Receptor-bearing Cells Research Articles

Vertebral endplate trauma induces disc cell apoptosis and promotes organ degeneration in vitro

There is a major controversy whether spinal trauma with vertebral endplate fractures can result in post-traumatic disc degeneration. Intervertebral discs, which are adjacent to burst endplates, are frequently removed and an intercorporal spondylodesis is performed. In any case, the biological effects within the discs following endplate fractures are poorly elucidated to date. The aim of our investigations was therefore to establish a novel disc/endplate trauma culture model to reproducibly induce endplate fractures and investigate concurrent disc changes in vitro. This model is based on a full-organ disc/endplate culture system, which has been validated by the authors before. Intervertebral disc/endplate specimens were isolated from Burgundy rabbits and cultured in standard media (DMEM/F12, 10%FCS). Burst endplate fractures were induced in half of the specimens with a custom-made fracture device and subsequently cultured for 9 days. The biological effects such as necrotic or apoptotic cell death and the expression of pro-apoptotic genes and other genes involved in organ degeneration, e.g. matrix metalloproteinases (MMPs) were analyzed. Cell damage was assessed by quantification of the lactate dehydrogenase (LDH) activity in the supernatant. The expression of genes involved in the cellular apoptotic pathway (caspase 3) and the pro-apoptotic proteins FasL and TNF-alpha were monitored. The results demonstrate that LDH levels increased significantly post trauma compared to the control and remained elevated for 3 days. Furthermore, a constant up-regulation of the caspase 3 gene in both disc compartments was present. The pro-apoptotic proteins FasL and TNF-alpha were up regulated predominantly in the nucleus whereas the MMP-1 and -13 transcripts (collagenases) were increased in both disc structures. From this study we can conclude that endplate burst fractures result in both necrotic and apoptotic cell death in nucleus and annulus tissue. Moreover, FasL and TNF-alpha expression by nucleus cells may lead to continued apoptosis induced by Fas- and TNF-alpha receptor bearing cells. In addition TNF-alpha over-expression has potentially deleterious effects on disc metabolism such as over-expression of matrix proteinases. Taken together, the short term biological response of the disc following endplate fracture exhibits characteristics, which may initiate the degeneration of the organ.

Avian influenza receptor expression in H5N1‐infected and noninfected human tissues

Avian and human influenza viruses preferentially bind to alpha-2,3-linked and alpha-2,6-linked sialic acids, respectively. Until today, the distributions of these two receptor types had never been investigated in H5N1-infected human tissue samples. Here, the expression of avian (AIV-Rs) and human influenza receptors (HuIV-Rs) is studied in various organs (upper and lower respiratory tracts, brain, placenta, liver, kidney, heart, intestines, and spleen) of two H5N1 cases and 14 control cases. Histochemical stains using biotinylated Maackia amurensis lectin II and Sambucus nigra agglutinin were performed to localize AIV-Rs and HuIV-Rs, respectively. Immunohistochemical stainings were performed to identify the receptor-bearing cells. AIV-Rs were detected on type II pneumocytes; a limited number of epithelial cells of the upper respiratory tract; and the bronchi, bronchioli, and trachea; as well as on Kupffer cells, glomerular cells, splenic T cells, and neurons in the brain and intestines. HuIV-Rs were abundantly present in the respiratory tract and lungs. They were also detected on Hofbauer cells, glomerular cells, splenic B cells, and in the liver. Moreover, endothelial cells of all organs examined expressed both receptor types. In conclusion, the distribution pattern of AIV-Rs is partially inconsistent with the pattern of infected cells as detected in previous studies, which suggests there may be other receptors or mechanisms involved in H5N1 infection. In addition, the diffuse presence of receptors on endothelial cells may account for the multiple organ involvement in H5N1 influenza. Finally, the relative lack of AIV-Rs in the upper airway may be a one of the factors preventing efficient human-to-human transmission of H5N1 influenza.

