Search

The use of fluorescence is a valuable and increasingly accessible means of probing the pharmacology and physiology of cells and their receptors. To date, the use of fluorescence-based methods for 5-HT3 receptor research has been quite limited and, although a variety of approaches have been described, these are broadly distributed throughout the literature. In this review we condense these findings into a single, accessible source of reference with the hope of promoting the use of these valuable molecular probes. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Priming of the mucosal immune system during the postnatal period substantially influences host-microbial interaction and susceptibility to immune-mediated diseases in adult life. The underlying mechanisms are ill defined. Here we show that shortly after birth, CD4 T cells populate preformed lymphoid structures in the small intestine and quickly acquire a distinct transcriptional profile. T-cell recruitment is independent of microbial colonization and innate or adaptive immune stimulation but requires β7 integrin expression. Surprisingly, neonatal CD4 T cells remain immature throughout the postnatal period under homeostatic conditions but undergo maturation and gain effector function on barrier disruption. Maternal SIgA and regulatory T cells act in concert to prevent immune stimulation and maintain the immature phenotype of CD4 T cells in the postnatal intestine during homeostasis. Active suppression of CD4 T-cell maturation during the postnatal period might contribute to prevent auto-reactivity, sustain a broad TCR repertoire and establish life-long immune homeostasis.

Lymph node stromal cells are known to be immunorelevant during inflammation and tolerance. Differences between peripheral lymph nodes and mesenteric lymph nodes are important for an efficient and effective immune defense. Stromal cells were considered to be perfectly adapted to their draining area and not changeable concerning their expression pattern. Here we show that stromal cells can change their profile after isolation and transplantation into a different draining area. Subsequently, these newly organized lymph nodes are able to induce not only a region-specific but also an antigen-specific immune response. Thus, stromal cells are trend-setters for immune cells in producing a microenvironment that allows an optimized immune defense.

IL-17-producing Th17 cells mediate immune responses against a variety of fungal and bacterial infections. Signaling via NF-κB has been linked to the development and maintenance of Th17 cells. We analyzed the role of the unusual inhibitor of NF-κB, IκBNS, in the proliferation and effector cytokine production of murine Th17 cells. Our study demonstrates that nuclear IκBNS is crucial for murine Th17 cell generation. IκBNS is highly expressed in Th17 cells; in the absence of IκBNS, the frequencies of IL-17A-producing cells are drastically reduced. This was measured in vitro under Th17-polarizing conditions and confirmed in two colitis models. Mechanistically, murine IκBNS (-/-) Th17 cells were less proliferative and expressed markedly reduced levels of IL-2, IL-10, MIP-1α, and GM-CSF. Citrobacter rodentium was used as a Th17-inducing infection model, in which IκBNS (-/-) mice displayed an increased bacterial burden and diminished tissue damage. These results demonstrate the important function of Th17 cells in pathogen clearance, as well as in inflammation-associated pathology. We identified IκBNS to be crucial for the generation and function of murine Th17 cells upon inflammation and infection. Our findings may have implications for the therapy of autoimmune conditions, such as inflammatory bowel disease, and for the treatment of gut-tropic infections., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The pharmacological characterization of ligands depends upon the ability to accurately measure their binding properties. Fluorescence provides an alternative to more traditional approaches such as radioligand binding. Here we describe the binding and spectroscopic properties of eight fluorescent 5-HT3 receptor ligands. These were tested on purified receptors, expressed receptors on live cells, or in vivo. All compounds had nanomolar affinities with fluorescent properties extending from blue to near infra-red emission. A fluorescein-derivative had the highest affinity as measured by fluorescence polarization (FP; 1.14 nM), flow cytometry (FC; 3.23 nM) and radioligand binding (RB; 1.90 nM). Competition binding with unlabeled 5-HT3 receptor agonists (5-HT, mCPBG, quipazine) and antagonists (granisetron, palonosetron, tropisetron) yielded similar affinities in all three assays. When cysteine substitutions were introduced into the 5-HT3 receptor binding site the same changes in binding affinity were seen for both granisetron and the fluorescein-derivative, suggesting that they both adopt orientations that are consistent with co-crystal structures of granisetron with a homologous protein (5HTBP). As expected, in vivo live imaging in anaesthetized mice revealed staining in the abdominal cavity in intestines, but also in salivary glands. The unexpected presence of 5-HT3 receptors in mouse salivary glands was confirmed by Western blots. Overall, these results demonstrate the wide utility of our new high-affinity fluorescently-labeled 5-HT3 receptor probes, ranging from in vitro receptor pharmacology, including FC and FP ligand competition, to live imaging of 5-HT3 expressing tissues., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Activated naive CD4(+) T cells are highly plastic cells that can differentiate into various T helper (Th) cell fates characterized by the expression of effector cytokines like IFN-γ (Th1), IL-4 (Th2) or IL-17A (Th17). Although previous studies have demonstrated that epigenetic mechanisms including DNA demethylation can stabilize effector cytokine expression, a comprehensive analysis of the changes in the DNA methylation pattern during differentiation of naive T cells into Th cell subsets is lacking. Hence, we here performed a genome-wide methylome analysis of ex vivo isolated naive CD4(+) T cells, Th1 and Th17 cells. We could demonstrate that naive CD4(+) T cells share more demethylated regions with Th17 cells when compared to Th1 cells, and that overall Th17 cells display the highest number of demethylated regions, findings which are in line with the previously reported plasticity of Th17 cells. We could identify seven regions located in Il17a, Zfp362, Ccr6, Acsbg1, Dpp4, Rora and Dclk1 showing pronounced demethylation selectively in ex vivo isolated Th17 cells when compared to other ex vivo isolated Th cell subsets and in vitro generated Th17 cells, suggesting that this unique epigenetic signature allows identifying and functionally characterizing in vivo generated Th17 cells., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The specific regulation of cellular metabolic processes is of major importance for directing immune cell differentiation and function. We review recent evidence indicating that changes in basic cellular lipid metabolism have critical effects on T cell proliferation and cell fate decisions. While induction of de novo fatty acid (FA) synthesis is essential for activation-induced proliferation and differentiation of effector T cells, FA catabolism via β-oxidation is important for the development of CD8(+) T cell memory as well as for the differentiation of CD4(+) regulatory T cells. We consider the influence of lipid metabolism and metabolic intermediates on the regulation of signaling and transcriptional pathways via post-translational modifications, and discuss how an improved understanding of FA metabolism may reveal strategies for manipulating immune responses towards therapeutic outcomes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

