to 0.85 kUTo assess the biological relevance of anti- mutant IgG, the ability to inhibit Fel d 1-mediated degranulation of basophils was investigated. Briefly, 10-fold serial dilutions of rFel d 1 (1000 ng/ml to 0.01 ng/ml) or medium was pre-incubated over night with the four normalized mouse anti-mutant IgG serum pools and the rFel d 1 pool as control. The mixes of antigen and serum pool were then added to fresh venous blood from three cat allergic patients as described above.For real-time analysis of the different mouse antisera with respect to binding properties to rFel d 1 an Attana A200 system  was used. rFel d 1 (5 µg/mL in 10 mM sodium actetate pH 4.5) was immobilized on a LNB sensor chip by amine coupling according to the manufacturer's instructions. All experiments were performed in running buffer, HBS-T , at 37°C. Dissociation rate constants for the 5 different antisera were determined at 25 µL/min using the Attester Evaluation off-rate screening tool, between time points 150 and 450 seconds. All sera were normalized as determined by ELISA, and diluted 1∶100 in HBS-T. The surface was regenerated using a 41 s pulse of 10 mM glycine pH 1.5. Non-specific binding to the activated and deactivated LNB carboxyl surface was recorded using sera from mice immunized with PBS.Animals were obtained and housed as described above. Mice were sensitized s.c. with 1 µg, rFel d 1 adsorbed to 1 mg alum in 200 µl PBS or PBS only (non-sensitized negative control) on day 0, 14 and 28 to obtain high titers of anaphylactic antibodies. Groups of mice (n = 7–10) were then vaccinated s.c. with 10 µg of rFel d 1, 3.4.7) or 6.7.1 respectively in 200 µl PBS or 200 µl PBS alone (control) on day 30, 32 and 34. The mice were sacrificed on day 42 and peripheral blood was collected and stored at −80°CLevels of Fel d 1-specific IgE, IgG1 and IgG2a was analyzed by ELISA. For analysis of IgE, 96-well plates (Nunc) were coated with 0.2 µg/100 µl rat anti-mouse IgE (BD Biosciences), and IgG1 and IgG2a plates were coated with 0.5 µg rFel d 1. Sera were diluted 1∶20 for IgE measurement, 1∶25000, 1∶100000 or 1∶1000000 for IgG1 and 1∶1000 for IgG2a. Levels of IgG 1 anti rFel d 1 was analysed using an in-house reference curve and presented in units/ml, U/ml. Bound IgE levels was measured using 0.2 µg/100 µl and well of biotinylated rFel d 1 followed by alkaline phosphatase (AP)-conjugated streptavidine . IgG1 and IgG2a levels were detected using goat anti-mouse AP-conjugated IgG1 (1∶5000) and IgG2a (1∶1000) respectively , followed by OD measurements at 405 nm.The IgE blocking capability of the antibodies obtained after treatment was investigated using a competitive inhibition sandwich ELISA as described above. Pooled sera from the group of mice treated with PBS only were selected and added to wells coated with rat anti-mouse IgE. Fresh serum pools from the respective groups were prepared, diluted 1∶10 and pre-incubated over night with biotinylated rFel d 1 protein to a final concentration of 2 µg/ml. The pre-incubated protein mixes were then added to wells and detection of bound biotinylated rFel d 1 was carried out.Proliferation data was analysed by ANOVA with Newman-Keuls multiple comparisons test for pair wise analysis and the ELISA and mouse study analysed by Kruskal-Wallis with Dunn's multiple comparisons test. All statistics were performed using Graphpad Prism 5.02 software . P-values<0.05 were considered significant.All animal research has been approved by the local ethics committee at Karolinska University Hospital, “Stockholms norra djurförsöksetiska nämnd”, with the approval ID [DNR: N245/08]. All human research has been approved by the local ethics committee at Karolinska Institutet, “Regionala etikprövningsnämnden i Stockholm”. Written consent was obtained from all participants involved in the present study.Figure S1Size and purity of rFel d 1 and mutants. (A) SDS-PAGE of purified rFel d 1 mutants under reducing conditions with comassie blue staining. Molecular weight markers , rFel d 1 (lane 2), 3.4.7 (lane 3), 6.7.1 (lane 4), 6.7.3 (lane 5), 11.5.2 (lane 6). (B) Analytical size exclusion chromatography of rFel d 1 and mutants. The dots denote bovine serum albumin (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen A (25 kDa) used as molecular weight markers.(TIF)Click here for additional data file.Figure S2Comparison of immunogenicity of rFel d 1 and mutants. Serial dilutions (x-axis) of pooled mouse sera from mice immunized with either rFel d 1 or each respective mutant. Pre-diluted sera were added to 96-well microtiter plates coated with either (a) respective allergen or rFel d 1 only (b), and IgG responses (y-axis) measured by ELISA.(TIF)Click here for additional data file.


The present results show that this ancient herbal decoction benefits endothelial function through increased activity of Akt kinase and eNOS; this effect is causally via a rise of intracellular Ca2+ and a reduction of ROS.Fo Shou San (FSS) is an ancient herbal decoction comprised of Chuanxiong Rhizoma  and Angelicae Sinensis Radix  in a ratio of 2∶3. Previous studies indicate that FSS promotes blood circulation and dissipates blood stasis, thus which is being used widely to treat vascular diseases. Here, we aim to determine the cellular mechanism for the vascular benefit of FSS. The treatment of FSS reversed homocysteine-induced impairment of acetylcholine (ACh)-evoked endothelium-dependent relaxation in aortic rings, isolated from rats. Like radical oxygen species (ROS) scavenger tempol, FSS attenuated homocysteine-stimulated ROS generation in cultured human umbilical vein endothelial cells (HUVECs), and it also stimulated the production of nitric oxide (NO) as measured by fluorescence dye and biochemical assay. In addition, the phosphorylation levels of both Akt kinase and endothelial NO synthases (eNOS) were markedly increased by FSS treatment, which was abolished by an Akt inhibitor triciribine. Likewise, triciribine reversed FSS-induced NO production in HUVECs. Finally, FSS elevated intracellular Ca Fo Shou San (FSS) composed of Angelicae Sinensis Radix  and Chuanxiong Rhizoma  in a weight ratio of 3∶2, is an ancient Chinese herbal decoction. The first description of FSS was recorded by Xu Shuwei in Puji Benshi in Song Dynasty (AD 1132) of China, and indeed this decoction was being used clinically for hundreds of years. According to traditional Chinese medicine (TCM) theory and practice, FSS should be functioned in “Nourishing Blood” and “Promoting Blood Circulation”. Historically, FSS was originally prescribed for treating women aliments, especially for those suffering obstetric diseases, such as dystocia, vaginal bleeding with fetal movement, dead fetus in uterus and post-partum anemic fainting. Our recent reports suggested that FSS was capable of inducing the differentiation of erythropoietic precursor cells and the expression of erythropoietin (EPO), an erythrocyte-specific hematopoietic growth factor, in cultured liver cells endothelium-derived relaxing factor (EDRF), nitric oxide (NO) dilates blood vessels, increases blood flow, and inhibits platelet activation and angiogenesis endothelial nitric oxide synthases , in endothelial cells has been well studied. The activation of eNOS could be achieved by its phosphorylation at Ser 1177, and this eNOS phosphorylation can be triggered by the activation of protein kinase Akt  and phosphoinositide-3-kinase (PI3 kinase) 2•−) to form the potent oxidant peroxynitrite (ONOO2•−) The endothelium is a thin layer of cells covering the interior surface of blood vessels, as to form an interface