Inherent to sea ice, like other high latitude environments, is the strong seasonality driven by changes in insolation throughout the year. Sea-ice organisms are exposed to shifting, sometimes limiting, conditions of temperature and salinity. An array of adaptations to survive these and other challenges has been acquired by those organisms that inhabit the ice. One key adaptive response is the production of extracellular polymeric substances (EPS), which play multiple roles in the entrapment, retention and survival of microorganisms in sea ice. In this concept paper we consider two main areas of sea-ice microbiology: the physico-chemical properties that define sea ice as a microbial habitat, imparting particular advantages and limits; and extracellular responses elicited in microbial inhabitants as they exploit or survive these conditions. Emphasis is placed on protective strategies used in the face of fluctuating and extreme environmental conditions in sea ice. Gaps in knowledge and testable hypotheses are identified for future research. Sea ice is a dynamic, porous matrix that harbors within its interior network of brine pores and channels an active e.g., ,2) and d and d2])The seasonal  decrease in temperature that leads to the formation of sea ice in polar waters progressively reduces the liquid phase of the ice—the brine volume fraction—and consequently increases the concentration of solutes and particles in the brine. Phase equations of sea ice ,21,22 ar2 and O2, decreases as the salinity of brines increases, translating to a general degassing effect throughout the winter season [2 consumption can further reduce O2 concentration in some sections of the sea-ice column leading to the development of microbial communities and processes that are favored by low oxygen conditions [Other properties of sea-ice brines also show temperature-dependent changes. For instance, the solubility of biologically relevant gases, including COr season . Subsequnditions ,15,24.2) or decrease in pH (by consumption of CO2 and consequent shifts in the carbonate chemistry of brine).Summer brings a new set of changes. Melting of snow and then surface ice, and the consequent formation of melt ponds, exposes sea-ice microorganisms to salinities close to that of freshwater, or else flushes them back to the ocean through brine drainage ,25. Alth“It is a curious fact, and yet a well-known experience, to find that bacteria may live dormant in ice for prolonged periods, and that infection may be carried through ice, but it is not so generally recognized that some bacteria prefer to grow on ice. Microorganisms, as a rule, are capable of resisting a low temperature when their ordinary activities cease, and they tend, either as single units or in clusters, to throw out a mucilaginous protein substance for their protection.”Some microorganisms have constitutive adaptations (expressed constantly) that allow them to thrive or survive under specific conditions of high salinity or low temperature. In a variable environment such as sea ice, however, acclimation mechanisms that allow a microorganism to function across a range of conditions may have an advantage over constitutive mechanisms finely tuned to an extreme, but relatively constant, environment. Microbial adaptations to sea ice may involve intracellular processes, membrane proteins or the secretion of extracellular polymeric substances (EPS). The latter can modify the immediate surroundings of the organism and its neighbors. The protective role of EPS, which tends to be gelatinous in nature, has been recognized since the earliest stages of sea-ice microbiology, as this account from 1918 by McLean  relates:This review addresses some of the physico-chemical parameters that define the sea-ice environment on a seasonal basis and the protective responses they elicit in microorganisms, particularly bacteria, the category of microorganisms known to inhabit all depths, forms and seasons of sea ice . It diveSea ice formation starts in the fall. When water reaches its freezing point, ice crystals, known as frazil ice, form throughout the upper water column and rise to the surface accumulating in a slush. This surface slush continues to freeze, consolidating into an upper layer of ice. Ensuing sea-ice growth depends on the conditions of the ocean: calm conditions promote the formation of congelation ice; turbulent conditions lead to frazil ice growth . The groDifferent processes can contribute to the retention of some of the microorganisms, particles and organic substances entrained into the ice from the source water . PhysicaColwellia psychrerythraea strain 34H, a model marine psychrophilic bacterium [In contrast, enrichment of smaller bacterial  cells in sea ice does not appear to occur directly by physical processes. Bacterial incorporation can be facilitated by the presence of algae, through bacterial association with algal cells or aggregated algal EPS, that are then concentrated by physical processes ,42,43. Aacterium , was fouacterium . Marine As sea ice consolidates, brines in the upper layers of the ice are expelled upwards to the ice surface forming a surface skim layer . Sea-iceA defining characteristic of the sea-ice environment is temperatures below, and sometimes well below, 0 °C. Meltponds, the accumulated meltwater from snow and surface ice during the summer season, are the only ice-associated environments with temperatures above 0 °C . Sea-icPsychrobacter cryohalolentis K5, a psychrotolerant bacterium isolated from permafrost, showed important changes in its proteome with up to 30% of the proteins having significantly different levels of expression when exposed to low temperatures. Among these proteins were cold-shock chaperones to facilitate translation, cold-adapted alleles that would allow a same function be performed at two different temperatures, and an increase in the expression of certain transporters. Bacteria also respond to low temperatures by changing the type of fatty acids and carotenoids present in their membranes and altering the membrane protein content [Low temperatures impose constraints at different levels in the single-cell microorganism and, consequently, elicit responses spanning different aspects of its physiology. Reaction and transport rates decrease with temperature, slowing most physiological processes. Protein folding is affected by a decrease in hydrophobic forces and changes in hydration . Membran content ,52,53. Ii.e., uniquely adapted to low temperatures [Given the prevailing low temperatures, organisms inhabiting sea ice and associated environments can be expected to use many of these strategies to cope with low-temperature constraints. When compared with the underlying water, the sea-ice bacterial community is enriched in culturable taxa considered psychrophilic, eratures . Also, peratures . Extraceeratures , especiaeratures .3·6H2O) at −2.2 °C, mirabilite (Na2SO4·10H2O) at −8.2 °C, and hydrohalite (NaCl ·2H2O) at −22.9 °C [The brine channel network containing the liquid fraction of the ice has a complex three-dimensional structure . For rel−22.9 °C ,60. SaltTemperature-dependent reduction of brine volume also increases the percentage of brine channel area covered by organisms , as wellObservations by Krembs and collaborators  confirmeFragilariopsis cylindrus can alter the microscopic and macroscopic structure of saline ice, opening the possibility for this protein and similar ones to play an important role in shaping the sea-ice microbial environment if produced in sufficient quantities [in situ.Sea-ice microorganisms can also modify the brine channel network through their antifreeze proteins, another type of extracellular substance produced by both sea-ice diatoms  and bactantities ,70. The antities  equationThe high salinity characteristic of sea-ice brine imposes at least two types of constraints on resident microorganisms. First, high concentrations of salts tend to affect the functioning of proteins, including precipitating them. Bacteria and archaea inhabiting high salinity environments tend to have, as a response, acidic proteins that, given their abundant negative charges, remain soluble and functional at higher salinities than basic proteins. Second, high environmental salinity exposes organisms to high osmotic pressure that drives water out of the cell, resulting in potential dehydration, loss of turgor pressure and reduction of cell volume. To counteract this water efflux, microorganisms of all types compensate for excessive concentrations of external solutes by accumulating compatible solutes in the interior of the cell. The general microbial ability to tolerate and even thrive in sea-ice brines comes with the added benefit of refuge against metazoan predators more susceptible to increases in salinity, such as those reported by Krembs and collaborators .+ and glutamate, accompanied by a release of putrescine to balance intracellular charges [+ with compatible solutes, which are either imported or synthesized directly in the cell [The osmotic up-shift that occurs with ice formation happens quickly as the temperature of the ice drops b. Sea-ic charges ,72. Avoithe cell . Compatithe cell . Dozens the cell . A suitethe cell .Halobacterium salinarum, which grows optimally at 26% NaCl, compensate high external concentration of solutes by incorporating salts in their cytoplasm. To keep cytoplasmic proteins functioning after the accumulation of salts, H. salinarum expresses an unusually high ratio of acidic to basic proteins . Membraytoplasm .Halobacterium, contrary to its name, is an archaeal genus), we hypothesize that the salting-in strategy used by H. salinarum, with long term accumulation of K+ ions in the cytoplasm and the majority of cytoplasmic proteins being acidic, has not been adopted by microorganisms from the sea-ice environment despite exposure to high brine salinities. Greater physiological flexibility will be provided by use of the compatible solutes strategy in the face of strong seasonal fluctuations in salinity inherent to sea-ice brines. Note that Psychromonas ingrahamii, which has the lowest ratio of acidic to basic proteins in either its membrane or cytoplasmic proteome compared with other marine isolates considered , also pnsidered , was isoFragilariopsis cylindrus has been shown to increase the production of all types of EPS  when frozen under high-salinity conditions [Pseudoalteromonas were shown to extend the range of salinities at which this strain could grow, while also providing protection against freeze-thaw cycles [Extracellular polymeric substances are also used by sea-ice microorganisms as a response to elevated salinities. The sea-ice diatom nditions . Likewisw cycles .Osmotic down-shift will be experienced in the late spring and the summer as the ice warms and melts. The extent of the osmotic down-shift depends on summer brine drainage. If melting prompts the brine to be flushed back into the ocean, microorganisms may not likely experience salinities much lower than seawater. If melting results in the formation of surface meltponds, then one of two conditions will follow: meltponds connected to seawater will have salinities close to 29, similar to those in nearby surface water; unconnected meltponds will have salinities below 5, reaching values as low as 0.1 . In the Solar radiation drives numerous reactions, biotic and abiotic, including the alteration and destruction of biologically relevant molecules. Processes driven by solar radiation, and the responses they trigger in microorganisms, are of particular relevance in polar regions where strong seasonal changes in insolation occur. At the organism level, UV radiation (UVR) can damage DNA and other nucleic acids