The Murine Gammaherpesvirus-68 gp150 Acts as an Immunogenic Decoy to Limit Virion Neutralization

Herpesviruses maintain long-term infectivity without marked antigenic variation. They must therefore evade neutralization by other means. Immune sera block murine gammaherpesvirus-68 (MHV-68) infection of fibroblasts, but fail to block and even enhance its infection of IgG Fc receptor-bearing cells, suggesting that the antibody response to infection is actually poor at ablating virion infectivity completely. Here we analyzed this effect further by quantitating the glycoprotein-specific antibody response of MHV-68 carrier mice. Gp150 was much the commonest glycoprotein target and played a predominant role in driving Fc receptor-dependent infection: when gp150-specific antibodies were boosted, Fc receptor-dependent infection increased; and when gp150-specific antibodies were removed, Fc receptor-dependent infection was largely lost. Neither gp150-specific monoclonal antibodies nor gp150-specific polyclonal sera gave significant virion neutralization. Gp150 therefore acts as an immunogenic decoy, distorting the MHV-68-specific antibody response to promote Fc receptor-dependent infection and so compromise virion neutralization. This immune evasion mechanism may be common to many non-essential herpesvirus glycoproteins.

Anthrax Toxin: Receptor Binding, Internalization, Pore Formation, and Translocation

Anthrax toxin consists of three nontoxic proteins that self-assemble at the surface of receptor-bearing mammalian cells or in solution, yielding a series of toxic complexes. Two of the proteins, called Lethal Factor (LF) and Edema Factor (EF), are enzymes that act on cytosolic substrates. The third, termed Protective Antigen (PA), is a multifunctional protein that binds to receptors, orchestrates the assembly and internalization of the complexes, and delivers them to the endosome. There, the PA moiety forms a pore in the endosomal membrane and promotes translocation of LF and EF to the cytosol. Recent advances in understanding the entry process include insights into how PA recognizes its two known receptors and its ligands, LF and EF; how the PA:receptor interaction influences the pH-dependence of pore formation; and how the pore functions in promoting translocation of LF and EF across the endosomal membrane.

A novel, simple bioactivity assay for relaxin based on inhibition of platelet aggregation

In humans, the relaxin hormone family includes H1, H2 and H3 isoforms and insulin-like peptides 3 to 6. The ever-increasing interest in relaxin as potential new drug requires reliable methods to compare bioactivity of different relaxins. The existing bioassays include in vivo or ex vivo methods evaluating the organ-specific responses to relaxin and in vitro methods based on measurement of cAMP increase in relaxin receptor-bearing cells. We previously demonstrated that relaxin dose-dependently inhibits platelet aggregation. On this basis, we have developed a simple, reliable bioassay for relaxin used to compare purified porcine relaxin, assumed as reference standard, with two recombinant human H2 relaxins, H3 relaxin, insulin-like peptides 3 and 5. Pre-incubation of platelets with relaxins (3, 10, 30,100, 300 ng/ml; 10 min.) caused the inhibition of ADP-induced platelet aggregation. Within the 10–100 ng/ml range, porcine relaxin showed the highest effects and a nearly linear dose–response correlation. Lower peptide concentrations were ineffective, as were insulin-like peptides 3 and 5 at any concentration assayed. Platelet inhibition was mediated by specific RXFP1 relaxin receptor and cGMP, whose intracellular levels dose-dependently increased upon relaxin. For comparison, we stimulated THP-1 cells, a relaxin receptor-bearing cell line, with porcine relaxin, human H2 and H3 relaxins at the above concentrations (15 min.). We observed a dose-related increase of intracellular cAMP similar to the trend of platelet inhibition. Insulin like peptide 5 was ineffective. In conclusion, this study shows that inhibition of platelet aggregation may be used to assess bioactivity of relaxin preparations for experimental and clinical purposes.

