Sample records for cell receptor variable
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Correlated receptor transport processes buffer single-cell heterogeneity
Kallenberger, Stefan M.; Unger, Anne L.; Legewie, Stefan; Lymperopoulos, Konstantinos; Eils, Roland
2017-01-01
Cells typically vary in their response to extracellular ligands. Receptor transport processes modulate ligand-receptor induced signal transduction and impact the variability in cellular responses. Here, we quantitatively characterized cellular variability in erythropoietin receptor (EpoR) trafficking at the single-cell level based on live-cell imaging and mathematical modeling. Using ensembles of single-cell mathematical models reduced parameter uncertainties and showed that rapid EpoR turnover, transport of internalized EpoR back to the plasma membrane, and degradation of Epo-EpoR complexes were essential for receptor trafficking. EpoR trafficking dynamics in adherent H838 lung cancer cells closely resembled the dynamics previously characterized by mathematical modeling in suspension cells, indicating that dynamic properties of the EpoR system are widely conserved. Receptor transport processes differed by one order of magnitude between individual cells. However, the concentration of activated Epo-EpoR complexes was less variable due to the correlated kinetics of opposing transport processes acting as a buffering system. PMID:28945754
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Parham, Peter; Moffett, Ashley
2013-02-01
Natural killer (NK) cells have roles in immunity and reproduction that are controlled by variable receptors that recognize MHC class I molecules. The variable NK cell receptors found in humans are specific to simian primates, in which they have progressively co-evolved with MHC class I molecules. The emergence of the MHC-C gene in hominids drove the evolution of a system of NK cell receptors for MHC-C molecules that is most elaborate in chimpanzees. By contrast, the human system of MHC-C receptors seems to have been subject to different selection pressures that have acted in competition on the immunological and reproductive functions of MHC class I molecules. We suggest that this compromise facilitated the development of the bigger brains that enabled archaic and modern humans to migrate out of Africa and populate other continents.
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NASA Astrophysics Data System (ADS)
Hilyard, Katherine L.; Reyburn, Hugh; Chung, Shan; Bell, John I.; Strominger, Jack L.
1994-09-01
An Escherichia coli expression system has been developed to produce milligram quantities of the variable domains of a human T-cell receptor from a cytotoxic T cell that recognizes the HLA-A2-influenza matrix peptide complex as a single polypeptide chain. The recombinant protein was purified by metal-chelate chromatography and then refolded in a redox buffer system. The refolded protein was shown to directly bind both Staphylococcus aureus enterotoxin B and the major histocompatibility complex protein-peptide complex using a BIAcore biosensor. Thus this preparation of a single-chain, variable-domain, T-cell receptor fragment can bind both of its natural ligands and some of it is therefore a functional fragment of the receptor molecule.
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Sayegh, Camil E.; Demaries, Sandra L.; Iacampo, Sandra; Ratcliffe, Michael J. H.
1999-01-01
Immunoglobulin gene rearrangement in avian B cell precursors generates surface Ig receptors of limited diversity. It has been proposed that specificities encoded by these receptors play a critical role in B lineage development by recognizing endogenous ligands within the bursa of Fabricius. To address this issue directly we have introduced a truncated surface IgM, lacking variable region domains, into developing B precursors by retroviral gene transfer in vivo. Cells expressing this truncated receptor lack endogenous surface IgM, and the low level of endogenous Ig rearrangements that have occurred within this population of cells has not been selected for having a productive reading frame. Such cells proliferate rapidly within bursal epithelial buds of normal morphology. In addition, despite reduced levels of endogenous light chain rearrangement, those light chain rearrangements that have occurred have undergone variable region diversification by gene conversion. Therefore, although surface expression of an Ig receptor is required for bursal colonization and the induction of gene conversion, the specificity encoded by the prediversified receptor is irrelevant and, consequently, there is no obligate ligand for V(D)J-encoded determinants of prediversified avian cell surface IgM receptor. PMID:10485907
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Goicochea, Nancy L; Garnovskaya, Maria; Blanton, Mary G; Chan, Grace; Weisbart, Richard; Lilly, Michael B
2017-12-01
Castration-resistant prostate cancer cells exhibit continued androgen receptor signaling in spite of low levels of ligand. Current therapies to block androgen receptor signaling act by inhibiting ligand production or binding. We developed bispecific antibodies capable of penetrating cells and binding androgen receptor outside of the ligand-binding domain. Half of the bispecific antibody molecule consists of a single-chain variable fragment of 3E10, an anti-DNA antibody that enters cells. The other half is a single-chain variable fragment version of AR441, an anti-AR antibody. The resulting 3E10-AR441 bispecific antibody enters human LNCaP prostate cells and accumulates in the nucleus. The antibody binds to wild-type, mutant and splice variant androgen receptor. Binding affinity of 3E10-AR441 to androgen receptor (284 nM) was lower than that of the parental AR441 mAb (4.6 nM), but could be improved (45 nM) through alternative placement of the affinity tags, and ordering of the VH and VK domains. The 3E10-AR441 bispecific antibody blocked genomic signaling by wild-type or splice variant androgen receptor in LNCaP cells. It also blocked non-genomic signaling by the wild-type receptor. Furthermore, bispecific antibody inhibited the growth of C4-2 prostate cancer cells under androgen-stimulated conditions. The 3E10-AR441 biAb can enter prostate cancer cells and inhibits androgen receptor function in a ligand-independent manner. It may be an attractive prototype agent for prostate cancer therapy. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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Yancopoulos, G D; Blackwell, T K; Suh, H; Hood, L; Alt, F W
1986-01-31
We have recently proposed that a common recombinase performs all of the many variable region gene assembly events in B and T cells, and that the specificity of these joining events is mediated by regulating the "accessibility" of the involved gene segments. To test this possibility, we have introduced "accessible" T cell receptor (TCR) variable region gene segments into a pre-B cell line capable of recombining endogenous and transfected immunoglobulin (Ig) variable region gene segments. Although the corresponding "inaccessible" endogenous TCR gene segments do not rearrange in this line or in B cells in general, the introduced TCR gene segments join very frequently and, in fact, closely resemble introduced Ig gene segments in their recombination characteristics. These observations suggest a new role for conventional Ig transcriptional enhancers--recombinational enhancement. Our studies provide insight into additional aspects of the joining mechanism such as N region insertion, aberrant joining, and recombination-recognition sequence requirements for joining.
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NMDA receptor content of synapses in stratum radiatum of the hippocampal CA1 area.
Racca, C; Stephenson, F A; Streit, P; Roberts, J D; Somogyi, P
2000-04-01
Glutamate receptors activated by NMDA (NMDARs) or AMPA (AMPARs) are clustered on dendritic spines of pyramidal cells. Both the AMPAR-mediated postsynaptic responses and the synaptic AMPAR immunoreactivity show a large intersynapse variability. Postsynaptic responses mediated by NMDARs show less variability. To assess the variability in NMDAR content and the extent of their coexistence with AMPARs in Schaffer collateral-commissural synapses of adult rat CA1 pyramidal cells, electron microscopic immunogold localization of receptors has been used. Immunoreactivity of NMDARs was detected in virtually all synapses on spines, but AMPARs were undetectable, on average, in 12% of synapses. A proportion of synapses had a very high AMPAR content relative to the mean content, resulting in a distribution more skewed toward larger values than that of NMDARs. The variability of synaptic NMDAR content [coefficient of variation (CV), 0.64-0.70] was much lower than that of the AMPAR content (CV, 1.17-1.45). Unlike the AMPAR content, the NMDAR content showed only a weak correlation with synapse size. As reported previously for AMPARs, the immunoreactivity of NMDARs was also associated with the spine apparatus within spines. The results demonstrate that the majority of the synapses made by CA3 pyramidal cells onto spines of CA1 pyramids express both NMDARs and AMPARs, but with variable ratios. A less-variable NMDAR content is accompanied by a wide variability of AMPAR content, indicating that the regulation of expression of the two receptors is not closely linked. These findings support reports that fast excitatory transmission at some of these synapses is mediated by activation mainly of NMDARs.
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Duty, J Andrew; Szodoray, Peter; Zheng, Nai-Ying; Koelsch, Kristi A; Zhang, Qingzhao; Swiatkowski, Mike; Mathias, Melissa; Garman, Lori; Helms, Christina; Nakken, Britt; Smith, Kenneth; Farris, A Darise; Wilson, Patrick C
2009-01-16
Self-reactive B cells not controlled by receptor editing or clonal deletion may become anergic. We report that fully mature human B cells negative for surface IgM and retaining only IgD are autoreactive and functionally attenuated (referred to as naive IgD(+)IgM(-) B cells [B(ND)]). These B(ND) cells typically make up 2.5% of B cells in the peripheral blood, have antibody variable region genes in germline (unmutated) configuration, and, by all current measures, are fully mature. Analysis of 95 recombinant antibodies expressed from the variable genes of single B(ND) cells demonstrated that they are predominantly autoreactive, binding to HEp-2 cell antigens and DNA. Upon B cell receptor cross-linkage, B(ND) cells have a reduced capacity to mobilize intracellular calcium or phosphorylate tyrosines, demonstrating that they are anergic. However, intense stimulation causes B(ND) cells to fully respond, suggesting that these cells could be the precursors of autoantibody secreting plasma cells in autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis. This is the first identification of a distinct mature human B cell subset that is naturally autoreactive and controlled by the tolerizing mechanism of functional anergy.
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Variability in Beta-Adrenergic Receptor Population in Cultured Chicken Muscle Cells
NASA Technical Reports Server (NTRS)
Young, Ronald B; Bridge, Kristin Y.; Vaughn, Jeffrey R.
1998-01-01
Investigations into expression of the beta-adrenergic receptor (bAR) in chicken skeletal muscle cells in culture were initiated because several beta-adrenergic receptor agonists are known to increase skeletal muscle protein deposition in avian and mammalian species. During initial attempts to study the bAR population on the surface of chicken skeletal muscle cells, we observed a high degree of variability that was later found to be the result of using different batches of horse serum in the cell culture media. The separation between total binding and nonspecific binding in cells grown in two serum samples was approximately two-fold The number of nuclei within multinucleated myotubes was not significantly different in cells grown in the two serum samples. To investigate whether these two sera had an effect on coupling efficiency between bAR population and cAMP production, the ability of these cells to synthesize cAMP was also assessed. Despite the two-fold difference in receptor population, the ability of these cells to synthesize cAMP was not significantly different. Because of the possible link between bAR population and muscle protein, we also determined if the quantity of the major skeletal muscle protein, myosin, was affected by conditions that so drastically affected the bAR population. The quantity of myosin heavy chain was not significantly different.
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Lipowska-Bhalla, Grazyna; Gilham, David E; Hawkins, Robert E; Rothwell, Dominic G
2013-12-01
The clinical potential of chimeric antigen receptors in adoptive cellular therapy is beginning to be realized with several recent clinical trials targeting CD19 showing promising results in advanced B cell malignancies. This increased efficacy corresponds with improved engineering of the chimeric receptors with the latest-generation receptors eliciting greater signaling and proliferation potential. However, the antigen-binding single-chain variable fragment (scFv) domain of the receptors is critical in determining the activity of the chimeric receptor-expressing T cells, as this determines specificity and affinity to the tumor antigen. In this study, we describe a mammalian T cell line screening protocol employing a 2A-based bicistronic retroviral vector to isolate functional scFvs. This approach involves expression of the scFv library in a chimeric antigen receptor, and is based on selection of clones capable of stimulating CD69 upregulation in a T cell line and has a number of advantages over previously described methods in that the use of a 2A cassette ensures the exclusion of nonexpressing scFvs and the screening using a chimeric receptor in a mammalian T cell line ensures selection in the optimum context for therapeutic use. Proof-of-principle experiments show that the protocol was capable of a 10(5)-fold enrichment of positive clones after three rounds of selection. Furthermore, an antigen-specific clone was successfully isolated from a partially enriched scFv library, confirming the strength of the protocol. This approach has the potential to identify novel scFvs of use in adoptive T cell therapy and, potentially, wider antibody-based applications.
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Ng, KY; Yeung, BHS; Wong, YH; Wise, H
2013-01-01
Background and Purpose Hyper-nociceptive PGE2 EP4 receptors and prostacyclin (IP) receptors are present in adult rat dorsal root ganglion (DRG) neurones and glial cells in culture. The present study has investigated the cell-specific expression of two other Gs-protein coupled hyper-nociceptive receptor systems: β-adrenoceptors and calcitonin gene-related peptide (CGRP) receptors in isolated DRG cells and has examined the influence of neurone–glial cell interactions in regulating adenylyl cyclase (AC) activity. Experimental Approach Agonist-stimulated AC activity was determined in mixed DRG cell cultures from adult rats and compared with activity in DRG neurone-enriched cell cultures and pure DRG glial cell cultures. Key Results Pharmacological analysis showed the presence of Gs-coupled β2-adrenoceptors and CGRP receptors, but not β1-adrenoceptors, in all three DRG cell preparations. Agonist-stimulated AC activity was weakest in DRG neurone-enriched cell cultures. DRG neurones inhibited IP receptor-stimulated glial cell AC activity by a process dependent on both cell–cell contact and neurone-derived soluble factors, but this is unlikely to involve purine or glutamine receptor activation. Conclusions and Implications Gs-coupled hyper-nociceptive receptors are readily expressed on DRG glial cells in isolated cell cultures and the activity of CGRP, EP4 and IP receptors, but not β2-adrenoceptors, in glial cells is inhibited by DRG neurones. Studies using isolated DRG cells should be aware that hyper-nociceptive ligands may stimulate receptors on glial cells in addition to neurones, and that variable numbers of neurones and glial cells will influence absolute measures of AC activity and affect downstream functional responses. PMID:22924655
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Nyíri, G; Stephenson, F A; Freund, T F; Somogyi, P
2003-01-01
Pyramidal cells receive input from several types of GABA-releasing interneurons and innervate them reciprocally. Glutamatergic activation of interneurons involves both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) type glutamate receptors expressed in type I synapses, mostly on their dendritic shafts. On average, the synaptic AMPA receptor content is several times higher on interneurons than in the spines of pyramidal cells. To compare the NMDA receptor content of synapses, we used a quantitative postembedding immunogold technique on serial electron microscopic sections, and analysed the synapses on interneuron dendrites and pyramidal cell spines in the CA1 area. Because all NMDA receptors contain the obligatory NR1 subunit, receptor localisation was carried out using antibodies recognising all splice variants of the NR1 subunit. Four populations of synapse were examined: i). on spines of pyramidal cells in stratum (str.) radiatum and str. oriens; ii). on parvalbumin-positive interneuronal dendritic shafts in str. radiatum; iii). on randomly found dendritic shafts in str. oriens and iv). on somatostatin-positive interneuronal dendritic shafts and somata in str. oriens. On average, the size of the synapses on spines was about half of those on interneurons. The four populations of synapse significantly differed in labelling for the NR1 subunit. The median density of NR1 subunit labelling was highest on pyramidal cell spines. It was lowest in the synapses on parvalbumin-positive dendrites in str. radiatum, where more than half of these synapses were immunonegative. In str. oriens, synapses on interneurons had a high variability of receptor content; some dendrites were similar to those in str. radiatum, including the proximal synapses of somatostatin-positive cells, whereas others had immunoreactivity for the NR1 subunit similar to or higher than synapses on pyramidal cell spines. These results show that synaptic NMDA receptor density differs between pyramidal cells and interneurons. Some interneurons may have a high NMDA receptor content, whereas others, like some parvalbumin-expressing cells, a particularly low synaptic NMDA receptor content. Consequently, fast glutamatergic activation of interneurons is expected to show cell type-specific time course and state-dependent dynamics.
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Evolution of Alternative Adaptive Immune Systems in Vertebrates.
Boehm, Thomas; Hirano, Masayuki; Holland, Stephen J; Das, Sabyasachi; Schorpp, Michael; Cooper, Max D
2018-04-26
Adaptive immunity in jawless fishes is based on antigen recognition by three types of variable lymphocyte receptors (VLRs) composed of variable leucine-rich repeats, which are differentially expressed by two T-like lymphocyte lineages and one B-like lymphocyte lineage. The T-like cells express either VLRAs or VLRCs of yet undefined antigen specificity, whereas the VLRB antibodies secreted by B-like cells bind proteinaceous and carbohydrate antigens. The incomplete VLR germline genes are assembled into functional units by a gene conversion-like mechanism that employs flanking variable leucine-rich repeat sequences as templates in association with lineage-specific expression of cytidine deaminases. B-like cells develop in the hematopoietic typhlosole and kidneys, whereas T-like cells develop in the thymoid, a thymus-equivalent region at the gill fold tips. Thus, the dichotomy between T-like and B-like cells and the presence of dedicated lymphopoietic tissues emerge as ancestral vertebrate features, whereas the somatic diversification of structurally distinct antigen receptor genes evolved independently in jawless and jawed vertebrates.
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[Production, specificity and structure of immunoglobulins].
Goujard, C; Delfraissy, J F
1991-03-21
Immunoglobulin is a key factor of the immune response resulting from B-cell activation and associated with T-cell stimulation. Because of its structure, this antibody has a dual function: it specifically recognizes the inducer antigen in the variable region and eliminates it by a constant portion which is responsible for effector properties. Surface immunoglobulin, therefore, is the B-cell antigen receptor; it differs from the T-cell receptor in that it recognizes the antigen unbound to the major istocompatibility complex; binding the antigen results in direct signal transduction first in the cytoplasm, then in the nucleus. This receptor can be secreted in the body: it is made up of circulating immunoglobulins. Human immunoglobulins are divided into 5 classes, each of them with its own response kinetics, distribution and functions. The variability of the antibody response accounts for a genetic organization involving numerous genes which may be associated with each other, or mutate, or recombine during maturation of the lymphocytes. Altogether, this system has a theoretical capacity of response to three hundred million different antigens.
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Isolation and characterization of anti c-met single chain fragment variable (scFv) antibodies.
Qamsari, Elmira Safaie; Sharifzadeh, Zahra; Bagheri, Salman; Riazi-Rad, Farhad; Younesi, Vahid; Abolhassani, Mohsen; Ghaderi, Sepideh Safaei; Baradaran, Behzad; Somi, Mohammad Hossein; Yousefi, Mehdi
2017-12-01
The receptor tyrosine kinase (RTK) Met is the cell surface receptor for hepatocyte growth factor (HGF) involved in invasive growth programs during embryogenesis and tumorgenesis. There is compelling evidence suggesting important roles for c-Met in colorectal cancer proliferation, migration, invasion, angiogenesis, and survival. Hence, a molecular inhibitor of an extracellular domain of c-Met receptor that blocks c-Met-cell surface interactions could be of great thera-peutic importance. In an attempt to develop molecular inhibitors of c-Met, single chain variable fragment (scFv) phage display libraries Tomlinson I + J against a specific synthetic oligopeptide from the extracellular domain of c-Met receptor were screened; selected scFv were then characterized using various immune techniques. Three c-Met specific scFv (ES1, ES2, and ES3) were selected following five rounds of panning procedures. The scFv showed specific binding to c-Met receptor, and significantly inhibited proliferation responses of a human colorectal carcinoma cell line (HCT-116). Moreover, anti- apoptotic effects of selected scFv antibodies on the HCT-116 cell line were also evaluated using Annexin V/PI assays. The results demonstrated rates of apoptotic cell death of 46.0, 25.5, and 37.8% among these cells were induced by use of ES1, ES2, and ES3, respectively. The results demonstrated ability to successfully isolate/char-acterize specific c-Met scFv that could ultimately have a great therapeutic potential in immuno-therapies against (colorectal) cancers.
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Dopamine D2 receptor expression in the corticotroph cells of the human normal pituitary gland.
Pivonello, Rosario; Waaijers, Marlijn; Kros, Johan M; Pivonello, Claudia; de Angelis, Cristina; Cozzolino, Alessia; Colao, Annamaria; Lamberts, Steven W J; Hofland, Leo J
2017-08-01
The dopamine D 2 receptor is the main dopamine receptor expressed in the human normal pituitary gland. The aim of the current study was to evaluate dopamine D 2 receptor expression in the corticotroph cell populations of the anterior lobe and pars intermedia, as well as posterior lobe of the human normal pituitary gland by immunohistochemistry. Human normal pituitary gland samples obtained from routine autopsies were used for the study. In all cases, histology together with immunostaining for adrenocorticotropic hormone, melanocyte-stimulating hormone, prolactin, and neurofilaments were performed and compared to the immunostaining for D 2 receptor. D 2 receptor was heterogeneously expressed in the majority of the cell populations of the anterior and posterior lobe as well as in the area localized between the anterior and posterior lobe, and arbitrary defined as "intermediate zone". This zone, characterized by the presence of nerve fibers included the residual pars intermedia represented by the colloid-filled cysts lined by the remnant melanotroph cells strongly expressing D 2 receptors, and clusters of corticotroph cells, belonging to the anterior lobe but localized within the cysts and adjacent to the posterior lobe, variably expressing D 2 receptors. D 2 dopamine receptor is expressed in the majority of the cell populations of the human normal pituitary gland, and particularly, in the different corticotroph cell populations localized in the anterior lobe and the intermediate zone of the pituitary gland.
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Wang, Jueqiong; Lu, Liu; Kok, Chung H.; Saunders, Verity A.; Goyne, Jarrad M.; Dang, Phuong; Leclercq, Tamara M.; Hughes, Timothy P.; White, Deborah L.
2017-01-01
Imatinib is actively transported by organic cation transporter-1 (OCT-1) influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Herein we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor γ agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor γ antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to BCR-ABL kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor γ-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor γ transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; P<0.0001), suggesting that peroxisome proliferator-activated receptor γ activation has a negative impact on the intracellular uptake of imatinib and consequent BCR-ABL kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor γ activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor γ agonist pioglitazone was reported to act synergistically with imatinib, targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor γ ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor γ activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in patients with high peroxisome proliferator-activated receptor γ transcriptional activity. PMID:28154092
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Rakhmanov, Mirzokhid; Keller, Baerbel; Gutenberger, Sylvia; Foerster, Christian; Hoenig, Manfred; Driessen, Gertjan; van der Burg, Mirjam; van Dongen, Jacques J.; Wiech, Elisabeth; Visentini, Marcella; Quinti, Isabella; Prasse, Antje; Voelxen, Nadine; Salzer, Ulrich; Goldacker, Sigune; Fisch, Paul; Eibel, Hermann; Schwarz, Klaus; Peter, Hans-Hartmut; Warnatz, Klaus
2009-01-01
The homeostasis of circulating B cell subsets in the peripheral blood of healthy adults is well regulated, but in disease it can be severely disturbed. Thus, a subgroup of patients with common variable immunodeficiency (CVID) presents with an extraordinary expansion of an unusual B cell population characterized by the low expression of CD21. CD21low B cells are polyclonal, unmutated IgM+IgD+ B cells but carry a highly distinct gene expression profile which differs from conventional naïve B cells. Interestingly, while clearly not representing a memory population, they do share several features with the recently defined memory-like tissue, Fc receptor-like 4 positive B cell population in the tonsils of healthy donors. CD21low B cells show signs of previous activation and proliferation in vivo, while exhibiting defective calcium signaling and poor proliferation in response to B cell receptor stimulation. CD21low B cells express decreased amounts of homeostatic but increased levels of inflammatory chemokine receptors. This might explain their preferential homing to peripheral tissues like the bronchoalveolar space of CVID or the synovium of rheumatoid arthritis patients. Therefore, as a result of the close resemblance to the gene expression profile, phenotype, function and preferential tissue homing of murine B1 B cells, we suggest that CD21low B cells represent a human innate-like B cell population. PMID:19666505
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Stone, Jennifer D.; Harris, Daniel T.; Soto, Carolina M.; Chervin, Adam S.; Aggen, David H.; Roy, Edward J.; Kranz, David M.
2014-01-01
Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: 1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or 2) introduction of a chimeric antigen receptor (CAR), including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vβ-linker-Vα) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains, and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins. PMID:25082071
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Comparison of the cattle leukocyte receptor complex with related livestock species
USDA-ARS?s Scientific Manuscript database
The natural killer (NK) cell receptor gene complexes are highly variable between species, and their repetitive nature makes genomic assembly and characterization problematic. As a result, most reference genome assemblies are heavily fragmented and/or misassembled over these regions. However, new lon...
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Pyo, Suhkneung; Kang, Chung Hyo; Lee, Chong Ock; Lee, Heung Kyoung; Choi, Sang Un; Park, Chi Hoon
2018-01-01
Gastric cancer is a malignancy that has a high mortality rate. Although progress has been made in the treatment of gastric cancer, many patients experience cancer recurrence and metastasis. Folate receptor 1 (FOLR1) is overexpressed on the cell surface in over one-third of gastric cancer patients, but rarely is expressed in normal tissue. This makes FOLR1 a potential target for chimeric antigen receptor (CAR) T cell immunotherapy, although the function of FOLR1 has not been elucidated. CAR are engineered fusion receptor composed of an antigen recognition region and signaling domains. T cells expressing CAR have specific activation and cytotoxic effects against cancer cells containing the target antigen. In this study, we generated a CAR that targets FOLR1 composed of a single-chain variable fragment (scFv) of FOLR1 antibody and signaling domains consisting of CD28 and CD3ζ. Both FOLR1-CAR KHYG-1, a natural killer cell line, and FOLR1-CAR T cells recognized FOLR1-positive gastric cancer cells in a MHC-independent manner and induced secretion of various cytokines and caused cell death. Conclusively, this is the first study to demonstrate that CAR KHYG-1/T cells targeting FOLR1 are effective against FOLR1-positive gastric cancer cells. PMID:29874279
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Muntasell, Aura; López-Montañés, María; Vera, Andrea; Heredia, Gemma; Romo, Neus; Peñafiel, Judith; Moraru, Manuela; Vila, Joan; Vilches, Carlos; López-Botet, Miguel
2013-12-01
Human cytomegalovirus (HCMV) infection promotes a persistent expansion of a functionally competent NK-cell subset expressing the activating CD94/NKG2C receptor. Factors underlying the wide variability of this effect observed in HCMV-seropositive healthy individuals and exacerbated in immunocompromized patients are uncertain. A deletion of the NKG2C gene has been reported, and an apparent relation of NKG2C genotype with circulating NKG2C(+) NK-cell numbers was observed in HCMV(+) children. We have assessed the influence of NKG2C gene dose on the NK-cell repertoire in a cohort of young healthy adults (N = 130, median age 19 years). Our results revealed a relation of NKG2C copy number with surface receptor levels and with NKG2C(+) NK-cell numbers in HCMV(+) subjects, independently of HLA-E dimorphism. Functional studies showed quantitative differences in signaling (i.e. iCa(2+) influx), degranulation, and IL-15-dependent proliferation, in response to NKG2C engagement, between NK cells from NKG2C(+/+) and hemizygous subjects. These observations provide a mechanistic interpretation on the way the NKG2C genotype influences steady-state NKG2C(+) NK-cell numbers, further supporting an active involvement of the receptor in the HCMV-induced reconfiguration of the NK-cell compartment. The putative implications of NKG2C zygosity over viral control and other clinical variables deserve attention. © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.
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Haynes, Nicole M; Trapani, Joseph A; Teng, Michele W L; Jackson, Jacob T; Cerruti, Loretta; Jane, Stephen M; Kershaw, Michael H; Smyth, Mark J; Darcy, Phillip K
2002-11-15
A new strategy to improve the therapeutic utility of redirected T cells for cancer involves the development of novel Ag-specific chimeric receptors capable of stimulating optimal and sustained T cell antitumor activity in vivo. Given that T cells require both primary and costimulatory signals for optimal activation and that many tumors do not express critical costimulatory ligands, modified single-chain Ab receptors have been engineered to codeliver CD28 costimulation. In this study, we have compared the antitumor potency of primary T lymphocytes expressing carcinoembryonic Ag (CEA)-reactive chimeric receptors that incorporate either TCR-zeta or CD28/TCR-zeta signaling. Although both receptor-transduced T cell effector populations demonstrated cytolysis of CEA(+) tumors in vitro, T cells expressing the single-chain variable fragment of Ig (scFv)-CD28-zeta chimera had a far greater capacity to control the growth of CEA(+) xenogeneic and syngeneic colon carcinomas in vivo. The observed enhanced antitumor activity of T cells expressing the scFv-CD28-zeta receptor was critically dependent on perforin and the production of IFN-gamma. Overall, this study has illustrated the ability of a chimeric scFv receptor capable of harnessing the signaling machinery of both TCR-zeta and CD28 to augment T cell immunity against tumors that have lost expression of both MHC/peptide and costimulatory ligands in vivo.
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NK cells and their receptors in naive and rituximab-treated patients with anti-MAG polyneuropathy.
Benedetti, Luana; Facco, Monica; Franciotta, Diego; Dalla Torre, Chiara; Campagnolo, Marta; Lucchetta, Marta; Boscaro, Elisa; Ermani, Mario; Del Sette, Massimo; Berno, Tamara; Candiotto, Laura; Zambello, Renato; Briani, Chiara
2013-08-15
Natural killer (NK) cells can bridge innate and acquired immunity, and play a role in autoimmunity. A few studies evaluated the distribution of NK cells and the expression of their receptors in chronic immune-mediated demyelinating polyneuropathies. We investigated NK cell distribution and NK cell receptor expression in 20 naïve patients with anti-MAG polyneuropathy (MAG-PN). Using flow cytometry, we analysed NK cells and a series of NK cell receptors in the peripheral blood of patients with MAG-PN, and, as controls, in patients with chronic inflammatory demyelinating peripheral polyradiculoneuropathy (CIDP) and in healthy subjects. Six MAG-PN patients were also tested after rituximab treatment. At baseline the percentage of NK cells did not differ among the groups. KIR2DL2 receptor expression in MAG-PN patients was higher, andCD94/NKG2A receptor expression in both MAG-PN and CIDP patients was lower than in healthy controls. These abnormalities did not correlate with any clinical or demographic variable. No modification was found after rituximab therapy. The data suggest that MAG-PN shows abnormalities in NK cell receptors that characterise other autoimmune diseases, and cannot help in differential diagnosis with CIDP. The impairment of the relevant CD94/NKG2A inhibitory pathway, which might play a central role in the development and perpetuation of MAG-PN, warrants further functional investigations. Copyright © 2013 Elsevier B.V. All rights reserved.
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A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells.
Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Hamana, Hiroshi; Nakagawa, Hidetoshi; Jin, Aishun; Lin, Zhezhu; Muraguchi, Atsushi
2014-10-31
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy. Copyright © 2014 Elsevier Inc. All rights reserved.
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Co-evolution of MHC class I and variable NK cell receptors in placental mammals.
Guethlein, Lisbeth A; Norman, Paul J; Hilton, Hugo G; Parham, Peter
2015-09-01
Shaping natural killer (NK) cell functions in human immunity and reproduction are diverse killer cell immunoglobulin-like receptors (KIRs) that recognize polymorphic MHC class I determinants. A survey of placental mammals suggests that KIRs serve as variable NK cell receptors only in certain primates and artiodactyls. Divergence of the functional and variable KIRs in primates and artiodactyls predates placental reproduction. Among artiodactyls, cattle but not pigs have diverse KIRs. Catarrhine (humans, apes, and Old World monkeys) and platyrrhine (New World monkeys) primates, but not prosimians, have diverse KIRs. Platyrrhine and catarrhine systems of KIR and MHC class I are highly diverged, but within the catarrhines, a stepwise co-evolution of MHC class I and KIR is discerned. In Old World monkeys, diversification focuses on MHC-A and MHC-B and their cognate lineage II KIR. With evolution of C1-bearing MHC-C from MHC-B, as informed by orangutan, the focus changes to MHC-C and its cognate lineage III KIR. Evolution of C2 from C1 and fixation of MHC-C drove further elaboration of MHC-C-specific KIR, as exemplified by chimpanzee. In humans, the evolutionary trajectory changes again. Emerging from reorganization of the KIR locus and selective attenuation of KIR avidity for MHC class I are the functionally distinctive KIR A and KIR B haplotypes. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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Armour, Kathryn L; Smith, Cheryl S; Clark, Michael R
2010-03-31
The efficacy of a therapeutic IgG molecule may be as dependent on the optimisation of the constant region to suit its intended indication as on the selection of its variable regions. A crucial effector function to be maximised or minimised is antibody-dependent cell-mediated cytotoxicity by natural killer cells. Traditional assays of ADCC activity suffer from considerable inter-donor and intra-donor variability, which makes the measurement of antibody binding to human FcgammaRIIIa, the key receptor for ADCC, an attractive alternative method of assessment. Here, we describe the development of cell lines and assays for this purpose. The transmembrane receptor, FcgammaRIIIa, requires co-expression with signal transducing subunits to prevent its degradation, unlike the homologous receptor FcgammaRIIIb that is expressed as a GPI-anchored molecule. Therefore, to simplify the production of cell lines as reliable assay components, we expressed FcgammaRIIIa as a GPI-anchored molecule. Separate, stable CHO cell lines that express either the 158F or the higher-affinity 158V allotype of FcgammaRIIIa were isolated using fluorescence-activated cell sorting. The identities of the expressed receptors were confirmed using a panel of monoclonal antibodies that distinguish between subclasses and allotypes of FcgammaRIII and the cell lines were shown to have slightly higher levels of receptor than FcgammaRIII-positive peripheral blood mononuclear cells. Because the affinity of FcgammaRIIIa for IgG is intermediate amongst the receptors that bind IgG, we were able to use these cell lines to develop flow cytometric assays to measure the binding of both complexed and monomeric immunoglobulin. Thus, by choosing the appropriate method, weakly- or strongly-binding IgG can be efficiently compared. We have quantified the difference in the binding of wildtype IgG1 and IgG3 molecules to the two functional allotypes of the receptor and report that the FcgammaRIIIa-158V-antibody interaction is 3- to 4-fold stronger that the interaction with FcgammaRIIIa-158F. Overall, these robust assays should be valuable for batch-testing clinical material as well as providing tools for improving the design of therapeutic IgG. 2010 Elsevier B.V. All rights reserved.
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Variability and Order in Cytoskeletal Dynamics of Motile Amoeboid Cells
NASA Astrophysics Data System (ADS)
Hsu, Hsin-Fang; Bodenschatz, Eberhard; Westendorf, Christian; Gholami, Azam; Pumir, Alain; Tarantola, Marco; Beta, Carsten
2017-10-01
The chemotactic motion of eukaryotic cells such as leukocytes or metastatic cancer cells relies on membrane protrusions driven by the polymerization and depolymerization of actin. Here we show that the response of the actin system to a receptor stimulus is subject to a threshold value that varies strongly from cell to cell. Above the threshold, we observe pronounced cell-to-cell variability in the response amplitude. The polymerization time, however, is almost constant over the entire range of response amplitudes, while the depolymerization time increases with increasing amplitude. We show that cell-to-cell variability in the response amplitude correlates with the amount of Arp2 /3 , a protein that enhances actin polymerization. A time-delayed feedback model for the cortical actin concentration is consistent with all our observations and confirms the role of Arp2 /3 in the observed cell-to-cell variability. Taken together, our observations highlight robust regulation of the actin response that enables a reliable timing of cell movement.
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Nogueira, R L M; Faria, M H G; Osterne, R L V; Cavalcante, R B; Ribeiro, R A; Rabenhorst, S H B
2012-08-01
Central giant cell lesion is an uncommon benign jaw lesion, with uncertain aetiology, and variable clinical behaviour. Studies of molecular markers may help to understand the nature and behaviour of this lesion, and eventually may represent a target for pharmacological approaches to treatment. The aim of this study was to analyse the expression of glucocorticoid and calcitonin receptors in central giant cell lesions before and after treatment with intralesional steroid. Paraffin-embedded blocks from patients who underwent treatment with intralesional triamcinolone hexacetonide injections were stained immunohistochemically. Biological material from patients who underwent a surgical procedure after treatment were tested immunohistochemically. 18 cases (9 aggressive and 9 non-aggressive) were included. The difference in calcitonin receptor expression was not statistically significant between the aggressive and non-aggressive lesions and between the patients with a good response and those with a moderate/negative response to treatment. Glucocorticoid receptor expression in the multinucleated giant cells was higher in patients with a good response. It can be postulated that immunohistochemical staining for glucocorticoid receptors may provide a tool for selecting the therapeutic strategy. An H-score greater than 48 for glucocorticoid receptors in multinucleated giant cells predicted a good response in this study. Copyright © 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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Phenotype Variation in Human Immunodeficiency virus Type 1 Transmission and Disease Progression
Cavarelli, Mariangela; Scarlatti, Gabriella
2009-01-01
Human immunodeficiency virus type I (HIV-1) infects target cells through interaction with the CD4 molecule and chemokine receptors, mainly CCR5 and CXCR4. Viral isolates can be phenotypically classified based on the co-receptor they utilize to infect target cells. Thus, R5 and X4 virus use respectively CCR5 and CXCR4, whereas R5X4 virus can use either CCR5 or CXCR4. This review describes the central role played by co-receptor expression and usage for HIV-1 cell tropism, transmission and pathogenesis. We discuss various hypotheses proposed to explain the preferential transmission of R5 viruses and the mechanisms driving the change of HIV-1 co-receptor usage in the course of infection. Recent insights in the intrinsic variability of R5 viruses and their role in influencing disease progression in both adults and children are also discussed. PMID:19893208
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Phenotype variation in human immunodeficiency virus type 1 transmission and disease progression.
Cavarelli, Mariangela; Scarlatti, Gabriella
2009-01-01
Human immunodeficiency virus type I (HIV-1) infects target cells through interaction with the CD4 molecule and chemokine receptors, mainly CCR5 and CXCR4. Viral isolates can be phenotypically classified based on the co-receptor they utilize to infect target cells. Thus, R5 and X4 virus use respectively CCR5 and CXCR4, whereas R5X4 virus can use either CCR5 or CXCR4. This review describes the central role played by co-receptor expression and usage for HIV-1 cell tropism, transmission and pathogenesis. We discuss various hypotheses proposed to explain the preferential transmission of R5 viruses and the mechanisms driving the change of HIV-1 co-receptor usage in the course of infection. Recent insights in the intrinsic variability of R5 viruses and their role in influencing disease progression in both adults and children are also discussed.
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Allcock, Richard J N; Barrow, Alexander D; Forbes, Simon; Beck, Stephan; Trowsdale, John
2003-02-01
We have characterized a cluster of single immunoglobulin variable (IgV) domain receptors centromeric of the major histocompatibility complex (MHC) on human chromosome 6. In addition to triggering receptor expressed on myeloid cells (TREM)-1 and TREM2, the cluster contains NKp44, a triggering receptor whose expression is limited to NK cells. We identified three new related genes and two gene fragments within a cluster of approximately 200 kb. Two of the three new genes lack charged residues in their transmembrane domain tails. Further, one of the genes contains two potential immunotyrosine Inhibitory motifs in its cytoplasmic tail, suggesting that it delivers inhibitory signals. The human and mouse TREM clusters appear to have diverged such that there are unique sequences in each species. Finally, each gene in the TREM cluster was expressed in a different range of cell types.
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Role of the B-cell receptor in chronic lymphocytic leukemia: where do we stand?
Fais, Franco; Bruno, Silvia; Ghiotto, Fabio
2010-01-01
The past 15 years have witnessed an enormous effort in studying B-cell Chronic Lymphocytic Leukemia. A great number of researches brought significant novel information and a better understanding of the natural history of this disease. This mini review will focus on the studies related to the Immunoglobulin variable (IgV) genes rearrangements that compose the B-cell receptor (BcR) of the leukemic clones. These studies have defined a role for the antigen(s) in the paths that lead to leukemic clone generation/expansion and underscore the informative value represented by BcR analyses.
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Surface receptors on human haematopoietic cell lines.
Huber, C; Sundström, C; Nilsson, K; Wigzell, H
1976-01-01
The expression of complement receptors, of Fc receptors, of SRBC receptors and of S-Ig was investigated on human haematopoietic cell lines of proved malignant derivation. According to their origin and to a panel of phenotypic markers these lines have been classified into lymphoma lines, myeloma lines and leukemia lines. Results were compared with those obtained on non-malignant EBV carrying lymphoblastoid cell lines (LCL). Among the lymphoid cell lines the LCL showed a pattern of B-lymphocyte surface markers, i.e. surface immunoglobulins, C3 receptors but low density of Fc receptors. The non-Burkitt lymphoma lines bore in varying degree these B-lymphocyte markers. The lines U-698 M and DG-75 were exceptional in having only surface immunoglobulin. The Burkitt lymphoma lines had all B-lymphocyte markers. The myeloma lines differed from the lymphoid lines in lacking C3 and Fc receptors and showed only trace amounts of surface immunoglobulins. In contrast to lymphoid and myeloma lines, the leukaemia lines were completely lacking surface immunoglobulins, but showed C3 and Fc receptors in variable densities. On line, the ALL derived line MOLT-3 showed the capacity to spontaneous rosette formation with SRBC. The findings that LCL presented a homogeneous pattern of B-lymphocyte surface markers may be of value in order to discriminate between these lines and lines derived from haematopoietic malignancies other than Burkitt lymphomas. PMID:963908
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Riet, Tobias; Holzinger, Astrid; Dörrie, Jan; Schaft, Niels; Schuler, Gerold; Abken, Hinrich
2013-01-01
Redirecting T cells with a chimeric antigen receptor (CAR) of predefined specificity showed remarkable efficacy in the adoptive therapy trials of malignant diseases. The CAR consists of a single chain fragment of variable region (scFv) antibody targeting domain covalently linked to the CD3ζ signalling domain of the T cell receptor complex to mediate T cell activation upon antigen engagement. By using an antibody-derived targeting domain a CAR can potentially redirect T cells towards any target expressed on the cell surface as long as a binding domain is available. Antibody-mediated targeting moreover circumvents MHC restriction of the targeted antigen, thereby broadening the potential of applicability of adoptive T cell therapy. While T cells were so far genetically modified by viral transduction, transient modification with a CAR by RNA transfection gained increasing interest during the last years. This chapter focuses on methods to modify human T cells from peripheral blood with a CAR by electroporation of in vitro transcribed RNA and to test modified T cells for function for use in adoptive immunotherapy.
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Primer sets for cloning the human repertoire of T cell Receptor Variable regions.
Boria, Ilenia; Cotella, Diego; Dianzani, Irma; Santoro, Claudio; Sblattero, Daniele
2008-08-29
Amplification and cloning of naïve T cell Receptor (TR) repertoires or antigen-specific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V) regions are encoded by several genes that recombine during T cell development. The cloning of expressed genes as large diverse libraries from natural sources relies upon the availability of primers able to amplify as many V genes as possible. Here, we present a list of primers computationally designed on all functional TR V and J genes listed in the IMGT, the ImMunoGeneTics information system. The list consists of unambiguous or degenerate primers suitable to theoretically amplify and clone the entire TR repertoire. We show that it is possible to selectively amplify and clone expressed TR V genes in one single RT-PCR step and from as little as 1000 cells. This new primer set will facilitate the creation of more diverse TR libraries than has been possible using currently available primer sets.
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Clinico-pathological and biological prognostic variables in squamous cell carcinoma of the vulva.
Gadducci, Angiolo; Tana, Roberta; Barsotti, Cecilia; Guerrieri, Maria Elena; Genazzani, Andrea Riccardo
2012-07-01
Several clinical-pathological parameters have been related to survival of patients with invasive squamous cell carcinoma of the vulva, whereas few studies have investigated the ability of biological variables to predict the clinical outcome of these patients. The present paper reviews the literature data on the prognostic relevance of lymph node-related parameters, primary tumor-related parameters, FIGO stage, blood variables, and tissue biological variables. Regarding these latter, the paper takes into account the analysis of DNA content, cell cycle-regulatory proteins, apoptosis-related proteins, epidermal growth factor receptor [EGFR], and proteins that are involved in tumor invasiveness, metastasis and angiogenesis. At present, the lymph node status and FIGO stage according to the new 2009 classification system are the main predictors for vulvar squamous cell carcinoma, whereas biological variables do not have yet a clinical relevance and their role is still investigational. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
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Zarin, Payam; In, Tracy S H; Chen, Edward L Y; Singh, Jastaranpreet; Wong, Gladys W; Mohtashami, Mahmood; Wiest, David L; Anderson, Michele K; Zúñiga-Pflücker, Juan Carlos
2018-05-13
γδ T-cells perform a wide range of tissue and disease specific functions that are dependent on the effector cytokines produced by these cells. However, the aggregate signals required for the development of interferon-γ (IFNγ) and interleukin-17 (IL-17) producing γδ T-cells remain unknown. Here, we define the cues involved in the functional programming of γδ T-cells, by examining the roles of T-cell receptor (TCR), Notch, and cytokine-receptor signaling. KN6 γδTCR-transduced Rag2 -/- T-cell progenitors were cultured on stromal cells variably expressing TCR and Notch ligands, supplemented with different cytokines. We found that distinct combinations of these signals are required to program IFNγ versus IL-17 producing γδ T cell subsets, with Notch and weak TCR ligands optimally enabling development of γδ17 cells in the presence of IL-1β, IL-21 and IL-23. Notably, these cytokines were also shown to be required for the intrathymic development of γδ17 cells. Together, this work provides a framework of how signals downstream of TCR, Notch and cytokine receptors integrate to program the effector function of IFNγ and IL-17 producing γδ T-cell subsets. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
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Statistical inference of the generation probability of T-cell receptors from sequence repertoires.
Murugan, Anand; Mora, Thierry; Walczak, Aleksandra M; Callan, Curtis G
2012-10-02
Stochastic rearrangement of germline V-, D-, and J-genes to create variable coding sequence for certain cell surface receptors is at the origin of immune system diversity. This process, known as "VDJ recombination", is implemented via a series of stochastic molecular events involving gene choices and random nucleotide insertions between, and deletions from, genes. We use large sequence repertoires of the variable CDR3 region of human CD4+ T-cell receptor beta chains to infer the statistical properties of these basic biochemical events. Because any given CDR3 sequence can be produced in multiple ways, the probability distribution of hidden recombination events cannot be inferred directly from the observed sequences; we therefore develop a maximum likelihood inference method to achieve this end. To separate the properties of the molecular rearrangement mechanism from the effects of selection, we focus on nonproductive CDR3 sequences in T-cell DNA. We infer the joint distribution of the various generative events that occur when a new T-cell receptor gene is created. We find a rich picture of correlation (and absence thereof), providing insight into the molecular mechanisms involved. The generative event statistics are consistent between individuals, suggesting a universal biochemical process. Our probabilistic model predicts the generation probability of any specific CDR3 sequence by the primitive recombination process, allowing us to quantify the potential diversity of the T-cell repertoire and to understand why some sequences are shared between individuals. We argue that the use of formal statistical inference methods, of the kind presented in this paper, will be essential for quantitative understanding of the generation and evolution of diversity in the adaptive immune system.
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Enterovirus 71 Uses Cell Surface Heparan Sulfate Glycosaminoglycan as an Attachment Receptor
Tan, Chee Wah; Poh, Chit Laa; Sam, I-Ching
2013-01-01
Enterovirus 71 (EV-71) infections are usually associated with mild hand, foot, and mouth disease in young children but have been reported to cause severe neurological complications with high mortality rates. To date, four EV-71 receptors have been identified, but inhibition of these receptors by antagonists did not completely abolish EV-71 infection, implying that there is an as yet undiscovered receptor(s). Since EV-71 has a wide range of tissue tropisms, we hypothesize that EV-71 infections may be facilitated by using receptors that are widely expressed in all cell types, such as heparan sulfate. In this study, heparin, polysulfated dextran sulfate, and suramin were found to significantly prevent EV-71 infection. Heparin inhibited infection by all the EV-71 strains tested, including those with a single-passage history. Neutralization of the cell surface anionic charge by polycationic poly-d-lysine and blockage of heparan sulfate by an anti-heparan sulfate peptide also inhibited EV-71 infection. Interference with heparan sulfate biosynthesis either by sodium chlorate treatment or through transient knockdown of N-deacetylase/N-sulfotransferase-1 and exostosin-1 expression reduced EV-71 infection in RD cells. Enzymatic removal of cell surface heparan sulfate by heparinase I/II/III inhibited EV-71 infection. Furthermore, the level of EV-71 attachment to CHO cell lines that are variably deficient in cell surface glycosaminoglycans was significantly lower than that to wild-type CHO cells. Direct binding of EV-71 particles to heparin-Sepharose columns under physiological salt conditions was demonstrated. We conclude that EV-71 infection requires initial binding to heparan sulfate as an attachment receptor. PMID:23097443
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Multiple P2Y receptor subtypes in the apical membranes of polarized epithelial cells
McAlroy, H L; Ahmed, S; Day, S M; Baines, D L; Wong, H Y; Yip, C Y; Ko, W H; Wilson, S M; Collett, A
2000-01-01
Apical ATP, ATP, UTP and UDP evoked transient increases in short circuit current (ISC, a direct measure of transepithelial ion transport) in confluent Caco-2 cells grown on permeable supports. These responses were mediated by a population of at least three pharmacologically distinct receptors. Experiments using cells grown on glass coverslips showed that ATP and UTP consistently increased intracellular free calcium ([Ca2+]i) whilst sensitivity to UDP was variable. Cross desensitization experiments suggested that the responses to UTP and ATP were mediated by a common receptor population. Messenger RNA transcripts corresponding to the P2Y2, P2Y4 and P2Y6 receptors genes were detected in cells grown on Transwell membranes by the reverse transcriptase–polymerase chain reaction. Identical results were obtained for cells grown on glass. Experiments in which ISC and [Ca2+]i were monitored simultaneously in cells on Transwell membranes, confirmed that apical ATP and UTP increased both parameters and showed that the UDP-evoked increase in ISC was accompanied by a [Ca2+]i-signal. Ionomycin consistently increased [Ca2+]i in such polarized cells but caused no discernible change in ISC. However, subsequent application of apical ATP or UTP evoked a small rise in ISC but no rise in [Ca2+]i. UDP evoked no such response. As well as evoking increases in [Ca2+]i, the ATP/UTP-sensitive receptors present in Caco-2 cells thus allow direct control over ion channels in the apical membrane. The UDP-sensitive receptors, however, appear to simply evoke a rise in [Ca2+]i. PMID:11139443
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Aghebati-Maleki, Leili; Younesi, Vahid; Jadidi-Niaragh, Farhad; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi
2017-01-01
Receptor tyrosine kinase-like orphan receptor (ROR1) belongs to one of the families of receptor tyrosine kinases (RTKs). RTKs are involved in the various physiologic cellular functions including proliferation, migration, survival, signaling and differentiation. Several RTKs are deregulated in various cancers implying the targeting potential of these molecules in cancer therapy. ROR1 has recently been shown to be expressed in various types of cancer cells but not in normal adult cells. Hence a molecular inhibitor of extracellular domain of ROR1 that inhibits ROR1-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of ROR1, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I + J, against one specific synthetic oligopeptide from extracellular domain of ROR1 and selected scFvs were characterized using various immunological techniques. Several ROR1 specific scFvs were selected following five rounds of panning procedure. The scFvs showed specific binding to ROR1 using immunological techniques. Our results demonstrate successful isolation and characterization of specific ROR1 scFvs that may have great therapeutic potential in cancer immunotherapy.
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Telesmanich, N R; Goncharenko, E V; Chaika, S O; Chaika, I A; Telicheva, V O
2016-01-01
Study mechanisms of interaction of diagnostic bacteriophage El Tor with sensitive strain Vibrio cholerae El Tor 18507 using direct protein profiling, identification of constant and variable proteins, taking part in interaction of the phage and cell, as well as carbohydrate-specific phage receptors. . A commercial preparation of cholera diagnostic bacteriophage El Tor, strain V. cholerae El Tor 18507 were used. Effect of carbohydrates on bacteriophage activity was determined in experiments with phage by a classic and modified by us method. Protein profiles of the studied objects were studied using MSP-analysis method. Sucrose was shown to inhibit lytic activity of bacteriophage. Proteome profiles of El Tor bacteriophage and sensitive indicator strains were studied, identification of constant and variable proteins of the studied objects by MSP Peak-list program was carried out. Analysis of changes of profiles of phage and microbial cell during interaction with sucrose gave a basis for assuming, that sucrose in the mixture of culture-phage enters interaction namely with phage protein receptors, blocking receptors specific for cholera vibrio, that subsequently manifests in a sharp decrease of phage activity against the sensitive strain.
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NASA Astrophysics Data System (ADS)
Xu, Xiaochun; Sinha, Lagnojita; Singh, Aparna; Yang, Cynthia; Xiang, Jialing; Tichauer, Kenneth M.
2015-03-01
Immunofluorescence staining is a robust way to visualize the distribution of targeted biomolecules invasively in in fixed tissues and tissue culture. Despite the fact that these methods has been a well-established method in fixed tissue imaging for over 70 years, quantification of receptor concentration still simply assumes that the signal from the targeted fluorescent marker after incubation and sufficient rinsing is directly proportional to the concentration of targeted biomolecules, thus neglecting the experimental inconsistencies in incubation and rinsing procedures and assuming no, nonspecific binding of the fluorescent markers. This work presents the first imaging approach capable of quantifying the concentration of cell surface receptor on cancer cells grown in vitro based on compartment modeling in a nondestructive way. The approach utilizes a dual-tracer protocol where any non-specific retention or variability in incubation and rinsing of a receptor-targeted imaging agent is corrected by simultaneously imaging the retention of a chemically similar, "untargeted" imaging agent. Various different compartment models were used to analyze the data in order to find the optimal procedure for extracting estimates of epidermal growth factor receptor (EGFR) concentration (a receptor overexpressed in many cancers and a key target for emerging molecular therapies) in tissue cultures with varying concentrations of human glioma cells (U251). Preliminary results demonstrated a need to model nonspecific binding of both the targeted and untargeted imaging agents used. The approach could be used to carry out the first repeated measures of cell surface receptor dynamics during 3D tumor mass development, in addition to the receptor response to therapies.
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Cao, Pengbo; Wall, Daniel
2017-04-04
The ability to recognize close kin confers survival benefits on single-celled microbes that live in complex and changing environments. Microbial kinship detection relies on perceptible cues that reflect relatedness between individuals, although the mechanisms underlying recognition in natural populations remain poorly understood. In myxobacteria, cells identify related individuals through a polymorphic cell surface receptor, TraA. Recognition of compatible receptors leads to outer membrane exchange among clonemates and fitness consequences. Here, we investigated how a single receptor creates a diversity in recognition across myxobacterial populations. We first show that TraA requires its partner protein TraB to function in cell-cell adhesion. Recognition is shown to be traA allele-specific, where polymorphisms within TraA dictate binding selectivity. We reveal the malleability of TraA recognition, and seemingly minor changes to its variable region reprogram recognition outcomes. Strikingly, we identify a single residue (A/P205) as a molecular switch for TraA recognition. Substitutions at this position change the specificity of a diverse panel of environmental TraA receptors. In addition, we engineered a receptor with unique specificity by simply creating an A205P substitution, suggesting that modest changes in TraA can lead to diversification of new recognition groups in nature. We hypothesize that the malleable property of TraA has allowed it to evolve and create social barriers between myxobacterial populations and in turn avoid adverse interactions with relatives.
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Yoon, Dok Hyun; Osborn, Mark J.; Tolar, Jakub; Kim, Chong Jai
2018-01-01
Chimeric antigen receptor (CAR) T cell therapy represents the first U.S. Food and Drug Administration approved gene therapy and these engineered cells function with unprecedented efficacy in the treatment of refractory CD19 positive hematologic malignancies. CAR translation to solid tumors is also being actively investigated; however, efficacy to date has been variable due to tumor-evolved mechanisms that inhibit local immune cell activity. To bolster the potency of CAR-T cells, modulation of the immunosuppressive tumor microenvironment with immune-checkpoint blockade is a promising strategy. The impact of this approach on hematological malignancies is in its infancy, and in this review we discuss CAR-T cells and their synergy with immune-checkpoint blockade. PMID:29364163
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Liu, Yanling; McDaniel, Jonathan R; Khan, Srijit; Campisi, Paolo; Propst, Evan J; Holler, Theresa; Grunebaum, Eyal; Georgiou, George; Ippolito, Gregory C; Ehrhardt, Götz R A
2018-06-15
FCRL4, a low-affinity IgA Ab receptor with strong immunoregulatory potential, is an identifying feature of a tissue-based population of memory B cells (Bmem). We used two independent approaches to perform a comparative analysis of the Ag receptor repertoires of FCRL4 + and FCRL4 - Bmem in human tonsils. We determined that FCRL4 + Bmem displayed lower levels of somatic mutations in their Ag receptors compared with FCRL4 - Bmem but had similar frequencies of variable gene family usage. Importantly, Abs with reactivity to commensal microbiota were enriched in FCRL4 + cells, a phenotype not due to polyreactive binding characteristics. Our study links expression of the immunoregulatory FCRL4 molecule with increased recognition of commensal microbial Ags. Copyright © 2018 by The American Association of Immunologists, Inc.
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Pivonello, Claudia; Rousaki, Panagoula; Negri, Mariarosaria; Sarnataro, Maddalena; Napolitano, Maria; Marino, Federica Zito; Patalano, Roberta; De Martino, Maria Cristina; Sciammarella, Concetta; Faggiano, Antongiulio; Rocco, Gaetano; Franco, Renato; Kaltsas, Gregory A; Colao, Annamaria; Pivonello, Rosario
2017-06-01
Somatostatin analogues and mTOR inhibitors have been used as medical therapy in lung carcinoids with variable results. No data are available on dopamine agonists as treatment for lung carcinoids. The main aim of the current study was to evaluate the effect of the combined treatment of somatostatin analogue octreotide and the dopamine agonist cabergoline with mTOR inhibitors in an in vitro model of typical lung carcinoids: the NCI-H727 cell line. In NCI-H727 cell line, reverse transcriptase-quantitative polymerase chain reaction and immunofluorescence were assessed to characterize the expression of the somatostatin receptor 2 and 5, dopamine receptor 2 and mTOR pathway components. Fifteen typical lung carcinoids tissue samples have been used for somatostatin receptor 2, dopamine receptor 2, and the main mTOR pathway component p70S6K expression and localization by immunohistochemistry. Cell viability, fluorescence-activated cell sorting analysis and western blot have been assessed to test the pharmacological effects of octreotide, cabergoline and mTOR inhibitors, and to evaluate the activation of specific cell signaling pathways in NCI-H727 cell line. NCI-H727 cell line expressed somatostatin receptor 2, somatostatin receptor 5 and dopamine receptor 2 and all mTOR pathway components at messenger and protein levels. Somatostatin receptor 2, dopamine receptor 2, and p70S6K (non phosphorylated and phosphorylated) proteins were expressed in most typical lung carcinoids tissue samples. Octreotide and cabergoline did not reduce cell viability as single agents but, when combined with mTOR inhibitors, they potentiate mTOR inhibitors effect after long-term exposure, reducing Akt and ERK phosphorylation, mTOR escape mechanisms, and increasing the expression DNA-damage-inducible transcript 4, an mTOR suppressor. In conclusion, the single use of octreotide and cabergoline is not sufficient to block cell viability but the combined approach of these agents with mTOR inhibitors might reduce the mTOR inhibitors-induced escape mechanisms and/or activate the endogenous mTOR suppressor, potentiating the effect of the mTOR inhibitors in an in vitro model of typical lung carcinoids.
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Hammer, Quirin; Rückert, Timo; Borst, Eva Maria; Dunst, Josefine; Haubner, André; Durek, Pawel; Heinrich, Frederik; Gasparoni, Gilles; Babic, Marina; Tomic, Adriana; Pietra, Gabriella; Nienen, Mikalai; Blau, Igor Wolfgang; Hofmann, Jörg; Na, Il-Kang; Prinz, Immo; Koenecke, Christian; Hemmati, Philipp; Babel, Nina; Arnold, Renate; Walter, Jörn; Thurley, Kevin; Mashreghi, Mir-Farzin; Messerle, Martin; Romagnani, Chiara
2018-05-01
Natural killer (NK) cells are innate lymphocytes that lack antigen-specific rearranged receptors, a hallmark of adaptive lymphocytes. In some people infected with human cytomegalovirus (HCMV), an NK cell subset expressing the activating receptor NKG2C undergoes clonal-like expansion that partially resembles anti-viral adaptive responses. However, the viral ligand that drives the activation and differentiation of adaptive NKG2C + NK cells has remained unclear. Here we found that adaptive NKG2C + NK cells differentially recognized distinct HCMV strains encoding variable UL40 peptides that, in combination with pro-inflammatory signals, controlled the population expansion and differentiation of adaptive NKG2C + NK cells. Thus, we propose that polymorphic HCMV peptides contribute to shaping of the heterogeneity of adaptive NKG2C + NK cell populations among HCMV-seropositive people.
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Eaton, Megan M.; Bracamontes, John; Shu, Hong-Jin; Li, Ping; Mennerick, Steven; Steinbach, Joe Henry
2014-01-01
Native γ-aminobutyric acid (GABA)A receptors consisting of α4, β1–3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4βδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4β2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct β2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, β2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4βδ receptors may assemble in a similar configuration in neurons and oocytes. PMID:25238745
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Sridharan, Rajashri; Connelly, Sara M; Naider, Fred; Dumont, Mark E
2016-11-11
We report here on the relationship between ligand binding and signaling responses in the yeast pheromone response pathway, a well characterized G protein-coupled receptor system. Responses to agonist (α-factor) by cells expressing widely varying numbers of receptors depend primarily on fractional occupancy, not the absolute number of agonist-bound receptors. Furthermore, the concentration of competitive antagonist required to inhibit α-factor-dependent signaling is more than 10-fold higher than predicted based on the known ligand affinities. Thus, responses to a particular number of agonist-bound receptors can vary greatly, depending on whether there are unoccupied or antagonist-bound receptors present on the same cell surface. This behavior does not appear to be due to pre-coupling of receptors to G protein or to the Sst2p regulator of G protein signaling. The results are consistent with a signaling response that is determined by the integration of positive signals from agonist-occupied receptors and inhibitory signals from unoccupied receptors, where the inhibitory signals can be diminished by antagonist binding. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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2014-01-01
Background Aberrant epigenetic profiles are concomitant with a spectrum of developmental defects and diseases. Role of methylation is an increasingly accepted factor in the pathophysiology of diabetes and its associated complications. This study aims to examine the correlation between oxidative stress and methylation of β1, β2 and β3-adrenergic receptors and to analyze the differential variability in the expression of these genes under hyperglycemic conditions. Methods Human retinal endothelial cells were cultured in CSC complete medium in normal (5 mM) or high (25 mM) glucose to mimic a diabetic condition. Reverse transcription PCR and Western Blotting were performed to examine the expression of β1, β2 and β3-adrenergic receptors. For detections, immunocytochemistry was used. Bisulfite sequencing method was used for promoter methylation analysis. Apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used to measure reactive oxygen species (ROS) production in the cells. Results β1 and β3-adrenergic receptors were expressed in retinal endothelial cells while β2-adrenergic receptor was not detectable both at protein and mRNA levels. Hyperglycemia had no significant effect on β1 and β2-adrenergic receptors methylation and expression however β3-adrenergic receptors showed a significantly higher expression (p < 0.05) and methylation (p < 0.01) in high and low glucose concentration respectively. Apoptosis and oxidative stress were inversely correlated with β3-adrenergic receptors methylation with no significant effect on β1 and β2-adrenergic receptors. β2-adrenergic receptor was hypermethylated with halted expression. Conclusion Our study demonstrates that β1 and β3-adrenergic receptors expressed in human retinal endothelial cells. Oxidative stress and apoptosis are inversely proportional to the extent of promoter methylation, suggesting that methylation loss might be due to oxidative stress-induced DNA damage. PMID:24885710
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Variable ligand- and receptor-binding hot spots in key strains of influenza neuraminidase
Votapka, Lane; Demir, Özlem; Swift, Robert V; Walker, Ross C; Amaro, Rommie E
2012-01-01
Influenza A continues to be a major public health concern due to its ability to cause epidemic and pandemic disease outbreaks in humans. Computational investigations of structural dynamics of the major influenza glycoproteins, especially the neuraminidase (NA) enzyme, are able to provide key insights beyond what is currently accessible with standard experimental techniques. In particular, all-atom molecular dynamics simulations reveal the varying degrees of flexibility for such enzymes. Here we present an analysis of the relative flexibility of the ligand- and receptor-binding area of three key strains of influenza A: highly pathogenic H5N1, the 2009 pandemic H1N1, and a human N2 strain. Through computational solvent mapping, we investigate the various ligand- and receptor-binding “hot spots” that exist on the surface of NA which interacts with both sialic acid receptors on the host cells and antiviral drugs. This analysis suggests that the variable cavities found in the different strains and their corresponding capacities to bind ligand functional groups may play an important role in the ability of NA to form competent reaction encounter complexes with other species of interest, including antiviral drugs, sialic acid receptors on the host cell surface, and the hemagglutinin protein. Such considerations may be especially useful for the prediction of how such complexes form and with what binding capacity. PMID:22872804
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Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don’t Forget the Fuel
Irving, Melita; Vuillefroy de Silly, Romain; Scholten, Kirsten; Dilek, Nahzli; Coukos, George
2017-01-01
T-cells play a critical role in tumor immunity. Indeed, the presence of tumor-infiltrating lymphocytes is a predictor of favorable patient prognosis for many indications and is a requirement for responsiveness to immune checkpoint blockade therapy targeting programmed cell death 1. For tumors lacking immune infiltrate, or for which antigen processing and/or presentation has been downregulated, a promising immunotherapeutic approach is chimeric antigen receptor (CAR) T-cell therapy. CARs are hybrid receptors that link the tumor antigen specificity and affinity of an antibody-derived single-chain variable fragment with signaling endodomains associated with T-cell activation. CAR therapy targeting CD19 has yielded extraordinary clinical responses against some hematological tumors. Solid tumors, however, remain an important challenge to CAR T-cells due to issues of homing, tumor vasculature and stromal barriers, and a range of obstacles in the tumor bed. Protumoral immune infiltrate including T regulatory cells and myeloid-derived suppressor cells have been well characterized for their ability to upregulate inhibitory receptors and molecules that hinder effector T-cells. A critical role for metabolic barriers in the tumor microenvironment (TME) is emerging. High glucose consumption and competition for key amino acids by tumor cells can leave T-cells with insufficient energy and biosynthetic precursors to support activities such as cytokine secretion and lead to a phenotypic state of anergy or exhaustion. CAR T-cell expansion protocols that promote a less differentiated phenotype, combined with optimal receptor design and coengineering strategies, along with immunomodulatory therapies that also promote endogenous immunity, offer great promise in surmounting immunometabolic barriers in the TME and curing solid tumors. PMID:28421069
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Modification of Expanded NK Cells with Chimeric Antigen Receptor mRNA for Adoptive Cellular Therapy.
Chu, Yaya; Flower, Allyson; Cairo, Mitchell S
2016-01-01
NK cells are bone marrow-derived cytotoxic lymphocytes that play a major role in the rejection of tumors and cells infected by viruses. The regulation of NK activation vs inhibition is regulated by the expression of a variety of NK receptors (NKRs) and specific NKRs' ligands expressed on their targets. However, factors limiting NK therapy include small numbers of active NK cells in unexpanded peripheral blood and lack of specific tumor targeting. Chimeric antigen receptors (CAR) usually include a single-chain Fv variable fragment from a monoclonal antibody, a transmembrane hinge region, and a signaling domain such as CD28, CD3-zeta, 4-1BB (CD137), or 2B4 (CD244) endodimers. Redirecting NK cells with a CAR will circumvent the limitations of the lack of NK targeting specificity. This chapter focuses on the methods to expand human NK cells from peripheral blood by co-culturing with feeder cells and to modify the expanded NK cells efficiently with the in vitro transcribed CAR mRNA by electroporation and to test the functionality of the CAR-modified expanded NK cells for use in adoptive cellular immunotherapy.
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Primer sets for cloning the human repertoire of T cell Receptor Variable regions
Boria, Ilenia; Cotella, Diego; Dianzani, Irma; Santoro, Claudio; Sblattero, Daniele
2008-01-01
Background Amplification and cloning of naïve T cell Receptor (TR) repertoires or antigen-specific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V) regions are encoded by several genes that recombine during T cell development. The cloning of expressed genes as large diverse libraries from natural sources relies upon the availability of primers able to amplify as many V genes as possible. Results Here, we present a list of primers computationally designed on all functional TR V and J genes listed in the IMGT®, the ImMunoGeneTics information system®. The list consists of unambiguous or degenerate primers suitable to theoretically amplify and clone the entire TR repertoire. We show that it is possible to selectively amplify and clone expressed TR V genes in one single RT-PCR step and from as little as 1000 cells. Conclusion This new primer set will facilitate the creation of more diverse TR libraries than has been possible using currently available primer sets. PMID:18759974
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Meiotic recombination generates rich diversity in NK cell receptor genes, alleles, and haplotypes
Norman, Paul J.; Abi-Rached, Laurent; Gendzekhadze, Ketevan; Hammond, John A.; Moesta, Achim K.; Sharma, Deepti; Graef, Thorsten; McQueen, Karina L.; Guethlein, Lisbeth A.; Carrington, Christine V.F.; Chandanayingyong, Dasdayanee; Chang, Yih-Hsin; Crespí, Catalina; Saruhan-Direskeneli, Güher; Hameed, Kamran; Kamkamidze, Giorgi; Koram, Kwadwo A.; Layrisse, Zulay; Matamoros, Nuria; Milà, Joan; Park, Myoung Hee; Pitchappan, Ramasamy M.; Ramdath, D. Dan; Shiau, Ming-Yuh; Stephens, Henry A.F.; Struik, Siske; Tyan, Dolly; Verity, David H.; Vaughan, Robert W.; Davis, Ronald W.; Fraser, Patricia A.; Riley, Eleanor M.; Ronaghi, Mostafa; Parham, Peter
2009-01-01
Natural killer (NK) cells contribute to the essential functions of innate immunity and reproduction. Various genes encode NK cell receptors that recognize the major histocompatibility complex (MHC) Class I molecules expressed by other cells. For primate NK cells, the killer-cell immunoglobulin-like receptors (KIR) are a variable and rapidly evolving family of MHC Class I receptors. Studied here is KIR3DL1/S1, which encodes receptors for highly polymorphic human HLA-A and -B and comprises three ancient allelic lineages that have been preserved by balancing selection throughout human evolution. While the 3DS1 lineage of activating receptors has been conserved, the two 3DL1 lineages of inhibitory receptors were diversified through inter-lineage recombination with each other and with 3DS1. Prominent targets for recombination were D0-domain polymorphisms, which modulate enhancer function, and dimorphism at position 283 in the D2 domain, which influences inhibitory function. In African populations, unequal crossing over between the 3DL1 and 3DL2 genes produced a deleted KIR haplotype in which the telomeric “half” was reduced to a single fusion gene with functional properties distinct from its 3DL1 and 3DL2 parents. Conversely, in Eurasian populations, duplication of the KIR3DL1/S1 locus by unequal crossing over has enabled individuals to carry and express alleles of all three KIR3DL1/S1 lineages. These results demonstrate how meiotic recombination combines with an ancient, preserved diversity to create new KIR phenotypes upon which natural selection acts. A consequence of such recombination is to blur the distinction between alleles and loci in the rapidly evolving human KIR gene family. PMID:19411600
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Reher, David; Klink, Barbara; Deutsch, Andreas; Voss-Böhme, Anja
2017-08-11
Cancer cell invasion, dissemination, and metastasis have been linked to an epithelial-mesenchymal transition (EMT) of individual tumour cells. During EMT, adhesion molecules like E-cadherin are downregulated and the decrease of cell-cell adhesion allows tumour cells to dissociate from the primary tumour mass. This complex process depends on intracellular cues that are subject to genetic and epigenetic variability, as well as extrinsic cues from the local environment resulting in a spatial heterogeneity in the adhesive phenotype of individual tumour cells. Here, we use a novel mathematical model to study how adhesion heterogeneity, influenced by intrinsic and extrinsic factors, affects the dissemination of tumour cells from an epithelial cell population. The model is a multiscale cellular automaton that couples intracellular adhesion receptor regulation with cell-cell adhesion. Simulations of our mathematical model indicate profound effects of adhesion heterogeneity on tumour cell dissemination. In particular, we show that a large variation of intracellular adhesion receptor concentrations in a cell population reinforces cell dissemination, regardless of extrinsic cues mediated through the local cell density. However, additional control of adhesion receptor concentration through the local cell density, which can be assumed in healthy cells, weakens the effect. Furthermore, we provide evidence that adhesion heterogeneity can explain the remarkable differences in adhesion receptor concentrations of epithelial and mesenchymal phenotypes observed during EMT and might drive early dissemination of tumour cells. Our results suggest that adhesion heterogeneity may be a universal trigger to reinforce cell dissemination in epithelial cell populations. This effect can be at least partially compensated by a control of adhesion receptor regulation through neighbouring cells. Accordingly, our findings explain how both an increase in intra-tumour adhesion heterogeneity and the loss of control through the local environment can promote tumour cell dissemination. This article was reviewed by Hanspeter Herzel, Thomas Dandekar and Marek Kimmel.
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Romero-Fernandez, W.; Borroto-Escuela, D.O.; Vargas-Barroso, V.; Narváez, M.; Di Palma, M.; Agnati, L.F.; Sahd, J. Larriva
2014-01-01
Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tuberoinfundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region. Dopamine D1 and D2 receptors may therefore directly and differentially modulate the activity and/or Dopamine synthesis of substantial numbers of tubero-infundibular dopamine neurons at the somatic and terminal level. The immunohistochemical work also gives support to the view that dopamine D1 receptors and/or dopamine D2 receptors in the lateral palisade zone by mediating dopamine volume transmission may contribute to the inhibition of luteinizing hormone releasing hormone release from nerve terminals in this region. PMID:25308843
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Romero-Fernandez, W; Borroto-Escuela, D O; Vargas-Barroso, V; Narváez, M; Di Palma, M; Agnati, L F; Larriva Sahd, J; Fuxe, K
2014-07-18
Dopamine D1 and D2 receptor immunohistochemistry and Golgi techniques were used to study the structure of the adult rat arcuate-median eminence complex, and determine the distribution of the dopamine D1 and D2 receptor immunoreactivities therein, particularly in relation to the tubero-infundibular dopamine neurons. Punctate dopamine D1 and D2 receptor immunoreactivities, likely located on nerve terminals, were enriched in the lateral palisade zone built up of nerve terminals, while the densities were low to modest in the medial palisade zone. A codistribution of dopamine D1 receptor or dopamine D2 receptor immunoreactive puncta with tyrosine hydroxylase immunoreactive nerve terminals was demonstrated in the external layer. Dopamine D1 receptor but not dopamine D2 receptor immnunoreactivites nerve cell bodies were found in the ventromedial part of the arcuate nucleus and in the lateral part of the internal layer of the median eminence forming a continuous cell mass presumably representing neuropeptide Y immunoreactive nerve cell bodies. The major arcuate dopamine/ tyrosine hydroxylase nerve cell group was found in the dorsomedial part. A large number of tyrosine hydroxylase immunoreactive nerve cell bodies in this region demonstrated punctate dopamine D1 receptor immunoreactivity but only a few presented dopamine D2 receptor immunoreactivity which were mainly found in a substantial number of tyrosine hydroxylase cell bodies of the ventral periventricular hypothalamic nucleus, also belonging to the tubero-infundibular dopamine neurons. Structural evidence for projections of the arcuate nerve cells into the median eminence was also obtained. Distal axons formed horizontal axons in the internal layer issuing a variable number of collaterals classified into single or multiple strands located in the external layer increasing our understanding of the dopamine nerve terminal networks in this region. Dopamine D1 and D2 receptors may therefore directly and differentially modulate the activity and /or Dopamine synthesis of substantial numbers of tubero-infundibular dopamine neurons at the somatic and terminal level. The immunohistochemical work also gives support to the view that dopamine D1 receptors and/or dopamine D2 receptors in the lateral palisade zone by mediating dopamine volume transmission may contribute to the inhibition of luteinizing hormone releasing hormone release from nerve terminals in this region.
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Muntasell, Aura; Vilches, Carlos; Angulo, Ana; López-Botet, Miguel
2013-05-01
As discussed in this review, human cytomegalovirus (HCMV) infection in healthy individuals is associated with a variable and persistent increase of NK cells expressing the CD94/NKG2C activating receptor. The expansion of NKG2C(+) NK cells reported in other infectious diseases is systematically associated with HCMV co-infection. The functionally mature NKG2C(bright) NK-cell subset expanding in HCMV(+) individuals displays inhibitory Ig-like receptors (KIR and LILRB1) specific for self HLA class I, and low levels of NKp46 and NKp30 activating receptors. Such reconfiguration of the NK-cell compartment appears particularly marked in immunocompromised patients and in children with symptomatic congenital infection, thus suggesting that its magnitude may be inversely related with the efficiency of the T-cell-mediated response. This effect of HCMV infection is reminiscent of the pattern of response of murine Ly49H(+) NK cells against murine CMV (MCMV), and it has been hypothesized that a cognate interaction of the CD94/NKG2C receptor with HCMV-infected cells may drive the expansion of the corresponding NK-cell subset. Yet, the precise role of NKG2C(+) cells in the control of HCMV infection, the molecular mechanisms underlying the NK-cell compartment redistribution, as well as its putative influence in the response to other pathogens and tumors remain open issues. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis
2013-01-01
A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536
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CSF1R mutations in hereditary diffuse leukoencephalopathy with spheroids are loss of function
NASA Astrophysics Data System (ADS)
Pridans, Clare; Sauter, Kristin A.; Baer, Kristin; Kissel, Holger; Hume, David A.
2013-10-01
Hereditary diffuse leukoencephalopathy with spheroids (HDLS) in humans is a rare autosomal dominant disease characterized by giant neuroaxonal swellings (spheroids) within the CNS white matter. Symptoms are variable and can include personality and behavioural changes. Patients with this disease have mutations in the protein kinase domain of the colony-stimulating factor 1 receptor (CSF1R) which is a tyrosine kinase receptor essential for microglia development. We investigated the effects of these mutations on Csf1r signalling using a factor dependent cell line. Corresponding mutant forms of murine Csf1r were expressed on the cell surface at normal levels, and bound CSF1, but were not able to sustain cell proliferation. Since Csf1r signaling requires receptor dimerization initiated by CSF1 binding, the data suggest a mechanism for phenotypic dominance of the mutant allele in HDLS.
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Immunological aspects in chronic lymphocytic leukemia (CLL) development.
García-Muñoz, Ricardo; Galiacho, Verónica Roldan; Llorente, Luis
2012-07-01
Chronic lymphocytic leukemia (CLL) is unique among B cell malignancies in that the malignant clones can be featured either somatically mutated or unmutated IGVH genes. CLL cells that express unmutated immunoglobulin variable domains likely underwent final development prior to their entry into the germinal center, whereas those that express mutated variable domains likely transited through the germinal center and then underwent final development. Regardless, the cellular origin of CLL remains unknown. The aim of this review is to summarize immunological aspects involved in this process and to provide insights about the complex biology and pathogenesis of this disease. We propose a mechanistic hypothesis to explain the origin of B-CLL clones into our current picture of normal B cell development. In particular, we suggest that unmutated CLL arises from normal B cells with self-reactivity for apoptotic bodies that have undergone receptor editing, CD5 expression, and anergic processes in the bone marrow. Similarly, mutated CLL would arise from cells that, while acquiring self-reactivity for autoantigens-including apoptotic bodies-in germinal centers, are also still subject to tolerization mechanisms, including receptor editing and anergy. We believe that CLL is a proliferation of B lymphocytes selected during clonal expansion through multiple encounters with (auto)antigens, despite the fact that they differ in their state of activation and maturation. Autoantigens and microbial pathogens activate BCR signaling and promote tolerogenic mechanisms such as receptor editing/revision, anergy, CD5+ expression, and somatic hypermutation in CLL B cells. The result of these tolerogenic mechanisms is the survival of CLL B cell clones with similar surface markers and homogeneous gene expression signatures. We suggest that both immunophenotypic surface markers and homogenous gene expression might represent the evidence of several attempts to re-educate self-reactive B cells.
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Palikhe, Nami Shrestha; Sin, Hye Jung; Kim, Seung Hyun; Sin, Hyun Jung; Hwang, Eui Kyung; Ye, Young Min; Park, Hae-Sim
2012-08-01
Prostaglandin E2 receptor subtype EP4 (PTGER4) is one of the four subtypes of receptors for prostaglandin E2 (PGE2). Overproduction of cysteinyl leukotriene in mast cells may be related with suppression of PGE2 in patients with aspirin hypersensitivity. Considering the association of PTGER4 in mast cells, urticaria- and aspirin-related disease, we hypothesized the genetic variability of PTGER4 may be associated with aspirin-intolerant chronic urticaria (AICU). The case-control study was performed in 141 with AICU, 153 with aspirin-tolerant chronic urticaria (ATCU) and 174 with normal controls (NCs). PTGER4 promoter single-nucleotide polymorphism was genotyped using a primer extension method with the SNAPshot ddNTP primer extension kit. The functional variability of PTGER4 promoter polymorphism was carried out by dual-luciferase system and electrophoretic mobility shift assay (EMSA) in human mast cells (HMC-1). Furthermore, the effect of aspirin was performed for PTGER4 mRNA expression using real-time PCR, and PGE2 production was checked in HMC-1 cells using ELISA. AICU patients carrying GG genotype at -1254 G>A showed significantly higher frequency compared with NC (P=0.032). Similarly, the minor allele frequency, G allele was significantly higher in AICU compared with NC (P=0.031). In vitro functional study demonstrated that the -1254 G allele had lower luciferase activity (P<0.001) in HMC-1 cells. EMSA finding showed that PTGER4 -1254 G produced a specific band. Significantly decreased PTGER4 expression (P=0.008) and PGE2 production by aspirin exposure was confirmed in in vitro HMC cell line model (P=0.001). The PTGER4 -1254 G allele demonstrated a higher frequency in AICU patients and lower promoter activity with decreased expression of PTGER4 and contributes to the development of AICU.
