The blood endothelial cell chamber - An innovative system to study immune responses in drug development.

The risk for adverse immune-mediated reactions, associated with the administration of certain immunotherapeutic agents, should be mitigated early. Infusion reactions to monoclonal antibodies and other biopharmaceuticals, known as cytokine release syndrome, can arise from the release of cytokines via the drug target cell, as well as the recruitment of immune effector cells. While several in vitro cytokine release assays have been proposed up to date, many of them lack important blood components, required for this response to occur. The blood endothelial cell chamber model is an in vitro assay, composed of freshly drawn human whole blood and cultured human primary endothelial cells. Herein, its potential to study the compatibility of immunotherapeutics with the human immune system was studied by evaluating three commercially available monoclonal antibodies and bacterial endotoxin lipopolysaccharide. We demonstrate that the anti-CD28 antibody TGN1412 displayed an adaptive cytokine release profile and a distinct IL-2 response, accompanied with increased CD3+ cell recruitment. Alemtuzumab exhibited a clear cytokine response with a mixed adaptive/innate source (IFNγ, TNFα and IL-6). Its immunosuppressive nature is observed in depleted CD3+ cells. Cetuximab, associated with low infusion reactions, showed a very low or absent stimulatory effect on proinflammatory cytokines. In contrast, bacterial endotoxin demonstrated a clear innate cytokine response, defined by TNFα, IL-6 and IL-1ß release, accompanied with a strong recruitment of CD14+CD16+ cells. Therefore, the blood endothelial cell chamber model is presented as a valuable in vitro tool to investigate therapeutic monoclonal antibodies with respect to cytokine release and vascular immune cell recruitment.

Combined Inhibition of C5 and CD14 Attenuates Systemic Inflammation in a Piglet Model of Meconium Aspiration Syndrome.

BACKGROUND: Meconium aspiration syndrome (MAS) is a severe lung condition affecting newborns and it can lead to a systemic inflammatory response. We previously documented complement activation and cytokine release in a piglet MAS model. Additionally, we showed ex vivo that meconium-induced inflammation was dependent on complement and Toll-like receptors. OBJECTIVES: To assess the efficacy of the combined inhibition of complement (C5) and CD14 on systemic inflammation induced in a forceful piglet MAS model. METHODS: Thirty piglets were randomly allocated to a treatment group receiving the C5-inhibitor SOBI002 and anti-CD14 (n = 15) and a nontreated control group (n = 15). MAS was induced by intratracheal meconium instillation, and the piglets were observed for 5 h. Complement, cytokines, and myeloperoxidase (MPO) were measured by ELISA. RESULTS: SOBI002 ablated C5 activity and the formation of the terminal complement complex in vivo. The combined inhibition attenuated the inflammasome cytokines IL-1ß and IL-6 by 60 (p = 0.029) and 44% (p = 0.01), respectively, and also MPO activity in the bronchoalveolar fluid by 42% (p = 0.017). Ex vivo experiments in human blood revealed that the combined regimen attenuated meconium-induced MPO release by 64% (p = 0.008), but there was only a negligible effect with single inhibition, indicating a synergic cross-talk between the key molecules C5 and CD14. CONCLUSION: Combined inhibition of C5 and CD14 attenuates meconium-induced inflammation in vivo and this could become a future therapeutic regimen for MAS.

Stratification of responders towards eculizumab using a structural epitope mapping strategy.

The complement component 5 (C5)-binding antibody eculizumab is used to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome (aHUS). As recently reported there is a need for a precise classification of eculizumab responsive patients to allow for a safe and cost-effective treatment. To allow for such stratification, knowledge of the precise binding site of the drug on its target is crucial. Using a structural epitope mapping strategy based on bacterial surface display, flow cytometric sorting and validation via haemolytic activity testing, we identified six residues essential for binding of eculizumab to C5. This epitope co-localizes with the contact area recently identified by crystallography and includes positions in C5 mutated in non-responders. The identified epitope also includes residue W917, which is unique for human C5 and explains the observed lack of cross-reactivity for eculizumab with other primates. We could demonstrate that Ornithodorus moubata complement inhibitor (OmCI), in contrast to eculizumab, maintained anti-haemolytic function for mutations in any of the six epitope residues, thus representing a possible alternative treatment for patients non-responsive to eculizumab. The method for stratification of patients described here allows for precision medicine and should be applicable to several other diseases and therapeutics.