Recombinant gp120 Vaccine-Induced Antibodies Inhibit Clinical Strains of HIV-1 in the Presence of Fc Receptor-Bearing Effector Cells and Correlate Inversely with HIV Infection Rate

Nonneutralizing Abs may play a role in protecting animals and humans from lentiviral infections. We explored the Ab-dependent, cell-mediated virus inhibition (ADCVI) Ab response to recombinant gp120 (rgp120) vaccination in sera from 530 participants in the Vax 004 trial. Serum ADCVI activity was measured against a clinical R5 strain of HIV-1 using peripheral blood mononuclear effector cells from healthy donors. The level of vaccine-induced ADCVI activity correlated inversely with the rate of acquiring HIV infection following vaccination, such that for every 10% increase in ADCVI activity, there was a 6.3% decrease in the hazard rate of infection (p=0.019). Some vaccinated individuals also mounted an ADCVI response against two other clinical R5 strains of HIV-1. However, ADCVI activity correlated poorly with neutralizing or CD4-gp120-blocking Ab activity measured against laboratory strains. Finally, the degree to which the ADCVI Ab response predicted the rate of infection was influenced by polymorphisms at the FcgammaRIIa and FcgammaRIIIa gene loci. These data indicate that rgp120 vaccination can elicit Abs with antiviral activity against clinical strains of HIV-1. However, such activity requires the presence of FcR-bearing effector cells. Our results provide further evidence that ADCVI may play a role in preventing HIV infection.

Inhibition of CXCL16 Attenuates Inflammatory and Progressive Phases of Anti-Glomerular Basement Membrane Antibody-Associated Glomerulonephritis

Chemokines recruit and activate leukocytes during inflammation. CXCL16 is a recently discovered chemokine that is expressed as a transmembrane protein that is cleaved to form the active, soluble chemokine. We analyzed the role of CXCL16 in the development of inflammation and in the progression of the anti-glomerular basement membrane (GBM) antibody-induced experimental glomerulonephritis in Wistar-Kyoto rats. CXCL16 was expressed in glomerular endothelial cells and mediated adhesion of macrophages expressing CXCL16 and its cognate receptor, CXCR6. Glomerular infiltrates displayed a strong migratory response to soluble CXCL16. Soluble CXCL16 and its receptor CXCR6 were induced in nephritic glomeruli throughout the disease, and CXCL16 expression correlated with the up-regulation of ADAM10, suggesting that this disintegrin and metalloproteinase mediates the chemokine activity of CXCL16. Blocking CXCL16 in the acute inflammatory phase or progressive phase of established glomerulonephritis significantly attenuated monocyte/macrophage infiltration and glomerular injury; proteinuria also improved. We conclude that CXCL16/CXCR6 plays a critical role in stimulating leukocyte influx, which causes glomerular damage during anti-GBM glomerulonephritis. Blocking CXCL16 actions limits the progression of anti-GBM glomerulonephritis even when the disease is established.

Selection of Anthrax Toxin Protective Antigen Variants That Discriminate between the Cellular Receptors TEM8 and CMG2 and Achieve Targeting of Tumor Cells

Anthrax toxin, a three-component protein toxin secreted by Bacillus anthracis, assembles into toxic complexes at the surface of receptor-bearing eukaryotic cells. The protective antigen (PA) protein binds to receptors, either tumor endothelial cell marker 8 (TEM8) or CMG2 (capillary morphogenesis protein 2), and orchestrates the delivery of the lethal and edema factors into the cytosol. TEM8 is reported to be overexpressed during tumor angiogenesis, whereas CMG2 is more widely expressed in normal tissues. To extend prior work on targeting of tumor with modified anthrax toxins, we used phage display to select PA variants that preferentially bind to TEM8 as compared with CMG2. Substitutions were randomly introduced into residues 605-729 of PA, within the C-terminal domain 4 of PA, which is the principal region that contacts receptor. Candidates were characterized in cellular cytotoxicity assays with Chinese hamster ovary (CHO) cells expressing either TEM8 or CMG2. A PA mutant having the substitutions R659S and M662R had enhanced specificity toward TEM8-overexpressing CHO cells. This PA variant also displayed broad and potent tumoricidal activity to various human tumor cells, especially to HeLa and A549/ATCC cells. By contrast, the substitution N657Q significantly reduced toxicity to TEM8 but not CMG2-overexpressing CHO cells. Our results indicate that certain amino acid substitutions within PA domain 4 create anthrax toxins that selectively kill human tumor cells. The PA R659S/M662R protein may be useful as a therapeutic agent for cancer treatment.

Hypoallergens for Allergen-specific Immunotherapy by Directed Molecular Evolution of Mite Group 2 Allergens

Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wild-type allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.