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Baculovirus-mediated expression of GPCRs in insect cells.
Saarenpää, Tuulia; Jaakola, Veli-Pekka; Goldman, Adrian
2015-01-01
G-protein-coupled receptors (GPCRs) are a large family of seven transmembrane proteins that influence a considerable number of cellular events. For this reason, they are one of the most studied receptor types for their pharmacological and structural properties. Solving the structure of several GPCR receptor types has been possible using almost all expression systems, including Escherichia coli, yeast, mammalian, and insect cells. So far, however, most of the GPCR structures solved have been done using the baculovirus insect cell expression system. The reason for this is mainly due to cost-effectiveness, posttranslational modification efficiency, and overall effortless maintenance. The system has evolved so much that variables starting from vector type, purification tags, cell line, and growth conditions can be varied and optimized countless ways to suit the needs of new constructs. Here, we present the array of techniques that enable the rapid and efficient optimization of expression steps for maximal protein quality and quantity, including our emendations. © 2015 Elsevier Inc. All rights reserved.
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Pede, Valerie; Rombout, Ans; Vermeire, Jolien; Naessens, Evelien; Mestdagh, Pieter; Robberecht, Nore; Vanderstraeten, Hanne; Van Roy, Nadine; Vandesompele, Jo; Speleman, Frank; Philippé, Jan; Verhasselt, Bruno
2013-01-01
Chronic lymphocytic leukemia (CLL) is a disease with variable clinical outcome. Several prognostic factors such as the immunoglobulin heavy chain variable genes (IGHV) mutation status are linked to the B-cell receptor (BCR) complex, supporting a role for triggering the BCR in vivo in the pathogenesis. The miRNA profile upon stimulation and correlation with IGHV mutation status is however unknown. To evaluate the transcriptional response of peripheral blood CLL cells upon BCR stimulation in vitro, miRNA and mRNA expression was measured using hybridization arrays and qPCR. We found both IGHV mutated and unmutated CLL cells to respond with increased expression of MYC and other genes associated with BCR activation, and a phenotype of cell cycle progression. Genome-wide expression studies showed hsa-miR-132-3p/hsa-miR-212 miRNA cluster induction associated with a set of downregulated genes, enriched for genes modulated by BCR activation and amplified by Myc. We conclude that BCR triggering of CLL cells induces a transcriptional response of genes associated with BCR activation, enhanced cell cycle entry and progression and suggest that part of the transcriptional profiles linked to IGHV mutation status observed in isolated peripheral blood are not cell intrinsic but rather secondary to in vivo BCR stimulation. PMID:23560086
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Salmikangas, Paula; Kinsella, Niamh; Chamberlain, Paul
2018-05-31
The first CD19 CAR T-cell products, Kymriah and Yescarta, are entering the US market and also being evaluated for marketing authorization in the EU. This breakthrough has expanded the interest and also investments towards novel chimeric antigen receptor (CAR) designs, both for hematological malignancies and solid tumors. At the same time, there is active development in moving from autologous products to allogeneic, off-the-shelf -products. New manufacturing technologies are also emerging for production of these complex genetically-modified cells and even decentralized manufacturing in hospitals is under consideration. However, the high potency of CAR T-cells is associated with toxicity and not all patients respond to the treatment. In addition, the number of patient and product variables impacting the clinical outcome is high. The race towards novel CAR T treatment options for cancer patients has begun, but without careful design of the constructs and overall understanding of the factors that impact the ultimate outcome in each case, the road towards commercial success may be long and winding. This review discusses the product- and patient-related variables that may pose challenges for the industry and developers both from the scientific and regulatory perspective.
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Chen, Pei-Wen; Kroog, Glenn S
2004-12-01
G protein-coupled receptors activate extracellular signal-regulated kinases (ERKs) via different pathways in different cell types. In this study, we demonstrate that gastrin-releasing peptide receptor (GRPr) regulates ERK through multiple pathways in a single cell type depending upon receptor expression and agonist concentration. We examined stably transfected BALB/c 3T3 fibroblasts expressing GRPr constructs at different levels and treated the cells with several concentrations of bombesin (BN, a GRPr agonist) to activate a variable number of GRPr per cell. GRPr induced two waves of ERK activation and one wave of ERK inhibition. One wave of activation required an intact GRPr carboxyl-terminal domain (CTD). It peaked 6 min after addition of high BN concentration ([BN]) in cells with high GRPr expression. Another wave of activation was CTD-independent. It peaked 2 to 4 min after BN addition in cells when [BN] and/or GRPr expression were lower. The early wave of ERK activation was more sensitive than the later one to pretreatment with Bisindolylmaleimide I (GF 109203X) (a protein kinase C inhibitor) or hypertonic sucrose. Because these two waves of activation differ in time course, dose-response curve, requirement for GRPr CTD, and sensitivity to inhibitors, they result from different signaling pathways. A third pathway in these cells inhibited ERK phosphorylation 2 min after addition of high [BN] in cells with high GRPr expression. Furthermore, a GRPr-expressing human duodenal cancer cell line showed differential sensitivity to GF 109203X throughout BN-induced ERK activation, indicating that GRPr may activate ERK via multiple pathways in cells expressing endogenous GRPr.
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Rossotti, Martín; Tabares, Sofía; Alfaya, Lucía; Leizagoyen, Carmen; Moron, Gabriel; González-Sapienza, Gualberto
2015-01-01
BACKGROUND Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domain (nanobody) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. METHODS Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct used for pull-down/MS target identification. RESULTS The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. CONCLUSIONS This strategy streamline the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. GENERAL SIGNIFICANCE This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets. PMID:25819371
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Neuhaus, Jochen; Oberbach, Andreas; Schwalenberg, Thilo; Stolzenburg, Jens-Uwe
2006-05-01
To compare histamine receptor expression in cultured smooth muscle cells from the human detrusor and internal sphincter using receptor-specific agonists. Smooth muscle cells from the bladder dome and internal sphincter were cultured from 5 male patients undergoing cystectomy for bladder cancer therapy. Calcium transients in cells stimulated with carbachol, histamine, histamine receptor 1 (H1R)-specific heptanecarboxamide (HTMT), dimaprit (H2R), and R-(alpha)-methylhistamine (H3R) were measured by calcium imaging. Histamine receptor proteins were detected by Western blot analysis and immunocytochemistry. H1R, H2R, and H3R expression was found in tissue and cultured cells. Carbachol stimulated equal numbers of detrusor and sphincter cells (60% and 51%, respectively). Histamine stimulated significantly more cells than carbachol in detrusor (100%) and sphincter (99.34%) cells. Calcium responses to carbachol in detrusor and sphincter cells were comparable and did not differ from those to histamine in detrusor cells. However, histamine and specific agonists stimulated more sphincter cells than did carbachol (P <0.001), and the calcium increase was greater in sphincter cells than in detrusor cells. Single cell analysis revealed comparable H2R responses in detrusor and sphincter cells, but H1R and H3R-mediated calcium reactions were significantly greater in sphincter cells. Histamine very effectively induces calcium release in smooth muscle cells. In sphincter cells, histamine is even more effective than carbachol regarding the number of reacting cells and the intracellular calcium increase. Some of the variability in the outcome of antihistaminic interstitial cystitis therapies might be caused by the ineffectiveness of the chosen antihistaminic or unintentional weakening of sphincteric function.
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Chemodetection in fluctuating environments: receptor coupling, buffering, and antagonism.
Lalanne, Jean-Benoît; François, Paul
2015-02-10
Variability in the chemical composition of the extracellular environment can significantly degrade the ability of cells to detect rare cognate ligands. Using concepts from statistical detection theory, we formalize the generic problem of detection of small concentrations of ligands in a fluctuating background of biochemically similar ligands binding to the same receptors. We discover that in contrast with expectations arising from considerations of signal amplification, inhibitory interactions between receptors can improve detection performance in the presence of substantial environmental variability, providing an adaptive interpretation to the phenomenon of ligand antagonism. Our results suggest that the structure of signaling pathways responsible for chemodetection in fluctuating and heterogeneous environments might be optimized with respect to the statistics and dynamics of environmental composition. The developed formalism stresses the importance of characterizing nonspecific interactions to understand function in signaling pathways.
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Cellular Decision Making by Non-Integrative Processing of TLR Inputs.
Kellogg, Ryan A; Tian, Chengzhe; Etzrodt, Martin; Tay, Savaş
2017-04-04
Cells receive a multitude of signals from the environment, but how they process simultaneous signaling inputs is not well understood. Response to infection, for example, involves parallel activation of multiple Toll-like receptors (TLRs) that converge on the nuclear factor κB (NF-κB) pathway. Although we increasingly understand inflammatory responses for isolated signals, it is not clear how cells process multiple signals that co-occur in physiological settings. We therefore examined a bacterial infection scenario involving co-stimulation of TLR4 and TLR2. Independent stimulation of these receptors induced distinct NF-κB dynamic profiles, although surprisingly, under co-stimulation, single cells continued to show ligand-specific dynamic responses characteristic of TLR2 or TLR4 signaling rather than a mixed response, comprising a cellular decision that we term "non-integrative" processing. Iterating modeling and microfluidic experiments revealed that non-integrative processing occurred through interaction of switch-like NF-κB activation, receptor-specific processing timescales, cell-to-cell variability, and TLR cross-tolerance mediated by multilayer negative feedback. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
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Kerekov, Nikola S; Ivanova, Iva I; Mihaylova, Nikolina M; Nikolova, Maria; Prechl, Jozsef; Tchorbanov, Andrey I
2014-10-01
Highly purified, subunit, or synthetic viral antigens are known to be weakly immunogenic and potentate only the antibody, rather than cell-mediated immune responses. An alternative approach for inducing protective immunity with small viral peptides would be the direct targeting of viral epitopes to the immunocompetent cells by DNA vaccines encoding antibody fragments specific to activating cell surface co-receptor molecules. Here, we are exploring as a new genetic vaccine, a DNA chimeric molecule encoding a T and B cell epitope-containing influenza A virus hemagglutinin peptide joined to sequences encoding a single-chain variable fragment antibody fragment specific for the costimulatory B cell complement receptors 1 and 2. This recombinant DNA molecule was inserted into eukaryotic expression vector and used as a naked DNA vaccine in WT and CR1/2 KO mice. The intramuscular administration of the DNA construct resulted in the in vivo expression of an immunogenic chimeric protein, which cross-links cell surface receptors on influenza-specific B cells. The DNA vaccination was followed by prime-boosting with the protein-engineered replica of the DNA construct, thus delivering an activation intracellular signal. Immunization with an expression vector containing the described construct and boosting with the protein chimera induced a strong anti-influenza cytotoxic response, modulation of cytokine profile, and a weak antibody response in Balb/c mice. The same immunization scheme did not result in generation of influenza-specific response in mice lacking the target receptor, underlining the molecular adjuvant effect of receptor targeting.
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RNA-binding proteins ZFP36L1 and ZFP36L2 promote cell quiescence.
Galloway, Alison; Saveliev, Alexander; Łukasiak, Sebastian; Hodson, Daniel J; Bolland, Daniel; Balmanno, Kathryn; Ahlfors, Helena; Monzón-Casanova, Elisa; Mannurita, Sara Ciullini; Bell, Lewis S; Andrews, Simon; Díaz-Muñoz, Manuel D; Cook, Simon J; Corcoran, Anne; Turner, Martin
2016-04-22
Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-μ at the pre-BCR checkpoint. Copyright © 2016, American Association for the Advancement of Science.
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Biomarkers of Progression after HIV Acute/Early Infection: Nothing Compares to CD4+ T-cell Count?
Ghiglione, Yanina; Hormanstorfer, Macarena; Coloccini, Romina; Salido, Jimena; Trifone, César; Ruiz, María Julia; Falivene, Juliana; Caruso, María Paula; Figueroa, María Inés; Salomón, Horacio; Giavedoni, Luis D.; Pando, María de los Ángeles; Gherardi, María Magdalena; Rabinovich, Roberto Daniel; Sued, Omar
2018-01-01
Progression of HIV infection is variable among individuals, and definition disease progression biomarkers is still needed. Here, we aimed to categorize the predictive potential of several variables using feature selection methods and decision trees. A total of seventy-five treatment-naïve subjects were enrolled during acute/early HIV infection. CD4+ T-cell counts (CD4TC) and viral load (VL) levels were determined at enrollment and for one year. Immune activation, HIV-specific immune response, Human Leukocyte Antigen (HLA) and C-C chemokine receptor type 5 (CCR5) genotypes, and plasma levels of 39 cytokines were determined. Data were analyzed by machine learning and non-parametric methods. Variable hierarchization was performed by Weka correlation-based feature selection and J48 decision tree. Plasma interleukin (IL)-10, interferon gamma-induced protein (IP)-10, soluble IL-2 receptor alpha (sIL-2Rα) and tumor necrosis factor alpha (TNF-α) levels correlated directly with baseline VL, whereas IL-2, TNF-α, fibroblast growth factor (FGF)-2 and macrophage inflammatory protein (MIP)-1β correlated directly with CD4+ T-cell activation (p < 0.05). However, none of these cytokines had good predictive values to distinguish “progressors” from “non-progressors”. Similarly, immune activation, HIV-specific immune responses and HLA/CCR5 genotypes had low discrimination power. Baseline CD4TC was the most potent discerning variable with a cut-off of 438 cells/μL (accuracy = 0.93, κ-Cohen = 0.85). Limited discerning power of the other factors might be related to frequency, variability and/or sampling time. Future studies based on decision trees to identify biomarkers of post-treatment control are warrantied. PMID:29342870
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Biomarkers of Progression after HIV Acute/Early Infection: Nothing Compares to CD4⁺ T-cell Count?
Turk, Gabriela; Ghiglione, Yanina; Hormanstorfer, Macarena; Laufer, Natalia; Coloccini, Romina; Salido, Jimena; Trifone, César; Ruiz, María Julia; Falivene, Juliana; Holgado, María Pía; Caruso, María Paula; Figueroa, María Inés; Salomón, Horacio; Giavedoni, Luis D; Pando, María de Los Ángeles; Gherardi, María Magdalena; Rabinovich, Roberto Daniel; Pury, Pedro A; Sued, Omar
2018-01-13
Progression of HIV infection is variable among individuals, and definition disease progression biomarkers is still needed. Here, we aimed to categorize the predictive potential of several variables using feature selection methods and decision trees. A total of seventy-five treatment-naïve subjects were enrolled during acute/early HIV infection. CD4⁺ T-cell counts (CD4TC) and viral load (VL) levels were determined at enrollment and for one year. Immune activation, HIV-specific immune response, Human Leukocyte Antigen (HLA) and C-C chemokine receptor type 5 (CCR5) genotypes, and plasma levels of 39 cytokines were determined. Data were analyzed by machine learning and non-parametric methods. Variable hierarchization was performed by Weka correlation-based feature selection and J48 decision tree. Plasma interleukin (IL)-10, interferon gamma-induced protein (IP)-10, soluble IL-2 receptor alpha (sIL-2Rα) and tumor necrosis factor alpha (TNF-α) levels correlated directly with baseline VL, whereas IL-2, TNF-α, fibroblast growth factor (FGF)-2 and macrophage inflammatory protein (MIP)-1β correlated directly with CD4⁺ T-cell activation ( p < 0.05). However, none of these cytokines had good predictive values to distinguish "progressors" from "non-progressors". Similarly, immune activation, HIV-specific immune responses and HLA/CCR5 genotypes had low discrimination power. Baseline CD4TC was the most potent discerning variable with a cut-off of 438 cells/μL (accuracy = 0.93, κ-Cohen = 0.85). Limited discerning power of the other factors might be related to frequency, variability and/or sampling time. Future studies based on decision trees to identify biomarkers of post-treatment control are warrantied.
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The zipper mechanism in phagocytosis: energetic requirements and variability in phagocytic cup shape
2010-01-01
Background Phagocytosis is the fundamental cellular process by which eukaryotic cells bind and engulf particles by their cell membrane. Particle engulfment involves particle recognition by cell-surface receptors, signaling and remodeling of the actin cytoskeleton to guide the membrane around the particle in a zipper-like fashion. Despite the signaling complexity, phagocytosis also depends strongly on biophysical parameters, such as particle shape, and the need for actin-driven force generation remains poorly understood. Results Here, we propose a novel, three-dimensional and stochastic biophysical model of phagocytosis, and study the engulfment of particles of various sizes and shapes, including spiral and rod-shaped particles reminiscent of bacteria. Highly curved shapes are not taken up, in line with recent experimental results. Furthermore, we surprisingly find that even without actin-driven force generation, engulfment proceeds in a large regime of parameter values, albeit more slowly and with highly variable phagocytic cups. We experimentally confirm these predictions using fibroblasts, transfected with immunoreceptor FcγRIIa for engulfment of immunoglobulin G-opsonized particles. Specifically, we compare the wild-type receptor with a mutant receptor, unable to signal to the actin cytoskeleton. Based on the reconstruction of phagocytic cups from imaging data, we indeed show that cells are able to engulf small particles even without support from biological actin-driven processes. Conclusions This suggests that biochemical pathways render the evolutionary ancient process of phagocytic highly robust, allowing cells to engulf even very large particles. The particle-shape dependence of phagocytosis makes a systematic investigation of host-pathogen interactions and an efficient design of a vehicle for drug delivery possible. PMID:21059234
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Pearson, Helen; Britt, Rodney D; Pabelick, Christine M; Prakash, Y S; Amrani, Yassine; Pandya, Hitesh C
2015-12-01
Adult human airway smooth muscle (ASM) produce cytokines involved in recruitment and survival of leukocytes within airway walls. Cytokine generation by adult ASM is glucocorticoid-sensitive. Whether developing lung ASM produces cytokines in a glucocorticoid-sensitive fashion is unknown. Cultured fetal human ASM cells stimulated with TNF-α (0-20 ng/ml) were incubated with TNF-α receptor-blocking antibodies, fluticasone (1 and 100 nm), or vehicle. Supernatants and cells were assayed for the production of CCL5, CXCL10, and CXCL8 mRNA and protein and glucocorticoid receptor phosphorylation. CCL5, CXCL10, and CXCL8 mRNA and protein production by fetal ASM cell was significantly and dose-dependently following TNF-α treatment. Cytokine mRNA and protein production were effectively blocked by TNF-α R1 and R2 receptor neutralizing antibodies but variably inhibited by fluticasone. TNF-α-induced TNF-R1 and R2 receptor mRNA expression was only partially attenuated by fluticasone. Glucocorticoid receptor phosphorylation at serine (Ser) 211 but not at Ser 226 was enhanced by fluticasone. Production of CCL5, CXCL10, and CXCL8 by fetal ASM appears to involve pathways that are both qualitatively and mechanistically distinct to those described for adult ASM. The findings imply developing ASM has potential to recruit leukocyte into airways and, therefore, of relevance to childhood airway diseases.
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Pearson, Helen; Britt, Rodney D.; Pabelick, Christine M.; Prakash, Y.S.; Amrani, Yassine; Pandya, Hitesh C.
2016-01-01
Background Adult human airway smooth muscle (ASM) produce cytokines involved in recruitment and survival of leukocytes within airway walls. Cytokine generation by adult ASM is glucocorticoid-sensitive. Whether developing lung ASM produces cytokines in a glucocorticoid-sensitive fashion is unknown. Methods Cultured fetal human ASM cells stimulated with TNF-α (0–20 ng/ml) were incubated with TNF-α receptor-blocking antibodies, fluticasone (1 and 100 nm), or vehicle. Supernatants and cells were assayed for the production of CCL5, CXCL10, and CXCL8 mRNA and protein and glucocorticoid receptor phosphorylation. Results CCL5, CXCL10, and CXCL8 mRNA and protein production by fetal ASM cell was significantly and dose-dependently following TNF-α treatment. Cytokine mRNA and protein production were effectively blocked by TNF-α R1 and R2 receptor neutralizing antibodies but variably inhibited by fluticasone. TNF-α-induced TNF-R1 and R2 receptor mRNA expression was only partially attenuated by fluticasone. Glucocorticoid receptor phosphorylation at serine (Ser) 211 but not at Ser 226 was enhanced by fluticasone. Conclusion Production of CCL5, CXCL10, and CXCL8 by fetal ASM appears to involve pathways that are both qualitatively and mechanistically distinct to those described for adult ASM. The findings imply developing ASM has potential to recruit leukocyte into airways and, therefore, of relevance to childhood airway diseases. PMID:26331770
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Kuramitsu, S; Ohno, M; Ohka, F; Shiina, S; Yamamichi, A; Kato, A; Tanahashi, K; Motomura, K; Kondo, G; Kurimoto, M; Senga, T; Wakabayashi, T; Natsume, A
2015-10-01
The epidermal growth factor receptor variant III (EGFRvIII) is exclusively expressed on the cell surface in ~50% of glioblastoma multiforme (GBM). This variant strongly and persistently activates the phosphatidylinositol 3-kinase-Akt signaling pathway in a ligand-independent manner resulting in enhanced tumorigenicity, cellular motility and resistance to chemoradiotherapy. Our group generated a recombinant single-chain variable fragment (scFv) antibody specific to the EGFRvIII, referred to as 3C10-scFv. In the current study, we constructed a lentiviral vector transducing the chimeric antigen receptor (CAR) that consisted of 3C10-scFv, CD3ζ, CD28 and 4-1BB (3C10-CAR). The 3C10-CAR-transduced peripheral blood mononuclear cells (PBMCs) and CD3(+) T cells specifically lysed the glioma cells that express EGFRvIII. Moreover, we demonstrated that CAR CD3(+) T cells migrated to the intracranial xenograft of GBM in the mice treated with 3C10-CAR PBMCs. An important and novel finding of our study was that a thalidomide derivative lenalidomide induced 3C10-CAR PBMC proliferation and enhanced the persistent antitumor effect of the cells in vivo. Lenalidomide also exhibited enhanced immunological synapses between the effector cells and the target cells as determined by CD11a and F-actin polymerization. Collectively, lentiviral-mediated transduction of CAR effectors targeting the EGFRvIII showed specific efficacy, and lenalidomide even intensified CAR cell therapy by enhanced formation of immunological synapses.
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Watkins, Stephan; Pichler, Werner J.
2013-01-01
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. PMID:24116097
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1987-02-06
MICROCOPY RESOLUTION TEST CHART NA7 UNAL hu AT ’N Ad . . 0 0 10 PROCEEDINGS THE TWENTY-SIXTH MIDWINTER CONFERENCE OF IMMUNOLOGISTS January 17-20, 1987...monoclonal BA5 has also been used to study the abnormal regulation of expression of BCGF receptors in common variable ilmmunodeficiency, SLE , and B cell...antigen, we have shown that membrane Ig-mediated antigen presentation by small B cells to rabbit globulin - specific T cell lines results in a vigorous
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Forsell, Mattias N E; Dey, Barna; Mörner, Andreas; Svehla, Krisha; O'dell, Sijy; Högerkorp, Carl-Magnus; Voss, Gerald; Thorstensson, Rigmor; Shaw, George M; Mascola, John R; Karlsson Hedestam, Gunilla B; Wyatt, Richard T
2008-10-03
The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3). Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4) rabbits with envelope glycoprotein (Env) trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT) rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity) primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.
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Bagheri, Salman; Yousefi, Mehdi; Safaie Qamsari, Elmira; Riazi-Rad, Farhad; Abolhassani, Mohsen; Younesi, Vahid; Dorostkar, Ruhollah; Movassaghpour, Ali Akbar; Sharifzadeh, Zahra
2017-03-01
The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor-receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.
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Bolin, Lisa L; Chandhasin, Chandtip; Lobelle-Rich, Patricia A; Albritton, Lorraine M; Levy, Laura S
2011-05-13
Feline leukemia virus (FeLV)-945, a member of the FeLV-A subgroup, was previously isolated from a cohort of naturally infected cats. An unusual multicentric lymphoma of non-T-cell origin was observed in natural and experimental infection with FeLV-945. Previous studies implicated the FeLV-945 surface glycoprotein (SU) as a determinant of disease outcome by an as yet unknown mechanism. The present studies demonstrate that FeLV-945 SU confers distinctive properties of binding to the cell surface receptor. Virions bearing the FeLV-945 Env protein were observed to bind the cell surface receptor with significantly increased efficiency, as was soluble FeLV-945 SU protein, as compared to the corresponding virions or soluble protein from a prototype FeLV-A isolate. SU proteins cloned from other cohort isolates exhibited increased binding efficiency comparable to or greater than FeLV-945 SU. Mutational analysis implicated a domain containing variable region B (VRB) to be the major determinant of increased receptor binding, and identified a single residue, valine 186, to be responsible for the effect. The FeLV-945 SU protein binds its cell surface receptor, feTHTR1, with significantly greater efficiency than does that of prototype FeLV-A (FeLV-A/61E) when present on the surface of virus particles or in soluble form, demonstrating a 2-fold difference in the relative dissociation constant. The results implicate a single residue, valine 186, as the major determinant of increased binding affinity. Computational modeling suggests a molecular mechanism by which residue 186 interacts with the receptor-binding domain through residue glutamine 110 to effect increased binding affinity. Through its increased receptor binding affinity, FeLV-945 SU might function in pathogenesis by increasing the rate of virus entry and spread in vivo, or by facilitating entry into a novel target cell with a low receptor density.
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The EphA2 Receptor and EphrinA1 Ligand in Solid Tumors: Function and Therapeutic Targeting
Wykosky, Jill; Debinski, Waldemar
2013-01-01
The Eph receptor tyrosine kinases and ephrin ligands have been studied extensively for their roles in developmental processes. In recent years, Eph receptors and ephrins have been found to be integral players in cancer formation and progression. Among these are EphA2 and ephrinA1, which are involved in the development and maintenance of many different types of solid tumors. The function of EphA2 and ephrinA1 in tumorigenesis and tumor progression is complex and seems to be dependent on cell type and microenvironment. These variables affect the expression of the EphA2 and ephrinA1 proteins, the pathways through which they induce signaling, and the functional consequences of that signaling on the behavior of tumor cells and tumor-associated cells. This review will specifically focus on the roles that EphA2 and ephrinA1 play in the different cell types that contribute to the malignancy of solid tumors, with emphasis on the opportunities for therapeutic targeting. PMID:19074825
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Fattahi, Sadegh; Ghadami, Elham; Asouri, Mohsen; Motevalizadeh Ardekanid, Ali; Akhavan-Niaki, Haleh
2018-02-28
Breast cancer is a heterogeneous and multifactorial disease with variable disease progression risk, and treatment response. Urtica dioica is a traditional herb used as an adjuvant therapeutic agent in cancer. In the present study, we have evaluated the effects of the aqueous extract of Urtica dioica on Adenosine deaminase (ADA) and Ornithine decarboxylase (ODC1) gene expression in MCF-7, MDA-MB-231, two breast cancer cell lines being estrogen receptor positive and estrogen receptor negative, respectively. Cell lines were cultured in suitable media. After 24 h, different concentrations of the extract were added and after 72 h, ADA and ODC1 gene expression as well as BCL2 and BAX apoptotic genes were assessed by Taqman real time PCR assay. Cells viability was assessed by MTT assay, and apoptosis was also evaluated at cellular level. The intra and extracellular levels of ODC1 and ADA enzymes were evaluated by ELISA. Results showed differential expression of ADA and ODC1 genes in cancer cell lines. In MCF-7 cell line, the expression level of ADA was upregulated in a dose-dependent manner but its expression did not change in MDA-MB cell line. ODC1 expression was increased in both examined cell lines. Also, increased level of the apoptotic BAX/BCL-2 ratio was detected in MCF-7 cells. These results demonstrated that Urtica dioica induces apoptosis in breast cancer cells by influencing ODC1 and ADA genes expression, and estrogen receptors. The different responses observed with these cell lines could be due to the interaction of Urtica dioica as a phytoestrogen with the estrogen receptor.
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Smith, Eric L; Staehr, Mette; Masakayan, Reed; Tatake, Ishan J; Purdon, Terence J; Wang, Xiuyan; Wang, Pei; Liu, Hong; Xu, Yiyang; Garrett-Thomson, Sarah C; Almo, Steven C; Riviere, Isabelle; Liu, Cheng; Brentjens, Renier J
2018-06-06
B cell maturation antigen (BCMA) has recently been identified as an important multiple myeloma (MM)-specific target for chimeric antigen receptor (CAR) T cell therapy. In CAR T cell therapy targeting CD19 for lymphoma, host immune anti-murine CAR responses limited the efficacy of repeat dosing and possibly long-term persistence. This clinically relevant concern can be addressed by generating a CAR incorporating a human single-chain variable fragment (scFv). We screened a human B cell-derived scFv phage display library and identified a panel of BCMA-specific clones from which human CARs were engineered. Despite a narrow range of affinity for BCMA, dramatic differences in CAR T cell expansion were observed between unique scFvs in a repeat antigen stimulation assay. These results were confirmed by screening in a MM xenograft model, where only the top preforming CARs from the repeat antigen stimulation assay eradicated disease and prolonged survival. The results of this screening identified a highly effective CAR T cell therapy with properties, including rapid in vivo expansion (>10,000-fold, day 6), eradication of large tumor burden, and durable protection to tumor re-challenge. We generated a bicistronic construct including a second-generation CAR and a truncated-epithelial growth factor receptor marker. CAR T cell vectors stemming from this work are under clinical investigation. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.
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Predina, Jarrod D.; Newton, Andrew D.; Xia, Leilei; Corbett, Christopher; Connolly, Courtney; Shin, Michael; Sulyok, Lydia Frezel; Litzky, Leslie; Deshpande, Charuhas; Nie, Shuming; Kularatne, Sumith A.; Low, Philip S.; Singhal, Sunil
2018-01-01
Background Clinical applicability of folate receptor-targeted intraoperative molecular imaging (FR-IMI) has been established for surgically resectable pulmonary adenocarcinoma. A role for FR-IMI in other lung cancer histologies has not been studied. In this study, we evaluate feasibility of FR-IMI in patients undergoing pulmonary resection for squamous cell carcinomas (SCCs). Methods In a human clinical trial (NCT02602119), twelve subjects with pulmonary SCCs underwent FR-IMI with a near-infrared contrast agent that targets the folate receptor-α (FRα), OTL38. Near-infrared signal from tumors and benign lung was quantified to calculate tumor-to-background ratios (TBR). Folate receptor-alpha expression was characterized, and histopathologic correlative analyses were performed to evaluate patterns of OTL38 accumulation. An exploratory analysis was performed to determine patient and histopathologic variables that predict tumor fluorescence. Results 9 of 13 SCCs (in 9 of 12 of subjects) displayed intraoperative fluorescence upon NIR evaluation (median TBR, 3.9). OTL38 accumulated within SCCs in a FRα-dependent manner. FR-IMI was reliable in localizing nodules as small as 1.1 cm, and prevented conversion to thoracotomy for nodule localization in three subjects. Upon evaluation of patient and histopathologic variables, in situ fluorescence was associated with distance from the pleural surface, and was independent of alternative variables including tumor size and metabolic activity. Conclusions This work demonstrates that FR-IMI is potentially feasible in 70% of SCC patients, and that molecular imaging can improve localization during minimally invasive pulmonary resection. These findings complement previous data demonstrating that ∼98% of pulmonary adenocarcinomas are localized during FR-IMI and suggest broad applicability for NSCLC patients undergoing resection. PMID:29568374
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Wang, Enxiu; Wang, Liang-Chuan; Tsai, Ching-Yi; Bhoj, Vijay; Gershenson, Zack; Moon, Edmund; Newick, Kheng; Sun, Jing; Lo, Albert; Baradet, Timothy; Feldman, Michael D.; Barrett, David; Puré, Ellen; Albelda, Steven; Milone, Michael C.
2015-01-01
Chimeric antigen receptors (CAR) bearing an antigen-binding domain linked in cis to the cytoplasmic domains of CD3ζ and costimulatory receptors have provided a potent method for engineering T-cell cytotoxicity towards B-cell leukemia and lymphoma. However, resistance to immunotherapy due to loss of T-cell effector function remains a significant barrier, especially in solid malignancies. We describe an alternative chimeric immunoreceptor design in which we have fused a single-chain variable fragment for antigen recognition to the transmembrane and cytoplasmic domains of KIR2DS2, a stimulatory killer immunoglobulin-like receptor (KIR). We show that this simple, KIR-based CAR (KIR-CAR) triggers robust antigen-specific proliferation and effector function in vitro when introduced into human T cells with DAP12, an immunotyrosine-based activation motifs (ITAM)-containing adaptor. T cells modified to express a KIR-CAR and DAP12 exhibit superior antitumor activity compared to standard first and second generation CD3ζ-based CARs in a xenograft model of mesothelioma highly resistant to immunotherapy. The enhanced antitumor activity is associated with improved retention of chimeric immunoreceptor expression and improved effector function of isolated tumor-infiltrating lymphocytes. These results support the exploration of KIR-CARs for adoptive T-cell immunotherapy, particularly in immunotherapy-resistant solid tumors. PMID:25941351
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Borek, Aleksandra; Sokolowska-Wedzina, Aleksandra; Chodaczek, Grzegorz; Otlewski, Jacek
2018-01-01
Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.
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Baliakas, Panagiotis; Hadzidimitriou, Anastasia; Sutton, Lesley-Ann; Minga, Eva; Agathangelidis, Andreas; Nichelatti, Michele; Tsanousa, Athina; Scarfò, Lydia; Davis, Zadie; Yan, Xiao-Jie; Shanafelt, Tait; Plevova, Karla; Sandberg, Yorick; Vojdeman, Fie Juhl; Boudjogra, Myriam; Tzenou, Tatiana; Chatzouli, Maria; Chu, Charles C; Veronese, Silvio; Gardiner, Anne; Mansouri, Larry; Smedby, Karin E; Pedersen, Lone Bredo; van Lom, Kirsten; Giudicelli, Véronique; Francova, Hana Skuhrova; Nguyen-Khac, Florence; Panagiotidis, Panagiotis; Juliusson, Gunnar; Angelis, Lefteris; Anagnostopoulos, Achilles; Lefranc, Marie-Paule; Facco, Monica; Trentin, Livio; Catherwood, Mark; Montillo, Marco; Geisler, Christian H; Langerak, Anton W; Pospisilova, Sarka; Chiorazzi, Nicholas; Oscier, David; Jelinek, Diane F; Darzentas, Nikos; Belessi, Chrysoula; Davi, Frederic; Rosenquist, Richard; Ghia, Paolo; Stamatopoulos, Kostas
2014-11-01
About 30% of cases of chronic lymphocytic leukaemia (CLL) carry quasi-identical B-cell receptor immunoglobulins and can be assigned to distinct stereotyped subsets. Although preliminary evidence suggests that B-cell receptor immunoglobulin stereotypy is relevant from a clinical viewpoint, this aspect has never been explored in a systematic manner or in a cohort of adequate size that would enable clinical conclusions to be drawn. For this retrospective, multicentre study, we analysed 8593 patients with CLL for whom immunogenetic data were available. These patients were followed up in 15 academic institutions throughout Europe (in Czech Republic, Denmark, France, Greece, Italy, Netherlands, Sweden, and the UK) and the USA, and data were collected between June 1, 2012, and June 7, 2013. We retrospectively assessed the clinical implications of CLL B-cell receptor immunoglobulin stereotypy, with a particular focus on 14 major stereotyped subsets comprising cases expressing unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain variable genes. The primary outcome of our analysis was time to first treatment, defined as the time between diagnosis and date of first treatment. 2878 patients were assigned to a stereotyped subset, of which 1122 patients belonged to one of 14 major subsets. Stereotyped subsets showed significant differences in terms of age, sex, disease burden at diagnosis, CD38 expression, and cytogenetic aberrations of prognostic significance. Patients within a specific subset generally followed the same clinical course, whereas patients in different stereotyped subsets-despite having the same immunoglobulin heavy variable gene and displaying similar immunoglobulin mutational status-showed substantially different times to first treatment. By integrating B-cell receptor immunoglobulin stereotypy (for subsets 1, 2, and 4) into the well established Döhner cytogenetic prognostic model, we showed these, which collectively account for around 7% of all cases of CLL and represent both U-CLL and M-CLL, constituted separate clinical entities, ranging from very indolent (subset 4) to aggressive disease (subsets 1 and 2). The molecular classification of chronic lymphocytic leukaemia based on B-cell receptor immunoglobulin stereotypy improves the Döhner hierarchical model and refines prognostication beyond immunoglobulin mutational status, with potential implications for clinical decision making, especially within prospective clinical trials. European Union; General Secretariat for Research and Technology of Greece; AIRC; Italian Ministry of Health; AIRC Regional Project with Fondazione CARIPARO and CARIVERONA; Regione Veneto on Chronic Lymphocytic Leukemia; Nordic Cancer Union; Swedish Cancer Society; Swedish Research Council; and National Cancer Institute (NIH). Copyright © 2014 Elsevier Ltd. All rights reserved.
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Characterization of MHC-II antigen presentation by B cells and monocytes from older individuals
HL, Clark; R, Banks; L, Jones; TR, Hornick; PA, Higgins; CJ, Burant; DH, Canaday
2012-01-01
In this study we examine the effects of aging on antigen presentation of B cells and monocytes. We compared the antigen presentation function of peripheral blood B cells from young and old subjects using a system that specifically measures the B cell receptor (BCR)-mediated MHC-II antigen presentation. Monocytes were studied as well. Overall the mean magnitude of antigen presentation of soluble antigen and peptide was not different in older and younger subjects for both B cells and monocytes. Older subjects, however, showed increased heterogeneity of BCR-mediated antigen presentation by their B cells. The magnitude and variability of peptide presentation, which does not require uptake and processing, was the same between groups. Presentation by monocytes had similar variability between the older and younger subjects. These data suggest that poor B cell antigen processing, which results in diminished presentation in some older individuals may contribute to poor vaccine responses. PMID:22797466
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Mutations and polymorphisms in FSH receptor: functional implications in human reproduction.
Desai, Swapna S; Roy, Binita Sur; Mahale, Smita D
2013-12-01
FSH brings about its physiological actions by activating a specific receptor located on target cells. Normal functioning of the FSH receptor (FSHR) is crucial for follicular development and estradiol production in females and for the regulation of Sertoli cell function and spermatogenesis in males. In the last two decades, the number of inactivating and activating mutations, single nucleotide polymorphisms, and spliced variants of FSHR gene has been identified in selected infertile cases. Information on genotype-phenotype correlation and in vitro functional characterization of the mutants has helped in understanding the possible genetic cause for female infertility in affected individuals. The information is also being used to dissect various extracellular and intracellular events involved in hormone-receptor interaction by studying the differences in the properties of the mutant receptor when compared with WT receptor. Studies on polymorphisms in the FSHR gene have shown variability in clinical outcome among women treated with FSH. These observations are being explored to develop molecular markers to predict the optimum dose of FSH required for controlled ovarian hyperstimulation. Pharmacogenetics is an emerging field in this area that aims at designing individual treatment protocols for reproductive abnormalities based on FSHR gene polymorphisms. The present review discusses the current knowledge of various genetic alterations in FSHR and their impact on receptor function in the female reproductive system.
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Lopez, M; Maroc, N; Kerangueven, F; Bardin, F; Courcoul, M; Lavezzi, C; Birg, F; Mannoni, P
1991-09-01
Leukemic cells isolated from patients with either acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL) were screened for their capacity to express the interleukin 6 (IL-6) and IL-6 receptor genes, both at the RNA and protein levels. Variable levels (10 to greater than 600 U/ml) of an IL-6 activity, inhibited by neutralizing anti-IL-6 antibodies, were detected in AML cell supernatants using the B9 cell bioassay. High levels (greater than 100 U/ml) were observed in differentiated (M4 and M5 stages) AML, as well as in less mature (M1 and M2 stages) AML. Detection of the IL-6 transcript correlated with the biological activity. In addition, both IL-6 activity and IL-6 mRNA were detected in "fresh" leukemic cells, indicating that the glycoprotein was actually synthesized in vivo. In contrast, the IL-6 gene was less frequently expressed in ALL. The IL-6 receptor gene was transcribed in both AML and ALL; binding experiments showed that the protein was present at the cell surface. The spontaneous in vitro proliferation of leukemic cells coexpressing the transcripts for IL-6 and its receptor was not significantly inhibited by a neutralizing anti-IL-6 antibody, suggesting that IL-6 is not primarily implicated in the proliferation of the leukemic clone via an autocrine loop. Synthesis of IL-6 could, however, confer on leukemic cells a selective growth advantage through activation of the cytokine cascade.
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González, Sergio; Rangel-Barajas, Claudia; Peper, Marcela; Lorenzo, Ramiro; Moreno, Estefanía; Ciruela, Francisco; Borycz, Janusz; Ortiz, Jordi; Lluís, Carme; Franco, Rafael; McCormick, Peter J.; Volkow, Nora D.; Rubinstein, Marcelo; Floran, Benjamin; Ferré, Sergi
2011-01-01
Polymorphic variants of the dopamine D4 receptor have been consistently associated with attention-deficit hyperactivity disorder (ADHD). However the functional significance of the risk polymorphism (variable number of tandem repeats in exon 3) is still unclear. Here we show that whereas the most frequent 4-repeat (D4.4) and the 2-repeat (D4.2) variants form functional heteromers with the short isoform of the dopamine D2 receptor (D2S), the 7-repeat risk allele (D4.7) does not. D2 receptor activation in the D2S-D4 receptor heteromer potentiates D4 receptor-mediated MAPK signaling in transfected cells and in the striatum, which did not occur in cells expressing D4.7 or in the striatum of knock-in mutant mice carrying the 7 repeats of the human D4.7 in the third intracellular loop of the D4 receptor. In the striatum D4 receptors are localized in cortico-striatal glutamatergic terminals, where they selectively modulate glutamatergic neurotransmission by interacting with D2S receptors. This interaction shows the same qualitative characteristics than the D2S-D4 receptor heteromer-mediated MAPK signaling and D2S receptor activation potentiates D4 receptor-mediated inibition of striatal glutamate release. It is therefore postulated that dysfunctional D2S-D4.7 heteromers may impair presynaptic dopaminergic control of corticostriatal glutamatergic neurotransmission and explain functional deficits associated with ADHD. PMID:21844870
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Stroncek, David F; Ren, Jiaqiang; Lee, Daniel W; Tran, Minh; Frodigh, Sue Ellen; Sabatino, Marianna; Khuu, Hanh; Merchant, Melinda S; Mackall, Crystal L
2016-07-01
Autologous chimeric antigen receptor (CAR) T-cell therapies have shown promising clinical outcomes, but T-cell yields have been variable. CD19- and GD2-CAR T-cell manufacturing records were reviewed to identify sources of variability. CD19-CAR T cells were used to treat 43 patients with acute lymphocytic leukemia or lymphoma and GD2-CAR T cells to treat eight patients with osteosarcoma and three with neuroblastoma. Both types of CAR T cells were manufactured using autologous peripheral blood mononuclear cells (PBMC) concentrates and anti-CD3/CD28 beads for T-cell enrichment and simulation. A comparison of the first 6 GD2- and the first 22 CD19-CAR T-cell products manufactured revealed that GD2-CAR T-cell products contained fewer transduced cells than CD19-CAR T-cell products (147 ± 102 × 10(6) vs 1502 ± 1066 × 10(6); P = 0.0059), and their PBMC concentrates contained more monocytes (31.4 ± 12.4% vs 18.5 ± 13.7%; P = 0.019). Among the first 28 CD19-CAR T-cell products manufactured, four had poor expansion yielding less than 1 × 10(6) transduced T cells per kilogram. When PBMC concentrates from these four patients were compared with the 24 others, PBMC concentrates of poorly expanding products contained greater quantities of monocytes (39.8 ± 12.9% vs. 15.3 ± 10.8%, P = 0.0014). Among the patients whose CD19-CAR T cells expanded poorly, manufacturing for two patients was repeated using cryopreserved PBMC concentrates but incorporating a monocyte depleting plastic adherence step, and an adequate dose of CAR T cells was produced for both patients. Variability in CAR T-cell expansion is due, at least in part, to the contamination of the starting PBMC concentrates with monocytes. Published by Elsevier Inc.
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Park, Jin; Jeon, Won Il; Lee, So Yeong; Ock, Kwang-Su; Seo, Ji Hye; Park, Jinho; Ganbold, Erdene-Ochir; Cho, Keunchang; Song, Nam Woong; Joo, Sang-Woo
2012-05-01
We investigate the cellular uptake behaviors and efficacy of folate-coated gold nanoparticles (AuNPs) for the targeted drug delivery system in human cancer cells. Folate-conjugated AuNPs embedded with a purine analogue cancer drug of 6-mercaptopurine (6MP) were assembled via a 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) coupling reaction between the amino group of 4-aminobenzenethiol (ABT) and the carboxyl group of folic acid. The assembly of folate and 6MP on AuNPs has been examined by absorption spectroscopy, transmission electron microscopy (TEM), and confocal Raman spectroscopy. The internalization of the conjugated AuNPs inside the folate receptor-positive HeLa and KB cells was checked by TEM and dark-field microscopy (DFM) combined with label-free confocal spectroscopy over the depth variable z at a micrometer resolution. DFM live cell imaging of folate-conjugated AuNPs in HeLa cells indicated that the targeted AuNPs appeared to attach on the cell surfaces and enter into the cell with an hour. The cell viability was also compared to estimate the efficacy of folate-conjugated AuNP delivery systems. Folate receptor-targeted AuNP systems appeared to decrease cancer cell viability both in vitro and in vivo more than did the use of the 6MP-coated AuNPs drug without any targeting systems. Copyright © 2012 Wiley Periodicals, Inc.
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STCRDab: the structural T-cell receptor database
de Oliveira, Saulo H P; Krawczyk, Konrad
2018-01-01
Abstract The Structural T–cell Receptor Database (STCRDab; http://opig.stats.ox.ac.uk/webapps/stcrdab) is an online resource that automatically collects and curates TCR structural data from the Protein Data Bank. For each entry, the database provides annotations, such as the α/β or γ/δ chain pairings, major histocompatibility complex details, and where available, antigen binding affinities. In addition, the orientation between the variable domains and the canonical forms of the complementarity-determining region loops are also provided. Users can select, view, and download individual or bulk sets of structures based on these criteria. Where available, STCRDab also finds antibody structures that are similar to TCRs, helping users explore the relationship between TCRs and antibodies. PMID:29087479
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Ochnik, Aleksandra M; Moore, Nicole L; Jankovic-Karasoulos, Tanja; Bianco-Miotto, Tina; Ryan, Natalie K; Thomas, Mervyn R; Birrell, Stephen N; Butler, Lisa M; Tilley, Wayne D; Hickey, Theresa E
2014-01-01
Medroxyprogesterone acetate (MPA), a component of combined estrogen-progestin therapy (EPT), has been associated with increased breast cancer risk in EPT users. MPA can bind to the androgen receptor (AR), and AR signaling inhibits cell growth in breast tissues. Therefore, the aim of this study was to investigate the potential of MPA to disrupt AR signaling in an ex vivo culture model of normal human breast tissue. Histologically normal breast tissues from women undergoing breast surgical operation were cultured in the presence or in the absence of the native AR ligand 5α-dihydrotestosterone (DHT), MPA, or the AR antagonist bicalutamide. Ki67, bromodeoxyuridine, B-cell CLL/lymphoma 2 (BCL2), AR, estrogen receptor α, and progesterone receptor were detected by immunohistochemistry. DHT inhibited the proliferation of breast epithelial cells in an AR-dependent manner within tissues from postmenopausal women, and MPA significantly antagonized this androgenic effect. These hormonal responses were not commonly observed in cultured tissues from premenopausal women. In tissues from postmenopausal women, DHT either induced or repressed BCL2 expression, and the antiandrogenic effect of MPA on BCL2 was variable. MPA significantly opposed the positive effect of DHT on AR stabilization, but these hormones had no significant effect on estrogen receptor α or progesterone receptor levels. In a subset of postmenopausal women, MPA exerts an antiandrogenic effect on breast epithelial cells that is associated with increased proliferation and destabilization of AR protein. This activity may contribute mechanistically to the increased risk of breast cancer in women taking MPA-containing EPT.
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Mechanical regulation of T-cell functions
Chen, Wei; Zhu, Cheng
2013-01-01
Summary T cells are key players of the mammalian adaptive immune system. They experience different mechanical microenvironments during their life cycles, from the thymus, secondary lymph organs, and peripheral tissues that are free of externally applied force but display variable substrate rigidities, to the blood and lymphatic circulation systems where complicated hydrodynamic forces are present. Regardless of whether T cells are subject to external forces or generate their own internal forces, they response and adapt to different biomechanical cues to modulate their adhesion, migration, trafficking, and triggering of immune functions through mechanical regulation of various molecules that bear force. These include adhesive receptors, immunoreceptors, motor proteins, cytoskeletal proteins, and their associated molecules. Here we discuss the forces acting on various surface and cytoplasmic proteins of a T cell in different mechanical milieus. We review existing data on how force regulates protein conformational changes and interactions with counter molecules, including integrins, actin, and the T-cell receptor, and how each relates to T-cell functions. PMID:24117820
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Peters, Derek T.; Henderson, Christopher A.; Warren, Curtis R.; Friesen, Max; Xia, Fang; Becker, Caroline E.; Musunuru, Kiran; Cowan, Chad A.
2016-01-01
ABSTRACT Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro, but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal, we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray, and tested their ability to perform mature hepatocyte functions (albumin and urea secretion, cytochrome activity). By these measures, ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. PMID:27143754
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Neuhaus, J; Heinrich, M; Schlichting, N; Oberbach, A; Fitzl, G; Schwalenberg, T; Horn, L-C; Stolzenburg, J-U
2007-09-01
Myofibroblasts play a pivotal role in numerous pathological alterations. Clarification of the structure and function and of the cellular plasticity of this cell type in the bladder may lead to new insights into the pathogenesis of lower urinary tract disorders. Bladder biopsies from patients with bladder carcinoma and interstitial cystitis were used to analyse the morphology and receptor expression using confocal immunofluorescence and electron microscopy. Cytokine effects and coupling behavior were tested in cultured myofibroblasts and detrusor smooth muscle cells. Myofibroblasts are in close contact with the suburothelial capillary network. They express Cx43 and form functional syncytia. The expression of muscarinic and purinergic receptors is highly variable. Dye coupling experiments showed differences to detrusor myocytes. Upregulation of smooth muscle cell alpha-actin and/or transdifferentiation into smooth muscle cells may contribute to the etiology of urge incontinence. A multi-step model is presented as a working hypothesis.
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Yin, Ling-Ling; Ruan, Su-Hong; Tian, Yu; Zhao, Kai; Xu, Kai Lin
2015-10-01
To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs). The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells. The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired. Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.
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Phenotypic Intratumoral Heterogeneity of Endometrial Carcinomas.
Silva, Cátia; Pires-Luís, Ana S; Rocha, Eduardo; Bartosch, Carla; Lopes, José M
2018-03-01
Intratumoral heterogeneity has been shown to play an important role in diagnostic accuracy, development of treatment resistance, and prognosis of cancer patients. Recent studies have proposed quantitative measurement of phenotypic intratumoral heterogeneity, but no study is yet available in endometrial carcinomas. In our study we evaluated the phenotypic intratumoral heterogeneity of a consecutive series of 10 endometrial carcinomas using measures of dispersion and diversity. Morphometric architectural (%tumor cells, %solid tumor, %differentiated tumor, and %lumens) and nuclear [volume-weighted mean nuclear volume ((Equation is included in full-text article.))] parameters, as well as estrogen receptor, progesterone receptor, p53, vimentin, and beta-catenin immunoexpression (H-score) were digitally analyzed in 20 microscopic fields per carcinoma. Quantitative measures of intratumoral heterogeneity included coefficient of variation (CV) and relative quadratic entropy (rQE). In each endometrial carcinoma there was slight variation of architecture from field to field, resulting in globally low levels of heterogeneity measures (mean CV %tumor cells: 0.10, %solid tumor: 0.73, %differentiated tumor: 0.19, %lumens: 0.61 and mean rQE %tumor cells: 18.5, %solid tumor: 20.3, %differentiated tumor: 25.6, %lumens: 21.8). Nuclear intratumoral heterogeneity was also globally low (mean (Equation is included in full-text article.)CV: 0.23 and rQE: 27.3), but significantly higher than the heterogeneity of architectural parameters within most carcinomas. In general, there was low to moderate variability of immunoexpression markers within each carcinoma, but estrogen receptor (mean CV: 0.56 and rQE: 46.2) and progesterone receptor (mean CV: 0.60 and rQE: 39.3) displayed the highest values of heterogeneity measures. Intratumoral heterogeneity of immunoexpression was significantly higher than that observed for morphometric parameters. In conclusion, our study indicates that endometrial carcinomas present a variable but predominantly low degree of phenotypic intratumoral heterogeneity.
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Song, De-Gang; Ye, Qunrui; Poussin, Mathilde; Liu, Lin; Figini, Mariangela; Powell, Daniel J.
2015-01-01
Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors in an HLA-independent manner. To date, various CARs have been constructed using mouse single chain antibody variable fragments (scFvs) of high affinity that are immunogenic in humans and have the potential to mediate “on-target” toxicity. Here, we developed and evaluated a fully human CAR comprised of the human C4 folate receptor-alpha (αFR)-specific scFv coupled to intracellular T cell signaling domains. Human T cells transduced to express the C4 CAR specifically secreted proinflammatory cytokine and exerted cytolytic functions when cultured with αFR-expressing tumors in vitro. Adoptive transfer of C4 CAR T cells mediated the regression of large, established human ovarian cancer in a xenogeneic mouse model. Relative to a murine MOv19 scFv-based αFR CAR, C4 CAR T cells mediated comparable cytotoxic tumor activity in vitro and in vivo but had lower affinity for αFR protein and exhibited reduced recognition of normal cells expressing low levels of αFR. Thus, T cells expressing a fully human CAR of intermediate affinity can efficiently kill antigen-expressing tumors in vitro and in vivo and may overcome issues of transgene immunogenicity and “on-target off-tumor” toxicity that plague trials utilizing CARs containing mouse-derived, high affinity scFvs. PMID:26101914
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Campbell, Jacquelyn A.; Schelling, Pierre; Wetzel, J. Denise; Johnson, Elizabeth M.; Forrest, J. Craig; Wilson, Greame A. R.; Aurrand-Lions, Michel; Imhof, Beat A.; Stehle, Thilo; Dermody, Terence S.
2005-01-01
Reovirus infections are initiated by the binding of viral attachment protein σ1 to receptors on the surface of host cells. The σ1 protein is an elongated fiber comprised of an N-terminal tail that inserts into the virion and a C-terminal head that extends from the virion surface. The prototype reovirus strains type 1 Lang/53 (T1L/53) and type 3 Dearing/55 (T3D/55) use junctional adhesion molecule A (JAM-A) as a receptor. The C-terminal half of the T3D/55 σ1 protein interacts directly with JAM-A, but the determinants of receptor-binding specificity have not been identified. In this study, we investigated whether JAM-A also mediates the attachment of the prototype reovirus strain type 2 Jones/55 (T2J/55) and a panel of field-isolate strains representing each of the three serotypes. Antibodies specific for JAM-A were capable of inhibiting infections of HeLa cells by T1L/53, T2J/55, and T3D/55, demonstrating that strains of all three serotypes use JAM-A as a receptor. To corroborate these findings, we introduced JAM-A or the structurally related JAM family members JAM-B and JAM-C into Chinese hamster ovary cells, which are poorly permissive for reovirus infection. Both prototype and field-isolate reovirus strains were capable of infecting cells transfected with JAM-A but not those transfected with JAM-B or JAM-C. A sequence analysis of the σ1-encoding S1 gene segment of the strains chosen for study revealed little conservation in the deduced σ1 amino acid sequences among the three serotypes. This contrasts markedly with the observed sequence variability within each serotype, which is confined to a small number of amino acids. Mapping of these residues onto the crystal structure of σ1 identified regions of conservation and variability, suggesting a likely mode of JAM-A binding via a conserved surface at the base of the σ1 head domain. PMID:15956543
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N-Glycosylation Determines Ionic Permeability and Desensitization of the TRPV1 Capsaicin Receptor*
Veldhuis, Nicholas A.; Lew, Michael J.; Abogadie, Fe C.; Poole, Daniel P.; Jennings, Ernest A.; Ivanusic, Jason J.; Eilers, Helge; Bunnett, Nigel W.; McIntyre, Peter
2012-01-01
The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca2+]i) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1−/− mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission. PMID:22570472
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Kachko, Alla; Costafreda, Maria Isabel; Zubkova, Iryna; Jacques, Jerome; Takeda, Kazuyo; Wells, Frances; Kaplan, Gerardo; Major, Marian E
2018-03-15
Hepatitis C virus (HCV) is the leading cause of chronic hepatitis in humans. Several host molecules participate in HCV cell entry, but this process remains unclear. The complete unraveling of the HCV entry process is important to further understand viral pathogenesis and develop therapeutics. Human hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, functions as a phospholipid receptor involved in cell entry of several enveloped viruses. Here, we studied the role of HAVCR1 in HCV infection. HAVCR1 antibody inhibited entry in a dose-dependent manner. HAVCR1 soluble constructs neutralized HCV, which did not require the HAVCR1 mucinlike region and was abrogated by a mutation of N to A at position 94 (N94A) in the Ig variable (IgV) domain phospholipid-binding pocket, indicating a direct interaction of the HAVCR1 IgV domain with HCV virions. However, knockout of HAVCR1 in Huh7 cells reduced but did not prevent HCV growth. Interestingly, the mouse HAVCR1 ortholog, also a phospholipid receptor, did not enhance infection and a soluble form failed to neutralize HCV, although replacement of the mouse IgV domain with the human HAVCR1 IgV domain restored the enhancement of HCV infection. Mutations in the cytoplasmic tail revealed that direct HAVCR1 signaling is not required to enhance HCV infection. Our data show that the phospholipid-binding function and other determinant(s) in the IgV domain of human HAVCR1 enhance HCV infection. Although the exact mechanism is not known, it is possible that HAVCR1 facilitates entry by stabilizing or enhancing attachment, leading to direct interactions with specific receptors, such as CD81. IMPORTANCE Hepatitis C virus (HCV) enters cells through a multifaceted process. We identified the human hepatitis A virus cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, as a facilitator of HCV entry. Antibody blocking and silencing or knockout of HAVCR1 in hepatoma cells reduced HCV entry. Our findings that the interaction of HAVCR1 with HCV early during infection enhances entry but is not required for infection support the hypothesis that HAVCR1 facilitates entry by stabilizing or enhancing virus binding to the cell surface membrane and allowing the correct virus-receptor positioning for interaction with the main HCV receptors. Furthermore, our data show that in addition to the phospholipid-binding function of HAVCR1, the enhancement of HCV infection involves other determinants in the IgV domain of HAVCR1. These findings expand the repertoire of molecules that HCV uses for cell entry, adding to the already complex mechanism of HCV infection and pathogenesis. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
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Górski, A; Castronovo, V; Stepień-Sopniewska, B; Grieb, P; Ryba, M; Mrowiec, T; Korczak-Kowalska, G; Wierzbicki, P; Matysiak, W; Dybowska, B
1994-07-01
Although T cells infiltrate malignant tumors, the local immune response is usually inefficient and tumors escape destruction. While extracellular matrix proteins strongly costimulate T cell responses in normal individuals, our studies indicate that peripheral blood T cells from cancer patients and tumor infiltrating cells respond poorly or are resistant to stimulative signals mediated by collagen I and IV and fibronectin. Moreover, the adhesive properties of cancer T cells are markedly depressed. Those functional deficiencies are paralleled by variable deficits in integrin and non-integrin T cell receptors for extracellular matrix. Immunotherapy with BCG causes a dramatic but transient increase in T cell: ECM interactions.
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Beck, Christina M.; Willett, Julia L. E.; Kim, Jeff J.; Low, David A.; Hayes, Christopher S.
2016-01-01
Many Gram-negative bacterial pathogens express contact-dependent growth inhibition (CDI) systems that promote cell-cell interaction. CDI+ bacteria express surface CdiA effector proteins, which transfer their C-terminal toxin domains into susceptible target cells upon binding to specific receptors. CDI+ cells also produce immunity proteins that neutralize the toxin domains delivered from neighboring siblings. Here, we show that CdiAEC536 from uropathogenic Escherichia coli 536 (EC536) uses OmpC and OmpF as receptors to recognize target bacteria. E. coli mutants lacking either ompF or ompC are resistant to CDIEC536-mediated growth inhibition, and both porins are required for target-cell adhesion to inhibitors that express CdiAEC536. Experiments with single-chain OmpF fusions indicate that the CdiAEC536 receptor is heterotrimeric OmpC-OmpF. Because the OmpC and OmpF porins are under selective pressure from bacteriophages and host immune systems, their surface-exposed loops vary between E. coli isolates. OmpC polymorphism has a significant impact on CDIEC536 mediated competition, with many E. coli isolates expressing alleles that are not recognized by CdiAEC536. Analyses of recombinant OmpC chimeras suggest that extracellular loops L4 and L5 are important recognition epitopes for CdiAEC536. Loops L4 and L5 also account for much of the sequence variability between E. coli OmpC proteins, raising the possibility that CDI contributes to the selective pressure driving OmpC diversification. We find that the most efficient CdiAEC536 receptors are encoded by isolates that carry the same cdi gene cluster as E. coli 536. Thus, it appears that CdiA effectors often bind preferentially to "self" receptors, thereby promoting interactions between sibling cells. As a consequence, these effector proteins cannot recognize nor suppress the growth of many potential competitors. These findings suggest that self-recognition and kin selection are important functions of CDI. PMID:27723824
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Sullivan, Lucy C; Clements, Craig S; Beddoe, Travis; Johnson, Darryl; Hoare, Hilary L; Lin, Jie; Huyton, Trevor; Hopkins, Emma J; Reid, Hugh H; Wilce, Matthew C J; Kabat, Juraj; Borrego, Francisco; Coligan, John E; Rossjohn, Jamie; Brooks, Andrew G
2007-12-01
The CD94-NKG2 receptor family that regulates NK and T cells is unique among the lectin-like receptors encoded within the natural killer cell complex. The function of the CD94-NKG2 receptors is dictated by the pairing of the invariant CD94 polypeptide with specific NKG2 isoforms to form a family of functionally distinct heterodimeric receptors. However, the structural basis for this selective pairing and how they interact with their ligand, HLA-E, is unknown. We describe the 2.5 A resolution crystal structure of CD94-NKG2A in which the mode of dimerization contrasts with that of other homodimeric NK receptors. Despite structural homology between the CD94 and NKG2A subunits, the dimer interface is asymmetric, thereby providing a structural basis for the preferred heterodimeric assembly. Structure-based sequence comparisons of other CD94-NKG2 family members, combined with extensive mutagenesis studies on HLA-E and CD94-NKG2A, allows a model of the interaction between CD94-NKG2A and HLA-E to be established, in which the invariant CD94 chain plays a more dominant role in interacting with HLA-E in comparison to the variable NKG2 chain.
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The role of NK cells in HIV-1 protection: autologous, allogeneic or both?
Hens, Jef; Jennes, Wim; Kestens, Luc
2016-01-01
Natural killer (NK) cells specialize in killing virally infected- or tumor cells and are part of the innate immune system. The activational state of NK cells is determined by the balance of incoming activating and inhibitory signals mediated by receptor-ligand binding with the target cell. These receptor-ligand bonds mainly consist of the killer immunoglobulin-like receptors (KIR), which are expressed at the cell surface of NK cells, and their ligands: the highly variable human leukocyte antigen -class I molecules (HLA). Absence of an inhibitory receptor-ligand bond lowers the NK cell activation threshold, whereas an activating receptor-ligand bond stimulates the cell, potentially overcoming this threshold and triggering NK cell activation. NK cells influence the course of infection as well as the acquisition of HIV-1. Several lines of evidence relate the activating NK cell receptor KIR3DS1, in the presence or absence of its putative ligand HLA-Bw4, with slower disease progression as well as resistance to HIV-1 infection. Overall, resistance to HIV-1 infection predominantly correlates with activating KIR/HLA profiles, consisting of e.g. activating KIRs, group B haplotypes, or inhibitory KIRs in absence of their ligands. Such a conclusion is less evident for studies of HIV-1 disease progression, with studies reporting beneficial as well as detrimental effects of activating KIR/HLA genotypes. It is likely that KIR/HLA association studies are complicated by the complexity of the KIR and HLA loci and their mutual interactions, as well as by additional factors like route of HIV exposure, immune activation, presence of co-infections, and the effect of anti-HIV-1 antibodies. One newly discovered NK cell activation pathway associated with resistance to HIV-1 infection involves the presence of an iKIR/HLA mismatch between partners. The absence of such an iKIR/HLA bond renders donor-derived allogeneic HIV-1 infected cells vulnerable to NK cell responses during HIV-1 transmission. Therefore, theoretically, HIV-1 would be eliminated before it has the chance to infect the autologous cells in the recipient. While this "alloreactive" NK cell mechanism is especially relevant to HIV transmission in monogamous couples, it would be interesting to investigate how it could influence resistance to HIV in other settings. The objective of this review is to summarize the knowledge about these autologous and alloreactive NK cell responses with regard to HIV-1 outcome.
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McIntosh, K R; Linsley, P S; Drachman, D B
1995-11-01
The pathogenic antibody response to acetylcholine receptor (AChR) in experimental autoimmune myasthenia gravis (EAMG) is T cell dependent. Therefore, it should be possible to design specific immunotherapeutic approaches to treat EAMG (and human MG) by interfering with AChR-specific helper T cells. Productive T cell activation by antigen requires at least two signals: one signal delivered through the T cell receptor by antigen and a second costimulatory signal delivered through the CD28 receptor via the B7 counterreceptor expressed on antigen-presenting cells. Here we show that interference with the B7 costimulatory signal, using a soluble CD28 analogue, CTLA4Ig, resulted in a profound decrease in IL2 production and significantly decreased lymphoproliferative responses and antibody responses by primed lymph node cells from rats with EAMG, when stimulated with AChR in vitro. Nonclonal AChR-specific T cell lines, when stimulated with AChR in the presence of CTLA4Ig, were also inhibited in their ability to proliferate and to produce the cytokines IL2 and IFN-gamma. They remained deficient in their ability to produce IL2 when restimulated with AChR plus fresh antigen-presenting cells and showed variable inhibition of proliferation. The induction of hyporesponsiveness was accompanied by the expression of functional IL2 receptors, as shown by vigorous proliferative responses to addition of exogenous IL2. These results indicate that specific antigen stimulation in the presence of CTLA4Ig can induce certain features typical of anergy. CTLA4Ig provides a promising approach for the immunomodulation of MG and other antibody-mediated autoimmune diseases.
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Chemokines and their receptors in whiplash injury: elevated RANTES and CCR-5.
Kivioja, J; Rinaldi, L; Ozenci, V; Kouwenhoven, M; Kostulas, N; Lindgren, U; Link, H
2001-07-01
The human sufferings and socioeconomic burden due to whip-lash-associated disorders (WAD) are obvious but the pathogenesis of WAD is obscure. The possible involvement of the immune system during the disease process in WAD is not known. Effector molecules including chemokines and their receptors could play a role in WAD. In a prospective study using flow cytometry, we examined percentages of blood mononuclear cells (MNC) expressing the chemokines RANTES, MCP-1, MIP-1alpha, MIP-1beta, and IL-8, the chemokine receptor CCR-5, the T cell activation marker CD25, and the T cell chemoattractant IL-16 in patients with WAD and, for reference, in healthy controls. Higher percentages of RANTES-expressing blood MNC and T cells were observed in patients with WAD examined within 3 days compared to 14 days after the whiplash injury and, likewise, compared with healthy controls. The patients with WAD examined within 3 days after the accident also had higher percentages of CCR-5-expressing blood MNC, T cells, and CD45RO+ T cells compared to healthy controls. In contrast, there were no differences for any of these variables between patients with WAD examined 14 days after injury and healthy controls. In conclusion, WAD is associated with a systemic but transient dysregulation in percentages of RANTES and CCR-5 expressing MNC and T cells.
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Molecular mechanism of acetylcholine receptor-controlled ion translocation across cell membranes
Cash, Derek J.; Hess, George P.
1980-01-01
Two molecular processes, the binding of acetylcholine to the membrane-bound acetylcholine receptor protein and the receptor-controlled flux rates of specific inorganic ions, are essential in determining the electrical membrane potential of nerve and muscle cells. The measurements reported establish the relationship between the two processes: the acetylcholine receptor-controlled transmembrane ion flux of 86Rb+ and the concentration of carbamoylcholine, a stable analog of acetylcholine. A 200-fold concentration range of carbamoylcholine was used. The flux was measured in the millisecond-to-minute time region by using a quench flow technique with membrane vesicles prepared from the electric organ of Electrophorus electricus in eel Ringer's solution at pH 7.0 and 1°C. The technique makes possible the study of the transmembrane transport of specific ions, with variable known internal and external ion concentrations, in a system in which a determinable number of receptors is exposed to a known concentration of ligand. The response curve of ion flux to ligand was sigmoidal with an average maximum rate of 84 sec-1. Carbamoylcholine induced inactivation of the receptor with a maximum rate of 2.7 sec-1 and a different ligand dependence so that it was fast relative to ion flux at low ligand concentration but slow relative to ion flux at high ligand concentration. The simplest model that fits the data consists of receptor in the active and inactive states in ligand-controlled equilibria. Receptor inactivation occurs with one or two ligand molecules bound. For channel opening, two ligand molecules bound to the active state are required, and cooperativity results from the channel opening process itself. With carbamoylcholine, apparently, the equilibrium position for the channel opening step is only one-fourth open. The integrated rate equation, based on the model, predicts the time dependence of receptor-controlled ion flux over the concentration range of carbamoylcholine investigated. The values of the constants in the rate equation form the basis for predicting receptor-controlled changes in the transmembrane potential of cells and the conditions leading to transmission of signals between cells. PMID:6928684
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Roschmann, E; Wienker, T F; Gerok, W; Volk, B A
1993-12-01
Genetic susceptibility of celiac disease is primarily associated with a particular combination of and HLA-DQA1/DQB1 gene; however, this does not fully account for the genetic predisposition. Therefore, the aim of this study was to examine whether T-cell receptor (TCR) genes may be susceptibility genes in celiac disease. HLA class II typing was performed by polymerase chain reaction amplification in combination with sequence-specific oligonucleotide hybridization. TCR alpha (TCRA), TCR gamma (TCRG), and TCR beta (TCRB) loci were investigated by restriction fragment length polymorphism analysis. Allelic frequencies of TCRA, TCRG, and TCRB variable genes were compared between patients with celiac disease (n = 53) and control patients (n = 67), and relative risk (RR) estimates were calculated. The RR was 1.67 for allele C1 at TCRA1, 3.35 for allele D2 at TCRA2, 1.66 for allele B2 at TCRG, and 1.35 for allele B at TCRB, showing no significant association. Additionally, linkage analysis was performed in 23 families. The logarithm of odd scores for celiac disease vs. the TCR variable genes at TCRA, TCRG, and TCRB showed no significant linkage. These data suggest that the analyzed TCR variable gene segments V alpha 1.2, V gamma 11, and V beta 8 do not play a major role in susceptibility to celiac disease.
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Streich, Roswita; Breysach, Caroline; Raddatz, Dirk; Oniga, Septimia; Peccerella, Teresa; Findeisen, Peter; Kzhyshkowska, Julia; Gratchev, Alexei; Schweyer, Stefan; Saunders, Bernadette; Wessels, Johannes T.; Möbius, Wiebke; Keane, Joseph; Becker, Heinz; Ganser, Arnold; Neumaier, Michael; Kaminski, Wolfgang E.
2011-01-01
Macrophages play a central role in host defense against mycobacterial infection and anti- TNF therapy is associated with granuloma disorganization and reactivation of tuberculosis in humans. Here, we provide evidence for the presence of a T cell receptor (TCR) αβ based recombinatorial immune receptor in subpopulations of human and mouse monocytes and macrophages. In vitro, we find that the macrophage-TCRαβ induces the release of CCL2 and modulates phagocytosis. TNF blockade suppresses macrophage-TCRαβ expression. Infection of macrophages from healthy individuals with mycobacteria triggers formation of clusters that express restricted TCR Vβ repertoires. In vivo, TCRαβ bearing macrophages abundantly accumulate at the inner host-pathogen contact zone of caseous granulomas from patients with lung tuberculosis. In chimeric mouse models, deletion of the variable macrophage-TCRαβ or TNF is associated with structurally compromised granulomas of pulmonary tuberculosis even in the presence of intact T cells. These results uncover a TNF-regulated recombinatorial immune receptor in monocytes/macrophages and demonstrate its implication in granuloma formation in tuberculosis. PMID:22114556
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Aghebati-Maleki, Leili; Younesi, Vahid; Baradaran, Behzad; Abdolalizadeh, Jalal; Motallebnezhad, Morteza; Nickho, Hamid; Shanehbandi, Dariush; Majidi, Jafar; Yousefi, Mehdi
2017-04-01
Receptor tyrosine kinase-like orphan receptor (ROR) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including cell survival, differentiation, cell migration, cell communication, cell polarity, proliferation, metabolism, and angiogenesis. ROR1 has recently been shown to be expressed in various types of cancer cells but not normal cells. Pharmacokinetics and pharmacodynamics of single-chain Fragment variable (scFv) antibodies provide potential therapeutic advantages over whole antibody molecules. In the present study, scFvs against a specific peptide from the extracellular domain of ROR1 were selected using phage display technology. The selected scFvs were further characterized using polyclonal and monoclonal phage enzyme-linked immunosorbent assay (ELISA), soluble monoclonal ELISA, colony PCR, and sequencing. Antiproliferative and apoptotic effects of selected scFv antibodies were also evaluated in lymphoma and myeloma cancer cell lines using MTT and annexin V/PI assays. The results of ELISA indicated specific reactions of the isolated scFvs against the ROR1 peptide. Colony PCR confirmed the presence of full-length V H and Vκ inserts. The percentages of cell growth after 24 h of treatment of cells with individual scFv revealed that the scFv significantly inhibited the growth of the RPMI8226 and chronic lymphocytic leukemia (CLL) cells in comparison with the untreated cells ( p < 0.05). Interestingly, 24-h treatment with specific scFv induced apoptosis cell death in the RPMI8226 and CLL cells. Taken together, our results demonstrate that targeting of ROR1 using peptide-specific scFv can be an effective immunotherapy strategy in hematological malignancies.
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Variability and repertoire size of T-cell receptor V alpha gene segments.
Becker, D M; Pattern, P; Chien, Y; Yokota, T; Eshhar, Z; Giedlin, M; Gascoigne, N R; Goodnow, C; Wolf, R; Arai, K
The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta.
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Adnectin-Based Design of Chimeric Antigen Receptor for T Cell Engineering.
Han, Xiaolu; Cinay, Gunce E; Zhao, Yifan; Guo, Yunfei; Zhang, Xiaoyang; Wang, Pin
2017-11-01
Although chimeric antigen receptor (CAR)-engineered T cell therapy has achieved encouraging clinical trial results for treating hematological cancers, further optimization can likely expand this therapeutic success to more patients and other cancer types. Most CAR constructs used in clinical trials incorporate single chain variable fragment (scFv) as the extracellular antigen recognition domain. The immunogenicity of nonhuman scFv could cause host rejection against CAR T cells and compromise their persistence and efficacy. The limited availability of scFvs and slow discovery of new monoclonal antibodies also limit the development of novel CAR constructs. Adnectin, a class of affinity molecules derived from the tenth type III domain of human fibronectin, can be an alternative to scFv as an antigen-binding moiety in the design of CAR molecules. We constructed adnectin-based CARs targeting epithelial growth factor receptor (EGFR) and found that compared to scFv-based CAR, T cells engineered with adnectin-based CARs exhibited equivalent cell-killing activity against target H292 lung cancer cells in vitro and had comparable antitumor efficacy in xenograft tumor-bearing mice in vivo. In addition, with optimal affinity tuning, adnectin-based CAR showed higher selectivity on target cells with high EGFR expression than on those with low expression. This new design of adnectin CARs can potentially facilitate the development of T cell immunotherapy for cancer and other diseases. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.
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Genetic variants of dopamine D2 receptor impact heterodimerization with dopamine D1 receptor.
Błasiak, Ewa; Łukasiewicz, Sylwia; Szafran-Pilch, Kinga; Dziedzicka-Wasylewska, Marta
2017-04-01
The human dopamine D2 receptor gene has three polymorphic variants that alter its amino acid sequence: alanine substitution by valine in position 96 (V96A), proline substitution by serine in position 310 (P310S) and serine substitution by cysteine in position 311 (S311C). Their functional role has never been the object of extensive studies, even though there is some evidence that their occurrence correlates with schizophrenia. The HEK293 cell line was transfected with dopamine D1 and D2 receptors (or genetic variants of the D2 receptor), coupled to fluorescent proteins which allowed us to measure the extent of dimerization of these receptors, using a highly advanced biophysical approach (FLIM-FRET). Additionally, Fluoro-4 AM was used to examine changes in the level of calcium release after ligand stimulation of cells expressing different combinations of dopamine receptors. Using FLIM-FRET experiments we have shown that in HEK 293 expressing dopamine receptors, polymorphic mutations in the D2 receptor play a role in dimmer formation with the dopamine D1 receptor. The association level of dopamine receptors is affected by ligand administration, with variable effects depending on polymorphic variant of the D2 dopamine receptor. We have found that the level of heteromer formation is reflected by calcium ion release after ligand stimulation and have observed variations of this effect dependent on the polymorphic variant and the ligand. The data presented in this paper support the hypothesis on the role of calcium signaling regulated by the D1-D2 heteromer which may be of relevance for schizophrenia etiology. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
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Berendeeva, T A; Ponomarev, S A; Antropova, E N; Rykova, M P
2015-01-01
Studies of Toll-like receptors (TLR) in 20 cosmonauts-members of long-duration (124-199-day) missions to the International space station evidenced changes in relative and absolute counts of peripheral blood monocytes with TLR2, TLR4 and TLR6 on the surface, expression of TLR2 and TLR6 genes, and genes of molecules involved in the TLR signaling pathway and TLR-related NF-KB-, JNK/p38- and IRF pathways on the day of return to Earth. The observed changes displayed individual variability.
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The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors
DOE Office of Scientific and Technical Information (OSTI.GOV)
Lin, David Yin-wei; Tanaka, Yoshimasa; Iwasaki, Masashi
2008-07-29
Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives 'exhausted' virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains onmore » the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.« less
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Maciaszek, Jamie L; Partola, Kostyantyn; Zhang, Jing; Andemariam, Biree; Lykotrafitis, George
2014-12-18
Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding. Copyright © 2014 Elsevier Ltd. All rights reserved.
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GBM heterogeneity as a function of variable epidermal growth factor receptor variant III activity.
Lindberg, Olle R; McKinney, Andrew; Engler, Jane R; Koshkakaryan, Gayane; Gong, Henry; Robinson, Aaron E; Ewald, Andrew J; Huillard, Emmanuelle; David James, C; Molinaro, Annette M; Shieh, Joseph T; Phillips, Joanna J
2016-11-29
Abnormal activation of the epidermal growth factor receptor (EGFR) due to a deletion of exons 2-7 of EGFR (EGFRvIII) is a common alteration in glioblastoma (GBM). While this alteration can drive gliomagenesis, tumors harboring EGFRvIII are heterogeneous. To investigate the role for EGFRvIII activation in tumor phenotype we used a neural progenitor cell-based murine model of GBM driven by EGFR signaling and generated tumor progenitor cells with high and low EGFRvIII activation, pEGFRHi and pEGFRLo. In vivo, ex vivo, and in vitro studies suggested a direct association between EGFRvIII activity and increased tumor cell proliferation, decreased tumor cell adhesion to the extracellular matrix, and altered progenitor cell phenotype. Time-lapse confocal imaging of tumor cells in brain slice cultures demonstrated blood vessel co-option by tumor cells and highlighted differences in invasive pattern. Inhibition of EGFR signaling in pEGFRHi promoted cell differentiation and increased cell-matrix adhesion. Conversely, increased EGFRvIII activation in pEGFRLo reduced cell-matrix adhesion. Our study using a murine model for GBM driven by a single genetic driver, suggests differences in EGFR activation contribute to tumor heterogeneity and aggressiveness.
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Ramírez-Anguita, Juan Manuel; Rodríguez-Espigares, Ismael; Guixà-González, Ramon; Bruno, Agostino; Torrens-Fontanals, Mariona; Varela-Rial, Alejandro; Selent, Jana
2018-01-01
The serotonin 5-hydroxytryptamine 2A (5-HT 2A ) receptor is a G-protein-coupled receptor (GPCR) relevant for the treatment of CNS disorders. In this regard, neuronal membrane composition in the brain plays a crucial role in the modulation of the receptor functioning. Since cholesterol is an essential component of neuronal membranes, we have studied its effect on the 5-HT 2A receptor dynamics through all-atom MD simulations. We find that the presence of cholesterol in the membrane increases receptor conformational variability in most receptor segments. Importantly, detailed structural analysis indicates that conformational variability goes along with the destabilization of hydrogen bonding networks not only within the receptor but also between receptor and lipids. In addition to increased conformational variability, we also find receptor segments with reduced variability. Our analysis suggests that this increased stabilization is the result of stabilizing effects of tightly bound cholesterol molecules to the receptor surface. Our finding contributes to a better understanding of membrane-induced alterations of receptor dynamics and points to cholesterol-induced stabilizing and destabilizing effects on the conformational variability of GPCRs. © 2017 International Union of Biochemistry and Molecular Biology, Inc.
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Devi, Suma Priya Sudarsana; Howe, James R.