A truncated analogue of CCL2 mediates anti-fibrotic effects on murine fibroblasts independently of CCR2.

The truncated [1+9-76] CCL2 analogue, also known as 7ND, has been described in numerous reports as an anti-inflammatory and anti-fibrotic agent in a wide spectrum of animal models, e.g. models of cardiovascular disease, graft versus host disease and bleomycin-induced pulmonary fibrosis. 7ND has been reported to function as a competitive inhibitor of CCL2 signaling via CCR2 in human in vitro systems. In contrast, the mechanistic basis of 7ND action in animal models has not been previously reported. Here we have studied how 7ND interacts with CCL2 and CCR2 of murine origin. Surprisingly, 7ND was shown to be a weak inhibitor of murine CCL2/CCR2 signaling and displaced murine CCL2 (JE) from the receptor with a K(i)>1 µM. Using surface plasmon resonance, we found that 7ND binds murine CCL2 with a K(d) of 670 nM, which may indicate that 7ND inhibits murine CCL2/CCR2 signaling by a dominant negative mechanism rather than by competitive binding to the CCR2 receptor. In addition we observed that sub-nanomolar levels of 7ND mediate anti-fibrotic effects in CCR2 negative fibroblasts cultured from fibrotic lung of bleomycin-induced mice. Basal levels of extracellular matrix proteins were reduced (collagen type 1 and fibronectin) as well as expression levels of α-smooth muscle actin and CCL2. Our conclusion from these data is that the previously reported effects of 7ND in murine disease models most probably are mediated via mechanisms independent of CCR2.

SAR studies of capsazepinoid bronchodilators 3: The thiourea part (coupling region) and the 2-(4-chlorophenyl)ethyl moiety (C-region).

Certain derivatives and analogues of capsazepine are potent in vitro inhibitors of bronchoconstriction in human small airways. During an investigation of the dependency of the potency on the structural features of the capsazepinoids in the thiourea moiety (coupling region) and the 2-(4-chlorophenyl)ethyl moiety (C-region), it was revealed that capsazepinoids with a thiourea or an amide link between the B-ring and the C-region in general have a good bronchorelaxing activity, while urea is a less attractive choice. Further, it was shown that 1,2,3,4-tetrahydroisoquinolines with a 2-(phenyl)ethyl derivative as the C-region are considerably more potent than those with an octyl group, while 2,3,4,5-tetrahydro-1H-2-benzazepines were found to be more insensitive to the nature of the C-region.

SAR studies of capsazepinoid bronchodilators. Part 2: Chlorination and catechol replacement in the A-ring.

Capsazepine as well as its derivatives and analogues are general inhibitors of constriction of human small airways. From a systematic variation of the capsazepine structure, divided into four regions, SARs were established. This paper concerns the chlorination of the A-ring as well as the replacement of the catechol with bioisosteric groups. It is revealed that chlorination of the A-ring has a profound effect on activity. Moreover, di-chlorination of the 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline structure results in a 10-fold increase in potency compared to capsazepine.

SAR studies of capsazepinoid bronchodilators. Part 1: The importance of the catechol moiety and aspects of the B-ring structure.

Capsazepine as well as its derivatives and analogues are general inhibitors of constriction of human small airways. From a systematic variation of the capsazepine structure, divided into four regions, SARs were established. This part concerns the catechol moiety of the A-ring as well as the 2,3,4,5-tetrahydro-1H-2-azepine moiety (the B-ring) of capsazepine. It is revealed that a conformational constrain (as a fused ring) is important and that compounds with a six-membered B-ring (as a 1,2,3,4-tetrahydroisoquinoline) in general are more potent than the corresponding isoindoline, 2,3,4,5-tetrahydro-1H-2-benzazepine and 2,3,4,5-tetrahydro-1H-3-benzazepine derivatives.

Discovery of a potent and long-acting bronchorelaxing capsazepinoid, RESPIR 4-95.

