Allergies represent the most prevalent non infective diseases worldwide. Approaching IgE-mediated sensitizations improved much by adopting allergenic molecules instead of extracts, and by using the micro-technology for multiplex testing.To provide a proof-of-concept that a flow cytometric bead array is a feasible mean for the detection of specific IgE reactivity to allergenic molecules in a multiplex-like way. A flow cytometry Allergenic Molecule-based micro-bead Array system (ABA) was set by coupling allergenic molecules with commercially available micro-beads. Allergen specific polyclonal and monoclonal antibodies, as well as samples from 167 allergic patients, characterized by means of the ISAC microarray system, were used as means to show the feasibility of the ABA. Three hundred and thirty-six sera were tested for 1 or more of the 16 selected allergens, for a total number of 1,519 tests on each of the two systems.Successful coupling was initially verified by detecting the binding of rabbit polyclonal IgG, mouse monoclonal, and pooled human IgE toward three allergens, namely nDer s 1, nPen m 1, and nPru p 3. The ABA assay showed to detect IgE to nAct d 1, nAct d 11, rAln g 1, nAmb a 1, nArt v 3, rBet v 1, rCor a 1, nCup a 1, nDer p 1, nDer s 1, rHev b 5, nOle e 1, rPar j 2, nPen m 1, rPhl p 1, and nPru p 3. Results obtained by ABA IgE testing were highly correlated to ISAC testing . No unspecific binding was recorded because of high total IgE values.The ABA assay represents a useful and flexible method for multiplex IgE detection using allergenic molecules. As also shown by our initial experiments with monoclonals and polyclonals, ABA is suitable for detecting other human and non-human immunoglobulins. It seemed to us then that there could be the need for a more flexible but anyhow multiplexed immunoassay platform, allowing the choice of allergens and control molecules  was developed to detect soluble factors, like cytokines or mediators, released Drawing inspiration from the early reports of the method developer Dermatophagoides siboney, was provided by Biocen Penaeus monodon) was purchased from BIAL-Aristegui , and nPru p 3, a lipid transfer protein (LTP), was purified from peaches (Prunus persica) as previously reported Allergenic molecules selected for micro-beads conjugation, derived from animal, pollen, and fruit allergenic sources were used. For the first set of experiments, lyophilized purified natural (n) Der s 1, a group 1 mite allergen from Actinidia deliciosa) Ambrosia artemisiifolia) as reported in literature with minor modifications Artemisia vulgaris), purified recombinant (r) Aln g 1, rBet v 1, and rCor a 1, marker allergens from alder pollen (Alnus glutinosa), birch pollen (Betula verrucosa), hazel tree pollen (Corylus avellana), respectively ; nCup a 1, the Arizona cypress pollen allergen (Cupressus arizonica), nOle e 1, an olive tree pollen allergen (Olea europaea), rPar j 2, the parietaria pollen allergen (Parietaria judaica), and rPhl p 1, a grass pollen allergen (Phleum pratense) (BIAL-Aristegui); nDer p 1, a house dust mite major allergen (Dermatophagoides pteronyssinus) ; rHev b 5, a marker of natural latex allergy (Hevea brasiliensis) . Further details on single molecule characteristics are available on the Allergome web site  For the second part of the study, using a broader panel of molecules including the three mentioned above, the following additional thirteen allergens were considered: nAct d 1, a cystein protease, and nAct d 11, a ripening-related protein, recently reported as allergen, both purified from green kiwifruit  of distinct and non-overlapping fluorescence emission intensities The conjugation procedure consisted of four steps: micro-bead preparation, protein modification, buffer exchange, and protein conjugation.g for 3 minutes. Afterwards, supernatant removal micro-beads were washed three times and the pellet re-suspended in 20 µl of coupling buffer.The BD CBA Functional Beads (Becton Dickinson) were used. Selected micro-beads chosen for conjugation were vortexed vigorously for 30 seconds; 75 µl of micro-beads were taken for 500 test reactions; 1 M 1.9 µl dithiothreitol stored at −20°C  were added to a micro-centrifuge tube containing the micro-beads and vortexed again for 5 seconds. The mix was incubated on an orbital shaker for 1 h at R.T. in the dark. After incubation, 1 ml of coupling buffer (Becton Dickinson) was added and vortexed for 5 seconds. Afterwards, the micro-beads were centrifuged at 900× Ninety µl of selected allergens at a concentration of 1 mg/ml were used for preparing 500 tests. Sulfo-SMCC (Pierce) stock solution (2 mg/ml water solution) was prepared immediately before use, and 2 µl were added to the protein preparation. The mixture was incubated on an orbital shaker for 1 h at R.T. in the dark.g. After centrifugation, the columns were placed in a new tube and the entire volume of protein/sulfo-SMCC solution was transferred to the spin columns and centrifuged again at 1,000× g for 2 minutes.The Bio-Rad Spin Columns  were washed with coupling buffer twice and then placed in a tube and centrifuged for 2 minutes at 1,000× g. Washes with storage buffer were repeated three times. Afterwards, the micro-bead pellet, re-suspended in 0.5 ml of storage buffer, was stored at +4°C, at a concentration of 6×106 micro-beads/ml.Activated proteins in the eluate were transferred into the tube containing the previously prepared functional micro-beads and vortexed for 5 seconds. Proteins and micro-beads were incubated for 1 h at R.T. on an orbital shaker in the dark. Two µl of N-ethylmaleimide  were added to the tube containing functional micro-beads and activated protein, vortexed for 5 seconds and incubated for 15 minutes on an orbital shaker. After incubation, 1 ml of the storage buffer was added into the tube and the micro-beads were centrifuged for 3 minutes at 900× To verify the coupling of selected allergens, specific mouse monoclonal (MoAb) and rabbit polyclonal (PoAb) antibodies were used as probes. To detect nDer s 1 onto the micro-beads, two different MoAbs were used, an IgG1 isotype (4E10/E10) and an IgG2b isotype (5F7/H8F2) (Biocen) a/l. Selected subjects for the Der s 1 ABA testing reported a reliable history of respiratory symptoms of rhinitis and/or asthma suggesting mite allergy and all had IgE positive result using the Dermatophagoides pteronyssinus ImmunoCAP test . A reliable clinical history of food reactions  or a positive double blind food challenge test for peach or shrimps, and a positive ImmunoCAP IgE testing for Pru p 3 or Pen a 1, peach LTP and shrimp tropomyosin respectively, were the basis for selecting patients for Pru p 3 and Pen m 1 ABA testing. All subjects were recruited for previously published collaborative studies within our research Center. Allergic control subjects did not report symptoms in relation to exposure to any of the three allergens and had negative allergy in vitro tests.Initially, sera from a total of 167 clinically allergic subjects presenting for a consult at the Center for Molecular Allergology  were selected after testing on the ISAC system, bearing recombinant and natural allergens immobilized in triplicate on a glass slide a/l were mixed together creating the Der s 1 sera pool. The 12 samples were also reactive to other ISAC tested group 1 mite allergens, namely nDer p 1 or nDer f 1. Eighteen nPru p 3 positive patients , and 21 nPen m 1 positive patients  were selected for the Pru p 3 and the Pen m 1 sera pools, respectively. In all three cases, although sera were selected on the basis of one of the three specificities - but then used for detecting coupled molecules and ABA optimization - the other two specificities could be present at a lower IgE level, and not avoided on purpose. Three serum samples were selected from patients with one of the three specificities included in our study and not the other two. A forth one had no ISAC detectable nPru p 3 specific IgE. The selection was made in the attempt to mimic the use of discrete antigen specific non-human antibodies, and setting the multiplex approach to verify ABA specificity. Not relevant, but anyway tested allergenic molecules in the same tube, acted as negative controls when the targeted one was tested.Human sera to create pools were selected to define the ABA experimental conditions. In order to obtain the highest IgE signal after ABA testing, three distinct pools of sera were set. Sera from twelve patients having nDer s 1 specific IgE on ISAC testing, ranging between 8 and 139 kUAll other human sera used in the study were tested as single non-pooled samples. The number of samples is reported in the Result section for each allergen specificity and detailed in Total IgE were measured using the ImmunoCAP system (Phadia). As the assay defines IgE concentrations in a range between 1 and 2000 IU/l, sera having very high total IgE levels  were tested diluted 2 or higher folds in order to get the correct estimation of their total IgE values.Oral informed consent for blood sampling was obtained from patients or caregivers during the allergy consult as a normal routine procedure during the allergic disease diagnostic work-up. This procedure was approved by our Institutional ethical committee as it is part of a routine procedure where no consent is required by law. Due to the limited amount of serum to be used in the test, no additional blood drawing was necessary. The study was approved by our Institutional ethical committee, at the Istituto Dermopatico dell'Immacolata, Rome, Italy. Informed signed consent was obtained by patients and caregivers when the serum samples were used for the initial part of our study where the new system was set up.g. After removing the supernatant, the micro-beads were re-suspended in capture micro-bead diluent for serum and plasma to a final volume of 50 µl/test, then vortexed and incubated for 15 minutes in the dark at R.T. Fifty µl of micro-bead mix was combined with 50 µl of the biological sample to be tested, being either non-human or human sera, vortexed, and then incubated for 1 hour at R.T. in the dark. PoAbs, MoAbs, human serum samples were used in each given experiment as reported in the ×g. The supernatant was aspirated and the pellet re-suspended in 100 µl capture micro-bead diluent for serum and plasma (Becton Dickinson), then 50 µl of the detection diluent containing phycoerythrin-  or fluorescein isothiocyanate-conjugated secondary antibody  were added to each tube, vortexed, and then incubated for 1 h at room temperature in the dark. Secondary antibodies used in our ABA assay were as follows: a FITC conjugated goat anti-rabbit IgG  was used for rabbit primary PoAbs (10 µl for each sample); MoAbs were detected using 5 µl of PE-conjugated goat anti-mouse ; human IgE were detected using 20 µl of PE-conjugated anti-human IgE (Becton Dickinson). The detection diluent was added to all secondary antibodies to reach the final volume of 50 µl. Samples were then washed once as described above and finally re-suspended in 300 µl of washing buffer.One µl of conjugated micro-beads was used for each test, for each specific allergen. The micro-beads, washed with 0.5 ml of washing buffer as provided by the manufacturer, were centrifuged for 5 minutes at 200× Specimens were analyzed according with the CBA assay for cytokine detection vs SSC-A dot plot a region was created that includes the singlet population of micro-beads. The statistics view was edited to display the PE, FITC, APC and APC-Cy7 median fluorescence values. Through the singlet gate, the A9 setup micro-beads were run and the voltage of APC and APC-Cy7 were adjusted until obtaining a 160,000±2,000 mean for each parameter. Through the singlet gate the PE-F1 tube was run and the PE voltage was adjusted until a mean value of 65±5 was reached.Five tubes labelled