T helper 17 (Th17) cells play an essential role in the clearance of extracellular pathogenic bacteria and fungi. However, this subset is critically involved in the pathology of many autoimmune diseases, e.g., psoriasis, multiple sclerosis, allergy, rheumatoid arthritis, and inflammatory bowel diseases in humans. Therefore, Th17 responses need to be tightly regulated in vivo to mediate effective host defenses against pathogens without causing excessive host tissue damage. Foxp3(+) regulatory T (Treg) cells play an important role in maintaining peripheral tolerance to self-antigens and in counteracting the inflammatory activity of effector T helper cell subsets. Although Th17 and Treg cells represent two CD4(+) T cell subsets with opposing principal functions, these cell types are functionally connected. In this review, we will first give an overview on the biology of Th17 cells and describe their development and in vivo function, followed by an account on the special developmental relationship between Th17 and Treg cells. We will describe the identification of Treg/Th17 intermediates and consider their lineage stability and function in vivo. Finally, we will discuss how Treg cells may regulate the Th17 cell response in the context of infection and inflammation, and elude on findings demonstrating that Treg cells can also have a prominent function in promoting the differentiation of Th17 cells., (© 2015 Elsevier Inc. All rights reserved.)

Proguanil is an antimalarial prodrug that is metabolized to 4-chlorophenyl-1-biguanide (CPB) and the active metabolite cycloguanil (CG). These compounds are structurally related to meta-chlorophenyl biguanide (mCPBG), a 5-hydroxytryptamine 3 (5-HT3) receptor agonist. Here we examine the effects of proguanil and its metabolites on the electrophysiology and ligand-binding properties of human 5-HT3A receptors expressed in Xenopus oocytes and human embryonic kidney 293 cells, respectively. 5-HT3 receptor responses were reversibly inhibited by proguanil, with an IC50 of 1.81 μM. Competitive antagonism was shown by a lack of voltage-dependence, Schild plot (Kb = 1.70 μM), and radioligand competition (Ki = 2.61 μM) with the 5-HT3 receptor antagonist [(3)H]granisetron. Kinetic measurements (kon = 4.0 × 10(4) M(-1) s(-1) ; koff = 0.23 s(-1)) were consistent with a simple bimolecular reaction scheme with a Kb of 4.35 μM. The metabolites CG and CPB similarly inhibited 5-HT3 receptors as assessed by IC50 (1.48 and 4.36 μM, respectively), Schild plot (Kb = 2.97 and 11.4 μM), and radioligand competition (Ki = 4.89 and 0.41 μM). At higher concentrations, CPB was a partial agonist (EC50 = 14.1 μM; I/Imax = 0.013). These results demonstrate that proguanil competitively inhibits 5-HT3 receptors, with an IC50 that exceeds whole-blood concentrations following its oral administration. They may therefore be responsible for the occasional gastrointestinal side effects, nausea, and vomiting reported following its use. Clinical development of related compounds should therefore consider effects at 5-HT3 receptors as an early indication of possible unwanted gastrointestinal side effects., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Intestinal infection with the mouse pathogen Citrobacter rodentium induces a strong local Th17 response in the colon. Although this inflammatory immune response helps to clear the pathogen, it also induces inflammation-associated pathology in the gut and thus, has to be tightly controlled. In this project, we therefore studied the impact of Foxp3(+) regulatory T cells (Treg) on the infectious and inflammatory processes elicited by the bacterial pathogen C. rodentium. Surprisingly, we found that depletion of Treg by diphtheria toxin in the Foxp3(DTR) (DEREG) mouse model resulted in impaired bacterial clearance in the colon, exacerbated body weight loss, and increased systemic dissemination of bacteria. Consistent with the enhanced susceptibility to infection, we found that the colonic Th17-associated T-cell response was impaired in Treg-depleted mice, suggesting that the presence of Treg is crucial for the establishment of a functional Th17 response after the infection in the gut. As a consequence of the impaired Th17 response, we also observed less inflammation-associated pathology in the colons of Treg-depleted mice. Interestingly, anti-interleukin (IL)-2 treatment of infected Treg-depleted mice restored Th17 induction, indicating that Treg support the induction of a protective Th17 response during intestinal bacterial infection by consumption of local IL-2.