between circulating blood in the lumen and the rest of vascular wall. The best characterized : this fingerprint served a purpose of herbal identification. By determining the amounts of ferulic acid and Z-ligustilide in the decoction, we were able to standardize FSS for the subsequent studies on their biological activities described below. A standardized FSS should contain no less than 120±7 mg of ferulic acid and 30±6 mg of Z-ligustilide in 100 g of the dry herbs. The extracting yield of FSS was about 45±5% (n = 3). These parameters established the chemical standards of FSS, as reported previously To standardize FSS chemically, a typical HPLC fingerprint of FSS at an absorbance of 280 nm was developed radical oxygen species (ROS) , suggesting that homocysteine was effective in impairing endothelial function. Pre-treatment with FSS for 30 min after the challenge of homocysteine, the ACh-induced relaxations were largely restored , i.e. showing a vaso-protective effect of FSS. The effect of homocysteine in aortic ring was mainly mediated by the formation of ROS, because different ROS inhibitors could regulate the effect . Tempol, a ROS scavenger, served as a positive control. In addition, the application of L-NAME attenuated the ACh-induced vasodilation . The time-dependent internal control of rat aortic ring relaxation to homocysteine alone, or the co-treatment of homocysteine with FSS, tempol, apocynin and catalase were tested without being induced by the application of ACh .Endothelial dysfunction refers mainly to the reduced NO bioavailability or/and the over-produced es (ROS) , suggestes (ROS) , suggestumbilical vein endothelial cell (HUVEC) is a commonly used cell line for investigating the vascular pathology. Here, the application of homocysteine at 300 µM induced a significant rise of ROS formation in cultured HUVECs . Tempol, a ROS scavenger, used as a positive control, inhibited homocysteine-stimulated ROS formation. Application of FSS reversed homocysteine-induced ROS production in cultured HUVECs , and this inhibition was stronger than that caused by tempol . Besides, the treatment of FSS, or tempol, also suppressed the basal level of ROS in HUVECs. The vaso-protective effect of FSS in cultured HUVECs was revealed by the detection of lucigenin-enhanced chemiluminescence , but the MTT results showed that 1 mg/ml FSS, the commonly used concentration here, increased the proliferation of HUVECs by ∼50% . FSS also produced a dose-dependent stimulation of NO production in HUVECs . The stimulated NO production was confirmed by laser confocal fluorescent microscopy using a specific dye: 4-amino-5-methylamino-20, 70-difluorofluorescein diacetate (DAF-FM DA). Application of FSS triggered a progressive rise in intracellular NO production in cultured HUVECs, as reflected by the increase of fluorescence intensity, which peaked at around 10 min after FSS addition . A23187, a calcium ionophore, was employed as a positive control to evoke NO production . The present results strongly indicate that FSS could elevate the NO production in human endothelial cells. Since FSS could rescue the homocysteine-induced endothelial dysfunction, therefore, HUVECs were first challenged with homocysteine for 45 min to induce the endothelial dysfunction. . These results further suggested that FSS could protect endothelial dysfunction, which was caused by oxidative stress.The production of NO was determined by using Griess reaction assay in cultured HUVECs. Application of FSS (1 mg/ml) stimulated a time-dependent rise in the intracellular NO production, which reached a steady level at around 10 min . FSS als. FSS als. FSS als. FSS als. FSS als. Vascular endothelial growth factor (VEGF), serving as positive control, robustly elevated eNOS phosphorylation. Either FSS-induced or VEGF-induced eNOS phosphorylation was fully abolished by 100 µM L-NAME, a NOS inhibitor . Likewise, L-NAME abolished partially the FSS-stimulated NO production in HUVECs , thus suggesting that the FSS-induced NO production was mediated by eNOS. The downstream effector of eNOS, cGMP, was regulated in parallel to the NO production in responding to different drug applications .To investigate the possible role of eNOS and its upstream regulators, the phosphorylation level of eNOS was first determined to reflect the eNOS activity. The phosphorylation of eNOS at S1177 (at ∼135 kDa) in HUVECs was increased by a 5-min treatment of 1 mg/ml FSS . Vascula. Vascula. VEGF, as a positive control, produced similar effects. To confirm this positive role, the PKB/Akt specific inhibitor, triciribine . Similarly, the phosphorylation of eNOS at S1177 (at ∼135 kDa) was induced by FSS, or VEGF, to over 2-fold. Triciribine also inhibited FSS-stimulated, or VEGF-stimulated, eNOS phosphorylation at S1177 without affecting the total eNOS (∼135 kDa) . These results suggested that the FSS-induced eNOS phosphorylation was likely to be mediated through Akt/PKB signaling pathway.The Akt kinase is one of the major regulators for acute NO production . VEGF, a. VEGF, a2+ activates calmodulin, which then binds to the calmodulin-binding domain in eNOS leading to the activation of calmodulin kinase II 2+ indicator, was used to test whether the FSS-induced Ca2+ increase could regulate the phosphorylation of eNOS. The application of FSS alone to HUVECs increased the level of Ca2+ by about ∼20% . In addition, the pre-treatment of a Ca2+ chelator BAPTA-AM inhibited completely the FSS-induced eNOS phosphorylation . These results therefore suggested that the FSS-induced eNOS phosphorylation could be partially explained by an increased intracellular Ca2+ levels in HUVECs.Calmodulin kinase II is playing an important role in rapid activation of eNOS out ∼20% . In addiAlthough FSS is widely used to treat vascular diseases, such as cerebral apoplexy, cerebral thrombosis, and myocardial infarction in TCM clinic in China, its pharmacological properties and cellular mechanisms of its actions are incompletely understood. We have recently started examining both chemical and biological properties of FSS 2+ concentration in endothelial cells because both Akt inhibitor triciribine and Ca2+ chelator BAPTA-AM inhibited the FSS-stimulated eNOS phosphorylation.We previously reported that FSS possessed the ability to inhibit the ADP-induced platelet aggregation Panax ginseng) and Astragali Radix , Citri Reticulatae Pericarpium (fruit peel of Citrus reticulata) and Pinelliae Rhizoma (rhizome of Pinellia ternata), Zingiberis Recens Rhizoma  and Pinelliae Rhizoma are frequently used together. The making up of FSS having these two chemically similar herbs is not known. According to the basic theory of TCM prescription, the purpose of TCM paired-herbs is to produce synergistic effects aiming to enhance therapeutic efficacy and/or to minimize toxicity and adverse effects. For FSS, the reduction of side effects might be a possible main reason of using ASR and CR together FSS decoction is composed of two herbs: CR and ASR, and the constituents in ASR and CR are quite similar, including alkylphthalides (e.g. Z-ligustilide and senkyunolide A), phthalide dimers (e.g. levistolide A and tokinolide B), phenolic constituents (e.g. ferulic acid and coniferyl ferulate) 2+ influx in endothelial cells. In HVUECs, the application of ferulic acid protected the cells against radiation-induced oxidative stress Although the active components in FSS have not been revealed in the present study, Z-Ligustilide and ferulic acid are considered to be active constituents in both ASR and CR. Ferulic acid possesses a vasodilator and