by the formation of thymine dimers. UVR is known to decrease viability in bacteria from aquatic ecosystems  and damaGiven the potential detrimental effects of high irradiation, microorganisms have developed multiple protective responses, including the production of shading pigments, antioxidant compounds, and the performance of rapid DNA repair. For instance, in sediment-associated diatoms, exposure to UV-B prompts motility (away from radiation) and the production of carotenoid pigments able to function as quenching agents . The synSolar radiation further influences the sea-ice ecosystem by driving reactions that modify the dissolved organic carbon (DOC) pool. The potential of organic compounds to participate in photochemical reactions can be inferred from their absorption of visible radiation and UVR. DOC from spring sea ice, known to absorb UVR, has been shown to undergo varied photochemical reactions including changes in bioavailability and photooxidation to CO2 ,88. EPS Possible effects of solar radiation on EPS specific to sea-ice environments have been considered  but not Extracellular polymeric substances (EPS), composed primarily of polysaccharides, are commonly produced by a wide range of microorganisms from both terrestrial and marine environments. EPS differ amongst organisms and producing conditions in sugar chain length and branching, sugar composition, type of sugar linkages, and the presence of additional chemical groups such as sulfates, proteins, lipids and even nucleic acids ,93. DiffEPS can be either tightly bound to the cell surface, loosely attached, or cell free . Cell-frFragilariopsis curta and F. cylindrus, produced complex polysaccharides of higher molecular mass, with low relative abundance of glucose but high relative content of galactose, xylose and fucose. In contrast, a species of Synedropsis from the ice-water interface produced EPS dominated by low-molecular weight polysaccharides with low complexity and high relative content of glucose [Most of the EPS in sea ice can be attributed to production by ice algae, either before or after entrainment into the ice. Even in regions of the ice dominated by bacteria, the trail of algal EPS is expected to overwhelm the amount produced by sea-ice bacteria ,66. Diat glucose .EPS abundance in sea ice and associated environments has been quantified in numerous studies beginning with those by Krembs and collaborators in 2002  and by Min situ as a stress response, a process inferred from the increase in EPS concentration in winter sea ice [The EPS pool in sea ice is established during ice formation ,46,102 b sea ice ,101. On  sea ice .Colwellia psychrerythraea strain 34H, whose immediate relatives are found in sea ice, increased the amount of EPS produced per cell when exposed to low salinities not permissive of growth [The widespread, yet heterogeneous , presenf growth . The surFurther insight into the protective role of EPS comes from experiments by Krembs and collaborators , who obsThe salinity of the brine pockets, however, is conventionally described as a function of temperature only and does not depend on the bulk salinity of ice. Following earlier work ,112, we 2+ determines self-assembly of marine gels, which can in turn increase the availability of nutrients for the microbial population [2+ may figure in the fate of carbonates in sea ice [3 precipitation were not evident in Antarctic sea ice [3 precipitation. Data on the interactions between EPS and calcium in other environments [Divalent cations present in sea-ice brines can also interact with charged groups in the backbone of EPS. In the marine environment, this interaction has been suggested to play a role in the binding of key nutrients for the cell such as iron . Likewispulation . In the  sea ice . Relatio sea ice , but exp sea ice  includedronments  may info2+ ions is evident. Xanthan, being an ionic polysaccharide, has a titration curve with a clear offset due to its conductive properties and the presence of associated counter-ions. Its curve also shows two segments with distinctive slopes, indicating that Ca2+ ions interacted with the polysaccharide until all binding sites were occupied [Bergmann and collaborators used conductometric titrations to estimate the amount of binding sites for divalent cations present in ionic and nonionic bacterial extracellular polysaccharides . Conductoccupied .Colwellia psychrerythraea strain 34H. A blank with no polysaccharide added was also included  and multiple binding sites for Ca2+ (no change in slope over the tested range of CaCl2 concentrations). The presence of charged EPS from this Arctic marine psychrophile has implications for the dynamics of carbonates in the sea-ice environment given that charged polysaccharides, unlike non-ionic polysaccharides, have known effects on the precipitation of carbonates [Following this approach, conductometric titrations were performed on solutions (in de-ionized water) of dextran, xanthan, and EPS obtained from a culture of uded see  agree wirbonates .2+ [2+ [2+ [2+ concentrations [Interactions between cations and polysaccharides also confer both algal and bacterial EPS with the potential to adsorb heavy metal contaminants such as Cd2+ , Pb2+ [12+ [2+  and Hg2+ [2+ [2+ , which c [2+ [2+ ,125. Dis [2+ [2+ ; EPS frotrations ,123,124,C. psychrerythraea 34H [in situ conditions relevant to microbial life in sea ice remain largely unexplored.EPS can also interact with other extracellular macromolecules, most relevantly with proteins. Non-covalent interactions between EPS and proteins allow the formation of complexes, coacervates and aggregates, increasing the range of pH in which a protein is soluble . In the raea 34H . The intraea 34H  to the b2+, were to be as significant in sea ice as they are in other environments, then the implications for carbon transport through the sea-ice cover could be quantitatively important. As much as has been learned over the past decades about sea-ice microorganisms and their self-protective responses to the constraints of their habitat, more awaits discovery.In focusing on the extracellular responses of sea-ice microorganisms to the sometimes severe and fluctuating environmental conditions of their habitat, our goal has been to highlight a number of features, particularly regarding EPS, that have not been fully explored or that raise new research directions. The extracellular products of microorganisms entrapped in sea ice are known to influence the microstructure of the ice, and thus its habitability, but how they may influence the effectiveness of traditional equations for calculating key parameters of sea ice or function at the micrometer scale within an ice pore is not clear. Unanswered questions involve the potential role of EPS in mitigating the salt concentration directly experienced by the cell, contributing to the adaptive strategy of compatible solutes, and blocking viral attack. EPS interactions with other exudates, including ice-active proteins and hydrolytic enzymes that serve a substrate-acquisition function for the cell, are poorly known. If the unexplored interactions of EPS with inorganic ions, particularly Ca


Synthetic contiguous overlapping peptides (COPs) may represent an alternative to allergen extracts or recombinant allergens for allergen specific immunotherapy. In combination, COPs encompass the entire allergen sequence, providing all potential T cell epitopes, while preventing IgE conformational epitopes of the native allergen.in vitro in competition ELISA and basophil degranulation assays. Their in vivo reactivity was determined by intraperitoneal challenge in rBet v 1 sensitized mice as well as by skin prick tests in volunteers with allergic rhinoconjunctivitis to birch pollen.Individual COPs were derived from the sequence of Bet v 1, the major allergen of birch pollen, and its known crystal structure, and designed to avoid IgE binding. Three sets of COPs were tested in vitro was unable to induce anaphylaxis in sensitized mice in contrast to rBet v 1. In addition no positive reactivity to AllerT was observed in skin prick tests in human volunteers allergic to birch pollen. In contrast, a second set of COPs, AllerT4-T5 displayed some residual IgE binding in competition ELISA and a weak subliminal reactivity to skin prick testing.The combination, named AllerT, of three COPs selected for undetectable IgE binding in competition assays and for the absence of basophil activation in vitro, by the absence of basophil activation and the absence of in vivo induction of allergic reactions in mouse and human. The hypoallergenicity of contiguous overlapping peptides was confirmed by low, if any, IgE binding activity NCT01719133ClinicalTrials.gov  Allergen specific immunotherapy (SIT) is the only etiologic treatment available for IgE-mediated allergy . SIT conFagales order and at the origin of crossreactive hypersensitivities to a large array of food allergens belonging to Rosaceae, Juglandaceae, Corylaceae, Apiaceae or Solanaceae families [We previously performed a phase I trial based on the subcutaneous injection of synthetic contiguous overlapping peptides (COPs) from the major bee venom allergen phospholipase A2 (PLA2) . Clinicafamilies -24.in vitro (based on their IgE binding capacity) and in vivo  as a preliminary study to a therapeutic phase I clinical trial in patients with allergic rhinitis to birch pollen.Our aim in this study was to evaluate COPs hypoallergenicity Three sets of COPs composed of peptides T1-T2-T3, T4-T5 and T6-T7-T8 respectively (Table d) were obtained from Harlan  and used at the age of 6–8 weeks.Four weeks-old female BALB/c mice  up to six times at 2 weeks intervals as previously described . Two weeMouse serum IgE, IgG1 and IgG2a antibody responses were determined by ELISA as previously described ,27. Brie4 cells/well) overnight. Passive sensitization of RBL-2H3 cells was carried out with sera from rBet v 1-sensitized mice at a final dilution of 1:30 for 2 h. Unbound antibodies were removed by washing the cell layer twice in Hanks’ balanced salt solution . Degranulation of RBL cells was induced by adding rBet v 1, individual COP T1, T2, T3 or AllerT diluted in HBH buffer (HBSS with 1% BSA and 10 mM HEPES) for 1h at 37°C at indicated concentrations. β-hexosaminidase activity was analyzed by incubating 50 μl of cell supernatant with 50 μl of 1.3 mg/ml 4-Nitrophenyl N-acetyl-β-D-glucosaminide (Sigma) in citrate buffer  for 1 h at 37°C. The reaction was stopped by addition of 100 μl glycine buffer  and optical densities (OD) measured at 405 nm.Degranulation assay was performed as previously described . RBL-2H3-5 M, then distributed to rBet v 1 coated and BSA 1%-blocked 96-well immunoplates  for 2 h RT or overnight at 4°C. Successively and with washings between steps, plates were incubated 1 h at RT with biotinylated mouse anti-huIgE , Streptavidin-HRP or -alkaline phosphatase  and TMB  or 4-NPP. OD were read at 450 nm, 630 nm and 405 nm, respectively. Inhibition of IgE binding to Betv 1 was calculated as: (OD with competitor minus ODmin)/(OD without competitor minus ODmin) × 100, where ODmin was determined in wells without serum.Dilutions (1:5 to 1:10) of sera from 6 birch pollen allergic patients  were incubated 1h with tenfold serial dilutions of either individual COPs , COPs combinations AllerT, T4-T5, T6-T7-T8 or rBet v 1, starting at 103 basophils for each sample were acquired within 1 h on a FASCalibur, Becton-Dickinson. Results were expressed as the percentage of basophils (IgE+ cells) expressing CD63. A cut-off ≥15% CD63+ IgE+ basophils was chosen as the lower limit of positivity according to the manufacturer. In indirect assay, 100 μl of heparinized blood from a non-allergic