Consumption of C.E.R.A. and Epoetin Beta in a Cellular Assay: UT-7 Consumption Model.

Continuous Erythropoietin Receptor Activator (C.E.R.A.), an innovative erythropoietic agent with unique receptor activity, is currently in development to provide correction of anemia and stable maintenance of hemoglobin (Hb) levels at extended administration intervals up to once monthly in patients with all stages of chronic kidney disease, and is also in development for the treatment of chemotherapy-induced anemia. C.E.R.A. has an equivalent half-life in humans of approximately 130 h following subcutaneous or intravenous administration. Clearance of erythropoietic agents may occur by binding, internalization and degradation in receptor bearing cells in the bone marrow. A functional assay was developed to evaluate the consumption of C.E.R.A. and epoetin beta in a UT-7 cellular system (a human acute myeloid leukemia cell line whose growth is dependent on the presence erythropoietic and other growth factors). UT-7 cells were incubated in the presence of C.E.R.A. (1000 pM) or epoetin beta (100 pM) for 72, 96 or 120 h. The cell supernatants were then harvested and the amounts of remaining growth factor were determined by a validated erythropoietin ELISA. As controls, the growth factors were incubated for the same times in medium only. Five independent identical experiments were conducted for each compound. Mean percentages of C.E.R.A. and epoetin beta were calculated relative to the control values (set at 100%). C.E.R.A. 1000 pM and epoetin beta 100 pM were shown to be equipotent, stimulating the proliferation of UT-7 cells to the same degree. There was a highly significant difference (P=1.9×10−9) between C.E.R.A. and epoetin beta for the time course of concentrations relative to controls. For epoetin beta 100 pM, the relative concentration decreased significantly over time, while for C.E.R.A. 1000 pM no statistically significant changes were observed. Thus, C.E.R.A. was still present in the culture supernatant at close to 100% after 4–5 days' incubation, while the epoetin beta concentration had declined to approximately 20%. In conclusion, at the end of the incubation time the C.E.R.A. concentration was still sufficient to maintain the initial level of cell stimulation. However, at the end of the 96 h incubation the epoetin beta concentration dropped to a level that was insufficient to produce further stimulation. The UT-7 consumption model thus emulates the in vivo situation in humans with regard to receptor mediated elimination of the compounds and stimulation of erythroid progenitors.This study provides further evidence that C.E.R.A. interacts differently with the erythropoietin receptor relative to epoetin beta. Preclinical studies have shown that these properties translate into more continuous stimulation of erythropoiesis in vivo compared with epoetin beta and Phase III data indicate that C.E.R.A. allows predictable Hb maintenance in direct conversion from more frequently dosed agents.

Targeting and Penetration of Virus Receptor Bearing Cells by Nanoparticles Coated with Envelope Proteins of Moloney Murine Leukemia Virus

One of the most important steps in a productive viral infection is when the virus fuses to a cell membrane and delivers its genome into the cell cytosol. This dynamic event is mediated by interactions between specific virus envelope proteins with their cell-bound receptors. This process is exemplified by Moloney murine leukemia virus (Mo-MLV) where envelope protein interaction with its receptor, mCAT-1, leads to virus-cell membrane fusion and infection of cells. Here, fluorescent nanoparticles (NPs) were coated with Mo-MLV derived membranes (Mo-NPs) by extrusion. Electron microscopy and biochemical analysis showed tight association of the virus membranes and NPs. The coated NPs mimic native virus by binding and entering only cells expressing the virus receptor. Confocal microscopy revealed that the coated NPs were taken up into endocytic compartments containing receptor and were also seen associated with caveolin, a marker of caveolae. To demonstrate that the Mo-NPs could escape endosomes and deliver a protein cargo into the cell cytosol, beta-lactamase (betalac) was covalently coupled to the Mo-NP cores and incubated with cells. betalac activity was only detected in the cytosol of mCAT-1-expressing cells. This is the first time that virus proteins have been used to specifically target NPs to receptor-bearing cells as well as penetration into the cell cytosol. Extrusion provides a rapid, detergent-free method to couple virus membranes to NPs and should be readily applicable for many other virus and NP types.