BACKGROUND: Current drugs including beta-agonists have limited smooth muscle relaxant effects on human small airways. Yet this is a major site of obstruction in asthma and chronic obstructive pulmonary disease (COPD). OBJECTIVE: This study explores human small airway relaxant effects of RESPIR 4-95, a novel chemical analogue (capsazepinoid) to capsazepine. Capsazepine was recently shown to relax small airways in a way which was independent of its TRPV1 antagonism and independent of current bronchodilator drug mechanisms. METHOD: In vitro preparations of human small airways, 0.5-1.5mm in diameter and responding with reproducible contractions to leukotriene D4 (LTD4) for 12h, were used. RESULTS: RESPIR 4-95 reversibly prevented LTD4-induced contractions as well as relaxed the established tonic contraction by LTD4. RESPIR 4-95 exhibited marked improvements over the reference capsazepinoid, capsazepine, by being 10 times more potent, exhibiting twice as long duration of action after wash-out (9h), and inhibiting equally well LTD4-, histamine-, prostaglandin D2 (PGD2)-, and acetylcholine (ACh)-induced contractions. RESPIR 4-95 was distinguished from l-type calcium channel antagonist nifedipine by its greater efficacy and potency and by exhibiting increased relaxant effect by repeated exposures. Furthermore, RESPIR 4-95 was more efficacious and longer acting than the long-acting beta-agonist formoterol. CONCLUSION: Efficacy, potency, duration of action, and inexhaustibility of its relaxation of human small airways make RESPIR 4-95 an interesting lead compound for further developments aiming at drug treatment of small airway obstruction in asthma and COPD. Further work is warranted to unveil the molecular biology behind its relaxant actions.

Co-expression of neuropeptide Y Y1 and Y5 receptors results in heterodimerization and altered functional properties.

Centrally administered neuropeptide Y (NPY) produces anxiolytic and orexigenic effects by interacting with Y1 and Y5 receptors that are colocalized in many brain regions. Therefore, we tested the hypothesis that co-expression of Y1 and Y5 receptors results in heterodimerization, altered pharmacological properties and altered desensitization. To accomplish this, the carboxyl-termini of Y1 and Y5 receptors were fused with Renilla luciferase and green fluorescent protein and the proximity of the tagged receptors assessed using bioluminescent resonance energy transfer. Under basal conditions, cotransfection of tagged Y1 receptor and Y5 produced a substantial dimerization signal that was unaffected by the endogenous, nonselective agonists, NPY and peptide YY (PYY). Selective Y5 agonists produced an increase in the dimerization signal while Y5 antagonists also produced a slight but significant increase. In the absence of agonists, selective antagonists decreased dimerization. In functional studies, Y5 agonists produced a greater inhibition of adenylyl cyclase activity in Y1/Y5 cells than cells expressing Y5 alone while NPY and PYY exhibited no difference. With PYY stimulation, the Y1 antagonist became inactive and the Y5 antagonist exhibited uncompetitive kinetics in the Y1/Y5 cell line. In confocal microscopy studies, Y1/Y5 co-expression resulted in increased Y5 signaling following PYY stimulation. Addition of both Y1 and Y5 receptor antagonists was required to significantly decrease PYY-induced internalization. Therefore, Y1/Y5 co-expression results in heterodimerization, altered agonist and antagonist responses and reduced internalization rate. These results may account for the complex pharmacology observed when assessing the responses to NPY and analogs in vivo.

Nurses' and nurse students' demands of functions and usability in a PDA.

INTRODUCTION: Healthcare organisations are nowadays expecting the support of IT in the daily routines. Personal digital assistants (PDAs) are in use in some healthcare organisations but in an irregular and unplanned way. The aim of the present study was to describe nurses' and nurse students' demands of functions and usability in a PDA. METHODS: Interviews were made with 12 nurses at the County Hospital of Kalmar and a questionnaire was given to nurse students (n=84) in their last, i.e. third, year at the Department of Health and Behavioural Sciences, University of Kalmar. RESULTS: There was a need for nurses to make the information in general more rich and efficient by means of a PDA. In a PDA, the nurses and nurse students expected access to information about the patients, knowledge resources and functions for their daily work. CONCLUSIONS: The nurses and nurse students had high expectations of a PDA for information retrieval. A PDA has the potential to be accepted as a supportive tool in healthcare organisations if it contains the demanded content which must be adapted to the users' needs and the general IT-system used, and the PDA must have a user friendly design.