hypotensive effect in spontaneously hypertensive rats 2+; and (iii) reduced ROS generation. Our experimental findings shall enhance the prospects of using FSS in combating against certain forms of cardiovascular diseases in oriental populations.The herbal decoction generally has multiple components acting on multiple targets. Combining appropriate experimental models such as blood vessels and endothelial cells in the present study with the theory of TCM, the mechanisms of actions of herbal decoction can be gradually revealed. On the other hand, better understanding of molecular pathways involved in TCM actions shall provide useful information relevant to therapeutic applications of TCM. In summary, the present study elucidates that the cellular and molecular mechanisms of FSS in blood vessels and in human endothelial cells. The primary vascular benefit is attributed to FSS’s ability to stimulate the eNOS-derived NO production via: (i) PKB/Akt signaling pathway; (ii) increased intracellular CaAngelica sinensis (Oliv.) Diels.) from Minxian of Gansu province and 3-year-old CR (rhizomes of Ligusticum chuanxiong Hort.) from Guanxian of Sichuan province. These areas were known to produce the best quality of ASR and CR, respectively Fresh roots were obtained from China from September to October of 2008, listed as follows: 2-year-old ASR  with 1% acetate acid in water (as solvent A): acetonitrile (as solvent B) in the mobile phase at a flow rate of 1.0 ml/min at room temperature. A gradient elution was applied from 0 to 81% of solvent A starting from 0 to 18 min, from 81% to 0 of solvent A starting from 18 to 60 min, and 0 of A starting from 60 to 75 min. Samples were filtered through a 0.45 µm Millipore syringe filter unit. Ten µl samples were injected for HPLC analysis. For the calibration of ferulic acid and Z-ligustilide, the standards were weighed and dissolved in methanol to give serial concentrations from 1 to 100 mg/l, and two injections onto HPLC were performed for each dilution. The concentrations of these compounds in the samples were calculated according to the regression parameters derived from the standard curve (Table S1).Ferulic acid was purchased from Sigma . Ligustilide (Z-isoform) was kindly provided by Prof. Pengfei Tu from Peking University; their purities, confirmed by HPLC, were higher than 99.0%. AR- and High Performance Liquid Chromatography (HPLC)-grade reagents were from Merck . A Waters  HPLC system consisting of a 600 pump, a 717 auto-sampler, and a UV/VIS Photodiode Array 2996 Detector was used for all analyses. Chromatographic separations were carried out on a Phenomenex C3, 2.5 mM CaCl2, 1 mM MgCl2, 1.2 mM KH2PO4, and 11 mM D-glucose. The bathing solution was gassed with 95% O2–5% CO2 at 37°C (pH = 7.4). The rings were placed under an optimal basal tone of 20 mN, determined from previous length–tension experiments. Changes in isometric tension were measured with a Grass force transducer and stored on MacLab software  for later data analysis. Twenty minutes after mounting in organ baths, the rings were first contracted with 0.5 µM phenylephrine to test the contractility and then relaxed by the application of ACh. They were rinsed several times until baseline tone was restored. The rings were thereafter allowed to equilibrate for 60 min. Baseline tone was re-adjusted to 20 mN when necessary. Each set of experiments was performed on rings prepared from different rats. The use of laboratory animals was approved by the Animal Research Ethical Committee of the Chinese University of Hong Kong.Male Sprague–Dawley rats (∼250–300 g) were sacrificed by cervical dislocation and bled. The thoracic aorta was excised. After surrounding connective tissue had been carefully cleaned off, four 3-mm-wide and 2-mm-long ring segments were prepared from each aorta. The rings were suspended between two stainless wire hooks in a 10-ml organ bath 2. HUVECs between passages 3 and 8 were used in these studies as to ensure the genetic stability of the culture. All culture reagents were purchased from Lonza.HUVEC cell line was obtained from Lonza  and cultured on 0.2% gelatin-coated tissue culture plate in Lonza Endothelial Growth Medium (EGM) Bulletkit in a humidified incubator at 37°C with 95% air, 5% COCultured HUVECs were seeded onto a 96-well plate at a density of 2,000 cells/well in 0.1 ml of EGM Bulletkit medium. The medium was replaced by 0.1 ml of serum and growth factor free medium containing FSS every day. The concentrations of NO in the culture medium were measured with the NO Detection Kit  according to the manufacturer’s instructions. NO is rapidly oxidized to nitrite and nitrate, which are the mean to determine the NO production. The amount of nitrate was determined by converting it to nitrite, followed by the colorimetric determination of the total concentration of nitrite as a colored azo dye product of the Griess reaction that absorbed visible light at 540 nm using a microplate reader 2, 1 mM MgCl2, 10 mM glucose, and 5 mM HEPES (pH 7.4). To measure the Ca2+ influx, cultured HUVECs were labeled with 5 µM of Fluo-4 AM in NPSS buffer. The Ca2+ influx could be indicated by the fluorescence intensity excited at 488 nm and emitted at 525 nm Fluorimetric measurements were performed on cultured HUVECs using an Olympus Fluoview FV1000 laser scanning confocal system  mounted on an inverted I ×81 Olympus microscope, equipped with a 10× objective (NA 0.5). Intracellular NO production was monitored using fluorescent NO indicator 4-amino-5-methylamino-20, 70-difluorofluorescein diacetate . Cultured HUVECs seeded on glass coverslips were incubated for 30 min at room temperature in normal physiological saline solution (NPSS) containing 1 µM DAF-FM DA. The amount of NO was evaluated by measuring the fluorescence intensity excited at 495 nm and emitted at 515 nm. Changes in intracellular NO production were displayed as a ratio of fluorescent intensity at any time relative to control at time 0 (Fn/F0) Cultured HUVECs were treated with different drugs, and then which were collected to perform western blot analysis. For the determination of eNOS and Akt proteins, the cell lysate was collected in lysis buffer  and then denatured at 100°C for 10 min, which was then subjected to 8% SDS-PAGE and western blotting. The phospho-eNOS S1177, total eNOS (∼135 kDa), phospho-AKT S473 and total Akt (∼60 kDa) were identified by specific antibodies. The anti-eNOS, anti-phospho-eNOS S1177, anti-Akt, anti-phospho-Akt S473 antibodies  and HRP-conjugated anti-rabbit secondary antibody  were employed here. The eNOS dimers were assayed using low-temperature SDS-PAGE under non-reducing conditions, as described previously 2•− was measured using a lucigenin -enhanced chemiluminescence technique 6/ml in HEPEs buffer containing (mM): NaCl 140, KCl 5, MgCl2 0.8, CaCl2 1.8, Na2HPO4 1, HEPEs 25 and 1% glucose, pH 7.4). Cells were distributed at 5×104 per well on a 96-well white plate. Before recording the chemiluminescence, NADPH (100 µM) and lucigenin (25 µM) was added via the auto-dispenser. H2O2 was used as an assay control. The light emission between O2•− and lucigenin was detected and quantified by FLUOstar Optima (BMG Labtech) over 20 min. This time point was determined to be optimized The generation of OThe concentration of protein was determined following the instructions of the manufacturer. The analysis was done on 96-well microtiter plate. Essentially, one part dye reagent concentrate was diluted with 4 parts of double distilled water before use. Five dilutions of BSA standard (0.1–0.5 µg/ml) were used for the test. The samples were assayed in triplicate readings. Ten µl of each standard and sample solution were added with 0.2 ml of diluted dye reagent into separate wells and mixed well. After 5-min incubation at room temperature, the absorbance at 595 nm was taken. The concentration of protein was determined from the standard curve.All data were analyzed by one-way ANOVA  using the SPSS version 11 statistical analysis program. Statistical significance was considered as ** for p<0.01 and *** for p<0.001.Figure S1(A): The structures and HPLC chromatography of the chemical markers, ferulic acid senkyunolide I, senkyunolide H and Z-ligustilide, were shown. (B): The HPLC analysis (at an absorbance of 280 nm) was performed to illustrate the standard chemical markers, ferulic acid, senkyunolide I, senkyunolide H and Z-ligustilide. Results are Means ± SEM, n = 3.