Synthesis and characterization of chitosan-g-poly(ethylene glycol)-folate as a non-viral carrier for tumor-targeted gene delivery

Poor water solubility and low transfection efficiency of chitosan are major drawbacks for its use as a gene delivery carrier. PEGylation can increase its solubility, and folate conjugation may improve gene transfection efficiency due to promoted uptake of folate receptor-bearing tumor cells. The aim of this study was to synthesize and characterize folate-poly(ethylene glycol)-grafted chitosan (FA-PEG-Chi) for targeted plasmid DNA delivery to tumor cells. Gel electrophoresis study showed strong DNA binding ability of modified chitosan. The pH(50) values, defined as the pH when the transmittance of a polymer solution at 600 nm has reached 50% of the original value, suggested that the water solubility of PEGylated chitosan had improved significantly. Regression analysis of pH(50) value as a function of substitution degree of PEG yielded an almost linear correlation for PEG-Chi and FA-PEG-Chi. The solubility of PEGylated chitosan decreased slightly by further conjugation of folic acid due to the relatively more hydrophobic nature of folic acid when compared to PEG. In addition, the chitosan-based DNA complexes did not induce remarkable cytotoxicity against HEK 293 cells. FA-PEG-Chi can be a promising gene carrier due to its solubility in physiological pH, efficiency in condensing DNA, low cytotoxicity and targeting ability.

Fluorescent Indicators Distributed throughout the Pharmacophore of Cholecystokinin Provide Insights into Distinct Modes of Binding and Activation of Type A and B Cholecystokinin Receptors

Ligand probes with fluorescent indicators positioned throughout the pharmacophoric domain can provide important insights into the molecular basis of receptor binding and activation as reflected in the microenvironment of each indicator while docked at a receptor. We developed three cholecystokinin-like probes with Aladan situated at the N terminus, in the mid-region, and at the C terminus (positions 24, 29, and 33, respectively). These were studied in solution and docked at type A and B cholecystokinin receptors. This study demonstrated clear differences in mechanisms of cholecystokinin binding and activation of these structurally related receptors with distinct agonist structure-activity relationships. The fluorescence characteristics of Aladan are highly sensitive to the polarity of its microenvironment. The mid-region probe was least accessible to the aqueous milieu as determined by fluorescence emission spectra and iodide quenching, which was not altered by changes in conformation from active to inactive. Accessibility of the N- and C-terminal probes was affected by receptor conformation. The position 24 probe was more easily quenched in the active than in the G protein-uncoupled conformation for both receptors. However, the position 33 probe docked at the type A cholecystokinin receptor was more easily quenched in the active conformation, whereas the same probe docked at the type B cholecystokinin receptor was more easily quenched in the inactive conformation. Fluorescence anisotropy and red edge excitation shift determinations confirmed these observations and supported the proposed movements. Although both type A and B cholecystokinin receptors bind cholecystokinin with high affinity, resulting in fully efficacious biological responses, these receptors utilize distinct molecular modes of binding.

Rhesus Macaque Polyclonal and Monoclonal Antibodies Inhibit Simian Immunodeficiency Virus in the Presence of Human or Autologous Rhesus Effector Cells

Although antibodies can prevent or modulate lentivirus infections in nonhuman primates, the biological functions of antibody responsible for such effects are not known. We sought to determine the role of antibody-dependent cell-mediated virus inhibition (ADCVI), an antibody function that inhibits virus yield from infected cells in the presence of Fc receptor-bearing effector cells, in preventing or controlling SIVmac251 infection in rhesus macaques (Macaca mulatta). Using CEMx174 cells infected with simian immunodeficiency virus mac251 (SIVmac251), both polyclonal and monoclonal anti-SIV antibodies were capable of potent virus inhibition in the presence of human peripheral blood mononuclear cell (PBMC) effector cells. In the absence of effector cells, virus inhibition was generally very poor. PBMCs from healthy rhesus macaques were also capable of mediating virus inhibition either against SIVmac251-infected CEMx174 cells or against infected, autologous rhesus target cells. We identified both CD14(+) cells and, to a lesser extent, CD8(+) cells as the effector cell population in the rhesus PBMCs. Finally, pooled, nonneutralizing SIV-antibody-positive serum, shown in a previous study to prevent infection of neonatal macaques after oral SIVmac251 challenge, had potent virus-inhibitory activity in the presence of effector cells; intact immunoglobulin G, rather than F(ab')(2), was required for such activity. This is the first demonstration of both humoral and cellular ADCVI functions in the macaque-SIV model. ADCVI activity in nonneutralizing serum that prevents SIV infection suggests that ADCVI may be a protective immune function. Finally, our data underscore the potential importance of Fc-Fc receptor interactions in mediating biological activities of antibody.

