Alkylamides from Echinacea are a new class of cannabinomimetics. Cannabinoid type 2 receptor-dependent and -independent immunomodulatory effects

Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor alpha mRNA expression in human monocytes/macrophages via the cannabinoid type 2 (CB2) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563-569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) bind to the CB2 receptor more strongly than the endogenous cannabinoids. The Ki values of A1 and A2 (CB2 approximately 60 nM; CB1 >1500 nM) were determined by displacement of the synthetic high affinity cannabinoid ligand [3H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB2, directed by H-bonding and pi-pi interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB2 and CB1. A1 and A2 elevated total intracellular Ca2+ in CB2-positive but not in CB2-negative promyelocytic HL60 cells, an effect that was inhibited by the CB2 antagonist SR144528. At 50 nM, A1, A2, and the endogenous cannabinoid anandamide (CB2 Ki >200 nM) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB2-dependent manner. A1, A2, anandamide, the CB2 antagonist SR144528 (Ki <10 nM), and also the non-CB2-binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor alpha, IL-1beta, and IL-12p70 expression (5-500 nM) in a CB2-independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB2 binding.

The immune geography of IgA induction and function

The production of immunoglobulin A (IgA) in mammals exceeds all other isotypes, and it is mostly exported across mucous membranes. The discovery of IgA and the realization that it dominates humoral mucosal immunity, in contrast to the IgG dominance of the systemic immune system, was early evidence for the distinct nature of mucosal immunology. It is now clear that IgA can function in high-affinity modes for neutralization of toxins and pathogenic microbes, and as a low-affinity system to contain the dense commensal microbiota within the intestinal lumen. The basic map of induction of IgA B cells in the Peyer's patches, which then circulate through the lymph and bloodstream to seed the mucosa with precursors of plasma cells that produce dimeric IgA for export through the intestinal epithelium, has been known for more than 30 years. In this review, we discuss the mechanisms underlying selective IgA induction of mucosal B cells for IgA production and the immune geography of their homing characteristics. We also review the functionality of secretory IgA directed against both commensal organisms and pathogens.

Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody

BACKGROUND: beta(3)-Integrins are involved in platelet aggregation via alpha(IIb)beta(3) [glycoprotein (GP)IIb-GPIIIa], and in angiogenesis via endothelial alpha(V)beta(3). Cross-reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. OBJECTIVES: In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on beta(3)-integrins. METHODS: Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC(50) were determined by immunodot assays, enzyme-linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb-GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. RESULTS: We observed specific and high-affinity binding to an intact GPIIb-GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose-dependent fibrinogen binding to GPIIb-GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross-reactivity with alpha(V)beta(3), and also demonstrated proangiogenic effects in tube formation and CAM assays. CONCLUSIONS: These Fab fragments are the first entirely human anti-GPIIb-GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual-specificity anti-beta(3)-integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.

Highly efficient in vivo agonist-induced internalization of sst2 receptors in somatostatin target tissues

The successful peptide receptor imaging of tumors, as exemplified for somatostatin receptors, is based on the overexpression of peptide receptors in selected tumors and the high-affinity binding to these tumors of agonist radioligands that are subsequently internalized into the tumor cells in which they accumulate. Although in vitro studies have shown ample evidence that the ligand-receptor complex is internalized, in vivo evidence of agonist-induced internalization of peptide receptors, such as somatostatin receptors, is missing. METHODS: Rats subcutaneously transplanted with the somatostatin receptor subtype 2 (sst(2))-expressing AR42J tumor cells were treated with intravenous injections of various doses of the sst(2) agonist [Tyr(3), Thr(8)]-octreotide (TATE) or of the sst(2) antagonist 1,4,7,10-tetraazacyclododecane-N,N',N'',N''',-tetraacetic acid (DOTA)-Bass and were sacrificed at various times ranging from 2.5 min to 24 h after injection. The tumors and pancreas were then removed from each animal. All tissue samples were processed for sst(2) immunohistochemistry using sst(2)-specific antibodies. RESULTS: Compared with the sst(2) receptors in untreated animals, which localized at the plasma membrane in pancreatic and AR42J tumor cells, the sst(2) receptors in treated animals are detected intracellularly after an intravenous injection of the agonist TATE. Internalization is fast, as the receptors are already internalizing 2.5 min after TATE injection. The process is extremely efficient, as most of the cell surface receptors internalize into the cell and are found in endosomelike structures after TATE injection. The internalization is most likely reversible, because 24 h after injection the receptors are again found at the cell surface. The process is also agonist-dependent, because internalization is seen with high-affinity sst(2) agonists but not with high-affinity sst(2) antagonists. The same internalization properties are seen in pancreatic and AR42J tumor cells. They can further be confirmed in vitro in human embryonic kidney-sst(2) cells, with an immunofluorescence microscopy-based sst(2) internalization assay. CONCLUSION: These animal data strongly indicate that the process of in vivo sst(2) internalization after agonist stimulation is fast, extremely efficient, and fully functional under in vivo conditions in neoplastic and physiologic sst(2) target tissues. This molecular process is, therefore, likely to be responsible for the high and long-lasting uptake of sst(2) radioligands seen in vivo in sst(2)-expressing tumors.