A9, PE-F1, F1, F9 and A1 were prepared. After vortexing, 200 µl of washing buffer was added to each tube followed by 25 µl of the corresponding setup micro-beads. The following parameters were edited: FSC-A, FSC-W, SSC-A, SSC-W, PE-A, APC-A and APC-Cy7-Then, the following plots were created on a global worksheet : FSC-A/SSC-A dot plot, APC/APC-Cy7 dot plot and a PE histogram. The FSC-A and SSC-A were set to log and a statistic view was created showing the FSC-A and the SSC-A means. Measurements were performed counting at least 500 events for each analysis. The FSC and SSC thresholds were adjusted using the mean channel as a guideline. In the FSC-A ABA-measured IgE values were expressed as MFI as given by the final readout of the cytometer. No standard curves, as reported in other studies Demographic and clinical data were recorded for all patients at admission by using the InterAll software, a customized allergy electronic record for diagnostic and clinical data storing . All laboratory data obtained in the present study along with all ISAC and ABA testing results were stored in the patients' personal records and analyzed.2 test was used for the two by two contingency tables. The non-parametric Spearman correlation coefficient (r) was used to compare the IgE values obtained by the two testing systems. All tests were used with two-sided options and the statistically significant level was set at a p value<0.05.Graphpad Prism version 5.02 was used for statistics and graphs . The frequencies of events were compared by using the Fisher's exact test and the χTo begin validation of the ABA system, the proof of a successful allergen conjugation on the blank micro-beads was searched in three independent experiments. The recognition of nDer s 1, nPen m 1 and nPru p 3 by specific non-human antibodies was tested with both a singleplex and a multiplex approach. In the singleplex assays, every molecule coupled with the micro-beads was tested with its specific antibody. In the multiplex approach, the three molecules, coupled with their respective micro-beads, were tested simultaneously with each of the antibodies to check any reciprocal interference.Two different MoAbs to nDer s 1 were tested; the 4E10/E10 showed that the allergen was specifically coupled with the micro-bead, with a higher performance when compared to the MoAb 5F7/H8F2 , panel AIn order to exclude a possible interference, the three different micro-beads conjugated with nDer s1, nPen m 1, nPru p 3, were mixed in the same tube to be tested. The best performing antibodies among those reported above were used, namely the anti-Der s 1 MoAb, clone 4E10/E10, the anti-tropomyosin and the anti-Pru p 3 PoAb supplied by Alk-Abelló. None of the control MoAbs or PoAbs produced any unspecific binding with the three allergen-conjugated beads, since the recorded fluorescence signal was always within the lowest background range (data not shown).To validate data obtained by non-human antibodies and obtain the first proof of feasibility of the new testing, IgE recognitions of the three molecules were analysed. Pooled sera from individual donors reactive to nDer s 1, nPen m 1 and nPru p 3 were used in three independent experiments. As for non-human antibodies, the reactivity profiles of the pooled sera were evaluated both in ABA singleplex and multiplex. Singleplex results, obtained by testing the single micro-bead with the specific serum pool, reflected the specific IgE recognition pattern obtained using the ISAC system and detailed in When the three micro-beads were tested together, they produced the following results , panel BAdditional experiments according to this validation approach included the evaluation of three mono-reactive patients to either nDer s 1, or to nPen m 1, or to nPru p 3, and an additional individual having IgE to both nDer s 1 and nPen m 1. As shown in One hundred and thirty-seven allergic individuals were selected on the basis of their ISAC IgE reactivity to one of the three allergens of our study and not to the other two. Twenty-nine nDer s 1 positive patients , 28 nPen m 1 positive , and 55 nPru p 3 positive  were selected. Twenty-five allergic subjects , without any of the three above positive IgE determinations on ISAC acted as negative controls. Without a set cut off value for the ABA IgE detection system, any value above the no fluorescence signal was considered as positive.When comparing ABA IgE reactivity to the ISAC IgE testing in the 137 samples (411 determinations), a statistically significant high correlation was found  , panel ATo show the feasibility of the system as a flexible multiplex array and to explore if we introduced any bias in previous experiments due to the limited number of sera and allergens, we additionally tested 336 sera from allergic patients, 171 females (50.9%), median age 27 years, range 2–80 years, 70 of them were younger than 12 years old, 17 in pre-school age. Full demographics are reported in 2 1096.1, p<0.0001) were found  and a very high agreement of positive and negative tests  dilution curves of selected non-human sera. All dilution curves started with the undiluted antibody preparation as provided by the manufacturer. Panel A: nDer s 1 and monoclonal antibody from Biocen 4E10E10; 1∶10 dilution factor; Panel B: nPen m 1 and the polyclonal antibody from BIAL; 1∶2 dilution factor; Panel C: nPen m 1 and the polyclonal antibody from ALK-Abelló; 1∶10 dilution factor.(TIF)Click here for additional data file.Figure S2Allergen micro-Bead Array (ABA) reproducibility experiment on non-randomized sera. Sera have been selected among those reported in (TIF)Click here for additional data file.Table S1Allergen micro-Bead Array (ABA) for IgE detection in human sera. Aggregated and raw patient IgE data showing the flexibility of the system. The 16 tested allergens are listed and hyperlinks are available for each of them cross-referencing to www.allergome.org. Panel A: Summary of the (XLS)Click here for additional data file.


A receptor-mediated tonic inhibition in regulating CNS function. However, outside of pathological conditions, there is relatively little evidence that the magnitude of tonic inhibition is itself under regulation. Here we review the mechanisms by which tonic inhibition is known to be modulated, and outline the potential behavioral consequences of this modulation. Specifically, we address the ability of protein kinase A and C to phosphorylate the extrasynaptic receptors responsible for the tonic GABAA current, and how G-protein coupled receptors can regulate tonic inhibition through these effectors. We then speculate about the possible functional consequences of regulating the magnitude of the tonic GABAA current.A large body of work now shows the importance of GABA A receptors, GABA works to increase membrane permeability to Cl- (and to a lesser extent HCO3-) thereby reducing membrane impedance and potentially hyperpolarizing the membrane potential. The role of GABAA receptor-mediated inhibition in the control of neural function is undeniable, and can be seen in nearly every aspect of neural function , generally as a combination of α, β and γ subunits. Other combinations exist, where δ, ε, θ or π subunits replace the γ subunit. Finally, other permutations have been described, such as ρ homopentamers and receptors containing solely α and β subunits . They are expressed at a high density in the extrasynaptic compartment of dentate gyrus granule cells, cerebellar granule cells and thalamocortical cells   . Due to Phosphorylation is one of the most well understood post-translational modifications a protein can undergo. This reaction is catalyzed by kinases, and involves the transfer of a phosphate group from ATP to a serine, threonine or tyrosine residue in the target polypeptide. This phorphorylation changes the structure of the protein, and potentially its function. Due to the residues they target, kinases are generally subdivided into serine/threonine kinases such as calcium-dependent protein kinase (PKC) or cyclic AMP dependent protein kinase (PKA), tyrosine kinases such a v-Src, dual specificity kinases and histidine kinases . FurtherA receptors can be modulated by kinases directly was provided by A receptors with a variety of subunit compositions. Soon afterward, this effect was shown to be mediated by phosphorylation of both β and γ subunits, with serine 409 (S409) being the target on the β1 and β3 subunits and S410 being the target on β2 subunits, while S327 and S343 are the target on the γ2 subunit   appears to be facilitated by the receptor for activated C kinase  . It is aigure 1) . Similarigure 1) . The β2Ligure 1) . The α4  RACK-1; .A receptors is diverse, and appears to be dependent on the subunit composition in question. For instance, in hippocampal pyramidal cells, PKC appeared to have no effect on miniature inhibitory postsynaptic potentials (mIPSCs), while in dentate gyrus granule cells, PKC enhanced mIPSC amplitudes  was not elucidated  is a serine/threonine that has been demonstrated to be able to modulate synaptic inhibition in a wide variety of cell types .Caes e.g., . Saliba -/- animals or in animals treated with L-655, 708, an inverse agonist selective for α5-containin GABAA receptors. Acute systemic IL-1β injections or in vitro application of IL-1β both caused an increase in the tonic current measured in hippocampal CA1 cells, where there was a concurrent increase in α5 subunit surface expression. This effect was dependent on the activity of serine/threonine kinase, p38 mitogen-activated protein kinase (MAPK), though how it causes increased cell-surface expression of α5 subunit containing receptors is still unclear.A receptors appear to sense ambient GABA and/or GABA which spills over from the synaptic cleft, it seems likely that manipulations that increase the release of GABA will increase the magnitude of the tonic current. Indeed, it appears that blocking action potential dependent release of GABA can reduce the size of the tonic current  cannot. That is to say, hyperpolarizing inhibition alters the offset (the excitatory input needed to bring the cell to fire) while not greatly affecting the gain, i.e., the relationship between input excitation and firing rate. Shunting inhibition is often suggested to largely have the opposite effect, reducing the gain, while not affecting the offset. However, it appears that the situation is more complex, and also depends on the nature of the excitatory drive, specifically, during tonic excitation shunting inhibition affects only the offset, while during noisy trains of excitation shunting inhibition mainly alters the gains , there are a multitude of pathways by which the magnitude of the tonic current can be modulated, with most of them largely independent of synaptic GABA release. This means that, as opposed to regulation of the potassium channels responsible for the resting membrane potential, modulating tonic GABAA inhibition affects neuronal excitability largely independently of the resting membrane potential.If the tonic GABAA system is susceptible to modulation . While there have been some studies showing a role of dynamic modulation of the tonic current, for instance in response to ethanol abuse or in response to epilepsy, these effects are generally seen to be due to changes in expression  can modulate the tonic current through PKC (though see A receptors remains attractive, yet elusive.The results cited above clearly demonstrate that the tonic GABApression . It woulThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this work, we explored convergent evidence supporting the fronto-striatal model of obsessive-compulsive disorder (FSMOCD) and the contribution of event-related potential (ERP) studies to this model. First, we considered minor modifications to the FSMOCD model based on neuroimaging and neuropsychological data. We noted