Interleukin-17 (IL-17)-secreting T cells of the T helper 17 (TH17) lineage play a pathogenic role in multiple inflammatory and autoimmune conditions and thus represent a highly attractive target for therapeutic intervention. We report that inhibition of acetyl-CoA carboxylase 1 (ACC1) restrains the formation of human and mouse TH17 cells and promotes the development of anti-inflammatory Foxp3(+) regulatory T (Treg) cells. We show that TH17 cells, but not Treg cells, depend on ACC1-mediated de novo fatty acid synthesis and the underlying glycolytic-lipogenic metabolic pathway for their development. Although TH17 cells use this pathway to produce phospholipids for cellular membranes, Treg cells readily take up exogenous fatty acids for this purpose. Notably, pharmacologic inhibition or T cell-specific deletion of ACC1 not only blocks de novo fatty acid synthesis but also interferes with the metabolic flux of glucose-derived carbon via glycolysis and the tricarboxylic acid cycle. In vivo, treatment with the ACC-specific inhibitor soraphen A or T cell-specific deletion of ACC1 in mice attenuates TH17 cell-mediated autoimmune disease. Our results indicate fundamental differences between TH17 cells and Treg cells regarding their dependency on ACC1-mediated de novo fatty acid synthesis, which might be exploited as a new strategy for metabolic immune modulation of TH17 cell-mediated inflammatory diseases.

The 5-HT3 receptor is one of several ion channels responsible for the transmission of nerve impulses in the peripheral and central nervous systems. Until now, it has been difficult to characterize transmembrane receptors with classical structural biology approaches like X-ray crystallography. The use of photoaffinity probes is an alternative approach to identify regions in the protein where small molecules bind. To this end, we present two photoaffinity probes based on granisetron, a well known antagonist of the 5-HT3 receptor. These new probes show nanomolar binding affinity for the orthosteric binding site. In addition, we investigated their reactivity using irradiation experiments.

2-Aminoethyl diphenylborinate (2-APB) is a known modulator of the IP3 receptor, the calcium ATPase SERCA, the calcium release-activated calcium channel Orai and TRP channels. More recently, it was shown that 2-APB is an efficient inhibitor of the epithelial calcium channel TRPV6 which is overexpressed in prostate cancer. We have conducted a structure-activity relationship study of 2-APB congeners to understand their inhibitory mode of action on TRPV6. Whereas modifying the aminoethyl moiety did not significantly change TRPV6 inhibition, substitution of the phenyl rings of 2-APB did. Our data show that the diaryl borinate moiety is required for biological activity and that the substitution pattern of the aryl rings can influence TRPV6 versus SOCE inhibition. We have also discovered that 2-APB is hydrolyzed and transesterified within minutes in solution., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Glutamate transporters play important roles in the termination of excitatory neurotransmission and in providing cells throughout the body with glutamate for metabolic purposes. The high-affinity glutamate transporters EAAC1 (SLC1A1), GLT1 (SLC1A2), GLAST (SLC1A3), EAAT4 (SLC1A6), and EAAT5 (SLC1A7) mediate the cellular uptake of glutamate by the co-transport of three sodium ions (Na(+)) and one proton (H(+)), with the counter-transport of one potassium ion (K(+)). Thereby, they protect the CNS from glutamate-induced neurotoxicity. Loss of function of glutamate transporters has been implicated in the pathogenesis of several diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. In addition, glutamate transporters play a role in glutamate excitotoxicity following an ischemic stroke, due to reversed glutamate transport. Besides glutamate transporters, the SLC1 family encompasses two transporters of neutral amino acids, ASCT1 (SLC1A4) and ASCT2 (SLC1A5). Both transporters facilitate electroneutral exchange of amino acids in neurons and/or cells of the peripheral tissues. Some years ago, a high resolution structure of an archaeal homologue of the SLC1 family was determined, followed by the elucidation of its structure in the presence of the substrate aspartate and the inhibitor d,l-threo-benzyloxy aspartate (d,l-TBOA). Historically, the first few known inhibitors of SLC1 transporters were based on constrained glutamate analogs which were active in the high micromolar range but often also showed off-target activity at glutamate receptors. Further development led to the discovery of l-threo-β-hydroxyaspartate derivatives, some of which effectively inhibited SLC1 transporters at nanomolar concentrations. More recently, small molecule inhibitors have been identified whose structures are not based on amino acids. Activators of SLC1 family members have also been discovered but there are only a few examples known., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Extrapyramidal symptoms may appear in Alzheimer's disease (AD). In the present study, using morphometric immunohistochemistry in 34 cases with AD-related pathology, we evaluated whether nigral burden of tau pathology or striatal burden of amyloid-β deposition correlates with dopamine transporter (DAT) expression in the striatum. Our observations show a lack of correlation between these variables and support the notion that lower striatal DAT expression in AD patients suggests concomitant nigral α-synuclein pathology. Extrapyramidal symptoms may have a complex background in AD.

The synthesis, photophysical and biological characterization of a small library of fluorescent 5-HT(3) receptor ligands is described. Several of these novel granisetron conjugates have high quantum yields and show high affinity for the human 5-HT(3)AR., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The mammalian intestine provides a unique niche for a large community of bacterial symbionts that complements the host in digestive and anabolic pathways, as well as in protection from pathogens. Only a few bacterial phyla have adapted to this predominantly anaerobic environment, but hundreds of different species create an ecosystem that affects many facets of the host's physiology. Recent data show how particular symbionts are involved in the maturation of the immune system, in the intestine and beyond, and how dysbiosis, or alteration of that community, can deregulate immunity and lead to immunopathology. The extensive and dynamic interactions between the symbionts and the immune system are key to homeostasis and health, and require all the blends of so-called regulatory and pro-inflammatory immune reactions. Unfortunately, pro-inflammatory immunity leading to the generation of Th17 cells has been mainly associated with its role in immunopathology. Here we discuss the view that the immune system in general, and type 17 immunity in particular, develop to maintain the equilibrium of the host with its symbionts., (© 2011 Blackwell Publishing Ltd.)