Stable Association of Herpes Simplex Virus with Target Membranes Is Triggered by Low pH in the Presence of the gD Receptor, HVEM

Using a liposome-binding assay, we investigated the requirements for activation of herpes simplex virus (HSV) into a state capable of membrane interaction. Virions were mixed with liposomes along with the ectodomain of one of three gD receptors (HVEMt, nectin-1t, or nectin-2t) and incubated under different pH and temperature conditions. Virions failed to associate with liposomes in the presence of nectin-1 or nectin-2 at any temperature or pH tested. In contrast, HVEMt triggered association of HSV with liposomes at pH 5.3 or 5.0 when incubated at 37 degrees C, suggesting that HVEM binding and mildly acidic pH at a physiological temperature provide coactivation signals, allowing virus association with membranes. Virions incubated with HVEMt at 37 degrees C without liposomes rapidly lost infectivity upon exposure to pH 5.0, suggesting that these conditions lead to irreversible virus inactivation in the absence of target membranes. Consistent with the idea that soluble receptor molecules provide a trigger for HSV entry, HVEMt promoted virus entry into receptor-deficient CHO K1 cells. However, in B78H1 cells, HVEMt promoted virus entry with markedly lower efficiency. Interestingly, HSV entry into receptor-bearing CHO K1 cells has been shown to proceed via a pH-dependent manner, whereas HSV entry into receptor-bearing B78H1 cells is pH independent. Based on these observations, we propose that the changes triggered by HVEM and mildly acidic pH that allow liposome association are similar or identical to changes that occur during pH-dependent HSV entry.

Possible Endogenous Agonist Mechanism for the Activation of Secretin Family G Protein-Coupled Receptors

The class B family of G protein-coupled receptors contains several potentially important drug targets, yet our understanding of the molecular basis of ligand binding and receptor activation remains incomplete. Although a key role is recognized for the cysteine-rich, disulfide-bonded amino-terminal domain of these receptors, detailed insights into ligand docking and resultant conformational changes are not clear. We postulate that binding natural ligands to this domain results in a conformational change that exposes an endogenous ligand which interacts with the body of the receptor to activate it. In this work, we examined whether a synthetic peptide corresponding to a candidate region between the first and third conserved cysteines could act as an agonist. Indeed, this peptide was a weakly potent but fully efficacious agonist, stimulating a concentration-dependent cAMP response in secretin receptor-bearing cells. This effect was maintained as the peptide length was reduced from 30 to 5, and ultimately, three residues focused on the conserved residue Asp49. The agonist potency was enhanced by cyclization through a diaminopropionic acid linker and by amino-terminal fatty acid acylation. Both ends of the cyclic peptide were shown to interact with the top of transmembrane segment 6 of the receptor, using probes with a photolabile benzoyl-phenylalanine on each end. Analogous observations were also made for two other members of this family, the vasoactive intestinal polypeptide type 1 and calcitonin receptors. These data may provide a unique molecular mechanism and novel leads for the development of small-molecule agonists acting at potential drug targets within this physiologically important receptor family.