the brain areas most affected in this disorder -anterior cingulate cortex (ACC), basal ganglia (BG), and orbito-frontal cortex (OFC) and their related cognitive functions, such as monitoring and inhibition. Then, we assessed the ERPs that are directly related to the FSMOCD, including the error-related negativity (ERN), N200, and P600. Several OCD studies present enhanced ERN and N2 responses during conflict tasks as well as an enhanced P600 during working memory (WM) tasks. Evidence from ERP studies , neuroimaging and neuropsychological findings suggests abnormal activity in the OFC, ACC, and BG in OCD patients. Moreover, additional findings from these analyses suggest dorsolateral prefrontal and parietal cortex involvement, which might be related to executive function (EF) deficits. Thus, these convergent results suggest the existence of a self-monitoring imbalance involving inhibitory deficits and executive dysfunctions. OCD patients present an impaired ability to monitor, control, and inhibit intrusive thoughts, urges, feelings, and behaviors. In the current model, this imbalance is triggered by an excitatory role of the BG  and inhibitory activity of the OFC as well as excessive monitoring of the ACC to block excitatory impulses. This imbalance would interact with the reduced activation of the parietal-DLPC network, leading to executive dysfunction. ERP research may provide further insight regarding the temporal dynamics of action monitoring and executive functioning in OCD. Obsessive-compulsive disorder (OCD) affects approximately 50 million people worldwide , anterior cingulate cortex (ACC), and basal ganglia (BG) during rest or symptom provocation  studies assessing action monitoring, inhibition, and other related executive functions, such as WM. The purpose of this review is to integrate the ERP findings  hyperactivation of the OFC, ACC, and BG involved in the control, monitoring and inhibition of behaviors and thoughts; and (2) decreased activity of the DLPC/parietal network involved in executive functions (attention and WM). Thus, an extended FSMOCD involving OFC/ACC/BG hyperactivation as well as DLPC/parietal deactivation suggests an intertwining of monitoring/inhibitory control with executive functions. These abnormalities in different areas might underlie the variety of OCD symptoms.ERPs constitute a millisecond-by-millisecond record of neural information processing that can be associated with particular operations, such as sensory encoding, motor control or higher cognitive processes   as ERN  that have been found to be impaired during electrophysiological investigations of OCD patients.OCD presents a highly variable neurocognitive profile regarding neuropsychological and neuroimaging assessments. This finding is not unexpected given the heterogeneity of symptoms and high co-morbidity involved in OCD . This higher N200 amplitude could be a sign of attention readiness to sustain effective top–down inhibitory control to avoid the interference of distracters. Thus, convergent evidence highlights a core inhibitory control impairment in this disorder  with action monitoring and inhibition, particularly because they are modulated by the ACC, OFC, and BG, which are intimately linked to OCD.ERP studies offer several advantages. One of the most relevant is a temporal resolution that allows a more precise analysis of different stages of cognitive processes during the performance of a motor or cognitive task. Another advantage is its lower cost compared to other techniques, such as neuroimaging. The integration of neuropsychological studies, neuroimaging techniques and ERP findings might become a powerful strategy for obtaining more precise and complete knowledge of disorders such as OCD. This integrative approach could also be useful in analyzing the possible role of conflict monitoring and inhibition  as well as executive functions such as WM (P600) as biomarkers or endophenotypes of this disorder.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium vivax Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s) lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of  Plasmodium, which have limited N-glycosylation machinery and therefore may provide a better platform to explore this strategy. Here, we used glycan masking to investigate the binding interaction between Plasmodium vivax Duffy binding protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC). This study showed that addition of an N-glycan site in a predicted host interaction surface abolished binding and potentially covered up an inhibitory antibody epitope. In contrast, addition of multiple N-glycan sites distant from predicted interaction surfaces did not inhibit binding but did slightly enhance elicitation of inhibitory antibodies. This analysis shows that glycan resurfacing offers an integrated approach to characterize protein function and immunogenicity and that glycan resurfacing of PvDBPII immunogens may have utility in P. vivax-malaria vaccine development.An important goal of many vaccine efforts is to inhibit pathogen invasion of host cells, but few approaches exist to target vaccine antibodies on invasion blocking epitopes. Glycan masking is a vaccine design strategy to hide protein surfaces with carbohydrates and focus antibodies on exposed surfaces. This approach has mostly been evaluated on the heavily glycosylated HIV envelope glycoprotein, but it has never been tested on eukaryotic pathogens, such as  Plasmodium vivax invasion of human reticulocytes is strongly dependent on an interaction between the P. vivax Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) on the reticulocyte surface P. vivax infection P. vivax invasion has recently been described P. vivax infection P. vivax infection making it an attractive vaccine target.P. knowlesi (Pkα-DBL) The molecular mechanisms of PvDBP-DARC binding and immune evasion are only partially understood. PvDBP is a member of the Erythrocyte Binding-Like (EBL) protein superfamily Although the PvDBP structure has been solved, the precise extent of the DARC binding “footprint” remains unclear P. vivax invasion of reticulocytes. High titer and broadly neutralizing antibodies to PvDBP are only found after repeated infection P. vivax dependence on DARC for invasion and its restrictive preference for reticulocytes supports PvDBP vaccine development. However, the rapid kinetics of parasite invasion (less than one minute) These two binding models are not necessarily exclusive but lead to different predictions about how antibodies may inhibit Plasmodium and Giardia, have limited N-glycosylation machinery Structural vaccinology is an emerging strategy that uses three dimensional structure to guide immunogen design to improve functional antibody responses. Viral enveloped pathogens, like HIV, use a number of mechanisms to evade immune surveillance, one of them being glycan shielding of epitopes P. knowlesi α DBL domain The monomeric crystal structure of the P. vivax Salvador 1 strain  contains three potential N-glycosylation sites at positions 255, 351 and 420  or HEK293 cells (3 native N-glycosylation sites).To investigate immunogenicity, mice were immunized with wild-type and DBPII glycosylation variants. Since the yield of PvDBP recombinant proteins was low (between 0.1 to 0.5 mg/L) we employed a DNA prime–protein boost strategy . In thisE. coli recombinant protein that lacks N-glycosylation and therefore has no potential antibody epitopes hidden. One-way analysis of variance (ANOVA) of individual mouse ELISA titers showed no significant difference in antibody titer between any of the six immunization groups . Moreover, the percent inhibition by P1 or Max plasma was significantly better than wild-type at multiple plasma dilutions  while in the COS-7 assay, antibodies are challenged to inhibit an interaction between two cells (RBCs displaying DARC and COS-7 cells displaying PvDBPII) that is likely to be multi-valent and potentially highly multi-valent . We initP. vivax is highly restricted to reticulocytes and theoretically poses fewer challenges for blood stage vaccine development than P. falciparum, PvDBP is polymorphic Efforts to make a blood stage malaria vaccine have proven extremely challenging because of antigen polymorphism, redundant pathways of RBC invasion, and the rapid kinetics of parasite invasion P. falciparum encodes an identifiable Alg7 glycosyltransferase and SST3 oligosaccharyltransferase, and it is predicted that only a short GlcNAc2 group would be transferred to proteins Plasmodium proteins may be better platforms to evaluate this vaccine design strategy because they are not already highly evolved to evade antibody responses via glycan masking.Glycan masking is an emerging vaccine design strategy to target antibodies to specific antibody epitopes. A glycan shield is employed by HIV and other enveloped viruses, such as influenza, to hide regions of viral surface proteins from neutralizing antibodies Here, glycan masking was used both to probe PvDBP binding function and to test a vaccine strategy to focus antibodies on critical interaction surfaces. Although several different PvDBP-DARC binding mutagenesis studies have been done, there is still controversy surrounding the PvDBPII-DARC binding model In mouse immunization studies, glycoengineered proteins were equally immunogenic to wild-type proteins by ELISA. This finding is encouraging because it suggests that substantial glycoengineering did not inadvertently blunt T helper cell priming P. vivax infections In contrast, consistent and significant enhancement of PvDBP∶DARC binding inhibition was observed in the yeast display assay used in this study. Of particular interest is the observed increase in inhibitory potency elicited by the P1 and Max constructs with increased glycosylation distal to predicted inhibitory antibody binding areas, as well as correspondingly decreased inhibition elicited by the STBP construct. The extra N-glycosylation site in the STBP construct covers a linear inhibitory antibody epitope from natural If the P1 and Max variants successfully enhanced inhibitory binding titers as implied by the yeast display assay, critical questions for further study are whether the increased potency observed in the yeast assay is indeed clinically relevant, and whether the glycosylated constructs developed in this study are amenable to further engineering and concomitant improvement in elicited immune response. One possibility is that in designing the current constructs we may have inadvertently masked inhibitory epitopes that have recently been localized to subdomain 3 P. vivax-malaria vaccine development.In conclusion, this analysis shows that PvDBPII recombinant proteins retain immunogenicity despite considerable glycoengineering and that glycan resurfacing offers an integrated approach to characterize protein function and immunogenicity. Glycan resurfacing of PvDBPII immunogens may have utility in PvDBPII and PvDBPII glycosylated variants were synthesized at Genscript with mammalian codon optimization and cloned into a modified version of the pTT3 vector  using HindIII and BamHI restriction sites. The PvDBPII is in frame with a signal sequence from murine IgG kappa chain at the N-terminus and 6× His tag at the C-terminus P. knowlesi PkDBPII (2C6J) N-linked glycosylation site selection for PvDBPII  was performed by fixed-backbone redesign in Rosetta, using Rosetta-generated homology models derived from the crystal structure of the 2 for 4 h, then made up to 400 ml with Freestyle media (Invitrogen) and placed back in the shaker. Tissue culture supernatant was harvested 5 days after transfection and spun at 1000 rpm for 20 min to remove cell debris. Following centrifugation, the supernatant was balanced with 20 mM HEPES pH 7 and 200 mM sodium chloride. Recombinant PvDBPII proteins were affinity purified