2016-01-01
Key points Purkinje cells of the cerebellum receive ∼180,000 parallel fibre synapses, which have often been viewed as a homogeneous synaptic population and studied using single action potentials.Many parallel fibre synapses might be silent, however, and granule cells in vivo fire in bursts. Here, we used trains of stimuli to study parallel fibre inputs to Purkinje cells in rat cerebellar slices.Analysis of train EPSCs revealed two synaptic components, phase 1 and 2. Phase 1 is initially large and saturates rapidly, whereas phase 2 is initially small and facilitates throughout the train. The two components have a heterogeneous distribution at dendritic sites and different pharmacological profiles.The differential sensitivity of phase 1 and phase 2 to inhibition by pentobarbital and NBQX mirrors the differential sensitivity of AMPA receptors associated with the transmembrane AMPA receptor regulatory protein, γ‐2, gating in the low‐ and high‐open probability modes, respectively. Abstract Cerebellar granule cells fire in bursts, and their parallel fibre axons (PFs) form ∼180,000 excitatory synapses onto the dendritic tree of a Purkinje cell. As many as 85% of these synapses have been proposed to be silent, but most are labelled for AMPA receptors. Here, we studied PF to Purkinje cell synapses using trains of 100 Hz stimulation in rat cerebellar slices. The PF train EPSC consisted of two components that were present in variable proportions at different dendritic sites: one, with large initial EPSC amplitude, saturated after three stimuli and dominated the early phase of the train EPSC; and the other, with small initial amplitude, increased steadily throughout the train of 10 stimuli and dominated the late phase of the train EPSC. The two phases also displayed different pharmacological profiles. Phase 2 was less sensitive to inhibition by NBQX but more sensitive to block by pentobarbital than phase 1. Comparison of synaptic results with fast glutamate applications to recombinant receptors suggests that the high‐open‐probability gating mode of AMPA receptors containing the auxiliary subunit transmembrane AMPA receptor regulatory protein γ‐2 makes a substantial contribution to phase 2. We argue that the two synaptic components arise from AMPA receptors with different functional signatures and synaptic distributions. Comparisons of voltage‐ and current‐clamp responses obtained from the same Purkinje cells indicate that phase 1 of the EPSC arises from synapses ideally suited to transmit short bursts of action potentials, whereas phase 2 is likely to arise from low‐release‐probability or ‘silent’ synapses that are recruited during longer bursts. PMID:27094216
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Calcineurin-dependent negative regulation of CD94/NKG2A expression on naive CD8+ T cells.
Cho, Jae-Ho; Kim, Hee-Ok; Webster, Kylie; Palendira, Mainthan; Hahm, Bumsuk; Kim, Kyu-Sik; King, Cecile; Tangye, Stuart G; Sprent, Jonathan
2011-07-07
Immune responses lead to expression of immunoregulatory molecules on T cells, including natural killer (NK) receptors, such as CD94/NKG2A on CD8(+) T cells; these receptors restrain CD8(+) responses, thereby preventing T-cell exhaustion in chronic infections and limiting immunopathology. Here, we examined the requirements for inducing CD94/NKG2A on T cells responding to antigen. In vitro, moderate induction of CD94/NKG2A expression occurred after exposure of naive CD8(+) (but not CD4(+)) cells to CD3 ligation or specific peptide. Surprisingly, expression was inhibited by CD28/B7 costimulation. Such inhibition applied only to CD94/NKG2A and not other NK receptors (NKG2D) and was mediated by IL-2. Inhibition by IL-2 occurred via a NFAT cell-independent component of the calcineurin pathway, and CD94/NKG2A induction was markedly enhanced in the presence of calcineurin blockers, such as FK506 or using calcineurin-deficient T cells, both in vitro and in vivo. In addition to CD28-dependent inhibition by IL-2, CD94/NKG2A expression was impaired by several other cytokines (IL-4, IL-23, and transforming growth factor-β) but enhanced by others (IL-6, IL-10, and IL-21). The complex interplay between these various stimuli may account for the variable expression of CD94/NKG2A during responses to different pathogens in vivo.
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Shalev, Zvi; Duffy, Simon P; Adema, Karen W; Prasad, Rati; Hussain, Naveen; Willett, Brian J; Tailor, Chetankumar S
2009-07-01
The pathogenic subgroup C feline leukemia virus (FeLV-C) arises in infected cats as a result of mutations in the envelope (Env) of the subgroup A FeLV (FeLV-A). To better understand emergence of FeLV-C and potential FeLV intermediates that may arise, we characterized FeLV Env sequences from the primary FY981 FeLV isolate previously derived from an anemic cat. Here, we report the characterization of the novel FY981 FeLV Env that is highly related to FeLV-A Env but whose variable region A (VRA) receptor recognition sequence partially resembles the VRA sequence from the prototypical FeLV-C/Sarma Env. Pseudotype viruses bearing FY981 Env were capable of infecting feline, human, and guinea pig cells, suggestive of a subgroup C phenotype, but also infected porcine ST-IOWA cells that are normally resistant to FeLV-C and to FeLV-A. Analysis of the host receptor used by FY981 suggests that FY981 can use both the FeLV-C receptor FLVCR1 and the feline FeLV-A receptor THTR1 for infection. However, our results suggest that FY981 infection of ST-IOWA cells is not mediated by the porcine homologue of FLVCR1 and THTR1 but by an alternative receptor, which we have now identified as the FLVCR1-related protein FLVCR2. Together, our results suggest that FY981 FeLV uses FLVCR1, FLVCR2, and THTR1 as receptors. Our findings suggest the possibility that pathogenic FeLV-C arises in FeLV-infected cats through intermediates that are multitropic in their receptor use.
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Baker, Salah A; Drumm, Bernard T; Saur, Dieter; Hennig, Grant W; Ward, Sean M; Sanders, Kenton M
2016-06-15
Interstitial cells of Cajal at the level of the deep muscular plexus (ICC-DMP) in the small intestine generate spontaneous Ca(2+) transients that consist of localized Ca(2+) events and limited propagating Ca(2+) waves. Ca(2+) transients in ICC-DMP display variable characteristics: from discrete, highly localized Ca(2+) transients to regionalized Ca(2+) waves with variable rates of occurrence, amplitude, duration and spatial spread. Ca(2+) transients fired stochastically, with no cellular or multicellular rhythmic activity being observed. No correlation was found between the firing sites in adjacent cells. Ca(2+) transients in ICC-DMP are suppressed by the ongoing release of inhibitory neurotransmitter(s). Functional intracellular Ca(2+) stores are essential for spontaneous Ca(2+) transients, and the sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump is necessary for maintenance of spontaneity. Ca(2+) release mechanisms involve both ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3 Rs). Release from these channels is interdependent. ICC express transcripts of multiple RyRs and InsP3 Rs, with Itpr1 and Ryr2 subtypes displaying the highest expression. Interstitial cells of Cajal in the deep muscular plexus of the small intestine (ICC-DMP) are closely associated with varicosities of enteric motor neurons and generate responses contributing to neural regulation of intestinal motility. Responses of ICC-DMP are mediated by activation of Ca(2+) -activated Cl(-) channels; thus, Ca(2+) signalling is central to the behaviours of these cells. Confocal imaging was used to characterize the nature and mechanisms of Ca(2+) transients in ICC-DMP within intact jejunal muscles expressing a genetically encoded Ca(2+) indicator (GCaMP3) selectively in ICC. ICC-DMP displayed spontaneous Ca(2+) transients that ranged from discrete, localized events to waves that propagated over variable distances. The occurrence of Ca(2+) transients was highly variable, and it was determined that firing was stochastic in nature. Ca(2+) transients were tabulated in multiple cells within fields of view, and no correlation was found between the events in adjacent cells. TTX (1 μm) significantly increased the occurrence of Ca(2+) transients, suggesting that ICC-DMP contributes to the tonic inhibition conveyed by ongoing activity of inhibitory motor neurons. Ca(2+) transients were minimally affected after 12 min in Ca(2+) free solution, indicating these events do not depend immediately upon Ca(2+) influx. However, inhibitors of sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump and blockers of inositol triphosphate receptor (InsP3 R) and ryanodine receptor (RyR) channels blocked ICC Ca(2+) transients. These data suggest an interdependence between RyR and InsP3 R in the generation of Ca(2+) transients. Itpr1 and Ryr2 were the dominant transcripts expressed by ICC. These findings provide the first high-resolution recording of the subcellular Ca(2+) dynamics that control the behaviour of ICC-DMP in situ. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.
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Baker, Salah A.; Drumm, Bernard T.; Saur, Dieter; Hennig, Grant W.; Ward, Sean M.
2016-01-01
Key points Interstitial cells of Cajal at the level of the deep muscular plexus (ICC‐DMP) in the small intestine generate spontaneous Ca2+ transients that consist of localized Ca2+ events and limited propagating Ca2+ waves.Ca2+ transients in ICC‐DMP display variable characteristics: from discrete, highly localized Ca2+ transients to regionalized Ca2+ waves with variable rates of occurrence, amplitude, duration and spatial spread.Ca2+ transients fired stochastically, with no cellular or multicellular rhythmic activity being observed. No correlation was found between the firing sites in adjacent cells.Ca2+ transients in ICC‐DMP are suppressed by the ongoing release of inhibitory neurotransmitter(s).Functional intracellular Ca2+ stores are essential for spontaneous Ca2+ transients, and the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) pump is necessary for maintenance of spontaneity.Ca2+ release mechanisms involve both ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3Rs). Release from these channels is interdependent.ICC express transcripts of multiple RyRs and InsP3Rs, with Itpr1 and Ryr2 subtypes displaying the highest expression. Abstract Interstitial cells of Cajal in the deep muscular plexus of the small intestine (ICC‐DMP) are closely associated with varicosities of enteric motor neurons and generate responses contributing to neural regulation of intestinal motility. Responses of ICC‐DMP are mediated by activation of Ca2+‐activated Cl− channels; thus, Ca2+ signalling is central to the behaviours of these cells. Confocal imaging was used to characterize the nature and mechanisms of Ca2+ transients in ICC‐DMP within intact jejunal muscles expressing a genetically encoded Ca2+ indicator (GCaMP3) selectively in ICC. ICC‐DMP displayed spontaneous Ca2+ transients that ranged from discrete, localized events to waves that propagated over variable distances. The occurrence of Ca2+ transients was highly variable, and it was determined that firing was stochastic in nature. Ca2+ transients were tabulated in multiple cells within fields of view, and no correlation was found between the events in adjacent cells. TTX (1 μm) significantly increased the occurrence of Ca2+ transients, suggesting that ICC‐DMP contributes to the tonic inhibition conveyed by ongoing activity of inhibitory motor neurons. Ca2+ transients were minimally affected after 12 min in Ca2+ free solution, indicating these events do not depend immediately upon Ca2+ influx. However, inhibitors of sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) pump and blockers of inositol triphosphate receptor (InsP3R) and ryanodine receptor (RyR) channels blocked ICC Ca2+ transients. These data suggest an interdependence between RyR and InsP3R in the generation of Ca2+ transients. Itpr1 and Ryr2 were the dominant transcripts expressed by ICC. These findings provide the first high‐resolution recording of the subcellular Ca2+ dynamics that control the behaviour of ICC‐DMP in situ. PMID:26824875
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Peters, Derek T; Henderson, Christopher A; Warren, Curtis R; Friesen, Max; Xia, Fang; Becker, Caroline E; Musunuru, Kiran; Cowan, Chad A
2016-05-01
Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro, but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal, we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray, and tested their ability to perform mature hepatocyte functions (albumin and urea secretion, cytochrome activity). By these measures, ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation. © 2016. Published by The Company of Biologists Ltd.
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Seki, Akiko; Rutz, Sascha
2018-03-05
CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell-based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will greatly extend the feasibly of target gene discovery and validation in primary T cells and simplify the gene editing process for next-generation immunotherapies. © 2018 Genentech.
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USDA-ARS?s Scientific Manuscript database
Channel catfish, Ictalurus punctatus, T cell receptors (TCR) gamma and delta were identified by mining of expressed sequence tag databases and full length sequences were obtained by 5'-RACE and RT-PCR protocols. cDNAs for each of these TCR chains encode typical variable (V), (diversity; D), joining ...
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Ghamari-Langroudi, M; Glavinovíc, M I
1998-01-01
Spontaneous miniature excitatory postsynaptic currents (mEPSCs) in rat hippocampal pyramidal neurones in slices (CA1 region) were recorded at 35-37 degrees C using the whole-cell patch-clamp technique before and after addition of aniracetam (1 mM) to determine how a partial blockade of desensitization alters the relationship between the amplitude (A) and kinetics of mEPSCs, and to evaluate the factors that determine their variability. The rise time (taur) and the time constant of decay of mEPSCs (taud) are essentially amplitude independent in control conditions, but become clearly amplitude dependent in the presence of aniracetam. The slopes of the best fitting lines to taud:A and taur:A data pairs were (+/- SD; ms/pA; n = 5): (1) (control) 0.07 +/- 0.02 and 0.008 +/- 0.003; (2) (aniracetam) 0.40 +/- 0.19 and 0.22 +/- 0.22. The amplitude-dependent prolongation of taud is explained by the concentration dependence of two related processes, the buffering of glutamate molecules by AMPA receptor channels, and the occupancy of the double-bound activatable states. A slower deactivation makes an amplitude-independent contribution. Desensitization reduces the amplitude dependence of taud by minimizing repeated openings of alpha-amino-3-hydroxy-methyl-isoxazole (AMPA) receptor channels. A greater amplitude dependence of taur probably involves both pre- and postsynaptic factors. The variability of A and taud values did not change significantly, but the factors underlying the variability of taud values were much affected. The greater amplitude dependence and the greater scatter about the best fitting lines to taud:A data pairs are approximately balanced by the greater mean values. The greater scatter of taud about the best fitting lines probably occurs because the saturation of AMPA receptors is not the same at different synapses with different numbers of AMPA receptors.
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Gorzkiewicz, Michał; Sztandera, Krzysztof; Jatczak-Pawlik, Izabela; Zinke, Robin; Appelhans, Dietmar; Klajnert-Maculewicz, Barbara; Pulaski, Łukasz
2018-05-14
Poly(propyleneimine) dendrimers fully surface-modified with disaccharide moieties (maltose, cellobiose, and lactose) designed to mimic natural lectin receptor ligands were tested for their bioactivity in two myeloid cell lines: THP-1 and HL-60. Depending on the sugar modification, we observed variable activation of NF-κB, AP-1, and NF-AT signaling pathways: lactose-coated dendrimers had the strongest impact on marker gene expression and most signaling events with the notable exception of NF-κB activation in THP-1 cells. The two cell lines showed an overall similar pattern of transcription factor and gene expression activation upon treatment with glycodendrimers, suggesting the involvement of galectin and C-type lectin receptor types. An important result of this action was the overexpression of CD40 and IL8 genes, potentially leading to an activated, proinflammatory phenotype in the monocyte/macrophage cell lineage. These pharmacodynamic characteristics of glycodendrimers need to be taken into account during their pharmaceutical applications both in drug delivery and direct immunomodulation.
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The expression of Toll-like receptors 2, 4, 5, 7 and 9 in Merkel cell carcinoma.
Jouhi, Lauri; Koljonen, Virve; Böhling, Tom; Haglund, Caj; Hagström, Jaana
2015-04-01
We sought to clarify whether the expression of toll-like receptors (TLR) in Merkel cell carcinoma (MCC) is linked to tumor and patient characteristics, especially the presence of Merkel cell polyoma virus (MCV). The study comprised of 128 patients with data on Merkel cell polyomavirus (MCV) status and clinical features were included in the study. Immunohistochemistry for TLR expression was performed on tissue microarray (TMA) slides. TLR 2, 4, 5, 7 and 9 expression was noted in most of the tumor specimens. Decreased expression of TLR 9 correlated strongly with MCV positivity. Cytoplasmic TLR 2 expression correlated with small tumor size, while nuclear TLR 2 and TLR 5 expressions with larger tumors. Increased nuclear TLR 4 expression and decreased TLR 7 expression were associated with older age. TLR 2, 4, 5, 7 and 9 appear to reflect certain clinicopathological variables and prognostic markers of MCC tumors. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
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A structural basis for antigen recognition by the T cell-like lymphocytes of sea lamprey
DOE Office of Scientific and Technical Information (OSTI.GOV)
Deng, Lu; Velikovsky, C. Alejandro; Xu, Gang
Adaptive immunity in jawless vertebrates is mediated by leucine-rich repeat proteins called 'variable lymphocyte receptors' (VLRs). Two types of VLR (A and B) are expressed by mutually exclusive lymphocyte populations in lamprey. VLRB lymphocytes resemble the B cells of jawed vertebrates; VLRA lymphocytes are similar to T cells. We determined the structure of a high-affinity VLRA isolated from lamprey immunized with hen egg white lysozyme (HEL) in unbound and antigen-bound forms. The VLRA-HEL complex demonstrates that certain VLRAs, like {gamma}{delta} T-cell receptors (TCRs) but unlike {alpha}{beta} TCRs, can recognize antigens directly, without a requirement for processing or antigen-presenting molecules. Thus,more » these VLRAs feature the nanomolar affinities of antibodies, the direct recognition of unprocessed antigens of both antibodies and {gamma}{delta} TCRs, and the exclusive expression on the lymphocyte surface that is unique to {alpha}{beta} and {gamma}{delta} TCRs.« less
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Hoppe, T; Kraus, D; Novak, N; Probstmeier, R; Frentzen, M; Wenghoefer, M; Jepsen, S; Winter, J
2016-10-01
The impact of oral pathogens onto the generation and variability of oral tumors has only recently been investigated. To get further insights, oral cancer cells were treated with pathogens and additionally, as a result of this bacterial cellular infection, with human defensins, which are as anti-microbial peptide members of the innate immune system. After cell stimulation, proliferation behavior, expression analysis of oncogenic relevant defensin genes, and effects on EGFR signaling were investigated. The expression of oncogenic relevant anti-microbial peptides was analyzed with real-time PCR and immunohistochemistry. Cell culture experiments were performed to examine cellular impacts caused by stimulation, i.e., altered gene expression, proliferation rate, and EGF receptor-dependent signaling. Incubation of oral tumor cells with an oral pathogen (Porphyromonas gingivalis) and human α-defensins led to an increase in cell proliferation. In contrast, another oral bacterium used, Aggregatibacter actinomycetemcomitans, enhanced cell death. The bacteria and anti-microbial peptides exhibited diverse effects on the transcript levels of oncogenic relevant defensin genes and epidermal growth factor receptor signaling. These two oral pathogens exhibited opposite primary effects on the proliferation behavior of oral tumor cells. Nevertheless, both microbe species led to similar secondary impacts on the proliferation rate by modifying expression levels of oncogenic relevant α-defensin genes. In this respect, oral pathogens exerted multiplying effects on tumor cell proliferation. Additionally, human defensins were shown to differently influence epidermal growth factor receptor signaling, supporting the hypothesis that these anti-microbial peptides serve as ligands of EGFR, thus modifying the proliferation behavior of oral tumor cells.
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Zheng, Tingting; Fu, Jia-Ju; Hu, Lihui; Qiu, Fan; Hu, Minjin; Zhu, Jun-Jie; Hua, Zi-Chun; Wang, Hui
2013-06-04
The variable susceptibility to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment observed in various types of leukemia cells is related to the difference in the expression levels of death receptors, DR4 and DR5, on the cell surfaces. Quantifying the DR4/DR5 expression status on leukemia cell surfaces is of vital importance to the development of diagnostic tools to guide death receptor-based leukemia treatment. Taking the full advantages of novel nanobiotechnology, we have developed a robust electrochemical cytosensing approach toward ultrasensitive detection of leukemia cells with detection limit as low as ~40 cells and quantitative evaluation of DR4/DR5 expression on leukemia cell surfaces. The optimization of electron transfer and cell capture processes at specifically tailored nanobiointerfaces and the incorporation of multiple functions into rationally designed nanoprobes provide unique opportunities of integrating high specificity and signal amplification on one electrochemical cytosensor. The high sensitivity and selectivity of this electrochemical cytosensing approach also allows us to evaluate the dynamic alteration of DR4/DR5 expression on the surfaces of living cells in response to drug treatments. Using the TRAIL-resistant HL-60 cells and TRAIL-sensitive Jurkat cells as model cells, we have further verified that the TRAIL susceptibility of various types of leukemia cells is directly correlated to the surface expression levels of DR4/DR5. This versatile electrochemical cytosensing platform is believed to be of great clinical value for the early diagnosis of human leukemia and the evaluation of therapeutic effects on leukemia patients after radiation therapy or drug treatment.
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Cell specific, variable density, polymer microspheres
NASA Technical Reports Server (NTRS)
Yen, Shiao-Ping S. (Inventor); Rembaum, Alan (Inventor); Molday, Robert S. (Inventor)
1977-01-01
Biocompatible polymeric microspheres having an average diameter below about 3 microns and having density at least 15% greater or lesser than organic cells and having covalent binding sites are provided in accordance with this invention. The microspheres are obtained by copolymerizing a hydroxy or amine substituted acrylic monomer such as hydroxyethylmethacrylate with a light or dense comonomer such as a fluoromonomer. A lectin or antibody is bound to the hydroxy or amine site of the bead to provide cell specificity. When added to a cell suspension the marked bead will specifically label the cell membrane by binding to specific receptor sites thereon. The labelled membrane can then be separated by density gradient centrifugation.
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Abken, Hinrich
2017-11-01
Adoptive therapy with chimeric antigen receptor (CAR) redirected T cells achieved lasting remissions in hematologic malignancies, even in terminal stages of the disease. Exploring CAR T cell therapy in the treatment of solid tumors has just begun, balancing efficacy versus toxicity in early phase trials. In contrast to leukemia/lymphoma, solid tumors display a tremendously variable biology demanding different strategies to make a T cell attack successful in the long term. This article summarizes current developments, discusses the hurdles, and considers some modifications to improve the CAR T cell therapy in the treatment of solid tumors.
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2013-01-01
Background Expression of miR-17-92 enhances T-cell survival and interferon (IFN)-γ production. We previously reported that miR-17-92 is down-regulated in T-cells derived from glioblastoma (GBM) patients. We hypothesized that transgene-derived co-expression of miR17-92 and chimeric antigen receptor (CAR) in T-cells would improve the efficacy of adoptive transfer therapy against GBM. Methods We constructed novel lentiviral vectors for miR-17-92 (FG12-EF1a-miR-17/92) and a CAR consisting of an epidermal growth factor receptor variant III (EGFRvIII)-specific, single-chain variable fragment (scFv) coupled to the T-cell receptor CD3ζ chain signaling module and co-stimulatory motifs of CD137 (4-1BB) and CD28 in tandem (pELNS-3C10-CAR). Human T-cells were transduced with these lentiviral vectors, and their anti-tumor effects were evaluated both in vitro and in vivo. Results CAR-transduced T-cells (CAR-T-cells) exhibited potent, antigen-specific, cytotoxic activity against U87 GBM cells that stably express EGFRvIII (U87-EGFRvIII) and, when co-transduced with miR-17-92, exhibited improved survival in the presence of temozolomide (TMZ) compared with CAR-T-cells without miR-17-92 co-transduction. In mice bearing intracranial U87-EGFRvIII xenografts, CAR-T-cells with or without transgene-derived miR-17-92 expression demonstrated similar levels of therapeutic effect without demonstrating any uncontrolled growth of CAR-T-cells. However, when these mice were re-challenged with U87-EGFRvIII cells in their brains, mice receiving co-transduced CAR-T-cells exhibited improved protection compared with mice treated with CAR-T-cells without miR-17-92 co-transduction. Conclusion These results warrant the development of novel CAR-T-cell strategies that incorporate miR-17-92 to improve therapeutic potency, especially in patients with GBM. PMID:24829757
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Ohno, Masasuke; Ohkuri, Takayuki; Kosaka, Akemi; Tanahashi, Kuniaki; June, Carl H; Natsume, Atsushi; Okada, Hideho
2013-01-01
Expression of miR-17-92 enhances T-cell survival and interferon (IFN)-γ production. We previously reported that miR-17-92 is down-regulated in T-cells derived from glioblastoma (GBM) patients. We hypothesized that transgene-derived co-expression of miR17-92 and chimeric antigen receptor (CAR) in T-cells would improve the efficacy of adoptive transfer therapy against GBM. We constructed novel lentiviral vectors for miR-17-92 (FG12-EF1a-miR-17/92) and a CAR consisting of an epidermal growth factor receptor variant III (EGFRvIII)-specific, single-chain variable fragment (scFv) coupled to the T-cell receptor CD3ζ chain signaling module and co-stimulatory motifs of CD137 (4-1BB) and CD28 in tandem (pELNS-3C10-CAR). Human T-cells were transduced with these lentiviral vectors, and their anti-tumor effects were evaluated both in vitro and in vivo. CAR-transduced T-cells (CAR-T-cells) exhibited potent, antigen-specific, cytotoxic activity against U87 GBM cells that stably express EGFRvIII (U87-EGFRvIII) and, when co-transduced with miR-17-92, exhibited improved survival in the presence of temozolomide (TMZ) compared with CAR-T-cells without miR-17-92 co-transduction. In mice bearing intracranial U87-EGFRvIII xenografts, CAR-T-cells with or without transgene-derived miR-17-92 expression demonstrated similar levels of therapeutic effect without demonstrating any uncontrolled growth of CAR-T-cells. However, when these mice were re-challenged with U87-EGFRvIII cells in their brains, mice receiving co-transduced CAR-T-cells exhibited improved protection compared with mice treated with CAR-T-cells without miR-17-92 co-transduction. These results warrant the development of novel CAR-T-cell strategies that incorporate miR-17-92 to improve therapeutic potency, especially in patients with GBM.
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Krug, Christian; Wiesinger, Manuel; Abken, Hinrich; Schuler-Thurner, Beatrice; Schuler, Gerold; Dörrie, Jan; Schaft, Niels
2014-10-01
Chimeric antigen receptors (CARs), which combine an antibody-derived binding domain (single chain fragment variable) with T-cell-activating signaling domains, have become a promising tool in the adoptive cellular therapy of cancer. Retro- and lenti-viral transductions are currently the standard methods to equip T cells with a CAR; permanent CAR expression, however, harbors several risks like uncontrolled auto-reactivity. Modification of T cells by electroporation with CAR-encoding RNA to achieve transient expression likely circumvents these difficulties. We here present a GMP-compliant protocol to activate and expand T cells for clinical application. The protocol is optimized in particular to produce CAR-modified T cells in clinically sufficient numbers under full GMP-compliance from late-stage cancer patients. This protocol allows the generation of 6.7 × 10(8) CAR-expressing T cells from one patient leukapheresis. The CAR-engineered T cells produced pro-inflammatory cytokines after stimulation with antigen-bearing tumor cells and lysed tumor cells in an antigen-specific manner. This functional capacity was maintained after cryopreservation. Taken together, we provide a clinically applicable protocol to transiently engineer sufficient numbers of antigen-specific patient T cells for use in adoptive cell therapy of cancer.
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Organization of GABAergic synaptic circuits in the rat ventral tegmental area.
Ciccarelli, Alessandro; Calza, Arianna; Panzanelli, Patrizia; Concas, Alessandra; Giustetto, Maurizio; Sassoè-Pognetto, Marco
2012-01-01
The ventral tegmental area (VTA) is widely implicated in drug addiction and other psychiatric disorders. This brain region is densely populated by dopaminergic (DA) neurons and also contains a sparse population of γ-aminobutyric acid (GABA)ergic cells that regulate the activity of the principal neurons. Therefore, an in-depth knowledge of the organization of VTA GABAergic circuits and of the plasticity induced by drug consumption is essential for understanding the mechanisms by which drugs induce stable changes in brain reward circuits. Using immunohistochemistry, we provide a detailed description of the localization of major GABA(A) and GABA(B) receptor subunits in the rat VTA. We show that DA and GABAergic cells express both GABA(A) and GABA(B) receptors. However VTA neurons differ considerably in the expression of GABA(A) receptor subunits, as the α1 subunit is associated predominantly with non-DA cells, whereas the α3 subunit is present at low levels in both types of VTA neurons. Using an unbiased stereological method, we then demonstrate that α1-positive elements represent only a fraction of non-DA neurons and that the ratio of DA and non-DA cells is quite variable throughout the rostro-caudal extent of the VTA. Interestingly, DA and non-DA cells receive a similar density of perisomatic synapses, whereas axo-dendritic synapses are significantly more abundant in non-DA cells, indicating that local interneurons receive prominent GABAergic inhibition. These findings reveal a differential expression of GABA receptor subtypes in the two major categories of VTA neurons and provide an anatomical basis for interpreting the plasticity of inhibitory circuits induced by drug exposure.
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Meyer, F R L; Steinborn, R; Grausgruber, H; Wolfesberger, B; Walter, I
2015-10-01
The discovery of expression of the erythropoietin receptor (EPO-R) on neoplastic cells has led to concerns about the safety of treating anaemic cancer patients with EPO. In addition to its endocrine function, the receptor may play a role in tumour progression through an autocrine mechanism. In this study, the expression of EPO, EPO-R and platelet-derived growth factor BB (PDGF-BB) was analysed in five feline and 13 canine osteosarcomas using immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR). EPO expression was positive in all tumours by IHC, but EPO mRNA was only detected in 38% of the canine and 40% of the feline samples. EPO-R was expressed in all samples by quantitative RT-PCR (RT-qPCR) and IHC. EPO-R mRNA was expressed at higher levels in all feline tumours, tumour cell lines, and kidney when compared to canine tissues. PDGF-BB expression was variable by IHC, but mRNA was detected in all samples. To assess the functionality of the EPO-R on tumour cells, the proliferation of canine and feline osteosarcoma cell lines was evaluated after EPO administration using an alamarBlue assay and Ki67 immunostaining. All primary cell lines responded to EPO treatment in at least one of the performed assays, but the effect on proliferation was very low indicating only a weak responsiveness of EPO-R. In conclusion, since EPO and its receptor are expressed by canine and feline osteosarcomas, an autocrine or paracrine tumour progression mechanism cannot be excluded, although in vitro data suggest a minimal role of EPO-R in osteosarcoma cell proliferation. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
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Meyer, F.R.L.; Steinborn, R.; Grausgruber, H.; Wolfesberger, B.; Walter, I.
2015-01-01
The discovery of expression of the erythropoietin receptor (EPO-R) on neoplastic cells has led to concerns about the safety of treating anaemic cancer patients with EPO. In addition to its endocrine function, the receptor may play a role in tumour progression through an autocrine mechanism. In this study, the expression of EPO, EPO-R and platelet-derived growth factor BB (PDGF-BB) was analysed in five feline and 13 canine osteosarcomas using immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR). EPO expression was positive in all tumours by IHC, but EPO mRNA was only detected in 38% of the canine and 40% of the feline samples. EPO-R was expressed in all samples by quantitative RT-PCR (RT-qPCR) and IHC. EPO-R mRNA was expressed at higher levels in all feline tumours, tumour cell lines, and kidney when compared to canine tissues. PDGF-BB expression was variable by IHC, but mRNA was detected in all samples. To assess the functionality of the EPO-R on tumour cells, the proliferation of canine and feline osteosarcoma cell lines was evaluated after EPO administration using an alamarBlue assay and Ki67 immunostaining. All primary cell lines responded to EPO treatment in at least one of the performed assays, but the effect on proliferation was very low indicating only a weak responsiveness of EPO-R. In conclusion, since EPO and its receptor are expressed by canine and feline osteosarcomas, an autocrine or paracrine tumour progression mechanism cannot be excluded, although in vitro data suggest a minimal role of EPO-R in osteosarcoma cell proliferation. PMID:26189892
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WC1 is a hybrid γδ TCR coreceptor and pattern recognition receptor for pathogenic bacteria.
Hsu, Haoting; Chen, Chuang; Nenninger, Ariel; Holz, Lauren; Baldwin, Cynthia L; Telfer, Janice C
2015-03-01
WC1 proteins are uniquely expressed on γδ T cells and belong to the scavenger receptor cysteine-rich (SRCR) superfamily. While present in variable, and sometimes high, numbers in the genomes of mammals and birds, in cattle there are 13 distinct genes (WC1-1 to WC1-13). All bovine WC1 proteins can serve as coreceptors for the TCR in a tyrosine phosphorylation dependent manner, and some are required for the γδ T cell response to Leptospira. We hypothesized that individual WC1 receptors encode Ag specificity via coligation of bacteria with the γδ TCR. SRCR domain binding was directly correlated with γδ T cell response, as WC1-3 SRCR domains from Leptospira-responsive cells, but not WC1-4 SRCR domains from Leptospira-nonresponsive cells, bound to multiple serovars of two Leptospira species, L. borgpetersenii, and L. interrogans. Three to five of eleven WC1-3 SRCR domains, but none of the eleven WC1-4 SRCR domains, interacted with Leptospira spp. and Borrelia burgdorferi, but not with Escherichia coli or Staphylococcus aureus. Mutational analysis indicated that the active site for bacterial binding in one of the SRCR domains is composed of amino acids in three discontinuous regions. Recombinant WC1 SRCR domains with the ability to bind leptospires inhibited Leptospira growth. Our data suggest that WC1 gene arrays play a multifaceted role in the γδ T cell response to bacteria, including acting as hybrid pattern recognition receptors and TCR coreceptors, and they may function as antimicrobials. Copyright © 2015 by The American Association of Immunologists, Inc.
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van Schouwenburg, Pauline A; Davenport, Emma E; Kienzler, Anne-Kathrin; Marwah, Ishita; Wright, Benjamin; Lucas, Mary; Malinauskas, Tomas; Martin, Hilary C; Lockstone, Helen E; Cazier, Jean-Baptiste; Chapel, Helen M; Knight, Julian C; Patel, Smita Y
2015-10-01
Common Variable Immunodeficiency Disorders (CVIDs) are the most prevalent cause of primary antibody failure. CVIDs are highly variable and a genetic causes have been identified in <5% of patients. Here, we performed whole genome sequencing (WGS) of 34 CVID patients (94% sporadic) and combined them with transcriptomic profiling (RNA-sequencing of B cells) from three patients and three healthy controls. We identified variants in CVID disease genes TNFRSF13B, TNFRSF13C, LRBA and NLRP12 and enrichment of variants in known and novel disease pathways. The pathways identified include B-cell receptor signalling, non-homologous end-joining, regulation of apoptosis, T cell regulation and ICOS signalling. Our data confirm the polygenic nature of CVID and suggest individual-specific aetiologies in many cases. Together our data show that WGS in combination with RNA-sequencing allows for a better understanding of CVIDs and the identification of novel disease associated pathways. Copyright © 2015. Published by Elsevier Inc.
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Mori, Masayuki X; Itsuki, Kyohei; Hase, Hideharu; Sawamura, Seishiro; Kurokawa, Tatsuki; Mori, Yasuo; Inoue, Ryuji
2015-01-01
Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that carry receptor-operated Ca(2+) currents (ROCs) triggered by receptor-induced, phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Within the vasculature, TRPC channel ROCs contribute to smooth muscle cell depolarization, vasoconstriction, and vascular remodeling. However, TRPC channel ROCs exhibit a variable response to receptor-stimulation, and the regulatory mechanisms governing TRPC channel activity remain obscure. The variability of ROCs may be explained by their complex regulation by PI(4,5)P2 and its metabolites, which differentially affect TRPC channel activity. To resolve the complex regulation of ROCs, the use of voltage-sensing phosphoinositide phosphatases and model simulation have helped to reveal the time-dependent contribution of PI(4,5)P2 and the possible role of PI(4,5)P2 in the regulation of ROCs. These approaches may provide unprecedented insight into the dynamics of PI(4,5)P2 regulation of TRPC channels and the fundamental mechanisms underlying transmembrane ion flow. Within that context, we summarize the regulation of TRPC channels and their coupling to receptor-mediated signaling, as well as the application of voltage-sensing phosphoinositide phosphatases to this research. We also discuss the controversial bidirectional effects of PI(4,5)P2 using a model simulation that could explain the complicated effects of PI(4,5)P2 on different ROCs.
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Chen, C; Li, J; Micko, C J; Pierce, G F; Cunningham, M R; Lumsden, A B
1998-02-15
Vascular smooth muscle cell (SMC) proliferation is an integral component of intimal lesion formation. In this study we compared the mitogenic effects of basic fibroblast growth factor (bFGF) and heparin binding epidermal growth factor (HBEGF) and the cytotoxic effects of bFGF and HBEGF conjugated with plant cytotoxin saporin (SAP) on vascular cell cultures. Human vascular SMCs and endothelial cells were cultured and FGF receptor-1 (FGFR-1) and EGF receptor (EGFR) expression were detected by immunohistochemical staining. Cells were grown in 24-well plates. Variable amounts of testing drugs (bFGF, HBEGF, SAP, bFGF-SAP, or HBEGF-SAP) were added to quadruplicate wells after 24 h. Cells without drugs were used as control. The total number of cells was counted at 72 h using a hemocytometer. The cultured human vascular SMCs and endothelial cells expressed both FGFR-1 and EGFR with predominant perinuclear localization. bFGF and HBEGF demonstrated equally potent mitogenic effects on SMC proliferation. SAP alone showed a limited cytotoxic effect on both SMCs and endothelial cells. bFGF had a more potent effect on endothelial cell proliferation than HBEGF. bFGF-SAP was equally cytotoxic for both SMCs and endothelial cells, while HBEGF-SAP had a more selectively cytotoxic effect on SMCs than on endothelial cells. These data suggest that the mitogenic effects of bFGF and HBEGF and the cytotoxic effects of bFGF-SAP and HBEGF-SAP may both be mediated by their corresponding growth factor receptors. Because of its selective cytotoxic effect on SMCs, HBEGF-SAP may become a more attractive agent for controlling intimal lesion formation.
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Sanderson, Nicholas D.; Norman, Paul J.; Guethlein, Lisbeth A.; Ellis, Shirley A.; Williams, Christina; Breen, Matthew; Park, Steven D. E.; Magee, David A.; Babrzadeh, Farbod; Warry, Andrew; Watson, Mick; Bradley, Daniel G.; MacHugh, David E.; Parham, Peter
2014-01-01
Under selection pressure from pathogens, variable NK cell receptors that recognize polymorphic MHC class I evolved convergently in different species of placental mammal. Unexpectedly, diversified killer cell Ig–like receptors (KIRs) are shared by simian primates, including humans, and cattle, but not by other species. Whereas much is known of human KIR genetics and genomics, knowledge of cattle KIR is limited to nine cDNA sequences. To facilitate comparison of the cattle and human KIR gene families, we determined the genomic location, structure, and sequence of two cattle KIR haplotypes and defined KIR sequences of aurochs, the extinct wild ancestor of domestic cattle. Larger than its human counterpart, the cattle KIR locus evolved through successive duplications of a block containing ancestral KIR3DL and KIR3DX genes that existed before placental mammals. Comparison of two cattle KIR haplotypes and aurochs KIR show the KIR are polymorphic and the gene organization and content appear conserved. Of 18 genes, 8 are functional and 10 were inactivated by point mutation. Selective inactivation of KIR3DL and activating receptor genes leaves a functional cohort of one inhibitory KIR3DL, one activating KIR3DX, and six inhibitory KIR3DX. Functional KIR diversity evolved from KIR3DX in cattle and from KIR3DL in simian primates. Although independently evolved, cattle and human KIR gene families share important function-related properties, indicating that cattle KIR are NK cell receptors for cattle MHC class I. Combinations of KIR and MHC class I are the major genetic factors associated with human disease and merit investigation in cattle. PMID:25398326
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Sharma, P; Postel, S; Sundberg, E J; Kranz, D M
2013-12-01
Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection.
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Sharma, P.; Postel, S.; Sundberg, E.J.; Kranz, D.M.
2013-01-01
Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection. PMID:24167300
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Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.
Chaudhary, Anu; Leite, Mara; Kulasekara, Bridget R; Altura, Melissa A; Ogahara, Cassandra; Weiss, Eli; Fu, Wenqing; Blanc, Marie-Pierre; O'Keeffe, Michael; Terhorst, Cox; Akey, Joshua M; Miller, Samuel I
2016-07-25
Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Girard, Tanya; Gaucher, Denis; El-Far, Mohamed; Breton, Gaëlle; Sékaly, Rafick-Pierre
2014-09-01
CD86 and CD80, the ligands for the co-stimulatory molecules CD28 and CTLA-4, are members of the Ig superfamily. Their structure includes Ig variable-like (IgV) domains, Ig constant-like (IgC) domains and intracellular domains. Although crystallographic studies have clearly identified the IgV domain to be responsible for receptor interactions, earlier studies suggested that both Ig domains are required for full co-signaling function. Herein, we have used deletion and chimeric human CD80 and CD86 molecules in co-stimulation assays to study the impact of the multimeric state of IgV and IgC domains on receptor binding properties and on co-stimulatory function in a peptide-specific T cell activation model. We report for the first time the presence of CD80 dimers and CD86 monomers in living cells. Moreover, we show that the IgC domain of both molecules inhibits multimer formation and greatly affects binding to the co-receptors CD28 and CTLA-4. Finally, both IgC and intracellular domains are required for full co-signaling function. These findings reveal the distinct but complementary roles of CD80 and CD86 IgV and IgC domains in T cell activation. Copyright © 2014 Elsevier B.V. All rights reserved.
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Duchmann, R; Jaffe, J; Ehrhardt, R; Alling, D W; Strober, W
1996-01-01
In this study, we report that differences between T-cell receptor (TCR) V beta gene family usage in CD4+ and CD8+ T cells are significantly greater in a subgroup of patients with common variable immunodeficiency (CVI) and high levels of activated CD8+ T cells (CD8hi CVI) than in controls (P < 0.001). In CD8hi CVI patients, such differences were also significantly greater for V beta 12 than for other V beta families. As the causes of the differential usage of V beta gene families by CD4+ and CD8+ T cells are under investigation, it was interesting that the combined differences between V beta gene family usage in the CD4+ and CD8+ T-cell subpopulations as a whole were significantly lower than the combined differences between individual V beta gene family usage in either CD4+ or CD8+ T-cell subpopulations (P < 0.001 in both control and CD8hi CVI patients). Further, the pattern of V beta gene family usage in CD4+ T cells was remarkably similar to that in CD8+ T cells in both groups. These data strongly suggest that differences in V beta gene family usage arising from coselection by major histocompatibility complex (MHC) class I versus MHC class II restriction elements do not fundamentally distort 'basic' V beta gene family usage patterns. They also support the concept that differences in CD4+ and CD8+ T-cell V beta gene family usage, which were increased in CD8hi CVI, can arise from high-affinity interactions between disease-associated antigens or superantigens and T cells in the post-thymic T-cell compartment. Images Figure 6 PMID:8666443
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Intraclonal Cell Expansion and Selection Driven by B Cell Receptor in Chronic Lymphocytic Leukemia
Colombo, Monica; Cutrona, Giovanna; Reverberi, Daniele; Fabris, Sonia; Neri, Antonino; Fabbi, Marina; Quintana, Giovanni; Quarta, Giovanni; Ghiotto, Fabio; Fais, Franco; Ferrarini, Manlio
2011-01-01
The mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes utilized by chronic lymphocytic leukemia (CLL) clones defines two disease subgroups. Patients with unmutated IGHV have a more aggressive disease and a worse outcome than patients with cells having somatic IGHV gene mutations. Moreover, up to 30% of the unmutated CLL clones exhibit very similar or identical B cell receptors (BcR), often encoded by the same IG genes. These “stereotyped” BcRs have been classified into defined subsets. The presence of an IGHV gene somatic mutation and the utilization of a skewed gene repertoire compared with normal B cells together with the expression of stereotyped receptors by unmutated CLL clones may indicate stimulation/selection by antigenic epitopes. This antigenic stimulation may occur prior to or during neoplastic transformation, but it is unknown whether this stimulation/selection continues after leukemogenesis has ceased. In this study, we focused on seven CLL cases with stereotyped BcR Subset #8 found among a cohort of 700 patients; in six, the cells expressed IgG and utilized IGHV4-39 and IGKV1-39/IGKV1D-39 genes, as reported for Subset #8 BcR. One case exhibited special features, including expression of IgM or IgG by different subclones consequent to an isotype switch, allelic inclusion at the IGH locus in the IgM-expressing cells and a particular pattern of cytogenetic lesions. Collectively, the data indicate a process of antigenic stimulation/selection of the fully transformed CLL cells leading to the expansion of the Subset #8 IgG-bearing subclone. PMID:21541442
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Friedman, Kevin M; Garrett, Tracy E; Evans, John W; Horton, Holly M; Latimer, Howard J; Seidel, Stacie L; Horvath, Christopher J; Morgan, Richard A
2018-05-01
B-cell maturation antigen (BCMA) expression has been proposed as a marker for the identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Consistent BCMA expression was confirmed on MM biopsies (29/29 BCMA+), and it was further demonstrated that BCMA is expressed in a substantial number of lymphoma samples, as well as primary chronic lymphocytic leukemia B cells. To target BCMA using redirected autologous T cells, lentiviral vectors (LVV) encoding chimeric antigen receptors (CARs) were constructed with four unique anti-BCMA single-chain variable fragments, fused to the CD137 (4-1BB) co-stimulatory and CD3ζ signaling domains. One LVV, BB2121, was studied in detail, and BB2121 CAR-transduced T cells (bb2121) exhibited a high frequency of CAR + T cells and robust in vitro activity against MM cell lines, lymphoma cell lines, and primary chronic lymphocytic leukemia peripheral blood. Based on receptor quantification, bb2121 recognized tumor cells expressing as little as 222 BCMA molecules per cell. The in vivo pharmacology of anti-BCMA CAR T cells was studied in NSG mouse models of human MM, Burkitt lymphoma, and mantle cell lymphoma, where mice received a single intravenous administration of vehicle, control vector-transduced T cells, or anti-BCMA CAR-transduced T cells. In all models, the vehicle and control CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors and 100% survival in all treatment models. Together, these data support the further development of anti-BCMA CAR T cells as a potential treatment for not only MM but also some lymphomas.
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Li, Shu-Jing; Vaughan, Alexander; Sturgill, James Fitzhugh; Kepecs, Adam
2018-06-06
Retrogradely transported neurotropic viruses enable genetic access to neurons based on their long-range projections and have become indispensable tools for linking neural connectivity with function. A major limitation of viral techniques is that they rely on cell-type-specific molecules for uptake and transport. Consequently, viruses fail to infect variable subsets of neurons depending on the complement of surface receptors expressed (viral tropism). We report a receptor complementation strategy to overcome this by potentiating neurons for the infection of the virus of interest-in this case, canine adenovirus type-2 (CAV-2). We designed AAV vectors for expressing the coxsackievirus and adenovirus receptor (CAR) throughout candidate projection neurons. CAR expression greatly increased retrograde-labeling rates, which we demonstrate for several long-range projections, including some resistant to other retrograde-labeling techniques. Our results demonstrate a receptor complementation strategy to abrogate endogenous viral tropism and thereby facilitate efficient retrograde targeting for functional analysis of neural circuits. Copyright © 2018 Elsevier Inc. All rights reserved.
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Reddy, Venkat; Cambridge, Geraldine; Isenberg, David A; Glennie, Martin J; Cragg, Mark S; Leandro, Maria
2015-05-01
Rituximab, a type I anti-CD20 monoclonal antibody (mAb), induces incomplete B cell depletion in some patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), thus contributing to a poor clinical response. The mechanisms of this resistance remain elusive. The purpose of this study was to determine whether type II mAb are more efficient than type I mAb at depleting B cells from RA and SLE patients, whether internalization influences the efficiency of depletion, and whether Fcγ receptor type IIb (FcγRIIb) and the B cell receptor regulate this internalization process. We used an in vitro whole blood B cell-depletion assay to assess the efficiency of depletion, flow cytometry to study cell surface protein expression, and surface fluorescence-quenching assays to assess rituximab internalization, in samples from patients with RA and patients with SLE. Paired t-test or Mann-Whitney U test was used to compare groups, and Spearman's rank correlation test was used to assess correlation. We found that type II mAb internalized significantly less rituximab than type I mAb and depleted B cells from patients with RA and SLE at least 2-fold more efficiently than type I mAb. Internalization of rituximab was highly variable between patients, was regulated by FcγRIIb, and inversely correlated with cytotoxicity in whole blood B cell-depletion assays. The lowest levels of internalization were seen in IgD- B cells, including postswitched (IgD-CD27+) memory cells. Internalization of type I anti-CD20 mAb was also partially inhibited by anti-IgM stimulation. Variability in internalization of rituximab was observed and was correlated with impaired B cell depletion. Therefore, slower-internalizing type II mAb should be considered as alternative B cell-depleting agents for the treatment of RA and SLE. © 2015 The Authors. Arthritis & Rheumatology is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.
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Muntasell, Aura; Cabo, Mariona; Servitja, Sonia; Tusquets, Ignasi; Martínez-García, María; Rovira, Ana; Rojo, Federico; Albanell, Joan; López-Botet, Miguel
2017-01-01
Overexpression of the human epidermal growth factor receptor 2 (HER2) defines a subgroup of breast tumors with aggressive behavior. The addition of HER2-targeted antibodies (i.e., trastuzumab, pertuzumab) to chemotherapy significantly improves relapse-free and overall survival in patients with early-stage and advanced disease. Nonetheless, considerable proportions of patients develop resistance to treatment, highlighting the need for additional and co-adjuvant therapeutic strategies. HER2-specific antibodies can trigger natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity and indirectly enhance the development of tumor-specific T cell immunity; both mechanisms contributing to their antitumor efficacy in preclinical models. Antibody-dependent NK cell activation results in the release of cytotoxic granules as well as the secretion of pro-inflammatory cytokines (i.e., IFNγ and TNFα) and chemokines. Hence, NK cell tumor suppressive functions include direct cytolytic killing of tumor cells as well as the regulation of subsequent antitumor adaptive immunity. Albeit tumors with gene expression signatures associated to the presence of cytotoxic lymphocyte infiltrates benefit from trastuzumab-based treatment, NK cell-related biomarkers of response/resistance to HER2-specific therapeutic antibodies in breast cancer patients remain elusive. Several variables, including (i) the configuration of the patient NK cell repertoire; (ii) tumor molecular features (i.e., estrogen receptor expression); (iii) concomitant therapeutic regimens (i.e., chemotherapeutic agents, tyrosine kinase inhibitors); and (iv) evasion mechanisms developed by progressive breast tumors, have been shown to quantitatively and qualitatively influence antibody-triggered NK cell responses. In this review, we discuss possible interventions for restoring/enhancing the therapeutic activity of HER2 therapeutic antibodies by harnessing NK cell antitumor potential through combinatorial approaches, including immune checkpoint blocking/stimulatory antibodies, cytokines and toll-like receptor agonists. PMID:29181007
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Cell specific, variable density, polymer microspheres
NASA Technical Reports Server (NTRS)
Yen, Shiao-Ping S. (Inventor); Rembaum, Alan (Inventor); Molday, Robert S. (Inventor)
1978-01-01
Biocompatible polymeric microspheres having an average diameter below about 3 microns and having a density at least 15% greater or lesser than organic cells and having covalent binding sites are provided in accordance with this invention. The microspheres are obtained by copolymerizing a hydroxy or amine substituted acrylic monomer such as hydroxyethylmethacrylate with a light or dense comonomer such as a fluoromonomer. A lectin or antibody is bound to the hydroxy or amine site of the bead to provide cell specificity. When added to a cell suspension the marked bead will specifically label the cell membrane by binding to specific receptor sites thereon. The labelled membrane can then be separated by density gradient centrifugation.
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Vitamin D is a determinant of mouse intestinal Lgr5 stem cell functions.
Peregrina, Karina; Houston, Michele; Daroqui, Cecilia; Dhima, Elena; Sellers, Rani S; Augenlicht, Leonard H
2015-01-01
Lgr5+ intestinal crypt base columnar cells function as stem cells whose progeny populate the villi, and Lgr5+ cells in which Apc is inactivated can give rise to tumors. Surprisingly, these Lgr5+ stem cell properties were abrogated by the lower dietary vitamin D and calcium in a semi-purified diet that promotes both genetically initiated and sporadic intestinal tumors. Inactivation of the vitamin D receptor in Lgr5+ cells established that compromise of Lgr5 stem cell function was a rapid, cell autonomous effect of signaling through the vitamin D receptor. The loss of Lgr5 stem cell function was associated with presence of Ki67 negative Lgr5+ cells at the crypt base. Therefore, vitamin D, a common nutrient and inducer of intestinal cell maturation, is an environmental factor that is a determinant of Lgr5+ stem cell functions in vivo. Since diets used in reports that establish and dissect mouse Lgr5+ stem cell activity likely provided vitamin D levels well above the range documented for human populations, the contribution of Lgr5+ cells to intestinal homeostasis and tumor formation in humans may be significantly more limited, and variable in the population, then suggested by published rodent studies. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Moreira, Thiago S; Takakura, Ana C; Colombari, Eduardo; West, Gavin H; Guyenet, Patrice G
2007-01-01
The retrotrapezoid nucleus (RTN) contains CO2-activated interneurons with properties consistent with central respiratory chemoreceptors. These neurons are glutamatergic and express the transcription factor Phox2b. Here we tested whether RTN neurons receive an input from slowly adapting pulmonary stretch receptors (SARs) in halothane-anaesthetized ventilated rats. In vagotomized rats, RTN neurons were inhibited to a variable extent by stimulating myelinated vagal afferents using the lowest intensity needed to inhibit the phrenic nerve discharge (PND). In rats with intact vagus nerves, RTN neurons were inhibited, also to a variable extent, by increasing positive end-expiratory pressure (PEEP; 2–6 cmH2O). The cells most sensitive to PEEP were inhibited during each lung inflation at rest and were instantly activated by stopping ventilation. Muscimol (GABA-A agonist) injection in or next to the solitary tract at area postrema level desynchronized PND from ventilation, eliminated the lung inflation-synchronous inhibition of RTN neurons and their steady inhibition by PEEP but did not change their CO2 sensitivity. Muscimol injection into the rostral ventral respiratory group eliminated PND but did not change RTN neuron response to either lung inflation, PEEP increases, vagal stimulation or CO2. Generalized glutamate receptor blockade with intracerebroventricular (i.c.v.) kynurenate eliminated PND and the response of RTN neurons to lung inflation but did not change their CO2 sensitivity. PEEP-sensitive RTN neurons expressed Phox2b. In conclusion, RTN chemoreceptors receive an inhibitory input from myelinated lung stretch receptors, presumably SARs. The lung input to RTN may be di-synaptic with inhibitory pump cells as sole interneurons. PMID:17255166
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Ligand-targeted theranostic nanomedicines against cancer
Yao, Virginia J.; D'Angelo, Sara; Butler, Kimberly S.; ...
2016-01-06
Nanomedicines have significant potential for cancer treatment. Although the majority of nanomedicines currently tested in clinical trials utilize simple, biocompatible liposome-based nanocarriers, their widespread use is limited by non-specificity and low target site concentration and thus, do not provide a substantial clinical advantage over conventional, systemic chemotherapy. In the past 20 years, we have identified specific receptors expressed on the surfaces of tumor endothelial and perivascular cells, tumor cells, the extracellular matrix and stromal cells using combinatorial peptide libraries displayed on bacteriophage. These studies corroborate the notion that unique receptor proteins such as IL-11Rα, GRP78, EphA5, among others, are differentiallymore » overexpressed in tumors and present opportunities to deliver tumor-specific therapeutic drugs. By using peptides that bind to tumor-specific cell-surface receptors, therapeutic agents such as apoptotic peptides, suicide genes, imaging dyes or chemotherapeutics can be precisely and systemically delivered to reduce tumor growth in vivo, without harming healthy cells. Given the clinical applicability of peptide-based therapeutics, targeted delivery of nanocarriers loaded with therapeutic cargos seems plausible. We propose a modular design of a functionalized protocell in which a tumor-targeting moiety, such as a peptide or recombinant human antibody single chain variable fragment (scFv), is conjugated to a lipid bilayer surrounding a silica-based nanocarrier core containing a protected therapeutic cargo. The functionalized protocell can be tailored to a specific cancer subtype and treatment regimen by exchanging the tumor-targeting moiety and/or therapeutic cargo or used in combination to create unique, theranostic agents. In this review, we summarize the identification of tumor-specific receptors through combinatorial phage display technology and the use of antibody display selection to identify recombinant human scFvs against these tumor-specific receptors. We compare the characteristics of different types of simple and complex nanocarriers, and discuss potential types of therapeutic cargos and conjugation strategies. As a result, the modular design of functionalized protocells may improve the efficacy and safety of nanomedicines for future cancer therapy.« less
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Natural killer cell receptor genes in the family Equidae: not only Ly49.
Futas, Jan; Horin, Petr
2013-01-01
Natural killer (NK) cells have important functions in immunity. NK recognition in mammals can be mediated through killer cell immunoglobulin-like receptors (KIR) and/or killer cell lectin-like Ly49 receptors. Genes encoding highly variable NK cell receptors (NKR) represent rapidly evolving genomic regions. No single conservative model of NKR genes was observed in mammals. Single-copy low polymorphic NKR genes present in one mammalian species may expand into highly polymorphic multigene families in other species. In contrast to other non-rodent mammals, multiple Ly49-like genes appear to exist in the horse, while no functional KIR genes were observed in this species. In this study, Ly49 and KIR were sought and their evolution was characterized in the entire family Equidae. Genomic sequences retrieved showed the presence of at least five highly conserved polymorphic Ly49 genes in horses, asses and zebras. These findings confirmed that the expansion of Ly49 occurred in the entire family. Several KIR-like sequences were also identified in the genome of Equids. Besides a previously identified non-functional KIR-Immunoglobulin-like transcript fusion gene (KIR-ILTA) and two putative pseudogenes, a KIR3DL-like sequence was analyzed. In contrast to previous observations made in the horse, the KIR3DL sequence, genomic organization and mRNA expression suggest that all Equids might produce a functional KIR receptor protein molecule with a single non-mutated immune tyrosine-based inhibition motif (ITIM) domain. No evidence for positive selection in the KIR3DL gene was found. Phylogenetic analysis including rhinoceros and tapir genomic DNA and deduced amino acid KIR-related sequences showed differences between families and even between species within the order Perissodactyla. The results suggest that the order Perissodactyla and its family Equidae with expanded Ly49 genes and with a potentially functional KIR gene may represent an interesting model for evolutionary biology of NKR genes.
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Ligand-targeted theranostic nanomedicines against cancer
DOE Office of Scientific and Technical Information (OSTI.GOV)
Yao, Virginia J.; D'Angelo, Sara; Butler, Kimberly S.
Nanomedicines have significant potential for cancer treatment. Although the majority of nanomedicines currently tested in clinical trials utilize simple, biocompatible liposome-based nanocarriers, their widespread use is limited by non-specificity and low target site concentration and thus, do not provide a substantial clinical advantage over conventional, systemic chemotherapy. In the past 20 years, we have identified specific receptors expressed on the surfaces of tumor endothelial and perivascular cells, tumor cells, the extracellular matrix and stromal cells using combinatorial peptide libraries displayed on bacteriophage. These studies corroborate the notion that unique receptor proteins such as IL-11Rα, GRP78, EphA5, among others, are differentiallymore » overexpressed in tumors and present opportunities to deliver tumor-specific therapeutic drugs. By using peptides that bind to tumor-specific cell-surface receptors, therapeutic agents such as apoptotic peptides, suicide genes, imaging dyes or chemotherapeutics can be precisely and systemically delivered to reduce tumor growth in vivo, without harming healthy cells. Given the clinical applicability of peptide-based therapeutics, targeted delivery of nanocarriers loaded with therapeutic cargos seems plausible. We propose a modular design of a functionalized protocell in which a tumor-targeting moiety, such as a peptide or recombinant human antibody single chain variable fragment (scFv), is conjugated to a lipid bilayer surrounding a silica-based nanocarrier core containing a protected therapeutic cargo. The functionalized protocell can be tailored to a specific cancer subtype and treatment regimen by exchanging the tumor-targeting moiety and/or therapeutic cargo or used in combination to create unique, theranostic agents. In this review, we summarize the identification of tumor-specific receptors through combinatorial phage display technology and the use of antibody display selection to identify recombinant human scFvs against these tumor-specific receptors. We compare the characteristics of different types of simple and complex nanocarriers, and discuss potential types of therapeutic cargos and conjugation strategies. As a result, the modular design of functionalized protocells may improve the efficacy and safety of nanomedicines for future cancer therapy.« less
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Natural Killer Cell Receptor Genes in the Family Equidae: Not only Ly49
Futas, Jan; Horin, Petr
2013-01-01
Natural killer (NK) cells have important functions in immunity. NK recognition in mammals can be mediated through killer cell immunoglobulin-like receptors (KIR) and/or killer cell lectin-like Ly49 receptors. Genes encoding highly variable NK cell receptors (NKR) represent rapidly evolving genomic regions. No single conservative model of NKR genes was observed in mammals. Single-copy low polymorphic NKR genes present in one mammalian species may expand into highly polymorphic multigene families in other species. In contrast to other non-rodent mammals, multiple Ly49-like genes appear to exist in the horse, while no functional KIR genes were observed in this species. In this study, Ly49 and KIR were sought and their evolution was characterized in the entire family Equidae. Genomic sequences retrieved showed the presence of at least five highly conserved polymorphic Ly49 genes in horses, asses and zebras. These findings confirmed that the expansion of Ly49 occurred in the entire family. Several KIR-like sequences were also identified in the genome of Equids. Besides a previously identified non-functional KIR-Immunoglobulin-like transcript fusion gene (KIR-ILTA) and two putative pseudogenes, a KIR3DL-like sequence was analyzed. In contrast to previous observations made in the horse, the KIR3DL sequence, genomic organization and mRNA expression suggest that all Equids might produce a functional KIR receptor protein molecule with a single non-mutated immune tyrosine-based inhibition motif (ITIM) domain. No evidence for positive selection in the KIR3DL gene was found. Phylogenetic analysis including rhinoceros and tapir genomic DNA and deduced amino acid KIR-related sequences showed differences between families and even between species within the order Perissodactyla. The results suggest that the order Perissodactyla and its family Equidae with expanded Ly49 genes and with a potentially functional KIR gene may represent an interesting model for evolutionary biology of NKR genes. PMID:23724088
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Morphology of the olfactory system in the predatory mite Phytoseiulus persimilis.
van Wijk, Michiel; Wadman, Wytse J; Sabelis, Maurice W
2006-01-01
The predatory mite Phytoseiulus persimilis locates its prey, the two-spotted spider mite, by means of herbivore-induced plant volatiles. The olfactory response to this quantitatively and qualitatively variable source of information is particularly well documented. The mites perform this task with a peripheral olfactory system that consists of just five putative olfactory sensilla that reside in a dorsal field at the tip of their first pair of legs. The receptor cells innervate a glomerular olfactory lobe just ventral of the first pedal ganglion. We have made a 3D reconstruction of the caudal half of the olfactory lobe in adult females. The glomerular organization as well as the glomerular innervation appears conserved across different individuals. The adult females have, by approximation, a 1:1 ratio of olfactory receptor cells to olfactory glomeruli.
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Central muscarinic cholinergic regulation of the systemic inflammatory response during endotoxemia
Pavlov, Valentin A.; Ochani, Mahendar; Gallowitsch-Puerta, Margot; Ochani, Kanta; Huston, Jared M.; Czura, Christopher J.; Al-Abed, Yousef; Tracey, Kevin J.
2006-01-01
TNF has a critical mediator role in inflammation and is an important therapeutic target. We recently discovered that TNF production is regulated by neural signals through the vagus nerve. Activation of this “cholinergic antiinflammatory pathway” inhibits the production of TNF and other cytokines and protects animals from the inflammatory damage caused by endotoxemia and severe sepsis. Here, we describe a role for central muscarinic acetylcholine receptors in the activation of the cholinergic antiinflammatory pathway. Central muscarinic cholinergic activation by muscarine, the M1 receptor agonist McN-A-343, and the M2 receptor antagonist methoctramine inhibited serum TNF levels significantly during endotoxemia. Centrally administered methoctramine stimulated vagus-nerve activity measured by changes in instantaneous heart-rate variability. Blockade of peripheral muscarinic receptors did not abolish antiinflammatory signaling through the vagus nerve, indicating that peripheral muscarinic receptors on immune cells are not required for the cytokine-regulating activities of the cholinergic antiinflammatory pathway. The role of central muscarinic receptors in activating the cholinergic antiinflammatory pathway is of interest for the use of centrally acting muscarinic cholinergic enhancers as antiinflammatory agents. PMID:16549778
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Mraz, Marek; Chen, Liguang; Rassenti, Laura Z.; Ghia, Emanuela M.; Li, Hongying; Jepsen, Kristen; Smith, Erin N.; Messer, Karen; Frazer, Kelly A.; Kipps, Thomas J.
2014-01-01
We examined the microRNAs (miRNAs) expressed in chronic lymphocytic leukemia (CLL) and identified miR-150 as the most abundant, but with leukemia cell expression levels that varied among patients. CLL cells that expressed ζ-chain–associated protein of 70 kDa (ZAP-70) or that used unmutated immunoglobulin heavy chain variable (IGHV) genes, each had a median expression level of miR-150 that was significantly lower than that of ZAP-70–negative CLL cells or those that used mutated IGHV genes. In samples stratified for expression of miR-150, CLL cells with low-level miR-150 expressed relatively higher levels of forkhead box P1 (FOXP1) and GRB2-associated binding protein 1 (GAB1), genes with 3′ untranslated regions having evolutionary-conserved binding sites for miR-150. High-level expression of miR-150 could repress expression of these genes, which encode proteins that enhance B-cell receptor signaling, a putative CLL-growth/survival signal. Also, high-level expression of miR-150 was a significant independent predictor of longer treatment-free survival or overall survival, whereas an inverse association was observed for high-level expression of GAB1 or FOXP1 for overall survival. This study demonstrates that expression of miR-150 can influence the relative expression of GAB1 and FOXP1 and the signaling potential of the B-cell receptor, thereby possibly accounting for the noted association of expression of miR-150 and disease outcome. PMID:24787006
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Mraz, Marek; Chen, Liguang; Rassenti, Laura Z; Ghia, Emanuela M; Li, Hongying; Jepsen, Kristen; Smith, Erin N; Messer, Karen; Frazer, Kelly A; Kipps, Thomas J
2014-07-03
We examined the microRNAs (miRNAs) expressed in chronic lymphocytic leukemia (CLL) and identified miR-150 as the most abundant, but with leukemia cell expression levels that varied among patients. CLL cells that expressed ζ-chain-associated protein of 70 kDa (ZAP-70) or that used unmutated immunoglobulin heavy chain variable (IGHV) genes, each had a median expression level of miR-150 that was significantly lower than that of ZAP-70-negative CLL cells or those that used mutated IGHV genes. In samples stratified for expression of miR-150, CLL cells with low-level miR-150 expressed relatively higher levels of forkhead box P1 (FOXP1) and GRB2-associated binding protein 1 (GAB1), genes with 3' untranslated regions having evolutionary-conserved binding sites for miR-150. High-level expression of miR-150 could repress expression of these genes, which encode proteins that enhance B-cell receptor signaling, a putative CLL-growth/survival signal. Also, high-level expression of miR-150 was a significant independent predictor of longer treatment-free survival or overall survival, whereas an inverse association was observed for high-level expression of GAB1 or FOXP1 for overall survival. This study demonstrates that expression of miR-150 can influence the relative expression of GAB1 and FOXP1 and the signaling potential of the B-cell receptor, thereby possibly accounting for the noted association of expression of miR-150 and disease outcome. © 2014 by The American Society of Hematology.
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Maghazachi, Azzam A
2005-09-01
Human natural killer (NK) cells are important cells of the innate immune system. These cells perform two prominent functions: the first is recognizing and destroying virally infected cells and transformed cells; the second is secreting various cytokines that shape up the innate and adaptive immune re-sponses. For these cells to perform these activities, they express different sets of receptors. The receptors used by NK cells to extravasate into sites of injury belong to the seven transmembrane (7TM) family of receptors, which characteristically bind heterotrimeric G proteins. These receptors allow NK cells to sense the chemotactic gradients and activate second messengers, which aid NK cells in polarizing and migrating toward the sites of injured tissues. In addition, these receptors determine how and why human resting NK cells are mainly found in the bloodstream, whereas activated NK cells extravasate into inflammatory sites. Receptors for chemokines and lysophospholipids belong to the 7TM family. On the other hand, NK cells recognize invading or transformed cells through another set of receptors that belong to the single transmembrane-spanning domain family. These receptors are either inhibitory or activating. Inhibitory receptors contain the immune receptor tyrosine-based inhibitory motif, and activating receptors belong to either those that associate with adaptor molecules containing the immune receptor tyrosine-based activating motif (ITAM) or those that associate with adaptor molecules containing motifs other than ITAM. This article will describe the nature of these receptors and examine the intracellular signaling pathways induced in NK cells after ligating both types of receptors. These pathways are crucial for NK cell biology, development, and functions.
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CD10/NEP in non-small cell lung carcinomas. Relationship to cellular proliferation.
Ganju, R K; Sunday, M; Tsarwhas, D G; Card, A; Shipp, M A
1994-01-01
The cell surface metalloproteinase CD10/neutral endopeptidase 24.11 (NEP) hydrolyzes a variety of peptide substrates and reduces cellular responses to specific peptide hormones. Because CD10/NEP modulates peptide-mediated proliferation of small cell carcinomas of the lung (SCLC) and normal fetal bronchial epithelium, we evaluated the enzyme's expression in non-small cell lung carcinomas (NSCLC). Bronchoalveolar and large cell carcinoma cell lines had low levels of CD10/NEP expression whereas squamous, adenosquamous, and adenocarcinoma cell lines had higher and more variable levels of the cell surface enzyme. Regional variations in CD10/NEP immunostaining in primary NSCLC specimens prompted us to correlate CD10/NEP expression with cell growth. In primary carcinomas of the lung, clonal NSCLC cell lines and SV40-transformed fetal airway epithelium, subsets of cells expressed primarily CD10/NEP or the proliferating cell nuclear antigen (PCNA). Cultured airway epithelial cells had the lowest levels of CD10/NEP expression when the highest percentage of cells were actively dividing; in addition, these cells grew more rapidly when cell surface CD10/NEP was inhibited. NSCLC cell lines had receptors for a variety of mitogenic peptides known to be CD10/NEP substrates, underscoring the functional significance of growth-related variability in CD10/NEP expression. Images PMID:7962523
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Schmetterer, Klaus G; Haiderer, Daniela; Leb-Reichl, Victoria M; Neunkirchner, Alina; Jahn-Schmid, Beatrice; Küng, Hans J; Schuch, Karina; Steinberger, Peter; Bohle, Barbara; Pickl, Winfried F
2011-01-01
Regulatory T (Treg) cells establish and maintain tolerance to self-antigens and many foreign antigens, such as allergens, by suppressing effector T-cell proliferation and function. We have previously shown that human T-cell receptor (TCR) αβ-chains specific for allergen-derived epitopes confer allergen specificity on peripheral blood T cells of individuals with and without allergy. To study the feasibility of generating allergen-specific human Treg cells by retroviral transduction of a transcription unit encoding forkhead box protein 3 (FOXP3) and allergen-specific TCR αβ-chains. cDNAs encoding the α and β-chains of a Bet v 1(142-153)-specific TCR (TCR alpha variable region 6/TCR beta variable region 20) and human FOXP3 were linked via picornaviral 2A sequences and expressed as single translational unit from an internal ribosomal entry site-green fluorescence protein-containing retroviral vector. Retrovirally transduced peripheral blood T cells were tested for expression of transgenes, Treg phenotype, and regulatory capacity toward allergen-specific effector T cells. Transduced T cells displayed a Treg phenotype with clear-cut upregulation of CD25, CD39, and cytotoxic T-lymphocyte antigen 4. The transduced cells were hyporesponsive in cytokine production and secretion and, like naturally occurring Treg cells, did not proliferate after antigen-specific or antigen-mimetic stimulation. However, proliferation was inducible upon exposure to exogenous IL-2. In coculture experiments, TRAV6(+)TRBV20(+)FOXP3(+) transgenic T cells, unlike FOXP3(+) single transgenic T cells or naturally occurring Treg cells, highly significantly suppressed T cell cytokine production and proliferation of corresponding allergen-specific effector T cells in an allergen-specific, dose-dependent manner. We demonstrate a transgenic approach to engineer human allergen-specific Treg cells that exert their regulatory function in an activation-dependent manner. Customized Treg cells might become useful for tolerance induction therapies in individuals with allergic and other immune-mediated diseases. Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.
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BINDING OF SOLUBLE IMMUNE COMPLEXES TO HUMAN LYMPHOBLASTOID CELLS
Theofilopoulos, Argyrios N.; Dixon, Frank J.; Bokisch, Viktor A.
1974-01-01
In the present work we studied the expression of membrane-bound Ig (MBIg) as well as receptors for IgG Fc and complement on nine human lymphoblastoid cell lines. When MBIg and receptors for IgG Fc were compared, four categories of cell lines could be distinguished: (a) cell lines having both MBIg and receptors for IgG Fc, (b) cell lines having MBIg but lacking receptors for IgG Fc, (c) cell lines lacking MBIg but having receptors for IgG Fc, and (d) cell lines lacking both MBIg and receptors for IgG Fc. Two types of receptors for complement could be detected on the cell lines studied, one for C3-C3b and one for C3d. When sensitized red cells carrying C3b or C3d were used for rosette tests, three categories of cell lines could be distinguished: (a) cell lines having receptors for C3b and C3d, (b) cell lines having receptors only for C3d and (c) cell lines lacking both receptors. However, when a more sensitive immunofluorescent method was used instead of the rosette technique, it was found that cell lines unable to form rosettes with EAC1423bhu were able to bind soluble C3 or C3b which indicated the presence of these receptors on the cell surface. Inhibition experiments showed that receptors for C3-C3b and receptors for C3d are distinct and that receptors for C3-C3b and C3d are different from receptors for IgG Fc. A cell line (Raji) without MBIg but with receptors for IgG Fc, C3-C3b, and C3d was selected for use in studying the binding mechanism of soluble immune complexes to cell surface membrane. Aggregated human gamma globulin was used in place of immune complexes. Immune complexes containing complement bind to Raji cells only via receptors for complement, namely receptors for C3-C3b and C3d. Binding of immune complexes containing complement to cells is much greater than that of complexes without complement. Immune complexes bound to cells via receptors for complement can be partially released from the cell surface by addition of normal human serum as well as isolated human C3 or C3b. We postulate that such release is due to competition of immune complex bound C3b and free C3 or C3b for the receptors on Raji cells. PMID:4139225
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Cyclic AMP-receptor proteins in heart muscle of rats flown on Cosmos 1887
NASA Technical Reports Server (NTRS)
Mednieks, Maija I.; Popova, Irina A.; Grindeland, Richard E.
1991-01-01
The cellular compartmentalization of the cyclic AMP-receptor proteins in heart ventricular tissue obtained from rats flown on the Cosmos 1887 is determined. Photoaffinity labeling of soluble and particular cell fractions with a (32P)-8-azido analog of cyclic AMP is followed by electrophoretic separation of the proteins and by autoradiographic identification of the labeled isoforms of cAPK R subunits. It is shown that RII in the particulate subcellular fraction was significantly decreased in heart cells from rats in the flight group when compared to controls. Protein banding patterns in both the cytoplasmic fraction and in a fraction enriched in chromatin-bound proteins exhibited some variability in tissues of individual animals, but showed no changes that could be directly attributed to flight conditions. No significant change was apparent in the distribution of RI or RII cyclic AMP binding in the soluble fractions. It is inferred that the cardiac cell integrity or its protein content is not compromised under flight conditions.
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A semi high-throughput method for screening small bispecific antibodies with high cytotoxicity.
Sugiyama, Aruto; Umetsu, Mitsuo; Nakazawa, Hikaru; Niide, Teppei; Onodera, Tomoko; Hosokawa, Katsuhiro; Hattori, Shuhei; Asano, Ryutaro; Kumagai, Izumi
2017-06-06
Small bispecific antibodies that induce T-cell-mediated cytotoxicity have the potential to damage late-stage tumor masses to a clinically relevant degree, but their cytotoxicity is critically dependent on their structural and functional properties. Here, we constructed an optimized procedure for identifying highly cytotoxic antibodies from a variety of the T-cell-recruiting antibodies engineered from a series of antibodies against cancer antigens of epidermal growth factor receptor family and T-cell receptors. By developing and applying a set of rapid operations for expression vector construction and protein preparation, we screened the cytotoxicity of 104 small antibodies with diabody format and identified some with 10 3 -times higher cytotoxicity than that of previously reported active diabody. The results demonstrate that cytotoxicity is enhanced by synergistic effects between the target, epitope, binding affinity, and the order of heavy-chain and light-chain variable domains. We demonstrate the importance of screening to determine the critical rules for highly cytotoxic antibodies.
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Kipps, Thomas J.; Stevenson, Freda K.; Wu, Catherine J.; Croce, Carlo M.; Packham, Graham; Wierda, William G.; O’Brien, Susan; Gribben, John; Rai, Kanti
2017-01-01
Chronic lymphocytic leukaemia (CLL) is a malignancy of CD5+ B cells that is characterized by the accumulation of small, mature-appearing lymphocytes in the blood, marrow and lymphoid tissues. Signalling via surface immunoglobulin, which constitutes the major part of the B cell receptor, and several genetic alterations play a part in CLL pathogenesis, in addition to interactions between CLL cells and other cell types, such as stromal cells, T cells and nurse-like cells in the lymph nodes. The clinical progression of CLL is heterogeneous and ranges from patients who require treatment soon after diagnosis to others who do not require therapy for many years, if at all. Several factors, including the immunoglobulin heavy-chain variable region gene (IGHV) mutational status, genomic changes, patient age and the presence of comorbidities, should be considered when defining the optimal management strategies, which include chemotherapy, chemoimmunotherapy and/or drugs targeting B cell receptor signalling or inhibitors of apoptosis, such as BCL-2. Research on the biology of CLL has profoundly enhanced our ability to identify patients who are at higher risk for disease progression and our capacity to treat patients with drugs that selectively target distinctive phenotypic or physiological features of CLL. How these and other advances have shaped our current understanding and treatment of patients with CLL is the subject of this Primer. PMID:28102226
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Hexamethonium sensitivity of the swim musculature of the pteropod mollusc, Clione limacina.
Satterlie, Richard A; Courtney, Christopher
2008-12-01
Swimming in reduced electrophysiological preparations of the pteropod mollusc, Clione limacina, was blocked by bath application of hexamethonium even though pattern generator activity continued with this treatment. Neuromuscular recordings indicated that hexamethonium blocked synaptic input from Pd-3 and Pd-4 motoneurons to slow-twitch muscle cells, while connections from Pd-1A and Pd-2A motoneurons to fast-twitch muscle cells were variable in their response to hexamethonium-synaptic inputs were suppressed in most cases and occasionally blocked, but the latter only with high concentrations and long incubations. Acutely dissociated wing muscle cells showed a concentration-dependency in the percentage of contracted cells with bath application of acetylcholine, and this contractile activity was blocked in preparations that were first bathed in hexamethonium. Intracellular recordings from dissociated slow-twitch muscle cells showed conductance-increase depolarizations of approximately 20 mV following 1 s pressure ejections of 10(-4) M acetylcholine from micropipettes placed immediately adjacent to the muscle cells. These responses were blocked when hexamethonium was bath applied prior to the pressure-applied acetylcholine. The results suggest the Pd-3/Pd-4 motoneuron to slow-twitch muscle cell junctions are cholinergic with nicotinic-like receptors, while the Pd-1A/Pd-2A to fast-twitch muscle cell connections are likely cholinergic, but with a different receptor type.
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Shaheen, Safa; Fawaz, Febin; Shah, Shaheen; Büsselberg, Dietrich
2018-06-19
Triple-negative breast cancer (TNBC) is among the most notorious types of breast cancer, the treatment of which does not give consistent results due to the absence of the three receptors (estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) as well as high amount of molecular variability. Drug resistance also contributes to treatment unresponsiveness. We studied differentially expressed genes, their biological roles, as well as pathways from RNA-Seq datasets of two different TNBC drug-resistant cell lines of Basal B subtype SUM159 and MDA-MB-231 treated with drugs JQ1 and Dexamethasone, respectively, to elucidate the mechanism of drug resistance. RNA sequencing(RNA-Seq) data analysis was done using edgeR which is an efficient program for determining the most significant Differentially Expressed Genes (DEGs), Gene Ontology (GO) terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. iPathway analysis was further used to obtain validated results using analysis that takes into consideration type, function, and interactions of genes in the pathway. The significant similarities and differences throw light into the molecular heterogeneity of TNBC, giving clues into the aspects that can be focused to overcome drug resistance. From this study, cytokine-cytokine receptor interaction pathway appeared to be a key factor in TNBC drug resistance.
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Misfolded rhodopsin mutants display variable aggregation properties.
Gragg, Megan; Park, Paul S-H
2018-06-08
The largest class of rhodopsin mutations causing autosomal dominant retinitis pigmentosa (adRP) is mutations that lead to misfolding and aggregation of the receptor. The misfolding mutants have been characterized biochemically, and categorized as either partial or complete misfolding mutants. This classification is incomplete and does not provide sufficient information to fully understand the disease pathogenesis and evaluate therapeutic strategies. A Förster resonance energy transfer (FRET) method was utilized to directly assess the aggregation properties of misfolding rhodopsin mutants within the cell. Partial (P23H and P267L) and complete (G188R, H211P, and P267R) misfolding mutants were characterized to reveal variability in aggregation properties. The complete misfolding mutants all behaved similarly, forming aggregates when expressed alone, minimally interacting with the wild-type receptor when coexpressed, and were unresponsive to treatment with the pharmacological chaperone 9-cis retinal. In contrast, variability was observed between the partial misfolding mutants. In the opsin form, the P23H mutant behaved similarly as the complete misfolding mutants. In contrast, the opsin form of the P267L mutant existed as both aggregates and oligomers when expressed alone and formed mostly oligomers with the wild-type receptor when coexpressed. The partial misfolding mutants both reacted similarly to the pharmacological chaperone 9-cis retinal, displaying improved folding and oligomerization when expressed alone but aggregating with wild-type receptor when coexpressed. The observed differences in aggregation properties and effect of 9-cis retinal predict different outcomes in disease pathophysiology and suggest that retinoid-based chaperones will be ineffective or even detrimental. Copyright © 2018 Elsevier B.V. All rights reserved.