Novel, potent, and radio-iodinatable somatostatin receptor 1 (sst1) selective analogues

The proposed sst(1) pharmacophore (J. Med. Chem. 2005, 48, 523-533) derived from the NMR structures of a family of mono- and dicyclic undecamers was used to design octa-, hepta-, and hexamers with high affinity and selectivity for the somatostatin sst(1) receptor. These compounds were tested for their in vitro binding properties to all five somatostatin (SRIF) receptors using receptor autoradiography; those with high SRIF receptor subtype 1 (sst(1)) affinity and selectivity were shown to be agonists when tested functionally in a luciferase reporter gene assay. Des-AA(1,4-6,10,12,13)-[DTyr(2),DAgl(NMe,2naphthoyl)(8),IAmp(9)]-SRIF-Thr-NH(2) (25) was radio-iodinated ((125)I-25) and specifically labeled sst(1)-expressing cells and tissues. 3D NMR structures were calculated for des-AA(1,4-6,10,12,13)-[DPhe(2),DTrp(8),IAmp(9)]-SRIF-Thr-NH(2) (16), des-AA(1,2,4-6,10,12,13)-[DAgl(NMe,2naphthoyl)(8),IAmp(9)]-SRIF-Thr-NH(2) (23), and des-AA(1,2,4-6,10,12,13)-[DAgl(NMe,2naphthoyl)(8),IAmp(9),Tyr(11)]-SRIF-NH(2) (27) in DMSO. Though the analogues have the sst(1) pharmacophore residues at the previously determined distances from each other, the positioning of the aromatic residues in 16, 23, and 27 is different from that described earlier, suggesting an induced fit mechanism for sst(1) binding of these novel, less constrained sst(1)-selective family members.

Synthesis and in vitro characterization of radioiodinatable benzodiazepines selective for type 1 and type 2 cholecystokinin receptors

Radiolabeled antagonists of specific peptide receptors identify a higher number of receptor binding sites than agonists and may thus be preferable for in vivo tumor targeting. In this study, two novel radioiodinated 1,4-benzodiazepines, (S)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (9) and (R)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (7), were developed. They were characterized in vitro as high affinity selective antagonists at cholecystokinin types 1 and 2 (CCK(1) and CCK(2)) receptors using receptor binding, calcium mobilization, and internalization studies. Their binding to human tumor tissues was assessed with in vitro receptor autoradiography and compared with an established peptidic CCK agonist radioligand. The (125)I-labeled CCK(1) receptor-selective compound 9 often revealed a substantially higher amount of CCK(1) receptor binding sites in tumors than the agonist (125)I-CCK. Conversely, the radioiodinated CCK(2) receptor-selective compound 7 showed generally weaker tumor binding than (125)I-CCK. In conclusion, compound 9 is an excellent radioiodinated nonpeptidic antagonist ligand for direct and selective labeling of CCK(1) receptors in vitro. Moreover, it represents a suitable candidate to test antagonist binding to CCK(1) receptor-expressing tumors in vivo.