Use of Probes with Fluorescence Indicator Distributed throughout the Pharmacophore to Examine the Peptide Agonist-binding Environment of the Family B G Protein-coupled Secretin Receptor

Fluorescence techniques can provide insight into the environment of fluorescence indicators situated at distinct sites within a ligand as it is bound to its receptor. Here, we have developed a series of analogues of the 27-amino acid hormone, secretin, that incorporate a fluorescent Alexa Fluor 488 into the amino terminus, the carboxyl terminus, and positions 13 and 22. Each probe bound with high affinity and was biologically active, stimulating full cAMP responses in receptor-bearing Chinese hamster ovary-SecR cells. Treatment with 10 mum guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp) shifted the agonist-bound receptor into a G protein-uncoupled low affinity state. Fluorescence spectra for the probes in solution and bound to the receptor demonstrated maximal emission at 521 nm after excitation at 481 nm. Collisional quenching of fluorescence with potassium iodide revealed that Alexa at the amino terminus of secretin was more accessible than at the other three positions within the probes. Of note, quenching constants for each probe were higher when bound in the active state than in the G protein-uncoupled, low affinity state of the receptor, with the most marked changes occurring for the two midregion probes. Anisotropy values and fluorescence lifetimes confirmed this, with higher anisotropy and longer lifetimes observed for position 13 and 22 probes bound to the receptor in its uncoupled state than in its active state. These observations suggest that the amino terminus of secretin as docked to the receptor is most exposed to the hydrophilic aqueous milieu, and that the major changes in conformation and exposure to the medium occur in the midregion of secretin. Photoaffinity labeling studies have demonstrated approximation of each of these ligand residues with distinct receptor residues. Combining the fluorescence data with photoaffinity labeling data provides insights into the conformation and dynamics of a natural peptide ligand docked to a Family B G protein-coupled receptor.

SARS Coronavirus, but Not Human Coronavirus NL63, Utilizes Cathepsin L to Infect ACE2-expressing Cells

Viruses require specific cellular receptors to infect their target cells. Angiotensin-converting enzyme 2 (ACE2) is a cellular receptor for two divergent coronaviruses, SARS coronavirus (SARS-CoV) and human coronavirus NL63 (HCoV-NL63). In addition to hostcell receptors, lysosomal cysteine proteases are required for productive infection by some viruses. Here we show that SARS-CoV, but not HCoV-NL63, utilizes the enzymatic activity of the cysteine protease cathepsin L to infect ACE2-expressing cells. Inhibitors of cathepsin L blocked infection by SARS-CoV and by a retrovirus pseudotyped with the SARS-CoV spike (S) protein but not infection by HCoV-NL63 or a retrovirus pseudotyped with the HCoV-NL63 S protein. Expression of exogenous cathepsin L substantially enhanced infection mediated by the SARS-CoV S protein and by filovirus GP proteins but not by the HCoV-NL63 S protein or the vesicular stomatitis virus G protein. Finally, an inhibitor of endosomal acidification had substantially less effect on infection mediated by the HCoV-NL63 S protein than on that mediated by the SARS-CoV S protein. Our data indicate that two coronaviruses that utilize a common receptor nonetheless enter cells through distinct mechanisms.

IVIG Down-Regulates LPS-Induced TNFα Production Via Inhibition of IκBα Activation: A Possible Mechanism of Action in the Treatment of Bacterial Infections.

Intravenous IgG (IVIG) preparations, originally developed as a substitution therapy for patients with low plasma IgG, are used in the treatment of various immune and nonimmune disorders including hemolytic uremic syndrome, necrotizing fasciitis, and idiopathic thrombocytopenic purpura (ITP); an autoimmune pro-inflammatory condition. However, the pleotropic effects of IVIG on Fcγ receptor-bearing immune cells are not fully understood. Bacterial lipopolyssacharide (LPS) is a principal pro-inflammatory signaling molecule recognized by the innate immune system. LPS binds to monocyte/macrophage Toll-like receptor 4 (TLR4) to activate transcription factors such as NF-κB/IκBα. In turn, NF-κB activation results in cytokine secretion such as tumor necrosis factor alpha (TNFα); a key pro-inflammatory cytokine that leads to monocyte apoptosis. Since IVIG reduces pro-inflammatory responses, we hypothesized that IVIG may down-regulate the LPS-induced pro-inflammatory response in monocytes. Using the human monocyte-like cell line THP-1 that expresses FcγRs and TLR4, we examined LPS-induced intracellular signaling after in vitro treatment with IVIG. Enzyme-linked immunosorbent assay (ELISA) on culture supernatants showed that 30 minute pre-treatment of THP-1 cells with IVIG (5 mg/mL final) downregulated subsequent LPS (200 ng/mL)-induced secretion of TNFα. Western blotting showed that IVIG increased Akt phosphorylation and resulted in phosphorylation of S6 ribosomal protein and Erk1/2 signaling molecules. The activation of S6 ribosomal protein was blocked by the PI3K inhibitor LY294002. More importantly, IVIG treatment inhibited LPS-induced IκBα activation; the likely reason for the decrease in LPS-induced TNFα secretion. In conclusion, IVIG down-regulates LPS-induced signal transduction and proinflammatory cytokine secretion, an action that is likely responsible for its usefulness in the treatment of bacterial infections and in other diseases where down-regulation of NF-κB is desirable.