In mammals, a variety of different lymphoid tissues have evolved as an integral part of the immune system that allows the host to survive in a sometimes hostile environment. While the development of secondary lymphoid organs is programmed in the fetus, the induction of other lymphoid structures like isolated lymphoid follicles (ILFs) in the gut or tertiary lymphoid tissues (tLT) need additional external triggers after birth. It is well established that for the development of secondary lymphoid organs, as well as ILFs, RORgt expressing lymphoid tissue inducer (LTi) cells play an important role. Yet, the requirement of these cells for tLT induction, especially in the gut, was less clear. Here, I will discuss recent data demonstrating that RORgt expressing LTi cells are not required for the development of tLT in the colon. In contrast, such structures even develop spontaneously in the absence of RORgt. In RORgt (-/-) mice, this is part of the host's strategy to establish a viable homeostasis between the intestinal microbiota and the host, despite the loss of important components of the intestinal immune system in these mice. Although this highlights the amazing capacity of the immune system for adaptation, I will also discuss the adverse effects of such a compensatory immune mechanism for the host.

Lymphoid cells that express the nuclear hormone receptor RORγt are involved in containment of the large intestinal microbiota and defense against pathogens through the production of interleukin 17 (IL-17) and IL-22. They include adaptive IL-17-producing helper T cells (T(H)17 cells), as well as innate lymphoid cells (ILCs) such as lymphoid tissue-inducer (LTi) cells and IL-22-producing NKp46+ cells. Here we show that in contrast to T(H)17 cells, both types of RORγt+ ILCs constitutively produced most of the intestinal IL-22 and that the symbiotic microbiota repressed this function through epithelial expression of IL-25. This function was greater in the absence of adaptive immunity and was fully restored and required after epithelial damage, which demonstrates a central role for RORγt+ ILCs in intestinal homeostasis. Our data identify a finely tuned equilibrium among intestinal symbionts, adaptive immunity and RORγt+ ILCs.

Retinoic acid-related orphan receptor (ROR)γt(+) TCRαβ(+) cells expressing IL-17, termed Th17 cells, are most abundant in the intestinal lamina propria. Symbiotic microbiota are required for the generation of Th17 cells, but the requirement for microbiota-derived Ag is not documented. In this study, we show that normal numbers of Th17 cells develop in the intestine of mice that express a single TCR in the absence of cognate Ag, whereas the microbiota remains essential for their development. However, such mice, or mice monocolonized with the Th17-inducing segmented filamentous bacteria, fail to induce normal numbers of Foxp3(+) RORγt(+) T cells, the regulatory counterpart of IL-17(+)RORγt(+) T cells. These results demonstrate that a complex microbiota and cognate Ag are required to generate a properly regulated set of RORγt(+) T cells and Th17 cells.

Rejection of solid organ allograft involves alloreactive T-cell expansion. The importance of NF-κB and NFAT in this process is underscored by the therapeutic efficacy of immunosuppressive agents, which target the two transcription factors. Since calpains, calcium-activated proteases, are involved in the activation of NF-κB and NFAT, we investigated the role of calpains in allograft rejection. In human transplant kidneys undergoing acute or chronic rejection, we show an increased expression of CAPN 1 gene encoding μ-calpain, associated with a marked expression of μ-calpain, mainly in infiltrating T cells. To address the role of calpain in rejection, we used a skin transplant model in transgenic mice expressing high levels of calpastatin, a calpain-specific inhibitor. We show that calpain inhibition extended skin allograft survival, from 11 to 20 days. This delay was associated with a limitation in allograft infiltration by T cells. In vitro, calpain inhibition by calpastatin transgene expression limited dramatically T-cell migration but, unexpectedly, increased slightly T-cell proliferation. Amplification of IL-2 signaling via the stabilization of IL-2R common γ-chain provided an explanation for the proliferation response. This is the first study establishing that calpain inhibition delays allograft rejection by slowing down T-cell migration rather than proliferation., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The programmed development of lymph nodes and Peyer's patches during ontogeny requires lymphoid tissue inducer (LTi) cells that express the nuclear hormone receptor RORγt. After birth, LTi cells in the intestine cluster into cryptopatches, the precursors of isolated lymphoid follicles (ILFs), which are induced to form by symbiotic bacteria and maintain intestinal homeostasis. We show that in RORγt-deficient mice, which lack LTi cells, programmed lymphoid tissues, ILFs, and Th17 cells, bacterial containment requires the generation of large numbers of tertiary lymphoid tissues (tLTs) through the activity of B cells. However, upon epithelial damage, these mice develop severe intestinal inflammation characterized by extensive recruitment of neutrophils and IgG(+) B cells, high expression of activation-induced deaminase in tLTs, and wasting disease. The pathology was prevented by antibiotic treatment or inhibition of lymphoid tissue formation and was significantly decreased by treatment with intravenous immunoglobulin G (IVIG). Our data show that intestinal immunodeficiency, such as an absence in RORγt-mediated proinflammatory immunity, can be compensated by increased lymphoid tissue genesis. However, this comes at a high cost for the host and can lead to a deregulated B cell response and aggravated inflammatory pathology.