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Bhaskaran, S; Jay, C M; Berghman, L R; Wagner, G G; Waghela, S D
2005-08-01
Bovine colibacillosis caused by enterotoxigenic Escherichia coli (ETEC) is a worldwide problem. Adhesion of ETEC to intestinal cell receptors mediated by the surface protein F5 fimbriae is the initial step in the establishment of colibacillosis. Prevention of ETEC F5(+) adhesion to enterocytes protects newborn calves against collibacillosis. On the enterocytes, the F5 fimbriae bind to a ganglioside that is also found on horse red blood cells. Thus, the presence of F5 fimbriae induces haemagglutination, which is useful as an indicator in a functional assay system. In this study, recombinant anti-F5 scFv antibody fragment produced in E. coli HB2151 reacted with F5 fimbriae in ELISA and Western immunoblot, and prevented haemagglutination induced by the binding of the F5 fimbriae to its natural host receptors on horse red blood cells. Given the ease with which recombinant antibodies can be mass-produced, the presently described scFv may hold promise as a prophylactic agent for colibacillosis.
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Neutrophil formyl-peptide receptors. Relationship to peptide-induced responses and emphysema.
Stockley, R A; Grant, R A; Llewellyn-Jones, C G; Hill, S L; Burnett, D
1994-02-01
A reproducible assay was established to assess the number of formyl-peptide receptors expressed on the surface of human polymorphonuclear leukocytes (PMN). Using this assay the number of receptors was shown to demonstrate wide within- and between-subject variability. However, the receptor numbers were related to the chemotactic response (r = 0.572) and degranulation response (r = 0.512) to the peptide formyl-methionyl-leucyl-phenylalanine. Subsequent studies showed increased receptor numbers on PMN from patients with emphysema (median, 459 x 10(3)/cell; range, 207 to 1,080) as compared with age-matched control subjects (median, 288; range, 168 to 519; p < 0.02), which may explain the increased chemotactic response of the PMN to formyl peptides. This difference was not observed in patients with bronchiectasis, suggesting that the increased receptor number is a feature of emphysema. Furthermore, the increase was largely a feature of smokers with emphysema (median, 463; range, 362 to 1,080), whereas age-matched smokers without emphysema had lower numbers of receptors (p < 0.001; median, 332; range, 243 to 411). This observation suggests a mechanism that may explain the susceptibility of some smokers to the development of emphysema.
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Expansion of inflammatory innate lymphoid cells in patients with common variable immune deficiency.
Cols, Montserrat; Rahman, Adeeb; Maglione, Paul J; Garcia-Carmona, Yolanda; Simchoni, Noa; Ko, Huai-Bin M; Radigan, Lin; Cerutti, Andrea; Blankenship, Derek; Pascual, Virginia; Cunningham-Rundles, Charlotte
2016-04-01
Common variable immunodeficiency (CVID) is an antibody deficiency treated with immunoglobulin; however, patients can have noninfectious inflammatory conditions that lead to heightened morbidity and mortality. Modular analyses of RNA transcripts in whole blood previously identified an upregulation of many interferon-responsive genes. In this study we sought the cell populations leading to this signature. Lymphoid cells were measured in peripheral blood of 55 patients with CVID (31 with and 24 without inflammatory/autoimmune complications) by using mass cytometry and flow cytometry. Surface markers, cytokines, and transcriptional characteristics of sorted innate lymphoid cells (ILCs) were defined by using quantitative PCR. Gastrointestinal and lung biopsy specimens of subjects with inflammatory disease were stained to seek ILCs in tissues. The linage-negative, CD127(+), CD161(+) lymphoid population containing T-box transcription factor, retinoic acid-related orphan receptor (ROR) γt, IFN-γ, IL-17A, and IL-22, all hallmarks of type 3 innate lymphoid cells, were expanded in the blood of patients with CVID with inflammatory conditions (mean, 3.7% of PBMCs). ILCs contained detectable amounts of the transcription factors inhibitor of DNA binding 2, T-box transcription factor, and RORγt and increased mRNA transcripts for IL-23 receptor (IL-23R) and IL-26, demonstrating inflammatory potential. In gastrointestinal and lung biopsy tissues of patients with CVID, numerous IFN-γ(+)RORγt(+)CD3(-) cells were identified, suggesting a role in these mucosal inflammatory states. An expansion of this highly inflammatory ILC population is a characteristic of patients with CVID with inflammatory disease; ILCs and the interferon signature are markers for the uncontrolled inflammatory state in these patients. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
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Carbachol dimers as homobivalent modulators of muscarinic receptors.
Matucci, Rosanna; Nesi, Marta; Martino, Maria Vittoria; Bellucci, Cristina; Manetti, Dina; Ciuti, Elisa; Mazzolari, Angelica; Dei, Silvia; Guandalini, Luca; Teodori, Elisabetta; Vistoli, Giulio; Romanelli, Maria Novella
2016-05-15
A series of homodimers of the well-known cholinergic agonist carbachol have been synthesized, showing the two agonist units symmetrically connected through a methylene chain of variable length. The new compounds have been tested on the five cloned muscarinic receptors (hM1-5) expressed in CHO cells by means of equilibrium binding studies, showing an increase in affinity by rising the number of methylene units up to 7 and 9. Functional experiments on guinea-pig ileum and assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 on CHO cells have shown that the new compounds are endowed with muscarinic antagonistic properties. Kinetic binding studies have revealed that some of the tested compounds are able to slow the rate of dissociation of NMS, suggesting a bitopic behavior. Docking simulations, performed on the hM1 and hM2 receptors, give a sound rationalization of the experimental data revealing how these compounds are able to interact with both orthosteric and allosteric binding sites depending on the length of their connecting chain. Copyright © 2016 Elsevier Inc. All rights reserved.
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Xie, Jiasen; Zhou, Zishan; Jiao, Shunchang; Li, Xiaokun
2018-01-01
A chimeric antigen receptor (CAR) is a type of fusion protein that comprises an antigen-recognition domain and signaling domains. In the present study, a programmed death-ligand 1 (PD-L1)-specific CAR, comprised of a single-chain variable fragment (scFv) derived from a monoclonal antibody, co-stimulatory domains of cluster of differentiation (CD) 28 and 4-1BB and a T-cell-activation domain derived from CD3ζ, was designed. The construction was cloned and packaged into the lentiviral vector pLVX. Flow cytometry confirmed that peripheral blood mononuclear cells were efficiently transduced and that the CAR was successfully expressed on T cells. The cytotoxicity of transduced T cells was detected using PD-L1-positive NCI-H358 bronchioalveolar carcinoma cells and A549 lung adenocarcinoma cells (with a low expression of PD-L1, only in the A549 cells). The results demonstrated mild cytotoxicity at an effector-to-target ratio of 10:1. An ELISA revealed a significant increase in the level of interferon-γ released from T cells transduced with scFv-28Bz when the cells were co-cultured with PD-L1-positive NCI-H358 cells, while interkeukin-2 and tumor necrosis factor-α levels remained unchanged. These data indicated a potential method for the treatment of solid tumors. PMID:29928397
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Anti-proliferative and gene expression actions of resveratrol in breast cancer cells in vitro
Yang, Sheng-Huei; Tsai, Po-Wei; Wang, Shwu-Huey; Wang, Ching-Chiung; Lee, Yee-Shin; Cheng, Guei-Yun; HuangFu, Wei-Chun; London, David; Tang, Heng-Yuan; Fu, Earl; Yen, Yun; Liu, Leroy F.; Lin, Hung-Yun; Davis, Paul J.
2014-01-01
We have used a perfusion bellows cell culture system to investigate resveratrolinduced anti-proliferation/apoptosis in a human estrogen receptor (ER)-negative breast cancer cell line (MDA-MB-231). Using an injection system to perfuse media with stilbene, we showed resveratrol (0.5 – 100 μM) to decrease cell proliferation in a concentration-dependent manner. Comparison of influx and medium efflux resveratrol concentrations revealed rapid disappearance of the stilbene, consistent with cell uptake and metabolism of the agent reported by others. Exposure of cells to 10 μM resveratrol for 4 h daily × 6 d inhibited cell proliferation by more than 60%. Variable extracellular acid-alkaline conditions (pH 6.8 – 8.6) affected basal cell proliferation rate, but did not alter anti-proliferation induced by resveratrol. Resveratrol-induced gene expression, including transcription of the most up-regulated genes and pro-apoptotic p53-dependent genes, was not affected by culture pH changes. The microarray findings in the context of induction of anti-proliferation with brief daily exposure of cells to resveratrol—and rapid disappearance of the compound in the perfusion system—are consistent with existence of an accessible initiation site for resveratrol actions on tumor cells, e.g., the cell surface receptor for resveratrol described on integrin αvβ3. PMID:25436977
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Global Manufacturing of CAR T Cell Therapy.
Levine, Bruce L; Miskin, James; Wonnacott, Keith; Keir, Christopher
2017-03-17
Immunotherapy using chimeric antigen receptor-modified T cells has demonstrated high response rates in patients with B cell malignancies, and chimeric antigen receptor T cell therapy is now being investigated in several hematologic and solid tumor types. Chimeric antigen receptor T cells are generated by removing T cells from a patient's blood and engineering the cells to express the chimeric antigen receptor, which reprograms the T cells to target tumor cells. As chimeric antigen receptor T cell therapy moves into later-phase clinical trials and becomes an option for more patients, compliance of the chimeric antigen receptor T cell manufacturing process with global regulatory requirements becomes a topic for extensive discussion. Additionally, the challenges of taking a chimeric antigen receptor T cell manufacturing process from a single institution to a large-scale multi-site manufacturing center must be addressed. We have anticipated such concerns in our experience with the CD19 chimeric antigen receptor T cell therapy CTL019. In this review, we discuss steps involved in the cell processing of the technology, including the use of an optimal vector for consistent cell processing, along with addressing the challenges of expanding chimeric antigen receptor T cell therapy to a global patient population.
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A novel pair of immunoglobulin-like receptors expressed by B cells and myeloid cells
Kubagawa, Hiromi; Burrows, Peter D.; Cooper, Max D.
1997-01-01
An Fcα receptor probe of human origin was used to identify novel members of the Ig gene superfamily in mice. Paired Ig-like receptors, named PIR-A and PIR-B, are predicted from sequence analysis of the cDNAs isolated from a mouse splenic library. Both type I transmembrane proteins possess similar ectodomains with six Ig-like loops, but have different transmembrane and cytoplasmic regions. The predicted PIR-A protein has a short cytoplasmic tail and a charged Arg residue in the transmembrane region that, by analogy with the FcαR relative, suggests the potential for association with an additional transmembrane protein to form a signal transducing unit. In contrast, the PIR-B protein has an uncharged transmembrane region and a long cytoplasmic tail containing four potential immunoreceptor tyrosine-based inhibitory motifs. These features are shared by the related killer inhibitory receptors. PIR-A proteins appear to be highly variable, in that predicted peptide sequences differ for seven randomly selected PIR-A clones, whereas PIR-B cDNA clones are invariant. Southern blot analysis with PIR-B and PIR-A-specific probes suggests only one PIR-B gene and multiple PIR-A genes. The PIR-A and PIR-B genes are expressed in B lymphocytes and myeloid lineage cells, wherein both are expressed simultaneously. The characteristics of the highly-conserved PIR-A and PIR-B genes and their coordinate cellular expression suggest a potential regulatory role in humoral, inflammatory, and allergic responses. PMID:9144225
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López-Botet, Miguel; Muntasell, Aura; Vilches, Carlos
2014-04-01
Human cytomegalovirus (HCMV) causes a highly prevalent and lifelong infection, with a multifaceted impact in human health. NK cells play an important role in the immune response to HCMV and the virus has reciprocally developed a variety of immune evasion strategies. We originally reported that HCMV infection promotes, to a variable degree in healthy individuals, a redistribution of the NK-cell receptor (NKR) repertoire which persists under steady-state conditions. Its hallmark is an expansion of a mature NK-cell subset displaying high surface levels of the CD94/NKG2C activating receptor, with additional distinctive phenotypic and functional features. Such adaptation of host NK cells to HCMV infection, confirmed in different clinical settings, is particularly magnified in immunocompromised patients and influenced by NKG2C gene copy number. The mechanism(s) underlying the differentiation and proliferation of NKG2C+ NK cells, the basis for the individual differences in the magnitude of their expansion, and their precise role in anti-viral defence remain open issues. Moreover, the possibility that the impact of HCMV infection on the NK-cell compartment may exert a broader influence on immunity deserves further attention. Copyright © 2014 Elsevier Ltd. All rights reserved.
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De Vita, Serena; Schneider, Rebekka K.; Garcia, Michael; Wood, Jenna; Gavillet, Mathilde; Ebert, Benjamin L.; Gerbaulet, Alexander; Roers, Axel; Levine, Ross L.; Mullally, Ann; Williams, David A.
2014-01-01
Systemic Mastocytosis (SM) is a clonal disease characterized by abnormal accumulation of mast cells in multiple organs. Clinical presentations of the disease vary widely from indolent to aggressive forms, and to the exceedingly rare mast cell leukemia. Current treatment of aggressive SM and mast cell leukemia is unsatisfactory. An imatinib-resistant activating mutation of the receptor tyrosine kinase KIT (KIT D816V) is most frequently present in transformed mast cells and is associated with all clinical forms of the disease. Thus the etiology of the variable clinical aggressiveness of abnormal mast cells in SM is unclear. TET2 appears to be mutated in primary human samples in aggressive types of SM, suggesting a possible role in disease modification. In this report, we demonstrate the cooperation between KIT D816V and loss of function of TET2 in mast cell transformation and demonstrate a more aggressive phenotype in a murine model of SM when both mutations are present in progenitor cells. We exploit these findings to validate a combination treatment strategy targeting the epigenetic deregulation caused by loss of TET2 and the constitutively active KIT receptor for the treatment of patients with aggressive SM. PMID:24788138
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Ishida, Sachiko; Matsu-ura, Toru; Fukami, Kiyoko; Michikawa, Takayuki; Mikoshiba, Katsuhiko
2014-01-01
A uniform extracellular stimulus triggers cell-specific patterns of Ca2+ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca2+ oscillations in terms of the time constant of Ca2+ spike amplitude decay and the Ca2+ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca2+ signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca2+ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca2+ oscillations, such as the time constant of the temporal changes in the Ca2+ spike amplitude and the Ca2+ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca2+ release, can cause cell-to-cell variability in the patterns of Ca2+ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca2+ signals evoked by G protein-coupled receptor stimulation. PMID:24475116
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Rohani, Nazanin; Parmeggiani, Andrea; Winklbauer, Rudolf; Fagotto, François
2014-01-01
Ephrins and Eph receptors are involved in the establishment of vertebrate tissue boundaries. The complexity of the system is puzzling, however in many instances, tissues express multiple ephrins and Ephs on both sides of the boundary, a situation that should in principle cause repulsion between cells within each tissue. Although co-expression of ephrins and Eph receptors is widespread in embryonic tissues, neurons, and cancer cells, it is still unresolved how the respective signals are integrated into a coherent output. We present a simple explanation for the confinement of repulsion to the tissue interface: Using the dorsal ectoderm–mesoderm boundary of the Xenopus embryo as a model, we identify selective functional interactions between ephrin–Eph pairs that are expressed in partial complementary patterns. The combined repulsive signals add up to be strongest across the boundary, where they reach sufficient intensity to trigger cell detachments. The process can be largely explained using a simple model based exclusively on relative ephrin and Eph concentrations and binding affinities. We generalize these findings for the ventral ectoderm–mesoderm boundary and the notochord boundary, both of which appear to function on the same principles. These results provide a paradigm for how developmental systems may integrate multiple cues to generate discrete local outcomes. PMID:25247423
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Gacerez, Albert T; Hua, Casey K; Ackerman, Margaret E; Sentman, Charles L
2018-05-01
B7H6 is emerging as a promising tumor antigen that is known to be expressed on a wide array of tumors and is reported to stimulate anti-tumor responses from the immune system. As such, B7H6 presents a good target for tumor-specific immunotherapies. B7H6-specific chimeric antigen receptors (CAR) based on a murine antibody showed successful targeting and elimination of tumors expressing B7H6. However, mouse single chain variable fragments (scFvs) have the potential to induce host anti-CAR responses that may limit efficacy, so human scFvs specific for B7H6 were selected by yeast surface display. In this study, we validate the functionality of these human scFvs when formatted into chimeric antigen receptors. The data indicate that T cells expressing these B7H6-specific human scFvs as CARs induced potent anti-tumor activity in vitro and in vivo against tumors expressing high amounts of B7H6. Importantly, these human scFv-based CARs are sensitive to changes in B7H6 expression which may potentially spare non-tumor cells that express B7H6 and provides the foundation for future clinical development.
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Rohani, Nazanin; Parmeggiani, Andrea; Winklbauer, Rudolf; Fagotto, François
2014-09-01
Ephrins and Eph receptors are involved in the establishment of vertebrate tissue boundaries. The complexity of the system is puzzling, however in many instances, tissues express multiple ephrins and Ephs on both sides of the boundary, a situation that should in principle cause repulsion between cells within each tissue. Although co-expression of ephrins and Eph receptors is widespread in embryonic tissues, neurons, and cancer cells, it is still unresolved how the respective signals are integrated into a coherent output. We present a simple explanation for the confinement of repulsion to the tissue interface: Using the dorsal ectoderm-mesoderm boundary of the Xenopus embryo as a model, we identify selective functional interactions between ephrin-Eph pairs that are expressed in partial complementary patterns. The combined repulsive signals add up to be strongest across the boundary, where they reach sufficient intensity to trigger cell detachments. The process can be largely explained using a simple model based exclusively on relative ephrin and Eph concentrations and binding affinities. We generalize these findings for the ventral ectoderm-mesoderm boundary and the notochord boundary, both of which appear to function on the same principles. These results provide a paradigm for how developmental systems may integrate multiple cues to generate discrete local outcomes.
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Sarkar, Mohosin; Liu, Yun; Qi, Junpeng; Peng, Haiyong; Morimoto, Jumpei; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas
2016-04-01
Chronic lymphocytic leukemia (CLL) is a disease in which a single B-cell clone proliferates relentlessly in peripheral lymphoid organs, bone marrow, and blood. DNA sequencing experiments have shown that about 30% of CLL patients have stereotyped antigen-specific B-cell receptors (BCRs) with a high level of sequence homology in the variable domains of the heavy and light chains. These include many of the most aggressive cases that haveIGHV-unmutated BCRs whose sequences have not diverged significantly from the germ line. This suggests a personalized therapy strategy in which a toxin or immune effector function is delivered selectively to the pathogenic B-cells but not to healthy B-cells. To execute this strategy, serum-stable, drug-like compounds able to target the antigen-binding sites of most or all patients in a stereotyped subset are required. We demonstrate here the feasibility of this approach with the discovery of selective, high affinity ligands for CLL BCRs of the aggressive, stereotyped subset 7P that cross-react with the BCRs of several CLL patients in subset 7p, but not with BCRs from patients outside this subset. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Cambridge, Geraldine; Isenberg, David A.; Glennie, Martin J.; Cragg, Mark S.; Leandro, Maria
2015-01-01
Objective Rituximab, a type I anti‐CD20 monoclonal antibody (mAb), induces incomplete B cell depletion in some patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), thus contributing to a poor clinical response. The mechanisms of this resistance remain elusive. The purpose of this study was to determine whether type II mAb are more efficient than type I mAb at depleting B cells from RA and SLE patients, whether internalization influences the efficiency of depletion, and whether Fcγ receptor type IIb (FcγRIIb) and the B cell receptor regulate this internalization process. Methods We used an in vitro whole blood B cell–depletion assay to assess the efficiency of depletion, flow cytometry to study cell surface protein expression, and surface fluorescence–quenching assays to assess rituximab internalization, in samples from patients with RA and patients with SLE. Paired t‐test or Mann‐Whitney U test was used to compare groups, and Spearman's rank correlation test was used to assess correlation. Results We found that type II mAb internalized significantly less rituximab than type I mAb and depleted B cells from patients with RA and SLE at least 2‐fold more efficiently than type I mAb. Internalization of rituximab was highly variable between patients, was regulated by FcγRIIb, and inversely correlated with cytotoxicity in whole blood B cell–depletion assays. The lowest levels of internalization were seen in IgD– B cells, including postswitched (IgD–CD27+) memory cells. Internalization of type I anti‐CD20 mAb was also partially inhibited by anti‐IgM stimulation. Conclusion Variability in internalization of rituximab was observed and was correlated with impaired B cell depletion. Therefore, slower‐internalizing type II mAb should be considered as alternative B cell–depleting agents for the treatment of RA and SLE. PMID:25916583
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2018-01-01
Plants have many, highly variable resistance (R) gene loci, which provide resistance to a variety of pathogens. The first R gene to be cloned, maize (Zea mays) Hm1, was published over 25 years ago, and since then, many different R genes have been identified and isolated. The encoded proteins have provided clues to the diverse molecular mechanisms underlying immunity. Here, we present a meta-analysis of 314 cloned R genes. The majority of R genes encode cell surface or intracellular receptors, and we distinguish nine molecular mechanisms by which R proteins can elevate or trigger disease resistance: direct (1) or indirect (2) perception of pathogen-derived molecules on the cell surface by receptor-like proteins and receptor-like kinases; direct (3) or indirect (4) intracellular detection of pathogen-derived molecules by nucleotide binding, leucine-rich repeat receptors, or detection through integrated domains (5); perception of transcription activator-like effectors through activation of executor genes (6); and active (7), passive (8), or host reprogramming-mediated (9) loss of susceptibility. Although the molecular mechanisms underlying the functions of R genes are only understood for a small proportion of known R genes, a clearer understanding of mechanisms is emerging and will be crucial for rational engineering and deployment of novel R genes. PMID:29382771
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Valdivieso-Torres, Leonardo; Sarangi, Anindita; Whidby, Jillian; Marcotrigiano, Joseph; Roth, Monica J
2015-12-30
Retargeting of gammaretroviral envelope proteins has shown promising results in the isolation of novel isolates with therapeutic potential. However, the optimal conditions required to obtain high-affinity retargeted envelope proteins with narrow tropism are not understood. This study highlights the advantage of constrained peptides within receptor binding domains and validates the random library screening technique of obtaining novel retargeted Env proteins. Using a modified vector backbone to screen the envelope libraries on 143B osteosarcoma cells, three novel and unique retargeted envelopes were isolated. The use of complex disulfide bonds within variable regions required for receptor binding is found within natural gammaretroviral envelope isolates. Interestingly, two of the isolates, named AII and BV2, have a pair of cysteines located within the randomized region of 11 amino acids similar to that identified within the CP Env, an isolate identified in a previous Env library screen on the human renal carcinoma Caki-1 cell line. The amino acids within the randomized region of AII and BV2 envelopes that are essential for viral infection have been identified in this study and include these cysteine residues. Through mutagenesis studies, the putative disulfide bond pairs including and beyond the randomized region were examined. In parallel, the disulfide bonds of CP Env were identified using mass spectrometry. The results indicate that this pair of cysteines creates the structural context to position key hydrophobic (F and W) and basic (K and H) residues critical for viral titer and suggest that AII, BV2, and CP internal cysteines bond together in distinct ways. Retargeted gammaretroviral particles have broad applications for therapeutic use. Although great advances have been achieved in identifying new Env-host cell receptor pairs, the rules for designing optimal Env libraries are still unclear. We have found that isolates with an additional pair of cysteines within the randomized region have the highest transduction efficiencies. This emphasizes the importance of considering cysteine pairs in the design of new libraries. Furthermore, our data clearly indicate that these cysteines are essential for viral infectivity by presenting essential residues to the host cell receptor. These studies facilitate the screening of Env libraries for functional entry into target cells, allowing the identification of novel gammaretroviral Envs targeting alternative host cell receptors for gene and protein delivery. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Gleason, Evanna
2012-01-01
Amacrine cells receive glutamatergic input from bipolar cells and GABAergic, glycinergic, cholinergic, and dopaminergic input from other amacrine cells. Glutamate, GABA, glycine, and acetylcholine (ACh) interact with ionotropic receptors and it is these interactions that form much of the functional circuitry in the inner retina. However, glutamate, GABA, ACh, and dopamine also activate metabotropic receptors linked to second messenger pathways that have the potential to modify the function of individual cells as well as retinal circuitry. Here, the physiological effects of activating dopamine receptors, metabotropic glutamate receptors, GABAB receptors, and muscarinic ACh receptors on amacrine cells will be discussed. The retina also expresses metabotropic receptors and the biochemical machinery associated with the synthesis and degradation of endocannabinoids and sphingosine-1-phosphate (S1P). The effects of activating cannabinoid receptors and S1P receptors on amacrine cell function will also be addressed. Copyright © Cambridge University Press, 2012
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Microscopic lymph node tumor burden quantified by macroscopic dual-tracer molecular imaging
Tichauer, Kenneth M.; Samkoe, Kimberley S.; Gunn, Jason R.; Kanick, Stephen C.; Hoopes, P. Jack; Barth, Richard J.; Kaufman, Peter A.; Hasan, Tayyaba; Pogue, Brian W.
2014-01-01
Lymph node biopsy (LNB) is employed in many cancer surgeries to identify metastatic disease and stage the cancer, yet morbidity and diagnostic delays associated with LNB could be avoided if non-invasive imaging of nodal involvement was reliable. Molecular imaging has potential in this regard; however, variable delivery and nonspecific uptake of imaging tracers has made conventional approaches ineffective clinically. A method of correcting for non-specific uptake with injection of a second untargeted tracer is presented, allowing tumor burden in lymph nodes to be quantified. The approach was confirmed in an athymic mouse model of metastatic human breast cancer targeting epidermal growth factor receptor, a cell surface receptor overexpressed by many cancers. A significant correlation was observed between in vivo (dual-tracer) and ex vivo measures of tumor burden (r = 0.97, p < 0.01), with an ultimate sensitivity of approximately 200 cells (potentially more sensitive than conventional LNB). PMID:25344739
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Murata, Yoshihiro; Mashiko, Masashi; Ozaki, Mamiko; Amakawa, Taisaku; Nakamura, Tadashi
2004-01-01
The taste organ in insects is a hair-shaped taste sensory unit having four functionally differentiated contact chemoreceptor cells. In the blowfly, Phormia regina, cGMP has been suggested to be a second messenger for the sugar receptor cell. Generally, cGMP is produced by membranous or soluble guanylyl cyclase (sGC), which can be activated by nitric oxide (NO). In the present paper, we electrophysiologically showed that an NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO), an NO donor, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC 7) or an NO synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) specifically affected the response in the sugar receptor cell, but not in other receptor cells. PTIO, when introduced into the receptor cells in a sensillum aided by sodium deoxycholate (DOC, pH 7.2), depressed the response of sugar receptor cells to sucrose but did not affect those of the salt or water receptor cells. NOC 7, given extracellularly, latently induced the response of sugar receptor cells; and L-NAME, when introduced into the receptor cells, depressed the response of sugar receptor cells. The results clearly suggest that NO, which may be produced by intrinsic NOS in sugar receptor cells, participates in the transduction cascade of these cells in blowfly.
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Chen, Ken; Deng, Kun; Wang, Xiaoyan; Wang, Zhen; Zheng, Shuo; Ren, Hongmei; He, Duofen; Han, Yu; Asico, Laureano D.; Jose, Pedro A.; Zeng, Chunyu
2014-01-01
The dopaminergic and renin angiotensin systems interact to regulate blood pressure. Disruption of the D4 dopamine receptor gene in mice produces hypertension that is associated with increased renal AT1 receptor expression. We hypothesize that the D4 receptor can inhibit AT1 receptor expression and function in renal proximal tubules (RPTs) cells from Wistar-Kyoto (WKY) rats but the D4 receptor regulation of AT1 receptor is aberrant in RPT cells from spontaneously hypertensive rats (SHRs). The D4 receptor agonist, PD168077, decreased AT1 receptor protein expression in a time and concentration-dependent manner in WKY cells. By contrast, in SHR cells, PD168077 increased AT1 receptor protein expression. The inhibitory effect of D4 receptor on AT1 receptor expression in WKY cells was blocked by a calcium channel blocker, nicardipine, or calcium-free medium, indicating that calcium is involved in the D4 receptor-mediated signaling pathway. Angiotensin II increased Na+-K+ ATPase activity in WKY cells. Pretreatment with PD168077 decreased the stimulatory effect of angiotensin II on Na+-K+ ATPase activity in WKY cells. In SHR cells, the inhibitory effect of D4 receptor on angiotensin II-mediated stimulation of Na+-K+ ATPase activity was aberrant; pretreatment with PD168077 augmented the stimulatory effect of AT1 receptor on Na+-K+ ATPase activity in SHR cells. This was confirmed in vivo; pre-treatment with PD128077 for one week augmented the anti-hypertensive and natriuretic effect of losartan in SHRs but not in WKY rats. We suggest that an aberrant interaction between D4 and AT1 receptors may play a role in the abnormal regulation of sodium excretion in hypertension. PMID:25368031
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T cell costimulation by chemokine receptors.
Molon, Barbara; Gri, Giorgia; Bettella, Monica; Gómez-Moutón, Concepción; Lanzavecchia, Antonio; Martínez-A, Carlos; Mañes, Santos; Viola, Antonella
2005-05-01
Signals mediated by chemokine receptors may compete with T cell receptor stop signals and determine the duration of T cell-antigen-presenting cell interactions. Here we show that during T cell stimulation by antigen-presenting cells, T cell chemokine receptors coupled to G(q) and/or G(11) protein were recruited to the immunological synapse by a G(i)-independent mechanism. When chemokine receptors were sequestered at the immunological synapse, T cells became insensitive to chemotactic gradients, formed more stable conjugates and finally responded with enhanced proliferation and cytokine production. We suggest that chemokine receptor trapping at the immunological synapse enhances T cell activation by improving T cell-antigen-presenting cell attraction and impeding the 'distraction' of successfully engaged T cells by other chemokine sources.
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Lozić, Maja; Greenwood, Michael; Šarenac, Olivera; Martin, Andrew; Hindmarch, Charles; Tasić, Tatjana; Paton, Julian; Murphy, David; Japundžić-Žigon, Nina
2014-01-01
Background and Purpose The paraventricular nucleus (PVN) of the hypothalamus is an important integrative site for neuroendocrine control of the circulation. We investigated the role of oxytocin receptors (OT receptors) in PVN in cardiovascular homeostasis. Experimental Approach Experiments were performed in conscious male Wistar rats equipped with a radiotelemetric device. The PVN was unilaterally co-transfected with an adenoviral vector (Ad), engineered to overexpress OT receptors, and an enhanced green fluorescent protein (eGFP) tag. Control groups: PVN was transfected with an Ad expressing eGFP alone or untransfected, sham rats (Wt). Recordings were obtained without and with selective blockade of OT receptors (OTX), during both baseline and stressful conditions. Baroreceptor reflex sensitivity (BRS) and cardiovascular short-term variability were evaluated using the sequence method and spectral methodology respectively. Key Results Under baseline conditions, rats overexpressing OT receptors (OTR) exhibited enhanced BRS and reduced BP variability compared to control groups. Exposure to stress increased BP, BP variability and HR in all rats. In control groups, but not in OTR rats, BRS decreased during stress. Pretreatment of OTR rats with OTX reduced BRS and enhanced BP and HR variability under baseline and stressful conditions. Pretreatment of Wt rats with OTX, reduced BRS and increased BP variability under baseline and stressful conditions, but only increased HR variability during stress. Conclusions and Implications OT receptors in PVN are involved in tonic neural control of BRS and cardiovascular short-term variability. The failure of this mechanism could critically contribute to the loss of autonomic control in cardiovascular disease. PMID:24834854
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Expression of NK cell receptors on decidual T cells in human pregnancy.
Tilburgs, Tamara; van der Mast, Barbara J; Nagtzaam, Nicole M A; Roelen, Dave L; Scherjon, Sicco A; Claas, Frans H J
2009-06-01
Specific receptors enable NK cells to discriminate between cells with normal expression of MHC class I and cells that have low or absent expression of MHC class I molecules. In addition to NK cells, these receptors can be expressed on T cell subsets, mainly on CD8+ T cells but also on gammadeltaTCR+ T cells and CD4+ T cells. Although the function of NK cell receptor expression on T cells is not completely understood, various studies have shown that they are involved in down regulation of T cell receptor (TCR)-mediated activation and influence effector functions, like cytotoxicity and cytokine production. The aim of this study was to analyze expression of NK cell receptors on peripheral blood and decidual T cells during human pregnancy using flow cytometry. We demonstrate that a proportion of decidual T cells express HLA-C specific killer immunoglobulin-like receptors (KIRs). Furthermore, a small proportion of decidual T cells express the HLA-E specific CD94-NKG2A inhibitory and CD94-NKG2C activating receptors. Decidual KIR+ and CD94-NKG2+ T cells mainly display a CD3+CD4-CD8- phenotype. However, decidual tissue also contains higher percentages of KIR and CD94-NKG2 expressing CD4+ and CD8+ T cells compared to peripheral blood. So far, the functional capacities of decidual T cells expressing the NK cell receptors are unknown but NK cell receptor expression on decidual T cells may provide an alternative means by which decidual T cells distinguish self (maternal) cells from allogeneic fetal cells, and act to modulate the decidual immune response.
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Bombesin-like peptide receptors in human bronchial epithelial cells.
Kane, M A; Toi-Scott, M; Johnson, G L; Kelley, K K; Boose, D; Escobedo-Morse, A
1996-01-01
Northern blot and RNAse protection assays previously failed to detect bombesin-like peptide (BLP) receptors in normal human lung tissue, but by RT/PCR cultured human bronchial epithelial (HBE) cells expressed all three BLP receptor subtypes, predominantly neuromedin B (NMB) receptor. By RT/PCR, we found expression of all three BLP receptor subtypes by human lung tissue and confirmed NMB receptor expression in six out of six HBE samples. However, transformed HBE BEAS B2B cells expressed only gastrin-releasing peptide (GRP) receptors; saturable, high-affinity (Kd = 3.5 nM) specific [125I]GRP binding confirmed functional GRP receptor, with M(r) = 75 kDa and immunologic cross-reactivity with GRP receptor from human small-cell lung carcinoma (SCLC) NCI-H345 cells. Altered regulation of BLP receptors may accompany transformation of normal lung cells to cancer.
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T cell receptor alpha variable 12-2 bias in the immunodominant response to Yellow fever virus.
Bovay, Amandine; Zoete, Vincent; Dolton, Garry; Bulek, Anna M; Cole, David K; Rizkallah, Pierre J; Fuller, Anna; Beck, Konrad; Michielin, Olivier; Speiser, Daniel E; Sewell, Andrew K; Fuertes Marraco, Silvia A
2018-02-01
The repertoire of human αβ T-cell receptors (TCRs) is generated via somatic recombination of germline gene segments. Despite this enormous variation, certain epitopes can be immunodominant, associated with high frequencies of antigen-specific T cells and/or exhibit bias toward a TCR gene segment. Here, we studied the TCR repertoire of the HLA-A*0201-restricted epitope LLWNGPMAV (hereafter, A2/LLW) from Yellow Fever virus, which generates an immunodominant CD8 + T cell response to the highly effective YF-17D vaccine. We discover that these A2/LLW-specific CD8 + T cells are highly biased for the TCR α chain TRAV12-2. This bias is already present in A2/LLW-specific naïve T cells before vaccination with YF-17D. Using CD8 + T cell clones, we show that TRAV12-2 does not confer a functional advantage on a per cell basis. Molecular modeling indicated that the germline-encoded complementarity determining region (CDR) 1α loop of TRAV12-2 critically contributes to A2/LLW binding, in contrast to the conventional dominant dependence on somatically rearranged CDR3 loops. This germline component of antigen recognition may explain the unusually high precursor frequency, prevalence and immunodominance of T-cell responses specific for the A2/LLW epitope. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Ly49 Receptors: Innate and Adaptive Immune Paradigms
Rahim, Mir Munir A.; Tu, Megan M.; Mahmoud, Ahmad Bakur; Wight, Andrew; Abou-Samra, Elias; Lima, Patricia D. A.; Makrigiannis, Andrew P.
2014-01-01
The Ly49 receptors are type II C-type lectin-like membrane glycoproteins encoded by a family of highly polymorphic and polygenic genes within the mouse natural killer (NK) gene complex. This gene family is designated Klra, and includes genes that encode both inhibitory and activating Ly49 receptors in mice. Ly49 receptors recognize class I major histocompatibility complex-I (MHC-I) and MHC-I-like proteins on normal as well as altered cells. Their functional homologs in humans are the killer cell immunoglobulin-like receptors, which recognize HLA class I molecules as ligands. Classically, Ly49 receptors are described as being expressed on both the developing and mature NK cells. The inhibitory Ly49 receptors are involved in NK cell education, a process in which NK cells acquire function and tolerance toward cells that express “self-MHC-I.” On the other hand, the activating Ly49 receptors recognize altered cells expressing activating ligands. New evidence shows a broader Ly49 expression pattern on both innate and adaptive immune cells. Ly49 receptors have been described on multiple NK cell subsets, such as uterine NK and memory NK cells, as well as NKT cells, dendritic cells, plasmacytoid dendritic cells, macrophages, neutrophils, and cells of the adaptive immune system, such as activated T cells and regulatory CD8+ T cells. In this review, we discuss the expression pattern and proposed functions of Ly49 receptors on various immune cells and their contribution to immunity. PMID:24765094
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Chemokine receptor binding and signal transduction in native cells of the central nervous system.
Davis, Christopher N; Chen, Shuzhen; Boehme, Stefen A; Bacon, Kevin B; Harrison, Jeffrey K
2003-04-01
Chemokine receptors belong to the superfamily of seven-transmembrane-spanning, G-protein-coupled receptors, and their expression by central nervous system cells is clearly documented. As this gene family has become the target of novel therapeutic development, the analysis of these receptors requires radioligand binding techniques as well as methods that entail assessing receptor stimulation of signal transduction pathways. Herein, we describe specific protocols for measuring radiolabeled chemokine binding to their cognate receptors on cultured glial cells as well as to receptors expressed in heterologous cell systems. Multiple downstream signaling pathways, including intracellular calcium influx and receptor-dependent kinase activation, are associated with chemokine receptor stimulation. Protocols for measuring these signaling events in chemokine-receptor-expressing cells are also presented.
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Nika, Lisa; Wallner, Jakob; Palmberger, Dieter; Koczka, Krisztina; Vorauer-Uhl, Karola; Grabherr, Reingard
2017-08-01
Biomarkers of cancer are often glycosylated membrane receptor proteins present on the cellular surface. In order to develop new antibodies for cancer diagnostics or treatment, it is a main pre-requisite that these target proteins are available in a native conformation. However, membrane receptor proteins are notoriously difficult to produce due to their hydrophobic nature and complex architecture. Here, we used the baculovirus-insect cell expression system to produce budded virus-like particles (VLPs) as the scaffold for the presentation of complex membrane proteins. Since the human epidermal growth factor receptor 2 (HER2) is known to be overexpressed in a number of cancers it was chosen as model for a tumor antigen. VLPs displaying full-length HER2 on the surface were produced in Spodoptera frugiperda 9 (Sf9) insect cells and purified by sucrose gradient ultracentrifugation. The number of secreted particles was quantified by nanoparticle tracking analysis. To confirm the presence of HER2 protein on the surface, VLPs were labeled with gold-conjugated antibodies and analyzed by transmission electron microscopy. Functionality of displayed HER2 was investigated by ELISA and a newly established biolayer interferometry based technique. Detection was accomplished using the specific monoclonal antibody Herceptin and filamentous phages displaying a single-chain variable fragment of an anti-HER2 antibody. Significant stronger binding of Herceptin and anti-HER2 phages to HER2-displaying VLPs as compared to control VLPs was demonstrated. Thus, we suggest that Sf9 insect cells are highly feasible for the fast and easy production of various budded VLPs that serve as a platform for full-length membrane receptor presentation. Copyright © 2017. Published by Elsevier Inc.
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Reassessing ecdysteroidogenic cells from the cell membrane receptors' perspective.
Alexandratos, Alexandros; Moulos, Panagiotis; Nellas, Ioannis; Mavridis, Konstantinos; Dedos, Skarlatos G
2016-02-05
Ecdysteroids secreted by the prothoracic gland (PG) cells of insects control the developmental timing of their immature life stages. These cells have been historically considered as carrying out a single function in insects, namely the biochemical conversion of cholesterol to ecdysteroids and their secretion. A growing body of evidence shows that PG cells receive multiple cues during insect development so we tested the hypothesis that they carry out more than just one function in insects. We characterised the molecular nature and developmental profiles of cell membrane receptors in PG cells of Bombyx mori during the final larval stage and determined what receptors decode nutritional, developmental and physiological signals. Through iterative approaches we identified a complex repertoire of cell membrane receptors that are expressed in intricate patterns and activate previously unidentified signal transduction cascades in PG cells. The expression patterns of some of these receptors explain precisely the mechanisms that are known to control ecdysteroidogenesis. However, the presence of receptors for the notch, hedgehog and wingless signalling pathways and the expression of innate immunity-related receptors such as phagocytosis receptors, receptors for microbial ligands and Toll-like receptors call for a re-evaluation of the role these cells play in insects.
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Biopsy variability of lymphocytic infiltration in breast cancer subtypes and the ImmunoSkew score
NASA Astrophysics Data System (ADS)
Khan, Adnan Mujahid; Yuan, Yinyin
2016-11-01
The number of tumour biopsies required for a good representation of tumours has been controversial. An important factor to consider is intra-tumour heterogeneity, which can vary among cancer types and subtypes. Immune cells in particular often display complex infiltrative patterns, however, there is a lack of quantitative understanding of the spatial heterogeneity of immune cells and how this fundamental biological nature of human tumours influences biopsy variability and treatment resistance. We systematically investigate biopsy variability for the lymphocytic infiltrate in 998 breast tumours using a novel virtual biopsy method. Across all breast cancers, we observe a nonlinear increase in concordance between the biopsy and whole-tumour score of lymphocytic infiltrate with increasing number of biopsies, yet little improvement is gained with more than four biopsies. Interestingly, biopsy variability of lymphocytic infiltrate differs considerably among breast cancer subtypes, with the human epidermal growth factor receptor 2-positive (HER2+) subtype having the highest variability. We subsequently identify a quantitative measure of spatial variability that predicts disease-specific survival in HER2+ subtype independent of standard clinical variables (node status, tumour size and grade). Our study demonstrates how systematic methods provide new insights that can influence future study design based on a quantitative knowledge of tumour heterogeneity.
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HLA-E regulatory and coding region variability and haplotypes in a Brazilian population sample.
Ramalho, Jaqueline; Veiga-Castelli, Luciana C; Donadi, Eduardo A; Mendes-Junior, Celso T; Castelli, Erick C
2017-11-01
The HLA-E gene is characterized by low but wide expression on different tissues. HLA-E is considered a conserved gene, being one of the least polymorphic class I HLA genes. The HLA-E molecule interacts with Natural Killer cell receptors and T lymphocytes receptors, and might activate or inhibit immune responses depending on the peptide associated with HLA-E and with which receptors HLA-E interacts to. Variable sites within the HLA-E regulatory and coding segments may influence the gene function by modifying its expression pattern or encoded molecule, thus, influencing its interaction with receptors and the peptide. Here we propose an approach to evaluate the gene structure, haplotype pattern and the complete HLA-E variability, including regulatory (promoter and 3'UTR) and coding segments (with introns), by using massively parallel sequencing. We investigated the variability of 420 samples from a very admixed population such as Brazilians by using this approach. Considering a segment of about 7kb, 63 variable sites were detected, arranged into 75 extended haplotypes. We detected 37 different promoter sequences (but few frequent ones), 27 different coding sequences (15 representing new HLA-E alleles) and 12 haplotypes at the 3'UTR segment, two of them presenting a summed frequency of 90%. Despite the number of coding alleles, they encode mainly two different full-length molecules, known as E*01:01 and E*01:03, which corresponds to about 90% of all. In addition, differently from what has been previously observed for other non classical HLA genes, the relationship among the HLA-E promoter, coding and 3'UTR haplotypes is not straightforward because the same promoter and 3'UTR haplotypes were many times associated with different HLA-E coding haplotypes. This data reinforces the presence of only two main full-length HLA-E molecules encoded by the many HLA-E alleles detected in our population sample. In addition, this data does indicate that the distal HLA-E promoter is by far the most variable segment. Further analyses involving the binding of transcription factors and non-coding RNAs, as well as the HLA-E expression in different tissues, are necessary to evaluate whether these variable sites at regulatory segments (or even at the coding sequence) may influence the gene expression profile. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Variability in H9N2 haemagglutinin receptor-binding preference and the pH of fusion.
Peacock, Thomas P; Benton, Donald J; Sadeyen, Jean-Remy; Chang, Pengxiang; Sealy, Joshua E; Bryant, Juliet E; Martin, Stephen R; Shelton, Holly; McCauley, John W; Barclay, Wendy S; Iqbal, Munir
2017-03-22
H9N2 avian influenza viruses are primarily a disease of poultry; however, they occasionally infect humans and are considered a potential pandemic threat. Little work has been performed to assess the intrinsic biochemical properties related to zoonotic potential of H9N2 viruses. The objective of this study, therefore, was to investigate H9N2 haemagglutinins (HAs) using two well-known correlates for human adaption: receptor-binding avidity and pH of fusion. Receptor binding was characterized using bio-layer interferometry to measure virus binding to human and avian-like receptor analogues and the pH of fusion was assayed by syncytium formation in virus-infected cells at different pHs. We characterized contemporary H9N2 viruses of the zoonotic G1 lineage, as well as representative viruses of the zoonotic BJ94 lineage. We found that most contemporary H9N2 viruses show a preference for sulphated avian-like receptor analogues. However, the 'Eastern' G1 H9N2 viruses displayed a consistent preference in binding to a human-like receptor analogue. We demonstrate that the presence of leucine at position 226 of the HA receptor-binding site correlated poorly with the ability to bind a human-like sialic acid receptor. H9N2 HAs also display variability in their pH of fusion, ranging between pH 5.4 and 5.85 which is similar to that of the first wave of human H1N1pdm09 viruses but lower than the pH of fusion seen in zoonotic H5N1 and H7N9 viruses. Our results suggest possible molecular mechanisms that may underlie the relatively high prevalence of human zoonotic infection by particular H9N2 virus lineages.
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Variability in H9N2 haemagglutinin receptor-binding preference and the pH of fusion
Peacock, Thomas P; Benton, Donald J; Sadeyen, Jean-Remy; Chang, Pengxiang; Sealy, Joshua E; Bryant, Juliet E; Martin, Stephen R; Shelton, Holly; McCauley, John W; Barclay, Wendy S; Iqbal, Munir
2017-01-01
H9N2 avian influenza viruses are primarily a disease of poultry; however, they occasionally infect humans and are considered a potential pandemic threat. Little work has been performed to assess the intrinsic biochemical properties related to zoonotic potential of H9N2 viruses. The objective of this study, therefore, was to investigate H9N2 haemagglutinins (HAs) using two well-known correlates for human adaption: receptor-binding avidity and pH of fusion. Receptor binding was characterized using bio-layer interferometry to measure virus binding to human and avian-like receptor analogues and the pH of fusion was assayed by syncytium formation in virus-infected cells at different pHs. We characterized contemporary H9N2 viruses of the zoonotic G1 lineage, as well as representative viruses of the zoonotic BJ94 lineage. We found that most contemporary H9N2 viruses show a preference for sulphated avian-like receptor analogues. However, the ‘Eastern' G1 H9N2 viruses displayed a consistent preference in binding to a human-like receptor analogue. We demonstrate that the presence of leucine at position 226 of the HA receptor-binding site correlated poorly with the ability to bind a human-like sialic acid receptor. H9N2 HAs also display variability in their pH of fusion, ranging between pH 5.4 and 5.85 which is similar to that of the first wave of human H1N1pdm09 viruses but lower than the pH of fusion seen in zoonotic H5N1 and H7N9 viruses. Our results suggest possible molecular mechanisms that may underlie the relatively high prevalence of human zoonotic infection by particular H9N2 virus lineages. PMID:28325922
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Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment.
Iezzi, María Elena; Policastro, Lucía; Werbajh, Santiago; Podhajcer, Osvaldo; Canziani, Gabriela Alicia
2018-01-01
Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs), in particular those engineered from the variable heavy-chain fragment (VHH gene) found in Camelidae heavy-chain antibodies (or IgG2 and IgG3), are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR) fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition "modules" to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo . Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads.
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Influence of human cytomegalovirus infection on the NK cell receptor repertoire in children.
Monsiváis-Urenda, Adriana; Noyola-Cherpitel, Daniel; Hernández-Salinas, Alba; García-Sepúlveda, Christian; Romo, Neus; Baranda, Lourdes; López-Botet, Miguel; González-Amaro, Roberto
2010-05-01
Human cytomegalovirus (hCMV) infection is usually asymptomatic but may cause disease in immunocompromised hosts. It has been reported that hCMV infection may shape the NK cell receptor (NKR) repertoire in adult individuals, promoting a variable expansion of the CD94/NKG2C+ NK cell subset. We explored the possible relationship between this viral infection and the expression pattern of different NKR including CD94/NKG2C, CD94/NKG2A, immunoglobulin-like transcript 2 (ILT2, CD85j), KIR2DL1/2DS1, KIR3DL1, and CD161 in peripheral blood lymphocytes from healthy children, seropositive (n=21) and seronegative (n=20) for hCMV. Consistent with previous observations in adults, a positive serology for hCMV was associated with increased numbers of NKG2C+ NK and T cells as well as with ILT2+ T lymphocytes. Moreover, the proportions of CD161+ and NKG2C+CD56-CD3- NK cells also tended to be increased in hCMV+ individuals. Excretion of the virus was associated with higher proportions of NKG2C+ NK cells. Altogether, these data reveal that hCMV may have a profound influence on the NKR repertoire in early childhood.
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Paolini, R; Kinet, J P
1993-01-01
Multiubiquitination of proteins is a critical step leading to selective degradation for many polypeptides. Therefore, activation-induced multiubiquitination of cell surface receptors, such as the platelet-derived growth factor (PDGF) receptor and the T cell antigen (TCR) receptor, may correspond to a degradation pathway for ligand-receptor complexes. Here we show that the antigen-induced engagement of high-affinity immunoglobulin E receptors (Fc epsilon RI) results in the immediate multiubiquitination of Fc epsilon RI beta and gamma chains. This ubiquitination is independent of receptor phosphorylation and is restricted to activated receptors. Surprisingly, receptor multiubiquitination is immediately reversible when receptors are disengaged. Therefore, multiubiquitination and deubiquitination of Fc epsilon RI receptors is controlled at the cell surface by receptor engagement and disengagement. The rapidity, specificity and, most importantly, the reversibility of the activation-induced receptor multiubiquitination suggest that this process may turn on/off a cell surface receptor signaling function thus far unsuspected. Images PMID:8382611
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GABA, its receptors, and GABAergic inhibition in mouse taste buds
Dvoryanchikov, Gennady; Huang, Yijen A; Barro-Soria, Rene; Chaudhari, Nirupa; Roper, Stephen D.
2012-01-01
Taste buds consist of at least three principal cell types that have different functions in processing gustatory signals — glial-like Type I cells, Receptor (Type II) cells, and Presynaptic (Type III) cells. Using a combination of Ca2+ imaging, single cell RT-PCR, and immunostaining, we show that γ-amino butyric acid (GABA) is an inhibitory transmitter in mouse taste buds, acting on GABA-A and GABA-B receptors to suppress transmitter (ATP) secretion from Receptor cells during taste stimulation. Specifically, Receptor cells express GABA-A receptor subunits β2, δ, π, as well as GABA-B receptors. In contrast, Presynaptic cells express the GABA-Aβ3 subunit and only occasionally GABA-B receptors. In keeping with the distinct expression pattern of GABA receptors in Presynaptic cells, we detected no GABAergic suppression of transmitter release from Presynaptic cells. We suggest that GABA may serve function(s) in taste buds in addition to synaptic inhibition. Finally, we also defined the source of GABA in taste buds: GABA is synthesized by GAD65 in Type I taste cells as well as by GAD67 in Presynaptic (Type III) taste cells and is stored in both those two cell types. We conclude that GABA is released during taste stimulation and possibly also during growth and differentiation of taste buds. PMID:21490220
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GABA, its receptors, and GABAergic inhibition in mouse taste buds.
Dvoryanchikov, Gennady; Huang, Yijen A; Barro-Soria, Rene; Chaudhari, Nirupa; Roper, Stephen D
2011-04-13
Taste buds consist of at least three principal cell types that have different functions in processing gustatory signals: glial-like (type I) cells, receptor (type II) cells, and presynaptic (type III) cells. Using a combination of Ca2+ imaging, single-cell reverse transcriptase-PCR and immunostaining, we show that GABA is an inhibitory transmitter in mouse taste buds, acting on GABA(A) and GABA(B) receptors to suppress transmitter (ATP) secretion from receptor cells during taste stimulation. Specifically, receptor cells express GABA(A) receptor subunits β2, δ, and π, as well as GABA(B) receptors. In contrast, presynaptic cells express the GABA(A) β3 subunit and only occasionally GABA(B) receptors. In keeping with the distinct expression pattern of GABA receptors in presynaptic cells, we detected no GABAergic suppression of transmitter release from presynaptic cells. We suggest that GABA may serve function(s) in taste buds in addition to synaptic inhibition. Finally, we also defined the source of GABA in taste buds: GABA is synthesized by GAD65 in type I taste cells as well as by GAD67 in presynaptic (type III) taste cells and is stored in both those two cell types. We conclude that GABA is an inhibitory transmitter released during taste stimulation and possibly also during growth and differentiation of taste buds.
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Vines, Douglass C.; Scollard, Deborah A.; Komal, Teesha; Ganguly, Milan; Do, Trevor; Wu, Bing; Alexander, Natasha; Besanger, Travis
2017-01-01
Peptide-receptor imaging and therapy with radiolabeled somatostatin analogs such as 68Ga-DOTA-TATE and 177Lu-DOTA-TATE have become an effective treatment option for SSTR-positive neuroendocrine tumors. The purpose of this study was to evaluate the correlation of somatostatin receptor-2 (SSTR2) expression with 68Ga-DOTA-TATE uptake and 177Lu-DOTA-TATE therapy in neuroblastoma (NB) xenograft models. We demonstrated variable SSTR2 expression profiles in eight NB cell lines. From micro-PET imaging and autoradiography, a higher uptake of 68Ga-DOTA-TATE was observed in SSTR2 high-expressing NB xenografts (CHLA-15) compared to SSTR2 low-expressing NB xenografts (SK-N-BE(2)). Combined autoradiography-immunohistochemistry revealed histological colocalization of SSTR2 and 68Ga-DOTA-TATE uptake in CHLA-15 tumors. With a low dose of 177Lu-DOTA-TATE (20 MBq/animal), tumor growth inhibition was achieved in the CHLA-15 high SSTR2 expressing xenograft model. Although, in vitro, NB cells showed variable expression levels of norepinephrine transporter (NET), a molecular target for 131I-MIBG therapy, low 123I-MIBG uptake was observed in all selected NB xenografts. In conclusion, SSTR2 expression levels are associated with 68Ga-DOTA-TATE uptake and antitumor efficacy of 177Lu-DOTA-TATE. 68Ga-DOTA-TATE PET is superior to 123I-MIBG SPECT imaging in detecting NB tumors in our model. Radiolabeled DOTA-TATE can be used as an agent for NB tumor imaging to potentially discriminate tumors eligible for 177Lu-DOTA-TATE therapy. PMID:29097943
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Zhang, Libo; Vines, Douglass C; Scollard, Deborah A; McKee, Trevor; Komal, Teesha; Ganguly, Milan; Do, Trevor; Wu, Bing; Alexander, Natasha; Vali, Reza; Shammas, Amer; Besanger, Travis; Baruchel, Sylvain
2017-01-01
Peptide-receptor imaging and therapy with radiolabeled somatostatin analogs such as 68 Ga-DOTA-TATE and 177 Lu-DOTA-TATE have become an effective treatment option for SSTR-positive neuroendocrine tumors. The purpose of this study was to evaluate the correlation of somatostatin receptor-2 (SSTR2) expression with 68 Ga-DOTA-TATE uptake and 177 Lu-DOTA-TATE therapy in neuroblastoma (NB) xenograft models. We demonstrated variable SSTR2 expression profiles in eight NB cell lines. From micro-PET imaging and autoradiography, a higher uptake of 68 Ga-DOTA-TATE was observed in SSTR2 high-expressing NB xenografts (CHLA-15) compared to SSTR2 low-expressing NB xenografts (SK-N-BE(2)). Combined autoradiography-immunohistochemistry revealed histological colocalization of SSTR2 and 68 Ga-DOTA-TATE uptake in CHLA-15 tumors. With a low dose of 177 Lu-DOTA-TATE (20 MBq/animal), tumor growth inhibition was achieved in the CHLA-15 high SSTR2 expressing xenograft model. Although, in vitro , NB cells showed variable expression levels of norepinephrine transporter (NET), a molecular target for 131 I-MIBG therapy, low 123 I-MIBG uptake was observed in all selected NB xenografts. In conclusion, SSTR2 expression levels are associated with 68 Ga-DOTA-TATE uptake and antitumor efficacy of 177 Lu-DOTA-TATE. 68 Ga-DOTA-TATE PET is superior to 123 I-MIBG SPECT imaging in detecting NB tumors in our model. Radiolabeled DOTA-TATE can be used as an agent for NB tumor imaging to potentially discriminate tumors eligible for 177 Lu-DOTA-TATE therapy.
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Nakayama, Toshiyuki; Hatachi, Go; Wen, Chun-Yang; Yoshizaki, Ayumi; Yamazumi, Kazuyuki; Niino, Daisuke; Sekine, Ichiro
2005-01-01
AIM: There is strong evidence that tyrosine kinases are involved in the regulation of tumor progression, cellular growth and differentiation. Recently, many kinds of tyrosine kinase receptors have been reported, among them Tie-1 and Tie-2 receptors constitute a major class. Angiopoietin (Ang)-1 is known as a ligand of Tie-2 tyrosine kinase receptor. The objective of this study was to establish a comprehensive Tie-1 and Tie-2 and Ang-1, 2 and 4 expression profile in human colorectal adenocarcinomas. METHODS: We examined 96 cases of surgically resected human colorectal adenocarcinoma by immunohistochemistry and investigated the statistical correlation between the expressions of Ties and Angs and clinicopathological factors. RESULTS: Among the 96 cases of adenocarcinoma, 87 (90.6%), 92 (95.8%), 83 (86.5%), 89 (92.7%), and 76 cases (79.2%) showed positive staining in the cytoplasm of carcinoma cells for the Tie-1 and Tie-2 and Ang-1, 2 and 4 proteins, respectively. Histologically, the expressions of Ties and Angs were variable. The expressions of Ties and Angs were correlated with several clinicopathological factors, but did not correlate with the presence of lymph node metastasis. Ties and Angs were highly expressed in human colorectal adenocarcinoma cells. CONCLUSION: These findings suggest that the Tie-Ang receptor-ligand complex is one of the factors involved in the cellular differentiation and progression of human colorectal adenocarcinoma. PMID:15742397
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Tomé, Angelo R; Castro, Enrique; Santos, Rosa M; Rosário, Luís M
2007-06-14
Adrenal chromaffin cells mediate acute responses to stress through the release of epinephrine. Chromaffin cell function is regulated by several receptors, present both in adrenergic (AD) and noradrenergic (NA) cells. Extracellular ATP exerts excitatory and inhibitory actions on chromaffin cells via ionotropic (P2X) and metabotropic (P2Y) receptors. We have taken advantage of the actions of the purinergic agonists ATP and UTP on cytosolic free Ca2+ concentration ([Ca2+]i) to determine whether P2X and P2Y receptors might be asymmetrically distributed among AD and NA chromaffin cells. The [Ca2+]i and the [Na+]i were recorded from immunolabeled bovine chromaffin cells by single-cell fluorescence imaging. Among the ATP-sensitive cells ~40% did not yield [Ca2+]i responses to ATP in the absence of extracellular Ca2+ (Ca2+o), indicating that they expressed P2X receptors and did not express Ca2+- mobilizing P2Y receptors; the remainder expressed Ca2+-mobilizing P2Y receptors. Relative to AD-cells approximately twice as many NA-cells expressed P2X receptors while not expressing Ca2+- mobilizing P2Y receptors, as indicated by the proportion of cells lacking [Ca2+]i responses and exhibiting [Na+]i responses to ATP in the absence and presence of Ca2+o, respectively. The density of P2X receptors in NA-cells appeared to be 30-50% larger, as suggested by comparing the average size of the [Na+]i and [Ca2+]i responses to ATP. Conversely, approximately twice as many AD-cells expressed Ca2+-mobilizing P2Y receptors, and they appeared to exhibit a higher (~20%) receptor density. UTP raised the [Ca2+]i in a fraction of the cells and did not raise the [Na+]i in any of the cells tested, confirming its specificity as a P2Y agonist. The cell density of UTP-sensitive P2Y receptors did not appear to vary among AD- and NA-cells. Although neither of the major purinoceptor types can be ascribed to a particular cell phenotype, P2X and Ca2+-mobilizing P2Y receptors are preferentially located to noradrenergic and adrenergic chromaffin cells, respectively. ATP might, in addition to an UTP-sensitive P2Y receptor, activate an UTP-insensitive P2Y receptor subtype. A model for a short-loop feedback interaction is presented whereby locally released ATP acts upon P2Y receptors in adrenergic cells, inhibiting Ca2+ influx and contributing to terminate evoked epinephrine secretion.
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Immunobiological Aspects of erbB Receptors in Breast Cancer
2000-08-01
receptor . The proliferation of cells expressing these chimeric receptors was EGF-dependent, and cells expressing EGFR/Y882F chimeric receptors were...determine Cells were washed twice with cold phosphate-buffered saline which cellular substrates couple with the receptor complex. (PBS) and lysed with 1...turnover, receptor proteins suggests that these substrates are properly lo- and cellular transformation in NEN757 cells (Qian et al., cated for
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Landowski, Matthew; Dabundo, Jeffrey; Liu, Qian; Nicola, Anthony V; Aguilar, Hector C
2014-12-01
Virus-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual virus-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of virus-cell membrane fusion. Drastic differences between viral and cellular membrane lipid and protein compositions and curvatures exist. For biosafety level 4 (BSL4) pathogens such as the deadly Nipah virus (NiV), virus-cell fusion mechanistic studies are notably cumbersome. To circumvent these limitations, we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. NiV's attachment (G) and fusion (F) envelope glycoproteins mediate viral binding to the ephrinB2/ephrinB3 cell receptors and virus-cell membrane fusion, respectively. The NiV matrix protein (M) can autonomously induce NiV assembly and budding. Using a β-lactamase (βLa) reporter/NiV-M chimeric protein, we produced NiVLPs expressing NiV-G and wild-type or mutant NiV-F on their surfaces. By preloading target cells with the βLa fluorescent substrate CCF2-AM, we obtained viral entry kinetic curves that correlated with the NiV-F fusogenic phenotypes, validating NiVLPs as suitable viral entry kinetic tools and suggesting overall relatively slower viral entry than cell-cell fusion kinetics. Additionally, the proportions of F and G on individual NiVLPs and the extent of receptor-induced conformational changes in NiV-G were measured via flow virometry, allowing the proper interpretation of the viral entry kinetic phenotypes. The significance of these findings in the viral entry field extends beyond NiV to other paramyxoviruses and enveloped viruses. Virus-cell membrane fusion is essential for enveloped virus infections. However, mechanistic viral membrane fusion studies have predominantly focused on cell-cell fusion models, largely due to the low availability of technologies capable of characterizing actual virus-cell membrane fusion. Although cell-cell fusion assays are valuable, they do not fully recapitulate all the variables of virus-cell membrane fusion. For example, drastic differences between viral and cellular membrane lipid and protein compositions and curvatures exist. For biosafety level 4 (BSL4) pathogens such as the deadly Nipah virus (NiV), virus-cell fusion mechanistic studies are especially cumbersome. To circumvent these limitations, we used enzymatic Nipah virus-like-particles (NiVLPs) and developed new flow virometric tools. Our new tools allowed us the high-throughput measurement of viral entry kinetics, glycoprotein proportions on individual viral particles, and receptor-induced conformational changes in viral glycoproteins on viral surfaces. The significance of these findings extends beyond NiV to other paramyxoviruses and enveloped viruses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Poliovirus Mutants Resistant to Neutralization with Soluble Cell Receptors
NASA Astrophysics Data System (ADS)
Kaplan, Gerardo; Peters, David; Racaniello, Vincent R.
1990-12-01
Poliovirus mutants resistant to neutralization with soluble cellular receptor were isolated. Replication of soluble receptor-resistant (srr) mutants was blocked by a monoclonal antibody directed against the HeLa cell receptor for poliovirus, indicating that the mutants use this receptor to enter cells. The srr mutants showed reduced binding to HeLa cells and cell membranes. However, the reduced binding phenotype did not have a major impact on viral replication, as judged by plaque size and one-step growth curves. These results suggest that the use of soluble receptors as antiviral agents could lead to the selection of neutralization-resistant mutants that are able to bind cell surface receptors, replicate, and cause disease.
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Allain, F; Denys, A; Spik, G
1996-07-15
Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein located within intracellular vesicles and released in biological fluids. We recently reported the specific binding of this protein to T-cell surface receptor which is internalized even in the presence of CsA. These results suggest that CyPB might target the drug to lymphocytes and consequently modify its activity. To verify this hypothesis, we have first investigated the binding capacity and internalization of the CsA-CyPB complex in human peripheral blood T-lymphocytes and secondly compared the inhibitory effect of both free and CyPB-complexed CsA on the CD3-induced activation and proliferation of T-cells. Here, we present evidence that both the CsA-CyPB complex and free CyPB bind to the T-lymphocyte surface, with similar values of Kd and number of sites. At 37 degrees C, the complex is internalized but, in contrast to the protein, the drug is accumulated within the cell. Moreover, CyPB receptors are internalized together with the ligand and rapidly recycled to the cell surface. Finally, we demonstrate that CyPB-complexed CsA remains as efficient as uncomplexed CsA and that CyPB enhances the immunosuppressive activity of the drug. Taken together, our results support the hypothesis that surface CyPB receptors may be related to the selective and variable action of CsA, through specific binding and targeting of the CyPB-CsA complex to peripheral blood T-lymphocytes.
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Breast carcinoma metastasis to the lacrimal gland: Two case reports
NICKELSEN, MARIE N.; VON HOLSTEIN, SARAH; HANSEN, ALASTAIR B.; PRAUSE, JAN U.; HEEGAARD, STEFFEN
2015-01-01
A 77-year-old female, with proptosis, reduced eye motility and diplopia which had developed over two to three months and a 69-year-old female with proptosis, oedema of the eyelid, reduced motility and ptosis, which had developed over three weeks, are presented in the present study. Computed tomography scans revealed irregular lacrimal gland tumours in the two patients. The two patients had history of breast cancer. The first breast cancer metastasis in the lacrimal gland demonstrated a cribriform growth pattern containing ductal elements. The epithelial tumour cells stained positive for cytokeratin (1–8, 10, 14–16, 18 and 19), oestrogen receptor, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA) and gross cystic disease fluid protein 15 (GCDFP-15). The second metastatic tumour was positive for EMA and estrogen receptor, but variably positive for CEA and GCDFP-15. The metastasis in the lacrimal gland was a pleomorphic tumour. The tumour cells were positive for EMA and variably positive for oestrogen and CEA. Metastases to the lacrimal gland are extremely rare, and metastases to the lacrimal gland should be considered in the diagnoses of lacrimal gland tumours. The present study aimed to describe two such cases and draw attention to breast carcinomas as a differential diagnosis and the most frequent cause of lacrimal gland metastasis. PMID:26622620
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Cell death sensitization of leukemia cells by opioid receptor activation
Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich
2013-01-01
Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472
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Palomba, M. Lia; Piersanti, Kelly; Ziegler, Carly G. K.; Decker, Hugo; Cotari, Jesse W.; Bantilan, Kurt; Rijo, Ivelise; Gardner, Jeff R.; Heaney, Mark; Bemis, Debra; Balderas, Robert; Malek, Sami N.; Seymour, Erlene; Zelenetz, Andrew D.
2014-01-01
Purpose Chronic Lymphocytic Leukemia (CLL) is defined by a perturbed B-cell receptor-mediated signaling machinery. We aimed to model differential signaling behavior between B cells from CLL and healthy individuals to pinpoint modes of dysregulation. Experimental Design We developed an experimental methodology combining immunophenotyping, multiplexed phosphospecific flow cytometry, and multifactorial statistical modeling. Utilizing patterns of signaling network covariance, we modeled BCR signaling in 67 CLL patients using Partial Least Squares Regression (PLSR). Results from multidimensional modeling were validated using an independent test cohort of 38 patients. Results We identified a dynamic and variable imbalance between proximal (pSYK, pBTK) and distal (pPLCγ2, pBLNK, ppERK) phosphoresponses. PLSR identified the relationship between upstream tyrosine kinase SYK and its target, PLCγ2, as maximally predictive and sufficient to distinguish CLL from healthy samples, pointing to this juncture in the signaling pathway as a hallmark of CLL B cells. Specific BCR pathway signaling signatures that correlate with the disease and its degree of aggressiveness were identified. Heterogeneity in the PLSR response variable within the B cell population is both a characteristic mark of healthy samples and predictive of disease aggressiveness. Conclusion Single-cell multidimensional analysis of BCR signaling permitted focused analysis of the variability and heterogeneity of signaling behavior from patient-to-patient, and from cell-to-cell. Disruption of the pSYK/pPLCγ2 relationship is uncovered as a robust hallmark of CLL B cell signaling behavior. Together, these observations implicate novel elements of the BCR signal transduction as potential therapeutic targets. PMID:24489640
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Su, Yan-Ye; Chen, Chang-Han; Chien, Chih-Yen; Lin, Wei-Che; Huang, Wan-Ting; Li, Shau-Hsuan
2017-01-01
Recent evidence suggests that the local renin-angiotensin system has been implicated in various malignancies. The mitochondrial assembly receptor is a newly identified receptor for angiotensin peptides, angiotensin-(1-7), and has an important role in the renin-angiotensin system. However, the role of the mitochondrial assembly receptor in the prognosis of cancer patients remains unclear. The aim of this study was to evaluate the significance of mitochondrial assembly receptor signaling in the prognosis of oral tongue squamous cell carcinoma. Mitochondrial assembly receptor immunohistochemistry was examined in 151 oral tongue squamous cell carcinoma patients and was correlated with treatment outcome. The functional relevance of the mitochondrial assembly receptor in oral tongue squamous cell carcinoma cell lines was evaluated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide reduction and bromodeoxyuridine incorporation assays. Mitochondrial assembly receptor overexpression was significantly correlated with early pathological T classification ( p=0.029) and the absence of extracapsular spread ( p=0.039). Univariate analyses demonstrated that mitochondrial assembly receptor overexpression was significantly associated with superior overall survival ( p=0.012). In multivariate comparison, mitochondrial assembly receptor overexpression remained independently associated with superior overall survival ( p=0.008, hazard ratio=1.862). In vitro, angiotensin-(1-7) suppressed the cell growth in oral tongue squamous cell carcinoma cells, and this response was reversed by the mitochondrial assembly receptor antagonist, A779. Mitochondrial assembly receptor expression is independently associated with the prognosis of oral tongue squamous cell carcinoma patients. These findings suggest that mitochondrial assembly receptor signaling may be a promising novel target for oral tongue squamous cell carcinoma.
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Muscarinic Control of MIN6 Pancreatic β Cells Is Enhanced by Impaired Amino Acid Signaling*
Guerra, Marcy L.; Wauson, Eric M.; McGlynn, Kathleen; Cobb, Melanie H.
2014-01-01
We have shown recently that the class C G protein-coupled receptor T1R1/T1R3 taste receptor complex is an early amino acid sensor in MIN6 pancreatic β cells. Amino acids are unable to activate ERK1/2 in β cells in which T1R3 has been depleted. The muscarinic receptor agonist carbachol activated ERK1/2 better in T1R3-depleted cells than in control cells. Ligands that activate certain G protein-coupled receptors in pancreatic β cells potentiate glucose-stimulated insulin secretion. Among these is the M3 muscarinic acetylcholine receptor, the major muscarinic receptor in β cells. We found that expression of M3 receptors increased in T1R3-depleted MIN6 cells and that calcium responses were altered. To determine whether these changes were related to impaired amino acid signaling, we compared responses in cells exposed to reduced amino acid concentrations. M3 receptor expression was increased, and some, but not all, changes in calcium signaling were mimicked. These findings suggest that M3 acetylcholine receptors are increased in β cells as a mechanism to compensate for amino acid deficiency. PMID:24695728
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The effect of interferon on the receptor sites to rabies virus on mouse neuroblastoma cells
DOE Office of Scientific and Technical Information (OSTI.GOV)
Briggs, D.J.
1989-01-01
The binding of rabies virus to mouse neuroblastoma cells (MNA) primed with alpha interferon (IFN-{alpha}), beta interferon (IFN-{beta}), or alpha bungarotoxin (BTX) was examined. A saturable number of receptor sites to rabies virus was calculated by increasing the amount of {sup 3}H-CVS added to a constant number of untreated MNA cells. MNA cells were then exposed to 20 I.U. of IFN-{alpha}, IFN-{beta}, or 1 {mu}g of BTX and assayed to determine if these treatments had an effect on the number of receptor sites to rabies virus. Total amount of {sup 3}H-CVS bound to MNA cells was determined during a threemore » hour incubation period. Cold competition assays using 1,000 fold excess unlabeled CVS were used to determine non-specific binding for each treatment. Specific binding was then calculated by subtracting non-specific binding from the total amount of CVS bound to MNA cells. A similar amount of total viral protein bound to untreated and IFN-{beta}, and BTX treated cells after 180 minutes of incubation. The bound protein varied by only 0.07 {mu}g. However, the amount of specific and non-specific binding varied a great deal between treatments. BTX caused an increase in non-specific and a decrease in specific binding of rabies virus. IFN-{beta} produced variable results in non-specific and specific binding while IFN-{alpha} caused mainly specific binding to occur. The most significant change brought about by IFN-{alpha} was an increase in the rate of viral attachment. At 30 minutes post-infection, IFN-{alpha} treated cells had bound 90% of the total amount of virus bound to untreated cells after 180 minutes. The increased binding rate did not cause a productive infection of rabies virus. No viral production was evident after an incubation period of 48 hours in either IFN-{alpha} or IFN-{beta} treated cells.« less
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Ligand-targeted theranostic nanomedicines against cancer.
Yao, Virginia J; D'Angelo, Sara; Butler, Kimberly S; Theron, Christophe; Smith, Tracey L; Marchiò, Serena; Gelovani, Juri G; Sidman, Richard L; Dobroff, Andrey S; Brinker, C Jeffrey; Bradbury, Andrew R M; Arap, Wadih; Pasqualini, Renata
2016-10-28
Nanomedicines have significant potential for cancer treatment. Although the majority of nanomedicines currently tested in clinical trials utilize simple, biocompatible liposome-based nanocarriers, their widespread use is limited by non-specificity and low target site concentration and thus, do not provide a substantial clinical advantage over conventional, systemic chemotherapy. In the past 20years, we have identified specific receptors expressed on the surfaces of tumor endothelial and perivascular cells, tumor cells, the extracellular matrix and stromal cells using combinatorial peptide libraries displayed on bacteriophage. These studies corroborate the notion that unique receptor proteins such as IL-11Rα, GRP78, EphA5, among others, are differentially overexpressed in tumors and present opportunities to deliver tumor-specific therapeutic drugs. By using peptides that bind to tumor-specific cell-surface receptors, therapeutic agents such as apoptotic peptides, suicide genes, imaging dyes or chemotherapeutics can be precisely and systemically delivered to reduce tumor growth in vivo, without harming healthy cells. Given the clinical applicability of peptide-based therapeutics, targeted delivery of nanocarriers loaded with therapeutic cargos seems plausible. We propose a modular design of a functionalized protocell in which a tumor-targeting moiety, such as a peptide or recombinant human antibody single chain variable fragment (scFv), is conjugated to a lipid bilayer surrounding a silica-based nanocarrier core containing a protected therapeutic cargo. The functionalized protocell can be tailored to a specific cancer subtype and treatment regimen by exchanging the tumor-targeting moiety and/or therapeutic cargo or used in combination to create unique, theranostic agents. In this review, we summarize the identification of tumor-specific receptors through combinatorial phage display technology and the use of antibody display selection to identify recombinant human scFvs against these tumor-specific receptors. We compare the characteristics of different types of simple and complex nanocarriers, and discuss potential types of therapeutic cargos and conjugation strategies. The modular design of functionalized protocells may improve the efficacy and safety of nanomedicines for future cancer therapy. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.
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Acetylcholine is released from taste cells, enhancing taste signalling
Dando, Robin; Roper, Stephen D
2012-01-01
Acetylcholine (ACh), a candidate neurotransmitter that has been implicated in taste buds, elicits calcium mobilization in Receptor (Type II) taste cells. Using RT-PCR analysis and pharmacological interventions, we demonstrate that the muscarinic acetylcholine receptor M3 mediates these actions. Applying ACh enhanced both taste-evoked Ca2+ responses and taste-evoked afferent neurotransmitter (ATP) secretion from taste Receptor cells. Blocking muscarinic receptors depressed taste-evoked responses in Receptor cells, suggesting that ACh is normally released from taste cells during taste stimulation. ACh biosensors confirmed that, indeed, taste Receptor cells secrete acetylcholine during gustatory stimulation. Genetic deletion of muscarinic receptors resulted in significantly diminished ATP secretion from taste buds. The data demonstrate a new role for acetylcholine as a taste bud transmitter. Our results imply specifically that ACh is an autocrine transmitter secreted by taste Receptor cells during gustatory stimulation, enhancing taste-evoked responses and afferent transmitter secretion. PMID:22570381
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The G-Protein-Coupled Estrogen Receptor (GPER/GPR30) in Ovarian Granulosa Cell Tumors
Heublein, Sabine; Mayr, Doris; Friese, Klaus; Jarrin-Franco, Maria Cristina; Lenhard, Miriam; Mayerhofer, Artur; Jeschke, Udo
2014-01-01
Ovarian granulosa cell tumors (GCTs) are thought to arise from cells of the ovarian follicle and comprise a rare entity of ovarian masses. We recently identified the G-protein-coupled estrogen receptor (GPER/GPR30) to be present in granulosa cells, to be regulated by gonadotropins in epithelial ovarian cancer and to be differentially expressed throughout folliculogenesis. Thus, supposing a possible role of GPER in GCTs, this study aimed to analyze GPER in GCTs. GPER immunoreactivity in GCTs (n = 26; n (primary diagnosis) = 15, n (recurrence) = 11) was studied and correlated with the main clinicopathological variables. Positive GPER staining was identified in 53.8% (14/26) of GCTs and there was no significant relation of GPER with tumor size or lymph node status. Those cases presenting with strong GPER intensity at primary diagnosis showed a significant reduced overall survival (p = 0.002). Due to the fact that GPER is regulated by estrogens, as well as gonadotropins, GPER may also be affected by endocrine therapies applied to GCT patients. Moreover, with our data supposing GPER to be associated with GCT prognosis, GPER might be considered as a possible confounder when assessing the efficacy of hormone-based therapeutic approaches in GCTs. PMID:25167139
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Yaniv, Yael; Ahmet, Ismayil; Tsutsui, Kenta; Behar, Joachim; Moen, Jack M; Okamoto, Yosuke; Guiriba, Toni-Rose; Liu, Jie; Bychkov, Rostislav; Lakatta, Edward G
2016-08-01
We aimed to determine how age-associated changes in mechanisms extrinsic and intrinsic to pacemaker cells relate to basal beating interval variability (BIV) reduction in vivo. Beating intervals (BIs) were measured in aged (23-25 months) and adult (3-4 months) C57BL/6 male mice (i) via ECG in vivo during light anesthesia in the basal state, or in the presence of 0.5 mg mL(-1) atropine + 1 mg mL(-1) propranolol (in vivo intrinsic conditions), and (ii) via a surface electrogram, in intact isolated pacemaker tissue. BIV was quantified in both time and frequency domains using linear and nonlinear indices. Although the average basal BI did not significantly change with age under intrinsic conditions in vivo and in the intact isolated pacemaker tissue, the average BI was prolonged in advanced age. In vivo basal BIV indices were found to be reduced with age, but this reduction diminished in the intrinsic state. However, in pacemaker tissue BIV indices increased in advanced age vs. adults. In the isolated pacemaker tissue, the sensitivity of the average BI and BIV in response to autonomic receptor stimulation or activation of mechanisms intrinsic to pacemaker cells by broad-spectrum phosphodiesterase inhibition declined in advanced age. Thus, changes in mechanisms intrinsic to pacemaker cells increase the average BIs and BIV in the mice of advanced age. Autonomic neural input to pacemaker tissue compensates for failure of molecular intrinsic mechanisms to preserve average BI. But this compensation reduces the BIV due to both the imbalance of autonomic neural input to the pacemaker cells and altered pacemaker cell responses to neural input. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
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Saini, Jasmine; Hershberg, Uri
2015-01-01
The exceptional ability of B cells to diversify through somatic mutation and improve affinity of the repertoire towards the antigens is the cornerstone of adaptive immunity. Somatic mutation is not evenly distributed and exhibits certain micro-sequence specificities. We show here that the combination of somatic mutation targeting and the codon usage in human B cell receptor (BCR) Variable (V) genes create expected patterns of mutation and post mutation changes that are focused on their complementarity determining regions (CDR). T cell V genes are also skewed in targeting mutations but to a lesser extent and are lacking the codon usage bias observed in BCRs. This suggests that the observed skew in T cell receptors is due to their amino acid usage, which is similar to that of BCRs. The mutation targeting and the codon bias allow B cell CDRs to diversify by specifically accumulating nonconservative changes. We counted the distribution of mutations to CDR in 4 different human datasets. In all four cases we found that the number of actual mutations in the CDR correlated significantly with the V gene mutation biases to the CDR predicted by our models. Finally, it appears that the mutation bias in V genes indeed relates to their long-term survival in actual human repertoires. We observed that resting repertoires of B cells overexpressed V genes that were especially biased towards focused mutation and change in the CDR. This bias in V gene usage was somewhat relaxed at the height of the immune response to a vaccine, presumably because of the need for a wider diversity in a primary response. However, older patients did not retain this flexibility and were biased towards using only highly skewed V genes at all stages of their response. PMID:25660968
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Saini, Jasmine; Hershberg, Uri
2015-05-01
The exceptional ability of B cells to diversify through somatic mutation and improve affinity of the repertoire toward the antigens is the cornerstone of adaptive immunity. Somatic mutation is not evenly distributed and exhibits certain micro-sequence specificities. We show here that the combination of somatic mutation targeting and the codon usage in human B cell receptor (BCR) Variable (V) genes create expected patterns of mutation and post mutation changes that are focused on their complementarity determining regions (CDR). T cell V genes are also skewed in targeting mutations but to a lesser extent and are lacking the codon usage bias observed in BCRs. This suggests that the observed skew in T cell receptors is due to their amino acid usage, which is similar to that of BCRs. The mutation targeting and the codon bias allow B cell CDRs to diversify by specifically accumulating nonconservative changes. We counted the distribution of mutations to CDR in 4 different human datasets. In all four cases we found that the number of actual mutations in the CDR correlated significantly with the V gene mutation biases to the CDR predicted by our models. Finally, it appears that the mutation bias in V genes indeed relates to their long-term survival in actual human repertoires. We observed that resting repertoires of B cells overexpressed V genes that were especially biased toward focused mutation and change in the CDR. This bias in V gene usage was somewhat relaxed at the height of the immune response to a vaccine, presumably because of the need for a wider diversity in a primary response. However, older patients did not retain this flexibility and were biased toward using only highly skewed V genes at all stages of their response. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Human IL-21 and IL-21R deficiencies: two novel entities of primary immunodeficiency.