subject was pre- incubated 2 h at 37°C with 25 μl of plasma or sera from allergic subjects before performing the Basotest™ assay as described above.The direct assay was performed using the Basotest™ assay according to manufacturer’s instructions . Heparinized blood (100 μl) was first incubated with stimulation buffer for 10 min at 37°C, and then with 100 μl allergen solution diluted in a saline solution for 20 min at 37°C. A dose–response curve was performed with ten-fold dilutions of rBet v 1 or AllerT. Formyl methionyl leucyl phenylalanine was used as a positive control and PBS as a negative control. The activation process was stopped by incubating blood samples at 4°C for 10 min. Samples were stained for 20 min at 4°C with 20 μl anti-IgE-PE mAb and anti-CD63-FITC. Erythrocytes were subsequently removed by adding 2 ml lysis solution (Becton-Dickinson) and two washes with PBS. Cells were resuspended in 200 μl PBS and at least 105 cells/well in medium (MEM containing glutamine and penicillin/streptomycin) and incubated overnight with sera from subjects with class 4, 5 and 6 IgE levels to birch pollen extract as determined by ImmunoCAP . Human serum was diluted 1/2 with 4M glucose and incubated for 45 min at 56°C, centrifuged and diluted 1/5 with MEM medium before addition to RBL703/21 cells. After washing with Tyrode’s buffer , cells were incubated for one hour at 37°C with various concentrations of rBet v 1, COPs or 0.5 μg/ml goat anti-human IgE heavy chain antiserum  in Tyrode’s buffer containing 50% deuterium oxide or Tyrode’s buffer alone as control. Supernatants (30 μl) were incubated in duplicates with 50 μl of substrate solution (1.3 mg/ml p-nitrophenyl-b-D-2-acetamido-2-deoxyglucopyranozide from Sigma in 0.1 M citrate pH 4.5) for 1 hour at 37°C. Reaction was stopped by the addition of 100 μl of 0.2 M glycin (pH 10.7). OD was measured at 405 nm (reference filter at 620 nm). β-hexosaminidase release was calculated using the following formula: (OD sample-ODbuffer) / (ODanti-IgE-ODbuffer) × 100.The assay was adapted from Ladics et al.  and BlanThe AN002 phase I clinical trial was a single center, open and controlled study conducted at the Centre Hospitalier Universitaire Vaudois (CHUV) in Lausanne. Volunteers (18–45 years) with at least one season history of birch pollen rhinoconjunctivitis or asthma were recruited outside of the birch pollen season. Volunteers treated with antihistamines or medication with antihistamine activity less than 15 days prior to skin prick testing were excluded as well as volunteers with peak expiratory flow (PEF) <20% of volunteer’s best PEF value. Birch pollen sensitization was documented 1) by positive skin prick test to birch pollen extract  and to rBet v 1 , and 2) positive birch pollen and rBet v 1-specific serum IgE (>0.35 kU/L by ImmunoCAP assay). Inferior limits of specific IgE to determine allergy classes 1 to 6 are 0.35, 0.7, 3.5, 17.5, 50 and 100 kU/L respectively. The clinical research protocol was approved by the Ethical Review Board of the Faculty of Biology and Medicine, Lausanne, and by the Swiss National Regulatory Authority (Swissmedic) and posted on the ClinicalTrials.gov site under the study number NCT01719133. The primary objective of the study was to determine the reactogenicity of selected COPs by skin prick tests.in vivo by skin prick tests following EAACI guidelines [Individual T1 to T5 COPs, AllerT and T4-T5 mix were evaluated idelines . For indt test. * = p<0.05; **= p<0.01; *** = p<0.001. Analysis was done using the Prism 4.00 software for Windows . Data analysis of clinical trial AN002 was descriptive.In animal studies, results are expressed as mean ± SD of 6–10 mice/group. Each experiment was done at least three times. Significance of differences between means was determined using Student’s or non-parametric Fagales species showing regions of particularly high sequence conservation that may possibly contain IgE epitopes as proposed by Spangfort et al. [Bet v 1 is a 159 amino acid (aa) long protein present as a mix of isoforms in pollen extracts and differing by a few amino acids . Based ot et al. . Three ct et al. . On the t et al. . The setin vitro for birch specific IgE binding by competition ELISA. Soluble rBet v 1 inhibited serum IgE binding with EC50 ranging from 10-14 to 10-10 M,  . Upon stMice were sensitized by repeated injections of rBet v 1 (0.1 μg in Aluminum Hydroxide) as described previously . Bet v 14-fold lower capacity to activate basophils from allergic patients.In preparation for application to human subjects, AllerT COPs and rBet v 1 were compared for their capacity to induce human basophil degranulation. In a direct Basotest™ assay, blood was collected from patients allergic to birch pollen and degranulation due to IgE crosslinking was tested in response to COPs mixes, namely T1-T2-T3, T4-T5 or T6-T7-T8  , whereas the i.p. injection of rBet v 1 induced a sharp drop in rectal temperature, a surrogate marker of anaphylaxis. These Although we cannot fully insure that anaphylactic reaction was dependent on the induction of IgE, anti-rBet v 1 specific murine IgE reached high titers after the fourth subcutaneous injection as measured by ELISA Figure A. In parin vivo assays, twenty volunteers were tested by skin prick tests for reactivity to Bet v 1, to birch pollen extract as well as to the five COPs and their mixes . As shown in Table in vivo injection as exemplified by the skin prick tests reported in this study.The low, if any, IgE binding activity of COPs as compared to rBet v 1 was confirmed by a functional human basophil activation assay as well as in an assay based on huFcϵRI transfected RBL. Using individual peptides as well as their mixes, only background basophil or RBL activation was noted in presence of sera from birch pollen hypersensitive patients. Finally in Taken together, these experiments demonstrate that an allergen specific immunotherapy strategy based on the injection of selected COPs derived from Bet v 1 may be applied to patients with hypersensitivity to birch pollen. The presented data, performed both in murine and human models, indicate that the binding of the selected COPs to IgE, if any, is extremely limited. COPs were a) safe after subcutaneous injection in sensitized mice since they did not induce anaphylaxis, b) unable to activate human basophils and c) did not induce prick test reactions. As compared to birch pollen derived COPs T4 and T5, the mix of COPs T1-T2-T3 (AllerT) appears as an optimal candidate mix for a phase I clinical trial in birch pollen hypersensitive patients with allergic rhinoconjunctivitis.Céline Pellaton, Yannick Perrin, Caroline Boudousquié, Nathalie Barbier, Jacqueline Wassenberg, Giampietro Corradin, Anne-Christine Thierry, and Régine Audran have no conflict of interest. Christophe Reymond is employee of Anergis SA, Epalinges, Switzerland. François Spertini and Christophe Reymond are founder members of Anergis SA and possess shares in the society.All authors have been involved in drafting the manuscript and revising it critically; all authors have given approval of the submitted version of the paper. Furthermore, FS and CR contributed to acquisition of funding, GC to peptide synthesis, CB, NB, and ACT to establishing key laboratory techniques and revising illustrations.


The mechanisms whereby H2O2–modulated phosphorylation pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) in these cells are incompletely understood. We show here that H2O2 treatment of adult mouse cardiac myocytes leads to increases in intracellular Ca2+ ([Ca2+]i), and document that activity of the L-type Ca2+ channel is necessary for the H2O2-promoted increase in sarcomere shortening and of [Ca2+]i. Using the chemical NO sensor Cu2(FL2E), we discovered that the H2O2-promoted increase in cardiac myocyte NO synthesis requires activation of the L-type Ca2+ channel, as well as phosphorylation of the AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase kinase 1/2 (MEK1/2). Moreover, H2O2-stimulated phosphorylations of eNOS, AMPK, MEK1/2, and ERK1/2 all depend on both an increase in [Ca2+]i as well as the activation of protein kinase C (PKC). We also found that H2O2-promoted cardiac myocyte eNOS translocation from peripheral membranes to internal sites is abrogated by the L-type Ca2+ channel blocker nifedipine. We have previously shown that kinase Akt is also involved in H2O2-promoted eNOS phosphorylation. Here we present evidence documenting that H2O2-promoted Akt phosphorylation is dependent on activation of the L-type Ca2+ channel, but is independent of PKC. These studies establish key roles for Ca2+- and PKC-dependent signaling pathways in the modulation of cardiac myocyte eNOS activation by H2O2.Nitric oxide (NO) and hydrogen peroxide (H We have recently shown that hydrogen peroxide (H2O2) is a critical intracellular mediator that modulates eNOS phosphorylation and enzyme activation in cardiac myocytes 2O2 in modulation of cardiac myocyte Ca2+ metabolism is less well understood, and there are major gaps in our understanding of the pathways connecting H2O2–dependent phosphorylation pathways, intracellular Ca2+ signaling, and eNOS activation.The endothelial isoform of nitric oxide synthase (eNOS) is robustly expressed in cardiac myocytes, and nitric oxide (NO) has been shown to play key roles in modulating cardiac function 2O2. Some protein kinase C (PKC) isoforms are activated by H2O2, yet little is known about the modulation of eNOS by PKC in the heart. Other protein kinases expressed in cardiac myocytes that have been implicated in eNOS regulation include ERK1/2, MEK1/2, kinase Akt, AMPK, and the cyclic AMP-dependent protein kinase (PKA). Since abnormalities in PKC-modulated signaling pathways and alterations in intracellular Ca2+ metabolism have been implicated in cardiomyopathy and heart failure 2O2 in control of PKC activation, intracellular Ca2+ pathways, and eNOS phosphorylation responses in cardiac myocytes. Here we provide data that establish roles for Ca2+, PKC and PKA in modulating eNOS phosphorylation in response to H2O2, and identify the key protein kinase pathways that modulate H2O2–dependent NO synthesis in cardiac myocytes.Cardiac myocytes contain an astonishingly broad array of protein kinases, several of which may be modulated by H2+ indicator Fura-2 was used to measure [Ca2+]i in electrically stimulated  cardiac myocytes that had been freshly isolated from adult mice. We found that H2O2 (25 µM) promotes an increase in [Ca2+]i, measured as the ratio of F340/F380 , which we previously used to explore the agonist-modulated regulation of cardiac myocyte NO synthesis 2O2-promoted NO synthesis is completely blocked by pre-treatment of the cells with nifedipine . Here we show that the AMPK inhibitor Compound C blocks H2O2-induced cardiac myocyte NO synthesis, measured with the NO chemical sensor Cu2(FL2E)  prior to H2O2 treatment   . In orde 15 min) . Nifediprylation . Importa and ACC . H2O2 ald ERK1/2  and of kynthesis , and alsd ERK1/2 . The MEKd ERK1/2 .2O2 appears to be differentially regulated: while H2O2–promoted Akt phosphorylation is blocked by nifedipine and BAPTA , calphostin C fails to attenuate Akt phosphorylation  to visualize NO synthesis in cardiac myocytes, we demonstrated that the L-type Ca2+ channel activity is required for H2O2-promoted NO synthesis , we demonstrate here that activation of AMPK is required for the H2O2-promoted increase in cardiac myocyte NO synthesis  is a serine/threonine protein kinase that has been characterized as a sensor of cellular energy balance in mammalian cells ynthesis . Similarependent . Nifedipependent . A23187 ependent . These frylation . Althougpathways .2O2-promoted eNOS phosphorylation, and