Interleukin(IL)-18 is a pleiotrophic cytokine with functions in immune modulation, angiogenesis and bone metabolism. In this study, the potential of IL-18 as an immunotherapy for prostate cancer (PCa) was examined using the murine model of prostate carcinoma, RM1 and a bone metastatic variant RM1(BM)/B4H7-luc. RM1 and RM1(BM)/B4H7-luc cells were stably transfected to express bioactive IL-18. These cells were implanted into syngeneic immunocompetent mice, with or without an IL-18-neutralising antibody (αIL-18, SK113AE4). IL-18 significantly inhibited the growth of both subcutaneous and orthotopic RM1 tumors and the IL-18 neutralizing antibody abrogated the tumor growth-inhibition. In vivo neutralization of interferon-gamma (IFN-γ) completely eliminated the anti-tumor effects of IL-18 confirming an essential role of IFN-γ as a down-stream mediator of the anti-tumor activity of IL-18. Tumors from mice in which IL-18 and/or IFN-γ was neutralized contained significantly fewer CD4(+) and CD8(+) T cells than those with functional IL-18. The essential role of adaptive immunity was demonstrated as tumors grew more rapidly in RAG1(-/-) mice or in mice depleted of CD4(+) and/or CD8(+) cells than in normal mice. The tumors in RAG1(-/-) mice were also significantly smaller when IL-18 was present, indicating that innate immune mechanisms are involved. IL-18 also induced an increase in tumor infiltration of macrophages and neutrophils but not NK cells. In other experiments, direct injection of recombinant IL-18 into established tumors also inhibited tumor growth, which was associated with an increase in intratumoral macrophages, but not T cells. These results suggest that local IL-18 in the tumor environment can significantly potentiate anti-tumor immunity in the prostate and clearly demonstrate that this effect is mediated by innate and adaptive immune mechanisms.

Neuronal nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels that mediate fast synaptic transmission. Methyllycaconitine (MLA) is a selective and potent antagonist of the α7 nAChR, and its anthranilate ester side-chain is important for its activity. Here we report the influence of structure on nAChR inhibition for a series of novel MLA analogs, incorporating either an alcohol or anthranilate ester side-chain to an azabicyclic or azatricyclic core against rat α7, α4β2, and α3β4 nAChRs expressed in Xenopus oocytes. The analogs inhibited ACh (EC(50)) within an IC(50) range of 2.3-26.6 μM. Most displayed noncompetitive antagonism, but the anthranilate ester analogs exerted competitive behavior at the α7 nAChR. At α4β2 nAChRs, inhibition by the azabicyclic alcohol was voltage-dependent suggesting channel block. The channel-lining residues of α4 subunits were mutated to cysteine and the effect of azabicyclic alcohol was evaluated by competition with methanethiosulfonate ethylammonium (MTSEA) and a thiol-reactive probe in the open, closed, and desensitized states of α4β2 nAChRs. The azabicyclic alcohol was found to compete with MTSEA between residues 6' and 13' in a state-dependent manner, but the reactive probe only bonded with 13' in the open state. The data suggest that the 13' position is the dominant binding site. Ligand docking of the azabicyclic alcohol into a (α4)(3)(β2)(2) homology model of the closed channel showed that the ligand can be accommodated at this location. Thus our data reveal distinct pharmacological differences between different nAChR subtypes and also identify a specific binding site for a noncompetitive channel blocker.

Lymphoid tissue-inducer (LTi) cells initiate the development of lymphoid tissues through the activation of local stromal cells in a process similar to inflammation. LTi cells express the nuclear hormone receptor RORγt, which also directs the expression of the proinflammatory cytokine interleukin-17 in T cells. We show here that LTi cells are part of a larger family of proinflammatory RORγt(+) innate lymphoid cells (ILCs) that differentiate from distinct fetal liver RORγt(+) precursors. The fate of RORγt(+) ILCs is determined by mouse age, and after birth, favors the generation of cells involved in intestinal homeostasis and defense. Contrary to RORγt(+) T cells, however, RORγt(+) ILCs develop in the absence of microbiota. Our study indicates that RORγt(+) ILCs evolve to preempt intestinal colonization by microbial symbionts.

The 5-HT3 receptor is a member of the Cys-loop family of transmitter receptors. It can function as a homopentamer (5-HT3A-only subunits) or as a heteropentamer. The 5-HT3AB receptor is the best characterized heteropentamer. This receptor differs from a homopentamer in its kinetics, voltage dependence, and single-channel conductance, but its pharmacology is similar. To understand the contribution of the 5-HT3B subunit to the binding site, we created homology models of 5-HT3AB receptors and docked 5-HT and granisetron into AB, BA, and BB interfaces. To test whether ligands bind in any or all of these interfaces, we mutated amino acids that are important for agonist and antagonist binding in the 5-HT3A subunit to their corresponding residues in the 5-HT3B subunit and vice versa. Changes in [3H]granisetron binding affinity (Kd) and 5-HT EC50 were determined using receptors expressed in HEK-293 cells and Xenopus oocytes, respectively. For all A-to-B mutant receptors, except T181N, antagonist binding was altered or eliminated. Functional studies revealed that either the receptors were nonfunctional or the EC50 values were increased. In B-to-A mutant receptors there were no changes in Kd, although EC50 values and Hill slopes, except for N170T mutant receptors, were similar to those for 5-HT3A receptors. Thus, the experimental data do not support a contribution of the 5-HT3B subunit to the binding pocket, and we conclude that both 5-HT and granisetron bind to an AA binding site in the heteromeric 5-HT3AB receptor., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

This report describes the synthesis and biological characterization of novel granisetron derivatives that are antagonists of the human serotonin (5-HT(3)A) receptor. Some of these substituted granisetron derivatives showed low nanomolar binding affinity and allowed the identification of positions on the granisetron core that might be used as attachment points for biophysical tags. A BODIPY fluorophore was appended to one such position and specifically bound to 5-HT(3)A receptors in mammalian cells.