Kotlarz, Daniel; Ziętara, Natalia; Milner, Joshua D; Klein, Christoph
2014-12-01
This review highlights the recent identification of human interleukin-21 (IL-21) and interleukin-21 receptor (IL-21R) deficiencies as novel entities of primary immunodeficiency. We recently described the first patients with IL-21R deficiency who had cryptosporidial infections associated with chronic cholangitis and liver disease. All IL-21R-deficient patients suffered from recurrent respiratory tract infections. Immunological work-up revealed impaired B cell proliferation and immunoglobulin class-switch, reduced T cell effector functions, and variable natural killer cell dysfunctions. Recently, these findings have been extended by the discovery of one patient with a mutation in the IL21 gene. This patient predominantly manifested with very early onset inflammatory bowel disease and recurrent respiratory infections. Laboratory examination showed reduced circulating B cells and impaired B cell class-switch. Human IL-21 and IL-21R deficiencies cause severe, primary immunodeficiency reminiscent of common variable immunodeficiency. Early diagnosis is critical to prevent life-threatening complications, such as secondary liver failure. In view of the critical role of IL-21 in controlling immune homeostasis, early hematopoietic stem cell transplantation might be considered as therapeutic intervention in affected children.
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Ohshima, Koichi; Karube, Kennosuke; Hamasaki, Makoto; Tutiya, Takeshi; Yamaguchi, Takahiro; Suefuji, Hiroaki; Suzuki, Keiko; Suzumiya, Junji; Ohga, Shouichi; Kikuchi, Masahiro
2003-08-01
T cell immunity plays an important role in the clinicopathology of Epstein-Barr virus (EBV)-associated diseases. Acute EBV-induced infectious mononucleosis (IM) is a common self-limiting disease, however, other EBV-associated diseases, including chronic active EBV infection (CAEBV), NK cell lymphoma (NKL), and Hodgkin's lymphoma (HL), exhibit distinct clinical features. Chemokines are members of a family of small-secreted proteins. The relationships between chemokines and the chemokine receptor (R) are thought to be important for selectivity of local immunity. Some chemokines, chemokine R and cytokines closely associate with the T cell subtypes, Th1 and Th2 T cells and cytotoxic cells. To clarify the role of T cell immunity in EBV-associated diseases, we conducted gene expression profiling, using chemokine, chemokine R and cytokine DNA chips. Compared to EBV negative non-specific lymphadenitis, CAEBV and NKL exhibited diffuse down- and up-regulation, respectively, of these gene profiles. IM had a predominantly Th1-type profile, whereas HL had a mixed Th1/Th2-type profile. Reduction of the Th1-type cytokine interferon gamma (IFN-gamma) in CAEBV was confirmed by Reverse transcriptase-polymerase chain reaction, whereas IFN-gamma expression was markedly enhanced in NKL, and moderately enhanced in IM. Compared to IM, CAEBV showed slight elevation of "regulated upon activation, normal T expressed and secreted" (RANTES), but almost all other genes assayed were down-regulated. NKL exhibited elevated expression of numerous genes, particularly IFN-gamma-inducible-10 (IP-10) and monokine induced by IFN-gamma (MIG). HL showed variable elevated and reduced expression of various genes, with increased expression of IL-13 receptor and MIG. Our study demonstrated the enormous potential of gene expression profiling for clarifying the pathogenesis of EBV-associated diseases.
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Allelic polymorphism in the T cell receptor and its impact on immune responses.
Gras, Stephanie; Chen, Zhenjun; Miles, John J; Liu, Yu Chih; Bell, Melissa J; Sullivan, Lucy C; Kjer-Nielsen, Lars; Brennan, Rebekah M; Burrows, Jacqueline M; Neller, Michelle A; Khanna, Rajiv; Purcell, Anthony W; Brooks, Andrew G; McCluskey, James; Rossjohn, Jamie; Burrows, Scott R
2010-07-05
In comparison to human leukocyte antigen (HLA) polymorphism, the impact of allelic sequence variation within T cell receptor (TCR) loci is much less understood. Particular TCR loci have been associated with autoimmunity, but the molecular basis for this phenomenon is undefined. We examined the T cell response to an HLA-B*3501-restricted epitope (HPVGEADYFEY) from Epstein-Barr virus (EBV), which is frequently dominated by a TRBV9*01(+) public TCR (TK3). However, the common allelic variant TRBV9*02, which differs by a single amino acid near the CDR2beta loop (Gln55-->His55), was never used in this response. The structure of the TK3 TCR, its allelic variant, and a nonnaturally occurring mutant (Gln55-->Ala55) in complex with HLA-B*3501(HPVGEADYFEY) revealed that the Gln55-->His55 polymorphism affected the charge complementarity at the TCR-peptide-MHC interface, resulting in reduced functional recognition of the cognate and naturally occurring variants of this EBV peptide. Thus, polymorphism in the TCR loci may contribute toward variability in immune responses and the outcome of infection.
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Wang, Shi Qiang; Stern, Michael D; Ríos, Eduardo; Cheng, Heping
2004-03-16
Intracellular Ca(2+) release in many types of cells is mediated by ryanodine receptor Ca(2+) release channels (RyRCs) that are assembled into two-dimensional paracrystalline arrays in the endoplasmic/sarcoplasmic reticulum. However, the in situ operating mechanism of the RyRC array is unknown. Here, we found that the elementary Ca(2+) release events, Ca(2+) sparks from individual RyRC arrays in rat ventricular myocytes, exhibit quantized Ca(2+) release flux. Analysis of the quantal property of Ca(2+) sparks provided a view of unitary Ca(2+) current and gating kinetics of the RyRC in intact cells and revealed that spark activation involves dynamic recruitment of small, variable cohorts of RyRCs. Intriguingly, interplay of RyRCs in multichannel sparks renders an unusual, thermodynamically irreversible mode of channel gating that is unshared by an RyRC acting solo, nor by RyRCs in vitro. Furthermore, an array-based inhibitory feedback, overriding the regenerative Ca(2+)-induced Ca(2+) release of RyRCs, provides a supramolecular mechanism for the microscopic stability of intracellular Ca(2+) signaling.
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Wang, Pei-Chen; Ho, Ing-Kang; Lee, Cynthia Wei-Sheng
2015-01-01
Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via κ- (KOP), μ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium-labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N-linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists— U-69593, DAMGO and nociceptin— inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration-dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP-expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration-dependent AC superactivation elicited by chronic buprenorphine exposure. PMID:26153065
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Lindner, Diana; Walther, Cornelia; Tennemann, Anja; Beck-Sickinger, Annette G
2009-01-01
The N terminus is the most variable element in G protein-coupled receptors (GPCRs), ranging from seven residues up to approximately 5900 residues. For family B and C GPCRs it is described that at least part of the ligand binding site is located within the N terminus. Here we investigated the role of the N terminus in the neuropeptide Y receptor family, which belongs to the class A of GPCRs. We cloned differentially truncated Y receptor mutants, in which the N terminus was partially or completely deleted. We found, that eight amino acids are sufficient for full ligand binding and signal transduction activity. Interestingly, we could show that no specific amino acids but rather the extension of the first transmembrane helix by any residues is sufficient for receptor activity but also for membrane integration in case of the hY(1) and the hY(4) receptors. In contrast, the complete deletion of the N terminus in the hY(2) receptors resulted in a mutant that is fully integrated in the membrane but does not bind the ligand very well and internalizes much slower compared to the wild type receptor. Interestingly, also these effects could be reverted by any N-terminal extension. Accordingly, the most important function of the N termini seems to be the stabilization of the first transmembrane helix to ensure the correct receptor structure, which obviously is essential for ligand binding, integration into the cell membrane and receptor internalization.
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Recent Observations on Australian Bat Lyssavirus Tropism and Viral Entry
Weir, Dawn L.; Annand, Edward J.; Reid, Peter A.; Broder, Christopher C.
2014-01-01
Australian bat lyssavirus (ABLV) is a recently emerged rhabdovirus of the genus lyssavirus considered endemic in Australian bat populations that causes a neurological disease in people indistinguishable from clinical rabies. There are two distinct variants of ABLV, one that circulates in frugivorous bats (genus Pteropus) and the other in insectivorous microbats (genus Saccolaimus). Three fatal human cases of ABLV infection have been reported, the most recent in 2013, and each manifested as acute encephalitis but with variable incubation periods. Importantly, two equine cases also arose recently in 2013, the first occurrence of ABLV in a species other than bats or humans. Similar to other rhabdoviruses, ABLV infects host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion facilitated by its single fusogenic envelope glycoprotein (G). Recent studies have revealed that proposed rabies virus (RABV) receptors are not sufficient to permit ABLV entry into host cells and that the unknown receptor is broadly conserved among mammalian species. However, despite clear tropism differences between ABLV and RABV, the two viruses appear to utilize similar endocytic entry pathways. The recent human and horse infections highlight the importance of continued Australian public health awareness of this emerging pathogen. PMID:24556791
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Recent observations on Australian bat lyssavirus tropism and viral entry.
Weir, Dawn L; Annand, Edward J; Reid, Peter A; Broder, Christopher C
2014-02-19
Australian bat lyssavirus (ABLV) is a recently emerged rhabdovirus of the genus lyssavirus considered endemic in Australian bat populations that causes a neurological disease in people indistinguishable from clinical rabies. There are two distinct variants of ABLV, one that circulates in frugivorous bats (genus Pteropus) and the other in insectivorous microbats (genus Saccolaimus). Three fatal human cases of ABLV infection have been reported, the most recent in 2013, and each manifested as acute encephalitis but with variable incubation periods. Importantly, two equine cases also arose recently in 2013, the first occurrence of ABLV in a species other than bats or humans. Similar to other rhabdoviruses, ABLV infects host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion facilitated by its single fusogenic envelope glycoprotein (G). Recent studies have revealed that proposed rabies virus (RABV) receptors are not sufficient to permit ABLV entry into host cells and that the unknown receptor is broadly conserved among mammalian species. However, despite clear tropism differences between ABLV and RABV, the two viruses appear to utilize similar endocytic entry pathways. The recent human and horse infections highlight the importance of continued Australian public health awareness of this emerging pathogen.
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Xin, Xiu; Wang, Hailong; Han, Lingling; Wang, Mingzhen; Fang, Hui; Hao, Yao; Li, Jiadai; Zhang, Hu; Zheng, Congyi; Shen, Chao
2018-05-01
Viral infection and replication are affected by host cell heterogeneity, but the mechanisms underlying the effects remain unclear. Using single-cell analysis, we investigated the effects of host cell heterogeneity, including cell size, inclusion, and cell cycle, on foot-and-mouth disease virus (FMDV) infection (acute and persistent infections) and replication. We detected various viral genome replication levels in FMDV-infected cells. Large cells and cells with a high number of inclusions generated more viral RNA copies and viral protein and a higher proportion of infectious cells than other cells. Additionally, we found that the viral titer was 10- to 100-fold higher in cells in G 2 /M than those in other cell cycle phases and identified a strong correlation between cell size, inclusion, and cell cycle heterogeneity, which all affected the infection and replication of FMDV. Furthermore, we demonstrated that host cell heterogeneity influenced the adsorption of FMDV due to differences in the levels of FMDV integrin receptors expression. Collectively, these results further our understanding of the evolution of a virus in a single host cell. IMPORTANCE It is important to understand how host cell heterogeneity affects viral infection and replication. Using single-cell analysis, we found that viral genome replication levels exhibited dramatic variability in foot-and-mouth disease virus (FMDV)-infected cells. We also found a strong correlation between heterogeneity in cell size, inclusion number, and cell cycle status and that all of these characteristics affect the infection and replication of FMDV. Moreover, we found that host cell heterogeneity influenced the viral adsorption as differences in the levels of FMDV integrin receptors' expression. This study provided new ideas for the studies of correlation between FMDV infection mechanisms and host cells. Copyright © 2018 American Society for Microbiology.
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Allegra, J C; Korat, O; Do, H M; Lippman, M
1981-01-01
The regulation of progesterone receptor by 17 beta estradiol and tamoxifen in the ZR-75-1 human breast cancer cell line in defined medium is described. ZR-75-1 cells maintained in serum free hormone supplemented medium minus estradiol lack progesterone receptor activity. Readdition of estradiol to these cells leads to a marked stimulation of progesterone receptor activity (0 to greater than 100 fmols of specifically bound progesterone per million cells). Tamoxifen (10(-6)M-10(-8)M) does not stimulate progesterone receptor activity in this cell line. The presence of progesterone receptor activity is not directly related to growth. Withdrawal of insulin in the continued presence of estradiol has no effect on progesterone receptor concentration although net cell growth ceases. Conversely, withdrawal of estradiol in the continued presence of insulin induces a cessation of net cell growth accompanied by a loss of all progesterone receptor activity within 3-5 days.
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Gray, J A; Sheffler, D J; Bhatnagar, A; Woods, J A; Hufeisen, S J; Benovic, J L; Roth, B L
2001-11-01
The effect of endocytosis inhibitors on 5-hydroxytryptamine(2A) (5-HT(2A)) receptor desensitization and resensitization was examined in transiently transfected human embryonic kidney (HEK) 293 cells and in C6 glioma cells that endogenously express 5-HT(2A) receptors. In HEK-293 cells, 5-HT(2A) receptor desensitization was unaffected by cotransfection with a dominant-negative mutant of dynamin (DynK44A), a truncation mutant of arrestin-2 [Arr2(319-418)], or by two well-characterized chemical inhibitors of endocytosis: concanavalin A (conA) and phenylarsine oxide (PAO). In contrast, beta 2-adrenergic receptor desensitization was significantly potentiated by each of these treatments in HEK-293 cells. In C6 glioma cells, however, DynK44A, Arr2(319-418), conA, and PAO each resulted in the potentiation of 5-HT(2A) and beta-adrenergic receptor desensitization. The cell-type-specific effect of Arr2(319-418) on 5-HT(2A) receptor desensitization was not related to the level of GRK2 or GRK5 expression. Interestingly, although beta 2-adrenergic receptor resensitization was potently blocked by cotransfection with DynK44A, 5-HT(2A) receptor resensitization was enhanced, suggesting the existence of a novel cell-surface mechanism for 5-HT(2A) receptor resensitization in HEK-293 cells. In addition, Arr2(319-418) had no effect on 5-HT(2A) receptor resensitization in HEK-293 cells, although it attenuated the resensitization of the beta 2-adrenergic receptor. However, in C6 glioma cells, both DynK44A and Arr2(319-418) significantly reduced 5-HT(2A) receptor resensitization. Taken together, these results provide the first convincing evidence of cell-type-specific roles for endocytosis inhibitors in regulating GPCR activity. Additionally, these results imply that novel GRK and arrestin-independent mechanisms of 5-HT(2A) receptor desensitization and resensitization exist in HEK-293 cells.
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NASA Astrophysics Data System (ADS)
Sears, Amy E.; McGwire, Bradford S.; Roizman, Bernard
1991-06-01
Herpes simplex virus 1 attaches to at least two cell surface receptors. In polarized epithelial (Madin-Darby canine kidney; MDCK) cells one receptor is located in the apical surface and attachment to the cells requires the presence of glycoprotein C in the virus. The second receptor is located in the basal surface and does not require the presence of glycoprotein C. Exposure of MDCK cells at either the apical or basal surface to wild-type virus yields plaques and viral products whereas infection by a glycoprotein C-negative mutant yields identical results only after exposure of MDCK cells to virus at the basal surface. Multiple receptors for viral entry into cells expand the host range of the virus. The observation that glycoprotein C-negative mutants are infectious in many nonpolarized cell lines suggests that cells in culture may express more than one receptor and explains why genes that specify the viral proteins that recognize redundant receptors, like glycoprotein C, are expendable.
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Cambiaghi, Valeria; Vitali, Eleonora; Morone, Diego; Peverelli, Erika; Spada, Anna; Mantovani, Giovanna; Lania, Andrea Gerardo
2017-04-01
Somatostatin exerts inhibitory effects on hormone secretion and cell proliferation via five receptor subtypes (SST1-SST5), whose internalization is regulated by β-arrestins. The receptor domains involved in these effects have been only partially elucidated. The aim of the study is to characterize the molecular mechanism and determinants responsible for somatostatin receptor 2 internalization and signaling in pancreatic neuroendocrine QGP-1 cell line, focusing on the third intracellular loop and carboxyl terminal domains. We demonstrated that in cells transfected with somatostatin receptor 2 third intracellular loop mutant, no differences in β-arrestins recruitment and receptor internalization were observed after somatostatin receptor 2 activation in comparison with cells bearing wild-type somatostatin receptor 2. Conversely, the truncated somatostatin receptor 2 failed to recruit β-arrestins and to internalize after somatostatin receptor 2 agonist (BIM23120) incubation. Moreover, the inhibitory effect of BIM23120 on cell proliferation, cyclin D1 expression, P-ERK1/2 levels, apoptosis and vascular endothelial growth factor secretion was completely lost in cells transfected with either third intracellular loop or carboxyl terminal mutants. In conclusion, we demonstrated that somatostatin receptor 2 internalization requires intact carboxyl terminal while the effects of SS on cell proliferation, angiogenesis and apoptosis mediated by somatostatin receptor 2 need the integrity of both third intracellular loop and carboxyl terminal.
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The neuropeptides CCK and NPY and the changing view of cell-to-cell communication in the taste bud.
Herness, Scott; Zhao, Fang-Li
2009-07-14
The evolving view of the taste bud increasingly suggests that it operates as a complex signal processing unit. A number of neurotransmitters and neuropeptides and their corresponding receptors are now known to be expressed in subsets of taste receptor cells in the mammalian bud. These expression patterns set up hard-wired cell-to-cell communication pathways whose exact physiological roles still remain obscure. As occurs in other cellular systems, it is likely that neuropeptides are co-expressed with neurotransmitters and function as neuromodulators. Several neuropeptides have been identified in taste receptor cells including cholecystokinin (CCK), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and glucagon-like peptide 1 (GLP-1). Of these, CCK and NPY are the best studied. These two peptides are co-expressed in the same presynaptic cells; however, their postsynaptic actions are both divergent and antagonistic. CCK and its receptor, the CCK-1 subtype, are expressed in the same subset of taste receptor cells and the autocrine activation of these cells produces a number of excitatory physiological actions. Further, most of these cells are responsive to bitter stimuli. On the other hand, NPY and its receptor, the NPY-1 subtype, are expressed in different cells. NPY, acting in a paracrine fashion on NPY-1 receptors, results in inhibitory actions on the cell. Preliminary evidence suggests the NPY-1 receptor expressing cell co-expresses T1R3, a member of the T1R family of G-protein coupled receptors thought to be important in detection of sweet and umami stimuli. Thus the neuropeptide expressing cells co-express CCK, NPY, and CCK-1 receptor. Neuropeptides released from these cells during bitter stimulation may work in concert to both modulate the excitation of bitter-sensitive taste receptor cells while concurrently inhibiting sweet-sensitive cells. This modulatory process is similar to the phenomenon of lateral inhibition that occurs in other sensory systems.
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A Tupaia paramyxovirus vector system for targeting and transgene expression.
Engeland, Christine E; Bossow, Sascha; Hudacek, Andrew W; Hoyler, Birgit; Förster, Judith; Veinalde, Rūta; Jäger, Dirk; Cattaneo, Roberto; Ungerechts, Guy; Springfeld, Christoph
2017-09-01
Viruses from the diverse family of Paramyxoviridae include important pathogens and are applied in gene therapy and for cancer treatment. The Tupaia paramyxovirus (TPMV), isolated from the kidney of a tree shrew, does not infect human cells and neutralizing antibodies against other Paramyxoviridae do not cross-react with TPMV. Here, we present a vector system for de novo generation of infectious TPMV that allows for insertion of additional genes as well as targeting using antibody single-chain variable fragments. We show that the recombinant TPMV specifically infect cells expressing the targeted receptor and replicate in human cells. This vector system provides a valuable tool for both basic research and therapeutic applications.
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Kardava, Lela; Moir, Susan; Wang, Wei; Ho, Jason; Buckner, Clarisa M.; Posada, Jacqueline G.; O’Shea, Marie A.; Roby, Gregg; Chen, Jenny; Sohn, Hae Won; Chun, Tae-Wook; Pierce, Susan K.; Fauci, Anthony S.
2011-01-01
Chronic immune activation in HIV-infected individuals leads to accumulation of exhausted tissue-like memory B cells. Exhausted lymphocytes display increased expression of multiple inhibitory receptors, which may contribute to the inefficiency of HIV-specific antibody responses. Here, we show that downregulation of B cell inhibitory receptors in primary human B cells led to increased tissue-like memory B cell proliferation and responsiveness against HIV. In human B cells, siRNA knockdown of 9 known and putative B cell inhibitory receptors led to enhanced B cell receptor–mediated (BCR-mediated) proliferation of tissue-like memory but not other B cell subpopulations. The strongest effects were observed with the putative inhibitory receptors Fc receptor–like–4 (FCRL4) and sialic acid–binding Ig-like lectin 6 (Siglec-6). Inhibitory receptor downregulation also led to increased levels of HIV-specific antibody-secreting cells and B cell–associated chemokines and cytokines. The absence of known ligands for FCRL4 and Siglec-6 suggests these receptors may regulate BCR signaling through their own constitutive or tonic signaling. Furthermore, the extent of FCLR4 knockdown effects on BCR-mediated proliferation varied depending on the costimulatory ligand, suggesting that inhibitory receptors may engage specific pathways in inhibiting B cell proliferation. These findings on HIV-associated B cell exhaustion define potential targets for reversing the deleterious effect of inhibitory receptors on immune responses against persistent viral infections. PMID:21633172
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Kourelis, Jiorgos; van der Hoorn, Renier A L
2018-02-01
Plants have many, highly variable resistance ( R ) gene loci, which provide resistance to a variety of pathogens. The first R gene to be cloned, maize ( Zea mays ) Hm1 , was published over 25 years ago, and since then, many different R genes have been identified and isolated. The encoded proteins have provided clues to the diverse molecular mechanisms underlying immunity. Here, we present a meta-analysis of 314 cloned R genes. The majority of R genes encode cell surface or intracellular receptors, and we distinguish nine molecular mechanisms by which R proteins can elevate or trigger disease resistance: direct (1) or indirect (2) perception of pathogen-derived molecules on the cell surface by receptor-like proteins and receptor-like kinases; direct (3) or indirect (4) intracellular detection of pathogen-derived molecules by nucleotide binding, leucine-rich repeat receptors, or detection through integrated domains (5); perception of transcription activator-like effectors through activation of executor genes (6); and active (7), passive (8), or host reprogramming-mediated (9) loss of susceptibility. Although the molecular mechanisms underlying the functions of R genes are only understood for a small proportion of known R genes, a clearer understanding of mechanisms is emerging and will be crucial for rational engineering and deployment of novel R genes. © 2018 American Society of Plant Biologists. All rights reserved.
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Kim, Aeyung; Shin, Tae-Hwan; Shin, Seung-Min; Pham, Chuong D; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung
2012-01-01
Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005 ≈ 0.01%) than that of TAT-M13 (0.001 ≈ 0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.
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Shin, Seung-Min; Pham, Chuong D.; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung
2012-01-01
Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs. PMID:23251631
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Rajalin, Ann-Marie; Pollock, Hanna; Aarnisalo, Piia, E-mail: piia.aarnisalo@helsinki.fi
The orphan nuclear receptor estrogen-related receptor-{alpha} (ERR{alpha}) has been reported to have both a positive and a negative regulatory role in osteoblastic and adipocytic differentiation. We have studied the role of ERR{alpha} in osteoblastic and adipogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells were isolated from ERR{alpha} deficient mice and their differentiation capacities were compared to that of the wild-type cells. ERR{alpha} deficient cultures displayed reduced cellular proliferation, osteoblastic differentiation, and mineralization. In the complementary experiment, overexpression of ERR{alpha} in MC3T3-E1 cells increased the expression of osteoblastic markers and mineralization. Alterations in the expression of bone sialoproteinmore » (BSP) may at least partially explain the effects on mineralization as BSP expression was reduced in ERR{alpha} deficient MSCs and enhanced upon ERR{alpha} overexpression in MC3T3-E1 cells. Furthermore, a luciferase reporter construct driven by the BSP promoter was efficiently transactivated by ERR{alpha}. Under adipogenic conditions, ERR{alpha} deficient cultures displayed reduced adipocytic differentiation. Our data thus propose a positive role for ERR{alpha} in osteoblastic and adipocytic differentiation. The variability in the results yielded in the different studies implies that ERR{alpha} may play different roles in bone under different physiological conditions.« less
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Sarkar, Dipak K; Sengupta, Amitabha; Zhang, Changqing; Boyadjieva, Nadka; Murugan, Sengottuvelan
2012-05-11
In the natural killer (NK) cells, δ-opiate receptor (DOR) and μ-opioid receptor (MOR) interact in a feedback manner to regulate cytolytic function with an unknown mechanism. Using RNK16 cells, a rat NK cell line, we show that MOR and DOR monomer and dimer proteins existed in these cells and that chronic treatment with a receptor antagonist reduced protein levels of the targeted receptor but increased levels of opposing receptor monomer and homodimer. The opposing receptor-enhancing effects of MOR and DOR antagonists were abolished following receptor gene knockdown by siRNA. Ethanol treatment increased MOR and DOR heterodimers while it decreased the cellular levels of MOR and DOR monomers and homodimers. The opioid receptor homodimerization was associated with an increased receptor binding, and heterodimerization was associated with a decreased receptor binding and the production of cytotoxic factors. Similarly, in vivo, opioid receptor dimerization, ligand binding of receptors, and cell function in immune cells were promoted by chronic treatment with an opiate antagonist but suppressed by chronic ethanol feeding. Additionally, a combined treatment of an MOR antagonist and a DOR agonist was able to reverse the immune suppressive effect of ethanol and reduce the growth and progression of mammary tumors in rats. These data identify a role of receptor dimerization in the mechanism of DOR and MOR feedback interaction in NK cells, and they further elucidate the potential for the use of a combined opioid antagonist and agonist therapy for the treatment of immune incompetence and cancer and alcohol-related diseases.
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Toy-Miou-Leong, Mireille; Cortes, Catherine Llorens; Beaudet, Alain; Rostène, William; Forgez, Patricia
2004-03-26
Most G protein-coupled receptors are internalized after interaction with their respective ligand, a process that subsequently contributes to cell desensitization, receptor endocytosis, trafficking, and finally cell resensitization. Although cellular mechanisms leading to cell desensitization have been widely studied, those responsible for cell resensitization are still poorly understood. We examined here the traffic of the high affinity neurotensin receptor (NT1 receptor) following prolonged exposure to high agonist concentration. Fluorescence and confocal microscopy of Chinese hamster ovary, human neuroblastoma (CHP 212), and murine neuroblastoma (N1E-115) cells expressing green fluorescent protein-tagged NT1 receptor revealed that under prolonged treatment with saturating concentrations of neurotensin (NT) agonist, NT1 receptor and NT transiently accumulated in the perinuclear recycling compartment (PNRC). During this cellular event, cell surface receptors remained markedly depleted as detected by both confocal microscopy and (125)I-NT binding assays. In dividing cells, we observed that following prolonged NT agonist stimulation, NT1 receptors were removed from the PNRC, accumulated in dispersed vesicles inside the cytoplasm, and subsequently reappeared at the cell surface. This NT binding recovery allowed for constant cell sensitization and led to a chronic activation of mitogen-activated protein kinases p42 and p44. Under these conditions, the constant activation of NT1 receptor generates an oncogenic regulation. These observations support the potent role for neuropeptides, such as NT, in cancer progression.
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Kaplan, S A
1984-03-01
Cells are endowed with specific cognitive molecules that function as receptors for hormones, neurotransmitters, and other intercellular messengers. The receptor molecules may be present in the plasma membrane, cytoplasm, or nucleus. When occupied by the messenger, the receptor is coupled to the cellular machinery that responds to the message-bearing molecules. For some hormones the events following attachment of the messenger to the receptor are well known. An example is the generation of cAMP after combination of glucagon with its receptor and the series of steps culminating in activation of phosphorylase. In the case of many other messengers, including insulin, the nature of these coupling steps is not known. Receptors are subject to the regulatory processes of synthesis, degradation, and conformational change; alterations in receptor properties may have significant effects on the qualitative and quantitative responses of the cell to the extracellular messenger. The insulin receptor is located in the plasma membrane, is composed of two pairs of subunits, and has a molecular weight of about 350,000. It is located in cells such as adipocytes, hepatocytes, and skeletal muscle cells as well as in cells not considered to be typical target organ cells. Insulin receptors in nonfetal cells are downregulated by exposure of the cells to high concentrations of insulin. Other factors that regulate insulin binding include muscular exercise, diet, thyroid hormones, glucocorticoids, androgens, estrogens, and cyclic nucleotides. The fetus has high concentrations of insulin receptors in several tissues. These begin to appear early in fetal life and may outnumber those found in adult tissues. Fetal insulin receptors are unusual in that they may not undergo downregulation but may experience the opposite when exposed to insulin in high concentrations. Thus the offspring of a mother with poorly controlled diabetes may be placed in double jeopardy by fetal hyperinsulinemia and augmented insulin binding by the receptors. Many disorders in children and adults are associated with changes in the properties of the insulin receptor. In general, the alterations have been measured in receptor-bearing cells that are readily accessible, such as circulating monocytes and erythrocytes. The receptors on these cells generally reflect the status of receptors on the major target organs of insulin, although exceptions are known, and conclusions drawn from studies of receptors on circulating cells must be made with caution.(ABSTRACT TRUNCATED AT 400 WORDS)
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Functional importance of GLP-1 receptor species and expression levels in cell lines.
Knudsen, Lotte Bjerre; Hastrup, Sven; Underwood, Christina Rye; Wulff, Birgitte Schjellerup; Fleckner, Jan
2012-04-10
Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands. Copyright © 2011 Elsevier B.V. All rights reserved.
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NK-92: an 'off-the-shelf therapeutic' for adoptive natural killer cell-based cancer immunotherapy.
Suck, Garnet; Odendahl, Marcus; Nowakowska, Paulina; Seidl, Christian; Wels, Winfried S; Klingemann, Hans G; Tonn, Torsten
2016-04-01
Natural killer (NK) cells are increasingly considered as immunotherapeutic agents in particular in the fight against cancers. NK cell therapies are potentially broadly applicable and, different from their T cell counterparts, do not cause graft-versus-host disease. Efficacy and clinical in vitro or in vivo expansion of primary NK cells will however always remain variable due to individual differences of donors or patients. Long-term storage of clinical NK cell lots to allow repeated clinical applications remains an additional challenge. In contrast, the established and well-characterized cell line NK-92 can be easily and reproducibly expanded from a good manufacturing practice (GMP)-compliant cryopreserved master cell bank. Moreover, no cost-intensive cell purification methods are required. To date, NK-92 has been intensively studied. The cells displayed superior cytotoxicity against a number of tumor types tested, which was confirmed in preclinical mouse studies. Subsequent clinical testing demonstrated safety of NK-92 infusions even at high doses. Despite the phase I nature of the trials conducted so far, some efficacy was noted, particularly against lung tumors. Furthermore, to overcome tumor resistance and for specific targeting, NK-92 has been engineered to express a number of different chimeric antigen receptors (CARs), including targeting, for example, CD19 or CD20 (anti-B cell malignancies), CD38 (anti-myeloma) or human epidermal growth factor receptor 2 (HER2; ErbB2; anti-epithelial cancers). The concept of an NK cell line as an allogeneic cell therapeutic produced 'off-the-shelf' on demand holds great promise for the development of effective treatments.
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Naumenko, Vladimir S; Bazovkina, Daria V; Semenova, Alina A; Tsybko, Anton S; Il'chibaeva, Tatyana V; Kondaurova, Elena M; Popova, Nina K
2013-12-01
The effect of glial cell line-derived neurotrophic factor (GDNF) on behavior and on the serotonin (5-HT) system of a mouse strain predisposed to depressive-like behavior, ASC/Icg (Antidepressant Sensitive Cataleptics), in comparison with the parental "nondepressive" CBA/Lac mice was studied. Within 7 days after acute administration, GDNF (800 ng, i.c.v.) decreased cataleptic immobility but increased depressive-like behavioral traits in both investigated mouse strains and produced anxiolytic effects in ASC mice. The expression of the gene encoding the key enzyme for 5-HT biosynthesis in the brain, tryptophan hydroxylase-2 (Tph-2), and 5-HT1A receptor gene in the midbrain as well as 5-HT2A receptor gene in the frontal cortex were increased in GDNF-treated ASC mice. At the same time, GDNF decreased 5-HT1A and 5-HT2A receptor gene expression in the hippocampus of ASC mice. GDNF failed to change Tph2, 5-HT1A , or 5-HT2A receptor mRNA levels in CBA mice as well as 5-HT transporter gene expression and 5-HT1A and 5-HT2A receptor functional activity in both investigated mouse strains. The results show 1) a GDNF-induced increase in the expression of key genes of the brain 5-HT system, Tph2, 5-HT1A , and 5-HT2A receptors, and 2) significant genotype-dependent differences in the 5-HT system response to GDNF treatment. The data suggest that genetically defined cross-talk between neurotrophic factors and the brain 5-HT system underlies the variability in behavioral response to GDNF. Copyright © 2013 Wiley Periodicals, Inc.
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Samuel, Marcus A; Mudgil, Yashwanti; Salt, Jennifer N; Delmas, Frédéric; Ramachandran, Shaliny; Chilelli, Andrea; Goring, Daphne R
2008-08-01
The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses.
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Medjakovic, Svjetlana; Hobiger, Stefanie; Ardjomand-Woelkart, Karin; Bucar, Franz; Jungbauer, Alois
2016-04-01
Pumpkin seeds have been known in folk medicine as remedy for kidney, bladder and prostate disorders since centuries. Nevertheless, pumpkin research provides insufficient data to back up traditional beliefs of ethnomedical practice. The bioactivity of a hydro-ethanolic extract of pumpkin seeds from the Styrian pumpkin, Cucurbita pepo L. subsp. pepo var. styriaca, was investigated. As pumpkin seed extracts are standardized to cucurbitin, this compound was also tested. Transactivational activity was evaluated for human androgen receptor, estrogen receptor and progesterone receptor with in vitro yeast assays. Cell viability tests with prostate cancer cells, breast cancer cells, colorectal adenocarcinoma cells and a hyperplastic cell line from benign prostate hyperplasia tissue were performed. As model for non-hyperplastic cells, effects on cell viability were tested with a human dermal fibroblast cell line (HDF-5). No transactivational activity was found for human androgen receptor, estrogen receptor and progesterone receptor, for both, extract and cucurbitin. A cell growth inhibition of ~40-50% was observed for all cell lines, with the exception of HDF-5, which showed with ~20% much lower cell growth inhibition. Given the receptor status of some cell lines, a steroid-hormone receptor independent growth inhibiting effect can be assumed. The cell growth inhibition for fast growing cells together with the cell growth inhibition of prostate-, breast- and colon cancer cells corroborates the ethnomedical use of pumpkin seeds for a treatment of benign prostate hyperplasia. Moreover, due to the lack of androgenic activity, pumpkin seed applications can be regarded as safe for the prostate. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.
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Bernier, M; Laferrere, B; Jaillard, C; Clerget, M; Saez, J M
1986-06-01
We have examined the role of the NaK-ATPase pump activity on the ligand-induced down-regulation of gonadotropin receptors in cultured porcine Leydig and Sertoli cells. In both cells, inhibition of the NaK pump by ouabain produced a depletion of intracellular K+ levels (ID50, 10(-7) M) after a lag period of about 8 h. In the absence of ligand, the number of FSH receptors in ouabain-treated Sertoli cells was unaffected or slightly reduced, whereas a 2-fold increase in the number of human CG (hCG)/LH receptors with small changes in the binding affinity was observed in Leydig cells treated by ouabain. The effect of ouabain was dose dependent. Differences were also observed in the down-regulation process of gonadotropin receptors in ouabain-treated cells. The hCG-induced receptor loss in Leydig cells was completely reversed by ouabain whereas the drug had no effect on ligand-induced loss of FSH receptors in Sertoli cells. Similar results were observed when the cells were incubated in K+-free medium. Kinetics studies with labeled hCG have shown that ouabain treatment slows down significantly the rate of [125I]iodo-hCG internalization (t 1/2, 18 h; control cells, t 1/2, 6 h), but had no effect on the degradation of internalized hormone. The internalization of receptor-bound [125I]iodo-hCG was also reduced when Leydig cells were incubated in K+-free medium, but was restored when this medium was supplemented with rubidium. The influence of the NaK pump on the receptor regulation of a ligand common to both types of cells, such as epidermal growth factor, was studied under the same experimental conditions. Neither ouabain nor K+-free medium were able to prevent the epidermal growth factor-induced reduction of receptor levels in Leydig and Sertoli cells. Thus, it appears that modulation of ligand-induced receptor loss by depletion of cellular K+ levels is not dependent on the cell type, but on the ligand-receptor complex. The data also show a striking difference in the dynamics of gonadotropin-receptor interaction of two structurally related hormones.
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Lee, Jong Do; You, Myung Hee; Kim, Young Seol; Kim, Jin Woo; Kim, Kwang Won; Kim, Sun Woo; Choi, Young Kil
1986-01-01
Although it has been well established that thyroid hormones increase β-adrenergic receptors of various tissues in the animal studies, there are controversies about the β-adrenergic receptor changes of human mononuclear cells and polymorphonuclear cells. The present study was performed to analyze the change of β-adrenergic receptor of those cells according to the thyroid functional status and to evaluate their usefulness in assessment of sympathetic hyperactivity. We measured [3H]-dihydroalprenolol binding to circulating mononuclear and polymorphonuclear cells from 18 patients with hyperthyrodism, 7 with hypothyroidism, 8 with euthyroid goiter and 21 normal controls. Only with polymorphonuclear cells the receptor concentration was significantly higher (P<0.01) in hyperthyroidism (46.07±4.78 fmol/mg protein) than in the normal control (28.42±2.06 fmol/mg protein) and the affinity constants of both cells were comparable to normal control values. And serum concentrations of T3 were not correlated well with the changes of receptor concentrations in hyperthyroidism. The patients with hypothyroidism and euthyroid goiter showed no significant difference in the receptor concentration and the affinity constants with both cell binding assays. These results indicate that thyroid hormones increase the receptor concentration in polymorphonuclear cells which might be responsible for the symptoms of sympathetic hyperactivity and the polymorphornuclear cells are useful for β-adrenergic receptor assay. PMID:15759381
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Evolution of olfactory receptors.
Hoover, Kara C
2013-01-01
Olfactory receptors are a specialized set of receptor cells responsible for the detection of odors. These cells are G protein-coupled receptors and expressed in the cell membranes of olfactory sensory neurons. Once a cell is activated by a ligand, it initiates a signal transduction cascade that produces a nerve impulse to the brain where odor perception is processed. Vertebrate olfactory evolution is characterized by birth-and-death events, a special case of the stochastic continuous time Markov process. Vertebrate fish have three general types of receptor cells (two dedicated to pheromones). Terrestrial animals have different epithelial biology due to the specialized adaptation to detecting airborne odors. Two general classes of olfactory receptor gene reflect the vertebrate marine heritage (Class I) and the derived amphibian, reptile, and mammal terrestrial heritage (Class II). While we know much about olfactory receptor cells, there are still areas where our knowledge is insufficient, such as intra-individual diversity throughout the life time, epigenetic processes acting on olfactory receptors, and association of ligands to specific cells.
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Construction and evaluation of a novel humanized HER2-specific chimeric receptor
2014-01-01
Introduction The human epidermal growth factor receptor 2 (HER2) represents one of the most studied tumor-associated antigens (TAAs) for cancer immunotherapy. The monoclonal antibody (mAb) trastuzumab has improved the outcomes of patients with HER2+ breast cancer. However, a large number of HER2+ tumors are not responsive to, or become resistant to, trastuzumab-based therapy, and thus more effective therapies targeting HER2 are needed. Methods HER2-specific T cells were generated by the transfer of genes that encode chimeric antigen receptor (CAR). Using a multistep overlap extension PCR method, we constructed a novel, humanized HER2 CAR-containing, chA21 single-chain variable fragment (scFv) region of antigen-specific mAb and T-cell intracellular signaling chains made up of CD28 and CD3ζ. An interferon γ and interleukin 2 enzyme-linked immunosorbent assay and a chromium-51 release assay were used to evaluate the antitumor immune response of CAR T cells in coculture with tumor cells. Furthermore, SKBR3 tumor–bearing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were treated with HER2 CAR T cells to evaluate antitumor activity. Human CD3+ T cell accumulation in tumor xenograft was detected by immunohistochemistry. Results chA21-28z CAR was successfully constructed, and both CD4+ and CD8+ T cells were transduced. The expanded HER2 CAR T cells expressed a central memory phenotype and specifically reacted against HER2+ tumor cell lines. Furthermore, the SKBR3 tumor xenograft model revealed that HER2 CAR T cells significantly inhibited tumor growth in vivo. Immunohistochemical analysis showed robust accumulation of human CD3+ T cells in regressing SKBR3 lesions. Conclusions The results of this study show that novel chA21 scFv-based, HER2-specific CAR T cells not only recognized and killed HER2+ breast and ovarian cancer cells ex vivo but also induced regression of experimental breast cancer in vivo. Our data support further exploration of the HER2 CAR T-cell therapy for HER2-expressing cancers. PMID:24919843
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Interneuron- and GABAA receptor-specific inhibitory synaptic plasticity in cerebellar Purkinje cells
NASA Astrophysics Data System (ADS)
He, Qionger; Duguid, Ian; Clark, Beverley; Panzanelli, Patrizia; Patel, Bijal; Thomas, Philip; Fritschy, Jean-Marc; Smart, Trevor G.
2015-07-01
Inhibitory synaptic plasticity is important for shaping both neuronal excitability and network activity. Here we investigate the input and GABAA receptor subunit specificity of inhibitory synaptic plasticity by studying cerebellar interneuron-Purkinje cell (PC) synapses. Depolarizing PCs initiated a long-lasting increase in GABA-mediated synaptic currents. By stimulating individual interneurons, this plasticity was observed at somatodendritic basket cell synapses, but not at distal dendritic stellate cell synapses. Basket cell synapses predominantly express β2-subunit-containing GABAA receptors; deletion of the β2-subunit ablates this plasticity, demonstrating its reliance on GABAA receptor subunit composition. The increase in synaptic currents is dependent upon an increase in newly synthesized cell surface synaptic GABAA receptors and is abolished by preventing CaMKII phosphorylation of GABAA receptors. Our results reveal a novel GABAA receptor subunit- and input-specific form of inhibitory synaptic plasticity that regulates the temporal firing pattern of the principal output cells of the cerebellum.
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T-cell receptor repertoire variation may be associated with type 2 diabetes mellitus in humans.
Frankl, Joseph A; Thearle, Marie S; Desmarais, Cindy; Bogardus, Clifton; Krakoff, Jonathan
2016-03-01
Recent work in Pima Indians, a population with high rates of obesity and type 2 diabetes mellitus (T2DM), demonstrated that human leukocyte antigen haplotype DRB1*02 carriers have an increased acute insulin response and decreased risk for the development of T2DM, implicating loss of self-tolerance in the pathogenesis of T2DM. Advances in genomic sequencing have made T-cell receptor repertoire analysis a practical mode of investigation. High-throughput sequencing of T-cell receptor complementarity-determining region 3 was carried out in male Pima Indians with normal glucose regulation (n = 11; age = 31 ± 8 years; %fat = 30.2 ± 8.7%) and the protective DRB1*02 haplotype versus those with T2DM without DRB1*02 (n = 7; age = 34 ± 8 years; %fat = 31.2 ± 4.7%). Findings were partially replicated in another cohort by assessing the predictive ability of T-cell receptor variation on risk of T2DM in Pima Indian men (n = 27; age = 28.9 ± 7.1 years; %fat = 28.8 ± 7.1%) and women (n = 20; age = 29 ± 7.0 years; %fat = 37.1 ± 6.8%) with baseline normal glucose regulation but without the protective haplotype who were invited to follow-up examinations as frequently as every 2 years where diabetes status was assessed by a 75-g oral glucose tolerance test. Of these subjects, 13 developed diabetes. T-cell receptor complementarity-determining region 3 length was shorter in those with T2DM, and a one-nucleotide decrease in complementarity-determining region 3 length was associated with a nearly threefold increase in risk for future diabetes. The frequency of one variable gene, TRBV7-8, was higher in those with T2DM. A 1% increase in TRBV7-8 frequency was associated with a greater than threefold increase in diabetes risk. These results indicate that T-cell autoimmunity may be an important component in progression to T2DM in Pima Indians. Copyright © 2015 John Wiley & Sons, Ltd.
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Expression profiling of G-protein-coupled receptors in human urothelium and related cell lines.
Ochodnický, Peter; Humphreys, Sian; Eccles, Rachel; Poljakovic, Mirjana; Wiklund, Peter; Michel, Martin C
2012-09-01
What's known on the subject? and What does the study add? Urothelium emerged as a crucial integrator of sensory inputs and outputs in the bladder wall, and urothelial G-protein-coupled receptors (GPCRs) may represent plausible targets for treatment of various bladder pathologies. Urothelial cell lines provide a useful tool to study urothelial receptor function, but their validity as models for native human urothelium remains unclear. We characterize the mRNA expression of genes coding for GPCRs in human freshly isolated urothelium and compare the expression pattern with those in human urothelial cell lines. To characterize the mRNA expression pattern of genes coding for G-protein-coupled receptors (GPCRs) in human freshly isolated urothelium. To compare GPCR expression in human urothelium-derived cell lines to explore the suitability of these cell lines as model systems to study urothelial function. Native human urothelium (commercially sourced) and human urothelium-derived non-cancer (UROtsa and TERT-NHUC) and cancer (J82) cell lines were used. For mRNA expression profiling we used custom-designed real-time polymerase chain reaction array for 40 receptors and several related genes. Native urothelium expressed a wide variety of GPCRs, including α(1A), α(1D) and all subtypes of α(2) and β adrenoceptors. In addition, M(2) and M(3) cholinergic muscarinic receptors, angiotensin II AT(1) receptor, serotonin 5-HT(2A) receptor and all subtypes of bradykinin, endothelin, cannabinoid, tachykinin and sphingosine-1-phosphate receptors were detected. Nerve growth factor and both its low- and high-affinity receptors were also expressed in urothelium. In all cell lines expression of most GPCRs was markedly downregulated, with few exceptions. In UROtsa cells, but much less in other cell lines, the expression of β(2) adrenoceptors, M(3) muscarinic receptors, B(1) and B(2) bradykinin receptors, ET(B) endothelin receptors and several subtypes of sphingosine-1-phosphate receptors was largely retained. Human urothelium expresses a wide range of receptors which enables sensing and integration of various extracellular signals. Human urothelium-derived cell lines, especially UROtsa cells, show comparable mRNA expression to native tissue for several physiologically relevant GPCRs, but lose expression of many other receptors. The use of cell lines as model systems of human urothelium requires careful validation of suitability for the genes of interest. © 2012 BJU INTERNATIONAL.
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NASA Astrophysics Data System (ADS)
McCoy, Michael J.; Habermann, Timothy J.; Hanke, Craig J.; Adar, Fran; Campbell, William B.; Nithipatikom, Kasem
1999-04-01
We developed a confocal Raman microspectroscopic technique to study ligand-receptor bindings in single cells using Raman-labeled ligands and surface-enhanced Raman scattering (SERS). The adrenal zona glomerulosa (ZG) cells were used as a model in this study. ZG cells have a high density of angiotensin II (AII) receptors on the cellular membrane. There are two identified subtypes of AII receptors,namely AT1 and AT2 receptors. AII is a peptidic hormone, which upon binding to its receptors, stimulates the release of aldosterone from ZG cells. The cellular localization of these receptors subtypes was detected in single ZG cells by using immunocomplexation of receptors with specific antibodies and confocal Raman microspectroscopy. In the binding study, we used biotin-labeled AII to bind to its receptors in ZG cells. Then, avidin and Raman-labeled AII. The binding was measure directly on the single ZG cells. The results showed that the binding was displaced with unlabeled AII and specific AII antagonists. This is a rapid and sensitive technique for detection of cellular ligand bindings as well as antagonists screening in drug discovery.
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Naturally occurring glucagon-like peptide-2 (GLP-2) receptors in human intestinal cell lines.
Sams, Anette; Hastrup, Sven; Andersen, Marie; Thim, Lars
2006-02-17
Although clinical trials with GLP-2 receptor agonists are currently ongoing, the mechanisms behind GLP-2-induced intestinal epithelial growth remain to be understood. To approach the GLP-2 mechanism of action this study aimed to identify intestinal cell lines endogenously expressing the GLP-2 receptor. Here we report the first identification of a cell line endogenously expressing functional GLP-2 receptors. The human intestinal epithelial cell line, FHC, expressed GLP-2 receptor encoding mRNA (RT-PCR) and GLP-2 receptor protein (Western blot). In cultured FHC cells, GLP-2 induced concentration dependent cAMP accumulation (pEC(50)=9.7+/-0.04 (mean+/-S.E.M., n=4)). In addition, a naturally occurring human intestinal fibroblast cell line, 18Co, endogenously expressing GLP-2 receptor encoding mRNA (RT-PCR) and protein (Western blot) was identified. No receptor functionality (binding or G-protein signalling) could be demonstrated in 18Co cells. The identified gut-relevant cell lines provide tools for future clarification of the mechanisms underlying GLP-2-induced epithelial growth.
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Functional Selectivity and Antidepressant Activity of Serotonin 1A Receptor Ligands
Chilmonczyk, Zdzisław; Bojarski, Andrzej Jacek; Pilc, Andrzej; Sylte, Ingebrigt
2015-01-01
Serotonin (5-HT) is a monoamine neurotransmitter that plays an important role in physiological functions. 5-HT has been implicated in sleep, feeding, sexual behavior, temperature regulation, pain, and cognition as well as in pathological states including disorders connected to mood, anxiety, psychosis and pain. 5-HT1A receptors have for a long time been considered as an interesting target for the action of antidepressant drugs. It was postulated that postsynaptic 5-HT1A agonists could form a new class of antidepressant drugs, and mixed 5-HT1A receptor ligands/serotonin transporter (SERT) inhibitors seem to possess an interesting pharmacological profile. It should, however, be noted that 5-HT1A receptors can activate several different biochemical pathways and signal through both G protein-dependent and G protein-independent pathways. The variables that affect the multiplicity of 5-HT1A receptor signaling pathways would thus result from the summation of effects specific to the host cell milieu. Moreover, receptor trafficking appears different at pre- and postsynaptic sites. It should also be noted that the 5-HT1A receptor cooperates with other signal transduction systems (like the 5-HT1B or 5-HT2A/2B/2C receptors, the GABAergic and the glutaminergic systems), which also contribute to its antidepressant and/or anxiolytic activity. Thus identifying brain specific molecular targets for 5-HT1A receptor ligands may result in a better targeting, raising a hope for more effective medicines for various pathologies. PMID:26262615
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Flood, Michael Daniel; Moore-Dotson, Johnnie M; Eggers, Erika D
2018-05-30
Dopamine modulation of retinal signaling has been shown to be an important part of retinal adaptation to increased background light levels but the role of dopamine modulation of retinal inhibition is not clear. We previously showed that light adaptation causes a large reduction in inhibition to rod bipolar cells, potentially to match the decrease in excitation after rod saturation. In this study we determined how dopamine D1 receptors in the inner retina contribute to this modulation. We found that D1 receptor activation significantly decreased the magnitude of inhibitory light responses from rod bipolar cells, while D1 receptor blockade during light adaptation partially prevented this decline. To determine what mechanisms were involved in the modulation of inhibitory light responses, we measured the effect of D1 receptor activation on spontaneous currents and currents evoked from electrically stimulating amacrine cell inputs to rod bipolar cells. D1 receptor activation decreased the frequency of spontaneous inhibition with no change in event amplitudes, suggesting a presynaptic change in amacrine cell activity in agreement with previous reports that rod bipolar cells lack D1 receptors. Additionally, we found that D1 receptor activation reduced the amplitude of electrically-evoked responses, showing that D1 receptors can modulate amacrine cells directly. Our results suggest that D1 receptor activation can replicate a large portion, but not all of the effects of light adaptation, likely by modulating release from amacrine cells onto rod bipolar cells.
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Confinement of activating receptors at the plasma membrane controls natural killer cell tolerance.
Guia, Sophie; Jaeger, Baptiste N; Piatek, Stefan; Mailfert, Sébastien; Trombik, Tomasz; Fenis, Aurore; Chevrier, Nicolas; Walzer, Thierry; Kerdiles, Yann M; Marguet, Didier; Vivier, Eric; Ugolini, Sophie
2011-04-05
Natural killer (NK) cell tolerance to self is partly ensured by major histocompatibility complex (MHC) class I-specific inhibitory receptors on NK cells, which dampen their reactivity when engaged. However, NK cells that do not detect self MHC class I are not autoreactive. We used dynamic fluorescence correlation spectroscopy to show that MHC class I-independent NK cell tolerance in mice was associated with the presence of hyporesponsive NK cells in which both activating and inhibitory receptors were confined in an actin meshwork at the plasma membrane. In contrast, the recognition of self MHC class I by inhibitory receptors "educated" NK cells to become fully reactive, and activating NK cell receptors became dynamically compartmentalized in membrane nanodomains. We propose that the confinement of activating receptors at the plasma membrane is pivotal to ensuring the self-tolerance of NK cells.
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Platelet dysfunction associated with the novel Trp29Cys thromboxane A₂ receptor variant.
Mumford, A D; Nisar, S; Darnige, L; Jones, M L; Bachelot-Loza, C; Gandrille, S; Zinzindohoue, F; Fischer, A-M; Mundell, S J; Gaussem, P
2013-03-01
Genetic variations that affect the structure of the thromboxane A2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are very rare in unselected populations. To determine the functional consequences of the TP receptor Trp29Cys (W29C) substitution. We performed a detailed phenotypic analysis of an index case (P1) with reduced platelet aggregation and secretion responses to TP receptor pathway activators, and a heterozygous TP receptor W29C substitution. An analysis of the variant W29C TP receptor expressed in heterologous cells was performed. Total TP receptor expression in platelets from P1 was similar to that of controls, but there was reduced maximum binding and reduced affinity of binding to the TP receptor antagonist [(3) H]SQ29548. HEK293 cells transfected with W29C TP receptor cDNA showed similar total TP receptor expression to wild-type (WT) controls. However, the TP receptor agonist U46619 was less potent at inducing rises in cytosolic free Ca(2+) in HEK293 cells expressing the W29C TP receptor than in WT controls, indicating reduced receptor function. Immunofluorescence microscopy and cell surface ELISA showed intracellular retention and reduced cell surface expression of the W29C TP receptor in HEK293 cells. Consistent with the platelet phenotype, both maximum binding and the affinity of binding of [(3) H]SQ29548 to the W29C TP receptor were reduced compared to WT controls. These findings extend the phenotypic description of the very rare disorder TP receptor deficiency, and show that the W29C substitution reduces TP receptor function by reducing surface receptor expression and by disrupting ligand binding. © 2012 International Society on Thrombosis and Haemostasis.
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Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment
Iezzi, María Elena; Policastro, Lucía; Werbajh, Santiago; Podhajcer, Osvaldo; Canziani, Gabriela Alicia
2018-01-01
Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs), in particular those engineered from the variable heavy-chain fragment (VHH gene) found in Camelidae heavy-chain antibodies (or IgG2 and IgG3), are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR) fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition “modules” to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo. Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads. PMID:29520274
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Vekony, M A; Holder, J E; Lee, A J; Horrocks, C; Eperon, I C; Camp, R D
1997-07-01
Several groups have investigated the role of T cells in the pathogenesis of psoriasis by determination of T-cell receptor (TCR) B-chain variable (V) region usage, both in chronic plaque (psoriasis vulgaris) and guttate forms, with various results. Because there are no data on TCR expression in early psoriasis vulgaris, when specific cellular immune events may be expected to be most pronounced, we have analyzed early lesions (less than 3 wk old) of ten patients, with highly reproducible results. We have developed a highly controlled anchored polymerase chain reaction (PCR) method in which TCR beta chain species are all amplified with the same primer pair and products are quantified by dot blot hybridization with BV family-specific oligonucleotide probes. Overexpression of certain TCR BV genes was observed in the majority of lesional biopsies, but in samples in which the expanded BV family formed more than 10% of total lesional BV (half of the samples analyzed), BV2 and BV6 predominated. The consistency of overexpression of these BV species between patients was much less than in previous studies of TCRBV usage in established chronic plaque psoriasis lesions. Complementarity-determining region 3 (CDR3) size spectratyping demonstrated evidence for selective clonal T cell accumulation in less than half of the lesional samples showing BV expansion. These results indicate that selective amplification of TCRBV species occurs in early psoriasis vulgaris but is not essential to the pathogenic process and may be more important in the maintenance or expansion of chronic lesions.
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Queirós, Maria Luís; Gonçalves, Marta; Fonseca, Sónia; Moura, João
2015-01-01
Studies of chemokine receptors (CKR) in natural killer- (NK-) cells have already been published, but only a few gave detailed information on its differential expression on blood NK-cell subsets. We report on the expression of the inflammatory and homeostatic CKR on normal blood CD56+low CD16+ and CD56+high CD16−/+low NK-cells. Conventional CD56+low and CD56+high NK-cells present in the normal PB do express CKR for inflammatory cytokines, although with different patterns CD56+low NK-cells are mainly CXCR1/CXCR2+ and CXCR3/CCR5−/+, whereas mostly CD56+high NK-cells are CXCR1/CXCR2− and CXCR3/CCR5+. Both NK-cell subsets have variable CXCR4 expression and are CCR4− and CCR6−. The CKR repertoire of the CD56+low NK-cells approaches to that of neutrophils, whereas the CKR repertoire of the CD56+high NK-cells mimics that of Th1+ T cells, suggesting that these cells are prepared to migrate into inflamed tissues at different phases of the immune response. In addition, we describe a subpopulation of NK-cells with intermediate levels of CD56 expression, which we named CD56+int NK-cells. These NK-cells are CXCR3/CCR5+, they have intermediate levels of expression of CD16, CD62L, CD94, and CD122, and they are CD57− and CD158a−. In view of their phenotypic features, we hypothesize that they correspond to a transitional stage, between the well-known CD56+high and CD56+low NK-cells populations. PMID:26543875
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Saleh, Noureldin; Ibrahim, Passainte; Saladino, Giorgio; Gervasio, Francesco Luigi; Clark, Timothy
2017-05-22
A generally applicable metadynamics scheme for predicting the free energy profile of ligand binding to G-protein-coupled receptors (GPCRs) is described. A common and effective collective variable (CV) has been defined using the ideally placed and highly conserved Trp6.48 as a reference point for ligand-GPCR distance measurement and the common orientation of GPCRs in the cell membrane. Using this single CV together with well-tempered multiple-walker metadynamics with a funnel-like boundary allows an efficient exploration of the entire ligand binding path from the extracellular medium to the orthosteric binding site, including vestibule and intermediate sites. The protocol can be used with X-ray structures or high-quality homology models (based on a high-quality template and after thorough refinement) for the receptor and is universally applicable to agonists, antagonists, and partial and reverse agonists. The root-mean-square error (RMSE) in predicted binding free energies for 12 diverse ligands in five receptors (a total of 23 data points) is surprisingly small (less than 1 kcal mol -1 ). The RMSEs for simulations that use receptor X-ray structures and homology models are very similar.
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Salari, Pooneh; Abdollahi, Mohammad
2011-01-01
Several drugs are used in the treatment of irritable bowel syndrome (IBS) but all have side effects and variable efficacy. Considering the role of the gut-brain axis, immune, neural, and endocrine pathways in the pathogenesis of IBS and possible beneficial effects of benzodiazepines (BZD) in this axis, the present systematic review focuses on the efficacy of BZD receptor modulators in human IBS. For the years 1966 to February 2011, all literature was searched for any articles on the use of BZD receptor modulators and IBS. After thorough evaluation and omission of duplicate data, 10 out of 69 articles were included. BZD receptor modulators can be helpful, especially in the diarrhea-dominant form of IBS, by affecting the inflammatory, neural, and psychologic pathways, however, controversies still exist. Recently, a new BZD receptor modulator, dextofisopam was synthesized and studied in human subjects, but the studies are limited to phase IIb clinical trials. None of the existing trials considered the neuroimmunomodulatory effect of BZDs in IBS, but bearing in mind the concentration-dependent effect of BZDs on cytokines and cell proliferation, future studies using pharmacodynamic and pharmacokinetic approaches are highly recommended. PMID:22090780
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Exact solutions to a spatially extended model of kinase-receptor interaction.
Szopa, Piotr; Lipniacki, Tomasz; Kazmierczak, Bogdan
2011-10-01
B and Mast cells are activated by the aggregation of the immune receptors. Motivated by this phenomena we consider a simple spatially extended model of mutual interaction of kinases and membrane receptors. It is assumed that kinase activates membrane receptors and in turn the kinase molecules bound to the active receptors are activated by transphosphorylation. Such a type of interaction implies positive feedback and may lead to bistability. In this study we apply the Steklov eigenproblem theory to analyze the linearized model and find exact solutions in the case of non-uniformly distributed membrane receptors. This approach allows us to determine the critical value of receptor dephosphorylation rate at which cell activation (by arbitrary small perturbation of the inactive state) is possible. We found that cell sensitivity grows with decreasing kinase diffusion and increasing anisotropy of the receptor distribution. Moreover, these two effects are cooperating. We showed that the cell activity can be abruptly triggered by the formation of the receptor aggregate. Since the considered activation mechanism is not based on receptor crosslinking by polyvalent antigens, the proposed model can also explain B cell activation due to receptor aggregation following binding of monovalent antigens presented on the antigen presenting cell.
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1991-01-01
The effect of receptor occupancy on insulin receptor endocytosis was examined in CHO cells expressing normal human insulin receptors (CHO/IR), autophosphorylation- and internalization-deficient receptors (CHO/IRA1018), and receptors which undergo autophosphorylation but lack a sequence required for internalization (CHO/IR delta 960). The rate of [125I]insulin internalization in CHO/IR cells at 37 degrees C was rapid at physiological concentrations, but decreased markedly in the presence of increasing unlabeled insulin (ED50 = 1-3 nM insulin, or 75,000 occupied receptors/cell). In contrast, [125I]insulin internalization by CHO/IRA1018 and CHO/IR delta 960 cells was slow and was not inhibited by unlabeled insulin. At saturating insulin concentrations, the rate of internalization by wild-type and mutant receptors was similar. Moreover, depletion of intracellular potassium, which has been shown to disrupt coated pit formation, inhibited the rapid internalization of [125I]insulin at physiological insulin concentrations by CHO/IR cells, but had little or no effect on [125I]insulin uptake by CHO/IR delta 960 and CHO/IRA1018 cells or wild-type cells at high insulin concentrations. These data suggest that the insulin-stimulated entry of the insulin receptor into a rapid, coated pit-mediated internalization pathway is saturable and requires receptor autophosphorylation and an intact juxtamembrane region. Furthermore, CHO cells also contain a constitutive nonsaturable pathway which does not require receptor autophosphorylation or an intact juxtamembrane region; this second pathway is unaffected by depletion of intracellular potassium, and therefore may be independent of coated pits. Our data suggest that the ligand-stimulated internalization of the insulin receptor may require specific saturable interactions between the receptor and components of the endocytic system. PMID:1757462
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Gene Transfer and Molecular Cloning of the Human NGF Receptor
NASA Astrophysics Data System (ADS)
Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita
1986-04-01
Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.
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Meyer Zu Schwabedissen, Henriette E; Ferreira, Celio; Schaefer, Anima M; Oufir, Mouhssin; Seibert, Isabell; Hamburger, Matthias; Tirona, Rommel G
2018-07-01
Levothyroxine replacement therapy forms the cornerstone of hypothyroidism management. Variability in levothyroxine oral absorption may contribute to the well-recognized large interpatient differences in required dose. Moreover, levothyroxine-drug pharmacokinetic interactions are thought to be caused by altered oral bioavailability. Interestingly, little is known regarding the mechanisms contributing to levothyroxine absorption in the gastrointestinal tract. Here, we aimed to determine whether the intestinal drug uptake transporter organic anion transporting polypeptide 2B1 (OATP2B1) may be involved in facilitating intestinal absorption of thyroid hormones. We also explored whether thyroid hormones regulate OATP2B1 gene expression. In cultured Madin-Darby Canine Kidney II/OATP2B1 cells and in OATP2B1-transfected Caco-2 cells, thyroid hormones were found to inhibit OATP2B1-mediated uptake of estrone-3-sulfate. Competitive counter-flow experiments evaluating the influence on the cellular accumulation of estrone-3-sulfate in the steady state indicated that thyroid hormones were substrates of OATP2B1. Additional evidence that thyroid hormones were OATP2B1 substrates was provided by OATP2B1-dependent stimulation of thyroid hormone receptor activation in cell-based reporter assays. Bidirectional transport studies in intestinal Caco-2 cells showed net absorptive flux of thyroid hormones, which was attenuated by the presence of the OATP2B1 inhibitor, atorvastatin. In intestinal Caco-2 and LS180 cells, but not in liver Huh-7 or HepG2 cells, OATP2B1 expression was induced by treatment with thyroid hormones. Reporter gene assays revealed thyroid hormone receptor α -mediated transactivation of the SLCO2B1 1b and the SLCO2B1 1e promoters. We conclude that thyroid hormones are substrates and transcriptional regulators of OATP2B1. These insights provide a potential mechanistic basis for oral levothyroxine dose variability and drug interactions. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.
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Heterogeneity of chemokine cell-surface receptor expression in triple-negative breast cancer
Norton, Kerri-Ann; Popel, Aleksander S; Pandey, Niranjan B
2015-01-01
Introduction: Tumor heterogeneity is a well-established concept in cancer research. In this paper, we examine an additional type of tumor cell heterogeneity - tumor cell-surface receptor heterogeneity. Methods: We use flow cytometry to measure the frequency and numbers of cell-surface receptors on triple negative breast cancer cell lines. Results: We find two distinct populations of human triple-negative breast cancer cells MDA-MB-231 when they are grown in culture, one with low surface levels of various chemokine receptors and a second with much higher levels. The population with high surface levels of these receptors is increased in the more metastatic MDA-MB-231-luc-d3h2ln cell line. Conclusion: We hypothesize that this high cell-surface receptor population is involved in metastasis. We find that the receptor high populations can be modulated by tumor conditioned media and IL6 treatment indicating that the tumor microenvironment is important for the maintenance and sizes of these populations. PMID:26101698
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Bakali, Evangelia; Elliott, Ruth A; Taylor, Anthony H; Lambert, David G; Willets, Jonathon M; Tincello, Douglas G
2014-06-01
To characterize human urothelial cell lines' cannabinoid receptor expression and evaluate their possible use for studying signalling interactions with purinergic and muscarinic receptor activation. PCR was used to detect cannabinoid (CB), muscarinic and purinergic receptor transcripts in HCV29 and UROtsa cells, whilst immunofluorescence evaluated protein expression and localization of cannabinoid receptors. The effect of CB1 agonist (ACEA) on carbachol- and ATP-induced changes in intracellular calcium ([Ca(2+)]i) levels was measured using fluorimetry. The ability of ACEA to reduce intracellular cAMP was investigated in HCV29 cells. CB1 and GPR55 receptor transcripts were detected in HCV29 and UROtsa cells, respectively. Immunofluorescence showed positive staining for CB1 in the HCV29 cells. Both cell lines expressed transcript levels for muscarinic receptors, but carbachol did not raise [Ca(2+)]i levels indicating a lack or low expression of G(q)-coupled muscarinic receptors. Transcripts for purinergic receptors were detected; ATP significantly increased [Ca(2+)]i in HCV29 and UROtsa cells by 395 ± 61 and 705 ± 100 nM (mean ± SEM, n = 6), respectively. ACEA did not alter ATP-induced [Ca(2+)]i or cAMP levels in HCV29 cells. Whilst HCV29 cells expressed CB1 and UROtsa cells expressed GPR55 receptors, these were not functionally coupled to the existing purinergic-driven increase in Ca2+ as such they do not represent a good model to study signalling interactions.
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Spontaneous endometriosis in a mandrill (Mandrillus sphinx).
Nakamura, S; Ochiai, K; Ochi, A; Ito, M; Kamiya, T; Yamamoto, H
2012-01-01
A 25-year-old female mandrill (Mandrillus sphinx) died after exhibiting weakness and recumbency with serosanguineous ascites. Gross findings included haemoperitoneum and multifocal to diffuse serosal thickening with petechiae and ecchymoses throughout the peritoneum. The uterus was covered entirely with large blood clots and was adherent to the ovaries and pelvic wall. Microscopical and immunohistochemical examination revealed extra- and intra-uterine growth of ectopic endometrial tissue with marked fibrosis. The ectopic endometrial tissues predominantly consisted of stromal cells expressing CD10 and progesterone receptor and variably-sized glands lined by the epithelium with occasional slight expression of oestrogen receptor α. A diagnosis of endometriosis was made. This is the first report of naturally occurring endometriosis in a mandrill. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells
DOE Office of Scientific and Technical Information (OSTI.GOV)
Hyatt, Dustin C.; Ceresa, Brian P.
2008-11-01
The epidermal growth factor (EGF) receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase that regulates diverse cell functions that are dependent upon cell type, the presence of downstream effectors, and receptor density. In addition to activating biochemical pathways, ligand stimulation causes the EGFR to enter the cell via clathrin-coated pits. Endocytic trafficking influences receptor signaling by controlling the duration of EGFR phosphorylation and coordinating the receptor's association with downstream effectors. To better understand the individual contributions of cell surface and cytosolic EGFRs on cell physiology, we used EGF that was conjugated to 900 nm polystyrene beads (EGF-beads). EGF-beads canmore » stimulate the EGFR and retain the activated receptor at the plasma membrane. In MDA-MB-468 cells, a breast cancer cell line that over-expresses the EGFR, only internalized, activated EGFRs stimulate caspase-3 and induce cell death. Conversely, signaling cascades triggered from activated EGFR retained at the cell surface inhibit caspase-3 and promote cell proliferation. Thus, through endocytosis, the activated EGFR can differentially regulate cell growth in MDA-MB-468 cells.« less
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Brynychova, Veronika; Hlavac, Viktor; Ehrlichova, Marie; Vaclavikova, Radka; Nemcova-Furstova, Vlasta; Pecha, Vaclav; Trnkova, Marketa; Mrhalova, Marcela; Kodet, Roman; Vrana, David; Gatek, Jiri; Bendova, Marie; Vernerova, Zdenka; Kovar, Jan; Soucek, Pavel
2017-01-01
Apoptosis plays a critical role in cancer cell survival and tumor development. We provide a hypothesis-generating screen for further research by exploring the expression profile and genetic variability of caspases (2, 3, 7, 8, 9, and 10) in breast carcinoma patients. This study addressed isoform-specific caspase transcript expression and genetic variability in regulatory sequences of caspases 2 and 9. Gene expression profiling was performed by quantitative real-time PCR in tumor and paired non-malignant tissues of two independent groups of patients. Genetic variability was determined by high resolution melting, allelic discrimination, and sequencing analysis in tumor and peripheral blood lymphocyte DNA of the patients. CASP3 A+B and S isoforms were over-expressed in tumors of both patient groups. The CASP9 transcript was down-regulated in tumors of both groups of patients and significantly associated with expression of hormonal receptors and with the presence of rs4645978-rs2020903-rs4646034 haplotype in the CASP9 gene. Patients with a low intratumoral CASP9A/B isoform expression ratio (predicted to shift equilibrium towards anti-apoptotic isoform) subsequently treated with adjuvant chemotherapy had a significantly shorter disease-free survival than those with the high ratio (p=0.04). Inheritance of CC genotype of rs2020903 in CASP9 was associated with progesterone receptor expression in tumors (p=0.003). Genetic variability in CASP9 and expression of its splicing variants present targets for further study.
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Effects of selective type I and II adrenal steroid agonists on immune cell distribution.
Miller, A H; Spencer, R L; hassett, J; Kim, C; Rhee, R; Ciurea, D; Dhabhar, F; McEwen, B; Stein, M
1994-11-01
Adrenal steroids exert their effects through two distinct adrenal steroid receptor subtypes; the high affinity type I, or mineralocorticoid, receptor and the lower affinity type II, or glucocorticoid, receptor. Adrenal steroids have well known effects on immune cell distribution, and although both type I and II receptors are expressed in immune cells and tissues, few data exist on the relative effects mediated through these two receptor subtypes. Accordingly, we administered selective type I and II adrenal steroid receptor agonists to young adult male Sprague-Dawley rats for 7 days and then measured immune cell distribution in the peripheral blood and spleen. Results were compared with those of similar studies using the naturally occurring glucocorticoid of the rat, corticosterone, which binds both type I and II receptors. The majority of the well characterized effects of adrenal steroids on peripheral blood immune cells (increased neutrophils and decreased lymphocytes and monocytes) were reproduced by the type II receptor agonist, RU28362. RU28362 decreased the numbers of all lymphocyte subsets [T-cells, B-cells, and natural killer (NK) cells] to very low absolute levels. The largest relative decrease (i.e. in percentage) was seen in B-cells, whereas NK cells exhibited the least relative decrease and actually showed a 2-fold increase in relative percentage during RU28362 treatment. Similar to RU28362, the type I receptor agonist, aldosterone, significantly reduced the number of lymphocytes and monocytes. In contrast to RU28362, however, aldosterone significantly decreased the number of neutrophils. Moreover, aldosterone decreased the number of T-helper cells and NK cells, while having no effect on the number of B-cells or T-suppressor/cytotoxic cells. Corticosterone at physiologically relevant concentrations had potent effects on immune cell distribution, which were indistinguishable from those of the type II receptor agonist, RU28362. Taken together, these results indicate that effects of adrenal steroids on immune cell distribution are dependent on the receptor subtype involved as well as the specific cell type targeted. These factors allow for varied and complex effects of adrenal steroids on the immune system under physiological conditions.
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LeBaron, Matthew J; Coady, Katie K; O'Connor, John C; Nabb, Diane L; Markell, Lauren K; Snajdr, Suzanne; Sue Marty, M
2014-02-01
Tier 1 of the U.S. EPA Endocrine Disruptor Screening Program comprises 11 studies: five in vitro assays, four in vivo mammalian assays, and two in vivo nonmammalian assays. The battery is designed to detect compounds with the potential to interact with the estrogen, androgen, or thyroid signaling pathways. This article examines the procedures, results, and data interpretation for the five Tier 1 in vitro assays: estrogen receptor (ER) and androgen receptor binding assays, an ER transactivation assay, an aromatase assay, and a steroidogenesis assay. Data are presented from two laboratories that have evaluated approximately 11 compounds in the Tier 1 in vitro assays. Generally, the ER and androgen receptor binding assays and the aromatase assay showed good specificity and reproducibility. As described in the guideline for the ER transactivation assay, a result is considered positive when the test compound induces a reporter gene signal that reaches 10% of the response seen with 1 nM 17β-estradiol (positive control). In the experience of these laboratories, this cutoff criterion may result in false-positive responses. For the steroidogenesis assay, there is variability in the basal and stimulated production of testosterone and estradiol by the H295R cells. This variability in responsiveness, coupled with potential cell stress at high concentrations of test compound, may make it difficult to discern whether hormone alterations are specific steroidogenesis alterations (i.e., endocrine active). Lastly, both laboratories had difficulty meeting some recommended performance criteria for each Tier 1 in vitro assay. Data with only minor deviations were deemed valid. © 2014 Wiley Periodicals, Inc.
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Shivali, B.; S., Kataria; Chandramouleeswari, K.; Anita, S.
2013-01-01
Myofibroblastoma (MFB) is a rare mesenchymal tumour, derived from mammary stromal fibro-myofibroblasts, with diverse biological and morphological behaviour. Large and cellular myofibroblastomas, especially those with epitheliod like cells, can mimic various spindle cell lesions and metaplastic carcinomas, thus posing diagnostic challenge. A 50–year woman presented with slow growing, painless lump in the left breast. Fine Needle Aspiration (FNA) smears showed predominant atypical spindle cell population, pleomorphic epithelial like cells and giant cells. Cytodiagnosis of atypical spindle cell lesion with the possibility of metaplastic carcinoma was suggested. Histopathological examination showed fascicles of spindle cell population admixed with epithelial like cells, atypical cells and tumour giant cells, thus raising differential diagnosis of metaplastic carcinoma, low grade spindle cell sarcoma and myofibroblastic tumour. Lymph nodes were negative for metastatic deposits. Immunohistochemistry revealed variable coexpression of markers for vimentin, fibronectin, CD34, SMA (smooth muscle actin), but negative expression for , S-100, CD99, CK7 (cytokeratin 7), HMWK (high molecular weight keratin), ER (oestrogen receptor) and PR(progesterone receptors). Diagnosis of cellular myofibroblastoma with mixed unusual morphological features was defined, based on both histological and immunohistochemical features. MFB may cause a potential diagnostic pitfall while interpreting FNA and histopathological sections due to its wide differential diagnosis. The distinction of MFB from its cytohistological mimics of malignancy is crucial to avoid unnecessary extensive procedures. The case report emphasizes the role of immunohistochemistry as gold standard in diagnosis of MFB. The case is also being presented because of its large size and rare mixed unusual morphological features. PMID:24298520
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Wieder, Robert; Shafiq, Basit; Adam, Nabil
2016-01-01
BACKGROUND: African American race negatively impacts survival from localized breast cancer but co-variable factors confound the impact. METHODS: Data sets were analyzed from the Surveillance, Epidemiology and End Results (SEER) directories from 1973 to 2011 consisting of patients with designated diagnosis of breast adenocarcinoma, race as White or Caucasian, Black or African American, Asian, American Indian or Alaskan Native, Native Hawaiian or Pacific Islander, age, stage I, II or III, grade 1, 2 or 3, estrogen receptor or progesterone receptor positive or negative, marital status as single, married, separated, divorced or widowed and laterality as right or left. The Cox Proportional Hazards Regression model was used to determine hazard ratios for survival. Chi square test was applied to determine the interdependence of variables found significant in the multivariable Cox Proportional Hazards Regression analysis. Cells with stratified data of patients with identical characteristics except African American or Caucasian race were compared. RESULTS: Age, stage, grade, ER and PR status and marital status significantly co-varied with race and with each other. Stratifications by single co-variables demonstrated worse hazard ratios for survival for African Americans. Stratification by three and four co-variables demonstrated worse hazard ratios for survival for African Americans in most subgroupings with sufficient numbers of values. Differences in some subgroupings containing poor prognostic co-variables did not reach significance, suggesting that race effects may be partly overcome by additional poor prognostic indicators. CONCLUSIONS: African American race is a poor prognostic indicator for survival from breast cancer independent of 6 associated co-variables with prognostic significance. PMID:27698895
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Cattle NK Cell Heterogeneity and the Influence of MHC Class I
Allan, Alasdair J.; Sanderson, Nicholas D.; Gubbins, Simon; Ellis, Shirley A.