Effects of capsazepine on human small airway responsiveness unravel a novel class of bronchorelaxants.

Capsazepine is known as a transient receptor potential channel vanilloid subfamily 1 (TRPV(1)) antagonist that inhibits bronchoconstriction evoked in animals by TRPV(1) agonists. In this study, effects of capsazepine and chemically related analogues, so called capsazepinoids, were examined in vitro on contractile effects in human small airway preparations. Repeated cycles with 1h of LTD(4)-free physiological saline solution followed by 30min exposure to LTD(4) (10nM) demonstrated that the contractile responsiveness of the preparations exhibited little change over time despite repeated challenges (>12h). Capsazepine (1-100microM) reversibly and concentration-dependently inhibited the contractile response to LTD(4) with EC(50) approximately 10microM and approximately 90% relaxation at 100microM. Capsazepine (10microM) was approximately equally effective to attenuate the contractions evoked by several different inflammatory contractile agonists (LTD(4), PGD(2), histamine), and it relaxed preparations with established tonic contraction due to LTD(4). Higher concentrations of capsazepine were needed to relax ACh-contractions. The effect of capsazepine on LTD(4)-induced contractions was not significantly reduced by pre-treating the preparations with either of propranolol (10microM)+atropine (1microM), L-NAME (1mM), indomethacin (1microM), iberiotoxin (0.1microM), capsaicin (10microM), and nifedipine (10microM). Although the mechanism of action of the present capsazepine-induced bronchorelaxation remains unknown it emerged here that they represent a generally effective principle exerting a functional antagonism against contractile mediators but distinct from beta receptor agonists and inhibitors of L-type calcium channels. The inhibitory effect of capsazepine is shared by chemical analogues, but not with other TRPV(1) antagonists, suggesting the possibility that capsazepine represents a novel class of bronchorelaxants effective in human small airways. These findings were not predicted by previous observations that have concerned quite limited effects of capsazepine on airway tone in different animal test systems. If potency can be further increased and the results translated to in vivo, compounds representing the capsazepinoid class of bronchorelaxants might become useful in the treatment of patients suffering from asthma and COPD.

Re-evaluation of receptor-ligand interactions of the human neuropeptide Y receptor Y1: a site-directed mutagenesis study.

Interactions of the human NPY (neuropeptide Y) receptor Y1 with the two endogenous agonists NPY and peptide YY and two non-peptide antagonists were investigated using site-directed mutagenesis at 17 positions. The present study was triggered by contradictions among previously published reports and conclusions that seemed inconsistent with sequence comparisons across species and receptor subtypes. Our results show that Asp287, at the border between TM (transmembrane) region 6 and EL3 (extracellular loop 3) influences peptide binding, while two aspartic residues in EL2 do not, in agreement with some previous studies but in disagreement with others. A hydrophobic pocket of the Y1 receptor consisting of Tyr100 (TM2), Phe286 (TM6) and His298 (EL3) has been proposed to interact with the amidated C-terminus of NPY, a theory that is unsupported by sequence comparisons between Y1, Y2 and Y5. Nevertheless, our results confirm that these amino acid residues are critical for peptide binding, but probably interact with NPY differently than proposed previously. Studies with the Y1-selective antagonist SR120819A identified a new site of interaction at Asn116 in TM3. Position Phe173 in TM4 is also important for binding of this antagonist. In contrast with previous reports, we found that Phe173 is not crucial for the binding of BIBP3226, another selective Y1 receptor antagonist. Also, we found that position Thr212 (TM5) is important for binding of both antagonists. Our mutagenesis results and our three-dimensional model of the receptor based on the high-resolution structure of bovine rhodopsin suggest new interactions for agonist as well as antagonist binding to the Y1 receptor.

Exploring the context of the lung proteome within the airway mucosa following allergen challenge.