Ion channels are transmembrane protein complexes that are found in virtually all cells. They fulfill a crucial physiological function by facilitating communication between and within cells. Consequently, impaired channel function, e.g. due to mutations, often has profound physiological effects. Their central role in cell-to-cell communication makes ion channels formidable drug targets, albeit their transmembrane nature often hampers efforts to obtain high resolution structures and hence impedes drug discovery. Decades of electrophysiology and molecular biology studies have made critical contributions to our understanding of ion channel structure and function. Small organic compounds, acting as either agonist or antagonist, have played vital roles in such studies and in recent years these molecular tools have become more sophisticated. Decorated with fluorescent, photoaffinity and/or affinity tags small molecular tools enable imaging, binding site mapping and isolation of biomolecular targets. Here, some of the methodologies employed in the context of ion channels are discussed and highlighted with representative examples.

Intestinal lymphoid tissues face the challenging task of inducing adaptive immunity to pathogens, yet maintaining homeostasis with the enormous commensal microbiota. To that aim, the ancient partnership between self and flora has resulted in the generation of a unique set of lymphoid tissues capable of constant large-scale reformatting. A first set of lymphoid tissues, the mesenteric lymph nodes and Peyer's patches, are programmed to develop in the sterile environment of the fetus, whereas a second set of lymphoid tissues, the tertiary lymphoid tissues, are induced to form by the microbiota and inflammation. The diversity of intestinal lymphoid tissues confers the flexibility required to adapt the number of immune inductive sites to the size of the flora and the extent of the pathogenic threat. The result is a functional superorganism combining self and microbes for the best possible symbiosis.

Stromal cells in lymphoid tissues regulate lymphocyte recruitment and survival through the expression of specific chemokines and cytokines. During inflammation, the same signals recruit lymphocytes to the site of injury; however, the "lymphoid" stromal (LS) cells producing these signals remain poorly characterized. We find that mouse inflammatory lesions and tumors develop gp38(+) LS cells, in recapitulation of the development of LS cells early during the ontogeny of lymphoid organs and the intestine, and express a set of genes that promotes the development of lymphocyte-permissive tissues. These gp38(+) LS cells are induced by a robust pathway that requires myeloid cells but not known Toll- or NOD-like receptors, the inflammasome, or adaptive immunity. Parabiosis and inducible genetic cell fate mapping experiments indicate that local precursors, presumably resident fibroblasts rather that circulating precursors, massively proliferate and give rise to LS cells during inflammation. Our results show that LS cells are both programmed during ontogeny and reinduced during inflammation.

Cryptosporidium parvum is an intracellular parasite causing enteritis which can become life-threatening in the immunocompromised host. CD4+ T cells and interferon (IFN)-gamma play dominant roles in host immune response to infection. However, effector mechanisms that are responsible for recovery from infection are poorly understood. In the present study we analyzed mice deficient in IFN-gamma or interleukin (IL)-12 in parallel to C57BL/6 wild type mice as models for murine cryptosporidiosis. Our results identified IFN-gamma as the key cytokine in the innate as well as adaptive immunity during primary and also challenge C. parvum infection. Furthermore, both Th1 and Th2 cytokines appear to contribute to the resolution of a primary infection, the former being dominant over the latter. Dramatic changes in the expression of cytokine genes were seen in the ileum (the site of infection) but not in the mesenteric lymph nodes and spleen. During re-challenge, a significant increase of IFN-gamma was recorded in IL-12 deficient mice (IL-12KO). Additionally, we present data suggesting a contribution of IL-18 in resistance of C. parvum infection even in the absence of IFN-gamma. Anti-IL-18 antibody treatment led to increased susceptibility to infection in both strains of immunodeficient mice. Besides its function in inducing IFN-gamma in IL-12 knock out mice, IL-18 appears to be involved in the regulation of the Th1/Th2 responses in C. parvum. Neutralization resulted in a cytokine imbalance with up regulation of systemic (spleen) Th2 cytokine genes, notably IL-4 and IL-13. These data demonstrate that susceptibility or resistance to C. parvum infection depends on a delicate balance between the production of Th1 cytokines, needed to control parasite growth, and Th2 cytokines, to limit pathology.

Natural killer (NK) cells are innate lymphocytes with spontaneous antitumor activity, and they produce interferon-gamma (IFN-gamma) that primes immune responses. Whereas T helper cell subsets differentiate from naive T cells via specific transcription factors, evidence for NK cell diversification is limited. In this report, we characterized intestinal lymphocytes expressing the NK cell natural cytotoxicity receptor NKp46. Gut NKp46+ cells were distinguished from classical NK cells by limited IFN-gamma production and absence of perforin, whereas several subsets expressed the nuclear hormone receptor retinoic acid receptor-related orphan receptor t (RORgammat) and interleukin-22 (IL-22). Intestinal NKp46+IL-22+ cells were generated via a local process that was conditioned by commensal bacteria and required RORgammat. Mice lacking IL-22-producing NKp46+ cells showed heightened susceptibility to the pathogen Citrobacter rodentium, consistent with a role for intestinal NKp46+ cells in immune protection. RORgammat-driven diversification of intestinal NKp46+ cells thereby specifies an innate cellular defense mechanism that operates at mucosal surfaces.

Invariant natural killer T (iNKT) cells constitute a subpopulation of T cells that recognize glycolipids presented by CD1d molecules. They are characterized by their prompt production of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma), which enables them to modulate diverse immune responses. Recently, we enlarged this concept by identifying a distinct IL-17-producing iNKT cell subset, named iNKT17 cells. The mechanisms leading to the acquisition of this new iNKT cell activity are unknown. Herein we show that IL-17-producing iNKT cells are already present in the thymus, predominantly among a subset regarded so far as an immature stage of thymic iNKT cell development, the CD1d tetramer(pos)CD44(pos)NK1.1(neg)CD4(neg) cells. Using EGFP reporter mice, we demonstrate that the transcription factor ROR-gammat is critical for the thymic differentiation of this subset because only ROR-gammat(pos) iNKT cells are capable of massively secreting IL-17. Moreover, IL-17-producing CD1d tetramer(pos)CD44(pos)NK1.1(neg)CD4(neg) thymic iNKT cells have reached a mature differentiation stage because they fail to generate other cell subsets in fetal thymic organ culture. Conversely, thymic ROR-gammat(neg) iNKT cell precursors give rise to progeny, but acquire neither ROR-gammat expression nor the ability to secrete IL-17. In conclusion, our findings demonstrate an alternative thymic pathway leading to the development of iNKT17 cells that requires ROR-gammat expression.