using Nickel Sepharose 6 Fast Flow resin , dialyzed into 1× PBS and stored. To assess purity, recombinant PvDBPII and glycosylated variants were run on 4 to 20% SDS gel (Invitrogen) and stained with GelCode Blue solution (Invitrogen) or transferred to PVDF membranes and probed with anti-His antibody or with rabbit anti-PvDBP plasma for Western blots.HEK-293F cells were cultured in SFM4TRNSFX media (Hyclone). For expression and purification, 400 million cells were suspended in 20 ml of fresh SFM4TRNSFX media. Cells were transfected with 500 µg of DNA and 1 mg of PEI MAX (Polysciences.com) using a previously described procedure This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Rabbit immunizations were performed by custom vendor at R&R Rabbitry. Mice immunizations were performed by Seattle Biomedical Research Institute personnel. Animals were housed under controlled laboratory conditions. The protocols were approved by the Institute Animal and Care Use Committee at Seattle Biomedical Research Institute (Protocols JS-ABP and JS-04).E. coli generated refolded PvDBPII protein at R&R Rabbitry in accordance with current guidelines on animal immunizations. Two animals received 50 µg of protein in Freund's complete adjuvant for the first injection followed by protein in incomplete Freund's adjuvant for the subsequent 5 boosts. Two others received 50 µg of protein in TiterMax gold adjuvant (TiterMax.com) for the immunization and the boosts. Boosts were carried out every 3 weeks. For mice immunizations, 6 groups of 10 mice each were subjected to immunization according to animal immunization guidelines. Group 1 and 2 mice received protein only for the immunization and three boosts. Mice in groups 3 to 6 received DNA immunization followed by three DNA boosts and two protein boosts at three week intervals. Protein immunizations were given as intramuscular injection into the left tibialis muscle. DNA immunization was done by DNA electroporation in the anterior tibialis muscle using Trigrid delivery system (ICHOR). Group 1 mice received 10 µg of E. coli generated, refolded PvDBPII. Group 2 mice received 5 µg of PvDBPII generated in HEK-293F cells. Groups 3–6 mice received 20 µg of DNA and 2.5 µg of PvDBPII, PvDBPII STBP glycan, PvDBPII partial glycosylated variant (P1) and PvDBPII maximum glycosylated variant (Max), respectively  by overnight incubation at 4°C. Mice plasma dilutions ranging from 1∶500 to 1∶512,000 were added to the plate and probed with ELISA kit  according to manufacturer's protocol. The results were analyzed using SoftMaxpro 5.0, graph fitted to 4-pt fit curve and OD determined at 0.1.100 ng of recombinant PvDBPII produced in COS-7 cells were grown in 60 mm culture dishes containing one coverslip each. Cells were transfected using GeneJuice transfection reagent and 1 µg plasmid DNA of wild-type PvDBPII-GFP fusion  or PvDBPII glycosylated variants   in pEGFPTo the coverslips generated above, 0.5% hematocrit blood was added and left for 1 h in a humidified chamber at 37°C. The culture dish was then filled with complete DMEM, a glass stand was placed in the dish, and the submerged coverslip was flipped on the stand for 10 min to allow unbound RBC to settle by gravity. Coverslips were then washed with 1× PBS and fixed with 1% glutaraldehyde for 20 min. The coverslips were moved into 1× PBS and 10 fields were counted at 200× magnification using a fluroscence microscope. Transfected cells bearing 5 or more RBCs were counted as rosettes.COS-7 cells were cultured and transfected as described above. Coverslips were incubated with varying dilutions of final bleed mouse plasma or a 1∶200 dilution of pre-bleed mouse plasma in incomplete DMEM for 1 h at 37°C in a humidified chamber S. cerevisiae and all asparagines in potential N-linked glycosylation sites were mutated to glutamines. Viable plasmid was generated by homologous recombination following electroporation into EBY100 yeast as reported by Chao et al. Yeast display of PvDBPII constructs was performed as per Binding and flow cytometry assays were modeled on the primary-secondary antibody protocol of Chao et al. 5′-CTTCGGTAAAATCT CTACTTGCTGGAAAGC-3′ and FYPdn 5′-CCATGGCACCGTTTGGTTCAGG-3′) and the coding sequence with (FYup 5′-GACTCTTCCGGTGTAACTCTGATG-3′ and FY851[-] 5′GGCCAAGACGGGCACCACAATG-3′). The thermocycling program used for both amplifications was 30 seconds at 95°C, 30 seconds at 58°C, and 30 seconds at 68°C for 30 to 45 cycles. The promoter-specific fragment was digested with StyI, and the coding-specific fragment digested with BanI for 2 h at 37°C. Restriction fragments were visualized after electrophoresis on 4% agarose (Lonza) gels stain. BanI digestion of the coding-specific PCR produces segments of 212 and 151 bp  and/or 363 bp . StyI digestion of the Fy promoter-specific fragment produces segments of 144, 108, and 77 bp (erythrocyte active promoter) or 144, 108, 65 bp (erythrocyte silent promoter).The following protocol was adapted from The amino acid variation at single multiple alignment position of a line-up of PvDBP sequences  was calciP represents the observed frequency of a residue type i in the aligned column 2 20) occurs at positions where all amino acids are observed with equal frequency.Fractional Shannon entropy values Figure S1Sequence of PvDBPII wild type and glycoengineered variants. N-glycan sites are highlighted in yellow. The PvDBP sequence indicates the amino acid boundaries for the DNA and protein immunization constructs. The PvDBP construct boundaries for COS-7 assay are underlined and those for yeast display are shown in red.(PDF)Click here for additional data file.Figure S2Alignment of PvDBP sequences. A multiple alignment was generated by Clustal 2.1 from 129 PvDBP sequences (PDF)Click here for additional data file.Figure S3Transfection of COS-7 cells with PvDBPII-GFP fusion proteins. The gating strategy for GFP and anti-PvDBPII doubly positive transfected COS-7 cells is shown. The gate for GFP-positive cells was set by comparison to untransfected cells. The gate for anti-PvDBPII-positive (PE-Texas Red) was set by comparison to cells labeled with secondary antibody alone.(PDF)Click here for additional data file.Figure S4Western blot of DBPII glycosylation variants expressed in COS-7 cells with or without PNGaseF treatment. COS-7 cells transfected with recombinant DBPII glycosylation variants were lysed 48 h post transfection and immunoprecipitated with anti-GFP agarose resin. Half of the sample was subjected to PNGaseF treatment and the other half was untreated to observe glycosylation modifications.(PDF)Click here for additional data file.Figure S5Inhibition of PvDBPII binding to DARC in different assay formats. Mice were immunized with wild-type DBPII protein produced in E. coli or HEK293 cells. Antibody inhibition of PvDBPII-DARC interaction in COS-7-RBC binding inhibition assay (A) and yeast display binding inhibition assay format (B).(PDF)Click here for additional data file.Figure S6Yeast display antibody binding inhibition assay. Histograms showing DARC-Fc binding to yeast. (A) Uninduced yeast with no PvDBPII surface expression plus wild-type DARC-Fc (negative control). (B) Induced yeast with wild-type DARC-Fc (positive control). (C) Induced yeast with an inactive DARC-Fc mutant (negative control).(PDF)Click here for additional data file.Table S1Adhesion of PvDBP mutants to DARC.(PDF)Click here for additional data file.Table S2Immunization schedule.(PDF)Click here for additional data file.Table S3IC50 of DBPII glycosylation variants.(PDF)Click here for additional data file.


Forty years after the initial discovery of light-dependent protein phosphorylation at the thylakoid membrane system, we are now beginning to understand the roles of chloroplast phosphorylation networks in their function to decode and mediate information on the metabolic status of the organelle to long-term adaptations in plastid and nuclear gene expression. With the help of genetics and functional genomics tools, chloroplast kinases and several hundred phosphoproteins were identified that now await detailed functional characterization. The regulation and the target protein spectrum of some kinases are understood, but this information is fragmentary with respect to kinase and target protein crosstalk in a changing environment. In this review, we will highlight the most recent advances in the field and discuss approaches that might lead to a comprehensive understanding of plastid signal integration by protein phosphorylation. With the help of genetics, the “state transition” kinase was first identified in Chlamydomonas and then – by homology – in Arabidopsis thaliana, and it was named Stt7 and STN7, respectively ” carries a functional N-terminal transit peptide that guides a truncated version of CIPK13 into chloroplasts ” mutant is unable to initiate the known redox-induced changes in the expression of nuclear encoded light-harvesting complex (LHC) proteins. PRIN2 associates with the plastid encoded RNA polymerase (PEP) complex, and its absence affects plastid transcription in a way that is characteristic for a defect in the plastid transcription machinery  and long-term responses (LTR) of the two photosystems and the metabolic state of the chloroplast to changing light conditions. Both responses are controlled by the functional status of the photosynthetic electron transport chain, in part by means of reduction/oxidation (redox) properties of redox sensors. At least the LTR is accompanied by changes in plastid and nuclear gene expression that result in the stable adaptation of plastid metabolism to environmental conditions. Redox regulation of nuclear and plastid gene expression is channeled through the plastid transcription system. For example, the “plastid redox insensitive 2 -binding site of the cytochrome b6/f complex  and pTAC16 (At3g46780) as new potential STN7 targets. TAC16 is a subunit of the “transcriptionally active chromosome,” a high molecular mass complex comprising the PEP polymerase, and as such a potential mediator between STN7 phosphorylation activity and the regulation of plastid transcription. However, TAC16 is not a core component of the plastid transcription machinery and it is distributed between the TAC and thylakoid membranes .The recent identification of a redox-regulated protein kinase that resembles two-component sensor kinases from prokaryotic systems opened up a new perspective on photosynthetic signaling. The CSK may represent the postulated redox sensor that directly regulates plastid transcription via phosphorylation, as predicted in the CoRR hypothesis ( regulon that controls plastid transcription activity. Plastid CKII is a pleiotropic kinase that phosphorylates numerous target proteins among them RNA-binding proteins (RNPs), components of the PEP complex, and sigma factors .Chloroplast sensor kinase, as a reminiscent two-component sensor kinase is expected to operate in conjunction with a response regulator. However, because CSK lacks the histidine residue required for sensor kinase activation, the signal transduction chain may differ from the common sensor kinase/response regulator-type that is prevailing in prokaryotes. In search for response regulator proteins that interact with CSK, two-hybrid assays were performed and CSK was found to interact with two proteins involved in transcriptional regulation, “sigma factor 1 (SIG1)” and the plastid transcription kinase cpCKII . Based o factors . Phospho factors . Notably factors . In a rein vitro . Two outregulon components: overexcitation of PSI is sensed as oxidized plastoquinone pool that activates CSK resulting in SIG1 phosphorylation and down-regulation of transcription of PSI subunits . Although phosphorylation control of cpCKII activity was only observed in vitro with “protein kinase A (PKA)” from bovine heart, this model could explain the specificity