2015-01-01
Primate and rodent NK cells form highly heterogeneous lymphocyte populations owing to the differential expression of germline-encoded receptors. Many of these receptors are polymorphic and recognize equally polymorphic determinants of MHC class I. This diversity can lead to individuals carrying NK cells with different specificities. Cattle have an unusually diverse repertoire of NK cell receptor genes predicted to encode receptors that recognize MHC class I. To begin to examine whether this genetic diversity leads to a diverse NK cell population, we isolated peripheral NK cells from cattle with different MHC homozygous genotypes. Cytokine stimulation differentially influenced the transcription of five receptors at the cell population level. Using dilution cultures, we found that a further seven receptors were differentially transcribed, including five predicted to recognize MHC class I. Moreover, there was a statistically significant reduction in killer cell lectin-like receptor mRNA expression between cultures with different CD2 phenotypes and from animals with different MHC class I haplotypes. This finding confirms that cattle NK cells are a heterogeneous population and reveals that the receptors creating this diversity are influenced by the MHC. The importance of this heterogeneity will become clear as we learn more about the role of NK cells in cattle disease resistance and vaccination. PMID:26216890
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Ecke, Denise; Hanck, Theodor; Tulapurkar, Mohan E; Schäfer, Rainer; Kassack, Matthias; Stricker, Rolf; Reiser, Georg
2008-01-01
Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y1 and P2Y11, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y11 receptor and the P2Y1 receptor where this interaction promotes agonist-induced internalization of the P2Y11 receptor. This is remarkable because the P2Y11 receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y11 and P2Y1 receptors, upon stimulation with ATP or the P2Y1 receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y11 receptor was found to functionally associate with endogenous P2Y1 receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y1 receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP-P2Y11 receptor. Pharmacological characteristics of the P2Y11 receptor expressed in HEK293 cells were determined by recording Ca2+ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y11 receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y1 and P2Y11 receptors allows novel functions of the P2Y11 receptor in response to extracellular nucleotides.
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B cell receptor editing in tolerance and autoimmunity
Luning Prak, Eline T.; Monestier, Marc; Eisenberg, Robert A.
2010-01-01
Receptor editing is the process of ongoing antibody gene rearrangement in a lymphocyte that already has a functional antigen receptor. The expression of a functional antigen receptor will normally terminate further rearrangement (allelic exclusion). However, lymphocytes with autoreactive receptors have a chance at escaping negative regulation by “editing” the specificities of their receptors with additional antibody gene rearrangements. Nemazee points out, “receptor editing separates receptor selection from cellular selection.”1 As such, editing complicates the Clonal Selection Hypothesis, because edited cells are not simply endowed for life with a single, invariant antigen receptor.2 For example, an edited B cell changes the specificity of its B cell receptor (BCR), and if the initial immunoglobulin gene is not inactivated during the editing process, allelic exclusion is violated, and the B cell can exhibit two specificities. Here we will describe the discovery of editing, the pathways of receptor editing at the heavy (H) and light (L) chain loci, and current evidence regarding how and where editing happens and what effects it has on the antibody repertoire. PMID:21251012
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Teitelbaum, A P; Silve, C M; Nyiredy, K O; Arnaud, C D
1986-02-01
Exposure of cultured embryonic chicken bone cells to the PTH agonists bovine (b) PTH-(1-34) and [8Nle, 18Nle, 34Tyr]bPTH-(1-34)amide [bPTH-(1-34)A] reduces the subsequent cAMP response to the hormone and decreases the specific binding of 125I-labeled PTH to these cultures. To determine whether PTH receptor down-regulation in cultured bone cells is mediated by cellular internalization of PTH-receptor complexes, we measured the uptake of [125I]bPTH-(1-34) into an acid-resistant compartment. Uptake of radioactivity into this compartment was inhibited by incubating cells at 4 C with phenylarsineoxide and unlabeled bPTH-(1-34). Tracer uptake into the acid-resistant compartment at any time was directly proportional to total cell binding at 22 C. Thus, it is likely that PTH-receptor complexes are internalized by bone cells. This mechanism may explain the loss of cell surface receptors after PTH pretreatment. To determine whether internalized PTH-receptor complexes are reinserted into the plasma membrane, we measured PTH binding and PTH stimulation of cAMP production after cells were exposed to monensin, a known inhibitor of receptor recycling. Monensin (25 microM) had no effect on PTH receptor number or affinity and did not alter PTH-stimulated cAMP accumulation. However, monensin (25 microM) incubated with cells pretreated with various concentrations of bPTH-(1-34) for 1 h potentiated the effect of the hormone to reduce subsequent [125I]bPTH-(1-34) binding and PTH-stimulated cAMP accumulation by more than 2 orders of magnitude. Chloroquine also potentiated PTH-induced down-regulation of PTH receptors. By contrast, neither agent influenced PTH binding or PTH-stimulated cAMP production in cells pretreated with the antagonist bPTH-(3-34)A. Thus, monensin potentiated PTH receptor loss only in cells pretreated with PTH agonists, indicating that antagonist-occupied receptors may be processed differently from agonist-occupied receptors in bone cells. The data further suggest that the attenuation of PTH stimulation of cAMP production in treated bone cells may be, at least in part, due to receptor-mediated endocytosis of the hormone.
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Morgan, Kevin; Stavrou, Emmanouil; Leighton, Samuel P; Miller, Nicola; Sellar, Robin; Millar, Robert P
2011-06-15
Human metastatic prostate cancer cell growth can be inhibited by GnRH analogs but effects on virus-immortalized prostate cells have not been investigated. Virus-immortalized prostate cells were stably transfected with rat GnRH receptor cDNA and levels of GnRH binding were correlated with GnRH effects on signaling, cell cycle, growth, exosome production, and apoptosis. High levels of cell surface GnRH receptor occurred in transfected papillomavirus-immortalized WPE-1-NB26 epithelial cells but not in non-tumourigenic RWPE-1, myoepithelial WPMY-1 cells, or SV40-immortalized PNT1A. Endogenous cell surface GnRH receptor was undetectable in non-transfected cells or cancer cell lines LNCaP, PC3, and DU145. GnRH receptor levels correlated with induction of inositol phosphates, elevation of intracellular Ca(2+) , cytoskeletal actin reorganization, modulation of ERK activation and cell growth-inhibition with GnRH agonists. Hoechst 33342 DNA staining-cell sorting indicated accumulation of cells in G2 following agonist treatment. Release of exosomes from transfected WPE-1-NB26 was unaffected by agonists, unlike induction observed in HEK293([SCL60]) cells. Increased PARP cleavage and apoptotic body production were undetectable during growth-inhibition in WPE-1-NB26 cells, contrasting with HEK293([SCL60]) . EGF receptor activation inhibited GnRH-induced ERK activation in WPE-1-NB26 but growth-inhibition was not rescued by EGF or PKC inhibitor Ro320432. Growth of cells expressing low levels of GnRH receptor was not affected by agonists. Engineered high-level GnRH receptor activation inhibits growth of a subset of papillomavirus-immortalized prostate cells. Elucidating mechanisms leading to clone-specific differences in cell surface GnRH receptor levels is a valuable next step in developing strategies to exploit prostate cell anti-proliferation using GnRH agonists. Copyright © 2010 Wiley-Liss, Inc.
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Insulation of a G protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cell
1995-01-01
Receptor desensitization is a key process for the protection of the cell from continuous or repeated exposure to high concentrations of an agonist. Well-established mechanisms for desensitization of guanine nucleotide-binding protein (G protein)-coupled receptors include phosphorylation, sequestration/internalization, and down-regulation. In this work, we have examined some mechanisms for desensitization of the cholecystokinin (CCK) receptor which is native to the pancreatic acinar cell, and have found the predominant mechanism to be distinct from these recognized processes. Upon fluorescent agonist occupancy of the native receptor, it becomes "insulated" from the effects of acid washing and becomes immobilized on the surface of the plasma membrane in a time- and temperature-dependent manner. This localization was assessed by ultrastructural studies using a colloidal gold conjugate of CCK, and lateral mobility of the receptor was assessed using fluorescence recovery after photobleaching. Of note, recent application of the same morphologic techniques to a CCK receptor-bearing Chinese hamster ovary cell line demonstrated prominent internalization via the clathrin-dependent endocytic pathway, as well as entry into caveolae (Roettger, B.F., R.U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J.M. Larkin, and L.J. Miller, 1995, J. Cell Biol. 128: 1029-1041). These organelles are not observed to represent prominent compartments for the same receptor to traverse in the acinar cell, although fluorescent insulin is clearly internalized in these cells via receptor-mediated endocytosis. In this work, the rate of lateral mobility of the CCK receptor is observed to be similar in both cell types (1-3 x 10(-10) cm2/s), while the fate of the agonist-occupied receptor is quite distinct in each cell. This supports the unique nature of desensitization processes which occur in a cell-specific manner. A plasmalemmal site of insulation of this important receptor on the pancreatic acinar cell could be particularly effective to protect the cell from processes which might initiate pancreatitis, while providing for the rapid resensitization of this receptor to ensure appropriate pancreatic secretion to aid in nutrient assimilation for the organism. PMID:7622559
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Chemokine Receptor Signatures in Allogeneic Stem Cell Transplantation
2014-08-01
versus-host disease (GHVD). We use T-cell receptor deep sequencing to characterize the repertoire of effector T-cells in allogeneic hematopoietic stem ... cell transplant (HSCT) recipients and identify the role of chemokine receptors in effector cell infiltration of target organs. In the recent funding
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Sivori, Simona; Falco, Michela; Marcenaro, Emanuela; Parolini, Silvia; Biassoni, Roberto; Bottino, Cristina; Moretta, Lorenzo; Moretta, Alessandro
2002-01-01
In this study we analyzed the progression of cell surface receptor expression during the in vitro-induced human natural killer (NK) cell maturation from CD34+ Lin− cell precursors. NKp46 and NKp30, two major triggering receptors that play a central role in natural cytotoxicity, were expressed before the HLA class I-specific inhibitory receptors. Moreover, their appearance at the cell surface correlated with the acquisition of cytolytic activity by developing NK cells. Although the early expression of triggering receptors may provide activating signals required for inducing further cell differentiation, it may also affect the self-tolerance of developing NK cells. Our data show that a fail-safe mechanism preventing killing of normal autologous cells may be provided by the 2B4 surface molecule, which, at early stages of NK cell differentiation, functions as an inhibitory rather than as an activating receptor. PMID:11917118
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Newsom-Davis, J.; Willcox, N.; Calder, L.
1981-11-26
We investigated the role of the thymus in 16 patients with myasthenia gravis without thymoma by studying the production of anti-acetylcholine-receptor antibody by thymic and blood lymphocytes cultured alone or together. In 10 responders (with the highest receptor-antibody titers in their plasma), cultured thymic cells spontaneously produced measurable receptor antibody. Receptor-antibody production by autologous blood lymphocytes was enhanced by the addition of responder's thymic cells, irradiated to abrogate antibody production and suppression (P<0.01). This enhancement was greater and more consistent than that by pokeweed mitogen; it depended on viable thymic cells, appeared to be selective for receptor antibody, and correlatedmore » with the ratio of thymic helper (OKT4-positive or OKT4+) to suppressor (OKT8+) T cells (P<0.01). These results suggest that myasthenic thymus contains cell-bound acetylcholine-receptor-like material or specific T cells (or both) that can aid receptor-antibody production. This may be relevant to the benefits of thymectomy in myasthenia and to the breakdown in self-tolerance in this and other autoimmune diseases.« less
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PAR-2 receptor-induced effects on human eccrine sweat gland cells.
L Bovell, Douglas; Kofler, Barbara; Lang, Roland
2009-01-01
Serine proteases can induce cell signaling by stimulating G-protein-coupled receptors, called proteinase-activated receptors (PAR's) on a variety of epithelial cells. While PAR-2, one such receptor, activates cell signaling in a secretory cell line derived from human sweat glands, there was no information on their presence and effects on intact sweat glands. PAR-2 presence and activation of eccrine sweat glands isolated from human skin samples was investigated using Western blot analysis, immunohistochemistry, electron microscopy (EM) and Ca(2+) imaging. Anti-human PAR-2 antibody demonstrated the presence of these receptors in eccrine sweat glands. EM showed that PAR-2 activation resulted in degranulation of secretory cells. Ca(2+) imaging using PAR-2 activators demonstrated a two phase increase in [Ca(2+)](i) which was dependent on extracellular Ca(2+) for the second phase, and that the response could be blocked by prior incubation with xestospongin, the IP(3) receptor blocker. The results demonstrated that PAR-2 receptors are present in human sweat gland secretory cells and that these receptors are functionally active and can induce changes associated with secretory events in eccrine glands.
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Multiple melanocortin receptors are expressed in bone cells
NASA Technical Reports Server (NTRS)
Zhong, Qing; Sridhar, Supriya; Ruan, Ling; Ding, Ke-Hong; Xie, Ding; Insogna, Karl; Kang, Baolin; Xu, Jianrui; Bollag, Roni J.; Isales, Carlos M.
2005-01-01
Melanocortin receptors belong to the seven transmembrane domain, G-protein coupled family of receptors. There are five members of this receptor family labeled MC1R-MC5R. These receptors are activated by fragments derived from a larger molecule, proopiomelanocortin (POMC) and include ACTH, alpha beta and gamma-MSH and beta-endorphin. Because of in vitro and in vivo data suggesting direct effects of these POMC molecules on bone and bone turnover, we examined bone and bone derived cells for the presence of the various members of the melanocortin receptor family. We report that the five known melanocortin receptors are expressed to varying degrees in osteoblast-like and osteoclastic cells. POMC fragments increased proliferation and expression of a variety of genes in osteoblastic cells. Furthermore, POMC mRNA was detected in osteoclastic cells. These data demonstrate that POMC-derived peptide hormones acting through high affinity melanocortin receptors have specific effects on bone cells. Thus, in addition to the indirect effects of POMC-derived hormones on bone turnover through their modulation of steroid hormone secretion, POMC fragments may have direct and specific effects on bone cell subpopulations.
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A novel system of polymorphic and diverse NK cell receptors in primates.
Averdam, Anne; Petersen, Beatrix; Rosner, Cornelia; Neff, Jennifer; Roos, Christian; Eberle, Manfred; Aujard, Fabienne; Münch, Claudia; Schempp, Werner; Carrington, Mary; Shiina, Takashi; Inoko, Hidetoshi; Knaust, Florian; Coggill, Penny; Sehra, Harminder; Beck, Stephan; Abi-Rached, Laurent; Reinhardt, Richard; Walter, Lutz
2009-10-01
There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in "higher" primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire.
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A Novel System of Polymorphic and Diverse NK Cell Receptors in Primates
Rosner, Cornelia; Neff, Jennifer; Roos, Christian; Eberle, Manfred; Aujard, Fabienne; Münch, Claudia; Schempp, Werner; Carrington, Mary; Shiina, Takashi; Inoko, Hidetoshi; Knaust, Florian; Coggill, Penny; Sehra, Harminder; Beck, Stephan; Abi-Rached, Laurent; Reinhardt, Richard; Walter, Lutz
2009-01-01
There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in “higher” primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire. PMID:19834558
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Cell-specific targeting by heterobivalent ligands.
Josan, Jatinder S; Handl, Heather L; Sankaranarayanan, Rajesh; Xu, Liping; Lynch, Ronald M; Vagner, Josef; Mash, Eugene A; Hruby, Victor J; Gillies, Robert J
2011-07-20
Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.
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Cell-Specific Targeting by Heterobivalent Ligands
Josan, Jatinder S.; Handl, Heather L.; Sankaranarayanan, Rajesh; Xu, Liping; Lynch, Ronald M.; Vagner, Josef; Mash, Eugene A.; Hruby, Victor J.; Gillies, Robert J.
2012-01-01
Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach—to specifically target combinations of cell-surface receptors using heteromultivalent ligands (“receptor combination approach”). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle4, DPhe7]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20–50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH2. Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging. PMID:21639139
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Insulin release: the receptor hypothesis.
Malaisse, Willy J
2014-07-01
It is currently believed that the stimulation of insulin release by nutrient secretagogues reflects their capacity to act as fuel in pancreatic islet beta cells. In this review, it is proposed that such a fuel concept is not incompatible with a receptor hypothesis postulating the participation of cell-surface receptors in the recognition of selected nutrients as insulinotropic agents. Pursuant to this, attention is drawn to such matters as the anomeric specificity of the beta cell secretory response to D-glucose and its perturbation in diabetes mellitus, the insulinotropic action of artificial sweeteners, the possible role of bitter taste receptors in the stimulation of insulin secretion by L-glucose pentaacetate, the recently documented presence of cell-surface sweet taste receptors in insulin-producing cells, the multimodal signalling process resulting from the activation of these latter receptors, and the presence in beta cells of a sweet taste receptor mediating the fructose-induced potentiation of glucose-stimulated insulin secretion.
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Moth pheromone binding proteins contribute to the excitation of olfactory receptor cells
NASA Astrophysics Data System (ADS)
Pophof, Blanka
2002-10-01
Pheromone binding proteins (PBPs) occur in high concentrations in the sensillum lymph surrounding the sensory dendrites of moth pheromone-sensitive sensilla. They were shown to transport the lipophilic odorants through the aqueous sensillum lymph to the receptor cells. The sensilla trichodea of the silkmoth Antheraea polyphemus are supplied with three types of receptor cells responding specifically to three pheromone components. The sensillum lymph of these sensilla contains three different types of PBPs. In this study, recombinant PBPs in various combinations with pheromone components were applied to the receptor cells via tip-opened sensilla during electrophysiological recordings. The responses of receptor cells were shown to depend on both the pheromone component and the PBP. Pheromone components artificially bound to particular PBPs elicited nerve impulses in receptor cell types which they do not activate under natural conditions. This is the first electrophysiological study to suggest that the PBPs contribute to the activation of receptor molecules.
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Effect of propofol on androgen receptor activity in prostate cancer cells.
Tatsumi, Kenichiro; Hirotsu, Akiko; Daijo, Hiroki; Matsuyama, Tomonori; Terada, Naoki; Tanaka, Tomoharu
2017-08-15
Androgen receptor is a nuclear receptor and transcription factor activated by androgenic hormones. Androgen receptor activity plays a pivotal role in the development and progression of prostate cancer. Although accumulating evidence suggests that general anesthetics, including opioids, affect cancer cell growth and impact patient prognosis, the effect of those drugs on androgen receptor in prostate cancer is not clear. The purpose of this study was to investigate the effect of the general anesthetic propofol on androgen receptor activity in prostate cancer cells. An androgen-dependent human prostate cancer cell line (LNCaP) was stimulated with dihydrotestosterone (DHT) and exposed to propofol. The induction of androgen receptor target genes was investigated using real-time reverse transcription polymerase chain reaction, and androgen receptor protein levels and localization patterns were analyzed using immunoblotting and immunofluorescence assays. The effect of propofol on the proliferation of LNCaP cells was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Propofol significantly inhibited DHT-induced expression of androgen receptor target genes in a dose- and time-dependent manner, and immunoblotting and immunofluorescence assays indicated that propofol suppressed nuclear levels of androgen receptor proteins. Exposure to propofol for 24h suppressed the proliferation of LNCaP cells, whereas 4h of exposure did not exert significant effects. Together, our results indicate that propofol suppresses nuclear androgen receptor protein levels, and inhibits androgen receptor transcriptional activity and proliferation in LNCaP cells. Copyright © 2017 Elsevier B.V. All rights reserved.
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Sales, Kurt J; Boddy, Sheila C; Williams, Alistair R W; Anderson, Richard A; Jabbour, Henry N
2007-08-01
Prostaglandin (PG) F(2alpha) is a potent bioactive lipid in the female reproductive tract, and exerts its function after coupling with its heptahelical G-protein-coupled receptor [F-series-prostanoid (FP) receptor] to initiate cell signaling and target gene transcription. In the present study, we found elevated expression of fibroblast growth factor (FGF) 2, FGF receptor 1 (FGFR1), and FP receptor, colocalized within the neoplastic epithelial cells of endometrial adenocarcinomas. We investigated a role for PGF(2alpha)-FP receptor interaction in modulating FGF2 expression and signaling using an endometrial adenocarcinoma cell line stably expressing the FP receptor to the levels detected in endometrial adenocarcinomas (FPS cells) and endometrial adenocarcinoma tissue explants. PGF(2alpha)-FP receptor activation rapidly induced FGF2 mRNA expression, and elevated FGF2 protein expression and secretion into the culture medium in FPS cells and endometrial adenocarcinoma explants. The effect of PGF(2alpha) on the expression and secretion of FGF2 could be abolished by treatment of FPS cells and endometrial tissues with an FP receptor antagonist (AL8810) and inhibitor of ERK (PD98059). Furthermore, we have shown that FGF2 can promote the expression of FGF2 and cyclooxygenase-2, and enhance proliferation of endometrial adenocarcinoma cells via the FGFR1 and ERK pathways, thereby establishing a positive feedback loop to regulate neoplastic epithelial cell function in endometrial adenocarcinomas.
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Endothelial atypical cannabinoid receptor: do we have enough evidence?
Bondarenko, Alexander I
2014-01-01
Cannabinoids and their synthetic analogues affect a broad range of physiological functions, including cardiovascular variables. Although direct evidence is still missing, the relaxation of a vast range of vascular beds induced by cannabinoids is believed to involve a still unidentified non-CB1, non-CB2 Gi/o protein-coupled receptor located on endothelial cells, the so called endothelial cannabinoid receptor (eCB receptor). Evidence for the presence of an eCB receptor comes mainly from vascular relaxation studies, which commonly employ pertussis toxin as an indicator for GPCR-mediated signalling. In addition, a pharmacological approach is widely used to attribute the relaxation to eCB receptors. Recent findings have indicated a number of GPCR-independent targets for both agonists and antagonists of the presumed eCB receptor, warranting further investigations and cautious interpretation of the vascular relaxation studies. This review will provide a brief historical overview on the proposed novel eCB receptor, drawing attention to the discrepancies between the studies on the pharmacological profile of the eCB receptor and highlighting the Gi/o protein-independent actions of the eCB receptor inhibitors widely used as selective compounds. As the eCB receptor represents an attractive pharmacological target for a number of cardiovascular abnormalities, defining its molecular identity and the extent of its regulation of vascular function will have important implications for drug discovery. This review highlights the need to re-evaluate this subject in a thoughtful and rigorous fashion. More studies are needed to differentiate Gi/o protein-dependent endothelial cannabinoid signalling from that involving the classical CB1 and CB2 receptors as well as its relevance for pathophysiological conditions. PMID:25073723
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Kupffer cell complement receptor clearance function and host defense.
Loegering, D J
1986-01-01
Kupffer cells are well known to be important for normal host defense function. The development of methods to evaluate the in vivo function of specific receptors on Kupffer cells has made it possible to assess the role of these receptors in host defense. The rationale for studying complement receptors is based on the proposed important role of these receptors in host defense and on the observation that the hereditary deficiency of a complement receptor is associated with recurrent severe bacterial infections. The studies reviewed here demonstrate that forms of injury that are associated with depressed host defense including thermal injury, hemorrhagic shock, trauma, and surgery also cause a decrease in complement receptor clearance function. This decrease in Kupffer cell receptor clearance function was shown not to be the result of depressed hepatic blood flow or depletion of complement components. Complement receptor function was also depressed following the phagocytosis of particulates that are known to depress Kupffer cell host defense function. Endotoxemia and bacteremia also were associated with a depression of complement receptor function. Complement receptor function was experimentally depressed in uninjured animals by the phagocytosis of IgG-coated erythrocytes. There was a close association between the depression of complement receptor clearance function and increased susceptibility to the lethal effects of endotoxin and bacterial infection. These studies support the hypotheses that complement receptors on Kupffer cells are important for normal host defense and that depression of the function of these receptors impairs host defense.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Mouledous, Lionel; Merker, Soren; Neasta, Jeremie
*: Mu opioid (MOP) receptor activation can be functionally modulated by stimulation of Neuropeptide FF 2 (NPFF{sub 2}) G protein-coupled receptors. Fluorescence recovery after photobleaching experiments have shown that activation of the NPFF{sub 2} receptor dramatically reduces the fraction of MOP receptors confined in microdomains of the plasma membrane of SH-SY5Y neuroblastoma cells. The aim of the present work was to assess if the direct observation of receptor compartmentation by fluorescence techniques in living cells could be related to indirect estimation of receptor partitioning in lipid rafts after biochemical fractionation of the cell. Our results show that MOP receptor distributionmore » in lipid rafts is highly dependent upon the method of purification, questioning the interpretation of previous data regarding MOP receptor compartmentation. Moreover, the NPFF analogue 1DMe does not modify the distribution profile of MOP receptors, clearly demonstrating that membrane fractionation data do not correlate with direct measurement of receptor compartmentation in living cells.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Sonier, Brigitte; Arseneault, Madeleine; Institut National de la Recherche Scientifique-Institut Armand-Frappier, Montreal, Que.
2006-05-19
Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT{sub 2A} serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTTmore » proliferation assay. We have demonstrated that the 5-HT{sub 2A} receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT{sub 2A} receptor present in this cell line is identical to the 5-HT{sub 2A} receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT{sub 2A} receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT{sub 2A} receptor subtype, which is fully expressed in this cell line.« less
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NMDA Receptor Activity in Circulating Red Blood Cells: Methods of Detection.
Makhro, Asya; Kaestner, Lars; Bogdanova, Anna
2017-01-01
Abundance and activity of N-methyl-D-aspartate (NMDA) in circulating red blood cells contributes to the maintenance of intracellular Ca 2+ in these cells and, by doing that, controls red cell volume, membrane stability, and O 2 carrying capacity. Detection of the NMDA receptor activity in red blood cells is challenging as the number of its copies is low and shows substantial cell-to-cell heterogeneity. Receptor abundance is reliably assessed using the radiolabeled antagonist ([ 3 H]MK-801) binding technique. Uptake of Ca 2+ following the NMDA receptor activation is detected in cells loaded with Ca 2+ -sensitive fluorescent dye Fluo-4 AM. Both microfluorescence live-cell imaging and flow cytometry may be used for fluorescence intensity detection. Automated patch clamp is currently used for recording of electric currents triggered by the stimulation of the NMDA receptor. These currents are mediated by the Ca 2+ -sensitive K + (Gardos) channels that open upon Ca 2+ uptake via the active NMDA receptor. Furthermore, K + flux through the Gardos channels induced by the NMDA receptor stimulation in red blood cells may be detected using unidirectional K + ( 86 Rb + ) influx.
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Audsley, Neil; Vandersmissen, Hans Peter; Weaver, Robert; Dani, Paulina; Matthews, June; Down, Rachel; Vuerinckx, Kristel; Kim, Young-Joon; Vanden Broeck, Jozef
2013-01-01
The insect PISCF/allatostatins (ASTs) are pleiotropic peptides that are involved in the regulation of juvenile hormone biosynthesis, are myoinhibitory on the gut and the heart, and suppress feeding in various insects, but their roles in beetles are poorly understood. To provide further insight into the significance of PISCF/ASTs in beetles, the PISCF/AST receptor from Tribolium castaneum has been characterised and its tissue distribution determined. The biological activity of the T. castaneum PISCF/AST (Trica-AS) was also investigated. The Trica-AS receptor shows high sequence homology to other insect PISCF/AST receptors, which are related to the mammalian somatostatin/opioid receptors, a family of G protein-coupled receptors. The Trica-AS receptor was activated in a dose-dependent manner by both Trica-AS and T. castaneum allatostatin double C (Trica-ASTCC) as well as Manduca sexta-allatostatin (Manse-AS). Other allatoregulatory peptides (a FLG/AST, a MIP/AST and an allatotropin) and somatostatin(14) were inactive on this receptor. Receptor transcript levels in tissues, determined by qRT-PCR, were highest in the head and the gut, with variable amounts in the fat body and reproductive organs. There were measurable differences in receptor levels of the head, fat body and reproductive organs between males and females. There was also a widespread distribution of Trica-AS in various tissues of T. castaneum. The Trica-AS peptide precursor was most abundant in the head and there was a significant difference between levels in the heads and reproductive organs of males and females. Whole mount immunocytochemistry localised Trica-AS in the median and lateral neurosecretory cells of the brain, in the corpus cardiacum and throughout the ventral nerve cord. The peptide was also present in midgut neurosecretory cells, but no immunostaining was detected in the reproductive organs or Malpighian tubules. The widespread distribution of both Trica-AS and its receptor suggest this peptide may have multiple roles in beetles. However, Trica-AS had no effect on the spontaneous contractions of the gut or ovaries of T. castaneum but this peptide did stimulate the release of proteases from the anterior midgut of another beetle, Tenebrio molitor. The activation of the Trica-AS receptor by Trica-ASTCC implies a physiological role for this peptide in beetles, which remains to be identified. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.
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Sabri, F; Tresoldi, E; Di Stefano, M; Polo, S; Monaco, M C; Verani, A; Fiore, J R; Lusso, P; Major, E; Chiodi, F; Scarlatti, G
1999-11-25
Human immunodeficiency virus type 1 (HIV-1) infection of the brain is associated with neurological manifestations both in adults and in children. The primary target for HIV-1 infection in the brain is the microglia, but astrocytes can also be infected. We tested 26 primary HIV-1 isolates for their capacity to infect human fetal astrocytes in culture. Eight of these isolates, independent of their biological phenotype and chemokine receptor usage, were able to infect astrocytes. Although no sustained viral replication could be demonstrated, the virus was recovered by coculture with receptive cells such as macrophages or on stimulation with interleukin-1beta. To gain knowledge into the molecular events that regulate attachment and penetration of HIV-1 in astrocytes, we investigated the expression of several chemokine receptors. Fluorocytometry and calcium-mobilization assay did not provide evidence of expression of any of the major HIV-1 coreceptors, including CXCR4, CCR5, CCR3, and CCR2b, as well as the CD4 molecule on the cell surface of human fetal astrocytes. However, mRNA transcripts for CXCR4, CCR5, Bonzo/STRL33/TYMSTR, and APJ were detected by RT-PCR. Furthermore, infection of astrocytes by HIV-1 isolates with different chemokine receptor usage was not inhibited by the chemokines SDF-1beta, RANTES, MIP-1beta, or MCP-1 or by antibodies directed against the third variable region or the CD4 binding site of gp120. These data show that astrocytes can be infected by primary HIV-1 isolates via a mechanism independent of CD4 or major chemokine receptors. Furthermore, astrocytes are potential carriers of latent HIV-1 and on activation may be implicated in spreading the infection to other neighbouring cells, such as microglia or macrophages. Copyright 1999 Academic Press.
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Pramanik, Subrata; Sulistio, Yanuar Alan; Heese, Klaus
2017-11-01
Neurotrophins (NTs) are members of a neuronal growth factor protein family whose action is mediated by the tropomyosin receptor kinase (TRK) receptor family receptors and the p75 NT receptor (p75NTR), a member of the tumor necrosis factor (TNF) receptor family. Although NTs were first discovered in neurons, recent studies have suggested that NTs and their receptors are expressed in various types of stem cells mediating pivotal signaling events in stem cell biology. The concept of stem cell therapy has already attracted much attention as a potential strategy for the treatment of neurodegenerative diseases (NDs). Strikingly, NTs, proNTs, and their receptors are gaining interest as key regulators of stem cells differentiation, survival, self-renewal, plasticity, and migration. In this review, we elaborate the recent progress in understanding of NTs and their action on various stem cells. First, we provide current knowledge of NTs, proNTs, and their receptor isoforms and signaling pathways. Subsequently, we describe recent advances in the understanding of NT activities in various stem cells and their role in NDs, particularly Alzheimer's disease (AD) and Parkinson's disease (PD). Finally, we compile the implications of NTs and stem cells from a clinical perspective and discuss the challenges with regard to transplantation therapy for treatment of AD and PD.
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Amin, Rada; Mourcin, Frédéric; Uhel, Fabrice; Pangault, Céline; Ruminy, Philippe; Dupré, Loic; Guirriec, Marion; Marchand, Tony; Fest, Thierry; Lamy, Thierry
2015-01-01
Follicular lymphoma (FL) results from the accumulation of malignant germinal center (GC) B cells leading to the development of an indolent and largely incurable disease. FL cells remain highly dependent on B-cell receptor (BCR) signaling and on a specific cell microenvironment, including T cells, macrophages, and stromal cells. Importantly, FL BCR is characterized by a selective pressure to retain surface immunoglobulin M (IgM) BCR despite an active class-switch recombination process, and by the introduction, in BCR variable regions, of N-glycosylation acceptor sites harboring unusual high-mannose oligosaccharides. However, the relevance of these 2 FL BCR features for lymphomagenesis remains unclear. In this study, we demonstrated that IgM+ FL B cells activated a stronger BCR signaling network than IgG+ FL B cells and normal GC B cells. BCR expression level and phosphatase activity could both contribute to such heterogeneity. Moreover, we underlined that a subset of IgM+ FL samples, displaying highly mannosylated BCR, efficiently bound dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN), which could in turn trigger delayed but long-lasting BCR aggregation and activation. Interestingly, DC-SIGN was found within the FL cell niche in situ. Finally, M2 macrophages induced a DC-SIGN–dependent adhesion of highly mannosylated IgM+ FL B cells and triggered BCR-associated kinase activation. Interestingly, pharmacologic BCR inhibitors abolished such crosstalk between macrophages and FL B cells. Altogether, our data support an important role for DC-SIGN–expressing infiltrating cells in the biology of FL and suggest that they could represent interesting therapeutic targets. PMID:26272216
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D'Antonio, Maurizio; Droggiti, Anna; Feltri, M Laura; Roes, Jürgen; Wrabetz, Lawrence; Mirsky, Rhona; Jessen, Kristján R
2006-08-16
During development, Schwann cell numbers are precisely adjusted to match the number of axons. It is essentially unknown which growth factors or receptors carry out this important control in vivo. Here, we tested whether the type II transforming growth factor (TGF) beta receptor has a role in this process. We generated a conditional knock-out mouse in which the type II TGFbeta receptor is specifically ablated only in Schwann cells. Inactivation of the receptor, evident at least from embryonic day 18, resulted in suppressed Schwann cell death in normally developing and injured nerves. Notably, the mutants also showed a strong reduction in Schwann cell proliferation. Consequently, Schwann cell numbers in wild-type and mutant nerves remained similar. Lack of TGFbeta signaling did not appear to affect other processes in which TGFbeta had been implicated previously, including myelination and response of adult nerves to injury. This is the first in vivo evidence for a growth factor receptor involved in promoting Schwann cell division during development and the first genetic evidence for a receptor that controls normal developmental Schwann cell death.
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Burger, Jan A
2010-12-01
Chemokines and their receptors organize the recruitment and positioning of cells at each stage of the immune response, a system critically dependent upon coordination to get the right cells to the right place at the right time. Chemokine receptors expressed on CLL B cells are thought to function in a similar fashion, regulating the trafficking of the leukemia cells between blood, lymphoid organs, and the bone marrow, and within sub compartments within these tissues, in concert with adhesion molecules and other guidance cues. CLL cells not only respond to chemokines secreted in the microenvironment, the leukemia cells also secrete chemokines in response to external signals, such as B cell receptor engagement. These CLL cell-derived chemokines facilitate interactions between CLL cells, T cells, and other immune cells that shape the CLL microenvironment. CXCR4, the most prominent chemokine receptor in CLL, is now targeted in a first clinical trial, emphasizing that chemokines and their receptors have become a highly dynamic translational research field. Copyright © 2010 Elsevier Ltd. All rights reserved.
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Chaste, Pauline; Clement, Nathalie; Mercati, Oriane; Guillaume, Jean-Luc; Delorme, Richard; Botros, Hany Goubran; Pagan, Cécile; Périvier, Samuel; Scheid, Isabelle; Nygren, Gudrun; Anckarsäter, Henrik; Rastam, Maria; Ståhlberg, Ola; Gillberg, Carina; Serrano, Emilie; Lemière, Nathalie; Launay, Jean Marie; Mouren-Simeoni, Marie Christine; Leboyer, Marion; Gillberg, Christopher; Jockers, Ralf; Bourgeron, Thomas
2010-07-15
Melatonin is a powerful antioxidant and a synchronizer of many physiological processes. Alteration of the melatonin pathway has been reported in circadian disorders, diabetes and autism spectrum disorders (ASD). However, very little is known about the genetic variability of melatonin receptors in humans. Here, we sequenced the melatonin receptor MTNR1A and MTNR1B, genes coding for MT1 and MT2 receptors, respectively, in a large panel of 941 individuals including 295 patients with ASD, 362 controls and 284 individuals from different ethnic backgrounds. We also sequenced GPR50, coding for the orphan melatonin-related receptor GPR50 in patients and controls. We identified six non-synonymous mutations for MTNR1A and ten for MTNR1B. The majority of these variations altered receptor function. Particularly interesting mutants are MT1-I49N, which is devoid of any melatonin binding and cell surface expression, and MT1-G166E and MT1-I212T, which showed severely impaired cell surface expression. Of note, several mutants possessed pathway-selective signaling properties, some preferentially inhibiting the adenylyl cyclase pathway, others preferentially activating the MAPK pathway. The prevalence of these deleterious mutations in cases and controls indicates that they do not represent major risk factor for ASD (MTNR1A case 3.6% vs controls 4.4%; MTNR1B case 4.7% vs 3% controls). Concerning GPR50, we detected a significant association between ASD and two variations, Delta502-505 and T532A, in affected males, but it did not hold up after Bonferonni correction for multiple testing. Our results represent the first functional ascertainment of melatonin receptors in humans and constitute a basis for future structure-function studies and for interpreting genetic data on the melatonin pathway in patients.
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Bremel, Robert D.; Homan, E. Jane
2015-01-01
T-cell receptor binding to MHC-bound peptides plays a key role in discrimination between self and non-self. Only a subset, typically a pentamer, of amino acids in a MHC-bound peptide form the motif exposed to the T-cell receptor. We categorize and compare the T-cell exposed amino acid motif repertoire of the total proteomes of two groups of bacteria, comprising pathogens and gastrointestinal microbiome organisms, with the human proteome and immunoglobulins. Given the maximum 205, or 3.2 million of such motifs that bind T-cell receptors, there is considerable overlap in motif usage. We show that the human proteome, exclusive of immunoglobulins, only comprises three quarters of the possible motifs, of which 65.3% are also present in both composite bacterial proteomes. Very few motifs are unique to the human proteome. Immunoglobulin variable regions carry a broad diversity of T-cell exposed motifs (TCEMs) that provides a stratified random sample of the motifs found in pathogens, microbiome, and the human proteome. Individual bacterial genera and species vary in the content of immunoglobulin and human proteome matched motifs that they carry. Mycobacteria and Burkholderia spp carry a particularly high content of such matched motifs. Some bacteria retain a unique motif signature and motif sharing pattern with the human proteome. The implication is that distinguishing self from non-self does not depend on individual TCEMs, but on a complex and dynamic overlay of signals wherein the same TCEM may play different roles in different organisms, and the frequency with which a particular TCEM appears influences its effect. The patterns observed provide clues to bacterial immune evasion and to strategies for intervention, including vaccine design. The breadth and distinct frequency patterns of the immunoglobulin-derived peptides suggest a role of immunoglobulins in maintaining a broadly responsive T-cell repertoire. PMID:26557118
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Eph receptor interclass cooperation is required for the regulation of cell proliferation
DOE Office of Scientific and Technical Information (OSTI.GOV)
Jurek, Aleksandra; Genander, Maria; Kundu, Parag
2016-10-15
Cancer often arises by the constitutive activation of mitogenic pathways by mutations in stem cells. Eph receptors are unusual in that although they regulate the proliferation of stem/progenitor cells in many adult organs, they typically fail to transform cells. Multiple ephrins and Eph receptors are often co-expressed and are thought to be redundant, but we here describe an unexpected dichotomy with two homologous ligands, ephrin-B1 and ephrin-B2, regulating specifically migration or proliferation in the intestinal stem cell niche. We demonstrate that the combined activity of two different coexpressed Eph receptors of the A and B class assembled into common signalingmore » clusters in response to ephrin-B2 is required for mitogenic signaling. The requirement of two different Eph receptors to convey mitogenic signals identifies a new type of cooperation within this receptor family and helps explain why constitutive activation of a single receptor fails to transform cells. - Highlights: • We demonstrate that ephrin-B1 and ephrin-B2 have largely non-overlapping functions in the intestinal stem cell niche. • Ephrin-B1 regulates cell positioning and ephrin-B2 regulates cell proliferation in the intestinal stem cell niche. • EphA4/B2 receptor cooperation in response to ephrin-B2 binding is obligatory to convey mitogenic signals in the intestine. • EphA4 facilitates EphB2 phosphorylation in response to ephrin-B2 in SW480 adenocarcinoma cells. • Ephrin-B1 and ephrin-B2 induce phosphorylation and degradation of the EphB2 receptor with different kinetics.« less
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Shah, Arpeet; Farooq, Asim V; Tiwari, Vaibhav; Kim, Min-Jung; Shukla, Deepak
2010-11-20
The human cornea is a primary target for herpes simplex virus-1 (HSV-1) infection. The goals of the study were to determine the cellular modalities of HSV-1 entry into human corneal epithelial (HCE) cells. Specific features of the study included identifying major entry receptors, assessing pH dependency, and determining trends of re-infection. A recombinant HSV-1 virus expressing beta-galactosidase was used to ascertain HSV-1 entry into HCE cells. Viral replication within cells was confirmed using a time point plaque assay. Lysosomotropic agents were used to test for pH dependency of entry. Flow cytometry and immunocytochemistry were used to determine expression of three cellular receptors--nectin-1, herpesvirus entry mediator (HVEM), and paired immunoglobulin-like 2 receptor alpha (PILR-a). The necessity of these receptors for viral entry was tested using antibody-blocking. Finally, trends of re-infection were investigated using viral entry assay and flow cytometry post-primary infection. Cultured HCE cells showed high susceptibility to HSV-1 entry and replication. Entry was demonstrated to be pH dependent as blocking vesicular acidification decreased entry. Entry receptors expressed on the cell membrane include nectin-1, HVEM, and PILR-α. Receptor-specific antibodies blocked entry receptors, reduced viral entry and indicated nectin-1 as the primary receptor used for entry. Cells re-infected with HSV-1 showed a decrease in entry, which was correlated to decreased levels of nectin-1 as demonstrated by flow cytometry. HSV-1 is capable of developing an infection in HCE cells using a pH dependent entry process that involves primarily nectin-1 but also the HVEM and PILR-α receptors. Re-infected cells show decreased levels of entry, correlated with a decreased level of nectin-1 receptor expression.
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Uemura, Yu; Isobe, Yasushi; Uchida, Akiko; Asano, Junko; Nishio, Yuji; Sakai, Hirotaka; Hoshikawa, Masahiro; Takagi, Masayuki; Nakamura, Naoya; Miura, Ikuo
2018-04-01
Peripheral T- or natural killer (NK)-cell lymphomas are rare and difficult-to-recognize diseases. It remains arduous to distinguish between NK cell- and cytotoxic T-lymphocyte-derived lymphomas through routine histological evaluation. To clarify the cells of origin, we focused on NK-cell receptors and examined the expression using immunohistochemistry in 22 cases with T- and NK-cell neoplasms comprising angioimmunoblastic T-cell lymphoma, anaplastic lymphoma kinase (ALK)-positive and -negative anaplastic large-cell lymphomas, extranodal NK/T-cell lymphoma, nasal type, monomorphic epitheliotropic intestinal T-cell lymphoma, aggressive NK-cell leukemia, and other peripheral T-cell lymphomas. Inhibitory receptor leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) was detected in 14 (64%) cases, whereas activating receptors DNAM1, NKp46, and NKG2D were expressed in 7 (32%), 9 (41%), and 5 (23%) cases, respectively. Although LILRB1 was detected regardless of the disease entity, the activating NK-cell receptors were expressed predominantly in TIA-1-positive neoplasms (DNAM1, 49%; NKp46, 69%; and NKG2D, 38%). In addition, NKp46 and NKG2D were detected only in NK-cell neoplasms and cytotoxic T-lymphocyte-derived lymphomas including monomorphic epitheliotropic intestinal T-cell lymphoma. One Epstein-Barr virus-harboring cytotoxic T-lymphocyte-derived lymphoma mimicking extranodal NK/T-cell lymphoma, nasal type lacked these NK-cell receptors, indicating different cell origin from NK and innate-like T cells. Furthermore, NKG2D expression showed a negative impact on survival among the 22 examined cases, which mainly received the standard chemotherapy regimen (log-rank test, P = .024). We propose that the presence of activating NK-cell receptors may provide new insights into understanding peripheral T-cell lymphomas and characterizing them as innate-like T-cell neoplasm. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.
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Capello, Daniela; Cerri, Michaela; Muti, Giuliana; Lucioni, Marco; Oreste, Pierluigi; Gloghini, Annunziata; Berra, Eva; Deambrogi, Clara; Franceschetti, Silvia; Rossi, Davide; Alabiso, Oscar; Morra, Enrica; Rambaldi, Alessandro; Carbone, Antonino; Paulli, Marco; Gaidano, Gianluca
2006-12-01
Post-transplant lymphoproliferative disorders (PTLD) derive from antigen-experienced B-cells and represent a major complication of solid organ transplantation. We characterized usage, mutation frequency and mutation pattern of immunoglobulin variable (IGV) gene rearrangements in 50 PTLD (polymorphic PTLD, n=10; diffuse large B-cell lymphoma, n=35; and Burkitt/Burkitt-like lymphoma, n=5). Among PTLD yielding clonal IGV amplimers, a functional IGV heavy chain (IGHV) rearrangement was found in 40/50 (80.0%) cases, whereas a potentially functional IGV light chain rearrangement was identified in 36/46 (78.3%) PTLD. By combining IGHV and IGV light chain rearrangements, 10/50 (20.0%) PTLD carried crippling mutations, precluding expression of a functional B-cell receptor (BCR). Immunohistochemistry showed detectable expression of IG light chains in only 18/43 (41.9%) PTLD. Failure to detect a functional IGV rearrangement associated with lack of IGV expression. Our data suggest that a large fraction of PTLD arise from germinal centre (GC)-experienced B-cells that display impaired BCR. Since a functional BCR is required for normal B-cell survival during GC transit, PTLD development may implicate rescue from apoptosis and expansion of B-cells that have failed the GC reaction. The high frequency of IGV loci inactivation appears to be a peculiar feature of PTLD among immunodeficiency-associated lymphoproliferations.
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Zheng, Yingfeng; Murphy, Leigh C.
2016-01-01
Cell cycle progression is tightly controlled by several kinase families including Cyclin-Dependent Kinases, Polo-Like Kinases, and Aurora Kinases. A large amount of data show that steroid hormone receptors and various components of the cell cycle, including cell cycle regulated kinases, interact, and this often results in altered transcriptional activity of the receptor. Furthermore, steroid hormones, through their receptors, can also regulate the transcriptional expression of genes that are required for cell cycle regulation. However, emerging data suggest that steroid hormone receptors may have roles in cell cycle progression independent of their transcriptional activity. The following is a review of how steroid receptors and their coregulators can regulate or be regulated by the cell cycle machinery, with a particular focus on roles independent of transcription in G2/M. PMID:26778927
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del Pliego, Margarita González; Aguirre-Benítez, Elsa; Paisano-Cerón, Karina; Valdovinos-Ramírez, Irene; Rangel-Morales, Carlos; Rodríguez-Mata, Verónica; Solano-Agama, Carmen; Martín-Tapia, Dolores; de la Vega, María Teresa; Saldoval-Balanzario, Miguel; Camacho, Javier; Mendoza-Garrido, María Eugenia
2013-01-01
Pituitary adenomas can invade surrounded tissue, but the mechanism remains elusive. Ether à go-go-1 (Eag1) potassium channel and epidermal growth factor receptors (ErbB1 and ErbB2) have been associated to invasive phenotypes or poor prognosis in cancer patients. However, cells arrange their cytoskeleton in order to acquire a successful migration pattern. We have studied ErbBs and Eag1 expression, and cytoskeleton arrangements in 11 human pituitary adenomas. Eag1, ErbB1 and ErbB2 expression were studied by immunochemistry in tissue and cultured cells. The cytoskeleton arrangement was analyzed in cultured cells by immunofluorescence. Normal pituitary tissue showed ErbB2 expression and Eag1 only in few cells. However, Eag1 and ErbB2 were expressed in all the tumors analyzed. ErbB1 expression was observed variable and did not show specificity for a tumor characteristic. Cultured cells from micro- and macro-adenomas clinically functional organize their cytoskeleton suggesting a mesenchymal pattern, and a round leucocyte/amoeboid pattern from invasive clinically silent adenoma. Pituitary tumors over-express EGF receptors and the ErbB2 repeated expression suggests is a characteristic of adenomas. Eag 1 was express, in different extent, and could be a therapeutic target. The cytoskeleton arrangements observed suggest that pituitary tumor cells acquire different patterns: mesenchymal, and leucocyte/amoeboid, the last observed in the invasive adenomas. Amoeboid migration pattern has been associated with high invasion capacity.
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Rectification properties and Ca2+ permeability of glutamate receptor channels in hippocampal cells.
Lerma, J; Morales, M; Ibarz, J M; Somohano, F
1994-07-01
Excitatory amino acids exert a depolarizing action on central nervous system cells through an increase in cationic conductances. Non-NMDA receptors have been considered to be selectively permeable to Na+ and K+, while Ca2+ influx has been thought to occur through the NMDA receptor subtype. Recently, however, the expression of cloned non-NMDA receptor subunits has shown that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are permeable to Ca2+ whenever the receptor lacks a particular subunit (edited GluR-B). The behaviour of recombinant glutamate receptor channels predicts that Ca2+ would only permeate through receptors that show strong inward rectification and vice versa, i.e. AMPA receptors with linear current-voltage relationships would be impermeable to Ca2+. Using the whole-cell configuration of the patch-clamp technique, we have studied the Ca2+ permeability and the rectifying properties of AMPA receptors, when activated by kainate, in hippocampal neurons kept in culture or acutely dissociated from differentiated hippocampus. Cells were classified according to whether they showed outward rectifying (type I), inward rectifying (type II) or almost linear (type III) current-voltage relationships for kainate-activated responses. AMPA receptors of type I cells (52.2%) were mostly Ca(2+)-impermeable (PCa/PCs = 0.1), while type II cells (6.5%) expressed Ca(2+)-permeable receptors (PCa/PCs = 0.9). Type III cells (41.3%) showed responses with low but not negligible Ca2+ permeability (PCa/PCs = 0.18). The degree of Ca2+ permeability and inward rectification were well correlated in cultured cells, i.e. more inward rectification corresponded to higher Ca2+ permeability.(ABSTRACT TRUNCATED AT 250 WORDS)
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B Cell Antigen Receptor Signaling and Internalization Are Mutually Exclusive Events
Hou, Ping; Araujo, Elizabeth; Zhao, Tong; Zhang, Miao; Massenburg, Don; Veselits, Margaret; Doyle, Colleen; Dinner, Aaron R; Clark, Marcus R
2006-01-01
Engagement of the B cell antigen receptor initiates two concurrent processes, signaling and receptor internalization. While both are required for normal humoral immune responses, the relationship between these two processes is unknown. Herein, we demonstrate that following receptor ligation, a small subpopulation of B cell antigen receptors are inductively phosphorylated and selectively retained at the cell surface where they can serve as scaffolds for the assembly of signaling molecules. In contrast, the larger population of non-phosphorylated receptors is rapidly endocytosed. Each receptor can undergo only one of two mutually exclusive fates because the tyrosine-based motifs that mediate signaling when phosphorylated mediate internalization when not phosphorylated. Mathematical modeling indicates that the observed competition between receptor phosphorylation and internalization enhances signaling responses to low avidity ligands. PMID:16719564
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Buitendijk, Maarten; Eszterhas, Susan K; Howell, Alexandra L
2014-05-01
Innate immune responses to microbial pathogens are initiated following the binding of ligand to specific pattern recognition receptors. Each pattern recognition receptor, which includes members of the Toll-like receptor (TLR) family, is specific for a particular type of pathogen associated molecular pattern ensuring that the organism can respond rapidly to a wide range of pathogens including bacteria, viruses, and fungi. We studied the extent to which agonists to endosomal TLR could induce anti-HIV-1 activity in peripheral blood mononuclear cells (PBMCs). When agonists to TLR3, TLR7, TLR8 and TLR9 were added prior to infection with HIV-1, they significantly reduced infection of peripheral blood mononuclear cells. Interestingly, agonists to TLR8 and TLR9 were highly effective at blocking HIV replication even when added as late as 48 h or 72 h, respectively, after HIV-1 infection, indicating that the anti-viral effect was durable and long lasting. Analysis of the induction of anti-viral genes after agonist activation of TLR indicated that all of the agonists induced expression of the type I interferons and interferon stimulated genes, although to variable levels that depended on the agonist used. Interestingly, only the agonist to TLR9, ODN2395 DNA, induced expression of type II interferon and the anti-HIV proteins Apobec3G and SAMHD1. By blocking TLR activity using an inhibitor to the MyD88 adaptor protein, we demonstrated that, at least for TLR8 and TLR9, the anti-HIV activity was not entirely mediated by TLR activation, but likely by the activation of additional anti-viral sensors in HIV target cells. These findings suggest that agonists to the endosomal TLR function to induce expression of anti-HIV molecules by both TLR-mediated and non-TLR-mediated mechanisms. Moreover, the non-TLR-mediated mechanisms induced by these agonists could potentially be exploited to block HIV-1 replication in recently HIV-exposed individuals.
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Strekalova, Elena; Malin, Dmitry; Rajanala, Harisha; Cryns, Vincent L
2017-06-01
Despite robust antitumor activity in diverse preclinical models, TNF-related apoptosis-inducing ligand (TRAIL) receptor agonists have not demonstrated efficacy in clinical trials, underscoring the need to identify agents that enhance their activity. We postulated that the metabolic stress induced by the diabetes drug metformin would sensitize breast cancer cells to TRAIL receptor agonists. Human triple (estrogen receptor, progesterone receptor, and HER2)-negative breast cancer (TNBC) cell lines were treated with TRAIL receptor agonists (monoclonal antibodies or TRAIL peptide), metformin, or the combination. The effects on cell survival, caspase activation, and expression of TRAIL receptors and the antiapoptotic protein XIAP were determined. In addition, XIAP was silenced by RNAi in TNBC cells and the effects on sensitivity to TRAIL were determined. The antitumor effects of metformin, TRAIL, or the combination were evaluated in an orthotopic model of metastatic TNBC. Metformin sensitized diverse TNBC cells to TRAIL receptor agonists. Metformin selectively enhanced the sensitivity of transformed breast epithelial cells to TRAIL receptor agonist-induced caspase activation and apoptosis with little effect on untransformed breast epithelial cells. These effects of metformin were accompanied by robust reductions in the protein levels of XIAP, a negative regulator of TRAIL-induced apoptosis. Silencing XIAP in TNBC cells mimicked the TRAIL-sensitizing effects of metformin. Metformin also enhanced the antitumor effects of TRAIL in a metastatic murine TNBC model. Our findings indicate that metformin enhances the activity of TRAIL receptor agonists, thereby supporting the rationale for additional translational studies combining these agents.
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Lynch, Adam; Hawk, William; Nylen, Emily; Ober, Sean; Autin, Pierre; Barber, Amorette
2017-11-01
Adoptive transfer of T cells is a promising cancer therapy and expression of chimeric antigen receptors can enhance tumour recognition and T-cell effector functions. The programmed death protein 1 (PD1) receptor is a prospective target for a chimeric antigen receptor because PD1 ligands are expressed on many cancer types, including lymphoma. Therefore, we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 extracellular domain fused to the cytoplasmic domain of CD3ζ. Additionally, chimeric antigen receptor therapies use various co-stimulatory domains to enhance efficacy. Hence, the inclusion of a Dap10 or CD28 co-stimulatory domain in the chPD1 receptor was compared to determine which domain induced optimal anti-tumour immunity in a mouse model of lymphoma. The chPD1 T cells secreted pro-inflammatory cytokines and lysed RMA lymphoma cells. Adoptive transfer of chPD1 T cells significantly reduced established tumours and led to tumour-free survival in lymphoma-bearing mice. When comparing chPD1 receptors containing a Dap10 or CD28 domain, both receptors induced secretion of pro-inflammatory cytokines; however, chPD1-CD28 T cells also secreted anti-inflammatory cytokines whereas chPD1-Dap10 T cells did not. Additionally, chPD1-Dap10 induced a central memory T-cell phenotype compared with chPD1-CD28, which induced an effector memory phenotype. The chPD1-Dap10 T cells also had enhanced in vivo persistence and anti-tumour efficacy compared with chPD1-CD28 T cells. Therefore, adoptive transfer of chPD1 T cells could be a novel therapy for lymphoma and inclusion of the Dap10 co-stimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile and T-cell differentiation phenotype for anti-tumour therapies. © 2017 John Wiley & Sons Ltd.
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2010-01-01
Background Rheumatoid Arthritis (RA) is a chronic autoimmune inflammatory disorder. Although the pathogenesis of disease is unclear, it is well known that T cells play a major role in both development and perpetuation of RA through activating macrophages and B cells. Since the lack of TNF-Related Apoptosis Inducing Ligand (TRAIL) expression resulted in defective thymocyte apoptosis leading to an autoimmune disease, we explored evidence for alterations in TRAIL/TRAIL receptor expression on peripheral T lymphocytes in the molecular mechanism of RA development. Methods The expression of TRAIL/TRAIL receptors on T cells in 20 RA patients and 12 control individuals were analyzed using flow cytometry. The correlation of TRAIL and its receptor expression profile was compared with clinical RA parameters (RA activity scored as per DAS28) using Spearman Rho Analysis. Results While no change was detected in the ratio of CD4+ to CD8+ T cells between controls and RA patient groups, upregulation of TRAIL and its receptors (both death and decoy) was detected on both CD4+ and CD8+ T cells in RA patients compared to control individuals. Death Receptor-4 (DR4) and the decoy receptors DcR1 and DcR2 on CD8+ T cells, but not on CD4+ T cells, were positively correlated with patients' DAS scores. Conclusions Our data suggest that TRAIL/TRAIL receptor expression profiles on T cells might be important in revelation of RA pathogenesis. PMID:20799941
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T Cell Calcium Signaling Regulation by the Co-Receptor CD5
Freitas, Claudia M. Tellez
2018-01-01
Calcium influx is critical for T cell effector function and fate. T cells are activated when T cell receptors (TCRs) engage peptides presented by antigen-presenting cells (APC), causing an increase of intracellular calcium (Ca2+) concentration. Co-receptors stabilize interactions between the TCR and its ligand, the peptide-major histocompatibility complex (pMHC), and enhance Ca2+ signaling and T cell activation. Conversely, some co-receptors can dampen Ca2+ signaling and inhibit T cell activation. Immune checkpoint therapies block inhibitory co-receptors, such as cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) and programmed death 1 (PD-1), to increase T cell Ca2+ signaling and promote T cell survival. Similar to CTLA-4 and PD-1, the co-receptor CD5 has been known to act as a negative regulator of T cell activation and to alter Ca2+ signaling and T cell function. Though much is known about the role of CD5 in B cells, recent research has expanded our understanding of CD5 function in T cells. Here we review these recent findings and discuss how our improved understanding of CD5 Ca2+ signaling regulation could be useful for basic and clinical research. PMID:29701673
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Brederlau, A.; Faigle, R.; Elmi, M.; Zarebski, A.; Sjöberg, S.; Fujii, M.; Miyazono, K.; Funa, K.
2004-01-01
Bone morphogenetic proteins (BMPs) act as growth regulators and inducers of differentiation. They transduce their signal via three different type I receptors, termed activin receptor-like kinase 2 (Alk2), Alk3, or bone morphogenetic protein receptor Ia (BMPRIa) and Alk6 or BMPRIb. Little is known about functional differences between the three type I receptors. Here, we have investigated consequences of constitutively active (ca) and dominant negative (dn) type I receptor overexpression in adult-derived hippocampal progenitor cells (AHPs). The dn receptors have a nonfunctional intracellular but functional extracellular domain. They thus trap BMPs that are endogenously produced by AHPs. We found that effects obtained by overexpression of dnAlk2 and dnAlk6 were similar, suggesting similar ligand binding patterns for these receptors. Thus, cell survival was decreased, glial fibrillary acidic protein (GFAP) expression was reduced, whereas the number of oligodendrocytes increased. No effect on neuronal differentiation was seen. Whereas the expression of Alk2 and Alk3 mRNA remained unchanged, the Alk6 mRNA was induced after impaired BMP signaling. After dnAlk3 overexpression, cell survival and astroglial differentiation increased in parallel to augmented Alk6 receptor signaling. We conclude that endogenous BMPs mediate cell survival, astroglial differentiation and the suppression of oligodendrocytic cell fate mainly via the Alk6 receptor in AHP culture. PMID:15194807
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Cargo binding promotes KDEL receptor clustering at the mammalian cell surface
Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.
2016-01-01
Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface. PMID:27353000
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Cargo binding promotes KDEL receptor clustering at the mammalian cell surface
NASA Astrophysics Data System (ADS)
Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.
2016-06-01
Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface.
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Increased Accuracy of Ligand Sensing by Receptor Internalization and Lateral Receptor Diffusion
NASA Astrophysics Data System (ADS)
Aquino, Gerardo; Endres, Robert
2010-03-01
Many types of cells can sense external ligand concentrations with cell-surface receptors at extremely high accuracy. Interestingly, ligand-bound receptors are often internalized, a process also known as receptor-mediated endocytosis. While internalization is involved in a vast number of important functions for the life of a cell, it was recently also suggested to increase the accuracy of sensing ligand as overcounting of the same ligand molecules is reduced. A similar role may be played by receptor diffusion om the cell membrane. Fast, lateral receptor diffusion is known to be relevant in neurotransmission initiated by release of neurotransmitter glutamate in the synaptic cleft between neurons. By binding ligand and removal by diffusion from the region of release of the neurotransmitter, diffusing receptors can be reasonably expected to reduce the local overcounting of the same ligand molecules in the region of signaling. By extending simple ligand-receptor models to out-of-equilibrium thermodynamics, we show that both receptor internalization and lateral diffusion increase the accuracy with which cells can measure ligand concentrations in the external environment. We confirm this with our model and give quantitative predictions for experimental parameters values. We give quantitative predictions, which compare favorably to experimental data of real receptors.
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Modulating Estrogen Receptor-related Receptor-α Activity Inhibits Cell Proliferation*
Bianco, Stéphanie; Lanvin, Olivia; Tribollet, Violaine; Macari, Claire; North, Sophie; Vanacker, Jean-Marc
2009-01-01
High expression of the estrogen receptor-related receptor (ERR)-α in human tumors is correlated to a poor prognosis, suggesting an involvement of the receptor in cell proliferation. In this study, we show that a synthetic compound (XCT790) that modulates the activity of ERRα reduces the proliferation of various cell lines and blocks the G1/S transition of the cell cycle in an ERRα-dependent manner. XCT790 induces, in a p53-independent manner, the expression of the cell cycle inhibitor p21waf/cip1 at the protein, mRNA, and promoter level, leading to an accumulation of hypophosphorylated Rb. Finally, XCT790 reduces cell tumorigenicity in Nude mice. PMID:19546226
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Garrigues, H Jacques; Rubinchikova, Yelena E; Rose, Timothy M
2014-03-01
Cell surface structures initiating attachment of Kaposi's sarcoma-associated herpesvirus (KSHV) were characterized using purified hapten-labeled virions visualized by confocal microscopy with a sensitive fluorescent enhancement using tyramide signal amplification (TSA). KSHV attachment sites were present in specific cellular domains, including actin-based filopodia, lamellipodia, ruffled membranes, microvilli and intercellular junctions. Isolated microdomains were identified on the dorsal surface, which were heterogeneous in size with a variable distribution that depended on cellular confluence and cell cycle stage. KSHV binding domains ranged from scarce on interphase cells to dense and continuous on mitotic cells, and quantitation of bound virus revealed a significant increase on mitotic compared to interphase cells. KSHV also bound to a supranuclear domain that was distinct from microdomains in confluent and interphase cells. These results suggest that rearrangement of the cellular membrane during mitosis induces changes in cell surface receptors implicated in the initial attachment stage of KSHV entry. Copyright © 2014 Elsevier Inc. All rights reserved.
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Utz, U; Banks, D; Jacobson, S; Biddison, W E
1996-02-01
Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neurological disease characterized by marked degeneration of the spinal cord and the presence of antibodies against HTLV-1. Patients with HAM/TSP, but not asymptomatic carriers, show very high precursor frequencies of HTLV-1-specific CD8+ T cells in peripheral blood and cerebrospinal fluid, suggestive of a role of these T cells in the pathogenesis of the disease. In HLA-A2+ HAM/TSP patients, HTLV-1-specific T cells were demonstrated to be directed predominantly against one HTLV-1 epitope, namely, Tax11-19. In the present study, we analyzed HLA-A2-restricted HTLV-1 Tax11-19-specific cytotoxic T cells from three patients with HAM/TSP. An analysis of the T-cell receptor (TCR) repertoire of these cells revealed an absence of restricted variable (V) region usage. Different combinations of TCR V alpha and V beta genes were utilized between, but also within, the individual patients for the recognition of Tax11-19. Sequence analysis of the TCR showed evidence for an oligoclonal expansion of few founder T cells in each patient. Apparent structural motifs were identified for the CDR3 regions of the TCR beta chains. One T-cell clone could be detected within the same patient over a period of 3 years. We suggest that these in vivo clonally expanded T cells might play a role in the pathogenesis of HAM/TSP and provide information on HTLV-1-specific TCR which may elucidate the nature of the T cells that infiltrate the central nervous system in HAM/TSP patients.
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Glucagon Like Peptide-1 Receptor Expression in the Human Thyroid Gland
Gier, Belinda; Butler, Peter C.; Lai, Chi K.; Kirakossian, David; DeNicola, Matthew M.
2012-01-01
Background: Glucagon like peptide-1 (GLP-1) mimetic therapy induces medullary thyroid neoplasia in rodents. We sought to establish whether C cells in human medullary thyroid carcinoma, C cell hyperplasia, and normal human thyroid express the GLP-1 receptor. Methods: Thyroid tissue samples with medullary thyroid carcinoma (n = 12), C cell hyperplasia (n = 9), papillary thyroid carcinoma (n = 17), and normal human thyroid (n = 15) were evaluated by immunofluorescence for expression of calcitonin and GLP-1 receptors. Results: Coincident immunoreactivity for calcitonin and GLP-1 receptor was consistently observed in both medullary thyroid carcinoma and C cell hyperplasia. GLP-1 receptor immunoreactivity was also detected in 18% of papillary thyroid carcinoma (three of 17 cases). Within normal human thyroid tissue, GLP-1 receptor immunoreactivity was found in five of 15 of the examined cases in about 35% of the total C cells assessed. Conclusions: In humans, neoplastic and hyperplastic lesions of thyroid C cells express the GLP-1 receptor. GLP-1 receptor expression is detected in 18% papillary thyroid carcinomas and in C cells in 33% of control thyroid lobes. The consequence of long-term pharmacologically increased GLP-1 signaling on these GLP-1 receptor-expressing cells in the thyroid gland in humans remains unknown, but appropriately powered prospective studies to exclude an increase in medullary or papillary carcinomas of the thyroid are warranted. PMID:22031513
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Burastero, Samuele E.; Frigerio, Barbara; Lopalco, Lucia; Sironi, Francesca; Breda, Daniela; Longhi, Renato; Scarlatti, Gabriella; Canevari, Silvana; Figini, Mariangela; Lusso, Paolo
2011-01-01
To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs) directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env) bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ) was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1. PMID:21818294
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Burastero, Samuele E; Frigerio, Barbara; Lopalco, Lucia; Sironi, Francesca; Breda, Daniela; Longhi, Renato; Scarlatti, Gabriella; Canevari, Silvana; Figini, Mariangela; Lusso, Paolo
2011-01-01
To penetrate susceptible cells, HIV-1 sequentially interacts with two highly conserved cellular receptors, CD4 and a chemokine receptor like CCR5 or CXCR4. Monoclonal antibodies (MAbs) directed against such receptors are currently under clinical investigation as potential preventive or therapeutic agents. We immunized Balb/c mice with molecular complexes of the native, trimeric HIV-1 envelope (Env) bound to a soluble form of the human CD4 receptor. Sera from immunized mice were found to contain gp120-CD4 complex-enhanced antibodies and showed broad-spectrum HIV-1-inhibitory activity. A proportion of MAbs derived from these mice preferentially recognized complex-enhanced epitopes. In particular, a CD4-specific MAb designated DB81 (IgG1Κ) was found to preferentially bind to a complex-enhanced epitope on the D2 domain of human CD4. MAb DB81 also recognized chimpanzee CD4, but not baboon or macaque CD4, which exhibit sequence divergence in the D2 domain. Functionally, MAb DB81 displayed broad HIV-1-inhibitory activity, but it did not exert suppressive effects on T-cell activation in vitro. The variable regions of the heavy and light chains of MAb DB81 were sequenced. Due to its broad-spectrum anti-HIV-1 activity and lack of immunosuppressive effects, a humanized derivative of MAb DB81 could provide a useful complement to current preventive or therapeutic strategies against HIV-1.
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Ochsner, Scott A; Watkins, Christopher M; LaGrone, Benjamin S; Steffen, David L; McKenna, Neil J
2010-10-01
Nuclear receptors (NRs) are ligand-regulated transcription factors that recruit coregulators and other transcription factors to gene promoters to effect regulation of tissue-specific transcriptomes. The prodigious rate at which the NR signaling field has generated high content gene expression and, more recently, genome-wide location analysis datasets has not been matched by a committed effort to archiving this information for routine access by bench and clinical scientists. As a first step towards this goal, we searched the MEDLINE database for studies, which referenced either expression microarray and/or genome-wide location analysis datasets in which a NR or NR ligand was an experimental variable. A total of 1122 studies encompassing 325 unique organs, tissues, primary cells, and cell lines, 35 NRs, and 91 NR ligands were retrieved and annotated. The data were incorporated into a new section of the Nuclear Receptor Signaling Atlas Molecule Pages, Transcriptomics and Cistromics, for which we designed an intuitive, freely accessible user interface to browse the studies. Each study links to an abstract, the MEDLINE record, and, where available, Gene Expression Omnibus and ArrayExpress records. The resource will be updated on a regular basis to provide a current and comprehensive entrez into the sum of transcriptomic and cistromic research in this field.
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Tsai, Y-S; Shiau, A-L; Chen, Y-F; Tsai, H-T; Tzai, T-S; Wu, C-L
2010-01-01
The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-gamma, IL-1beta, and tumor necrosis factor-alpha proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.
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Transfer factor - hypotheses for its structure and function.
Shifrine, M; Scibienski, R
1975-01-01
Transfer factor (TF) is a dialyzable extract from primed lymphocytes that is able to transfer specific delayed hypersensitivity from one animal to another. On the basis of available data we suggest that TF is a polypeptide with a molecular weight below 15,000 daltons. We hypothesize that TF is the variable light or heavy chain domain of immunoglobulin: such a molecule conforms with the accepted properties of TF and also has the necessary specificity requirements. We also hypothesize that TF is part of a receptor site. beta-2-microglobulin, a molecule that is an integral part of cell surfaces, could be the anchor for TF. beta-2-microglobulin has homologies with the constant portion of immunoglobulin light or heavy chain and thus would combine with the variable domain (TF) to form a complete receptor site for a specific antigen. The properties of TF suggest its mode of action, which is discussed in detail in the text. The biologic advantages of TF is its ability to confer immediate (immunologie specific) protection while the 'normal' immune response develops.
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Stevenson, M; Zhang, X H; Volsky, D J
1987-01-01
Noncytopathic infection of human T-lymphoid cell line CR-10 with human immunodeficiency virus (HIV) (CEM-N1T isolate) resulted in a gradual loss of cell surface receptors for OKT4/OKT4A (HIV receptor), OKT8, OKT3, and OKT11 but not for OKT9 (transferrin receptor) within 10 days after infection. Surface receptor decline was accompanied by a rapid increase in HIV antigens and mRNA expression. Multireceptor downregulation was also observed in three T-lymphoid cell lines (MT-4, CEM, and HBD-1) cytopathically infected with the HIV/N1T virus and in HUT-78 cells infected with the HIV/SF-2 isolate. HIV-infected and uninfected CR-10 cells contained similar levels of mRNAs coding for T3, T8, T9, T11, HLA-A2, and HLA-B7 proteins. By densitometry, fully infected CR-10 cells showed approximately 75% reduction in T4 and tubulin (beta chain) mRNA levels when compared with uninfected CR-10 cells. No such reduction was detected in HIV-infected MT-4 and HBD-1 cells. A T-cell receptor gene (beta chain) rearrangement study revealed that no distinct CR-10 subpopulation was selected out upon infection with HIV. Our results suggest that the reduction in cell surface receptors observed between 1 and 2 weeks postinfection cannot be directly attributed to similar reductions in mRNA levels coding for these receptor proteins. We conclude that HIV infection induces posttranscriptional downregulation of several T-cell surface receptors. Images PMID:3500327
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Quast, Robert B.; Ballion, Biljana; Stech, Marlitt; Sonnabend, Andrei; Varga, Balázs R.; Wüstenhagen, Doreen A.; Kele, Péter; Schiller, Stefan M.; Kubick, Stefan
2016-01-01
Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system. PMID:27670253
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Paquet, J -L; Luccarini, J -M; Fouchet, C; Defrêne, E; Loillier, B; Robert, C; Bélichard, P; Cremers, B; Pruneau, D
1999-01-01
The present study addresses the differences in binding profiles and functional properties of the human and rat bradykinin (BK) B2 receptor using various kinin receptor peptide derivatives as well as the non-peptide receptor antagonists WIN 64338 (phosphonium, [[4-[[2-[[bis(cyclohexylamino)methylene]amino]-3-(2-naphtalenyl)1-oxopropyl]amino]-phenyl]-methyl]tributyl, chloride, monohydro-chloride), and FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[-N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]-phenyl]N-methylamino carbonyl methyl] acrylamide. [3H]-BK bound with a similar affinity to membranes of Chinese hamster ovary cells (CHO-K1) expressing the cloned human (hB2-CHO) or rat (rB2-CHO) B2 receptor, human embryonic intestine cells (INT407) expressing the native B2 receptor, human umbilical vein (HUV) and rat uterus (RU). WIN 64338 and FR173657 bound with a 3.8–6.6 fold and 7.0–16.3 fold higher affinity the rat than the human B2 receptor, respectively. The affinity values of BK derivatives as well as non-peptide antagonists were reduced by 6–23 fold in physiological HBSS compared to low ionic strength TES binding buffer. BK (0.01–3000 nM) increased inositol triphosphates (IP3) levels in hB2-CHO, rB2-CHO and INT407 cells. The B2 receptor antagonist, Hoe 140 (D-Arg0-[ Hyp3, Thi5, D-Tic7, Oic8]-BK) at 10−7 M, significantly shifted to the right the IP3 response curves to BK giving apparent pKB values of 8.56, 9.79 and 8.84 for hB2-CHO, rB2-CHO and INT407 cells, respectively. In human isolated umbilical vein, Hoe 140, D-Arg0-[Hyp3, D-Phe7, Leu8]-BK and NPC 567 had a lower potency in functional assays (pKB 8.18, 5.77 and 5.60, respectively) than expected from their affinity in binding studies (pKi 10.52, 8.64 and 8.27, respectively). FR173657 behaved as a high affinity ligand with pKi values of 8.59 and 9.81 and potent competitive antagonist with pKB values of 7.80 and 8.17 in HUV and RU, respectively. FR173657 bound with a similar affinity the cloned and native bradykinin B2 receptor in human (pKi of 8.66 and 8.59, respectively) and in rat (pKi 9.67 and 9.81, respectively). In conclusion, we suggest that the binding buffer composition has to be taken into account when screening new compounds and that inter-species differences should be considered when setting up animal models with the aim of developing bradykinin B2 receptor antagonists as therapeutic agents. PMID:10204994
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Receptor-based differences in human aortic smooth muscle cell membrane stiffness
NASA Technical Reports Server (NTRS)
Huang, H.; Kamm, R. D.; So, P. T.; Lee, R. T.
2001-01-01
Cells respond to mechanical stimuli with diverse molecular responses. The nature of the sensory mechanism involved in mechanotransduction is not known, but integrins may play an important role. The integrins are linked to both the cytoskeleton and extracellular matrix, suggesting that probing cells via integrins should yield different mechanical properties than probing cells via non-cytoskeleton-associated receptors. To test the hypothesis that the mechanical properties of a cell are dependent on the receptor on which the stress is applied, human aortic smooth muscle cells were plated, and magnetic beads, targeted either to the integrins via fibronectin or to the transferrin receptor by use of an IgG antibody, were attached to the cell surface. The resistance of the cell to deformation ("stiffness") was estimated by oscillating the magnetic beads at 1 Hz by use of single-pole magnetic tweezers at 2 different magnitudes. The ratio of bead displacements at different magnitudes was used to explore the mechanical properties of the cells. Cells stressed via the integrins required approximately 10-fold more force to obtain the same bead displacements as the cells stressed via the transferrin receptors. Cells stressed via integrins showed stiffening behavior as the force was increased, whereas this stiffening was significantly less for cells stressed via the transferrin receptor (P<0.001). Mechanical characteristics of vascular smooth muscle cells depend on the receptor by which the stress is applied, with integrin-based linkages demonstrating cell-stiffening behavior.
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Receptor-mediated cell mechanosensing
Chen, Yunfeng; Ju, Lining; Rushdi, Muaz; Ge, Chenghao; Zhu, Cheng
2017-01-01
Mechanosensing describes the ability of a cell to sense mechanical cues of its microenvironment, including not only all components of force, stress, and strain but also substrate rigidity, topology, and adhesiveness. This ability is crucial for the cell to respond to the surrounding mechanical cues and adapt to the changing environment. Examples of responses and adaptation include (de)activation, proliferation/apoptosis, and (de)differentiation. Receptor-mediated cell mechanosensing is a multistep process that is initiated by binding of cell surface receptors to their ligands on the extracellular matrix or the surface of adjacent cells. Mechanical cues are presented by the ligand and received by the receptor at the binding interface; but their transmission over space and time and their conversion into biochemical signals may involve other domains and additional molecules. In this review, a four-step model is described for the receptor-mediated cell mechanosensing process. Platelet glycoprotein Ib, T-cell receptor, and integrins are used as examples to illustrate the key concepts and players in this process. PMID:28954860
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Bridgeman, J S; Ladell, K; Sheard, V E; Miners, K; Hawkins, R E; Price, D A; Gilham, D E
2014-01-01
Chimeric antigen receptors (CARs) can mediate redirected lysis of tumour cells in a major histocompatibility complex (MHC)-independent manner, thereby enabling autologous adoptive T cell therapy for a variety of malignant neoplasms. Currently, most CARs incorporate the T cell receptor (TCR) CD3ζ signalling chain; however, the precise mechanisms responsible for CAR-mediated T cell activation are unclear. In this study, we used a series of immunoreceptor tyrosine-based activation motif (ITAM)-mutant and transmembrane-modified receptors to demonstrate that CARs activate T cells both directly via the antigen-ligated signalling chain and indirectly via associated chains within the TCR complex. These observations allowed us to generate new receptors capable of eliciting polyfunctional responses in primary human T cells. This work increases our understanding of CAR function and identifies new avenues for the optimization of CAR-based therapeutic interventions. PMID:24116999
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High throughput functional assays for P2X receptors.
Namovic, Marian T; Jarvis, Michael F; Donnelly-Roberts, Diana
2012-06-01
Adenosine triphosphate (ATP) activates two receptor superfamilies, metabotropic P2Y and ionotropic P2X receptors. The P2X receptors are nonselective cation channels that are widely expressed on excitable cells including neurons, glia, and smooth muscle cells. The protocols in this unit are useful for evaluating ligands that interact with P2X receptors on native cells or that are cloned and expressed in recombinant heterologous cell systems. Calcium imaging methods are described for the pharmacological characterization of fast or slowly desensitizing P2X receptors. While these methods are readily applicable to a wide variety of ligand-gated ion channels, the protocols provided herein detail how they can be used to measure activation of homomeric P2X3 (fast desensitizing) and heteromeric P2X2/3 (slowly desensitizing) receptors. Appropriate agonists and/or calcium dyes can be substituted to assess activity at other P2X receptor subtypes. An additional protocol is provided for measuring P2X7 receptor-mediated pore formation in THP-1, a native human acute monocytic leukemia cell line that can be used to study homomeric P2X7 (non-desensitizing) receptors that are expressed on macrophages and microglial cells. © 2012 by John Wiley & Sons, Inc.
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Borghuis, Bart G; Looger, Loren L; Tomita, Susumu; Demb, Jonathan B
2014-04-30
A fundamental question in sensory neuroscience is how parallel processing is implemented at the level of molecular and circuit mechanisms. In the retina, it has been proposed that distinct OFF cone bipolar cell types generate fast/transient and slow/sustained pathways by the differential expression of AMPA- and kainate-type glutamate receptors, respectively. However, the functional significance of these receptors in the intact circuit during light stimulation remains unclear. Here, we measured glutamate release from mouse bipolar cells by two-photon imaging of a glutamate sensor (iGluSnFR) expressed on postsynaptic amacrine and ganglion cell dendrites. In both transient and sustained OFF layers, cone-driven glutamate release from bipolar cells was blocked by antagonists to kainate receptors but not AMPA receptors. Electrophysiological recordings from bipolar and ganglion cells confirmed the essential role of kainate receptors for signaling in both transient and sustained OFF pathways. Kainate receptors mediated responses to contrast modulation up to 20 Hz. Light-evoked responses in all mouse OFF bipolar pathways depend on kainate, not AMPA, receptors.