(TIF)Click here for additional data file.Figure S2Rat aortic ring was isolated. The tension responsible for the vascular relaxation and constriction was tested after the treatment of different drugs as in n = 4.(TIF)Click here for additional data file.Figure S3Rat aortic ring was isolated (as in) and scratch to get rid of the intact of endothelium. The ring was suspended between two stainless steelwires in a 10-ml chamber on a Multi Myograph, which was used to measure the tension. A concentration-response curve for FSS (0.1–10 mg/ml) was tested.(TIF)Click here for additional data file.Figure S4Rat aortic ring was isolated. The tension responsible for the vascular relaxation and constriction was tested after the treatment of different drugs as in (TIF)Click here for additional data file.Figure S5Cultured HUVECs were seeded in 12-well plates. The cultures were serum starved for 3 hours before the pre-treatment with homocysteine (300 µM) for another 1 hour. The cells were then treated with FSS (1 mg/ml), or VEGF , or control (without drug treatment), at different time points. Total and phosphorylated eNOS was revealed by using specific antibodies, as in (TIF)Click here for additional data file.Figure S6Cultured HUVECs were treated with FSS (1 mg/ml) for 24 hours and cell viability was tested using MTT assay. Data are expressed as percentage of increase compared to control (without FSS treatment). Results are Means ± SEM, n = 4.(TIF)Click here for additional data file.Figure S7Rat aortas were isolated  and cut into spiral strips (2 mm×1 cm), which were then mounted in 5 ml myograph bath containing Krebs solution and gassed with 95% O After exposure to different drugs as indicated, the tissues were quickly frozen in liquid nitrogen, and cGMP levels were assayed as described in Direct cGMP ELISA kit . The aortas were treated with Phe (0.5 µM) first for about 3 min, and then add FSS (3 mg/ml). For the pre-treatment, L-NAME (100 µM) was added for 45 min. Values of cGMP are expressed as X Basal, where the control was set as 1. Mean ± SEM, n = 4.(TIF)Click here for additional data file.Table S1Calibration curves the chemical markers.a These calibration curves were constructed by plotting the peak area versus the concentration of each analyte. Each calibration curve was derived from four data points, n = 3, and the SD was <5% of the Mean.(TIF)Click here for additional data file.


Information processing within neuronal networks is determined by a dynamic partnership between principal neurons and local circuit inhibitory interneurons. The population of GABAergic interneurons is extremely heterogeneous and comprises, in many brain regions, cells with divergent morphological and physiological properties, distinct molecular expression profiles, and highly specialized functions. GABAergic interneurons have been studied extensively during the past two decades, especially in the hippocampus, which is a relatively simple cortical structure. Different types of hippocampal inhibitory interneurons control spike initiation  and synaptic integration  within pyramidal neurons and synchronize local network activity, providing a means for functional segregation of neuronal ensembles and proper routing of hippocampal information. Thus, it is thought that, at least in the hippocampus, GABAergic inhibitory interneurons represent critical regulating elements at all stages of information processing, from synaptic integration and spike generation to large-scale network activity. However, this raises an important question: if inhibitory interneurons are fundamental for network computations, what are the mechanisms that control the activity of the interneurons themselves? Given the essential role of synaptic inhibition in the regulation of neuronal activity, it would be logical to expect that specific inhibitory mechanisms have evolved to control the operation of interneurons. Here, we review the mechanisms of synaptic inhibition of interneurons and discuss their role in the operation of hippocampal inhibitory circuits. GABAergic inhibitory neurons. These cells represent only 10–20% of the total neuronal population; however, they are able to efficiently control the information flow within cortical circuits  form a high number of synapses (7–20) onto each other , BCs, SC-ACs, and perforant path-ACs in the LM , which, in addition to principal cells, target interneurons  form a small fraction of synapses with each other. Similarly, interneurons targeting the distal dendrites of principal neurons  tend to also control other interneurons that are responsible for distal dendritic inhibition. Finally, the populations of soma- and distal-dendrite-targeting interneurons can be also interconnected. For example, O–LMs make monosynaptic connection with LM BCs  interneurons. First, a combination of immunohistochemistry and anatomical analysis identified three distinct subtypes of IS interneurons in the rat hippocampus  have a soma located in the stratum oriens/alveus (O/A), PYR, or RAD and express CR. In addition to innervating other interneurons exclusively, these cells show further preferences, as they avoid PV-expressing BCs and axo-axonic cells and contact calbindin (CB)- and CR-positive interneurons Figure  Acsady . The proIS interneurons type II (IS-IIs) have been found at the border between the RAD and the LM. These interneurons express VIP, but lack CR. They have a vertically oriented cell body, dendrites restricted to the LM, and an axon that arborizes in the RAD. It has been shown that the VIPergic projections in the RAD form synapses onto CB- or VIP-positive dendrites, with a preference for CCK/VIP-coexpressing BCs Figure  Acsady .two-photon glutamate uncaging-based photostimulation and patch-clamp recordings in slices obtained from VIP-eGFP mice, we found that the activation of VIP-positive interneurons located at the border between the PYR and the RAD produces small-amplitude and kinetically slow inhibitory postsynaptic currents (IPSCs) in O–LMs. After the uncaging experiments, VIP-positive interneurons connected to O–LMs were patched and filled with biocytin. Further electrophysiological, anatomical, and neurochemical analyses revealed that putative IS-IIIs exhibit a high input resistance and an irregularly spiking firing pattern, establish several synaptic contacts onto dendrites of O–LMs, and coexpress CR  are typically located at the PYR and RAD border and has a vertically oriented cell body with dendrites extending into the LM. Their axons form a dense plexus in the O/A  (Freund and Antal, Second, a bidirectional GABAergic connection between the MEC and the hippocampus has been identified (Germroth et al., In summary, hippocampal interneurons receive GABAergic inputs from three main