Time-dependent interactions of the two porphyrinic compounds chlorin e6 and mono-L-aspartyl-chlorin e6 with phospholipid vesicles probed by NMR spectroscopy

The distribution processes of chlorin e6 (CE) and monoaspartyl-chlorin e6 (MACE) between the outer and inner phospholipid monolayers of 1,2-dioleoyl-phosphatidylcholine (DOPC) vesicles were monitored by 1H NMR spectroscopy through analysis of chemical shifts and line widths of the DOPC vesicle resonances. Chlorin adsorption to the outer vesicle monolayer induced changes in the DOPC 1H NMR spectrum. Most pronounced was a split of the N-methyl choline resonance, allowing for separate analysis of inner and outer vesicle layers. Transbilayer distribution of the chlorin compounds was indicated by time-dependent characteristic spectral changes of the DOPC resonances. Kinetic parameters for the flip-flop processes, that is, half-lives and rate constants, were obtained from the experimental data points. In comparison to CE, MACE transbilayer movement was significantly reduced, with MACE remaining more or less attached to the outer membrane layer. The distribution coefficients for CE and MACE between the vesicular and aqueous phase were determined. Both CE and MACE exhibited a high affinity for the vesicular phase. For CE, a positive correlation was found between transfer rate and increasing molar ratio CE/DOPC. Enhanced membrane rigidity induced by increasing amounts of cholesterol into the model membrane was accompanied by a decrease of CE flip-flop rates across the membrane. The present study shows that the movement of porphyrins across membranes can efficiently be investigated by 1H NMR spectroscopy and that small changes in porphyrin structure can have large effects on membrane kinetics.

Marked improvement in nail psoriasis during treatment with adalimumab

BACKGROUND: Nail involvement is known as a common finding in psoriatic patients and represents a significant impact on patients' quality of life. The treatment of nail psoriasis is often challenging, and there is a need for new therapeutic options. Biologicals effective in the treatment of moderate to severe chronic plaque psoriasis may represent a new therapeutic modality for this disease. Adalimumab is a fully human IgG1 monoclonal antibody that binds to tumor necrosis factor alpha with high affinity and specificity. OBSERVATIONS: We report two cases of rapid improvement in nail psoriasis under adalimumab monotherapy with maintained effectiveness despite intermittent treatment as well as long remission after therapy discontinuation. CONCLUSION: The marked improvement of our two cases indicates that adalimumab may also help ameliorate nail psoriasis and warrants further controlled studies to establish the effectiveness and therapeutic regimes.

The basophil activation test in immediate-type drug allergy

Diagnosis of drug allergy involves first the recognition of sometimes unusual symptoms as drug allergy and, second, the identification of the eliciting drug. This is an often difficult task, as the clinical picture and underlying pathomechanisms are heterogeneous. In clinical routine, physicians frequently have to rely upon a suggestive history and eventual provocation tests, both having their specific limitations. For this reason both in vivo (skin tests) and in vitro tests are investigated intensively as tools to identify the disease-eliciting drug. One of the tests evaluated in drug allergy is the basophil activation test (BAT). Basophils with their high-affinity IgE receptors are easily accessible and therefore can be used as indicator cells for IgE-mediated reactions. Upon allergen challenge and cross-linking of membrane-bound IgE antibodies (via Fc-epsilon-RI) basophils up-regulate certain activation markers on their surface such as CD63 and CD203c, as well as intracellular markers (eg, phosphorylated p38MAPK). In BAT, these alterations can be detected rapidly on a single-cell basis by multicolor flow cytometry using specific monoclonal antibodies. Combining this technique with in vitro passive sensitization of donor basophils with patients' serum, one can prove the IgE dependence of a drug reaction. This article summarizes the authors' current experience with the BAT in the diagnostic management of immediate-type drug allergy mediated by drug-specific IgE antibodies.