of CKII-mediated regulation of chloroplast transcription. It should be noted, however, that the suggested phosphorylation events in this regulatory system are just anticipated and biochemical proof for any of these in vivo is missing. An excellent review with a detailed discussion is available from Allen and colleagues .The large number of cpCKII targets requires fine-tuned control of kinase activity. In fact, some regulatory functions of cpCKII are mutually exclusive. For example, there is evidence for light-induced phosphorylation of RNPs during etioplast to chloroplast conversion . PhosphoFigure 1; Chlamydomonas are both destabilized under prolonged “state 1” conditions, i.e., when phosphorylation of the LHC proteins does not occur. Under these conditions, phospho-mimic mutants in which the phosphorylated threonines are exchanged with aspartate are stabilized compared to their wild-type counterpart suggesting that the negative charge at the C-terminus of the kinase may protect it from degradation  STN7 stability was unchanged compared to wild-type . Phosphatase-deficient plants remain longer in “state 2” compared to wild-type. Biochemical analyses showed that the phosphorylation status of LHC complex proteins correlates inversely with the abundance of the PPH1 gene product. Together, these data suggest that PPH1 specifically counteracts the activity of STN7  the irreversible phosphoprotein degradation by proteases and (ii) the reversible release of the phosphate group by phosphatases. Early reports showed that different phosphatase activities with distinct kinetic properties act on thylakoid phosphoproteins. LHCII proteins are most rapidly dephosphorylated, followed by D1 and D2, and then by CP43 and PsbH . Consist of STN7 . Surpris of STN7 , suggestFigure 1). PBCP dephosphorylates PSII core proteins and is involved in thylakoid stacking. Other targets of STN8 such as the Ca2+ sensing protein Cas and PGRL1 were either not affected in the mutant or not analyzed (This situation is different for the “PSII core phosphatase (PBCP)” and its counteracting kinase STN8 ”, cocktails of synthetic peptides are used to measure phosphorylation activity of a purified kinase. Following the phosphorylation reaction, the peptide mixture is analyzed by mass spectrometry and phosphorylated peptides are identified and quantified by spectral counting or ion intensity measurements. A proof-of-concept study using a mixture of 79 peptides (11–20 mers) and purified pyruvate dehydrogenase kinase (PDK) showed a surprisingly high specificity of this assay system  for relative quantification. Because phosphopeptide elution times may be shifted between different LC runs, this method requires an algorithm for chromatographic alignment (reviewed in 14N/15N labeling was used to study the abscisic acid (ABA)-dependent dynamic changes in the phosphorylation network (Theunbiased . Such a  network  and the  network . These tThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pneumonia represents a major health burden. Previous work demonstrated that although the induction of inflammation is important for adequate host defense against pneumonia, an inability to regulate the host's inflammatory response within the lung later during infection can be detrimental. Intracellular signaling pathways commonly rely on activation of kinases, and kinases play an essential role in the regulation of the inflammatory response of immune cells.Streptococcus (S.) pneumoniae. Kinomics peptide arrays, exhibiting 1024 specific consensus sequences for protein kinases, were used to produce a systems biology analysis of cellular kinase activity during the course of pneumonia. Several differences in kinase activity revealed by the arrays were validated in lung homogenates of individual mice using western blot. We identified cascades of activated kinases showing that chemotoxic stress and a T helper 1 response were induced during the course of pneumococcal pneumonia. In addition, our data point to a reduction in WNT activity in lungs of S. pneumoniae infected mice. Moreover, this study demonstrated a reduction in overall CDK activity implying alterations in cell cycle biology.Pneumonia was induced in mice via intranasal instillation of This study utilizes systems biology to provide insight into the signaling events occurring during lung infection with the common cause of community acquired pneumonia, and may assist in identifying novel therapeutic targets in the treatment of bacterial pneumonia. S. pneumoniae is the main causative pathogen in community-acquired pneumonia (CAP), responsible for an estimated ten million deaths annually worldwide Due to its unique relationship with the environment, the lung must defend itself from infection by numerous inhaled micro-organisms. Although in general the lung is successful in doing so, bacterial pneumonia remains a major health burden. The Gram-positive bacterium Recognition of invading bacteria by the host is considered to occur mainly through toll-like receptors (TLRs). After interacting with their ligands, TLRs signal via adaptor proteins and kinases to ultimately activate Nuclear factor-κB (NF-κB) inducing inflammatory responses S. pneumoniae pneumonia in mice and furthermore attempt to elucidate complex interactions occurring during the course of the infection. To our surprise, we did not detect signaling pathways belonging to the TLR signaling cascades. In contrast, we detected pathways that induce chemotoxic stress and promoted the T helper 1 (Th1) response. In addition we found an overall reduction in WNT signaling. Canonical WNT signaling, named after the homology of WNT-genes with int-1 and wingless in Drosophila, is important in developmental signaling Kinases comprise an important part of the intracellular responses mediated by a variety of receptors. Although it is highly likely that kinases mediate lung inflammation during pneumonia, knowledge about the activation of kinases during pneumonia is limited. Microarray-based kinome profiling approaches have been subject of development over the last years and an interesting tool to integral study signaling events S. pneumoniae pneumonia. This study is the first to apply kinome profiling using kinomics chip arrays in infectious diseases.Moreover, we found a reduction in cell cycle activity during the course of S. pneumoniae bacterial loads remained similar at 3 and 6 hours  signaling was inhibited, as demonstrated by the reduced activity of pyruvate dehydrogenase kinase  at 6, 24 and 48 hours, AMP-activated protein kinase  at 48 hours and the dynamic profiles of AKT , which has events at all time points, and glycogen synthase kinase-3β  at 3 and 24 hours with both an increase and decrease in activity early during infection and a subsequent decrease/stabilization in the late phases of pneumococcal pneumonia.A decrease in transforming growth factor- β  activity was seen at 3 hours. Casein kinase-2  is involved and needed for WNT activation The found GSK-3β primary activation and reduction could also attribute to WNT signaling. In line, we found overall reduced activity of ephrin  at 3 hours and β-catenin(spots 187 and 321) at 3, 24 and 48 hours.Interestingly, kinome array analysis revealed reduced activity for numeral kinases involved in regulation of the cell cycle (Cyclin-dependent kinase (CDK)1: spots 105, 3112, 542, 796, 554, 615/CDK2 spot 309/CDK4 spots: 274, 374, 688/CDK5 spot 420).S. pneumoniae infected animals . However inhibition of GSK-3β at Ser9 results in reduced GS-1 phosphorylation. Indeed, GS-1 on the kinomics chip showed reduced phosphorylation at all time points except for 24 hours after infection. As demonstrated by S. pneumoniae infection was decreased.In general, CDK activity was decreased over time during pneumonia . Since aUpon phosphorylation, β-catenin is rapidly degraded, reflecting the off state of WNT S. pneumoniae. The most strongly affected pathways identified during pneumococcal pneumonia were associated with enhanced chemotoxic stress, a developing Th1 response, reduced WNT signaling and down regulated cell cycle activity.Current mass spectrometry techniques and novel proteomics approaches like antibody microarrays determine protein phosphorylation levels rather than the enzymatic activity resulting from it, while measurement of kinase activity using the peptide microarray provides a direct view on the extent of enzymatic activity leading to specific signal transduction. We here utilized kinome substrate arrays to obtain insight in alterations in kinase activity in the lungs during respiratory tract infection caused by the most common causative agent in CAP, S. pneumoniae chosen causes lethality in mice from 48 hours onward with an overall mortality rate of 90%–100% after 4 to 6 days The infectious dose of S. pneumoniae entering the airways likely affect kinome profiling patterns, since these were generated from whole lung homogenates. Kinome studies on specific leukocyte subsets purified from lung tissue at several time points after induction of pneumonia might circumvent this shortcoming. However, cell isolation procedures potentially influence phosphorylation states and thus we chose to utilize total lung homogenates, generated from snap frozen material. One also has to consider that, when searching for new therapies, it is the whole lung that will be exposed to a potential drug, e.g. administered via nebulization. Therefore, we chose to determine kinase profiles of total lung lysates without isolating different cell types.The kinome profile dendogram  generateS. pneumoniae gave rise to p38MAPK/c-Jun N-terminal kinase and extracellular signal-regulated kinase (ERK) phosphorylation Analysis of our chip data revealed several significant ongoing processes in pneumonia. Much to our surprise specific MAPKs and other kinases classically related with the immune response, like those involved with TLR signaling (e.g. inhibitory κβ (IΚΚ)-α/IΚΚβ, TANK-binding kinase 1 and MAPK-kinase 1 (MEK-1)) were not prominently present in the results obtained from the kinomics chip, not even at the earliest time point. A general MAPK substrate however was shared between time points, but no links to its specific contexts could be made. Ex vivo stimulation of bone marrow derived macrophages with serotype 2 PKA was also shared in all time points. This kinase is involved in regulation of proliferation and differentiation, microtubule dynamics, chromatin condensation and decondensation, nuclear envelope disassembly and reassembly, as well as regulation of intracellular transport mechanisms and ion fluxes In context of an immune response to bacterial infection, the Th1 response signaling was the only prominent pathway that appeared in our analysis. Th1 responses were mirrored by the decreased activity of BCR and NFAT. De-phosphorylation of NFAT (as a substrate) enables its translocation to the nucleus inducing IL-2 transcription + T-cell depleted wild type mice displayed an increased susceptibility to serotype 3 pneumococcal pneumonia, while adoptive transfer of CD8+ T-cell to knockout mice improved survival Moreover, in recent studies CD8 knockout mice or CD8S. pneumoniae meningitis S. pneumoniae pneumonia remains to be established.A variety of CDKs and CDK associated kinases displayed a reduced activity during the course of pneumococcal pneumonia. These kinases regulate the progression through the cell cycle Several kinases not primarily associated with inflammation or infection, were abundant in our findings. Nonetheless, most of these kinases have been implicated to also contribute to an inflammatory response. AMPK-α and GSK-3β mainly are known in metabolic context, specifically in insulin and glucose signaling It should be noted, however, that differences in pneumococcal serotype can illicit differential immune responses. Serotype 11a (M10) murine