sources: non-specific local inhibitory connections, IS interneurons, and long-range inhibitory projections. Some inhibitory microcircuits, which represent a single class of interconnected interneurons, can be considered as relatively autonomous. For example, dentate gyrus PV-positive BCs are connected in a tight network that is sufficient for generating oscillations in the gamma range (Bartos et al., A receptor activation operate in principal cells and interneurons, input- and target-cell specificities of synaptic inhibition have been reported. The inhibitory postsynaptic responses recorded in interneurons can be significantly faster than those recorded in principal cells (Ali et al., Although the same basic mechanisms of GABAA receptor channel is determined by the subunit composition of the receptor. For example, the presence of the α1 or α6 GABAA receptor subunits is associated with a faster current (Tia et al., A receptor subunits, supporting the heterogeneity of the properties of their inhibitory synapses (Gao and Fritschy, A receptor subunit at their synapses, which may account for the faster IPSC kinetics observed in these cells (Gao and Fritschy, A receptor subunit can be incorporated into synapses later during development and is responsible for the age-dependent slowing of IPSCs (Salesse et al., A receptor (Nusser et al., First, different location of various inhibitory synapses along the dendritic tree and associated electrotonic attenuation can explain kinetic variations of inhibitory currents in different targets. In fact, even in a given interneuron the rise time and decay of inhibitory currents can vary over 10-fold as a result of dendritic filtering (Hajos and Mody, A receptor, the dendritic filtering and the intracellular chloride concentration shape the dynamics of transmission at different inhibitory synapses. The mechanisms of synaptic inhibition in interneurons and, in particular, the great diversity of the properties of inhibitory synapses within and between different inhibitory circuits remain to be explored.Together, these findings highlight the input- and target-specific organization of synaptic inhibition in hippocampal interneurons. In particular, the properties and spatiotemporal profile of transmitter release, the composition of the GABAB receptors and group I/II metabotropic glutamate receptors (Patenaude et al., B receptors or group I/II metabotropic glutamate receptors. Furthermore, high-frequency stimulation or postsynaptic depolarization alone produces a short-term depression of IPSCs in principal cells, but not in interneurons (Patenaude et al., The efficacy of GABAergic synapses can be regulated in an activity-dependent manner (Stelzer et al., A receptor subunit and LTP was blocked by an α5-GABAA receptor subunit inverse agonist, highlighting the primary role of this subunit in LTP at local synapses.These findings highlight the similarities and major differences between specific forms of plasticity at GABAergic synapses formed onto different targets. Such heterogeneity in plasticity mechanisms may result from the cellular and molecular heterogeneity of interneurons and the distinct types of inhibitory inputs that contact these cells. Obviously, understanding these mechanisms will require careful examination of synapses formed by specific inhibitory projections onto the same GABAergic target. To this end, recent data from our laboratory demonstrate the selective strengthening of GABAergic synapses formed by different inputs onto O–LM interneurons (Chamberland et al., +/Cl− cotransporter KCC2, the effect of GABA can be depolarizing (Rivera et al., There is growing evidence that the activity of hippocampal inhibitory circuits is tightly regulated by multiple inhibitory mechanisms. First, distinct classes of interneurons are reciprocally interconnected and form closely interacting networks. As proposed by Buzsaki , such neWhy does a dedicated population of interneurons specialized in the exclusive innervation of other GABAergic cells exist? First, it appears that IS interneurons are highly selective in choosing their targets, suggesting that not all classes of hippocampal interneurons are controlled by IS cells. Interestingly, both IS-Is and IS-IIs prefer to contact CCK-positive BCs, whereas IS-IIIs prefer O–LMs. The reasons underlying such preferences remain to be determined. It is important to note, however, that IS-IIIs are strategically positioned to detect the changes in network activity that are transmitted by the perforant path and, therefore, to adjust the strength of inhibition exerted by the O–LMs on perforant path integration. As O–LMs are not contacted by the perforant path, such an indirect mechanism of regulation of their activity seems functionally plausible. Unfortunately, very little is currently known about the physiological organization of IS interneurons. Thus, even though IS-IIIs exhibit extensively branching dendrites in the LM and are likely to be recruited via activation of the perforant path, this hypothesis has not been tested directly. Therefore, from the functional point of view, IS interneurons may be positioned to control selective inhibitory circuits. Second, IS cells may sense and communicate specific changes in network activity and the environment depending on the brain state. For example, the coexpression of enkephalins (Blasco-Ibanez et al., Finally, given that the strength and time course of inhibitory transmission serve to control the recruitment and synchronization of interneurons during network activity, a better understanding of the properties of synaptic transmission between individual inhibitory circuit elements is required. The efficiency of the communication between inhibitory circuits given a relatively sparse connection pattern, the manner via which it may change during different forms of activity, and the manner via which the balance between excitatory and inhibitory inputs is achieved to provide coordinated interneuron activity remain largely unknown. The view that coordinated inhibition is required for the proper routing of excitatory trajectories and for the spatiotemporal organization of hippocampal cell assemblies should inspire further research on the targeting of interconnected inhibitory circuits.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This work expands the current knowledge on the biological properties of naphthoquinones, highlighting naphthazarin, diospyrin and menadione as potential lead compounds for structural modification in the process of improving and developing novel anti-allergic drugs.Allergic disorders are characterized by an abnormal immune response towards non-infectious substances, being associated with life quality reduction and potential life-threatening reactions. The increasing prevalence of allergic disorders demands for new and effective anti-allergic treatments. Here we test the anti-allergic potential of monomeric  and dimeric (diospyrin and diosquinone) naphthoquinones. Inhibition of RBL-2H3 rat basophils' degranulation by naphthoquinones was assessed using two complementary stimuli: IgE/antigen and calcium ionophore A23187. Additionally, we tested for the inhibition of leukotrienes production in IgE/antigen-stimulated cells, and studied hyaluronidase and lipoxidase inhibition by naphthoquinones in cell-free assays. Naphthazarin (0.1 µM) decreased degranulation induced by IgE/antigen but not A23187, suggesting a mechanism upstream of the calcium increase, unlike diospyrin (10 µM) that reduced degranulation in A23187-stimulated cells. Naphthoquinones were weak hyaluronidase