Antibody responses induced by long-term vaccination with an octovalent conjugate Pseudomonas aeruginosa vaccine in children with cystic fibrosis

We assessed the serological responses over 10 years to repeated immunization of cystic fibrosis (CF) patients with an O-polysaccharide (OPS)-toxin A conjugate vaccine against Pseudomonas aeruginosa. A retrospective analysis was performed with sera from 25 vaccinated and 25 unvaccinated children treated at the same CF centre and matched for clinical management, age and gender. Yearly immunization led to sustained elevations of serum immunoglobulin G (IgG) antibody levels to all vaccine components. Eighteen unvaccinated patients but only eight vaccinated ones developed chronic pseudomonal lung infections. Infection rapidly caused further marked elevations of polysaccharide- but not toxin A-specific serum IgG in both immunized and nonimmunized patients, indicating that protection did not depend on the quantity of IgG present. However, qualitative analyses revealed that the protective capacity of specific serum IgG antibodies was linked to high affinity and to specificity for OPS serotypes rather than for lipopolysaccharide core epitopes.

DARPins as bispecific receptor antagonists analyzed for immunoglobulin E receptor blockage

The concept of multispecific antibodies is of high therapeutic interest but has failed to produce pharmaceutical products due to the poor biophysical properties of such molecules. Here, we propose an alternative and simple way to generate bispecific binding molecules using designed ankyrin repeat proteins (DARPins). For this purpose, monovalent DARPins with different epitope specificities were selected against the alpha chain of the high-affinity receptor for human immunoglobulin E (IgE) (FcepsilonRIalpha). Two of the isolated binders interfering with IgE binding to the receptor were joined to each other or to themselves via a flexible protein linker. The resulting bivalent and bispecific DARPins were tested for their ability to prevent allergen-induced cell degranulation using rat basophilic leukemia cells stably transfected with human FcepsilonRIalpha. The bispecific DARPin construct was the most potent one, efficiently blocking the IgE-FcepsilonRI interaction and preventing the release of proinflammatory mediators. Noteworthy, the multivalent and multispecific DARPin construct did not show any alteration of the beneficial biophysical properties of the monovalent parental DARPins. Hence, bispecific DARPins may be used to generate receptor antagonists simultaneously targeting different epitopes on the same molecule. Moreover, they easily overcome the limiting immunoglobulin binding paradigm (one binding molecule=one epitope) and thereby represent an alternative to monoclonal antibodies in cases where the immunoglobulin scaffold is unsuitable.

RNA-Seq-based analysis of the physiologic cold shock-induced changes in Moraxella catarrhalis gene expression

BACKGROUND Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. The prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis is greatest in winter. We investigated how M. catarrhalis uses the physiologic exposure to cold air to regulate pivotal survival systems that may contribute to M. catarrhalis virulence. RESULTS In this study we used the RNA-seq techniques to quantitatively catalogue the transcriptome of M. catarrhalis exposed to a 26 °C cold shock or to continuous growth at 37 °C. Validation of RNA-seq data using quantitative RT-PCR analysis demonstrated the RNA-seq results to be highly reliable. We observed that a 26 °C cold shock induces the expression of genes that in other bacteria have been related to virulence a strong induction was observed for genes involved in high affinity phosphate transport and iron acquisition, indicating that M. catarrhalis makes a better use of both phosphate and iron resources after exposure to cold shock. We detected the induction of genes involved in nitrogen metabolism, as well as several outer membrane proteins, including ompA, m35-like porin and multidrug efflux pump (acrAB) indicating that M. catarrhalis remodels its membrane components in response to downshift of temperature. Furthermore, we demonstrate that a 26 °C cold shock enhances the induction of genes encoding the type IV pili that are essential for natural transformation, and increases the genetic competence of M. catarrhalis, which may facilitate the rapid spread and acquisition of novel virulence-associated genes. CONCLUSION Cold shock at a physiologically relevant temperature of 26 °C induces in M. catarrhalis a complex of adaptive mechanisms that could convey novel pathogenic functions and may contribute to enhanced colonization and virulence.