we also found that these inhibitors do not attenuate H2O2-promoted AMPK phosphorylation 2O2-promoted increase in Akt phosphorylation, suggesting that AMPK may lie upstream of Akt in cardiac myocytes, as previously shown in vascular endothelial cells 2O2-promoted eNOS phosphorylation by the PKA inhibitor H89  was synthesized as previously reported Polyclonal antibodies directed against phospho-eNOS (Ser1177), phospho-PKC (pan) (βII Ser660), phospho-AMPK (Thr172), phospho-Akt (Ser473), phospho-ACC (Ser79), phospho-MEK1/2 (Ser217/212), phospho-ERK1/2 (Thr202/Tyr204), AMPK, Akt, ACC, MEK1/2, ERK1/2, phospho-VASP (Ser157), and H-89 were from Cell Signaling Technologies . Antibodies against total eNOS, total VASP, Caveolin-3, and phospho-eNOS (Ser633) were from BD Transduction Laboratories . Collagenase type 2 was from Worthington Biochemical . Compound C, PD98059 (a selective cell-permeable inhibitor of MAP kinase kinase [MEK]), “MEK1/2 inhibitor” (a cell-permeable vinylogous cyanamide that acts as a selective inhibitor of MEK1/2), and Calphostin C were from Calbiochem. Super Signal substrate for chemiluminescence detection and secondary antibodies conjugated with horseradish peroxidase were from Pierce. Tris-buffered saline and phosphate-buffered saline were from Boston Bioproducts . Laminin was from BD Bioscience . Minimum essential medium with Hank’s balanced salt solution and glutamine were from Gibco-BRL. Calf serum was from HyClone . The AlexaFluor488 Annexin V/Dead cell apoptosis kit, Alexa Fluor-Green (488)-tagged goat anti-rabbit antibody and Alexa Fluor red (568)-tagged goat anti-mouse antibody were from Invitrogen/Molecular Probes. All other reagents were from Sigma . Mouse line C57BL6/J was from Jackson Labs . Cu2, after which the heart tissue was minced and the cells were dispersed by trituration. Subsequently, the cardiac myocytes were allowed to settle, and then washed, pelleted, counted, and plated.All animal experimentation was performed according to protocols approved by the Harvard Medical School Committee on Use of Animals in Research. For these studies, 8–10-week-old C57BL6/J, mice were lightly anesthetized with isofluorane, heparinized , and sacrificed. The heart was quickly removed from the chest and retrogradely perfused through the aorta as described Cardiac myocytes were plated in laminin-coated 6-well culture dishes  in plating medium consisting of Minimum Essential Medium with Hank’s balanced salt solution, supplemented with calf serum (10% v/v), 2,3-butanedione monoxime (10 mM), penicillin-streptomycin (100 U/ml), glutamine (2 mM), and ATP (2 mM). After the cells were attached (∼1 hour), the plating medium was changed to culture medium consisting of Minimum Essential Medium with Hank’s balanced salt solution, supplemented with bovine serum albumin (1 mg/ml), penicillin-streptomycin (100 U/ml), and glutamine (2 mM) and the cells were cultured for 4 hours.2 added). A two-point Fura2 calibration was performed according to the method of Grynkiewicz et al. Intracellular calcium concentrations were monitored using electrically stimulated freshly isolated cardiac myocytes. In brief, coverslips of cardiac myocytes loaded with Fura-2AM  were used to monitor intracellular calcium transients. Fura-2 fluorescence was measured using an IonOptix spectrophotometer . Fura-2 was excited by light at 340-nm and 380-nm. A photomultiplier tube detected the emitted fluorescence at 510 nm. Experiments were performed at room temperature on the stage of an inverted microscope , and myocytes were visualized using an air objective (S Fluor 40X). Field stimulation  was accomplished using the MyoPacer (IonOptix). In all experiments, myocytes were kept in Tyrode’s solution  at 1 Hz, 5–10 Volts. Sarcomere length was recorded with a video edge detector coupled to a camera . Sarcomere shortening analyses was performed using IonWizard Core Analysis software (IonOptix) in myocytes without any treatment and after 5 to 10 min of H2O2. In some studies, myocytes were pre-treated with nifedipine (100 µM) for at least 15 min before and during H2O2 treatment. Sarcomere shortening was expressed as percent shortening relative to the resting diastolic length.Myocytes were placed in a rapid change stimulation chamber on an inverted Nikon microscope stage and continuously bathed in Tyrode's solution at room temperature, pH 7.45 with 1.0 mM CaCl2(FL2E) 2. Cover slips were then placed in an onstage incubator  on an Olympus IX81 inverted microscope equipped with an UPlan 40X/1.3 oil objective in a low-volume glass-covered recording chamber. Fluorescence signals were analyzed by using a Hamamatsu Orca CCD camera  at 470 nm. Viable rod-shaped cardiac myocytes with rectangular ends were selected by differential interface contrast imaging and then subjected to fluorescence imaging, following treatments as indicated.Cardiac myocytes harvested from at least three independent preparations were analyzed. The signal from the NO sensor was analyzed as the slope of the fluorescence increase observed following the addition of agonist or vehicle. Cells were cultured on cover slips and loaded with 5 µM Cu3VO4; 1 mM NaF; 2 µg/mL leupeptin; 2 µg/ml antipain; 2 µg/ml soybean trypsin inhibitor; and 2 µg/mL lima trypsin inhibitor). Cells were harvested by scraping. After separation by SDS-PAGE, proteins were electroblotted onto nitrocellulose membranes. After incubating the membranes in 5% nonfat dry milk in Tris-buffered saline with 0.1% (vol/vol) Tween 20 (TBST), membranes were incubated overnight in TBST containing 5% bovine serum albumin plus the specified primary antibody. After four washes (10 min each) with TBST, the membranes were incubated for one hour with a horseradish peroxidase-labeled goat anti-rabbit or anti-mouse immunoglobulin secondary antibody in TBST containing 1% milk. The membranes were washed four additional times in TBST, then incubated with a chemiluminescent reagent according to the manufacturer's protocols , and digitally imaged in a chemiluminescence imaging system . Quantitative analyses of the chemiluminescent signals were performed using an AlphaEase®FC software . For quantitative analyses of immunoblot experiments, the signal is normalized to the value obtained in the absence of added drug.After drug treatments, cardiac myocytes were washed with PBS buffer and incubated for 10 minutes in lysis buffer  were fixed in 4% paraformaldehyde for 20 min, rinsed twice with PBS, permeabilized in 0.1% Triton X-100 for 45 min, and blocked with 10% goat serum overnight. Immunoreactive eNOS and caveolin-3 were co-localized using confocal microscopy. After incubating with both primary antibodies , samples were washed three times in PBS for 10 min. The eNOS primary antibody was localized by immunofluorescent detection with a secondary Alexa Fluor-Green (488)-tagged goat anti-rabbit antibody , and Cav-3 primary antibody was detected with a secondary Alexa Fluor red (568)-tagged goat anti-mouse antibody . Samples were washed three times in PBS for 10 min to remove excess secondary antibody and then mounted on slides using medium containing 4',6-diamidino-2-phenylindole as nuclear counter stain. Microscopic analysis of samples was performed using an Olympus IX81 inverted microscope in conjunction with a DSU spinning disk confocal system equipped with a Hamamatsu Orca ER cooled-CCD camera. Images were acquired using a 40X/1.3 differential interference contrast oil immersion objective lens and analyzed using Metamorph software from Universal Imaging, Inc. .2 added. Cardiac myocytes were treated with varying concentrations of H2O2 for 15 minutes. Cell viability was determined by the ratio of rod-shaped to total cells. Apoptosis and necrosis were detected using an AlexaFluor488 annexin V/propidium iodide detection kit (Invitrogen/Molecular Probes). Briefly, cardiac myocytes were incubated with annexin V and propidium iodide for 10 minutes at room temperature. Dishes were photographed under both phase-contrast and fluorescence microcopy, and rod-shaped (viable), rounded (non-viable), and total cells were counted. Apoptotic cardiac myocytes were defined as annexin V-positive (green-stained cells) and necrotic myocytes as annexin V plus propidium iodide-positive cells (green- and red-stained cells).Cardiac myocytes were plated on laminin-coated culture dishes in Tyrode's solution at room temperature, pH 7.45 with 1.0 mM CaClANOVA. A p value of less than 0.05 was considered significant.Mean values for individual experiments were expressed as means ± S.E. Statistical differences were assessed by Figure S12O2, 25 µM) and analyzed in immunoblots probed with antibodies as shown. The immunoblots shown are representative of three independent experiments that yielded similar results. Panel B shows immunoblot analyses from cardiac myocytes incubated with PD98959  or vehicle, then treated with H2O2 . Panel C shows representative immunoblot analyses from cells incubated with MEK1/2 inhibitor  or vehicle, then treated with H2O2. The immunoblots in panel B and C were probed with antibodies against phospho-Akt (Ser 473) or phospho-AMPK (Ser172). Panels D and E show results of pooled data corresponding to representative experiments shown in 2O2 (25 µM), or H2O2 (500 µM) for 15 min and stained with annexin V and propidium iodide as described in the text. The two fluorescence channels were obtained sequentially; overlaying of the differential interference contrast image (DIC) and both fluorescence channels (annexin V and propidium iodide) is shown. Panel G shows the percentage of apoptotic (annexin V positive) and necrotic (annexin V + propidium iodide positive) cardiac myocytes. Panel H shows the percentage of viable (rod-shaped) and nonviable (round) cardiac myocytes. *indicates p<0.05; **indicates p<0.01; and ***indicates p<0.001 (ANOVA).In Panel A, cardiac myocytes were treated with hydrogen peroxide (H(TIF)Click here for additional data file.


One of the most frequent food allergies in childhood is cow's milk allergy (CMA) that manifest with diverse symptoms. Cow's milk has four main proteins that their recognition can help to physician for patients treatment. The aim of this study was to determine the most common cow's milk allergenic proteins in children with CMA.All of children with the positive history of CMA and positive prick test &/or specific IgE were entered in this study. Specific IgE were determined for Cow's milk protein, α-lactalbumin, β-lactoglobulin, and casein and bovin serum albumin with RIDA Allergy Screen test. The specific IgE more than Class 2+ was acceptable.Eighty six patients with CMA (3month to 18 yrs) were entered this study Fifty three patients were males (61.1%). Median of age was 2.5 yrs (Mean=3.98) and most of CMA patients was under 2 yrs (45.1 %). Mean of total IgE was 343.39±93.63 (SE)(0.2-5000IU/ml). The symptoms of patients was as following: . Totally, positive Specific IgE to cow's milk protein, α-lactalbumin, casein, β-lactoglobulin and bovin serum albumin were 89.2%, 78.3%, 75%, 63.4%, & 45.5%, respectively. Although α-lactalbumin & casein were the most allergenic protein, mean of specific IgE concentration to β-lactoglobulin was highest in patients with CMA. There was a significant correlation between positive specific IgE to β-lactalbumin & anaphylaxis (P=0.05) and also between respiratory symptoms and positive specific IgE to casein (P=0.01). There was significant difference between mean of specific IgE to casein between males and females (P=0.001) that was the more frequent in males.In this study, α-lactalbumin and casein seems to be the most allergenic protein in cow's milk but it seems that specific IgE positivity to β-lactoglobulin is related to anaphylaxis to CM.