pneumococcal pneumonia resulted in an early local levels of TNF-α and IL-6 in the pulmonary compartment, whereas serotype 3 (ATCC6303) displayed a more delayed response, which remained until death occurred Notably, kinome profiles were determined in homogenates prepared from right (whole) lungs. As such, since the extent of pneumonia and the associated inflammation likely are not equally distributed in all lung segments in our model of intranasal infection, our data are not representative for specific areas of infection. In addition, analyses of both (whole) lungs would have provided more definitive information about pulmonary kinome profiles, since this approach would have excluded bias due to unequal left-right distribution of the infection.S. pneumoniae infection. These data may pave the way to future drug interventions seeking to interfere with specific signaling pathways e.g. activating WNT signaling or reducing CDK activity.Here we demonstrate the use of a systems biology approach on kinome activity in the lung during pneumococcal pneumonia. We uncovered pathways that induce chemotoxic stress and promote the Th1 response. Moreover, we found an overall reduction in WNT signaling and reduction in cell cycle activity during the course of severe This study was carried out in accordance with the Dutch Experiment on Animals Act. The Animal Care and Use Committee of the University of Amsterdam approved all experiments (Permit number: DIX100121).For all experiments female C57Bl/6 mice (aged 10 weeks) were purchased from Charles River . The Animal Care and Use Committee of the University of Amsterdam approved all experiments.S. pneumoniae serotype 3 (ATCC 6303) was grown to a mid-logarithmic phase at 37°C in Todd-Hewitt broth supplemented with 0.5% Yeast extract . Bacteria were harvested by centrifugation at 4000 rpm for 15 minutes; washed twice and resuspended in sterile saline at a concentration of 5×104 colony forming units (CFU)/50 µl. Mice were inoculated with 50 µl of bacterial suspensions intranasally under isoflurane inhalation anesthesia .Pneumonia was induced as previously described Three, six, 24 and 48 hours after infection, 3 mice per time point were sacrificed by cardiac puncture under Domitor  and Nimatek  anesthesia. Left lungs were harvested and homogenized in 4 volumes of sterile saline with a tissue homogenizer . CFUs were determined from serial dilutions of samples, plated on blood agar plates and incubated at 37°C for 16 hours before colonies were counted.2, 1 mM -glycerophosphate, 1 mM Na3VO4, 1 mM NaF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM phenylmethylsulphonyl fluoride). Homogenates were centrifuged at 14,000 RPM for 5 minutes and pellets were discarded. As a control, right lungs were harvested from 3 mice administered with sterile isotonic saline 3 hours earlier and treated as described above. For the kinomics chip kinome profile assay samples were pooled and diluted to a protein concentration of 1 mg/ml. Of these lysates, 80 µl was added to 12 µl of Activation mix  and this mixture was applied to a kinomics chip  per pool. The chips were incubated at 37°C for 2 hours. The arrays were washed twice in 2 M NaCl (1% TWEEN 20), once in PBS (1% SDS) and rinsed twice in demineralised H2O. Subsequently the chips were air-dried and exposed to a phosphor imager plate for 72 hours. Radioactive signals were measured using a phosphor imager .Right lungs were snap frozen in liquid nitrogen, after which they were homogenized in three volumes of lysis buffer  enriched with 1 mM MgClhttp://rana.lbl.gov/EisenSoftware.htm). These data were exported to a spreadsheet  for further analysis. Spot intensities were normalized to 90 percentile, as previously described http://bioinfogp.cnb.csic.es/tools/venny/index.html).For analysis, Spot density and individual background were analyzed using Scanalyse  and β-actin . Samples for western blotting were boiled at 95°C for 5 minutes in laemmli buffer and loaded onto SDS-PAGE gels. After electrophoresis the content of the gels was transferred onto Immobilon-PVDF membranes . The membranes were blocked in 5% BSA  in TBS-T at room temperature for 60 minute. All primary antibodies were diluted 1:500 with exception of β-actin, which was diluted 1:4000. The membranes were incubated overnight at 4°C. Next, the membranes were incubated for 60 minutes with 1:1000 anti-rabbit-HRP conjugated secondary antibody  and blots were imaged using LumiLight Plus ECL  on a GeneGnome chemiluminescence imager .2, 2 mM CaCl2, 2% Triton X-100, AEBSF(4-(2-aminoethyl)benzeensolfonyl fluoride, EDTA-Na2, 8 µg/ml pepstatin and leupeptin, pH 7.4) and incubated on ice for 30 minutes. Homogenates were centrifuges at 3600 rpm at 4°C for 10 minutes and stored at -20°C until use. IL-1β, IL-6, TNF-α, KC and MIP-2 were measured in lung homogenates using ELISAs  according to the manufacturer's instructions. Detection limits were: TNF-α: 62.5 pg/ml, MIP-2 and KC: 15 pg/ml and 31.25 pg/ml for IL-1β, IL-6.For cytokine and chemokine measurements, left lungs were excised, weighted and homogenized in saline four volumes of saline. Homogenates were diluted 1:2 in lysis buffer (300 mM NaCl, 30 mM Tris, 2 mM MgCl


This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH , Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin's cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable P-gp substrates such as amprenavir, quinidine, ketoconazole and verapamil do not, regardless of whether they actually use the basolateral transporter.We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC Inhibition of digoxin transport is often determined using confluent polarized cell lines expressing high levels of P-gp such as Caco-2 It is well established that transporters play an important role in absorption, distribution, metabolism and elimination of drugs. Inhibition of drug transporters can affect drug safety and efficacy. The International Transporter Consortium published a white paper reviewing the clinically important drug transporters and summarizing which In the past, it has been assumed that when an investigational drug inhibits transport of digoxin across these cell lines, it is due to inhibition of P-gp. However, Acharya et al. The putative digoxin and loperamide uptake transporters were identified kinetically by virtue of the fact that they are inhibitable by low concentrations of GF120918. Prototypical inhibitors of organic anion transporters  and organic cation transporters (OCTs) did not affect digoxin or loperamide uptake transport, therefore the identity of the putative digoxin uptake transporter remains unknown. The existence of an as yet unidentified digoxin uptake transporter has also been proposed in Caco-2 cells 50 determinations using digoxin as probe substrate, since the observed overall IC50 could well be a convolution of inhibition of both uptake transport as well as P-gp. We demonstrate here by kinetic analysis that the GF120918 IC50 value for inhibition of digoxin transport across MDCK-MDR1-NKI cells is indeed a convolution of inhibition of P-gp and basolateral digoxin uptake transport.Digoxin uptake transport may have important implications for P-gp IC50 curves for GF120918, cyclosporine, ketoconazole, and verapamil. Kinetic analysis showed that each of these cell lines contain a basolateral digoxin uptake mechanism that was inhibited by GF120918 and cyclosporine-A.The investigation into the presence of GF120918 inhibitable digoxin uptake transporters has now been extended to three additional P-gp expressing cell lines: MDCK-MDR1-NIH , Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). For each of cell lines, we obtained ICSince we have not yet been able to identify a basolateral digoxin uptake transporter, we have examined three different hypotheses to identify the source(s) of the basolateral inhibitable digoxin uptake mechanism:The cell line has a basolateral digoxin uptake transporter that is inhibited.Partitioning of inhibitor into the basolateral membrane causes the membrane to become less permeable.There is hetero-dimerization of digoxin with the inhibitor in the basolateral donor chamber that reduces the free digoxin concentration, thereby inhibiting digoxin uptake into the cell.Hypothesis 3, the consideration of a physical interaction between inhibitor and digoxin, is based on studies of inhibitors of enzymes, primarily kinases, in high-throughput screens that found several “promiscuous” inhibitors, i.e. inhibitors that were able to inhibit multiple and quite different enzymes In addition, we have added vinblastine to the list of drugs that kinetically need a basolateral uptake transport mechanism in the MDCK-MDR1-NKI cells. This led to the question of how small must a drug's passive permeability be in order that a kinetic need for an uptake transporter can be justified. By simulations, we have found that a kinetic need for a basolateral uptake transporter can only be observed using low concentrations of drugs with passive permeability values below about 320 nm/s, e.g. digoxin, loperamide and vinblastine. Highly permeable compounds, e.g. amprenavir, quinidine, ketoconazole and verapamil, will show no clear kinetic need for uptake transport to explain their trans-cellular transport, due to the overwhelming transport signal from passive permeability. They may access the transport mechanism, but that would not be kinetically indentifiable. This matches our observations.3H-verapamil (80 Ci/mmol) was from Perkin Elmer. 3H-ketoconazole (10 Ci/mmol) and 3H-vinblastine (20 Ci/mmol) were from American Radiolabelled Chemicals Inc. . Dimethyl sulfoxide (DMSO) was from Sigma-Aldrich . Dulbecco's modified Eagle's medium (DMEM) with 25 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer, high glucose (4.5 g/L), L-glutamine, without sodium pyruvate, and with phenol red was from Invitrogen . The same medium without phenol red was used for transport experiments. Transwell 12-well plates with polycarbonate inserts (0.4 µm pore size and 12 mm in diameter) were obtained from Costar .For experiments performed at GSK  materials were obtained from the following sources. Amprenavir and GF120918 were from GlaxoSmithKline ; verapamil hydrochloride, vinblastine sulfate, ketoconazole and cylcosporin A were from Sigma . Transwell@ plates (12 well) were purchased from Corning Incorporated . EndOhm for TEER measurements was purchased from World Precision Instruments .For the experiments performed at Absorption Systems  with the three cell lines  and the four inhibitors , materials were obtained from the following sources. Cyclosporin A (CsA) was obtained from CalBiochem . Digoxin, ketoconazole, verapamil, and D-glucose were obtained from Sigma . GF120918 was provided by GSK. Lucifer yellow (LY), HEPES, Hanks Balanced Salt Solution (HBSS), Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's phosphate buffered saline (DPBS), and fetal bovine serum (FBS) were obtained from Invitrogen . Dimethylsulfoxide (DMSO) was obtained from EMT . Penicillin-streptomycin (PEST) and trypsin were obtained from CelGro . For transport and inhibition studies performed at GSK . P-gp cell line and culture conditions have been described previously 2. All transport and inhibition assays were performed with cells between passage 30–55. P-gp mediated transport was measured in 12-well transwell plates fitted with polycarbonate membrane inserts. Cells were seeded at a density of 175,000–200,000 cells per insert and grown for four days in culture media. Cells were given fresh media one day after seeding.Cells were split twice a week and maintained in culture media . Cells were kept at 37°C in 5% CO3H-verapamil, 3H-ketoconazole, 3H-vinblastine or 3H-cyclosporin was added to each respective drug concentration to allow quantitation of transport from donor to receiver chambers in both directions over time, i.e. apical to basolateral (A>B) and basolateral to apical (B>A) in the presence and absence of GF120918. At the indicated time points, 25 µL samples were taken from both these chambers and counted by TopCount Model 9912.Prior to the transport experiments, culture media was removed and cells were preincubated for 30 minutes with either transport medium alone or transport medium supplemented with 2 µM GF120918 to inhibit P-gp and measure the substrate's passive permeability. For incubations in the presence of GF120918, the inhibitor was added to both chambers. In addition, 100 µM Lucifer yellow was added to the donor chamber to monitor cell monolayer integrity. 