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A unique proteomic profile on surface IgM ligation in unmutated chronic lymphocytic leukemia
Perrot, Aurore; Pionneau, Cédric; Nadaud, Sophie; Davi, Frédéric; Leblond, Véronique; Jacob, Frédéric; Merle-Béral, Hélène; Herbrecht, Raoul; Béné, Marie-Christine; Gribben, John G.; Vallat, Laurent
2011-01-01
Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course with 2 extreme subsets: indolent, ZAP70− and mutated immunoglobulin heavy chain gene (M-CLL); and aggressive, ZAP70+ and unmutated immunoglobulin heavy chain (UM-CLL). Given the long-term suspicion of antigenic stimulation as a primum movens in the disease, the role of the B-cell receptor has been extensively studied in various experimental settings; albeit scarcely in a comparative dynamic proteomic approach. Here we use a quantitative 2-dimensional fluorescence difference gel electrophoresis technology to compare 48 proteomic profiles of the 2 CLL subsets before and after anti-IgM ligation. Differentially expressed proteins were subsequently identified by mass spectrometry. We show that unstimulated M- and UM-CLL cells display distinct proteomic profiles. Furthermore, anti-IgM stimulation induces a specific proteomic response, more pronounced in the more aggressive CLL. Statistical analyses demonstrate several significant protein variations according to stimulation conditions. Finally, we identify an intermediate form of M-CLL cells, with an indolent profile (ZAP70−) but sharing aggressive proteomic profiles alike UM-CLL cells. Collectively, this first quantitative and dynamic proteome analysis of CLL further dissects the complex molecular pathway after B-cell receptor stimulation and depicts distinct proteomic profiles, which could lead to novel molecular stratification of the disease. PMID:21602524
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Yu, Chuan-Jiang; Butt, Christopher M.; Debski, Elizabeth A.
2008-01-01
Cholinergic input to the optic tectum is necessary for visual map maintenance. To understand why, we examined the effects of activation of the different cholinergic receptor subtypes in tectal brain slices and determined whether the retinotectal map was affected by manipulations of their activity in vivo. Both α-bungarotoxin sensitive and insensitive nicotinic receptor agonists increased spontaneous postsynaptic currents (sPSCs) in a subpopulation of patch-clamped tectal cells; application of subtype selective receptor antagonists reduced nicotine-induced increases in sPSCs. Activation of α-bungarotoxin insensitive nicotinic receptors also induced substantial inward current in some cells. Muscarinic receptor mediated outward current responses were blocked by the M2-like muscarinic receptor antagonists himbacine or AF-DX 384 and mimicked by application of the M2-like agonist oxotremorine. A less frequently observed muscarinic response involving a change in sPSC frequency appeared to be mediated by M1-like muscarinic receptors. In separate experiments, pharmacological manipulation of cholinergic receptor subtype activation led to changes in the activity-dependent visual map created in the tectum by retinal ganglion cell terminals. Chronic exposure of the tectum to either α-bungarotoxin insensitive, α-bungarotoxin sensitive or M1-like receptor antagonists resulted in map disruption. However, treatment with the M2-like receptor antagonist, AF-DX 384, compressed the map. We conclude that nicotinic or M1-like muscarinic receptors control input to tectal cells while α-bungarotoxin insensitive nicotinic receptors and M2-like muscarinic receptors change tectal cell responses to that input. Blockade of the different cholinergic receptor subtypes can have opposing effects on map topography that are consistent with expected effects on tectal cell activity levels. PMID:12670313
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Raufman, Jean-Pierre, E-mail: jraufman@medicine.umaryland.edu; Cheng, Kunrong; Saxena, Neeraj
2011-11-18
Highlights: Black-Right-Pointing-Pointer Muscarinic receptor agonists stimulated robust human colon cancer cell invasion. Black-Right-Pointing-Pointer Anti-matrix metalloproteinase1 antibody pre-treatment blocks cell invasion. Black-Right-Pointing-Pointer Bile acids stimulate MMP1 expression, cell migration and MMP1-dependent invasion. -- Abstract: Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasionmore » of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers - this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre-treatment with anti-MMP1 antibody. This study contributes to understanding mechanisms underlying muscarinic receptor agonist-induced promotion of colon cancer and, more importantly, indicates that blocking MMP1 expression and activation has therapeutic promise to stop or retard colon cancer invasion and dissemination.« less
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Kasikara, Canan; Kumar, Sushil; Kimani, Stanley; Tsou, Wen-I; Geng, Ke; Davra, Viralkumar; Sriram, Ganapathy; Devoe, Connor; Nguyen, Khanh-Quynh N; Antes, Anita; Krantz, Allen; Rymarczyk, Grzegorz; Wilczynski, Andrzej; Empig, Cyril; Freimark, Bruce; Gray, Michael; Schlunegger, Kyle; Hutchins, Jeff; Kotenko, Sergei V; Birge, Raymond B
2017-06-01
Tyro3, Axl, and Mertk (collectively TAM receptors) are three homologous receptor tyrosine kinases that bind vitamin K-dependent endogenous ligands, Protein S (ProS), and growth arrest-specific factor 6 (Gas6), and act as bridging molecules to promote phosphatidylserine (PS)-mediated clearance of apoptotic cells (efferocytosis). TAM receptors are overexpressed in a vast array of tumor types, whereby the level of expression correlates with the tumor grade and the emergence of chemo- and radioresistance to targeted therapeutics, but also have been implicated as inhibitory receptors on infiltrating myeloid-derived cells in the tumor microenvironment that can suppress host antitumor immunity. In the present study, we utilized TAM-IFNγR1 reporter lines and expressed TAM receptors in a variety of epithelial cell model systems to show that each TAM receptor has a unique pattern of activation by Gas6 or ProS, as well as unique dependency for PS on apoptotic cells and PS liposomes for activity. In addition, we leveraged this system to engineer epithelial cells that express wild-type TAM receptors and show that although each receptor can promote PS-mediated efferocytosis, AKT-mediated chemoresistance, as well as upregulate the immune checkpoint molecule PD-L1 on tumor cells, Mertk is most dominant in the aforementioned pathways. Functionally, TAM receptor-mediated efferocytosis could be partially blocked by PS-targeting antibody 11.31 and Annexin V, demonstrating the existence of a PS/PS receptor (i.e., TAM receptor)/PD-L1 axis that operates in epithelial cells to foster immune escape. These data provide a rationale that PS-targeting, anti-TAM receptor, and anti-PD-L1-based therapeutics will have merit as combinatorial checkpoint inhibitors. Implications: Many tumor cells are known to upregulate the immune checkpoint inhibitor PD-L1. This study demonstrates a role for PS and TAM receptors in the regulation of PD-L1 on cancer cells. Mol Cancer Res; 15(6); 753-64. ©2017 AACR . ©2017 American Association for Cancer Research.
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Pone, Egest J.; Zan, Hong; Zhang, Jinsong; Al-Qahtani, Ahmed; Xu, Zhenming; Casali, Paolo
2011-01-01
Differentiation of naïve B cells, including immunoglobulin (Ig) class switch DNA recombination (CSR), is critical for the immune response and depends on the extensive integration of signals from the B cell receptor (BCR), tumor necrosis factor (TNF) receptor family members, Toll-like receptors (TLRs) and cytokine receptors. TLRs and BCR synergize to induce CSR in T cell-dependent and T cell-independent antibody responses to microbial pathogens. BCR triggering together with simultaneous endosomal TLR engagement leads to enhanced B cell differentiation and antibody responses. The requirement of both BCR and TLR engagement would ensure appropriate antigen-specific activation in an infection. Co-stimulation of TLRs and BCR likely plays a significant role in anti-microbial antibody responses to contain pathogen loads until the T cell-dependent antibody responses peak. Furthermore, the temporal sequence of different signals is also critical for optimal B cell responses, as exemplified by the activation of B cells by initial TLR engagement, leading to the upregulation of co-stimulatory CD80 and MHC-II receptors, which, in turn, result in more efficient interactions with T cells, thereby enhancing the germinal center (GC) reaction and antibody affinity maturation. Overall, BCR and TLR stimulation and the integration with signals from the pathogen or immune cells and their products, determine the ensuing B cell antibody response. PMID:20370617
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Heterogeneity of cell firing properties and opioid sensitivity in the thalamic reticular nucleus.
Brunton, J; Charpak, S
1997-05-01
The thalamic reticular nucleus receives afferents from the dorsal thalamus, cortex and brainstem, and projects back onto most cortically projecting thalamic nuclei thus playing a key role in the synchronization of the thalamocortical network. Although this nucleus was initially thought to consist of a homogeneous population of cells using GABA as a transmitter, and sharing identical intrinsic membrane properties, some heterogeneity was subsequently reported. The morphological diversity is generally acknowledged, but only two studies have shown functional differences between two classes of cells which vary in their ability to discharge in bursts. However, the location of the non-bursting cells was not characterized with anatomical techniques. Our recent work on the action of mu-opioid agonists in the thalamus revealed a widespread K+-mediated inhibition of most, if not all, thalamic relay and diffuse projection neurons. However, in the reticular nucleus, preliminary experiments suggested that the opioid sensitivity was variable. Based on these results and on observations of a discrete localization of mu-opioid receptors in the reticular nucleus, we investigated cellular heterogeneity within the nucleus using opioid agonists as markers. Using the whole cell patch clamp technique in young rat thalamic slices, we tested the responses of 28 neurons to opioids, the intrinsic membrane properties of each cell, and their relative location within the nucleus. Two types of intrinsic membrane properties underlying distinct discharge behaviours were seen in neurobiotin-labelled cells clearly located in the reticular nucleus: type I with the typical bursting behaviour previously reported in reticularis neurons, and type II in which bursting was greatly reduced or absent. Each class of cell could be further divided into subpopulations based on their opioid sensitivity. About half of both bursting (20) and non-bursting or tonic (8) cells were strongly inhibited by the mu-opioid receptor agonist D-Ala2,N-Me-Phe4,glycinol5-enkephalin, an effect mediated by an increase in K+ conductance. At no time was inhibition by delta- or kappa-receptor agonists seen. Our work therefore further demonstrates that the reticular nucleus is functionally heterogeneous, although the role of such cell diversity has still to be determined.
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Willerslev-Olsen, Andreas; Krejsgaard, Thorbjørn; Lindahl, Lise M; Litvinov, Ivan V; Fredholm, Simon; Petersen, David L; Nastasi, Claudia; Gniadecki, Robert; Mongan, Nigel P; Sasseville, Denis; Wasik, Mariusz A; Bonefeld, Charlotte M; Geisler, Carsten; Woetmann, Anders; Iversen, Lars; Kilian, Mogens; Koralov, Sergei B; Odum, Niels
2016-03-10
Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient-derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region β chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk-dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis. © 2016 by The American Society of Hematology.
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Morandini, R; Süli-Vargha, H; Libert, A; Loir, B; Botyánszki, J; Medzihradszky, K; Ghanem, G
1994-01-02
Four alpha-MSH drug conjugates have been synthesized, 2 C-terminal (Pep 3 and 4) and 2 central fragments (Pep 1 and 2), the latter being the 4-10 sequence known to be the main alpha-MSH-receptor-recognition site. Melphalan was introduced into each sequence at different locations. Their ability to recognize alpha-MSH receptors as well as their cytotoxic effects were compared in 3 cell lines: melanoma, carcinoma and fibroblast cells. Pep 1 and 2 were able to specifically bind to MSH receptors on melanoma cells by displacing labelled alpha-MSH from its binding sites at concentrations similar to the 4-10 heptapeptide sequence known to contain the main receptor-recognition site. They subsequently penetrate the cell, most probably by a receptor internalization mechanism, since about half of their effect could be inhibited by competition at the receptor level. Significant and selective cytotoxic effects to melanoma cells could be observed after only 2 hr exposure to the drug conjugates. Interestingly, these 2 conjugates, differing only in melphalan position, showed completely different cytotoxicity in terms of IC50 values, Pep 1 being 24 times more toxic to all cells; but the 2 were equally specific to melanoma cells. However, they both were less toxic to all cells than melphalan itself. Furthermore, Pep 1 and 2 were able to block the receptor and, unlike Pep 3 and 4, their cytotoxic effect could be significantly inhibited by an alpha-MSH agonist. Pep 3 and 4 were 5 to 10 times less toxic than melphalan to melanoma and carcinoma cells and 50 times less to fibroblast cells, and did not show any cell-type selectivity. They were less toxic than Pep 1 to melanoma and carcinoma cells by a factor of 2, but equally toxic to fibroblasts. In contrast, they were more toxic than Pep 2 to fibroblasts, melanoma and carcinoma by a factor of 3, 10 and 24 respectively. Our data strongly suggest a receptor-mediated cytotoxicity mechanism occurring with alpha-MSH central fragments in human melanoma cells due to the presence of alpha-MSH-specific receptors. This mechanism appeared to be both peptide- and cell-type-specific.
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Differential regulation of the cell cycle by alpha1-adrenergic receptor subtypes.
Gonzalez-Cabrera, Pedro J; Shi, Ting; Yun, June; McCune, Dan F; Rorabaugh, Boyd R; Perez, Dianne M
2004-11-01
Alpha(1)-Adrenergic receptors have been implicated in growth-promoting pathways. A microarray study of individual alpha(1)-adrenergic receptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) expressed in Rat-1 fibroblasts revealed that epinephrine altered the transcription of several cell cycle regulatory genes in a direction consistent with the alpha(1A)- and alpha(1D)-adrenergic receptors mediating G(1)-S cell cycle arrest and the alpha(1B-)mediating cell-cycle progression. A time course indicated that in alpha(1A) cells, epinephrine stimulated a G(1)-S arrest, which began after 8 h of stimulation and maximized at 16 h, at which point was completely blocked with cycloheximide. The alpha(1B)-adrenergic receptor profile also showed unchecked cell cycle progression, even under low serum conditions and induced foci formation. The G(1)-S arrest induced by alpha(1A)- and alpha(1D)-adrenergic receptors was associated with decreased cyclin-dependent kinase-6 and cyclin E-associated kinase activities and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1), all of which were blocked by prazosin. There were no differences in kinase activities and/or expression of p27(Kip1) in epinephrine alpha(1B)-AR fibroblasts, although the microarray did indicate differences in p27(Kip1) RNA levels. Cell counts proved the antimitotic effect of epinephrine in alpha(1A) and alpha(1D) cells and indicated that alpha(1B)-adrenergic receptor subtype expression was sufficient to cause proliferation of Rat-1 fibroblasts independent of agonist stimulation. Analysis in transfected PC12 cells also confirmed the alpha(1A)- and alpha(1B)-adrenergic receptor effect. The alpha(1B)-subtype native to DDT1-MF2 cells, a smooth muscle cell line, caused progression of the cell cycle. These results indicate that the alpha(1A)- and alpha(1D)-adrenergic receptors mediate G(1)-S cell-cycle arrest, whereas alpha(1B)-adrenergic receptor expression causes a cell cycle progression and may induce transformation in sensitive cell lines.
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NASA Astrophysics Data System (ADS)
Garcia, Marcel; Derocq, Danielle; Freiss, Gilles; Rochefort, Henri
1992-12-01
Breast cancers containing estrogen receptors are responsive to antiestrogen treatment and have a better prognosis than estrogen receptor-negative tumors. The loss of estrogen and progesterone receptors appears to be associated with a progression to less-differentiated tumors. We transfected the human estrogen receptor into the estrogen receptor-negative metastatic breast cancer cell line MDA-MB-231 in an attempt to restore their sensitivity to antiestrogens. Two stable sublines of MDA-MB-231 cells (HC1 and HE5) expressing functional estrogen receptors were studied for their ability to grow and invade in vitro and to metastasize in athymic nude mice. The number and size of lung metastases developed by these two sublines in ovariectomized nude mice was not markedly altered by tamoxifen but was inhibited 3-fold by estradiol. Estradiol also significantly inhibited in vitro cell proliferation of these sublines and their invasiveness in Matrigel, a reconstituted basement membrane, whereas the antiestrogens 4-hydroxytamoxifen and ICI 164,384 reversed these effects. These results show that estradiol inhibits the metastatic ability of estrogen receptornegative breast cancer cells following transfection with the estrogen receptor, whereas estrogen receptor-positive breast cancers are stimulated by estrogen, indicating that factors other than the estrogen receptor are involved in progression toward hormone independence. Reactivation or transfer of the estrogen receptor gene can therefore be considered as therapeutic approaches to hormone-independent cancers
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Jing, Xin; Infante, Jorge; Nachtman, Ronald G.; Jurecic, Roland
2008-01-01
Objective FLRF (Rnf41) gene was identified through screening of subtracted cDNA libraries form murine hematopoietic stem cells and progenitors. Subsequent work has revealed that FLRF acts as E3 ubiquitin ligase, and that it regulates steady-state levels of neuregulin receptor ErbB3, and participates in degradation of IAP protein BRUCE and parkin. The objective of this study was to start exploring the role of FLRF during hematopoiesis. Methods FLRF was over-expressed in a murine multipotent hematopoietic progenitor cell line EML, which can differentiate into almost all blood cell lineages, and in pro-B progenitor cell line BaF3. The impact of FLRF over-expression on EML cell differentiation into myelo-erythroid lineages was studied using hematopoietic colony-forming assays. The interaction of FLRF with cytokine receptors and receptor levels in control cells and EML and BaF3 cells over-expressing FLRF were examined with Western and immunoprecipitation. Results Remarkably, over-expression of FLRF significantly attenuated erythroid and myeloid differentiation of EML cells in response to cytokines Epo and IL-3, and retinoic acid (RA), and resulted in significant and constitutive decrease of steady-state levels of IL-3, Epo and RA receptor RARα in EML and BaF3 cells. Immunoprecipitation has revealed that FLRF interacts with IL-3, Epo and RARα receptors in EML and BaF3 cells, and that FLRF-mediated down-regulation of these receptors is ligand binding-independent. Conclusions The results of this study have revealed new FLRF-mediated pathway for ligand-independent receptor level regulation, and support the notion that through maintaining basal levels of cytokine receptors, FLRF is involved in the control of hematopoietic progenitor cell differentiation into myelo-erythroid lineages. PMID:18495327
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Jing, Xin; Infante, Jorge; Nachtman, Ronald G; Jurecic, Roland
2008-09-01
FLRF (Rnf41) gene was identified through screening of subtracted cDNA libraries form murine hematopoietic stem cells and progenitors. Subsequent work has revealed that FLRF acts as E3 ubiquitin ligase, and that it regulates steady-state levels of neuregulin receptor ErbB3 and participates in degradation of IAP protein BRUCE and parkin. The objective of this study was to start exploring the role of FLRF during hematopoiesis. FLRF was overexpressed in a murine multipotent hematopoietic progenitor cell line EML, which can differentiate into almost all blood cell lineages, and in pro-B progenitor cell line BaF3. The impact of FLRF overexpression on EML cell differentiation into myeloerythroid lineages was studied using hematopoietic colony-forming assays. The interaction of FLRF with cytokine receptors and receptor levels in control cells and EML and BaF3 cells overexpressing FLRF were examined with Western and immunoprecipitation. Remarkably, overexpression of FLRF significantly attenuated erythroid and myeloid differentiation of EML cells in response to cytokines erythropoietin (EPO) and interleukin-3 (IL-3), and retinoic acid (RA), and resulted in significant and constitutive decrease of steady-state levels of IL-3, EPO, and RA receptor-alpha (RARalpha) in EML and BaF3 cells. Immunoprecipitation has revealed that FLRF interacts with IL-3, EPO, and RARalpha receptors in EML and BaF3 cells, and that FLRF-mediated downregulation of these receptors is ligand binding-independent. The results of this study have revealed new FLRF-mediated pathway for ligand-independent receptor level regulation, and support the notion that through maintaining basal levels of cytokine receptors, FLRF is involved in the control of hematopoietic progenitor cell differentiation into myeloerythroid lineages.
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Freeman, Spencer A; Jaumouillé, Valentin; Choi, Kate; Hsu, Brian E; Wong, Harikesh S; Abraham, Libin; Graves, Marcia L; Coombs, Daniel; Roskelley, Calvin D; Das, Raibatak; Grinstein, Sergio; Gold, Michael R
2015-02-03
Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection.
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Freeman, Spencer A.; Jaumouillé, Valentin; Choi, Kate; Hsu, Brian E.; Wong, Harikesh S.; Abraham, Libin; Graves, Marcia L.; Coombs, Daniel; Roskelley, Calvin D.; Das, Raibatak; Grinstein, Sergio; Gold, Michael R.
2015-01-01
Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection. PMID:25644899
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Accili, D; Frapier, C; Mosthaf, L; McKeon, C; Elbein, S C; Permutt, M A; Ramos, E; Lander, E; Ullrich, A; Taylor, S I
1989-01-01
Insulin binds to a receptor on the cell surface, thereby triggering a biological response within the target cell. Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. We have studied a family in which two sisters have a genetic form of insulin-resistant diabetes mellitus. The technique of homozygosity mapping has been used to demonstrate that the mutation causing diabetes in this consanguineous family is genetically linked to the insulin receptor gene. The two insulin-resistant sisters are homozygous for a mutation encoding substitution of valine for phenylalanine at position 382 in the alpha-subunit of the insulin receptor. Transfection of mutant insulin receptor cDNA into NIH3T3 cells demonstrated that the Val382 mutation impaired post-translational processing and retarded transport of the insulin receptor to the plasma membrane. Thus, the mutation causes insulin resistance by decreasing the number of insulin receptors on the surface of the patients' cells. Images PMID:2573522
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Lidocaine Stimulates the Function of Natural Killer Cells in Different Experimental Settings.
Cata, Juan P; Ramirez, Maria F; Velasquez, Jose F; Di, A I; Popat, Keyuri U; Gottumukkala, Vijaya; Black, Dahlia M; Lewis, Valerae O; Vauthey, Jean N
2017-09-01
One of the functions of natural killer (NK) cells is to eliminate cancer cells. The cytolytic activity of NK cells is tightly regulated by inhibitory and activation receptors located in the surface membrane. Lidocaine stimulates the function of NK cells at clinically relevant concentrations. It remains unknown whether this effect of lidocaine has an impact on the expression of surface receptors of NK cells, can uniformly stimulate across different cancer cell lines, and enhances the function of cells obtained during oncological surgery. NK cells from healthy donors and 43 patients who had undergone surgery for cancer were isolated. The function of NK cells was measured by lactate dehydrogenase release assay. NK cells were incubated with clinically relevant concentrations of lidocaine. By flow cytometry, we determined the impact of lidocaine on the expression of galactosylgalactosylxylosylprotein3-beta-glucuronosytranferase 1, marker of cell maturation (CD57), killer cell lectin like receptor A, inhibitory (NKG2A) receptors and killer cell lectin like receptor D, activation (NKG2D) receptors of NK cells. Differences in expression at p<0.05 were considered statistically significant. Lidocaine increased the expression of NKG2D receptors and stimulated the function of NK cells against ovarian, pancreatic and ovarian cancer cell lines. Lidocaine also increased the cytolytic activity of NK cells from patients who underwent oncological surgery, except for those who had orthopedic procedures. Lidocaine showed an important stimulatory activity on NK cells. Our findings suggest that lidocaine might be used perioperatively to minimize the impact of surgery on NK cells. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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Umami Responses in Mouse Taste Cells Indicate More than One Receptor
Maruyama, Yutaka; Pereira, Elizabeth; Margolskee, Robert F.; Chaudhari, Nirupa; Roper, Stephen D.
2013-01-01
A number of gustatory receptors have been proposed to underlie umami, the taste of L-glutamate, and certain other amino acids and nucleotides. However, the response profiles of these cloned receptors have not been validated against responses recorded from taste receptor cells that are the native detectors of umami taste. We investigated umami taste responses in mouse circumvallate taste buds in an intact slice preparation, using confocal calcium imaging. Approximately 5% of taste cells selectively responded to L-glutamate when it was focally applied to the apical chemosensitive tips of receptor cells. The concentration–response range for L-glutamate fell approximately within the physiologically relevant range for taste behavior in mice, namely 10 mM and above. Inosine monophosphate enhanced taste cell responses to L-glutamate, a characteristic feature of umami taste. Using pharmacological agents, ion substitution, and immunostaining, we showed that intracellular pathways downstream of receptor activation involve phospholipase C β2. Each of the above features matches those predicted by studies of cloned and expressed receptors. However, the ligand specificity of each of the proposed umami receptors [taste metabotropic glutamate receptor 4, truncated metabotropic glutamate receptor 1, or taste receptor 1 (T1R1) and T1R3 dimers], taken alone, did not appear to explain the taste responses observed in mouse taste cells. Furthermore, umami responses were still observed in mutant mice lacking T1R3. A full explanation of umami taste transduction may involve novel combinations of the proposed receptors and/or as-yet-undiscovered taste receptors. PMID:16495449
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Lin, Liang-Tzung; Richardson, Christopher D.
2016-01-01
The hemagglutinin (H) protein of measles virus (MeV) interacts with a cellular receptor which constitutes the initial stage of infection. Binding of H to this host cell receptor subsequently triggers the F protein to activate fusion between virus and host plasma membranes. The search for MeV receptors began with vaccine/laboratory virus strains and evolved to more relevant receptors used by wild-type MeV. Vaccine or laboratory strains of measles virus have been adapted to grow in common cell lines such as Vero and HeLa cells, and were found to use membrane cofactor protein (CD46) as a receptor. CD46 is a regulator that normally prevents cells from complement-mediated self-destruction, and is found on the surface of all human cells, with the exception of erythrocytes. Mutations in the H protein, which occur during adaptation and allow the virus to use CD46 as a receptor, have been identified. Wild-type isolates of measles virus cannot use the CD46 receptor. However, both vaccine/laboratory and wild-type strains can use an immune cell receptor called signaling lymphocyte activation molecule family member 1 (SLAMF1; also called CD150) and a recently discovered epithelial receptor known as Nectin-4. SLAMF1 is found on activated B, T, dendritic, and monocyte cells, and is the initial target for infections by measles virus. Nectin-4 is an adherens junction protein found at the basal surfaces of many polarized epithelial cells, including those of the airways. It is also over-expressed on the apical and basal surfaces of many adenocarcinomas, and is a cancer marker for metastasis and tumor survival. Nectin-4 is a secondary exit receptor which allows measles virus to replicate and amplify in the airways, where the virus is expelled from the body in aerosol droplets. The amino acid residues of H protein that are involved in binding to each of the receptors have been identified through X-ray crystallography and site-specific mutagenesis. Recombinant measles “blind” to each of these receptors have been constructed, allowing the virus to selectively infect receptor specific cell lines. Finally, the observations that SLAMF1 is found on lymphomas and that Nectin-4 is expressed on the cell surfaces of many adenocarcinomas highlight the potential of measles virus for oncolytic therapy. Although CD46 is also upregulated on many tumors, it is less useful as a target for cancer therapy, since normal human cells express this protein on their surfaces. PMID:27657109
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Lin, Liang-Tzung; Richardson, Christopher D
2016-09-20
The hemagglutinin (H) protein of measles virus (MeV) interacts with a cellular receptor which constitutes the initial stage of infection. Binding of H to this host cell receptor subsequently triggers the F protein to activate fusion between virus and host plasma membranes. The search for MeV receptors began with vaccine/laboratory virus strains and evolved to more relevant receptors used by wild-type MeV. Vaccine or laboratory strains of measles virus have been adapted to grow in common cell lines such as Vero and HeLa cells, and were found to use membrane cofactor protein (CD46) as a receptor. CD46 is a regulator that normally prevents cells from complement-mediated self-destruction, and is found on the surface of all human cells, with the exception of erythrocytes. Mutations in the H protein, which occur during adaptation and allow the virus to use CD46 as a receptor, have been identified. Wild-type isolates of measles virus cannot use the CD46 receptor. However, both vaccine/laboratory and wild-type strains can use an immune cell receptor called signaling lymphocyte activation molecule family member 1 (SLAMF1; also called CD150) and a recently discovered epithelial receptor known as Nectin-4. SLAMF1 is found on activated B, T, dendritic, and monocyte cells, and is the initial target for infections by measles virus. Nectin-4 is an adherens junction protein found at the basal surfaces of many polarized epithelial cells, including those of the airways. It is also over-expressed on the apical and basal surfaces of many adenocarcinomas, and is a cancer marker for metastasis and tumor survival. Nectin-4 is a secondary exit receptor which allows measles virus to replicate and amplify in the airways, where the virus is expelled from the body in aerosol droplets. The amino acid residues of H protein that are involved in binding to each of the receptors have been identified through X-ray crystallography and site-specific mutagenesis. Recombinant measles "blind" to each of these receptors have been constructed, allowing the virus to selectively infect receptor specific cell lines. Finally, the observations that SLAMF1 is found on lymphomas and that Nectin-4 is expressed on the cell surfaces of many adenocarcinomas highlight the potential of measles virus for oncolytic therapy. Although CD46 is also upregulated on many tumors, it is less useful as a target for cancer therapy, since normal human cells express this protein on their surfaces.
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Zhu, Jie; Bandi, Venkata; Qiu, Shengyang; Figueroa, David J.; Evans, Jilly F.; Barnes, Neil; Guntupalli, Kay K.
2012-01-01
Background: Cysteinyl leukotriene 1 (CysLT1) receptor expression is known to be increased in the airway mucosa of patients with asthma, especially during exacerbations; however, nothing is known of its expression in COPD. Methods: We applied immunohistochemistry and in situ hybridization to endobronchial biopsies to determine inflammatory cell CysLT1 receptor protein and mRNA expression in the following: (1) 15 nonsmoker control subjects (NSC), (2) 16 smokers with moderate to severe COPD in its stable phase (S-COPD), and (3) 15 smokers with COPD hospitalized for a severe exacerbation (SE-COPD). Results: The total number of bronchial mucosal inflammatory cells (CD45+) and those expressing CysLT1 receptor protein were significantly greater in SE-COPD (CysLT1 receptor protein: median [range] = 139 [31-634]) as compared with S-COPD (32 [6-114]) or NSC (16 [4-66]) (P < .001 for both). CysLT1 receptor gene expression showed similar differences. A greater proportion of CD451 cells expressed CysLT1 receptor protein in SE-COPD (median [range] = 22% [8-81]) compared with S-COPD (10% [4-32]) (P < .03) or NSC (7% [1-19]) (P < .002). In SE-COPD, the relative frequencies of CysLT1 receptor-expressing cells were as follows: tryptase1 mast cells > CD681 monocytes/macrophage > neutrophils > CD201 B lymphocytes = EG21 eosinophils. Moreover, there were positive correlations between the numbers of cells expressing CysLT1 receptor protein and the numbers of CD451 cells (r = 0.78; P < .003) and tryptase1 mast cells (r = 0.62; P < .02). Conclusions: Bronchial mucosal CysLT1 receptor-positive inflammatory cells are present in the bronchial mucosa in COPD in greatest number in those experiencing a severe exacerbation. PMID:22871757
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Jiang, Ninghong; Xie, Feng; Guo, Qisang; Li, Ming-Qing; Xiao, Jingjing; Sui, Long
2017-06-01
Toll-like receptor 4 is overexpressed in various tumors, including cervical carcinoma. However, the role of Toll-like receptor 4 in cervical cancer remains controversial, and the underlying mechanisms are largely elusive. Therefore, Toll-like receptor 4 in cervical cancer and related mechanisms were investigated in this study. Quantitative reverse transcription polymerase chain reaction and western blot analyses were used to detect messenger RNA and protein levels in HeLa, Caski, and C33A cells with different treatments. Proliferation was quantified using Cell Counting Kit-8. Cell cycle distribution and apoptosis were assessed by flow cytometry. Higher levels of Toll-like receptor 4 expression were found in human papillomavirus-positive cells compared to human papillomavirus-negative cells. Proliferation of HeLa and Caski cells was promoted in lipopolysaccharide-stimulated groups but suppressed in short hairpin RNA-transfected groups. Apoptosis rates were lower in lipopolysaccharide-stimulated groups relative to short hairpin RNA-transfected groups. In addition, G2-phase distribution was enhanced when Toll-like receptor 4 was downregulated. Moreover, the pNF-κBp65 level was positively correlated with the Toll-like receptor 4 level in HeLa and Caski cells, though when an nuclear factor-κB inhibitor was applied to lipopolysaccharide-stimulated groups, the patterns of proliferation and apoptosis were opposite to those of the lipopolysaccharide-stimulated groups without inhibitor treatment. In conclusion, these data suggest that Toll-like receptor 4 promotes proliferation and apoptosis resistance in human papillomavirus-related cervical cancer cells at least in part through the Toll-like receptor 4/nuclear factor-κB pathway, which may be correlated with the occurrence and development of cervical carcinoma.
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Harnessing tumor necrosis factor receptors to enhance antitumor activities of drugs.
Muntané, Jordi
2011-10-17
Cancer is the second-leading cause of death in the U.S. behind heart disease and over stroke. The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors. The inhibition of cell death pathways is one of these tumor characteristics which also include sustained proliferative signaling, evading growth suppressor signaling, replicative immortality, angiogenesis, and promotion of invasion and metastasis. Cell death is mediated through death receptor (DR) stimulation initiated by specific ligands that transmit signaling to the cell death machinery or through the participation of mitochondria. Cell death involving DR is mediated by the superfamily of tumor necrosis factor receptor (TNF-R) which includes TNF-R type I, CD95, DR3, TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 (TRAIL-R1) and -2 (TRAIL-R2), DR6, ectodysplasin A (EDA) receptor (EDAR), and the nerve growth factor (NGF) receptor (NGFR). The expression of these receptors in healthy and tumor cells induces treatment side effects that limit the systemic administration of cell death-inducing therapies. The present review is focused on the different therapeutic strategies such as targeted antibodies or small molecules addressed to selective stimulated DR-mediated apoptosis or reduce cell proliferation in cancer cells.
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Chimeric antigen receptors: driving immunology towards synthetic biology
Sadelain, Michel
2017-01-01
The advent of second generation CARs and the CD19 paradigm have ushered a new therapeutic modality in oncology. In contrast to earlier forms of adoptive cell therapy, which were based on the isolation and expansion of naturally occurring T cells, CAR therapy is based on the design and manufacture of engineered T cells with optimized properties. A new armamentarium, comprising not only CARs but also chimeric costimulatory receptors, chimeric cytokine receptors, inhibitory receptors and synthetic Notch receptors, expressed in naïve, central memory or stem cell-like memory T cells, is being developed for clinical use in a wide range of cancers. Immunological principles are thus finding a new purpose thanks to advances in genetic engineering, synthetic biology and cell manufacturing sciences. PMID:27372731
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Binding of phycoerythrin and its conjugates to murine low affinity receptors for immunoglobulin G.
Takizawa, F; Kinet, J P; Adamczewski, M
1993-06-18
Conjugates of R-phycoerythrin are widely used for immunohistochemistry, especially for two-color flow cytometry. Their use is however limited by their apparent tendency to bind non-specifically. Using cells transfected with cDNAs for the murine low affinity receptors for immunoglobulin G (Fc gamma RII and -III) and cells naturally expressing these receptors, we demonstrate that R-phycoerythrin and its conjugates bind specifically and inhibitably to Fc gamma RII and -III. Immunofluorescence stainings of cells bearing these receptors, such as macrophages, monocytes, neutrophils, mast cells, subsets of T cells, and natural killer cells, may therefore not reflect the binding of antibody to antigen, but rather the binding of R-phycoerythrin to the receptors.
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Lanemo Myhrinder, Anna; Hellqvist, Eva; Bergh, Ann-Charlotte; Jansson, Mattias; Nilsson, Kenneth; Hultman, Per; Jonasson, Jon; Buhl, Anne Mette; Bredo Pedersen, Lone; Jurlander, Jesper; Klein, Eva; Weit, Nicole; Herling, Marco; Rosenquist, Richard; Rosén, Anders
2013-08-01
Chronic lymphocytic leukemia (CLL) B-cells resemble self-renewing CD5 + B-cells carrying auto/xeno-antigen-reactive B-cell receptors (BCRs) and multiple innate pattern-recognition receptors, such as Toll-like receptors and scavenger receptors. Integration of signals from BCRs with multiple surface membrane receptors determines whether the cells will be proliferating, anergic or apoptotic. To better understand the role of antigen in leukemogenesis, CLL cell lines producing monoclonal antibodies (mAbs) will facilitate structural analysis of antigens and supply DNA for genetic studies. We present here a comprehensive genotypic and phenotypic characterization of available CLL and normal B-cell-derived lymphoblastoid cell lines (LCLs) from the same individuals (n = 17). Authenticity and verification studies of CLL-patient origin were done by IGHV sequencing, fluorescence in situ hybridization (FISH) and DNA/short tandem repeat (STR) fingerprinting. Innate B-cell features, i.e. natural Ab production and CD5 receptors, were present in most CLL cell lines, but in none of the normal LCLs. This panel of immortalized CLL-derived cell lines is a valuable reference representing a renewable source of authentic Abs and DNA.
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Direct Force Measurements of Receptor-Ligand Interactions on Living Cells
NASA Astrophysics Data System (ADS)
Eibl, Robert H.
The characterization of cell adhesion between two living cells at the level of single receptor-ligand bonds is an experimental challenge. This chapter describes how the extremely sensitive method of atomic force microscopy (AFM) based force spectroscopy can be applied to living cells in order to probe for cell-to-cell or cell-to-substrate interactions mediated by single pairs of adhesion receptors. In addition, it is outlined how single-molecule AFM force spectroscopy can be used to detect physiologic changes of an adhesion receptor in a living cell. This force spectroscopy allows us to detect in living cells rapidly changing, chemokine SDF-1 triggered activation states of single VLA-4 receptors. This recently developed AFM application will allow for the detailed investigation of the integrin-chemokine crosstalk of integrin activation mechanisms and on how other adhesion receptors are modulated in health and disease. As adhesion molecules, living cells and even bacteria can be studied by single-molecule AFM force spectroscopy, this method is set to become a powerful tool that can not only be used in biophysics, but in cell biology as well as in immunology and cancer research.
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Georgescu, Maria-Magdalena; Kirsch, Kathrin H.; Shishido, Tomoyuki; Zong, Chen; Hanafusa, Hidesaburo
1999-01-01
The c-Mer receptor tyrosine kinase (RTK) is most closely related to chicken c-Eyk and belongs to the Axl RTK subfamily. Although not detected in normal lymphocytes, c-Mer is expressed in B- and T-cell leukemia cell lines, suggesting an association with lymphoid malignancies. To gain an understanding of the role of this receptor in lymphoid cells, we expressed in murine interleukin-3 (IL-3)-dependent Ba/F3 pro-B-lymphocyte cells a constitutively active receptor, CDMer, formed from the CD8 extracellular domain and the c-Mer intracellular domain. Cells transfected with a plasmid encoding the CDMer receptor became IL-3 independent. When tyrosine (Y)-to-phenylalanine (F) mutations were introduced into c-Mer, only the Y867 change significantly reduced the IL-3-independent cell proliferation. The Y867 residue in the CDMer receptor mediated the binding of Grb2, which recruited the p85 phosphatidylinositol 3-kinase (PI 3-kinase). Despite the difference in promotion of proliferation, both the CDMer and mutant F867 receptors activated Erk in transfected cells. On the other hand, we found that both transcriptional activation of NF-κB and activation of PI 3-kinase were significantly suppressed with the F867 mutant receptor, suggesting that the activation of antiapoptotic pathways is the major mechanism for the observed phenotypic difference. Consistent with this notion, apoptosis induced by IL-3 withdrawal was strongly prevented by CDMer but not by the F867 mutant receptor. PMID:9891051
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Functional dynamics of cell surface membrane proteins
NASA Astrophysics Data System (ADS)
Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio
2014-04-01
Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.
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Functional dynamics of cell surface membrane proteins.
Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio
2014-04-01
Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.
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Cassiman, David; Libbrecht, Louis; Sinelli, Nicoletta; Desmet, Valeer; Denef, Carl; Roskams, Tania
2002-01-01
In the rat the hepatic branch of the nervus vagus stimulates proliferation of hepatocytes after partial hepatectomy and growth of bile duct epithelial cells after bile duct ligation. We studied the effect of hepatic vagotomy on the activation of the hepatic progenitor cell compartment in human and rat liver. The number of hepatic progenitor cells and atypical reactive ductular cells in transplanted (denervated) human livers with hepatitis was significantly lower than in innervated matched control livers and the number of oval cells in vagotomized rat livers with galactosamine hepatitis was significantly lower than in livers of sham-operated rats with galactosamine hepatitis. The expression of muscarinic acetylcholine receptors (M1-M5 receptor) was studied by immunohistochemistry and reverse transcriptase-polymerase chain reaction. In human liver, immunoreactivity for M3 receptor was observed in hepatic progenitor cells, atypical reactive ductules, intermediate hepatocyte-like cells, and bile duct epithelial cells. mRNA for the M1-M3 and the M5 receptor, but not the M4 receptor, was detected in human liver homogenates. In conclusion, the hepatic vagus branch stimulates activation of the hepatic progenitor cell compartment in diseased liver, most likely through binding of acetylcholine to the M3 receptor expressed on these cells. These findings may be of clinical importance for patients with a transplant liver. PMID:12163377
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P2 receptor subtypes in the cardiovascular system.
Kunapuli, S P; Daniel, J L
1998-01-01
Extracellular nucleotides have been implicated in a number of physiological functions. Nucleotides act on cell-surface receptors known as P2 receptors, of which several subtypes have been cloned. Both ATP and ADP are stored in platelets and are released upon platelet activation. Furthermore, nucleotides are also released from damaged or broken cells. Thus during vascular injury nucleotides play an important role in haemostasis through activation of platelets, modulation of vascular tone, recruitment of neutrophils and monocytes to the site of injury, and facilitation of adhesion of leucocytes to the endothelium. Nucleotides also moderate these functions by generating nitric oxide and prostaglandin I2 through activation of endothelial cells, and by activating different receptor subtypes on vascular smooth muscle cells. In the heart, P2 receptors regulate contractility through modulation of L-type Ca2+ channels, although the molecular mechanisms involved are still under investigation. Classical pharmacological studies have identified several P2 receptor subtypes in the cardiovascular system. Molecular pharmacological studies have clarified the nature of some of these receptors, but have complicated the picture with others. In platelets, the classical P2T receptor has now been resolved into three P2 receptor subtypes: the P2Y1, P2X1 and P2TAC receptors (the last of these, which is coupled to the inhibition of adenylate cyclase, is yet to be cloned). In peripheral blood leucocytes, endothelial cells, vascular smooth muscle cells and cardiomyocytes, the effects of classical P2X, P2Y and P2U receptors have been found to be mediated by more than one P2 receptor subtype. However, the exact functions of these multiple receptor subtypes remain to be understood, as P2-receptor-selective agonists and antagonists are still under development. PMID:9841859
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Herpes B Virus Utilizes Human Nectin-1 but Not HVEM or PILRα for Cell-Cell Fusion and Virus Entry
Fan, Qing; Amen, Melanie; Harden, Mallory; Severini, Alberto; Griffiths, Anthony
2012-01-01
To investigate the requirements of herpesvirus entry and fusion, the four homologous glycoproteins necessary for herpes simplex virus (HSV) fusion were cloned from herpes B virus (BV) (or macacine herpesvirus 1, previously known as cercopithecine herpesvirus 1) and cercopithecine herpesvirus 2 (CeHV-2), both related simian simplexviruses belonging to the alphaherpesvirus subfamily. Western blots and cell-based enzyme-linked immunosorbent assay (ELISA) showed that glycoproteins gB, gD, and gH/gL were expressed in whole-cell lysates and on the cell surface. Cell-cell fusion assays indicated that nectin-1, an HSV-1 gD receptor, mediated fusion of cells expressing glycoproteins from both BV and CeHV-2. However, herpesvirus entry mediator (HVEM), another HSV-1 gD receptor, did not facilitate BV- and CeHV-2-induced cell-cell fusion. Paired immunoglobulin-like type 2 receptor alpha (PILRα), an HSV-1 gB fusion receptor, did not mediate fusion of cells expressing glycoproteins from either simian virus. Productive infection with BV was possible only with nectin-1-expressing cells, indicating that nectin-1 mediated entry while HVEM and PILRα did not function as entry receptors. These results indicate that these alphaherpesviruses have differing preferences for entry receptors. The usage of the HSV-1 gD receptor nectin-1 may explain interspecies transfer of the viruses, and altered receptor usage may result in altered virulence, tropism, or pathogenesis in the new host. A heterotypic cell fusion assay resulting in productive fusion may provide insight into interactions that occur to trigger fusion. These findings may be of therapeutic significance for control of deadly BV infections. PMID:22345445
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Norris, J S; Kohler, P O
1978-01-01
Two hamster cell lines have been isolated from androgen target tissue. The DDT1 cells derived from ductus deferens tissue exhibit a growth response to androgens, while the HVP cells derived from ventral prostate are androgen unresponsive. Both cell lines contain androgen receptors, that are similar when compared by kinetic methods, sedimentation velocity, chromatographic procedures or nuclear translocation ability. The forms of the high salt extracted nuclear receptors are indistinguishable chromatographically. Therefore, we postulate that the lesion preventing androgen induced growth in the HVP cell line is subseqent to nuclear translocation of the steroid receptor complex.
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Janefjord, Emelie; Mååg, Jesper L V; Harvey, Benjamin S; Smid, Scott D
2014-01-01
Cannabinoid (CB) ligands have demonstrated neuroprotective properties. In this study we compared the effects of a diverse set of CB ligands against β amyloid-mediated neuronal toxicity and activated microglial-conditioned media-based neurotoxicity in vitro, and compared this with a capacity to directly alter β amyloid (Aβ) fibril or aggregate formation. Neuroblastoma (SH-SY5Y) cells were exposed to Aβ1-42 directly or microglial (BV-2 cells) conditioned media activated with lipopolysaccharide (LPS) in the presence of the CB1 receptor-selective agonist ACEA, CB2 receptor-selective agonist JWH-015, phytocannabinoids Δ(9)-THC and cannabidiol (CBD), the endocannabinoids 2-arachidonoyl glycerol (2-AG) and anandamide or putative GPR18/GPR55 ligands O-1602 and abnormal-cannabidiol (Abn-CBD). TNF-α and nitrite production was measured in BV-2 cells to compare activation via LPS or albumin with Aβ1-42. Aβ1-42 evoked a concentration-dependent loss of cell viability in SH-SY5Y cells but negligible TNF-α and nitrite production in BV-2 cells compared to albumin or LPS. Both albumin and LPS-activated BV-2 conditioned media significantly reduced neuronal cell viability but were directly innocuous to SH-SY5Y cells. Of those CB ligands tested, only 2-AG and CBD were directly protective against Aβ-evoked SH-SY5Y cell viability, whereas JWH-015, THC, CBD, Abn-CBD and O-1602 all protected SH-SY5Y cells from BV-2 conditioned media activated via LPS. While CB ligands variably altered the morphology of Aβ fibrils and aggregates, there was no clear correlation between effects on Aβ morphology and neuroprotective actions. These findings indicate a neuroprotective action of CB ligands via actions at microglial and neuronal cells.
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Kaartinen, Tanja; Luostarinen, Annu; Maliniemi, Pilvi; Keto, Joni; Arvas, Mikko; Belt, Heini; Koponen, Jonna; Loskog, Angelica; Mustjoki, Satu; Porkka, Kimmo; Ylä-Herttuala, Seppo; Korhonen, Matti
2017-06-01
Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory. We have developed a cost-effective expansion protocol for chimeric antigen receptor (CAR) T cells with an early memory phenotype. Lymphocytes were transduced with third-generation lentiviral vectors and expanded using CD3/CD28 microbeads. The effects of altering the IL-2 supplementation (0-300 IU/mL) and length of expansion (10-20 days) on the phenotype of the T-cell products were analyzed. High IL-2 levels led to a decrease in overall generation of early memory T cells by both decreasing central memory T cells and augmenting effectors. T memory stem cells (T SCM , CD95 + CD45RO - CD45RA + CD27 + ) were present variably during T-cell expansion. However, their presence was not IL-2 dependent but was linked to expansion kinetics. CD19-CAR T cells generated in these conditions displayed in vitro antileukemic activity. In summary, production of CAR T cells without any cytokine supplementation yielded the highest proportion of early memory T cells, provided a 10-fold cell expansion and the cells were functionally potent. The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost-effective T-cell manufacturing. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
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Kouzel, Ivan U; Pohlentz, Gottfried; Storck, Wiebke; Radamm, Lena; Hoffmann, Petra; Bielaszewska, Martina; Bauwens, Andreas; Cichon, Christoph; Schmidt, M Alexander; Mormann, Michael; Karch, Helge; Müthing, Johannes
2013-03-01
Glycosphingolipids (GSLs) of the globo-series constitute specific receptors for Shiga toxins (Stxs) released by certain types of pathogenic Escherichia coli strains. Stx-loaded leukocytes may act as transporter cells in the blood and transfer the toxin to endothelial target cells. Therefore, we performed a thorough investigation on the expression of globo-series GSLs in serum-free cultivated Raji and Jurkat cells, representing B- and T-lymphocyte descendants, respectively, as well as THP-1 and HL-60 cells of the monocyte and granulocyte lineage, respectively. The presence of Stx-receptors in GSL preparations of Raji and THP-1 cells and the absence in Jurkat and HL-60 cells revealed high compliance of solid-phase immunodetection assays with the expression profiles of receptor-related glycosyltransferases, performed by qRT-PCR analysis, and Stx2-caused cellular damage. Canonical microdomain association of Stx GSL receptors, sphingomyelin, and cholesterol in membranes of Raji and THP-1 cells was assessed by comparative analysis of detergent-resistant membrane (DRM) and nonDRM fractions obtained by density gradient centrifugation and showed high correlation based on nonparametric statistical analysis. Our comprehensive study on the expression of Stx-receptors and their subcellular distribution provides the basis for exploring the functional role of lipid raft-associated Stx-receptors in cells of leukocyte origin.
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Anton, Olga M.; Vielkind, Susina; Peterson, Mary E.; Tagaya, Yutaka; Long, Eric O.
2015-01-01
IL-15 bound to the IL-15 receptor α chain (IL-15Rα) is presented in trans to cells bearing the IL-2 receptor β and γc chains. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, it has the potential for regulation by and of other receptor–ligand interactions. In this study, human NK cells were tested for the sensitivity of IL-15 transpresentation to inhibitory receptors. Human cells expressing HLA class I ligands for inhibitory receptors KIR2DL1, KIR2DL2/3, or CD94-NKG2A were transfected with IL-15Rα. Proliferation of primary NK cells in response to transpresented IL-15 was reduced by engagement of either KIR2DL1 or KIR2DL2/3 by cognate HLA-C ligands. Inhibitory KIR–HLA-C interactions did not reduce the proliferation induced by soluble IL-15. Therefore, transpresentation of IL-15 is subject to down-regulation by MHC class I-specific inhibitory receptors. Similarly, proliferation of the NKG2A+ cell line NKL induced by IL-15 transpresentation was inhibited by HLA-E. Co-engagement of inhibitory receptors, either KIR2DL1 or CD94-NKG2A, did not inhibit phosphorylation of Stat5 but inhibited selectively phosphorylation of Akt and S6 ribosomal protein. IL-15Rα was not excluded from, but was evenly distributed across inhibitory synapses. These findings demonstrate a novel mechanism to attenuate IL-15 dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis. PMID:26453750
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Agui, T; Yamada, T; Legros, G; Nakajima, T; Clark, M; Peschel, C; Matsumoto, K
1992-05-01
Atrial natriuretic peptide (ANP) receptors were identified on both murine bone marrow-derived stromal cell lines A-3 and ALC and primary cultured cells using [125I]ANP binding assays and Northern blot analyses. The binding of [125I] ANP to the stromal cells was rapid, saturable, and of high affinity. The dissociation constants between ANP and its receptors on these cells showed no difference among cell types, while maximal binding capacity values were different among cell types. Competitive inhibition of [125I]ANP binding with C-atrial natriuretic factor, specific for ANP clearance receptor (ANPR-C), revealed that most of [125I]ANP-binding sites corresponded to ANPR-C. Northern blotting data corroborated that bone marrow-derived stromal cells expressed ANPR-C. However, in ALC cells, ANP biological receptors (either ANPR-A or ANPR-B), the mol wt of which is approximately 130K, were detected, and cGMP was accumulated after stimulation with ANP. On the other hand, in another stromal cell clone, A-3 cells, the expression of biological receptor was not detected in the affinity cross-linking and competitive inhibition experiments using [125I]ANP. However, A-3 cells accumulated cGMP by responding to ANPR-B-specific ligand, C-type natriuretic peptide. These results suggest that ALC cells equally express ANPR-A and ANPR-B, while A-3 cells express ANPR-B dominantly. Although the physiological roles of these receptors in the bone marrow is still not resolved, ANP is expected to play a role in the regulation of stromal cell functions in bone marrow.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kellokumpu, S.
1987-02-01
The metabolic fate of LH/hCG receptors after exposure to human chorionic gonadotropin (hCG) was examined in cultured rat luteal cells and murine Leydig tumor cells (MLTC-1). Kinetic studies performed after pulse-labelling of the cells with (/sup 125/I)hCG indicated that the bound hormone was lost much more rapidly from the tumor cells than from the luteal cells. The tumor cells were also found to internalize and degrade the hormone more effectively than the luteal cells. Chemical cross-linking and analyses by SDS-PAGE of this material revealed that both cell types also released, in addition to intact hCG, two previously characterized receptor fragment-(/supmore » 125/I)hCG complexes (M/sub r/ 96,000 and 74,000) into the medium, although their amount was negligible in MLTC-1 cells. Possibly due to rapid discharge of the ligand from its receptor, no similar complexes could be detected inside the MLTC-1 cells, suggesting that they were released directly from the cell surface. However, the M/sub r/ 74,000 complex was observed inside MLTC-1 cells if chloroquine, a lysosomotropic agent, was present during the incubations. This suggests that the internalized receptor also becomes degraded, at least when complexed to hCG. The results thus provide evidence that there exist two different mechanisms for proteolytic processing of LH/hCG receptors in these target cells. In tumor cells, the degradation seems to occur almost exclusively intracellularly, whereas in luteal cells a substantial portion of the receptors is also degraded at the cell surface.« less
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2008-01-01
Mast cells have been recognized for well over 100 years. With time, human mast cells have been documented to originate from CD34+ cells, and have been implicated in host responses in both innate and acquired immunity. In clinical immunology, they are recognized for their central role in IgE-mediated degranulation and allergic inflammation by virtue of their expression of the high-affinity receptor for IgE and release of potent proinflammatory mediators. In hematology, the clinical disease of mastocytosis is characterized by a pathologic increase of mast cells in tissues, often associated with mutations in KIT, the receptor for stem cell factor. More recently, and with increased understanding of how human mast cells are activated through receptors including the high-affinity receptor for IgE and KIT, specific tyrosine kinase inhibitors have been identified with the potential to interrupt signaling pathways and thus limit the proliferation of mast cells as well as their activation through immunoglobulin receptors. PMID:18684881
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Faridi, R M; Agrawal, S
2011-02-01
Decidual natural killer (NK) cells play key developmental roles at the feto-maternal interface. Individual differences in NK-cell interactions are dependent on the combinations of variable killer immunoglobulin-like receptor (KIR) and HLA class-I gene products. As different receptor-ligand interactions may result in altered NK-cell-mediated immunity against pathogens, it is proposed that the relationship between these genes may be important in a state such as recurrent miscarriage (RM). We had earlier reported that the predisposition to RM is influenced by the maternal KIR gene content. In the present study, we have attempted to extend our findings in the light of contribution from the paternal antigens on the outcome of pregnancy, since maternal NK cells may potentially encounter non-self-paternal HLA-C alleles on trophoblasts. All HLA-C allotypes fall into two major KIR epitopes--C1 (HLA-C*01/*03/*07/*08/*12/*14/*16) and C2 (HLA-C*02/*04/*05/*06/*15/*17/*18)--on the basis of a dimorphism at position 80 of the α1 domain. PCR-sequence specific primer-based genotyping was used to determine the maternal KIR gene content and HLA-C genotypes down to allele level in couples experiencing RM and controls. KIR2DL1 with both partners homozygous for HLA C2 was significantly higher in control couples when compared with the patients [P = 0.0004, odds ratio (OR) = 0.28, 95% confidence interval (CI) = 0.13-0.58]. The activating KIR2DS2 with both partners homozygous for HLA C1 was significantly higher in patients when compared with the controls (P = 0.002, OR = 2.83, 95% CI = 1.47-5.40). Our results represented the 'top-end' of the activation spectrum of KIR-HLA-C compound genotype for NK cells and this may contribute to the immunological etiology of RM.
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The Sigma-2 (σ2) Receptor: A Novel Protein for the Imaging and Treatment of Cancer
Mach, Robert H.; Zeng, Chenbo; Hawkins, William G.
2014-01-01
The sigma-2 (σ2) receptor is an important target for the development of molecular probes in oncology because of its 10-fold higher density in proliferating tumor cells than in quiescent tumor cells, and the observation that σ2 receptor agonists are able to kill tumor cells via apoptotic and non-apoptotic mechanisms. Although recent evidence indicates the σ2 receptor binding site is localized within the progesterone receptor membrane component 1 (PGRMC1), most information regarding this protein has been obtained using either radiolabeled or fluorescent receptor-based probes, and from biochemical analysis of the effect of σ2 selective ligands on cells grown in culture. This article reviews the development of σ2 receptor ligands, and presents an overview of how they have been used in vitro and in vivo to increase our understanding of the role of the σ2 receptor in cancer and proliferation. PMID:23734634
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Cellular progesterone receptor phosphorylation in response to ligands activating protein kinases
DOE Office of Scientific and Technical Information (OSTI.GOV)
Rao, K.V.; Peralta, W.D.; Greene, G.L.
1987-08-14
Progesterone receptors were immunoprecipitated with monoclonal antibodies KD68 from lysates of human breast carcinoma T47D cells labelled to steady state specific activity with /sup 32/Pi. The 120 kDa /sup 32/P-labelled progesterone receptor band was resolved by polyacrylamide gel electrophoresis and identified by autoradiography. Phosphoamino acid analysis revealed serine phosphorylation, but no threonine or tyrosine phosphorylation. Treatment of the /sup 32/Pi-labelled cells with EGF, TPA or dibutyryl cAMP had no significant quantitative effect on progesterone receptor phosphorylation, though the EGF receptor and the cAMP-dependent protein kinases have been reported to catalyze phosphorylation of purified avian progesterone receptor preparations in cell freemore » systems. Progesterone receptor phosphorylation on serine residues was increased by 2-fold in cells treated with 10 nM progesterone; EGF had no effect on progesterone-mediated progesterone receptor phosphorylation.« less
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Pathophysiological roles of P2 receptors in glial cells.
Abbracchio, Maria P; Verderio, Claudia
2006-01-01
Extracellular nucleotides act through specific receptors on target cells: the seven ionotropic P2X and the eight G protein-coupled P2Y receptors. All these receptors are expressed by brain astroglia and microglia. In astrocytes, P2 receptors have been implicated in short-term calcium-dependent cell-cell communication. Upon mechanical stimulation or activation by other transmitters, astrocytes release ATP and respond to ATP with a propagating wave of intracellular calcium increases, allowing a homotypic astrocyte-astrocyte communication, as well as an heterotypic signalling which also involves neurons, oligodendrocytes and microglia. Astrocytic P2 receptors also mediate reactive astrogliosis, a reaction contributing to neuronal death in neurodegenerative diseases. Signalling leading to inflammatory astrogliosis involves induction of cyclo-oxygenase 2 through stimulation of ERK1,2 and of the transcriptional factors AP-1 and NF-kappaB. Microglia also express several P2 receptors linked to intracellular calcium increases. P2 receptor subtypes are differentially regulated by typical proinflammatory signals for these cells (e.g. lipopolysaccharide), suggesting specific roles in brain immune responses. Globally, these findings highlight the roles of P2 receptors in glial cell pathophysiology suggesting a contribution to neurodegenerative diseases characterized by excessive gliosis and neuro-inflammation. They also open up the possibility of modulating brain damage by ligands selectively targeting the specific P2 receptor subtypes involved in the gliotic response.
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Androgen responsiveness of the new human endometrial cancer cell line MFE-296.
Hackenberg, R; Beck, S; Filmer, A; Hushmand Nia, A; Kunzmann, R; Koch, M; Slater, E P; Schulz, K D
1994-04-01
MFE-296 endometrial cancer cells express androgen receptors in vitro. These cells, which are tumorigenic in nude mice, are derived from a moderately differentiated human endometrial adenocarcinoma. They express vimentin and the cytokeratins 7, 8, 18, and 19. Karyotyping revealed near-tetraploidy for most of the cells. No marker chromosomes were observed. DNA analyses confirmed the genetic identity of the cell line and the patient from whom the cell line was derived. Proliferation of MFE-296 cells was inhibited by the progestin R5020 and the androgen dihydrotestosterone (DHT). The inhibition of proliferation by DHT was antagonized by the antiandrogen Casodex, demonstrating the involvement of the androgen receptor. Androgen binding was determined at 22,000 binding sites per cell using a whole-cell assay (KD = 0.05 nM) and 30 fmol/mg protein with the dextran charcoal method; 7 fmol/mg protein of progesterone receptors were found, whereas estrogen receptors were below 5 fmol/mg protein. The androgen receptor was functionally intact, as demonstrated by transfection experiments with a reporter-gene construct, containing an androgen-responsive element. In MFE-296 cells the content of the androgen receptor was up-regulated by its own ligand.
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Variability in apoptotic response to poliovirus infection.
Romanova, Lyudmila I; Belov, George A; Lidsky, Peter V; Tolskaya, Elena A; Kolesnikova, Marina S; Evstafieva, Alexandra G; Vartapetian, Andrey B; Egger, Denise; Bienz, Kurt; Agol, Vadim I
2005-01-20
In several cell types, poliovirus activates the apoptotic program, implementation of which is suppressed by viral antiapoptotic functions. In such cells, productive infection leads to a necrotic cytopathic effect (CPE), while abortive reproduction, associated with inadequate viral antiapoptotic functions, results in apoptosis. Here, we describe two other types of cell response to poliovirus infection. Murine L20B cells expressing human poliovirus receptor responded to the infection by both CPE and apoptosis concurrently. Interruption of productive infection decreased rather than increased the proportion of apoptotic cells. Productive infection was accompanied by the early efflux of cytochrome c from the mitochondria in a proportion of cells and by activation of DEVD-specific caspases. Inactivation of caspase-9 resulted in a marked, but incomplete, prevention of the apoptotic response of these cells to viral infection. Thus, the poliovirus-triggered apoptotic program in L20B cells was not completely suppressed by the viral antiapoptotic functions. In contrast, human rhabdomyosarcoma RD cells did not develop appreciable apoptosis during productive or abortive infection, exhibiting inefficient efflux of cytochrome c from mitochondria and no marked activation of DEVD-specific caspases. The cells were also refractory to several nonviral apoptosis inducers. Nevertheless, typical caspase-dependent signs of apoptosis in a proportion of RD cells were observed after cessation of viral reproduction. Such "late" apoptosis was also observed in productively infected HeLa cells. In addition, a tiny proportion of all studied cells were TUNEL positive even in the presence of a caspase inhibitor. Degradation of DNA in such cells appeared to be a postmortem phenomenon. Biological relevance of variable host responses to viral infection is discussed.
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Coke, Christopher J.; Scarlett, Kisha A.; Chetram, Mahandranauth A.; Jones, Kia J.; Sandifer, Brittney J.; Davis, Ahriea S.; Marcus, Adam I.
2016-01-01
The G-protein-coupled chemokine receptor CXCR4 generates signals that lead to cell migration, cell proliferation, and other survival mechanisms that result in the metastatic spread of primary tumor cells to distal organs. Numerous studies have demonstrated that CXCR4 can form homodimers or can heterodimerize with other G-protein-coupled receptors to form receptor complexes that can amplify or decrease the signaling capacity of each individual receptor. Using biophysical and biochemical approaches, we found that CXCR4 can form an induced heterodimer with cannabinoid receptor 2 (CB2) in human breast and prostate cancer cells. Simultaneous, agonist-dependent activation of CXCR4 and CB2 resulted in reduced CXCR4-mediated expression of phosphorylated ERK1/2 and ultimately reduced cancer cell functions such as calcium mobilization and cellular chemotaxis. Given that treatment with cannabinoids has been shown to reduce invasiveness of cancer cells as well as CXCR4-mediated migration of immune cells, it is plausible that CXCR4 signaling can be silenced through a physical heterodimeric association with CB2, thereby inhibiting subsequent functions of CXCR4. Taken together, the data illustrate a mechanism by which the cannabinoid system can negatively modulate CXCR4 receptor function and perhaps tumor progression. PMID:26841863
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Functional somatostatin receptors on a rat pancreatic acinar cell line
DOE Office of Scientific and Technical Information (OSTI.GOV)
Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.
1988-07-01
Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of {sup 125}I-(Tyr{sup 11})Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 {plus minus} 20 fmol/10{sup 6} cells. Somatostatin receptor structure was analyzed by covalently cross-linking {sup 125}I-(Tyr{sup 11})somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibitionmore » of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N{sub i} to inhibit adenylate cyclase.« less
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Tachykinin receptors and the airways.
Frossard, N; Advenier, C
1991-01-01
The tachykinins, substance P, neurokinin A and neurokinin B, belong to a structural family of peptides. In mammalian airways, substance P and neurokinin A are colocalized to afferent C-fibres. Substance P-containing fibres are close to bronchial epithelium, smooth muscle, mucus glands and blood vessels. Sensory neuropeptides may be released locally, possibly as a result of a local reflex, and produce bronchial obstruction through activation of specific receptors on these various tissues. Three types of tachykinin receptors, namely NK-1, NK-2 and NK-3 receptors, have been characterized by preferential activation by substance P, neurokinin A and neurokinin B respectively. NK-1 and NK-2 receptors were recently cloned. The determination of receptor types involved in the effects of tachykinins in the airways has been done with synthetic agonists and antagonists binding specifically to NK-1, NK-2 and NK-3 receptors. Although the existence of species differences, the conclusion that bronchial smooth muscle contraction is mainly related to activation of NK-2 receptors on bronchial smooth muscle cell has been drawn. The hypothesis of a NK-2 receptor subclassification has been proposed with NK-2A receptor subtype in the guinea-pig airways. Other effects in the airways are related to stimulation of NK-1 receptors on mucus cells, vessels, epithelium and inflammatory cells. A non-receptor-mediated mechanism is also involved in the effect of substance P on inflammatory cells and mast cells.
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Taste Receptor Cells That Discriminate Between Bitter Stimuli
Caicedo, Alejandro; Roper, Stephen D.
2013-01-01
Recent studies showing that single taste bud cells express multiple bitter taste receptors have reignited a long-standing controversy over whether single gustatory receptor cells respond selectively or broadly to tastants. We examined calcium responses of rat taste receptor cells in situ to a panel of bitter compounds to determine whether individual cells distinguish between bitter stimuli. Most bitter-responsive taste cells were activated by only one out of five compounds tested. In taste cells that responded to multiple stimuli, there were no significant associations between any two stimuli. Bitter sensation does not appear to occur through the activation of a homogeneous population of broadly tuned bitter-sensitive taste cells. Instead, different bitter stimuli may activate different subpopulations of bitter-sensitive taste cells. PMID:11222863
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Short-chain fatty acid receptors inhibit invasive phenotypes in breast cancer cells
Thirunavukkarasan, Madhumathi; Wang, Chao; Rao, Angad; Hind, Tatsuma; Teo, Yuan Ru; Siddiquee, Abrar Al-Mahmood; Goghari, Mohamed Ally Ibrahim; Kumar, Alan Prem
2017-01-01
Short chain fatty acids (2 to 6 carbons in length) are ubiquitous lipids that are present in human plasma at micromolar concentrations. In addition to serving as metabolic precursors for lipid and carbohydrate synthesis, they also act as cognate ligands for two known G protein-coupled receptors (GPCRs), FFAR2 and FFAR3. While there is evidence that these receptors may inhibit the progression of colorectal cancer, their roles in breast cancer cells are largely unknown. We evaluated the effects of enforced overexpression of these receptors in two phenotypically distinct breast cancer cell lines: MCF7 and MDA-MD-231. Our results demonstrate that both receptors inhibit cell invasiveness, but through different signaling processes. In invasive, mesenchymal-like MDA-MB-231 cells, FFAR2 inhibits the Hippo-Yap pathway and increases expression of adhesion protein E-cadherin, while FFAR3 inhibits MAPK signaling. Both receptors have the net effect of reducing actin polymerization and invasion of cells through a Matrigel matrix. These effects were absent in the less invasive, epithelial-like MCF7 cells. Correspondingly, there is reduced expression of both receptors in invasive breast carcinoma and in aggressive triple-negative breast tumors, relative to normal breast tissue. Cumulatively, our data suggest that the activation of cognate receptors by short chain fatty acids drives breast cancer cells toward a non-invasive phenotype and therefore may inhibit metastasis. PMID:29049318
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Li, Wenhui; Xu, Jiachao; Kou, Xiaolong; Zhao, Rong; Zhou, Wei; Fang, Xiaohong
2018-05-01
Angiotensin II type 1 receptor (AT1R), a typical G protein-coupled receptor, plays a key role in regulating many cardiovascular functions. Different ligands can bind with AT1R to selectively activate either G protein (Gq) or β-arrestin (β-arr) pathway, or both pathways, but the molecular mechanism is not clear yet. In this work, we used, for the first time, atomic force microscopy-based single molecule force spectroscopy (SMFS) to study the interactions of AT1R with three types of ligands, balanced ligand, Gq-biased ligand, and β-arr-biased ligand, in living cells. The results revealed their difference in binding force and binding stability. The complex of the Gq-biased ligand-AT1R overcame two energy barriers with an intermediate state during dissociation, whereas that of β-arr-biased ligand-AT1R complex overcame one energy barrier. This indicated that AT1R had different ligand-binding conformational substates and underwent different structural changes to activate downstream signaling pathways with variable agonist efficacies. Quantitative analysis of AT1R-ligand binding in living cells at the single-molecule level offers a new tool to study the molecular mechanism of AT1R biased activation. Graphical Abstract Single-molecule force measurement on the living cell expressing AT1R-eGFP with a ligand modified AFM tip (left), the dynamic force spectra of β-arrestin biased ligands-AT1R (middle), and Gq-biased ligands-AT1R (right). The complexes of β-arr-biased ligand-AT1R overcame one energy barrier, with one linear region in the spectra, whereas the Gq-biased ligand-AT1R complexes overcame two energy barriers with two linear regions.
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Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways
Willett, Julia L. E.; Gucinski, Grant C.; Fatherree, Jackson P.; Low, David A.; Hayes, Christopher S.
2015-01-01
Contact-dependent growth inhibition (CDI) systems function to deliver toxins into neighboring bacterial cells. CDI+ bacteria export filamentous CdiA effector proteins, which extend from the inhibitor-cell surface to interact with receptors on neighboring target bacteria. Upon binding its receptor, CdiA delivers a toxin derived from its C-terminal region. CdiA C-terminal (CdiA-CT) sequences are highly variable between bacteria, reflecting the multitude of CDI toxin activities. Here, we show that several CdiA-CT regions are composed of two domains, each with a distinct function during CDI. The C-terminal domain typically possesses toxic nuclease activity, whereas the N-terminal domain appears to control toxin transport into target bacteria. Using genetic approaches, we identified ptsG, metI, rbsC, gltK/gltJ, yciB, and ftsH mutations that confer resistance to specific CdiA-CTs. The resistance mutations all disrupt expression of inner-membrane proteins, suggesting that these proteins are exploited for toxin entry into target cells. Moreover, each mutation only protects against inhibition by a subset of CdiA-CTs that share similar N-terminal domains. We propose that, following delivery of CdiA-CTs into the periplasm, the N-terminal domains bind specific inner-membrane receptors for subsequent translocation into the cytoplasm. In accord with this model, we find that CDI nuclease domains are modular payloads that can be redirected through different import pathways when fused to heterologous N-terminal “translocation domains.” These results highlight the plasticity of CDI toxin delivery and suggest that the underlying translocation mechanisms could be harnessed to deliver other antimicrobial agents into Gram-negative bacteria. PMID:26305955
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USDA-ARS?s Scientific Manuscript database
Abundance of IGF-2 receptor (IGF2R), FSH receptor (FSHR), and LH receptor (LHCGR) mRNA in granulosa cells (GCs) or theca cells (TCs) or both cells as well as estradiol (E2), progesterone (P4), and androstenedione concentrations in follicular fluid were compared in cows genetically selected (Twinner)...
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Nuclear receptors in pancreatic tumor cells.
Damaskos, Christos; Garmpis, Nikolaos; Karatzas, Theodore; Kostakis, Ioannis D; Nikolidakis, Lampros; Kostakis, Alkiviadis; Kouraklis, Gregory
2014-12-01
This review focuses on nuclear receptors expressed in pancreatic cancer. An extensive search of articles published up to March 2013 was conducted using the MEDLINE database. The key words used were "pancreatic cancer", "molecular receptors" and "growth factors". A total of 112 articles referred to pancreatic cancer, molecular receptors and/or growth factors were included. Receptors of growth factors, such as the epithelial growth factor receptor, insulin-like growth factor-1 receptor, vascular endothelial growth factor receptor and others, such as integrin α5β1, somatostatin receptors, the death receptor 5, claudin, notch receptors, mesothelin receptors, follicle-stimulating hormone receptors, the MUC1 receptor, the adrenomedullin receptor, the farnesoid X receptor, the transferrin receptor, sigma-2 receptors, the chemokine receptor CXCR4, the urokinase plasminogen activator receptor, the ephrine A2 receptor, the GRIA3 receptor, the RON receptor and the angiotensin II receptor AT-1 are expressed in pancreatic tumor cells. These molecules are implicated in tumor growth, apoptosis, angiogenesis, metastasis etc. After identifying the molecular receptors associated with the pancreatic cancer, many more target molecules playing important roles in tumor pathophysiology and senescence-associated signal transduction in cancer cells will be identified. This may have a significant influence on diagnosis, therapy and prognosis of pancreatic cancer. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
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Gene transfer of Hodgkin cell lines via multivalent anti-CD30 scFv displaying bacteriophage.
Chung, Yoon-Suk A; Sabel, Katja; Krönke, Martin; Klimka, Alexander
2008-04-16
The display of binding ligands, such as recombinant antibody fragments, on the surface of filamentous phage makes it possible to specifically attach these phage particles to target cells. After uptake of the phage, their internal single-stranded DNA is processed by the host cell, which allows transient expression of an encoded eukaryotic gene cassette. This opens the possibility to use bacteriophage as vectors for targeted gene therapy, although the transduction efficiency is very low. Here we demonstrate the display of an anti-CD30 single chain variable fragment fused to the major coat protein pVIII on the surface of bacteriophage. These phage particles showed an improved binding and transduction efficiency of CD30 positive Hodgkin-lymphoma cells, compared to bacteriophage with the anti-CD30 single chain variable fragment fused to the minor coat protein pIII. We can conclude from the results that the postulated multivalency of the anti-CD30-pVIII displaying bacteriophage combined with disseminated display of the anti-CD30 scFv on the whole particle surface is responsible for the improved gene transfer rate. These results mark an important step towards the use of phage particles as a cheap and safe gene transfer vehicle for the gene delivery of the desired target cells via their specific surface receptors.
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Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.
Grupp, Stephan A; Kalos, Michael; Barrett, David; Aplenc, Richard; Porter, David L; Rheingold, Susan R; Teachey, David T; Chew, Anne; Hauck, Bernd; Wright, J Fraser; Milone, Michael C; Levine, Bruce L; June, Carl H
2013-04-18
Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.
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Reagan, L P; Ye, X; Maretzski, C H; Fluharty, S J
1993-01-01
Murine neuroblastoma N1E-115 cells possess membranous receptors for the octapeptide angiotensin II (AngII) whose density is substantially increased by in vitro differentiation. Incubation of differentiated N1E-115 cells with AngII produced a rapid decrease in receptor density, but did not alter the affinity of these receptors for either 125I-AngII or the high-affinity antagonist 125I-[Sarc1,Ile8]-AngII. This apparent down-regulation was dose related with an ED50 of 1 nM, and maximal decreases of approximately 90% were obtained with 100 nM AngII. Receptor loss from differentiated cell membranes was unaffected by incubations of membranes obtained from agonist-exposed cells with non-hydrolyzable analogues of GTP for 60 min at 37 degrees C to ensure dissociation of the ligand. Partial loss of AngII receptors was apparent within 5 min of agonist exposure, whereas maximal declines were not observed until 30 min. This temporal pattern resulted from a preferential decrease in the AT1 receptor subtype during the first 5 min, followed by a decline in both AT1 and AT2 receptors with longer periods of agonist exposure. The loss of membranous receptors was reversible with partial recovery observed after 4 h, and with nearly full recovery observed 18 h after exposure of the cells to AngII. However, the long-term recovery of receptor density was blocked by the protein synthesis inhibitor, cycloheximide. The heptapeptide angiotensin III produced a similar down-regulation of receptors, and the high-affinity antagonist [Sarc1,Thr8]-AngII blocked agonist-induced down-regulation. Finally, the apparent loss of cell surface AngII receptors decreased the ability of AngII to stimulate cyclic GMP production within intact N1E-115 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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Vyas, Ashish Kumar; Ramakrishna, Usha; Sen, Bijoya; Islam, Mojahidul; Ramakrishna, Gayatri; Patra, Sharda; Rastogi, Archana; Sarin, Shiv Kumar; Trehanpati, Nirupma
2018-04-30
Asialoglycoprotein receptor expression on hepatocytes has been associated with endocytosis, binding and uptake of hepatitis B virus. The role of asialoglycoprotein receptor in hepatitis B virus vertical transmission and its expression on placenta has not yet been studied. Thirty-four HBsAg+ve and 13 healthy pregnant mothers along with their newborns were enrolled. The former were categorized into transmitting and non-transmitting mothers based on their newborns being hepatitis B surface antigen and hepatitis B virus DNA positive. Expression of asialoglycoprotein receptor and hepatitis B surface antigen in placenta and isoform of asialoglycoprotein receptor on dendritic cell in peripheral and cord blood dendritic cells were analysed using flowcytometry, immune histochemistry, immune florescence and qRT-PCR. Twelve HBsAg+ve mothers transmitted hepatitis B virus to their newborns whereas the rest (n = 22) did not. Hepatitis B virus-transmitting mothers showed increased expression of asialoglycoprotein receptor in trophoblasts of placenta. Immunofluorescence microscopy revealed colocalization of hepatitis B surface antigen and asialoglycoprotein receptor in placenta as well as in DCs of transmitting mothers. There was no significant difference in the expression of asialoglycoprotein receptor on peripheral blood mononuclear cells or chord blood mononuclear cells between the 2 groups. However, hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed increased mRNA levels of isoform of asialoglycoprotein receptor on dendritic cell in peripheral blood mononuclear cells. Hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed an increased expression of isoform of asialoglycoprotein receptor on dendritic cell on circulating dendritic cells compared to hepatitis B virus non-transmitting mothers and their negative newborns. This study revealed that increased expression of asialoglycoprotein receptor in placenta and colocalization with hepatitis B surface antigen strongly indicates its role in intrauterine transmission of hepatitis B virus. Asialoglycoprotein receptor-blocking strategy can be used for therapeutic intervention of vertical transmission. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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Wang, Heng; Pu, Yong; Luo, Lei; Li, Yunsheng; Zhang, Yunhai; Cao, Zubing
2018-06-01
Excessive secretion of androgens including androstenedione and testosterone in theca cells frequently causes female infertility in mammals. Melatonin is a potent inhibitor of androgen production in gonadal cells of several species in a membrane receptor-dependent manner. However, the function of melatonin in steroidogenesis of porcine theca cells remains unclear. Here we report that melatonin inhibits androgen biosynthesis independently of its membrane receptors in pigs. Using flow cytometry, immunofluorescence and RT-PCR we showed that the vast majority of cells isolated from the theca layer of antral follicles are indeed theca cells. Furthermore, we demonstrated that of the two of melatonin membrane receptors encoded in the porcine genome, theca cells exclusively express melatonin receptor 1B. Cell counting analysis indicated that different concentrations of melatonin did not alter the normal viability and proliferation of theca cells. Additionally, hormone radioimmunoassay and qPCR respectively showed that a high concentration of melatonin significantly repressed both androgen production and expression of steroidogenic genes involving StAR, CYP11A1, HSD3β and SET (P < 0.05), but did not impair progesterone production. Interestingly, these effects were not reversed by N-acetyl-2-benzyltryptamin, a melatonin membrane receptor antagonist. Overall, these results demonstrate that melatonin inhibits androgen production in porcine theca cells independently of its membrane receptor. Copyright © 2018 Elsevier Inc. All rights reserved.
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Divide, Conquer, and Sense: CD8+CD28− T Cells in Perspective
Arosa, Fernando A.; Esgalhado, André J.; Padrão, Carolina A.; Cardoso, Elsa M.
2017-01-01
Understanding the rationale for the generation of a pool of highly differentiated effector memory CD8+ T cells displaying a weakened capacity to scrutinize for peptides complexed with major histocompatibility class I molecules via their T cell receptor, lacking the “signal 2” CD28 receptor, and yet expressing a highly diverse array of innate receptors, from natural killer receptors, interleukin receptors, and damage-associated molecular pattern receptors, among others, is one of the most challenging issues in contemporary human immunology. The prevalence of these differentiated CD8+ T cells, also known as CD8+CD28−, CD8+KIR+, NK-like CD8+ T cells, or innate CD8+ T cells, in non-lymphoid organs and tissues, in peripheral blood of healthy elderly, namely centenarians, but also in stressful and chronic inflammatory conditions suggests that they are not merely end-of-the-line dysfunctional cells. These experienced CD8+ T cells are highly diverse and capable of sensing a variety of TCR-independent signals, which enables them to respond and fine-tune tissue homeostasis. PMID:28096804
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Divide, Conquer, and Sense: CD8+CD28- T Cells in Perspective.
Arosa, Fernando A; Esgalhado, André J; Padrão, Carolina A; Cardoso, Elsa M
2016-01-01
Understanding the rationale for the generation of a pool of highly differentiated effector memory CD8 + T cells displaying a weakened capacity to scrutinize for peptides complexed with major histocompatibility class I molecules via their T cell receptor, lacking the "signal 2" CD28 receptor, and yet expressing a highly diverse array of innate receptors, from natural killer receptors, interleukin receptors, and damage-associated molecular pattern receptors, among others, is one of the most challenging issues in contemporary human immunology. The prevalence of these differentiated CD8 + T cells, also known as CD8 + CD28 - , CD8 + KIR + , NK-like CD8 + T cells, or innate CD8 + T cells, in non-lymphoid organs and tissues, in peripheral blood of healthy elderly, namely centenarians, but also in stressful and chronic inflammatory conditions suggests that they are not merely end-of-the-line dysfunctional cells. These experienced CD8 + T cells are highly diverse and capable of sensing a variety of TCR-independent signals, which enables them to respond and fine-tune tissue homeostasis.