Previous studies have shown that enhanced accumulation of contractile proteins such as smooth muscle myosin light chain kinase (smMLCK) plays a major role in human airway smooth muscle cells (HASM) cell hypercontractility and hypertrophy. Furthermore, serum IgE levels play an important role in smooth muscle hyperreactivity. However, the effect of IgE on smMLCK expression has not been investigated. In this study, we demonstrate that IgE increases the expression of smMLCK at mRNA and protein levels. This effect was inhibited significantly with neutralizing abs directed against FcεRI but not with anti-FcεRII/CD23. Furthermore, Syk knock down and pharmacological inhibition of mitogen activated protein kinases (MAPK)  and phosphatidylinositol 3-kinase (PI3K) significantly diminished the IgE-mediated upregulation of smMLCK expression in HASM cells. Taken together, our data suggest a role of IgE in regulating smMLCK in HASM cells. Therefore, targeting the FcεRI activation on HASM cells may offer a novel approach in controlling the bronchomotor tone in allergic asthma. Asthma is a chronic inflammatory disease of the airways, clinically characterized by airway obstruction, inflammation, and hyperresponsiveness. Inflammatory components of this disease include an increased infiltration of activated T lymphocytes, mast cells, eosinophils, and neutrophils within the airway lumen and bronchial submucosa It is well recognized that most asthma in children and adults is associated with atopy, characterized by an increased synthesis of IgE against common allergens. Indeed, two-thirds of asthmatics are allergic and more than 50% of patients with severe asthma have allergy et al2+) mobilization Previously, we and others have shown that HASM cells express the high and low affinity IgE receptor (FcεRI) and (FcεRII/CD23)All the experimental procedures were approved by the Human Research Ethics Board of the University of Manitoba, Winnipeg, MB, Canada. Written informed consent for HASM harvesting was obtained from all patients.Purified human IgE  was obtained from Antibody shop . Fetal bovine serum (FBS) and sodium pyruvate were purchased from HyClone . 100X L-glutamine, DMEM, Ham’s F-12, trypsin-EDTA, and antibiotics  were purchased from Invitrogen Canada Inc. . All other reagents were purchased from Sigma unless specified. Monoclonal anti Human FcεRI α chain (mAb15-1) was kindly provided by Dr. Franz Kriceck, . Anti CD23/FcεRII mAb (clone M-L233) was purchased from BD biosciences.2+ in response to acetylcholine, a physiologically relevant contractile agonist HASM cells were obtained from macroscopically healthy segments of second to fourth generation lobar or main bronchus of patients undergoing surgery for lung adenocarcinoma as we described previously Sub-confluent HASM cells were serum deprived for 48 h in Ham’s F-12 medium containing 5 μg/ml human recombinant insulin, 5 μg/ml human transferrin, 5 ng/ml selenium, and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). Cells were then stimulated with IgE  in fresh FBS-free medium for the specified time. We found similar preliminary observations with all doses tested; therefore we performed experiments with 5 μg/ml. In neutralizing experiments, cells were pre-treated with mouse anti-human FcεRI , mouse anti-human CD23/FcεRII (M-L233), or mouse IgG1 isotype control (MOPC21)  for 1 hr prior to IgE stimulation. In some experiments, cells were also pretreated with Erk1/2 inhibitor U-0126 (10 μM), P38 inhibitor SB-203580 (10 μM), JNK inhibitor SP600125 (40 nM) or phosphatidylinositol-3-kinase (PI3K) inhibitor Wortmannin (100nM) for 45 minutes prior to stimulation with IgE.5′-AGCAATGCCTCCTGCACCACCAAC-3′ and reverse primer 5′-CCGGAGGGGCCATCCACAGTCT-3′. Primers for smMLCK are forward Primer: 5′ GACTGCAAGATTGAAGGATAC 3′ and Reverse Primer: 5′ GTTTCCACAATGAGCTCTGC 3′. Real-time quantitative PCR was carried out using ABI 7500 Real-Time PCR System and analyzed by 7500 System SDS software version 1.3.1 , following manufacturer’s instructions. Product specificity was determined by melting curve analysis and by visualization of PCR products on agarose gels. Calculation of the relative amount of each cDNA species was performed according to standard protocols. Briefly, the amplification of smMLCK gene in stimulated cells was calculated first as the copy number ratio of smMLCK to GAPDH, and then expressed as normalized values of fold increase over the value obtained with unstimulated (control) cells.Serum-deprived confluent HASM cell cultures were stimulated for the specified time, harvested, and total cellular RNA was extracted using TRIzol method . Reverse transcription was performed by using 2 μg of total RNA in a first-strand cDNA synthesis reaction with High Capacity cDNA Reverse transcriptase kit as recommended by the supplier  are forward primer For western blots, HASM cells were lysed for 2 min on ice in M-PER lysis buffer (Thermo Scientific) supplemented with a cocktail of protease inhibitors (Sigma-Aldrich) and centrifuged for 20 min to collect protein lysate. For immunoblotting, 10 μg of lysate from each sample was separated on 6% SDS polyacrylamide gel and electro-transferred onto PVDF membrane . The membrane was blocked at room temperature for 2 h with 5% skim milk, incubated with mouse anti-MLCK (K36 clone) polyclonal Ab (Sigma-Aldrich), or mouse anti-calponin antibody (Sigma-Aldrich) at room temperature for 2 h, followed by secondary antibody HRP-goat anti-mouse IgG prepared in 1% skim milk. All the blots were developed by enhanced chemiluminiscence as recommended by the supplier . β-actin was used as internal control. The intensity of smMLCK, myosin, calponin and β-actin bands was determined by using AlphaEase FC software version 3.1.2 relative to control loading levels. For signaling, the intensity of phosphorylated forms of ERK, JNK, P38, myosin and smMLCK was normalized with the intensity of total ERK1/2, JNK and P38, respectively.) expressing specific Syk and Lyn shRNA, respectively . 293T cells used for virus production and titration, were cultured in Dulbecco’s medium  supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin/glutamate  as described in Lyn and Syk kinases were silenced by transducing HASM cells with pseudotyped lentiviral vector . P values <0.05 were considered statistically significant.All the data were performed from at least three experiments. Statistical analysis was performed by doing Previous reports indicated that IgE-rich atopic serum induces smooth muscle contractile response We then investigated the involvement of FcεRI and FcεRII/CD23 receptor in the regulation of smMLCK expression in HASM cells using FcεRI blocking monoclonal Ab (mAb) (15/1) directed against the IgE binding site + , P38 (SB203580), JNK (SP600125) and PI3 K (Wortmannin), and then stimulated with IgE for 48 h. Inhibition of MAPK (ERK1/2) and PI3K pathway resulted in a significant reduction of IgE-mediated smMLCK protein expression  . Pre-treIgE binding to FcεRI in inflammatory cells was earlier thought to be a “passive sensitization” step requiring subsequent allergen/antigen cross-linking. Recent data suggests that IgE binding alone (sensitization) have critical effector functions on cellular activation. IgE sensitization induces pro-survival effects in mast cells, monocytes and neutrophils IgE-induced upregulation of smMLCK was found to involve FcεRI activation as an FcεRI-α chain-specific blocking antibody mAb 15/1, but not anti CD23/FcεRII mAb, significantly inhibited the IgE-mediated upregulation of smMLCK expression in HASM cells. The mAb 15/1 is an anti- human FcεRI alpha chain mouse antibody which inhibits the binding of IgE to membrane bound FcεRI 2+ levels −/− mice displays increased serum IgE levels and increased expression of surface FcεRI on mast cells in vivo that account for the exacerbated allergic phenotype as compared to their WT littermates −/− BMMCs results in greater increase in mRNAs encoding Th2 cytokines and chemokines IgE-antigen complex aggregates and activates the FcεRI which induces Lyn kinase phosphorylation. Lyn then phosphorylates immunoreceptor tyrosine activating motif (ITAM) on FcεRI providing a docking site for the activation of Syk kinase. These events subsequently result in phosphatidylinositol-3-phosphate (PIP3) production, activation of phospholipase C and thus increase in cytosolic CaSly et al. Kalesnikoff et alMAPKs play a crucial role in modulating the cellular functions such as cell proliferation, survival, muscle contraction and cell migration Prevalent notion suggests that smMLCK is activated by the calcium-calmodulin complex leading to myosin RLC phosphorylation, which induces contraction in HASM cells Collectively, our data supports the concept that IgE induced the expression of smMCLK in HASM cells via Syk, MAPK and PI3K signaling pathways suggesting a plausible role of IgE in modulating HASM cell contraction.Figure S1Cell cycle analysis (A) and calponin expression (B) in HASM cells upon serum deprivation.(TIF)Click here for additional data file.Figure S2FcεRII/CD23 blocking mAb fails to inhbit IgE mediated smMLCK expression in HASM cells. Cells were pretreated with anti-FcεRII/CD23 mAb (Clone M-L233) for 1 h before stimulation with IgE. MOPC21 was used as an isotype control. Western blot is a representative of three different experiments showing smMLCK protein content in different treatment groups. Fold change in the level of smMLCK upon different treatment as compared to untreated control is shown in the graphs. One way ANOVA was performed to determine the significance of data. P<0.05 (*), (n = 4).(TIF)Click here for additional data file.