Sulfamethoxazole induces a switch mechanism in T cell receptors containing TCRVβ20-1, altering pHLA recognition

T cell receptors (TCR) containing Vβ20-1 have been implicated in a wide range of T cell mediated disease and allergic reactions, making it a target for understanding these. Mechanics of T cell receptors are largely unexplained by static structures available from x-ray crystallographic studies. A small number of molecular dynamic simulations have been conducted on TCR, however are currently lacking either portions of the receptor or explanations for differences between binding and non-binding TCR recognition of respective peptide-HLA. We performed molecular dynamic simulations of a TCR containing variable domain Vβ20-1, sequenced from drug responsive T cells. These were initially from a patient showing maculopapular eruptions in response to the sulfanilamide-antibiotic sulfamethoxazole (SMX). The CDR2β domain of this TCR was found to dock SMX with high affinity. Using this compound as a perturbation, overall mechanisms involved in responses mediated by this receptor were explored, showing a chemical action on the TCR free from HLA or peptide interaction. Our simulations show two completely separate modes of binding cognate peptide-HLA complexes, with an increased affinity induced by SMX bound to the Vβ20-1. Overall binding of the TCR is mediated through a primary recognition by either the variable β or α domain, and a switch in recognition within these across TCR loops contacting the peptide and HLA occurs when SMX is present in the CDR2β loop. Large binding affinity differences are induced by summed small amino acid changes primarily by SMX modifying only three critical CDR2β loop amino acid positions. These residues, TYRβ57, ASPβ64, and LYSβ65 initially hold hydrogen bonds from the CDR2β to adjacent CDR loops. Effects from SMX binding are amplified and traverse longer distances through internal TCR hydrogen bonding networks, controlling the overall TCR conformation. Thus, the CDR2β of Vβ20-1 acts as a ligand controlled switch affecting overall TCR binding affinity.

Peptide transporter DtpA has two alternate conformations, one of which is promoted by inhibitor binding

Peptide transporters (PTRs) of the large PTR family facilitate the uptake of di- and tripeptides to provide cells with amino acids for protein synthesis and for metabolic intermediates. Although several PTRs have been structurally and functionally characterized, how drugs modulate peptide transport remains unclear. To obtain insight into this mechanism, we characterize inhibitor binding to the Escherichia coli PTR dipeptide and tripeptide permease A (DtpA), which shows substrate specificities similar to its human homolog hPEPT1. After demonstrating that Lys[Z-NO2]-Val, the strongest inhibitor of hPEPT1, also acts as a high-affinity inhibitor for DtpA, we used single-molecule force spectroscopy to localize the structural segments stabilizing the peptide transporter and investigated which of these structural segments change stability upon inhibitor binding. This characterization was done with DtpA embedded in the lipid membrane and exposed to physiologically relevant conditions. In the unbound state, DtpA adopts two main alternate conformations in which transmembrane α-helix (TMH) 2 is either stabilized (in ∼43% of DtpA molecules) or not (in ∼57% of DtpA molecules). The two conformations are understood to represent the inward- and outward-facing conformational states of the transporter. With increasing inhibitor concentration, the conformation characterized by a stabilized TMH 2 becomes increasingly prevalent, reaching ∼92% at saturation. Our measurements further suggest that Lys[Z-NO2]-Val interacts with discrete residues in TMH 2 that are important for ligand binding and substrate affinity. These interactions in turn stabilize TMH 2, thereby promoting the inhibited conformation of DtpA.

Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Na v1.5 expressed in HEK293 cells

Background: The pore-forming subunit of the cardiac sodium channel, Na v1.5, has been previously found to be mutated in genetically determined arrhythmias. Na v1.5 associates with many proteins that regulate its function and cellular localisation. In order to identify more in situ Na v1.5 interacting proteins, genetically-modified mice with a high-affinity epitope in the sequence of Na v1.5 can be generated. Methods: In this short study, we (1) compared the biophysical properties of the sodium current (I Na) generated by the mouse Na v1.5 (mNa v1.5) and human Na v1.5 (hNa v1.5) constructs that were expressed in HEK293 cells, and (2) investigated the possible alterations of the biophysical properties of the human Na v1.5 construct that was modified with specific epitopes. Results: The biophysical properties of mNa v1.5 were similar to the human homolog. Addition of epitopes either up-stream of the N-terminus of hNa v1.5 or in the extracellular loop between the S5 and S6 transmembrane segments of domain 1, significantly decreased the amount of I Na and slightly altered its biophysical properties. Adding green fluorescent protein (GFP) to the N-terminus did not modify any of the measured biophysical properties of hNa v1.5. Conclusions: These findings have to be taken into account when planning to generate genetically-modified mouse models that harbour specific epitopes in the gene encoding mNa v1.5.