Humoral Immune Response and Immunoglobulin G Fc Receptor Genotype Are Associated with Better Clinical Outcome Following Idiotype Vaccination in Follicular Lymphoma Patients Regardless of Their Response to Induction Chemotherapy.

Recently, we found that anti-idiotype humoral immune response and FcγRIIIa 158 Valine/Valine (V/V) correlated with clinical outcome in follicular lymphoma patients receiving idiotype vaccination (JCO 22:4717). This result highlighted the importance of developing anti-tumor antibodies and hosting an efficient ADCC machinery. In the previous study, all 136 patients received induction chemotherapy and were in clinical remission at the time of idiotype vaccination. One important question is whether the outcome of these patients was correlated with their clinical response to induction chemotherapy. We, therefore, analyzed the impact of chemotherapy response on clinical outcome to determine if the better outcome associated with humoral immune response and V/V genotype applies to patients with different chemotherapy response. Induction cheomtherapies included chlorambucil, CVP, an adriamycin-containing regimen, or alternating CVP and fludarabine. Ninety patients (66%) achieved complete response or unconfirmed complete response (CR/CRu) and 46 patient (34%) achieved partial response (PR). The progression free survival at 4 years was 59% for CR/CRu patients and 41% for PR patients. The median time to progression (TTP) of 5.21 for CR/CRu patients was significantly longer than the 1.62 years for PR patients (p=0.004). Multi-variant analysis using the Cox Proportional Hazards Model showed that humoral IR, V/V genotype and CR/CRu were three independent positive predictors for progression free survival, while FcγRIIa genotype, FcγRIIb genotype, cellular IR, gender, age, B symptoms, time from diagnosis to therapy, use of adriamycin or use of fludarabine during induction chemotherapy had no impact. The relative benefit was 2.26 (95% CI, 1.36–3.76, p=0.0016) for humoral IR, 5.03 (95% CI, 2.06–12.25, p=0.0004) for V/V genotype and 2.01 (95% CI, 1.22–3.29, p=0.0057) for CR/CRu. The impact of humoral immune response and V/V genotype on CR/CRu or PR patients were then examined. In CR/CRu patients, the progression free survival at 4 years was 74% for patients with humoral immune response and/or V/V genotype while only 45% for patients without either, with median TTP not reached and of 3.46 years for the two groups, respectively (p=0.007). In PR patients, the benefit of having humoral immune response and/or V/V genotype was even more pronounced. The progression free survival at 4 years was 63% for patients with humoral immune response and/or V/V genotype and 21% for patients without either, with median TTP not reached after 8 years and of 1.31 years for the two groups, respectively (p=0.0004). This result suggests that having humoral immune response and/or V/V genotype was associated with longer TTP regardless of their response to induction chemotherapy. In fact, there is no difference in the TTP between CR/CRu and PR patients if they had humoral immune response and/or V/V genotype (TTP not reached in both groups, p=0.338). In contrast, in the group with neither humoral immune response nor V/V genotype, CR/CRu patients enjoyed much longer TTP than PR patients (TTP 3.46 year vs 1.31 years, p=0.0005). This result further confirms the strong association of clinical outcome with humoral immune response and with the functional activity of Fc receptor bearing cells in patients receiving idiotype vaccination. Our results also argue for the inclusion of PR as well as CR patients in idiotype vaccine trials, since the apparent benefit of immune response is even greater in PR patients.