The lung proteome is a dynamic collection of specialized proteins related to pulmonary function. Many cells of different derivations, activation states, and levels of maturity contribute to the changing environment, which produces the lung proteome. Inflammatory cells reacting to environmental challenge, for example from allergens, produce and secrete proteins which have profound effects on both resident and nonresident cells located in airways, alveoli, and the vascular tree which provides blood cells to the parenchyma alveolar bed for gas exchange. In an experimental model of allergic airway inflammation, we have compared control and allergen challenged lung compartments to determine global protein expression patterns using 2D-gel electrophoresis and subsequent spot identification by MS/MS mass spectrometry. We have then specifically isolated the epithelial mucosal layer, which lines conducting airways, from control and allergen challenged lungs, using laser capture technology and performed proteome identification on these selected cell samples. A central component of our investigations has been to contextually relate the histological features of the dynamic pulmonary environment to the changes in protein expression observed following challenge. Our results provide new information of the complexity of the submucosa/epithelium interface and the mechanisms behind the transformation of airway epithelium from normal steady states to functionally activated states.

Targeting hepatocytes from liver tissue by laser capture microdissection and proteomics expression profiling.

A tissue proteomics process is presented where hepatocyte cell isolation in combination with two-dimensional (2-D) gel electrophoresis and mass spectrometric identification were used to annotate the liver proteome. Laser microdissection of 8 microm liver tissue sections was performed and protein expression profiling was compared using a variety of quantities of input cells, and gel separation conditions. The 30 microm diameter laser generated the highest protein yields from the polymer coated caps following microsolubilization. We found that 6000 laser pulses (approximately 7200 hepatocytes) were required in order to generate high-resolution gel maps. Within homogeneous tissue samples, this could be accomplished in a total cycle time of 20 min using an automated dissection procedure. Close to 1000 high-quality gel annotations were generated from the corresponding 2-D gel expression profiles which matched closely the corresponding patterns of analytical-scale liver preparations detected by silver staining.

High affinity agonistic metal ion binding sites within the melanocortin 4 receptor illustrate conformational change of transmembrane region 3.

We created a molecular model of the human melanocortin 4 receptor (MC4R) and introduced a series of His residues into the receptor protein to form metal ion binding sites. We were able to insert micromolar affinity binding sites for zinc between transmembrane region (TM) 2 and TM3 where the metal ion alone was able to activate this peptide binding G-protein-coupled receptor. The exact conformation of the metal ion interactions allowed us to predict the orientation of the helices, and remodeling of the receptor protein indicated that Glu100 and Ile104 in TM2 and Asp122 and Ile125 in TM3 are directed toward a putative area of activation of the receptor. The molecular model suggests that a rotation of TM3 may be important for activation of the MC4R. Previous models of G-protein-coupled receptors have suggested that unlocking of a stabilizing interaction between the DRY motif, in the cytosolic part of TM3, and TM6 is important for the activation process. We suggest that this unlocking process may be facilitated through creation of a new interaction between TM3 and TM2 in the MC4R.

Neuropeptide Y Y4 receptor homodimers dissociate upon agonist stimulation.

The pancreatic polypeptide-fold family of peptides consists of three 36-amino acid peptides, namely neuropeptide Y (NPY), peptide YY, and pancreatic polypeptide (PP). These peptides regulate important functions, including food intake, circadian rhythms, mood, blood pressure, intestinal secretion, and gut motility, through four receptors: Y1, Y2, Y4, and Y5. Additional receptor subtypes have been proposed based on pharmacology observed in native tissues. Recent studies with other G-protein-coupled receptors have shown that homo- and heterodimerization may be important in determining receptor function and pharmacology. In the present study, the recently cloned rhesus (rh) Y4 receptor was evaluated using radioligand binding, and the pharmacological profile was found to be very similar to the human Y4 receptor. To study homo- and heterodimerization involving the Y4 receptor using bioluminescence resonance energy transfer 2 (BRET(2)), the carboxy termini of the rhesus Y1, Y2, Y4, and Y5 receptors were fused to Renilla luciferase, and rhY4 was also fused to green fluorescent protein. Dimerization was also studied using Western blot analysis. Using both BRET(2) and Western analysis, we found that the rhY4 receptor is present at the cell surface as a homodimer. Furthermore, agonist stimulation using the Y4-selective agonists PP and 1229U91 can dissociate these dimers in a concentration-dependent manner. In contrast, rhY4 did not heterodimerize with other members of the NPY receptor family or with human opioid delta and mu receptors. Therefore, homodimerization is an important component in the regulation of the Y4 receptor.

The use of bioluminescence resonance energy transfer 2 to study neuropeptide Y receptor agonist-induced beta-arrestin 2 interaction.