Sea ice is a fundamental component of the climate system and plays a key role in polar trophic food webs. Nonetheless sea ice biogeochemical dynamics at large temporal and spatial scales are still rarely described. Numerical models may potentially contribute integrating among sparse observations, but available models of sea ice biogeochemistry are still scarce, whether their relevance for properly describing the current and future state of the polar oceans has been recently addressed. A general methodology to develop a sea ice biogeochemical model is presented, deriving it from an existing validated model application by extension of generic pelagic biogeochemistry model parameterizations. The described methodology is flexible and considers different levels of ecosystem complexity and vertical representation, while adopting a strategy of coupling that ensures mass conservation. We show how to apply this methodology step by step by building an intermediate complexity model from a published realistic application and applying it to analyze theoretically a typical season of first-year sea ice in the Arctic, the one currently needing the most urgent understanding. The aim is to (1) introduce sea ice biogeochemistry and address its relevance to ocean modelers of polar regions, supporting them in adding a new sea ice component to their modelling framework for a more adequate representation of the sea ice-covered ocean ecosystem as a whole, and (2) extend our knowledge on the relevant controlling factors of sea ice algal production, showing that beyond the light and nutrient availability, the duration of the sea ice season may play a key-role shaping the algal production during the on going and upcoming projected changes. More recently the biogeochemical importance of sea ice in global biogeochemical cycles has been reviewed A major aim of this paper is to formulate a theoretical background for the construction of applicative models of sea ice ecosystems. The presented conceptual study stems from a previous application that was thoroughly tested against observations Among the ice-covered oceans, the Arctic is the one facing the most dramatic changes: the sea ice pack has decreased by more than 40% in the last three decades The limited number of biogeochemical models implemented for the sea ice ecosystem with respect to those implemented for the adjacent pelagic ecosystem is generally attributed to large uncertainties in sea ice biogeochemical processes. While this may be true for specific processes that are still difficult to quantify in a general framework  availability, and those are the major physical constrains to microalgae's growth, making the sea ice bottom a more similar habitat to that of seawater, thus more suitable for the biological community. On the other hand, light is strongly attenuated from surface to bottom sea ice, especially if sea ice is snow-covered. Thus photosynthetic organisms require adaptation/acclimation to low temperature, high salinity and/or low light intensities in order to survive and/or bloom in sea ice. In general, rates of physiological processes such as photosynthesis increase with temperature up to some point and this is true also for polar species Dissolved nutrients are found in the brines, thus their concentration is usually higher at the bottom of sea ice than at the surface. Occasionally, heavy snow loads lower the freeboard bringing seawater at the interface between snow and ice where surface communities might flourish. Similarly, during rafting events, some seawater can be placed in the interior of sea ice where the internal communities can develop. Thus, depending on their ability to adapt/acclimate, photosynthetic organisms are differently distributed in the bottom, interior and surface of sea ice . Those dBiogeochemical reactions occur in brines, which are the liquid fraction of sea ice. Brines are not uniformly distributed in sea ice. In an ideal sea ice season, during which first-year ice does not deform and does not depress, sea ice brines are constantly larger at the bottom - and are likely to exceed the permeability threshold - and become smaller towards the surface. In this ideal ice season the development of the biological community is only at the sea ice bottom and a 2-layers model may be considered sufficient to realistically represent the biological and non-biological fraction of sea ice. The simplest approach is to consider a single sea ice layer of constant thickness where biology is active , as for This simple method tends to underestimate primary production in first-year ice, as for example shown in In other cases, sea ice can depress and seawater can flood due to snow-ice formation, and deform due to converging or diverging of ice floes. Those are typical features of Antarctic sea ice. In such conditions brines may be temporarily large also at the surface or at intermediate depths, allowing the development of the so-called surface and internal communities, respectively, as described in While sea ice and seawater are certainly characterized by similar biogeochemical processes, there are relevant physical differences between the two systems. In the ocean, particles move in all directions in space, while in sea ice particles are trapped in a semi-solid matrix and organisms are distributed in the available volume. Because the volume of brines changes with time, sea ice models are better described with one or more layers of variable thickness, where only the last one is connected with the underlying ocean model. One dimensional  sea ice algae models have represented the coupling and the boundary fluxes at the ice-ocean interface in several ways. The more intuitive approaches are to prescribe a constant diffusivity value at the sea ice-ocean interface Based on a published time series −2 s−1 as in −3 for snow, 900 kg m−3 for sea ice and 1000 kg m−3 for seawater. The surface temperature linearly decreases from freezing temperature on day 1 to −20°C on day 60, then linearly increases until day 120 when it reaches again the freezing point. The surface temperature is then fixed at the freezing point until the ocean is ice-free , 2 functional groups of algae (adapted and survivors), particulate and dissolved organic and inorganic matter, and gases such as carbon dioxide and oxygen, for a total number of 22 state variables. The simplified version of the model presented here features one single limiting macronutrient (SiO4) and one single group of sea ice algae, i.e. diatoms, generally dominant in the sea ice habitat We also choose an intermediate approach for the ecosystem complexity, simplifying the comprehensive model used in −3 of dissolved silicate and 1 mg C m−3 of diatoms in seawater. The sea ice model did not need to be initialized because it is controlled by the exchanges of dissolved and particulate matter between sea ice and seawater. As a coupling method between the ocean and the sea ice habitat we choose again a method of intermediate complexity We coupled the sea ice model with a simple slab ocean, which is meant to represent the mixed layer depth under sea ice (15 m). We defined in seawater the same constituents and processes that we find in sea ice with the addition of bacteria and microzooplankton, as in The reference simulation (S0) reproduces an enrichment of dissolved silica during the sea ice growth season, followed by a sharp decrease due to the combined action of silica uptake by algae and brine loss due to melting . The alg−3), while for nutrients initialization we compared: (i) a Redfield-like initialization 15 Si: 16 C: 1 P given 8 mmol m−3 of initial silicate as in S0; (ii) non-Redfield typical concentrations of the open Barents Sea −3, 12 mmol m−3 of nitrate and 0.85 mmol m−3 of phosphate (iii) average concentrations reported for the whole Arctic in the Hydrochemical Atlas of the Arctic Ocean −3 of silicate, 3.28 mmol m−3 of nitrate and 0.83 mmol m−3 of phosphate). All simulations reproduce a similar bloom timing but different bloom magnitude  and the time of the peak (mt) of an algal bloom may be identified with frequent observations, the exact extent is a matter of definition. Time of initiation and termination may be individuated according to a threshold criterion that can be set in absolute terms (concentration) or in relative terms  and postponing it to day 170 (S3b). As described in the previous scenario, light attenuation by snow is proportional to the amount of light that reaches the surface and at the same latitude this depends only on the day of the year. The sooner snow completely melts, the sooner larger amount of light penetrates sea ice and reaches its bottom. And the longer the ice season the larger will be the amount of incoming short-wave radiation until the maximum reached at the summer solstice. By changing this parameter we do not observe any significant difference in the peak timing . We attrIt was pointed out in the Introduction that ice seasons in the Arctic are generally projected to be shorter. We look in scenario S4 at the response of sea ice algae to shorter ice seasons, while keeping the same amount of maximum snow cover (0.10 m) as in the reference simulation S0. We do that by reducing the ice season length of an equal amount of days at the beginning and at the end of the season. In scenario 4a the ice season is shorter of about 2587 days, . Accordier bloom . The 50 ne in S0 . The Chlne in S0  clearly ne in S0 , space  and vertical representation (from single to multi-layer models). This list of options is intended to help the modeller to choose the most appropriate set up under different conditions of applicability. In large-scale simulations and coupled configurations, compromises have to be made and model complexity may be reduced for computational reasons. The applied model will be a compromise between resolving the vertical variability of the sea ice biota, the complexity of the food web, the extent of the spatial and temporal scales and the overall computational constraints. In this work we applied this methodology to build an intermediate complex sea ice biogeochemical model from a previously validated realistic application and using a dynamic single-layer vertical representation together with a simplified plankton functional type model with variable stoichiometry. We coupled the sea ice model to the ocean by computing the boundary fluxes as function of growth/melt velocities and sea ice thickness. We presented a coupling strategy between the bottom sea ice layer and the first ocean level that relies on simple concepts of boundary fluxes and ensures strict mass conservation. We emphasize the importance of developing models that clearly obey to such law.We used in this work some ecological indicators for sea ice algae commonly used to describe phytoplankton phenology, such as the time of initial growth, the time of maximum amplitude, the maximum amplitude value, and the bloom duration. We proposed a modified criteria to detect the timing of the bloom in terms of initiation and duration, based on arguments that refer to the specific seasonal nature of first-year ice. Ecological indicators are objective-oriented tools that can help quantifying temporal and spatial variations, which are very much needed at this time of rapid changes. More studies to test different indices suitable for sea ice biogeochemical modelling are thus envisaged.Polar regions are rapidly changing and the Arctic is currently facing unpredicted changes. First-year ice is rapidly replacing multi-year ice and our understanding of the changes in primary production patterns in sea ice and in the oceans is mostly a speculative topic These considerations are based on the biological production of sea ice diatoms, which we earlier pointed out to be the most abundant and productive group of algae found in sea ice. However, sea ice is inhabited also by other algal groups that have been demonstrated to respond differently to varying light regimes e.g.. Given eFile S1(PDF)Click here for additional data file.