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NASA Technical Reports Server (NTRS)
Gao, Wenyuan; Wiederhold, Michael L.
1997-01-01
The structure of the statocyst of the freshwater snail Biomphalaria glabrata has been examined by light and electron microscopy. The two statocysts are located on the dorsal-lateral side of the left and right pedal ganglion. The statocysts are spherical, fluid-filled capsules with a diameter of approximately 60 microns for young and 110 microns for adult snails. The wall of the cyst is composed of large receptor cells and many smaller supporting cells. The receptor cells bear cilia which are evenly distributed on the apical surface. The cilia have the typical 9+2 internal tubule configuration. Striate rootlets originate from the base of the basal body and run downward into the cytoplasm. Side-roots arise from one side of the basal body and a basal foot from the other. For each receptor cell, the basal foot always points to the periphery of the surface, indicating that the receptor cell is non-polarized. The receptor cells contain cytoplasmic organelles such as mitochondria, ribosomes, rough and smooth endoplasmic reticulum, compact Golgi bodies and multivesicular bodies. Supporting cells bearing microvilli are interposed between the receptor cells. The junction complex between the supporting cells and the receptor cells is composed of adherens and septate junctions, while between supporting cells only the adherens junctions are present. The static nerve arises from the lateral side of the cyst and contains axons in which parallel neurotubules and mitochondria are found. The axons arise directly from the base of the receptor cells without synapse. In the cyst lumen there are unattached statoconia. The statoconia have a plate-like or concentric membranous ring structure. Based on the morphology, the function of the statocyst in Biomphalaria is discussed.
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Uchida, Hiroaki; Shah, Waris A; Ozuer, Ali; Frampton, Arthur R; Goins, William F; Grandi, Paola; Cohen, Justus B; Glorioso, Joseph C
2009-04-01
Both initial infection and cell-to-cell spread by herpes simplex virus type 1 (HSV-1) require the interaction of the viral glycoprotein D (gD) with an entry receptor on the cell surface. The two major HSV entry receptors, herpesvirus entry mediator (HVEM) and nectin-1, mediate infection independently but are coexpressed on a variety of cells. To determine if both receptors are active in these instances, we have established mutant viruses that are selectively impaired for recognition of one or the other receptor. In plaque assays, these viruses showed approximately 1,000-fold selectivity for the matched receptor over the mismatched receptor. Separate assays showed that each virus is impaired for both infection and spread through the mismatched receptor. We tested several human tumor cell lines for susceptibility to these viruses and observed that HT29 colon carcinoma cells are susceptible to infection by nectin-1-restricted virus but are highly resistant to HVEM-restricted virus infection, despite readily detectable HVEM expression on the cell surface. HVEM cDNA isolated from HT29 cells rendered HSV-resistant cells permissive for infection by the HVEM-restricted virus, suggesting that HT29 cells lack a cofactor for HVEM-mediated infection or express an HVEM-specific inhibitory factor. Passaging of HVEM-restricted virus on nectin-1-expressing cells yielded a set of gD missense mutations that each restored functional recognition of nectin-1. These mutations identify residues that likely play a role in shaping the nectin-1 binding site of gD. Our findings illustrate the utility of these receptor-restricted viruses in studying the early events in HSV infection.
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Kusumi, Maki; Yamashita, Takahiro; Fujii, Tomoyuki; Nagamatsu, Takeshi; Kozuma, Shiro; Taketani, Yuji
2006-06-01
The balance of inhibitory and activating natural killer (NK) receptors on maternal decidual NK cells, most of which are CD56bright, is thought to be crucial for the proper growth of trophoblasts in placenta. A lectin-like NK receptor, CD94/NKG2, is the receptor for human leukocyte antigen (HLA)-E, which is expressed on trophoblasts. To clarify the mechanism regulating the activity of decidual NK cells during pregnancy, we investigated the expression patterns of inhibitory NK receptor, CD94/NKG2A, and activating receptor, CD94/NKG2C, on decidual NK cells in an early stage of normal pregnancy and compared them with those on peripheral NK cells, most of which are CD56dim. The rate of NKG2A-positive cells was significantly higher for decidual CD56bright NK cells than for peripheral CD56dim NK cells, but the rates of NKG2C-positive cells were comparable between the two cell types. Interestingly, peripheral CD56dim NK cells reciprocally expressed inhibitory NKG2A and activating NKG2C, but decidual CD56bright NK cells that expressed activating NKG2C simultaneously expressed inhibitory NKG2A. The co-expression of inhibitory and activating NKG2 receptors may fine-tune the immunoregulatory functions of the decidual NK cells to control the trophoblast invasion in constructing placenta.
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Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import
Buchanan, Susan K; Lukacik, Petra; Grizot, Sylvestre; Ghirlando, Rodolfo; Ali, Maruf M U; Barnard, Travis J; Jakes, Karen S; Kienker, Paul K; Esser, Lothar
2007-01-01
Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 Å above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane. PMID:17464289
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Quantitative Impact of Plasma Clearance and Down-regulation on GLP-1 Receptor Molecular Imaging.
Zhang, Liang; Thurber, Greg M
2016-02-01
Quantitative molecular imaging of beta cell mass (BCM) would enable early detection and treatment monitoring of type 1 diabetes. The glucagon-like peptide-1 (GLP-1) receptor is an attractive target due to its beta cell specificity and cell surface location. We quantitatively investigated the impact of plasma clearance and receptor internalization on targeting efficiency in healthy B6 mice. Four exenatide-based probes were synthesized that varied in molecular weight, binding affinity, and plasma clearance. The GLP-1 receptor internalization rate and in vivo receptor expression were quantified. Receptor internalization (54,000 receptors/cell in vivo) decreased significantly within minutes, reducing the benefit of a slower-clearing agent. The multimers and albumin binding probes had higher kidney and liver uptake, respectively. Slow plasma clearance is beneficial for GLP-1 receptor peptide therapeutics. However, for exendin-based imaging of islets, down-regulation of the GLP-1 receptor and non-specific background uptake result in a higher target-to-background ratio for fast-clearing agents.
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Quantitative Impact of Plasma Clearance and Down-regulation on GLP-1 Receptor Molecular Imaging
Zhang, Liang; Thurber, Greg M.
2016-01-01
Purpose Quantitative molecular imaging of beta cell mass (BCM) would enable early detection and treatment monitoring of type-1 diabetes. The glucagon like peptide-1 (GLP-1) receptor is an attractive target due to its beta cell specificity and cell surface location. We quantitatively investigated the impact of plasma clearance and receptor internalization on targeting efficiency in healthy B6 mice. Procedures Four exenatide-based probes were synthesized that varied in molecular weight, binding affinity, and plasma clearance. The GLP-1 receptor internalization rate and in vivo receptor expression were quantified. Results Receptor internalization (54,000 receptors/cell in vivo) decreased significantly within minutes, reducing the benefit of a slower clearing agent. The multimers and albumin binding probes had higher kidney and liver uptake, respectively. Conclusions Slow plasma clearance is beneficial for GLP-1 receptor peptide therapeutics. However, for exendin-based imaging of islets, downregulation of the GLP-1 receptor and non-specific background uptake result in a higher TBR for fast-clearing agents. PMID:26194012
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Watanabe, H.; Grubb, J.H.; Sly, W.S.
1990-10-01
The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functionalmore » receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.« less
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Visperas, Patrick R.; Wilson, Christopher G.; Winger, Jonathan A.; ...
2016-12-13
ZAP-70 is a critical molecule in the transduction of T cell antigen receptor signaling and the activation of T cells. Upon activation of the T cell antigen receptor, ZAP-70 is recruited to the intracellular ζ-chains of the T cell receptor, where ZAP-70 is activated and colocalized with its substrates. Inhibitors of ZAP-70 could potentially function as treatments for autoimmune diseases or organ transplantation. In this work, we present the design, optimization, and implementation of a screen for inhibitors that would disrupt the interaction between ZAP-70 and the T cell antigen receptor. Finally, the screen is based on a fluorescence polarizationmore » assay for peptide binding to ZAP-70.« less
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The BCL11A Transcription Factor Directly Activates RAG Gene Expression and V(D)J Recombination
Lee, Baeck-seung; Dekker, Joseph D.; Lee, Bum-kyu; Iyer, Vishwanath R.; Sleckman, Barry P.; Shaffer, Arthur L.; Ippolito, Gregory C.
2013-01-01
Recombination-activating gene 1 protein (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining (VDJ) segment recombination, an essential process for antigen receptor expression and lymphocyte development. The transcription factor BCL11A is required for B cell development, but its molecular function(s) in B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds the RAG1 promoter and Erag enhancer to activate RAG1 and RAG2 transcription in pre-B cells. We employed BCL11A overexpression with recombination substrates in a cultured pre-B cell line as well as Cre recombinase-mediated Bcl11alox/lox deletion in explanted murine pre-B cells to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination. PMID:23438597
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Sciammas, Roger; Li, Ying; Warmflash, Aryeh; Song, Yiqiang; Dinner, Aaron R; Singh, Harinder
2011-01-01
The B-lymphocyte lineage is a leading system for analyzing gene regulatory networks (GRNs) that orchestrate distinct cell fate transitions. Upon antigen recognition, B cells can diversify their immunoglobulin (Ig) repertoire via somatic hypermutation (SHM) and/or class switch DNA recombination (CSR) before differentiating into antibody-secreting plasma cells. We construct a mathematical model for a GRN underlying this developmental dynamic. The intensity of signaling through the Ig receptor is shown to control the bimodal expression of a pivotal transcription factor, IRF-4, which dictates B cell fate outcomes. Computational modeling coupled with experimental analysis supports a model of ‘kinetic control', in which B cell developmental trajectories pass through an obligate transient state of variable duration that promotes diversification of the antibody repertoire by SHM/CSR in direct response to antigens. More generally, this network motif could be used to translate a morphogen gradient into developmental inductive events of varying time, thereby enabling the specification of distinct cell fates. PMID:21613984
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Trošt, Nina; Hevir, Neli; Rižner, Tea Lanišnik; Debeljak, Nataša
2013-03-01
Erythropoietin (EPO) receptor (EPOR) expression in breast cancer has been shown to correlate with the expression of estrogen receptor (ESR) and progesterone receptor (PGR) and to be associated with the response to tamoxifen in ESR+/PGR+ tumors but not in ESR- tumors. In addition, the correlation between EPOR and G protein-coupled estrogen receptor 1 [GPER; also known as G protein-coupled receptor 30 (GPR30)] has been reported, suggesting the prognostic potential of EPOR expression. Moreover, the involvement of colony stimulating factor 2 receptor, β, low‑affinity (CSF2RB) and ephrin type-B receptor 4 (EPHB4) as EPOR potential receptor partners in cancer has been indicated. This study analyzed the correlation between the expression of genes for EPO, EPOR, CSF2RB, EPHB4, ESR, PGR and GPER in the MCF-7, MDA-MB-361, T-47D, MDA-MB-231, Hs578Bst, SKBR3, MCF-10A and Hs578T cell lines. The cell lines were also treated with recombinant human EPO (rHuEPO) in order to determine its ability to activate the Jak/STAT5, MAPK and PI3K signaling pathways and modify cell growth characteristics. Expression analysis stratified the cell lines in 2 main clusters, hormone-dependent cell lines expressing ESR and PGR and a hormone-independent cluster. A significant correlation was observed between the expression levels of ESR and PGR and their expression was also associated with that of GPER. Furthermore, the expression of GPER was associated with that of EPOR, suggesting the connection between this orphan G protein and EPO signaling. A negative correlation between EPOR and CSF2RB expression was observed, questioning the involvement of these two receptors in the hetero-receptor formation. rHuEPO treatment only influenced the hormone-independent cell lines, since only the MDA-MB-231, SKBR3 and Hs578T cells responded to the treatment. The correlation between the expression of the analyzed receptors suggests that the receptors may interact in order to activate signaling pathways or to evade their inhibition. Therefore, breast cancer classification upon ESR, PGR and human epidermal growth factor receptor 2 (HER2) may not be sufficient for the selection of suitable treatment protocol. The expression of EPOR, GPER and EPHB4 may be considered as additional classification factors.
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Cadet, Patrick; Mantione, Kirk J; Stefano, George B
2003-05-15
Studies from our laboratory have revealed a novel mu opiate receptor, mu 3, which is expressed in both vascular tissues and leukocytes. The mu 3 receptor is selective for opiate alkaloids and is insensitive to opioid peptides. We now identify the mu 3 receptor at the molecular level using a 441-bp conserved region of the mu 1 receptor. Sequence analysis of the isolated cDNA suggests that it is a novel, alternatively spliced variant of the mu opiate receptor gene. To determine whether protein expressed from this cDNA exhibits the biochemical characteristics expected of the mu 3 receptor, the cDNA clone was expressed in a heterologous system. At the functional level, COS-1 cells transfected with the mu 3 receptor cDNA exhibited dose-dependent release of NO following treatment with morphine, but not opioid peptides (i.e., Met-enkephalin). Naloxone was able to block the effect of morphine on COS-1 transfected cells. Nontransfected COS-1 cells did not produce NO in the presence of morphine or the opioid peptides at similar concentrations. Receptor binding analysis with [(3)H]dihydromorphine further supports the opiate alkaloid selectivity and opioid peptide insensitivity of this receptor. These data suggest that this new mu opiate receptor cDNA encodes the mu 3 opiate receptor, since it exhibits biochemical characteristics known to be unique to this receptor (opiate alkaloid selective and opioid peptide insensitive). Furthermore, using Northern blot, RT-PCR, and sequence analysis, we have demonstrated the expression of this new mu variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells.
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Wetter, Justin A; Revankar, Chetana; Hanson, Bonnie J
2009-10-01
Cellular assay development for the endothelial differentiation gene (EDG) family of G-protein-coupled receptors (GPCRs) and related lysophospholipid (LP) receptors is complicated by endogenous receptor expression and divergent receptor signaling. Endogenously expressed LP receptors exist in most tissue culture cell lines. These LP receptors, along with other endogenously expressed GPCRs, contribute to off-target signaling that can complicate interpretation of second-messenger-based cellular assay results. These receptors also activate a diverse and divergent set of cellular signaling pathways, necessitating the use of a variety of assay formats with mismatched procedures and functional readouts. This complicates examination and comparison of these receptors across the entire family. The Tango technology uses the conserved beta-arrestin-dependent receptor deactivation process to allow interrogation of the EDG and related receptors with a single functional assay. This method also isolates the target receptor signal, allowing the use of tissue culture cell lines regardless of their endogenous receptor expression. The authors describe the use of this technique to build cell-based receptor-specific assays for all 8 members of the EDG receptor family as well as the related LPA receptors GPR23, GPR92, and GPR87. In addition, they demonstrate the value of this technology for identification and investigation of functionally selective receptor compounds as demonstrated by the immunosuppressive compound FtY720-P and its action at the EDG(1) and EDG(3) receptors.
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Clustering of adhesion receptors following exposure of insect blood cells to foreign surfaces.
Nardi, James B; Zhuang, Shufei; Pilas, Barbara; Bee, Charles Mark; Kanost, Michael R
2005-05-01
Cell-mediated immune responses of insects involve interactions of two main classes of blood cells (hemocytes) known as granular cells and plasmatocytes. In response to a foreign surface, these hemocytes suddenly transform from circulating, non-adherent cells to cells that interact and adhere to each other and the foreign surface. This report presents evidence that during this adhesive transformation the extracellular matrix (ECM) proteins lacunin and a ligand for peanut agglutinin (PNA) lectin are released by granular cells and bind to surfaces of both granular cells and plasmatocytes. ECM protein co-localizes on cell surfaces with the adhesive receptors integrin and neuroglian, a member of the immunoglobulin superfamily. The ECM protein(s) secreted by granular cells are hypothesized to interact with adhesion receptors such as neuroglian and integrin by cross linking and clustering them on hemocyte surfaces. This clustering of receptors is known to enhance the adhesiveness (avidity) of interacting mammalian immune cells. The formation of ring-shaped clusters of these adhesion receptors on surfaces of insect immune cells represents an evolutionary antecedent of the mammalian immunological synapse.
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Bender, Ruben R; Muth, Anke; Schneider, Irene C; Friedel, Thorsten; Hartmann, Jessica; Plückthun, Andreas; Maisner, Andrea; Buchholz, Christian J
2016-06-01
Receptor-targeted lentiviral vectors (LVs) can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV) glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance). Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV) glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4) was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.
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Ma, Shi-Wu; Li, Yong-Yin; Zhang, Guang-Wen; Huang, Xuan; Sun, Jian; Li, Chris; Abbott, William G. H.; Hou, Jin-Lin
2011-01-01
An increased CD8+ T cell response to hepatitis B virus (HBV) peptides occurs between 12 and 24 weeks after starting antiviral therapy for chronic hepatitis B. It is not known whether these cells have antiviral function. The aim of this study was to determine whether clonal expansions of CD8+ T cells at these time points predict the virological response to therapy. Peripheral blood CD8+ T cells were obtained from 20 patients treated with lamivudine or telbivudine for chronic hepatitis B at baseline, 12 weeks, and 24 weeks. The CDR3 spectratype of each T cell receptor (TCR) β chain variable region (Vβ) gene family was analyzed, and the changes in the numbers of Vβ families with clonal expansions were compared in subjects with (n = 12) and without (n = 8) a virological response (52 week HBV DNA < 300 copies/ml). The number of CD8+ TCR Vβ families with clonal expansions at 12 weeks relative to baseline (median [10th to 90th percentile], +2.5 [0 to +7] versus +1 [0 to +2], P = 0.03) and at 24 weeks relative to 12 weeks (+1 [0 to +2] versus −1 [−3 to +4], P = 0.006) was higher in subjects with a virological response versus subjects without a virological response, as were interleukin-2 (IL-2) but not IL-21 mRNA levels in peripheral blood mononuclear cells. The duration of new expansions at 12 weeks was higher (P < 0.0001) in responders. Increased numbers of CD8+ T cell expansions after antiviral therapy are associated with a virological response to treatment. These CD8+ T cells are a potential target for a therapeutic vaccine for chronic hepatitis B. PMID:21098256
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2008-05-01
adoptive therapy using CD19- specific chimeric antigen receptor re-directed T cells for recurrent/refractory follicular lymphoma. Mol Ther...T- cell therapies for B- cell malignancies we have developed a chimeric antigen receptor (CAR) which when expressed on the cell surface redirects T...that both CD4+ and CD8+ T cells expressing CD19-specific chimeric antigen receptor (CAR) can be generated usmg a novel non-viral gene
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Sgnaolin, V; Pereira, T C B; Bogo, M R; Zanin, R; Battastini, A M O; Morrone, F B; Campos, M M
2013-08-01
Kinins and their receptors have been recently implicated in cancer. Using functional and molecular approaches, we investigated the relevance of kinin B1 and B2 receptors in bladder cancer. Functional studies were conducted using bladder cancer cell lines, and human biopsies were employed for molecular studies. Both B1 des-Arg(9)-BK and B2 BK receptor agonists stimulated the proliferation of grade 3-derived T24 bladder cancer cells. Furthermore, treatment with B1 and B2 receptor antagonists (SSR240612 and HOE140) markedly inhibited the proliferation of T24 cells. Only higher concentrations of BK increased the proliferation of the grade 1 bladder cancer cell line RT4, while des-Arg(9)-BK completely failed to induce its proliferation. Real-time PCR revealed that the mRNA expression of kinin receptors, particularly B1 receptors, was increased in T24 cells relative to RT4 cells. Data from bladder cancer human biopsies revealed that B1 receptor expression was increased in all tumor samples and under conditions of chronic inflammation. We also show novel evidence demonstrating that the pharmacological inhibition of PI3Kγ (phosphatidylinositol 3-kinase) with AS252424, concentration-dependently reduced T24 cell proliferation induced by BK or des-Arg(9)-BK. Finally, the incubation of T24 cells with kinin agonists led to a marked activation of the PI3K/AKT and ERK 1/2 signaling pathways, whereas p38 MAP kinase remained unaffected. Kinin receptors, especially B1 receptors, appear to be implicated in bladder cancer progression. It is tempting to suggest that selective kinin antagonists might represent potential alternative therapies for bladder cancer.
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Compeer, Ewoud B; Janssen, Willemijn; van Royen-Kerkhof, Annet; van Gijn, Marielle; van Montfrans, Joris M; Boes, Marianne
2015-05-10
Common Variable Immunodeficiency (CVID) is the most prevalent primary antibody deficiency, and characterized by defective generation of high-affinity antibodies. Patients have therefore increased risk to recurrent infections of the respiratory and intestinal tract. Development of high-affinity antigen-specific antibodies involves two key actions of B-cell receptors (BCR): transmembrane signaling through BCR-complexes to induce B-cell differentiation and proliferation, and BCR-mediated antigen internalization for class-II MHC-mediated presentation to acquire antigen-specific CD4(+) T-cell help.We identified a variant (L3P) in the B-lymphoid tyrosine kinase (BLK) gene of 2 related CVID-patients, which was absent in healthy relatives. BLK belongs to the Src-kinases family and involved in BCR-signaling. Here, we sought to clarify BLK function in healthy human B-cells and its association to CVID.BLK expression was comparable in patient and healthy B-cells. Functional analysis of L3P-BLK showed reduced BCR crosslinking-induced Syk phosphorylation and proliferation, in both primary B-cells and B-LCLs. B-cells expressing L3P-BLK showed accelerated destruction of BCR-internalized antigen and reduced ability to elicit CD40L-expression on antigen-specific CD4(+) T-cells.In conclusion, we found a novel BLK gene variant in CVID-patients that causes suppressed B-cell proliferation and reduced ability of B-cells to elicit antigen-specific CD4(+) T-cell responses. Both these mechanisms may contribute to hypogammaglobulinemia in CVID-patients.
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Menezes, C A S; Sullivan, A K; Falta, M T; Mack, D G; Freed, B M; Rocha, M O C; Gollob, K J; Fontenot, A P; Dutra, W O
2012-01-01
Human infection with Trypanosoma cruzi leads to Chagas disease, which presents as several different clinical conditions ranging from an asymptomatic form to a severe dilated cardiomyopathy. Several studies have demonstrated that T cells play a critical role in the development of cardiac pathology, as well as in immunoregulation during chronic disease. However, the mechanisms that drive protective or pathogenic T cell response are not known. We have shown that CD4+ T cells from chagasic patients preferentially express T cell receptor (TCR) β-chain variable region (Vβ) 5. The aim of this work was to determine whether T cells expressing this particular Vβ region displayed variable or restricted CDR3 sequences, as an indicator of the nature of the stimulus leading to the activation of these T cells in vivo. Additionally, we aimed to evaluate phenotypic characteristics of these cells that might be associated with pathology. CDR3 junctional region sequencing of Vβ5·1 expressing CD4+ T cells revealed the occurrence of a highly homologous CDR3 region with conserved TCR Jβ region usage among patients with cardiac, but not indeterminate, Chagas disease. Moreover, correlation analysis indicated that the frequency of CD4+Vβ5·1+ cells is associated with granzyme A expression, suggesting that these cells might display cytotoxic function. Together these results provide new insight into T cell recognition of antigens involved in Chagas disease and suggest that these cells may be implicated in the pathogenesis of chagasic cardiomyopathy. PMID:22774985
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Mancardi, David A; Jönsson, Friederike; Iannascoli, Bruno; Khun, Huot; Van Rooijen, Nico; Huerre, Michel; Daëron, Marc; Bruhns, Pierre
2011-02-15
K/BxN serum-induced passive arthritis was reported to depend on the activation of mast cells, triggered by the activating IgG receptor FcγRIIIA, when engaged by IgG1 autoantibodies present in K/BxN serum. This view is challenged by the fact that FcγRIIIA-deficient mice still develop K/BxN arthritis and because FcγRIIIA is the only activating IgG receptor expressed by mast cells. We investigated the contribution of IgG receptors, IgG subclasses, and cells in K/BxN arthritis. We found that the activating IgG2 receptor FcγRIV, expressed only by monocytes/macrophages and neutrophils, was sufficient to induce disease. K/BxN arthritis occurred not only in mast cell-deficient W(sh) mice, but also in mice whose mast cells express no activating IgG receptors. We propose that at least two autoantibody isotypes, IgG1 and IgG2, and two activating IgG receptors, FcγRIIIA and FcγRIV, contribute to K/BxN arthritis, which requires at least two cell types other than mast cells, monocytes/macrophages, and neutrophils.
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Drent, Esther; Groen, Richard W J; Noort, Willy A; Themeli, Maria; Lammerts van Bueren, Jeroen J; Parren, Paul W H I; Kuball, Jürgen; Sebestyen, Zsolt; Yuan, Huipin; de Bruijn, Joost; van de Donk, Niels W C J; Martens, Anton C M; Lokhorst, Henk M; Mutis, Tuna
2016-05-01
Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies. Copyright© Ferrata Storti Foundation.
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Adenovirus receptors and their implications in gene delivery
Sharma, Anurag; Li, Xiaoxin; Bangari, Dinesh S.; Mittal, Suresh K.
2010-01-01
Adenoviruses (Ads) have gained popularity as gene delivery vectors for therapeutic and prophylactic applications. Ad entry into host cells involves specific interactions between cell surface receptors and viral capsid proteins. Several cell surface molecules have been identified as receptors for Ad attachment and entry. Tissue tropism of Ad vectors is greatly influenced by their receptor usage. A variety of strategies have been investigated to modify Ad vector tropism by manipulating the receptor-interacting moieties. Many such strategies are aimed at targeting and/or detargeting of Ad vectors. In this review, we discuss the various cell surface molecules that are implicated as receptors for virus attachment and internalization. Special emphasis is given to Ad types that are utilized as gene delivery vectors. Various strategies to modify Ad tropism using the knowledge of Ad receptors are also discussed. PMID:19647886
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Kimani, Stanley G; Kumar, Sushil; Davra, Viralkumar; Chang, Yun-Juan; Kasikara, Canan; Geng, Ke; Tsou, Wen-I; Wang, Shenyan; Hoque, Mainul; Boháč, Andrej; Lewis-Antes, Anita; De Lorenzo, Mariana S; Kotenko, Sergei V; Birge, Raymond B
2016-09-06
Tyro3, Axl, and Mertk (TAMs) are a family of three conserved receptor tyrosine kinases that have pleiotropic roles in innate immunity and homeostasis and when overexpressed in cancer cells can drive tumorigenesis. In the present study, we engineered EGFR/TAM chimeric receptors (EGFR/Tyro3, EGFR/Axl, and EGF/Mertk) with the goals to interrogate post-receptor functions of TAMs, and query whether TAMs have unique or overlapping post-receptor activation profiles. Stable expression of EGFR/TAMs in EGFR-deficient CHO cells afforded robust EGF inducible TAM receptor phosphorylation and activation of downstream signaling. Using a series of unbiased screening approaches, that include kinome-view analysis, phosphor-arrays, RNAseq/GSEA analysis, as well as cell biological and in vivo readouts, we provide evidence that each TAM has unique post-receptor signaling platforms and identify an intrinsic role for Axl that impinges on cell motility and invasion compared to Tyro3 and Mertk. These studies demonstrate that TAM show unique post-receptor signatures that impinge on distinct gene expression profiles and tumorigenic outcomes.
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McGrady, Nolan R; Minton, Alena Z; Stankowska, Dorota L; He, Shaoqing; Jefferies, Hayden B; Krishnamoorthy, Raghu R
2017-03-01
Primary open angle glaucoma is a heterogeneous group of optic neuropathies that results in optic nerve degeneration and a loss of retinal ganglion cells (RGCs) ultimately causing blindness if allowed to progress. Elevation of intraocular pressure (IOP) is the most attributable risk factor for developing glaucoma and lowering of IOP is currently the only available therapy. However, despite lowering IOP, neurodegenerative effects persist in some patients. Hence, it would be beneficial to develop approaches to promote neuroprotection of RGCs in addition to IOP lowering therapies. The endothelin system is a key target for intervention against glaucomatous neurodegeneration. The endothelin family of peptides and receptors, particularly endothelin-1 (ET-1) and endothelin B (ET B ) receptor, has been shown to have neurodegenerative roles in glaucoma. The purpose of this study was to examine changes in endothelin A (ET A ) receptor protein expression in the retinas of adult male Brown Norway rats following IOP elevation by the Morrison's model of ocular hypertension and the impact of ET A receptor overexpression on RGC viability in vitro. IOP elevation was carried out in one eye of Brown Norway rats by injection of hypertonic saline through episcleral veins. After 2 weeks of IOP elevation, immunohistochemical analysis of retinal sections from rat eyes showed an increasing trend in immunostaining for ET A receptors in multiple retinal layers including the inner plexiform layer, ganglion cell layer and outer plexiform layer. Following 4 weeks of IOP elevation, a significant increase in immunostaining for ET A receptor expression was found in the retina, primarily in the inner plexiform layer and ganglion cells. A modest increase in staining for ET A receptors was also found in the outer plexiform layer in the retina of rats with IOP elevation. Cell culture studies showed that overexpression of ET A receptors in 661W cells as well as primary RGCs decreases cell viability, compared to empty vector transfected cells. Adeno-associated virus mediated overexpression of the ET A receptor produced an increase in the ET B receptor in primary RGCs. Elevated IOP results in an appreciable change in ET A receptor expression in the retina. Overexpression of the ET A receptor results in an overall decrease in cell viability, accompanied by an increase in ET B receptor levels, suggesting the involvement of both ET A and ET B receptors in mediating cell death. These findings raise possibilities for the development of ET A /ET B dual receptor antagonists as neuroprotective treatments for glaucomatous neuropathy.
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Amsler, K; Kuwada, S K
1999-01-01
Signal transduction from receptors is mediated by the interaction of activated receptors with proximate downstream signaling proteins. In polarized epithelial cells, the membrane is divided into subdomains: the apical and basolateral membranes. Membrane receptors may be present in one or both subdomains. Using a combination of immunoprecipitation and Western blot analyses, we tested the hypothesis that a tyrosine kinase growth factor receptor, epidermal growth factor receptor (EGFR), interacts with distinct signaling proteins when present at the apical vs. basolateral membrane of a polarized renal epithelial cell. We report here that tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) was induced only when basolateral EGFR was activated. In contrast, tyrosine phosphorylation of several other signaling proteins was increased by activation of receptor at either surface. All signaling proteins were distributed diffusely throughout the cytoplasm; however, PLC-gamma protein also displayed a concentration at lateral cell borders. These results demonstrate that in polarized epithelial cells the array of signaling pathways initiated by activation of a membrane receptor is defined, at least in part, by the membrane location of the receptor.
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NASA Astrophysics Data System (ADS)
Imboden, John B.; Shoback, Dolores M.; Pattison, Gregory; Stobo, John D.
1986-08-01
The addition of monoclonal antibodies to the antigen receptor complex on the malignant human T-cell line Jurkat generates increases in inositol trisphosphate and in the concentration of cytoplasmic free calcium. Exposure of Jurkat cells to cholera toxin for 3 hr inhibited these receptor-mediated events and led to a selective, partial loss of the antigen receptor complex from the cellular surface. None of the effects of cholera toxin on the antigen receptor complex were mimicked by the B subunit of cholera toxin or by increasing intracellular cAMP levels with either forskolin or 8-bromo cAMP. These results suggest that a cholera toxin substrate can regulate signal transduction by the T-cell antigen receptor.
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Brené, S; Hall, H; Lindefors, N; Karlsson, P; Halldin, C; Sedvall, G
1995-07-01
Messenger RNAs for the D1 dopamine receptor and dopamine- and cyclic AMP-regulated phosphoprotein of relative mass 32,000 (DARPP-32) were examined by in situ hybridization in the cynomolgus monkey brain. The messenger RNA distribution was compared to the distribution of D1 dopamine receptors using [3H]SCH 23390 autoradiography. In the caudate nucleus and putamen, D1 dopamine receptor messenger RNA-positive cells were unevenly distributed. Clusters of cells with an approximately three-fold higher intensity of labeling, as compared to surrounding regions, were found. Some of these D1 dopamine receptor messenger RNA intensive cell clusters in the caudate nucleus appeared to some extent to be matched to regions of higher intensity of [3H]SCH 23390 binding. The distribution of cells expressing DARPP-32 messenger RNA in the caudate nucleus and putamen was found to be non-clustered. In neocortical regions, cells of different sizes expressing D1 dopamine receptor messenger RNA were present in layers II-VI. D1 dopamine receptor messenger RNA-positive cells were most abundant in layer V. Unexpectedly, no DARPP-32 messenger RNA signal was detected in neocortex. Chronic SCH 23390 administration did not change the relative levels of messenger RNAs for the D1 dopamine receptor and DARPP-32 or [3H]SCH 23390 binding as measured by quantitative image analysis. The clustered distribution of D1 dopamine receptor messenger RNA is in contrast to that of DARPP-32 messenger RNA. This suggests that D1 dopamine receptors may play a more significant role in regulating DARPP-32 function in patch regions as compared to matrix regions. D1 dopamine receptor messenger RNA-expressing cells could also be visualized in several layers of the primate neocortex, implying that dopamine acts through D1 dopamine receptors within functionally different neuronal circuits of the neocortex.
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Tsukada, Junko; Tahara, Atsuo; Tomura, Yuichi; Wada, Koh-ichi; Kusayama, Toshiyuki; Ishii, Noe; Yatsu, Takeyuki; Uchida, Wataru; Taniguchi, Nobuaki; Tanaka, Akihiro
2001-01-01
YM471, (Z)-4′-{4,4-difluoro-5-[2-(4-dimethylaminopiperidino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl}-2-phenylbenzanilide monohydrochloride, is a newly synthesized potent vasopressin (AVP) receptor antagonist. Its effects on binding to and signal transduction by cloned human AVP receptors (V1A, V1B and V2) stably expressed in Chinese hamster ovary (CHO) cells, and oxytocin receptors in human uterine smooth muscle cells (USMC) were studied. YM471 potently inhibited specific [3H]-AVP binding to V1A and V2 receptors with Ki values of 0.62 nM and 1.19 nM, respectively. In contrast, YM471 exhibited much lower affinity for V1B and oxytocin receptors with Ki values of 16.4 μM and 31.6 nM, respectively. In CHO cells expressing V1A receptors, YM471 potently inhibited AVP-induced intracellular Ca2+ concentration ([Ca2+]i) increase, exhibiting an IC50 value of 0.56 nM. However, in human USMC expressing oxytocin receptors, YM471 exhibited much lower potency in inhibiting oxytocin-induced [Ca2+]i increase (IC50=193 nM), and did not affect AVP-induced [Ca2+]i increase in CHO cells expressing V1B receptors. Furthermore, in CHO cells expressing V2 receptors, YM471 potently inhibited the production of cyclic AMP stimulated by AVP with an IC50 value of 1.88 nM. In all assays, YM471 showed no agonistic activity. These results demonstrate that YM471 is a potent, nonpeptide human V1A and V2 receptor antagonist which will be a valuable tool in defining the physiologic and pharmacologic actions of AVP. PMID:11429400
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Tsukada, J; Tahara, A; Tomura, Y; Wada Ki; Kusayama, T; Ishii, N; Yatsu, T; Uchida, W; Taniguchi, N; Tanaka, A
2001-07-01
YM471, (Z)-4'-[4,4-difluoro-5-[2-(4-dimethylaminopiperidino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl]-2-phenylbenzanilide monohydrochloride, is a newly synthesized potent vasopressin (AVP) receptor antagonist. Its effects on binding to and signal transduction by cloned human AVP receptors (V(1A), V(1B) and V(2)) stably expressed in Chinese hamster ovary (CHO) cells, and oxytocin receptors in human uterine smooth muscle cells (USMC) were studied. YM471 potently inhibited specific [(3)H]-AVP binding to V(1A) and V(2) receptors with K(i) values of 0.62 nM and 1.19 nM, respectively. In contrast, YM471 exhibited much lower affinity for V(1B) and oxytocin receptors with K(i) values of 16.4 microM and 31.6 nM, respectively. In CHO cells expressing V(1A) receptors, YM471 potently inhibited AVP-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) increase, exhibiting an IC(50) value of 0.56 nM. However, in human USMC expressing oxytocin receptors, YM471 exhibited much lower potency in inhibiting oxytocin-induced [Ca(2+)](i) increase (IC(50)=193 nM), and did not affect AVP-induced [Ca(2+)](i) increase in CHO cells expressing V(1B) receptors. Furthermore, in CHO cells expressing V(2) receptors, YM471 potently inhibited the production of cyclic AMP stimulated by AVP with an IC(50) value of 1.88 nM. In all assays, YM471 showed no agonistic activity. These results demonstrate that YM471 is a potent, nonpeptide human V(1A) and V(2) receptor antagonist which will be a valuable tool in defining the physiologic and pharmacologic actions of AVP.
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Schelman, William R; Andres, Robert; Ferguson, Paul; Orr, Brent; Kang, Evan; Weyhenmeyer, James A
2004-09-10
While angiotensin II (Ang II) plays a major role in the regulation of blood pressure, fluid homeostasis and neuroendocrine function, recent studies have also implicated the peptide hormone in cell growth, differentiation and apoptosis. In support of this, we have previously demonstrated that Ang II attenuates N-methyl-D-aspartate (NMDA) receptor signaling [Molec. Brain Res. 48 (1997) 197]. To further examine the modulatory role of Ang II on NMDA receptor function, we investigated the effect of angiotensin receptor (AT) activation on NMDA-mediated cell death and the accompanying decrease in Bcl-2 expression. The viability of differentiated N1E-115 and NG108-15 neuronal cell lines was reduced following exposure to NMDA in a dose-dependent manner. MTT analysis (mitochondrial integrity) revealed a decrease in cell survival of 49.4+/-12.3% in NG108 cells and 79.9+/-6.8% in N1E cells following treatment with 10 mM NMDA for 20 h. Cytotoxicity in N1E cells was inhibited by the noncompetitive NMDA receptor antagonist, MK-801. Further, NMDA receptor-mediated cell death in NG108 cells was attenuated by treatment with Ang II. The Ang II effect was inhibited by both AT1 and AT2 receptor antagonists, losartan and PD123319, respectively, suggesting that both receptor subtypes may play a role in the survival effect of Ang II. Since it has been shown that activation of NMDA receptors alters the expression of Bcl-2 family proteins, Western blot analysis was performed in N1E cells to determine whether Ang II alters the NMDA-induced changes in Bcl-2 expression. A concentration-dependent decrease of intracellular Bcl-2 protein levels was observed following treatment with NMDA, and this reduction was inhibited by MK801. Addition of Ang II suppressed the NMDA receptor-mediated reduction in Bcl-2. The Ang II effect on NMDA-mediated changes in Bcl-2 levels was blocked by PD123319, but was not significantly changed by losartan, suggesting AT2 receptor specificity. Taken together, these results suggest that Ang II attenuates NMDA receptor-mediated neurotoxicity and that this effect may be due, in part, to an alteration in Bcl-2 expression.
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Yang, Jian; Chen, Caiyu; Ren, Hongmei; Han, Yu; He, Duofen; Zhou, Lin; Hopfer, Ulrich; Jose, Pedro A.; Zeng, Chunyu
2013-01-01
Background The renin–angiotensin (Ang) system controls blood pressure, in part, by regulating renal tubular sodium transport. In the kidney, activation of the angiotensin II type 1 (AT1) receptor increases renal sodium reabsorption, whereas the angiotensin II type 2 (AT2) receptor produces the opposite effect. We hypothesized that the AT2 receptor regulates AT1 receptor expression and function in the kidney. Methods and results In immortalized renal proximal tubule (RPT) cells from Wistar–Kyoto rats, CGP42112, an AT2 receptor agonist, decreased AT1 receptor mRNA and protein expression (P < 0.05), as assessed by reverse transcriptase-polymerase chain reaction and immunoblotting. The inhibitory effect of the AT2 receptor on AT1 receptor expression was blocked by the AT2 receptor antagonist, PD123319 (10−6 mol/l), the nitric oxide synthase inhibitor Nw-nitro-l-arginine methyl ester (10−4 mol/l), or the nitric oxide-dependent soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (10−5 mol/l), indicating that both nitric oxide and cyclic guanosine monophosphate (cGMP) were involved in the signaling pathway. Furthermore, CGP42112 decreased Sp1 serine phosphorylation and reduced the binding of Sp1 to AT1 receptor DNA. Stimulation with Ang II (10−11 mol/l per 30 min) enhanced Na+-K+-ATPase activity in RPT cells, which was prevented by pretreatment with CGP42112 (10−7 mol/l per 24 h) (P < 0.05). The above-mentioned results were confirmed in RPT cells from AT2 receptor knockout mice; AT1 receptor expression and Ang II-stimulated Na+-K+-ATPase activity were greater in these cells than in RPT cells from wild-type mice (P < 0.05). AT1/AT2 receptors co-localized and co-immunoprecipitated in RPT cells; short-term CGP42112 (10−7 mol/l per 30 min) treatment increased AT1/AT2 receptor co-immunoprecipitation (P < 0.05). Conclusions These results indicate that the renal AT2 receptor, via nitric oxide/cGMP/Sp1 pathway, regulates AT1 receptor expression and function, which may be important in the regulation of sodium excretion and blood pressure. PMID:22504846
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Blocking Blood Supply to Breast Carcinoma With a DNA Vaccine Encoding VEGF Receptor-2
2006-03-01
recognize antigens in the form of 8 to 10 amino acid long peptides, presented to T- cell receptors (TCRs) on the cell surface as complexes with major... receptor , and providing tumor- associated antigens , our DNA vaccine can efficiently activate DCs, NK cells , and CTLs, presumably in Peyer’s patches. The... immunoreceptor in immune cell activation and natural killing. Immunity. 2002;17:19-29. (5) Snyder MR, Weyand CM, Goronzy JJ. The double life of NK receptors
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Analysis of odorant receptor protein function in the yellow fever mosquito, aedes aegypti
USDA-ARS?s Scientific Manuscript database
Odorant receptors (ORs) in insects are ligand-gated ion channels comprised of two subunits: a variable receptor and an obligatory co-receptor (Orco). This protein receptor complex of unknown stoichiometry interacts with an odor molecule leading to changes in permeability of the sensory dendrite, th...
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Knight, B L; Patel, D D; Soutar, A K
1983-01-01
Human blood monocytes cultured in medium containing 20% whole serum showed the greatest activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and [14C]acetate incorporation into non-saponifiable lipids around the 7th day after seeding, the period of greatest growth. Although there was enough low-density lipoprotein (LDL) in the medium to saturate the LDL receptors that were expressed by normal cells at that time, HMG-CoA reductase activity and acetate incorporation were as high in normal cells as in cells from familial-hypercholesterolaemic (FH) patients. Both the addition of extra LDL, which interacted with the cells by non-saturable processes, and receptor-mediated uptake of acetylated LDL significantly reduced reductase activity and increased incorporation of [14C]oleate into cholesteryl esters in normal cells and cells from FH patients ('FH cells'), and reduced the expression of LDL receptors in normal cells. Pre-incubation for 20h in lipoprotein-deficient medium apparently increased the number of LDL receptors expressed by normal cells but reduced the activity of HMG-CoA reductase in both normal and FH cells. During subsequent incubations the same rate of degradation of acetylated LDL and of non-saturable degradation of LDL by FH cells was associated with the same reduction in HMG-CoA reductase activity, although LDL produced a much smaller stimulation of oleate incorporation into cholesteryl esters. In normal cells pre-incubated without lipoproteins, receptor-mediated uptake of LDL could abolish reductase activity and the expression of LDL receptors. The results suggested that in these cells, receptor-mediated uptake of LDL might have a greater effect on reductase activity and LDL receptors than the equivalent uptake of acetylated LDL. It is proposed that endogenous synthesis is an important source of cholesterol for growth of normal cells, and that the site at which cholesterol is deposited in the cells may determine the nature and extent of the metabolic events that follow. PMID:6305342
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Wanecek, M; Oldner, A; Rudehill, A; Sollevi, A; Alving, K; Weitzberg, E
1997-05-01
In a porcine endotoxin shock model, the mixed nonpeptide endothelin receptor antagonist bosentan was administered 2 h after onset of endotoxemia (n = 8). Cardiopulmonary vascular changes, oxygen-related variables, and plasma levels of endothelin-1-like immunoreactivity were compared with a control group that received only endotoxin (n = 8). Bosentan abolished the progressive increase in mean pulmonary artery pressure and pulmonary vascular resistance seen in controls. Possible mechanisms include blockade of vasoconstrictive endothelin receptors, and a lesser degree of edema and inflammation indicated by less alveolar protein and a lower inflammatory cell count observed in bronchoalveolar lavage. Further, bosentan restored cardiac index to the pre-endotoxin level by an increase in stroke volume index, improved systemic oxygen delivery, and acid base balance. Because mean arterial blood pressure was unaffected, bosentan reduced systemic vascular resistance. Endotoxemia resulted in an increase in tumor necrosis factor-alpha and endothelin-1-like immunoreactivity plasma levels, the latter being further increased by bosentan. In conclusion, in porcine endotoxemia, treatment with the endothelin receptor antagonist bosentan, administered during fulminate shock, abolished pulmonary hypertension and restored cardiac index. These findings suggest that bosentan could be an effective treatment for reversing a deteriorated cardiopulmonary state during septic shock.
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NASA Astrophysics Data System (ADS)
Li, Long; Hu, Jinglei; Xu, Guangkui; Song, Fan
2018-01-01
Cell-cell adhesion and the adhesion of cells to tissues and extracellular matrix, which are pivotal for immune response, tissue development, and cell locomotion, depend sensitively on the binding constant of receptor and ligand molecules anchored on the apposing surfaces. An important question remains of whether the immobilization of ligands affects the affinity of binding with cell adhesion receptors. We have investigated the adhesion of multicomponent membranes to a flat substrate coated with immobile ligands using Monte Carlo simulations of a statistical mesoscopic model with biologically relevant parameters. We find that the binding of the adhesion receptors to ligands immobilized on the substrate is strongly affected by the ligand distribution. In the case of ligand clusters, the receptor-ligand binding constant can be significantly enhanced due to the less translational entropy loss of lipid-raft domains in the model cell membranes upon the formation of additional complexes. For ligands randomly or uniformly immobilized on the substrate, the binding constant is rather decreased since the receptors localized in lipid-raft domains have to pay an energetic penalty in order to bind ligands. Our findings help to understand why cell-substrate adhesion experiments for measuring the impact of lipid rafts on the receptor-ligand interactions led to contradictory results.
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IL-3 specifically inhibits GM-CSF binding to the higher affinity receptor
DOE Office of Scientific and Technical Information (OSTI.GOV)
Taketazu, F.; Chiba, S.; Shibuya, K.
1991-02-01
The inhibition of binding between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor by human interleukin-3 (IL-3) was observed in myelogenous leukemia cell line KG-1 which bore the receptors both for GM-CSF and IL-3. In contrast, this phenomenon was not observed in histiocytic lymphoma cell line U-937 or in gastric carcinoma cell line KATO III, both of which have apparent GM-CSF receptor but an undetectable IL-3 receptor. In KG-1 cells, the cross-inhibition was preferentially observed when the binding of GM-CSF was performed under the high-affinity binding condition; i.e., a low concentration of 125I-GM-CSF was incubated. Scatchard analysis of 125I-GM-CSF bindingmore » to KG-1 cells in the absence and in the presence of unlabeled IL-3 demonstrated that IL-3 inhibited GM-CSF binding to the higher-affinity component of GM-CSF receptor on KG-1 cells. Moreover, a chemical cross-linking study has revealed that the cross-inhibition of the GM-CSF binding observed in KG-1 cells is specific for the beta-chain, Mr 135,000 binding protein which has been identified as a component forming the high-affinity GM-CSF receptor existing specifically on hemopoietic cells.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Delpeut, Sebastien; Noyce, Ryan S.; IWK Health Centre, Canadian Center for Vaccinology, Goldbloom Pavilion, Halifax, Nova Scotia, Canada B3H 1X5
The entry of canine distemper virus (CDV) is a multistep process that involves the attachment of CDV hemagglutinin (H) to its cellular receptor, followed by fusion between virus and cell membranes. Our laboratory recently identified PVRL4 (nectin-4) to be the epithelial receptor for measles and canine distemper viruses. In this study, we demonstrate that the V domain of PVRL4 is critical for CDV entry and virus cell-to-cell spread. Furthermore, four key amino acid residues within the V domain of dog PVRL4 and two within the CDV hemagglutinin were shown to be essential for receptor-mediated virus entry. - Highlights: • PVRL4more » (nectin-4) is the epithelial cell receptor for measles and canine distemper viruses. • V domain of PVRL4 is critical for CDV entry, cell-to-cell spread, and syncytia formation. • Chimeric PVRL1 backbone substituted with the V domain of PVRL4 can function as a receptor. • Amino acids (F132/P133/A134/G135) within the V domain are essential for PVRL4 receptor activity. • Amino acids (P493/Y539) within CDV H protein are essential for PVRL4 receptor interaction.« less
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Documentation of angiotensin II receptors in glomerular epithelial cells
NASA Technical Reports Server (NTRS)
Sharma, M.; Sharma, R.; Greene, A. S.; McCarthy, E. T.; Savin, V. J.; Cowley, A. W. (Principal Investigator)
1998-01-01
Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial cells participate in angiotensin II-mediated control of the glomerular filtration barrier.
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Zhao, Yan-Dong; Cheng, Sai-Yu; Ou, Shan; Xiao, Zhi; He, Wen-Juan; Jian-Cui; Ruan, Huai-Zhen
2012-01-01
This study was designed to evaluate the effect of hypobaric hypoxia (HH) on the function and expression of P2X receptors in rat hippocampus CA1 pyramidal cells. The functional changes of P2X receptors were investigated through the cell HH model and the expressional alterations of P2X receptors were observed through the animal HH model. P2X receptors mediated currents were recorded from the freshly dissociated CA1 pyramidal cells of 7-day-old SD rats by whole cell patch clamp recording. The expression and distribution of P2X receptors were observed through immunohistochemistry and western blot at HH 3-day and 7-day. In acute HH conditions, the amplitudes of ATP evoked peak currents were decreased compared to control. The immunohistochemistry and western blot results reflected there was no change in P2X receptors expression after 3 days HH injury, while P2X receptors expression was up-regulated in response to 7 days HH injury. These findings supported the possibility that the function of P2X receptors was sensitive to HH damage and long-term function decrease should result in the expression increase of P2X receptors.
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Adenosine A2B and A3 receptor location at the mouse neuromuscular junction.
Garcia, Neus; Priego, Mercedes; Hurtado, Erica; Obis, Teresa; Santafe, Manel M; Tomàs, Marta; Lanuza, Maria Angel; Tomàs, Josep
2014-07-01
To date, four subtypes of adenosine receptors have been cloned (A(1)R, A(2A)R, A(2B)R, and A(3)R). In a previous study we used confocal immunocytochemistry to identify A(1)R and A(2A)R receptors at mouse neuromuscular junctions (NMJs). The data shows that these receptors are localized differently in the three cells (muscle, nerve and glia) that configure the NMJs. A(1)R localizes in the terminal teloglial Schwann cell and nerve terminal, whereas A(2A)R localizes in the postsynaptic muscle and in the axon and nerve terminal. Here, we use Western blotting to investigate the presence of A(2B)R and A(3)R receptors in striated muscle and immunohistochemistry to localize them in the three cells of the adult neuromuscular synapse. The data show that A(2B)R and A(3)R receptors are present in the nerve terminal and muscle cells at the NMJs. Neither A(2B)R nor A(3)R receptors are localized in the Schwann cells. Thus, the four subtypes of adenosine receptors are present in the motor endings. The presence of these receptors in the neuromuscular synapse allows the receptors to be involved in the modulation of transmitter release. © 2014 Anatomical Society.
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Maruyama, Yutaka; Yasuda, Reiko; Kuroda, Motonaka; Eto, Yuzuru
2012-01-01
Recently, we reported that calcium-sensing receptor (CaSR) is a receptor for kokumi substances, which enhance the intensities of salty, sweet and umami tastes. Furthermore, we found that several γ-glutamyl peptides, which are CaSR agonists, are kokumi substances. In this study, we elucidated the receptor cells for kokumi substances, and their physiological properties. For this purpose, we used Calcium Green-1 loaded mouse taste cells in lingual tissue slices and confocal microscopy. Kokumi substances, applied focally around taste pores, induced an increase in the intracellular Ca2+ concentration ([Ca2+]i) in a subset of taste cells. These responses were inhibited by pretreatment with the CaSR inhibitor, NPS2143. However, the kokumi substance-induced responses did not require extracellular Ca2+. CaSR-expressing taste cells are a different subset of cells from the T1R3-expressing umami or sweet taste receptor cells. These observations indicate that CaSR-expressing taste cells are the primary detectors of kokumi substances, and that they are an independent population from the influenced basic taste receptor cells, at least in the case of sweet and umami. PMID:22511946
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Mellor, J R; Wisden, W; Randall, A D
2000-07-10
Electrophysiological investigation of cultured cerebellar murine granule cells revealed differences between the GABA(A) receptors at inhibitory synapses and those on the cell body. Specifically, mIPSCs decayed more rapidly than cell body receptors deactivated, the mean single channel conductance at the synapse (32 pS) was greater than that at cell body (21 pS) and only cell body receptors were sensitive to Zn(2+) (150 microM), which depressed response amplitude by 82+/-5% and almost doubled the rate of channel deactivation. The GABA(A) receptor alpha6 subunit is selectively expressed in cerebellar granule cells. Although concentrated at synapses, it is also found on extrasynaptic membranes. Using a mouse line (Deltaalpha6lacZ) lacking this subunit, we investigated its role in the somato-synaptic differences in GABA(A) receptor function. All differences between cell body and synaptic GABA(A) receptors observed in wild-type (WT) granule cells persisted in Deltaalpha6lacZ cells, thus demonstrating that they are not specifically due to the cellular distribution of the alpha6 subunit. However, mIPSCs from WT and Deltaalpha6lacZ cells differed in both their kinetics (faster decay in WT cells) and underlying single channel conductance (32 pS WT, 25 pS Deltaalpha6lacZ). This provides good evidence for a functional contribution of the alpha6 subunit to postsynaptic GABA(A) receptors in these cells. Despite this, deactivation kinetics of mIPSCs in WT and Deltaalpha6lacZ granule cells exhibited similar benzodiazepene (BDZ) sensitivity. This suggests that the enhanced BDZ-induced ataxia seen in Deltaalpha6lacZ mice may reflect physiological activity at extrasynaptic receptors which, unlike those at synapses, display differential BDZ-sensitivity in WT and Deltaalpha6lacZ granule cells (Jones, A.M., Korpi, E.R., McKernan, R.M., Nusser, Z., Pelz, R., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H., Wisden, W., 1997. Ligand-gated ion channel partnerships: GABA(A) receptor alpha(6) subunit inactivation inhibits delta subunit expression. Journal of Neuroscience 17, 1350-1362).
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Melia, Mary M; Earle, John Philip; Abdullah, Haniah; Reaney, Katherine; Tangy, Frederic; Cosby, Sara Louise
2014-01-01
Signalling lymphocyte activation molecule (SLAM) has been identified as an immune cell receptor for the morbilliviruses, measles (MV), canine distemper (CDV), rinderpest and peste des petits ruminants (PPRV) viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4), also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV) remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt) MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG) and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF),for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO) cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.
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2009-05-01
adoptive therapy using CD19-specific chimeric antigen receptor re-directed T cells for recurrent/refrctory follicular lymphoma...Beauty (SB) transposon/transposase system to express a CD19-specific chimeric antigen receptor (CAR). T cells that have undergone transposition...accomplished using genetic engineering to express a chimeric antigen receptor (CAR) to redirect the specificity of T cells for CD19 on malignant B cells
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Badache, A; Hynes, N E
2001-01-01
Interleukin (IL)-6, a multifunctional regulator of immune response, hematopoiesis, and acute phase reactions, has also been shown to regulate cancer cell proliferation. We have investigated IL-6 signaling pathways and cellular responses in the T47D breast carcinoma cell line. The IL-6-type cytokines, IL-6 and oncostatin M, simultaneously inhibited cell proliferation and increased cell migration. In T47D cells, IL-6 stimulated the activation of Janus-activated kinase 1 tyrosine kinase and signal transducers and activators of transcription (STAT) 1 and STAT3 transcription factors. Expression of dominant negative STAT3 in the cells strongly reduced IL-6-mediated growth inhibition but did not prevent IL-6-induced cell migration. IL-6 treatment led to activation of the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3'-kinase (PI3K) pathways. Inhibition of MAPK or PI3K activity reversed IL-6- and oncostatin M-stimulated migration. Because cross-talk between cytokine receptors and members of the ErbB family of receptor tyrosine kinases has been described previously, we have examined their interaction in T47D cells. Down-regulation of ErbB receptor activity, through the use of specific pharmacological inhibitors or dominant negative receptor constructs, revealed that IL-6-induced MAPK activation was largely dependent on epidermal growth factor (EGF) receptor activity, but not on ErbB-2 activity. Using a monoclonal antibody that interferes with EGF receptor-ligand interaction, we have shown that in T47D cells, IL-6 cooperates with an EGF receptor autocrine activity loop for signaling through the MAPK and PI3K pathways and for cell migration. Both the tyrosine phosphatase SHP-2 and the multisubstrate docking molecule Gab1, which are potential links between IL-6 and the MAPK/PI3K pathways, were constitutively associated with the active EGF receptor. On IL-6 stimulation, SHP-2 and Gab1 were recruited to the gp130 subunit of the IL-6 receptor and tyrosine phosphorylated, allowing downstream signaling to the MAPK and PI3K pathways. Thus, in T47D breast carcinoma cells, IL-6 acts in synergy with EGF receptor autocrine activity to signal through the MAPK/PI3K pathways. Cooperation between IL-6 and the EGF receptor in T47D breast carcinoma cells illustrates how a combination of multiple stimuli, either exogenous or endogenous, may result in synergistic cellular responses.
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Costafreda, Maria Isabel; Kaplan, Gerardo
2018-05-01
The hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), classified as CD365, was initially discovered as an HAV cellular receptor using an expression cloning strategy. Due to the lack of HAV receptor-negative replication-competent cells, it was not possible to fully prove that HAVCR1 was a functional HAV receptor. However, biochemistry, classical virology, and epidemiology studies further supported the functional role of HAVCR1 as an HAV receptor. Here, we show that an anti-HAVCR1 monoclonal antibody that protected African green monkey kidney (AGMK) cells against HAV infection only partially protected monkey Vero E6 cells and human hepatoma Huh7 cells, indicating that these two cell lines express alternative yet unidentified HAV receptors. Therefore, we focused our work on AGMK cells to further characterize the function of HAVCR1 as an HAV receptor. Advances in clustered regularly interspaced short palindromic repeat/Cas9 technology allowed us to knock out the monkey ortholog of HAVCR1 in AGMK cells. The resulting AGMK HAVCR1 knockout (KO) cells lost susceptibility to HAV infection, including HAV-free viral particles (vpHAV) and exosomes purified from HAV-infected cells (exo-HAV). Transfection of HAVCR1 cDNA into AGMK HAVCR1 KO cells restored susceptibility to vpHAV and exo-HAV infection. Furthermore, transfection of the mouse ortholog of HAVCR1, mHavcr1, also restored the susceptibility of AGMK HAVCR1 KO cells to HAV infection. Taken together, our data clearly show that HAVCR1 and mHavcr1 are functional HAV receptors that mediate HAV infection. This work paves the way for the identification of alternative HAV receptors to gain a complete understanding of their interplay with HAVCR1 in the cell entry and pathogenic processes of HAV. IMPORTANCE HAVCR1, an HAV receptor, is expressed in different cell types, including regulatory immune cells and antigen-presenting cells. How HAV evades the immune response during a long incubation period of up to 4 weeks and the mechanism by which the subsequent necroinflammatory process clears the infection remain a puzzle that most likely involves the HAV-HAVCR1 interaction. Based on negative data, a recent paper from the S. M. Lemon and W. Maury laboratories (A. Das, A. Hirai-Yuki, O. Gonzalez-Lopez, B. Rhein, S. Moller-Tank, R. Brouillette, L. Hensley, I. Misumi, W. Lovell, J. M. Cullen, J. K. Whitmire, W. Maury, and S. M. Lemon, mBio 8:e00969-17, 2017, https://doi.org/10.1128/mBio.00969-17) suggested that HAVCR1 is not a functional HAV receptor, nor it is it required for HAV infection. However, our data, based on regain of the HAV receptor function in HAVCR1 knockout cells transfected with HAVCR1 cDNA, disagree with their findings. Our positive data show conclusively that HAVCR1 is indeed a functional HAV receptor and lays the ground for the identification of alternative HAV receptors and how they interact with HAVCR1 in cell entry and the pathogenesis of HAV. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
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Tutton, P J; Barkla, D H
1978-03-01
Cell proliferation in dimethylhydrazine-induced colonic carcinomata was stimulated by histamine and by the histamine H2 receptor agonist dimaprit and inhibited by the histamine H2 receptor antagonists Metiamide and Cimetidine but not by the histamine H1 receptor antagonist Mepyramine. In contrast histamine had no effect on colonic crypt cell proliferation in normal or dimethylhydrazine-treated rats.
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Yang, Jiezuan; Yan, Dong; Guo, Renyong; Chen, Jiajia; Li, Yongtao; Fan, Jun; Fu, Xuyan; Yao, Xinsheng; Diao, Hongyan; Li, Lanjuan
2017-03-01
Effective antiviral therapy plays a key role in slowing the progression of chronic hepatitis B (CHB). Identification of serum indices, including hepatitis B e antigen (HBeAg) expression and seroconversion, will facilitate evaluation of the efficacy of antiviral therapy in HBeAg-positive CHB patients. The biochemical, serological, virological parameters, and the frequency of circulating CD4CD25 regulatory T cell (Treg) in 32 patients were measured at baseline and every 12 weeks during 96 weeks of tenofovir disoproxil fumarate (TDF) treatment. The relationship between the hepatitis B virus (HBV) deoxyribonucleic acid (DNA) and Treg and alanine aminotransferase (ALT) levels was analyzed, respectively. The molecular profiles of T-cell receptor beta variable chain (TRBV) were determined using gene melting spectral pattern. For the seroconverted 12 patients, ALT declined to normal levels by week 24 and remained at this level in subsequent treatment; moreover, the predictive cutoff value of ALT for HBeAg seroconversion (SC) was 41.5 U/L at week 24. The positive correlation between HBV DNA and Treg and ALT was significant in SC patients, but not in non-SC patients. Six TRBV families (BV3, BV11, BV12, BV14, BV20, and BV24) were predominantly expressed in SC patients at baseline. The decline of ALT could be used to predict HBeAg seroconversion for CHB patients during TDF treatment. In addition, the profile of Tregs and TRBVs may be associated with HBeAg seroconversion and could also be a potential indicator for predicting HBeAg SC and treatment outcome for CHB patients.
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Klein, Ophir; Polack, Glenda W.; Surti, Toral; Kegler-Ebo, Deena; Smith, Steven O.; DiMaio, Daniel
1998-01-01
The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) β receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine 17 in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine 17 forms a hydrogen bond with threonine 513 of the PDGF β receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position 17 and examined the ability of these proteins to transform C127 fibroblasts, which express endogenous PDGF β receptor. Although several position 17 mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF β receptor or transform cells, in agreement with the proposed interaction between position 17 of the E5 protein and threonine 513 of the receptor. The nature of the residue at position 17 also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF β receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF β receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF β receptor in mediating E5 transformation and highlight the critical role of the residue at position 17 of the E5 protein in the productive interaction with the PDGF β receptor. On the basis of molecular modeling analysis and the known chemical properties of the amino acids, we suggest a structural basis for the role of the residue at position 17 in E5 dimerization and in complex formation between the E5 protein and the PDGF β receptor. PMID:9765437
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Klein, O; Polack, G W; Surti, T; Kegler-Ebo, D; Smith, S O; DiMaio, D
1998-11-01
The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine 17 in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine 17 forms a hydrogen bond with threonine 513 of the PDGF beta receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position 17 and examined the ability of these proteins to transform C127 fibroblasts, which express endogenous PDGF beta receptor. Although several position 17 mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF beta receptor or transform cells, in agreement with the proposed interaction between position 17 of the E5 protein and threonine 513 of the receptor. The nature of the residue at position 17 also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF beta receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF beta receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF beta receptor in mediating E5 transformation and highlight the critical role of the residue at position 17 of the E5 protein in the productive interaction with the PDGF beta receptor. On the basis of molecular modeling analysis and the known chemical properties of the amino acids, we suggest a structural basis for the role of the residue at position 17 in E5 dimerization and in complex formation between the E5 protein and the PDGF beta receptor.
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Shah, Girish V.; Muralidharan, Anbalagan; Gokulgandhi, Mitan; Soan, Kamal; Thomas, Shibu
2009-01-01
Calcitonin, a neuroendocrine peptide, and its receptor are localized in the basal epithelium of benign prostate but in the secretory epithelium of malignant prostates. The abundance of calcitonin and calcitonin receptor mRNA displays positive correlation with the Gleason grade of primary prostate cancers. Moreover, calcitonin increases tumorigenicity and invasiveness of multiple prostate cancer cell lines by cyclic AMP-dependent protein kinase-mediated actions. These actions include increased secretion of matrix metalloproteinases and urokinase-type plasminogen activator and an increase in prostate cancer cell invasion. Activation of calcitonin-calcitonin receptor autocrine loop in prostate cancer cell lines led to the loss of cell-cell adhesion, destabilization of tight and adherens junctions, and internalization of key integral membrane proteins. In addition, the activation of calcitonin-calcitonin receptor axis induced epithelial-mesenchymal transition of prostate cancer cells as characterized by cadherin switch and the expression of the mesenchymal marker, vimentin. The activated calcitonin receptor phosphorylated glycogen synthase kinase-3, a key regulator of cytosolic β-catenin degradation within the WNT signaling pathway. This resulted in the accumulation of intracellular β-catenin, its translocation in the nucleus, and transactivation of β-catenin-responsive genes. These results for the first time identify actions of calcitonin-calcitonin receptor axis on prostate cancer cells that lead to the destabilization of cell-cell junctions, epithelial-to-mesenchymal transition, and activation of WNT/β-catenin signaling. The results also suggest that cyclic AMP-dependent protein kinase plays a key role in calcitonin receptor-induced destabilization of cell-cell junctions and activation of WNT-β-catenin signaling. PMID:19001380
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Samuel, Marcus A.; Mudgil, Yashwanti; Salt, Jennifer N.; Delmas, Frédéric; Ramachandran, Shaliny; Chilelli, Andrea; Goring, Daphne R.
2008-01-01
The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses. PMID:18552232
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Jansen, Felix; Yang, Xiaoyan; Hoyer, Friedrich Felix; Paul, Kathrin; Heiermann, Nadine; Becher, Marc Ulrich; Abu Hussein, Nebal; Kebschull, Moritz; Bedorf, Jörg; Franklin, Bernardo S; Latz, Eicke; Nickenig, Georg; Werner, Nikos
2012-08-01
Endothelial microparticles (EMP) are released from activated or apoptotic cells, but their effect on target cells and the exact way of incorporation are largely unknown. We sought to determine the uptake mechanism and the biological effect of EMP on endothelial and endothelial-regenerating cells. EMP were generated from starved endothelial cells and isolated by ultracentrifugation. Caspase 3 activity assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that EMP protect target endothelial cells against apoptosis in a dose-dependent manner. Proteomic analysis was performed to identify molecules contained in EMP, which might be involved in EMP uptake. Expression of annexin I in EMP was found and confirmed by Western blot, whereas the corresponding receptor phosphatidylserine receptor was present on endothelial target cells. Silencing either annexin I on EMP or phosphatidylserine receptor on target cells using small interfering RNA showed that the uptake of EMP by human coronary artery endothelial cells is annexin I/phosphatidylserine receptor dependent. Annexin I-downregulated EMP abrogated the EMP-mediated protection against apoptosis of endothelial target cells. p38 activation was found to mediate camptothecin-induced apoptosis. Finally, human coronary artery endothelial cells pretreated with EMP inhibited camptothecin-induced p38 activation. EMP are incorporated by endothelial cells in an annexin I/phosphatidylserine receptor-dependent manner and protect target cells against apoptosis. Inhibition of p38 activity is involved in EMP-mediated protection against apoptosis.
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Altered receptor trafficking in Huntingtin Interacting Protein 1-transformed cells.
Rao, Dinesh S; Bradley, Sarah V; Kumar, Priti D; Hyun, Teresa S; Saint-Dic, Djenann; Oravecz-Wilson, Katherine; Kleer, Celina G; Ross, Theodora S
2003-05-01
The clathrin-associated protein, Huntingtin Interacting Protein 1 (HIP1), is overexpressed in multiple human epithelial tumors. Here, we report that HIP1 is a novel oncoprotein that transforms cells. HIP1-transformed cells, in contrast to RasV12-transformed cells, have dysregulation of multiple receptors involved in clathrin trafficking. Examples include upregulation of the epidermal growth factor receptor (EGFR) and the transferrin receptor. Furthermore, accumulation of transferrin and EGF in the HIP1-transformed cells was increased, and breast tumors that had EGFR expressed also had HIP1 upregulated. Thus, HIP1 overexpression promotes tumor formation and is associated with a general alteration in receptor trafficking. HIP1 is the first endocytic protein to be directly implicated in tumor formation.
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Patrussi, Laura; Capitani, Nagaja; Martini, Veronica; Pizzi, Marco; Trimarco, Valentina; Frezzato, Federica; Marino, Filippo; Semenzato, Gianpietro; Trentin, Livio; Baldari, Cosima T
2015-10-01
Lymphocyte trafficking is orchestrated by chemokine and sphingosine 1-phosphate (S1P) receptors that enable homing and egress from secondary lymphoid organs (SLO). These receptors undergo rapid internalization and plasma membrane recycling to calibrate cellular responses to local chemoattractants. Circulating chronic lymphocytic leukemia (CLL) cells display an abnormal increase in the surface levels of the homing receptors CCR7 and CXCR4 concomitant with low S1P receptor 1 (S1P1) expression. In this study, we investigated the role of receptor recycling on CXCR4/CCR7 surface levels in CLL cells and addressed the impact of quantitative alterations of these receptors and S1P1 on the ability of leukemic cells to accumulate in SLOs. We show that recycling accounts, to a major extent, for the high levels of surface CXCR4/CCR7 on CLL cells. In addition, increased expression of these receptors, together with S1P1 deficiency, is detectable not only in circulating leukemic cells, but also in SLOs of CLL patients with lymphoadenopathy. We further provide evidence that ibrutinib, a Btk inhibitor that promotes mobilization of leukemic cells from SLOs, normalizes the imbalance between CXCR4/CCR7 and S1P1. Taken together, our results highlight the relevance of chemokine and S1P receptor recycling in CLL pathogenesis and clinical outcome. ©2015 American Association for Cancer Research.
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Development and Function of CD94-Deficient Natural Killer Cells
Orr, Mark T.; Wu, Jun; Fang, Min; Sigal, Luis J.; Spee, Pieter; Egebjerg, Thomas; Dissen, Erik; Fossum, Sigbjørn; Phillips, Joseph H.; Lanier, Lewis L.
2010-01-01
The CD94 transmembrane-anchored glycoprotein forms disulfide-bonded heterodimers with the NKG2A subunit to form an inhibitory receptor or with the NKG2C or NKG2E subunits to assemble a receptor complex with activating DAP12 signaling proteins. CD94 receptors expressed on human and mouse NK cells and T cells have been proposed to be important in NK cell tolerance to self, play an important role in NK cell development, and contribute to NK cell-mediated immunity to certain infections including human cytomegalovirus. We generated a gene-targeted CD94-deficient mouse to understand the role of CD94 receptors in NK cell biology. CD94-deficient NK cells develop normally and efficiently kill NK cell-susceptible targets. Lack of these CD94 receptors does not alter control of mouse cytomegalovirus, lymphocytic choriomeningitis virus, vaccinia virus, or Listeria monocytogenes. Thus, the expression of CD94 and its associated NKG2A, NKG2C, and NKG2E subunits is dispensable for NK cell development, education, and many NK cell functions. PMID:21151939
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Development and function of CD94-deficient natural killer cells.
Orr, Mark T; Wu, Jun; Fang, Min; Sigal, Luis J; Spee, Pieter; Egebjerg, Thomas; Dissen, Erik; Fossum, Sigbjørn; Phillips, Joseph H; Lanier, Lewis L
2010-12-03
The CD94 transmembrane-anchored glycoprotein forms disulfide-bonded heterodimers with the NKG2A subunit to form an inhibitory receptor or with the NKG2C or NKG2E subunits to assemble a receptor complex with activating DAP12 signaling proteins. CD94 receptors expressed on human and mouse NK cells and T cells have been proposed to be important in NK cell tolerance to self, play an important role in NK cell development, and contribute to NK cell-mediated immunity to certain infections including human cytomegalovirus. We generated a gene-targeted CD94-deficient mouse to understand the role of CD94 receptors in NK cell biology. CD94-deficient NK cells develop normally and efficiently kill NK cell-susceptible targets. Lack of these CD94 receptors does not alter control of mouse cytomegalovirus, lymphocytic choriomeningitis virus, vaccinia virus, or Listeria monocytogenes. Thus, the expression of CD94 and its associated NKG2A, NKG2C, and NKG2E subunits is dispensable for NK cell development, education, and many NK cell functions.
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Wegner, Florian; Kraft, Robert; Busse, Kathy; Härtig, Wolfgang; Ahrens, Jörg; Leffler, Andreas; Dengler, Reinhard; Schwarz, Johannes
2012-01-01
Human fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A) receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+)-K(+)-Cl(-) co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.
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Wegner, Florian; Kraft, Robert; Busse, Kathy; Härtig, Wolfgang; Ahrens, Jörg; Leffler, Andreas; Dengler, Reinhard; Schwarz, Johannes
2012-01-01
Background Human fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson’s disease. While glutamate and GABAA receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. Methodology/Principal Findings Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na+-K+-Cl− co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. Conclusions/Significance These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro. PMID:22606311
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Adoptive cell therapy: genetic modification to redirect effector cell specificity.
Morgan, Richard A; Dudley, Mark E; Rosenberg, Steven A
2010-01-01
Building on the principals that the adoptive transfer of T cells can lead to the regression of established tumors in humans, investigators are now further manipulating these cells using genetic engineering. Two decades of human gene transfer experiments have resulted in the translation of laboratory technology into robust clinical applications. The purpose of this review is to give the reader an introduction to the 2 major approaches being developed to redirect effector T-cell specificity. Primary human T cells can be engineered to express exogenous T-cell receptors or chimeric antigen receptors directed against multiple human tumor antigens. Initial clinical trial results have demonstrated that both T-cell receptor- and chimeric antigen receptor-engineered T cells can be administered to cancer patients and mediate tumor regression.
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Kouzel, Ivan U.; Pohlentz, Gottfried; Storck, Wiebke; Radamm, Lena; Hoffmann, Petra; Bielaszewska, Martina; Bauwens, Andreas; Cichon, Christoph; Schmidt, M. Alexander; Mormann, Michael; Karch, Helge; Müthing, Johannes
2013-01-01
Glycosphingolipids (GSLs) of the globo-series constitute specific receptors for Shiga toxins (Stxs) released by certain types of pathogenic Escherichia coli strains. Stx-loaded leukocytes may act as transporter cells in the blood and transfer the toxin to endothelial target cells. Therefore, we performed a thorough investigation on the expression of globo-series GSLs in serum-free cultivated Raji and Jurkat cells, representing B- and T-lymphocyte descendants, respectively, as well as THP-1 and HL-60 cells of the monocyte and granulocyte lineage, respectively. The presence of Stx-receptors in GSL preparations of Raji and THP-1 cells and the absence in Jurkat and HL-60 cells revealed high compliance of solid-phase immunodetection assays with the expression profiles of receptor-related glycosyltransferases, performed by qRT-PCR analysis, and Stx2-caused cellular damage. Canonical microdomain association of Stx GSL receptors, sphingomyelin, and cholesterol in membranes of Raji and THP-1 cells was assessed by comparative analysis of detergent-resistant membrane (DRM) and nonDRM fractions obtained by density gradient centrifugation and showed high correlation based on nonparametric statistical analysis. Our comprehensive study on the expression of Stx-receptors and their subcellular distribution provides the basis for exploring the functional role of lipid raft-associated Stx-receptors in cells of leukocyte origin. PMID:23248329
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Meisenberg, Annika; Kaschuba, Dagmar; Balfanz, Sabine; Jordan, Nadine; Baumann, Arnd
2015-10-01
Calcium ions (Ca(2+)) play a pivotal role in cellular physiology. Often Ca(2+)-dependent processes are studied in commonly available cell lines. To induce Ca(2+) signals on demand, cells may need to be equipped with additional proteins. A prominent group of membrane proteins evoking Ca(2+) signals are G-protein coupled receptors (GPCRs). These proteins register external signals such as photons, odorants, and neurotransmitters and convey ligand recognition into cellular responses, one of which is Ca(2+) signaling. To avoid receptor cross-talk or cross-activation with introduced proteins, the repertoire of cell-endogenous receptors must be known. Here we examined the presence of histamine receptors in six cell lines frequently used as hosts to study cellular signaling processes. In a concentration-dependent manner, histamine caused a rise in intracellular Ca(2+) in HeLa, HEK 293, and COS-1 cells. The concentration for half-maximal activation (EC50) was in the low micromolar range. In individual cells, transient Ca(2+) signals and Ca(2+) oscillations were uncovered. The results show that (i) HeLa, HEK 293, and COS-1 cells express sufficient amounts of endogenous receptors to study cellular Ca(2+) signaling processes directly and (ii) these cell lines are suitable for calibrating Ca(2+) biosensors in situ based on histamine receptor evoked responses. Copyright © 2015 Elsevier Inc. All rights reserved.
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β-Cell Hyperplasia Induced by Hepatic Insulin Resistance
Escribano, Oscar; Guillén, Carlos; Nevado, Carmen; Gómez-Hernández, Almudena; Kahn, C. Ronald; Benito, Manuel
2009-01-01
OBJECTIVE Type 2 diabetes results from a combination of insulin resistance and impaired insulin secretion. To directly address the effects of hepatic insulin resistance in adult animals, we developed an inducible liver-specific insulin receptor knockout mouse (iLIRKO). RESEARCH DESIGN AND METHODS Using this approach, we were able to induce variable insulin receptor (IR) deficiency in a tissue-specific manner (liver mosaicism). RESULTS iLIRKO mice presented progressive hepatic and extrahepatic insulin resistance without liver dysfunction. Initially, iLIRKO mice displayed hyperinsulinemia and increased β-cell mass, the extent of which was proportional to the deletion of hepatic IR. Our studies of iLIRKO suggest a cause-and-effect relationship between progressive insulin resistance and the fold increase of plasma insulin levels and β-cell mass. Ultimately, the β-cells failed to secrete sufficient insulin, leading to uncontrolled diabetes. We observed that hepatic IGF-1 expression was enhanced in iLIRKO mice, resulting in an increase of circulating IGF-1. Concurrently, the IR-A isoform was upregulated in hyperplastic β-cells of iLIRKO mice and IGF-1–induced proliferation was higher than in the controls. In mouse β-cell lines, IR-A, but not IR-B, conferred a proliferative capacity in response to insulin or IGF-1, providing a potential explanation for the β-cell hyperplasia induced by liver insulin resistance in iLIRKO mice. CONCLUSIONS Our studies of iLIRKO mice suggest a liver-pancreas endocrine axis in which IGF-1 functions as a liver-derived growth factor to promote compensatory pancreatic islet hyperplasia through IR-A. PMID:19136656
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Back, J.; Malchiodi, E; Cho, S
2009-01-01
Certain cell-surface receptors engage ligands expressed on juxtaposed cells and ligands on the same cell. The structural basis for trans versus cis binding is not known. Here, we showed that Ly49 natural killer (NK) cell receptors bound two MHC class I (MHC-I) molecules in trans when the two ligand-binding domains were backfolded onto the long stalk region. In contrast, dissociation of the ligand-binding domains from the stalk and their reorientation relative to the NK cell membrane allowed monovalent binding of MHC-I in cis. The distinct conformations (backfolded and extended) define the structural basis for cis-trans binding by Ly49 receptors andmore » explain the divergent functional consequences of cis versus trans interactions. Further analyses identified specific stalk segments that were not required for MHC-I binding in trans but were essential for inhibitory receptor function. These data identify multiple distinct roles of stalk regions for receptor function.« less
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Sustained neurotensin exposure promotes cell surface recruitment of NTS2 receptors
DOE Office of Scientific and Technical Information (OSTI.GOV)
Perron, Amelie; Sharif, Nadder; Gendron, Louis
2006-05-12
In this study, we investigated whether persistent agonist stimulation of NTS2 receptors gives rise to down-regulation, in light of reports that their activation induced long-lasting effects. To address this issue, we incubated COS-7 cells expressing the rat NTS2 with neurotensin (NT) for up to 24 h and measured resultant cell surface [{sup 125}I]-NT binding. We found that NTS2-expressing cells retained the same surface receptor density despite efficient internalization mechanisms. This preservation was neither due to NTS2 neosynthesis nor recycling since it was not blocked by cycloheximide or monensin. However, it appeared to involve translocation of spare receptors from internal stores,more » as NT induced NTS2 migration from trans-Golgi network to endosome-like structures. This stimulation-induced regulation of cell surface NTS2 receptors was even more striking in rat spinal cord neurons. Taken together, these results suggest that sustained NTS2 activation promotes recruitment of intracellular receptors to the cell surface, thereby preventing functional desensitization.« less