inhibitors, but all inhibited soybean lipoxidase with the most lipophilic diospyrin, diosquinone and menadione being the most potent, thus suggesting a mechanism of competition with natural lipophilic substrates. Menadione was the only naphthoquinone reducing leukotriene C Allergy is an abnormal immune response against non-infectious environmental substances, named allergens Vitis vinifera L. Dioscorea membranacea Pierre ex Prain & Burkill, in which the main active compound was a quinone (dioscoreanone) Rhinacanthus nasutus Kuntze, which is rich in naphthoquinones R. nasutus were capable of inhibiting RBL-2H3 basophils' degranulation in the micromolar range, and decreasing tumour necrosis factor (TNF)-α and interleukin production Lawsonia inermis L.), where lawsone  is the main compound responsible for dye properties. Still, allergic reactions to henna have been attributed only to the occasional additive p-phenylenediamine Nature continues to be a rich source of novel bioactive molecules, and several plant extracts have been probed for anti-allergic properties. Namely, the grape seed extract of Diospyros species: diospyrin (DPR), diosquinone (DQN), juglone (JGL), menadione (MND), naphthazarin (NTZ) and plumbagin (PLB)  and the released β-hexosaminidase and histamine were quantified. Additionally, hyaluronidase and lipoxidase inhibition by naphthoquinones were evaluated, as well as the inhibition of leukotrienes production in IgE/antigen-exposed RBL-2H3 cells.In this work we studied the anti-allergic properties of naphthoquinones commonly produced by in (PLB) . SeveralDiospyros chamaethamnus Dinter ex. Mildbr. Plumbagin (PLB), naphthazarin (NTZ), menadione (MND) and juglone (JGL) were obtained from Sigma-Aldrich . Diospyrin (DPR) and diosquinone (DQN)  were isoMedium, buffers and supplements for cell culture, including Earle's Balanced Salt Solution (EBSS) were from Gibco, Invitrogen™  and bovine albumin fraction V solution 7.5% (BSA) was from Sigma-Aldrich .Streptococcus equi, hyaluronidase from bovine tests , lipoxidase from Glicine max (L.) Merr. , as well as degranulation stimuli, monoclonal anti-dinitrophenyl (DNP) antibody produced in mouse, dinitrophenyl albumin (DNP-BSA) and calcium ionophore A23187 were from Sigma-Aldrich . Leukotriene C4 EIA kit was from Abcam . All other chemicals were from Sigma-Aldrich , with the exception of 3--2,5-diphenyltetrazolium bromide (MTT), which was from Duchefa Biochemie .Hyaluronic acid sodium salt from 2, at 37°C, in humidified air.Rat basophilic leukaemia cell line, RBL-2H3, was from the American Type Culture Collection (ATCC®) . Cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) + GlutaMAX™- I supplemented with 15% heat inactivated foetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were maintained under 5% CO5 cells/mL in 24-wells plate (1 mL/well), and assayed after 24h at near-confluent stage (∼90%). Two different degranulation stimuli were used: calcium ionophore A23187 500 ng/mL (1 µM) and an immunologic stimulus (100 ng/mL IgE anti-DNP followed by 100 ng/mL DNP-BSA) that we refer to as IgE/antigen  increased by 65.4±6.8% above basal , whereas histamine increased from the basal value of 0.156±0.151 µM towards 0.521±0.186 µM . With A23187, β-hexosaminidase release increased by 187±18.9% above basal , whereas histamine increased towards 2.43±0.330 µM . Thus, the ability of naphthoquinones to reduce β-hexosaminidase release was quantified for both stimuli, whereas effects upon histamine were only quantified with the A23187 stimulus, given the low histamine release and poor signal to noise achieved with IgE/antigen.RBL-2H3 cells were seeded at 3.0×10/antigen . Both stQuercetin was used as positive anti-degranulation control A23187, quercetin and naphthoquinones stocks were dissolved in dimethyl sulfoxide (DMSO), aliquoted and stored at −20°C. We determined the maximal non-interfering solvent concentrations , cells were treated for 1 h, at 37°C, with 100 ng/ml DNP-BSA and with individual naphthoquinones diluted in EBSS supplemented with 0.1% BSA  [21]. Afβ-hexosaminidase and histamine release was quantified in supernatants, whereas the MTT cell viability assay was performed on adherent cells.Before treatment with A23187, cells were incubated with individual naphthoquinones during 15 min, at 37°C. After that, A23187 1 µM was added and cells incubated for 30 min, at 37°C . CompounCell viability was assessed by the cellular dehydrogenases' dependent reduction of MTT to formazan, which was quantified by the measurement of optical density at 550 nm using a microplate reader (Multiskan ASCENT Thermo®), as described before β-hexosaminidase from stimulated-RBL-2H3 cells was measured as previously described p-nitrophenyl N-acetyl-D-glucosamine 1.3 mg/ml in citrate buffer (pH 4.5)] were added to 30 µl of supernatant. The plate was incubated at 37°C, during 1 h. The reaction was stopped by the addition of 80 µl of NaOH 0.5 M and the reaction product, p-nitrophenolate, was measured spectrophotometrically at 405 nm, in a microplate reader (Multiskan ASCENT Thermo®).The release of β-hexosaminidase release, individual naphthoquinones may directly inhibit β-hexosaminidase enzymatic activity. For this, the inhibition of β-hexosaminidase enzymatic activity by naphthoquinones and quercetin was evaluated in an assay similar to the one described above: individual naphthoquinones (5 µl) were incubated with supernatant of degranulated cells where β-hexosaminidase is present (25 µl of supernatant of cells treated with A23187), in presence of 50 µl of substrate solution, during 1 h, at 37°C. The determination was made at 405 nm, in a microplate reader (Multiskan ASCENT Thermo®) Beyond avoiding o-phthalaldehyde (OPA) 1% (w/v) were added to 500 µl of supernatant to convert histamine into fluorescent histamine-OPA-products. After 4 min incubation at room temperature, the reaction was stopped with of 50 µl of HCl 3 M. Precipitated proteins were removed by centrifugation at 14,000 rpm, during 3 min. The fluorescent histamine-OPA-products were quantified in the supernatant using 360 nm excitation and 450 nm emission in a microplate reader (Biotek Synergy HT®) 100 µl of NaOH 1 M and 25 µl of 4 quantification was performed in the supernatant of IgE/antigen stimulated cells using a competitive enzyme immunoassay kit, according to the supplier's protocol  in a microplate reader (Multiskan ASCENT Thermo®) at 405 nm.Leukotriene CN-acetyl-sugar, was quantified according Morgan-Elson colour reaction with minor modifications. The Morgan-Elson reaction was started by addition of 25 µl disodium tetraborate 0.8 M and subsequent heating in a boiling water bath during 3 min. After cooling, 750 µl p-dimethylaminobenzaldehyde (DMAB) was added and the reaction mixture was incubated at 37°C for 20 min. DMAB stock solution was prepared by dissolving 2 g DMAB in glacial acetic acid with 12.5% of HCl 10 N. This solution was further diluted in glacial acetic acid (1∶2) immediately before use. The measurement was made spectrophotometrically, at 560 nm, in a microplate reader (Multiskan ASCENT Thermo®) The enzymatic reaction mixture was composed by 50 µl hyaluronic acid (5 mg/ml in water), 100 µl buffer pH 3.68 , 200 µl water, 50 µl individual naphthoquinones solution and 50 µl hyaluronidase 600 U. The enzymatic reaction occurred during 1 h, at 37°C. The reaction product, Lipoxidase catalyses the oxidation of linoleic acid to the conjugated diene, 13-hydroperoxy linoleic acid, which was measured spectrophotometrically at 234 nm on a UV/visible spectrophotometer (UNICAM Helios α) n), performed at least in duplicate. Results concerning cell viability, released histamine and released β-hexosaminidase are expressed in percentage of the respective control. The normality of the values was evaluated by Shapiro-Wilk test, which was performed using Statistical Package for the Social Sciences version 20.0 . Paired t-test or One-Way ANOVA with Bonferroni as post-hoc test were used, respectively, to compare two or more groups . P values under 0.05 were considered statistically significant.Values are presented as mean±standard error of mean (SEM) of at least three independent experiments (β-hexosaminidase release by 52.1±11.9% when IgE/antigen was used (P<0.05)  or the calcium ionophore A23187 ; Schematic protocols in (P<0.05) , but it ed cells , likely β-hexosaminidase release by 30.8±3.3%   was the only naphthoquinone able to significantly reduce degranulation, decreasing  P<0.05) . Jugloneminidase .