The 5-HT(3) receptor belongs to a family of therapeutically important neurotransmitter-gated receptors whose ligand binding sites are formed by the convergence of six peptide loops (A-F). Here we have mutated 15 amino acid residues in and around loop B of the 5-HT(3) receptor (Ser-177 to Asn-191) to Ala or a residue with similar chemical properties. Changes in [3H]granisetron binding affinity (K(d)) and 5-HT EC(50) were determined using receptors expressed in human embryonic kidney 293 cells. Substitutions at all but one residue (Thr-181) altered or eliminated binding for one or both mutants. Receptors were nonfunctional or EC(50) values were altered for all but two mutants (S182T, I190L). Homology modeling indicates that loop B contributes two residues to a hydrophobic core that faces into the beta-sandwich of the subunit, and the experimental data indicate that they are important for both the structure and the function of the receptor. The models also show that close to the apex of the loop (Ser-182 to Ile-190), loop B residues form an extensive network of hydrogen bonds, both with other loop B residues and with adjacent regions of the protein. Overall, the data suggest that loop B has a major role in maintaining the structure of the region by a series of noncovalent interactions that are easily disrupted by amino acid substitutions.

Th cells producing IL-17 play a pro-inflammatory role at mucosal surfaces. Treg at the same sites dampen inflammation and prevent immunopathology. Th cells producing IL-17 (Th17) and Treg are thought to be distinct populations defined by expression of the transcription factors ROR-gammat and Foxp3, respectively. Here, we show that mouse CD25(+)Foxp3(+) Treg can be converted into a hybrid T-cell population characterized by the expression of Foxp3 and ROR-gammat and the production of IL-17. Conversion was observed upon coculture with DC selectively activated via dectin-1, a C-type lectin receptor involved in fungal recognition, and depended on IL-23 produced by DC. Within the Foxp3(+) population, only Foxp3(+)ROR-gammat(+) T cells but not Foxp3(+)ROR-gammat(-)-T cells become Foxp3(+)IL-17(+) T cells. These results indicate that some Foxp3(+) T cells can produce IL-17 while retaining Foxp3 expression and suggest that Treg could play an unexpected pro-inflammatory role in some settings.

The induction of proinflammatory cytokines such as gamma interferon (IFN-gamma) and tumor necrosis factor alpha is crucial for the early control of bacterial infections. Since interleukin-18 (IL-18) acts as a potent inducer of IFN-gamma, it might play an important role in the induction of a protective immune response in listeriosis. We used a murine model of systemic Listeria monocytogenes infection to study the immune response to these intracellular bacteria in the absence of IL-18. For this purpose, IL-18-deficient mice and mice treated with anti-IL-18 neutralizing antibody were infected with L. monocytogenes, and their innate and adaptive immune responses were compared to those of control mice. Unexpectedly, we found that mice deficient in IL-18 were partially resistant to primary infection with L. monocytogenes. At day 3 after infection, the numbers of listeriae in the livers and spleens of control mice were up to 500 times higher than those in IL-18-deficient or anti-IL-18 antibody-treated mice. In addition, the level of proinflammatory cytokines was markedly reduced in IL-18-deficient mice. Enhanced resistance to L. monocytogenes infection in IL-18-deficient mice was accompanied by increased numbers of leukocytes and reduced apoptosis in the spleen 48 to 72 h after infection. In contrast, control and IL-18-deficient mice showed no significant differences in their abilities to mount a protective L. monocytogenes-specific T-cell response.

The nuclear hormone receptor retinoic acid receptor-related orphan receptor gamma t (RORgamma t) is required for the generation of T helper 17 cells expressing the proinflammatory cytokine interleukin (IL)-17. In vivo, however, less than half of RORgamma t(+) T cells express IL-17. We report here that RORgamma t(+) T alphabeta cells include Foxp3(+) cells that coexist with IL-17-producing RORgamma t(+) T alphabeta cells in all tissues examined. The Foxp3(+) RORgamma t(+) T alphabeta express IL-10 and CCL20, and function as regulatory T cells. Furthermore, the ratio of Foxp3(+) to IL-17-producing RORgamma t(+) T alphabeta cells remains remarkably constant in mice enduring infection and inflammation. This equilibrium is tuned in favor of IL-10 production by Foxp3 and CCL20, and in favor of IL-17 production by IL-6 and IL-23. In the lung and skin, the largest population of RORgamma t(+) T cells express the gammadelta T cell receptor and produce the highest levels of IL-17 independently of IL-6. Thus, potentially antagonistic proinflammatory IL-17-producing and regulatory Foxp3(+) RORgamma t(+) T cells coexist and are tightly controlled, suggesting that a perturbed equilibrium in RORgamma t(+) T cells might lead to decreased immunoreactivity or, in contrast, to pathological inflammation.