Protein export from the endoplasmic reticulum (ER) is an initial and rate-limiting step of molecular trafficking and secretion. This is mediated by coat protein II (COPII)-coated vesicles, whose formation requires small GTPase Sar1 and 6 Sec proteins including Sec23 and Sec31. Sec31 is a component of the outer layer of COPII coat and has been identified as a phosphoprotein. The initiation and promotion of COPII vesicle formation is regulated by Sar1; however, the mechanism regulating the completion of COPII vesicle formation followed by vesicle release is largely unknown. Hypothesizing that the Sec31 phosphorylation may be such a mechanism, we identified phosphorylation sites in the middle linker region of Sec31. Sec31 phosphorylation appeared to decrease its association with ER membranes and Sec23. Non-phosphorylatable mutant of Sec31 stayed longer at ER exit sites and bound more strongly to Sec23. We also found that CK2 is one of the kinases responsible for Sec31 phosphorylation because CK2 knockdown decreased Sec31 phosphorylation, whereas CK2 overexpression increased Sec31 phosphorylation. Furthermore, CK2 knockdown increased affinity of Sec31 for Sec23 and inhibited ER-to-Golgi trafficking. These results suggest that Sec31 phosphorylation by CK2 controls the duration of COPII vesicle formation, which regulates ER-to-Golgi trafficking. Molecular trafficking and secretion is initiated by the rate-limiting step of protein export from the endoplasmic reticulum (ER). This process is mediated by coat protein II (COPII)-coated vesicles, the formation of which is a crucial step. The COPII coat consists of small GTPase Sar1 and 6 Sec proteins. Coat assembly is initiated by the exchange of GDP for GTP on Sar1 It is well established that COPII vesicle formation is initiated by the activation of Sar1. However, the regulation of the subsequent process has not been elucidated completely. One way of achieving such a regulation could be Sec31 phosphorylation. Sec31 is phosphorylated in yeast, and its phosphorylation–dephosphorylation cycle is implicated in the budding of COPII vesicles Casein Kinase II (CK2) is a constitutively active serine/threonine kinase that regulates cellular events such as cell cycle and transcriptional regulation, cell survival, virus infection and tumor growth To determine the role of Sec31 phosphorylation in COPII-mediated ER-to-Golgi transport, we first examined whether Sec31 phosphorylation affects its membrane association by subcellular fractionation. Ultracentrifugation was used to separate the membranes from the cytosol. Sec31 was recovered from each fraction by immunoprecipitation with anti-Sec31 and its phospho-status was determined by western blotting using anti-phosphoserine/threonine antibodies. As shown in Since Sec31 phosphorylation appears to be important for its membrane dissociation, we determined its phosphorylation sites. Endogenous Sec31 was isolated from total cell lysates by immunoprecipitation and analyzed by mass spectrometry. Peptide fragments containing serines 527 (S527), 799 (S799), and 1163 (S1163) and threonine 1165 (T1165) were phosphorylated. These sites (indicated by the arrows in on/koffk (Membrane association of the WT Sec31 and the 4SA mutant was assessed by fluorescence recovery after photobleaching (FRAP). GFP-tagged WT Sec31 and the 4SA mutant were expressed transiently in HeLa cells. A GFP-positive dot (an ERES) was then photobleached, and the fluorescence recovery was monitored. As shown in kon/koff . To deteSec23 forms the inner layer of the COPII coat on ER membranes, whereas Sec31 is part of the outer layer. It is thought that Sec31 is recruited to the membrane through direct binding to Sec23 32P-ATP as well as 32P-GTP as a phosphate source (data not shown), which is consistent with the fact that CK2 and CK2-like kinases can use both ATP and GTP as a phosphate source in vitro  by measuring SEAP activity in the culture supernatants of mock or CK2 siRNA-treated cells. As shown in Finally, we measured the colocalization of wild type Sec31 (WT) or 4SA mutant (SA) with Sec24, which is known to colocalize with Sec31 and Sec23 at ERES. As shown in COPII vesicle formation is an essential part of protein export from the ER and thus membrane trafficking. However, the regulation of this vital process has not been elucidated completely. In this study, we demonstrated that Sec31 phosphorylation by CK2 plays a crucial role in COPII vesicle formation. Sec31 phosphorylation reduces its association with ER membranes as well as with Sec23. We also identified the Sec31 phosphorylation sites, namely serines 527, 799, and 1163 and threonine 1165, which are located in the middle linker region between the N-terminal WD-40 repeats and C-terminal extensin-like proline-rich domain, and near the C-terminus. The N-terminal WD-40 repeats and C-terminal proline-rich domain of Sec31 have been shown to interact with Sec13 and Sec23, respectively We also identified CK2 as a kinase capable of Sec31 phosphorylation. Furthermore, we demonstrated that CK2 facilitates ER-to-Golgi transport and protein secretion. Given that Sec31 membrane association is regulated by its phosphorylation and that CK2 can phosphorylate Sec31, we speculated that the functional role of CK2 in membrane trafficking may be related to its capacity to phosphorylate Sec31 and thus change Sec31 membrane associations. Further studies are required to obtain direct evidence to prove this speculation.CK2 or a CK2-like kinase has been suggested to be responsible for phosphorylation of the vesicle tethering factor p115 ++  responsible for Sec31 dephosphorylation will help in further understanding the molecular mechanisms underlying the regulation of COPII vesicle formation through Sec31 phosphorylation.The antibodies used in this study are as follows: monoclonal anti-casein kinase II, alpha-subunit , anti-gamma tubulin  and anti-FLAG tag , anti-GM130 (clone 35), anti-Sec31A (clone 32), anti-phospho-serine/threonine antibodies  and anti-calnexin , anti-Myc tag ), polyclonal anti-giantin CTGGTCGCTTACATCACTTTA  and siRNA2; TCAAGATGACTACCAGCTGT , and siRNA to eGFP: AAGACGTAAACGGCCACAAGTTC .The siRNAs used in this study are as follows: siRNA to human casein kinase II alpha 1: siRNA1; HeLa or HEK293 cells  were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS . Transient transfection with plasmids and siRNAs were performed using Lipofectamine LTX and RNAiMAX (Invitrogen), respectively, following the manufacturer’s instructions.Plasmids to express secretory alkaline phosphatases (SEAP) and VSV-G-tsO45-SP-YFP (VSVG-YFP), and YFP-Sec23a were kindly provided by Drs. Craig Roy and Derek Toomre , and Dr. David Stephens . Myc-CK2 alpha 1 was purchased from Origene . A cDNA of human Sec31a (clone KIAA 0905) was obtained from Kazusa DNA Research Institute . FLAG- or GFP-tagged Sec31 was constructed by subcloning of the human Sec31a cDNA into p3×FLAG-CMV (Sigma) or pQCXIP-GFP . Point mutations of Sec31 were introduced using QuikChange kit .HeLa cells stably expressing SEAP were established as previously described 2, 1% Triton X-100, protease inhibitor cocktails  and phosphatases inhibitor cocktails 1 and 2 (Sigma). After incubation for 10 min on ice, the lysate was clarified by centrifugation at 14,000 g for 20 min. For immunoprecipitation, the supernatants were incubated with M2-agarose (Sigma), or anti-Sec31 (Fred Gorelick) or anti-GFP bound to Protein A-Sepharose  for 30 min at 4°C. After washing three times with immunoprecipitation buffer, the samples were fractionated by SDS-PAGE followed by western blotting with antibodies indicated in the figures. Quantification of western blots was performed using Image J software. For some experiments, recombinant CK2 from New England BioLabs  was used to treat the immunoprecipitants for 30 min at 37°C. To identify the phosphorylation sites, immunoprecipitated endogenous Sec31 from total cell lysates was incubated with 32P-ATP and cytosol at 37°C for 30 min followed by a separation by SDS-PAGE. Peptides generated by trypsin were analyzed by mass-spectrometry performed by Yale Keck facility .Cell lysates were prepared with immunoprecipitation buffer , 2.5 mM MgCl2, protease inhibitor cocktail and phosphatases inhibitor cocktails 1 and 2) and passed through 25 G needle 6 times using 1 ml syringe, followed by a 4 seconds sonication. The samples were incubated on ice for 20 min, and then centrifuged at 4,400 rpm  for 5 min at 4°C. The supernatants were collected, layered onto 1.2 M sucrose containing Cell fractionation buffer and centrifuged at 55,000 rpm  in a TLS-55 rotor  for 30 min at 4°C. The supernatants and pellets of the 100,000 g spin were collected as the cytoplasmic and membrane fractions, respectively.For subcellular fractionation, cells in culture dishes were scraped, suspended in cell fractionation buffer  for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. The cells were blocked with 4% bovine serum albumin (BSA) in PBS for 15 min, and then incubated for 15 min with primary antibodies diluted in 4% BSA in PBS. The cells were washed three times with PBS, and incubated for 15 min with secondary antibodies conjugated to Alexa fluorophors (Invitrogen). After washing the cells, the coverslips were mounted on microscope slides and imaged using a FV1000 confocal microscope equipped with a 60× oil objectives . Image data were processed and quantified using Image J software and Adobe Photoshop.on/koffk of each experiment was carried out after best fitting of the experimental data obtained to a single curve using CurveExpert Pro software (curveexpert.com). The mobile fraction and t1/2 maximum recovery (s) were obtained by fitting the FRAP data to the following equation,1/2 is determined onk/offk was obtained by fitting the FRAP data to the reaction dominant model,kon” represents the on rate at the localized binding sites (association rate constant), and “koff” represents the off rate at the localized binding sites (dissociation rate constant) FRAP assay was performed as described previously SEAP transport assay was performed as previously described VSVG transport assay was performed as previously described Figure S1Depletion of CK2 by siRNA1. HeLa cells were transfected with a siRNA (siRNA1) for CK2 with indicated concentration and the efficiency of CK2 depletion was determined by western blotting. The CK2 protein levels among the samples were normalized by gamma Tubulin protein levels, and quantified by Image J software. The CK2 depletion by CK2 siRNA1 was efficient with 10∼15% remaining CK2 protein level in the experiments shown in (TIF)Click here for additional data file.Figure S2CK2 interacts with Sec31. HEK293 cells were co-transfected with Myc-CK2 and GFP-Sec31 or GFP and incubated for 48 h. In the presence of 1 mM Ca++ in immunoprecipitation buffer, cells were lysed and GFP-tagged protein was immunoprecipitated with anti-GFP beads and subjected to western blotting with anti-Myc and anti-GFP antibodies. The “input 1% CK2” is a western blot of 1% aliquots of total cell lysates to show the transfection and binding efficiency of CK2.(TIF)Click here for additional data file.Figure S3Other CK2 siRNA also reduces trafficking. (A) siRNA2 also reduced CK2 protein level. CK2 protein level was also decreased by siRNA2 but to a less lesser extent (∼50% reduction). (B) Secretory alkaline phosphatase (SEAP) secretion assay using cells transfected with siRNA2. Similar to siRNA1 shown in (TIF)Click here for additional data file.Figure S4Sec31 4SA mutant colocalized better with Sec24 than wild type Sec31. (A) HeLa cells were transfected with GFP-Sec24c and wild type Sec31 (WT) or 4SA mutant (SA). After incubation for 18 h, cells were fixed and stained with immunofluorescence with anti-FLAG shown in red and Hoechst shown in blue. The bottle panels are 4 times magnification of the boxed area of the top panels. Bar, 20 µm. The colocalization of Sec31 and Sec24 were measured using Image J software and shown in (B). Bar, SD.(TIF)Click here for additional data file.Table S1Phosphorylation sites of Sec31 and experimental conditions in published studies (PMID).(DOC)Click here for additional data file.