β-hexosaminidase release, when compared with the IgE/antigen assay. Diospirin (DPR) at the higher 10 µM concentration allowed by this assay, significantly decreased both β-hexosaminidase (56.8±14.6%) and histamine (51.4±12.8%) release, an amplitude of effect approaching that achieved with quercetin, and standing out amongst the other dimeric and monomeric naphthoquinones, none of which significantly affected degranulation at the maximal tested concentrations (β-hexosaminidase enzymatic activity (data not shown).In the A23187 assay, the positive control quercetin required a 10-fold higher concentration (100 µM) to reduce trations . None ofSodium cromoglycate was used as a positive control for hyaluronidase inhibition 50 values of 28.9 and 83.8 µM, whereas all monomeric naphthoquinones displayed IC50 values above 100 µM, with the most potent being menadione with an IC50 of 128 µM  or diosquinone (1 µM), but completely abrogated by menadione (5 µM). We thus performed a concentration response curve for menadione on LTC4 levels  decreased IgE/antigen-induced degranulation . HoweverDiospyrin (10 µM) reduced degranulation in calcium ionophore-stimulated cells . AnotherPlumbagin was previously reported to exhibit anti-allergic properties, namely, at 5 µM plumbagin inhibited cytokines production by phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMC) Naphthazarin and diospyrin, shown capable of inhibiting RBL-2H3 basophils' degranulation in the present study, warrant further investigation in models such as primary mast cells. Several questions about the membrane permeability of naphthoquinones may be drawn; however, the effects of both naphthazarin (the most hydrophilic), and diospyrin (the most lipophilic) on RBL-2H3 degranulation strongly suggest that naphthoquinones can cross cell membranes. Menadione in particular is well known for its mitochondrial effects β-N-acetyl-D-glucosaminidic linkages of hyaluronic acid 4, which is transformed to LTB4 or LTC4 according with the cell type and the enzymes present  synthase via ROS production with an IC50 of 5 µM Considering the results obtained with soybean lipoxidase, we tested the inhibition of leukotriene production by diospyrin, diosquinone and menadione. Menadione was the only naphthoquinone able to reduce leukotriene production, achieving full inhibition at 5 µM . Higher 4 production induced by allergic stimuli, as well as by the evaluation of inhibition of enzymes involved in allergic responses (main findings for each compound are summarized in Concluding, we evaluated the anti-allergic properties of monomeric and dimeric naphthoquinones by studying the inhibition of RBL-2H3 basophils' degranulation and LTC


S473) was transient only in the first few hours of the PP242 treatment. Receptor tyrosine kinase arrays further revealed that PP242 treatment increased the phosphorylated epidermal growth factor receptor (EGFR) at Tyr 1068 (EGFRT1068). The parallel increase of AKTS473 and EGFRT1068 in the cells following PP242 treatment raised the possibility that EGFR phosphorylation might contribute to the PP242 incomplete inhibition of mTORC2. To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity. In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death. A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice. This study suggests that the combination of mTOR kinase and EGFR inhibitors may provide an effective treatment of colorectal carcinoma.Mammalian target of rapamycin complex 1 and 2 (mTORC1/2) are overactive in colorectal carcinomas; however, the first generation of mTOR inhibitors such as rapamycin have failed to show clinical benefits in treating colorectal carcinoma in part due to their effects only on mTORC1. The second generation of mTOR inhibitors such as PP242 targets mTOR kinase; thus, they are capable of inhibiting both mTORC1 and mTORC2. To examine the therapeutic potential of the mTOR kinase inhibitors, we treated a panel of colorectal carcinoma cell lines with PP242. Western blotting showed that the PP242 inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT Colorectal carcinoma is the third most common cancer in men and women but the second leading cause of cancer-related deaths in the United States . Recent mTOR is a central integrator for upstream inputs from growth factors, nutrients and stress . InsulinThe mTOR pathway is overactive in cancers ; thus, mThe second generation of mTOR inhibitors has been developed to target the adenosine triphosphate (ATP)-binding site of mTOR kinase . These mTo examine the therapeutic potential of the mTOR kinase inhibitor PP242, we show here that PP242 can only transiently block the mTORC2 kinase activity in the first few hours of the treatment. In parallel, the return of mTORC2 kinase activity is associated with the increase of phosphorylated EGFR. The combination treatment of PP242 and erlotinib, an EGFR inhibitor, completely block both mTORC1 and mTORC2 activity, inhibits cell growth and suppresses the progression of colorectal carcinoma xenografts.2, Colo205, Colo320, DLD-1, HCT-8, HT29, HCT-116 and SW948 were purchased from American Type Collection  and grown in RPMI-1640 medium  supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified 5% CO2 and 37 oC incubator. Matched colorectal carcinoma and normal colon tissue samples were collected in the First Hospital Tissue Bank. All patients provided written informed consent for tissue sample collection in the bank. This study was approved by the First Hospital Ethical Committee of Jilin University.Human colorectal carcinoma cell lines CacooC. Epidermal growth factor (EGF) was purchased from Peprotech (RockyHillNJ). From Cell Signaling Technology  were the antibodies against human mTOR, phosphor-mTOR (Ser2448), AKT, phosphor-AKT (Ser473), S6, phophor-S6 (Ser235/236), 4E-BP1, phophor-4E-BP1 (Ser37/46), EGFR, phosphor-EGFR (Tyr1068). Also included in the study were the antibodies against DNF fragmentation factor (DFF45), poly (ADP-ribose) polymerase (PARP), caspase-3 , β-actin , horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit antibodies . Protease inhibitor mixture, Triton X-100 and other chemicals were from Sigma-Aldrich.PP242 , erlotinib, rapamycin and LY294002  were dissolved in dimethyl sulfoxide at the concentrations of 10 or 20 mM and stored in aliquots at -803 cells per well in 100 ul of growth medium. Cells were treated or untreated with PP242 and erlotinib, alone or in combination. After incubation for the times indicated in the Results, cells were washed with a phosphate buffer and each of the wells was added with 100 ul buffer 0.2M containing sodium acetate (pH 5.5), 0.1% (v/v) Triton X-100 and 20 mM p-nitrophenyl