2 on 12-well Transwell plates containing collagen-coated, micropore (0.4 µm pore size), polycarbonate filter membranes. The monolayers were grown to confluence on the filter membranes in a humidified atmosphere containing 5% CO2 at 37°C. MDCK-MDR1-NIH cells were incubated for six days, while Caco-2 and CPT-B2 cells were incubated for 20 days. The culture medium was changed three times per week. Monolayer integrity was assessed by measuring transepithelial electrical resistance (TEER) using an EndOhm, and determining apparent permeability (Papp) values of selected reference compounds .For experiments performed at Absorption Systems LP . Madin-Darby Canine Kidney cells overexpressing human MDR1 were obtained from the NIH (MDCK-MDR1-NIH), Caco 2 cells were from the ATTC and CPT-B2 cells were generated at Absorption Systems by knocking down the BCRP gene in Caco-2 cells (unpublished). Cell monolayers used in this study were prepared in accordance with the standard operating procedures (SOPs) of Absorption Systems. Briefly, stock MDCK-MDR1-NIH, Caco-2 and CPT-B2 cells were cultured in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 100 µM non-essential amino acids, 4 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. For MDCK-MDR1-NIH cells, colchicine (40 µg/mL) was added into the complete medium for selection of cells expressing MDR1. To maintain the properties of CPT-B2 cells, puromycin (10 µg/mL) was added to the normal growth medium. For preparation of Transwell assay plates, cells were harvested from T-75 or T-150 flasks using a solution of 0.25% trypsin and 2.21 mM EDTA (Gibco Life Technologies) and cells were seeded at a density of 60,000 cells/cmThese experiments were performed at GSK. Cholesterol and porcine brain lipids were from Avanti Polar Lipids, Inc. . Three different liposome types were prepared to roughly mimic the lipid compositions of the relevant monolayers of the plasma membrane AO.The apical membrane outer monolayer mimic was a (1∶1∶1) mixture of phosphotidylcholine/sphingomyelin/cholesterol, denoted PC/SM/chol. The partition coefficient to these liposomes was denoted KBO.The basolateral membrane outer monolayer mimic was a (2∶1) mixture of phosphotidylcholine/cholesterol, denoted PC/chol. The partition coefficient to these liposomes was denoted KPC.The plasma membrane inner monolayer mimic (facing the cytosol) was a (1∶1∶1) mixture of phosphotidylserine/phosphatidylethanolamine/cholesterol, denoted PS/PE/chol. The partition coefficient to these liposomes was denoted KLipid mixtures were dried into thin lipid films in round bottom flasks , and freeze-dried overnight on a Flexi-Dry MP freeze-dryer . The lipid films were then suspended in phosphate buffered saline, pH 7.4 (Gibco) and extruded through two 0.1 µm Nucleopore membranes for at least 10 cycles using a Lipex Thermal Extruder. The sphingomyelin-containing liposomes were extruded at 65°C to maintain their liquid-disordered state during extrusion.Partitioning of the drugs to the liposomes was determined in a 20-cell equilibrium dialyzer  using Spectra/Por 4 membrane with a 12–14 kD molecular weight cut off. Each half of the Teflon cells received 10 µM concentrations of the cold drug but the half cell with the liposomes (5 or 10 mM lipid) received an additional 0.25 µCi/ml of appropriate radiolabeled drug. The cells were allowed to equilibrate in a 37°C water bath. At various time points , 25 µL samples were removed from the donor and receiver chambers, placed in a 96 well luma plate and radioactivity was measured using the TopCount. These data showed that partitioning was complete by 6 hr and independent of lipid concentration at these concentrations.PC, was calculated using the relation KPC = CL/CW, where CL is the mol of drug per liter of aqueous buffer and CL is the mol of drug per liter of lipid in the liposomes, using the average molar volume of 1.6 µL/µmol of total lipid BO and KAO, were calculated the same way.The partition coefficient for the plasma membrane inner monolayer mimic, denoted KA, and the basolateral chamber, denoted CB. However, the concentration of substrate in the cytosol, denoted CC, and in the inner plasma membrane in contact with the P-gp binding site, denoted CPC, cannot be measured rigorously in real time. These intracellular and membrane concentrations are variables of the mass action model and are fitted by elementary rate constants for well-defined kinetic barriers and approximate partition coefficients according to the measured values of CB and CA over time We measure the concentration of substrate in the apical chamber, denoted C0 is the empty transporter, CPC is the substrate in the apical membrane inner monolayer, TC is the transporter bound by substrate and CA is the substrate after efflux into the apical chamber. For the inhibitor, QPC is the inhibitor in the apical membrane inner monolayer, TQ is the transporter bound by inhibitor and QA is the inhibitor after efflux into the apical chamber. The rate constant for both probe-substrate and inhibitor are k1 for association with P-gp, kr for dissociation from P-gp back into the membrane and k2 for efflux into the apical chamber. The concentrations of CPC and QPC are predicted from the fitted cytosolic concentrations CC and QC times the partition coefficients measured using the liposomes, respectively, We use the simplest competitive mass action reaction to model P-gp transport:For transport mediated by other digoxin transporters, shown to exist in MDCKII-MDR1-NKI cells The mass action kinetic equations generated by these reactions are shown in Appendix S1 in 1, fitted in Agnani et al. 1 remained fixed at the same value, since the cells have human P-gp and we have found for these drugs that k1 is essentially the same 1, kr & k2 are essentially the same for Caco-2 and MDCK-MDR1-NKI cells . For each cell line, T(0) was fitted as a consensus for all the data. The observed cell line dependent inhibition was due to differences in P-gp inhibition and the inhibition of the other digoxin uptake transport mechanism. The goodness of fits in the model was determined using coefficient of variation calculated between model simulations and the experimentally measured kinetic data.Kinetic parameters were optimized using the Particle Swarm algorithm In order to analyze the digoxin transport inhibition studies by GF120918, cyclosporin-A (CsA), ketoconazole and verapamil, as described below, we first needed to fit the basic kinetic rate constants for transport of these drugs, so we can calculate their inhibition constants for Pgp. To do this, we performed a series of bidirectional transport studies with radiolabelled verapamil and ketoconazole  and CsA . The smaller CsA concentrations were used since CsA is a potent inhibitor of P-gp. The concentration-time profile for the donor and receiver chambers in both the basolateral to apical and apical to basolateral transport direction for each P-gp substrate was then fitted to the mass action model using the recently published improved fitting algorithm optimized for simultaneous fitting of all substrate concentrations PCKC, where KPC is the partition coefficient of the drug to the cytosolic face of the plasma membrane and KC is the binding constant of the drug to P-gp from the plasma membrane. The estimate of the partition coefficient of a drug into the inner apical monolayer allows an estimate of the binding constant to P-gp from the lipid membrane.As shown in the mass action kinetic equations in Appendix S1 in Partition coefficients for amprenavir, cyclosporin-A, digoxin, ketoconazole, loperamide, quinidine, verapamil and vinblastine were measured using 0.1 µm extruded unilamellar liposomes for three different lipid compositions that mimic roughly the lipids in plasma membrane monolayers: (1) apical outer monolayer PC/SM/chol (1∶1∶1) mols; (2) basolateral outer monolayer PC/chol (2∶1) mols; and (3) inner cytosolic monolayer PS/PE/chol (1∶1∶1) mols While these liposome compositions are simple compared to the cell membranes, they give us a reasonable measure of the independent contributions to drug transport by the drug partitioning into the membrane and by the drug binding to P-gp from within the membrane. The cells cannot be used directly for these measurements, since the drugs are adequately permeable to allow binding throughout the cell for any defensible equilibrium measurement r (dissociation from P-gp to the membrane) and k2 , along with the drug-independent rate constant k1 (association to P-gp from the membrane) and the efflux active P-gp surface density for these cells, r∼1 s−1, and efflux was very slow, k2∼0.01 s−1, Verapamil, ketoconazole and CsA data were well fitted using only the drug specific P-gp rate constants kr∼1 s−1 and efflux was very slow, k2∼0.01 s−1 (data not shown).We could not look at transport of GF120918, because no radiolabelled compound was available. As a kinetic surrogate, we used the inhibition of amprenavir transport by GF120918. Since amprenavir has no kinetic need for other transporters besides P-gp r/KPC, with respect to these two parameters, and fits have shown that the ratio of kr/KPC is essentially constant. Fits for k2 are essentially unaffected by changes in the partition coefficient. So our conclusions here will not change once the partition coefficients for GF120918 are known. The permeability coefficients were set to 500 nm/s, but these parameters were only relevant for the inhibition studies described below, where GF120918 was added to both chambers for 30 min, so equilibrium binding could be assumed Additionally, these fits assumed that the partition coefficients of GF120918 were the same as those of CsA, QB) of the inhibitor to the basolateral uptake transporter inhibiting the digoxin uptake clearance, kB,Since digoxin is a substrate of uptake and efflux transport in MDCK-MDR1-NKI cells 50 was in the range of 0.01–0.02 µM GF120918, which agrees with The inhibition of 0.03 µM digoxin B>A transport across MDCK-MDR1-NKI confluent cell monolayers by GF120918 (0.005–2 µM) was measured, Clearly, the predicted inhibition of P-gp underestimates the measured GF120918 inhibition at all concentrations. The level of inhibition at all GF120918 concentrations was much larger than possible based on P-gp inhibition alone, showing substantial non-P-gp inhibition. The question is: what is the mechanism of this inhibition of digoxin uptake transport?B, B] is the inhibitor concentration in the basolateral donor chamber and kB([QB]) is the basolateral uptake clearance of digoxin in the presence of the inhibitor. QB combines both uptake transporter surface density and its binding constant to the inhibitor. If the inhibitor accesses the basolateral transporter binding site from the membrane, then KQB also embeds the partition coefficient of the inhibitor to the outer basolateral monolayer. Thus, for a given inhibitor, the KQB values for an inhibitor across different cell lines cannot be compared.Hypothesis 1 was that the non-P-gp inhibition could be due to the binding of the inhibitor to the basolateral digoxin uptake transporter. The first order basolateral uptake clearance for digoxin in the MDCK-MDR1-NKI cells is denoted kQB = 0, dashed