Background and Purpose: The antimalarial compounds quinine, chloroquine and mefloquine affect the electrophysiological properties of Cys-loop receptors and have structural similarities to 5-HT(3) receptor antagonists. They may therefore act at 5-HT(3) receptors., Experimental Approach: The effects of quinine, chloroquine and mefloquine on electrophysiological and ligand binding properties of 5-HT(3A) receptors expressed in HEK 293 cells and Xenopus oocytes were examined. The compounds were also docked into models of the binding site., Key Results: 5-HT(3) responses were blocked with IC (50) values of 13.4 microM, 11.8 microM and 9.36 microM for quinine, chloroquine and mefloquine. Schild plots indicated quinine and chloroquine behaved competitively with pA (2) values of 4.92 (K (B)=12.0 microM) and 4.97 (K (B)=16.4 microM). Mefloquine displayed weakly voltage-dependent, non-competitive inhibition consistent with channel block. On and off rates for quinine and chloroquine indicated a simple bimolecular reaction scheme. Quinine, chloroquine and mefloquine displaced [(3)H]granisetron with K (i) values of 15.0, 24.2 and 35.7 microM. Docking of quinine into a homology model of the 5-HT(3) receptor binding site located the tertiary ammonium between W183 and Y234, and the quinoline ring towards the membrane, stabilised by a hydrogen bond with E129. For chloroquine, the quinoline ring was positioned between W183 and Y234 and the tertiary ammonium stabilised by interactions with F226., Conclusions and Implications: This study shows that quinine and chloroquine competitively inhibit 5-HT(3) receptors, while mefloquine inhibits predominantly non-competitively. Both quinine and chloroquine can be docked into a receptor binding site model, consistent with their structural homology to 5-HT(3) receptor antagonists.

Oral infection of C57BL/6 mice with Toxoplasma gondii results in small intestinal Th1-type immunopathology mediated by local production of IFN-gamma, TNF-alpha, and NO. To analyze whether the proinflammatory cytokines IL-12 and IL-18 play a role in the induction of immunopathology, IL-12p35/p40(-/-) and IL-18(-/-) mice were orally infected with T. gondii. Wild-type mice developed massive necrosis in their small intestines and died 7-10 days post infection. Even though IL-12p35/40(-/-) mice did not develop the necrosis they all died between day 9 and 11 after infection. In contrast, 50% of IL-18(-/-) mice died during the acute phase of infection. Compared to wild-type mice, IL-12p35/p40(-/-) but not IL-18(-/-) mice showed significantly higher parasite numbers in their small intestines and significantly higher numbers of parasite-associated inflammatory foci in their livers. IFN-gamma production was similar in infected wild-type and IL-18(-/-) mice but significantly decreased in IL-12p35/p40(-/-) mice. Treatment of mice with anti-IL-12- or anti-IL-18 antibodies after infection prevented the development of intestinal necrosis. These results reveal that both IL-12 and IL-18 play an important role in the development of intestinal immunopathology following oral infection with T. gondii. However, IL-12 is dominant over IL-18 in the host defense against parasite replication. Therefore, neutralization of IL-18 (rather than TNF-alpha, IL-12, and IFN-gamma) may be a safe strategy for the treatment of Th1-associated diseases.

Crown-capped iron(S-) porphyrins 1 x H2O and 2 x H2O and their corresponding Ba2+ complexes have been prepared as active site analogues of the resting state of cytochrome P450cam. cw-EPR studies and electronic structure calculations at the density functional theory (DFT) level of model systems suggest a functional role of the water cluster of P450cam.

Interleukin (IL)-18 is a cytokine with a broad array of effector functions, the most prominent of which is to act synergistically with IL-12 in interferon-gamma production and the induction of a strong T-helper-1-mediated immune response. In addition, IL-18 also upregulates the production of proinflammatory cytokines such as IL-1 and tumor necrosis factor-alpha. Analysis of IL-18-deficient mice revealed an important role of IL-18 in the activation of macrophages and natural killer cells in the context of infection with intracellular bacteria or parasites. In humans, it was reported that IL-18 is elevated at sites of inflammation in inflammatory bowel disease (IBD), particularly in Crohn's disease, suggesting a possible role for IL-18 in the development and persistence of IBD. In this review we summarize recent findings on the functional role of IL-18 in the pathogenesis of colitis with a special focus on murine models of IBD. The neutralizing mouse anti-mouse IL-18 antibodies generated in our group should facilitate the evaluation of the efficiency of therapeutic blockade of endogenous IL-18 in chronic mouse models of colitis besides the use of recombinant forms of the inhibitory IL-18-binding protein., (Copyright 2003 S. Karger AG, Basel)

The proinflammatory cytokine IL-18 mediates IFN-gamma production as well as the induction of Th1 polarized immune responses in synergy with IL-12. In this study, we describe the production of isogeneic monoclonal antibodies (Mabs) directed against murine IL-18 (mIL-18). Immunization of IL-18-deficient mice with recombinant mIL-18 in the presence of CpG-oligodeoxynucleotides (CpG-ODN) and alum as adjuvant resulted in high anti-IL-18 serum titers. We could identify two Mabs, SK721-2 and SK113AE-4, which were able to bind to IL-18 and neutralize its IFN-gamma inducing effect in vitro with an IC(50) of 40-100 ng/ml. In vivo, LPS-induced IFN-gamma production was reduced by 60-85% following a single administration of Mabs SK113AE-4 or SK721-2. Since IL-18 is likely to be involved in the pathogenesis of inflammatory diseases such as rheumatoid arthritis or Crohn's disease, neutralizing mouse anti-mouse IL-18 Mabs have the potential to become valuable tools for the therapeutic exploration of long-term IL-18 blockade in vivo.

Several polyamine derivatives were synthesized in order to produce novel antagonists of muscular nicotinic acetylcholine receptors. Their affinities were compared with those of philanthotoxin PhTX-343.