The neuropeptide Y (NPY) family peptides NPY, peptide YY (PYY), and pancreatic polypeptide (PP) bind to four G protein-coupled receptors (GPCRs): Y1, Y2, Y4, and Y5. A key step in the desensitization and internalization of GPCRs is the association of the receptor with beta-arrestins. In the present study, these receptors were analyzed with respect to their ability to interact with GFP2-tagged beta-arrestin 2 using the new bioluminescence resonance energy transfer 2 method. Agonists induced a concentration-dependent association of beta-arrestin 2 with all four receptors. Whereas the Y1 receptor exhibited the highest maximum response and rapid association (t(1/2) = 3.4 min), the maximal signals for the association of Y2 and Y4 receptors were less than half of that of Y1, and the association rates were much slower. Interestingly, when evaluated at the Y4 receptor, the Y4 agonist 1229U91 [(Ile,Glu,Pro,Dpr,Tyr,Arg, Leu,Arg,Try-NH2)-2-cyclic(2,4'),(2',4)-diamide] was unable to provoke the same maximal response as human PP, suggesting that 1229U91 is a partial agonist. When stimulated by PYY, the Y5 receptor responded with a t(1/2) of 4.6 min and a maximal response approximately 60% of what was observed with Y1. Because beta-arrestins are key components in GPCR internalization, it is interesting to note that the receptor that is known to internalize rapidly (Y1) exhibits the most rapid association with beta-arrestin 2, whereas the receptor that is known to internalize slowly, or not at all (Y2) associates slowly with beta-arrestin 2.

Recent developments in our understanding of the physiological role of PP-fold peptide receptor subtypes.

The three peptides pancreatic polypeptide (PP), peptide YY (PYY), and neuropeptide Y (NPY) share a similar structure known as the PP-fold. There are four known human G-protein coupled receptors for the PP-fold peptides, namely Y1, Y2, Y4, and Y5, each of them being able to bind at least two of the three endogenous ligands. All three peptides are found in the circulation acting as hormones. Although NPY is only released from neurons, PYY and PP are primarily found in endocrine cells in the gut, where they exert such effects as inhibition of gall bladder secretion, gut motility, and pancreatic secretion. However, when PYY is administered in an experimental setting to animals, cloned receptors, or tissue preparations, it can mimic the effects of NPY in essentially all studies, making it difficult to study the effects of PP-fold peptides and to delineate what receptor and peptide accounts for a particular effect. Initial studies with transgenic animals confirmed the well-established action of NPY on metabolism, food-intake, vascular systems, memory, mood, neuronal excitability, and reproduction. More recently, using transgenic techniques and novel antagonists for the Y1, Y2, and Y5 receptors, NPY has been found to be a key player in the regulation of ethanol consumption and neuronal development.

Reciprocal mutations of neuropeptide Y receptor Y2 in human and chicken identify amino acids important for antagonist binding.

The neuropeptide Y (NPY) receptor Y2 antagonist BIIE0246 has sub-nanomolar affinity for the human Y2 (hY2) receptor but binds very poorly to chicken Y2 (chY2) with micromolar affinity. Sequence comparisons identified several amino acids for investigation by mutagenesis. Reciprocal mutagenesis between hY2 and chY2 revealed that three of these, individually and in combination, are important for BIIE0246 binding, namely positions Gln(135) in transmembrane (TM) 3, Leu(227) in TM5, and Leu(284) in TM6. Mutagenesis of hY2 to the corresponding amino in chY2 (generating hY2[Q135H,L227Q,L284F]) made the affinity of BIIE0246 as low as for chY2. Introduction into chY2 of the three human residues resulted in antagonist affinity almost as high as for hY2. To distinguish between direct and indirect effects, each of the three residues in hY2 was replaced with alanine. BIIE0246 bound with 28-fold lower affinity to hY2[L227A], suggesting the Leu(227) interacts directly with the antagonist. The other two alanine mutants bound with unaltered affinity, suggesting that the corresponding chY2 residues abolish binding through steric hindrance or charge repulsion. Thus, three amino acid residues can in an additive manner completely account for the difference in antagonist binding between the hY2 and chY2 receptors. These results will be useful for construction of three-dimensional models of the widely divergent NPY receptor subtypes.