line. The solid line is the predicted inhibition assuming inhibition of both P-gp and the uptake transporter when KQB = 2×108 M−1 for GF120918. All other kinetic parameters were fixed by the values in In Q is defined as the mols of inhibitor in the bilayer divided by the total number of mols of lipid and inhibitor in the bilayer. It depends upon the concentration of inhibitor in the basolateral compartment, [QB], the partition coefficient of the inhibitor into the basolateral outer membrane, KBO, and the average molar volume of the basolateral outer monolayer Vmol≈1.6 L/mol molKBO[QB].Hypothesis 2 was that the non-P-gp inhibition could be due to the partitioning of the inhibitor into the basolateral membrane and reducing the digoxin passive permeability by some mechanism. While partitioning would be linear with inhibitor concentration, a mechanism of permeation reduction could be non-linear with inhibitor concentration, e.g. sigmoidal. The mol fraction of inhibitor in the lipid bilayer XB] = 0.1 µM GF120918, if we assume a very high partition coefficient of GF120918 into the outer basolateral membrane of KBO = 1×104, which is more than 20-fold higher than any KBO observed value thus far . So, we have added the following equilibrium reaction to our standard mass action kinetic model for P-gp transport CQ = 0, i.e. no dimerization, and only inhibition of P-gp. A simple sigmoidal curve is seen with the small inhibitor concentration plateau, denoted negative control or NC plateau, showing the nmol transported after 2 h due to the combined action of digoxin's basolateral uptake transporter  cells shows a PC plateau above zero, One more control was done, which was the inhibition of digoxin transport in these cells by nicardipine up to 100 µM. The inhibition curve was sigmoidal and the PC plateau was significantly above zero (data not shown). So nicardipine inhibition was due to binding to P-gp and not due to binding to digoxin. This compound was identified as forming colloidal aggregates at about 30 µM Our primary goal for these inhibition studies is to investigate whether a basolateral uptake transport mechanism also exists in other additional cell lines. The inhibition assays were performed with confluent cell monolayers of the respective cell lines, using digoxin as probe substrate at a concentration of 10 µM. The amount of digoxin transported in the B>A direction in 1 hr was measured in triplicate.50 data in these additional cell lines, we assume that the P-gp specific kinetic parameters are system independent, i.e. equal to the values shown in 1, kr, and k2). We also have preliminary data that these P-gp specific rate constants extrapolate well between MDCK-MDR1-NKI cells and Caco-2 cells , so it is likely that the elementary rate constants for P-gp expressed in the additional cell lines are well estimated by the values in For fitting of the IC2) we have rigorously fitted for the MDCK-MDR1-NKI cell line, rel. So when we fit the efflux active P-gp surface density for the MDCK-MDR1-NIH cell line to be 60% of the value of T(0) shown in rel = 0.6.For simplicity and clarity, we define the efflux active P-gp surface density of a cell line relative to the number . Using Western blot analysis, it has been reported that Caco-2 cells express similar lower levels of P-gp than the MDCK-MDR1-NKI cells We first fitted the P-gp surface density on the three additional cell lines relative to that on the MDCK-MDR1-NKI cell line by using the average digoxin transport at the lowest inhibitor concentration in each cell line . We founrel = 0.6 and only P-gp is inhibited is shown by the dashed line. While the smaller inhibitor concentrations fit well enough, when [GF120918]>0.1 µM there was no fit. Much more inhibition was observed then was predicted by P-gp inhibition alone, indicating a GF120918 inhibitable digoxin uptake mechanism. A good fit to all the data was observed when Trel = 0.6 and hypothesis 1 was tested with a KQB = 1×107 M−1 (solid line). While the fitted KQB for GF120918 is larger in the MDCK-MDR1-NKI cell line, we do not know whether the basolateral digoxin uptake transporters in these two cell lines are related or not. Even if they are the same transporter, their surface densities could be quite different.Symbols in rel = 0.2 for the Caco-2 cells. The dashed curve shows the simulation when GF120918 does not bind to the basolateral transporter, i.e. KQB = 0 M−1 and the solid line shows the simulation for hypothesis 1 when GF120918 binds to the basolateral transporter with KQB = 2×107 M−1.Similar results were obtained with the Caco-2 cell line and its BCRP knockdown line (CPT-B2). rel was fixed at 0.3. The dashed curve shows the simulation when GF120918 does not bind to the basolateral transporter, KQB = 0 M−1 and the solid line shows the simulation for hypothesis 1 when GF120918 binds to the basolateral transporter with KQB = 2×107 M−1.Hypothesis 3 is unlikely, since the PC plateaus are all above zero and because there is 10 µM of digoxin and less than 2 µM GF120918. Hypothesis 2 is unlikely for the same reasons as given with rel was fixed at 0.6  did not have a kinetic need for a basolateral uptake transporter. We therefore investigated another compound, vinblastine, with a low passive permeability, close to that of digoxin, for it's kinetic need for an uptake transporter. We note that while CsA has a low passive permeability, its efflux transport is so small, 2 = 100 s−1. The problem is that vinblastine's passive permeability in the presence of GF120918 was so low that the measured transport in the absence of GF120918 could not be achieved by the fits, like digoxin and loperamide We performed a series of bidirectional transport studies with radiolabelled vinblastine . The vinblastine data could not be well fitted by P-gp alone. The dotted line above the B∶A>B data and below the A∶B>A data in r and k2 pairs. Curvature in the transport data over time is required to obtain unique fits Because the vinblastine transport was essentially linear in time up to 4 hours, the fit shown in To obtain data with curvature for vinblastine transport, we must monitor the transport over a much longer period of time, as we have done previously for digoxin We have added vinblastine to the list of drugs transported by a basolateral uptake transporter in the MDCK-MDR1-NKI cells. Given that both digoxin and vinblastine have passive permeabilities <100 nm/s, i.e. it would take over a week for passive permeability to achieve 50% of the final steady-state concentration in the receiver chamber B = 100 s−1, i.e. loperamide's value. We then fitted these simulations assuming that P-gp was the only transporter involved. The result is plotted on We addressed this question using simulations of a virtual drug. We simulated a P-gp substrate using the kinetic parameters of verapamil in the MDCK-MDR1-NKI cell line in 50 in these cells represent inhibition of P-gp, inhibition of the uptake transporters, or a combination of the two and (2) do other P-gp expressing cell lines also express a digoxin uptake transporter.Digoxin is a widely prescribed cardiovascular drug with a narrow therapeutic index and therefore not very tolerant of drug-drug interactions. Since digoxin is a P-gp substrate, digoxin drug-drug interactions are often ascribed to P-gp inhibition. It is common practice in the pharmaceutical industry to assess the risk for a clinically relevant digoxin drug-drug interaction by determining the in vitro P-gp inhibitory potency using P-gp expressing cell lines To investigate these questions, we used three other cell lines: MDCK-MDR1-NIH, Caco-2 and CPT-B2, which is the Caco-2 cell with a BCRP knockdown. Digoxin B>A transport across these cells was inhibited by four inhibitors: GF120918, cyclosporine-A (CsA), ketoconazole (KCZ) and verapamil (VRP). The inhibition curves were analyzed to answer two questions: (1) Can the curve be explained solely by the inhibition of P-gp and if not, (2) what is the source of the non-P-gp inhibition portion of the curve. Using our kinetic analysis based upon fitting elementary rate constants, rather than Michaelis-Menten steady-state parameters, we found that in all cases there was substantial non-P-gp inhibition.The next question was that of mechanism. We have examined three different hypotheses to identify the source of the non-P-gp inhibition:The cell line has a basolateral digoxin uptake transporter that is inhibited.Partitioning of inhibitor into the basolateral membrane causes the membrane to become less permeable.There is hetero-dimerization of digoxin with the inhibitor in the basolateral donor chamber that reduces the free digoxin concentration, thereby inhibiting digoxin uptake into the cell.Hypothesis 1 assumes that the cell line has a basolateral digoxin uptake transporter, which has only been clearly implicated for the MDCK-MDR1-NKI cell line BO.Hypothesis 2 appears simplest because nothing beyond partitioning of the inhibitor into the basolateral membrane is required, which is certain, but the mechanism of permeability reduction is unspecified. We found there was substantial non-P-gp inhibition at concentrations of CsA where the measured partition coefficient, K4, which is 20-fold larger than any partition coefficient measured in In For KCZ and VRP, we found substantial non-P-gp inhibition at concentrations where the measured partition coefficients, Hypothesis 3 is a simplified model extrapolated from work showing that at least one P-gp substrate, nicardipine, can form micron sized aggregates Hypothesis 3 would have the inhibitor reduce digoxin uptake by hetero-dimerization, so that the free digoxin concentration would go to zero. By simulations, Since the inhibition curve for GF120918 in all the cells is sigmoidal and the PC plateau is above zero, For KCZ and VRP, hypothesis 2 is unlikely, as discussed above. For these drugs, the PC plateau was not clearly articulated, We have added vinblastine to the list of drugs transported by a basolateral uptake transporter in the MDCK-MDR1-NKI cells. Given that both digoxin and vinblastine have passive permeabilities <100 nm/s, i.e. it would take over a week for passive permeability to achieve 50% of the final steady-state concentration in the receiver chamber B = 100 s−1, i.e. loperamide's value, and we varied it's passive permeability. We fitted these simulations assuming that P-gp was the only transporter involved. Our current working benchmark for fits that may require another transporter is a coefficient of variation CV>0.03 We addressed this question using simulations of a virtual drug. We simulated a P-gp substrate using the kinetic parameters of verapamil in the MDCK-MDR1-NKI cell line, 50 for inhibition of digoxin transport across MDCK-MDR1-NKI, MDCK-MDR1-NIH, Caco-2 and CPT-B2 cells is a convolution of inhibition of P-gp and of a kinetically identified basolateral digoxin uptake mechanism. Three possible sources are discussed for this kinetically identified uptake mechanism: (1) inhibition of an uptake transporter, (2) inhibitor binding to the basolateral outer membrane directly reducing digoxin passive permeability and (3) aggregation between digoxin and inhibitor in basolateral chamber resulting in reduced free digoxin concentrations. The weight of evidence presented here supports the existence of a basolateral digoxin uptake transporter in all cell lines and that GF120918 and CsA can bind to this uptake transporter, inhibiting digoxin uptake by the cells. This model is shown in The structural mass action kinetic analysis has been used to demonstrate that the ICFile S1Appendix S1, Mass Action differential equations for the kinetic model for transport from the mass action reactions shown in Glossary S1, Definitions of variables and parameters used in the Mass Action equations shown in Appendix S1.(DOCX)Click here for additional data file.