phosphate. The plates were incubated at 37 oC for 1.5 hours and the reaction was stopped by the addition of 10 ul 1M NaOH to each well and the color developed was measured at 405 nm by a microplate reader (BioRad).Cells were seeded and grown in 96-well plates at 8ⅹ10Cells in single-cell suspension were plated and grown in 6-wells plates at a density of 1000 cells per well for 24 hours. Cells were then treated or untreated with PP242 and erlotinib, alone or combination. The medium was replaced every 4 days with fresh medium containing PP242 and/or erlotinib. After 14 days, the medium was removed and cell colonies were stained with 0.5% crystal violet solution.Cells were treated with 1 uM PP242 and 2 uM erlotinib, alone or in combination, for 20 hours, harvested, fixed with 70% ethanol, and stained with propidium iodide. The data were acquired using a flow cytometry  and were analyzed using FlowJo software . Sub-G1 apoptotic cells were presented in percentage of the cells.oC. The supernatant was collected and protein concentrations were determined by the Bradford protein assay following the manufacturer’s protocol (Bio-Rad Laboratories). Equal amounts of protein were separated through SDS-PAGE gels and transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were incubated overnight at 4oC with primary antibody and then for 1 hour with HP-conjugated secondary antibody. The membranes were developed by chemiluminescence.Western blotting was carried out based on the protocols reported earlier . Cells wThe pathScan RTK Signaling Antibody Array Kit was purchased from Cell Signaling Technology . This slide-based antibody array included twenty-eight RTKs. The slides were incubated with cell lysates and revealed by chemiluminescence according to the manufacturer’s instructions.6) were injected subcutaneously into the flank regions of six-week old (about 20 g of body weight) female athymic (nu/nu) mice . The mice were allowed to develop subcutaneous xenografts and tumor volumes were measured using caliper measurements. When tumors reached approximately 150-200 mm3, mice were assigned randomly to 3 experimental groups (n=4 per group); the first group of mice were treated with saline (control), the second group were treated with PP242 (50mg/kg) and the third group were treated with the combination of PP242 50 mg/kg and erlotinib 75 mg/kg. The agents were administrated through oral gavages, twice per week for 21 days. Tumor volumes were measured once every 3 days and calculated based on the formula: V =4/3 x πx (length/2x[width/2]2). At the end of treatment, the mice were sacrificed and the tumors were removed and weighed at necropsy.The animal studies were approved by the Institutional Animal Care and Use Committee of Emory University. The DLD-1 cells . The differences in the means between two groups were analyzed with two-tailed unpaired Student’s S473) [S473 and determine whether the mTORC2 are activated in the carcinoma. Western blotting revealed that AKTS473 were elevated in the carcinoma tissues as compared with the matched normal tissues (S235/236) in the carcinoma but not matched normal tissues suggested the elevated mTORC1 activity in the carcinomas  –11, we t tissues , thus surcinomas .S235/236 and 4E-BP1 at T36/45 (4E-BP1T36/45) and the mTORC2 substrate AKTS473 in the cells after PP242 treatment at the high dose of 1 μM. Western blotting showed that PP242 treatment at the high dose completely abolished the S6S235/236 but partially reduced the 4E-BP1T36/45  in the cell lines (S473) kept in line with the report that rapamycin does not inhibit mTORC2 [RTKs activates mTOR kinase through PI3K-AKT pathway ; thus toll lines ; the datt mTORC2 .T1068 and AKTS473 in a timely manner in both cell lines (S473), the substrate of mTOCR2. LY294002 is not selective PI3K inhibitor and, to confirm the role of PI3K, we repeated the experiment with a selective PI3K inhibitor BKM120 [S473 under the PP242 treatment , alone or in the combination with the EGFR inhibitor, erlotinib (2 μM). The erlotinib treatment blocked the phosphorylation of EGFR, temporally inhibited S6 phosphorylation but had no effect on 4E-BP1 phosphorylation (T1068) and mTORC1 substrates ; thus, EGFR activation contributes to the incomplete inhibition of mTORC1 by PP242 and in combination with the EGFR inhibitor erlotinib, PP242 can completely block the mTORC1 kinase activity. The treatment of PP242  and PP242 (0.5 μM), alone and in combination, for 24 hours in the presence of EGF (50 ng/ml) according to the experimental design as described in PP242 has been reported to induce apoptosis in leukemia and breast cancer cells through inhibition of mTORC2 activity . The com3 sizes, the mice were treated for 21 days through oral gavage of saline as control or PP242 (50mg/kg), alone or in combination with erlotinib (75 mg/kg). Our earlier work [S235/236 and the mTORC2 substrate AKTS473. Collectively, these results indicate that the combination treatment of PP242 and erlotinib completely blocks the mTORC1 and mTORC2 kinase activity, induce apoptosis in colorectal carcinoma cells and suppresses the carcinoma xenograft progression.To evaluate therapeutic potential of the combination of PP242 and erlotinib in treating colorectal carcinoma, we generated subcutaneous xenografts by injecting subcutaneously DLD-1 cells in athymic (nu/nu) mice. Once the xenografts were formed at approximately 150-200 mmier work  and the Emerging evidence supports the notion that targeting of mTOR pathway may provide effective treatment of colorectal carcinoma . ImmunohIn contrast, the second generation of mTOR inhibitors targets the mTOR kinase activity, thus inhibits both mTORC1 and mTORC2 and are more potent than the first generation of the inhibitors . While tS473), the mTORC2 phosphorylation site on AKT, in colorectal carcinoma cells at 12 hour [S473 resumes gradually up to the untreated cell levels within 24 hours of PP242 treatment and inhibition of PI3K fails to block the AKTS473 phosphorylation. In contrast, rapamycin does not inhibit the AKTS473 phosphorylation through the time course, keeping in line with the notion that rapamycin does not inhibit mTORC2 activity [The results reported here indicate that PP242 only transiently inhibits mTORC2 kinase activity in colorectal carcinoma cells. Early studies show that PP242 treatment inhibits the AKT phosphorylation at S473 (AKT 12 hour  and panc 12 hour . In a tiactivity . Our resAnother salience of our results is that PP242 treatment leads to the increase of EGFR phosphorylation through PI3K-independent pathway in colorectal carcinoma cells. EGFR can activate downstream PI3K and ERK pathway and thus drives cancer progression . While iThe study presented here provides several lines of evidence in support of the notion that the combination of mTOR kinase and EGFR inhibitors may provide strategy in treatment of colorectal carcinoma. First, the combination treatment of erlotinib and PP242 results in the complete inhibition of mTORC1 and mTORC2 activity in the carcinoma cells. Second, the combination inhibits the colony formation and cell growth. Thirdly, the EGFR inhibitor enhances PP242-induced apoptosis. Finally, erlotinib enhances PP242 therapeutic efficacy in treatment of colorectal carcinoma xenografts. EGFR has been therapeutic targeted by small molecular inhibitors and neutralizing antibodies ; howeverPhase I and II trials of erlotinib have created controversial regarding the toxicity of the agent. As a single agent, erlotinib was tolerated by patients in phase I trial of treatment of metastatic colorectal carcinoma . Phase I