PPARγ is upregulated in T cells of septic patients, sensitizing these cells to PPARγ-dependent apoptosis and thus contributing to T-cell depletion ,2. In thsurvival . BecauseFirefly luciferase gene. These two vectors were co-transfected, in combination with a control vector encoding Renilla luciferase (pRL-CMV) to normalize Firefly luciferase activity for transfection efficiency. Following transfection, cells were incubated with the SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 for different times (2 to 48 hours) and at increasing doses (0.01 to 10 μM), with or without rosiglitazone (0.01 to 10 μM). Transactivation was analyzed using a 96-well plate format.A PPARγ-dependent transactivation assay was used in HEK293T cells. It is based on the vector pFA-PPARγ-LBD-GAL4-DBD encoding the hybrid protein PPARγ-LBD-GAL4-DBD and the reporter vector pFR-Luc, carrying a GAL4-responsive element in front of the Rosiglitazone transactivated PPARγ in a time-dependent and dose-dependent manner, the response gradually increasing to a maximum at 48 hours with 10 μM. Low concentrations (0.01 to 0.1 μM) of SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 all exerted dose-independent antagonistic effects at an early incubation time point (2 hours). From 10 hours onwards, MCC-555 and GW9662, given alone, both exerted PPARγ agonistic effects, MCC-555 in parallel to responses to rosiglitazone, but GW9662 with characteristics of partial antagonism. F-MOC showed no dose-dependent effect at any concentration at later time points. Only GW9662 (1 to 10 μM) was able to inhibit rosiglitazone (0.1 to 1 μM)-induced PPARγ transactivation after 10 hours.Our kinetic analysis reveals clear differences in the modulatory characteristics of PPARγ inhibitors, with previously unreported early inhibitory effects and late agonistic or partial agonistic activity. New SPPARγMs with extended inhibitory activity may prove useful in the therapy of sepsis.


Olanzapine inhibited CYP3A-catalyzed production of 1′, and 4′-hydroxymidazolam . In contrast, risperidone (IC50 = 20.7 μM) and levomepromazine (IC50 = 30 μM) showed selectivity towards the inhibition of midazolam 1′-hydroxylation reaction, and haloperidol did so towards 4′-hydroxylation (IC50 of 2.76 μM). Thioridazine displayed a Ki of 1.75 μM and an inhibitory potency of 1.57 on CYP2D6, suggesting a potential to induce in vivo interactions. However, with this exception, and given the observed Ki values, the potential of the assayed antipsychotics to produce clinically significant inhibitions of CYP450 isoforms in vivo seems limited.The goal of this study was to assess in human liver microsomes the inhibitory capacity of commonly used antipsychotics on the most prominent CYP450 drug metabolizing enzymes . Chlorpromazine was the only antipsychotic that inhibited CYP1A2 activity (IC In contrast to conventional neuroleptics, atypical antipsychotics have been shown to be effective against both positive and negative symptoms of schizophrenia while showing a reduced propensity to induce extrapyramidal effects . The cytin vitro inhibition techniques in human liver microsomes.In this paper we have assessed the potential for nine of the most commonly used atypical antipsychotics  and abaperidone -piperidin-1-yl]propoxy]-3-(hydroxymethyl)chromen-4-one; FI-8602), an underdevelopment compound with a potentially atypical antipsychotic profile , 16, to '-hydroxy-midazolam, and 4-hydroxymidazolam were supplied by Hoffman-La-Roche . Bufuralol and 1′-hydroxybufuralol were obtained from Ultrafine Chemicals , 4-hydroxydiclofenac from Gentest Corp , -quetiapine from Astra Zeneca , and olanzapine from Lilly Laboratories .Abaperidone and iloperidone were gifts from Centro de Investigación Farmacéutica, Grupo Ferrer . Chlorpromazine, levomepromazine, thioridazine, haloperidol, risperidone, clozapine, quinidine, sulphaphenazole, ketoconazole, diclofenac, furafylline, phenacetine, and acetaminophen were purchased from Sigma Chemical Co. . Midazolam, 1Methanol and acetonitrile were of HPLC grade . Acetic and formic acids were of analytical grade and supplied by Merck . Glucose-6-phosphatedehydrogenase, glucose-6-phosphate, and NADPH were purchased from Roche Diagnostics SL . All other chemicals and reagents used were of the highest commercially available quality. Water was filtered by a Milli Q water system from Millipore Ibérica, S.A. .Chromatographic separation was performed with a system consisting of a Hewlett-Packard (HP) 1100 series  that had a degasser (model G1322A), quaternary pump (model G1311A), autosampler (model G1313A), column oven compartment (model G1316A), ultraviolet/visible detector (model G1315A), and fluorescence detector (model G and a mass spectrometer engine (model G1946A). A computer-assisted HP G2710AA LC/MS ChemStation  was used to operate modules and facilitate data management.μm particle size, 100 cm × 3.0 mm reversed-phase column . The composition of the mobile phase was 18% acetonitrile: 82% acetic acid (0.1%) (vol/vol). The flow rate through the column at 30°C was 0.3 mL/minute and phenacetin and its oxidized metabolite paracetamol were monitored by ultraviolet absorbance at 247 nm.Phenacetin and its metabolite paracetamol were separated on a Hypersil BDS C18, 3 μm particle size, 7.5 cm × 4.6 mm reversed-phase column . The composition of the mobile phase was 40% acetonitrile: 60% acetic acid (0.1%) (vol/vol). The flow rate through the column at 30°C was 1 mL/minute. Bufuralol and its oxidized metabolite 1′-hydroxybufuralol were monitored by fluorescence detection .Bufuralol and its metabolite 1′-hydroxybufuralol were separated on an Ultrasphere ODS, 3 μm particle size, 100 cm × 3.0 mm reversed-phase column . The composition of the mobile phase was 50% acetonitrile : 50% acetic acid (0.1%) (vol/vol). The flow rate through the column at 30°C was 0.4 mL/minute, and diclofenac and its oxidized metabolite 4′-hydroxydiclofenac were monitored by ultraviolet absorbance at 220 nm Midazolam, 4-hydroxymidazolam and 1′-hydroxymidazolam were separated on an Ultrasphere ODS, 3 μm particle size, 7.5 cm × 4.6 mm reversed-phase column . The composition of the mobile phase was 39% acetonitrile : 61% formic acid (0.1%) (vol/vol). The flow rate through the column at 30°C was 0.5 mL/minute and midazolam, 4-hydroxymidazolam, and 1′-hydroxymidazolam were monitored by means of an HPLC/electrospray ionization-mass spectrometry (LC/ESI-MS) method with isocratic elution.Diclofenac and its metabolite 4′-hydroxydiclofenac were separated on a Hypersil BDS C18, 3 The mass spectrometer was run in the positive ion mode. Nitrogen as the drying gas was supplied at a flow of 10 L/minutes and at a temperature of 350°C. The capillary voltage was adjusted to 4100 V. To quantitate midazolam, 4-hydroxymidazolam, and 1′-hydroxymidazolam, the mass spectrometer was operated in the selected ion-monitoring mode (SIM) with a dwell time of 229 msec. The eluant from HPLC was introduced into the source via the electrospray interface, generating the positively charged pseudomolecular ions (MH+) at m/z 326 and 342.μM. Abaperidone was an exception and was dissolved in hot water up to a maximum concentration of 20 μM.Inhibition studies on four different CYP isoform-specific substrates were carried out in human liver microsomes . Assessed reactions were phenacetin O-deethylation for CYP1A2, (+/−)-bufuralol 1′-hydroxylation for CYP2D6, diclofenac 4′-hydroxylation for CYP2C9, and midazolam 1′- and 4′-hydroxylation for CYP3A. The different antipsychotics were dissolved in methanol and serially diluted with 100 mM tris buffer (pH = 7.5) to obtain final concentrations ranging from 0.05 to 50 μM phenacetin, 50 μM bufuralol, 15 μM diclofenac, or 10 μM midazolam) was incubated in the absence or presence of increasing concentrations of the tested antipsychotics. Reactions were started by adding microsomal protein and the mix was then vortexed and incubated at 37°C for 30 min. The process was stopped by the addition of acetonitrile and incubation on ice during 10 minutes. Samples were then centrifuged at 10,000 g for 3 minutes, and the supernatant was taken for subsequent HPLC analysis. Control inhibition studies were carried out with prototypic inhibitors in increasing concentrations up to 20 μM, namely, furafylline (CYP1A2), quinidine (CYP2D6), sulphaphenazole (CYP2C9), and ketoconazole (CYP3A).The conditions of the incubation reactions as well as the concentration of the substrate reaction probes utilized were established based on previous CYP450 inhibition studies by our group in human liver microsomes –24. In b50) was calculated by interpolation of a regression line of the log concentration versus percent inhibition plot over at least three concentrations of inhibitor. Ki  estimates were obtained as described previously [The effect of antipsychotics on the rate of CYP-catalyzed probe substrates was expressed as a percentage of the control activity with no antipsychotic present. The concentration of inhibitor causing 50% reduction of activity relative to the appropriate control value . The retention times (given in minutes) for the analyzed substances were as follows: paracetamol: 2.3; phenacetin: 11.2; 1′-hydroxybufuralol: 4.6; bufuralol: 27; 4′-hydroxydiclofenac: 2.5; diclofenac: 5.4; 4′-hydroxymidazolam: 4.9; 1′-hydroxymidazolam: 5.8; midazolam: 8.7.The chromatographic conditions efficiently separated the compounds of interest in all the assays. Calibration curves were linear (coefficients of correlation >0.99) within the range of concentrations established , risperidone (IC50 = 20.7 μM), and levomepromazine (IC50 = 30 μM) inhibited midazolam 1′-hydroxylation (50 = 42.20 μM) and haloperidol (IC50 = 2.76 μM)  .Ki estimates presented in in vivo inhibition of a CYP enzyme would be thioridazine on CYP2D6. Indeed, our group has previously reported that subjects taking thioridazine were more prone to display a CYP2D6 poor metabolizer phenotype [Ki values obtained for the other antipsychotics do not allow for anticipating a significant effect in vivo (in vivo interactions caused by these agents; for example, risperidone and olanzapine have been shown to modify the pharmacokinetics of aripiprazole, another antipsychotic metabolized by CYP450 isoforms [Given the henotype . Ki val in vivo . Howeverisoforms . A possiisoforms , 31 or iisoforms .Interestingly, abaperidone did not cause a significant inhibition of any of the CYP enzymes assayed. This is a pharmacologically active substance underdevelopment, which therefore appears to be a remarkably safe agent with regard to CYP-mediated drug interactions, the main mechanism underlying interactions involving atypical antipsychotics , 34. It in vitro inhibitory capacity of CYP450 enzymes. However, and with the exception of thioridazine, the potential to cause significant in vivo interactions appears to be limited.In summary, the results show that several commonly used atypical antipsychotics exerted a significant